Bispecific antigen binding molecules specifically binding to VEGF and ANG-2

Abstract

The invention relates to a bispecific antigen-binding molecule, which comprises a first antigen-binding domain that specifically binds to ANG-2 and a second antigen-binding domain that specifically binds to VEGF. The invention also relates to a monoclonal antibody that specifically binds to ANG-2, and the preparation and application of the antibody.

Description

Technical Field

[0001] The present disclosure belongs to the field of biopharmaceuticals, and specifically relates to the preparation and use of anti-ANG-2 antibodies and bispecific antigen-binding molecules of anti-ANG-2 and anti-VEGF. Background Art

[0002] The statements herein merely provide background information related to the present disclosure and do not necessarily constitute prior art.

[0003] The generation of new blood vessels provides tumor cells with oxygen and nutrients, allowing them to gain a growth advantage, transitioning from a slow, avascular growth phase to a rapid, vascularized growth phase. Therefore, inhibiting tumor growth by inhibiting angiogenesis is a promising and effective strategy. Among the many factors that promote angiogenesis, vascular endothelial growth factor (VEGF) is a crucial and important factor promoting angiogenesis. VEGF can promote angiogenesis in tumor cells by binding to VEGF receptors, promoting cell proliferation and migration, and increasing vascular permeability. Therefore, blocking VEGF can inhibit tumor angiogenesis, thereby inhibiting tumor growth and metastasis. Clinically, many biological agents that block VEGF through various strategies, such as Avastin, a monoclonal antibody targeting VEGF, soluble VEGF receptors that neutralize VEGF, and monoclonal antibodies targeting VEGF receptors, have demonstrated promising activity.

[0004] However, the generation of tumor blood vessels is a complex process involving numerous molecules and multiple signaling pathways. Blocking one pathway cannot completely inhibit the tumor, and other angiogenesis-related factors need to be blocked at the same time.

[0005] Tie2 is the second endothelial cell-specific tyrosine kinase receptor identified. Its binding to its ligands angiopoietin-1 (ANG1) and angiopoietin-2 (ANG2) also plays a crucial role in angiogenesis. Both ANG1 and ANG2 bind to Tie2. ANG1 supports endothelial cell (EC) survival and promotes vascular integrity and stability, while ANG2 has the opposite effect, causing peripheral cells to detach from the endothelium, leading to increased endothelial cell permeability and enabling VEGF to exert its angiogenesis-promoting effects. ANG2 and VEGF complement and coordinate their actions during tumor angiogenesis. Therefore, simultaneous blockade of VEGF and ANG2 can more effectively inhibit angiogenesis, promote vascular normalization, and ultimately suppress tumor growth and metastasis.

[0006] Currently, patent applications WO1998045332, WO2007095338A2, WO201004058, CN102250247A, WO2011117329, etc. disclose bispecific antibodies against ANG-2 and VEGF or VEGF antibodies. However, new and highly effective bispecific antibodies against ANG-2 and VEGF are still to be developed. Summary of the Invention

[0007] This disclosure provides a bispecific antibody targeting ANG-2 and VEGF. This bispecific antibody exhibits enhanced in vitro blocking activity against the binding of ANG-2 to its receptor Tie2, significantly inhibits VEGF-induced increases in phosphorylated VEGFR levels in HUVEC cells, and suppresses VEGF-induced HUVEC cell proliferation. Furthermore, this bispecific antibody exhibits superior tumor growth inhibition in mice and can be used to treat ocular diseases, such as AMD.

[0008] In some embodiments, 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 first antigen-binding domain that specifically binds ANG-2 comprises:

[0009] i) 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

[0010] 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:

[0011] The sequence of SEQ ID NO: 38 is: TINX1X2SSYTYYPDNVKG;

[0012] The sequence of SEQ ID NO: 39 is: X3X4ATGX5FDY

[0013] Among them, X1 is D or E, X2 is D or N, X3 is D or N, X4 is E or Q, and X5 is C, S or V;

[0014] or

[0015] ii) 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

[0016] The light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 15, SEQ ID NO: 11 and SEQ ID NO: 16, respectively.

[0017] In some embodiments, the aforementioned bispecific antigen-binding molecule, wherein the first antigen-binding domain that specifically binds to ANG-2 comprises:

[0018] a heavy chain variable region comprising a HCDR1 as set forth in SEQ ID NO: 7, a HCDR2 as set forth in SEQ ID NO: 8, 22 or 24, and a HCDR3 as set forth in SEQ ID NO: 9, 23, 25, 26 or 27; and

[0019] The light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively.

[0020] In some embodiments, the aforementioned bispecific antigen-binding molecule, wherein the first antigen-binding domain that specifically binds to ANG-2 comprises:

[0021] a) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 7, SEQ ID NO: 22 and SEQ ID NO: 23, respectively; and

[0022] 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;

[0023] b) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 7, SEQ ID NO: 24, and SEQ ID NO: 23, respectively; and

[0024] 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;

[0025] c) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 25, respectively; and

[0026] 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;

[0027] d) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 26, respectively; and

[0028] 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;

[0029] e) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 7, SEQ ID NO: 22, and SEQ ID NO: 25, respectively; and

[0030] 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;

[0031] f) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 7, SEQ ID NO: 22, and SEQ ID NO: 26, respectively; and

[0032] 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;

[0033] g) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 7, SEQ ID NO: 24, and SEQ ID NO: 25, respectively; and

[0034] 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;

[0035] h) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 7, SEQ ID NO: 24, and SEQ ID NO: 26, respectively; and

[0036] 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;

[0037] j) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 23, respectively; and

[0038] 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; or

[0039] k) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 7, SEQ ID NO: 22, and SEQ ID NO: 27, respectively; and

[0040] 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; or

[0041] 1) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively; and

[0042] The light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively.

[0043] In some embodiments, the aforementioned bispecific antigen binding molecules are present in an amount equal to or less than 10 -7 M binds to human ANG-2 with a dissociation equilibrium constant, in some embodiments, equal to or less than 10 -8 M or 10 -9 M dissociation equilibrium constant for binding to human ANG-2.

[0044] In some embodiments, the aforementioned bispecific antigen binding molecules are present in an amount equal to or less than 10 -7 M binds to human VEGF with a dissociation equilibrium constant, in some embodiments, equal to or less than 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M or 10-13 M dissociation equilibrium constant for binding to human VEGF.

[0045] In some embodiments, the aforementioned bispecific antigen binding molecules cross-bind to monkey ANG-2 and VEGF.

[0046] In some embodiments, the aforementioned bispecific antigen-binding molecules 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, less than 5 nM, less than 3 nM, less than 1 nM, or less than 0.5 nM, wherein the blocking activity is detected by the ELISA assay described in Test Example 2.

[0047] In some embodiments, the aforementioned bispecific antigen binding molecules inhibit ANG-2-induced Tie2 phosphorylation in Tie2-transfected CHO cells with an IC50 of less than 21.27 nM, less than 15 nM, less than 10 nM, less than 8 nM, or less than 5 nM.

[0048] In some embodiments, the aforementioned bispecific antigen binding molecules can significantly inhibit the increase in the level of intracellular phosphorylated VEGFR in HUVECs caused by VEGF.

[0049] In some embodiments, 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.

[0050] In some embodiments, the aforementioned bispecific antigen-binding molecule, wherein the first antigen-binding domain that specifically binds to ANG-2 comprises:

[0051] i) a heavy chain variable region comprising the amino acid sequence of any one of SEQ ID NOs: 3, 20, 21, 28-37, 72, or 73, or an amino acid sequence that has at least 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 3, 20, 21, 28-37, 72, or 73, respectively; and / or

[0052] a light chain variable region comprising the amino acid sequence of SEQ ID NO: 4, 17, 18, 19, or 74, or an amino acid sequence that has at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 4, 17, 18, 19, or 74, respectively; or

[0053] ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 5, 44, 45, 46 or 75, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 5, 44, 45, 46 or 75, respectively; and / or

[0054] A light chain variable region comprising the amino acid sequence of SEQ ID NO: 6, 40, 41, 42, 43 or 76, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 6, 40, 41, 42, 43 or 76, respectively.

[0055] In some embodiments, the aforementioned bispecific antigen-binding molecule, wherein the first antigen-binding domain that specifically binds to ANG-2 comprises:

[0056] i) a heavy chain variable region having an amino acid sequence as shown in any one of SEQ ID NOs: 3, 20, 21, 28-37, 72 or 73, or having at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NOs: 3, 20, 21, 28-37, 72 or 73, respectively; and / or

[0057] a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 4, 17, 18, 19, or 74, or having at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 4, 17, 18, 19, or 74, respectively; or

[0058] ii) a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 5, 44, 45, 46 or 75, or having at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 5, 44, 45, 46 or 75, respectively; and / or

[0059] A light chain variable region having an amino acid sequence as shown in SEQ ID NO: 6, 40, 41, 42, 43 or 76, or having a sequence identity of at least 95%, 96%, 97%, 98% or 99% to SEQ ID NO: 6, 40, 41, 42, 43 or 76, respectively.

[0060] In some embodiments, the aforementioned bispecific antigen-binding molecule, wherein the first antigen-binding domain that specifically binds to ANG-2 comprises:

[0061] a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 4;

[0062] b) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 20 or 21; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 17, 18 or 19;

[0063] c) a heavy chain variable region comprising the amino acid sequence of any one of SEQ ID NOs: 28-37; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 17;

[0064] d) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 72 or 73; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 74;

[0065] e) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 5; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 6;

[0066] f) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 44, 45, or 46; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 40, 41, 42, or 43; or

[0067] g) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 75; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 76. In some embodiments, the aforementioned bispecific antigen-binding molecule, wherein the first antigen-binding domain that specifically binds to ANG-2 comprises:

[0068] a) the heavy chain variable region set forth in SEQ ID NO: 3; and the light chain variable region set forth in SEQ ID NO: 4;

[0069] b) a heavy chain variable region as shown in SEQ ID NO: 20 or 21; and a light chain variable region as shown in SEQ ID NO: 17, 18 or 19;

[0070] c) a heavy chain variable region as shown in any one of SEQ ID NOs: 28-37; and a light chain variable region as shown in SEQ ID NO: 17;

[0071] d) a heavy chain variable region as set forth in SEQ ID NO: 72 or 73; and a light chain variable region as set forth in SEQ ID NO: 74;

[0072] e) the heavy chain variable region set forth in SEQ ID NO: 5; and the light chain variable region set forth in SEQ ID NO: 6;

[0073] f) a heavy chain variable region as shown in SEQ ID NO: 44, 45 or 46; and a light chain variable region as shown in SEQ ID NO: 40, 41, 42 or 43; or

[0074] g) the heavy chain variable region set forth in SEQ ID NO: 75; and the light chain variable region set forth in SEQ ID NO: 76.

[0075] In some embodiments, the aforementioned bispecific antigen-binding molecule, wherein the first antigen-binding domain that specifically binds to ANG-2 comprises:

[0076] a) a heavy chain variable region of SEQ ID NO: 20, 21, or 33 and a light chain variable region of SEQ ID NO: 17;

[0077] b) the heavy chain variable region of SEQ ID NO: 44 or 45 and the light chain variable region of SEQ ID NO: 40;

[0078] c) the heavy chain variable region of SEQ ID NO: 72 or 73 and the light chain variable region of SEQ ID NO: 74; or

[0079] d) the heavy chain variable region of SEQ ID NO: 75 and the light chain variable region of SEQ ID NO: 76.

[0080] In some embodiments, the aforementioned bispecific antigen-binding molecule, wherein the first antigen-binding domain that specifically binds to ANG-2 comprises:

[0081] 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

[0082] a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 45, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 40. In some embodiments, the aforementioned bispecific antigen-binding molecule, wherein the first antigen-binding domain that specifically binds to ANG-2 comprises:

[0083] The heavy chain variable region shown in SEQ ID NO: 33, and the light chain variable region shown in SEQ ID NO: 17; or

[0084] 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.

[0085] In some embodiments, the aforementioned bispecific antigen binding molecule, wherein the second antigen binding domain that specifically binds to VEGF comprises:

[0086] a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 61, SEQ ID NO: 62, and SEQ ID NO: 63, respectively; and

[0087] The light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 64, SEQ ID NO: 65 and SEQ ID NO: 66, respectively.

[0088] In some embodiments, the aforementioned bispecific antigen binding molecule, wherein the second antigen binding domain that specifically binds to VEGF comprises:

[0089] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 58, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 56. In some embodiments, the aforementioned bispecific antigen-binding molecule, wherein the second antigen-binding domain that specifically binds to VEGF comprises:

[0090] 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.

[0091] In some embodiments, the aforementioned bispecific antigen binding molecule, wherein the second antigen binding domain that specifically binds to human VEGF further comprises a heavy chain constant region and a light chain constant region.

[0092] In some embodiments, the aforementioned bispecific antigen binding molecule, wherein the heavy chain constant region of the second antigen binding domain that specifically binds to human VEGF comprises L234A, L235A, I253A, H310A, and H435A mutations.

[0093] In some embodiments, the aforementioned bispecific antigen-binding molecule, wherein the heavy chain constant region of the second antigen-binding domain that specifically binds to human VEGF is an IgG1 constant region and comprises L234A, L235A, I253A, H310A and H435A mutations (EU numbering).

[0094] In some embodiments, the aforementioned bispecific antigen binding molecule, wherein the second antigen binding domain that specifically binds to VEGF comprises:

[0095] 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; and

[0096] 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. In some embodiments, the aforementioned bispecific antigen-binding molecule, wherein the second antigen-binding domain that specifically binds to VEGF comprises:

[0097] a heavy chain having an amino acid sequence as set forth in SEQ ID NO: 59 or 60, or having at least 95%, 96%, 97%, 98% or 99% sequence identity thereto; and

[0098] A light chain having an amino acid sequence as set forth in SEQ ID NO: 57, or having at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 57.

[0099] In some embodiments, the aforementioned bispecific antigen binding molecule, wherein the second antigen binding domain that specifically binds to VEGF comprises:

[0100] The antigen binding molecule, wherein the second antigen binding domain that specifically binds to VEGF comprises:

[0101] The heavy chain shown in SEQ ID NO: 59; and

[0102] The light chain is shown in SEQ ID NO:57.

[0103] In some embodiments, the aforementioned bispecific antigen binding molecule, wherein the second antigen binding domain that specifically binds to VEGF comprises:

[0104] The heavy chain shown in SEQ ID NO: 60; and

[0105] The light chain is shown in SEQ ID NO:57.

[0106] The aforementioned bispecific antigen-binding molecule may have the molecular structure of any bispecific antibody known in the art. Specifically, the aforementioned bispecific antigen-binding molecule may have the molecular structure of a bispecific antibody containing an Fc fragment, or the molecular structure of a bispecific antibody without an Fc fragment. More specifically, the molecular structure of the bispecific antibody containing an Fc fragment includes but is not limited to TrioMab, Crossmab / KIH, DVD-Ig, IgG-scFv, FIT-Ig, mAb-Trap. The molecular structure of the bispecific antibody without an Fc fragment includes but is not limited to BiTE, DART, TandAb, ImmTAC, TriKE.

[0107] In some embodiments, the aforementioned bispecific antigen binding molecules are in the form of IgG-scFv.

[0108] In an exemplary embodiment of the bispecific antibody in the IgG-scFv format, the first antigen-binding domain that specifically binds to ANG-2 is an anti-ANG-2 single-chain antibody (scFv), and the second antigen-binding domain that specifically binds to VEGF is a full-length IgG antibody, wherein the anti-ANG-2 single-chain antibody is linked directly or via a linker to the C-terminus of the heavy chain or light chain of the full-length IgG antibody that specifically binds to VEGF.

[0109] In some embodiments, 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.

[0110] In some embodiments, 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, comprises:

[0111] a) a heavy chain variable region of SEQ ID NO: 20, 21, or 33 and a light chain variable region of SEQ ID NO: 17;

[0112] b) the heavy chain variable region of SEQ ID NO: 44 or 45 and the light chain variable region of SEQ ID NO: 40;

[0113] c) the heavy chain variable region of SEQ ID NO: 72 or 73 and the light chain variable region of SEQ ID NO: 74; or

[0114] d) the heavy chain variable region of SEQ ID NO: 75 and the light chain variable region of SEQ ID NO: 76.

[0115] In some embodiments, the aforementioned bispecific antigen-binding molecule, wherein the anti-ANG-2 single-chain antibody comprises the amino acid sequence of SEQ ID NO: 67, 68, 69, 70, 71, 77, 78, or 79.

[0116] In some embodiments, the aforementioned bispecific antigen-binding molecule, wherein the first antigen-binding domain that specifically binds to ANG-2 is linked directly or through a linker to the second antigen-binding domain that specifically binds to VEGF.

[0117] In some embodiments, the aforementioned bispecific antigen-binding molecule, wherein the first antigen-binding domain that specifically binds to ANG-2 is linked directly or through a linker to the heavy chain or light chain of the second antigen-binding domain that specifically binds to VEGF.

[0118] In some embodiments, the aforementioned bispecific antigen-binding molecule, wherein the N-terminus of the anti-ANG-2 single-chain antibody is linked to the C-terminus of the heavy chain of the second antigen-binding domain that specifically binds to VEGF via a linker.

[0119] In some embodiments, the aforementioned bispecific antigen binding molecule, wherein the linker is (GG) n , where n is an integer from 1 to 20.

[0120] In some embodiments, the aforementioned bispecific antigen binding molecule comprises:

[0121] i) a first chain comprising the amino acid sequence of SEQ ID NO: 80, 81, 82, or 83, or an amino acid sequence that has at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 80, 81, 82, or 83, respectively; and

[0122] ii) a second chain comprising the amino acid sequence of SEQ ID NO:57, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:57.

[0123] In some embodiments, the aforementioned bispecific antigen-binding molecule is a four-peptide structure having an IgG-scFv format, comprising two identical first chains and two identical second chains, wherein:

[0124] i) a first chain comprising the amino acid sequence of SEQ ID NO: 80, 81, 82, or 83, or an amino acid sequence that has at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 80, 81, 82, or 83, respectively; and

[0125] ii) a second chain comprising the amino acid sequence of SEQ ID NO:57, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:57.

[0126] In some embodiments, the aforementioned bispecific antigen binding molecule comprises:

[0127] i) a first chain having an amino acid sequence as set forth in SEQ ID NO: 80, 81, 82, or 83, or having at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 80, 81, 82, or 83, respectively; and

[0128] ii) a second chain having an amino acid sequence as set forth in SEQ ID NO: 57, or having at least 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0129] In some embodiments, the aforementioned bispecific antigen-binding molecule is a four-peptide structure having an IgG-scFv format, comprising two identical first chains and two identical second chains, wherein:

[0130] i) the first chain has an amino acid sequence as set forth in SEQ ID NO: 80, 81, 82, or 83, or has at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 80, 81, 82, or 83, respectively; and

[0131] ii) the second chain has an amino acid sequence 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.

[0132] In some embodiments, the aforementioned bispecific antigen-binding molecule is a four-peptide structure having an IgG-scFv format, comprising two identical first chains and two identical second chains, wherein:

[0133] i) the first chain comprises the amino acid sequence of SEQ ID NO: 80; and the second chain comprises the amino acid sequence of SEQ ID NO: 57;

[0134] ii) a first chain comprising the amino acid sequence of SEQ ID NO: 81; and a second chain comprising the amino acid sequence of SEQ ID NO: 57;

[0135] iii) a first chain comprising the amino acid sequence of SEQ ID NO: 82; and a second chain comprising the amino acid sequence of SEQ ID NO: 57; or

[0136] iv) a first chain comprising the amino acid sequence of SEQ ID NO: 83; and a second chain comprising the amino acid sequence of SEQ ID NO: 57.

[0137] In some embodiments, the aforementioned bispecific antigen-binding molecule is a four-peptide structure having an IgG-scFv format, comprising two identical first chains and two identical second chains, wherein:

[0138] i) the 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;

[0139] ii) the amino acid sequence of the first chain is set forth in SEQ ID NO: 81; and the amino acid sequence of the second chain is set forth in SEQ ID NO: 57;

[0140] iii) the amino acid sequence of the first chain is set forth in SEQ ID NO: 82; and the amino acid sequence of the second chain is set forth in SEQ ID NO: 57; or

[0141] iv) The 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.

[0142] In some embodiments, the aforementioned bispecific antigen-binding molecules have a 4-peptide structure in the Crossmab format.

[0143] In some embodiments, the bispecific antigen binding molecule in the form of Crossmab comprises:

[0144] a) a first light chain and a first heavy chain of a first full-length antibody that specifically binds to ANG-2;

[0145] b) a second light chain and a second heavy chain of a second full-length antibody that specifically binds to VEGF; wherein the constant domains CL and CH1 are replaced with each other.

[0146] In some embodiments, the aforementioned bispecific antigen binding molecule comprises:

[0147] a) a first light chain and a first heavy chain of a first full length antibody that specifically binds to ANG-2; and

[0148] b) a second light chain and a second heavy chain of a second full-length antibody that specifically binds to VEGF; wherein:

[0149] The constant domain CL of the first light chain and the constant domain CH1 of the first heavy chain are mutually replaced;

[0150] The constant domain CL of the second light chain is replaced by the constant domain CH1 of the second heavy chain;

[0151] The light chain variable region VL of the first light chain and the heavy chain variable region VH of the first heavy chain are replaced with each other; or

[0152] The light chain variable region VL of the second light chain and the heavy chain variable region VH of the second heavy chain are replaced with each other.

[0153] In some embodiments, the aforementioned bispecific antigen binding molecule comprises:

[0154] a) a first light chain and a first heavy chain of a first full length antibody that specifically binds to ANG-2; and

[0155] b) a second light chain and a second heavy chain of a second full-length antibody that specifically binds to VEGF; and,

[0156] The constant domain CL of the first light chain and the constant domain CH1 of the first heavy chain are replaced with each other.

[0157] In some embodiments, the aforementioned bispecific antigen binding molecule comprises:

[0158] a) a first light chain and a first heavy chain that specifically bind to ANG-2;

[0159] b) a second light chain and a second heavy chain that specifically bind to VEGF; wherein the constant domains CL and CH1 are replaced with each other.

[0160] In some embodiments, the aforementioned bispecific antigen binding molecule comprises:

[0161] a) a first light chain and a first heavy chain that specifically bind to ANG-2; and

[0162] b) a second light chain and a second heavy chain that specifically bind to VEGF; wherein:

[0163] The constant domain CL of the first light chain and the constant domain CH1 of the first heavy chain are mutually replaced;

[0164] The constant domain CL of the second light chain is replaced by the constant domain CH1 of the second heavy chain;

[0165] The light chain variable region VL of the first light chain and the heavy chain variable region VH of the first heavy chain are replaced with each other; or

[0166] The light chain variable region VL of the second light chain and the heavy chain variable region VH of the second heavy chain are replaced with each other.

[0167] In some embodiments, the aforementioned bispecific antigen binding molecule comprises:

[0168] a) a first light chain and a first heavy chain that specifically bind to ANG-2; and

[0169] b) a second light chain and a second heavy chain that specifically bind to VEGF; and,

[0170] The constant domain CL of the first light chain and the constant domain CH1 of the first heavy chain are replaced with each other.

[0171] In some embodiments, in the aforementioned bispecific antigen binding molecules:

[0172] a) the first light chain comprises a light chain variable region of SEQ ID NO: 17, and the first heavy chain comprises a heavy chain variable region of SEQ ID NO: 33; or

[0173] the first light chain comprises a light chain variable region of SEQ ID NO: 40, and the first heavy chain comprises a heavy chain variable region of SEQ ID NO: 45;

[0174] and

[0175] b) said second light chain comprises a light chain variable region of SEQ ID NO: 56, and said second heavy chain comprises a heavy chain variable region of SEQ ID NO: 58.

[0176] In some embodiments, the aforementioned bispecific antigen binding molecule, wherein:

[0177] a) 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 to SEQ ID NO:85, and

[0178] The first heavy chain comprises the amino acid sequence of SEQ ID NO:84, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:84; or

[0179] 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

[0180] 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; and

[0181] b) the first light chain comprises 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; and

[0182] 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. In some embodiments, in the aforementioned bispecific antigen-binding molecules:

[0183] a) the amino acid sequence of the first light chain is as shown in SEQ ID NO: 85, or has at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 85, and

[0184] The amino acid sequence of the first heavy chain is as shown in SEQ ID NO: 84, or has at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 84; or

[0185] The amino acid sequence of the first light chain is as shown in SEQ ID NO: 88, or has at least 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 88, and

[0186] The amino acid sequence of the first heavy chain is as shown in SEQ ID NO: 87, or has at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 87; and

[0187] b) the amino acid sequence of the second light chain is as shown in SEQ ID NO: 57, or has at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 57; and

[0188] The amino acid sequence of the second heavy chain is as shown in SEQ ID NO: 86, or has at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 86.

[0189] In some embodiments, in the aforementioned bispecific antigen binding molecules:

[0190] i) the amino acid sequence of the first light chain is shown in SEQ ID NO: 85, and the amino acid sequence of the first heavy chain is shown in SEQ ID NO: 84; and

[0191] The 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

[0192] ii) the amino acid sequence of the first light chain is shown in SEQ ID NO: 88, and the amino acid sequence of the first heavy chain is shown in SEQ ID NO: 87; and

[0193] The 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.

[0194] In some embodiments, the disclosure provides an antibody that specifically binds ANG-2.

[0195] In some embodiments, the aforementioned antibody that specifically binds to ANG-2 comprises:

[0196] i) 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

[0197] 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:

[0198] The sequence of SEQ ID NO: 38 is: TINX1X2SSYTYYPDNVKG;

[0199] The sequence of SEQ ID NO: 39 is: X3X4ATGX5FDY

[0200] Among them, X1 is D or E, X2 is D or N, X3 is D or N, X4 is E or Q, and X5 is C, S or V;

[0201] or

[0202] ii) 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

[0203] The light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 15, SEQ ID NO: 11 and SEQ ID NO: 16, respectively.

[0204] In some embodiments, the aforementioned antibody that specifically binds to ANG-2 comprises:

[0205] 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 HCDR3 as set forth in SEQ ID NO: 9, 23, 25, 26 or 27; and

[0206] The light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively.

[0207] In some embodiments, the aforementioned antibody that specifically binds to ANG-2 comprises:

[0208] a) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 7, SEQ ID NO: 22 and SEQ ID NO: 23, respectively; and

[0209] 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;

[0210] b) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 7, SEQ ID NO: 24, and SEQ ID NO: 23, respectively; and

[0211] 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;

[0212] c) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 25, respectively; and

[0213] 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;

[0214] d) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 26, respectively; and

[0215] 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;

[0216] e) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 7, SEQ ID NO: 22, and SEQ ID NO: 25, respectively; and

[0217] 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;

[0218] f) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 7, SEQ ID NO: 22, and SEQ ID NO: 26, respectively; and

[0219] 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;

[0220] g) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 7, SEQ ID NO: 24, and SEQ ID NO: 25, respectively; and

[0221] 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;

[0222] h) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 7, SEQ ID NO: 24, and SEQ ID NO: 26, respectively; and

[0223] 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;

[0224] j) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 23, respectively; and

[0225] 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; or

[0226] k) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 7, SEQ ID NO: 22, and SEQ ID NO: 27, respectively; and

[0227] 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; or

[0228] 1) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively; and

[0229] The light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively.

[0230] In some embodiments, the aforementioned antibody that specifically binds to ANG-2 is present at a concentration equal to or less than 10 -7 M binds to human ANG-2 with a dissociation equilibrium constant, in some embodiments, equal to or less than 10 -8 M or 10 -9 M dissociation equilibrium constant for binding to human ANG-2.

[0231] In some embodiments, the aforementioned antibodies that specifically bind to ANG-2 can cross-bind to monkey ANG-2.

[0232] In some embodiments, the aforementioned antibody that specifically binds to ANG-2 blocks 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 blocking activity is detected by the ELISA assay described in Test Example 2.

[0233] In some embodiments, the aforementioned antibodies that specifically bind to ANG-2 inhibit ANG-2-induced Tie2 phosphorylation in Tie2-transfected CHO cells with an IC50 of less than 21.27 nM, less than 15 nM, less than 10 nM, or less than 8 nM.

[0234] In some embodiments, the aforementioned antibody that specifically binds to ANG-2 is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0235] In some embodiments, the aforementioned antibody that specifically binds to ANG-2, wherein said antibody comprises a framework region, wherein:

[0236] i) the heavy chain framework region comprises one or more amino acid back mutations selected from 44R, 77S or 84S; and / or

[0237] The light chain framework region comprises one or more amino acid back mutations selected from 1N, 43S, 68A, 85D, 87H; or

[0238] ii) the heavy chain framework region comprises one or more amino acid back mutations selected from 2L, 44R, 74V, 82AS or 83K; and / or

[0239] The light chain framework region comprises one or more amino acid back mutations selected from 1N, 43S, 46V, 68A, 85D, and 87H.

[0240] In some embodiments, the aforementioned antibody that specifically binds to ANG-2 comprises:

[0241] i) 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 one or more amino acid backmutations selected from 44R, 77S or 84S; and

[0242] 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 a light chain framework region comprising one or more amino acid backmutations selected from 1N, 43S, 68A, 85D, and 87H; wherein:

[0243] The sequence of SEQ ID NO: 38 is: TINX1X2SSYTYYPDNVKG;

[0244] The sequence of SEQ ID NO: 39 is: X3X4ATGX5FDY

[0245] wherein X1 is D or E, X2 is D or N, X3 is D or N, X4 is E or Q, and X5 is C, S or V; or

[0246] ii) 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 a heavy chain framework region comprising a member selected from the group consisting of 2L, 44R, 74V, 82A One or more amino acid back mutations in S or 83K; and

[0247] 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 its light chain framework region comprises one or more amino acid back mutations selected from 1N, 43S, 46V, 68A, 85D, 87H.

[0248] In some embodiments, the aforementioned antibody that specifically binds to ANG-2 comprises:

[0249] a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 7, SEQ ID NO: 22, and SEQ ID NO: 26, respectively, and whose heavy chain framework region comprises one or more amino acid backmutations selected from 44R, 77S, or 84S; and

[0250] 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 the light chain framework region comprises one or more amino acid back mutations selected from 1N, 43S, 68A, 85D, 87H.

[0251] In some embodiments, the aforementioned antibody that specifically binds to ANG-2 comprises:

[0252] 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; and

[0253] A light chain variable region comprising the amino acid sequence of SEQ ID NO: 17, or an amino acid sequence comprising one or more amino acid backmutations selected from 1N, 43S, 68A, 85D, and 87H in the amino acid sequence of SEQ ID NO: 17.

[0254] In some embodiments, the aforementioned antibody that specifically binds to ANG-2 comprises:

[0255] A heavy chain variable region having an amino acid sequence as shown 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; and

[0256] The light chain variable region has an amino acid sequence as shown in SEQ ID NO: 17, or comprises one or more amino acid back mutations selected from 1N, 43S, 68A, 85D, and 87H in the amino acid sequence of SEQ ID NO: 17.

[0257] In some embodiments, the aforementioned antibody that specifically binds to ANG-2 comprises:

[0258] a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 33; and

[0259] A light chain variable region comprising the amino acid sequence of SEQ ID NO: 17, or an amino acid sequence comprising one or more amino acid backmutations selected from 1N, 43S, 68A, 85D, and 87H in the amino acid sequence of SEQ ID NO: 17.

[0260] In some embodiments, the aforementioned antibody that specifically binds to ANG-2 comprises:

[0261] The heavy chain variable region of SEQ ID NO: 33; and

[0262] The light chain variable region has an amino acid sequence as shown in SEQ ID NO: 17, or comprises one or more amino acid back mutations selected from 1N, 43S, 68A, 85D, and 87H in the amino acid sequence of SEQ ID NO: 17.

[0263] In some embodiments, the aforementioned antibody that specifically binds to ANG-2 comprises:

[0264] A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 44, or comprising a sequence selected from 2L, 44R, 74V, 82A An amino acid sequence in which one or more amino acids in S or 83K are reverse mutated; and

[0265] A light chain variable region comprising the amino acid sequence of SEQ ID NO: 40, or an amino acid sequence comprising one or more amino acid back mutations selected from 1N, 43S, 46V, 68A, 85D, and 87H in the amino acid sequence of SEQ ID NO: 40.

[0266] In some embodiments, the aforementioned antibody that specifically binds to ANG-2 comprises:

[0267] The heavy chain variable region has an amino acid sequence as shown in SEQ ID NO: 44, or comprises a sequence selected from 2L, 44R, 74V, 82AOne or more amino acid back mutations in S or 83K; and

[0268] The light chain variable region has an amino acid sequence as shown in SEQ ID NO: 40, or comprises one or more amino acid back mutations selected from 1N, 43S, 46V, 68A, 85D, and 87H in the amino acid sequence of SEQ ID NO: 40.

[0269] In some embodiments, the aforementioned antibody that specifically binds to ANG-2 comprises:

[0270] i) a heavy chain variable region comprising the amino acid sequence of any one of SEQ ID NOs: 3, 20, 21, 28-37, 72, or 73, or an amino acid sequence that has at least 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 3, 20, 21, 28-37, 72, or 73, respectively; and / or

[0271] a light chain variable region comprising the amino acid sequence of SEQ ID NO: 4, 17, 18, 19, or 74, or an amino acid sequence that has at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 4, 17, 18, 19, or 74, respectively; or

[0272] ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 5, 44, 45, 46 or 75, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 5, 44, 45, 46 or 75, respectively; and / or

[0273] A light chain variable region comprising the amino acid sequence of SEQ ID NO: 6, 40, 41, 42, 43 or 76, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 6, 40, 41, 42, 43 or 76, respectively.

[0274] In some embodiments, the aforementioned antibody that specifically binds to ANG-2 comprises:

[0275] i) a heavy chain variable region having an amino acid sequence as shown in any one of SEQ ID NOs: 3, 20, 21, 28-37, 72 or 73, or having at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NOs: 3, 20, 21, 28-37, 72 or 73, respectively; and / or

[0276] a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 4, 17, 18, 19, or 74, or having at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 4, 17, 18, 19, or 74, respectively; or

[0277] ii) a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 5, 44, 45, 46 or 75, or having at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 5, 44, 45, 46 or 75, respectively; and / or

[0278] A light chain variable region having an amino acid sequence as shown in SEQ ID NO: 6, 40, 41, 42, 43 or 76, or having a sequence identity of at least 95%, 96%, 97%, 98% or 99% to SEQ ID NO: 6, 40, 41, 42, 43 or 76, respectively.

[0279] In some embodiments, the aforementioned antibody that specifically binds to ANG-2 comprises:

[0280] a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 3; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 4;

[0281] b) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 20 or 21; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 17, 18 or 19;

[0282] c) a heavy chain variable region comprising the amino acid sequence of any one of SEQ ID NOs: 28-37; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 17;

[0283] d) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 72 or 73; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 74;

[0284] e) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 5; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 6;

[0285] f) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 44, 45, or 46; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 40, 41, 42, or 43; or

[0286] g) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 75; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 76.

[0287] In some embodiments, the aforementioned antibody that specifically binds to ANG-2 comprises:

[0288] a) the heavy chain variable region of SEQ ID NO: 3; and the light chain variable region of SEQ ID NO: 4;

[0289] b) a heavy chain variable region of SEQ ID NO: 20 or 21; and a light chain variable region of SEQ ID NO: 17, 18 or 19;

[0290] c) the heavy chain variable region set forth in any one of SEQ ID NOs: 28-37; and the light chain variable region of SEQ ID NO: 17;

[0291] d) the heavy chain variable region of SEQ ID NO: 72 or 73; and the light chain variable region of SEQ ID NO: 74;

[0292] e) the heavy chain variable region of SEQ ID NO: 5; and the light chain variable region of SEQ ID NO: 6;

[0293] f) the heavy chain variable region of SEQ ID NO: 44, 45, or 46; and the light chain variable region of SEQ ID NO: 40, 41, 42, or 43; or

[0294] g) the heavy chain variable region of SEQ ID NO: 75; and the light chain variable region of SEQ ID NO: 76.

[0295] In some embodiments, the aforementioned antibody that specifically binds to ANG-2 comprises:

[0296] a) a heavy chain variable region of SEQ ID NO: 20, 21, or 33 and a light chain variable region of SEQ ID NO: 17;

[0297] b) the heavy chain variable region of SEQ ID NO: 44 or 45 and the light chain variable region of SEQ ID NO: 40;

[0298] c) the heavy chain variable region of SEQ ID NO: 72 or 73 and the light chain variable region of SEQ ID NO: 74; or

[0299] d) the heavy chain variable region of SEQ ID NO: 75 and the light chain variable region of SEQ ID NO: 76.

[0300] In some embodiments, the aforementioned antibody that specifically binds to ANG-2 comprises:

[0301] 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

[0302] The heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 45, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 40.

[0303] In some embodiments, the aforementioned antibody that specifically binds to ANG-2 comprises:

[0304] The heavy chain variable region of SEQ ID NO: 33, and the light chain variable region of SEQ ID NO: 17; or

[0305] The heavy chain variable region is SEQ ID NO: 45, and the light chain variable region is SEQ ID NO: 40.

[0306] In some embodiments, the aforementioned antibody that specifically binds to ANG-2 comprises a constant region. In other embodiments, the heavy chain constant region is selected from human IgG1, IgG2, IgG3, and IgG4 constant regions and conventional variants thereof, and the light chain constant region is selected from human antibody kappa and lambda chain constant regions and conventional variants thereof.

[0307] In some embodiments, the aforementioned antibody that specifically binds to ANG-2 comprises the heavy chain constant region of SEQ ID NO: 47 and the light chain constant region of SEQ ID NO: 48.

[0308] In some embodiments, the aforementioned antibody that specifically binds to ANG-2 comprises:

[0309] a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 49, 51 or 52, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 49, 51 or 52, respectively, and / or

[0310] a light chain comprising the amino acid sequence of SEQ ID NO:50, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:50; or

[0311] b) a heavy chain comprising the amino acid sequence of SEQ ID NO: 53 or 55, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 53 or 55, respectively, and / or

[0312] A light chain comprising the amino acid sequence of SEQ ID NO: 54, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 54.

[0313] In some embodiments, the aforementioned antibody that specifically binds to ANG-2 comprises:

[0314] a) a heavy chain having an amino acid sequence as shown in SEQ ID NO: 49, 51 or 52, or having at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 49, 51 or 52, respectively, and / or

[0315] a light chain having an amino acid sequence as set forth in SEQ ID NO: 50, or having at least 95%, 96%, 97%, 98% or 99% sequence identity thereto; or

[0316] b) a heavy chain having an amino acid sequence as shown in SEQ ID NO: 53 or 55, or having at least 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 53 or 55, respectively, and / or

[0317] A light chain having an amino acid sequence as set forth in SEQ ID NO: 54, or having at least 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0318] In some embodiments, the aforementioned antibody that specifically binds to ANG-2 comprises:

[0319] a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 49, 51 or 52, and

[0320] A light chain comprising the amino acid sequence of SEQ ID NO: 50; or

[0321] b) a heavy chain comprising the amino acid sequence of SEQ ID NO: 53 or 55, and

[0322] The light chain comprises the amino acid sequence of SEQ ID NO: 54.

[0323] In some embodiments, the aforementioned antibody that specifically binds to ANG-2 comprises:

[0324] a) a heavy chain having an amino acid sequence as shown in SEQ ID NO: 49, 51 or 52, and

[0325] A light chain having an amino acid sequence as shown in SEQ ID NO: 50; or

[0326] b) a heavy chain having an amino acid sequence as shown in SEQ ID NO: 53 or 55, and

[0327] The light chain has an amino acid sequence as shown in SEQ ID NO: 54.

[0328] In some embodiments, the disclosure provides an isolated antibody that competes for binding to human ANG-2 with the aforementioned antibodies that specifically bind to ANG-2.

[0329] In some embodiments, the present disclosure provides a nucleic acid molecule encoding a bispecific antigen-binding molecule according to the foregoing, or an antibody that specifically binds to ANG-2 as described above.

[0330] In some embodiments, the present disclosure provides a vector comprising the aforementioned nucleic acid molecule.

[0331] In some embodiments, the present disclosure provides a host cell comprising the aforementioned vector.

[0332] In some embodiments, the present disclosure provides a pharmaceutical composition comprising an effective amount of the aforementioned bispecific antigen-binding molecule, or the aforementioned antibody that specifically binds to ANG-2, or the aforementioned nucleic acid molecule, and one or more pharmaceutically acceptable carriers, diluents, or excipients. In some embodiments, the therapeutically effective amount is 0.1-3000 mg of the aforementioned bispecific antigen-binding molecule or antibody that specifically binds to ANG-2 per unit dose of the composition.

[0333] In some embodiments, the present disclosure provides a method for producing the aforementioned bispecific antigen-binding molecule, or the aforementioned antibody that specifically binds to ANG-2, the method comprising culturing the aforementioned host cell to express the bispecific antigen-binding molecule or the antibody that specifically binds to ANG-2.

[0334] In some embodiments, the present disclosure provides a method for preventing or treating cancer or angiogenic eye diseases, comprising administering to a subject a therapeutically effective amount of the aforementioned bispecific antigen binding molecule, or the aforementioned antibody that specifically binds to ANG-2, or the aforementioned nucleic acid molecule, or the aforementioned pharmaceutical composition.

[0335] In some embodiments, the aforementioned methods of treatment, wherein the cancer is selected from breast cancer, adrenal tumors, 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. In some embodiments, wherein the angiogenic eye disease is selected from neovascular glaucoma, age-related macular degeneration (AMD), diabetic macular edema, corneal neovascularization, corneal transplant neovascularization, corneal transplant rejection, retinal / choroidal neovascularization, angiogenesis (iris rubeosis), ocular neovascular disease, vascular restenosis and arteriovenous malformation (AVM). In some embodiments, the aforementioned cancer or angiogenic eye disease is associated with VEGF or ANG-2.

[0336] In some embodiments, the present disclosure provides use of the aforementioned bispecific antigen-binding molecule, or the aforementioned antibody that specifically binds to ANG-2, or the aforementioned nucleic acid molecule, or the aforementioned pharmaceutical composition in the preparation of a medicament for treating or preventing cancer or angiogenic eye diseases.

[0337] In some embodiments, the aforementioned use, wherein the cancer is selected from breast cancer, adrenal tumors, 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. In some embodiments, wherein the angiogenic eye disease is selected from neovascular glaucoma, age-related macular degeneration (AMD), diabetic macular edema, corneal neovascularization, corneal transplant neovascularization, corneal transplant rejection, retinal / choroidal neovascularization, angiogenesis (iris rubeosis), ocular neovascular disease, vascular restenosis and arteriovenous malformation (AVM). In some embodiments, the aforementioned cancer or angiogenic eye disease is associated with VEGF or ANG-2.

[0338] In some embodiments, the present disclosure provides the aforementioned bispecific antigen binding molecule, or the aforementioned antibody that specifically binds to ANG-2, or the aforementioned nucleic acid molecule, or the aforementioned pharmaceutical composition for use as a medicament.

[0339] In some embodiments, the present disclosure provides the aforementioned bispecific antigen binding molecules, or the aforementioned antibodies that specifically bind to ANG-2, or the aforementioned nucleic acid molecules, or the aforementioned pharmaceutical compositions for use as a medicament, which can be used to treat or prevent cancer or angiogenic eye diseases. In some embodiments, the cancer is selected from breast cancer, adrenal tumors, 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. In some embodiments, the angiogenic eye disease is selected from neovascular glaucoma, age-related macular degeneration (AMD), diabetic macular edema, corneal neovascularization, corneal transplant neovascularization, corneal transplant rejection, retinal / choroidal neovascularization, angiogenesis (iris rubeosis), ocular neovascular disease, vascular restenosis, and arteriovenous malformation (AVM). In some embodiments, the aforementioned cancer or angiogenic eye disease is associated with VEGF or ANG-2. BRIEF DESCRIPTION OF THE DRAWINGS

[0340] Figure 1 The results show that the bispecific antibody inhibits the binding of ANG-2 to Tie2;

[0341] Figure 2 The results of bispecific antibody inhibition-induced Tie2 phosphorylation are shown;

[0342] Figure 3 The bispecific antibody significantly inhibited the increase in intracellular phosphorylated VEGFR levels in HUVECs caused by VEGF;

[0343] Figure 4 It was shown that the bispecific antibody can significantly inhibit the proliferation of HUVECs induced by VEGF;

[0344] Figure 5 It was shown that the bispecific antibodies disclosed herein can significantly inhibit the growth of PC-3 tumors in mice;

[0345] Figure 6 It was shown that the bispecific antibodies disclosed herein can significantly inhibit the growth of H460 transplanted tumors in mice;

[0346] Figure 7 It was shown that the bispecific antibodies disclosed herein can significantly inhibit the growth of A431 transplanted tumors in mice;

[0347] Figure 8A and Figure 8B The results of the inhibition of choroidal neovascularization in rhesus monkeys by the bispecific antibody disclosed herein are shown; wherein Figure 8A It is shown that the bispecific antibody of the present disclosure improves the fluorescence leakage area in rhesus monkey eyes. Figure 8BIt was shown that the bispecific antibodies disclosed herein can significantly reduce the number of fluorescent spots in the eyes of rhesus monkeys;

[0348] Figure 9 It was shown that the bispecific antibody of the present disclosure can significantly reduce the expression of VEGF in the aqueous humor of rhesus monkey eyes.

[0349] Figure 10A and Figure 10B shows the molecular structure of an exemplary bispecific antibody disclosed herein; Figure 10A Shows the structure of an IgG-scFv type bispecific antibody; Figure 10B The structure of a Crossmab-type bispecific antibody is shown. DETAILED DESCRIPTION

[0350] the term

[0351] In order to make the present disclosure more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise explicitly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present disclosure belongs.

[0352] The three letter and one letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

[0353] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-o-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids (i.e., an α-carbon bound to hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium). Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics refer to compounds that have a structure that is different from the general chemical structure of an amino acid but functions in a manner similar to naturally occurring amino acids.

[0354] The term "ANG-2" refers to angiopoietin-2 (ANGPT2 or ANG2), which is described, for example, in Maisonpierre, PC et al., Science 277 (1997) 55-60 and Cheung, AH et al., Genomics 48 (1998) 389-91. Angiopoietins-1 and -2 were found to be ligands for Tie, a family of tyrosine kinases selectively expressed in the vascular endothelium. Yancopoulos, GD et al., Nature 407 (2000) 242-48. The angiopoietin family has four identified members, and angiopoietins-3 and -4 (ANG-3 and ANG-4) may represent counterparts of a broad region of the same gene locus in mice and humans. Kim, I. et al., FEBS Let, 443 (1999) 353-56; Kim, I. et al., J Biol Chem 274 (1999) 26523-28. 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). All known angiopoietins bind primarily to Tie2, and both ANG1 and 2 bind to Tie2 with an affinity of 3 nM (Kd), Maisonpierre, PC et al., Science 277 (1997) 55-60.

[0355] The term "VEGF" refers to human vascular endothelial growth factor (VEGF / VEGF-A), which is described in, for example, 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 regulating 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. 91 (1993) 153-159; Brown, LF et al., Human Pathol. 26 (1995) 86-91; Brown, LF et al., Cancer Res. 53 (1993) 4727-4735; Mattern, J. et al., Brit. J. Cancer. 73 (1996) 931-934; and Dvorak, HF et al., Am. J. Pathol. 146 (1995) 1029-1039). VEGF is a homodimeric glycoprotein that promotes mitogenesis in endothelial cells.

[0356] As used herein, the term "antigen-binding molecule" refers in the broadest sense to a molecule that specifically binds to an antigen. Examples of antigen-binding molecules are antibodies, antibody fragments, antibody fusion proteins, or fusion proteins. Exemplarily, the bispecific antigen-binding molecules herein include bispecific antibodies and antibody fusion proteins. Exemplarily, the bispecific antibodies or antibody fusion proteins herein may comprise a first chain and a second chain, wherein the first chain is an antibody heavy chain or a polypeptide comprising an antibody heavy chain, and the second chain is an antibody light chain or a polypeptide comprising an antibody light chain. Exemplarily, in some embodiments, the bispecific antigen-binding molecules herein have the basic structure of a full-length antibody, comprising a tetrapeptide chain structure formed by two identical first chains and two identical second chains connected by interchain disulfide bonds; wherein the first chain comprises an anti-VEGF antibody heavy chain and an anti-ANG-2 single-chain antibody, and the second chain comprises an anti-VEGF antibody light chain.

[0357] As used herein, a "bispecific antigen-binding molecule" refers to an antigen-binding molecule capable of simultaneously binding two antigens or antigenic determinants, comprising a first antigen-binding domain that binds to a first antigen or antigenic determinant and a second antigen-binding domain that binds to a second antigen or antigenic determinant. In some embodiments of the present disclosure, the first antigen-binding domain specifically binds to ANG-2, and the second antigen-binding domain specifically binds to VEGF; alternatively, in some embodiments of the present disclosure, the first antigen-binding domain specifically binds to VEGF, and the second antigen-binding domain specifically binds to ANG-2.

[0358] In some embodiments, the bispecific antigen-binding molecules described herein are bispecific bivalent antibodies or bispecific tetravalent antibodies.

[0359] It is well known in the art that bispecific antigen-binding molecules can be divided into two major categories based on their different structures: bispecific antigen-binding molecules containing an Fc fragment and bispecific antigen-binding molecules without an Fc fragment. Bispecific antigen-binding molecules containing an Fc fragment include, but are not limited to, TrioMab, Crossmab / KIH, DVD-Ig, IgG-scFv, FIT-Ig, mAb-Trap, etc. Bispecific antigen-binding molecules without an Fc fragment include, but are not limited to, BiTE, DART, TandAb, ImmTAC, TriKE, etc.

[0360] As described herein, "Crossmab" is an IgG-like bispecific antigen-binding molecule with an Fc region. It is achieved by exchanging the heavy and light chain structures within the Fab region of the first antigen-binding domain, thereby changing the molecular structure of the VH-VL and CH1-CL interfaces. Due to the principle of mutual repulsion (VH and VH repel each other, and CL and CL repel each other), the exchanged antibody light chains are less likely to mispair with the heavy chains of the unmodified antibody, thereby achieving correct pairing of the light and heavy chains.

[0361] In other embodiments, the bispecific antigen-binding molecule of the present disclosure is in the form of Crossmab, which is 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 heavy chain and the first light chain constitute a first antigen-binding domain that binds to ANG-2, the second heavy chain and the second light chain constitute a second antigen-binding domain that binds to VEGF, and the CH1 of the first heavy chain is exchanged with the CL of the first light chain.

[0362] As described in the present disclosure, "IgG-scFv" is an IgG-like bispecific antigen-binding molecule structure with an Fc region, in which an scFv of one antigen-binding domain is fused to the C-terminus of the other antigen-binding domain IgG heavy chain or light chain to form an IgG-scFv bispecific antigen-binding molecule.

[0363] In other embodiments, the bispecific antigen-binding molecules of the present disclosure are in the form of IgG-scFv. In exemplary embodiments, a scFv that specifically binds to ANG-2 is linked directly or via a linker to the C-terminus of the heavy chain of an antibody that specifically binds to VEGF, forming an IgG-scFv bispecific antigen-binding molecule.

[0364] The term "valent" refers to the presence of a specified number of binding sites in an antigen-binding molecule. Thus, the terms "bivalent," "tetravalent," and "hexavalent" refer to the presence of two, four, and six binding sites, respectively, in an antigen-binding molecule.

[0365] As used herein, a "first antigen-binding domain capable of specifically binding to ANG-2" or a "second antigen-binding domain capable of specifically binding to VEGF" refers to a region of an antigen-binding molecule that specifically binds to all or part of ANG-2 or VEGF. For example, the antigen-binding domain may comprise one or more antibody variable regions. In particular, the antigen-binding domain capable of specifically binding to an antigen comprises an antibody light chain variable region and an antibody heavy chain variable region, which may form a configuration such as scFv or Fab.

[0366] The term "antibody fusion protein" refers to a biologically active fusion protein formed by linking a target protein (polypeptide) to an antibody. The fusion protein has the biological activity of the linked protein as well as immunoglobulin activity.

[0367] The term "antibody" is used in the broadest sense and encompasses various 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.

[0368] "Native antibodies" refer to naturally occurring immunoglobulin molecules with diverse structures. For example, natural IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two identical light chains and two identical heavy chains bonded by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also known as the variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also known as the variable light domain or light chain variable domain, followed by a constant light (CL) domain. Based on the amino acid sequence of their constant domains, antibody light chains include two types: kappa (κ) and lambda (λ). Based on the amino acid composition and arrangement order of the constant region of the antibody heavy chain, antibodies can be divided into five classes, or antibody isotypes: IgM, IgD, IgG, IgA, and IgE, with the corresponding heavy chains being μ, δ, γ, α, and ε, respectively. Igs of the same class can be further divided into different subclasses based on the amino acid composition of their hinge regions and the number and location of disulfide bonds in their heavy chains. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Each of the five Ig classes can have either kappa or lambda chains.

[0369] The approximately 110 amino acids near the N-terminus of an antibody's heavy and light chains vary greatly in sequence and constitute the variable region; the remaining amino acid sequence near the C-terminus is relatively stable and constitutes the constant region. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains (CH1, CH2, and CH3). Each light chain is composed 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, called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each light chain contains three CDR regions: LCDR1, LCDR2, and LCDR3; each heavy chain contains three CDR regions: HCDR1, HCDR2, and HCDR3. Each VH and VL is composed of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0370] The terms "complementarity determining region," "CDR," or "hypervariable region" refer to the regions within the variable domain that primarily contribute to antigen binding. Typically, there are three CDRs (HCDR1, HCDR2, HCDR3) in each heavy chain variable region and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable region. The amino acid sequence boundaries of the CDRs can be determined using various well-known schemes, such as the "Kabat" numbering convention (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD), the "Chothia" numbering convention (see Martin, ACR. Protein Sequence and Structure Analysis of Antibody Variable Domains [J]. 2001), and the ImMunoGenTics (IMGT) numbering convention (Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003); Front Immunol. 2018 Oct 16; 9: 2278, etc.). The relationship between numbering systems, including, for example, the Kabat numbering and the IMGT unique numbering system, is well known to those skilled in the art.

[0371] For example, for the classical format, following the Kabat rules, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3); the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3).

[0372] Following the Chothia convention, the CDR amino acids in VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the amino acid residues in VL are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3).

[0373] By combining the CDR definitions of both Kabat and Chothia, the CDRs consist of 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) in human VL.

[0374] Following the IMGT rules, the CDR amino acid residues in VH are numbered approximately as 27-38 (CDR1), 56-65 (CDR2), and 105-117 (CDR3), and the CDR amino acid residues in VL are numbered approximately as 27-38 (CDR1), 56-65 (CDR2), and 105-117 (CDR3). Following the IMGT rules, the CDR regions of an antibody can be determined using the program IMGT / DomainGapAlign.

[0375] Following AbM convention, the CDR amino acids in VH are numbered 26-35 (HCDR1), 50-58 (HCDR2), and 95-102 (HCDR3); and the amino acid residues in VL are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3).

[0376] Unless otherwise specified, the variable region and CDR sequences in the disclosed examples are all numbered using the "Kabat" convention.

[0377] The term "antibody framework" or "FR region" refers to a 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.

[0378] "Antibody constant region domain" refers to the domain of the constant region of the light chain and heavy chain of an antibody, including CL and CH1, CH2, CH3 and CH4 domains derived from different classes of antibodies. The constant region disclosed herein also includes "conventional variants" of the human antibody heavy chain constant region and the human antibody light chain constant region, which refers to variants of the heavy chain constant region or light chain constant region that do not change the structure and function of the antibody variable region disclosed in the prior art. Exemplary variants include IgG1, IgG2, IgG3 or IgG4 heavy chain constant region variants that undergo site-directed modification and amino acid substitutions in the heavy chain constant region, specifically replacing YTE mutations, L234A and / or L235A mutations, S228P mutations, and / or mutations that obtain a knob-into-hole structure (so that the antibody heavy chain has a knob-Fc and hole-Fc combination) known in the prior art. These mutations have been shown to give antibodies new properties without changing the function of the antibody variable region.

[0379] "Antibody fragments" refer to molecules other than intact antibodies that comprise a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, Fd, dAb; camelid VHH domains; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0380] A "single-chain variable fragment (scFv)", also known as a "single-chain antibody", is a fusion protein of the heavy chain variable region (VH) and the light chain variable region (VL) of an antibody connected by a linker. In particular, the linker is a short polypeptide of 10 to 25 amino acids and is generally rich in glycine for flexibility, and serine or threonine for solubility, and can connect the N-terminus of the VH to the C-terminus of the VL, or vice versa. Despite the removal of the constant region and the introduction of the linker, this protein retains the specificity of the original antibody. For a review of scFv fragments, see, for example, Pluckthün, in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds., (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93 / 16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458.

[0381] "Linker" or "connector" refers to a polypeptide sequence used to connect polypeptides (such as protein domains), which usually has a certain degree of flexibility. The use of the linker does not cause the loss of the original structure and function of the polypeptide.

[0382] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of a 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. Unless otherwise indicated herein, 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.

[0383] The term "binding site" or "antigen binding site" refers to the area where an antibody molecule actually binds to an antigen. The term "antigen binding site" comprises both the antibody heavy chain variable domain (VH) and the antibody light chain variable domain (VL), or comprises only the antibody heavy chain variable domain or only the light chain variable domain.

[0384] A "chimeric" antibody is one in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0385] The term "humanized" antibody is an antibody that retains the reactivity of a non-human antibody while having lower immunogenicity in humans. For example, this can be achieved by retaining the non-human CDR regions and replacing the rest of the antibody with their human counterparts (i.e., the constant region and the framework portion of the variable region). See, e.g., Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855, 1984; Morrison and Oi, Adv. Immunol., 44: 65-92, 1988; Verhoeyen et al., Science, 239: 1534-1536, 1988; Padlan, Molec. Immun., 28: 489-498, 1991; and Padlan, Molec. Immun., 31: 169-217, 1994. Other examples of human engineering technology include, but are not limited to, the Xoma technology disclosed in US 5,766,886.

[0386] In general, non-human antibodies are humanized to reduce immunogenicity to people while retaining the specificity and affinity of the parent non-human antibody. Generally, humanized antibodies include one or more variable domains, wherein HVR (heavy chain variable region), such as CDR (or part thereof) is derived from non-human antibodies, and FR (or part thereof) is derived from human antibody sequences. Optionally, humanized antibodies also include at least a portion of human constant region. In some embodiments, some FR residues in humanized antibodies are replaced with corresponding residues from non-human antibodies (such as antibodies derived from HVR residues), such as to restore or improve antibody specificity or affinity.

[0387] The term "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. In addition, if the antibody contains a constant region, the constant region is also derived from such human sequences, such as human germline sequences or mutant forms of human germline sequences. The human antibodies of the present disclosure may include amino acid residues that are not encoded by human sequences (e.g., mutations introduced by random mutagenesis or site-specific mutagenesis in vitro or by somatic mutation in vivo).

[0388] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody that has a structure substantially similar to a native antibody structure or has heavy chains that contain an Fc region as defined herein.

[0389] The term "antigen" refers to a molecule or portion of a molecule that can be bound by a selective binding agent such as an antigen binding protein (including, for example, an antibody) and that can additionally be used in an animal to produce antibodies that can bind to the antigen. An antigen can have one or more epitopes that can interact with different antigen binding proteins (e.g., antibodies).

[0390] The term "epitope" refers to an area or region on an antigen that is capable of specific binding to an antibody or its antigen-binding fragment. An epitope can be formed from a continuous string of amino acids (linear epitope) or comprise non-contiguous amino acids (conformational epitope), for example, brought into spatial proximity by folding of the antigen (i.e., by tertiary folding of the antigen in a proteinaceous manner). Conformational epitopes differ from linear epitopes in that antibody binding to the conformational epitope is lost in the presence of denaturing solvents. For example, 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.

[0391] Screening for antibodies that bind to a particular epitope (i.e., those that bind to the same epitope) can be performed using routine methods 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 Harb., NY)).

[0392] Competitive binding can also be used to identify whether an antibody binds to the same epitope or competes for binding with a reference antibody. For example, an "antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competition assay, and conversely, the reference antibody blocks the binding of the antibody to its antigen by 50% or more in a competition assay. 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 removing excess reference antibody, the ability of the test antibody to bind to the antigen is assessed. If the test antibody is able to bind to the antigen after saturation binding of the reference antibody, it can be concluded that the test antibody binds to a different epitope than the reference antibody. However, if the test antibody is unable to bind to the antigen after saturation binding of the reference antibody, then the test antibody may bind to the same epitope as the reference antibody. To confirm whether the test antibody binds to the same epitope or is merely sterically hindered from binding, routine experiments can be used (e.g., peptide mutations and binding analysis using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art). This assay should be performed in two settings, i.e., both antibodies are saturated antibodies. If, in both settings, only the first (saturated) antibody is able to bind to the antigen, then it can be concluded that the test antibody and the reference antibody compete for binding to the antigen.

[0393] In some embodiments, two antibodies are considered to bind to the same or overlapping epitope if a 1-fold, 5-fold, 10-fold, 20-fold, or 100-fold excess of one antibody inhibits the binding of the other antibody by at least 50%, at least 75%, at least 90%, or even 99% or more, as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 50 (1990) 1495-1502).

[0394] As used herein, "competition" between antibodies and / or antigen-binding fragments thereof means that two antibodies (or antigen-binding fragments thereof) bind to the same or overlapping epitope (e.g., as determined by any method known to those skilled in the art in a competitive binding assay). If the competing antibody or antigen-binding fragment thereof binds to the same epitope or an overlapping epitope as the antibody or antigen-binding fragment of the disclosure, then the antibody or antigen-binding fragment thereof also "competes" with the antibody or antigen-binding fragment of the disclosure. As used herein, competing antibodies or antigen-binding fragments thereof may also include the following: (i) competing antibodies or antigen-binding fragments thereof that sterically block the binding of the antibody or antigen-binding fragment of the disclosure to its target (e.g., if the competing antibody binds to an adjacent, non-overlapping and / or identical epitope and physically prevents the antibody or antigen-binding fragment of the disclosure from binding to its target); and / or (ii) competing antibodies or antigen-binding fragments thereof that bind to a different, non-overlapping epitope and induce a conformational change in the antigen such that the antigen no longer binds to the antibody or antigen-binding fragment of the disclosure in the same manner as it would in the absence of the conformational change.

[0395] "Specifically binds," "specific binding," or "binding" refers to an antibody that binds to an antigen or an epitope within the antigen with a higher affinity than for other antigens or epitopes. Typically, an antibody binds to an antigen or an epitope within an antigen with an equilibrium dissociation constant (KD) of about 1×10-7 M or less (e.g., about 1×10-8 M or less, about 1×10-9 M or less, about 1×10-10 M or less, about 1×10-11 M or less, or about 1×10-12 M or less), typically with a KD of at least one percent of the KD of the antibody binding to a nonspecific antigen (e.g., BSA, casein). KD can be measured using standard procedures. However, an antibody that specifically binds to an antigen or an epitope within an antigen may have cross-reactivity to other related antigens, for example, cross-reactivity to the same antigen from other species (homologous), such as humans or monkeys, e.g., Macaca fascicularis (cynomolgus, cyno), chimpanzee (Pan troglodytes) (chimpanzee, chimp), or marmoset (Callithrix jacchus) (common marmoset, marmoset).

[0396] "Affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). The affinity of a molecule X for its binding partner Y can generally be expressed in terms of the dissociation constant (KD). Affinity can be measured by conventional methods known in the art, including those described herein.

[0397] The term "kassoc" or "ka" is intended to refer to the on-rate of a specific antibody-antigen interaction, while the term "kdis" or "kd" as used herein is intended to refer to the off-rate of a specific antibody-antigen interaction. As used herein, the term "KD" is intended to refer to the dissociation constant, which is derived from the ratio of kd to ka (i.e., kd / ka) and is expressed as molar concentration (M). The KD value of an antibody can be measured using methods well established in the art. Methods for measuring antibody KD include using a biosensor system such as a system measuring surface plasmon resonance, or by solution equilibrium titration (SET) to measure affinity in solution.

[0398] The term "nucleic acid" is used interchangeably with the term "polynucleotide" in this article and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single-stranded or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or connections, which are synthetic, naturally occurring, and non-naturally occurring, with binding properties similar to reference nucleic acids, and metabolized in a manner similar to reference nucleotides. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).

[0399] Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as the explicitly indicated sequence. Specifically, as described in detail below, degenerate codon substitutions can be obtained by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081, 1991; Ohtsuka et al., J. Biol. Chem. 260:2605-2608, 1985; and Rossolini et al., Mol. Cell. Probes 8:91-98, 1994).

[0400] Sequence " identity " refers to, when two sequences are optimally aligned, introducing gaps when necessary, to obtain maximum sequence identity percentage, and not considering any conservative substitutions as part of sequence identity, the degree (percentage) to which the amino acids / nucleic acids of the two sequences are identical at equivalent positions. For determining percent sequence identity, alignment can be achieved in a variety of ways within the scope of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters applicable to measuring alignment, including any algorithm required for achieving maximum alignment over the full length of the compared sequences.

[0401] "Conservatively modified variants" or "conservative substitutions" refer to replacing an amino acid in a protein with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophilicity / hydrophobicity, backbone architecture, and rigidity, etc.), such that such changes can generally be made without altering the biological activity of the protein. It is known to those skilled in the art that, generally, 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.)). In addition, substitutions of structurally or functionally similar amino acids are less likely to destroy biological activity. Exemplary conservative substitutions are shown in the table below.

[0402] Table 1. Exemplary conservative amino acid substitutions

[0403]

[0404]

[0405] The term "conservatively modified variants" when applied to nucleic acid sequences refers to nucleic acids that encode identical or substantially identical amino acid sequences, or, where the nucleic acid does not encode an amino acid sequence, to substantially identical sequences. 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 every position where a codon specifies alanine, the codon can be changed to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are a type of conservatively modified variation. Each nucleic acid sequence encoding a polypeptide herein also describes every possible silent variation of the nucleic acid. One of skill will recognize that every codon in a nucleic acid (except AUG, which is generally the only codon for methionine, and TGG, which is generally the only codon for tryptophan) can be modified to produce functionally identical molecules. Thus, every silent variation of a nucleic acid encoding a polypeptide is implicit in each described sequence.

[0406] The term "vector" means a polynucleotide molecule capable of transporting another polynucleotide to which it is connected. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop, wherein additional DNA segments can be connected. Another type of vector is a viral vector, such as an adeno-associated viral vector (AAV or AAV2), in which additional DNA segments can be connected to the viral genome. Some vectors can replicate autonomously (for example, bacterial vectors and additional mammalian vectors with bacterial replication origins) in the host cell in which they are introduced. Other vectors (for example, non-additional mammalian vectors) can be integrated into the genome of the host cell after being introduced into the host cell, thereby replicating together with the host genome.

[0407] The term "expression vector" or "expression construct" refers to a vector suitable for transformation of a host cell and containing a nucleic acid sequence that directs and / or controls (along with the host cell) the expression of one or more heterologous coding regions operably linked thereto. An expression construct may include, but is not limited to, sequences that affect or control transcription, translation, and, when introns are present, RNA splicing of the coding region operably linked thereto.

[0408] As used herein, "operably linked" means that the components to which the term is applied are in a relationship that allows them to perform their inherent functions under appropriate conditions. For example, a control sequence in a vector that is "operably linked" to a protein coding sequence is linked thereto so as to achieve expression of the protein coding sequence under conditions compatible with the transcriptional activity of the control sequence.

[0409] The terms "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, including the offspring of such cells. Host cells include "transformants" and "transformed cells", which include primary transformed cells and offspring derived therefrom without considering the number of passages. Offspring may not be completely identical to the parent cell in nucleic acid content, but may contain mutations. Mutant offspring having the same function or biological activity as screened or selected in the initial transformed cells 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 (e.g., Spodopterafrugiperda or Trichoplusia ni), amphibian cells, bacterial cells, plant cells and fungal cells. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, cow, horse, and hamster cells, including but not limited to Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, and HEK-293 cells.Fungal cells include yeast and filamentous fungal cells, including, for example, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia akoclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia truncatulae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces cerevisiae, Hansenula polymorpha. polymorpha), Kluyveromyces spp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum, Physcomitrella patens, and Neurospora crassa. Pichia, any Saccharomyces spp., Hansenula polymorpha, any Kluyveromyces spp., Candida albicans, any Aspergillus spp., Trichoderma reesei, Chrysosporium lucknowense, any Fusarium spp., Yarrowia lipolytica, and Neurospora crassa. In some embodiments, the host cell is a non-human cell.

[0410] As used in this application, the expressions "cell," "cell line," and "cell culture" are used interchangeably, and all such designations include progeny. Thus, the terms "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom, regardless of the number of passages. It is also understood that not all progeny 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 cell from which they were selected are included.

[0411] "Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0412] A "pharmaceutical composition" refers to a mixture containing one or more antibodies or antigen-binding fragments thereof described herein together with other chemical components, such as physiologically / pharmaceutically acceptable carriers and excipients.

[0413] The term "pharmaceutically acceptable carrier" means any solvent, dispersion medium, coating, antibacterial and antifungal agent, isotonic and absorption enhancing or retarding agent, etc. that is physiologically compatible. Some examples of pharmaceutically acceptable carriers are water, saline, phosphate buffered saline, acetate buffer with sodium chloride, dextrose, glycerol, polyethylene glycol, ethanol, etc., and combinations thereof. In many cases, it is preferred to include an isotonic agent, such as a sugar, a polyol (e.g., mannitol, sorbitol), or sodium chloride in the composition. Other examples of pharmaceutically acceptable substances are surfactants, wetting agents, or small amounts of auxiliary substances such as wetting agents or emulsifiers, preservatives, or buffers, which enhance the shelf life or effectiveness of the antibody.

[0414] The pharmaceutical compositions of the present disclosure can be administered by various methods known in the art. The route and / or mode of administration vary depending on the desired outcome. Preferably, administration can be intravitreal, intravenous, intramuscular, intraperitoneal or subcutaneous or administered near the target site. The pharmaceutically acceptable carrier should be suitable for intravitreal, intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound (i.e., antibody, bispecific and multispecific molecules) can be coated in a material to protect the compound from the effects of acid and other natural conditions that may inactivate the compound.

[0415] The term "subject" includes humans and non-human animals. Non-human animals include all vertebrates (e.g., mammals and non-mammals) such as non-human primates (e.g., cynomolgus monkeys), sheep, dogs, cows, chickens, amphibians, and reptiles. Unless otherwise indicated, the terms "patient" or "subject" are used interchangeably herein. As used herein, the term "cynomolgus monkey" or "cynomolgus monkey" refers to cynomolgus monkeys (Macaca fascicularis). In certain embodiments, the individual or subject is a human.

[0416] "Administer," "administer," and "treat," as they apply to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refers to the contacting of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid.

[0417] "Sample" refers to a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present in a subject. Exemplary samples are biological fluids such as blood, serum and serosal fluid, plasma, lymph, urine, saliva, cystic fluid, tears, feces, sputum, mucosal secretions of secretory tissues and organs, vaginal secretions, ascites, pleura, pericardium, peritoneum, fluids of the abdominal cavity and other body cavities, fluids collected by bronchial lavage, synovial fluid, liquid solutions in contact with a subject or biological source, such as cell and organ culture media (including cell or organ conditioned media), lavage fluids, etc., tissue biopsy samples, fine needle aspirations, surgically resected tissues, organ cultures, or cell cultures.

[0418] "Treatment" (and grammatical variations thereof) refers to clinical intervention that attempts to alter the natural course of the individual being treated and can be performed for prevention or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the occurrence or recurrence of disease, alleviating symptoms, alleviating / reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or palliating the disease state, and regression or improved prognosis. In some embodiments, the antibodies of the present disclosure are used to delay the development of disease or slow the progression of disease.

[0419] "Effective amount" is generally an amount sufficient to reduce the severity and / or frequency of symptoms, eliminate these symptoms and / or potential causes, prevent symptoms and / or their potential causes from occurring and / or improve or ameliorate the damage (e.g., lung disease) caused by or associated with the disease state. In some embodiments, an effective amount is a therapeutically effective amount or a prophylactic effective amount. A "therapeutically effective amount" is an amount sufficient to treat a disease state or symptom, particularly a state or symptom associated with the disease state, or otherwise prevent, hinder, delay, or reverse the disease state or any other undesirable symptom associated with the disease in any way. A "prophylactic effective amount" is an amount that will have a predetermined preventive effect when administered to a subject, 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 may not occur with the administration of one dose, and may only occur after a series of doses are administered. Thus, a therapeutic or prophylactic 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 disease state, age, sex, and weight of the individual, and the ability of the therapeutic agent or combination of therapeutic agents to elicit a desired response in the individual. Exemplary indicators of an effective therapeutic agent or combination of therapeutic agents include, for example, improved health status of the patient.

[0420] The terms "cancer" and "cancerous" refer to or describe a physiological condition typically characterized by unregulated cell growth in mammals. Both benign and malignant cancers are included in this definition. "Early stage cancer" or "early stage tumor" refers to a cancer that is non-invasive or metastatic, or classified as a stage 0, stage I, or stage II cancer. Examples of cancer include, but are not limited to, lymphomas, blastomas (including medulloblastoma and retinoblastoma), sarcomas (including liposarcoma and synovial cell sarcoma), neuroendocrine tumors (including carcinoid tumors, gastrinomas, and islet cell carcinomas), mesotheliomas, Schwann cell tumors (including acoustic neuromas), meningiomas, adenocarcinomas, melanomas, adrenal tumors, and leukemias or lymphoid malignancies. More specific examples of such cancers include, but are not limited to, squamous cell carcinoma (e.g., epithelial squamous cell carcinoma); lung cancer, including 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; liver cancer (liver cancer or hepatic carcinoma), bladder cancer; hepatoma; breast cancer (including metastatic breast cancer); colon cancer; rectal cancer; colorectal cancer; endometrial cancer 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 and neck cancer and multiple myeloma.

[0421] Examples and Test Examples

[0422] The present disclosure is further described below with reference to the following examples and test cases, but these examples and test cases are not intended to limit the scope of the present disclosure. Experimental methods not specifying specific conditions in the examples or test cases of this disclosure generally follow conventional conditions, such as those in the Cold Spring Harbor Laboratory Manual of Antibody Techniques and the Molecular Cloning Manual, or the conditions recommended by the raw material or product manufacturer. Reagents and materials not specifying their sources were purchased commercially.

[0423] Example 1. Expression of ANG-2 and ANG-2 receptor Tie2

[0424] The sequences encoding the extracellular domain of human ANG-2 and human ANG-2 receptor Tie2 with human IgG1-Fc tag were inserted into the phr vector to construct an expression plasmid, which was then transfected into HEK293 cells. The specific transfection steps were as follows: the day before, HEK293E cells were cultured at 1×10 61 mL of the transfection plasmid was inoculated into freestyle expression medium (containing 1% FBS, Gibco, 12338-026) and cultured in a 37°C shaker (120 rpm) for 24 hours. After 24 hours, the transfection plasmid and transfection reagent PEI mixture was slowly added to 200 mL of HEK293E cells and incubated in a shaker at 8% CO2, 120 rpm, and 37°C. On the third day of transfection, 10% of the volume of feed medium (20 mM glucose + 2 mM L-glutamate) was added. On the sixth day of transfection, the cell supernatant was collected by centrifugation at 4500 rpm for 10 minutes. The recombinant ANG-2 and Tie2 receptor proteins were purified according to the method described in Example 2.

[0425] 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.

[0426] The relevant sequences are as follows:

[0427] (1) Human ANG-2 amino acid sequence with human Fc tag (huANG-2-Fc)

[0428]

[0429] Note: The underlined portion is the full-length sequence of ANG-2 protein, the dotted line is the linker, and the italicized portion is the human IgG1 Fc tag.

[0430] (2) Tie2 amino acid sequence with human Fc tag (huTie2-Fc)

[0431]

[0432]

[0433] Note: The underlined part is the extracellular region of Tie2, and the italicized part is the human IgG1 Fc tag.

[0434] Example 2. Purification of Fc-tagged recombinant proteins or antibodies by Protein A affinity chromatography

[0435] Supernatant samples from cells expressing antibodies or huANG-2-Fc or huTie2-Fc were centrifuged at high speed to remove impurities and then purified using a 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 sample was appropriately concentrated and further purified using a Superdex200 (GE) gel chromatography equilibrated with PBS. The resulting protein was confirmed to be positive by electrophoresis, peptide mapping, and LC-MS before being aliquoted for use.

[0436] Example 3. Construction and identification of cell lines expressing recombinant ANG-2 receptor Tie2

[0437] To screen functional antibodies, the present disclosure constructed a CHO-K1 / Tie2 cell line expressing Tie2.

[0438] The full-length human Tie2 gene was cloned into the mammalian cell expression vector pBABE. HEK293T cells (ATCC, CRL-3216) were co-transfected with the pVSV-G, pGag-pol, and pBABE-Tie2 plasmids to package the virus. 48 hours after transfection, the virus was harvested and infected into CHOK1 cells (ATCC, CRL-9618). 72 hours after infection, the cells were pressure-selected with 10 μg / mL puromycin. After the clones expanded and grew, the cells were digested and assayed for expression using FACS. The positive rate was approximately 40%. Single clones were then isolated to obtain the human Tie2-expressing monoclonal clone 1B11.

[0439] Example 4. Screening and identification of anti-human ANG-2 hybridoma antibodies

[0440] The present disclosure discloses the preparation of a monoclonal antibody against human ANG-2 using hybridoma technology. The resulting antibody has a high affinity for human ANG-2 and can cross-react with cynomolgus macaque ANG-2, can block the binding of ANG-2 to its receptor, and can inhibit ANG-2-induced Tie2 phosphorylation.

[0441] SJL mice were immunized with the recombinant protein huANG-2-Fc (100 / 50 / 50 μg) in TiterMax / Alum / CpG adjuvant. ANG-2-specific immune responses were assessed using serum titers using ELISA and ligand-receptor blocking assays. Mice exhibiting robust specific immune responses were sacrificed, and spleen cells were harvested and fused with myeloma cells.

[0442] Initial screening was performed using ELISA binding assays for human and mouse ANG-2 and an assay to block human ANG-2 binding to its receptor, Tie2. After hybridoma cells were transferred to 24-well plates, the cell supernatants were rescreened using ELISA binding assays for human and mouse ANG-2 and an ELISA-based receptor blocking assay to block ANG-2 binding to its receptor, Tie2. Positive clones screened underwent two rounds of subcloning to obtain hybridoma clones for antibody production and affinity purification.

[0443] The monoclonal hybridoma cell lines HR54 and CP33 with good activity were screened, and the hybridoma cells in the logarithmic growth phase were collected. RNA was extracted with NucleoZol (MN) (according to the kit instructions) and reverse transcribed (PrimeScript TM The cDNA obtained by reverse transcription was amplified by PCR using the Mouse Ig-Primer Set (Novagen, TB326 Rev. B 0503) and then sequenced by a sequencing company. Sequencing yielded the mouse anti-ANG-2 antibodies HR54 and CP33, whose variable region amino acid sequences are as follows:

[0444] >HR54 murine heavy chain variable region sequence:

[0445] EVHLEESGGGLVKPGGSLKLSCAASGFTFS SYVMS WVRQTPEKRLEWVA TINDDSSYTYYPDNVKG RFTISRDNAKSNLYLQMSHLKSEDTAMYYCAR DEATGCFDY WGQGTTLTVSS

[0446] SEQ ID NO: 3

[0447] >HR54 murine light chain variable region sequence:

[0448] NIVMTQSPKSMSISVGERVTLSC KASENVGTYVS WYQQKPDQSPKLLIY GASNRYT GVPDRFTGSGSATDFTLTISSVQAEDLADYHC GQSYINPT FGSGTKLEIK

[0449] SEQ ID NO: 4

[0450] >CP33 murine heavy chain variable region sequence:

[0451] ELQLVESGGNLVRPGGSLKLSCAASGFTFS SYVMS WVRQTPDKRLEWVA TINDDGTYTYYPDNLKG RFTISRDNVNNNLYLQMSHLKSDDTAIYYCAR DHSSSFFDF WGQGTTLTVSS

[0452] SEQ ID NO: 5

[0453] >CP33 murine light chain variable region sequence:

[0454] NIVMTQSPKSMSVSVGERVTLTC TASENVGRYVS WYQQKPDQSPNVLIY GASNRYT GVPDRFTGSGSATDFTLTISSVQAEDLADYHC GQTYINPT FGAGTKLEVK

[0455] SEQ ID NO: 6

[0456] Among them, the underline represents CDR, the italic part represents the linker, and the rest is FR.

[0457] The amino acid sequences of the CDR regions obtained according to the Kabat numbering rules are shown in the following table:

[0458] Table 2. CDR sequences of heavy and light chains of hybridoma clone-derived antibodies

[0459]

[0460] The light and heavy chain variable regions of the above-mentioned murine antibody were connected to the light and heavy chain constant regions of the human antibody (such as the heavy chain constant region shown in SEQ ID NO: 47 and the light chain constant region shown in SEQ ID NO: 48) to form a chimeric antibody. The chimeric antibody corresponding to the HR54 clone was named CHR54, and the same applies to other antibodies.

[0461] Example 5. Humanization of anti-human ANG-2 monoclonal antibodies

[0462] Humanization of mouse monoclonal antibodies can be performed according to methods published in numerous literatures in the field. Briefly, based on the typical VH / VL CDR structures of mouse antibodies, homologous sequences of the light chain variable region (VL) and heavy chain variable region (VH) are searched from the human germline database. A germline with high FR homology is selected as a template, and the CDR region of the mouse antibody is transplanted onto the human template. Certain amino acids in the FR region are backmutated, and the constant region of the mouse antibody is replaced with a human constant region to obtain the final humanized molecule.

[0463] 5.1 Humanization of HR54 Mouse Antibody

[0464] The humanized VH template of HR54 is IGHV3-7*01, and the humanized VL template is IGKV4-1*01. The CDRs of the HR54 mouse antibody were transplanted onto the human template, and the framework region amino acids were backmutated. The specific backmutations are shown in the following table:

[0465] Table 3. Reversion mutations of HR54 antibody

[0466]

[0467] Note: Grafted represents the mouse antibody CDR implanted into the human germline FR sequence. N84S indicates that position 84 was mutated back to S according to the Kabat numbering system.

[0468] The variable region sequence of the humanized mouse antibody HR54 is as follows:

[0469] >huHR54VL1(hu HR54 VL-CDR grafted)

[0470] DIVMTQSPDSLAVSLGERATINC KASENVGTYVS WYQQKPGQPPKLLIY GASNRYT GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC GQSYINPT FGGGTKVEIK

[0471] SEQ ID NO: 17

[0472] >huHR54VL2

[0473] NIVMTQSPDSLAVSLGERATINC KASENVGTYVS WYQQKPGQPPKLLIY GASNRYT GVPDRFSGSGSGTDFTLTISSLQAEDVADYHC GQSYINPT FGGGTKVEIK

[0474] SEQ ID NO: 18

[0475] >huHR54VL3

[0476] NIVMTQSPDSLAVSLGERATINC KASENVGTYVS WYQQKPGQSPKLLIY GASNRYTG VPDRFSGSGSATDFTLTISSLQAEDVADYHC GQSYINPT FGGGTKVEIK

[0477] SEQ ID NO: 19

[0478] >huHR54VH1(huHR54 VH-CDR Grafted)

[0479] EVQLVESGGGLVQPGGSLRLSCAASGFTFS SYVMS WVRQAPGKGLEWVA TINDDSSYTYYPDNVKG RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR DEATGCFDY WGQGTTVTVSS

[0480] SEQ ID NO: 20

[0481] >huHR54VH2

[0482] EVQLVESGGGLVQPGGSLRLSCAASGFTFS SYVMS WVRQAPGKRLEWVA TINDDSSYTYYPDNVKG RFTISRDNAKSSLYLQMSSLRAEDTAVYYCAR DEATGCFDY WGQGTTVTVSS

[0483] SEQ ID NO: 21

[0484] Among them, the underline represents CDR, the italic part represents the linker, and the rest is FR.

[0485] The above-described light and heavy chain variable regions are combined with human germline light chain and heavy constant region sequences to form the final complete light and heavy chain sequences, thereby obtaining a full-length antibody. Unless otherwise specified in this disclosure, when forming a full-length antibody, the heavy chain variable region is linked to the constant region set forth in SEQ ID NO: 47 to form the antibody heavy chain, and the light chain variable region is linked to the constant region set forth in SEQ ID NO: 48 to form the antibody light chain, resulting in a humanized antibody of HR54 (huHR54). The heavy and light chain variable regions of different huHR54 antibodies are shown in the table below.

[0486] Table 4. Variable regions of huHR54 antibody

[0487] variable region huHR54VH1 huHR54VH2 huHR54VL1 huHR54-01 huHR54-04 huHR54VL2 huHR54-02 huHR54-05 huHR54VL3 huHR54-03 huHR54-06

[0488] Note: For huHR54-01 antibody, its heavy chain variable region is huHR54VH1, its light chain variable region is huHR54VL1, its heavy chain constant region is SEQ ID NO: 47, and its light chain constant region is SEQ ID NO: 48, and so on.

[0489] The binding activity of HR54 humanized antibody or chimeric antibody to human ANG-2 was tested by ELISA. The method is as follows:

[0490] After coating the plate with 100 μL of streptavidin (abcam, ab123480) at a concentration of 1 ng / μL overnight at 4°C, the supernatant was removed and the plate was blocked with 250 μL of 5% nonfat dry milk at 37°C for 1 hour. The plate was then washed three times using a plate washer. 0.5 ng / μL biotin-hAng2-His (sinobiologican, 10691-H07H) was then added and incubated at 37°C for 1 hour. The plate was then washed three times using a plate washer. 100 μL of a 1:1 diluted phage supernatant was then added and incubated at 37°C for 1 hour. The plate was then washed three times using a plate washer. 100 μL of a 1:10,000 diluted anti-M13-HRP (GE, 27-9421-01) was then added to each well and incubated at 37°C for 1 hour. The plate was then washed three times using a plate washer. 100 μL of TMB was added to each well for color development. After 5-10 minutes, add 100 μL of 1M H2SO4 to each well to stop color development and measure the OD450 value with a microplate reader. The results are shown in the following table:

[0491] Table 5. ELISA results of antibody binding to human ANG-2

[0492] Antibody EC50 (nM) Antibody 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

[0493] The results showed that both the HR54 chimeric antibody and the humanized antibody disclosed herein can specifically bind to human ANG-2.

[0494] 5.2 Modification of huHR54 Humanized Antibody

[0495] D analysis of HCDR2 of huHR54-04 antibody 52A E or D53N mutations, and D95N, E96Q, C100S or C100V mutations were performed on HCDR3. The CDR sequences of the resulting huHR54-04 mutants are as follows:

[0496] Table 6. CDR sequences of huHR54 mutants

[0497]

[0498]

[0499]

[0500] Exemplarily, the mutated heavy chain variable region of huR54 is as follows:

[0501] >huHR54VH2a

[0502]

[0503] >huHR54VH2b

[0504]

[0505] >huHR54VH2c

[0506]

[0507] >huHR54VH2d

[0508]

[0509] >huHR54VH2e

[0510]

[0511] >huHR54VH2f

[0512]

[0513] >huHR54VH2g

[0514]

[0515]

[0516] >huHR54VH2k

[0517]

[0518] >huHR54VH2m

[0519]

[0520] >huHR54VH2n

[0521]

[0522] Among them, the underline represents CDR, the italic part represents the linker, and the rest is FR.

[0523] The heavy chain variable region and heavy chain constant region of the huHR54-04 mutant were combined to form a complete heavy chain sequence, which was then recombined with the light chain of the huHR54-04 humanized antibody to generate a full-length antibody. Unless otherwise specified herein, when generating a full-length antibody, the heavy chain variable region was linked to the constant region set forth in SEQ ID NO:47 to form the antibody heavy chain, and the light chain variable region was linked to the constant region set forth in SEQ ID NO:48 to form the antibody light chain. The heavy and light chain variable region combinations of the resulting humanized antibody are shown in the table below.

[0524] Table 7. Variable region combinations of huHR54a antibody

[0525] Mutant 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

[0526] Note: For huHR54-07 antibody, its heavy chain variable region is huHR54VH2a, its light chain variable region is huHR54VL1, its heavy chain constant region is SEQ ID NO: 47, and its light chain constant region is SEQ ID NO: 48. Other examples are similar.

[0527] The binding activity of the huHR54 antibody mutants to human ANG-2 was tested by ELISA. The results are shown in the following table:

[0528] Table 8. Binding activity of huHR54 antibody mutants to human ANG-2

[0529] Antibody 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

[0530] The results showed that point mutations in HCDR2 and HCDR3 of the huHR54 antibody did not affect the binding activity of the huHR54 antibody to human ANG-2.

[0531] The CDR regions of the huHR54 humanized antibody can be shown in the following table:

[0532] Table 9. CDR regions of huHR54 humanized antibody

[0533]

[0534] Wherein X1 is D or E, X2 is D or N, X3 is D or N, X4 is E or Q, and X5 is C, S or V.

[0535] 5.3 Humanization of CP33 Mouse Antibody

[0536] The humanized VH template of CP33 is IGHV3-7*01, and the VL template is IGKV4-1*01. The CDRs of CP33 were transplanted onto the humanized templates, and the framework region amino acids were backmutated. The specific backmutations are shown in the table below:

[0537] Table 10. Back mutations of CP33 humanized antibody

[0538]

[0539] Note: Grafted represents the mouse antibody CDR implanted into the human germline FR sequence. N84S indicates that position 84 was mutated back to S according to the Kabat numbering system.

[0540] The variable region sequence of the humanized mouse antibody CP33 is as follows:

[0541] >huCP33VL1(hu CP33 VL-CDR grafted)

[0542] DIVMTQSPDSLAVSLGERATINC TASENVGRYVS WYQQKPGQPPKLLIY GASNRYT GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC GQTYINPT FGGGTKVEIK

[0543] SEQ ID NO:40

[0544] >huCP33VL2

[0545] NIVMTQSPDSLAVSLGERATINC TASENVGRYVS WYQQKPGQPPKVLIY GASNRYT GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC GQTYINPT FGGGTKVEIK

[0546] SEQ ID NO:41

[0547] >huCP33VL3

[0548] NIVMTQSPDSLAVSLGERATINC TASENVGRYVS WYQQKPGQPPKVLIY GASNRYT GVPDRFSGSGSGTDFTLTISSLQAEDVADYHC GQTYINPT FGGGTKVEIK

[0549] SEQ ID NO:42

[0550] >huCP33VL4

[0551] NIVMTQSPDSLAVSLGERATINC TASENVGRYVS WYQQKPGQSPKVLIY GASNRYT GVPDRFSGSGSATDFTLTISSLQAEDVADYHC GQTYINP TFGGGTKVEIK

[0552] SEQ ID NO:43

[0553] >huCP33VH1(huCP33 VH-CDR Grafted)

[0554] EVQLVESGGGLVQPGGSLRLSCAASGFTFS SYVMS WVRQAPGKGLEWVA TINDDGTYTYYPDNLKG RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR DHSSSFFDF WGQGTTVTVSS

[0555] SEQ ID NO: 44

[0556] >huCP33VH2

[0557] EVQLVESGGGLVQPGGSLRLSCAASGFTFS SYVMS WVRQAPGKGLEWVA TINDDGTYTYYPDNLKG RFTISRDNVKNSLYLQMSSLRAEDTAVYYCAR DHSSSFFDF WGQGTTVTVSS

[0558] SEQ ID NO: 45

[0559] >huCP33VH3

[0560] ELQLVESGGGLVQPGGSLRLSCAASGFTFS SYVMS WVRQAPGKRLEWVA TINDDGTYTYYPDNLKG RFTISRDNVKNSLYLQMSSLKAEDTAVYYCAR DHSSSFFDF WGQGTTVTVSS

[0561] SEQ ID NO: 46

[0562] Among them, the underline represents CDR, the italic part represents the linker, and the rest is FR.

[0563] The above-described light and heavy chain variable regions are combined with human light and heavy constant region sequences to form the final complete light and heavy chain sequences, thereby obtaining a full-length antibody. Unless otherwise specified in this disclosure, when forming a full-length antibody, the heavy chain variable region is linked to the constant region set forth in SEQ ID NO: 47 to form the antibody heavy chain, and the light chain variable region is linked to the constant region set forth in SEQ ID NO: 48 to form the antibody light chain, resulting in a humanized antibody to CP33 (huCP33). The heavy and light chain variable regions of different huCP33 antibodies are shown in the table below.

[0564] Table 11. huCP33 antibody variable regions

[0565] variable region 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

[0566] Note: For huCP33-01 antibody, its heavy chain variable region is huCP33VH1, its light chain variable region is huCP33VL1, its heavy chain constant region is SEQ ID NO: 47, and its light chain constant region is SEQ ID NO: 48, and so on.

[0567] The binding activity of huCP33 antibody and chimeric antibody to human ANG-2 was tested by ELISA. The results are shown in the following table:

[0568] Table 12. ELISA results of ANG-2 antibody binding to human ANG-2

[0569] Antibody EC50 (nM) Antibody EC50 (nM) Antibody 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

[0570] The results showed that the affinity of the CP33 humanized antibody to human ANG-2 was comparable to that of ChCP33, and both could specifically recognize human ANG-2.

[0571] 5.4. Constant Region and Full-Length Antibody Sequences of Anti-ANG-2 Antibodies

[0572] The heavy and light chain constant regions of exemplary antibodies are as follows:

[0573] Heavy chain constant region:

[0574] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0575] SEQ ID NO: 47

[0576] Light chain constant region:

[0577] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0578] SEQ ID NO: 48

[0579] The sequences of exemplary huHR54 full-length antibodies and huCP33 full-length antibodies are as follows:

[0580] (a) huHR54-01 antibody

[0581] huHR54-01 heavy chain:

[0582]

[0583] huHR54-01 light chain:

[0584]

[0585] (b) huHR54-04 antibody

[0586] huHR54-04 heavy chain:

[0587]

[0588]

[0589] huHR54-04 light chain:

[0590]

[0591] (c) huHR54-12 antibody

[0592] huHR54-12 heavy chain:

[0593]

[0594] huHR54-12 light chain:

[0595]

[0596] (d) huCP33-01 antibody

[0597] huCP33-01 antibody heavy chain:

[0598]

[0599]

[0600] huCP33-01 antibody light chain:

[0601]

[0602] (e) huCP33-05 antibody

[0603] huCP33-05 heavy chain:

[0604]

[0605] huCP33-05 light chain:

[0606]

[0607] The dashed line represents CDR, the rest represents FR, and the dot-dashed line represents the constant region.

[0608] Example 6. Preparation and identification of anti-VEGF / ANG-2 bispecific antibodies

[0609] The present disclosure constructs different formats of bispecific antibodies against VEGF and ANG-2, including but not limited to IgG-scFv, DVD, and crosmab formats.

[0610] The anti-VEGF antibody can be any currently known antibody against VEGF, such as Avastin, RAZUMAB (Axxiom Inc), GNR-011 (Affitech A / S), R-TPR-024 (Reliance Life Sciences Group), ramucirumab (ImClone Systems), and the like.

[0611] An exemplary antibody is Genentech's marketed Fab antibody, 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 this anti-VEGF antibody is a complete IgG1 heavy chain composed of the heavy chain variable region of Lucentis (sequence shown in SEQ ID NO: 58) combined with different human IgG1 constant regions. The specific sequence is as follows:

[0612] (1) Light chain variable region of ranibizumab

[0613] DIQLTQSPSSLSASVGDRVTITC SASQDISNYLN WYQQKPGKAPKVLIY FTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQYSTVPWT FGQGTKVEIK

[0614] SEQ ID NO:56

[0615] (2) Light chain of ranibizumab

[0616]

[0617] (3) Heavy chain variable region of ranibizumab

[0618] EVQLVESGGGLVQPGGSLRLSCAASGYDFT HYGMN WVRQAPGKGLEWVG WINTYTGEPTYAADFKR RFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAK YPYYYGTSHWYFDV WGQGTLVTVSS

[0619] SEQ ID NO:58

[0620] (4) Ranibizumab heavy chain variable region + IgG1 constant region (herein referred to as Ranibizumab heavy chain 1)

[0621]

[0622] (5) Ranibizumab heavy chain variable region + IgG1 constant region variant (herein referred to as Ranibizumab heavy chain 2)

[0623]

[0624]

[0625] Among them, the underline represents CDR, the italic part represents the linker, and the rest is FR.

[0626] Table 13. Ranibizumab CDR region sequences

[0627]

[0628] To prepare a bispecific antibody with an IgG-scFv configuration, the heavy and light chain variable regions of the screened anti-ANG-2 antibodies were linked via a linker to produce an anti-ANG-2 single-chain antibody. The linker can be any known linker in the art, with an exemplary linker being (GGGGS)n, where n is an integer between 1 and 10. An exemplary ANG-2 single-chain antibody sequence is shown below:

[0629] huHR54-01-scFv

[0630] EVQLVESGGGLVQPGGSLRLSCAASGFTFS SYVMS WVRQAPGKGLEWVA TINDDSSYTYYPDNVKG RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR DEATGCFDY WGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINC KASENVGTYVS WYQQKPGQPPKLLIY GASNRYT GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC GQSYINPT FGGGTKVEIK

[0631] SEQ ID NO: 67

[0632] huHR54-04-scFv

[0633] EVQLVESGGGLVQPGGSLRLSCAASGFTFS SYVMS WVRQAPGKRLEWVA TINDDSSYTYYPDNVKG RFTISRDNAKSSLYLQMSSLRAEDTAVYYCAR DEATGCFDY WGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINC KASENVGTYVS WYQQKPGQPPKLLIY ​ GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC ​ FGGGTKVEIK

[0634] SEQ ID NO: 68

[0635] huHR54-12-scFv:

[0636]

[0637] huCP33-01-scFv:

[0638]

[0639] huCP33-05-scFv:

[0640]

[0641] Among them, the underlined parts indicate CDRs, the italicized parts indicate linkers, and the remaining parts are FRs.

[0642] In addition, cysteine ​​mutations were introduced into the variable regions of the heavy and light chains of the anti-ANG-2 single-chain antibody. Exemplary sequences are as follows:

[0643] huHR54VH2-C

[0644] EVQLVESGGGLVQPGGSLRLSCAASGFTFS ​ WVRQAPGKCLEWVA ​ RFTISRDNAKSSLYLQMSSLRAEDTAVYYCAR ​ WGQGTTVTVSS

[0645] SEQ ID NO: 72

[0646] huHR54VH2f-C

[0647] EVQLVESGGGLVQPGGSLRLSCAASGFTFS ​ WVRQAPGKCLEWVA ​ RFTISRDNAKSSLYLQMSSLRAEDTAVYYCAR ​ WGQGTTVTVSS

[0648] SEQ ID NO: 73

[0649] huHR54VL1-C

[0650] DIVMTQSPDSLAVSLGERATINC ​ WYQQKPGQPPKLLIY ​ GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC ​ FGCGTKVEIK

[0651] SEQ ID NO: 74

[0652] huCP33VH2-C

[0653] EVQLVESGGGLVQPGGSLRLSCAASGFTFS ​ WVRQAPGKCLEWVA ​ RFTISRDNVKNSLYLQMSSLRAEDTAVYYCAR ​ WGQGTTVTVSS

[0654] SEQ ID NO: 75huCP33VL1-C

[0655] DIVMTQSPDSLAVSLGERATINC ​ WYQQKPGQPPKLLIY ​ GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC ​ FGCGTKVEIK

[0656] SEQ ID NO: 76

[0657] The underline represents CDR, and the rest are FR.

[0658] Exemplary, the sequence of an anti-ANG-2 single-chain antibody is as follows:

[0659] huHR54-04-scFv-CC:

[0660]

[0661] huHR54-12-scFv-CC

[0662]

[0663] huCP33-05-scFv-CC

[0664]

[0665] Among them, the underline represents CDR, the italic part represents the linker, and the rest is FR.

[0666] The anti-ANG-2 single-chain antibody was directly (via a peptide bond) or indirectly (via a linker) linked to the heavy chain or light chain of an anti-VEGF antibody through genetic recombination technology and expressed using the 293 expression system to obtain a bispecific antibody.

[0667] For example, 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 via a linker (e.g., (GG)n, where n is 1-20) to obtain a bispecific antibody, the sequence of which is shown below:

[0668] (1) Bispecific antibody 1

[0669] > Bispecific antibody 1 first chain:

[0670]

[0671] The wavy underlined portion represents the HCDR of the anti-VEGF antibody, the dotted underline represents the constant region, the double underline represents the linker, the italic portion represents the ANG2-scFv, and the underlined portion represents the CDR of the ANG2-scFv.

[0672] >Bispecific antibody 1 second chain:

[0673]

[0674] The dotted underline indicates the constant region, and the underlined portion indicates the CDR.

[0675] (2) Bispecific Antibody 2

[0676] > Bispecific antibody 2 first chain:

[0677]

[0678]

[0679] The wavy underlined portion represents the HCDR of the anti-VEGF antibody, the dotted underline represents the constant region, the double underline represents the linker, the italic portion represents the ANG2-scFv, and the underlined portion represents the CDR of the ANG2-scFv.

[0680] > Bispecific antibody 2 second chain:

[0681]

[0682] The dotted underline indicates the constant region, and the underlined portion indicates the CDR.

[0683] (3) Bispecific Antibody 3

[0684] > Bispecific antibody 3 first chain:

[0685]

[0686]

[0687] The wavy underlined portion represents the HCDR of the anti-VEGF antibody, the dotted underline represents the constant region, the double underline represents the linker, the italic portion represents the ANG2-scFv, and the underlined portion represents the CDR of the ANG2-scFv.

[0688] > Bispecific antibody 3 second chain:

[0689]

[0690] The dotted underline indicates the constant region, and the underlined portion indicates the CDR.

[0691] (4) Bispecific Antibody 4

[0692] > Bispecific antibody 4 first chain:

[0693]

[0694] The wavy underlined portion represents the HCDR of the anti-VEGF antibody, the dotted underline represents the constant region, the double underline represents the linker, the italic portion represents the ANG2-scFv, and the underlined portion represents the CDR of the ANG2-scFv.

[0695] > Bispecific antibody 4 second chain:

[0696]

[0697]

[0698] The dotted underline indicates the constant region, and the underlined portion indicates the CDR.

[0699] At the same time, using the anti-ANG-2 antibody and VEGF antibody obtained through screening, the present disclosure constructs a bispecific antibody in the form of Crossmab. The sequence of an exemplary bispecific antibody in the form of Crossmab is as follows:

[0700] (5) Bispecific Antibody 5

[0701] > Bispecific antibody 5 first heavy chain: anti-ANG-2 heavy chain

[0702]

[0703] > Bispecific antibody 5 first light chain: anti-ANG-2 light chain

[0704]

[0705] > Bispecific antibody 5 second heavy chain: anti-VEGF heavy chain

[0706]

[0707] > Bispecific antibody 5 second light chain: anti-VEGF light chain

[0708]

[0709] The dotted underline indicates the constant region, and the underlined portion indicates the CDR.

[0710] (6) Bispecific Antibody 6

[0711] > Bispecific antibody 6 first heavy chain: anti-ANG-2 heavy chain

[0712]

[0713] > Bispecific antibody 6 first light chain: anti-ANG-2 light chain

[0714]

[0715] > Bispecific antibody 6 second heavy chain: anti-VEGF heavy chain

[0716]

[0717] > Bispecific antibody 6 second light chain: anti-VEGF light chain

[0718]

[0719] The dotted underline indicates the constant region, and the underlined portion indicates the CDR.

[0720] According to the purification method in Example 2, protein A affinity chromatography was used for purification to obtain a bispecific antibody molecule with a purity of >98%.

[0721] This disclosure also uses RG7716, Avastin, and RG7221 (vanucizumab) as control molecules, whose sequences are shown below:

[0722] >RG7716 anti-ANG-2 heavy chain:

[0723] QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARSPNPYYYDSSGYYYPGA FDIWGQGTMVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGECDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLAQDWLNGKEYKCKVSNKALGAP IEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGK

[0724] SEQ ID NO: 89

[0725] >RG7716 anti-ANG-2 light chain:

[0726] SYVLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHWVFGGGTKLTVLSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC

[0727] SEQ ID NO: 90

[0728] >RG7716 anti-VEGF heavy chain

[0729] EVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLAQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGK

[0730] SEQ ID NO: 91

[0731] >RG7716 anti-VEGF light chain

[0732] DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0733] SEQ ID NO: 92

[0734] > Bevacizumab heavy chain

[0735] EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0736] SEQ ID NO: 93

[0737] > Bevacizumab light chain

[0738] DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0739] SEQ ID NO: 94

[0740] >RG7221 anti-ANG2 heavy chain

[0741] ​ ​ ASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0742] SEQ ID NO: 95

[0743] >RG7221 anti-ANG2 light chain <>

[0744] ​ ​ ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC

[0745] SEQ ID NO: 96

[0746] >RG7221 anti-VEGF heavy chain

[0747] ​ ​ ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0748] SEQ ID NO: 97

[0749] >RG7221 anti-VEGF light chain

[0750] DIQMTQSPSSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGT DFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0751] SEQ ID NO: 98

[0752] In addition, the negative control (NC) used in this disclosure refers to an IgG monoclonal antibody against HIV.

[0753] In vitro biological evaluation of activity

[0754] Test Example 1. Biacore determination of the affinity of VEGF / ANG-2 bispecific antibody to VEGF / ANG-2 of different species

[0755] The affinity of the VEGF / ANG-2 bispecific antibody to human, monkey, and mouse VEGF and ANG-2 was determined using Biacore T200 (GE).

[0756] The antibody was affinity-captured using a Protein A biosensor chip. Antigens (human VEGF (R&D, 293-VE), monkey VEGF (Sinobiological, 11066), mouse VEGF (Sinobiological, 51059), human ANG-2 (Sinobiological, 10691-H08H), monkey ANG-2 (Sinobiological, 90026-C07H), and mouse ANG-2 (Sinobiological, 50298-M07H)) were then passed over the chip surface. Binding and dissociation curves were obtained using a Biacore T200 instrument for real-time signal monitoring. After each experimental cycle, the biosensor chip was washed and regenerated with 10 mM glycine-HCl regeneration solution (pH 1.5). Data were fitted to a (1:1) Langmuir model using BIA Evaluation Version 4.1 and GE Software. Affinity values ​​are shown in Table 14.

[0757] Table 14. Affinity of bispecific antibodies to ANG-2 from different species

[0758]

[0759] The results showed that bispecific antibody 1, bispecific antibody 5, bispecific antibody 2 and bispecific antibody 3 had high affinity to human and monkey VEGF and ANG-2.

[0760] Test Example 2. ELISA-based antibody blocking experiment of ANG-2 binding to Tie2 receptor

[0761] ANG-2 binds to the ANG-2 receptor Tie2 on the surface of vascular endothelial cells, triggering phosphorylation of the intracellular tyrosine kinase Tie2. This in turn transmits signals that cause peripheral cells to detach from the endothelial cells, rendering the blood vessels unstable and prone to proliferation. Therefore, blocking the binding of ANG-2 to Tie2 with antibodies can stabilize blood vessels and inhibit angiogenesis. The results of this experiment demonstrate that bispecific antibodies can block ANG-2 binding to the extracellular domain of the recombinantly expressed Tie2 protein.

[0762] Specific method: ELISA plates were coated with Tie2-Fc (SEQ ID NO: 2, 3 μg / mL in PBS, 100 μL / well) overnight at 4°C. After removing the coating solution, 200 μL / well of 5% skim milk blocking solution diluted in PBS was added and the plates were incubated at 37°C for 2 hours. After blocking, the blocking solution was discarded and the plates were washed five times with PBST buffer (PBS containing 0.05% Tween-20, pH 7.4). Then, 50 μL of huANG-2-Fc (SEQ ID NO: 1, bio-huANG-2-Fc) labeled with 1% BSA (Dongren Chemical, LK03) was added, with a final concentration of 0.15 μg / mL, and 50 μL of the test antibody (starting at 10 μg / mL, diluted 3-fold). After mixing, the plates were incubated at 37°C for 15 minutes. The plates were then added to the ELISA plates and incubated at 37°C for 1 hour. After incubation, the reaction solution in the ELISA plate was discarded, and the plate was washed five times with PBST. 100 μL / well of a 1:4000 dilution of streptavidin-peroxidase polymer (Sigma, S2438-250UG) was added and incubated at 37°C for 1 hour. After washing the plate five times with PBST, 100 μL / well of TMB chromogenic substrate (KPL, 52-00-03) was added and incubated at room temperature for 3-10 minutes. The reaction was terminated by adding 100 μL / well of 1 M H2SO4. The absorbance was read at 450 nm using a NOVOStar microplate reader, and the IC50 value of the antibody blocking the binding of ANG-2 to Tie2 was calculated.

[0763] The results showed that both bispecific antibodies could strongly inhibit the binding of ANG-2 to Tie2 (see Table 15 and Figure 1 ).

[0764] Table 15. Results of bispecific antibodies inhibiting the binding of ANG-2 to Tie2

[0765]

[0766] Test Example 3. Bispecific Antibody Inhibits ANG-2-Induced Tie2 Phosphorylation

[0767] The stable transgenic strain CHO-Tie2#1B11-1 overexpressing huTie2 was digested and resuspended, and the density was adjusted to 2.5×10 5 cells / mL, 100 μL / well was plated into a 96-well cell culture plate, i.e. 2.5×10 4100 μL / well of anti-huTie2 capture (R&D Systems, Cat#DYC2720E) was added to the plate. After 4-5 hours, the medium was changed (DME / F-12, HyClone, Cat#SH30023.01 + 0.1% BSA + 20 μg / mL puromycin, Gibco, Cat#A1113803) and starved overnight. The plate was coated with 100 μL / well of anti-huTie2 capture (R&D Systems, Cat#DYC2720E) at 4.0 μg / mL and incubated overnight at room temperature. The coating solution was removed and the plate was blocked with 250 μL / well of 1% BSA + 0.05% NaN3 blocking solution at room temperature for 2 hours. Mix 25 μL of huANG-2-Fc (final concentration 2.5 μg / mL) and 25 μL of the test antibody (3-fold dilution, maximum concentration 50.0 nM) in equal volumes. Incubate at 37°C for 15 minutes. Then add Na₃VO₄ (1.0 mM, Sigma, Cat#S6508) and mix thoroughly. For overnight cell cultures, discard 50 μL of the culture supernatant and add 50 μL of the prepared antigen-antibody mixture. Incubate at 37°C for 10 minutes. Wash twice with 200 μL / well of wash buffer (PBS + 2.0 mM Na₃VO₄). Add 90 μL of lysis buffer (1× lysis buffer + 10 μg / mL leupeptin hemisulfate (Tocris, Cat#1167) + 10.0 μg / mL aprotininin, Sigma, Cat#SRE0050) and lyse on ice for 10-15 minutes. Centrifuge at 4000 g for 5 minutes. Collect cell lysate and add to a blocked ELISA plate. Incubate at room temperature for 2 hours. Wash the plate five times with PBST and add a 1:1000 dilution of secondary antibody, anti-PY-HRP (R&D Systems, Cat# DYC2720E), and incubate at room temperature for 1–2 hours. Wash the plate five times with PBST and develop color with TMB for 15–30 minutes. Stop the color development with 1 M H₂SO₄. Read the OD₄50 value using a Versa Max microplate reader, and calculate the IC₅₅₀ value.

[0768] Results (see Figure 2 ) showed that the bispecific antibody exhibited a strong ability to inhibit ANG-2-mediated Tie2 phosphorylation.

[0769] Table 16. Results of bispecific antibody inhibition-induced Tie2 phosphorylation

[0770]

[0771] Test Example 4. Bispecific Antibody Inhibits VEGF-Induced VEGFR Phosphorylation

[0772] VEGF binds to VEGFR on vascular endothelial cells, phosphorylating the VEGFR intracellular domain, promoting endothelial cell proliferation and the formation of new blood vessels, thereby promoting tumor cell growth and metastasis. This experiment was used to identify bispecific antibodies that can block VEGF-induced VEGFR phosphorylation.

[0773] Specifically, HUVEC cells (PromoCell / Miaotong Biotechnology, C-12205) were digested and the cell density was adjusted to 1.5×10 cells per 500 μL using complete culture medium. 5 Cells were plated in 24-well plates, 500 μL per well. After overnight incubation at 37°C, the medium was discarded and the cells were washed once with 500 μL of ice-cold DPBS (Gibco, 14190-250). Each well was starved for 30 minutes by adding 200 μL of minimal medium containing 0.1% BSA. The test antibody and negative control antibody were diluted to 10 nM, 1 nM, and 0.1 nM (RG7221 and bispecific antibody 5 were diluted to 20 nM, 2 nM, and 0.2 nM) in minimal medium. PBS was used as a control (ctrl). VEGF (R&D system, Cat#293-VE) was diluted to 400 ng / mL in minimal medium. An equal volume of diluted VEGF and antibody was mixed, and 200 μL was added to the corresponding wells of the culture plate and incubated at 37°C for 5 minutes. 4× Lysis Buffer #1 (cisbio, 63ADK041PEG) was diluted to 1× with ddH2O. Dilute the blocking buffer 100-fold with 1× lysis buffer to prepare lysis buffer. Remove the cell culture plate, discard the culture medium, add 500 μL of ice-cold PBS, shake gently, and discard the PBS. Immediately add 50 μL of the prepared lysis buffer, place on a shaker, and incubate at room temperature for 30 minutes. Centrifuge at 2400 g for 10 minutes and collect the supernatant. Detect p-VEGFR in the supernatant using the Phospho-VEGFR2 (Tyr1175) kit (cisbio, 63ADK041PEG). To detect p-VEGFR in the supernatant, add 10 μL of phospho-VEGFR2 (Tyr1175) d2 antibody to 200 μL of detection buffer to prepare a working solution. Add 10 μL of phospho-VEGFR2 (Tyr1175) Cryptate antibody to 200 μL of detection buffer to prepare a working solution. Mix equal volumes of d2 antibody working solution and Cryptate antibody working solution. Add 16 μL of cell lysate to an HTRF 96-well microplate, add 4 μL of the d2 antibody and Cryptate antibody mixture, seal the plate with sealing film, centrifuge the microplate for 1 minute, incubate at room temperature in the dark for 4-24 hours, and read the fluorescence value at 340 nm excitation and 665 nm and 620 nm emission wavelengths using a PHERAstar multifunctional microplate reader.

[0774] Data processing: ratio = signal 665nm / signal 620nm×10000, and histogram was drawn using Graphpad Prism 5. The results are shown in Figure 5. Figure 3 shown.

[0775] The results showed that the bispecific antibody could significantly inhibit the increase in the level of intracellular phosphorylated VEGFR in HUVECs caused by VEGF.

[0776] Test Example 5. Bispecific Antibody Inhibits VEGF-Induced HUVEC Proliferation

[0777] VEGF binds to VEGFR on HUVEC, causing phosphorylation of the VEGFR intracellular domain and promoting HUVEC proliferation. This experiment was used to identify bispecific antibodies that can prevent VEGF-induced HUVEC proliferation.

[0778] The specific method is as follows:

[0779] HUVEC cells in the logarithmic growth phase were digested with 0.08% trypsin for about 1-2 minutes at room temperature and terminated by adding 10% FBS. The digested HUVECs were collected, centrifuged at 800 rpm / min for 5 minutes, and washed three times with PBS to remove the cytokines in the culture medium that stimulated HUVEC proliferation (800 rpm / min, centrifuged for 5 minutes). The HUVEC cells were resuspended in 6% FBS culture medium, and after cell counting, they were seeded in a white 96-well cell culture plate at 4000 cells / 50 μL / well and cultured in an incubator for 2 hours. The starting concentration of VEGF was adjusted to 300 ng / mL, and 120 μL / well was added to a sterile 96-well plate. The antibody to be tested was diluted stepwise: the starting concentration of the antibody was 600 nM, and a 4-fold gradient dilution was performed. An equal volume of the diluted antibody was added to the above 96-well plate and incubated at room temperature for 30 minutes. 100 μL / well of the incubated antibody and VEGF mixture was added to the attached HUVEC cells and cultured in an incubator for 5 days. After the culture was completed, the antibody was added (G7573, PROMEGA), 50 μL / well, incubate at room temperature in the dark for 10 min, and detect using the Luminescence program of Cytation5 cell imager. Figure 4 shown.

[0780] The results showed that the bispecific antibody could significantly inhibit the proliferation of HUVEC induced by VEGF.

[0781] Table 17. Results of bispecific antibodies inhibiting HUVEC proliferation

[0782]

[0783] In vivo biological evaluation

[0784] Test Example 6: Efficacy of bispecific antibodies in a subcutaneous prostate cancer cell transplant model in mice

[0785] 5×10 6 PC-3 cells (ATCC) were inoculated subcutaneously in the right rib of Balb / c nude mice. When the tumor volume of the tumor-bearing mice reached 100 mm 3 When the mice reached 1000 mm, they were randomly divided into 6 groups, each with 8 mice. The day of grouping was defined as day 0, and equimolar amounts of each antibody were injected intraperitoneally starting on the day of grouping. The injection was repeated twice a week for a total of 6 doses. Tumor volume and animal weight were monitored and recorded twice a week. When the tumor volume exceeded 1000 mm, the mice were randomly divided into 6 groups, each with 8 mice. 3 The experimental endpoint was euthanasia of the tumor-bearing animals when most tumors ruptured or when the body weight decreased by 20%. All data were plotted and statistically analyzed using Excel and GraphPad Prism 5 software.

[0786] The formula for calculating tumor volume (V) is: V = 1 / 2 × a × b 2 , where a and b represent length and width respectively.

[0787] Relative tumor growth rate 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.

[0788] Tumor inhibition rate TGI (%) = 1-T / C (%).

[0789] The grouping and dosing regimen are shown in Table 18, and the tumor growth curve and tumor inhibition rate are shown in Figure 5 and Table 19.

[0790] Table 18. Grouping and dosing regimen

[0791] Group Test substance Dosage (mg / kg) Dosage regimen 1 PBS (solvent) 3 ipBIW*3 weeks 2 Avastin 3 ipBIW*3 weeks 3 RG7221 6 ipBIW*3 weeks 4 Bispecific antibody 5 6 ipBIW*3 weeks 5 Bispecific Antibody 2 3.75 ipBIW*3 weeks

[0792] Table 19. Tumor inhibition rate results

[0793]

[0794]

[0795] The results showed that both bispecific antibody 5 and bispecific antibody 2 disclosed herein could significantly inhibit the growth of PC-3 tumors. The tumor inhibition effect of bispecific antibody 5 was significantly better than that of Avastin monoclonal antibody and RG7221 bispecific antibody.

[0796] Test Example 7: Efficacy of a bispecific antibody in a BALB / c nude mouse subcutaneous transplant tumor model of highly metastatic non-small cell lung cancer H460-Luc cell line

[0797] This study evaluated the efficacy of ANG-2 / VEGF bispecific antibody in inhibiting the growth and metastasis of human non-small cell lung cancer H460 xenografts after intraperitoneal injection.

[0798] Female BALB / c nude mice, 4-5 weeks old, weighing 18-20 g, were purchased from Shanghai Lingchang Biotechnology Co., Ltd. Human non-small cell lung cancer H460-Luc cells (stable transfected with the luciferase gene) were cultured in RPMI 1640 medium supplemented with 10% FBS. The cells were cultured for five consecutive passages and inoculated subcutaneously in mice. Mice were anesthetized with 3-4% isoflurane prior to inoculation. Approximately 1×10 6 H460-Luc cells were resuspended in serum-free culture medium and Matrigel suspension (culture medium: Matrigel = 50%: 50%) and inoculated into mice by subcutaneous injection with an inoculation volume of 200 μL. When the tumors grew to an average of about 100-150 mm 3 At about 14 days, the animals were randomly divided into 3 groups, with 8 animals in each group. The day of group administration was defined as day 0. The grouping and administration schedule are shown in Table 20.

[0799] Table 20. Grouping, dosing regimen and tumor inhibition rate

[0800]

[0801] After grouping, tumor volume was measured twice a week for 3 consecutive weeks. The tumor volume (V) was calculated as follows:

[0802] V=(length×width 2 ) / 2.

[0803] The relative tumor volume (RTV) of each mouse was calculated as follows:

[0804] RTV = Vt / V0, where Vt is the daily measured volume and V0 is the volume at the start of treatment.

[0805] At the end of the experiment, all tumor-bearing animals were photographed, and all tumors were removed, weighed, and photographed.

[0806] Statistical analysis

[0807] The results are presented as mean ± SEM. Comparisons between the two groups were performed using Dunnett's multiple comparison test. Statistically significant differences were considered if p < 0.05.

[0808] The test results are as follows Figure 6 shown.

[0809] Figure 6 It was shown that both the bispecific antibody 1 and the bispecific antibody 2 disclosed herein can significantly inhibit tumor growth.

[0810] Test Example 8: Efficacy of bispecific antibodies in a subcutaneous transplant tumor model of human skin cancer cells in mice

[0811] A431 cells (ATCC) 2×10 6 Cells / mouse / 100 μL were inoculated subcutaneously in the right rib of Balb / c nude mice. When the tumor volume of the tumor-bearing mice reached 100 mm 3 At approximately 14 days, mice were randomly divided into three groups: vehicle (PBS), bispecific antibody 24 mpk, and bispecific antibody 5 6 mpk, with 8 mice in each group. The day of grouping was defined as day 0, and each antibody was injected intraperitoneally starting on the day of grouping. The injection was repeated twice a week for a total of 6 times. Tumor volume and animal weight were monitored and recorded twice a week. When the tumor volume exceeded 1000 mm 3 The experimental endpoint was euthanasia of the tumor-bearing animals when most tumors ruptured or when the body weight decreased by 20%. All data were plotted and statistically analyzed using Excel and GraphPad Prism 5 software. The tumor volume (V) was calculated as follows:

[0812] V=1 / 2×a×b 2 , where a and b represent length and width respectively.

[0813] Relative tumor growth rate 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.

[0814] Tumor inhibition rate TGI (%) = 1-T / C (%).

[0815] The grouping and dosing regimen are shown in Table 21, and the tumor growth curve is shown in Figure 7 shown.

[0816] Table 21. Grouping and dosing regimen

[0817] Group Test substance Dosage (mg / kg) Dosage regimen 1 PBS 3 ipBIW*3 weeks 2 Bispecific antibody 5 6 ipBIW*3 weeks 3 Bispecific Antibody 2 4 ipBIW*3 weeks

[0818] See the results Figure 7 , showing that both bispecific antibody 2 and bispecific antibody 5 in the present disclosure can significantly inhibit the growth of A431 tumors.

[0819] Test Example 9. Testing of the Inhibitory Function of Bispecific Antibodies on Laser-Induced Choroidal Neovascularization in Rhesus Monkeys

[0820] Administer the drug through intravitreal injection, and detect its effect on laser-induced choroidal neovascularization leakage and growth in rhesus monkeys, so as to verify that the bispecific antibody can be used for the treatment of diseases such as age-related macular degeneration (AMD) through intravitreal injection. The specific methods are as follows:

[0821] Induce choroidal neovascularization in the fundus of rhesus monkeys by laser photocoagulation around the fovea centralis of the fundus macula, and establish an animal model similar to human choroidal neovascularization. Perform fluorescein fundus angiography before and 20 days after photocoagulation to determine the success of the model. Select 16 rhesus monkeys (Sichuan Greenhouse Biotechnology Co., Ltd., production license number: SCXK (Sichuan) 2014-013, experimental animal quality certificate number: No: 0022202) with successful model establishment. Randomly divide the rhesus monkeys into a solvent control group, a ranibizumab-IgG1 (Lucentis, 96 μg, 2 μM) group, an RG7716 (292 μg, 2 μM) group, and a bispecific antibody 3 (396 μg, 2 μM) group, a total of 4 groups, with 4 monkeys in each group.

[0822] On the 21st day after photocoagulation, in the Lucentis group, RG7716 group, and bispecific antibody 3 group, 50 μL of Lucentis with a concentration of 1.92 mg / mL, RG7716 with a concentration of 5.84 mg / mL, and bispecific antibody 3 with a concentration of 7.92 mg / mL were intravitreally injected into both eyes at 96 μg, 292 μg, and 396 μg / eye respectively. The solvent control group was given an equal volume of solvent. The intraocular pressure, fundus color photography, fluorescein fundus angiography, and optical coherence tomography (OCT) were performed on the animals in each group at 7, 14, and 28 days after administration to observe the inhibitory effect of the antibody on choroidal neovascularization. At 28 days after administration, 100 - 200 μL of aqueous humor was taken, and 100 μL was aliquoted for the determination of aqueous humor VEGF. On the 29th day after administration, after euthanasia, 3 eyeballs were taken from each group for HE staining histological examination. The results are as follows:

[0823] AMD model establishment

[0824] Twenty days after laser model establishment, in the fundus color photography of both eyes of the 16 monkeys included in the trial, 9 laser spots could be seen around the macula in each eye. High fluorescence could be seen around the fundus macula, obvious fluorescein leakage, and the leakage exceeded the edge of the spot. Twenty days after laser model establishment (before administration), the number of grade 4 fluorescence spots in the solvent control group, Lucentis group, RG7716 group, and bispecific antibody 3 group were 46, 42, 40, and 45 respectively. The above changes were similar to the changes of clinical choroidal neovascularization (CNV), indicating successful model establishment.

[0825] Fluorescein angiography examination

[0826] The fluorescent spot areas of the Lucentis, RG7716, and bispecific antibody groups 3 all showed a certain degree of reduction 7, 14, and 28 days after administration. The improvement rate of the fluorescent leakage area and the reduction in the fluorescein leakage area in each group were superior to those in the vehicle control group. The number of grade 4 fluorescent spots in each group was significantly lower than that in the vehicle control group.

[0827] After 28 days, the 2μM bispecific antibody group 3, the 2μM ranibizumab group, and the 2μM RG7716 group all reduced the fluorescence leakage area and the number of fluorescent spots. The results are shown in Figure 8A and 8B .

[0828] Aqueous humor VEGF

[0829] The expression of VEGF in aqueous humor of Lucentis group, RG7716 group and bispecific antibody group 3 was significantly lower than that in vehicle control group 28 days after administration. The expression of VEGF in aqueous humor of bispecific antibody group 3 was significantly lower than that in Lucentis group and RG7716 group. Figure 9 .

[0830] In summary, under the experimental conditions of this study, rhesus monkeys with a laser CNV model were given a single intravitreal injection of bispecific antibody 3 at a dose of 396 μg / eye. Retinal vascular fluorescence angiography, optical coherence tomography, aqueous humor VEGF, and ocular histopathology examinations showed that bispecific antibody 3 had a significant inhibitory effect on monkey CNV at a dose of 396 μg / eye. Sequence Listing <110> Jiangsu Hengrui Medicine Co., Ltd. Shanghai Hengrui Medicine Co., Ltd. <120> Bispecific antigen-binding molecules specifically binding to VEGF and ANG-2 <130> 721109CPCT <150> CN202010980869.2 <151> 2020-09-17 <160> 98 <170> SIPOSequenceListing 1.0 <210> 1 <211> 723 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <223> Human ANG-2 amino acid sequence with an Fc tag <400> 1 Tyr Asn Asn Phe Arg Lys Ser Met Asp Ser Ile Gly Lys Lys Gln Tyr 1 5 10 15 Gln Val Gln His Gly Ser Cys Ser Tyr Thr Phe Leu Leu Pro Glu Met 20 25 30 Asp Asn Cys Arg Ser Ser Ser Ser Pro Tyr Val Ser Asn Ala Val Gln 35 40 45 Arg Asp Ala Pro Leu Glu Tyr Asp Asp Ser Val Gln Arg Leu Gln Val 50 55 60 Leu Glu Asn Ile Met Glu Asn Asn Thr Gln Trp Leu Met Lys Leu Glu 65 70 75 80 Asn Tyr Ile Gln Asp Asn Met Lys Lys Glu Met Val Glu Ile Gln Gln 85 90 95 Asn Ala Val Gln Asn Gln Thr Ala Val Met Ile Glu Ile Gly Thr Asn 100 105 110 Leu Leu Asn Gln Thr Ala Glu Gln Thr Arg Lys Leu Thr Asp Val Glu 115 120 125 Ala Gln Val Leu Asn Gln Thr Thr Arg Leu Glu Leu Gln Leu Leu Glu 130 135 140 His Ser Leu Ser Thr Asn Lys Leu Glu Lys Gln Ile Leu Asp Gln Thr 145 150 155 160 Ser Glu Ile Asn Lys Leu Gln Asp Lys Asn Ser Phe Leu Glu Lys Lys 165 170 175 Val Leu Ala Met Glu Asp Lys His Ile Ile Gln Leu Gln Ser Ile Lys 180 185 190 Glu Glu Lys Asp Gln Leu Gln Val Leu Val Ser Lys Gln Asn Ser Ile 195 200 205 Ile Glu Glu Leu Glu Lys Lys Ile Val Thr Ala Thr Val Asn Asn Ser 210 215 220 Val Leu Gln Lys Gln Gln His Asp Leu Met Glu Thr Val Asn Asn Leu 225 230 235 240 Leu Thr Met Met Ser Thr Ser Asn Ser Ala Lys Asp Pro Thr Val Ala 245 250 255 Lys Glu Glu Gln Ile Ser Phe Arg Asp Cys Ala Glu Val Phe Lys Ser 260 265 270 Gly His Thr Thr Asn Gly Ile Tyr Thr Leu Thr Phe Pro Asn Ser Thr 275 280 285 Glu Glu Ile Lys Ala Tyr Cys Asp Met Glu Ala Gly Gly Gly Gly Trp 290 295 300 Thr Ile Ile Gln Arg Arg Glu Asp Gly Ser Val Asp Phe Gln Arg Thr 305 310 315 320 Trp Lys Glu Tyr Lys Val Gly Phe Gly Asn Pro Ser Gly Glu Tyr Trp 325 330 335 Leu Gly Asn Glu Phe Val Ser Gln Leu Thr Asn Gln Gln Arg Tyr Val 340 345 350 Leu Lys Ile His Leu Lys Asp Trp Glu Gly Asn Glu Ala Tyr Ser Leu 355 360 365 Tyr Glu His Phe Tyr Leu Ser Ser Glu Glu Leu Asn Tyr Arg Ile His 370 375 380 Leu Lys Gly Leu Thr Gly Thr Ala Gly Lys Ile Ser Ser Ile Ser Gln 385 390 395 400 Pro Gly Asn Asp Phe Ser Thr Lys Asp Gly Asp Asn Asp Lys Cys Ile 405 410 415 Cys Lys Cys Ser Gln Met Leu Thr Gly Gly Trp Trp Phe Asp Ala Cys 420 425 430 Gly Pro Ser Asn Leu Asn Gly Met Tyr Tyr Pro Gln Arg Gln Asn Thr 435 440 445 Asn Lys Phe Asn Gly Ile Lys Trp Tyr Tyr Trp Lys Gly Ser Gly Tyr 450 455 460 Ser Leu Lys Ala Thr Thr Met Met Ile Arg Pro Ala Asp Phe Asp Ile 465 470 475 480 Glu Gly Arg Met Asp Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys 485 490 495 Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 500 505 510 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 515 520 525 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 530 535 540 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 545 550 555 560 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 565 570 575 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 580 585 590 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 595 600 605 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 610 615 620 Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 625 630 635 640 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 645 650 655 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 660 665 670 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 675 680 685 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 690 695 700 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys His His His 705 710 715 720 His His His <210> 2 <211> 953 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <223> Tie2 amino acid sequence with human Fc tag <400> 2 Ala Met Asp Leu Ile Leu Ile Asn Ser Leu Pro Leu Val Ser Asp Ala 1 5 10 15 Glu Thr Ser Leu Thr Cys Ile Ala Ser Gly Trp Arg Pro His Glu Pro 20 25 30 Ile Thr Ile Gly Arg Asp Phe Glu Ala Leu Met Asn Gln His Gln Asp 35 40 45 Pro Leu Glu Val Thr Gln Asp Val Thr Arg Glu Trp Ala Lys Lys Val 50 55 60 Val Trp Lys Arg Glu Lys Ala Ser Lys Ile Asn Gly Ala Tyr Phe Cys 65 70 75 80 Glu Gly Arg Val Arg Gly Glu Ala Ile Arg Ile Arg Thr Met Lys Met 85 90 95 Arg Gln Gln Ala Ser Phe Leu Pro Ala Thr Leu Thr Met Thr Val Asp 100 105 110 Lys Gly Asp Asn Val Asn Ile Ser Phe Lys Lys Val Leu Ile Lys Glu 115 120 125 Glu Asp Ala Val Ile Tyr Lys Asn Gly Ser Phe Ile His Ser Val Pro 130 135 140 Arg His Glu Val Pro Asp Ile Leu Glu Val His Leu Pro His Ala Gln 145 150 155 160 Pro Gln Asp Ala Gly Val Tyr Ser Ala Arg Tyr Ile Gly Gly Asn Leu 165 170 175 Phe Thr Ser Ala Phe Thr Arg Leu Ile Val Arg Arg Cys Glu Ala Gln 180 185 190 Lys Trp Gly Pro Glu Cys Asn His Leu Cys Thr Ala Cys Met Asn Asn 195 200 205 Gly Val Cys His Glu Asp Thr Gly Glu Cys Ile Cys Pro Pro Gly Phe 210 215 220 Met Gly Arg Thr Cys Glu Lys Ala Cys Glu Leu His Thr Phe Gly Arg 225 230 235 240 Thr Cys Lys Glu Arg Cys Ser Gly Gln Glu Gly Cys Lys Ser Tyr Val 245 250 255 Phe Cys Leu Pro Asp Pro Tyr Gly Cys Ser Cys Ala Thr Gly Trp Lys 260 265 270 Gly Leu Gln Cys Asn Glu Ala Cys His Pro Gly Phe Tyr Gly Pro Asp 275 280 285 Cys Lys Leu Arg Cys Ser Cys Asn Asn Gly Glu Met Cys Asp Arg Phe 290 295 300 Gln Gly Cys Leu Cys Ser Pro Gly Trp Gln Gly Leu Gln Cys Glu Arg 305 310 315 320 Glu Gly Ile Pro Arg Met Thr Pro Lys Ile Val Asp Leu Pro Asp His 325 330 335 Ile Glu Val Asn Ser Gly Lys Phe Asn Pro Ile Cys Lys Ala Ser Gly 340 345 350 Trp Pro Leu Pro Thr Asn Glu Glu Met Thr Leu Val Lys Pro Asp Gly 355 360 365 Thr Val Leu His Pro Lys Asp Phe Asn His Thr Asp His Phe Ser Val 370 375 380 Ala Ile Phe Thr Ile His Arg Ile Leu Pro Pro Asp Ser Gly Val Trp 385 390 395 400 Val Cys Ser Val Asn Thr Val Ala Gly Met Val Glu Lys Pro Phe Asn 405 410 415 Ile Ser Val Lys Val Leu Pro Lys Pro Leu Asn Ala Pro Asn Val Ile 420 425 430 Asp Thr Gly His Asn Phe Ala Val Ile Asn Ile Ser Ser Glu Pro Tyr 435 440 445 Phe Gly Asp Gly Pro Ile Lys Ser Lys Lys Leu Leu Tyr Lys Pro Val 450 455 460 Asn His Tyr Glu Ala Trp Gln His Ile Gln Val Thr Asn Glu Ile Val 465 470 475 480 Thr Leu Asn Tyr Leu Glu Pro Arg Thr Glu Tyr Glu Leu Cys Val Gln 485 490 495 Leu Val Arg Arg Gly Glu Gly Gly Glu Gly His Pro Gly Pro Val Arg 500 505 510 Arg Phe Thr Thr Ala Ser Ile Gly Leu Pro Pro Pro Arg Gly Leu Asn 515 520 525 Leu Leu Pro Lys Ser Gln Thr Thr Leu Asn Leu Thr Trp Gln Pro Ile 530 535 540 Phe Pro Ser Ser Glu Asp Asp Phe Tyr Val Glu Val Glu Arg Arg Ser 545 550 555 560 Val Gln Lys Ser Asp Gln Gln Asn Ile Lys Val Pro Gly Asn Leu Thr 565 570 575 Ser Val Leu Leu Asn Asn Leu His Pro Arg Glu Gln Tyr Val Val Arg 580 585 590 Ala Arg Val Asn Thr Lys Ala Gln Gly Glu Trp Ser Glu Asp Leu Thr 595 600 605 Ala Trp Thr Leu Ser Asp Ile Leu Pro Pro Gln Pro Glu Asn Ile Lys 610 615 620 Ile Ser Asn Ile Thr His Ser Ser Ala Val Ile Ser Trp Thr Ile Leu 625 630 635 640 Asp Gly Tyr Ser Ile Ser Ser Ile Thr Ile Arg Tyr Lys Val Gln Gly 645 650 655 Lys Asn Glu Asp Gln His Val Asp Val Lys Ile Lys Asn Ala Thr Ile 660 665 670 Thr Gln Tyr Gln Leu Lys Gly Leu Glu Pro Glu Thr Ala Tyr Gln Val 675 680 685 Asp Ile Phe Ala Glu Asn Asn Ile Gly Ser Ser Asn Pro Ala Phe Ser 690 695 700 His Glu Leu Val Thr Leu Pro Glu Ser Gln Ala Pro Ala Asp Leu Gly 705 710 715 720 Gly Gly Lys Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro 725 730 735 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 740 745 750 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 755 760 765 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 770 775 780 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 785 790 795 800 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 805 810 815 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 820 825 830 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 835 840 845 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 850 855 860 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 865 870 875 880 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 885 890 895 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 900 905 910 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 915 920 925 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 930 935 940 Thr Gln Lys Ser Leu Ser Leu Ser Pro 945 950 <210> 3 <211> 118 <212> PRT <213> Mus musculus <220> <221> DOMAIN <223> HR54 mouse heavy chain variable region sequence <400> 3 Glu Val His Leu Glu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Asn Asp Asp Ser Ser Tyr Thr Tyr Tyr Pro Asp Asn Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Asn Leu Tyr 65 70 75 80 Leu Gln Met Ser His Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys[[ID=�4]] 85 90 95 Ala Arg Asp Glu Ala Thr Gly Cys Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Leu Thr Val Ser Ser 115 <210> 4 <211> 106 <212 PRT <213> Mus musculus <220> <221> DOMAIN <223> HR54 murine light chain variable region sequence <400> 4 Asn Ile Val Met Thr Gln Ser Pro Lys Ser Met Ser Ile Ser Val Gly 1 5 10 15 Glu Arg Val Thr Leu Ser Cys Lys Ala Ser Glu Asn Val Gly Thr Tyr 20 25 30 Val Ser Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Ala Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala 65 70 75 80 Glu Asp Leu Ala Asp Tyr His Cys Gly Gln Ser Tyr Ile Asn Pro Thr 85 90 95 Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 5 <211> 118 <212> PRT <213> Mus musculus <220> <221> DOMAIN <223> CP33 murine heavy chain variable region sequence <400> 5 Glu Leu Gln Leu Val Glu Ser Gly Gly Asn Leu Val Arg Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Thr Pro Asp Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Asn Asp Asp Gly Thr Tyr Thr Tyr Tyr Pro Asp Asn Leu 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Val Asn Asn Asn Leu Tyr 65 70 75 80 Leu Gln Met Ser His Leu Lys Ser Asp Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Arg Asp His Ser Ser Ser Phe Phe Asp Phe Trp Gly Gln Gly Thr 100​​​​​​​​​​​​​​​​​​​​​​​​​Glu Arg Val Thr Leu Thr Cys Thr Ala Ser Glu Asn Val Gly Arg Tyr 20 25 30 Val Ser Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Asn Val Leu Ile 35 40 45 Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Ala Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala 65 70 75 80 Glu Asp Leu Ala Asp Tyr His Cys Gly Gln Thr Tyr Ile Asn Pro Thr 85 90 95 Phe Gly Ala Gly Thr Lys Leu Glu Val Lys 100 105 <210> 7 <211> 5 <212> PRT <213> Mus musculus <220> <221> DOMAIN <223> HR54 HCDR1 <400> 7 Ser Tyr Val Met Ser 1 5 <210> 8 <211> 17 <212> PRT <213> Mus musculus <220> <221> DOMAIN <223> HR54 HCDR2 <400> 8 Thr Ile Asn Asp Asp Ser Ser Tyr Thr Tyr Tyr Pro Asp Asn Val Lys 1 5 10 15 Gly <210> 9 <211> 9 <212> PRT <213> Mus musculus <220> <221> DOMAIN <223> HR54 HCDR3 <400> 9 Asp Glu Ala Thr Gly Cys Phe Asp Tyr 1 5 <210> 10 <211> 11 <212> PRT <213> Mus musculus <220> <221> DOMAIN <223> HR54 LCDR1 <400> 10 Lys Ala Ser Glu Asn Val Gly Thr Tyr Val Ser 1 5 10 <210> 11 <211> 7 <212> PRT <213> Mus musculus <220> <221> DOMAIN <223> HR54 LCDR2 <400> 11 Gly Ala Ser Asn Arg Tyr Thr 1 5 <210> 12 <211> 8 <212> PRT <213> Mus musculus <220> <221> DOMAIN <223> HR54 LCDR3 <400> 12 Gly Gln Ser Tyr Ile Asn Pro Thr 1 5 <210> 13 <211> 17 <212> PRT <213> Mus musculus <220> <221> DOMAIN <223> CP33 HCDR2 <400> 13 Thr Ile Asn Asp Asp Gly Thr Tyr Thr Tyr Tyr Pro Asp Asn Leu Lys 1 5 10 15 Gly <210> 14 <211> 9 <212> PRT <213> Mus musculus <220> <221> DOMAIN <223> CP33 HCDR3 <400> 14 Asp His Ser Ser Ser Phe Phe Asp Phe 1 5 <210> 15 <211> 11 <212> PRT <213> Mus musculus <220> <221> DOMAIN <223> CP33 LCDR1 <400> 15 Thr Ala Ser Glu Asn Val Gly Arg Tyr Val Ser 1 5 10 <210> 16 <211> 8 <212> PRT <213> Mus musculus <220> <221> DOMAIN <223> CP33 LCDR3 <400> 16 Gly Gln Thr Tyr Ile Asn Pro Thr 1 5 <210> 17 <211> 106 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> hu HR54VL1 <400> 17 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Glu Asn Val Gly Thr Tyr 20 25 30 Val Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala 65 70 75 80 Glu Asp Val Ala Val Tyr Tyr Cys Gly Gln Ser Tyr Ile Asn Pro Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 18 <211> 106 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> hu HR54VL2 <400> 18 Asn Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Glu Asn Val Gly Thr Tyr 20 25 30 Val Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala 65 70 75 80 Glu Asp Val Ala Asp Tyr His Cys Gly Gln Ser Tyr Ile Asn Pro Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 19 <211> 106 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> huHR54VL3 <400> 19 Asn Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 1​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Thr Ile Asn Asp Asp Ser Ser Tyr Thr Tyr Tyr Pro Asp Asn Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Glu Ala Thr Gly Cys Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 21 <211> 118 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> huHR54VH2 <400> 21 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Asn Asp Asp Ser Ser Tyr Thr Tyr Tyr Pro Asp Asn Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Ser Leu Tyr 65 70 75 80 [[ID=2))Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Glu Ala Thr Gly Cys Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 22 <211> 17 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> HR54a HCDR2 <400> 22 Thr Ile Asn Glu Asp Ser Ser Tyr Thr Tyr Tyr Pro Asp Asn Val Lys 1 5 10 15 Gly <210> twenty three <211> 9 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> HR54a HCDR3 <400> twenty three Asp Glu Ala Thr Gly Ser Phe Asp Tyr 1 5 <210> twenty four <211> 17 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> HR54b HCDR2 <400> twenty four Thr Ile Asn Asp Asn Ser Ser Tyr Thr Tyr Tyr Pro Asp Asn Val Lys 1 5 10 15 Gly <210> 25 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> HR54c HCDR3 <400> 25 Asn Glu Ala Thr Gly Ser Phe Asp Tyr 1 5 <210> 26 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> HR54d HCDR3 <400> 26 Asp Gln Ala Thr Gly Ser Phe Asp Tyr 1 5 <210> 27 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> HR54n HCDR3 <400> 27 Asp Glu Ala Thr Gly Val Phe Asp Tyr 1 5 <210> 28 <211> 118 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> huHR54VH2a <400> 28 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Asn Glu Asp Ser Ser Tyr Thr Tyr Tyr Pro Asp Asn Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Ser Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Glu Ala Thr Gly Ser Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 29 <211> 118 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> huHR54VH2b<​​​​​​​​​​​​​​Ala Thr Ile Asn Asp Asn Ser Ser Tyr Thr Tyr Tyr Pro Asp Asn Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Ser Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Glu Ala Thr Gly Ser Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 30 <211> 118 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> huHR54VH2c <400> 30 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Asn Asp Asn Ser Ser Tyr Thr Tyr Tyr Pro Asp Asn Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Ser Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Glu Ala Thr Gly Ser Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 31 <211> 118 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> huHR54VH2d[[ID=3३]] <400> 31 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Asn Asp Asp Ser Ser Tyr Thr Tyr Tyr Pro Asp Asn Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Ser Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Gln Ala Thr Gly Ser Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 32 <211> 118 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> huHR54VH2e <400> 32 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Asn Glu Asp Ser Ser Tyr Thr Tyr Tyr Pro Asp Asn Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Ser Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asn Glu Ala Thr Gly Ser Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 33 <211> 118 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> huHR54VH2f <400> 33 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Asn Glu Asp Ser Ser Tyr Thr Tyr Tyr Pro Asp Asn Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Ser Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Gln Ala Thr Gly Ser Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 34 <211> 118 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> huHR54VH2g <400> 34 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Asn Asp Asn Ser Ser Tyr Thr Tyr Tyr Pro Asp Asn Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Ser Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asn Glu Ala Thr Gly Ser Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 35 <211> 118 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> huHR54VH2k <400> 35 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Asn Asp Asn Ser Ser Tyr Thr Tyr Tyr Pro Asp Asn Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Ser Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Gln Ala Thr Gly Ser Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 36 <211> 118 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> huHR54VH2m <400> 36 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Asn Asp Asp Ser Ser Tyr Thr Tyr Tyr Pro Asp Asn Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Ser Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Glu Ala Thr Gly Ser Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 37 <211> 118 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> huHR54VH2n <400> 37 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Asn Glu Asp Ser Ser Tyr Thr Tyr Tyr Pro Asp Asn Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Ser Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Glu Ala Thr Gly Val Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 38 <211> 17 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> huHR54 HCDR2 general formula <220> <221> DOMAIN <222> (4)..(4) <223> Xaa is selected from Asp or Glu. <220> <221> DOMAIN <222> (5)..(5) <223> X is selected from Asp or Asn. <400> 38 Thr Ile Asn Xaa Xaa Ser Ser Tyr Thr Tyr Tyr Pro Asp Asn Val Lys 1 5 10 15 Gly <210> 39 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> huHR54 HCDR3 general formula <220> <221> DOMAIN <222> (1)..(1) <223> Xaa is selected from Asp or Asn. <220> <221> DOMAIN <222> (2)..(2) <223> Xaa is selected from Glu or GIn. <220> <221> DOMAIN <222> (6) <223> Xaa is selected from Cys, Ser or Val. <400> 39 Xaa Xaa Ala Thr Gly Xaa Phe Asp Tyr 1 5 <210> 40 <211> 106 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> huCP33VL1 <400> 40 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Thr Ala Ser Glu Asn Val Gly Arg Tyr 20 25 30 Val Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala 65 70 75 80 Glu Asp Val Ala Val Tyr Tyr Cys Gly Gln Thr Tyr Ile Asn Pro Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 41 <211> 106 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> huCP33VL2 <400> 41 Asn Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Thr Ala Ser Glu Asn Val Gly Arg Tyr 20 25 30 Val Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Val Leu Ile 35 40 45 Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala 65 70 75 80 Glu Asp Val Ala Val Tyr Tyr Cys Gly Gln Thr Tyr Ile Asn Pro Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 42 <211> 106 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> huCP33VL3 <400> 42 Asn Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Thr Ala Ser Glu Asn Val Gly Arg Tyr 20 25 30 Val Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Val Leu Ile 35 40 45 Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala 65 70 75 80 Glu Asp Val Ala Asp Tyr His Cys Gly Gln Thr Tyr Ile Asn Pro Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 43 <211> 106 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> huCP33VL4 <400> 43 Asn Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Thr Ala Ser Glu Asn Val Gly Arg Tyr 20 25 30 Val Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Val Leu Ile 35 40 45 Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Ala Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala 65 70 75 80 Glu Asp Val Ala Asp Tyr His Cys Gly Gln Thr Tyr Ile Asn Pro Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 44 <211> 118 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> huCP33VH1 <400> 44 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Thr Ile Asn Asp Asp Gly Thr Tyr Thr Tyr Tyr Pro Asp Asn Leu 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp His Ser Ser Ser Phe Phe Asp Phe Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 45 <211> 118 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> huCP33VH2 <400> 45 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Thr Ile Asn Asp Asp Gly Thr Tyr Thr Tyr Tyr Pro Asp Asn Leu 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Val Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp His Ser Ser Ser Phe Phe Asp Phe Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 46 <211> 118 <212> PRT <213> Artificial Sequence <220>[[ID=十七]] <221> DOMAIN <223> huCP33VH3 <400> 46 Glu Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Asn Asp Asp Gly Thr Tyr Thr Tyr Tyr Pro Asp Asn Leu 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Val Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 It should be noted that in the above translation, the Chinese character "十七" should be the correct Arabic numeral "17" in the original text for a more accurate translation. This might be a misrepresentation in the original input. The translation is adjusted accordingly based on the overall context and the requirements of maintaining the original tags and text structure.Ala Arg Asp His Ser Ser Ser Phe Phe Asp Phe Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 47 <211> 3三百三十 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Heavy chain constant region <400> 47 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys It should be noted that in the translation of "<211> 330", it is assumed that "三百三十" is the correct translation based on the context, but it's possible there could be a more accurate rendering depending on the specific meaning in the patent context. Also, for the tags like - , they are left unchanged as per the requirement. 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 48 <211> 107 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Light chain constant region <400> 48 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45<Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 49 <211> 448 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> huHR54-01 heavy chain <400> 49 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Thr Ile Asn Asp Asp Ser Ser Tyr Thr Tyr Tyr Pro Asp Asn Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Glu Ala Thr Gly Cys Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 50 <211> 213 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> huHR54-01 light chain <400> 50 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Glu Asn Val Gly Thr Tyr 20 25 30 Val Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala 65 70 75 80 Glu Asp Val Ala Val Tyr Tyr Cys Gly Gln Ser Tyr Ile Asn Pro Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro 100 105 110 Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 115 120 125 Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135 140 Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu 145 150 155 160 Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170 175 Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185 190 Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200 205 Asn Arg Gly Glu Cys 210 <210> 51 <211> 448 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> huHR54-04 heavy chain <400> 51 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Asn Asp Asp Ser Ser Tyr Thr Tyr Tyr Pro Asp Asn Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Ser Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Glu Ala Thr Gly Cys Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 52 <211> 448 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> huHR54-12 heavy chain <400> 52 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Asn Glu Asp Ser Ser Tyr Thr Tyr Tyr Pro Asp Asn Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Ser Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Gln Ala Thr Gly Ser Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 53 <211> 448 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> huCP33-01 heavy chain <400> 53 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Thr Ile Asn Asp Asp Gly Thr Tyr Thr Tyr Tyr Pro Asp Asn Leu 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp His Ser Ser Ser Phe Phe Asp Phe Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 54 <211> 213 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> huCP33-01 antibody light chain <400> 54 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Thr Ala Ser Glu Asn Val Gly Arg Tyr 20 25 30 Val Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala 65 70 75 80 Glu Asp Val Ala Val Tyr Tyr Cys Gly Gln Thr Tyr Ile Asn Pro Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro 100 105 110 Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 115 120 125 Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135 140 Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu 145 150 155 160 Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170 175 Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185 190 Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200 205 Asn Arg Gly Glu Cys 210 <210> 55 <211> 448 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> huCP33-05 heavy chain <400> 55 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 It should be noted that in the translation process, the 7-digit tags , , etc. are preserved as they are according to the requirements. Also, the special placeholders [[ID=]], are not modified. If there are any specific errors or inaccuracies in the original text, they will be carried over to the translation as well. For example, the "5" in might be a typo in the original, and it remains the same in the translation. If this is an important error, it may need to be verified with the source of the original text.Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Thr Ile Asn Asp Asp Gly Thr Tyr Thr Tyr Tyr Pro Asp Asn Leu 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Val Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp His Ser Ser Ser Phe Phe Asp Phe Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 56 <211> 107 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Ranibizumab light chain variable region <400> 56 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile 35 40 45 Tyr Phe Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Thr Val Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 57 <211> 214 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> The light chain of ranibizumab <400> 57 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile 35 40 45 Tyr Phe Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Thr Val Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 58 <211> 123 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Ranibizumab heavy chain variable region <400> 58 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Asp Phe Thr His Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe 50 55 60 Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Tyr Pro Tyr Tyr Tyr Gly Thr Ser His Trp Tyr Phe Asp Val 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 59 <211> 453 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> Ranibizumab heavy chain 1 <400> 59 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 [[ID=2�]]Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Asp Phe Thr His Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe 50 55 60 Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Tyr Pro Tyr Tyr Tyr Gly Thr Ser His Trp Tyr Phe Asp Val 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 225 230 235 240 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290 295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350 Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 355 360 365 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410 415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 435 440 445 Leu Ser Pro Gly Lys 450 <210> 60 <211> 453 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Ranibizumab heavy chain 2 <400> 60 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Asp Phe Thr His Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe 50 55 60 Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Tyr Pro Tyr Tyr Tyr Gly Thr Ser His Trp Tyr Phe Asp Val 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala 225 230 235 240 Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met Ala Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290 295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu Ala Gln Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350 Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 355 360 365 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410 415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430 Val Met His Glu Ala Leu His Asn Ala Tyr Thr Gln Lys Ser Leu Ser 435 440 445 Leu Ser Pro Gly Lys 450 <210> 61 <211> 5 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Ranibizumab HCDR1 <400> 61 His Tyr Gly Met Asn 1 5 <210> 62 <211> 17 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Ranibizumab HCDR2 <400> 62 Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe Lys 1 5 10 15 Arg <210> 63 <211> 14 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Ranibizumab HCDR3 <400> 63 Tyr Pro Tyr Tyr Tyr Gly Thr Ser His Trp Tyr Phe Asp Val 1 5 10 <210> 64 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Ranibizumab LCDR1 <400> 64 Ser Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn 1 5 10 <210> 65 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Ranibizumab LCDR2 <400> 65 Phe Thr Ser Ser Leu His Ser 1 5 <210> 66 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> Ranibizumab LCDR3 <400> 66 Gln Gln Tyr Ser Thr Val Pro Trp Thr Phe 1 5 10 <210> 67 <211> 254 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <223> huHR54 - 01 - scFv <400> 67 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Thr Ile Asn Asp Asp Ser Ser Tyr Thr Tyr Tyr Pro Asp Asn Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Glu Ala Thr Gly Cys Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala 145 150 155 160 Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Glu Asn 165 170 175 Val Gly Thr Tyr Val Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 180 185 190 Lys Leu Leu Ile Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp 195 200 205 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 210 215 220 Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gly Gln Ser Tyr<000​​​​​​​​​​​​​​​​​​​ <400> 68 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Asn Asp Asp Ser Ser Tyr Thr Tyr Tyr Pro Asp Asn Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Ser Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Glu Ala Thr Gly Cys Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala 145 150 155 160 Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Glu Asn 165 170 175 Val Gly Thr Tyr Val Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 180 185 190 Lys Leu Leu Ile Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp 195 200 205 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 210 215 220 Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gly Gln Ser Tyr 225 230 235 240 Ile Asn Pro Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 245 250 <210> 69 <211> 254 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <223> huHR54-12-scFv <400> 69 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Asn Glu Asp Ser Ser Tyr Thr Tyr Tyr Pro Asp Asn Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Ser Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Gln Ala Thr Gly Ser Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala 145 150 155 160 Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Glu Asn 165 170 175 Val Gly Thr Tyr Val Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 180 185 190 Lys Leu Leu Ile Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp 195 200 205 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 210 215 220 Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gly Gln Ser Tyr 225 230 235 240 Ile Asn Pro Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 245 250 <210> 70 <211> 254 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <223> huCP33-01-scFv <400> 70 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Thr Ile Asn Asp Asp Gly Thr Tyr Thr Tyr Tyr Pro Asp Asn Leu 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp His Ser Ser Ser Phe Phe Asp Phe Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala 145 150 155 160 Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Thr Ala Ser Glu Asn 165 170 175 Val Gly Arg Tyr Val Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 180 185 190 Lys Leu Leu Ile Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp 195 200 205 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 210 215 220 Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gly Gln Thr Tyr 225 230 235 240 Ile Asn Pro Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 245 250 <210> 71 <211> 254 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <223> huCP33-05-scFv <400> 71 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Thr Ile Asn Asp Asp Gly Thr Tyr Thr Tyr Tyr Pro Asp Asn Leu 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Val Lys Asn Ser Leu Tyr '65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp His Ser Ser Ser Phe Phe Asp Phe Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala 145 150 155 160 Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Thr Ala Ser Glu Asn 165 170 175 Val Gly Arg Tyr Val Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 180 185 190 Lys Leu Leu Ile Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp 195 200 205 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 210 215 220 Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gly Gln Thr Tyr 225 230 235 240 Ile Asn Pro Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 245 250 <210> 72 <211> 118 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> huHR54VH2-C <400> 72 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Val 35 40 45 Ala Thr Ile Asn Asp Asp Ser Ser Tyr Thr Tyr Tyr Pro Asp Asn Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Ser Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Glu Ala Thr Gly Cys Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 73 <211> 118 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> huHR54VH2f-C <400> 73 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Val 35 40 45 Ala Thr Ile Asn Glu Asp Ser Ser Tyr Thr Tyr Tyr Pro Asp Asn Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Ser Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Gln Ala Thr Gly Ser Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 74 <211> 106 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> huHR54VL1-C <400> 74 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Glu Asn Val Gly Thr Tyr 20 25 30 Val Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala 65 70 75 80 Glu Asp Val Ala Val Tyr Tyr Cys Gly Gln Ser Tyr Ile Asn Pro Thr 85 90 95 Phe Gly Cys Gly Thr Lys Val Glu Ile Lys 100 105 <210> 75 <211> 118 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN <223> huCP33VH2-C <400> 75 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Val 35 40 45 Ala Thr Ile Asn Asp Asp Gly Thr Tyr Thr Tyr Tyr Pro Asp Asn Leu 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Val Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp His Ser Ser Ser Phe Phe Asp Phe Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 76 <211> 106 <212> PRT <213> Artificial Sequence <220> <221> DOMAIN[[ID=4​​​​Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Thr Ala Ser Glu Asn Val Gly Arg Tyr 20 25 30 Val Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala 65 70 75 80 Glu Asp Val Ala Val Tyr Tyr Cys Gly Gln Thr Tyr Ile Asn Pro Thr 85 90 95 Phe Gly Cys Gly Thr Lys Val Glu Ile Lys 100 105 <210> 77 <211> 254 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE [[ID=​​​​​​Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Val 35 40 45 Ala Thr Ile Asn Asp Asp Ser Ser Tyr Thr Tyr Tyr Pro Asp Asn Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Ser Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Glu Ala Thr Gly Cys Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala 145 150 155 160 Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Glu Asn 165 170 175 Val Gly Thr Tyr Val Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro The positions 180, 185, 190 Lys Leu Leu Ile Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp The positions 195, 200, 205 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser The positions 210, 215, 220 Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gly Gln Ser Tyr The positions 225, 230, 235, 240 Ile Asn Pro Thr Phe Gly Cys Gly Thr Lys Val Glu Ile Lys The positions 245, 250 <210> 78 <211> 254 <212> PRT ​​​​​​​​​​​​​​​​​​​​​​Ala Thr Ile Asn Glu Asp Ser Ser Tyr Thr Tyr Tyr Pro Asp Asn Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Ser Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Gln Ala Thr Gly Ser Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala 145 150 155 160 Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Glu Asn 165 170 175 Val Gly Thr Tyr Val Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 180 185 190 Lys Leu Leu Ile Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp 195 200 205 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 210 215 220 Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gly Gln Ser Tyr 225 230 235 240 Ile Asn Pro Thr Phe Gly Cys Gly Thr Lys Val Glu Ile Lys 245 250 <210> 79 <211> 254 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <223> huCP33-05-scFv-CC <400> 79 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Val 35 40 45 Ala Thr Ile Asn Asp Asp Gly Thr Tyr Thr Tyr Tyr Pro Asp Asn Leu 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Val Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp His Ser Ser Ser Phe Phe Asp Phe Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala 145 150 155 160 Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Thr Ala Ser Glu Asn 165 170 175 Val Gly Arg Tyr Val Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 180 185 190 Lys Leu Leu Ile Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp 195 200 205 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 210 215 220 Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gly Gln Thr Tyr 225 230 235 240 Ile Asn Pro Thr Phe Gly Cys Gly Thr Lys Val Glu Ile Lys 245 250 <210> 80 <211> 708 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> The first chain of bispecific antibody 1 <400> 80 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Asp Phe Thr His Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe 50 55 60 Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Tyr Pro Tyr Tyr Tyr Gly Thr Ser His Trp Tyr Phe Asp Val 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 225 230 235 240 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290 295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350 Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 355 360 365 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410 415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 435 440 445 Leu Ser Pro Gly Gly Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly 450 455 460 Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 465 470 475 480 Phe Thr Phe Ser Ser Tyr Val Met Ser Trp Val Arg Gln Ala Pro Gly 485 490 495 Lys Cys Leu Glu Trp Val Ala Thr Ile Asn Glu Asp Ser Ser Tyr Thr 500 505 510 Tyr Tyr Pro Asp Asn Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 515 520 525 Ala Lys Ser Ser Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp 530 535 540 Thr Ala Val Tyr Tyr Cys Ala Arg Asp Gln Ala Thr Gly Ser Phe Asp 545 550 555 560 Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly 565 570 575 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 580 585 590 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln 595 600 605 Ser Pro Asp Ser Leu Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn 610 615 620 Cys Lys Ala Ser Glu Asn Val Gly Thr Tyr Val Ser Trp Tyr Gln Gln 625 630 635 640 Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Asn Arg 645 650 655 Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 660 665 670 Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr 675 680 685 Tyr Cys Gly Gln Ser Tyr Ile Asn Pro Thr Phe Gly Cys Gly Thr Lys 690 695 700 Val Glu Ile Lys 705 <210> 81 <211> 708 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> The first chain of bispecific antibody 2 <400> 81 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Asp Phe Thr His Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe 50 55 60 Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Tyr Pro Tyr Tyr Tyr Gly Thr Ser His Trp Tyr Phe Asp Val 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 225 230 235 240 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290 295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350 Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 355 360 365 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410 415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 435 440 445 Leu Ser Pro Gly Gly Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly 450 455 460 Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 465 470 475 480 Phe Thr Phe Ser Ser Tyr Val Met Ser Trp Val Arg Gln Ala Pro Gly 485 490 495 Lys Cys Leu Glu Trp Val Ala Thr Ile Asn Asp Asp Gly Thr Tyr Thr 500 505 510 Tyr Tyr Pro Asp Asn Leu Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 515 520 525 Val Lys Asn Ser Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp 530 535 540 Thr Ala Val Tyr Tyr Cys Ala Arg Asp His Ser Ser Ser Phe Phe Asp 545 550 555 560 Phe Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly 565 570 575 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 580 585 590 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln 595 600 605 Ser Pro Asp Ser Leu Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn 610 615 620 Cys Thr Ala Ser Glu Asn Val Gly Arg Tyr Val Ser Trp Tyr Gln Gln 625 630 635 640 Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Asn Arg 645 650 655 Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 660 665 670 Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr 675 680 685 Tyr Cys Gly Gln Thr Tyr Ile Asn Pro Thr Phe Gly Cys Gly Thr Lys 690 695 700 Val Glu Ile Lys 705 <210> 82 <211> 708 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> The first chain of bispecific antibody 3 <400> 82 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Asp Phe Thr His Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe 50 55 60 Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Tyr Pro Tyr Tyr Tyr Gly Thr Ser His Trp Tyr Phe Asp Val 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala 225 230 235 240 Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met Ala Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290 295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu Ala Gln Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350 Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 355 360 365 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410 415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430 Val Met His Glu Ala Leu His Asn Ala Tyr Thr Gln Lys Ser Leu Ser 435 440 445 Leu Ser Pro Gly Gly Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly 450 455 460 Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 465 470 475 480 Phe Thr Phe Ser Ser Tyr Val Met Ser Trp Val Arg Gln Ala Pro Gly 485 490 495 Lys Cys Leu Glu Trp Val Ala Thr Ile Asn Glu Asp Ser Ser Tyr Thr 500 505 510 Tyr Tyr Pro Asp Asn Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 515 520 525 Ala Lys Ser Ser Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp 530 535 540 Thr Ala Val Tyr Tyr Cys Ala Arg Asp Gln Ala Thr Gly Ser Phe Asp 545 550 555 560 Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly 565 570 575 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 580 585 590 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln 595 600 605 Ser Pro Asp Ser Leu Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn 610 615 620 Cys Lys Ala Ser Glu Asn Val Gly Thr Tyr Val Ser Trp Tyr Gln Gln 625 630 635 640 Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Asn Arg 645 650 655 Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 660 665 670 Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr 675 680 685 Tyr Cys Gly Gln Ser Tyr Ile Asn Pro Thr Phe Gly Cys Gly Thr Lys 690 695 700 Val Glu Ile Lys 705 <210> 83 <211> 708 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> The first chain of bispecific antibody 4 <400> 83 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Asp Phe Thr His Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe 50 55 60 Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Tyr Pro Tyr Tyr Tyr Gly Thr Ser His Trp Tyr Phe Asp Val 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala 225 230 235 240 Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met Ala Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290 295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu Ala Gln Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350 Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 355 360 365 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410 415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430 Val Met His Glu Ala Leu His Asn Ala Tyr Thr Gln Lys Ser Leu Ser 435 440 445 Leu Ser Pro Gly Gly Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly 450 455 460 Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 465 470 475 480 Phe Thr Phe Ser Ser Tyr Val Met Ser Trp Val Arg Gln Ala Pro Gly 485 490 495 Lys Cys Leu Glu Trp Val Ala Thr Ile Asn Asp Asp Gly Thr Tyr Thr 500 505 510 Tyr Tyr Pro Asp Asn Leu Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 515 520 525 Val Lys Asn Ser Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp 530 535 540 Thr Ala Val Tyr Tyr Cys Ala Arg Asp His Ser Ser Ser Phe Phe Asp 545 550 555 560 Phe Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly 565 570 575 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 580 585 590 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln 595 600 605 Ser Pro Asp Ser Leu Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn 610 615 620 Cys Thr Ala Ser Glu Asn Val Gly Arg Tyr Val Ser Trp Tyr Gln Gln 625 630 635 640 Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Asn Arg 645 650 655 Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 660 665 670 Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr 675 680 685 Tyr Cys Gly Gln Thr Tyr Ile Asn Pro Thr Phe Gly Cys Gly Thr Lys 690 695 700 Val Glu Ile Lys 705 <210> 84 <211> 452 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> Bispecific antibody 5 first heavy chain: anti-ANG-2 heavy chain <400> 84 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Ala Pro Gly Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Asn Glu Asp Ser Ser Tyr Thr Tyr Tyr Pro Asp Asn Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Ser Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Gln Ala Thr Gly Ser Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro Ser Val Phe Ile 115 120 125 Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val 130 135 140 Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys 145 150 155 160 Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu 165 170 175 Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu 180 185 190 Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr 195 200 205 His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 355 360 365 Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser Pro Gly Lys 450 <210> 85 <211> 211 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> Bispecific antibody 5 first light chain: anti-ANG-2 light chain <400> 85 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ala Ser Glu Asn Val Gly Thr Tyr 20 25 30 Val Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala 65 70 75 80 Glu Asp Val Ala Val Tyr Tyr Cys Gly Gln Ser Tyr Ile Asn Pro Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Ser Ser Ala Ser Thr Lys 100 105 110 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 115 120 125 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 130 135 140 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 145 150 155 160 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 165 170 175 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 180 185 190 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro 195 200 205 Lys Ser Cys 210 <210> 86 <211> 453 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> Bispecific antibody 5 second heavy chain: anti-VEGF heavy chain <400> 86 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Asp Phe Thr His Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe 50 55 60 Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Tyr Pro Tyr Tyr Tyr Gly Thr Ser His Trp Tyr Phe Asp Val 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 225 230 235 240 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290 295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350 Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln 355 360 365 Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410 415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 435 440 445 Leu Ser Pro Gly Lys 450 <210> 87 <211> 452 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> Bispecific antibody 6 first heavy chain: anti-ANG-2 heavy chain <400> 87 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Thr Ile Asn Asp Asp Gly Thr Tyr Thr Tyr Tyr Pro Asp Asn Leu 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Val Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp His Ser Ser Ser Phe Phe Asp Phe Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro Ser Val Phe Ile 115 120 125 Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val 130 135 140 Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys 145 150 155 160 Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu 165 170 175 Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu 180 185 190 Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr 195 200 205 His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 355 360 365 Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser Pro Gly Lys 450 <210> 88 <211> 211 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> Bispecific antibody 6 first light chain: anti-ANG-2 light chain <400> 88 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Thr Ala Ser Glu Asn Val Gly Arg Tyr 20 25 30 Val Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala 65 70 75 80 Glu Asp Val Ala Val Tyr Tyr Cys Gly Gln Thr Tyr Ile Asn Pro Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Ser Ser Ala Ser Thr Lys 100 105 110 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 115 120 125 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 130 135 140 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 145 150 155 160 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 165 170 175 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 180 185 190 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro 195 200 205 Lys Ser Cys 210 <210> 89 <211> 463 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> RG7716 anti-ANG-2 heavy chain <400> 89 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Pro Asn Pro Tyr Tyr Tyr Asp Ser Ser Gly Tyr Tyr Tyr 100 105 110 Pro Gly Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser 115 120 125 Ser Ala Ser Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 130 135 140 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 145 150 155 160 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 165 170 175 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 180 185 190 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 195 200 205 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 210 215 220 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Asp Lys Thr His 225 230 235 240 Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val 245 250 255 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ala Ser Arg Thr 260 265 270 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 275 280 285 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 290 295 300 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 305 310 315 320 Val Leu Thr Val Leu Ala Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 325 330 335 Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile 340 345 350 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro 355 360 365 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala 370 375 380 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 385 390 395 400 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 405 410 415 Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg 420 425 430 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 435 440 445 His Asn Ala Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 450 455 460 <210> 90 <211> 213 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> RG7716 anti-ANG-2 light chain <400> 90 Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr 35 40 45 Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His 85 90 95 Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Ser Ser Ala Ser 100 105 110 Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr 115 120 125 Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro 130 135 140 Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val 145 150 155 160 His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser 165 170 175 Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile 180 185 190 Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val 195 200 205 Glu Pro Lys Ser Cys 210 <210> 91 <211> 453 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> RG7716 anti-VEGF heavy chain <400> 91 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Asp Phe Thr His Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe 50 55 60 Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Tyr Pro Tyr Tyr Tyr Gly Thr Ser His Trp Tyr Phe Asp Val 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala 225 230 235 240 Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met Ala Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290 295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu Ala Gln Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350 Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln 355 360 365 Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410 415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430 Val Met His Glu Ala Leu His Asn Ala Tyr Thr Gln Lys Ser Leu Ser 435 440 445 Leu Ser Pro Gly Lys 450 <210> 92 <211> 214 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> RG7716 anti-VEGF light chain <400> 92 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr 2 \* 20 \* 25 \* 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile 35 40 45 Tyr Phe Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Thr Val Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 93 <211> 453 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> Bevacizumab heavy chain <400> 93 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe 50 55 60 Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Tyr Pro His Tyr Tyr Gly Ser Ser His Trp Tyr Phe Asp Val 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 225 230 235 240 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290 295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350 Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln 355 360 365 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410 415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 435 440 445 Leu Ser Pro Gly Lys 450 <210> 94 <211> 214 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> Bevacizumab light chain <400> 94 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile 35 40 45 Tyr Phe Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Thr Val Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205<​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Pro Asn Pro Tyr Tyr Tyr Asp Ser Ser Gly Tyr Tyr Tyr 100 105 110 Pro Gly Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser 115 120 125 Ser Ala Ser Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 130 135 140 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 145 150 155 160 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 165 170 175 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 180 185 190 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 195 200 205 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 210 215 220 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Asp Lys Thr His 225 230 235 240 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 245 250 255 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 260 265 270 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 275 280 285 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 290 295 300 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 305 310 315 320 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 325 330 335 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 340 345 350 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro 355 360 365 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala 370 375 380 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 385 390 395 400 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 405 410 415 Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg 420 425 430 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 435 440 445 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 450 455 460 <210> 96 <211> 213 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> RG7221 anti - ANG2 light chain <400> 96 Gln Pro Gly Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr 35 40 45 Asp Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His 85 90 95 Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Ser Ser Ala Ser 100 105 110 Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr 115 120 125 Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro 130 135 140 Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val 145 150 155 160 His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser 165 170 175 Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile 180 185 190 Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val 195 200 205 Glu Pro Lys Ser Cys 210 <210> 97 <211> 453 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> RG7221 anti-VEGF heavy chain <400> 97 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe 50 55 60 Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Tyr Pro His Tyr Tyr Gly Ser Ser His Trp Tyr Phe Asp Val 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 225 230 235 240 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290 295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350 Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln 355 360 365 Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410 415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 435 440 445 Leu Ser Pro Gly Lys 450 <210> 98 <211> 214 <212> PRT <213> Artificial Sequence <220> <221> CHAIN <223> RG7221 Anti-VEGF Light Chain <400> 98 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile 35 40 45 Tyr Phe Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Thr Val Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210

Claims

1. A bispecific antigen-binding molecule comprising a first antigen-binding domain that specifically binds to ANG-2 and a second antigen-binding domain that specifically binds to VEGF, wherein the first antigen-binding domain that specifically binds to ANG-2 comprises: 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 The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 7, SEQ ID NO: 22 and SEQ ID NO: 26, respectively.

2. The bispecific antigen binding molecule of claim 1, wherein the first antigen binding domain that specifically binds to ANG-2 comprises a heavy chain variable region and a light chain variable region, wherein: c) the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 33; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 17; or d) the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 73; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

74.

3. The bispecific antigen binding molecule of claim 1, wherein the second antigen binding domain that specifically binds to VEGF comprises: a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 61, SEQ ID NO: 62 and SEQ ID NO: 63, respectively; and The light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 64, SEQ ID NO: 65 and SEQ ID NO: 66, respectively.

4. The bispecific antigen binding molecule of claim 3, wherein the second antigen binding domain that specifically binds to VEGF comprises a heavy chain variable region and a light chain variable region, wherein: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 58, and The amino acid sequence of the light chain variable region is shown in SEQ ID NO:

56. 5 . The bispecific antigen binding molecule of claim 4 , wherein the second antigen binding domain that specifically binds to human VEGF further comprises a heavy chain constant region and a light chain constant region.

6. The bispecific antigen binding molecule of claim 5, wherein the heavy chain constant region comprises L234A, L235A, I253A, H310A and H435A mutations.

7. The bispecific antigen binding molecule of claim 1, wherein the second antigen binding domain that specifically binds to VEGF comprises a heavy chain and a light chain, wherein: The amino acid sequence of the heavy chain is shown in SEQ ID NO: 59 or 60; and The amino acid sequence of the light chain is shown in SEQ ID NO:

57.

8. The bispecific antigen-binding molecule of claim 7, wherein the first antigen-binding domain that specifically binds to ANG-2 is an anti-ANG-2 single-chain antibody.

9. The bispecific antigen binding molecule of claim 8, wherein the anti-ANG-2 single chain antibody comprises: The heavy chain variable region of SEQ ID NO:73 and the light chain variable region of SEQ ID NO:

74.

10. The bispecific antigen binding molecule of claim 9, wherein the amino acid sequence of the anti-ANG-2 single chain antibody is as shown in SEQ ID NO:

78.

11. The bispecific antigen-binding molecule of claim 10, wherein the first antigen-binding domain that specifically binds to ANG-2 is linked directly or via a linker to the second antigen-binding domain that specifically binds to VEGF.

12. The bispecific antigen-binding molecule of claim 11, wherein the N-terminus of the anti-ANG-2 single-chain antibody is linked to the C-terminus of the heavy chain of the second antigen-binding domain that specifically binds to VEGF via a linker.

13. The bispecific antigen binding molecule according to claim 12, wherein the linker is (GG) n , where n is an integer from 1 to 20.

14. The bispecific antigen binding molecule of claim 1, comprising a first chain and a second chain, wherein: i) the amino acid sequence of the first chain is shown in SEQ ID NO: 80 or 82; and ii) The amino acid sequence of the second chain is shown in SEQ ID NO:

57.

15. The bispecific antigen binding molecule of claim 14, comprising two identical first and second chains, wherein: The amino acid sequence of the first chain is shown in SEQ ID NO: 80 or 82; and The amino acid sequence of the second chain is shown in SEQ ID NO:

57.

16. The bispecific antigen binding molecule of claim 1, comprising: a) a first light chain and a first heavy chain that specifically bind to ANG-2; b) a second light chain and a second heavy chain that specifically bind to VEGF; wherein: The constant domains CL and CH1 are interchangeable. The bispecific antigen binding molecule according to claim 16 , wherein the constant domain CL of the first light chain is replaced with the constant domain CH1 of the first heavy chain.

18. The bispecific antigen binding molecule of claim 17, wherein: a) the first light chain comprises a light chain variable region of SEQ ID NO: 17, and the first heavy chain comprises a heavy chain variable region of SEQ ID NO: 33; and b) the second light chain comprises the light chain variable region of SEQ ID NO:56, and the second heavy chain comprises the heavy chain variable region of SEQ ID NO:

58.

19. The bispecific antigen binding molecule of claim 18, wherein: a) the amino acid sequence of the first light chain is shown in SEQ ID NO: 85, and The amino acid sequence of the first heavy chain is shown in SEQ ID NO: 84; and b) the 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.

20. An antibody that specifically binds to ANG-2, comprising: i) 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 A heavy chain variable region as shown in any one of a) to l) below: a) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 7, SEQ ID NO: 22 and SEQ ID NO: 23, respectively; b) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 7, SEQ ID NO: 24 and SEQ ID NO: 23, respectively; c) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 25, respectively; d) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 26, respectively; e) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 7, SEQ ID NO: 22 and SEQ ID NO: 25, respectively; f) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 7, SEQ ID NO: 22 and SEQ ID NO: 26, respectively; g) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 7, SEQ ID NO: 24 and SEQ ID NO: 25, respectively; h) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 7, SEQ ID NO: 24 and SEQ ID NO: 26, respectively; j) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 23, respectively; k) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 7, SEQ ID NO: 22 and SEQ ID NO: 27, respectively; and l) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively; or ii) 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 The light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 15, SEQ ID NO: 11 and SEQ ID NO: 16, respectively.

21. The antibody that specifically binds to ANG-2 according to claim 20, which is a murine antibody, a chimeric antibody, a humanized antibody or a fully human antibody.

22. The antibody that specifically binds to ANG-2 according to claim 21, wherein said antibody comprises a framework region, wherein: i) the heavy chain framework region comprises one or more amino acid back mutations selected from 44R, 77S and 84S; and / or The light chain framework region comprises one or more amino acid back mutations selected from 1N, 43S, 68A, 85D and 87H; or ii) the heavy chain framework region comprises 2L, 44R, 74V, 82A One or more amino acid reversion mutations in S and 83K; and / or The light chain framework region comprises one or more amino acid back mutations selected from 1N, 43S, 46V, 68A, 85D and 87H.

23. The antibody that specifically binds to ANG-2 according to claim 22, comprising a heavy chain variable region and a light chain variable region, wherein: a) the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 3; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 4; b) the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 20 or 21; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 17, 18 or 19; c) the amino acid sequence of the heavy chain variable region is as shown in any one of SEQ ID NOs: 28-37; and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 17; d) the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 72 or 73; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 74; e) the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 5; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 6; f) the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 44, 45 or 46; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 40, 41, 42 or 43; or g) the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 75; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

76.

24. The antibody that specifically binds to ANG-2 according to claim 23, further comprising a constant region.

25. The antibody that specifically binds to ANG-2 according to claim 24, comprising the heavy chain constant region of SEQ ID NO: 47 and the light chain constant region of SEQ ID NO:

48.

26. The antibody that specifically binds to ANG-2 according to claim 20, comprising a heavy chain and a light chain, wherein: a) the amino acid sequence of the heavy chain is shown in SEQ ID NO: 49, 51 or 52, and The amino acid sequence of the light chain is shown in SEQ ID NO: 50; or b) the amino acid sequence of the heavy chain is shown in SEQ ID NO: 53 or 55, and the amino acid sequence of the light chain is shown in SEQ ID NO:

54.

27. A nucleic acid molecule encoding the bispecific antigen binding molecule of any one of claims 1 to 19, or the antibody specifically binding to ANG-2 of any one of claims 20 to 26.

28. A vector comprising the nucleic acid molecule according to claim 27.

29. A host cell comprising the vector according to claim 28.

30. A pharmaceutical composition comprising a therapeutically effective amount of the bispecific antigen binding molecule according to any one of claims 1 to 19, or the antibody specifically binding to ANG-2 according to any one of claims 20 to 26, or the nucleic acid molecule according to claim 27, and one or more pharmaceutically acceptable carriers, diluents or excipients.

31. A method of producing a bispecific antigen binding molecule according to any one of claims 1 to 19, or an antibody that specifically binds to ANG-2 according to any one of claims 20 to 26, the method comprising culturing the host cell according to claim 29 to express the bispecific antigen binding molecule or the antibody that specifically binds to ANG-2.

32. Use of the bispecific antigen binding molecule of any one of claims 1 to 19, or the antibody that specifically binds to ANG-2 according to any one of claims 20 to 26, or the nucleic acid molecule of claim 27, or the pharmaceutical composition of claim 30 in the preparation of a medicament for preventing or treating cancer or angiogenic eye disease associated with VEGF or ANG-2, wherein the cancer is selected from breast cancer, adrenal tumors, 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; wherein the angiogenic eye disease is an ocular neovascular disease.

33. The use according to claim 32, wherein the angiogenic eye disease is selected from neovascular glaucoma, age-related macular degeneration (AMD) and diabetic macular edema, corneal neovascularization, corneal transplant neovascularization, retinal / choroidal neovascularization and angle neovascularization (rubeoma iris).

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