ANG-2 / vegf antibodies and uses thereof

Abstract

Described herein are antibodies that bind to VEGFA or ANG-2, or bispecific antibodies that bind to VEGFA and ANG-2. The antibodies of the present disclosure are useful in the treatment of eye diseases, including macular degeneration.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 388,538, filed on July 12, 2022, which is incorporated herein by reference. Background Art

[0003] Eye diseases including macular degeneration and retinal vein occlusion affect thousands of people each year and can lead to permanent vision loss or blindness. Vascular endothelial growth factor (VEGF) and angiopoietin-2 (ANG2) proteins play a role in the development of these diseases and therefore represent potential therapeutic targets. Summary of the invention

[0004] Described herein are antibodies and bispecific antibodies that bind to VEGF and / or ANG-2. Methods of treating eye diseases by administering the antibodies and bispecific antibodies to individuals in need thereof are also described. Such diseases include macular degeneration, retinal vein occlusion, branch vein occlusion, and central vein occlusion. The bispecific antibodies described herein have advantages over previously described bispecific antibodies because the binding moieties that bind to VEGF and ANG-2 both bind to their respective antigens with high affinity. In addition, the surrogate light chain used as the common light chain is from the human germline and is therefore less immunogenic than other common light chains, allowing for safer molecules that are less likely to be subject to anti-therapeutic antibody responses. A low anti-therapeutic antibody immune response is desirable because it allows the therapeutic antibody to maintain efficacy after repeated administration.

[0005] In one aspect, as described herein is a vascular endothelial growth factor (VEGF) binding antibody or antigen-binding fragment thereof, comprising: (a) a heavy chain complementary determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO:3 (GYTFSIYT); (b) a heavy chain complementary determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO:4 (INPYNGNT); and (c) a heavy chain complementary determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO:5 (AKAPAVFWWTGLDY); wherein the antibody binds to VEGF. In certain embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO:1. In certain embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region comprising an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 2. In certain embodiments, the antibody or antigen-binding fragment thereof comprises an alternative light chain. In certain embodiments, the alternative light chain comprises: (a) a light chain complementary determining region 1 (LCDR1) comprising the amino acid sequence shown in SEQ ID NO: 23 (NDHDIGVYS); (b) a light chain complementary determining region 2 (LCDR2) comprising the amino acid sequence shown in SEQ ID NO: 24 (YFSQSDK); and (c) a light chain complementary determining region 3 (LCDR3) comprising the amino acid sequence shown in SEQ ID NO: 25 (AMGARSSVTH). In certain embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 21. In certain embodiments, the antibody or antigen-binding fragment thereof comprises a light chain constant region comprising an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 22. In certain embodiments, the antibody binds to a peptide comprising a platelet-derived growth factor (PDGF) domain of VEGF-A. In certain embodiments, the PDGF domain of VEGF_A comprises a VEGF_A fragment ranging from amino acids 50 to 132. In certain embodiments, the antibody binds to a peptide comprising a VEGF-C heparin domain of VEGF-A.In certain embodiments, the VEGF_C heparin domain of VEGF_A comprises a VEGF-A fragment ranging from the 143rd to the 191st amino acid. In certain embodiments, the antibody binds to VEGF-A or a fragment thereof. In certain embodiments, the antibody binds to VEGF_A or a fragment thereof with an EC50 of about 60 picomoles or less. In certain embodiments, the antibody inhibits the interaction of VEGF-A or a fragment thereof with a VEGF receptor. In certain embodiments, the antibody inhibits the interaction of VEGF_A or a fragment thereof with the VEGF receptor with an IC50 of about 2000 picomoles or less. In certain embodiments, the antibody inhibits the interaction of VEGF_A or a fragment thereof with the VEGF receptor at least 3 times stronger than Faricimab inhibits the interaction. In certain embodiments, the VEGF receptor is a VEGF receptor 2 / kinase insert domain receptor (KDR). In certain embodiments, the antibody or its antigen-binding fragment is Fab, F(ab)2, a single domain antibody or a single chain variable fragment (scFv). In certain embodiments, the antibody or its antigen-binding fragment is an IgG antibody. In certain embodiments, the antibody or its antigen-binding fragment is human, chimeric or humanized. In certain embodiments, the antibody or its antigen-binding fragment is humanized. A pharmaceutical composition is also described, comprising the antibody or its antigen-binding fragment and a pharmaceutically acceptable carrier, excipient or diluent. In certain embodiments, the pharmaceutical composition is formulated for intravenous administration. In certain embodiments, the pharmaceutical composition is formulated for intravitreal administration. A nucleic acid encoding the antibody is also described herein. In certain embodiments, the nucleic acid is an expression vector. In certain embodiments, the expression vector is a lentiviral vector, an adenoviral vector, an adeno-associated viral vector or a plasmid vector. In certain embodiments, the expression vector is formulated for intravenous administration. In certain embodiments, the expression vector is formulated for intravitreal administration. A cell is also described herein, comprising the nucleic acid or the expression vector. In certain embodiments, the cell is a eukaryotic cell suitable for producing an antibody. In certain embodiments, the bispecific antibody is used in a method of treating an eye disease in an individual, the method comprising administering the antibody, the nucleic acid or nucleic acids encoding the antibody, or a pharmaceutical composition comprising the antibody to the individual, thereby treating the disease of the eye. In certain embodiments, the disease of the eye is macular degeneration. In certain embodiments, the macular degeneration is age-related. In certain embodiments, the macular degeneration is associated with diabetes. In certain embodiments, the macular degeneration is wet macular degeneration. In certain embodiments, the disease of the eye is branch retinal vein occlusion.In certain embodiments, said disease of said eye is central retinal vein occlusion.

[0006] In one aspect, as described herein is an angiopoietin 2 (ANG-2) binding antibody or antigen-binding fragment thereof comprising: (a) a heavy chain complementary determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 13 (GFTFSSYG); (b) a heavy chain complementary determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 14 (ISADSGDK); and (c) a heavy chain complementary determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 15 (AKEFISWIYTFDYLDY), wherein the antibody binds to ANG-2. In certain embodiments, the antibody or antigen-binding fragment thereof comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 11. In certain embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region comprising an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 12. In certain embodiments, the antibody or antigen-binding fragment thereof comprises an alternative light chain. In certain embodiments, the alternative light chain comprises: (a) a light chain complementary determining region 1 (LCDR1) comprising the amino acid sequence shown in SEQ ID NO: 23 (NDHDIGVYS); (b) a light chain complementary determining region 2 (LCDR2) comprising the amino acid sequence shown in SEQ ID NO: 24 (YFSQSDK); and (c) a light chain complementary determining region 3 (LCDR3) comprising the amino acid sequence shown in SEQ ID NO: 25 (AMGARSSVTH). In certain embodiments, the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 21. In certain embodiments, the antibody or antigen-binding fragment thereof comprises a light chain constant region comprising an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 22. In certain embodiments, the antibody binds to ANG-2. In certain embodiments, the antibody binds to ANG-2 with an EC50 of about 55 pmolar or less. In certain embodiments, the antibody binds to ANG-2 at least 50-fold more strongly than the binding of fareciximab to ANG-2. In certain embodiments, the antibody inhibits the interaction of ANG-2 with an ANG-2 receptor.In certain embodiments, the antibody inhibits the interaction of ANG-2 with the ANG-2 receptor with an IC50 of about 800 picomolar or less. In certain embodiments, the antibody inhibits the interaction of ANG-2 with the ANG-2 receptor at least 15 times more potently than farecimab. In certain embodiments, the ANG-2 receptor is a tyrosine kinase with immunoglobulin-like and EGF-like domains 2 receptor (Tie-2 receptor). In certain embodiments, the antibody or antigen-binding fragment thereof is a Fab, F(ab)2, single domain antibody, or single chain variable fragment (scFv). In certain embodiments, the antibody or antigen-binding fragment thereof is an IgG antibody. In certain embodiments, the antibody or antigen-binding fragment thereof is human, chimeric, or humanized. In certain embodiments, the antibody or antigen-binding fragment thereof is humanized. Also described is a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof and a pharmaceutically acceptable carrier, excipient, or diluent. In certain embodiments, the pharmaceutical composition is formulated for intravenous administration. In certain embodiments, the pharmaceutical composition is formulated for intravitreal administration. Also described herein are nucleic acids encoding the antibodies. In certain embodiments, the nucleic acid is an expression vector. In certain embodiments, the expression vector is a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, or a plasmid vector. In certain embodiments, the expression vector is formulated for intravenous administration. In certain embodiments, the expression vector is formulated for intravitreal administration. Also described herein is a cell comprising the nucleic acid or the expression vector. In certain embodiments, the cell is a eukaryotic cell suitable for producing antibodies. In certain embodiments, the antibody is used in a method for treating an individual eye disease, the method comprising administering the antibody, the nucleic acid or nucleic acids encoding the antibody, or a pharmaceutical composition comprising the antibody to the individual, thereby treating the disease of the eye. In certain embodiments, the disease of the eye is macular degeneration. In certain embodiments, the macular degeneration is age-related. In certain embodiments, the macular degeneration is associated with diabetes. In certain embodiments, the macular degeneration is wet macular degeneration. In certain embodiments, the disease of the eye is branch retinal vein occlusion. In certain embodiments, the disease of the eye is central retinal vein occlusion.

[0007] In another aspect, described herein is a bispecific antibody comprising: a vascular endothelial growth factor (VEGF) binding portion comprising: (a) a heavy chain complementary determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 3 (GYTFSIYT); (b) a heavy chain complementary determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 4 (INPYNGNT); and (c) a heavy chain complementary determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 5 (AKAPAVFWWTGLDY); and an angiopoietin 2 (ANG-2) binding portion comprising: (a) a heavy chain complementary determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 13 (GFTFSSYG); (b) a heavy chain complementary determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 14 (ISADSGDK); and (c) a heavy chain complementary determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 15 NO:15 (AKEFISWIYTFDYLDY); wherein the bispecific antibody binds to VEGF and ANG-2. In certain embodiments, the VEGF binding portion comprises a heavy chain variable region, the heavy chain variable region comprising an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:1. In certain embodiments, the VEGF binding portion further comprises a heavy chain constant region. In certain embodiments, the heavy chain constant region comprises an engineered protrusion or an engineered cavity such that homodimerization of the VEGF binding portion is inhibited. In certain embodiments, the engineered protrusion comprises a T366W substitution according to EU numbering. In certain embodiments, the engineered cavity comprises a T366S / L368A / Y407V substitution according to EU numbering. In certain embodiments, the heavy chain constant region comprises one or more substitutions in the Fc region that reduce antibody effector function. In certain embodiments, the one or more substitutions of the Fc region that reduce antibody effector function comprise L234A and / or L235A according to EU numbering. In certain embodiments, the heavy chain constant region comprises one or more substitutions of cysteine ​​residues. In certain embodiments, the one or more substitutions of cysteine ​​residues are located at serine 354 according to EU numbering. In certain embodiments, the VEGF binding portion comprises a heavy chain constant region comprising an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 2.In certain embodiments, the ANG-2 binding portion comprises a heavy chain variable region comprising an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence shown in SEQ ID NO: 11. In certain embodiments, the ANG-2 binding portion further comprises a heavy chain constant region. In certain embodiments, the heavy chain constant region comprises an engineered protrusion or an engineered cavity such that homodimerization of the ANG-2 binding portion is inhibited. In certain embodiments, the engineered protrusion comprises a T366W substitution according to EU numbering. In certain embodiments, the engineered cavity comprises a T366S / L368A / Y407V substitution according to EU numbering. In certain embodiments, the heavy chain constant region comprises one or more substitutions of the Fc region that reduce antibody effector function. In certain embodiments, the one or more substitutions of the Fc region that reduce antibody effector function comprise L234A and / or L235A according to EU numbering. In certain embodiments, the heavy chain constant region comprises one or more replacements of cysteine ​​residues. In certain embodiments, one or more replacements of the cysteine ​​residues are located at serine 354 according to EU numbering. In certain embodiments, the bispecific antibody is bound to a peptide comprising a platelet-derived growth factor (PDGF) domain of VEGF-A. In certain embodiments, the PDGF domain of VEGF_A comprises a VEGF_A fragment ranging from the 50th to the 132nd amino acid. In certain embodiments, the bispecific antibody is bound to a peptide comprising a VEGF-C heparin domain of VEGF-A. In certain embodiments, the VEGF_C heparin domain of VEGF_A comprises a VEGF-A fragment ranging from the 143rd to the 191st amino acid. In certain embodiments, the bispecific antibody is bound to VEGF-A or its fragment. In certain embodiments, the bispecific antibody is bound to VEGF_A or its fragment with an EC50 of about 60 picomoles or less. In certain embodiments, the bispecific antibody inhibits the interaction of VEGF-A or its fragment with a VEGF receptor. In certain embodiments, the bispecific antibody inhibits the interaction of VEGF_A or its fragment with the VEGF receptor with an IC50 of about 2000 picomoles or less. In certain embodiments, the bispecific antibody inhibits the interaction of VEGF_A or its fragment with the VEGF receptor at least 3 times stronger than farecimab inhibits the interaction. In certain embodiments, the VEGF receptor is VEGF receptor 2 / kinase insert domain receptor (KDR).In certain embodiments, the ANG-2 binding portion comprises a heavy chain constant region comprising an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 12. In certain embodiments, the bispecific antibody comprises a common light chain. In certain embodiments, the common light chain is a surrogate light chain. In certain embodiments, the surrogate light chain comprises: (a) a light chain complementary determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 23 (NDHDIGVYS); (b) a light chain complementary determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 24 (YFSQSDK); and (c) a light chain complementary determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 25 (AMGARSSVTH). In certain embodiments, the surrogate light chain comprises a light chain variable region comprising an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 21. In certain embodiments, the surrogate light chain comprises a light chain constant region comprising an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 22. In certain embodiments, the bispecific antibody comprises: (a) a first heavy chain variable region comprising an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence set forth in SEQ ID NO: 1; (b) a second heavy chain variable region comprising an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence set forth in SEQ ID NO: 11; and (c) a common light chain variable region, wherein the common light chain variable region comprises a light chain variable region comprising an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence set forth in SEQ ID NO: 21, wherein the bispecific antibody binds to VEGF and ANG-2. In certain embodiments, the bispecific antibody binds to ANG-2. In certain embodiments, the bispecific antibody binds to ANG-2 with an EC50 of about 55 picomolar or less. In certain embodiments, the bispecific antibody binds to ANG-2 at least 50-fold stronger than the binding of farecximab to ANG-2.In certain embodiments, the bispecific antibody inhibits the interaction of ANG-2 with an ANG-2 receptor. In certain embodiments, the bispecific antibody inhibits the interaction of ANG-2 with the ANG-2 receptor with an IC50 of about 800 picomolar or less. In certain embodiments, the bispecific antibody inhibits the interaction of ANG-2 with the ANG-2 receptor at least 15 times more potently than farecimab. In certain embodiments, the ANG-2 receptor is a tyrosine kinase with immunoglobulin-like and EGF-like domains 2 receptor (Tie-2 receptor). In certain embodiments, the bispecific antibody or antigen-binding fragment thereof is human, chimeric or humanized. In certain embodiments, the bispecific antibody or antigen-binding fragment thereof is humanized. Also described is a pharmaceutical composition comprising the bispecific antibody or antigen-binding fragment thereof and a pharmaceutically acceptable carrier, excipient or diluent. In certain embodiments, the pharmaceutical composition is formulated for intravenous administration. In certain embodiments, the pharmaceutical composition is formulated for intravitreal administration. Also described herein are nucleic acids encoding the bispecific antibodies. In certain embodiments, the nucleic acid is an expression vector. In certain embodiments, the expression vector is a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, or a plasmid vector. In certain embodiments, the expression vector is formulated for intravenous administration. In certain embodiments, the expression vector is formulated for intravitreal administration. A cell is also described herein, comprising the nucleic acid or the expression vector. In certain embodiments, the cell is a eukaryotic cell suitable for producing a bispecific antibody. In certain embodiments, the bispecific antibody is used in a method for treating an individual eye disease, the method comprising administering the bispecific antibody, the nucleic acid or nucleic acids encoding the bispecific antibody, or a pharmaceutical composition comprising the bispecific antibody to the individual, thereby treating the disease of the eye. In certain embodiments, the disease of the eye is macular degeneration. In certain embodiments, the macular degeneration is age-related. In certain embodiments, the macular degeneration is associated with diabetes. In certain embodiments, the macular degeneration is wet macular degeneration. In certain embodiments, the disease of the eye is branch retinal vein occlusion. In certain embodiments, the disease of the eye is central retinal vein occlusion.

[0008] In another aspect, described herein is a bispecific antibody comprising: (a) a first heavy chain comprising an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity to the amino acid sequence shown in SEQ ID NO: 2; (b) a second heavy chain comprising an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity to the amino acid sequence shown in SEQ ID NO: 12; and (c) a common light chain, wherein the common light chain comprises a light chain comprising an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identity to the amino acid sequence shown in SEQ ID NO: 22, wherein the bispecific antibody binds to VEGF and ANG-2. In certain embodiments, the bispecific antibody binds to VEGF with an EC50 of about 60 picomolar or less. In certain embodiments, the bispecific antibody inhibits the interaction of VEGF_A or its fragment with the VEGF receptor with an IC50 of about 2000 picomolar or less. In certain embodiments, the binding of the bispecific antibody to ANG-2 is at least 50 times stronger than the binding of fareximab to ANG-2. In certain embodiments, the bispecific antibody inhibits the interaction of ANG-2 with the ANG-2 receptor. In certain embodiments, the bispecific antibody inhibits the interaction of ANG-2 with the ANG-2 receptor with an IC50 of about 800 picomolar or less. In certain embodiments, the bispecific antibody inhibits the interaction of ANG-2 with the ANG-2 receptor at least 15 times stronger than fareximab. In certain embodiments, the bispecific antibody inhibits the interaction of VEGF_A or its fragment with the VEGF receptor at least 3 times stronger than fareximab inhibits the interaction. In certain embodiments, the bispecific antibody binds to ANG-2 with an EC50 of about 55 picomolar or less. In certain embodiments, the bispecific antibody or its antigen-binding fragment is human, chimeric or humanized. In certain embodiments, the bispecific antibody or antigen-binding fragment thereof is humanized. A pharmaceutical composition is also described, comprising the bispecific antibody or antigen-binding fragment thereof and a pharmaceutically acceptable carrier, excipient or diluent. In certain embodiments, the pharmaceutical composition is formulated for intravenous administration. In certain embodiments, the pharmaceutical composition is formulated for intravitreal administration. Also described herein is a nucleic acid encoding the bispecific antibody. In certain embodiments, the nucleic acid is an expression vector. In certain embodiments, the expression vector is a lentiviral vector, an adenoviral vector, an adeno-associated viral vector or a plasmid vector. In certain embodiments, the expression vector is formulated for intravenous administration.In certain embodiments, the expression vector is formulated for intravitreal administration. A cell is also described herein, comprising the nucleic acid or the expression vector. In certain embodiments, the cell is a eukaryotic cell suitable for producing a bispecific antibody. In certain embodiments, the bispecific antibody is used in a method for treating an individual eye disease, the method comprising administering the bispecific antibody, the nucleic acid or nucleic acids encoding the bispecific antibody, or a pharmaceutical composition comprising the bispecific antibody to the individual, thereby treating the disease of the eye. In certain embodiments, the disease of the eye is macular degeneration. In certain embodiments, the macular degeneration is age-related. In certain embodiments, the macular degeneration is associated with diabetes. In certain embodiments, the macular degeneration is wet macular degeneration. In certain embodiments, the disease of the eye is branch retinal vein occlusion. In certain embodiments, the disease of the eye is central retinal vein occlusion.

[0009] In another aspect, described herein is a bispecific antibody comprising: (a) a first heavy chain comprising an amino acid sequence as shown in SEQ ID NO: 2; (b) a second heavy chain comprising an amino acid sequence as shown in SEQ ID NO: 12; and (c) a common light chain, wherein the common light chain comprises a light chain comprising an amino acid sequence as shown in SEQ ID NO: 22, wherein the bispecific antibody binds to VEGF and ANG-2. In certain embodiments, the bispecific antibody binds to VEGF with an EC50 of about 60 picomolar or less. In certain embodiments, the bispecific antibody inhibits the interaction of VEGF-A or a fragment thereof with a VEGF receptor. In certain embodiments, the bispecific antibody inhibits the interaction of VEGF_A or a fragment thereof with the VEGF receptor with an IC50 of about 2000 picomolar or less. In certain embodiments, the bispecific antibody inhibits the interaction of VEGF_A or a fragment thereof with the VEGF receptor at least 3 times stronger than farecimab inhibits the interaction. In certain embodiments, the bispecific antibody binds to ANG-2 with an EC50 of about 55 picomolar or less. In certain embodiments, the bispecific antibody binds to ANG-2 at least 50 times stronger than the binding of fareximab to ANG-2. In certain embodiments, the bispecific antibody inhibits the interaction of ANG-2 with an ANG-2 receptor. In certain embodiments, the bispecific antibody inhibits the interaction of ANG-2 with the ANG-2 receptor with an IC50 of about 800 picomolar or less. In certain embodiments, the bispecific antibody inhibits the interaction of ANG-2 with the ANG-2 receptor at least 15 times stronger than fareximab. In certain embodiments, the bispecific antibody or antigen-binding fragment thereof is human, chimeric or humanized. In certain embodiments, the bispecific antibody or antigen-binding fragment thereof is humanized. Also described is a pharmaceutical composition comprising the bispecific antibody or antigen-binding fragment thereof and a pharmaceutically acceptable carrier, excipient or diluent. In certain embodiments, the pharmaceutical composition is formulated for intravenous administration. In certain embodiments, the pharmaceutical composition is formulated for intravitreal administration. A nucleic acid encoding the bispecific antibody is also described herein. In certain embodiments, the nucleic acid is an expression vector. In certain embodiments, the expression vector is a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, or a plasmid vector. In certain embodiments, the expression vector is formulated for intravenous administration. In certain embodiments, the expression vector is formulated for intravitreal administration. A cell is also described herein, comprising the nucleic acid or the expression vector. In certain embodiments, the cell is a eukaryotic cell suitable for producing a bispecific antibody.In certain embodiments, the bispecific antibody is used in a method for treating an eye disease in an individual, the method comprising administering the bispecific antibody, the nucleic acid or nucleic acids encoding the bispecific antibody, or a pharmaceutical composition comprising the bispecific antibody to the individual, thereby treating the disease in the eye. In certain embodiments, the disease in the eye is macular degeneration. In certain embodiments, the macular degeneration is age-related. In certain embodiments, the macular degeneration is associated with diabetes. In certain embodiments, the macular degeneration is wet macular degeneration. In certain embodiments, the disease in the eye is branch retinal vein occlusion. In certain embodiments, the disease in the eye is central retinal vein occlusion. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The novel features described herein are set forth with particularity in the appended claims. A better understanding of the features and advantages of the features described herein will be obtained by referring to the following detailed description and the accompanying drawings, which set forth illustrative examples of the principles in which the features described herein are utilized, wherein:

[0011] Figure 1A-1B Shown are SDS-PAGE and SE-HPLC analyses of bispecific surrobodies produced in CHO cells having amino acid sequences corresponding to SEQ ID NO: 2, SEQ ID NO: 12, and SEQ ID NO: 22. Figure 1A The SDS-PAGE results of reduced and non-reduced samples are shown. Lane 1, MW ladder; Lane 2, IgG control (non-reduced); Lane 3, Sample 1 (non-reduced); Lane 4, Sample 2 (non-reduced); Lane 5, Sample 3 (non-reduced); Lane 6, IgG control (reduced); Lane 7, Sample 1 (reduced); Lane 8, Sample 2 (reduced); Lane 5, Sample 3 (reduced). Figure 1B Shown are the SE-HPLC results of three samples.

[0012] Figures 2A-2F The graph shows the binding of the control antibody RO-101 to VEGF and its inhibition of the interaction between VEGF and its receptor compared to fareciximab. Figure 2A VEGF is shown 121 VEGF 165 The sequence comparison between . Figure 2B Diagram showing RO-101 and VEGF 121 VEGF 165 Combined ELISA binding results. Figure 2C RO-101 or farecimab and VEGF 121 Combined ELISA binding results. Figure 2DRO-101 or farecimab and VEGF 165 Combined ELISA binding results. Figure 2E An assay diagram for testing surrogate antibodies for their ability to inhibit the interaction between VEGF and its receptor is shown. Figure 2F Figure 3 shows the effect of RO-101 or farecximab on VEGF 165 ELISA binding results for inhibition of interaction with its receptor.

[0013] Figures 3A-3C Graph showing the binding of the surrogate antibody RO-101 to ANG-2 and its inhibition of the interaction between ANG-2 and its receptor compared to farecximab. Figure 3A ELISA binding results of RO-101 or farecximab binding to ANG-2 are shown. Figure 3B An assay diagram for testing the ability of surrogate antibodies to inhibit the interaction between ANG-2 and its receptor is shown. Figure 3C Graphed are the ELISA binding results for inhibition of the interaction between ANG-2 and its receptor by RO-101 or farecximab.

[0014] Figures 4A-4B The binding of different forms of RO-101 to VEGF and ANG-2 is shown. Figure 4A Graphed are the ELISA binding results of RO-101SgG, scSv or Sab derived from RO-101 to VEGF. Figure 4B Graphed are the ELISA binding results of RO-101 SgG, scSv or Sab derived from RO-101 to ANG-2.

[0015] Figures 5A-5D The binding of RO-101 to other PDGF / VEGF family members is shown. Figure 5A The sequence alignment between VEGF_B, VEGF_C, VEGF_D, PIGF-1 and PIGF-3 is shown. Figure 5B VEGF 165 Compared to the ELISA binding results of RO-101 binding to VEGF_B from different suppliers. Figure 5C VEGF 165 Compared with the binding of RO-101 to VEGF_C or VEGF_D. Figure 5D VEGF 165 Compared to the binding of RO-101 to PIGF-1 or PIGF-3.

[0016] Figures 6A-6D Binding of RO-101 to non-human orthologs of VEGF or ANG-2 is shown. Fig. 6AShown is a sequence alignment between VEGF orthologs from human, rabbit, dog and pig. Figure 6B Graphed are ELISA binding results for RO-101 binding to VEGF orthologs from human, rabbit, dog and pig. Figure 6C Diagram showing sequence alignment between ANG-2 orthologs from human, rabbit, dog and pig Fig.6D Graphed are ELISA binding results for RO-101 binding to ANG-2 orthologs from human, rabbit and dog. DETAILED DESCRIPTION

[0017] In one aspect, the present invention describes a vascular endothelial growth factor (VEGF) binding antibody or an antigen-binding fragment thereof, which comprises: a heavy chain complementary determining region 1 (HCDR1) comprising the amino acid sequence shown in SEQ ID NO: 3 (GYTFSIYT); a heavy chain complementary determining region 2 (HCDR2) comprising the amino acid sequence shown in SEQ ID NO: 4 (INPYNGNT); and a heavy chain complementary determining region 3 (HCDR3) comprising the amino acid sequence shown in SEQ ID NO: 5 (AKAPAVFWWTGLDY); wherein the antibody binds to VEGF.

[0018] In another aspect, described herein is an angiopoietin 2 (ANG-2) binding antibody or antigen-binding fragment thereof comprising: a heavy chain complementary determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 13 (GFTFSSYG); a heavy chain complementary determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 14 (ISADSGDK); and a heavy chain complementary determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 15 (AKEFISWIYTFDYLDY), wherein the antibody binds to ANG-2.

[0019] In another aspect, the present invention provides a bispecific antibody comprising: a vascular endothelial growth factor (VEGF) binding portion comprising: a heavy chain complementary determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 3 (GYTFSIYT); a heavy chain complementary determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 4 (INPYNGNT); and a heavy chain complementary determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 5 (AKAPAVFWWTGLDY); and an angiopoietin 2 (ANG-2) binding portion comprising: a heavy chain complementary determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 13 (GFTFSSYG); a heavy chain complementary determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 14 (ISADSGDK); and a heavy chain complementary determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 15 (ISADSGDK). The amino acid sequence shown in NO: 15 (AKEFISWIYTFDYLDY); wherein the bispecific antibody binds to VEGF and ANG-2.

[0020] In another aspect, described herein is a bispecific antibody comprising: (a) a first heavy chain variable region comprising an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence shown in SEQ ID NO: 1; (b) a second heavy chain variable region comprising an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence shown in SEQ ID NO: 11; and (c) a common light chain variable region, wherein the common light chain variable region comprises a light chain variable region comprising an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence shown in SEQ ID NO: 21, wherein the bispecific antibody binds to VEGF and ANG-2.

[0021] In another aspect, described herein is a bispecific antibody comprising: (a) a first heavy chain comprising an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence shown in SEQ ID NO:2; (b) a second heavy chain comprising an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence shown in SEQ ID NO:12; and (c) a common light chain, wherein the common light chain comprises a light chain comprising an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence shown in SEQ ID NO:22, wherein the bispecific antibody binds to VEGF and ANG-2.

[0022] In another aspect, described herein is a bispecific antibody comprising: (a) a first heavy chain comprising the amino acid sequence as shown in SEQ ID NO: 2; (b) a second heavy chain comprising the amino acid sequence as shown in SEQ ID NO: 12; and (c) a common light chain, wherein the common light chain comprises a light chain comprising the amino acid sequence as shown in SEQ ID NO: 22, wherein the bispecific antibody binds to VEGF and ANG-2.

[0023] In the following description, certain specific details are set forth in order to fully understand the various embodiments. However, those skilled in the art will appreciate that the embodiments provided can be implemented without these details. Unless the context otherwise requires, throughout the specification and the claims that follow, the word "comprise" and its variants, such as "comprises" and "comprising" should be interpreted in an open, inclusive sense, i.e., "including but not limited to". As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural indicators unless the content clearly provides otherwise. It should also be noted that the term "or" is generally used in a sense that includes "and / or", unless the content clearly provides otherwise. In addition, the titles provided herein are for convenience only and do not explain the scope or meaning of the claimed embodiments.

[0024] As used herein, the term "about" refers to an amount that is approximately 10% or less of the stated amount.

[0025] As used herein, "vascular endothelial growth factor" or "VEGF" refers to a polypeptide encoded by the VEGFA gene, the amino acid sequence of which can be obtained at www.uniprot.org / uniprot / P15692. Such sequences include post-translational modifications, naturally occurring amino acid variants, and naturally occurring splice variants that do not affect the binding of the antibodies and bispecific antibodies described herein.

[0026] As used herein, "Angiopoietin 2" or "ANG-2" refers to a polypeptide encoded by the ANGPT2 gene, the amino acid sequence of which can be obtained at www.uniprot.org / uniprot / 015123. Such sequences include post-translational modifications, naturally occurring amino acid variants, and naturally occurring splice variants that do not affect the binding of the antibodies and bispecific antibodies described herein.

[0027] As used herein, "surrogate light chain" refers to a light chain formed by the fusion of a VpreB polypeptide and a λ5 polypeptide, and can be promiscuously associated with two or more structurally different heavy chains. Such surrogate light chains are described in US8,114,967, US10,214,580 and 8,969,082, each of which is incorporated herein in its entirety.

[0028] As used herein, the terms "individual," "patient," or "subject" refer to an individual diagnosed with, suspected of having, or at risk of developing at least one disease for which the compositions and methods can be used to treat. In certain embodiments, the individual is a mammal. In certain embodiments, the mammal is a mouse, rat, rabbit, dog, cat, horse, cow, sheep, pig, goat, llama, alpaca, or yak. ​​In certain embodiments, the individual is a human.

[0029] The antibodies provided include monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies and multireactive antibodies) and antibody fragments. Antibodies include antibody conjugates and molecules including antibodies, such as chimeric molecules. Therefore, antibodies include but are not limited to full-length antibodies and natural antibodies, and fragments and parts thereof that retain their binding specificity, such as any specific binding portion thereof, including those with any number of immunoglobulin classes and / or isotypes (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD, IgE, and IgM); and biologically relevant (antigen binding) fragments or specific binding portions thereof, including but not limited to Fab, F(ab')2, Fv, and scFv (single chain or related entities). Monoclonal antibodies are generally one of substantially homogeneous antibody compositions; therefore, except for possible natural mutations that may be present in small amounts, any single antibody included in a monoclonal antibody composition is identical. Monoclonal antibodies may include human IgG1 constant regions. Monoclonal antibodies may include human IgG4 constant regions.

[0030] The term "antibody" herein is used in the broadest sense and includes monoclonal antibodies, bispecific antibodies, and includes complete antibodies and functional (antigen binding) antibody fragments thereof, including fragment antigen binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, single-chain antibody fragments (including single-chain variable fragments (sFv or scFv)) and single-domain antibodies (e.g., sdAb, sdFv, nanobody) fragments. The term encompasses genetically engineered and / or otherwise modified immunoglobulin forms such as intracellular antibodies, peptide antibodies, chimeric antibodies, fully human antibodies, humanized antibodies and heterologous conjugate antibodies, multispecific (e.g., bispecific) antibodies, double antibodies, three antibodies and four antibodies, tandem double scFv, tandem three scFv. Unless otherwise indicated, the term "antibody" should be understood to encompass its functional antibody fragments. The term also encompasses intact or full-length antibodies, including antibodies of any class or subclass, including IgG and its subclasses, IgM, IgE, IgA and IgD. The antibody may comprise a human IgG1 constant region. The antibody may comprise a human IgG4 constant region.

[0031] As used herein, molecules, peptides, polypeptides, antibodies or antibody fragments may be referred to as "bispecific" or "dual specific", including grammatical equivalents. Bispecific molecules have the ability to specifically bind to at least two structurally distinct targets. Specific binding can be the result of two different binding moieties that are structurally distinct at the molecular level, including but not limited to different, non-identical amino acid sequences; or can be capable of binding with high affinity (e.g., KD less than about 1x10 -6 ) a single binding moiety that specifically binds to two structurally distinct targets. A molecule, peptide, polypeptide, antibody, or antibody fragment that is referred to as "multispecific" is one that has the ability to specifically bind to at least three structurally distinct targets. "Bispecific antibodies," including grammatical equivalents, refer to bispecific molecules that retain at least one antibody fragment that is capable of specifically binding to a target, such as a variable region, heavy chain, or light chain, or one or more complementarity determining regions from an antibody molecule. "Multispecific antibodies," including grammatical equivalents, refer to multispecific molecules that retain at least one antibody fragment that is capable of specifically binding to a target, such as a variable region, heavy chain, or light chain, or complementarity determining regions from an antibody molecule.

[0032] The terms "complementarity determining region" and "CDR" are synonymous with "hypervariable region" or "HVR", and are known in the art to refer to discontinuous amino acid sequences within an antibody variable region that confer antigen specificity and / or binding affinity. Typically, there are three CDRs in each heavy chain variable region (CDR-H1, CDR-H2, CDR-H3), and three CDRs in each light chain variable region (CDR-L1, CDR-L2, CDR-L3). "Framework region" and "FR", as known in the art, refer to the non-CDR portions of the heavy and light chain variable regions. In general, there are four FRs (FR-H1, FR-H2, FR-H3, and FR-H4) in each full-length heavy chain variable region, and four FRs (FR-L1, FR-L2, FR-L3, and FR-L4) in each full-length light chain variable region. The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of a number of well-known schemes, including Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed., Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme), Al-Lazikani et al., (1997) JMB 273, 927-948 ("Chothia" numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), "Antibody-antigen interactions: Contact analysis and binding site topography," J. Mol. Biol. 262, 732-745.(“Contact” numbering scheme); Lefranc MP et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Igsuperfamily V-like domains,” Dev Comp Immunol, 2003 Jan;27(1):55-77 (“IMGT” numbering scheme); Honegger A and Plückthun A, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” J Mol Biol, 2001 Jun 8;309(3):657-70, (“Aho” numbering scheme); and Whitelegg NR and Rees AR, “WAM: an improved algorithm for modelling antibodies on the WEB," Protein Eng. 2000 Dec; 13(12): 819-24 ("AbM" numbering scheme. In certain embodiments, the CDRs of the antibodies described herein can be defined by a method selected from Kabat, Chothia, IMGT, Aho, AbM, or a combination thereof.

[0033] The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignments, while the Chothia scheme is based on structural information. The numbering of both the Kabat and Chothia schemes is based on the most common antibody region sequence lengths, wherein insertions are represented by inserting letters such as "30a", and deletions may occur in some antibodies. These two schemes place certain insertions and deletions ("indels") in different positions, resulting in different numbering. The Contact scheme is based on the analysis of complex crystal structures, and is similar to the Chothia numbering scheme in many respects.

[0034] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of a native antibody (V H and V L) generally have a similar structure, each domain comprising four conserved framework regions (FRs) and three CDRs (see, e.g., Kindt et al., Kuby Immunology, 6th edition, WH Freeman and Co., p. 91 (2007)). A single V H or V L A domain may be sufficient to confer antigen binding specificity. In addition, a V domain from an antibody that binds to a specific antigen may be used. H or V L domain to separate antibodies that bind to the antigen and to screen for complementary V L or V H Domain libraries (see, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991)).

[0035] Specific binding or binding of antibody molecules described herein refers to binding mediated by one or more CDR portions of the antibody. Not all CDRs may be required for specific binding. Specific binding can be demonstrated, for example, by ELISA for a specific target or antigen, where binding is significantly increased compared to an isotype control antibody.

[0036] As used herein, "epitope" refers to the minimum binding determinant of the antibody or fragment described herein that is necessary for the specific binding of the antibody or fragment thereof to the target antigen. When the target antigen is a polypeptide, the epitope will be a continuous or discontinuous epitope. A continuous epitope is formed by one region of the target antigen, while a discontinuous epitope can be formed by two or more separate regions. For example, a discontinuous epitope can be formed when the target antigen adopts a tertiary structure that binds two amino acid sequences together and forms a three-dimensional structure bound by the antibody. When the target antigen is a polypeptide, the epitope is generally a plurality of amino acids connected into a polypeptide chain. A continuous epitope may contain 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 consecutive amino acids. Although an epitope may contain a continuous amino acid polymer, not every amino acid of the polymer may contact the amino acid residues of the antibody. Such uncontacted amino acids will still constitute part of the epitope because they may be important for the structure and connection of the contacting amino acids. A skilled artisan can determine whether any given antibody binds to an epitope of a reference antibody, for example, by performing a cross-blocking experiment with the reference antibody. In certain embodiments, described herein are antibodies that bind to the same epitope of the antibody. In certain embodiments, described herein are antibodies that are competitively blocked by the antibody. In certain embodiments, described herein are antibodies that compete for binding with the antibody.

[0037] The antibodies provided include antibody fragments. "Antibody fragments" refer to molecules other than intact antibodies that include a portion of an intact antibody that binds to an 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; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv or sFv); and multispecific antibodies formed from antibody fragments. In a specific embodiment, the antibody is a single-chain antibody fragment comprising a variable heavy chain region and / or a variable light chain region, such as scFv.

[0038] Antibody fragments can be prepared by various techniques, including but not limited to proteolytic digestion of intact antibodies and production of recombinant host cells. In some embodiments, antibodies are recombinantly produced fragments, such as fragments comprising non-naturally occurring arrangements, such as fragments having two or more antibody regions or chains connected by synthetic linkers (e.g., polypeptide linkers), and / or fragments not produced by enzymatic digestion of naturally occurring intact antibodies. In some aspects, antibody fragments are scFv.

[0039] "Humanized" antibody is an antibody in which all or substantially all CDR amino acid residues are derived from non-human CDR and all or substantially all FR amino acid residues are derived from human FR. Humanized antibodies may alternatively include at least a portion of an antibody constant region derived from a human antibody. "Humanized form" of a non-human antibody refers to a variant of a humanized non-human antibody, usually to reduce immunogenicity to people while retaining specificity and affinity of the parent non-human antibody. In some embodiments, some FR residues in a humanized antibody are replaced by corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve antibody specificity or affinity.

[0040] The antibodies provided include human antibodies. A "human antibody" is an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell or a non-human source utilizing a human antibody library or other human antibody encoding sequences (including a human antibody library). The term excludes humanized non-human antibody forms containing non-human antigen binding regions, such as those in which all or substantially all CDRs are non-human.

[0041] Human antibodies can be prepared by administering immunogens to transgenic animals, which have been modified to produce complete human antibodies or complete antibodies with human variable regions in response to antigenic attack. Such animals generally contain all or part of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or are present outside the chromosomes or randomly integrated into the animal chromosomes. In such transgenic animals, the endogenous immunoglobulin loci are generally inactivated. Human antibodies can also be derived from human antibody libraries, including phage display libraries and cell-free libraries, which contain antibody coding sequences derived from human libraries.

[0042] The terms "polypeptide" and "protein" are used interchangeably and refer to polymers of amino acid residues and are not limited to a minimum length. Polypeptides, including the antibodies and antibody chains provided, and other peptides, such as linkers and binding peptides, may include amino acid residues including natural and / or non-natural amino acid residues. These terms also include post-expression modifications of polypeptides, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. In some aspects, as long as the protein maintains the desired activity, the polypeptide may contain modifications relative to the natural or native sequence. These modifications may be intentional, such as by site-directed mutagenesis, or accidental, such as by mutations of the host producing the protein or errors due to PCR amplification. In some embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. Variants are generally different from the polypeptides specifically disclosed herein in one or more substitutions, deletions, additions, and / or insertions. Such variants may be naturally occurring or synthetically produced, for example, by modifying one or more of the above-mentioned polypeptide sequences of the present invention and evaluating one or more biological activities of the polypeptides described herein and / or using any of a number of known techniques. For example, it may be necessary to improve the binding affinity and / or other biological properties of the antibody. The amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues in the amino acid sequence of the antibody. Any combination of deletions, insertions and substitutions can be performed to obtain the final construct, provided that the final construct has the desired properties, such as antigen binding.

[0043] The percentage of sequence identity (%) relative to a reference polypeptide sequence is the percentage of the amino acid residues in the candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, after the alignment sequence and the introduction of a gap (if necessary) to achieve the maximum percentage of sequence identity, any conservative substitution is not considered as a part of the sequence identity. The comparison for determining the percentage of amino acid sequence identity can be achieved by a variety of known means, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Suitable parameters for aligning sequences can be determined, including the algorithm required for achieving maximum alignment over the full length of the compared sequences. However, for the purposes of this article, the amino acid sequence identity percentage values ​​are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program is written by Genentech, Inc., and the source code has been submitted to the U.S. Copyright Office (Washington, D.C., 20559) together with user documentation, and the program is registered in the U.S. Copyright Office with registration number TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc. (South San Francisco, California) and can also be compiled from source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not change.

[0044] In the case of using ALIGN-2 for amino acid sequence comparison, the percentage of amino acid sequence identity between a given amino acid sequence A and a given amino acid sequence B (which can also be alternatively expressed as a given amino acid sequence A having or comprising a certain percentage of amino acid sequence identity with a given amino acid sequence B) is calculated as follows: 100 multiplied by a fraction X / Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in the program alignment of A and B, and where Y is the total number of amino acid residues in B. It is understood that when the length of amino acid sequence A is not equal to the length of amino acid sequence B, the percentage of amino acid sequence identity between A and B will not be equal to the percentage of amino acid sequence identity between B and A. Unless explicitly stated otherwise, all amino acid sequence identity percentage values ​​used herein are obtained using the ALIGN-2 computer program as described in the previous paragraph.

[0045] Amino acid sequence insertions and deletions include amino-terminal and / or carboxyl-terminal fusions ranging in length from one residue to a polypeptide containing one hundred or more residues, and intrasequence insertions and deletions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with N-terminal methionyl residues. Other insertion variants of antibody molecules include fusing the N-terminal or C-terminal of the antibody to an enzyme (e.g., for ADEPT) or a polypeptide that increases the serum half-life of the antibody. Examples of intrasequence insertion variants of antibody molecules include inserting 3 amino acids in the light chain. Examples of terminal deletions include antibodies with 7 or fewer amino acids missing at the light chain end.

[0046] In some embodiments, the antibodies provided herein have an affinity for the antibody target of about 1 μM, 100 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, 2 nM, 1 nM, 0.5 nM, 0.1 nM, 0.05 nM, 0.01 nM or less (e.g., 10 -8 M or lower, for example, from 10 -8 M to 10 -13 M, for example, from 10 -9 M to 10 -13 The dissociation constant (K D In some embodiments, the antibodies provided herein have an affinity for the antibody target of about 100 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, 2 nM, 1 nM, 0.5 nM, 0.1 nM, 0.05 nM, 0.01 nM, or 0.001 nM or greater (e.g., 10 -8 M or lower, for example, from 10 -8 M to 10 -13 M, for example, from 10 -9 M to 10 -13 The dissociation constant (K D ). The antibody target may be an anti-VEGF or anti-ANG-2 antibody. D Can be measured by any suitable assay. In certain embodiments, surface plasmon resonance assay (e.g., using -2000, -3000 or Octet) to measure KD.

[0047] In certain embodiments, the antibodies disclosed herein bind to VEGF-A. VEGF-A may have several different splice sites and may therefore have various isoforms depending on the splice site. Thus, in certain embodiments, the antibodies disclosed herein bind to VEGF 165 In certain embodiments, the antibodies disclosed herein bind to VEGF 121 In certain embodiments, the antibody binds to VEGF121 The binding affinity of can predict the efficacy of antibodies in treating eye diseases. In certain embodiments, antibodies disclosed herein bind to peptides comprising a platelet-derived growth factor (PDGF) domain of VEGF_A. In some embodiments, the PDGF domain of VEGF_A comprises a fragment of VEGF_A ranging from about the 40th, 41st, 42nd, 43rd, 44th, 45th, 46th, 46th, 47th, 48th, 49th or 50th to about the 130th, 131st, 132nd, 133rd, 134th, 135th, 136th, 137th, 138th, 139th or 140th amino acid. In certain embodiments, antibodies disclosed herein bind to peptides comprising a VEGF_C heparin domain of VEGF_A. In some embodiments, the VEGF_C heparin domain of VEGF_A comprises a fragment of VEGF_A ranging from about amino acid 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, or 150 to about amino acid 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 120. In certain embodiments, the antibodies disclosed herein bind to VEGF_A from a human or non-human species, e.g., rabbit, canine (dog) or porcine (pig)).

[0048] In certain embodiments, the antibody is an anti-human VEGF antibody that has a K of 100, 75, 60, 50, 40, 30, or 25 pmolar or less. D or EC 50 In certain embodiments, the antibody is an anti-human VEGF-A antibody that binds to human VEGF with a K of 100, 75, 60, 50, 40, 30 or 25 picomolar or less. D or EC 50 In certain embodiments, the antibody is an anti-human VEGF-A antibody that binds to human VEGF with a K of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 picomolar or higher. D or EC 50 Binds to human VEGF. In certain embodiments, the EC binding of the antibodies disclosed herein to VEGF_A or a fragment thereof 50 EC between farecximab and VEGF_A or its fragments 50 In certain embodiments, the EC binding sites of the antibodies disclosed herein and VEGF_A or its fragments are 50 EC between bifacitinib and VEGF_A or its fragments 50Low 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500pM.

[0049] In certain embodiments, the antibody is an anti-human VEGF_A antibody that inhibits the interaction of VEGF-A or its fragments with a VEGF receptor. In certain embodiments, the antibody is an anti-human VEGF_A antibody that inhibits the interaction of VEGF-A or its fragments with a VEGF receptor. 50 5nM, 4nM, 3nM, 2nM, 1nM, 900pM, 800pM, 700pM, 600pM or less. In certain embodiments, the antibody is an anti-human VEGF-A antibody that inhibits the interaction of VEGF-A or its fragment with a VEGF receptor, IC 50 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 picomoles or more. In certain embodiments, the antibodies disclosed herein inhibit the interaction of VEGF_A or its fragments with the VEGF receptor at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times stronger than farecimab inhibits the interaction. In certain embodiments, the VEGF receptor is VEGF receptor 2 / kinase insert domain receptor (KDR).

[0050] In certain embodiments, the antibodies disclosed herein bind significantly less to VEGF_B than to VEGF_A. In certain embodiments, the antibodies disclosed herein bind significantly less to VEGF_C than to VEGF_A. In certain embodiments, the antibodies disclosed herein bind significantly less to VEGF_D than to VEGF_A. In certain embodiments, the antibodies disclosed herein bind significantly less to PIGF-1 than to VEGF_A. In certain embodiments, the antibodies disclosed herein bind significantly less to PIGF-3 than to VEGF_A.

[0051] In certain embodiments, the antibody is an anti-human ANG-2 antibody that has a K of 100, 75, 60, 55, 50, 40, 30, or 25 pmolar or less. D or EC 50 In certain embodiments, the antibody is an anti-human ANG-2 antibody having a K of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 picomolar or more. D or EC 50 Binding to human ANG-2. In certain embodiments, the antibody binds to ANG-2 at least 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, or 70-fold stronger than the binding of farecximab to ANG-2.

[0052] In certain embodiments, the antibody is an anti-human ANG-2 antibody that inhibits the interaction of ANG-2 with an ANG-2 receptor. In certain embodiments, the antibody is an anti-human VEGF_A antibody that inhibits the interaction of ANG-2 with an ANG-2 receptor. 50 In some embodiments, the antibody is an anti-human VEGF-A antibody that inhibits the interaction of ANG-2 with the ANG-2 receptor, IC 50 In some embodiments, the antibodies disclosed herein inhibit the interaction of ANG-2 with the ANG-2 receptor at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 picomolar or more. In some embodiments, the antibodies disclosed herein inhibit the interaction of ANG-2 with the ANG-2 receptor at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 times more potently than fareciximab inhibits the interaction. In some embodiments, the ANG-2 receptor is a tyrosine kinase with immunoglobulin-like and EGF-like domains 2 receptor (Tie-2 receptor).

[0053] In certain embodiments, the antibody is an anti-human VEGF / ANG-2 bispecific antibody that has a K of 100, 75, 60, 50, 40, 30, or 25 pmolar or less. D or EC 50 In certain embodiments, the antibody is an anti-human VEGF / ANG-2 bispecific antibody that binds to human VEGF with a K of 100, 75, 60, 50, 40, 30, or 25 pmolar or less. D or EC 50 Binds to human VEGF_A. In certain embodiments, the antibody is an anti-human VEGF / ANG-2 bispecific antibody that binds to human VEGF_A with a K of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 picomolar or higher. D or EC 50 Binds to human VEGF_A.

[0054] In certain embodiments, the antibody is an anti-human VEGF / ANG-2 bispecific antibody that has a K of 100, 75, 60, 50, 40, 30, or 25 pmolar or less. D or EC 50 In certain embodiments, the antibody is an anti-human VEGF / ANG-2 bispecific antibody that binds to human ANG-2 with a K of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 picomolar or more. D or EC 50 Binds to human ANG-2.

[0055] In some embodiments, the antibodies disclosed herein are IgG antibodies. In some embodiments, the antibodies disclosed herein are IgE antibodies. In some embodiments, the antibodies disclosed herein are IgM antibodies. In some embodiments, the antibodies disclosed herein are IgA antibodies. In some embodiments, the antibodies disclosed herein are Fab, F(ab)2, single domain antibodies or single chain variable fragments (scFv).

[0056] In some embodiments, paired with one or more alternative light chains, the antibodies disclosed herein are alternative antibodies. In some embodiments, the antigen binding fragment thereof is an alternative antibody antigen binding fragment (Sab) or a single chain variable fragment (scSv) of an alternative antibody.

[0057] In some embodiments, one or more amino acid modifications can be introduced into the Fc region of the antibody provided herein, thereby producing Fc region variants. The Fc region herein is the C-terminal region of the immunoglobulin heavy chain comprising at least a portion of the constant region. The Fc region includes a native sequence Fc region and a variant Fc region. The Fc region variant can be included in a human Fc region sequence (e.g., human IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g., replacement) at one or more amino acid positions.

[0058] In some embodiments, one or more amino acid modifications can be introduced into the Fc region of the antibody provided herein, thereby producing Fc region variants. The Fc region herein is the C-terminal region of the immunoglobulin heavy chain comprising at least a portion of the constant region. The Fc region includes a native sequence Fc region and a variant Fc region. The Fc region variant can be included in a human Fc region sequence (e.g., human IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g., replacement) at one or more amino acid positions.

[0059] In some cases, the Fc region of an immunoglobulin is important for many important antibody functions (e.g., effector functions), such as antigen-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cell-mediated phagocytosis (ADCP), resulting in the killing of target cells, albeit by different mechanisms. Therefore, in some embodiments, the antibodies described herein comprise variable domains of the present invention in combination with constant domains comprising different Fc regions, and the different Fc regions are selected based on the biological activity of the antibodies for the intended use. In some cases, for example, human IgG can be divided into four subclasses, IgG1, IgG2, IgG3, and IgG4, and each of these subclasses comprises an Fc region with unique characteristics for binding to one or more Fcγ receptors (activating receptors FcγRI (CD64), FcγRIIA, FcγRIIC (CD32); FcγRIIIA and FcγRIIIB (CD16) and inhibitory receptor FcγRIIB) and the first component (C1q) of complement. Human IgG1 and IgG3 bind to all Fcγ receptors; IgG2 binds to FcγRIIA H131 Binding to FcγRIIA R131 FcγRIIIA V158 IgG4 has a lower affinity for FcγRI, FcγRIIA, FcγRIIB, FcγRIIC, and FcγRIIIA V158 Binding; and the inhibitory receptor FcγRIIB has a lower affinity for IgG1, IgG2, and IgG3 than all other Fcγ receptors. Studies have shown that FcγRI does not bind to IgG2, and FcγRIIIB does not bind to IgG2 or IgG4. Same as above. In general, in terms of ADCC activity, human IgG1≥IgG3>>IgG4≥IgG2.

[0060] In some embodiments, the antibodies of the present disclosure are variants with reduced effector functions, which make them ideal candidates for applications where certain effector functions (such as complement fixation and ADCC) are unnecessary or harmful. Such antibodies may have reduced complement dependent cytotoxicity (CDC), antibody dependent cellular toxicity (ADCC) or antibody dependent cellular phagocytosis (ADCP). In some embodiments, the antibodies of the present disclosure are variants with enhanced effector functions, which are useful for increasing immunogenicity. Such antibodies may have increased CDC, ADCC or ADCP, or a combination thereof. Non-limiting examples of in vitro assays for assessing the ADCC activity of target molecules are described in U.S. Patent Nos. 5,500,362 and 5,821,337. Alternatively, non-radioactive assays (e.g., ACTI TM and CytoTox Non-radioactive cytotoxicity assays). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs), monocytes, macrophages, and natural killer (NK) cells.

[0061] The antibody may have increased half-life and improved binding to the neonatal Fc receptor (FcRn) (see, e.g., US 2005 / 0014934). Such antibodies may comprise an Fc region having one or more substitutions therein that improve binding of the Fc region to FcRn, and include those having substitutions at one or more of the following Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434 (according to the EU numbering system) (see, e.g., U.S. Pat. No. 7,371,826). Other examples of Fc region variants are also contemplated (see, e.g., Duncan & Winter, Nature 322:738-40 (1988); U.S. Pat. Nos. 5,648,260 and 5,624,821; and WO 94 / 29351).

[0062] In some embodiments, it may be desirable to create cysteine ​​engineered antibodies, e.g., "thioMAbs," in which one or more residues of an antibody are replaced with cysteine ​​residues. In some embodiments, the replaced residues occur at accessible sites of the antibody. Reactive thiol groups can be located at sites that bind to other moieties, such as drug moieties or linker drug moieties, to produce immunoconjugates. In some embodiments, any one or more of the following residues can be replaced with cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region.

[0063] In some embodiments, the antibodies provided herein can be further modified to include known and available additional non-protein parts. Suitable parts for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), dextran or poly (n vinyl pyrrolidone) polyethylene glycol, polypropylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. Polymers can be of any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody can vary, and if two or more polymers are attached, they can be identical or different molecules.

[0064] The antibodies described herein can be encoded by nucleic acids. Nucleic acids are polynucleotide types comprising two or more nucleotide bases. In certain embodiments, nucleic acids are components of vectors and can be used to transfer polynucleotides encoding polypeptides into cells. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid connected thereto. One type of vector is a genomic integration vector or "integration vector", which can be integrated into the chromosomal DNA of a host cell. Another type of vector is an "episomal" vector, such as a nucleic acid capable of extrachromosomal replication. The vector capable of guiding the expression of genes operably connected to the vector is referred to herein as an "expression vector". Suitable vectors include plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, viral vectors, etc. In expression vectors, regulatory elements (such as promoters, enhancers, polyadenylation signals) for controlling transcription can be derived from mammals, microorganisms, viruses or insect genes. In addition, the ability to replicate in the host and the selection genes that promote transformant recognition, which are usually conferred by the origin of replication, can also be incorporated. Vectors derived from viruses (such as slow viruses, retroviruses, adenoviruses, adeno-associated viruses, etc.) can be used. Plasmid vectors can be linearized to be integrated into the genomic region. In certain embodiments, the expression vector is a plasmid. In certain embodiments, the expression vector is a lentivirus, an adenovirus, or an adeno-associated virus. In certain embodiments, the expression vector is an adenovirus. In certain embodiments, the expression vector is an adeno-associated virus. In certain embodiments, the expression vector is a lentivirus. In certain embodiments, the expression vector described herein is used for gene therapy. Therefore, in some embodiments, the expression vector described herein is formulated for intravenous administration. In some embodiments, the expression vector described herein is formulated for intravitreal administration.

[0065] As used herein, the terms "homology", "homology" or "percent homology" when used herein to describe an amino acid sequence or a nucleic acid sequence relative to a reference sequence can be determined using the formula described by Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87:2264-2268, 1990, modified as Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). This formula is incorporated into the Basic Local Alignment Search Tool (BLAST) program of Altschul et al. (J. Mol. Biol. 215:403-410, 1990). The percent homology of a sequence can be determined using the latest version of BLAST as of the date of filing of this application.

[0066] Nucleic acids encoding antibodies described herein can be used to infect, transfect, transform or otherwise transduce cells suitable for the nucleic acid, thereby enabling the production of antibodies for commercial or therapeutic use. Standard cell lines and methods for producing antibodies from large-scale cell cultures are known in the art. See, for example, Li et al., "Cell culture processes for monoclonal antibody production." Mabs. 2010 Sep-Oct; 2(5): 466-477. In certain embodiments, the cell is a eukaryotic cell. In certain embodiments, the eukaryotic cell is a mammalian cell. In certain embodiments, the mammalian cell is a cell line that can be used to produce antibodies, which is a Chinese hamster ovary cell (CHO) cell, an NS0 mouse myeloma cell, or a Cells. In certain embodiments, nucleic acids encoding antibodies are integrated into genomic sites of cells that can be used to produce antibodies. In certain embodiments, a method of preparing an antibody is described herein, comprising culturing cells containing nucleic acids encoding the antibody under in vitro conditions sufficient to allow production and secretion of the antibody.

[0067] In certain embodiments, a master cell bank is described herein, comprising: (a) a mammalian cell line comprising a nucleic acid encoding an antibody or bispecific antibody described herein integrated in a genomic position; and (b) a cryoprotectant. In certain embodiments, the cryoprotectant comprises glycerol or DMSO. In certain embodiments, the master cell bank comprises: (a) a CHO cell line comprising a nucleic acid encoding an antibody or bispecific antibody described herein integrated in a genomic position; and (b) a cryoprotectant. In certain embodiments, the cryoprotectant comprises glycerol or DMSO. In certain embodiments, the master cell bank is contained in a suitable vial or container capable of withstanding liquid nitrogen freezing.

[0068] Also described herein is a method for preparing antibodies described herein. Such methods include hatching cells or cell lines comprising nucleic acids encoding the antibodies in cell culture medium under conditions sufficient to allow antibody expression and secretion, and further harvesting the antibodies from the cell culture medium. Harvesting may further include one or more purification steps to remove living cells, cell debris, non-antibody proteins or polypeptides, unwanted salts, buffers, and culture medium components. In certain embodiments, additional purification steps include centrifugation, ultracentrifugation, protein A, protein G, protein A / G, or protein L purification and / or ion exchange chromatography.

[0069] As used herein, "treat," "treatment," or "treating" refers to an intentional intervention with a physiological disease state, such as to reduce the severity of the disease or condition; reduce the duration of the condition; improve or eliminate one or more symptoms associated with the disease or condition; or provide a beneficial effect to a subject suffering from the disease or condition. Treatment does not require a cure for the underlying disease or condition.

[0070] A "therapeutically effective amount," "effective dose," "effective amount," or "therapeutically effective dose" of a drug or therapeutic agent refers to any amount of a drug that, when used alone or in combination with another therapeutic agent, protects a subject from the onset of disease or promotes regression of disease, as evidenced by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or prevention of impairment or disability resulting from disease affliction. The ability of a therapeutic agent to promote regression of disease can be assessed using a variety of methods known to skilled practitioners, such as in human subjects during clinical trials, in animal model systems predictive of human efficacy, or by measuring the activity of the agent in in vitro assays.

[0071] As used herein, "pharmaceutically acceptable" with respect to a "carrier," "excipient," or "diluent" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. In some aspects, the carrier is suitable for 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) may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0072] The pharmaceutical compounds described herein may include one or more pharmaceutically acceptable salts. "Pharmaceutically acceptable salts" refers to salts that retain the desired biological activity of the parent compound and do not impart any adverse toxicological effects (see, for example, Berge, SM, et al. (1977) J. Pharm. Sci. 66: 1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include salts derived from non-toxic inorganic acids (such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphorous acid, etc.) and non-toxic organic acids (such as aliphatic monocarboxylic acids and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, etc.). Base addition salts include salts derived from alkaline earth metals such as sodium, potassium, magnesium, calcium, etc., and salts derived from non-toxic organic amines such as N, N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, etc.

[0073] VEGF Antibody

[0074] Described herein are antibodies that bind to VEGF proteins. In some embodiments, the VEGF protein is a human VEGF protein.

[0075] In certain embodiments, described herein is a vascular endothelial growth factor (VEGF) binding antibody or an antigen-binding fragment thereof, which comprises; a heavy chain complementary determining region 1 (HCDR1) comprising the amino acid sequence shown in SEQ ID NO: 3 (GYTFSIYT); a heavy chain complementary determining region 2 (HCDR2) comprising the amino acid sequence shown in SEQ ID NO: 4 (INPYNGNT); and a heavy chain complementary determining region 3 (HCDR3) comprising the amino acid sequence shown in SEQ ID NO: 5 (AKAPAVFWWTGLDY); wherein the antibody binds to VEGF.

[0076] In certain embodiments, described herein is a vascular endothelial growth factor (VEGF) binding antibody or an antigen-binding fragment thereof, comprising: a heavy chain complementary determining region 1 (HCDR1) comprising the amino acid sequence shown in SEQ ID NO: 3 (GYTFSIYT); a heavy chain complementary determining region 2 (HCDR2) comprising the amino acid sequence shown in SEQ ID NO: 4 (INPYNGNT); and a heavy chain complementary determining region 3 (HCDR3) comprising the amino acid sequence shown in SEQ ID NO: 5 (AKAPAVFWWTGLDY); a light chain complementary determining region 1 (LCDR1) comprising the amino acid sequence shown in SEQ ID NO: 23 (NDHDIGVYS); a light chain complementary determining region 2 (LCDR2) comprising the amino acid sequence shown in SEQ ID NO: 24 (YFSQSDK); and a light chain complementary determining region 3 (LCDR3) comprising the amino acid sequence shown in SEQ ID NO: 25 (AMGARSSVTH); wherein the antibody binds to VEGF.

[0077] In certain embodiments, the heavy chain variable region of the VEGF antibody comprises an amino acid sequence that is at least 85% identical to the amino acid sequence shown in SEQ ID NO: 1. In certain embodiments, the heavy chain variable region of the VEGF antibody comprises an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 1. In certain embodiments, the heavy chain variable region of the VEGF antibody comprises an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 1. In certain embodiments, the heavy chain variable region of the VEGF antibody comprises an amino acid sequence that is at least 96% identical to the amino acid sequence shown in SEQ ID NO: 1. In certain embodiments, the heavy chain variable region of the VEGF antibody comprises an amino acid sequence that is at least 97% identical to the amino acid sequence shown in SEQ ID NO: 1. In certain embodiments, the heavy chain variable region of the VEGF antibody comprises an amino acid sequence that is at least 98% identical to the amino acid sequence shown in SEQ ID NO: 1. In certain embodiments, the heavy chain variable region of the VEGF antibody comprises an amino acid sequence that is at least 99% identical to the amino acid sequence shown in SEQ ID NO: 1. In certain embodiments, the heavy chain variable region of the VEGF antibody comprises an amino acid sequence comprising the amino acid sequence shown in SEQ ID NO:1.

[0078] In certain embodiments, the heavy chain of the VEGF antibody comprises an amino acid sequence that is at least 85% identical to the amino acid sequence shown in SEQ ID NO: 2. In certain embodiments, the heavy chain of the VEGF antibody comprises an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 2. In certain embodiments, the heavy chain of the VEGF antibody comprises an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 2. In certain embodiments, the heavy chain of the VEGF antibody comprises an amino acid sequence that is at least 96% identical to the amino acid sequence shown in SEQ ID NO: 2. In certain embodiments, the heavy chain of the VEGF antibody comprises an amino acid sequence that is at least 97% identical to the amino acid sequence shown in SEQ ID NO: 2. In certain embodiments, the heavy chain of the VEGF antibody comprises an amino acid sequence that is at least 98% identical to the amino acid sequence shown in SEQ ID NO: 2. In certain embodiments, the heavy chain of the VEGF antibody comprises an amino acid sequence that is at least 99% identical to the amino acid sequence shown in SEQ ID NO: 2. In certain embodiments, the heavy chain of the VEGF antibody comprises an amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 2.

[0079] In certain embodiments, the light chain variable region of the VEGF antibody comprises an amino acid sequence that is at least 85% identical to the amino acid sequence shown in SEQ ID NO: 21. In certain embodiments, the light chain variable region of the VEGF antibody comprises an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 21. In certain embodiments, the light chain variable region of the VEGF antibody comprises an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 21. In certain embodiments, the light chain variable region of the VEGF antibody comprises an amino acid sequence that is at least 96% identical to the amino acid sequence shown in SEQ ID NO: 21. In certain embodiments, the light chain variable region of the VEGF antibody comprises an amino acid sequence that is at least 97% identical to the amino acid sequence shown in SEQ ID NO: 21. In certain embodiments, the light chain variable region of the VEGF antibody comprises an amino acid sequence that is at least 98% identical to the amino acid sequence shown in SEQ ID NO: 21. In certain embodiments, the light chain variable region of the VEGF antibody comprises an amino acid sequence that is at least 99% identical to the amino acid sequence shown in SEQ ID NO: 21. In certain embodiments, the light chain variable region of the VEGF antibody comprises the amino acid sequence set forth in SEQ ID NO:21.

[0080] In certain embodiments, the light chain of the VEGF antibody comprises an amino acid sequence that is at least 85% identical to the amino acid sequence shown in SEQ ID NO: 22. In certain embodiments, the light chain of the VEGF antibody comprises an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 22. In certain embodiments, the light chain of the VEGF antibody comprises an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 22. In certain embodiments, the light chain of the VEGF antibody comprises an amino acid sequence that is at least 96% identical to the amino acid sequence shown in SEQ ID NO: 22. In certain embodiments, the light chain of the VEGF antibody comprises an amino acid sequence that is at least 97% identical to the amino acid sequence shown in SEQ ID NO: 22. In certain embodiments, the light chain of the VEGF antibody comprises an amino acid sequence that is at least 98% identical to the amino acid sequence shown in SEQ ID NO: 22. In certain embodiments, the light chain of the VEGF antibody comprises an amino acid sequence that is at least 99% identical to the amino acid sequence shown in SEQ ID NO: 22. In certain embodiments, the light chain of the VEGF antibody comprises the amino acid sequence set forth in SEQ ID NO:22.

[0081] In certain embodiments, the VEGF antibody binds to VEGF at an EC50 of about 75 picomolar or less. In certain embodiments, the VEGF antibody binds to VEGF at an EC50 of about 60 picomolar or less. In certain embodiments, the VEGF antibody binds to VEGF at an EC50 of about 50 picomolar or less. In certain embodiments, the VEGF antibody binds to VEGF at an EC50 of about 40 picomolar or less.

[0082] ANG-2 Antibody

[0083] Described herein are antibodies that bind to an ANG-2 protein. In some embodiments, the ANG-2 protein is a human ANG-2 protein.

[0084] In certain embodiments, described herein is an angiopoietin 2 (ANG-2) binding antibody or antigen-binding fragment thereof comprising: a heavy chain complementary determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 13 (GFTFSSYG); a heavy chain complementary determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 14 (ISADSGDK); and a heavy chain complementary determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 15 (AKEFISWIYTFDYLDY); wherein the antibody binds to ANG-2.

[0085] In certain embodiments, described herein is an angiopoietin 2 (ANG-2) binding antibody or antigen-binding fragment thereof comprising: a heavy chain complementary determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 13 (GFTFSSYG); a heavy chain complementary determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 14 (ISADSGDK); and a heavy chain complementary determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 15 (AKEFISWIYTFDYLDY); a light chain complementary determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 23 (NDHDIGVYS); a light chain complementary determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 24 (YFSQSDK); and a light chain complementary determining region 3 (LCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 25 (AMGARSSVTH); wherein the antibody binds to ANG-2.

[0086] In certain embodiments, the heavy chain variable region of an ANG-2 antibody comprises an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 11. In certain embodiments, the heavy chain variable region of an ANG-2 antibody comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 11. In certain embodiments, the heavy chain variable region of an ANG-2 antibody comprises an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 11. In certain embodiments, the heavy chain variable region of an ANG-2 antibody comprises an amino acid sequence that is at least 96% identical to the amino acid sequence set forth in SEQ ID NO: 11. In certain embodiments, the heavy chain variable region of an ANG-2 antibody comprises an amino acid sequence that is at least 97% identical to the amino acid sequence set forth in SEQ ID NO: 11. In certain embodiments, the heavy chain variable region of an ANG-2 antibody comprises an amino acid sequence that is at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 11. In certain embodiments, the heavy chain variable region of an ANG-2 antibody comprises an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 11. In certain embodiments, the heavy chain variable region of the ANG-2 antibody comprises an amino acid sequence comprising the amino acid sequence shown in SEQ ID NO:11.

[0087] In certain embodiments, the heavy chain of the ANG-2 antibody comprises an amino acid sequence that is at least 85% identical to the amino acid sequence shown in SEQ ID NO: 12. In certain embodiments, the heavy chain of the ANG-2 antibody comprises an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 12. In certain embodiments, the heavy chain of the ANG-2 antibody comprises an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 12. In certain embodiments, the heavy chain of the ANG-2 antibody comprises an amino acid sequence that is at least 96% identical to the amino acid sequence shown in SEQ ID NO: 12. In certain embodiments, the heavy chain of the ANG-2 antibody comprises an amino acid sequence that is at least 97% identical to the amino acid sequence shown in SEQ ID NO: 12. In certain embodiments, the heavy chain of the ANG-2 antibody comprises an amino acid sequence that is at least 98% identical to the amino acid sequence shown in SEQ ID NO: 12. In certain embodiments, the heavy chain of the ANG-2 antibody comprises an amino acid sequence that is at least 99% identical to the amino acid sequence shown in SEQ ID NO: 12. In certain embodiments, the heavy chain of the ANG-2 antibody comprises an amino acid sequence comprising the amino acid sequence shown in SEQ ID NO:12.

[0088] In certain embodiments, the light chain variable region of an ANG-2 antibody comprises an amino acid sequence that is at least 85% identical to the amino acid sequence shown in SEQ ID NO: 21. In certain embodiments, the light chain variable region of an ANG-2 antibody comprises an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 21. In certain embodiments, the light chain variable region of an ANG-2 antibody comprises an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 21. In certain embodiments, the light chain variable region of an ANG-2 antibody comprises an amino acid sequence that is at least 96% identical to the amino acid sequence shown in SEQ ID NO: 21. In certain embodiments, the light chain variable region of an ANG-2 antibody comprises an amino acid sequence that is at least 97% identical to the amino acid sequence shown in SEQ ID NO: 21. In certain embodiments, the light chain variable region of an ANG-2 antibody comprises an amino acid sequence that is at least 98% identical to the amino acid sequence shown in SEQ ID NO: 21. In certain embodiments, the light chain variable region of an ANG-2 antibody comprises an amino acid sequence that is at least 99% identical to the amino acid sequence shown in SEQ ID NO: 21. In certain embodiments, the light chain variable region of the ANG-2 antibody comprises the amino acid sequence set forth in SEQ ID NO:21.

[0089] In certain embodiments, the light chain of an ANG-2 antibody comprises an amino acid sequence that is at least 85% identical to the amino acid sequence shown in SEQ ID NO: 22. In certain embodiments, the light chain of an ANG-2 antibody comprises an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 22. In certain embodiments, the light chain of an ANG-2 antibody comprises an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 22. In certain embodiments, the light chain of an ANG-2 antibody comprises an amino acid sequence that is at least 96% identical to the amino acid sequence shown in SEQ ID NO: 22. In certain embodiments, the light chain of an ANG-2 antibody comprises an amino acid sequence that is at least 97% identical to the amino acid sequence shown in SEQ ID NO: 22. In certain embodiments, the light chain of an ANG-2 antibody comprises an amino acid sequence that is at least 98% identical to the amino acid sequence shown in SEQ ID NO: 22. In certain embodiments, the light chain of an ANG-2 antibody comprises an amino acid sequence that is at least 99% identical to the amino acid sequence shown in SEQ ID NO: 22. In certain embodiments, the light chain of the ANG-2 antibody comprises the amino acid sequence set forth in SEQ ID NO:22.

[0090] VEGF / ANG-2 Bispecific Antibody

[0091] Described herein are bispecific antibodies that bind to VEGF and ANG-2 proteins. In some embodiments, the VEGF and / or ANG-2 protein is a human VEGF or ANG-2 protein. The bispecific antibodies of the present disclosure have a common light chain format, wherein the heavy chain of the VEGF binding portion is different from the heavy chain of the ANG-2 binding portion, and a common light chain is used that can associate with either heavy chain. This format prevents unwanted heavy and light chain pairing (because there is only one type of light chain), thereby making antibody formation more efficient during production. The resulting antibody comprises two heavy chains and two light chains arranged as a classical antibody (except that the two heavy chains form heterodimers). To prevent unwanted heavy chain homodimerization (e.g., two anti-ANG-2 heavy chains or two anti-VEGF heavy chains), the heavy chain may further comprise mutations or substitutions to the heavy chain constant region to promote correct heterodimer formation.

[0092] The common light chain bispecific structure may comprise a first heavy chain molecule and a second heavy chain molecule, further comprising a mutation within the CH3 domain that promotes the coupling of the first heavy chain and the second heavy chain and / or prevents the coupling of the first heavy chain to another first heavy chain or the second heavy chain to another second heavy chain. The mutation can physically (e.g., steric hindrance, "knob" into "hole" or biochemically (e.g., electrostatic interaction) prevent the coupling of two first heavy chain molecules or two second heavy chain molecules. Exemplary knob into hole mutations may include T366W (EU numbering) in one heavy chain and T366S / L368A / Y407V (EU numbering) in one second heavy chain. Exemplary mutations that promote the coupling of a first heavy chain molecule and a second heavy chain molecule are disclosed in, e.g., WO2009089004, U.S. Pat. No. 8,642,745, US PG-PUB: US20140322756, and "The making of bispecific antibodies" MAbs. 2017 Feb-Mar; 9(2): 182-212.

[0093] The anti-VEGF / ANG-2 bispecific antibody may comprise one or more substitutions to reduce effector function. Such substitutions produce variant Fc molecules. In some embodiments, the variant Fc region comprises IgG1 Fc region, and wherein the one or more mutations comprise (a) 297A, 297Q, 297G, or 297D, (b) 279F, 279K, or 279L, (c) 228P, (d) 235A, 235E, 235G, 235Q, 235R, or 235S, (e) 237A, 237E, 237K, 237N, or 237R, (f) 234A, 234V, or 234F, (g) 233P, (h) 328A, (i) 327Q or 327T, (j) 329A, 329G, 329Y, or 329R (k) 331S, (l) 236F or 236R, (m) )238A, 238E, 238G, 238H, 238I, 238V, 238W, or 238Y, (n) 248A, (o) 254D, 254E, 254G, 254H, 254I, 254N, 254P, 254Q, 254T, or 254V, (p) 255N, (q) 256H, 256K, 256R, or 256V, (r) 264S, (s) 265H, 265K, 265S, 265Y, or 265A, (t) 267G, 267H, 267I, or 267K, (u) 268K, (v) 269N or 269Q, (w) 270A, 270G, 270M, or 270N, (x) 271T, (y) 272N, (z) 292E, 292F, 292G, or 292I, (aa) 293S, (bb) 301W, (cc) 304E, (dd) 311E, 311G, or 311S, (ee) 316F, (ff) 328V, (gg) 330R, (hh) 339E or 339L, (ii) 343I or 343V, (jj) 373A, 373G, or 373S, (kk) 376E, 376W, or 376Y, (ll) 380D, (mm) 382D or 382P, (nn) 3 85P, (oo) 424H, 424M, or 424V, (pp) 434I, (qq) 438G, (rr) 439E, 439H, or 439Q, (ss) 440A, 440D, 440E, 440F, 440M, 440T, or 440V, (tt) K322A, (uu) L235E, (vv) L234A and L235A, (ww) L234A, L235A and G237A, (xx) L234A, L235A and P329G, (yy) L234F, L235E and P331S, (zz) L234A, L235E and G237A,(aaa), L234A, L235E, G237A and P331S (bbb) L234A, L235A, G237A, P238S, H268A, A330S and P331S, (ccc) L234A, L235A and P329A, (ddd) G236R and L328R, (eee) G237A, (fff) F241A, (ggg) V264A, (hhh )D265A, (iii) D265A and N297A, (jjj) D265A and N297G, (kkk) D270A, (lll) A330L, (mmm) P331A or P331S, or (nnn) E233P, (ooo) L234A, L235E, G237A, A330S and P331S or any combination of (ppp)(a)–(uu), according to EU numbering.

[0094] In certain embodiments, described herein is a bispecific antibody comprising: a vascular endothelial growth factor (VEGF) binding portion comprising: (a) a heavy chain complementary determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 3 (GYTFSIYT); (b) a heavy chain complementary determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 4 (INPYNGNT); and (c) a heavy chain complementary determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 5 (AKAPAVFWWTGLDY); and an angiopoietin 2 (ANG-2) binding portion comprising: (a) a heavy chain complementary determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 13 (GFTFSSYG); (b) a heavy chain complementary determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 14 (ISADSGDK); and (c) a heavy chain complementary determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 15 The amino acid sequence shown in NO: 15 (AKEFISWIYTFDYLDY); wherein the bispecific antibody binds to VEGF and ANG-2.

[0095] In certain embodiments, described herein is a bispecific antibody comprising: a vascular endothelial growth factor (VEGF) binding portion comprising: (a) a heavy chain complementary determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 3 (GYTFSIYT); (b) a heavy chain complementary determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 4 (INPYNGNT); and (c) a heavy chain complementary determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 5 (AKAPAVFWWTGLDY); and an angiopoietin 2 (ANG-2) binding portion comprising: (a) a heavy chain complementary determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 13 (GFTFSSYG); (b) a heavy chain complementary determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 14 (ISADSGDK); and (c) a heavy chain complementary determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 15 NO: 15 (AKEFISWIYTFDYLDY); a light chain complementary determining region 1 (LCDR1) comprising: (a) the amino acid sequence shown in SEQ ID NO: 23 (NDHDIGVYS); (b) a light chain complementary determining region 2 (LCDR2) comprising the amino acid sequence shown in SEQ ID NO: 24 (YFSQSDK); and (c) a light chain complementary determining region 3 (LCDR3) comprising the amino acid sequence shown in SEQ ID NO: 25 (AMGARSSVTH); wherein the bispecific antibody binds to VEGF and ANG-2.

[0096] In certain embodiments, described herein is a bispecific antibody comprising: a vascular endothelial growth factor (VEGF) binding portion comprising: (a) a heavy chain complementary determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 3 (GYTFSIYT); (b) a heavy chain complementary determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 4 (INPYNGNT); and (c) a heavy chain complementary determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 5 (AKAPAVFWWTGLDY); and an angiopoietin 2 (ANG-2) binding portion comprising: (a) a heavy chain complementary determining region 1 (HCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 13 (GFTFSSYG); (b) a heavy chain complementary determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 14 (ISADSGDK); and (c) a heavy chain complementary determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 15 NO: 15 (AKEFISWIYTFDYLDY); a common light chain complementary determining region 1 (LCDR1), which comprises: (a) the amino acid sequence shown in SEQ ID NO: 23 (NDHDIGVYS); (b) a light chain complementary determining region 2 (LCDR2), which comprises the amino acid sequence shown in SEQ ID NO: 24 (YFSQSDK); and (c) a light chain complementary determining region 3 (LCDR3), which comprises the amino acid sequence shown in SEQ ID NO: 25 (AMGARSSVTH); wherein the bispecific antibody binds to VEGF and ANG-2.

[0097] In certain embodiments, also described herein is a bispecific antibody comprising: (a) a first heavy chain variable region comprising an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence shown in SEQ ID NO: 1; (b) a second heavy chain variable region comprising an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence shown in SEQ ID NO: 11; and (c) a common light chain variable region, wherein the common light chain variable region comprises a light chain variable region comprising an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence shown in SEQ ID NO: 21, wherein the bispecific antibody binds to VEGF and ANG-2.

[0098] In certain embodiments, the VEGF / ANG-2 bispecific antibody comprises: (a) a first heavy chain comprising an amino acid sequence that is at least 85% identical to the amino acid sequence shown in SEQ ID NO:2; (b) a second heavy chain comprising an amino acid sequence that is at least 85% identical to the amino acid sequence shown in SEQ ID NO:12; and (c) a common light chain, wherein the common light chain comprises a light chain comprising an amino acid sequence that is at least 85% identical to the amino acid sequence shown in SEQ ID NO:22; wherein the bispecific antibody binds to VEGF and ANG-2.

[0099] In certain embodiments, the VEGF / ANG-2 bispecific antibody comprises: (a) a first heavy chain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO:2; (b) a second heavy chain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO:12; and (c) a common light chain, wherein the common light chain comprises a light chain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO:22; wherein the bispecific antibody binds to VEGF and ANG-2.

[0100] In certain embodiments, the VEGF / ANG-2 bispecific antibody comprises: (a) a first heavy chain comprising an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO:2; (b) a second heavy chain comprising an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO:12; and (c) a common light chain, wherein the common light chain comprises a light chain comprising an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO:22; wherein the bispecific antibody binds to VEGF and ANG-2.

[0101] In certain embodiments, the VEGF / ANG-2 bispecific antibody comprises: (a) a first heavy chain comprising an amino acid sequence that is at least 97% identical to the amino acid sequence shown in SEQ ID NO:2; (b) a second heavy chain comprising an amino acid sequence that is at least 97% identical to the amino acid sequence shown in SEQ ID NO:12; and (c) a common light chain, wherein the common light chain comprises a light chain comprising an amino acid sequence that is at least 97% identical to the amino acid sequence shown in SEQ ID NO:22; wherein the bispecific antibody binds to VEGF and ANG-2.

[0102] In certain embodiments, the VEGF / ANG-2 bispecific antibody comprises: (a) a first heavy chain comprising an amino acid sequence that is at least 98% identical to the amino acid sequence shown in SEQ ID NO:2; (b) a second heavy chain comprising an amino acid sequence that is at least 98% identical to the amino acid sequence shown in SEQ ID NO:12; and (c) a common light chain, wherein the common light chain comprises a light chain comprising an amino acid sequence that is at least 98% identical to the amino acid sequence shown in SEQ ID NO:22; wherein the bispecific antibody binds to VEGF and ANG-2.

[0103] In certain embodiments, the VEGF / ANG-2 bispecific antibody comprises: (a) a first heavy chain comprising an amino acid sequence that is at least 99% identical to the amino acid sequence shown in SEQ ID NO:2; (b) a second heavy chain comprising an amino acid sequence that is at least 99% identical to the amino acid sequence shown in SEQ ID NO:12; and (c) a common light chain, wherein the common light chain comprises a light chain comprising an amino acid sequence that is at least 99% identical to the amino acid sequence shown in SEQ ID NO:22; wherein the bispecific antibody binds to VEGF and ANG-2.

[0104] In certain embodiments, also described herein is a bispecific antibody comprising: (a) a first heavy chain comprising the amino acid sequence as shown in SEQ ID NO: 2; (b) a second heavy chain comprising the amino acid sequence as shown in SEQ ID NO: 12; and (c) a common light chain, wherein the common light chain comprises a light chain comprising the amino acid sequence as shown in SEQ ID NO: 22; wherein the bispecific antibody binds to VEGF and ANG-2.

[0105] Treatment

[0106] In certain embodiments, disclosed herein are antibodies that can be used to treat eye diseases. In certain embodiments, the eye disease is associated with age or other comorbidities (such as diabetes). In certain embodiments, the eye disease is macular degeneration. In certain embodiments, macular degeneration is associated with age. In certain embodiments, macular degeneration is associated with diabetes. In certain embodiments, macular degeneration is wet macular degeneration. In certain embodiments, the eye disease is branch retinal vein occlusion. In certain embodiments, the eye disease is central retinal vein occlusion.

[0107] In certain embodiments, the antibody can be administered to a subject in need thereof by any route suitable for administering a pharmaceutical composition comprising the antibody, such as, for example, topically, subcutaneously, intravenously, or intravitreally. In certain embodiments, the antibody or pharmaceutical composition comprising the antibody is administered topically. In certain embodiments, the antibody or pharmaceutical composition comprising the antibody is administered subcutaneously. In certain embodiments, the antibody or pharmaceutical composition comprising the antibody is administered intravenously. In certain embodiments, the antibody or pharmaceutical composition comprising the antibody is administered intravitreally.

[0108] In certain embodiments, the antibody is administered with a suitable dosage regimen, such as weekly, twice a week, monthly, twice a month, once every two weeks, once every three weeks, or once a month, once every two months, once every three months, once every four months, once every five months, or once every six months, etc. The antibody can be administered in any therapeutically effective amount. In certain embodiments, the therapeutically acceptable amount is between about 0.1 mg / kg and about 50 mg / kg. In certain embodiments, the therapeutically acceptable amount is between about 1 mg / kg and about 40 mg / kg. In certain embodiments, the therapeutically acceptable amount is between about 1 mg / kg and about 20 mg / kg. In certain embodiments, the therapeutically acceptable amount is between about 1 mg / kg and about 10 mg / kg. In certain embodiments, the therapeutically acceptable amount is between about 5 mg / kg and about 30 mg / kg. In certain embodiments, the therapeutically acceptable amount is between about 5 mg / kg and about 20 mg / kg. The intravitreal dose concentration range can be 50 mg / ml to 150 mg / ml for intravitreal use. A therapeutically effective amount comprises an amount sufficient to ameliorate one or more symptoms associated with the disease or disorder being treated.

[0109] Pharmaceutically acceptable excipients, carriers and diluents

[0110] In certain embodiments, the anti-VEGF / ANG-2 monospecific and bispecific antibodies of the present disclosure are contained in a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients, carriers, and diluents. Pharmaceutically acceptable excipients, carriers, and diluents may be included to increase the shelf life, stability, or applicability of the antibodies. Such compounds include salts, pH buffers, detergents, anticoagulants, and preservatives. In certain embodiments, the antibodies of the present disclosure are administered suspended in a sterile solution. In certain embodiments, the solution comprises about 0.9% NaCl. In certain embodiments, the solution comprises about 5.0% dextrose. In some embodiments, the solution further comprises one or more of: a buffer such as acetate, citrate, histidine, succinate, phosphate, bicarbonate, and hydroxymethylaminomethane (Tris); a surfactant such as polysorbate 80 (Tween 80), polysorbate 20 (Tween 20), and poloxamer 188; a polyol / disaccharide / polysaccharide such as glucose, dextrose, mannose, mannitol, sorbitol, sucrose, trehalose, and dextran 40; an amino acid such as glycine or arginine; an antioxidant such as ascorbic acid, methionine; or a chelating agent such as EDTA or EGTA.

[0111] In certain embodiments, the antibodies of the present disclosure can be freeze-dried for transportation / storage and reconstituted before administration. In certain embodiments, the freeze-dried antibody formulation comprises a filler such as mannitol, sorbitol, sucrose, trehalose, dextran 40 or a combination thereof. The freeze-dried formulation may be contained in a vial made of glass or other suitable non-reactive materials. The antibody, when formulated, whether reconstituted or not, can be buffered at a specific pH (generally less than 7.0). In certain embodiments, the pH value may be between 4.5 and 7.0, 4.5 and 6.5, 4.5 and 6.0, 4.5 and 5.5, 4.5 and 5.0 or 5.0 and 6.0.

[0112] Also described herein are kits comprising, in a suitable container, one or more antibodies described herein and one or more additional components selected from the group consisting of: instructions for use; diluents, excipients, carriers, and administration devices.

[0113] In certain embodiments, described herein is a method of preparing a treatment for an eye disease comprising mixing one or more pharmaceutically acceptable excipients, carriers, or diluents with an antibody of the present disclosure. In certain embodiments, described herein is a method of preparing a cancer treatment for storage or transport comprising lyophilizing one or more antibodies of the present disclosure.

[0114] Example

[0115] The following illustrative examples represent embodiments of the compositions and methods described herein and are not meant to be limiting in any way.

[0116] Example 1 - Production of VEGF / ANG-2 bispecific surrogate antibodies

[0117] Nucleic acid sequences encoding sequences corresponding to SEQ ID NO: 2, SEQ ID NO: 12, and SEQ ID NO: 22 were cloned into plasmid vectors to direct eukaryotic cell expression. Three types of expression plasmids were constructed. The first plasmid contained 1 copy of each surrogate light chain (SLC), VEGF-specific knob heavy chain, and ANG-2-specific hole heavy chain. The second plasmid contained 2 copies of SLC and 1 copy of each heavy chain in the orientation of SLC: HC-knob: HC: hole: SLC. The third plasmid contained 2 copies of SLC and 1 copy of each heavy chain in the orientation of SLC: SLC-: HC-knob: HC: hole. The products expressed and purified from the three stable cell lines ("RO-101") were named Sample 1, Sample 2, and Sample 3, respectively.

[0118] CHO cells were cultured in CD-CHO medium (Life Technologies) supplemented with 6 mM L-glutamine (Life Technologies). The cells were incubated in a shaking incubator at 36.5°C, 5% CO2, and 140 rpm.

[0119] Gene expression plasmids for transfection were prepared by linearization followed by ethanol precipitation and resuspension in EB buffer to a final concentration of 400 μg / ml. Transfections were performed by electroporation using Gene Pulse XCell. For each transfection, live cells were resuspended in pre-warmed CD-CHO medium to 1.43 x 10 7 cells / ml. 100μl of linearized DNA at a concentration of 400μg / ml was aliquoted into 0.4cm gap electroporation cuvettes and 700μl of cell suspension was added. Three cuvettes of cells and DNA were electroporated at 300V, 900μF and immediately transferred to 30ml preheated CD-CHO supplemented with 10ml / L SP4 to generate a stable pool. Selected stable transfectants were amplified and the surrogate antibody was isolated from the supernatant by protein A chromatography. A secondary polishing step was performed to remove unwanted fractions, i.e. aggregates. The purified protein was then analyzed by SE-HPLC and SDS-PAGE.

[0120] For SDS-PAGE analysis, reduced and non-reduced samples of RO-101 were electrophoresed. Figure 1A The presence of the product was confirmed and a good level of purity was observed. Under non-reducing conditions, multiple protein bands between 100 and 150 kDa were visible in the product ( Figure 1A: Lanes 3 to 5). Under reducing conditions, bands corresponding to the molecular weights of the light chain (~25 kDa) and heavy chain (~50 kDa) were observed, and all three products were consistent with expectations ( Figure 1A : Lanes 7 to 9). The purified RO-101 protein was also analyzed in SE-HPLC. All three sample proteins showed 100% single peak (see Figure 1B ).

[0121] Example 2 - Binding of VEGF / ANG-2 bispecific surrogate antibodies

[0122] The surrogate antibody RO-101 prepared according to Example 1 was tested for its ability to bind to the target antigen.

[0123] VEGF ELISA -Recombinant human VEGF 165 (Peprotech, catalog number 100-20) was used to coat the plates at 1 μg / mL. The product was diluted in blocking buffer at a starting concentration of 100 nM and then tested at a 1:3 dilution. Binding was detected by HRP-conjugated donkey anti-human IgG Fcγ antibody (Jackson ImmunoResearch Laboratories, catalog number 709-035-098). Herceptin was used as a nonspecific control for no binding. Farximab and Aflibercept were run with the product as reference materials. The results are shown in Table 1. The results show that the product has a higher binding affinity with EC50 values ​​between 0.040 and 0.056 nM, which is comparable to or slightly better than the binding of the Farximab and Aflibercept reference materials.

[0124]

[0125] ANG-2ELISARecombinant human ANG-2 (R&D Systems, catalog number 623-AN-025 / CF) was used to coat the plates at 1 μg / mL. The product was diluted in blocking buffer at a starting concentration of 100 nM and then tested at a 1:3 dilution. Binding was detected by HRP-conjugated donkey anti-human IgG Fcγ antibody (Jackson ImmunoResearch Laboratories, catalog number 709-035-098). Herceptin was used as a nonspecific control for no binding. The results are shown in Table 2. The results show that the product has a higher binding affinity with EC50 values ​​between 0.045 and 0.054 nM, while farecimab showed significantly lower binding affinity and the aflibercept control showed no binding at all.

[0126]

[0127] Therefore, when compared to existing VEGF antibodies or human VEGFR1 and VEGFR2 ectodomain fusion proteins, the tested bispecific RO-101 surrogate antibodies have a significant 165 showed similar affinity, and showed superior affinity to ANG-2 (e.g., binding affinity increased 42-65 fold).

[0128] Example 3 - VEGF Binding ELISA and Inhibition of VEGF Receptor Binding

[0129] The surrogate antibody product RO-101 was tested for binding to different isoforms of VEGF and its inhibition of VEGF receptor interaction. 165 is the most abundant and potent isoform of VEGF-A, while VEGF 121 is the major isoform in the eye. Therefore, both isoforms were tested in the binding assay. 121 (“rh VEGF 121 ”) and recombinant human VEGF 165 (“rhVEGF 165 ”) were obtained from Peprotech (see Table 10). Sequence alignment and analysis are shown in Figure 2A rhVEGF 121 It contains a retained platelet-derived growth factor (PDGF) domain, lacks the heparin-binding domain, and is freely diffusible.

[0130] Therefore, targeting VEGF 121 and VEGF 165 The binding affinity of RO-101_sample 1 was tested (see Figure 2B ). rh VEGF 121 (Peprotech, Cat. No. 100-20A) and rh VEGF 165 (Peprotech, catalog number 100-20) was used to coat Corning 9017 medium binding plates at 1 μg / mL and 100 μl per well. The product was diluted in blocking buffer at a starting concentration of 100 nM and then tested at a 1:3 dilution. Donkey anti-human IgG Fcγ-HRP (Jackson ImmunoResearchLaboratories, catalog number 709-035-098) was used for detection and visualized by TMB substrate. OD450 nm readings were measured by Victor 3 (PerkinElmer 1420 Multilabel Counter).

[0131] Table 3: RO-101 and VEGF121 and VEGF 165 EC50 of binding

[0132] Target EC50(nM) <![CDATA[R 2 ]]> <![CDATA[rhVEGF 121 ]]> 0.0146 0.9966 <![CDATA[rhVEGF 165 ]]> 0.0135 0.9922

[0133] like Figure 2B As shown in Table 3, RO-101 binds to rhVEGF with similar affinity. 121 and rhVEGF 165 Combine.

[0134] Next, RO-101_Sample 1 in the SgG1 form was compared with Farximab (IgG1) for their effects on rhVEGF in an ELISA binding assay. 121 The binding affinity of rhVEGF was compared. 121 Used to coat Corning 9017 medium binding plates. RO-101 was diluted in blocking buffer at a starting concentration of 10 nM and then tested at a 1:3 dilution. Donkey anti-human IgG Fcγ-HRP was used for detection (Jackson ImmunoResearch Laboratories, catalog number 709-035-098) and visualized by TMB substrate. OD450 nm readings were measured by Victor 3 (PerkinElmer 1420 Multilabel Counter).

[0135] Table 4: RO-101 or Fariximab and VEGF 121 EC50 of binding

[0136] SgG / IgG EC50 nM <![CDATA[R 2 ]]> RO-101_Sample 1 0.0086 0.9984 Fariximab 0.0113 0.9980

[0137] like Figure 2C As shown in Table 4, RO-101 and farecimab bind to rhVEGF with similar affinity in ELISA. 121 Combine.

[0138] Next, RO-101_Sample 1 was compared with Farximab for their effects on rhVEGF in an ELISA binding assay. 165 The binding affinity of VEGF was compared. 165Used to coat Corning 9017 medium binding plates. RO-101 was diluted in blocking buffer at a starting concentration of 10 nM and then tested at a 1:3 dilution. Donkey anti-human IgG Fcγ-HRP (Jackson ImmunoResearch Laboratories, catalog number 709-035-098) was used for detection and visualized by TMB substrate. OD450 nm readings were measured by Victor 3 using a PerkinElmer 1420 Multilabel Counter.

[0139] Table 5: RO-101 or Fariximab and VEGF 165 EC50 of binding

[0140] SgG / IgG EC50 nM <![CDATA[R 2 ]]> RO-101_Sample 1 0.0231 0.9995 Fariximab 0.0228 0.9989

[0141] like Figure 2D As shown in Table 5, RO-101 and farecximab bound to rhVEGF with similar affinity in ELISA. 165 Combine.

[0142] Next, RO-101_sample 1 was compared with farecximab for its effects on VEGF. 165 The inhibition of the interaction between VEGF and VEGF receptor 2 (VEGF R2), also known as kinase insert domain receptor (KDR). Figure 2E As indicated, 1:3 serial dilutions of RO-101 or farecimab (starting at 200 nM) were mixed with 2 nM biotinylated rhVEGF R2 / KDR receptor (AcroBiosystems, catalog # KDR-H82E5) at a 1:1 ratio. The mixture was added to a 1:1 plate containing rhVEGF 165 The ELISA binding plate was coated with 1% VEGF R2 / KDR. Biotinylated rhVEGF R2 / KDR without RO-101 or Farcimab was used as a binding control. Receptor binding was detected by HRP-conjugated streptavidin (Jackson ImmunoResearch Laboratories, catalog number 016-030-084) and visualized by TMB substrate. The results are shown in Figure 2F The corresponding IC50 values ​​are shown in Table 6.

[0143] Table 6: Effects of RO-101 or Fariximab on VEGF 165 -IC50 for inhibition of VEGF R2 / KDR receptor binding

[0144]

[0145] like Figure 2F As shown in Table 6, RO-101_Sample 1 inhibited rhVEGF 165 The interaction between VEGF and rhVEGF R2 / KDR receptor is more than 3 times stronger than that of facilisimab.

[0146] Example 4 - ANG-2 Binding ELISA and Inhibition of ANG-2 Receptor Binding

[0147] The surrogate antibody product RO-101 was tested for its binding to ANG-2 and its inhibition of the interaction between ANG-2 and its receptor.

[0148] In an ELISA binding assay, RO-101_sample 1 or RO-101 in concentrated solution ("RO-101_LX") was compared with farecximab for their binding affinity to rhANG-2. rhANG-2 (AcroBioSystems, catalog number AN2-H52H4) was used to coat Corning 9017 medium binding plates. RO-101 was diluted in blocking buffer at a starting concentration of 100 nM and then tested at a 1:3 dilution. Donkey anti-human IgG Fcγ-HRP (Jackson ImmunoResearchLaboratories, catalog number 709-035-098) was used for detection and visualized by TMB substrate. OD450 nm readings were measured by Victor 3 using a PerkinElmer 1420 Multilabel Counter.

[0149] Table 7: EC50 of RO-101 or Fariximab binding to ANG-2

[0150]

[0151] like Figure 3A As shown in Table 7, RO-101 binds rhANG-2 with 53-fold greater affinity than farecximab in the ELISA. This is a significant improvement over farecximab by increasing binding to one of the two antigens.

[0152] Importantly, an independent team performed similar ELISA binding experiments and obtained similar results. The EC50 values ​​of RO-101 were 0.0526, 0.0498, and 0.0449 nM, with an average of 0.0491 nM. On the other hand, the EC50 values ​​of farecximab were 3.508, 3.129, and 1.918 nM, with an average of 2.8517 nM. Therefore, under different experimental settings, RO-101 binds to rhANG-2 58 times higher than farecximab. This further confirms that RO-101 binds to ANG-2 with much better affinity than farecximab.

[0153] Next, RO-101_Sample 1 was compared with Farximab for their inhibition of the interaction between human ANG-2 and tyrosine kinase with immunoglobulin-like and EGF-like domain 2 receptor (Tie-2), an ANG-2 receptor. 1:3 serial dilutions of RO-101 or Farximab (starting at 200 nM) were mixed with 2 nM His-tagged rhANG-2 (AcroBiosystems, catalog number AN2-H52H4) at a 1:1 ratio. rhANG-2-His without RO-101 or Farximab was used as a binding control. Figure 3B As shown, RO-101 or farecimab was added to an ELISA binding plate coated with recombinant human Tie-2-Fc (AcroBiosystems, catalog number TI2-H5255). The binding of rhANG-2 to rhTie-2-Fc was detected by HRP-conjugated rabbit anti-His antibody (Bethyl Laboratories, catalog number A190-114P). The results are shown in Figure 3C The corresponding IC50 values ​​are shown in Table 8.

[0154] Table 8: IC50 of inhibition of ANG-2 binding to ANG-2 receptor by RO-101 or Fariximab

[0155]

[0156] like Figure 3C As shown in Table 8, RO-101_Sample 1 is more than 17 times more potent than farecximab in inhibiting the interaction between ANG-2 and Tie-2 receptor.

[0157] In conclusion, RO-101 exhibited superior binding properties to rhANG-2 when compared to farecximab.

[0158] Example 5 - Binding Affinity of Different Forms of RO-101

[0159] VEGF-A specific surrogate antibody antigen binding monomer (Sab), Fab equivalents and single chain fragments (scSv), scFv equivalents derived from RO-101 were expressed in HEK293 cells. Specifically, Sab (50 kDa form of surrogate antibody) contains a VH-CH1 domain associated with VpreB and λ5 (surrogate light chain). scSV contains a single chain fragment consisting of a VH domain connected to VpreB.

[0160] The resulting supernatant was purified by CaptureSelcet IgG-CH1 or Ni-NTA to purify His-tagged scSv proteins. Together with RO-101 SgG, these three formats were tested for their ability to bind to rhVEGF. 165 and rhANG-2 binding affinity.

[0161] RO-101 SgG1, Sab, and scSv were tested in duplicate. They were diluted in blocking buffer at a starting concentration of 10 nM and then tested at a 1:3 dilution. rhVEGF was detected by biotinylated anti-human VpreB1 antibody and HRP-conjugated streptavidin 165 Binding ELISA.

[0162] Table 9.1: Detection of RO-101SgG, Sab and scSv with rhVEGF by biotinylated anti-VpreB antibody and HRP-conjugated streptavidin 165 EC50 of binding

[0163] Test protein EC50(nM) RO-101_SgG 0.0057 RP-008_VEGF-A_scSv 0.009 RP-014_VEGF-A_Sab 0.0063

[0164] like Figure 4A As shown in Table 9.1, monomeric anti-VEGF-A Sab and scSv and rhVEG 165 The binding was good, with the extent of binding comparable to that of the SgG form. Thus, all three forms of VEGF-A specific surrogate antibodies were comparable to rhVEGF 165 have similar binding affinities.

[0165] RO-101 SgG1, Sab and scSv were tested in duplicate. SgG1 was diluted in blocking buffer at a starting concentration of 10 nM. Sab and scSv were diluted in blocking buffer at a starting concentration of 100 nM. They were then serially diluted 1:3. ANG-2 binding ELISA was detected by biotinylated anti-human VpreB1 antibody and HRP-conjugated streptavidin.

[0166] Table 9.2: EC50 of RO-101 SgG, Sab and scSv binding to ANG-2 detected by biotinylated anti-VpreB antibody and HRP-conjugated streptavidin

[0167] Test protein EC50(nM) RO-101_SgG 0.0257 RP-022_ANG-2_scSv 0.0442 RP-017_ANG-2_Sab 0.0485

[0168] like Figure 4B As shown in Table 9.2, monomeric anti-ANG-2 Sab and scSv bound well to ANG-2 and had picomolar affinity for rhANG-2.

[0169] Example 6 - Binding of RO-101 to PDGF / VEGF family members

[0170] PDGF / VEGF family members were obtained from three different suppliers: Peprotech, R&D Systems and AcroBiosystems (see Table 10). Sequence alignment and analysis were performed (see Figure 5A ). Specifically, the rhVEGF-B from Peprotech and R&D had the same sequence, while the rhVEGF-B sequence from AcroBiosystems had a different C-terminus. All three suppliers used the same rhVEGF-C sequence, except that the rhVEGF-C sequence from AcroBiosystems had an additional 9 residues at the N-terminus. All three suppliers used the same rhVEGF-D sequence, except that the rhVEGF-D sequence from Peprotech had an additional 4 residues at the N-terminus and 4 residues at the C-terminus. The cross-reactivity of RO-101 with two other human PDGF / VEGF family members, placental growth factor-1 (P1GF-1) and placental growth factor-3 (P1GF-3) containing a conserved cysteine ​​knob domain, was also examined.

[0171] Table 10 - Summary of basic information of the tested PDGF / VEGF family members

[0172] Target Supplier_Catalog Number source <![CDATA[rhVEGF 165 ]]> Peprotech_100-20 Escherichia coli <![CDATA[rhVEGF 121 ]]> Peprotech_100-20A Escherichia coli rhVEGF-B_Peprotech Peprotech_100-20B Escherichia coli rhVEGF-B_AcroBio AcroBio_VE6-H5225 His-tagged; HEK293 rhVEGF_C AcroBio_VEC-H4225 His-tagged; HEK293 rhVEGF-D AcroBio_VED-H5228 His-tagged; HEK293 rhP1GF-1 Peprotech_100-06 Escherichia coli rhP1GF-3 Peprotech_100-57 Escherichia coli

[0173] First, with rhVEGF 165 In comparison, the binding to rhVEGF-B was tested. rhVEGF-B or rhVEGF 165 Used to coat Corning 9017 medium binding plates at 1 μg / mL and 100 μl / well. Product RO-101_Sample 1 was diluted in blocking buffer at a starting concentration of 100 nM and then tested at a 1:3 dilution. Donkey anti-human IgG Fcγ-HRP was used for detection. OD450 nm readings were measured by Victor 3.

[0174] Table 11: RO-101_Sample 1 and rhVEGF 165EC50 of rhVEGF-B binding

[0175]

[0176] like Figure 5B As shown in Table 11, RO-101 had almost no binding to rhVEGF-B at 100 nM.

[0177] Secondly, with rhVEGF 165 In comparison, RO-101_Sample 1 was tested for binding to rhVEGF-C or rhVEGF-D. rhVEGF-C and rhVEGF-D were obtained from AcroBiosystems. 165 Obtained from Peprotech and used as positive control. Corning 9017 medium binding plates were coated with different target proteins at 1 μg / mL and 100 μl / well. Donkey anti-human IgG Fcγ-HRP was used for detection. OD450 nm readings were measured by Victor 3.

[0178] Table 12: RO-101_Sample 1 and rhVEGF 165 EC50 of rhVEGF-C or rhVEGF-D binding

[0179]

[0180] like Figure 5C As shown in Table 12, RO-101 did not bind to commercial rhVEGF-C or rhVEGF-D proteins containing only the PDGF domain.

[0181] Third, with rhVEGF 165 In comparison, binding to rhPIGF-1 or rhPIGF-3 was tested. Target protein was used to coat Corning 9017 medium binding plates at 1 μg / mL and 100 μl / well. Product RO-101_sample 1 was diluted in blocking buffer at a starting concentration of 100 nM and then tested at a 1:3 dilution. Donkey anti-human IgG Fcγ-HRP was used for detection. OD450 nm readings were measured by Victor 3.

[0182] Table 13: RO-101_Sample 1 and rhVEGF 165 EC50 of binding of rhPlGF-1 or rhPlGF-3

[0183]

[0184] like Figure 5DAs shown in Table 13, at >100 nM, RO-101 did not bind to PIGF-1 and bound weakly to PIGF-3.

[0185] Example 7 - Binding of RO-101 to VEGF-A and ANG-2 across species

[0186] RO-101 was tested with VEGF from different species 165 and ANG-2 orthologs (see Table 14). Sequence alignments were performed (for VEGF, see Fig. 6A , for ANG-2, see Figure 6C ) and homology comparisons (for VEGF, see Table 15.1, for ANG-2, see Table 15.2).

[0187] Table 14 - VEGF from different species 165 Summary of basic information about ANG-2 orthologs

[0188]

[0189]

[0190] Table 15.1 - VEGF from different species 165 Percent homology of orthologs

[0191]

[0192] Table 15.2 - Percent homology of ANG-2 orthologs from different species

[0193]

[0194] Similar binding assays were performed as described in previous examples. Figure 6B and Table 16 show that RO-101 binds to rabbit, dog and porcine VEGF-A targets with comparable affinity when compared to binding to human VEGF-A.

[0195] Table 16 - EC50 of RO-101 binding to VEGF-A from different species

[0196] X-species VEGF-A EC50(nM) <![CDATA[R 2 ]]> Human VEGF-A 0.0171 0.9967 Rabbit VEGF-A 0.0334 0.9967 Dog VEGF-A 0.0237 0.9979 Porcine VEGF-A 0.0198 0.9899

[0197] Similarly, Fig.6D and Table 17 show that RO-101 binds to rabbit and dog ANG-2 targets with comparable affinity to human ANG-2.

[0198] Table 17 - EC50 of RO-101 binding to ANG-2 from different species

[0199] X-Species ANG-2 EC50(nM) <![CDATA[R 2 ]]> ANG-2 0.007 0.9814 Rabbit ANG-2 0.011 0.9868 Dog ANG-2 0.0135 0.9376

[0200] In summary, RO-101 exhibits similar strong binding affinity to VEGF and ANG-2 orthologs across multiple species.

[0201] Although preferred embodiments of the present invention are shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided by way of example only. Those skilled in the art will now appreciate that many variations, changes and substitutions will not depart from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be employed in practicing the present invention.

[0202] All publications, patent applications, issued patents, and other documents mentioned in this specification are incorporated by reference into this specification as if each individual publication, patent application, issued patent, or other document was specifically and individually indicated to be incorporated by reference in its entirety. Definitions contained in text incorporated by reference are excluded if they conflict with definitions in this disclosure.

[0203]

[0204]

Claims

1. A vascular endothelial growth factor A (VEGF_A) binding antibody or an antigen-binding fragment thereof, comprising: a. a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence shown in SEQ ID NO: 3 (GYTFSIYT); b. a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 4 (INPYNGNT); and c. a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence shown in SEQ ID NO: 5 (AKAPAVFWWTGLDY); Wherein the antibody binds to VEGF_A.

2. The antibody or antigen-binding fragment thereof of claim 1, comprising a heavy chain variable region comprising an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:

1.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, comprising a heavy chain constant region comprising an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:

2.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, further comprising a surrogate light chain.

5. The antibody or antigen-binding fragment thereof of claim 4, wherein the surrogate light chain comprises: a. a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 23 (NDHDIGVYS); b. a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 24 (YFSQSDK); and c. Light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence shown in SEQ ID NO: 25 (AMGARSSVTH).

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, comprising a light chain variable region comprising an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:

21.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, comprising a light chain constant region comprising an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:

22.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein the antibody binds to a peptide comprising the platelet-derived growth factor (PDGF) domain of VEGF-A.

9. The antibody or antigen-binding fragment thereof according to claim 8, wherein the PDGF domain of VEGF_A comprises a VEGF_A fragment ranging from amino acid position 50 to amino acid position 132.

10. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, wherein the antibody binds to a peptide comprising the VEGF-C heparin domain of VEGF-A.

11. The antibody or antigen-binding fragment thereof according to claim 10, wherein the VEGF_C heparin domain of VEGF_A comprises a VEGF-A fragment ranging from amino acid 143 to amino acid 191.

12. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, wherein the antibody binds to VEGF-A or a fragment thereof.

13. The antibody or antigen-binding fragment thereof of claim 12, wherein the antibody binds to VEGF_A or a fragment thereof with an EC50 of about 60 picomolar or less.

14. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, wherein the antibody inhibits the interaction of VEGF-A or a fragment thereof with a VEGF receptor.

15. The antibody or antigen-binding fragment thereof of claim 14, wherein the antibody inhibits the interaction of VEGF_A or a fragment thereof with the VEGF receptor with an IC50 of about 2000 picomolar or less.

16. The antibody or antigen-binding fragment thereof according to claim 14, wherein the antibody inhibits the interaction of VEGF_A or its fragment with VEGF receptor at least 3 times stronger than farecximab inhibits the interaction.

17. The antibody or antigen-binding fragment thereof of claim 16, wherein the VEGF receptor is VEGF receptor 2 / kinase insert domain receptor (KDR).

18. An angiopoietin 2 (ANG-2) binding antibody or an antigen-binding fragment thereof, comprising: a. a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence shown in SEQ ID NO: 13 (GFTFSSYG); b. a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 14 (ISADSGDK); and c. a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 15 (AKEFISWIYTFDYLDY), wherein the antibody binds to ANG-2.

19. The antibody or antigen-binding fragment thereof of claim 18, comprising a heavy chain variable region comprising an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:

11.

20. The antibody or antigen-binding fragment thereof of claim 18 or 19, comprising a heavy chain constant region comprising an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:

12.

21. The antibody or antigen-binding fragment thereof according to any one of claims 18 to 20, further comprising a surrogate light chain.

22. The antibody or antigen-binding fragment thereof of claim 21, wherein the surrogate light chain comprises: a. a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 23 (NDHDIGVYS); b. a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 24 (YFSQSDK); and c. Light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence shown in SEQ ID NO: 25 (AMGARSSVTH).

23. An antibody or antigen-binding fragment thereof according to any one of claims 18 to 22, comprising a light chain variable region comprising an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:

21.

24. The antibody or antigen-binding fragment thereof of any one of claims 18 to 23, comprising a light chain constant region comprising an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:

22.

25. The antibody or antigen-binding fragment thereof according to any one of claims 18 to 24, wherein the antibody binds to ANG-2.

26. The antibody or antigen-binding fragment thereof of claim 25, wherein the antibody binds to ANG-2 with an EC50 of about 55 picomolar or less.

27. The antibody or antigen-binding fragment thereof according to any one of claims 18 to 26, wherein the antibody binds to ANG-2 at least 50 times more strongly than the binding of farecximab to ANG-2.

28. The antibody or antigen-binding fragment thereof according to any one of claims 18 to 27, wherein the antibody inhibits the interaction of ANG-2 with an ANG-2 receptor.

29. The antibody or antigen-binding fragment thereof of claim 28, wherein the antibody inhibits the interaction of ANG-2 with the ANG-2 receptor with an IC50 of about 800 pmolar or less.

30. The antibody or antigen-binding fragment thereof of claim 28, wherein the antibody inhibits the interaction of ANG-2 with the ANG-2 receptor at least 15-fold more potently than farecximab.

31. The antibody or antigen-binding fragment thereof according to any one of claims 28 to 30, wherein the ANG-2 receptor is a tyrosine kinase having immunoglobulin-like and EGF-like domains 2 receptor (Tie-2 receptor).

32. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 31, wherein the antibody is an IgG antibody.

33. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 31, wherein the antigen-binding fragment thereof is a Fab, F(ab)2, a single domain antibody or a single chain variable fragment (scFv).

34. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 31, wherein the antibody is a surrogate antibody and the antigen-binding fragment thereof is a surrogate antibody antigen-binding fragment (Sab) or a single-chain variable fragment (scSv) of a surrogate antibody.

35. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 34, wherein the antibody or antigen-binding fragment thereof is human, chimeric or humanized.

36. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 34, wherein the antibody or antigen-binding fragment thereof is humanized.

37. A pharmaceutical composition comprising the antibody according to any one of claims 1 to 36 and a pharmaceutically acceptable carrier, excipient or diluent.

38. The pharmaceutical composition of claim 37, formulated for intravenous administration.

39. The pharmaceutical composition of claim 37, formulated for intravitreal administration.

40. A nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 36.

41. An expression vector comprising the nucleic acid of claim 40.

42. The expression vector of claim 41, wherein the expression vector is a lentiviral vector, an adenoviral vector, an adeno-associated viral vector or a plasmid vector.

43. The expression vector of claim 41 or 42, wherein the expression vector is formulated for intravenous administration.

44. The expression vector of claim 41 or 42, wherein the expression vector is formulated for intravitreal administration.

45. A cell comprising the nucleic acid of claim 40 or the expression vector of any one of claims 41 to 44.

46. ​​The cell of claim 45, wherein the cell is a eukaryotic cell suitable for producing antibodies, and optionally wherein the cell is a Chinese Hamster Ovary (CHO) cell.

47. A bispecific antibody comprising: A vascular endothelial growth factor A (VEGF_A) binding portion comprising: a. a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence shown in SEQ ID NO: 3 (GYTFSIYT); b. a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 4 (INPYNGNT); and c. a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 5 (AKAPAVFWWTGLDY); and Angiopoietin 2 (ANG-2) binding portion comprising: a. a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence shown in SEQ ID NO: 13 (GFTFSSYG); b. a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 14 (ISADSGDK); and c. a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence set forth in SEQ ID NO: 15 (AKEFISWIYTFDYLDY); Wherein the bispecific antibody binds to VEGF_A and ANG-2.

48. The bispecific antibody of claim 47, wherein the VEGF_A binding portion comprises a heavy chain variable region comprising an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:

1.

49. The bispecific antibody of claim 47 or 48, wherein the VEGF_A binding portion further comprises a heavy chain constant region.

50. The bispecific antibody of claim 49, wherein the heavy chain constant region comprises an engineered protrusion or an engineered cavity such that homodimerization of the VEGF_A binding portion is inhibited.

51. The bispecific antibody of claim 50, wherein the engineered protrusion comprises a T366W substitution according to EU numbering.

52. The bispecific antibody of claim 50 or 51, wherein the engineered cavity comprises T366S / L368A / Y407V substitutions according to EU numbering.

53. The bispecific antibody of any one of claims 49 to 52, wherein the heavy chain constant region comprises one or more substitutions in the Fc region that reduce antibody effector function.

54. The bispecific antibody of claim 53, wherein the one or more substitutions in the Fc region that reduce antibody effector function comprise L234A and / or L235A according to EU numbering.

55. The bispecific antibody of any one of claims 49 to 54, wherein the heavy chain constant region comprises one or more substitutions of cysteine ​​residues.

56. The bispecific antibody of claim 55, wherein the one or more substitutions of the cysteine ​​residues are at serine 354 according to EU numbering.

57. A bispecific antibody according to any one of claims 47 to 56, wherein the VEGF_A binding portion comprises a heavy chain constant region, which comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:

2.

58. The bispecific antibody of any one of claims 47 to 57, wherein the bispecific antibody binds to a peptide comprising the platelet-derived growth factor (PDGF) domain of VEGF_A.

59. The bispecific antibody according to claim 58, wherein the PDGF domain of VEGF_A comprises a VEGF_A fragment ranging from amino acid position 50 to amino acid position 132.

60. The bispecific antibody of any one of claims 47 to 59, wherein the bispecific antibody binds to a peptide comprising the VEGF-C heparin domain of VEGF_A.

61. The bispecific antibody of claim 60, wherein the VEGF_C heparin domain of VEGF_A comprises a VEGF-A fragment ranging from amino acid 143 to amino acid 191.

62. The bispecific antibody of any one of claims 47 to 61, wherein the bispecific antibody binds to VEGF-A or a fragment thereof.

63. The bispecific antibody of claim 62, wherein the bispecific antibody binds to VEGF_A or a fragment thereof with an EC50 of about 60 picomolar or less.

64. The bispecific antibody of any one of claims 47 to 63, wherein the bispecific antibody inhibits the interaction of VEGF_A or a fragment thereof with a VEGF receptor.

65. The bispecific antibody of claim 64, wherein the bispecific antibody inhibits the interaction of VEGF_A or a fragment thereof with the VEGF receptor with an IC50 of about 2000 picomolar or less.

66. The bispecific antibody of claim 64, wherein the bispecific antibody inhibits the interaction of VEGF_A or a fragment thereof with the VEGF receptor at least 3 times more potently than farecximab inhibits the interaction.

67. The bispecific antibody of claim 66, wherein the VEGF receptor is VEGF receptor 2 / kinase insert domain receptor (KDR).

68. The bispecific antibody according to any one of claims 47 to 67, wherein the ANG-2 binding portion comprises a heavy chain variable region comprising an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:

11.

69. The bispecific antibody according to any one of claims 47 to 68, wherein the ANG-2 binding portion further comprises a heavy chain constant region.

70. The bispecific antibody of claim 69, wherein the heavy chain constant region comprises an engineered protrusion or an engineered cavity such that homodimerization of the ANG-2 binding moiety is inhibited.

71. The bispecific antibody of claim 70, wherein the engineered protrusion comprises a T366W substitution according to EU numbering.

72. The bispecific antibody of claim 70 or 71, wherein the engineered cavity comprises T366S / L368A / Y407V substitutions according to EU numbering.

73. The bispecific antibody of any one of claims 69 to 72, wherein the heavy chain constant region comprises one or more of the Fc region substitutions that reduce antibody effector function.

74. The bispecific antibody of claim 73, wherein the one or more of the Fc region substitutions that reduce antibody effector function comprise L234A and / or L235A according to EU numbering.

75. The bispecific antibody of any one of claims 69 to 74, wherein the heavy chain constant region comprises one or more substitutions of cysteine ​​residues.

76. The bispecific antibody of claim 75, wherein the one or more substitutions of the cysteine ​​residues are at serine 354 according to EU numbering.

77. The bispecific antibody according to any one of claims 47 to 76, wherein the ANG-2 binding portion comprises a heavy chain constant region comprising an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:

12.

78. The bispecific antibody of any one of claims 47 to 77, wherein said bispecific antibody binds to ANG-2.

79. The bispecific antibody of claim 78, wherein the bispecific antibody binds to ANG-2 with an EC50 of about 55 pmolar or less.

80. The bispecific antibody of any one of claims 47 to 79, wherein the binding of the bispecific antibody to ANG-2 is at least 50-fold stronger than the binding of farecximab to ANG-2.

81. The bispecific antibody of any one of claims 47 to 80, wherein the bispecific antibody inhibits the interaction of ANG-2 with an ANG-2 receptor.

82. The bispecific antibody of claim 81, wherein said bispecific antibody inhibits said interaction of ANG-2 with said ANG-2 receptor with an IC50 of about 800 pmolar or less.

83. The bispecific antibody of claim 81 , wherein said bispecific antibody inhibits said interaction of ANG-2 with said ANG-2 receptor at least 15-fold more potently than farecximab.

84. The bispecific antibody according to any one of claims 81 to 83, wherein the ANG-2 receptor is a tyrosine kinase with immunoglobulin-like and EGF-like domains 2 receptor (Tie-2 receptor).

85. The bispecific antibody of any one of claims 47 to 84, further comprising a common light chain.

86. The bispecific antibody of claim 85, wherein the common light chain is a surrogate light chain.

87. The bispecific antibody of claim 86, wherein the surrogate light chain comprises: a. a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence set forth in SEQ ID NO: 23 (NDHDIGVYS); b. a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence set forth in SEQ ID NO: 24 (YFSQSDK); and c. Light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence shown in SEQ ID NO: 25 (AMGARSSVTH).

88. The bispecific antibody of any one of claims 86 to 87, wherein the surrogate light chain comprises a light chain variable region comprising an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:

21.

89. The bispecific antibody of any one of claims 47 to 67, wherein the surrogate light chain comprises a light chain constant region comprising an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence shown in SEQ ID NO:

22.

90. A bispecific antibody or antigen-binding peptide comprising: (a) a first heavy chain variable region comprising an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence shown in SEQ ID NO: 1; (b) a second heavy chain variable region comprising an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence shown in SEQ ID NO: 11; and (c) a common light chain variable region, wherein the common light chain variable region comprises a light chain variable region comprising an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence shown in SEQ ID NO: 21, wherein the bispecific antibody binds to VEGF_A and ANG-2.

91. A bispecific antibody comprising: (a) a first heavy chain comprising an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence shown in SEQ ID NO: 2; (b) a second heavy chain comprising an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence shown in SEQ ID NO: 12; and (c) a common light chain, wherein the common light chain comprises a light chain comprising an amino acid sequence at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% identical to the amino acid sequence shown in SEQ ID NO: 22, wherein the bispecific antibody binds to VEGF_A and ANG-2.

92. A bispecific antibody comprising: (a) a first heavy chain comprising the amino acid sequence as shown in SEQ ID NO:2; (b) a second heavy chain comprising the amino acid sequence as shown in SEQ ID NO:12; and (c) a common light chain, wherein the common light chain comprises a light chain comprising the amino acid sequence as shown in SEQ ID NO:22, wherein the bispecific antibody binds to VEGF-A and ANG-2.

93. A pharmaceutical composition comprising the bispecific antibody according to any one of claims 47 to 92 and a pharmaceutically acceptable carrier, excipient or diluent.

94. The pharmaceutical composition of claim 93, formulated for intravenous administration.

95. The pharmaceutical composition of claim 93, formulated for intravitreal administration.

96. A nucleic acid or multiple nucleic acids encoding the bispecific antibody according to any one of claims 47 to 92.

97. An expression vector comprising the nucleic acid of claim 96.

98. The expression vector of claim 97, wherein the expression vector is a lentiviral vector, an adenoviral vector, an adeno-associated viral vector or a plasmid vector.

99. The expression vector of claim 97 or 98, wherein the expression vector is formulated for intravenous administration.

100. The expression vector of claim 97 or 98, wherein the expression vector is formulated for intravitreal administration. 101 . A pharmaceutical composition comprising the nucleic acid or nucleic acids encoding the bispecific antibody according to any one of claims 96 to 100 and a pharmaceutically acceptable carrier, excipient or diluent.

102. The pharmaceutical composition of claim 101, formulated for intravenous administration.

103. The pharmaceutical composition of claim 101 formulated for intravitreal administration.

104. A method of treating an eye disease in an individual, comprising administering to the individual the antibody, bispecific antibody, the nucleic acid or multiple nucleic acids encoding the antibody or bispecific antibody, or pharmaceutical composition according to any of the preceding claims, thereby treating the disease of the eye.

105. The method of claim 104, wherein the disease of the eye is macular degeneration.

106. The method of claim 105, wherein the macular degeneration is age-related.

107. The method of claim 105, wherein the macular degeneration is associated with diabetes.

108. The method of claim 105, wherein the macular degeneration is wet macular degeneration.

109. The method of claim 104, wherein the disease of the eye is branch retinal vein occlusion.

110. The method of claim 104, wherein the disease of the eye is central retinal vein occlusion.

111. Use of the antibody, bispecific antibody or pharmaceutical composition according to any one of claims 1 to 103 in a method for treating an eye disease.

112. The use according to claim 111, wherein the disease of the eye is macular degeneration.

113. The use according to claim 112, wherein the macular degeneration is age-related.

114. The use according to claim 112, wherein the macular degeneration is associated with diabetes.

115. The use according to claim 112, wherein the macular degeneration is wet macular degeneration.

116. The use according to claim 111, wherein the disease of the eye is branch retinal vein occlusion.

117. The use according to claim 111, wherein the disease of the eye is central retinal vein occlusion.

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