Antibody that binds to VEGF-a and ANG2 and methods of use

Abstract

To provide improved therapeutic antibodies that bind to VEGF and ANG2, by improving efficacy relative to standard-of-care and by improving duration of action and in turn, reducing frequency of intravitreal injections, leading to less administration burden for the patient.SOLUTION: The present invention relates to anti-VEGF-A / anti-ANG2 antibodies in the form of a bispecific Fab fragment, comprising a specific amino acid sequence.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to anti-VEGF-A / anti-ANG2 antibodies and methods of use thereof. [Background technology]

[0002] Bispecific antibodies that bind VEGF and ANG2 have been previously reported and have been suggested for the treatment of ocular vascular diseases such as age-related macular degeneration. Faricimab (INN), a full-length IgG1 antibody containing a Fab binding arm that specifically binds VEGF and a second Fab binding arm that specifically binds ANG2, is the most advanced bispecific therapeutic currently being evaluated in clinical Phase III trials for the treatment of DME and nAMD (Sharma, A., Kumar, N., Kuppermann, BD et al. Eye 34, 802-804 (2020)).

[0003] Additional combination treatments targeting VEGF and ANG2 for the treatment of ocular vascular diseases have been suggested in recent years (e.g., WO 2016 / 122996, WO 2018 / 037000, WO 2019 / 200006, U.S. Patent Application Publication No. 2020 / 0102381).

[0004] Multispecific antibodies comprising two paratopes in one pair of variable heavy (VH) and variable light (VL) domains are described in WO 2008 / 027236; WO 2010 / 108127 and Bostrom, J., et al., Science 323 (2009) 1610-1614 and WO 2012 / 163520.

[0005] WO 2012 / 163520 discloses bispecific antibodies that contain two paratopes in a pair of VH and VL domains ("DutaFab"). Each paratope of the bispecific antibodies of WO 2012 / 163520 comprises amino acids from heavy and light chain CDRs, with heavy chain CDR-H1 and CDR-H3 and light chain CDR-L2 contributing to the first paratope, and light chain CDR-L1 and CDR-L3 and heavy chain CDR-H2 contributing to the second paratope. Monospecific antibodies containing individual paratopes are independently isolated from different Fab libraries in which either the first or second paratope is diversified. The amino acid sequences of the monospecific antibodies are identified and fused to biparatopic VH and VL pairs. An example of an exemplary Fab fragment that specifically binds VEGF and IL-6 is disclosed in WO 2012 / 163520.

[0006] However, there is a need for improved therapeutic antibodies that bind VEGF and ANG2, for example, by improving efficacy and improving duration of action compared to standard therapies, thereby reducing the frequency of intravitreal injections and reducing the dosing burden for patients. Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention relates to bispecific anti-VEGF-A / anti-ANG2 antibodies and methods of use thereof. [Means for solving the problem]

[0008] In one aspect, the invention provides an antibody that binds to human VEGF-A and human ANG2, the antibody comprising a VEGF-A paratope and an ANG2 paratope within a cognate pair of a variable light domain (VL domain) and a variable heavy domain (VH domain), the VEGF-A paratope comprising amino acid residues from CDR-H2, CDR-L1 and CDR-L3 of the antibody, and the ANG2 paratope comprising amino acid residues from CDR-H1, CDR-H3 and CDR-L2 of the antibody.

[0009] In one aspect, the invention provides an antibody that binds to human VEGF-A and human ANG2, the antibody comprising a VEGF-A paratope and an ANG2 paratope within a cognate pair of a variable light domain (VL domain) and a variable heavy domain (VH domain), wherein the pair of variable light domain and variable heavy domain simultaneously binds to human VEGF-A and human ANG2.

[0010] In one aspect, the invention provides an antibody that binds to human VEGF-A and human ANG2, the antibody comprising a VEGF-A paratope and an ANG2 paratope within a cognate pair of a variable light domain (VL domain) and a variable heavy domain (VH domain), and which binds to the same epitope on human VEGF-A and the same epitope on human ANG2 as an antibody having a variable heavy domain of SEQ ID NO: 19 and a variable light domain of SEQ ID NO: 20.

[0011] In one aspect, the invention provides an antibody that binds to human VEGF-A and human ANG2, wherein the antibody Fab fragment of the antibody has (i) a K of less than 50 pM as measured by KinExA. D binds to human VEGF-A121 with a K of less than 50 pM as measured by KinExA; D The present invention provides an antibody that binds to human ANG2 at a

[0012] In one aspect, the invention provides an antibody that binds to human VEGF-A and human ANG2, wherein an antibody Fab fragment of the antibody exhibits an onset aggregation temperature of 70° C. or greater, as measured by static light scattering (SLS), in one embodiment as measured by SLS as described under "Thermal Stability" in the Materials and General Methods section.

[0013] In one aspect, the invention provides an antibody that binds to human VEGF-A and human ANG2, wherein an antibody Fab fragment of the antibody exhibits a melting temperature of greater than 80° C. as measured by static light scattering (SLS), in one embodiment as measured by SLS as described under "Thermal Stability" in the Materials and General Methods section.

[0014] In one aspect, the invention provides an antibody that binds to human VEGF-A and human ANG2, wherein a solution of 180 mg / ml of the antibody Fab fragment in 20 mM His / HisHCl, pH 6.0 has a viscosity of less than 20 cP at 20° C. as detected by dynamic light scattering using the latex-bead DLS method described in Example 8.

[0015] In one aspect, the invention provides an antibody that binds to human VEGF-A and human ANG2, comprising a VH domain comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 3, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 21, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14, and a VL domain comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 22, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 23, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8.

[0016] In one aspect, the invention provides an antibody that binds to human VEGF-A and human ANG2, the antibody comprising: (c) a VH domain comprising an amino acid sequence of SEQ ID NO: 21; and (d) a human heavy chain framework having (i) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14, (d) a VL domain comprising (e) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 22, (f) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 23, (g) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8, and (h) a VL domain comprising a human light chain framework having (i) a FR1 comprising amino acid residues I2 and Y3, (ii) a FR2 comprising amino acid residues L46 and F49, and (iii) a FR3 comprising amino acid residue E57, wherein the numbering of the VH and VL domains is according to the Kabat numbering system.

[0017] In one aspect, the invention provides an antibody that binds to human VEGF-A and human ANG2, the antibody comprising a VH domain comprising amino acid residues H3, D26, F27, E29, Y30, D35b, D35c, D55, H56, K57, Y58, T61, K62, F63, I64, G65, R66, R94, D95, V96, F98, and F99, and a VL domain comprising amino acid residues I2, Y3, Y27, W27a, E32, L46, F49, D50, F53, K54, V55, Y56, E57, Y91, R92, Y93, H94, and P95, wherein numbering of the VH and VL domains is according to the Kabat numbering system. In one embodiment, the antibody comprises a VEGF-A paratope comprising the following amino acid residues in the VH domain: D35c, D55, H56, K57, Y58, T61, K62, F63, I64, G65, R66, and D95, and the following amino acid residues in the VL domain: I2, Y3, Y27, W27a, E32, R92, Y93, H94, and P95; and an ANG2 paratope comprising the following amino acid residues in the VH domain: H3, D26, F27, E29, Y30, D35b, R94, V96, F98, and F99, and the following amino acid residues in the VL domain: E32, L46, F49, D50, F53, K54, V55, Y56, E57, and Y91.

[0018] In one aspect, the invention provides an antibody that binds to human VEGF-A and human ANG2, comprising: (a) a VH domain comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 19; and (b) a VL domain comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 20.

[0019] In one aspect, the invention provides an antibody that binds to human VEGF-A and human ANG2, comprising: (a) a VH domain comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 3, (b) a CDR-H2 having the amino acid sequence of SEQ ID NO: 21, and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 14, and a VL domain comprising (d) a CDR-L1 having the amino acid sequence of SEQ ID NO: 22, (e) a CDR-L2 having the amino acid sequence of SEQ ID NO: 23, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 8, wherein the antibody comprises: (a) a VH domain comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 19; and (b) a VL domain comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 20.

[0020] In one aspect, the invention provides an antibody that binds to human VEGF-A and human ANG2, comprising a VH domain comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 3, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 21, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14, and a VL domain comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 22, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 23, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8. and (b) a VL domain comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:20, comprising I2, Y3, L46, F49, and E57, wherein the numbering of the VH and VL domains is according to the Kabat numbering system.

[0021] In one aspect, the invention relates to an antibody that binds to human VEGF-A and human ANG2, comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 3; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 21; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14; (d) a VH domain comprising a human heavy chain framework having (i) FR1 comprising amino acid residues H3, D26, F27, E29, and Y30, (ii) FR3 comprising amino acid residues R66 and R94; and (e) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 22; (f) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 23; (g) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8. and (h) a VL domain comprising a human light chain framework having (i) FR1 comprising amino acid residues I2 and Y3, (ii) FR2 comprising amino acid residues L46 and F49, and (iii) FR3 comprising amino acid residue E57, wherein the numbering of the VH and VL domains is according to the Kabat numbering system, and wherein the antibody comprises (a) a VH domain comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 19, and (b) a VL domain comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 20.

[0022] In one aspect, the present invention provides an antibody that binds to human VEGF-A and human ANG2, comprising: (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 19 with up to 15 amino acid substitutions, the amino acid substitutions being at one or more positions 1, 2, 4 to 25, 28, 35d to 54, 59, 60, 67 to 93, 97, 101 to 113 of SEQ ID NO: 19; and (b) a variable light chain domain comprising the amino acid sequence of SEQ ID NO: 20 with up to 15 amino acid substitutions, the amino acid substitutions being at positions 1, 4 to 26, 27b to 27d, 33 to 45, 47, 48, 51, 52, 58 to 90, 96 to 107 of SEQ ID NO: 20, wherein the numbering of the VH and VL domains is according to the Kabat numbering system.

[0023] In one aspect, the invention provides an antibody that binds to human VEGF-A and human ANG2, comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 3, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 21, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14, and a VL domain (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 22, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 23, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8, wherein the antibody comprises: (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 19 with up to 15 amino acid substitutions; and (b) a variable light domain comprising the amino acid sequence of SEQ ID NO: 20 with up to 15 amino acid substitutions.

[0024] In one aspect, the invention relates to an antibody that binds to human VEGF-A and human ANG2, comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 3; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 21; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14; (d) a human heavy chain framework having (i) FR1 comprising amino acid residues H3, D26, F27, E29, and Y30, (ii) FR3 comprising amino acid residues R66 and R94; and (e) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 22; (f) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 23; (g) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 24; and (h) a VL domain comprising a human light chain framework having (i) FR1 comprising amino acid residues I2 and Y3, (ii) FR2 comprising amino acid residues L46 and F49, and (iii) FR3 comprising amino acid residue E57, wherein the numbering of the VH and VL domains is according to the Kabat numbering system, wherein the VH and VL domains comprise: (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 19 with up to 15 amino acid substitutions; and (b) a variable light chain domain comprising the amino acid sequence of SEQ ID NO: 20 with up to 15 amino acid substitutions.

[0025] In one aspect, the invention provides an antibody that binds to human VEGF-A and human ANG2, comprising the VH sequence of SEQ ID NO:19 and the VL sequence of SEQ ID NO:20.

[0026] In one aspect, the invention provides an antibody that binds human VEGF- and human ANG2, comprising a heavy chain amino acid sequence of SEQ ID NO:24 and a light chain amino acid sequence of SEQ ID NO:25.

[0027] In one aspect, the invention provides an antibody that binds to human VEGF-A and human ANG2, the antibody comprising a VL domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 3, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 21, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14, and (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 22, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 23, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8, wherein an antibody Fab fragment of the antibody has (i) a K of less than 50 pM as measured by KinExA. D binds to human VEGF-A121 with a K of less than 50 pM as measured by KinExA; D It binds to human ANG2.

[0028] In one aspect, the invention provides an antibody that binds to human VEGF-A and human ANG2, comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 3; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 21; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14; (d) a VH domain comprising a human heavy chain framework having (i) FR1 comprising amino acid residues H3, D26, F27, E29, and Y30, (ii) FR3 comprising amino acid residues R66 and R94; and (e) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 22; (f) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 23; (g) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8; and (h) a CDR-L4 comprising the amino acid sequence of SEQ ID NO: 10; (a) a VH domain comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 19; and (b) a VL domain comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 20; and an antibody Fab fragment of the antibody has (i) a K of less than 50 pM as measured by KinExA. D binds to human VEGF-A121 with a K of less than 50 pM as measured by KinExA; D It binds to human ANG2.

[0029] In one aspect, the present invention provides an antibody that binds to human VEGF-A and human ANG2, comprising a VL domain including (a) a CDR-H1 having the amino acid sequence of SEQ ID NO: 3, (b) a CDR-H2 having the amino acid sequence of SEQ ID NO: 21, and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 14, as well as (d) a CDR-L1 having the amino acid sequence of SEQ ID NO: 22, (e) a CDR-L2 having the amino acid sequence of SEQ ID NO: 23, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 8, wherein the antibody Fab fragment of the antibody exhibits an aggregation onset temperature of 70°C or higher.

[0030] In one aspect, the invention provides an antibody that binds to human VEGF-A and human ANG2, comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 3; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 21; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14; (d) a VH domain comprising a human heavy chain framework having (i) FR1 comprising amino acid residues H3, D26, F27, E29, and Y30, (ii) FR3 comprising amino acid residues R66 and R94; and (e) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 22; (f) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 23; (g) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8; and h) An antibody comprising a VL domain comprising a human light chain framework having (i) FR1 comprising amino acid residues I2 and Y3, (ii) FR2 comprising amino acid residues L46 and F49, and (iii) FR3 comprising amino acid residue E57, wherein the numbering of the VH and VL domains is according to the Kabat numbering system, the antibody comprising (a) a VH domain comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 19 and (b) a VL domain comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 20, wherein an antibody Fab fragment of the antibody exhibits an onset aggregation temperature of 70°C or higher.

[0031] In one aspect, the invention provides an antibody that binds to human VEGF-A and human ANG2, comprising a VL domain comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 3, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 21, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14, and (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 22, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 23, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8, wherein an antibody Fab fragment of the antibody exhibits a melting temperature of greater than 80°C as measured by dynamic light scattering.

[0032] In one aspect, the invention provides an antibody that binds to human VEGF-A and human ANG2, comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 3; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 21; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14; (d) a VH domain comprising a human heavy chain framework having (i) FR1 comprising amino acid residues H3, D26, F27, E29, and Y30, (ii) FR3 comprising amino acid residues R66 and R94; and (e) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 22; (f) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 23; (g) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8; and (h) (i) a CDR-L4 comprising the amino acid sequence of SEQ ID NO: 15; and (iii) a FR3 that comprises amino acid residues E57, wherein the numbering of the VH and VL domains is according to the Kabat numbering system, the antibody comprising (a) a VH domain comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 19 and (b) a VL domain comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 20, wherein an antibody Fab fragment of the antibody exhibits a melting temperature of greater than 80°C as measured by dynamic light scattering.

[0033] In one aspect, the invention provides an antibody that binds to human VEGF-A and human ANG2, the antibody comprising a VL domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:3, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:21, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:14, and (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:22, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:23, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:8, wherein a solution of 180 mg / ml of the antibody Fab fragment in 20 mM His / HisHCl, pH 6.0 has a viscosity of less than 20 cP at 20° C. as detected by dynamic light scattering using the latex-bead DLS method described in Example 8.

[0034] In one aspect, the invention provides an antibody that binds to human VEGF-A and human ANG2, comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 3; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 21; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14; (d) a VH domain comprising a human heavy chain framework having (i) an FR1 comprising amino acid residues H3, D26, F27, E29, and Y30, (ii) an FR3 comprising amino acid residues R66 and R94; and (e) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 22; (f) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 23; and (g) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8. and (h) a VL domain comprising a human light chain framework having (i) a FR1 comprising amino acid residues I2 and Y3, (ii) a FR2 comprising amino acid residues L46 and F49, and (iii) a FR3 comprising amino acid residue E57, wherein the numbering of the VH and VL domains is according to the Kabat numbering system, the antibody comprising (a) a VH domain comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 19, and (b) a VL domain comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 20, wherein a solution of 180 mg / ml of the antibody Fab fragment in 20 mM His / HisHCl, pH 6.0 has a viscosity of less than 20 cP at 20° C. as detected by dynamic light scattering using the latex-bead DLS method described in Example 8.

[0035] One embodiment of the present invention relates to an antibody fragment that binds to human VEGF-A and human ANG2. One embodiment of the present invention relates to a bispecific antibody fragment that binds to human VEGF-A and human ANG2. In one embodiment, the antibody fragment is selected from Fv, Fab, Fab', Fab'-SH, F(ab')2 or single chain antibodies derived therefrom. One embodiment of the present invention relates to a Fab fragment that binds to human VEGF-A and human ANG2. One embodiment of the present invention relates to an Fv fragment that binds to human VEGF-A and human ANG2.

[0036] One embodiment of the present invention relates to a full-length IgG antibody that binds to human VEGF-A and human ANG2.

[0037] In one aspect, the invention provides an isolated nucleic acid encoding an antibody of the invention.

[0038] In one aspect, the invention provides a host cell comprising a nucleic acid of the invention. In one embodiment, the host cell is a CHO cell. In one embodiment, the host cell is an E. coli cell.

[0039] In one aspect, the invention provides an expression vector comprising a nucleic acid of the invention.

[0040] In one aspect, the invention provides a method for producing an antibody that binds human VEGF-A and human ANG2, the method comprising culturing a host cell of the invention so that the antibody is produced.

[0041] In one aspect, the invention provides an antibody produced by the method of the invention.

[0042] In one aspect, the invention provides a pharmaceutical formulation comprising an antibody of the invention and a pharma- ceutically acceptable carrier.

[0043] In one aspect, the invention provides a pre-filled syringe comprising an antibody of the invention and a pharma- ceutically acceptable carrier.

[0044] In one aspect, the invention provides an ocular implant comprising an antibody of the invention and a pharma- ceutically acceptable carrier. In one embodiment, the invention includes a port delivery device comprising an antibody of the invention.

[0045] In one aspect of the invention, the antibody or pharmaceutical formulation is administered by a port delivery device.

[0046] In one aspect, the invention provides an antibody of the invention for use as a medicament, in one embodiment for use in the treatment of vascular disease.

[0047] In one aspect, the invention provides the use of an antibody of the invention, or a pharmaceutical composition of the invention, in the manufacture of a medicament, in one embodiment a medicament for treating a vascular disease.

[0048] In one aspect, the invention provides a method of treating an individual having a vascular disease comprising administering to the individual an effective amount of an antibody of the invention or a pharmaceutical composition of the invention.

[0049] In one aspect, the invention provides a method of inhibiting angiogenesis in an individual, comprising administering to the individual an antibody of the invention or a pharmaceutical composition of the invention in an amount effective to inhibit angiogenesis.

[0050] According to the present invention, therapeutic anti-VEGF-A / anti-ANG2 antibodies are provided that can bind to their target antigens independently, even when provided in the form of antibody Fab fragments. The antibodies of the present invention are suitable for the treatment of ocular vascular diseases. The antibodies of the present invention offer several beneficial properties that allow their therapeutic application, such as high affinity for both targets supporting a low effective dose, and high stability favorable for long periods of time. Compared to non-antibody approaches, the antibodies of the present invention are more likely to be tolerated due to their high human nature and lack of artificial domains and linkers. Also, the antibodies of the present invention are advantageously provided in a highly concentrated liquid formulation with a viscosity suitable for application to the eye. Treatment with the antibodies of the present invention is more tolerable to patients, since they can be provided in high concentrations, higher doses of the therapeutic agent can be applied in one treatment, allowing for longer treatment cycles. Furthermore, when used as bispecific Fab fragments for therapy, the antibodies of the present invention allow for more binding sites per dose, compared to bispecific full-length IgG antibodies. [Brief description of the drawings]

[0051] [Figure 1]Schematic diagram of the Fab fragment of an anti-VEGF-A / anti-ANG2 antibody of the invention. A top view of the cognate VH / VL pair including the arrangement of the CDR amino acids is shown (top image). The VH domain is shown in grey and the VL domain in white. Additionally, the spatial arrangement of the CDR regions is shown. The paratope regions of the antibody of the invention are highlighted (bottom image), with the VEGF-A paratope located in the regions H-CDR2, L-CDR1 and L-CDR2, and the ANG2 paratope located in the regions H-CDR1, H-CDR3 and L-CDR2. [Diagram 2] 1 shows the amino acid sequence of the VH domain of an exemplary anti-VEGF-A / anti-ANG2 antibody of the invention. Kabat numbering of amino acid positions and CDR and FR regions are shown. Amino acid positions contributing to the VEGF-A paratope and the ANG2 paratope identified in Example 13 are highlighted. [Diagram 3] 1 shows the amino acid sequence of the VL domain of an exemplary anti-VEGF-A / anti-ANG2 antibody of the invention. Kabat numbering of amino acid positions and CDR and FR regions are shown. Amino acid positions contributing to the VEGF-A paratope and the ANG2 paratope identified in Example 13 are highlighted. [Figure 4] Independent antigen binding of bispecific antibody P1AD9820 to VEGF-A and ANG2 assessed by SPR according to Example 5. [Diagram 5] VEGF-A121 and VEGF-A165 blocking activity of the indicated antibodies tested in Example 11. [Figure 6] VEGF-A121 and VEGF-A165 blocking activity of the indicated antibodies of the invention and prior art antibody analogs tested in Example 11. [Figure 7] Reporter gene assay (RGA) for analysis of VEGF-A inhibition of the indicated antibodies of the invention and prior art antibody analogs tested in Example 10. [Figure 8] pTie2 assay for analysis of ANG2 inhibition of the indicated antibodies of the invention and prior art antibody analogs tested in Example 10. [Figure 9]ANG1 inhibition mediated by P1AD9820, as tested in Example 12. [Figure 10] Viscosity measured by latex-bead DLS method for P1AA0902 (top left) and P1AD9820 (top right) Fab fragments produced in E. coli and P1AD9820 (center) produced in CHO, as tested in Example 8. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0052] 1.Definition Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those skilled in the art. Furthermore, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. The methods and techniques of the present disclosure are generally carried out according to conventional methods well known in the art. In general, the nomenclature and techniques used in connection with biochemistry, enzymology, molecular and cell biology, microbiology, genetics, and chemistry of proteins and nucleic acids, and hybridization described herein are well known and commonly used in the art.

[0053] Unless otherwise defined herein, the term "comprising" is intended to include the term "consisting of."

[0054] The term "about" as used herein in connection with a particular value (e.g., temperature, concentration, time, etc.) is intended to refer to a + / - 1% variation of the particular value to which the term "about" refers.

[0055] The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0056] An "isolated" antibody is one that is separated from components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or greater than 99% purity as determined by electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC) methods. For a review of methods for assessing antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0057] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies constituting the population are identical and / or bind the same epitope, except for variant antibodies that may, for example, contain naturally occurring mutations or arise during production of a monoclonal antibody preparation, such variants being generally present in minor amounts. In contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method.

[0058] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a heavy chain having a structure substantially similar to a native antibody structure or containing an Fc region as defined herein.

[0059] The "class" of an antibody refers to the type of constant domain or constant region carried by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into "subclasses" (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. In certain embodiments, the antibody is of the IgG1 isotype. In certain embodiments, the antibody is of the IgG1 isotype with P329G, L234A, and L235A mutations to reduce Fc region effector function. In other embodiments, the antibody is of the IgG2 isotype. In certain embodiments, the antibody is of the IgG4 isotype with S228P mutation in the hinge region to improve the stability of the IgG4 antibody. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The light chain of an antibody may be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

[0060] The term "Fc region" is used herein to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl terminus of the heavy chain. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also referred to as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0061] A "variable region" or "variable domain" is a domain of an antibody's heavy or light chain that is involved in binding the antibody to an antigen. The heavy and light chain variable domains (VH and VL, respectively) of natural antibodies generally have a similar structure, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR) (see, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007)). In the antibody of the present invention, a single pair of VH and VL domains, i.e., a cognate VH / VL pair, specifically binds to its two targets: VEGF-A and ANG2.

[0062] "DutaFab" is a bispecific antibody disclosed in WO 2012 / 163520. In DutaFab, a single pair of VH and VL domains specifically binds two different epitopes, one paratope containing amino acid residues from CDR-H2, CDR-L1 and CDR-L3, and the other paratope containing amino acid residues from CDR-H1, CDR-H3 and CDR-L2. DutaFab contains two non-overlapping paratopes within the cognate VH / VL pair and can bind two different epitopes simultaneously. DutaFab and methods for their production by screening libraries containing monospecific Fab fragments are disclosed in WO 2012 / 163520.

[0063] A "human antibody" is an antibody having an amino acid sequence that corresponds to an antibody produced by a human or human cell, or to an antibody of non-human origin that utilizes human antibody-encoding sequences, such as the human antibody repertoire. This definition of a human antibody specifically excludes humanized antibodies, which contain non-human antigen-binding residues. Antibodies or antibody fragments isolated from a human antibody library are considered human antibodies or human antibody fragments herein.

[0064] A "human consensus framework" is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In one embodiment, for VL, the subgroup is subgroup kappa I in Kabat et al. (see above). In one embodiment, for VH, the subgroup is subgroup III in Kabat et al. (see above).

[0065] An "antibody fragment" is a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0066] "Paratope" or "antigen-binding site" refers to the portion of an antibody that recognizes and binds to an antigen, and are used interchangeably herein. A paratope is formed by multiple individual amino acid residues from the heavy and light chain variable domains of an antibody that are spatially adjacent in the tertiary structure of the Fv region. The antibody of the present invention contains two paratopes in one cognate VH / VL pair.

[0067] As used herein, a "VEGF-A paratope" is a paratope or antigen binding site that binds VEGF-A. The VEGF-A paratope of an antibody of the invention comprises amino acid residues from CDR-H2, CDR-L1 and CDR-L3 of the antibody.

[0068] As used herein, an "ANG2 paratope" is a paratope or antigen-binding site that binds to ANG2. The ANG2 paratope of an antibody of the invention comprises amino acid residues from the CDR-H1, CDR-H3, and CDR-L2 of the antibody.

[0069] The term "vascular endothelial growth factor", or "VEGF" for short, as used herein, refers to any naturally occurring VEGF from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses "full-length" unprocessed VEGF as well as any form of VEGF that results from processing within a cell. The term also encompasses naturally occurring variants of VEGF, such as splice variants or allelic variants. An exemplary amino acid sequence of human VEGF is shown in SEQ ID NO:26.

[0070] The terms "anti-VEGF-A antibody" and "antibody that binds VEGF-A" refer to an antibody that is capable of binding to VEGF-A with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting VEGF-A. In one embodiment, the extent of binding of an anti-VEGF-A antibody to an unrelated, non-VEGF-A protein is less than about 10% of the binding of the antibody to VEGF-A as measured, for example, by surface plasmon resonance (SPR). In certain embodiments, an antibody that binds to VEGF-A has a dissociation constant (K D ) is 1 nM or less, 0.1 nM or less, or 0.01 nM or less. The antibody has a K D An antibody is said to "specifically bind" to VEGF-A if it has

[0071] The term "angiopoietin-2", or "ANG2" as used herein, refers to any native ANG2 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses "full-length" unprocessed ANG2 as well as any form of ANG2 that results from processing within a cell. The term also encompasses naturally occurring variants of ANG2, such as splice variants or allelic variants. An exemplary amino acid sequence of human ANG2 is shown in SEQ ID NO:27.

[0072] The terms "anti-ANG2 antibody" and "antibody that binds to anti-ANG2" refer to an antibody that is capable of binding to anti-ANG2 with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting anti-ANG2. In one embodiment, the extent of binding of an anti-anti-ANG2 antibody to an unrelated, non-anti-ANG2 protein is less than about 10% of the binding of the antibody to anti-ANG2 as measured, for example, by surface plasmon resonance (SPR). In certain embodiments, an antibody that binds to ANG2 has a dissociation constant (K D ) is 1 nM or less, 0.1 nM or less, or 0.03 nM or less. D An antibody is said to "specifically bind" to anti-ANG2 if it has

[0073] The antibodies of the invention "bind simultaneously to human VEGF-A and human ANG2," meaning that (a) an antibody Fab fragment of the invention that binds to human ANG2 (also) specifically binds to human VEGF-A, and (b) an antibody Fab fragment of the invention that binds to human VEGF-A (also) specifically binds to human ANG2. Simultaneous binding can be assessed by methods known in the art, for example, by surface plasmon resonance as described herein.

[0074] As used herein, the term "complementarity determining region" or "CDR" refers to each region of an antibody variable domain that is hypervariable in sequence and contains the residues that contact the antigen. Typically, antibodies contain six CDRs: three in the VH domain (CDR-H1, CDR-H2, CDR-H3) and three in the VL domain (CDR-L1, CDR-L2, CDR-L3). Unless otherwise noted, herein, CDR residues and other residues in the variable domain (e.g., FR residues) are numbered according to the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991).

[0075] As used herein, "framework" or "FR" refers to the amino acid residues of a variable domain other than the CDR residues. The framework of a variable domain generally consists of four framework domains: FR1, FR2, FR3 and FR4. Thus, the CDR and FR amino acid sequences generally appear in the following order: (a) in the VH domain: FR1-CDR-H1-FR2-CDR-H2-FR3-CDR-H3-FR4; and (b) in the VL domain: FR1-CDR-L1-FR2-CDR-L2-FR3-CDR-L3-FR4.

[0076] According to the Kabat numbering system, as used herein, the framework and CDR regions are located in the following regions of the variable domain: [Table 1]

[0077] The amino acid positions according to the Kabat numbering system referred to herein are shown aligned with the amino acid sequences of the antibodies of the invention in Figures 2 and 3. Reference to an amino acid at a particular position within an amino acid sequence is made herein as known in the art by stating the respective amino acid and amino acid position, e.g., "E2" refers to a glutamic acid residue located at Kabat position 2 of the amino acid sequence of the respective antibody domain.

[0078] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y is generally determined by the dissociation constant (K D ) Affinity can be measured by methods common in the art, including those described herein. Specific illustrative exemplary embodiments for measuring binding affinity are described herein.

[0079] The term "epitope" refers to a site on an antigen, either proteinaceous or non-proteinaceous, to which an antibody binds. Epitopes may be formed from a contiguous stretch of amino acids (linear epitopes) or may contain non-contiguous amino acids (structural epitopes), formed in spatial proximity, for example, due to antigen folding (i.e., by tertiary folding of a proteinaceous antigen). Linear epitopes are typically still bound by antibodies after exposure of a proteinaceous antigen to a denaturing agent, whereas conformational epitopes are typically destroyed by treatment with a denaturing agent. An epitope comprises at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial structure.

[0080] Screening for antibodies that bind to a specific epitope (i.e., antibodies that bind the same epitope) can be performed using methods routine in the art, such as, for example, but not limited to, alanine scanning, peptide blotting (Meth. Mol. Biol. 248 (2004) 443-463), peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of antigens (see Prot. Sci. 9 (2000) 487-496), and cross-blocking (see "Antibodies", Harlow and Lane, Cold Spring Harbor Press, Cold Spring Harb., NY).

[0081] Antigen Structure-based Antibody Profiling (ASAP), also known as Modification-Assisted Profiling (MAP), allows multiple monoclonal antibodies that specifically bind to VEGF-A or ANG2 to be classified based on the binding profiles of each of the multiple antibodies to chemically or enzymatically modified antigen surfaces (see, e.g., US 2004 / 0101920). Each classified antibody binds to the same epitope, which may be distinct from epitopes represented in other classifications or may be a unique epitope that overlaps in part.

[0082] Competitive binding can also be used to easily determine whether an antibody binds to or competes for binding to the same epitope on VEGF-A or ANG2 as a reference antibody of the present invention. For example, an "antigen that binds to the same epitope on VEGF-A and ANG2" as a reference antibody refers to an antibody that blocks the binding of the reference antibody to that antigen by 50% or more in the respective competitive assays, and conversely, the reference antibody blocks the binding of the antibody to that antigen by 50% or more in the respective competitive assays. Also, for example, to determine whether an antibody binds to the same epitope as a reference antibody, the reference antibody can be bound to VEGF-A or ANG2 at saturation. After removing excess reference antibody, the ability of the antibody in question to bind to VEGF-A or ANG2 is evaluated. If the antibody in question can bind to VEGF-A or ANG2 after saturation binding of the reference antibody, it can be concluded that the antibody of interest binds to a different epitope than the reference antibody. However, if the antibody in question cannot bind to VEGF-A or ANG2 after saturation binding of the reference antibody, it is possible that the antibody of interest binds to the same epitope as the epitope bound by the reference antibody. To confirm whether the antibody in question binds to the same epitope or whether binding is only hindered for steric reasons, routine experiments can be used (e.g. peptide mutations and binding analysis using ELISA, RIA, surface plasmon resonance, flow cytometry, or other quantitative or qualitative antibody binding assays available in the art). This assay should be performed in two setups, i.e., both antibodies are saturating antibodies. If in both setups, only the first (saturating) antibody can bind to VEGF-A or ANG2, it can be concluded that the antibody in question and the reference antibody compete for binding to VEGF-A or ANG2.

[0083] In some embodiments, two antibodies are considered to bind to the same or overlapping epitope if a 1, 5, 10, 20, or 100-fold excess of one antibody inhibits binding of the other by at least 50%, at least 75%, at least 90%, or even 99% or more, as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 50 (1990) 1495-1502).

[0084] In some embodiments, two antibodies are considered to bind to the same epitope if substantially all of the amino acid mutations in the antigen that reduce or eliminate binding of one antibody also reduce or eliminate binding of the other antibody. Two antibodies are considered to have "overlapping epitopes" if only a subset of the amino acid mutations that reduce or eliminate binding of one antibody also reduce or eliminate binding of the other antibody.

[0085] "Percentage of amino acid sequence identity" to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, without considering any conservative substitutions as part of the sequence identity, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percentage of sequence identity for the purpose of alignment. Alignment for determining percent amino acid sequence identity can be achieved in various ways within the skill of the art, for example, using publicly available computer software, such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software or the FASTA program package. Those skilled in the art can determine appropriate parameters for sequence alignment, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared. Alternatively, percent identity values ​​can be generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and the source code is on file in the user documentation at the US Copyright Office, Washington DC, 20559, registered under US Copyright Registration No. TXU510087, and is described in WO 2000 / 005319.

[0086] However, unless otherwise specified, for purposes herein, percent amino acid sequence identity values ​​are generated using the ggsearch program of the FASTA package version 36.3.8c, or followed by the BLOSUM50 comparison matrix. The FASTA program package is described by WR Pearson and DJ Lipman (1988), "Improved Tools for Biological Sequence Analysis," PNAS 85:2444-2448; WR Pearson (1996) "Effective protein sequence comparison," Meth. Enzymol. 266:227-258; and Pearson et.al. (1997) Genomics 46:24-36, and is publicly available at www.fasta.bioch.virginia.edu / fasta_www2 / fasta_down.shtml or www.ebi.ac.uk / Tools / sss / fasta. Alternatively, sequences can be compared using the public server accessible at fasta.bioch.virginia.edu / fasta_www2 / index.cgi using the ggsearch(global protein:protein) program and default options (BLOSUM50; open:-10; ext:-2; Ktup=2), ensuring a global rather than local alignment. The percentage amino acid identity is given in the output alignment header.

[0087] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules, including but not limited to, a cytotoxic agent.

[0088] The term "nucleic acid molecule" or "polynucleotide" includes any compound and / or substance that comprises a polymer of nucleotides. Each nucleotide is composed of a base, specifically a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Nucleic acid molecules are often described by the sequence of bases, where the bases represent the primary (linear) structure of the nucleic acid molecule. The sequence of bases is typically represented from 5' to 3'. As used herein, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA), e.g., complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers containing two or more of these molecules. Nucleic acid molecules may be linear or circular. In addition, the term nucleic acid molecule includes both sense and antisense strands and both single-stranded and double-stranded forms. Furthermore, the nucleic acid molecules described herein can include naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases with derivatized sugar or phosphate backbone linkages or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules suitable as vectors for direct expression of the antibodies of the invention in vitro and / or in vivo, for example in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors can be unmodified or modified. For example, the mRNA may be chemically modified to increase the stability of the RNA vector and / or the expression of the encoded molecule, such that the mRNA can be injected into a subject to produce antibodies in vivo (see, e.g., Stadler ert al, Nature Medicine 2017, published online 12 June 2017, doi:10.1038 / nm.4356, or EP 2101823 B1).

[0089] An "isolated" nucleic acid is a nucleic acid molecule that is separated from a component of its natural environment. Isolated nucleic acid includes a nucleic acid molecule contained within a cell that normally contains the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from the natural chromosomal location.

[0090] An "isolated nucleic acid encoding" an antibody refers to one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of an antibody, wherein such nucleic acid molecules are contained in a single vector or in separate vectors, and wherein such nucleic acid molecules are present in one or more locations in a host cell.

[0091] The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as autonomously replicating nucleic acid structures as well as vectors that are integrated into the genome of a host cell into which the vector is introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."

[0092] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and its progeny, regardless of the number of passages. The progeny may not have exactly the same nucleic acid content as the parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included in the invention.

[0093] The term "pharmaceutical composition" or "pharmaceutical formulation" refers to a preparation that is in a form such that the biological activity of the active ingredients contained in the preparation is effective, and that does not contain additional ingredients that are unacceptably toxic to a subject to which the pharmaceutical composition will be administered.

[0094] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical composition or formulation, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0095] An "effective amount" of an agent, e.g., a pharmaceutical composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0096] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates, such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, an individual or subject is a human.

[0097] As used herein, "treatment" (and grammatical variants thereof, e.g., "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of a disease in the individual being treated, and can be carried out for prophylaxis or during the course of clinical pathology. Desired effects of treatment include preventing the onset or recurrence of disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, remission or palliation of the disease, and ameliorating or improving prognosis. In some embodiments, the antibodies of the invention are used to delay the onset of disease or to slow the progression of the disease.

[0098] The term "ocular disease" as used herein includes any ocular disease associated with pathological neovascularization and / or atrophy. Ocular diseases can be characterized by alterations or disregulation of neovascular proliferation and / or infiltration into structures of ocular tissues, such as the retina or cornea. Ocular diseases can be characterized by atrophy of retinal tissues (photoreceptors and the underlying retinal pigment epithelium (RPE) and choriocapillaris). Non-limiting ocular diseases include, for example, AMD (e.g., wet AMD, dry AMD, intermediate AMD, advanced AMD, and geographic atrophy (GA)), macular degeneration, macular edema, DME (e.g., focal, non-central DME, and diffuse, centrally involved DME), retinopathies, diabetic retinopathy (DR) (e.g., proliferative DR (PDR), non-proliferative DR (NPDR), and high altitude DR), other ischemia-related retinopathies, ROP, retinal edema, and retinal edema. Retinal vein occlusion (RVO) (e.g., central (CRVO) and branched (BRVO) forms), CNV (e.g., myopic CNV), corneal neovascularization, diseases associated with corneal neovascularization, retinal neovascularization, diseases associated with retinal / choroidal neovascularization, central serous retinopathy (CSR), pathological myopia, von Hippel-Lindau disease, ocular histoplasmosis, FEVR, Coats' disease, Norrie's disease, osteoporotic pseudoglioma syndrome (OPPG) Retinal abnormalities associated with retinal disorders, subconjunctival hemorrhage, rubeosis, ocular neovascular disease, neovascular glaucoma, retinitis pigmentosa (RP), hypertensive retinopathy, retinal angiomatous proliferation, macular vascular ectasia, iris neovascularization, intraocular neovascularization, retinal degeneration, cystoid macular edema (CME), vasculitis, papilledema, retinitis including but not limited to: CMV retinitis, ocular melanoma, retinoblastoma, conjunctivitis (e.g., infectious conjunctivitis and non-infectious (e.g., arginine, erythrocyte sedimentation, retinal malform ... In some embodiments, the ocular conditions include, but are not limited to, ocular neovascularization, ocular vascular leakage, and / or retinal edema or atrophy.Further exemplary eye diseases include diseases associated with retinoschisis (abnormal division of the retinal neurosensory layer), rubeosis (neovascularization of the angle), and diseases caused by abnormal proliferation of fibrovascular or fibrous tissue (including all forms of proliferative vitreoretinopathy). Exemplary diseases associated with corneal neovascularization include, but are not limited to, epidemic keratoconjunctivitis, vitamin A deficiency, contact lens overwear, atopic keratitis, superior limbal keratitis, pterygium, keratitis sicca, Sjogren's syndrome, acne rosacea, phylectenulosis, syphilis, mycobacterial infections, fatty degeneration, chemical burns, bacterial ulcers, fungal ulcers, herpes simplex infections, herpes zoster infections, protozoal infections, Kaposi's sarcoma, Mooren's ulcer, Therrien's peripheral corneal degeneration, peripheral keratolysis, rheumatoid arthritis, generalized erythema, polyarteritis nodosa, trauma, Wegener's sarcoidosis, scleritis, Steven-Johnson syndrome, pemphigoid, radial corneal incision, and post-corneal transplant rejection. Exemplary diseases associated with choroidal neovascularization and defects in the retinal vasculature, including increased vascular leakage, aneurysms, and capillary dropout, include, but are not limited to, diabetic retinopathy, macular degeneration, sickle cell anemia, sarcoids, syphilis, pseudoxanthoma elasticum, Paget's disease, venous occlusion, arterial occlusion, carotid occlusive disease, chronic uveitis / vitreous inflammation, mycobacterial infections, Lyme disease, systemic lupus erythematosus, retinopathy of prematurity, retinal edema (including macular edema), Eales' disease, Behcet's disease, infections causing retinitis or choroiditis (e.g., multifocal choroidal), presumed ocular histoplasmosis, Best's disease (vitreous macular degeneration), myopia, optic disc, pars planitis, retinal detachment (e.g., chronic retinal detachment), hyperviscosity syndrome, toxoplasmosis, trauma, and post-laser complications. Exemplary diseases associated with atrophy of retinal tissue (photoreceptors and the underlying RPE) include, but are not limited to, atrophic or non-exudative AMD (e.g., geographic atrophy or advanced dry AMD), macular atrophy (e.g., atrophy associated with neovascularization and / or geographic atrophy), diabetic retinopathy, Stargardt's disease, Sorsby Fundus dystrophy, retinoschisis, and retinitis pigmentosa.

[0099] The term "package insert" is used to refer to instructions typically included in the commercial packaging of a therapeutic product, and may include information regarding the indications, uses, dosages, administration, concomitant therapy, contraindications and / or warnings relating to such therapeutic product.

[0100] 2. Detailed Description of the Embodiments of the Invention In one aspect, the invention is based, in part, on the provision of bispecific antibodies for therapeutic applications. In one particular aspect, an antibody is provided that binds to human VEGF-A and human ANG2. The antibodies of the invention are useful, for example, in the diagnosis or treatment of vascular diseases, such as ocular vascular diseases.

[0101] A. Exemplary Antibodies that Bind Human VEGF-A and Human ANG2 In one aspect, the invention provides an antibody that binds to human VEGF-A and human ANG2. In one aspect, an isolated antibody that binds to human VEGF-A and human ANG2 is provided. In one aspect, the invention provides an antibody that specifically binds to human VEGF-A and human ANG2.

[0102] In certain embodiments, an antibody that binds human VEGF-A and human ANG2 comprises a VEGF-A paratope (i.e., an antigen binding site that binds VEGF-A) and an ANG2 paratope (i.e., an antigen binding site that binds ANG2) within one cognate pair of a VL domain and a VH domain; the VEGF-A paratope comprises amino acid residues derived from CDR-H2, CDR-L1 and CDR-L3 of an antibody, and the ANG2 paratope comprises amino acid residues derived from CDR-H1, CDR-H3 and CDR-L2 of an antibody; and / or the pair of variable light and heavy chain domains simultaneously binds human VEGF-A and human ANG2; and / or binds to the same epitope on human VEGF-A and the same epitope on human ANG2 as an antibody having a variable heavy chain domain of SEQ ID NO: 19 and a variable light chain domain of SEQ ID NO: 20; and / or The antibody Fab fragment of the antibody has (i) a K of less than 50 pM as measured by KinExA.D binds to human VEGF-A121 with a K of less than 50 pM as measured by KinExA; D and / or The antibody Fab fragment of the antibody exhibits an onset aggregation temperature of 70° C. or higher; and / or the antibody Fab fragment of the antibody exhibits a melting temperature of greater than 80° C. as measured by dynamic light scattering; and / or Antibodies are provided, wherein a solution of 180 mg / ml of the antibody Fab fragment in 20 mM His / HisHCl, pH 6.0 has a viscosity of less than 20 cP at 20° C. as detected by dynamic light scattering using the latex-bead DLS method described in Example 8.

[0103] In another aspect, the invention provides an antibody that binds to human VEGF-A and human ANG2, comprising a VL domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 3, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 21, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14, and (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 22, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 23, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8.

[0104] In another aspect, the invention provides an antibody that binds to human VEGF-A and human ANG2, comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:3; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:21; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:14; (d) a human heavy chain framework having (i) FR1 comprising amino acid residues H3, D26, F27, E29, and Y30, (ii) FR3 comprising amino acid residues R66 and R94; and (e) an amino acid sequence of SEQ ID NO:22. (f) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:23; (g) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:8; and (h) a VL domain comprising a human light chain framework having (i) FR1 comprising amino acid residues I2 and Y3, (ii) FR2 comprising amino acid residues L46 and F49, and (iii) FR3 comprising amino acid residue E57, wherein the numbering of the VH and VL domains is according to the Kabat numbering system.

[0105] In another aspect, the invention provides an antibody that binds to human VEGF-A and human ANG2, comprising a VH domain comprising amino acid residues H3, D26, F27, E29, Y30, D35b, D35c, D55, H56, K57, Y58, T61, K62, F63, I64, G65, R66, R94, D95, V96, F98, and F99, and a VL domain comprising amino acid residues I2, Y3, Y27, W27a, E32, L46, F49, D50, F53, K54, V55, Y56, E57, Y91, R92, Y93, H94, and P95, wherein numbering of the VH and VL domains is according to the Kabat numbering system. In one embodiment, the antibody comprises a VEGF-A paratope comprising the following amino acid residues in the VH domain: D35c, D55, H56, K57, Y58, T61, K62, F63, I64, G65, R66, and D95, and the following amino acid residues in the VL domain: I2, Y3, Y27, W27a, E32, R92, Y93, H94, and P95; and an ANG2 paratope comprising the following amino acid residues in the VH domain: H3, D26, F27, E29, Y30, D35b, R94, V96, F98, and F99, and the following amino acid residues in the VL domain: E32, L46, F49, D50, F53, K54, V55, Y56, E57, and Y91.

[0106] In another aspect, the invention provides an antibody that binds to human VEGF-A and human ANG2, comprising: (a) a VH domain comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 19; and (b) a VL domain comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 20.

[0107] In another aspect, the invention provides an antibody that binds to human VEGF-A and human ANG2, comprising: (a) a VH domain comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 19, the VH domain comprising amino acid residues H3, D26, F27, E29, Y30, D35b, D35c, D55, H56, K57, Y58, T61, K62, F63, I64, G65, R66, R94, D95, V96, F98, and F99. and (b) a VL domain comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 20, wherein the VL domain comprises amino acid residues I2, Y3, Y27, W27a, E32, L46, F49, D50, F53, K54, V55, Y56, E57, Y91, R92, Y93, H94, and P95, wherein the numbering of the VH and VL domains is according to the Kabat numbering system.

[0108] In another aspect, the invention relates to an antibody that binds to human VEGF-A and human ANG2, comprising a VH domain comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 3, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 21, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14, and a VL domain comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 22, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 23, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8; (b) a VH domain comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:19; and (b) a VL domain comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:20.

[0109] In another aspect, the invention relates to an antibody that binds to human VEGF-A and human ANG2, comprising a VH domain comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 3, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 21, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14, and a VL domain comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 22, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 23, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8, wherein (a) the amino acid sequence of SEQ ID NO: 19 and at least 8 CDR-L1 comprise a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 24, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14. and (b) a VH domain comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:20, the VH domain comprising H3, D26, F27, E29, Y30, R66, and R94, wherein the VH and VL domains are numbered according to the Kabat numbering system.

[0110] In another aspect, the invention provides an antibody that binds to human VEGF-A and human ANG2, comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 3; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 21; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14; (d) a VH domain comprising a human heavy chain framework having (i) FR1 comprising amino acid residues H3, D26, F27, E29, and Y30, and (ii) FR3 comprising amino acid residues R66 and R94; and (e) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 22; (f) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 23; (g) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8; and (h) a CDR-L4 comprising (i) FR1 comprising amino acid residues I2 and Y3, (ii) amino acid residues L46 and F49, and (iii) a VL domain comprising a human light chain framework having FR3 comprising amino acid residue E57, wherein the numbering of the VH and VL domains is according to the Kabat numbering system, and wherein the VH and VL domains comprise (a) a VH domain comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 19; and (b) a VL domain comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 20.

[0111] In another aspect, the present invention provides an antibody that binds to human VEGF-A and human ANG2, comprising: (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 19 having 1 to 15, 1 to 10, or 1 to 5 amino acid substitutions; and (b) a variable light chain domain comprising the amino acid sequence of SEQ ID NO: 20 having 1 to 15, 1 to 10, or 1 to 5 amino acid substitutions.

[0112] In another aspect, the present invention provides an antibody that binds to human VEGF-A and human ANG2, comprising: (a) a VH domain comprising an amino acid sequence of SEQ ID NO: 19 having 1 to 15, 1 to 10, or 1 to 5 amino acid substitutions, the amino acid substitutions being at one or more positions 1, 2, 4 to 25, 28, 35d to 54, 59, 60, 67 to 93, 97, and 101 to 113 of SEQ ID NO: 19; and (b) a variable light chain domain comprising an amino acid sequence of SEQ ID NO: 20 having 1 to 15, 1 to 10, or 1 to 5 amino acid substitutions, the amino acid substitutions being at positions 1, 4 to 26, 27b to 27d, 33 to 45, 47, 48, 51, 52, 58 to 90, and 96 to 107 of SEQ ID NO: 20, wherein the VH and VL domains are numbered according to the Kabat numbering system.

[0113] In another aspect, the present invention provides an antibody that binds to human VEGF-A and human ANG2, comprising a VH domain comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 3, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 21, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14, and a VL domain comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 22, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 23, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8, wherein the antibody comprises: (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 19 having 1 to 15, 1 to 10, or 1 to 5 amino acid substitutions; and (b) a variable light chain domain comprising the amino acid sequence of SEQ ID NO: 20 having 1 to 15, 1 to 10, or 1 to 5 amino acid substitutions.

[0114] In another aspect, the invention provides an antibody that binds to human VEGF-A and human ANG2, comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:3; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:21; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:14; (d) a VH domain comprising a human heavy chain framework having (i) FR1 comprising amino acid residues H3, D26, F27, E29, and Y30, (ii) FR3 comprising amino acid residues R66 and R94; and (e) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:22; (f) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:23; (g) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:8. and (h) a VL domain comprising a human light chain framework having (i) FR1 comprising amino acid residues I2 and Y3, (ii) FR2 comprising amino acid residues L46 and F49, and (iii) FR3 comprising amino acid residue E57, wherein the numbering of the VH and VL domains is according to the Kabat numbering system, and wherein the VH and VL domains comprise: (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 19 having 1 to 15, 1 to 10, or 1 to 5 amino acid substitutions; and (b) a variable light chain domain comprising the amino acid sequence of SEQ ID NO: 20 having 1 to 15, 1 to 10, or 1 to 5 amino acid substitutions.

[0115] In one aspect, the present invention provides an antibody that binds human VEGF-A and human ANG2 comprising a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 19. In certain aspects, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity comprises substitutions (e.g., conservative substitutions), insertions or deletions compared to the reference sequence, but an antibody that binds human VEGF-A and human ANG2 comprising the sequence retains the ability to bind to human VEGF-A and human ANG2. In certain aspects, a total of 1-10 amino acids are substituted, inserted and / or deleted in SEQ ID NO: 19. In certain aspects, the substitutions, insertions or deletions occur in the regions outside the CDRs (i.e., in the FRs). In a particular aspect, the VH comprises a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 3, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 21, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14.

[0116] In one aspect, the present invention provides an antibody that binds human VEGF-A and human ANG2 comprising a VL domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 20. In certain aspects, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity comprises substitutions (e.g., conservative substitutions), insertions or deletions compared to the reference sequence, but an antibody that binds human VEGF-A and human ANG2 comprising the sequence retains the ability to bind to human VEGF-A and human ANG2. In certain aspects, a total of 1-10 amino acids are substituted, inserted and / or deleted in SEQ ID NO: 20. In certain aspects, the substitutions, insertions or deletions occur in the regions outside the CDRs (i.e., in the FRs). In a particular aspect, the VL comprises (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 22, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 23, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8.

[0117] In another aspect, an antibody is provided that binds to human VEGF-A and human ANG2, the antibody comprising the VH sequence of any of the aspects provided above and the VL sequence of any of the aspects provided above. In one aspect, the antibody comprises the VH and VL sequences of SEQ ID NO: 19 and SEQ ID NO: 20, respectively, including post-translational modifications of these sequences.

[0118] In another aspect, an antibody that binds to human VEGF-A and human ANG2 is provided, the antibody comprising a heavy chain amino acid sequence of SEQ ID NO:24 and a light chain amino acid sequence of SEQ ID NO:25.

[0119] In another aspect, an antibody that binds to human VEGF-A and human ANG2 is provided, the antibody comprising a heavy chain amino acid sequence of SEQ ID NO:17 and a light chain amino acid sequence of SEQ ID NO:18.

[0120] In a further aspect of the invention, the antibody that binds human VEGF-A and human ANG2 according to any of the above aspects is a monoclonal antibody. In one aspect, the antibody that binds human VEGF-A and human ANG2 is an antibody fragment, such as an Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment. In another aspect, the antibody is a full-length antibody.

[0121] In further aspects, an antibody that binds human VEGF-A and human ANG2 according to any of the above aspects may incorporate any of the features, either alone or in combination, as described in Sections 1-5 below.

[0122] 1. Antibody affinity In certain embodiments, the antibodies provided herein have a dissociation constant (K D In preferred embodiments, the antibodies provided herein bind to VEGF-A with a dissociation constant (K) of ≦10 pM, and in preferred embodiments, ≦5 pM. DIn preferred embodiments, the antibodies provided herein bind to human VEGF-A with a dissociation constant (K) of ≦10 pM, and in preferred embodiments, ≦5 pM. D In preferred embodiments, the antibodies provided herein bind to human VEGFA-121 with a dissociation constant (K) of ≦10 pM, and in preferred embodiments, ≦5 pM. D ) and binds to human VEGFA-165.

[0123] In certain embodiments, antibodies that bind to ANG2 have a dissociation constant (K D ) of 1 nM or less, 0.1 nM or less, or 0.03 nM or less. In preferred embodiments, the antibodies provided herein have a dissociation constant (K D ) binds to human ANG2.

[0124] In one embodiment, K D is measured using a BIACORE® surface plasmon resonance assay.

[0125] In another embodiment, K D is measured using a KinExA assay. D is the K of VEGF-A binding D Detection of ANG2 binding K D is measured using the KinExA assay under the conditions as described below in the Materials and General Methods section for the detection of

[0126] For example, the K Dis measured in an assay using a KinExA 3200 instrument from Sapidyne Instruments (Boise, ID), where 30 μg of anti-VEGF antibody MAB293 (R&D) in 1 ml of PBS (pH 7.4) is used to coat PMMA beads with antigen according to the KinExA handbook protocol (Adsorption coating, Sapidyne). KinExA equilibrium assays are performed at room temperature using PBS (pH 7.4) with 0.01% BSA and 0.01% Tween 20 as running buffer, with samples and beads prepared in LowCross buffer (Candor Bioscience). The flow rate is 0.25 ml / min. A constant amount of VEGFA-121-His (50 pM and 500 pM in a second experiment) is titrated with the test antibody, and the equilibrated mixture is aspirated onto a column of beads coupled with anti-VEGF antibody (Mab293) in the KinExA system in a volume of 750 μl for 50 pM constant VEGF and in a volume of 125 μl for 500 pM constant VEGF. Detection of bound VEGFA-121 is performed using a secondary biotinylated anti-VEGF antibody (BAF293) at a concentration of 250 ng / ml, followed by injection of 250 ng / ml of streptavidin Alexa Fluor™ 647 conjugate in sample buffer. D is obtained from nonlinear regression analysis of the data using a one-site homogeneous binding model included in the KinExA software (version 4.0.11) using the "standard analysis" method. D Calculate the data points and approximate them to the theoretical K D Determine the 95% confidence interval by fitting the curve. The 95% confidence interval is K D Low and K D is given as high.

[0127] For example, the K Dis measured in an assay using a KinExA 3200 instrument from Sapidyne Instruments (Boise, ID), where 20 μg of anti-Ang2 antibody MAB098 (R&D) in 1 ml of PBS (pH 7.4) is used to coat PMMA beads with antigen according to the KinExA handbook protocol (Adsorption coating, Sapidyne). KinExA equilibrium assays are performed at room temperature using PBS (pH 7.4) with 0.01% BSA and 0.01% Tween 20 as running buffer, with samples and beads prepared in LowCross buffer (Candor Bioscience). The flow rate is 0.25 ml / min. A constant amount of Ang2-RBD-muFc (50 pM and 500 pM in a second experiment) was titrated with the test antibody and the equilibrated mixture was aspirated onto a column of beads coupled with anti-Ang2 antibody (MAB098) in a KinExA system in a volume of 750 μl for 50 pM static Ang2 and in a volume of 188 μl for 500 pM static Ang2. Detection of bound Ang2 is performed using a secondary biotinylated anti-Ang2 antibody (BAM0981) at a concentration of 250 ng / ml, followed by injection of 250 ng / ml streptavidin Alexa Fluor™ 647 conjugate in sample buffer. D is obtained from nonlinear regression analysis of the data using a one-site homogeneous binding model included in the KinExA software (version 4.0.11) using the "standard analysis" method. D Calculate the data points and approximate them to the theoretical K D Determine the 95% confidence interval by fitting the curve. The 95% confidence interval is K D Low and K D is given as high.

[0128] 2. Antibody Fragments In certain aspects, the antibodies provided herein are antibody fragments.

[0129] In one embodiment, the antibody fragment is a Fab, Fab', Fab'-SH, or F(ab')2 fragment, in particular a Fab fragment. Papain digestion of an intact antibody results in two identical antigen-binding fragments (so-called "Fab" fragments), each of which contains the variable domains of the heavy and light chains (VH and VL, respectively), as well as the constant domain of the light chain (CL) and the first constant domain of the heavy chain (CH1). Thus, a "Fab fragment" is an antibody fragment having a light chain containing the VL and CL domains, and a heavy chain fragment containing the VH and CH1 domains. A "Fab' fragment" differs from a Fab fragment by the addition of residues at the carboxy terminus of the CH1 domain, which contains one or more cysteines from the antibody hinge region. Fab'-SH is a Fab' fragment in which the cysteine ​​residues (multivalent) of the constant domains bear a free thiol group. Pepsin treatment results in a F(ab')2 fragment with two antigen-binding sites (two Fab fragments) and part of the Fc region. See US Pat. No. 5,869,046 for a description of Fab and F(ab')2 fragments which contain salvage receptor binding epitope residues and have increased in vivo half-lives.

[0130] Antibody fragments can be produced by a variety of techniques, including but not limited to, proteolytic digestion of intact antibodies and recombinant production by recombinant host cells (e.g., E. coli, CHO), as described herein.

[0131] In a preferred embodiment, the antibodies provided herein are Fab fragments.

[0132] In one embodiment, the VH domain of an antibody provided herein comprises a human VH3 framework.

[0133] In one embodiment, the VL domain of an antibody provided herein comprises a human Vkappa1 framework.

[0134] In one embodiment, the CL domain of an antibody provided herein is of the kappa isotype.

[0135] In one embodiment, the CH1 domain of an antibody provided herein is of the human IgG1 isotype.

[0136] In a preferred embodiment, the antibody provided herein is a Fab fragment comprising a CL domain of the kappa isotype and a CH1 domain of the human IgG1 isotype.

[0137] 3.Thermal stability The antibodies provided herein exhibit excellent thermal stability. In certain embodiments, the Fab fragments of the antibodies provided herein exhibit an onset aggregation temperature of 70° C. or higher. In certain embodiments, the Fab fragments of the antibodies provided herein exhibit a melting temperature of more than 80° C. as measured by dynamic light scattering.

[0138] 4. Multispecific antibodies In certain embodiments, the antibodies provided herein are multispecific antibodies. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites, i.e., different epitopes on different antigens or different epitopes on the same antigen. In certain embodiments, multispecific antibodies have three or more binding specificities.

[0139] Multispecific antibodies with three or more binding specificities, including the antibodies provided herein, can also be provided in an asymmetric manner with domain crossover in one or more binding arms of the same antigen specificity, i.e. by exchanging VH / VL domains (see, e.g., WO 2009 / 080252 and WO 2015 / 150447), CH1 / CL domains (see WO 2009 / 080253) or complete Fab arms (see WO 2009 / 080251, WO 2016 / 016299; see also Schaefer et al, PNAS, 108 (2011) 1187-1191, and Klein at al., MAbs 8 (2016) 1010-20). A variety of additional molecular formats of multispecific antibodies are known in the art and are included herein (see, e.g., Spiess et al., Mol Immunol 67 (2015) 95-106).

[0140] 5. Antibody Variants In certain aspects, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to change the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, so long as the final construct possesses the desired characteristics (e.g., antigen binding).

[0141] In certain embodiments, antibody variants with one or more amino acid substitutions are provided. Sites of interest for substitution mutagenesis include CDRs and FRs. Conservative substitutions are shown in the table below under the heading of "preferred substitutions". More substantial changes are provided in Table 1 under the heading of "exemplary substitutions" and as further described below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into the antibody of interest and the products are screened for the desired activity, for example, retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0142] [Table 2]

[0143] Amino acids can be classified according to common side chain properties.

[0144] (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues affecting chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0145] Non-conservative substitutions will involve exchanging a member of one of these classes for a member of another class.

[0146] One type of substitution variant involves substituting one or more CDR residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant selected for further study will have a modification (e.g., an improvement) in a particular biological property (e.g., increased affinity, reduced immunogenicity) compared to the parent antibody and / or will substantially retain a particular biological property of the parent antibody. An exemplary substitution variant is an affinity matured antibody, which may be conveniently generated using, for example, phage display-based affinity maturation techniques as described herein. Briefly, one or more CDR residues are mutated and the variant antibodies displayed on phage are screened for a particular biological activity (e.g., binding affinity).

[0147] In certain aspects, substitutions, insertions, or deletions may occur within one or more CDRs, so long as such modifications do not substantially reduce the ability of the antibody to bind to the antigen. For example, conservative changes (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in the CDRs. Such modifications may, for example, be outside the antigen contact residues in the CDRs. In the above specific variant VH and VL sequences, each CDR is either unmodified or has no more than one, two, or three amino acid substitutions.

[0148] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis" as described in Cunningham and Wells (1989) Science, 244:1081-1085. In this method, residues or groups of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and substituted with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the antibody-antigen interaction is affected. Further substitutions may be introduced at amino acid positions that show functional sensitivity to the initial substitution. Alternatively, or additionally, a crystal structure of an antigen-antibody complex may be used to identify contact points between the antibody and the antigen. Such contact and adjacent residues may be targeted as candidates for substitution or removed. Mutants may be screened to determine whether they have the desired properties.

[0149] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of antibody molecules include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., for ADEPT (antibody-directed enzyme prodrug therapy) or a polypeptide which increases the serum half-life of the antibody.

[0150] a) Glycosylation mutants In certain aspects, the antibodies provided herein are altered to increase or decrease the extent of glycosylation of the antibody. Adding or deleting glycosylation sites to an antibody can be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

[0151] If the antibody contains an Fc region, the oligosaccharides attached thereto can be altered. Natural antibodies produced by mammalian cells typically contain branched, biantennary oligosaccharides that are generally attached to Asn297 of the CH2 domain of the Fc region by an N-linkage. See, for example, Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharides can include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to the GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharides in the antibodies of the invention can be made to generate antibody variants with specific improved properties.

[0152] In one embodiment, antibody variants are provided that have nonfucosylated oligosaccharides, i.e., oligosaccharide structures that lack fucose linkages (direct or indirect) to the Fc region. Such nonfucosylated oligosaccharides (also referred to as "afucosylated" oligosaccharides) are in particular N-linked oligosaccharides that lack a fucose residue attached to the first GlcNAc at the stem of the biantennary oligosaccharide structure. In one embodiment, antibody variants are provided that have an increased proportion of nonfucosylated oligosaccharides in the Fc region compared to the endogenous or parent antibody. For example, the proportion of nonfucosylated oligosaccharides may be at least about 20%, at least about 40%, at least about 60%, at least about 80%, or even about 100% (i.e., no fucosylated oligosaccharides are present). The percentage of nonfucosylated oligosaccharides is the (average) amount of oligosaccharides lacking a fucose residue relative to the sum of all oligosaccharides (e.g. complex, hybrid and high mannose structures) attached to Asn297, as determined, for example, by MALDI-TOF mass spectrometry as described in WO 2006 / 082515. Asn297 refers to an asparagine residue located at about position 297 of the Fc region (EU numbering of Fc region residues); however, Asn297 may also be located upstream or downstream of position 297, i.e., about ±3 amino acids between positions 294 and 300, due to minor sequence variations of the antibody. Such antibodies having an increased percentage of nonfucosylated oligosaccharides in the Fc region may have improved FcγRIIIa receptor binding and / or improved effector function, in particular improved ADCC function. See, e.g., U.S. Patent Application Publication Nos. 2003 / 0157108; 2004 / 0093621.

[0153] Examples of cell lines capable of producing antibodies with reduced fucosylation include Lec13 CHO cells, which are deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application Publication No. 2003 / 0157108; and WO 2004 / 056312, especially Example 11), and knockout cell lines, such as FUT8 knockout CHO cells of the alpha-1,6-fucosyltransferase gene (e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614-622 (2004); Kanda, Y. et al. al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO 2003 / 085107), or cells with reduced or abolished activity of GDP-fucose synthesis or transporter proteins (see, e.g., U.S. Patent Application Publication Nos. 2004259150, 2005031613, 2004132140, and 2004110282).

[0154] In a further embodiment, antibody variants are provided with bisected oligosaccharides, e.g., biantennary oligosaccharides attached to the Fc region of the antibody are bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function, as described above. Examples of such antibody variants are described, for example, in Umana et al., Nat Biotechnol 17, 176-180 (1999); Ferrara et al., Biotechn Bioeng 93, 851-861 (2006); WO 99 / 54342, WO 2004 / 065540, WO 2003 / 011878.

[0155] Also provided are antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in International Publication Nos. WO1997 / 30087, WO1998 / 58964, and WO1999 / 22764.

[0156] b) Fc Region Mutants In certain aspects, one or more amino acid modifications may be introduced into the Fc region of an antibody presented herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) containing an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0157] In certain aspects, the present invention contemplates antibody variants that possess some, but not all, effector functions, making them desirable candidates for applications where the in vivo half-life of the antibody is important, but where certain effector functions (e.g., complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC)) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / absent CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to confirm that the antibody lacks FcγR binding (and thus likely lacks ADCC activity) but retains FcRn binding ability. NK cells, the primary cells for mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. Expression of FcR on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see, Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be employed (see, e.g., ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc. Mountain View, Calif.), and CytoTox 96® Non-Radioactive Cytotoxicity Test Method (Promega, Madison, Wis.). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.Alternatively, or in addition, the desired ADCC activity can be assessed in vitro, for example in an animal model as disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay can also be performed to confirm that the antibody is unable to bind C1q and lacks CDC activity. See, for example, the C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)), and FcRn binding and in vitro clearance / half-life determinations can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006); WO2013 / 120929A1).

[0158] Antibodies with reduced effector function include those with substitutions at one or more of residues 238, 265, 269, 270, 297, 327, and 329 in the Fc region (U.S. Patent No. 6,737,056). Such Fc variants include Fc variants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variant in which residues 265 and 297 have been substituted with alanine (U.S. Patent No. 7,332,581).

[0159] Certain antibody variants with improved or diminished binding to FcRs have been described (see, e.g., U.S. Pat. No. 6,737,056; WO 2004 / 056312; and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001)). In certain aspects, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, e.g., substitutions at positions 298, 333, and / or 334 (EU numbering of residues) of the Fc region.

[0160] In certain embodiments, the antibody variant comprises an Fc region having one or more amino acid substitutions that reduce FcγR binding, e.g., Fc region positions 234 and 235 (EU numbering residues). In one embodiment, the substitutions are L234A and L235A (LALA). In certain embodiments, the antibody variant further comprises D265A and / or P329G in an Fc region derived from a human IgG1 Fc region. In one embodiment, the substitutions are L234A, L235A, and P329G (LALA-PG) in an Fc region derived from a human IgG1 Fc region. (See, e.g., WO 2012 / 130831.) In another embodiment, the substitutions are L234A, L235A, and D265A (LALA-DA) in an Fc region derived from a human IgG1 Fc region.

[0161] In some embodiments, changes are made within the Fc region that result in altered (i.e., either improved or decreased) C1q binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0162] Antibodies with increased half-lives and improved binding to fetal Fc receptors (FcRn) that play a role in transferring maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) have been described in U.S. Patent Application Publication No. 2005 / 0014934 (Hinton et al.). These antibodies comprise an Fc region having one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include those having a substitution at one or more of Fc region residues: 238, 252, 254, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, e.g., a substitution at Fc region residue 434 (see, e.g., U.S. Patent No. 7,371,826; Dall'Acqua, WF, et al. J. Biol. Chem. 281 (2006) 23514-23524).

[0163] The Fc region residues critical for mouse Fc mouse FcRn interaction have been identified by site-directed mutagenesis (see, e.g., Dall'Acqua, WF, et al. J. Immunol 169 (2002) 5171-5180). Residues I253, H310, H433, N434, and H435 (EU index numbering) are involved in the interaction (Medesan, C., et al., Eur. J. Immunol. 26 (1996) 2533; Firan, M., et al., Int. Immunol. 13 (2001) 993; Kim, JK, et al., Eur. J. Immunol. 24 (1994) 542). Residues I253, H310, and H435 were found to be critical for the interaction of human Fc with mouse FcRn (Kim, JK, et al., Eur. J. Immunol. 29 (1999) 2819). Studies of the human Fc-human FcRn complex have shown that residues I253, S254, H435, and Y436 are critical for the interaction (Firan, M., et al., Int. Immunol. 13 (2001) 993; Shields, RL, et al., J. Biol. Chem. 276 (2001) 6591-6604). In Yeung, YA, et al. (J. Immunol. 182 (2009) 7667-7671), various mutations of residues 248-259, 301-317, 376-382, and 424-437 have been reported and investigated.

[0164] In certain embodiments, the antibody variant comprises an Fc region having one or more amino acid substitutions that reduce FcRn binding, e.g., mutations at Fc region positions 253, and / or 310, and / or 435 (EU numbering residues). In certain embodiments, the antibody variant comprises an Fc region having amino acid substitutions at positions 253, 310, and 435. In one embodiment, the substitutions are I253A, H310A, and H435A in the Fc region derived from a human IgG1 Fc region. See, e.g., Grevys, A., et al., J. Immunol. 194 (2015) 5497-5508.

[0165] In certain aspects, the antibody variant comprises an Fc region with one or more amino acid substitutions that reduce FcRn binding, e.g., mutations at positions 310, and / or 433, and / or 436 (EU numbering residues) of the Fc region. In certain aspects, the antibody variant comprises an Fc region with amino acid substitutions at positions 310, 433, and 436. In one aspect, the substitutions are H310A, H433A, and Y436A in the Fc region derived from a human IgG1 Fc region. (See, e.g., WO 2014 / 177460.)

[0166] In certain embodiments, the antibody variant comprises an Fc region having one or more amino acid substitutions that increase FcRn binding, e.g., mutations at Fc region positions 252, and / or 254, and / or 256 (residues in EU numbering). In certain embodiments, the antibody variant comprises an Fc region having amino acid substitutions at positions 252, 254, and 256. In one embodiment, the substitutions are M252Y, S254T, and T256E in the Fc region derived from a human IgG1 Fc region. See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351 for other examples of Fc region variants.

[0167] The C-terminus of the heavy chain of an antibody as reported herein may be a complete C-terminus ending in amino acid residue PGK. The C-terminus of the heavy chain may be a shortened C-terminus in which one or two of the C-terminal amino acid residues are removed. In one preferred embodiment, the C-terminus of the heavy chain is a shortened C-terminus PG. In one embodiment of all embodiments reported herein, an antibody comprising a heavy chain comprising a C-terminal CH3 domain as specified herein comprises a C-terminal glycine-lysine dipeptide (G446 and K447, amino acid positions of the EU index numbering). In one embodiment of all embodiments reported herein, an antibody comprising a heavy chain comprising a C-terminal CH3 domain as specified herein comprises a C-terminal glycine residue (G446, amino acid position of the EU index numbering).

[0168] c) Cysteine ​​Engineered Antibody Variants In certain aspects, cysteine ​​engineered antibodies, e.g., THIOMABs, in which one or more residues of the antibody are replaced with a cysteine ​​residue. TM In certain aspects, the substituted residues occur at accessible sites on the antibody. By replacing these residues with cysteine, reactive thiol groups are thereby placed at accessible sites on the antibody, which can be used to conjugate the antibody to other sites, such as drug moieties or linker-drug moieties, to create immunoconjugates, as further described herein. Cysteine ​​engineered antibodies can be generated, for example, as described in U.S. Pat. Nos. 7,521,541, 8,30,930, 7,855,275, 9,000,130, or WO2016040856.

[0169] 6. Immunoconjugates The invention also provides immunoconjugates comprising an antibody provided herein conjugated (chemically linked) to one or more agents, which in one embodiment are a cytotoxic agent, a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., a protein toxin, an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof), or a radioactive isotope.

[0170] In one embodiment, the present invention provides an immunoconjugate comprising an antibody provided herein conjugated to a polymer. The term "polymer" as used herein includes chemical polymers and protein polymers. In one embodiment, the immunoconjugate comprises an antibody provided herein conjugated to an extended recombinant polypeptide (XTEN). "Extended recombinant polypeptides" are known in the art and are disclosed, for example, in US Patent Publication No. 20190083577. In one embodiment, the immunoconjugate comprises an XTEN that (a) comprises a sequence selected from GGSPAGSCTSP, GASASCAPSTG, TAEAAGCGTAEAA, and GPEPTCPAPSG, (b) is 36-3000 L-amino acid residues in length, and / or (c) comprises an XTEN in which the sum of glycine (G), alanine (A), serine (S), threonine (T), glutamate (E), and proline (P) residues constitute more than 90% of the total amino acid residues of the XTEN.

[0171] B. Recombinant Methods and Compositions Antibodies can be produced using recombinant methods and compositions, for example, as described in U.S. Patent No. 4,816,567. For these methods, one or more isolated nucleic acids encoding the antibody are provided.

[0172] In one aspect, an isolated nucleic acid encoding an antibody of the invention is provided.

[0173] In one aspect, a method is provided for making an antibody that binds to human VEGF-A and human ANG2, the method comprising culturing a host cell comprising nucleic acid encoding the antibody under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture).

[0174] For recombinant production of antibodies that bind human VEGF-A and human ANG2, for example, nucleic acids encoding the above-mentioned antibodies are isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids can be readily isolated and sequenced using standard procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of the antibody).

[0175] Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies may be produced in bacteria, particularly if glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, for example, US 5,648,237, US 5,789,199 and US 5,840,523. (See also Charlton, KA, In: Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2003), pp. 245-254, which describes the expression of antibody fragments in E. coli.) After expression, the antibody may be isolated from the bacterial cell paste in appropriate fractions and may be further purified. In one embodiment, the host cell is an E. coli cell.

[0176] Vertebrate cells may also be used as hosts. For example, mammalian cell lines adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed with SV40 (COS-7), human embryonic kidney lines (e.g., 293 cells or 293T cells as described in Graham, FL et al., J. Gen Virol. 36 (1977) 59-74, baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4 cells as described in Mather, JP, Biol. Reprod. 23 (1980) 243-252), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor cells (MMT 060562), TRI cells (e.g., Mather, JP et al., Annals NY Acad. Sci. 383 (1982) 44-68), MRC5 cells and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77 (1980) 4216-4220), and myeloma cell lines, such as Y0, NS0 and Sp2 / 0. For a review of certain mammalian host cells suitable for antibody production, see, for example, Yazaki, P. and Wu, AM, Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268.

[0177] In one aspect, the host cell is a eukaryotic cell, such as a Chinese Hamster Ovary (CHO) cell or a lymphocytic cell (e.g., Y0, NS0, Sp20 cell). In one preferred embodiment, the host cell is a CHO cell. Production of the antibody of the present invention in a CHO cell can improve the injectability of the antibody.

[0178] C. Pharmaceutical Compositions In a further aspect, a pharmaceutical composition is provided comprising any of the antibodies provided herein, e.g., for use in any of the following therapeutic methods. In one aspect, the pharmaceutical composition comprises any of the antibodies provided herein and a pharma- ceutically acceptable carrier. In another aspect, the pharmaceutical composition comprises any of the antibodies provided herein and at least one additional therapeutic agent, e.g., as described below.

[0179] Pharmaceutical compositions of antibodies that bind human VEGF-A and human ANG2 described herein are prepared by mixing such antibodies having the desired purity with one or more optionally present pharma- ceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)) in the form of a lyophilized composition or aqueous solution. Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed and include buffers such as histidine, phosphate, citrate, acetate and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polysaccharides; and the like. Examples of suitable pharmacopoeitic carriers include, but are not limited to, peptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes), and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmacopoeitic carriers herein further include intercalating drug dispersing agents, such as soluble neutral active hyaluronidase glycoproteins (sHASEGPs), e.g., human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Halozyme, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Application Publication Nos. 2005 / 0260186 and 2006 / 0104968.In one embodiment, a sHASEGP is combined with one or more additional glycosaminoglycanases (eg, chondroitinases).

[0180] Exemplary lyophilized antibody compositions are described in U.S. Patent No. 6,267,958. Aqueous antibody compositions include those described in U.S. Patent No. 6,171,586 and WO2006 / 044908, the latter compositions including a histidine-acetate buffer.

[0181] The pharmaceutical compositions herein may also contain multiple active ingredients as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Such active ingredients are suitably present in combination in amounts effective for the purpose intended.

[0182] The active ingredient may be encapsulated in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively), colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th Edition, Osol, A. Ed. (1980).

[0183] Pharmaceutical compositions can be prepared for sustained release. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

[0184] Pharmaceutical compositions used for in vivo administration are generally sterile. Sterilization may be readily accomplished, for example, by filtration through sterile filtration membranes.

[0185] D. Treatment Methods and Routes of Administration Any of the antibodies provided herein that bind to human VEGF-A and human ANG2 can be used in the therapeutic methods.

[0186] In one aspect, an antibody that binds human VEGF-A and human ANG2 is provided for use as a medicament. In a further aspect, an antibody that binds human VEGF-A and human ANG2 is provided for use in treating vascular disease. In certain aspects, an antibody that binds human VEGF-A and human ANG2 is provided for use in a method of treatment. In certain aspects, the invention provides an antibody that binds human VEGF-A and human ANG2 for use in a method of treating an individual with vascular disease, comprising administering to the individual an effective amount of an antibody that binds human VEGF-A and human ANG2. In one such aspect, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent (e.g., one, two, three, four, five, or six additional therapeutic agents), e.g., as described below. In a further aspect, the invention provides an antibody that binds human VEGF-A and human ANG2 for use in inhibiting angiogenesis. In certain aspects, the invention provides an antibody that binds human VEGF-A and human ANG2 for use in a method of inhibiting angiogenesis in an individual comprising administering to the individual an effective amount of an antibody that binds human VEGF-A and human ANG2 to inhibit angiogenesis. An "individual" according to any of the above aspects is preferably a human.

[0187] In a further aspect, an antibody that binds human VEGF-A and human ANG2 is provided for use in treating an ocular disease. In one embodiment, the ocular disease is AMD (in one embodiment, wet AMD, dry AMD, intermediate AMD, advanced AMD, and geographic atrophy (GA)), macular degeneration, macular edema, DME (in one embodiment, focal, non-central DME, and diffuse, centrally involved DME), retinopathy, diabetic retinopathy (DR) (in one embodiment, proliferative DR (PDR), non-proliferative DR (NPDR), and high altitude DR), other ischemia-related retinopathies, ROP, retinopathies ... Retinal vein occlusion (RVO) (in one embodiment, central (CRVO) and branched (BRVO) morphology), CNV (in one embodiment, myopic CNV), corneal neovascularization, diseases associated with corneal neovascularization, retinal neovascularization, diseases associated with retinal / choroidal neovascularization, central serous retinopathy (CSR), pathological myopia, von Hippel-Lindau disease, ocular histoplasmosis, FEVR, Coats' disease, Norrie's disease, osteoporotic pseudoglioma syndrome (OPPG) ), retinal abnormalities associated with glaucoma, subconjunctival hemorrhage, rubeosis, ocular neovascular disease, neovascular glaucoma, retinitis pigmentosa (RP), hypertensive retinopathy, retinal angiomatous proliferation, macular vascular ectasia, iris neovascularization, intraocular neovascularization, retinal degeneration, cystoid macular edema (CME), vasculitis, papilledema, retinal inflammation including, but not limited to: CMV retinitis, ocular melanoma, retinoblastoma, conjunctivitis (in one embodiment, infectious conjunctivitis and non-infectious (in one embodiment, The ocular disease is selected from: allergic (allergic) conjunctivitis), Leber's congenital black circle (also known as Leber's congenital black circle or LCA), uveitis (including infectious and non-infectious uveitis), choroiditis (in one embodiment, multifocal choroiditis), ocular histoplasmosis, blepharitis, dry eye, traumatic eye injury, Sjogren's disease, and other ocular diseases in which the disease or disorders are associated with ocular neovascularization, vascular leakage, and / or retinal edema or retinal atrophy. In one embodiment, the ocular disease is selected from: AMD (in one embodiment, wet AMD, dry AMD, intermediate AMD, advanced AMD, and geographic atrophy (GA)), macular degeneration, macular edema, DME (in one embodiment, focal, non-central DME, and diffuse, centrally involved DME), retinopathy, diabetic retinopathy (DR) (in one embodiment, proliferative DR (PDR), non-proliferative DR (NPDR), and high altitude DR).

[0188] In a further aspect, the invention provides for the use of an antibody that binds human VEGF-A and human ANG2 in the manufacture or preparation of a medicament. In one aspect, the medicament is for the treatment of a vascular disease. In a further aspect, the medicament is for use in a method of treating a vascular disease comprising administering an effective amount of the medicament to an individual having the vascular disease. In one such aspect, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, such as those described below.

[0189] In one embodiment, the medicament is for treating an ocular disease. In a further embodiment, the medicament is for use in a method for treating an ocular disease, comprising administering an effective amount of the medicament to an individual having the ocular disease. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, such as those described below.

[0190] In a further aspect, the invention provides a method of treating a vascular disease. In one aspect, the method comprises administering to an individual having such a vascular disease an effective amount of an antibody that binds human VEGF-A and human ANG2. In one such aspect, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, as described below.

[0191] In a further aspect, the invention provides a method of treating an ocular disease. In one aspect, the method comprises administering to an individual having such an ocular disease an effective amount of an antibody that binds human VEGF-A and human ANG2. In one such aspect, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, as described below.

[0192] An "individual" according to any of the above embodiments may be a human.

[0193] In a further aspect, the invention provides a pharmaceutical composition comprising any of the antibodies that bind human VEGF-A and human ANG2 provided herein, e.g., for use in any of the above methods of treatment. In one aspect, the pharmaceutical composition comprises any of the antibodies that bind human VEGF-A and human ANG2 provided herein and a pharma- ceutically acceptable carrier. In another aspect, the pharmaceutical composition comprises any of the antibodies that bind human VEGF-A and human ANG2 provided herein and at least one additional therapeutic agent, e.g., as described below.

[0194] The antibodies of the invention may be administered by intravitreal administration (e.g., intravitreal injection) or using a port delivery device. In one embodiment, the antibodies of the invention are administered using a port delivery device for a period of 6 months or more, in one embodiment 8 months or more, in one embodiment 9 months or more, and in one embodiment 12 months or more before the port delivery device is refilled. In one embodiment, the antibodies of the invention are administered using a port delivery device, and the antibody is applied to the port delivery device at a concentration of 150 mg / ml or more, and in one embodiment 200 mg / ml or more.

[0195] The antibodies of the invention can be administered alone or can be used in combination therapy, for example, the combination therapy includes administering an antibody of the invention and at least one additional therapeutic agent (e.g., 1, 2, 3, 4, 5, or 6 additional therapeutic agents).

[0196] In certain embodiments according to (or applied to) any of the above embodiments, the ocular disease is an intraocular neovascular disease selected from the group consisting of proliferative retinopathy, choroidal neovascularization (CNV), age-related macular degeneration (AMD), diabetic and other ischemia-related retinopathies, diabetic macular edema, pathological myopia, von Hippel-Lindau disease, ocular histoplasmosis, retinal vein occlusion (RVO), including CRVO and BRVO, corneal neovascularization, retinal neovascularization, and retinopathy of prematurity (ROP).

[0197] In some examples, the antibodies that bind human VEGF-A and human ANG2 provided herein may be administered in combination with at least one additional therapeutic agent for treating an ocular disorder, e.g., an ocular disorder described herein (e.g., AMD (e.g., wet AMD), DME, DR, RVO, or GA).

[0198] Any suitable AMD therapeutic can be administered as an additional therapeutic agent in combination with the antibodies that bind human VEGF-A and human ANG2 provided herein to treat an ocular disorder (e.g., AMD, DME, DR, RVO, or GA), including, but not limited to, a VEGF antagonist, such as an anti-VEGF antibody (e.g., LUCENTIS® (ranibizumab), RTH-258 (formerly ESB-1008, an anti-VEGF single chain antibody fragment; Novartis), or a bispecific anti-VEGF antibody (e.g., an anti-VEGF / anti-angiopoietin 2 bispecific antibody, e.g., faricimab; Roche)), a soluble VEGF receptor fusion protein (e.g., EYLEA® (aflibercept)), an anti-VEGF DARPin® (e.g., abicipar pegol; Molecular Partners), AG / Allergan), or anti-VEGF aptamers (e.g., MACUGEN® (pegaptanib sodium); platelet-derived growth factor (PDGF) antagonists, such as anti-PDGF antibodies, anti-PDGFR antibodies (e.g., REGN2176-3), anti-PDGF-BB pegylated aptamers (e.g., FOVISTA®; Ophthotech / Novartis), soluble PDGFR receptor fusion proteins, or dual PDGF / VEGF antagonists (e.g., small molecule inhibitors (e.g., DE-120 (Santen) or X-82 (TyrogeneX)) or bispecific anti-PDGF / anti-VEGF antibodies)); VIS in combination with photodynamic therapy UDYNE® (verteporfin); antioxidants; complement system antagonists, such as complement factor C5 antagonists (e.g., small molecule inhibitors (e.g., ARC-1905; Opthotech) or anti-C5 antibodies (e.g., LFG-316; Novartis), properdin antagonists (e.g., anti-properdin antibodies, e.g., CLG-561; Alcon), or complement factor D antagonists (e.g., anti-complement factor D antibodies, e.g., lampalizumab; Roche)); C3 blocking peptides (e.g., APL-2, Appellis); visual cycle regulators (e.g., emixustat hydrochloride); squalamine (e.g., OHR-102; Ohr Pharmaceutical);Vitamin and mineral supplements (e.g., those described in Age-Related Eye Disease Study 1 (AREDS1; zinc and / or antioxidants) and Study 2 (AREDS2; zinc, antioxidants, lutein, zeaxanthin, and / or omega-3 fatty acids); cell-based therapies, such as NT-501 (Renexus); PH-05206388 (Pfizer), huCNS-SC cell transplantation (StemCells), CNTO-2476 (umbilical cord stem cell line; Janssen), OpRegen (suspension of RPE cells; Cell Cure Neurosciences), or MA09-hRPE cell transplantation (Ocata Therapeutics); tissue factor antagonists (e.g., hI-con1; Iconic Therapeutics); alpha-adrenergic receptor agonists (e.g., brimonidine tartrate; Allergan); peptide vaccines (e.g., S-646240; Shionogi); amyloid beta antagonists (e.g., anti-beta amyloid monoclonal antibodies, e.g., GSK-933776); S1P antagonists (e.g., anti-S1P antibodies, e.g., iSONEP™; Lpath Inc); ROBO4 antagonists (e.g., anti-ROBO4 antibodies, e.g., DS-7080a; Daiichi Sankyo); lentiviral vectors expressing endostatin and angiostatin (e.g., RetinoStat); and any combination thereof. In some examples, the AMD therapeutics (including any of the aforementioned AMD therapeutics) can be co-formulated. For example, the anti-PDGFR antibody REGN2176-3 can be co-formulated with aflibercept (EYLEA®). In some examples, such co-formulations can be administered in combination with antibodies that bind human VEGF and human ANG2 of the present invention. In some examples, the ocular disorder is AMD (e.g., wet AMD);

[0199] Any suitable DME and / or DR therapeutic, including but not limited to, a VEGF antagonist (e.g., LUCENTIS® or EYLEA®), a corticosteroid (e.g., a corticosteroid implant (e.g., OZURDEX® (dexamethasone intravitreal implant) or ILUVIEN® (fluocinolone acetonide intravitreal implant)) or a corticosteroid formulated for administration by intravitreal injection (e.g., triamcinolone acetonide)), or a combination thereof, can be administered in combination with an antibody that binds human VEGF and human ANG2 of the present invention for the treatment of an ocular disorder (e.g., AMD, DME, DR, RVO, or GA). In some examples, the ocular disorder is DME and / or DR.

[0200] Antibodies that bind human VEGF and human ANG2 provided herein are useful in treating retinal and retinal diseases, including those that involve the treatment of retinal and retinal malformations, such as, for example, laser photocoagulation (e.g., panretinal photocoagulation (PRP)), drusen laser irradiation, macular hole surgery, macular translocation surgery, implantable miniscopes, PHI motion angiography (also known as microlaser therapy and feeder vessel procedures), proton therapy, microstimulation therapy, retinal detachment and vitreous surgery, scleral buckle, submacular surgery, transpapillary thermotherapy, photosystem I therapy, use of RNA interference (RNAi), extracorporeal rheopheresis (also known as membrane fractional filtration and rheotherapy), microchip transplantation, stem cell therapy, gene replacement therapy, ribozyme gene therapy (including hypoxia response element gene therapy, Oxford Biomedica; Lentipak, Genetix; and PDEF gene therapy, GenVec), photoreceptor / retinal cell transplantation (including transplantable retinal epithelial cells, Diacrin, Inc.; retinal cell transplantation, e.g., Astellas Pharma US, Inc., ReNeuron, CHA The present invention may be administered in combination with therapeutic or surgical procedures for the treatment of ocular disorders (e.g., AMD, DME, DR, RVO, or GA), including ocular therapy, ocular biotech), paracentesis, and combinations thereof.

[0201] Such combination therapy as described above encompasses combined administration (wherein two or more therapeutic agents are included in the same or separate formulations) and separate administration, where administration of an antibody of the invention that binds human VEGF and human ANG2 can occur prior to, simultaneously with, and / or following administration of the additional therapeutic agent or agent. In one embodiment, administration of an antibody of the invention that binds human VEGF and human ANG2 and administration of the additional therapeutic agent occur within about 1, 2, 3, 4, or 5 months, or within about 1, 2, or 3 weeks, or within about 1, 2, 3, 4, 5, or 6 days of each other.

[0202] The antibodies of the invention (and any additional therapeutic agents) can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, as well as intralesional administration if desired for localized treatment. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, for example, injections such as intravenous or subcutaneous injections, depending in part on whether administration is brief or chronic. A variety of dosing schedules are contemplated herein, including, but not limited to, single or multiple doses over various time points, bolus administration, and pulse infusion.

[0203] The antibodies of the invention will be formulated, administered, and administered in a manner consistent with good medical practice. Factors to consider in this regard include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the administration schedule, and other factors known to medical practitioners. The antibodies need not be, but are optionally, formulated with one or more agents currently used to prevent or treat the disease in question. The effective amount of such other agents will depend on the amount of antibody present in the pharmaceutical composition, the type of disease or treatment, and other factors discussed above. These will generally be used in the same dosages and by any route of administration as described herein, or about 1-99% of the dosages described herein, or in any dosage and by any route empirically / clinically determined to be appropriate.

[0204] For the prevention or treatment of disease, the appropriate dosage of the antibody of the invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease being treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for prophylactic or therapeutic purposes, previous therapy, the patient's medical history and response to the antibody, and the discretion of the attending physician. The antibody of the invention is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg to 10 mg / kg) of the antibody may be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations or by continuous infusion. A typical daily dosage may range from about 1 μg / kg to 100 mg / kg, depending on the factors mentioned above. In repeated administrations over several days or more, depending on the condition, treatment is usually continued until a desired suppression of disease symptoms occurs. One exemplary dosage of the antibody would be in the range of about 0.05 mg / kg to about 10 mg / kg. Thus, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, or 10 mg / kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, for example every week or every three weeks (e.g., such that the patient receives from about 2 to about 20, or for example about 6 doses of the antibody). An initial larger dose, followed by one or more smaller doses, may be administered. The progress of this therapy is easily monitored by conventional techniques and assays.

[0205] E. Manufactured articles In another aspect of the invention, an article of manufacture is provided that includes materials useful for the treatment, prevention, and / or diagnosis of the above-mentioned disorders. The article of manufacture includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, vials, syringes, and the like. The container may be formed from a variety of materials, such as glass or plastic. The container holds a composition to be used alone or in combination with another composition effective to treat, prevent, and / or diagnose a condition, and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an antibody of the invention. The label or package insert indicates that the composition is used to treat the condition of choice.

[0206] Additionally, the article of manufacture includes (a) a first container containing a composition, the composition comprising an antibody of the invention, and (b) a second container containing a composition, the composition further comprising a cytopathic or other therapeutic agent. The article of manufacture in this aspect of the invention may further include a package insert indicating that the composition can be used to treat a particular condition. Alternatively, or in addition, the article of manufacture may further comprise a second (or third) container containing a pharma- ceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, such as other buffers, diluents, filters, needles, syringes, etc.

[0207] F. Equipment The antibodies of the invention may be administered to the eye using an ocular implant, in one embodiment using a port delivery device.

[0208] A port delivery device is an implantable, rechargeable device that can release a therapeutic agent (e.g., an antibody of the invention) over a period of several months (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or more). Exemplary port delivery devices that may be used include those from ForSight Labs, LLC and / or ForSight VISION4, as described, for example, in International Patent Application Publication Nos. WO 2010 / 088548, WO 2015 / 085234, WO 2013 / 116061, WO 2012 / 019176, WO 2013 / 040247, and WO 2012 / 019047, which are incorporated by reference in their entireties.

[0209] For example, the present invention provides a port delivery device comprising a reservoir containing any of the antibodies described herein. The port delivery device may further comprise a proximal region, a tubular body coupled to the proximal region in fluid communication with the reservoir, and one or more outlets in fluid communication with the reservoir and configured to release the composition into the eye. The tubular body may have an outer diameter configured to be inserted through an incision or opening in the eye of about 0.5 mm or less. The device may be about 1 mm to about 15 mm in length (e.g., about 1 mm, about 2 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 9 mm, about 11 mm, about 13 mm, or about 15 mm in length). The reservoir may have any suitable volume. In some cases, the reservoir has a volume of about 1 μl to about 100 μl (e.g., about 1 μl, about 5 μl, about 10 μl, about 20 μl, about 50 μl, about 75 μl, or about 100 μl). The device or its components may be made of any suitable material, for example polyimide.

[0210] In some examples, the port delivery device comprises a reservoir containing any of the antibodies described herein and one or more additional compounds.

[0211] In some examples, the port delivery device comprises any of the antibodies or antibody conjugates described herein and an additional VEGF antagonist.

[0212] 3. Specific Embodiments of the Invention Specific embodiments of the present invention are listed below.

[0213] 1. An antibody that binds to human VEGF-A and human ANG2, comprising a VEGF-A paratope and an ANG2 paratope within a cognate pair of a variable light domain (VL domain) and a variable heavy domain (VH domain), wherein the VEGF-A paratope comprises amino acid residues from CDR-H2, CDR-L1 and CDR-L3 of the antibody, and the ANG2 paratope comprises amino acid residues from CDR-H1, CDR-H3 and CDR-L2 of the antibody.

[0214] 2. An antibody that binds to human VEGF-A and human ANG2, which comprises a VEGF-A paratope and an ANG2 paratope within a single cognate pair of a variable light domain (VL domain) and a variable heavy domain (VH domain), and the pair of variable light domain and variable heavy domain simultaneously binds to human VEGF-A and human ANG2.

[0215] 3. An antibody that binds to human VEGF-A and human ANG2, comprising a VEGF-A paratope and an ANG2 paratope within a cognate pair of a variable light domain (VL domain) and a variable heavy domain (VH domain), and which binds to the same epitope on human VEGF-A and the same epitope on human ANG2 as an antibody having a variable heavy domain of SEQ ID NO: 19 and a variable light domain of SEQ ID NO: 20.

[0216] 4. An antibody that binds to human VEGF-A and human ANG2, comprising a VEGF-A paratope and an ANG2 paratope within one cognate pair of a variable light domain (VL domain) and a variable heavy domain (VH domain); the VEGF-A paratope comprises amino acid residues derived from CDR-H2, CDR-L1 and CDR-L3 of an antibody, and the ANG2 paratope comprises amino acid residues derived from CDR-H1, CDR-H3 and CDR-L2 of an antibody; and / or the pair of variable light and heavy domains simultaneously binds human VEGF-A and human ANG2; and / or binds to the same epitope on human VEGF-A and the same epitope on human ANG2 as an antibody having a variable heavy chain domain of SEQ ID NO: 19 and a variable light chain domain of SEQ ID NO: 20; and / or The antibody Fab fragment of the antibody has (i) a K of less than 50 pM as measured by KinExA D binds to human VEGF-A121 with a K of less than 50 pM as measured by KinExA; D and / or the antibody Fab fragment exhibits an onset aggregation temperature of 70°C or higher; and / or the antibody Fab fragment of the antibody exhibits a melting temperature of greater than 80°C as measured by dynamic light scattering; and / or An antibody, wherein a solution of 180 mg / ml of the antibody Fab fragment in 20 mM His / HisHCl, pH 6.0 has a viscosity of less than 20 cP at 20° C. as detected by dynamic light scattering using the latex-bead DLS method described in Example 8.

[0217] 5. An antibody described in any one of the preceding embodiments, comprising a VH domain comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 3, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 21, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14, and a VL domain comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 22, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 23, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8.

[0218] 6. An antibody that binds to human VEGF-A and human ANG2, comprising a VH domain comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 3, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 21, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14, and a VL domain comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 22, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 23, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8.

[0219] 7. The antibody of any one of the preceding embodiments, comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 3; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 21; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14; (d) a VH domain comprising a human heavy chain framework having (i) FR1 comprising amino acid residues H3, D26, F27, E29, and Y30, (ii) FR3 comprising amino acid residues R66 and R94; and (e) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 22; (f) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 23; (g) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8; and (h) a VL domain comprising a human light chain framework having (i) FR1 comprising amino acid residues I2 and Y3, (ii) FR2 comprising amino acid residues L46 and F49, and (iii) FR3 comprising amino acid residue E57, wherein the numbering of the VH and VL domains is according to the Kabat numbering system.

[0220] 8. An antibody that binds to human VEGF-A and human ANG2, comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:3; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:21; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:14; (d) a VH domain comprising a human heavy chain framework having (i) an FR1 comprising amino acid residues H3, D26, F27, E29, and Y30, (ii) an FR3 comprising amino acid residues R66 and R94; and (e) a VH domain comprising the amino acid sequence of SEQ ID NO:22. (f) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 23; (g) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8; and (h) a VL domain comprising a human light chain framework having (i) FR1 comprising amino acid residues I2 and Y3, (ii) FR2 comprising amino acid residues L46 and F49, and (iii) FR3 comprising amino acid residue E57, wherein the numbering of the VH and VL domains is according to the Kabat numbering system.

[0221] 9. The antibody of any one of the preceding embodiments, comprising a VH domain comprising amino acid residues H3, D26, F27, E29, Y30, D35b, D35c, D55, H56, K57, Y58, T61, K62, F63, I64, G65, R66, R94, D95, V96, F98, and F99, and a VL domain comprising amino acid residues I2, Y3, Y27, W27a, E32, L46, F49, D50, F53, K54, V55, Y56, E57, Y91, R92, Y93, H94, and P95, wherein numbering of the VH and VL domains is according to the Kabat numbering system.

[0222] 10. An antibody that binds to human VEGF-A and human ANG2, comprising amino acid residues contained in the VEGF-A paratope and ANG2 paratope of an antibody having a VH domain of SEQ ID NO: 19 and a VL domain of SEQ ID NO: 20.

[0223] 11. The antibody according to embodiment 9 or 10, - a VEGF-A paratope comprising the following amino acid residues in the VH domain: D35c, D55, H56, K57, Y58, T61, K62, F63, I64, G65, R66, and D95, and in the VL domain: I2, Y3, Y27, W27a, E32, R92, Y93, H94, and P95; and - an antibody comprising an ANG2 paratope comprising the following amino acid residues in the VH domain: H3, D26, F27, E29, Y30, D35b, R94, V96, F98, and F99, and the following amino acid residues in the VL domain: E32, L46, F49, D50, F53, K54, V55, Y56, E57, and Y91.

[0224] 12. An antibody that binds to human VEGF-A and human ANG2, said antibody comprising a VH domain comprising amino acid residues H3, D26, F27, E29, Y30, D35b, D35c, D55, H56, K57, Y58, T61, K62, F63, I64, G65, R66, R94, D95, V96, F98, and F99, and a VL domain comprising amino acid residues I2, Y3, Y27, W27a, E32, L46, F49, D50, F53, K54, V55, Y56, E57, Y91, R92, Y93, H94, and P95, wherein the numbering of the VH and VL domains is according to the Kabat numbering system.

[0225] 13. The antibody of embodiment 12, - a VEGF-A paratope comprising the following amino acid residues in the VH domain: D35c, D55, H56, K57, Y58, T61, K62, F63, I64, G65, R66, and D95, and in the VL domain: I2, Y3, Y27, W27a, E32, R92, Y93, H94, and P95; and - an antibody comprising an ANG2 paratope comprising the following amino acid residues in the VH domain: H3, D26, F27, E29, Y30, D35b, R94, V96, F98, and F99, and the following amino acid residues in the VL domain: E32, L46, F49, D50, F53, K54, V55, Y56, E57, and Y91.

[0226] 14. An antibody described in any one of the preceding embodiments, comprising: (a) a VH domain comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 19; and (b) a VL domain comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 20.

[0227] 15. The antibody of any one of the preceding embodiments, comprising: (a) a VH domain comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 19, the VH domain comprising amino acid residues H3, D26, F27, E29, Y30, D35b, D35c, D55, H56, K57, Y58, T61, K62, F63, I64, G65, R66, R94, D95, V96, F98, and F99; and (b) SEQ ID NO: 20, wherein the numbering of the VH and VL domains is according to the Kabat numbering system.

[0228] 16. An antibody that specifically binds to human VEGF-A and human ANG2, comprising: (a) a VH domain comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 3, (b) a CDR-H2 having the amino acid sequence of SEQ ID NO: 21, and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 14, and a VL domain comprising (d) a CDR-L1 having the amino acid sequence of SEQ ID NO: 22, (e) a CDR-L2 having the amino acid sequence of SEQ ID NO: 23, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 8, wherein the antibody comprises: (a) a VH domain comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 19; and (b) a VL domain comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 20.

[0229] 17. An antibody that specifically binds to human VEGF-A and human ANG2, comprising a VH domain comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 3, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 21, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14, and a VL domain comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 22, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 23, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8. and (b) a VL domain comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:20, the VH domain comprising H3, D26, F27, E29, Y30, R66, and R94; and (b) a VL domain comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:20, the VH and VL domains comprising I2, Y3, L46, F49, and E57, wherein the numbering of the VH and VL domains is according to the Kabat numbering system.

[0230] 18. An antibody that specifically binds to human VEGF-A and human ANG2, comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:3; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:21; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:14; (d) a VH domain comprising a human heavy chain framework having (i) an FR1 comprising amino acid residues H3, D26, F27, E29, and Y30, and (ii) an FR3 comprising amino acid residues R66 and R94; and (e) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:22; (f) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:23; (g) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:8. and (h) a VL domain comprising a human light chain framework having (i) FR1 comprising amino acid residues I2 and Y3, (ii) FR2 comprising amino acid residues L46 and F49, and (iii) FR3 comprising amino acid residue E57, wherein the numbering of the VH and VL domains is according to the Kabat numbering system, and wherein the numbering of the VH and VL domains is according to the Kabat numbering system, and the antibody comprises: (a) a VH domain comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 19; and (b) a VL domain comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 20.

[0231] 19. The antibody of any one of the preceding embodiments, comprising (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 19 with up to 15 amino acid substitutions, and (b) a variable light domain comprising the amino acid sequence of SEQ ID NO: 20 with up to 15 amino acid substitutions.

[0232] 20. The antibody of any one of the preceding embodiments, comprising: (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 19 with up to 15 amino acid substitutions, the amino acid substitutions being at one or more positions 1, 2, 4 to 25, 28, 35d to 54, 59, 60, 67 to 93, 97, 101 to 113 of SEQ ID NO: 19; and (b) a variable light domain comprising the amino acid sequence of SEQ ID NO: 20 with up to 15 amino acid substitutions, the amino acid substitutions being at positions 1, 4 to 26, 27b to 27d, 33 to 45, 47, 48, 51, 52, 58 to 90, 96 to 107 of SEQ ID NO: 20, wherein numbering of the VH and VL domains is according to the Kabat numbering system.

[0233] 21. An antibody that specifically binds to human VEGF-A and human ANG2, comprising: (a) a VH domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO:3, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:21, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:14, and a VL domain comprising: (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:22, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:23, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:8, and comprising: (a) a VH domain comprising the amino acid sequence of SEQ ID NO:19 with up to 15 amino acid substitutions; and (b) a variable light chain domain comprising the amino acid sequence of SEQ ID NO:20 with up to 15 amino acid substitutions.

[0234] 22. An antibody that specifically binds to human VEGF-A and human ANG2, comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:3; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:21; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:14; (d) a VH domain comprising a human heavy chain framework having (i) an FR1 comprising amino acid residues H3, D26, F27, E29, and Y30, and (ii) an FR3 comprising amino acid residues R66 and R94; and (e) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:22; and (f) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:23. (g) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8; and (h) a VL domain comprising a human light chain framework having (i) FR1 comprising amino acid residues I2 and Y3, (ii) FR2 comprising amino acid residues L46 and F49, and (iii) FR3 comprising amino acid residue E57, wherein the numbering of the VH and VL domains is according to the Kabat numbering system, and comprising: (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 19 with up to 15 amino acid substitutions; and (b) a variable light chain domain comprising the amino acid sequence of SEQ ID NO: 20 with up to 15 amino acid substitutions.

[0235] 23. The antibody of any one of the preceding embodiments, comprising a VH sequence of SEQ ID NO: 19 and a VL sequence of SEQ ID NO: 20.

[0236] 24. An antibody that binds to human VEGF-A and human ANG2, comprising the VH sequence of SEQ ID NO: 19 and the VL sequence of SEQ ID NO: 20.

[0237] 25. The antibody of any one of the preceding embodiments, comprising a heavy chain amino acid sequence of SEQ ID NO:24 and a light chain amino acid sequence of SEQ ID NO:25.

[0238] 26. An antibody that binds to human VEGF-A and human ANG2, comprising the heavy chain amino acid sequence of SEQ ID NO: 24 and the light chain amino acid sequence of SEQ ID NO: 25.

[0239] 27. The antibody of any one of the preceding embodiments, comprising a heavy chain amino acid sequence of SEQ ID NO:17 and a light chain amino acid sequence of SEQ ID NO:18.

[0240] 28. An antibody that specifically binds to human VEGF-A and human ANG2, comprising the heavy chain amino acid sequence of SEQ ID NO: 17 and the light chain amino acid sequence of SEQ ID NO: 18.

[0241] 29. The antibody Fab fragment of the antibody has (i) a K of less than 50 pM as measured by KinExA. D binds to human VEGF-A121 with a K of less than 50 pM as measured by KinExA; D 2. The antibody of any one of the preceding embodiments, wherein the antibody binds to human ANG2 at

[0242] 30. An antibody that specifically binds to human VEGF-A and human ANG2, wherein the antibody Fab fragment of the antibody has (i) a K of less than 50 pM as measured by KinExA. D binds to human VEGF-A121 with a K of less than 50 pM as measured by KinExA; D An antibody that binds to human VEGF-A121.

[0243] 31. An antibody that binds to human VEGF-A and human ANG2, comprising a VH domain comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:3, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:21, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:14, and a VL domain comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:22, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:23, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:8, wherein an antibody Fab fragment of the antibody has (i) a K of less than 50 pM as measured by KinExA. D binds to human VEGF-A121 with a K of less than 50 pM as measured by KinExA; D An antibody that binds to human ANG2.

[0244] 32. An antibody that specifically binds to human VEGF-A and human ANG2, comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 3; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 21; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14; (d) a VH domain comprising a human heavy chain framework having (i) FR1 comprising amino acid residues H3, D26, F27, E29, and Y30, (ii) FR3 comprising amino acid residues R66 and R94; and (e) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 22; (f) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 23; (g) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8; and (h) a VL domain comprising a human light chain framework having (i) an FR1 comprising amino acid residues I2 and Y3, (ii) an FR2 comprising amino acid residues L46 and F49, and (iii) an FR3 comprising amino acid residue E57, wherein the numbering of the VH and VL domains is according to the Kabat numbering system, and D binds to human VEGF-A121 with a K of less than 50 pM as measured by KinExA; D An antibody that binds to human ANG2.

[0245] 33. An antibody that specifically binds to human VEGF-A and human ANG2, comprising in a pair of VH and VL domains: (i) a VH domain comprising amino acid residues H3, D26, F27, E29, Y30, D35b, D35c, D55, H56, K57, Y58, T61, K62, F63, I64, G65, R66, R94, D95, V96, F98, and F99; and (ii) an amino acid residue 1111 in the VH domain. and a VL domain comprising the amino acid residues I2, Y3, Y27, W27a, E32, L46, F49, D50, F53, K54, V55, Y56, E57, Y91, R92, Y93, H94, and P95, wherein the numbering of the VH and VL domains is according to the Kabat numbering system, and wherein the antibody Fab fragment of the antibody has (i) a K of less than 50 pM as measured by KinExA. D binds to human VEGF-A121 with a K of less than 50 pM as measured by KinExA; DAn antibody that binds to human ANG2.

[0246] 34. An antibody that specifically binds to human VEGF-A and human ANG2, comprising: (a) a VH domain comprising a CDR-H1 having the amino acid sequence of SEQ ID NO:3, (b) a CDR-H2 having the amino acid sequence of SEQ ID NO:21, and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO:14; and (d) a VL domain comprising a CDR-L1 having the amino acid sequence of SEQ ID NO:22, (e) a CDR-L2 having the amino acid sequence of SEQ ID NO:23, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO:8, wherein the antibody Fab fragment of the antibody comprises: (a) a VH domain comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:19; and (b) a VL domain comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:20, and (i) a K of less than 50 pM as measured by KinExA. D binds to human VEGF-A121 with a K of less than 50 pM as measured by KinExA; D An antibody that binds to human ANG2.

[0247] 35. The antibody of any one of the preceding embodiments, wherein the antibody Fab fragment exhibits an onset aggregation temperature of 70°C or greater.

[0248] 36. An antibody that specifically binds to human VEGF-A and human ANG2, wherein the antibody Fab fragment of the antibody exhibits an aggregation onset temperature of 70°C or higher.

[0249] 37. An antibody that binds to human VEGF-A and human ANG2, comprising a VH domain comprising (a) CDR-H1 having the amino acid sequence of SEQ ID NO: 3, (b) CDR-H2 having the amino acid sequence of SEQ ID NO: 21, and (c) CDR-H3 having the amino acid sequence of SEQ ID NO: 14, and a VL domain comprising (d) CDR-L1 having the amino acid sequence of SEQ ID NO: 22, (e) CDR-L2 having the amino acid sequence of SEQ ID NO: 23, and (f) CDR-L3 having the amino acid sequence of SEQ ID NO: 8, wherein the antibody Fab fragment of the antibody exhibits an aggregation onset temperature of 70°C or higher.

[0250] 38. An antibody that specifically binds to human VEGF-A and human ANG2, comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 3; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 21; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14; (d) a VH domain comprising a human heavy chain framework having (i) FR1 comprising amino acid residues H3, D26, F27, E29, and Y30, (ii) FR3 comprising amino acid residues R66 and R94; and (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 22. (f) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 23, (g) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8, and (h) a VL domain comprising a human light chain framework having (i) FR1 comprising amino acid residues I2 and Y3, (ii) FR2 comprising amino acid residues L46 and F49, and (iii) FR3 comprising amino acid residue E57, wherein the numbering of the VH and VL domains is according to the Kabat numbering system, and wherein the antibody Fab fragment of the antibody exhibits an aggregation onset temperature of 70°C or higher.

[0251] 39. An antibody that specifically binds to human VEGF-A and human ANG2, comprising in a pair of VH and VL domains: (i) a VH domain comprising amino acid residues H3, D26, F27, E29, Y30, D35b, D35c, D55, H56, K57, Y58, T61, K62, F63, I64, G65, R66, R94, D95, V96, F98, and F99; and (ii) ) An antibody comprising a VL domain comprising amino acid residues I2, Y3, Y27, W27a, E32, L46, F49, D50, F53, K54, V55, Y56, E57, Y91, R92, Y93, H94, and P95, wherein the numbering of the VH and VL domains is according to the Kabat numbering system, and wherein an antibody Fab fragment of the antibody exhibits an aggregation onset temperature of 70°C or greater.

[0252] 40. An antibody that specifically binds to human VEGF-A and human ANG2, comprising: (a) a VH domain comprising CDR-H1 having the amino acid sequence of SEQ ID NO: 3, (b) a CDR-H2 having the amino acid sequence of SEQ ID NO: 21, and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 14, and a VL domain comprising (d) a CDR-L1 having the amino acid sequence of SEQ ID NO: 22, (e) a CDR-L2 having the amino acid sequence of SEQ ID NO: 23, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 8, wherein the antibody comprises: (a) a VH domain comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 19; and (b) a VL domain comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 20, and an antibody Fab fragment of the antibody exhibits an onset aggregation temperature of 70°C or higher.

[0253] 41. The antibody of any one of the preceding embodiments, wherein the antibody Fab fragment of the antibody exhibits a melting temperature of greater than 80°C as measured by dynamic light scattering.

[0254] 42. An antibody that specifically binds to human VEGF-A and human ANG2, wherein an antibody Fab fragment of the antibody exhibits a melting temperature of greater than 80° C. as measured by dynamic light scattering.

[0255] 43. An antibody that binds to human VEGF-A and human ANG2, comprising a VH domain comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:3, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:21, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:14, and a VL domain comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:22, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:23, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:8, wherein an antibody Fab fragment of the antibody exhibits a melting temperature of greater than 80°C as measured by dynamic light scattering.

[0256] 44. An antibody that specifically binds to human VEGF-A and human ANG2, comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 3; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 21; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14; (d) a VH domain comprising a human heavy chain framework having (i) FR1 comprising amino acid residues H3, D26, F27, E29, and Y30, (ii) FR3 comprising amino acid residues R66 and R94; and (e) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 22; (f) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 23; 8; and (h) a VL domain comprising a human light chain framework having (i) FR1 comprising amino acid residues I2 and Y3, (ii) FR2 comprising amino acid residues L46 and F49, and (iii) FR3 comprising amino acid residue E57, wherein the numbering of the VH and VL domains is according to the Kabat numbering system, and wherein an antibody Fab fragment of the antibody exhibits a melting temperature of greater than 80° C. as measured by dynamic light scattering.

[0257] 45. An antibody that specifically binds to human VEGF-A and human ANG2, comprising in a pair of VH and VL domains: (i) a VH domain comprising amino acid residues H3, D26, F27, E29, Y30, D35b, D35c, D55, H56, K57, Y58, T61, K62, F63, I64, G65, R66, R94, D95, V96, F98, and F99; and (ii) amino acid residues 1. An antibody comprising a VL domain comprising groups I2, Y3, Y27, W27a, E32, L46, F49, D50, F53, K54, V55, Y56, E57, Y91, R92, Y93, H94, and P95, wherein the numbering of the VH and VL domains is according to the Kabat numbering system, and wherein an antibody Fab fragment of the antibody exhibits a melting temperature of greater than 80° C. as measured by dynamic light scattering.

[0258] 46. ​​An antibody that specifically binds to human VEGF-A and human ANG2, comprising: (a) a VH domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO:3, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:21, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:14, and a VL domain comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:22, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:23, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:8, wherein the antibody comprises: (a) a VH domain comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:19; and (b) a VL domain comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:20, wherein an antibody Fab fragment of the antibody exhibits a melting temperature of greater than 80°C as measured by dynamic light scattering.

[0259] 47. The antibody of any one of the preceding embodiments, wherein a solution of 180 mg / ml of the antibody Fab fragment in 20 mM His / HisHCl, pH 6.0 has a viscosity of less than 20 cP at 20° C. as detected by dynamic light scattering using the latex-bead DLS method described in Example 8.

[0260] 48. An antibody that specifically binds to human VEGF-A and human ANG2, wherein a solution of 180 mg / ml of the antibody Fab fragment in 20 mM His / HisHCl, pH 6.0 has a viscosity of less than 20 cP at 20° C. as detected by dynamic light scattering using the latex-bead DLS method described in Example 8.

[0261] 49. An antibody that binds to human VEGF-A and human ANG2, comprising a VH domain comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:3, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:21, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:14, and a VL domain comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:22, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:23, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:8, wherein a solution of 180 mg / ml of the antibody Fab fragment in 20 mM His / HisHCl, pH 6.0 has a viscosity of less than 20 cP at 20° C. as detected by dynamic light scattering using the latex-bead DLS method described in Example 8.

[0262] 50. An antibody that specifically binds to human VEGF-A and human ANG2, comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:3; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:21; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:14; (d) a VH domain comprising a human heavy chain framework having (i) an FR1 comprising amino acid residues H3, D26, F27, E29, and Y30, (ii) an FR3 comprising amino acid residues R66 and R94; and (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:22. (f) CDR-L1 comprising the amino acid sequence of SEQ ID NO:23, (g) CDR-L3 comprising the amino acid sequence of SEQ ID NO:8, and (h) a VL domain comprising a human light chain framework having (i) FR1 comprising amino acid residues I2 and Y3, (ii) FR2 comprising amino acid residues L46 and F49, and (iii) FR3 comprising amino acid residue E57, wherein the numbering of the VH and VL domains is according to the Kabat numbering system, and wherein a solution of 180 mg / ml of the antibody Fab fragment in 20 mM His / HisHCl, pH 6.0 has a viscosity of less than 20 cP at 20° C. as detected by dynamic light scattering using the latex-bead DLS method described in Example 8.

[0263] 51. An antibody that specifically binds to human VEGF-A and human ANG2, comprising in a pair of VH and VL domains: (i) a VH domain comprising amino acid residues H3, D26, F27, E29, Y30, D35b, D35c, D55, H56, K57, Y58, T61, K62, F63, I64, G65, R66, R94, D95, V96, F98, and F99; and (ii) a VL domain comprising amino acid residues I2, Y3, Y27, W27a, E32, L46, F49, D50, F53, K54, V55, Y56, E57, Y91, R92, Y93, H94, and P95, wherein the numbering of the VH and VL domains is according to the Kabat numbering system, and wherein a solution of 180 mg / ml of the antibody Fab fragment in 20 mM His / HisHCl, pH 6.0, has a viscosity of less than 20 cP at 20° C. as detected by dynamic light scattering using a latex-bead DLS method as described in Example 8.

[0264] 52. An antibody that specifically binds to human VEGF-A and human ANG2, comprising: a VH domain comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:3, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:21, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:14, and a VL domain comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:22, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:23, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:8, wherein the antibody comprises: (a) a VH domain comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:19; and (b) a VL domain comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:20, wherein a solution of 180 mg / ml of the antibody Fab fragment in 20 mM His / HisHCl, pH 6.0 has a viscosity of less than 20 cP at 20° C. as detected by dynamic light scattering using the latex-bead DLS method described in Example 8.

[0265] 53. The antibody of any one of the preceding embodiments, which is a monoclonal antibody.

[0266] 54. The antibody of any one of the preceding embodiments, which is an antibody fragment that binds to human VEGF-A and human ANG2.

[0267] 55. The antibody of any one of the preceding embodiments, which is bispecific.

[0268] 56. The antibody of any one of the preceding embodiments, which is a Fab fragment.

[0269] 57. The antibody of any one of the preceding embodiments, which is a bispecific antibody fragment.

[0270] 58. The antibody of any one of the preceding embodiments, which is a multispecific antibody.

[0271] 59. The antibody of any one of the preceding embodiments, which specifically binds to human VEGF-A.

[0272] 60. The antibody of any one of the preceding embodiments, which specifically binds to human ANG2.

[0273] 61. An isolated nucleic acid encoding an antibody according to any one of embodiments 1 to 58.

[0274] 62. A host cell comprising the nucleic acid of embodiment 59.

[0275] 63. An expression vector comprising the nucleic acid of embodiment 61.

[0276] 64. A method for producing an antibody that binds to human VEGF-A and human ANG2, comprising culturing a host cell according to embodiment 60 so that the antibody is produced.

[0277] 65. The method of embodiment 64, further comprising recovering the antibody from the host cell.

[0278] 66. The method of embodiment 64 or 65, wherein the host cell is an E. coli cell.

[0279] 67. The method of embodiment 64 or 65, wherein the host cell is a CHO cell.

[0280] 68. An antibody produced by the method of embodiment 64 or 65.

[0281] 69. A pharmaceutical formulation comprising an antibody according to any one of embodiments 1 to 60 and a pharma- ceutically acceptable carrier.

[0282] 70. The antibody of any one of embodiments 1 to 60 for use as a medicament.

[0283] 71. The antibody of any one of embodiments 1 to 60 for use in the treatment of a vascular disease.

[0284] 72. The antibody of any one of embodiments 1 to 60 for use in the treatment of an ocular vascular disease.

[0285] 73. Use of the antibody according to any one of embodiments 1 to 60 or the pharmaceutical composition according to embodiment 65 in the manufacture of a medicament.

[0286] 74. Use of the antibody according to any one of embodiments 1 to 60 or the pharmaceutical composition according to embodiment 65 in the manufacture of a medicament for inhibiting angiogenesis.

[0287] 75. A method for treating an individual having a vascular disease, comprising administering to the individual an effective amount of an antibody according to any one of embodiments 1 to 60 or a pharmaceutical preparation according to embodiment 69.

[0288] 76. A method for treating an individual having an ocular vascular disease, comprising administering to the individual an effective amount of an antibody according to any one of embodiments 1 to 60 or a pharmaceutical preparation according to embodiment 69.

[0289] 77. A method for inhibiting angiogenesis in an individual, comprising administering to the individual an antibody according to any of embodiments 1 to 60 or a pharmaceutical preparation according to embodiment 69 in an amount effective to inhibit angiogenesis.

[0290] 78. A port delivery device comprising an antibody according to any of embodiments 1 to 60 or a pharmaceutical formulation according to embodiment 69.

[0291] 79. The antibody according to any of embodiments 1 to 60 or the pharmaceutical formulation according to embodiment 69, for ocular administration by a port delivery device.

[0292] 80. The antibody of any of embodiments 1 to 60 or the pharmaceutical formulation of embodiment 69, for ocular administration by a port delivery device of embodiment 79, wherein administration is for a period of 6 months or more, in one embodiment 8 months or more, and in one embodiment 9 months or more, before the port delivery device is refilled.

[0293] 81. A pharmaceutical formulation as described in any of embodiments 1 to 60 or embodiment 69 for use as a medicament by administering the antibody or pharmaceutical formulation using a port delivery device, wherein the antibody is applied to the port delivery device at a concentration of 150 mg / ml or more, in one embodiment at a concentration of 200 mg / ml or more.

[0294] [Table 3] TIFF2025072509000004.tif234169 TIFF2025072509000005.tif199169 TIFF2025072509000006.tif200169 EXAMPLES

[0295] The following examples are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit or scope of the invention.

[0296] Materials and General Methods Human VEGF-A 121 affinity Kinexa: Equipment and materials: A KinExA 3200 instrument and autosampler from Sapidyne Instruments (Boise, ID) was used. Polymethylmethacrylate (PMMA) beads were purchased from Sapidyne, and anti-VEGF antibodies were purchased from R&D Systems (Mab293, BAF293). Streptavidin Alexa Fluor™ 647 conjugate was purchased from Thermo Fisher scientific (S21374). PBS (phosphate buffered saline), BSA (bovine serum albumin fraction V), VEGFA-121 (SEQ ID NO: 28) were prepared in-house (Roche).

[0297] Preparation of antigen-coated beads PMMA beads were coated according to the KinExA Handbook protocol (Adsorption coating, Sapidyne). First, 30 μg of anti-VEGF antibody MAB293 (R&D) in 1 ml of PBS (pH 7.4) was added per vial of beads (200 mg) for adsorption coating. After rotating at room temperature for 2 hours, the supernatant was removed and filled with 1 ml of blocking solution (10 mg / ml BSA in buffer) and rocked for 1 hour.

[0298] KinExA equilibrium assay All KinExA experiments were performed at room temperature (RT) using PBS pH 7.4 with 0.01% BSA and 0.01% Tween 20 (BioRad, #161-0781) as running buffer. Samples and beads were prepared in LowCross buffer (Candor Bioscience) to reduce non-specific binding observed in previous measurements. The flow rate was 0.25 ml / min. Constant amounts of VEGFA-121-His (50 pM and 500 pM in the second experiment) were titrated with test antibodies by 2-fold serial dilutions starting at 4 nM (concentration range 1.95 pM to 4000 pM). Antigen-antibody complexes were incubated at RT for at least 8 h to reach equilibrium. The equilibrated mixture was aspirated into a column of beads coupled with anti-VEGF antibody (Mab293) in the KinExA system in a volume of 750 μl for 50 pM constant VEGF and in a volume of 125 μl for 500 pM constant VEGF, allowing the beads to capture unbound VEGFA-121 without disturbing the equilibrium state of the solution. Bound VEGFA-121 was detected using a secondary biotinylated anti-VEGF antibody (BAF293) at a concentration of 250 ng / ml, followed by injection of 250 ng / ml of streptavidin Alexa Fluor™ 647 conjugate in sample buffer. Each sample was measured in duplicate for every equilibration experiment. K D was obtained from nonlinear regression analysis of the data using a one-site homogeneous binding model included in the KinExA software (version 4.0.11) using the "standard analysis" method. D Calculate the data points and approximate them to the theoretical K D Determine the 95% confidence interval by fitting the curve. The 95% confidence interval is K D Low and K D is given as high.

[0299] Final K D For the determination, an n-curve determination of two measurements with different constant VEGFA-121 concentrations was performed. Using n-curve analysis, multiple standard K D Analyzing the experiment, K D A more accurate determination of the

[0300] Kinexa Human ANG2 Affinity: Equipment and materials A KinExA 3200 instrument and autosampler from Sapidyne Instruments (Boise, ID) was used. Polymethylmethacrylate (PMMA) beads were purchased from Sapidyne, and anti-Ang2 antibody was purchased from R&D Systems (MAB098, BAM0981). Streptavidin Alexa Fluor™ 647 conjugate was purchased from Thermo Fisher scientific (S21374). PBS (phosphate buffered saline), BSA (bovine serum albumin fraction V), and Ang2 (SEQ ID NO: 27) were prepared in-house (Roche).

[0301] Preparation of antigen-coated beads PMMA beads were coated according to the KinExA Handbook protocol (Adsorption coating, Sapidyne). First, 20 μg of anti-Ang2 antibody MAB098 (R&D) in 1 ml of PBS (pH 7.4) was added per vial of beads (200 mg) for adsorption coating. After rotating at room temperature for 2 hours, the supernatant was removed and filled with 1 ml of blocking solution (10 mg / ml BSA in buffer) and rocked for 1 hour.

[0302] KinExA equilibrium assay All KinExA experiments were performed at room temperature (RT) using PBS pH 7.4 with 0.01% BSA and 0.01% Tween 20 (BioRad, #161-0781) as running buffer. Samples and beads were prepared in LowCross buffer (Candor Bioscience) to reduce non-specific binding observed in previous measurements. The flow rate was 0.25 ml / min. A constant amount of Ang2-RBD-muFc (50 pM and 500 pM in the second experiment) was titrated with the test antibody by 2-fold serial dilutions starting at 4 nM (concentration range 1.95 pM to 4000 pM). The antigen-antibody complex was incubated at RT for at least 8 h to reach equilibrium. The equilibrated mixture was aspirated into a column of beads coupled with anti-Ang2 antibody (MAB098) in the KinExA system in a volume of 750 μl for 50 pM constant Ang2 and 188 μl for 500 pM constant Ang2, allowing the unbound Ang2 to be captured by the beads without disturbing the equilibrium state of the solution. Bound Ang2 was detected using a secondary biotinylated anti-Ang2 antibody (BAM0981) at a concentration of 250 ng / ml, followed by injection of 250 ng / ml of streptavidin Alexa Fluor™ 647 conjugate in sample buffer. Each sample was measured in duplicate for every equilibration experiment. K D was obtained from nonlinear regression analysis of the data using a one-site homogeneous binding model included in the KinExA software (version 4.0.11) using the "standard analysis" method. D Calculate the data points and approximate them to the theoretical K D Determine the 95% confidence interval by fitting the curve. The 95% confidence interval is K D Low and K D is given as high.

[0303] Final K D For the determination, an n-curve determination of two measurements with different constant Ang2 concentrations was performed. Using n-curve analysis, multiple standard K D Analyzing the experiment, K D A more accurate determination of the

[0304] Human VEGF-A binding kinetics as assessed by surface plasmon resonance (SPR): An anti-His capturing antibody (GE Healthcare 28995056) was immobilized on a Series S Sensor Chip C1 (GE Healthcare BR100535) using standard amine coupling chemistry, resulting in a surface density of approximately 600 resonance units (RU). HBS-P+ (10 mM HEPES, 150 mM NaCl pH 7.4, 0.05% surfactant P20) was used as running and dilution buffer. Human VEGF-A121-His was captured on the surface, resulting in ligand densities of approximately 10 and 20 RU, respectively. Serial dilutions of the test antibodies (3.7-300 nM, 1:3 dilution) were injected consecutively for 60 s each and dissociation was monitored for 3600 s at a flow rate of 30 μl / min (single cycle kinetics). The surface was regenerated by injecting 10 mM glycine pH 1.5 for 60 s at a flow rate of 5 μl / min. Bulk refractive index differences were corrected by subtracting a blank injection and the response obtained from a control flow cell not capturing human VEGF-A121. Curve fitting was performed using a 1:1 Langmuir binding model within the Biacore evaluation software.

[0305] VEGF-A binding kinetics as assessed by surface plasmon resonance (SPR) for cross-reactivity studies: An anti-His capturing antibody (GE Healthcare 28995056) was immobilized on a Series S Sensor Chip C1 (GE Healthcare BR100535) using standard amine coupling chemistry, resulting in a surface density of approximately 600 resonance units (RU). HBS-P+ (10 mM HEPES, 150 mM NaCl pH 7.4, 0.05% surfactant P20) was used as running and dilution buffer. VEGF-A121-His from the indicated species was captured on the surface, resulting in ligand densities of approximately 10 and 20 RU, respectively. Serial dilutions of the test antibodies (3.7-300 nM, 1:3 dilution) were injected consecutively for 60 s each and dissociation was monitored for 3600 s at a flow rate of 30 μl / min (single cycle kinetics). The surface was regenerated by injecting 10 mM glycine pH 1.5 for 60 s at a flow rate of 5 μl / min. Bulk refractive index differences were corrected for by subtracting a blank injection and the response obtained from a control flow cell not capturing VEGF-A121. Curve fitting was performed using a 1:1 Langmuir binding model within the Biacore evaluation software.

[0306] Human ANG 2 binding kinetics as assessed by surface plasmon resonance (SPR): Anti-His capturing antibody (GE Healthcare 28958325) was immobilized on a Series S Sensor Chip C1 (GE Healthcare BR100535) using standard amine coupling chemistry to obtain a surface density of approximately 800 resonance units (RU). HBS-P+ (10 mM HEPES, 150 mM NaCl pH 7.4, 0.05% surfactant P20) was used as running and dilution buffer, and the measurement temperature was 25°C. Test antibodies were captured by injecting a 50 nM solution at a flow rate of 5 μl / min for 60 s. Association was measured by injecting various concentrations of human Ang2-RBD in solution at a flow rate of 30 μl / min (0.07 nM-50 nM, 1:3 dilution) for 180 s. The dissociation phase was monitored for up to 900 s and was triggered by switching from the sample solution to the running buffer at a flow rate of 30 μl / min. The surface was regenerated by injecting 10 mM glycine pH 2.1 for 60 seconds at a flow rate of 30 μl / min. Bulk refractive index differences were corrected by subtracting a blank injection and the response obtained from a reference flow cell without captured test antibody. Curve fitting was performed using a 1:1 Langmuir binding model within the Biacore evaluation software.

[0307] ANG2 binding kinetics as assessed by surface plasmon resonance (SPR) for cross-reactivity testing: Anti-His capturing antibody (GE Healthcare 28958325) was immobilized on a Series S Sensor Chip C1 (GE Healthcare BR100535) using standard amine coupling chemistry to obtain a surface density of approximately 800 resonance units (RU). HBS-P+ (10 mM HEPES, 150 mM NaCl pH 7.4, 0.05% surfactant P20) was used as running and dilution buffer, and the measurement temperature was 25°C. Test antibodies were captured by injecting a 50 nM solution at a flow rate of 5 μl / min for 60 s. Association was measured by injecting various concentrations of Ang2-RBD from the indicated species in solution at a flow rate of 30 μl / min (0.07 nM-50 nM, 1:3 dilution) for 180 s. The dissociation phase was monitored for up to 600 s and was triggered by switching from the sample solution to the running buffer at a flow rate of 30 μl / min. The surface was regenerated by injecting 10 mM glycine pH 2.1 for 60 seconds at a flow rate of 30 μl / min. Bulk refractive index differences were corrected by subtracting a blank injection and the response obtained from a reference flow cell with no captured antibody. Curve fitting was performed using a 1:1 Langmuir binding model within the Biacore evaluation software.

[0308] Assessment of independent binding to target antigens by surface plasmon resonance (SPR) Approximately 5000 resonance units (RU) of the capture system (anti-Fab capture antibody (GE Healthcare 28958325)) were coupled to a CM5 chip (GE Healthcare BR-1005-30) at pH 5.0 by using an amine coupling kit supplied by GE Healthcare. The sample and system buffer was HBS-P+ (10 mM HEPES, 150 mM NaCl pH 7.4, 0.05% surfactant P20). The flow cell temperature was set to 25° C. and the sample block temperature was set to 12° C. Prior to capture, the flow cell was primed three times with running buffer.

[0309] The bispecific Fab was captured by injecting a 6.5 μg / ml solution for 60 seconds at a flow rate of 5 μl / min. Independent binding of each ligand to the bispecific Fab was analyzed by determining the active binding capacity of each ligand added sequentially or simultaneously (flow rate of 10 μl / min).

[0310] 1) Human VEGFA-121 at a concentration of 1 μg / ml is injected for 60 seconds (to identify single binding of the antigen).

[0311] 2) Human Ang2 at a concentration of 5 μg / ml is injected for 60 seconds (to identify single binding of antigen).

[0312] 3) Human VEGFA-121 at a concentration of 1 μg / ml is injected for 60 seconds, followed by an additional 60 second injection of human Ang2 at a concentration of 5 μg / ml (to identify binding of Ang2 in the presence of VEGFA-121).

[0313] 4) Human Ang2 at a concentration of 5 μg / ml is injected for 60 seconds, followed by an additional 60 second injection of human VEGFA-121 at a concentration of 1 μg / ml (to identify binding of VEGFA-121 in the presence of Ang2).

[0314] 5) Co-injection of human VEGFA-121 at a concentration of 1 μg / ml and human Ang2 at a concentration of 5 μg / ml for 60 seconds (to simultaneously identify binding of VEGFA-121 and Ang2).

[0315] The surface was regenerated by injecting 10 mM glycine pH 2.1 for 60 seconds at a flow rate of 30 μl / min. Bulk refractive index differences were corrected by subtracting a blank injection and by subtracting the response obtained from a reference flow cell that did not capture the bispecific Fab. If the final signals resulting from methods 3, 4, and 5 are equal to the sum of the individual final signals from methods 1 and 2, then the bispecific antibody is capable of binding both antigens independently of each other.

[0316] thermal stability Bispecific antibody Fab fragment samples were prepared at 1 mg / mL in 20 mM histidine / histidine chloride, 140 mM NaCl, pH 6.0 and transferred to a 10 μL microcuvette array. Static light scattering data as well as fluorescence data upon excitation with a 266 nm laser were recorded using an UNcle instrument (Unchained Labs) as samples were heated from 30° C. to 90° C. at a rate of 0.1° C. / min. Samples were measured in triplicate.

[0317] The evaluation of the onset temperature was performed by UNcle analysis software. The aggregation onset temperature is defined as the temperature at which the scattered light intensity starts to increase. The unfolding of the protein was monitored by the shift of the centroid mean (BCM) of the fluorescence signal versus heat. The melting temperature is defined as the inflection point of the BCM (nm) versus temperature curve.

[0318] Physicochemical stability: Antibody samples were formulated in 20 mM His / HisCl, 140 mM NaCl, pH 6.0 and split into three aliquots, one aliquot each rebuffered in PBS and two aliquots maintained in the original formulation. The PBS aliquot and one His / HisCl aliquot were incubated at 1 mg / ml at 40° C. (His / NaCl) or 37° C. (PBS) for two weeks (2w), and the PBS sample was further incubated for a total of four weeks (4w). A third control aliquot sample was stored at −80° C. After incubation was completed, samples were analyzed for relative activity concentration (Biacore; activity concentration of each binder in both stressed aliquots is normalized to the unstressed 4° C. aliquot), aggregation (SEC) and fragmentation (capillary electrophoresis or SDS-PAGE, CE-SDS) and compared to untreated controls.

[0319] Human VEGF-A and human ANG2 binding kinetics assessed by SPR for functional stability testing VEGFA-121 (in-house), Protein A (Pierce / Thermo Scientific 21181) and anti-human Fab capture antibody (GE Healthcare 28958325) were immobilized on different flow cells on a Series S Sensor Chip CM5 (GE Healthcare 29104988) using standard amine coupling chemistry, resulting in a surface density of 3000 for hVEGFA-121 and Protein A and 12000 resonance units (RU) for anti-human Fab capture antibody. HBS-N (10 mM HEPES, 150 mM NaCl pH 7.4, GE Healthcare) was used as running buffer and dilution buffer. The sample and running buffer for the following concentration measurements was HBS-P (10 mM HEPES, 150 mM NaCl pH 7.4, 0.05% surfactant P20; GE Healthcare). The flow cell temperature was set at 25°C and the sample block temperature was set at 12°C. Prior to concentration measurements, the flow cell was primed twice with running buffer. First, human Ang2-RBD-hFc dimer was captured by injecting a 10 μg / ml solution at a flow rate of 10 μl / min for 120 s. The tested antibody was injected in solution at a concentration of 1 μg / ml at a flow rate of 5 μl / min for 60 s. The dissociation phase was monitored for up to 30 s and was triggered by switching from the sample solution to the running buffer.

[0320] The hVEGFA-121 surface was regenerated by washing with 10 mM glycine pH 2.0 at a flow rate of 30 μl / min for 30 seconds. The Protein A surface was regenerated by washing with 10 mM glycine pH 1.5 at a flow rate of 30 μl / min for 30 seconds. Finally, the anti-human Fab antibody surface was regenerated by washing with 10 mM glycine pH 2.1 at a flow rate of 30 μl / min for 60 seconds.

[0321] Bulk refractive index differences were corrected for by subtracting the response obtained from a blank surface. For evaluation, binding responses were obtained.

[0322] Relative activity concentrations were calculated by referencing each temperature stressed sample to the corresponding unstressed sample.

number

number

[0323] Example 1: Generation of bispecific anti-VEGF-A / anti-ANG2 Fab fragments Bispecific anti-VEGF-A / anti-ANG2 Fab fragments were generated by independent screening of monospecific antibodies that bind VEGF-A and ANG2, and then combining the amino acid sequences into a biparatopic HC / LC pair that forms a Fab fragment that binds VEGF-A and ANG2, for example by the methods described in WO 2012 / 163520.

[0324] Two different phage display libraries of synthetic Fab fragments were utilized, the first phage display library diversified residues within the CDR-H1, CDR-H3 and CDR-L2 regions of the Fab fragments, and the second phage display library diversified residues within the CDR-L1, CDR-L3 and CDR-H2 regions of the Fab fragments. In each library, the other three CDR regions were left undiversified as invariant dummy sequences. In both libraries, the CH1 domain of the Fab fragments was fused to a truncated gene-III protein via a linker to facilitate phage display.

[0325] The first library was enriched for binders to human ANG2 and the second library was enriched for binders to human VEGF-A by phage library panning. After panning, plasmid minipreps were made for both enriched pools of phagemid vectors. The minipreps were digested with restriction enzymes to excise the region encoding the truncated gene-III protein and recircularized by ligation to obtain pools of expression vectors encoding soluble Fab fragments enriched for ANG2 or VEGF-A binders, respectively. These vector pools were transformed into TG1 E. coli cells and individual colonies were picked and cultured for soluble expression of individual Fab clones in microtiter plates. The supernatants containing soluble Fab fragments were screened for binding to ANG2 or VEGF-A using standard ELISA methods, and TG1 clones producing specific binders were subjected to DNA plasmid preparation and sequencing to obtain pairs of VH and VL sequences that specifically bind to either ANG2 or VEGF-A, respectively.

[0326] Bispecific anti-VEGF-A / anti-ANG2 VH and VL sequence pairs were designed in silico by (1) replacing irrelevant VH residues 52b-65 in the VH sequence of an ANG2-specific Fab with selected VH residues 52b-65 of a VEGF-A-specific Fab, thereby replacing CDR-H2 residues potentially part of a VEGF-A-specific paratope with the ANG2 binder heavy chain, and (2) replacing irrelevant VL residues 49-57 in the VL sequence of a VEGF-A-specific Fab with selected VL residues 49-57 of an ANG2-specific Fab, thereby replacing CDR-L2 residues potentially part of an ANG2-specific paratope with the VEGF-A binder light chain.

[0327] Example 2: Expression of the bispecific anti-VEGF-A / anti-ANG2 Fab fragment P1AA8906 The resulting designed pair of bispecific anti-VEGF-A / anti-ANG2 VH and VL sequences were synthesized and cloned in frame with gene sequences encoding the CH1 and Ckappa domains into an E. coli expression vector. The vector was transformed into TG1 E. coli cells and individual colonies were cultured for soluble expression of the bispecific antibody Fab fragments. The bispecific antibody was purified from TG1 culture supernatants by affinity chromatography and specific binding to both ANG2 and VEGF-A was verified.

[0328] The bispecific anti-VEGF-A / anti-ANG2 antibody "P1AA8906" was selected and is characterized by a heavy chain of SEQ ID NO:9 and a light chain of SEQ ID NO:10.

[0329] For further analysis, the anti-VEGF-A / anti-ANG2 antibodies of the invention were transformed and expressed in CHO cells by standard recombinant methods.

[0330] Example 3: Characterization of the bispecific anti-VEGF-A / anti-ANG2 Fab fragment P1AA8906 The binding affinity of the bispecific antibody P1AA8906 was assessed by SPR and KinExA as described above in the "Materials and General Methods" section.

[0331] [Table 4]

[0332] KinExA analysis was performed under the following conditions: VEGF-A-121 concentrations: 100 pM / 1000 pM (CBP), P1AA8906 4 nM-0 pM (1:2 dilution, 12-fold), pre-incubation time approximately 8 h at RT.

[0333] [Table 5]

[0334] KinExA analysis was performed under the following conditions: VEGF-A-121 concentrations: 100 pM / 1000 pM (CBP), P1AA8906 4 nM-0 pM (1:2 dilution, 12-fold), pre-incubation time approximately 8 h at RT.

[0335] The thermal stability of the bispecific antibody P1AA 8906 was assessed as described above in the "Materials and General Methods" section.

[0336] [Table 6]

[0337] The physicochemical stability of the bispecific antibody P1AA 8906 was evaluated as described above in the "Materials and General Methods" section.

[0338] [Table 7]

[0339] [Table 8]

[0340] Example 4: Improvement of the bispecific anti-VEGF-A / anti-ANG2 Fab fragment P1AA8906 As mentioned above, P1AA8906 shows high thermal stability and favorable affinity for VEGF-A, while showing affinity for ANG2 in the nanomolar range and tendency to form high molecular weight impurities that increase under stress. For the treatment of ocular vascular diseases that require injection of therapeutic agents into the eye, it is desirable to provide therapeutic agents with high affinity for target antigens and very high concentrations to enhance the duration of therapeutic effect and minimize inconvenience to patients. Therefore, it is desirable to increase affinity and improve physicochemical stability for the intended purpose.

[0341] As a result, for clinical application, the antibody required further improvement, e.g., with regard to ANG2 binding (especially by improving the off-rate), and reduced susceptibility to stress. Several rounds of maturation were performed by introducing different amino acid substitutions in the VH and VL domains. During maturation, candidate antibodies derived from antibody P1AA8906 were screened and selected based on their desired properties with regard to yield, affinity, simultaneous antigen binding, hydrophilicity, stability, viscosity and other parameters.

[0342] Improved candidate antibody P1AA0902 was selected from multiple tested candidate antibody molecules.Starting from this molecule, further optimization rounds were carried out by reintroducing different amino acid substitutions in VH and VL domains.Candidate selection was based on its desired properties, especially improved ANG2 binding and guaranteed injectability at high concentration, while maintaining other advantageous properties such as VEGF-A affinity, simultaneous antigen binding and thermal stability.

[0343] A further improved candidate antibody, P1AD9820, was selected from multiple tested candidate antibody molecules.

[0344] [Table 9]

[0345] Figures 2 and 3 show alignments of the variable heavy and variable light domains of the generated bispecific fragments. The numbering of the amino acid positions within the VH and VL domains follows the Kabat numbering system. For simplicity, the numbering is included in the figures to further illustrate the framework and CDR amino acid positions.

[0346] Candidate antibodies were expressed as described in Example 2.

[0347] Example 5: Improved antigen-binding kinetics of anti-VEGF-A / anti-ANG2 Fab fragments The binding kinetics of the candidate antibodies to human VEGF-A and human ANG2 were evaluated as described above using the indicated Fab fragments (amino acid sequences shown in Table 4). To compare the antigen-binding kinetics of the antibodies of the present invention with the prior art molecules faricimab (INN, also previously called RG7716), the anti-VEGF binders aflibercept (INN) and brolucizumab (INN), and the anti-ANG2 binder nesbacumab (INN), the aforementioned prior art antibodies were prepared by recombinant expression of the identical amino acid sequences disclosed in the respective INNs. The reference molecules are also referred to herein as "analogs" to emphasize that they were in-house preparations.

[0348] [Table 10]

[0349] [Table 11]

[0350] [Table 12]

[0351] [Table 13]

[0352] [Table 14]

[0353] [Table 15]

[0354] Independent binding of candidate antibodies to human VEGF-A and human ANG2 was analyzed by SPR as described above in the "Materials and General Methods" section.

[0355] [Table 16]

[0356] Cross-reactivity to VEGF-A and ANG2 from other species was assessed as described above in the "Materials and General Methods" section.

[0357] [Table 17]

[0358] [Table 18]

[0359] Example 6 Improved thermal stability of anti-VEGF-A / anti-ANG2 Fab fragments The thermal stability of the indicated bispecific antibodies was evaluated under the same conditions as in Example 3, as described above in the "Materials and General Methods" section.

[0360] [Table 19]

[0361] Example 7: Biophysical properties (stability) of improved bispecific anti-VEGF-A / anti-ANG2 Fab fragments The physicochemical stability of the indicated bispecific antibodies was evaluated under the same conditions as in Example 3, as described above in the "Materials and General Methods" section.

[0362] [Table 20]

[0363] Example 8: Biophysical characterization of improved bispecific anti-VEGF-A / anti-ANG2 Fab fragments (viscosity assessed by dynamic light scattering (DLS)) The aforementioned P1AA0902 and P1AD9820 Fab fragments were expressed in E. coli cells by standard methods.

[0364] Viscosity was measured using the latex-bead DLS method as previously described (He F et al.; Anal Biochem. 2010 Apr 1; ​​399(1):141-3). Specifically, the following protocol was followed using the indicated materials.

[0365] Viscosity Rating: Equipment and Materials Wyatt DLS plate reader with Greiner Bio-One microplates 3000 Series Nanosphere™ size standard (Thermofisher catalogue-number 3300A) Tween 20 (Roche, Cat. No. 11332465001) and silicone oils, e.g. (Alfa Aesar Cat. No. A12728) · UV photometer for concentration determination (e.g. Nanodrop 8000).

[0366] Sample preparation The antibody samples were rebuffered and diluted with 20 mM His / HCl, pH 6.0 (buffer) and 0.02% Tween 20 (final concentration). A bead concentration of 0.03% solids was added. At least four different concentrations were prepared, with the highest possible concentration being approximately 200 mg / mL. Two blank samples were required as controls without antibody: one containing nanosphere beads resuspended in water and one containing nanosphere beads resuspended in buffer. The samples were transferred to a microplate and each well was covered with silicone oil.

[0367] Measurements using a Wyatt DLS plate reader All samples and blanks were analyzed at different temperatures from 15 °C to 35 °C in 5 °C steps. The acquisition time was 30 s and the number of acquisitions was 40 per sample and temperature.

[0368] 4. Data Analysis The raw data Dapp (apparent radius) in nm was shown in the overview of the software template (Microsoft Dynamics 7.0 or higher). The viscosity was calculated using the formula (ηreal=Dapp*ηH2O / Dreal), where Dreal is the bead size measured in a blank sample, which is equal to the bead size (300 nm). The calculated viscosity was shown in an Excel curve. Using a Mooney curve fit (Excel), it is possible to extrapolate the viscosity at a given concentration. Here, the maximum protein concentration where the viscosity is above 20 cP was calculated.

[0369] The maximum concentrations of the indicated antibodies to achieve a viscosity of 20 cP at 20° C. are shown below. The results are also shown in FIG.

[0370] [Table 21]

[0371] The results show that the antibodies of the invention can be formulated at high concentrations, including viscosities below the acceptable viscosity limit for injectability. Both tested antibodies have been shown to be highly concentratable, although the effect is more pronounced for the P1AD9820 antibody.

[0372] As a result, the antibodies of the invention are highly suitable for ocular applications since they allow to provide high molar doses in limited injection volumes, which when combined with high efficacy leads to high durability and therefore reduced dosing frequency, which is desirable to reduce patient discomfort.

[0373] In another set of experiments, the viscosity of P1AD9820, produced in CHO cells according to standard recombinant methods, was analyzed as described above.

[0374] The maximum concentrations of the indicated antibodies to achieve a viscosity of 20 cP at 20° C. are shown below. The results are also shown in FIG.

[0375] [Table 22]

[0376] Example 9: Improved functional stability of anti-VEGF-A / anti-ANG2 Fab fragments P1AD9820 Fab fragments were treated under different stress conditions as described above in the "Materials and General Methods" section (physicochemical stability). The antigen binding of the resulting stressed antibody samples was analyzed as described above ("Materials and General Methods" section: human VEGF-A and human ANG2 binding kinetics assessed by SPR for functional stability testing).

[0377] [Table 23]

[0378] Example 10: Functional characterization of improved anti-VEGF-A / anti-ANG2 Fab fragments Candidate antibodies were assessed for ANG2 and VEGF-A inhibition in cell-based assays: ANG2 inhibition: pTie2-assay Candidate antibodies and selected reference molecules (nesbacumab analogues of in-house preparations by recombinant expression according to SEQ ID NOs: 29 and 30) were pre-incubated at 11 concentrations ranging from 250 nM to 0.14 nM in MEM Alpha-Medium. 3x concentrated anti-Ang2 antibodies in 40 μl were mixed with 40 μl of Ang-2 at 3x concentration c=3.9 μg / ml and incubated for 30 min in a cell incubator. Then 40 μl / well of Hek293_HOMSA-Tie2-Clone22_B11 cells suspended in FreeStyle medium without selection at a density of 40000 cells per well were added for 10 min at room temperature. Ang2 phosphorylation was then stopped by addition of cold lysis buffer containing protease inhibitors. Tie2 was then immobilized from the cell lysate by anti-Tie2 antibodies bound to maxisorp-plates for 90 min at room temperature with shaking. After a washing step with PBS, 0.05% Tween 20, a biotinylated antibody specific for phosphorylated Tie2 was added to a final concentration of 3 μg / ml for 1 h at RT. After three washing steps, HRP-conjugated streptavidin was added to a final concentration of 100 mU / ml and the plates were incubated at room temperature for 30 min, during which POD was converted, for final colorimetric endpoint assessment after stopping with 1M H2SO2 at 405 / 690 nm using a Tecan Sunrise plate reader. The results are shown below and in Figure 8.

[0379] [Table 24]

[0380] VEGF-A Inhibition: Reporter Gene Assay (RGA) For the assay, 37.5 μl of 4-fold concentrated test antibody or reference molecule was pre-incubated with 37.5 μl of 4-fold concentrated VEGF-A121 (R&D, c=100 μg / ml) for 15 min at room temperature. The mixture was then added to 75 μl / well of Hek_NFAT_KDR_Luc cells at a density of 40000 cells / well and incubated for 5 h in a cell incubator. Finally, luminescence was evaluated using an Infinite Pro Platereader (Tecan) after adding 100 μl of BioGlo reagent per well. The results are shown below and in FIG. 7.

[0381] [Table 25]

[0382] Example 11: Blocking activity of hVEGF-A121 and hVEGF-A165 (VEGF baseline assay) Maxisorp 96-well plates (ThermoScientific #442404) were coated with 50 μL / well of hVEGFR-1-Fc in 200 mM NaHCO3, pH 9.4 at a final concentration of 1 μg / mL for 1 hour at room temperature. The indicated candidate Fab fragments were diluted to a concentration of 409.6 nM in 280 μL of PBST-1% BSA. For two-fold serial dilutions, 140 μL of this diluted Fab sample was mixed with 140 μL of PBST-1% BSA and mixed 7 times by gentle pipetting. This two-fold dilution step was repeated 9 more times. Round-bottom 96-well plates were pre-filled with 50 μL / well of 2 nM VEGF-A121 or 2 nM VEGF-A165 in PBST-1% BSA. 50 μL of the Fab dilutions were added to the VEGF plate, mixed 6 times, and incubated for 1.5 hours. The maxisorp plate was then washed twice with PBST, after which 200 μL of 2% MPBST was added, followed by incubation at room temperature for 45 min. The plate was then washed twice with PBST. 50 μl of Fab-VEGF premix was transferred to the Maxisorp plate and incubated at room temperature for 1.5 h. The plate was then washed twice with PBST, and 50 μL of anti-VEGF-bio antibody (diluted 1:2000 in PBST) and SA-HRP (diluted 1:2000 in PBST) were added and incubated at room temperature for 30 min. The plate was washed six times with PBST, and 50 μL of TMB substrate solution was added and incubated at room temperature for 30 min. Finally, the reaction was stopped by adding 50 μL of 1N sulfuric acid, and the absorbance was read at 450 nm. The results are shown in Figures 5 and 6.

[0383] Example 12: Selective binding of ANG2 over ANG1 Selectivity for Ang-2 versus Ang-1 may be an important attribute for ocular safety: ANG1 inhibition of P1AD9820 Fab was evaluated in a cell-based assay.

[0384] ANG-1 is a weak inducer of viability in HUVECs grown under starvation conditions. Therefore, viability can be inhibited with ANG-1 neutralizing antibodies. Antibody RO5314196 (formerly LC-08-Thomas M, Kienast Y, Scheuer W, et al. A novel angiopoietin-2 selective fully human antibody with potent anti-tumoral and anti-angiogenic efficacy and superior side effect profile compared to Pan-Angiopoietin-1 / -2 inhibitors. PLoS One. 2013;8(2):e54923. doi:10.1371 / journal.pone.0054923) inhibits HUVEC viability by neutralizing ANG-1 and was used as a positive control in the assay. Corning cell culture flasks T162 (cat. no. 3151) coated with AF (attachment factor) (cat. no. S-006-100) from Gibco were used to maintain HUVECs up to the 5th passage. For viability assays, HUVECs were detached with Accutase® followed by a washing step with PBS- / -. Cells were then seeded at a cell density of 10.000 cells / well in 100 μl in EBM-2 with 0.5% FBS on fibronectin-coated 96-well plates. Cells were incubated overnight at 37° C., 5% CO2. The next day, P1AD9820 or the Ang-1 binding positive control RO5314196 were diluted in EBM / 0.5% FBS to a 10x working concentration. The starting concentration was 1000 μg / ml followed by a 3x 8-step dilution series ending at 457.2 μg / ml. ANG-1 was set to a 20x working concentration equivalent to 2400ng / ml. Then, 10μl of 10x prediluted P1AD9820 was added to the cells in quadruplicate wells per plate, followed by 5μl of 20xANG-1 solution. On each experimental day, each condition was performed in duplicate plates. Cells were incubated at 37°C, 5% CO2 for 72 hours.For analysis, 11 μl of alamarBlue® was added to each well followed by incubation for 4 hours in a cell culture incubator. Absorbance was detected at 570 nm with a reference wavelength of 600 nm.

[0385] For each experiment, conditions were replicated using two assay plates with each condition performed in quadruplicate. The background signal of unstimulated cells was subtracted from the experimental wells to calculate the average signal per condition. The 100% response level was calculated from cells stimulated with ANG-1 (120 ng / ml) without additional treatment, and the signal from antibody-treated wells was expressed as percent inhibition of the 100% response. For P1AD9820 and RO5314196, the percent inhibition for each antibody treatment was determined in n=4 independent experiments, and the mean and standard error of the mean were calculated. IC 50 Values ​​were calculated from the mean data for each antibody concentration using ExcelXLfit software (IDBS). Concentration-response curves were fitted by nonlinear regression analysis using a five-parameter logistic model (A+((BA) / (1+((C / x)^D)))) calculated for basal and maximal inhibitory activity.

[0386] Cell-based assays did not detect ANG1 inhibition mediated by P1AD9820.

[0387] Example 13: Structural analysis of P1AD9820 Fab fragment Structural analysis of the P1AD9820 Fab was performed by X-ray crystallography as follows: Complex formation and purification of the ternary complex Angiopoietin 2-RBD-VEGF-A121-P1AD9820. For complex formation, P1AD9820 Fab and human VEGF-A121 (Peprotech) were mixed at a molar ratio of 1.1:1. After 45 min of incubation at 4°C, Angiopoietin 2-RBD was added at a molar ratio of 1.25:1 to P1AD9820, followed by another 60 min of incubation at 4°C. The resulting ternary complex was further deglycosylated with PNGaseF (NEB) at 37°C for 13 h and purified in the final step by gel filtration chromatography on a Superdex200 (16 / 600) column. The fractions containing the ternary complex were pooled and concentrated to 3.55 mg / ml.

[0388] Crystallization of ternary angiopoietin 2-RBD-VEGF-A121-P1AD9820. Initial crystallization trials were performed at 14.5mg / ml protein concentration in a sitting drop vapor diffusion apparatus at 21°C. Crystals appeared within a day out of 35% MPD, 0.1M Na / K phosphate pH 6.2. Plate-like crystals grew over a week, with a final size of 100x80x30μm. The crystals were picked directly from the screening plate without further optimization steps.

[0389] Data collection and structure determination. For data collection, crystals were flash-cooled at 100 K in the precipitant solution without additional cryoprotectant. Diffraction data were collected at beamline X10SA at the Swiss Light Source (Villigen, Switzerland) with a PILATUS 6M detector at a wavelength of 1.0000 Å. Data were processed with XDS (Kabsch, W. Acta Cryst. D66, 133-144 (2010)) and scaled with SADABS (BRUKER). The crystal belongs to space group C2 with cell axes a = 165.74 Å, b = 90.27 Å, c = 127.89 Å, β = 112.05°, and diffracts to a resolution of 2.08 Å. The structure was determined by molecular replacement in PHASER (McCoy, AJ, Grosse-Kunstleve, RW, Adams, PD, Stroni, LC, and Read, RJ J Appl. Cryst. 40, 658-674 (2007)) using the coordinates of a related in-house structure of the Ang2-VEGF-Fab ternary complex as a search model. The data were subsequently refined using programs from the CCP4 suite (Collaborative Computational Project, Number 4 Acta Cryst. D50, 760-763 (1994)) and Buster (Bricogne, G., Blanc, E., Brandl, M., Flensburg, C., Keller, P., Paciorek, W., Roversi, P., Sharff, A., Smart, O.S., Vonrhein, C., Womack, T.O. (2011). Buster version 2.9.5 Cambridge, United Kingdom: Global Phasing Ltd). Manual reconstruction of the protein using differential electron density was performed with COOT (Emsley, P., Lohkamp, ​​B., Scott, W.G. and Cowtan, K. Acta Cryst D66, 486-501 (2010)). Data collection and refinement statistics for both structures are summarized in Table 23. All graphical displays were generated in PYMOL (DeLano Scientific, Palo Alto, CA, 2002).

[0390] [Table 26]

[0391] The amino acid residues in contact with the respective antigens VEGF-A and ANG2 were identified from the crystal structure of the ternary complex angiopoietin 2-RBD-VEGF-A121-P1AD9820. Illustrative locations of the paratope amino acid residues within the VH and VL domains are shown in Figures 2 and 3.

[0392] The amino acid residues identified as contributing to antigen binding are identified in Table 24 (for variable heavy domain amino acid residues) and Table 25 (for variable light domain amino acid residues). The amino acid positions are numbered according to the Kabat numbering system (the same numbering is used in Figures 2 and 3). The amino acid positions involved in antigen binding are identified by their Kabat position in the VH or VL domain (see also the numbering in Figures 2 and 3).

[0393] [Table 27]

[0394] [Table 28]

Claims

1. An antibody that binds to human VEGF-A and human ANG2, comprising a VH domain comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 3, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 21, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14, and a VL domain comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 22, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 23, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:

8.

2. 1. An antibody that binds to human VEGF-A and human ANG2, comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:3; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:21; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:14; (d) a VH domain comprising a human heavy chain framework having (i) an FR1 comprising amino acid residues H3, D26, F27, E29, and Y30; (ii) an FR3 comprising amino acid residues R66 and R94; and (e) an amino acid sequence of SEQ ID NO:

22. (f) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:23; (g) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:8; and (h) a VL domain comprising a human light chain framework having (i) a FR1 comprising amino acid residues I2 and Y3, (ii) a FR2 comprising amino acid residues L46 and F49, and (iii) a FR3 comprising amino acid residue E57, wherein the numbering of said VH domain and said VL domain is according to the Kabat numbering system.

3. The antibody described in claim 1 or 2, comprising (a) a VH domain comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 19, and (b) a VL domain comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:

20.

4. (a) a VH domain comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 19, the VH domain comprising amino acid residues H3, D26, F27, E29, Y30, D35b, D35c, D55, H56, K57, Y58, T61, K62, F63, I64, G65, R66, R94, D95, V96, F98, and F99; and (b) a VH domain comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:

20.

4. The antibody of claim 1, wherein the numbering of the VH domain and the VL domain is according to the Kabat numbering system.

5. 5. An antibody according to any one of claims 1 to 4, comprising (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 19 with up to 15 amino acid substitutions, and (b) a variable light domain comprising the amino acid sequence of SEQ ID NO: 20 with up to 15 amino acid substitutions.

6. An antibody that binds to human VEGF-A and human ANG2, comprising the VH sequence of SEQ ID NO:19 and the VL sequence of SEQ ID NO:

20.

7. The antibody Fab fragment of the antibody has (i) a K of less than 50 pM as measured by KinExA. Ｄ binds to human VEGF-A121 with a K of less than 50 pM as measured by KinExA; Ｄ 7. The antibody of claim 1 , which binds to human ANG2 at

8. 8. The antibody of any one of claims 1 to 7, wherein a solution of 180 mg / ml of the antibody Fab fragment in 20 mM His / HisHCl, pH 6.0 has a viscosity of less than 20 cP at 20°C as detected by dynamic light scattering using the latex-bead DLS method described in Example 8.

9. The antibody of claim 1 , wherein the antibody is a Fab fragment.

10. An isolated nucleic acid encoding an antibody according to any one of claims 1 to 9.

11. A host cell comprising the nucleic acid of claim 10.

12. 12. A method for producing an antibody that binds to human VEGF-A and human ANG2, comprising culturing the host cell of claim 11 so that said antibody is produced.

13. 13. The method of claim 12, wherein the host cell is a CHO cell.

14. A pharmaceutical formulation comprising an antibody according to any one of claims 1 to 9 and a pharma- ceutically acceptable carrier.

15. A port delivery device comprising an antibody according to any one of claims 1 to 9.

16. An antibody according to any one of claims 1 to 9 for use as a medicament.

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