Antibodies that bind VEGF and PDGF-B and methods of use

Abstract

The present invention relates to anti-VEGF / anti-PDGF-B antibodies and methods of use thereof.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to anti-VEGF / anti-PDGF-B antibodies and methods of use thereof. [Background technology]

[0002] 2. Background of the Invention Combination therapy approaches using separate VEGF and PDGF antagonists to block VEGF and PDGF for the treatment of ocular neovascular diseases have been suggested previously (WO 2005 / 020972, WO 2010 / 127029, WO 2016 / 025313).

[0003] Bispecific antibodies that bind PDGF-B and VEGF have been previously reported and suggested for the treatment of ocular vascular disease (WO 2016 / 075036, antibody "0117"). The bispecific anti-VEGF / anti-PDGF-B antibody 0117 is a full-length IgG-like antibody with a domain cross-swap in one binding arm (Schaefer, W. et al, PNAS, 108 (2011) 11187-1191). The VEGF binding arm contains the VH and VL domains of the anti-VEGF antibody ranibizumab.

[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 non-overlapping paratopes in a pair of VH and VL domains ("DutaFab"). Each paratope of the bispecific antibodies of WO 2012 / 163520 contains 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 screened independently from two independent Fab libraries. 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] There is a need for improved therapeutic antibodies that bind to VEGF and PDGF-B. Summary of the Invention

[0007] Summary of the Invention The present invention relates to bispecific anti-VEGF / anti-PDGF-B antibodies and methods of use thereof.

[0008] In one aspect, the invention provides an antibody that binds to human VEGF and human PDGF-B, comprising a VEGF paratope and a PDGF-B paratope within a cognate pair of variable light domains (VL domains) and variable heavy domains (VH domains), wherein the VEGF paratope comprises amino acid residues from CDR-H2, CDR-L1 and CDR-L3 of the antibody, and the PDGF-B paratope comprises 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 and human PDGF-B, the antibody comprising a VEGF paratope and a PDGF-B paratope within a cognate pair of VL and VH domains, wherein the pair of variable light and variable heavy domains simultaneously binds to human VEGF and human PDGF-B.

[0010] In one aspect, the invention provides an antibody that binds to human VEGF and human PDGF-B, wherein the antibody comprises a VEGF paratope and a PDGF-B paratope within a cognate pair of VL and VH domains, and wherein none of the amino acids contained in the VEGF paratope are contained in the PDGF-B paratope.

[0011] In one aspect, the invention provides an antibody that binds to human VEGF and human PDGF-B, the antibody comprising a VEGF paratope and a PDGF-B paratope within a cognate pair of VL and VH domains, and which binds to the same epitope on human VEGF and the same epitope on human PDGF-B as an antibody having a variable heavy domain of SEQ ID NO:11 and a variable light domain of SEQ ID NO:21.

[0012] In one aspect, the invention provides an antibody that binds to human VEGF and human PDGF-B, wherein the antibody Fab fragment of the antibody has (i) a K of less than 50 pM as measured by surface plasmon resonance. D and (ii) binds to human VEGF121 with a K of less than 10 nM as measured by surface plasmon resonance. D The present invention provides an antibody that binds to human PDGF-B at a specific site.

[0013] In one aspect, the present invention provides an antibody that binds to human VEGF and human PDGF-B, wherein the antibody Fab fragment exhibits an aggregation onset temperature of 68° C. or higher.

[0014] In one aspect, the invention provides an antibody that binds to human VEGF and human PDGF-B, 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: 13, 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: 15, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17.

[0015] In one aspect, the invention provides an antibody that binds to human VEGF and human PDGF-B, 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: 29, 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: 15, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17.

[0016] In one aspect, the invention provides an antibody that binds human VEGF and human PDGF-B, 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: 13; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14; and (d) a VH domain comprising a human heavy chain framework having: (i) an FR1 comprising amino acid residues D1, L2, D25, G26, W27, and W28; and (ii) an FR3 comprising amino acid residues D73, D74, T75, N76, and R94. and (e) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 15, (f) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, (g) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17, and (h) a VL domain comprising a human light chain framework having (i) FR1 comprising amino acid residues A1, I2, and (ii) FR3 comprising amino acid residues H68 and E69, wherein the numbering of the VH domain and the VL domain is according to the Kabat numbering system.

[0017] In one aspect, the invention provides an antibody that binds human VEGF and human PDGF-B, 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: 29; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14; and (d) a VH domain comprising a human heavy chain framework having: (i) an FR1 comprising amino acid residues D1, L2, D25, G26, W27, and W28; and (ii) an FR3 comprising amino acid residues D73, D74, T75, N76, and R94. and (e) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 15, (f) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, (g) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17, and (h) a VL domain comprising a human light chain framework having (i) FR1 comprising amino acid residues A1, I2, and (ii) FR3 comprising amino acid residues H68 and E69, wherein the numbering of the VH domain and the VL domain is according to the Kabat numbering system.

[0018] In one aspect, the invention provides an antibody that binds human VEGF and human PDGF-B, comprising (ii) a VH domain comprising amino acid residues D1, L2, D25, G26, W27, W28, Y31, T35b, Y52a, G55, S56, T57, Y58, T61, K62, F63, I64, G65, R66, D73, D74, T75, N76, R94, D96, Y98, D101, and T102, and (iii) a VL domain comprising amino acid residues A1, I2, S26, Y27, W27a, L27b, S27c, E55, H56, H68, E69, R92, Y93, H94, P95 and Y96; wherein numbering of the VH domain and VL domain is according to the Kabat numbering system. In one embodiment, the antibody comprises a VEGF paratope comprising the following amino acid residues in the VH domain: G55, S56, T57, Y58, T61, K62, F63, I64, G65, and R66, and the following amino acid residues in the VL domain: A1, I2, S26, Y27, W27a, L27b, S27c, H68, E69, R92, Y93, H94, P95, and Y96; and a PDGF-B paratope comprising the following amino acid residues in the VH domain: D1, L2, D25, G26, W27, W28, Y31, T35b, D73, D74, T75, N76, R94, D96, D98, D101, and T102, and the following amino acid residues in the VL domain: E55 and H56.

[0019] In one aspect, the invention provides an antibody that binds human VEGF and human PDGF-B, comprising (ii) a VH domain comprising amino acid residues D1, L2, D25, G26, W27, W28, Y31, T35b, Y52a, G55, H56, K57, Y58, T61, K62, F63, I64, G65, R66, D73, D74, T75, N76, R94, D96, Y98, D101, and T102, and (iii) a VL domain comprising amino acid residues A1, I2, S26, Y27, W27a, L27b, S27c, E55, H56, H68, E69, R92, Y93, H94, P95 and Y96; wherein numbering of the VH domain and VL domain is according to the Kabat numbering system. In one embodiment, the antibody comprises a VEGF paratope comprising the following amino acid residues in the VH domain: G55, H56, K57, Y58, T61, K62, F63, I64, G65, and R66, and the following amino acid residues in the VL domain: A1, I2, S26, Y27, W27a, L27b, S27c, H68, E69, R92, Y93, H94, P95, and Y96; and a PDGF-B paratope comprising the following amino acid residues in the VH domain: D1, L2, D25, G26, W27, W28, Y31, T35b, D73, D74, T75, N76, R94, D96, D98, D101, and T102, and the following amino acid residues in the VL domain: E55 and H56.

[0020] In one aspect, the invention provides an antibody that binds to human VEGF and human PDGF-B, 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:11; 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:21.

[0021] In one aspect, the invention provides an antibody that binds to human VEGF and human PDGF-B, 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:11; 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:25.

[0022] In one aspect, the invention provides an antibody that binds to human VEGF and human PDGF-B, 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: 28; 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: 25.

[0023] In one aspect, the invention provides an antibody that binds to human VEGF and human PDGF-B, 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: 28; 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: 31.

[0024] In one aspect, the invention provides an antibody that binds to human VEGF and human PDGF-B, 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: 13, and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 14; and (d) a VL domain comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 15, (e) a CDR-L2 having the amino acid sequence of SEQ ID NO: 16, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 17, 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: 11; 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: 21.

[0025] In one aspect, the invention provides an antibody that binds to human VEGF and human PDGF-B, 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: 13, 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: 15, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17; 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: 11, the VH domain comprising D1, L2, D25, G26, W27, W28, R66, D73, D74, T75, N76, 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: 21, the VL domain comprising A1, I2, H68, and E69, wherein the numbering of the VH domain and the VL domain is according to the Kabat numbering system.

[0026] In one aspect, the invention provides an antibody that binds human VEGF and human PDGF-B, 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: 13; 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 D1, L2, D25, G26, W27, and W28, (ii) FR3 comprising amino acid residues D73, D74, T75, N76, and R94; and (e) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 15; and (f) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16. and (h) a VL domain comprising a human light chain framework having (i) an FR1 comprising amino acid residues A1 and I2, and (ii) an FR3 comprising amino acid residues H68 and E69, wherein the numbering of the VH domain and the VL domain is according to the Kabat numbering system, and wherein the numbering of the VH domain and the VL domain 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: 11, 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: 21.

[0027] In one aspect, the invention provides an antibody that binds to human VEGF and human PDGF-B, comprising: (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 11 with up to 15 amino acid substitutions; and (b) a variable light domain comprising the amino acid sequence of SEQ ID NO: 21 with up to 15 amino acid substitutions. In one embodiment, the antibody comprises: (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 11 with up to 15 amino acid substitutions located at positions 3 to 24, 29, 30, 35c to 52, 52b, 54, 59, 60, 67 to 72, 77 to 93, 95, 97, 99, or 103 to 113; (b) a variable light domain comprising the amino acid sequence of SEQ ID NO: 21 with up to 15 amino acid substitutions located at positions 3 to 25, 27d to 54, 57 to 67, 70 to 91, or 97 to 107 of SEQ ID NO: 21, wherein the numbering of the VH domain and the VL domain is according to the Kabat numbering system.

[0028] In one aspect, the invention provides an antibody that binds to human VEGF and human PDGF-B, 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: 13, 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: 15, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17, and comprising: (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 11 with up to 15 amino acid substitutions; and (b) a variable light domain comprising the amino acid sequence of SEQ ID NO: 21 with up to 15 amino acid substitutions.

[0029] In one aspect, the invention provides an antibody that binds human VEGF and human PDGF-B, 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: 13; 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 D1, L2, D25, G26, W27, and W28, (ii) FR3 comprising amino acid residues D73, D74, T75, N76, and R94; and (e) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 15; (f) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16. (g) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17; and (h) a VL domain comprising a human light chain framework having (i) FR1 comprising amino acid residues A1 and I2, and (ii) FR3 comprising amino acid residues H68 and E69, wherein numbering of the VH domain and the VL domain is according to the Kabat numbering system, wherein the antibody comprises: (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 11 with up to 15 amino acid substitutions; and (b) a variable light chain domain comprising the amino acid sequence of SEQ ID NO: 21 with up to 15 amino acid substitutions.

[0030] In one embodiment, an antibody of the invention comprises a light chain framework region 1 (L-FR-1) comprising the amino acid sequence of SEQ ID NO:22, SEQ ID NO:26, or SEQ ID NO:32.

[0031] In one aspect, the invention provides an antibody that binds human VEGF and human PDGF-B comprising the VH sequence of SEQ ID NO:11 and the VL sequence of SEQ ID NO:21.

[0032] In one aspect, the invention provides an antibody that binds human VEGF and human PDGF-B comprising a VH sequence of SEQ ID NO:11 and a VL sequence of SEQ ID NO:25.

[0033] In one aspect, the invention provides an antibody that binds human VEGF and human PDGF-B comprising the VH sequence of SEQ ID NO:28 and the VL sequence of SEQ ID NO:25.

[0034] In one aspect, the invention provides an antibody that binds human VEGF and human PDGF-B comprising the VH sequence of SEQ ID NO:28 and the VL sequence of SEQ ID NO:31.

[0035] In one aspect, the invention provides an antibody that binds human VEGF and human PDGF-B, comprising a heavy chain amino acid sequence of SEQ ID NO:18 and a light chain amino acid sequence of SEQ ID NO:20.

[0036] In one aspect, the invention provides an antibody that binds human VEGF and human PDGF-B, comprising a heavy chain amino acid sequence of SEQ ID NO:18 and a light chain amino acid sequence of SEQ ID NO:19.

[0037] In one aspect, the invention provides an antibody that binds human VEGF and human PDGF-B, comprising a heavy chain amino acid sequence of SEQ ID NO:18 and a light chain amino acid sequence of SEQ ID NO:27.

[0038] In one aspect, the invention provides an antibody that binds human VEGF and human PDGF-B, comprising a heavy chain amino acid sequence of SEQ ID NO:30 and a light chain amino acid sequence of SEQ ID NO:27.

[0039] In one aspect, the invention provides an antibody that binds human VEGF and human PDGF-B, comprising a heavy chain amino acid sequence of SEQ ID NO:30 and a light chain amino acid sequence of SEQ ID NO:33.

[0040] In one aspect, the invention provides an antibody that binds human VEGF and human PDGF-B, 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: 13; 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 D1, L2, D25, G26, W27, and W28, (ii) FR3 comprising amino acid residues D73, D74, T75, N76, and R94; and (e) an amino acid sequence of SEQ ID NO: 15. (f) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16; (g) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17; and (h) a VL domain comprising a human light chain framework having (i) FR1 comprising amino acid residues A1 and I2, and (ii) FR3 comprising amino acid residues H68 and E69, wherein the numbering of the VH domain and the VL domain is according to the Kabat numbering system, and wherein an antibody Fab fragment of the antibody has (i) a K of less than 50 pM as measured by surface plasmon resonance. D and (ii) binds to human VEGF121 with a K of less than 10 nM as measured by surface plasmon resonance. D The present invention provides an antibody that binds to human PDGF-B at a specific site.

[0041] In one aspect, the invention provides an antibody that binds to human VEGF and human PDGF-B, 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: 13, 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: 15, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17, wherein the antibody Fab fragment of 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: 11; 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: 21, Dand (ii) binds to human VEGF121 with a K of less than 10 nM as measured by surface plasmon resonance. D The present invention provides an antibody that binds to human PDGF-B at

[0042] In one aspect, the present invention provides an antibody that binds to human VEGF and human PDGF-B, 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: 13, 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: 15, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17, 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: 11; 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: 21, and an antibody Fab fragment of the antibody exhibits an aggregation onset temperature of 68°C or higher.

[0043] One embodiment of the present invention relates to an antibody fragment that binds to human VEGF and human PDGF-B. One embodiment of the present invention relates to a bispecific antibody fragment that binds to human VEGF and human PDGF-B. In one embodiment, the antibody fragment is an Fv, Fab, Fab', Fab'-SH, F(ab') 2 or single chain antibodies derived therefrom. One embodiment of the invention relates to a Fab fragment that binds to human VEGF and human PDGF-B. One embodiment of the invention relates to an Fv fragment that binds to human VEGF and human PDGF-B.

[0044] One embodiment of the present invention relates to a full-length IgG antibody that binds to human VEGF and human PDGF-B.

[0045] In one aspect, the invention provides an isolated nucleic acid encoding an antibody of the invention.

[0046] In one aspect, the invention provides a host cell comprising a nucleic acid of the invention.

[0047] In one aspect, the invention provides an expression vector comprising a nucleic acid of the invention.

[0048] In one aspect, the invention provides a method for producing an antibody that binds human VEGF and human PDGF-B, the method comprising culturing a host cell of the invention so that the antibody is produced.

[0049] In one aspect, the invention provides an antibody produced by the method of the invention.

[0050] In one aspect, the invention provides a pharmaceutical formulation comprising an antibody of the invention and a pharma- ceutically acceptable carrier.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] According to the present invention, therapeutic anti-VEGF / anti-PDGF-B antibodies are provided that can simultaneously bind to their target antigens even when provided as bispecific Fab fragments. Furthermore, the antibodies of the present invention offer several beneficial properties that allow their therapeutic application, such as high affinity, hydrophilicity, high stability and high VEGF121 and VEGF165 blocking activity. The antibodies of the present invention can be provided in high concentration liquid formulations with a viscosity suitable for application to the eye. The antibodies of the present invention are suitable for the treatment of ocular vascular diseases. [Brief description of the drawings]

[0056] [Figure 1] Schematic diagram of the Fab fragment of an anti-VEGF / anti-PDGF-B antibody of the invention. A top view of the cognate VH / VL pair including the arrangement of the CDR amino acids is shown (upper 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 (lower image), with the VEGF paratope located in the regions H-CDR2, L-CDR1 and L-CDR2 and the PDGF-B paratope located in the regions H-CDR1, H-CDR3 and L-CDR2. LN: N-terminus of the VL domain; HN: N-terminus of the VH domain; [Diagram 2] 1 shows the amino acid sequence of the VH domain of an exemplary anti-VEGF / anti-PDGF-B antibody of the invention. Kabat numbering of amino acid positions and CDR and FR regions are shown. Amino acid positions contributing to the VEGF paratope and the PDGF-B paratope identified in Example 8 are highlighted. [Diagram 3] 1 shows the amino acid sequence of the VL domain of an exemplary anti-VEGF / anti-PDGF-B antibody of the invention. Kabat numbering of amino acid positions and CDR and FR regions are shown. Amino acid positions contributing to the VEGF paratope and the PDGF-B paratope identified in Example 8 are highlighted. [Figure 4] VEGF121 and VEGF165 blocking activity of the indicated antibodies tested in Example 7. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0057] Detailed Description of the Invention 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.

[0058] Unless otherwise defined herein, the term "comprising of" is intended to include the term "consisting of."

[0059] 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.

[0060] 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.

[0061] 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).

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] "Effector functions" refer to biological activities attributable to the Fc region of an antibody and vary depending on the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor); and B cell activation.

[0067] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding of the antibody to an antigen. The variable domains of the heavy and light chains (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 antibodies 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 and PDGF-B.

[0068] "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.

[0069] A "human antibody" is an antibody having an amino acid sequence that corresponds to an antibody produced by a human or a 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.

[0070] 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).

[0071] 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.

[0072] "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 "non-overlapping" paratopes in one cognate VH / VL pair. "Non-overlapping" means that none of the amino acids contained in one of the two paratopes are contained in the other paratope.

[0073] As used herein, a "VEGF paratope" is a paratope or antigen binding site that binds to VEGF. The VEGF paratope of an antibody of the invention comprises amino acid residues from CDR-H2, CDR-L1 and CDR-L3 of the antibody.

[0074] As used herein, a "PDGF-B paratope" is a paratope or antigen binding site that binds PDGF-B. The PDGF-B paratope of an antibody of the invention comprises amino acid residues from CDR-H1, CDR-H3, and CDR-L2 of the antibody.

[0075] The term "VEGF" 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 not only "full-length" unprocessed VEGF, but also 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 VEGF121 is shown in SEQ ID NO:23.

[0076] The terms "anti-VEGF antibody" and "antibody that binds VEGF" refer to an antibody that can bind VEGF with sufficient affinity so as to be useful as a VEGF-targeted diagnostic and / or therapeutic agent. In one embodiment, the extent of binding of the anti-VEGF antibody to unrelated, non-VEGF proteins is less than about 10% of the binding of the antibody to VEGF as measured, for example, by surface plasmon resonance (SPR). In certain embodiments, an antibody that binds VEGF has a dissociation constant (K D ) is 1 nM or less, or 0.1 nM or less. The antibody has a K D An antibody is said to "specifically bind" to VEGF if it has

[0077] The term "PDGF-B" as used herein refers to any native PDGF-B from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term also encompasses "full-length," unprocessed PDGF-B and any form of PDGF-B that results from processing within a cell. The term also encompasses naturally occurring variants of PDGF-B, such as splice variants or allelic variants. An exemplary amino acid sequence of human PDGF-B is shown in SEQ ID NO:24.

[0078] The terms "anti-PDGF-B antibody" and "antibody that binds to anti-PDGF-B" refer to an antibody that is capable of binding to anti-PDGF-B with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting anti-PDGF-B. In one embodiment, the extent of binding of an anti-anti-PDGF-B antibody to an unrelated, non-anti-PDGF-B protein is less than about 10% of the binding of the antibody to anti-PDGF-B as measured, for example, by surface plasmon resonance (SPR). In certain embodiments, an antibody that binds to PDGF-B has a dissociation constant (K D ) The antibody has a K D An antibody is said to "specifically bind" to anti-PDGF-B if it has the following structure:

[0079] The antibodies of the invention "bind simultaneously to human VEGF and human PDGF-B," meaning that (a) an antibody Fab fragment of the invention that is bound to human PDGF-B specifically binds (also) to human VEGF, and (b) an antibody Fab fragment of the invention that is bound to human VEGF specifically binds (also) to human PDGF-B. Simultaneous binding can be assessed by methods known in the art, for example, by surface plasmon resonance.

[0080] 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).

[0081] 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.

[0082] 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]

[0083] 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 Figure 2. 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.

[0084] "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.

[0085] 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.

[0086] Screening for antibodies that bind to a specific epitope (i.e., antibodies that bind the same epitope) can be performed using methods conventional in the art, such as, 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).

[0087] Antigen Structure-based Antibody Profiling (ASAP), also known as Modification-Assisted Profiling (MAP), allows multiple monoclonal antibodies that specifically bind to VEGF or PDGF-B 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.

[0088] Competitive binding can also be used to easily determine whether an antibody binds to the same epitope of VEGF or PDGF-B as a reference antibody of the present invention, or competes for binding. For example, an "antigen that binds to the same epitope on VEGF and PDGF-B" 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 or PDGF-B at saturation. After removing excess reference antibody, the ability of the antibody in question to bind to VEGF or PDGF-B is evaluated. If the antibody in question can bind to VEGF or PDGF-B 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 or PDGF-B after saturation binding of the reference antibody, the antibody of interest may bind 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 or PDGF-B, it can be concluded that the antibody of interest and the reference antibody compete for binding to VEGF or PDGF-B.

[0089] 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).

[0090] 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.

[0091] "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.

[0092] 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 percent amino acid identity is given in the output alignment header.

[0093] 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).

[0094] 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.

[0095] 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.

[0096] 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."

[0097] 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.

[0098] 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.

[0099] 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.

[0100] 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.

[0101] 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.

[0102] 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.

[0103] 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.

[0104] 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.

[0105] 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 and human PDGF-B. The antibodies of the invention are useful, for example, in the diagnosis or treatment of vascular diseases, such as ocular vascular diseases.

[0106] A. Exemplary Antibodies that Bind Human VEGF and Human PDGF-B In one aspect, the invention provides an antibody that binds human VEGF and human PDGF-B. In one aspect, an isolated antibody that binds human VEGF and human PDGF-B is provided. In one aspect, the invention provides an antibody that specifically binds human VEGF and human PDGF-B.

[0107] In certain embodiments, an antibody that binds human VEGF and human PDGF-B, comprising a VEGF paratope (i.e., an antigen binding site that binds VEGF) and a PDGF-B paratope (i.e., an antigen binding site that binds PDGF-B) within one cognate pair of VL and VH domains; the VEGF paratope comprises amino acid residues derived from CDR-H2, CDR-L1 and CDR-L3 of an antibody, and the PDGF-B 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 and human PDGF-B; and / or ● none of the amino acids included in the VEGF paratope are included in the PDGF-B paratope; and / or binds to the same epitope on human VEGF and the same epitope on human PDGF-B as an antibody having a variable heavy domain of SEQ ID NO: 11 and a variable light domain of SEQ ID NO: 21; and / or The antibody Fab fragment of the antibody has (i) a K of less than 50 pM as measured by surface plasmon resonance. D and (ii) binds to human VEGF121 with a K of less than 10 nM as measured by surface plasmon resonance. D binds to human PDGF-B at ● The antibody Fab fragment exhibits an onset aggregation temperature of 68°C or higher.

[0108] In another aspect, the present invention provides an antibody 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: 13, 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: 15, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17; or ● A VH domain comprising (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: 29, 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: 15, (e) a CDR-L2 having the amino acid sequence of SEQ ID NO: 16, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 17.

[0109] In another aspect, the present invention provides an antibody 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: 13, 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 D1, L2, D25, G26, W27, and W28, and (ii) an FR3 comprising amino acid residues D73, D74, T75, N76, and R94, and (e) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 15, (f) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, (g) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17, and (h) a VL domain comprising a human light chain framework having (i) an FR1 comprising amino acid residues A1, and I2, and (ii) an FR3 comprising amino acid residues H68 and E69; or ● (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: 29, and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 14, (d) a VH domain comprising a human heavy chain framework having (i) an FR1 having amino acid residues D1, L2, D25, G26, W27, and W28, and (ii) an FR3 having amino acid residues D73, D74, T75, N76, and R94, and (e) a CDR-L1 having the amino acid sequence of SEQ ID NO: 15, (f) a CDR-L2 having the amino acid sequence of SEQ ID NO: 16, (g) a CDR-L3 having the amino acid sequence of SEQ ID NO: 17, and (h) a VL domain comprising a human light chain framework having (i) an FR1 having amino acid residues A1 and I2, and (ii) an FR3 having amino acid residues H68 and E69 (the VH domain and VL domain are according to the Kabat numbering system).

[0110] In another aspect, the invention provides an antibody comprising (ii) a VH domain comprising amino acid residues D1, L2, D25, G26, W27, W28, Y31, T35b, Y52a, G55, S56, T57, Y58, T61, K62, F63, I64, G65, R66, D73, D74, T75, N76, R94, D96, Y98, D101, and T102, and (iii) a VL domain comprising amino acid residues A1, I2, S26, Y27, W27a, L27b, S27c, E55, H56, H68, E69, R92, Y93, H94, P95 and Y96, wherein numbering of the VH domain and VL domain is according to the Kabat numbering system. In one embodiment, the antibody comprises a VEGF paratope comprising the following amino acid residues in the VH domain: G55, S56, T57, Y58, T61, K62, F63, I64, G65, and R66, and the following amino acid residues in the VL domain: A1, I2, S26, Y27, W27a, L27b, S27c, H68, E69, R92, Y93, H94, P95, and Y96; and a PDGF-B paratope comprising the following amino acid residues in the VH domain: D1, L2, D25, G26, W27, W28, Y31, T35b, D73, D74, T75, N76, R94, D96, D98, D101, and T102, and the following amino acid residues in the VL domain: E55 and H56.

[0111] In another aspect, the invention provides an antibody comprising (ii) a VH domain comprising amino acid residues D1, L2, D25, G26, W27, W28, Y31, T35b, Y52a, G55, H56, K57, Y58, T61, K62, F63, I64, G65, R66, D73, D74, T75, N76, R94, D96, Y98, D101, and T102, and (iii) a VL domain comprising amino acid residues A1, I2, S26, Y27, W27a, L27b, S27c, E55, H56, H68, E69, R92, Y93, H94, P95 and Y96, wherein numbering of the VH domain and VL domain is according to the Kabat numbering system. In one embodiment, the antibody comprises a VEGF paratope comprising the following amino acid residues in the VH domain: G55, H56, K57, Y58, T61, K62, F63, I64, G65, and R66, and the following amino acid residues in the VL domain: A1, I2, S26, Y27, W27a, L27b, S27c, H68, E69, R92, Y93, H94, P95, and Y96; and a PDGF-B paratope comprising the following amino acid residues in the VH domain: D1, L2, D25, G26, W27, W28, Y31, T35b, D73, D74, T75, N76, R94, D96, D98, D101, and T102, and the following amino acid residues in the VL domain: E55 and H56.

[0112] In another aspect, the present invention provides an antibody comprising: ● 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: 11; 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: 21; or ● 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: 11; 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: 25; or ● 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: 28; 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: 25; or ● (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: 28; 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: 31.

[0113] In another aspect, the present invention provides an antibody 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: 11, comprising amino acid residues D1, L2, D25, G26, W27, W28, Y31, T35b, Y52a, G55, S56, T57, Y58, T61, K62, F63, I64, G65, R66, D73, D74, T75, N76, R94, D96, Y9 (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: 21, the VL domain comprising amino acid residues A1, I2, S26, Y27, W27a, L27b, S27c, E55, H56, H68, E69, R92, Y93, H94, P95 and Y96; or (a) a VH domain comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 11, comprising amino acid residues D1, L2, D25, G26, W27, W28, Y31, T35b, Y52a, G55, S56, T57, Y58, T61, K62, F63, I64, G65, R66, D73, D74, T75, N76, R94, D96, Y (b) a VL domain comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:25, the VL domain comprising amino acid residues A1, I2, S26, Y27, W27a, L27b, S27c, E55, H56, H68, E69, R92, Y93, H94, P95 and Y96; or (a) a VH domain comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 28, comprising amino acid residues D1, L2, D25, G26, W27, W28, Y31, T35b, Y52a, G55, H56, K57, Y58, T61, K62, F63, I64, G65, R66, D73, D74, T75, N76, R94, D96, Y (b) a VL domain comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:25, the VL domain comprising amino acid residues A1, I2, S26, Y27, W27a, L27b, S27c, E55, H56, H68, E69, R92, Y93, H94, P95 and Y96; or (a) a VH domain comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 28, the VH domain comprising amino acid residues D1, L2, D25, G26, W27, W28, Y31, T35b, Y52a, G55, H56, K57, Y58, T61, K62, F63, I64, G65, R66, D73, D74, T75, N76, R94, D96, Y98, D101, and T102; main; (b) a VL domain comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 31, said VL domain comprising amino acid residues A1, I2, S26, Y27, W27a, L27b, S27c, E55, H56, H68, E69, R92, Y93, H94, P95 and Y96 (numbering of the VH domain and VL domain is according to the Kabat numbering system).

[0114] In another aspect, the present invention provides an antibody 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: 13, 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: 15, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17, wherein (a) the amino acid sequence of SEQ ID NO: 11 and / or (b) the CDR-H2 comprising the amino acid sequence of SEQ ID NO: 13 and / or (c) the CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14 are similar to those of SEQ ID NO: 15 and / or (f) the CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17, (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: 21; or ● 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: 13, 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: 15, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17, 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: 11; 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: 25; or ● 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: 29, 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: 15, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17, 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: 28; 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: 25; or ● (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: 29, 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: 15, (e) a CDR-L2 having the amino acid sequence of SEQ ID NO: 16, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 17, 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: 28; 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: 31.

[0115] In another aspect, the present invention relates to an antibody 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: 13, 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: 15, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17, wherein the antibody comprises (a) 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: 11, and wherein the amino acid residues D1, L2, D25, G26, W27, W28, Y31, T3 (b) a VH domain comprising at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 160%, 161%, 162%, 163%, 164%, 165%, 166%, 167%, 168%, 169%, 170%, 171%, 172%, 173%, 174%, 175%, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187 5, 98% or 99% sequence identity to the VH domain, and a VL domain comprising amino acid residues A1, I2, S26, Y27, W27a, L27b, S27c, E55, H56, H68, E69, R92, Y93, H94, P95 and Y96, wherein the numbering of the VH domain and the VL domain is according to the Kabat numbering system.

[0116] In another aspect, the present invention provides 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: 13; 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 D1, L2, D25, G26, W27, and W28; (ii) an FR3 comprising amino acid residues D73, D74, T75, N76, and R94; and (e) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 15; (f) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16; (g) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17; and (h) a CDR-L4 comprising a human heavy chain framework having (i) an FR1 comprising amino acid residues A1 and I2; (ii) an FR3 comprising amino acid residues A1 and I3; 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:21.

[0117] In another aspect, the present invention provides an antibody comprising: (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 11 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: 21 having 1 to 15, 1 to 10 or 1 to 5 amino acid substitutions; or (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 11, 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: 25, having 1 to 15, 1 to 10, or 1 to 5 amino acid substitutions; or (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 28 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: 25 having 1 to 15, 1 to 10 or 1 to 5 amino acid substitutions; or ● (a) a VH domain having the amino acid sequence of SEQ ID NO: 28 having 1 to 15, 1 to 10 or 1 to 5 amino acid substitutions; and (b) a variable light chain domain having the amino acid sequence of SEQ ID NO: 31 having 1 to 15, 1 to 10 or 1 to 5 amino acid substitutions.

[0118] In another aspect, the present invention provides an antibody comprising: ● (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 11 having 1 to 15, 1 to 10, or 1 to 5 amino acid substitutions, wherein the amino acid substitutions are located at positions 3 to 24, 29, 30, 35c to 52, 52b, 54, 59, 60, 67 to 72, 77 to 93, 95, 97, 99, or 103 to 113 of SEQ ID NO: 11; (b) a variable light chain domain comprising the amino acid sequence of SEQ ID NO: 21 having 1 to 15, 1 to 10, or 1 to 5 amino acid substitutions, wherein the amino acid substitutions are located at positions 3 to 25, 27d to 54, 57 to 67, 70 to 91, or 97 to 107 of SEQ ID NO: 21; or ● (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 11 having 1 to 15, 1 to 10, or 1 to 5 amino acid substitutions, wherein the amino acid substitutions are located at positions 3 to 24, 29, 30, 35c to 52, 52b, 54, 59, 60, 67 to 72, 77 to 93, 95, 97, 99, or 103 to 113 of SEQ ID NO: 11; (b) a variable light chain domain comprising the amino acid sequence of SEQ ID NO: 25 having 1 to 15, 1 to 10, or 1 to 5 amino acid substitutions, wherein the amino acid substitutions are located at positions 3 to 25, 27d to 54, 57 to 67, 70 to 91, or 97 to 107 of SEQ ID NO: 25; or ● (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 28 having 1 to 15, 1 to 10, or 1 to 5 amino acid substitutions, wherein the amino acid substitutions are located at positions 3 to 24, 29, 30, 35c to 52, 52b, 54, 59, 60, 67 to 72, 77 to 93, 95, 97, 99, or 103 to 113 of SEQ ID NO: 28; (b) a variable light chain domain comprising the amino acid sequence of SEQ ID NO: 25 having 1 to 15, 1 to 10, or 1 to 5 amino acid substitutions, wherein the amino acid substitutions are located at positions 3 to 25, 27d to 54, 57 to 67, 70 to 91, or 97 to 107 of SEQ ID NO: 25; or ● (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 28 having 1 to 15, 1 to 10, or 1 to 5 amino acid substitutions, wherein the amino acid substitutions are located at positions 3 to 24, 29, 30, 35c to 52, 52b, 54, 59, 60, 67 to 72, 77 to 93, 95, 97, 99, or 103 to 113 of SEQ ID NO: 28; (b) a variable light chain domain comprising the amino acid sequence of SEQ ID NO: 31 having 1 to 15, 1 to 10, or 1 to 5 amino acid substitutions, wherein the amino acid substitutions are located at positions 3 to 25, 27d to 54, 57 to 67, 70 to 91, or 97 to 107 of SEQ ID NO: 31 (numbering of the VH domain and VL domain is according to the Kabat numbering system).

[0119] In another aspect, the present invention provides an antibody 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: 13, 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: 15, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17, wherein the antibody comprises: (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 11 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: 21 having 1 to 15, 1 to 10, or 1 to 5 amino acid substitutions.

[0120] In another aspect, the present invention provides 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: 13; 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 D1, L2, D25, G26, W27, and W28, (ii) an FR3 comprising amino acid residues D72, D74, T75, N76, and R94; and (e) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 15; (f) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16; and (h) a VL domain comprising a human light chain framework having (i) FR1 comprising amino acid residue I2, and (ii) FR3 comprising amino acid residues H68 and E69, wherein the numbering of the VH domain and the VL domain is according to the Kabat numbering system, and comprising: (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 11 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: 21 having 1 to 15, 1 to 10, or 1 to 5 amino acid substitutions.

[0121] In another aspect, the present invention provides an antibody comprising: ● a VH domain comprising (b) 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: 13, 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: 15, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17, and (g) an L-FR-1 comprising the amino acid sequence of SEQ ID NO: 22; or ● a VH domain comprising (b) 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: 13, 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: 15, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17, and (g) an L-FR-1 comprising the amino acid sequence of SEQ ID NO: 26; or ● a VH domain comprising (b) 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: 29, 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: 15, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17, and (g) an L-FR-1 comprising the amino acid sequence of SEQ ID NO: 26; or ● A VH domain comprising (b) 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: 29, 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: 15, (e) a CDR-L2 having the amino acid sequence of SEQ ID NO: 16, (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 17, and (g) an L-FR-1 having the amino acid sequence of SEQ ID NO: 32.

[0122] In another aspect, the present invention provides an antibody comprising: ● A VH domain comprising (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: 13, 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: 15, (e) a CDR-L2 having the amino acid sequence of SEQ ID NO: 16, (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 17, and (g) an L-FR-1 having the amino acid sequence of SEQ ID NO: 22, (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:11; 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:21; or ● A VH domain comprising (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: 13, 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: 15, (e) a CDR-L2 having the amino acid sequence of SEQ ID NO: 16, (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 17, and (g) an L-FR-1 having the amino acid sequence of SEQ ID NO: 26, (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:11; 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:25; or ● A VH domain comprising (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: 13, 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: 15, (e) a CDR-L2 having the amino acid sequence of SEQ ID NO: 16, (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 17, and (g) an L-FR-1 having the amino acid sequence of SEQ ID NO: 26, (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:28; 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:25; or ● 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: 13, 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: 15, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17, and (g) an L-FR-1 comprising the amino acid sequence of SEQ ID NO: 32, 28; 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:31.

[0123] In one aspect, the invention provides an antibody 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: 11 or SEQ ID NO: 28. In certain aspects, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity contains substitutions (e.g., conservative substitutions), insertions or deletions compared to the reference sequence, but an antibody that binds to human VEGF and human PDGF-B comprising the sequence retains the ability to bind to human VEGF and human PDGF-B. In certain aspects, a total of 1-10 amino acids are substituted, inserted and / or deleted in SEQ ID NO: 11 or SEQ ID NO: 28. 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: 13 or SEQ ID NO: 29, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14.

[0124] In one aspect, the invention provides an antibody 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: 21, SEQ ID NO: 25 or SEQ ID NO: 31. In certain aspects, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity contains substitutions (e.g., conservative substitutions), insertions or deletions compared to the reference sequence, but an antibody that binds to human VEGF and human PDGF-B comprising the sequence retains the ability to bind to human VEGF and human PDGF-B. In certain aspects, a total of 1-10 amino acids are substituted, inserted and / or deleted in SEQ ID NO: 21, SEQ ID NO: 25 or SEQ ID NO: 31. 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: 15, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17.

[0125] In another aspect, an antibody is provided that binds human VEGF and human PDGF-B, the antibody comprising a VH sequence of any of the aspects provided above and a VL sequence of any of the aspects provided above. In one aspect, the antibody comprises the VH and VL sequences of SEQ ID NO: 11 and SEQ ID NO: 21, respectively, including post-translational modifications of those sequences. In one aspect, the antibody comprises the VH and VL sequences of SEQ ID NO: 11 and SEQ ID NO: 25, respectively, including post-translational modifications of those sequences. In one aspect, the antibody comprises the VH and VL sequences of SEQ ID NO: 28 and SEQ ID NO: 25, respectively, including post-translational modifications of those sequences. In one aspect, the antibody comprises the VH and VL sequences of SEQ ID NO: 28 and SEQ ID NO: 31, respectively, including post-translational modifications of those sequences.

[0126] In another aspect, an antibody that binds to human VEGF and human PDGF-B is provided, the antibody comprising a heavy chain amino acid sequence of SEQ ID NO:18 and a light chain amino acid sequence of SEQ ID NO:20.

[0127] In another aspect, an antibody that binds to human VEGF and human PDGF-B is provided, the antibody comprising a heavy chain amino acid sequence of SEQ ID NO:18 and a light chain amino acid sequence of SEQ ID NO:19.

[0128] In another aspect, an antibody that binds to human VEGF and human PDGF-B is provided, the antibody comprising a heavy chain amino acid sequence of SEQ ID NO:18 and a light chain amino acid sequence of SEQ ID NO:27.

[0129] In another aspect, an antibody that binds to human VEGF and human PDGF-B is provided, the antibody comprising a heavy chain amino acid sequence of SEQ ID NO:30 and a light chain amino acid sequence of SEQ ID NO:27.

[0130] In another aspect, an antibody that binds to human VEGF and human PDGF-B is provided, the antibody comprising a heavy chain amino acid sequence of SEQ ID NO:18 and a light chain amino acid sequence of SEQ ID NO:33.

[0131] In a further aspect of the invention, the antibody that binds human VEGF and human PDGF-B according to any of the above aspects is a monoclonal antibody. In one aspect, the antibody that binds human VEGF and human PDGF-B is an antibody fragment, such as an Fv, Fab, Fab', scFv, diabody, or F(ab') 2 In another embodiment, the antibody is a full-length antibody.

[0132] In further aspects, an antibody that binds human VEGF and human PDGF-B according to any of the above aspects may incorporate any of the features, either alone or in combination, as described in Sections 1-7 below.

[0133] 1. Antibody affinity In certain embodiments, the antibodies provided herein have a dissociation constant (K D In certain embodiments, antibodies that bind PDGF-B bind to VEGF with a dissociation constant (K ) of 1 μM or less, 100 nM or less, or 10 nM or less.D ).

[0134] In one embodiment, K D is measured using the BIACORE® surface plasmon resonance assay described in Example 3.

[0135] For example, the K D is measured in assays using a BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) performed at 25°C with immobilized VEGF121 on a C1 chip at approximately 10 response units (RU). For kinetic measurements, two-fold serial dilutions of Fab (1.2-100 nM) are injected in HBS-P+ (10 mM HEPES, 150 mM NaCl pH 7.4, 0.05% surfactant P20) at 25°C at a flow rate of approximately 30 μl / min. The association rate (k on ) and dissociation rate (k off ) is calculated using a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgrams. D ) is k off / k on It is calculated as a ratio. See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999).

[0136] 2. Antibody Fragments In certain aspects, the antibodies provided herein are antibody fragments.

[0137] In one embodiment, the antibody fragment is a Fab, Fab', Fab'-SH, or F(ab') 2The term "Fab fragment" refers to an antibody fragment that has a light chain containing the VL and CL domains and a heavy chain fragment containing the VH and CH1 domains. The term "Fab' ... 2 The resulting fragments are Fab and F(ab') fragments that contain the salvage receptor binding epitope residues and have increased in vivo half-lives. 2 For a description of fragments, see US Pat. No. 5,869,046.

[0138] 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.

[0139] 3.Thermal stability The antibodies provided herein exhibit excellent thermal stability, in certain embodiments, the Fab fragments of the antibodies provided herein exhibit an aggregation onset temperature of greater than 68°C.

[0140] 4. Library-derived antibodies In some aspects, the antibody provided herein is obtained from a library. The antibody of the present invention can be isolated by screening a combinatorial library for an antibody having one or more desired activities. Methods for screening a combinatorial library are reviewed, for example, in Lerner et al. in Nature Reviews 16:498-508 (2016). For example, various methods are known in the art for producing a phage display library and screening such a library for an antibody having desired binding properties. Such methods can be found, for example, in Frenzel et al., mAbs 8:1177-1194 (2016); Bazan et al., Human Vaccines and Immunotherapeutics 8:1817-1828 (2012), and Zhao et al., Critical Reviews in Biotechnology 36:276-289 (2016), and Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., eds., Human Press, Totowa, NJ, 2001), and Marks and Bradbury Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003).

[0141] In some phage display methods, repertoires of VH and VL genes can be cloned separately by polymerase chain reaction (PCR), randomly recombined in a phage library, and then screened for antigen-binding phages, as described by Winter et al. in Annual Review of Immunology 12:433-455 (1994). Phages typically display antibody fragments as either single-chain Fv (scFv) fragments or Fab fragments. Libraries from immunized sources provide high affinity antibodies to immunogens without the need to construct hybridomas. Alternatively, naive repertoires can be cloned (e.g., from humans), as described by Griffiths et al. in EMBO Journal 12:725-734 (1993), to obtain a single source of antibodies against a wide range of non-self and even self antigens without immunization. Finally, naive libraries can also be synthetically generated by cloning unrearranged V gene segments from stem cells and using PCR primers containing random sequences to encode highly variable CDR3 regions and achieve re-arrangement in vitro, as described by Hoogenboom and Winter in Journal of Molecular Biology 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example, U.S. Patent Nos. 5,750,373, 7,985,840, 7,785,903 and 8,679,490, and U.S. Patent Application Publication Nos. 2005 / 0079574, 2007 / 0117126, 2007 / 0237764 and 2007 / 0292936.

[0142] Further examples of methods known in the art for screening combinatorial libraries for antibodies with a desired activity or activities include ribosome and mRNA display, as well as methods for antibody display and selection in bacteria, mammalian cells, insect cells or yeast cells. Methods for yeast surface display are reviewed, for example, in Scholler et al. in Methods in Molecular Biology 503:135-56 (2012) and Cherf et al. in Methods in Molecular biology 1319:155-175 (2015) and Zhao et al. in Methods in Molecular Biology 889:73-84 (2012). Methods for ribosome display are described, for example, in He et al. Nucleic Acids Research 25:5132-5134 (1997) and in Hanes et al, PNAS94:4937-4942 (1997).

[0143] Antibodies or antibody fragments isolated from a human antibody library are considered herein to be human antibodies or human antibody fragments.

[0144] 5. 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.

[0145] 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).

[0146] 6. 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).

[0147] 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. [Table 2]

[0148] Amino acids can be classified according to common side chain properties. (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.

[0149] Non-conservative substitutions will involve exchanging a member of one of these classes for a member of another class.

[0150] Certain substitutional variants involve substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further testing have modified (e.g., improved) certain biological properties (e.g., increased affinity, reduced immunogenicity) compared to the parent antibody and / or have substantially retained certain biological properties of the parent antibody. An exemplary substitutional 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 are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

[0151] To improve antibody affinity, for example, modifications (e.g., substitutions) may be made in the CDRs. Such modifications may be made in CDR "hot spots," i.e., residues encoded by codons that undergo frequent mutation during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or within residues that contact the antigen, and the resulting variants VH or VL are tested for binding affinity. Affinity maturation by construction of and reselection from secondary libraries is described, for example, in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some aspects of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then generated. This library is then screened to identify antibody variants with the desired affinity. Another method of introducing diversity involves CDR-directed approaches, in which several CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding can be specifically identified, for example, using alanine-scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.

[0152] 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.

[0153] 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. The variants may be screened to determine whether they have the desired properties.

[0154] 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 one or more amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule 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.

[0155] a) Fc domain variants In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody presented herein, thereby creating an Fc region variant. An Fc region variant is a human Fc region sequence (e.g., human IgG) that contains an amino acid modification (e.g., substitution) at one or more amino acid positions. 1 , IgG 2 , IgG 3 , or IgG 4 Fc region).

[0156] 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).

[0157] 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).

[0158] Certain antibody variants have been described with improved or diminished binding to FcRs (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)).

[0159] 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 aspects, the antibody variant comprises an Fc region having one or more amino acid substitutions that reduce FcγR binding, e.g., an Fc region having positions 234 and 235 (EU numbering residues) of the Fc region. In one aspect, the substitutions are L234A and L235A (LALA). In certain aspects, the antibody variant comprises a human IgG 1 In one embodiment, the substitution is in the Fc region derived from a human IgG 1 In another embodiment, the substitutions are L234A, L235A, and P329G (LALA-PG) in the Fc region derived from the Fc region. (See, e.g., WO 2012 / 130831.) In another embodiment, the substitutions are 1 These are L234A, L235A, and D265A (LALA-DA) in the Fc region, which are derived from the 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 substitutions 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 aspects, 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 aspects, the antibody variant comprises an Fc region having amino acid substitutions at positions 252, 254, and 256. In one aspect, the substitutions are made to the FcRn-binding domain of a human IgG 1 The Fc region is derived from M252Y, S254T, and T256E in the Fc region. For other examples of Fc region variants, 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.

[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] b) Cysteine ​​Engineered Antibody Variants In certain aspects, it may be desirable to generate cysteine ​​engineered antibodies, e.g., THIOMAB™, in which one or more residues of an antibody are replaced with a cysteine ​​residue. In certain aspects, the replaced 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] 7. Immunoconjugates The present invention also provides immunoconjugates comprising an antibody that binds human VEGF and human PDGF-B as disclosed herein conjugated to one or more therapeutic agents, such as 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 immunoconjugate is an antibody drug conjugate (ADC) in which an antibody is conjugated to one or more of the aforementioned therapeutic agents. The antibody is typically connected to one or more of the therapeutic agents using a linker. An overview of ADC technology, including examples of therapeutic agents and agents and linkers, is provided in Pharmacol Review 68:3-19 (2016).

[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 human VEGF and human PDGF-B, 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 and human PDGF-B, for example, nucleic acids encoding the above-described 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, e.g., 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.

[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 by 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 (BRL3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor cells (MMT060562), 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 embodiment, the host cell is eukaryotic, such as a Chinese Hamster Ovary (CHO) cell or a lymphocytic cell (eg, Y0, NS0, Sp20 cell).

[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 and human PDGF-B 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 intermediate 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 and human PDGF-B can be used in the therapeutic methods.

[0186] In one aspect, an antibody that binds human VEGF and human PDGF-B is provided for use as a medicament. In a further aspect, an antibody that binds human VEGF and human PDGF-B is provided for use in treating vascular disease. In certain aspects, an antibody that binds human VEGF and human PDGF-B is provided for use in a method of treatment. In certain aspects, the invention provides an antibody that binds human VEGF and human PDGF-B for use in a method of treating an individual having vascular disease, comprising administering to the individual an effective amount of an antibody that binds human VEGF and human PDGF-B. 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 and human PDGF-B for use in inhibiting angiogenesis. In certain aspects, the invention provides an antibody that binds human VEGF and human PDGF-B 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 and human PDGF-B 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 and human PDGF-B 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 the use of an antibody that binds human VEGF and human PDGF-B 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 and human PDGF-B. 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 and human PDGF-B. 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 pharmaceutical compositions comprising any of the antibodies that bind human VEGF and human PDGF-B 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 and human PDGF-B provided herein and a pharma- ceutically acceptable carrier. In another aspect, the pharmaceutical composition comprises any of the antibodies that bind human VEGF and human PDGF-B provided herein and at least one additional therapeutic agent, e.g., as described below.

[0194] 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).

[0195] For example, in certain embodiments, any of the aforementioned methods further comprise administering one or more additional compounds. In certain embodiments, the antibody that binds human VEGF and human PDGF-B provided herein is administered simultaneously with the additional compound(s). In certain embodiments, the antibody that binds human VEGF and human PDGF-B is administered before or after the additional compound(s). In certain embodiments, the further compound binds to a second biological molecule selected from the group consisting of IL-6; IL-6R; IL-13; IL-13R; PDGF; angiopoietin; Ang2; Tie2; S1P; integrins αvβ3, αvβ5 and α5β1; betacellulin; apelin / APJ; erythropoietin; complement factor D; TNFα; HtrA1; VEGF receptor; ST-2 receptor; and proteins genetically associated with AMD risk, such as complement pathway components C2, factor B, factor H, CFHR3, C3b, C5, C5a and C3a; HtrA1; ARMS2; TIMP3; HLA; interleukin-8 (IL-8); CX3CR1; TLR3; TLR4; CETP; LIPC; COL10A1; and TNFRSF10A. In certain embodiments, the further compound is an antibody or an antigen-binding fragment thereof.

[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 and human PDGF-B 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).Exemplary additional therapeutic agents for combination therapy to treat ocular disorders include, but are not limited to, anti-angiogenic agents, such as anti-VEGF antibodies (e.g., anti-VEGF Fab LUCENTIS® (ranibizumab)), soluble receptor fusion proteins (e.g., recombinant soluble receptor fusion protein EYLEA® (also known as aflibercept, VEGF Trap Eye; Regeneron / Aventis)), aptamers (e.g., anti-VEGF pegylated aptamer MACUGEN® (pegaptanib sodium; NeXstar Pharmaceuticals / OSI Pharmaceuticals)) and VEGFR tyrosine kinase inhibitors (e.g., 4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(1-methylpiperidin-4-ylmethoxy)quinazoline (ZD6474), 4-(4-fluoro-2-methylindol-5-yloxy)-6-methoxy-7-(3-pyrrolidin-1-ylpropoxy)quinazoline (AZD2171), vatalanib (PTK787), semaxaminib (SU5416; SUGEN), and SUTENT® (sunitinib)); tryptophanyl-tRNA synthetase inhibitors (e.g., 4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(1-methylpiperidin-4-ylmethoxy)quinazoline (ZD6474), 4-(4-fluoro-2-methylindol-5-yloxy)-6-methoxy-7-(3-pyrrolidin-1-ylpropoxy)quinazoline (AZD2171), vatalanib (PTK787), semaxaminib (SU5416; SUGEN), and SUTENT® (sunitinib)). (TrpRS); squalamine; RETAANE® (acannortabe acetate for depot suspension; Alcon, Inc.); combretastatin A4 prodrug (CA4P); MIFEPREX® (mifepristone-ru486); subtenone triamcinolone acetonide; intravitreal crystalline triamcinolone acetonide; matrix metalloproteinase inhibitors (e.g., Prinomast (AG3340; Pfizer)); fluocinolone acetonide (including fluocinolone intraocular implants; Bausch & Lomb / Control Delivery Systems); linomide; inhibitors of integrin β3 function; VEGF antagonists, including angiostatin, and combinations thereof. These and other therapeutic agents that can be administered in combination with the antibodies that bind human VEGF and human PDGF-B of the present invention are described, for example, in U.S. Patent Application Publication No. 2014 / 0017244, which is incorporated herein by reference in its entirety.

[0198] Further examples of additional therapeutic agents that can be used in combination with the antibodies that bind human VEGF and human PDGF-Bas provided herein for the treatment of ocular disorders (e.g., AMD, DME, DR, RVO, or GA) include, but are not limited to, VISUDYNE® (verteporfin; a light-activated drug typically used in conjunction with photodynamic therapy with non-thermal lasers), PKC412, Endovion (NS 3728; NeuroSearch A / S), neurotrophic factors (e.g., glial-derived neurotrophic factor (GDNF) and ciliary neurotrophic factor (CNTF)), diltiazem, dorzolamide, PHOTOTROP®, 9-cis-retinal, eye drops (e.g., phosphodiesterase, echothiophate, or carbonic anhydrase inhibitors), beovastat (AE-941; AEterna Laboratories, Inc.), Silna-027 (AGF-745; Sima Therapeutics, Inc.), neurotrophins (including, by way of example only, NT-4 / 5, Genentech), Cand5 (Acuity Pharmaceuticals), INS-37217 (Inspire Pharmaceuticals), integrin antagonists (including those from Jerini AG and Abbott Laboratories), EG-3306 (Ark Therapeutics Ltd.), BDM-E (BioDiem Ltd.), thalidomide (e.g., EntreMed, Inc.(used by the University of Michigan), cardiotrophin-1 (Genentech), 2-methoxyestradiol (Allergan / Oculex), DL-8234 (Toray Industries), NTC-200 (Neurotech), tetrathiomolybdate (University of Michigan), LYN-002 (Lynkeus Biotech), microalgae compounds (Aquasearch / Albany, Mera Pharmaceuticals), D-9120 (Celltech Group plc), ATX-S10 (Hamamatsu Photonics), TGF-beta 2 (Genzyme / Celtrix), tyrosine kinase inhibitors (e.g., from Allergan, SUGEN, or Pfizer), NX-278-L (NeXstar Pharmaceuticals / Gilead Sciences), Opt-24 (OPTIS France SA), retinal ganglion neuroprotectants (Cogent Neurosciences), N-nitropyrazole derivatives (Texas A&M University System), KP-102 (Krenitsky Pharmaceuticals), cyclosporine A, therapeutic agents used in photodynamic therapy (e.g., VISUDYNE®, receptor-targeted PDT, Bristol-Myers Squibb, Co.; injectable porfimer sodium with PDT; verteporfin, QLT Inc.; rostaporfin with PDT, Miravent Medical Technologies; talaporfin sodium with PDT, Nippon Petroleum; and motecsurf lutetium, Pharmacyclics, Inc.), antisense oligonucleotides (examples include products tested by Novagali Pharma SA and ISIS-13650, Ionis Pharmaceuticals), and combinations thereof.

[0199] Antibodies that bind human VEGF and human PDGF-B provided herein can be used in a variety of procedures, including, for example, laser photocoagulation (e.g., panretinal photocoagulation (PRP)), drusen laser, 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 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 (e.g., ophthalmic solution ...

[0200] In some examples, antibodies that bind human VEGF and human PDGF-B can be administered in combination with an anti-angiogenic agent to treat ocular disorders (e.g., AMD, DME, DR, RVO, or GA). Any suitable anti-angiogenic agent can be used in combination with the antibodies that bind human VEGF and human PDGF-B of the present invention, including but not limited to those listed by Carmeliet et al. Nature 407:249-257,2000. In some embodiments, the anti-angiogenic agent can be, but is not limited to, an anti-VEGF antibody (e.g., anti-VEGF Fab LUCENTIS® (ranibizumab), RTH-258 (formerly ESBA-1008, an anti-VEGF single chain antibody fragment; Novartis), or a bispecific anti-VEGF antibody (e.g., anti-VEGF / anti-angiogenesis 2 bispecific antibody, e.g., faricimab; Roche)), a soluble recombinant receptor fusion protein (e.g., EYLEA® (aflibercept)), a VEGF variant, a soluble VEGFR fragment, an aptamer capable of blocking VEGF (e.g., pegaptanib) or VEGFR, a neutralizing anti-VEGFR antibody, a small molecule inhibitor of VEGFR tyrosine kinase, an anti-VEGF DARPin® (e.g., abicipar pegol, Molecular Partners), AG / Allergan), small interfering RNA that inhibits expression of VEGF or VEGFR, VEGF antagonists including VEGFR tyrosine kinase inhibitors (e.g., 4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(1-methylpiperidin-4-ylmethoxy)quinazoline (ZD6474), 4-(4-fluoro-2-methylindol-5-yloxy)-6-methoxy-7-(3-pyrrolidin-1-ylpropoxy)quinazoline (AZD2171), vatalanib (PTK787), semaxaminib (SU5416; SUGEN), and SUTENT® (sunitinib)), and combinations thereof.

[0201] Other suitable antiangiogenic agents that may be administered in combination with the antibodies that bind human VEGF and human PDGF-B provided herein for the treatment of ocular disorders (e.g., AMD, DME, DR, RVO, or GA) include corticosteroids, antiangiogenic steroids, acanthorub acetate, angiostatin, endostatin, tyrosine kinase inhibitors, matrix metalloproteinase (MMP) inhibitors, insulin-like growth factor binding protein 3 (IGFBP) inhibitors, and the like. 3), stromal derived factor (SDF-1) antagonists (e.g., anti-SDF-1 antibodies), pigment epithelium-derived factor (PEDF), gamma-secretase, Delta-like ligand 4, integrin antagonists, hypoxia-inducible factor (HIF)-1α antagonists, protein kinase CK2 antagonists, agents that inhibit stem cells (e.g., endothelial progenitor cells) homing to sites of neovascularization (e.g., anti-vascular endothelial cadherin (CD-144) antibodies and / or anti-SDF-1 antibodies, and combinations thereof.

[0202] In further examples, in some instances, antibodies that bind human VEGF and human PDGF-B, and / or polymeric formulations thereof, can be administered in combination with agents active against neovascularization for the treatment of ocular disorders (e.g., AMD, DME, DR, RVO, or GA), such as anti-inflammatory agents, mammalian target of rapamycin (mTOR) inhibitors (e.g., rapamycin, AFINITOR® (everolimus) and TORISEL® (temsirolimus)), cyclosporine, tumor necrosis factor (TNF) antagonists (e.g., anti-TNFα antibodies or antigen-binding fragments thereof (e.g., infliximab, adalimumab, certolizumab pegol, and golimumab) or soluble receptor fusion proteins (e.g., etanercept)), anti-complement agents, nonsteroidal anti-inflammatory agents (NSAIDs), or combinations thereof.

[0203] In yet a further example, in some instances, antibodies that bind human VEGF and human PDGF-B can be administered in combination with agents that are neuroprotective and can potentially reduce the progression of dry AMD to wet AMD, such as a class of drugs called "neurosteroids," which includes drugs such as dehydroepiandrosterone (DHEA) (trade names: PRASTERA™ and FIDELIN®), dehydroepiandrosterone sulfate, and pregnenolone sulfate.

[0204] Any suitable AMD therapeutic agent may be a VEGF antagonist, such as an anti-VEGF antibody (e.g., LUCENTIS® (ranibizumab), RTH-258 (formerly ESBA-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); α-adrenergic receptor agonists (e.g., brimonidine tartrate; Allergan); peptide vaccines (e.g., S-646240; Shionogi); amyloid β antagonists (e.g., anti-β amyloid monoclonal antibodies, such as GSK-933776); S1P antagonists (e.g., anti-S1P antibodies, such as 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, may be administered as additional therapeutic agents in combination with antibodies that bind human VEGF and human PDGF-B provided herein for the treatment of ocular diseases (e.g., AMD, DME, DR, RVO, or GA). In some examples, AMD therapeutics (including any of the aforementioned AMD therapeutics) may be co-formulated. For example, the anti-PDGFR antibody REGN2176-3 may be co-formulated with aflibercept (EYLEA®). In some examples, such co-formulations may be administered in combination with antibodies that bind human VEGF and human PDGF-B of the present invention. In some examples, the ocular disorder is AMD (e.g., wet AMD).

[0205] The antibodies of the present invention that bind human VEGF and human PDGF-B can be administered in combination with LUCENTIS® (ranibizumab) for the treatment of ocular disorders (e.g., AMD, DME, DR, RVO, or GA). In some examples, the ocular disorder is AMD (e.g., wet AMD). In some examples, the ocular disorder is GA.

[0206] The antibodies that bind human VEGF and human PDGF-B of the present invention can be administered in combination with EYLEA® (aflibercept) for the treatment of eye disorders (e.g., AMD, DME, DR, RVO, or GA). In some examples, the eye disorder is AMD (e.g., wet AMD). In some examples, the eye disorder is GA.

[0207] The antibodies that bind human VEGF and human PDGF-B of the present invention can be administered in combination with MACUGEN® (pegaptanib sodium) for the treatment of ocular disorders (e.g., AMD, DME, DR, RVO, or GA). In some examples, the ocular disorder is AMD (e.g., wet AMD). In some examples, the ocular disorder is GA.

[0208] The antibodies that bind human VEGF and human PDGF-B of the present invention can be administered in combination with VISUDYNE® (verteporfin) in combination with photodynamic therapy to treat ocular disorders (e.g., AMD, DME, DR, RVO, or GA). In some examples, the ocular disorder is AMD (e.g., wet AMD). In some examples, the ocular disorder is GA.

[0209] The antibodies that bind human VEGF and human PDGF-B of the present invention can be administered in combination with a PDGF antagonist for the treatment of ocular disorders (e.g., AMD, DME, DR, RVO, or GA). Exemplary PDGF antagonists that can be used in combination with the antibodies that bind human VEGF and human PDGF-B of the present invention include anti-PDGF antibodies, anti-PDGFR antibodies, small molecule inhibitors (e.g., squalamine), anti-PDGF-B pegylated aptamers such as FOVISTA® (E10030; Ophthotech / Novartis), 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). For example, FOVISTA® can be administered as an adjunct therapy to the antibodies that bind human VEGF and human PDGF-B of the present invention. OHR-102 can be administered in combination with a VEGF antagonist, such as LUCENTIS® or EYLEA®. In some embodiments, an antibody that binds human VEGF and human PDGF-B of the present invention can be administered in combination with OHR-102, LUCENTIS®, and / or EYLEA®. In some examples, the eye disorder is AMD (e.g., wet AMD). In some examples, the eye disorder is GA.

[0210] The antibodies that bind human VEGF and human PDGF-B of the present invention can be administered in combination with RTH-258 for the treatment of ocular disorders (e.g., AMD, DME, DR, RVO, or GA). RTH-258 can be administered, for example, by intravitreal injection or ocular injection. In some examples, the ocular disorder is AMD (e.g., wet AMD). In some examples, the ocular disorder is GA.

[0211] The antibodies that bind human VEGF and human PDGF-B of the present invention can be administered in combination with abicipar pegol for the treatment of an ocular disorder (e.g., AMD, DME, DR, RVO, or GA). In some examples, the ocular disorder is AMD (e.g., wet AMD). In some examples, the ocular disorder is GA.

[0212] 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 PDGF-B 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.

[0213] Antibodies of the present invention that bind human VEGF and human PDGF-B can be administered in combination with LUCENTIS® (ranibizumab) for the treatment of DME and / or DR (eg, NPDR or PDR).

[0214] Antibodies of the invention that bind human VEGF and human PDGF-B can be administered in combination with EYLEA® (aflibercept) for the treatment of DME and / or DR (eg, NPDR or PDR).

[0215] Antibodies of the present invention that bind human VEGF and human PDGF-B can be administered in combination with OZURDEX® (dexamethasone intravitreal implant) for the treatment of DME and / or DR.

[0216] Antibodies of the present invention that bind human VEGF and human PDGF-B can be administered in combination with ILUVIEN® (dexamethasone intravitreal implant) for the treatment of DME and / or DR.

[0217] In some cases, AMD therapeutic agents (e.g., ranibizumab or aflibercept) may be administered in combination with the antibody and / or polymer formulation thereof that binds to human VEGF and human PDGF-B of the present invention using TAO / PRN treatment regimen or TAE treatment regimen. In some examples, the eye disorder is AMD (e.g., wet AMD). In some examples, the eye disorder is GA.

[0218] 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 PDGF-B may precede, be concurrent with, and / or be subsequent to administration of the additional therapeutic agent or agent. In one embodiment, administration of an antibody of the invention that binds human VEGF and human PDGF-B 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.

[0219] 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.

[0220] 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.

[0221] 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.

[0222] 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 inserted into or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, 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 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 a selected condition. 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 cytotoxic 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.

[0223] 3. Specific Embodiments of the Invention Specific embodiments of the present invention are listed below.

[0224] 1. An antibody that binds to human VEGF and human PDGF-B, comprising a VEGF paratope and a PDGF-B paratope within a cognate pair of variable light domain (VL domain) and variable heavy domain (VH domain), wherein the VEGF paratope comprises amino acid residues from CDR-H2, CDR-L1 and CDR-L3 of the antibody, and the PDGF-B paratope comprises amino acid residues from CDR-H1, CDR-H3 and CDR-L2 of the antibody.

[0225] 2. An antibody that binds to human VEGF and human PDGF-B, which comprises a VEGF paratope and a PDGF-B 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 and human PDGF-B.

[0226] 3. An antibody that binds to human VEGF and human PDGF-B, which contains a VEGF paratope and a PDGF-B paratope within a single cognate pair of a variable light domain (VL domain) and a variable heavy domain (VH domain), and none of the amino acids contained in the VEGF paratope are contained in the PDGF-B paratope.

[0227] 4. An antibody that binds to human VEGF and human PDGF-B, comprising a VEGF paratope and a PDGF-B paratope within one 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 and the same epitope on human PDGF-B as an antibody having a variable heavy domain of SEQ ID NO: 11 and a variable light domain of SEQ ID NO: 21. (RO7113083).

[0228] 5. An antibody that binds to human VEGF and human PDGF-B, comprising a VEGF paratope and a PDGF-B paratope within one cognate pair of a variable light domain (VL domain) and a variable heavy domain (VH domain); the VEGF paratope comprises amino acid residues derived from CDR-H2, CDR-L1 and CDR-L3 of an antibody, and the PDGF-B 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 and human PDGF-B; and / or ● none of the amino acids included in the VEGF paratope are included in the PDGF-B paratope; and / or binds to the same epitope on human VEGF and the same epitope on human PDGF-B as an antibody having a variable heavy domain of SEQ ID NO: 11 and a variable light domain of SEQ ID NO: 21; and / or The antibody Fab fragment of the antibody has (i) a K of less than 50 pM as measured by surface plasmon resonance. D and (ii) binds to human VEGF121 with a K of less than 10 nM as measured by surface plasmon resonance. D binds to human PDGF-B at - An antibody whose Fab fragment exhibits an onset of aggregation temperature of 68°C or higher.

[0229] 6. 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: 13, 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: 15, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17.

[0230] 7. 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: 29, 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: 15, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17.

[0231] 8. An antibody that specifically binds to human VEGF and human PDGF-B, 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: 13, 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: 15, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17.

[0232] 9. An antibody that specifically binds to human VEGF and human PDGF-B, 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: 29, 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: 15, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17.

[0233] 10. 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: 13; 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 D1, L2, D25, G26, W27, and W28, and (ii) FR3 comprising amino acid residues D73, D74, T75, N76, and R94; and (e) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 15; (f) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16; (g) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17; and (h) a VL domain comprising a human light chain framework having (i) FR1 comprising amino acid residues A1, and I2, and (ii) FR3 comprising amino acid residues H68 and E69, wherein the VH domain and the VL domain are according to the Kabat numbering system.

[0234] 11. 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: 29; 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 D1, L2, D25, G26, W27, and W28, and (ii) FR3 comprising amino acid residues D73, D74, T75, N76, and R94; and (e) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 15; (f) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16; (g) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17; and (h) a VL domain comprising a human light chain framework having (i) FR1 comprising amino acid residues A1, and I2, and (ii) FR3 comprising amino acid residues H68 and E69, wherein the VH domain and the VL domain are numbered according to the Kabat numbering system.

[0235] 12. An antibody that specifically binds to human VEGF and human PDGF-B, 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: 13; and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 14; and (d) a VH domain comprising a human heavy chain framework having (i) an FR1 comprising amino acid residues D1, L2, D25, G26, W27, and W28; and (ii) an FR3 comprising amino acid residues D73, D74, T75, N76, and R94. (e) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 15, (f) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, (g) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17, and (h) a VL domain comprising a human light chain framework having (i) FR1 comprising amino acid residues A1 and I2, and (ii) FR3 comprising amino acid residues H68 and E69, wherein the numbering of the VH domain and VL domain is according to the Kabat numbering system.

[0236] 13. The antibody of any one of the preceding embodiments, comprising (ii) a VH domain comprising amino acid residues D1, L2, D25, G26, W27, W28, Y31, T35b, Y52a, G55, S56, T57, Y58, T61, K62, F63, I64, G65, R66, D73, D74, T75, N76, R94, D96, Y98, D101, and T102, and (iii) a VL domain comprising amino acid residues A1, I2, S26, Y27, W27a, L27b, S27c, E55, H56, H68, E69, R92, Y93, H94, P95 and Y96, wherein numbering of the VH domain and VL domain is according to the Kabat numbering system.

[0237] 14. - a VEGF paratope comprising the following amino acid residues in the VH domain: G55, S56, T57, Y58, T61, K62, F63, I64, G65, and R66, and the following amino acid residues in the VL domain: A1, I2, S26, Y27, W27a, L27b, S27c, H68, E69, R92, Y93, H94, P95, and Y96; and - the antibody of embodiment 13, which comprises a PDGF-B paratope comprising the following amino acid residues in the VH domain: D1, L2, D25, G26, W27, W28, Y31, T35b, D73, D74, T75, N76, R94, D96, D98, D101, and T102, and the following amino acid residues in the VL domain: E55 and H56.

[0238] 15. The antibody of any one of the preceding embodiments, comprising (ii) a VH domain comprising amino acid residues D1, L2, D25, G26, W27, W28, Y31, T35b, Y52a, G55, H56, K57, Y58, T61, K62, F63, I64, G65, R66, D73, D74, T75, N76, R94, D96, Y98, D101, and T102, and (iii) a VL domain comprising amino acid residues A1, I2, S26, Y27, W27a, L27b, S27c, E55, H56, H68, E69, R92, Y93, H94, P95 and Y96, wherein numbering of the VH domain and VL domain is according to the Kabat numbering system.

[0239] 16. - a VEGF paratope comprising the following amino acid residues in the VH domain: G55, H56, K57, Y58, T61, K62, F63, I64, G65 and R66, and the following amino acid residues in the VL domain: A1, I2, S26, Y27, W27a, L27b, S27c, H68, E69, R92, Y93, H94, P95 and Y96; and - the antibody of embodiment 15, which comprises a PDGF-B paratope comprising the following amino acid residues in the VH domain: D1, L2, D25, G26, W27, W28, Y31, T35b, D73, D74, T75, N76, R94, D96, D98, D101, and T102, and the following amino acid residues in the VL domain: E55 and H56.

[0240] 17. An antibody that specifically binds to human VEGF and human PDGF-B, comprising within a pair of VL and VH domains: (i) amino acid residues D1, L2, D25, G26, W27, W28, Y31, T35b, Y52a, G55, S56, T57, Y58, T61, K62, F63, I64, G65, R66, D73, D74, T75, N76, R94, D9 6, Y98, D101, and T102, and (ii) a VL domain comprising amino acid residues A1, 12, S26, Y27, W27a, L27b, S27c, E55, H56, H68, E69, R92, Y93, H94, P95 and Y96; wherein the numbering of the VH domain and VL domain is according to the Kabat numbering system.

[0241] 18. - a VEGF paratope comprising the following amino acid residues in the VH domain: G55, S56, T57, Y58, T61, K62, F63, I64, G65, and R66, and the following amino acid residues in the VL domain: A1, I2, S26, Y27, W27a, L27b, S27c, H68, E69, R92, Y93, H94, P95, and Y96; and - the antibody of embodiment 17, which comprises a PDGF-B paratope comprising the following amino acid residues in the VH domain: D1, L2, D25, G26, W27, W28, Y31, T35b, D73, D74, T75, N76, R94, D96, D98, D101, and T102, and the following amino acid residues in the VL domain: E55 and H56.

[0242] 19. An antibody that specifically binds to human VEGF and human PDGF-B, comprising within a pair of VL and VH domains, amino acid residues D1, L2, D25, G26, W27, W28, Y31, T35b, Y52a, G55, H56, K57, Y58, T61, K62, F63, I64, G65, R66, D73, D74, T75, N76, R94, D96 , Y98, D101, and T102, and (ii) a VL domain comprising amino acid residues A1, 12, S26, Y27, W27a, L27b, S27c, E55, H56, H68, E69, R92, Y93, H94, P95 and Y96; wherein the numbering of the VH domain and VL domain is according to the Kabat numbering system.

[0243] 20. - a VEGF paratope comprising the following amino acid residues in the VH domain: G55, H56, K57, Y58, T61, K62, F63, I64, G65 and R66, and the following amino acid residues in the VL domain: A1, I2, S26, Y27, W27a, L27b, S27c, H68, E69, R92, Y93, H94, P95 and Y96; and - the antibody of embodiment 19, which comprises a PDGF-B paratope comprising the following amino acid residues in the VH domain: D1, L2, D25, G26, W27, W28, Y31, T35b, D73, D74, T75, N76, R94, D96, D98, D101, and T102, and the following amino acid residues in the VL domain: E55 and H56.

[0244] 21. 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: 11; 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: 21.

[0245] 22. 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: 11; 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: 25.

[0246] 23. An antibody according to 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: 28; 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: 25.

[0247] 24. 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: 28; 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: 31.

[0248] 25. (a) a VH domain comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 11, the VH domain comprising amino acid residues D1, L2, D25, G26, W27, W28, Y31, T35b, Y52a, G55, S56, T57, Y58, T61, K62, F63, I64, G65, R66, D73, D74, T75, N76, R94, D96, Y98, D101, and T102; (b) a VH domain comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 11, the VH domain comprising amino acid residues D1, L2, D25, G26, W27, W28, Y31, T35b, Y52a, G55, S56, T57, Y58, T61, K62, F63, I64, G65, R66, D73, D74, T75, N76, R94, D96, Y98, D101, and T102; 21, wherein the numbering of the VH domain and the VL domain is according to the Kabat numbering system.

[0249] 26. (a) a VH domain comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 11, the VH domain comprising amino acid residues D1, L2, D25, G26, W27, W28, Y31, T35b, Y52a, G55, S56, T57, Y58, T61, K62, F63, I64, G65, R66, D73, D74, T75, N76, R94, D96, Y98, D101, and T102; (b) a VH domain comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 11, the VH domain comprising amino acid residues D1, L2, D25, G26, W27, W28, Y31, T35b, Y52a, G55, S56, T57, Y58, T61, K62, F63, I64, G65, R66, D73, D74, T75, N76, R94, D96, Y98, D101, and T102; 25, wherein the numbering of the VH domain and the VL domain is according to the Kabat numbering system.

[0250] 27. (a) a VH domain comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 28, the VH domain comprising amino acid residues D1, L2, D25, G26, W27, W28, Y31, T35b, Y52a, G55, H56, K57, Y58, T61, K62, F63, I64, G65, R66, D73, D74, T75, N76, R94, D96, Y98, D101, and T102; (b) a VH domain comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 28, the VH domain comprising amino acid residues D1, L2, D25, G26, W27, W28, Y31, T35b, Y52a, G55, H56, K57, Y58, T61, K62, F63, I64, G65, R66, D73, D74, T75, N76, R94, D96, Y98, D101, and T102; 25, wherein the numbering of the VH domain and the VL domain is according to the Kabat numbering system.

[0251] 28. (a) a VH domain comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 28, the VH domain comprising amino acid residues D1, L2, D25, G26, W27, W28, Y31, T35b, Y52a, G55, H56, K57, Y58, T61, K62, F63, I64, G65, R66, D73, D74, T75, N76, R94, D96, Y98, D101, and T102; (b) a VH domain comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 28, the VH domain comprising amino acid residues D1, L2, D25, G26, W27, W28, Y31, T35b, Y52a, G55, H56, K57, Y58, T61, K62, F63, I64, G65, R66, D73, D74, T75, N76, R94, D96, Y98, D101, and T102; 31, wherein the numbering of the VH domain and the VL domain is according to the Kabat numbering system.

[0252] 29. An antibody that specifically binds to human VEGF and human PDGF-B, 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:13, 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:15, (e) a CDR-L2 having the amino acid sequence of SEQ ID NO:16, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO:17, the antibody 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:11; 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:21.

[0253] 30. An antibody that specifically binds to human VEGF and human PDGF-B, 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:13, 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:15, (e) a CDR-L2 having the amino acid sequence of SEQ ID NO:16, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO:17, the antibody 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:11; 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:25.

[0254] 31. An antibody that specifically binds to human VEGF and human PDGF-B, 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:29, 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:15, (e) a CDR-L2 having the amino acid sequence of SEQ ID NO:16, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO:17, the antibody 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:28; 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:25.

[0255] 32. An antibody that specifically binds to human VEGF and human PDGF-B, 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:29, 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:15, (e) a CDR-L2 having the amino acid sequence of SEQ ID NO:16, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO:17, the antibody 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:28; 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:31.

[0256] 33. An antibody that specifically binds to human VEGF and human PDGF-B, 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: 13, 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: 15, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17; An 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:11, and comprising D1, L2, D25, G26, W27, W28, R66, D73, D74, T75, N76, 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:21, and comprising A1, I2, H68, and E69, wherein the numbering of the VH domain and VL domain is according to the Kabat numbering system.

[0257] 34. An antibody that specifically binds to human VEGF and human PDGF-B, 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: 13; 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 D1, L2, D25, G26, W27, and W28, (ii) an FR3 comprising amino acid residues D73, D74, T75, N76, and R94; and (e) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 15; and (f) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16. an antibody 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: 11; (b) a VL domain comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 21; (c) a VL domain comprising a human light chain framework having (i) FR1 comprising amino acid residues A1 and I2, and (ii) FR3 comprising amino acid residues H68 and E69, wherein the numbering of the VH domain and the VL domain is according to the Kabat numbering system.

[0258] 35. The antibody of any one of the preceding embodiments, comprising (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 11 with up to 15 amino acid substitutions, and (b) a variable light domain comprising the amino acid sequence of SEQ ID NO: 21 with up to 15 amino acid substitutions.

[0259] 36. The antibody of any one of the preceding embodiments, comprising (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 11 with up to 15 amino acid substitutions, and (b) a variable light domain comprising the amino acid sequence of SEQ ID NO: 25 with up to 15 amino acid substitutions.

[0260] 37. The antibody of any one of the preceding embodiments, comprising (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 28 with up to 15 amino acid substitutions, and (b) a variable light domain comprising the amino acid sequence of SEQ ID NO: 25 with up to 15 amino acid substitutions.

[0261] 38. The antibody of any one of the preceding embodiments, comprising (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 28 with up to 15 amino acid substitutions, and (b) a variable light domain comprising the amino acid sequence of SEQ ID NO: 31 with up to 15 amino acid substitutions.

[0262] 39. The antibody of any one of the preceding embodiments, comprising: (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 11 with up to 15 amino acid substitutions, wherein the amino acid substitutions are located at positions 3 to 24, 29, 30, 35c to 52, 52b, 54, 59, 60, 67 to 72, 77 to 93, 95, 97, 99, or 103 to 113; (b) a variable light chain domain comprising the amino acid sequence of SEQ ID NO: 25 with up to 15 amino acid substitutions, wherein the amino acid substitutions are located at positions 3 to 25, 27d to 54, 57 to 67, 70 to 91, or 97 to 107 of SEQ ID NO: 25, wherein the numbering of the VH domain and the VL domain is according to the Kabat numbering system.

[0263] 40. The antibody of any one of the preceding embodiments, comprising: (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 28 with up to 15 amino acid substitutions, wherein the amino acid substitutions are located at positions 3 to 24, 29, 30, 35c to 52, 52b, 54, 59, 60, 67 to 72, 77 to 93, 95, 97, 99, or 103 to 113; (b) a variable light chain domain comprising the amino acid sequence of SEQ ID NO: 25 with up to 15 amino acid substitutions, wherein the amino acid substitutions are located at positions 3 to 25, 27d to 54, 57 to 67, 70 to 91, or 97 to 107 of SEQ ID NO: 25, wherein the numbering of the VH domain and the VL domain is according to the Kabat numbering system.

[0264] 41. The antibody of any one of the preceding embodiments, comprising: (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 28 with up to 15 amino acid substitutions, wherein the amino acid substitutions are located at positions 3 to 24, 29, 30, 35c to 52, 52b, 54, 59, 60, 67 to 72, 77 to 93, 95, 97, 99, or 103 to 113; (b) a variable light chain domain comprising the amino acid sequence of SEQ ID NO: 25 with up to 15 amino acid substitutions, wherein the amino acid substitutions are located at positions 3 to 25, 27d to 54, 57 to 67, 70 to 91, or 97 to 107 of SEQ ID NO: 31, wherein the numbering of the VH domain and the VL domain is according to the Kabat numbering system.

[0265] 42. An antibody that specifically binds to human VEGF and human PDGF-B, 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:13, 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:15, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:16, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:17, the antibody comprising: (a) a VH domain comprising the amino acid sequence of SEQ ID NO:11 with up to 15 amino acid substitutions; and (b) a variable light domain comprising the amino acid sequence of SEQ ID NO:21 with up to 15 amino acid substitutions.

[0266] 43. An antibody that specifically binds to human VEGF and human PDGF-B, 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: 13; 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 D1, L2, D25, G26, W27, and W28, (ii) FR3 comprising amino acid residues D73, D74, T75, N76, and R94; and (e) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 15; (f) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16. (g) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17, (h) a VL domain comprising a human light chain framework having (i) FR1 comprising amino acid residues A1 and I2, and (ii) FR3 comprising amino acid residues H68 and E69, wherein the numbering of the VH domain and the VL domain is according to the Kabat numbering system, comprising: (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 11 with up to 15 amino acid substitutions; and (b) a variable light chain domain comprising the amino acid sequence of SEQ ID NO: 21 with up to 15 amino acid substitutions.

[0267] 44. The antibody of any one of the preceding embodiments, comprising L-FR-1 comprising the amino acid sequence of SEQ ID NO:22.

[0268] 45. The antibody of any one of the preceding embodiments, comprising L-FR-1 comprising the amino acid sequence of SEQ ID NO:26.

[0269] 46. ​​The antibody of any one of the preceding embodiments, comprising L-FR-1 comprising the amino acid sequence of SEQ ID NO: 32.

[0270] 47. The antibody of any one of the preceding embodiments, comprising a VH sequence of SEQ ID NO:11 and a VL sequence of SEQ ID NO:21.

[0271] 48. The antibody of any one of the preceding embodiments, comprising a VH sequence of SEQ ID NO:11 and a VL sequence of SEQ ID NO:25.

[0272] 49. The antibody of any one of the preceding embodiments, comprising a VH sequence of SEQ ID NO:28 and a VL sequence of SEQ ID NO:25.

[0273] 50. The antibody of any one of the preceding embodiments, comprising a VH sequence of SEQ ID NO:28 and a VL sequence of SEQ ID NO:31.

[0274] 51. An antibody that specifically binds to human VEGF and human PDGF-B, comprising the VH sequence of SEQ ID NO: 11 and the VL sequence of SEQ ID NO: 21.

[0275] 52. The antibody of any one of the preceding embodiments, comprising a heavy chain amino acid sequence of SEQ ID NO: 18 and a light chain amino acid sequence of SEQ ID NO: 20.

[0276] 53. An antibody that specifically binds to human VEGF and human PDGF-B, comprising the heavy chain amino acid sequence of SEQ ID NO: 18 and the light chain amino acid sequence of SEQ ID NO: 20.

[0277] 54. The antibody of any one of the preceding embodiments, comprising a heavy chain amino acid sequence of SEQ ID NO: 18 and a light chain amino acid sequence of SEQ ID NO: 19.

[0278] 55. An antibody that specifically binds to human VEGF and human PDGF-B, comprising the heavy chain amino acid sequence of SEQ ID NO: 18 and the light chain amino acid sequence of SEQ ID NO: 19.

[0279] 56. The antibody of any one of the preceding embodiments, comprising a heavy chain amino acid sequence of SEQ ID NO: 18 and a light chain amino acid sequence of SEQ ID NO: 27.

[0280] 57. The antibody of any one of the preceding embodiments, comprising a heavy chain amino acid sequence of SEQ ID NO: 30 and a light chain amino acid sequence of SEQ ID NO: 27.

[0281] 58. The antibody of any one of the preceding embodiments, comprising a heavy chain amino acid sequence of SEQ ID NO: 30 and a light chain amino acid sequence of SEQ ID NO: 33.

[0282] 59. The antibody Fab fragment of the antibody has (i) a K of less than 50 pM as measured by surface plasmon resonance. D and (ii) binds to human VEGF121 with a K of less than 10 nM as measured by surface plasmon resonance. D 2. The antibody of any one of the preceding embodiments, wherein the antibody binds to human PDGF-B at

[0283] 60. An antibody that specifically binds to human VEGF and human PDGF-B, wherein the antibody Fab fragment of the antibody has (i) a K of less than 50 pM as measured by surface plasmon resonance. D and (ii) binds to human VEGF121 with a K of less than 10 nM as measured by surface plasmon resonance. D An antibody that binds to human PDGF-B.

[0284] 61. An antibody that specifically binds to human VEGF and human PDGF-B, 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: 13, 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: 15, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17, wherein an antibody Fab fragment of the antibody has (i) a K of less than 50 pM as measured by surface plasmon resonance. D and (ii) binds to human VEGF121 with a K of less than 10 nM as measured by surface plasmon resonance. D An antibody that binds to human PDGF-B.

[0285] 62. An antibody that specifically binds to human VEGF and human PDGF-B, 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: 13; 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 D1, L2, D25, G26, W27, and W28, (ii) FR3 comprising amino acid residues D73, D74, T75, N76, and R94; and (e) an amino acid sequence of SEQ ID NO: 15. (f) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16; (g) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17; and (h) a VL domain comprising a human light chain framework having (i) FR1 comprising amino acid residues A1 and I2, and (ii) FR3 comprising amino acid residues H68 and E69, wherein the numbering of the VH domain and the VL domain is according to the Kabat numbering system, and wherein an antibody Fab fragment of the antibody has (i) a K of less than 50 pM as measured by surface plasmon resonance. D and (ii) binds to human VEGF121 with a K of less than 10 nM as measured by surface plasmon resonance. D An antibody that binds to human PDGF-B.

[0286] 63. An antibody that specifically binds to human VEGF and human PDGF-B, comprising a pair of VH and VL domains, (i) a VH domain comprising amino acid residues D1, L2, D25, G26, W27, W28, Y31, T35b, Y52a, G55, S56, T57, Y58, T61, K62, F63, I64, G65, R66, D73, D74, T75, N76, R94, D96, Y98, D101, and T102; and (ii) a VL domain comprising amino acid residues A1, I2, S26, Y27, W27a, L27b, S27c, E55, H56, H68, E69, R92, Y93, H94, P95 and Y96, wherein the numbering of the VH domain and the VL domain is according to the Kabat numbering system, and an antibody Fab fragment of the antibody has (i) a K of less than 50 pM as measured by surface plasmon resonance. Dand (ii) binds to human VEGF121 with a K of less than 10 nM as measured by surface plasmon resonance. D An antibody that binds to human PDGF-B.

[0287] 64. An antibody that specifically binds to human VEGF and human PDGF-B, 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: 13, 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: 15, (e) a CDR-L2 having the amino acid sequence of SEQ ID NO: 16, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 17; 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: 11; 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: 21; and (i) a K of less than 50 pM as measured by surface plasmon resonance. D and (ii) binds to human VEGF121 with a K of less than 10 nM as measured by surface plasmon resonance. D An antibody that binds to human PDGF-B.

[0288] 65. The antibody of any one of the preceding embodiments, wherein the antibody Fab fragment exhibits an onset aggregation temperature of 68°C or greater.

[0289] 66. An antibody that specifically binds to human VEGF and human PDGF-B, wherein the antibody Fab fragment of the antibody exhibits an aggregation onset temperature of 68°C or higher.

[0290] 67. An antibody that specifically binds to human VEGF and human PDGF-B, 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: 13, 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: 15, (e) CDR-L2 having the amino acid sequence of SEQ ID NO: 16, and (f) CDR-L3 having the amino acid sequence of SEQ ID NO: 17, wherein the antibody Fab fragment of the antibody exhibits an aggregation onset temperature of 68°C or higher.

[0291] 68. An antibody that specifically binds to human VEGF and human PDGF-B, 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: 13; 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 D1, L2, D25, G26, W27, and W28; (ii) an FR3 comprising amino acid residues D73, D74, T75, N76, and R94; and (e) a VH domain comprising the human heavy chain framework having the amino acid sequence of SEQ ID NO: 15. (f) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16; (g) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17; and (h) a VL domain comprising a human light chain framework having (i) FR1 comprising amino acid residues A1 and I2, and (ii) FR3 comprising amino acid residues H68 and E69, wherein the numbering of the VH domain and the VL domain is according to the Kabat numbering system, and an antibody Fab fragment of the antibody exhibits an aggregation onset temperature of 68°C or higher.

[0292] 69. An antibody that specifically binds to human VEGF and human PDGF-B, comprising in a pair of VH and VL domains: (i) amino acid residues D1, L2, D25, G26, W27, W28, Y31, T35b, Y52a, G55, S56, T57, Y58, T61, K62, F63, I64, G65, R66, D73, D74, T75, N76, R94, D96, Y98, D101, and T102; and (ii) a VL domain comprising amino acid residues A1, I2, S26, Y27, W27a, L27b, S27c, E55, H56, H68, E69, R92, Y93, H94, P95 and Y96, wherein the numbering of the VH domain and VL domain is according to the Kabat numbering system, and wherein an antibody Fab fragment of the antibody exhibits an aggregation onset temperature of 68°C or greater.

[0293] 70. An antibody that specifically binds to human VEGF and human PDGF-B, 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: 13, and (c) a CDR-H3 having the amino acid sequence of SEQ ID NO: 14; and (d) a VL domain comprising CDR-L1 having the amino acid sequence of SEQ ID NO: 15, (e) a CDR-L2 having the amino acid sequence of SEQ ID NO: 16, and (f) a CDR-L3 having the amino acid sequence of SEQ ID NO: 17, 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: 11; 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: 21, and an antibody Fab fragment of the antibody exhibits an aggregation onset temperature of 68°C or higher.

[0294] 71. The antibody of any one of the preceding embodiments, which is a monoclonal antibody.

[0295] 72. The antibody of any one of the preceding embodiments, which is an antibody fragment that binds to human VEGF and human PDGF-B.

[0296] 73. The antibody of any one of the preceding embodiments, which is bispecific.

[0297] 74. The antibody of any one of the preceding embodiments, which is a Fab fragment.

[0298] 75. The antibody of any one of the preceding embodiments, which is a bispecific antibody fragment.

[0299] 76. The antibody of any one of the preceding embodiments, which is a multispecific antibody.

[0300] 77. An isolated nucleic acid encoding an antibody according to any one of embodiments 1 to 76.

[0301] 78. A host cell comprising the nucleic acid of embodiment 77.

[0302] 79. An expression vector comprising the nucleic acid of embodiment 77.

[0303] 80. A method for producing an antibody that binds to human VEGF and human PDGF-B, comprising culturing a host cell according to embodiment 78 so that the antibody is produced.

[0304] 81. The method of embodiment 80, further comprising recovering the antibody from the host cell.

[0305] 82. An antibody produced by the method of embodiment 80 or 81.

[0306] 83. A pharmaceutical formulation comprising an antibody according to any one of embodiments 1 to 76 and a pharma- ceutically acceptable carrier.

[0307] 84. An antibody according to any one of embodiments 1 to 76 for use as a medicament.

[0308] 85. The antibody according to any one of embodiments 1 to 76 for use in the treatment of a vascular disease.

[0309] 86. The antibody according to any one of embodiments 1 to 76 for use in the treatment of an ocular vascular disease.

[0310] 87. Use of the antibody according to any one of embodiments 1 to 76 or the pharmaceutical composition according to embodiment 83 in the manufacture of a medicament.

[0311] 88. Use of the antibody according to any one of embodiments 1 to 76 or the pharmaceutical composition according to embodiment 83 in the manufacture of a medicament for inhibiting angiogenesis.

[0312] 89. A method for treating an individual having a vascular disease, comprising administering to the individual an effective amount of an antibody described in any one of embodiments 1 to 76 or a pharmaceutical composition described in embodiment 83.

[0313] 90. A method for treating an individual having an ocular vascular disease, comprising administering to the individual an effective amount of an antibody described in any one of embodiments 1 to 76 or a pharmaceutical composition described in embodiment 83.

[0314] 91. A method for inhibiting angiogenesis in an individual, comprising administering to the individual an antibody according to any of embodiments 1 to 76 or a pharmaceutical composition according to any of embodiments 83 in an amount effective to inhibit angiogenesis. [Table 3] TIFF0007674383000004.tif230170 TIFF0007674383000005.tif248170 TIFF0007674383000006.tif221170 TIFF0007674383000007.tif169170 EXAMPLES

[0315] 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 of the invention.

[0316] Example 1: Generation of bispecific anti-VEGF / anti-PDGF-B Fab fragments 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.

[0317] The first library was enriched for binders to human PDGF-B 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 PDGF-B 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. Supernatants containing soluble Fab fragments were screened for binding to PDGF-B 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 PDGF-B or VEGF-A, respectively.

[0318] Description of bispecific designs for VEGF-PDGF applications: A pair of bispecific VH and VL sequences were designed in silico by (1) placing VH residues 1-3 including the heavy chain N-terminal region, 23-35 including CDR-H1, 71-77 including the VH outer loop region, and 93-102 including CDR-H3 from the PDGF-B-specific Fab sequence, and VH residues 50-65 including CDR-H2 from the VEGF-A-specific Fab sequence in frame with the VH3 and JH framework sequences, and (2) placing VL residues 1-3 including the light chain N-terminal region, 24-34 including CDR-L1, 66-71 including the VL outer loop region, and 89-97 including CDR-L3 from the VEGF-A-specific Fab sequence, and VL residues 49-57 including CDR-L2 from the PDGF-B-specific Fab sequence in frame with the Vkappa1 and JK framework sequences.

[0319] The resulting bispecific anti-VEGF / anti-PDGF-B antibody "P1AE2845" is characterized by a heavy chain of SEQ ID NO:9 and a light chain of SEQ ID NO:10.

[0320] Example 2: Expression of the bispecific anti-VEGF / anti-PDGF-B Fab fragment P1AE2845 The designed bispecific DutaFab VH and VL sequence pairs were synthesized and cloned into an E. coli expression vector in the context of gene sequences encoding the CH1 and Ckappa domains. The vector was transformed into TG1 E. coli cells and individual colonies were cultured for soluble expression of the bispecific Fab fragments. The bispecific Fab fragments were purified from TG1 culture supernatants by affinity chromatography and specific binding to both PDGF-B and VEGF-A was verified.

[0321] Example 3: Characterization of the bispecific anti-VEGF / anti-PDGF-B Fab fragment P1AE2845 The binding affinity, hydrophilicity and thermal stability of the bispecific antibody P1AE2845 were evaluated as follows.

[0322] VEGF 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 29104990) using standard amine coupling chemistry, resulting in a surface density of approximately 500 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 VEGF121-His was captured on the surface, resulting in ligand densities of approximately 10 and 20 RU, respectively. A dilution series of bispecific anti-VEGF / anti-PDGF-B Fab fragments (1.2-100 nM, 1:3 dilution) were injected sequentially for 90 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. Bulk refractive index differences were corrected by subtracting a blank injection and the response obtained from a control flow cell not capturing human VEGF121. Curve fitting was performed using a 1:1 Langmuir binding model within the Biacore evaluation software. The Multiple Rmax option was selected for a global fit using both ligand densities to provide a more robust fit.

[0323] PDGF-B binding kinetics as assessed by surface plasmon resonance (SPR): Human PDGF-BB binding ELISA: Binding analysis was performed using an enzyme-linked immunosorbent assay (ELISA) based technique. The antigen human PDGF-BB (Cell Signaling, Cat. No. 8921BF) was immobilized on 384-well microtiter plates (Thermo Scientific, Cat. No. 464718) at a concentration of 125 ng / mL in 25 μL in PBS, 0.5% BSA and 0.05% Tween. Three washing routines of 90 μL PBS with dispensing and aspiration were performed after each of the following steps: 1) blocking step: saturating non-binding surface (1 h, 2% BSA); 2) increasing concentrations of anti-PDGF-BB antibodies for 1 h; 3) detection antibody, dilution = 1:3000 (ECL anti-rabbit IgG-POD, NA9340V + ECL anti-human IgG-POD, NA933V or, for mouse antibodies, ECL anti-mouse IgG-POD; NA9310V). Optical density was measured at 370 nm 20–30 min after addition of the substrate 3,3',5,5'-tetramethylbenzidine (TMB, Piercenet, Cat. No. 34021). EC50 values ​​were calculated with a four-parameter logistic model using GraphPadPrism 6.0 software.

[0324] Cynomolgus Monkey PDGF-BB Binding ELISA: Binding analysis was performed using an enzyme-linked immunosorbent assay (ELISA) based technique. The antigen human PDGF-BB was immobilized on 384-well microtiter plates (Thermo Scientific, Cat. No. 464718) at a concentration of 125ng / mL in 25μL in PBS, 0.5% BSA and 0.05% Tween. Three washing routines of 90μL PBS, 0.5% BSA, 0.05% Tween with dispensing and aspiration were performed after each of the following steps: 1) Blocking step: saturating non-binding surface (1h, 2% BSA); 2) increasing concentrations of anti-PDGF-BB antibody for 1h; 3) Detection antibody, dilution = 1:3000 (ECL anti-rabbit IgG-POD, NA9340V + ECL anti-human IgG-POD, NA933V or ECL anti-mouse IgG-POD; NA9310V for mouse antibodies). Optical density was measured at 370 nm 20–30 min after addition of the substrate 3,3',5,5'-tetramethylbenzidine (TMB, Piercenet, Cat. No. 34021). EC50 values ​​were calculated with a four-parameter logistic model using GraphPadPrism 6.0 software.

[0325] Hydrophobic Interaction Chromatography (HIC): Apparent hydrophobicity was determined by injecting 20 μg of bispecific anti-VEGF / anti-PDGF-B Fab fragments onto a HIC-Ether-5 PW (Tosoh) column equilibrated with 25 mM Na phosphate, 1.5 M ammonium sulfate, pH 7.0. Elution was performed with a linear gradient of 0 to 100% buffer B (25 mM sodium phosphate, pH 7.0) within 60 min. Retention times were compared to protein standards with known hydrophobicity.

[0326] Thermal stability: Samples of bispecific anti-VEGF / anti-PDGF-B Fab fragments were prepared at a concentration of 1 mg / mL in 20 mM histidine / histidine chloride, 140 mM NaCl, pH 6.0, centrifuged through a 0.4 μm filter plate, transferred to an optical 384-well plate, and overlaid with paraffin oil. Hydrodynamic radii were repeatedly measured by dynamic light scattering in a DynaPro plate reader (Wyatt) while samples were heated from 25° C. to 80° C. at a rate of 0.05° C. / min. Alternatively, samples were transferred to a 10 μL microcuvette array and heated from 25° C. to 90° C. at a rate of 0.1° C. / min. while static light scattering data and fluorescence data with excitation with a 266 nm laser were recorded on an Optim1000 instrument (Avacta Inc.).

[0327] The onset of aggregation temperature is defined as the temperature at which the hydrodynamic radius (DLS) or scattered light intensity (Optim1000) begins to increase.

[0328] The results are shown in Tables 1 and 2. [Table 4] [Table 5]

[0329] Example 4: Improvement of the bispecific anti-VEGF / anti-PDGF-B Fab fragment P1AE2845 For clinical applications, the antibody was further improved, for example with respect to PDGF-B and VEGF binding. 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 P1AE2845 were screened and selected based on their desired properties with respect to yield, affinity, simultaneous antigen binding, hydrophilicity, stability, viscosity and other parameters.

[0330] Improved candidate antibodies P1AA9124, as well as 28.01, 28.02, RO7113083 and 28.07 were selected from multiple tested candidate antibody molecules. The amino acid sequences of these improved bispecific anti-VEGF / anti-PDGF-B Fab fragments are identified in Table 3. [Table 6]

[0331] Figures 2 and 3 show the alignment of the variable heavy and variable light domains of the generated bispecific anti-VEGF / anti-PDGF-B Fab 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.

[0332] Example 5: Improved antigen-binding kinetics of bispecific anti-VEGF / anti-PDGF-B Fab fragments For the candidate antibodies, the binding kinetics to VEGF and PDGF-B were assessed as described in Example 3 using the indicated bispecific anti-VEGF / anti-PDGF-B Fab fragments (amino acid sequences shown in Table 3).

[0333] The PDGF-B binding kinetics results are shown in Tables 4a, 4b and Tables 5a, b and c. For comparison, the antigen binding kinetics of the prior art anti-VEGF / anti-PDGF-B antibody 0117, a full-length IgG antibody disclosed in WO 2016 / 075036, is shown. Also shown is the PDGF binding kinetics of the prior art PDGF inhibitor pegprelanib (Fovista®), a pegylated aptamer. [Table 7] [Table 8] [Table 9] [Table 10] [Table 11] [Table 12]

[0334] The VEGF binding kinetics results are shown in Tables 6a and b. [Table 13] [Table 14]

[0335] Example 6: Biophysical properties (stability and hydrophobicity) of improved bispecific anti-VEGF / anti-PDGF-B Fab fragments The indicated biophysical properties of the candidate antibodies were evaluated as described in Example 3 using the indicated bispecific anti-VEGF / anti-PDGF-B Fab fragments (amino acid sequences shown in Table 3).

[0336] Table 7 shows the thermal stability and hydrophobicity of the antibodies analyzed. For comparison, the thermal stability of the prior art anti-VEGF / anti-PDGF-B antibody 0117, a full-length IgG antibody disclosed in WO 2016 / 075036, is included. [Table 15]

[0337] Example 7: VEGF121 and VEGF165 Blocking Activity (VEGF Baseline Assay) Maxisorp 96-well plates (ThermoScientific #442404) were incubated with 200 mM NaHCO at a final concentration of 1 μg / mL for 1 h at room temperature. 3 The plates were coated with 50 μL / well of hVEGFR-1-Fc in PBST-1% BSA, pH 9.4. 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-loaded with 50 μL / well of 2 nM VEGF121 or 2 nM VEGF165 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, followed by the addition of 200 μL of 2% MPBST, followed by incubation for 45 minutes at room temperature. The plate was then washed twice with PBST. 50 μl of Fab-VEGF premix was transferred to a Maxisorp plate and incubated for 1.5 h at room temperature. Afterwards, the plate was washed twice with PBST and 50 μL of anti-VEGF-bio antibody (1:2000 dilution in PBST) and SA-HRP (1:2000 dilution in PBST) were added and incubated for 30 min at room temperature. The plate was washed six times with PBST and 50 μL of TMB substrate solution was added and incubated for 30 min at room temperature. 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 Figure 4.

[0338] Example 8: Structural analysis of the improved bispecific anti-VEGF / anti-PDGF-B Fab fragment P1AA9124 The crystal structure of the bispecific anti-VEGF / anti-PDGF-B Fab fragment P1AA9124 in complex with VEGF and PDGF-B identified amino acid residues that contact the respective antigens VEGF and PDGF-B.

[0339] Crystallization of Fab-VEGF complex: To obtain crystals of Fab fragment P1AA9124 complexed with VEGFA-121 (Peprotech - Cat. No. 100-20A), both proteins were mixed at a 1:1 molar ratio with respect to VEGF monomer. The complex was concentrated to 11 mg / ml and crystallized by dropwise vapor diffusion in 0.1 M MES pH 6.5 and 1.6 M magnesium sulfate at 20 °C. Needle-shaped crystals grew for approximately 120 days and were frozen in liquid nitrogen containing 20% ​​glycerol as cryoprotectant.

[0340] Data collection and crystal structure determination Fab-VEGF complex: Data were collected at a temperature of 100 K on station I911-3 (MAX IV Laboratory, Lund, Sweden) equipped with a MarMosaic 225 detector. A total of 200 diffraction images were collected with an exposure time of 30 s and an oscillation range of 1° per image. Data were integrated and scaled using XDS (J. Appl. Cryst. (1993). 26, 795-800), then merged and converted to MTZ format using XDSCONV. The structure of the DutaFab:VEGF complex was solved and refined by molecular replacement with Phaser (J. Appl. Cryst. (2007). 40, 658-674) using the CCP4 suite (Acta Cryst. (2011). D67, 235-242). The solvent content and Matthews' coefficient were 43.5% and 2.18 Å, respectively. 3 The calculated Δ ...free This resulted in a final structure with values.

[0341] Crystallization of Fab-PDGF complex: To obtain crystals of Fab fragment P1AA9124 complexed with PDGF-BB (Peprotech - Cat. No. 100-13A), both proteins were mixed at a 1:1 molar ratio with respect to PDGF monomer. The complex was concentrated to 12 mg / ml and crystallized by dropwise vapor diffusion in 0.1 M Tris pH 7.5, 42% (+ / -)-2-methyl-2,4-pentanediol (MPD) at 20 °C. Plate-shaped crystals grew within a few weeks and were frozen in liquid nitrogen containing 20% ​​glycerol as cryoprotectant.

[0342] Data collection and crystal structure determination Fab-PDGF complex: Data were collected at a temperature of 100 K on station I03 (Diamond Light Source, UK) equipped with a Pilatus3 6M detector. A total of 800 diffraction images were collected with an exposure time of 0.1 s and an oscillation range of 0.2° per image. Data were integrated and scaled using XDS (J.Appl.Cryst.(1993).26,795-800), then merged and converted to MTZ format using XDSCONV. The structure of DutaFab:PDGF-BB complex was solved and refined by molecular replacement with Phaser (J.Appl.Cryst.(2007).40,658-674) using the CCP4 suite (Acta Cryst.(2011).D67,235-242). The solvent content and Matthews' coefficient were 53.2% and 2.63 Å, respectively. 3The Fab was calculated to be 1 / Da, corresponding to two DutaFab:PDGF complexes in the asymmetric unit. As search models, monomeric PDGF (PDB 4QCI) and the Fab from the DutaFab:VEGF structure were used to search for two copies of each in the asymmetric unit. Phaser was able to localize the two Fab molecules. After molecular replacement, rigid-body refinement was performed in Refmac5 (Acta Cryst. (1997). D53, 240-255). A first round of constrained refinement resulted in a sufficiently clear map that the dimeric PDGF-BB could be manually positioned. Iterative constrained refinement in Refmac5 and model building in Coot (Acta Cryst. (2004). D60, 2126-2132) gave R values ​​and R values ​​of 26.7% and 30.9%, respectively. free This resulted in a final structure with values. [Table 16]

[0343] An example of the location of paratope amino acid residues within the VH and VL domains is shown in Figure 2. As shown, the VEGF and PDGF-B paratopes do not overlap. Amino acids that contribute to the VEGF paratope do not contribute to the PDGF-B paratope. Conversely, amino acids that contribute to the PDGF-B paratope do not contribute to the VEGF paratope.

[0344] Amino acids from the light chain CDR1 and CDR3 and the heavy chain CDR2 contribute to the VEGF paratope. The VEGF paratope does not include amino acids from the light chain CDR2, the heavy chain CDR1, and the heavy chain CDR3.

[0345] The amino acid residues identified as contributing to antigen binding are identified below in Table 8 (for variable heavy domain amino acid residues) and Table 9 (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 1 and 2). 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 1 and 2). [Table 17] [Table 18]

Claims

1. An antibody that binds to human VEGF and human PDGF-B, - (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: 13, 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 D1, L2, D25, G26, W27, and W28, (ii) an FR3 comprising amino acid residues D73, D74, T75, N76, and R94, and (e) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 15, (f) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, (g) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17, and (h) a VL domain comprising a human light chain framework having (i) an FR1 comprising amino acid residues A1, I2, and (ii) an FR3 comprising amino acid residues H68, and E69; or (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: 29, 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 D1, L2, D25, G26, W27, and W28, and (ii) an FR3 comprising amino acid residues D73, D74, T75, N76, and R94, and (e) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 15, (f) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, (g) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17, and (h) a VL domain comprising a human light chain framework having (i) an FR1 comprising amino acid residues A1, I2, and (ii) an FR3 comprising amino acid residues H68, and E69. wherein the VH domain and the VL domain are according to the Kabat numbering system.

2. (a) a VH domain comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 11; 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: 21; or (a) a VH domain comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 11; 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: 25; or (a) a VH domain comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 28; 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: 25; or (a) a VH domain comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 28; 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:

31. The antibody of claim 1 , comprising:

3. The antibody of claim 1 or 2, comprising an L-FR-1 comprising the amino acid sequence of SEQ ID NO:22; SEQ ID NO:26; or SEQ ID NO:

32.

4. - the VH sequence according to SEQ ID NO: 11 and the VL sequence according to SEQ ID NO: 21; - the VH sequence according to SEQ ID NO: 11 and the VL sequence according to SEQ ID NO: 25; - a VH sequence according to SEQ ID NO: 28 and a VL sequence according to SEQ ID NO: 25; or - the VH sequence according to SEQ ID NO: 28 and the VL sequence according to SEQ ID NO: 31 The antibody of any one of claims 1 to 3, comprising:

5. An antibody that specifically binds to human VEGF and human PDGF-B, - the VH sequence according to SEQ ID NO: 11 and the VL sequence according to SEQ ID NO: 21; - the VH sequence according to SEQ ID NO: 11 and the VL sequence according to SEQ ID NO: 25; - a VH sequence according to SEQ ID NO: 28 and a VL sequence according to SEQ ID NO: 25; or - the VH sequence according to SEQ ID NO: 28 and the VL sequence according to SEQ ID NO: 31 An antibody comprising:

6. The antibody Fab fragment of the antibody has (i) a K of less than 50 pM as measured by surface plasmon resonance. Ｄ and (ii) binds to human VEGF121 with a K of less than 10 nM as measured by surface plasmon resonance. Ｄ The antibody of any one of claims 1 to 5, which binds to human PDGF-B at

7. The antibody according to claim 1 , wherein the antibody Fab fragment exhibits an aggregation onset temperature of 68° C. or higher.

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

9. An isolated nucleic acid encoding an antibody according to any one of claims 1 to 8.

10. A host cell comprising the nucleic acid of claim 9.

11. An expression vector comprising the nucleic acid of claim 9.

12. 11. A method for producing an antibody that binds to human VEGF and human PDGF-B, comprising culturing the host cell of claim 10 so that said antibody is produced, and further comprising recovering said antibody from said host cell.

Images