Antibodies binding to VEGF and il-1 beta and methods of use thereof

Abstract

The present invention relates to anti-VEGF / anti-IL-1 [beta] antibodies and methods of use thereof.

Description

[0001] This application is a divisional application of international application number PCT / EP2019 / 086529, international application date December 20, 2019, Chinese application date June 18, 2021, Chinese application number 201980084480.5, and invention name “Antibodies binding to VEGF and IL-1β and methods of use thereof”. Technical Field

[0002] The present invention relates to anti-VEGF / anti-IL-1β antibodies and methods of using the same. Background Art

[0003] Bispecific antibodies that bind to IL-1β and VEGF have been previously reported and suggested for use in treating ocular vascular diseases (WO2016 / 075034, antibody "0032"). The bispecific anti-VEGF / anti-IL-1β antibody 0032 is a full-length IgG-like antibody with a VH / VL domain exchange in one binding arm (WO2009 / 080252, Schaefer, W et al., PNAS, 108 (2011) 11187-1191), wherein the binding arm with the wild-type antibody domain arrangement specifically binds to IL-1β, and the binding arm containing the VH / VL domain exchange specifically binds to VEGF. The VEGF binding arm comprises the VH and VL domains of the anti-VEGF antibody ranibizumab.

[0004] Multispecific antibodies comprising two paratopes in a pair of variable heavy chain domains (VH) and variable light chain domains (VL) have been described in WO2008 / 027236; WO2010 / 108127 and Bostrom, J., et al., Science 323 (2009) 1610-1614 and in WO2012 / 163520.

[0005] WO2012 / 163520 discloses bispecific antibodies comprising two non-overlapping paratopes in a pair of VH and VL domains ("DutaFab"). Each paratope of the bispecific antibody of WO2012 / 163520 comprises amino acids from the heavy chain and from the light chain CDR, wherein the heavy chain CDR-H1 and CDR-H3 and the light chain CDR-L2 contribute to the first paratope, while the light chain CDR-L1 and CDR-L3 and the heavy chain CDR-H2 contribute to the second paratope. Monospecific antibodies comprising each paratope are independently isolated from different Fab libraries, and the Fab libraries are diversified in the first or second paratope. The amino acid sequences of the monospecific antibodies are identified and combined into dual paratopic VH and VL pairs. WO2012 / 163520 discloses an exemplary Fab fragment that specifically binds to VEGF and IL-6.

[0006] There is a need for improved therapeutic antibodies that bind to VEGF and IL-1β. Summary of the invention

[0007] The present invention relates to bispecific anti-VEGF / anti-IL-1β antibodies and methods of using the same.

[0008] On the one hand, the present invention provides an antibody that binds to human VEGF and human IL-1β, comprising a VEGF paratope and an IL-1β paratope within a cognate pair of a variable light chain domain (VL domain) and a variable heavy chain domain (VH domain), wherein the VEGF paratope comprises amino acid residues from CDR-H2, CDR-L1 and CDR-L3 of the antibody, wherein the IL-1β paratope comprises amino acid residues from CDR-H1, CDR-H3 and CDR-L2 of the antibody.

[0009] In one aspect, the present invention provides an antibody that binds to human VEGF and to human IL-1β, comprising a VEGF paratope and an IL-1β paratope within a cognate pair of a VL domain and a VH domain, wherein the variable light chain domain and the variable heavy chain domain pair simultaneously bind to human VEGF and human IL-1β.

[0010] In one aspect, the present invention provides an antibody that binds to human VEGF and to human IL-1β, comprising a VEGF paratope and an IL-1β paratope within a cognate pair of a VL domain and a VH domain, wherein none of the amino acids contained in the VEGF paratope are contained in the IL-1β paratope.

[0011] In one aspect, the present invention provides an antibody that binds to human VEGF and to human IL-1β, comprising a VEGF paratope and an IL-1β paratope within a cognate pair of a VL domain and a VH domain, wherein the antibody binds to the same epitope on human VEGF and to the same epitope on human IL-1β as an antibody having a variable heavy chain domain of SEQ ID NO: 11 and a variable light chain domain of SEQ ID NO: 12.

[0012] In one aspect, the present invention provides an antibody that binds to human VEGF and to human IL-1β, wherein the antibody Fab fragment of the antibody binds to: (i) human VEGF121, wherein the K measured by surface plasmon resonance is D less than 10 pM, and (ii) human IL-1β, where the K measured by surface plasmon resonance is D Less than 30pM.

[0013] In one aspect, the present invention provides an antibody that binds to human VEGF and to human IL-1β, wherein an antibody Fab fragment of the antibody exhibits an aggregation onset temperature greater than 70°C.

[0014] In one aspect, the invention provides an antibody that binds to human VEGF and to human IL-1β, wherein an antibody Fab fragment of the antibody exhibits a melting temperature greater than 80°C as measured by dynamic light scattering.

[0015] On the one hand, the present invention provides an antibody that binds to human VEGF and to human IL-1β, wherein the binding of the antibody Fab fragment of the antibody to human VEGF inhibits the binding of VEGF to VEGFR2, wherein the IC50 measured by surface plasmon resonance is less than 50nM; and wherein the binding of the antibody Fab fragment of the antibody to human IL-1β inhibits the binding of IL-1β to IL-1βR1, wherein the IC50 measured by surface plasmon resonance is less than 30nM.

[0016] On the one hand, the present invention provides an antibody that binds to human VEGF and human IL-1β, wherein the antibody comprises a VH domain and a VL domain, the VH domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15, and the VL domain comprises (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8.

[0017] In one aspect, the present invention provides an antibody that binds to human VEGF and human IL-1β, wherein the antibody comprises a VH domain and a VL domain, the VH domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14, (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15, (d) a human heavy chain framework having (i) a FR1 comprising amino acid residues E2, G26, V28 and K30, (ii) a FR3 comprising amino acid residues R66, R83 and K94; the VL domain comprising: (e) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, (f) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17, (g) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 18 NO:8, and (h) a human light chain framework having (i) a FR1 comprising amino acid residue I2, (ii) a FR2 comprising amino acid residue Y49, and (iii) a FR3 comprising amino acid residues G57, E67, D68 and Q69, 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 to human VEGF and to human IL-1β, comprising a VH domain and a VL domain, wherein the VH domain comprises amino acid residues E2, G26, V28, K30, W31, N35b, D35c, K52a, D55, H56, Y58, T61, K62, F63, I64, R66, R83, K94, D95, V96, F98 and D101, and the VL domain comprises amino acid residues I2, Y27, W27a, S27c, S27d, L32, Y49, D50, Y53, K54, L56, G57, E67, D68, Q69, Y91, R92, Y93, H94 and Y96, wherein the numbering of the VH domain and the 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: D55, H56, Y58, T61, K62, F63, I64, R66, and R83, and the following amino acid residues in the VL domain: I2, Y27, W27a, S27c, S27d, E67, D68, Q69, R92, Y93, H94, and Y96; and an IL-1 β paratope comprising the following amino acid residues in the VH domain: E2, G26, V28, K30, W31, N35b, D35c, K52a, K94, D95, V96, F98, and D101, and the following amino acid residues in the VL domain: L32, Y49, D50, Y53, K54, L56, G57, Y91.

[0019] In one aspect, the invention provides an antibody that binds to human VEGF and to human IL-1β, comprising a VH domain and a VL domain, wherein the VH domain comprises amino acid residues E2, G26, V28, K30, W31, N35b, D35c, K52a, D55, H56, Y58, T61, K62, F63, I64, R66, R83, K94, D95, V96, F98 and D101, and the VL domain comprises amino acid residues I2, Y27, W27a, S27c, S27d, L32, Y49, D50, Y53, K54, L56, G57, S67, H68, E69, Y91, R92, Y93, H94 and Y96, wherein the numbering of the VH domain and the 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: D55, H56, Y58, T61, K62, F63, I64, R66, and R83, and the following amino acid residues in the VL domain: I2, Y27, W27a, S27c, S27d, S67, H68, E69, R92, Y93, H94, and Y96; and an IL-1 β paratope comprising the following amino acid residues in the VH domain: E2, G26, V28, K30, W31, N35b, D35c, K52a, K94, D95, V96, F98, and D101, and the following amino acid residues in the VL domain: L32, Y49, D50, Y53, K54, L56, G57, Y91.

[0020] On the one hand, the present invention provides an antibody that binds to human VEGF and to human IL-1β, 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:12.

[0021] On the one hand, the present invention provides an antibody that binds to human VEGF and to human IL-1β, wherein the antibody comprises a VH domain and a VL domain, the VH domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15, the VL domain comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8, 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: 12.

[0022] In one aspect, the present invention provides an antibody that binds to human VEGF and to human IL-1β, wherein the antibody comprises a VH domain and a VL domain, the VH domain comprising: (a) a CDR-H1 comprising an amino acid sequence of SEQ ID NO: 13, (b) a CDR-H2 comprising an amino acid sequence of SEQ ID NO: 14, and (c) a CDR-H3 comprising an amino acid sequence of SEQ ID NO: 15; the VL domain comprises: (d) a CDR-L1 comprising an amino acid sequence of SEQ ID NO: 16, (e) a CDR-L2 comprising an amino acid sequence of SEQ ID NO: 17, and (f) a CDR-L3 comprising an amino acid sequence of SEQ ID NO: 8; the antibody comprises (a) a VH domain comprising an amino acid sequence of SEQ ID NO: 14, and (b) a CDR-H2 comprising an amino acid sequence of SEQ ID NO: 14, and (c) a CDR-H3 comprising an amino acid sequence of SEQ ID NO: 15; the VL domain comprises: (d) a CDR-L1 comprising an amino acid sequence of SEQ ID NO: 16, (e) a CDR-L2 comprising an amino acid sequence of SEQ ID NO: 17, and (f) a CDR-L3 comprising an amino acid sequence of SEQ ID NO: 8 NO:11 has an amino acid sequence with at least 90% sequence identity, wherein the VH domain comprises amino acid residues E2, G26, V28, K30, R66, R83 and K94; and (b) a VL domain comprising an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO:12, wherein the VL domain comprises amino acid residues I2, Y49, G57, E67, D68 and Q69, wherein the numbering of the VH domain and the VL domain is according to the Kabat numbering system.

[0023] In one aspect, the present invention provides an antibody that binds to human VEGF and human IL-1β, wherein the antibody comprises a VH domain and a VL domain, the VH domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14, (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15, (d) a human heavy chain framework having (i) a FR1 comprising amino acid residues E2, G26, V28 and K30, (ii) a FR3 comprising amino acid residues R66, R83 and K94; the VL domain comprising: (e) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, (f) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17, (g) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 18 NO:8, and (h) a human light chain framework having (i) FR1 comprising amino acid residue I2, (ii) FR2 comprising amino acid residue Y49, and (iii) FR3 comprising amino acid residues G57, E67, D68 and Q69, wherein the numbering of the VH domain and the VL domain is according to the Kabat numbering system, the antibody comprising (a) a VH domain comprising an amino acid sequence having at least 90% sequence identity 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:12.

[0024] In one aspect, the present invention provides an antibody that binds to human VEGF and to human IL-1β, comprising (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 11 having up to 15 amino acid substitutions; and (b) a variable light chain domain comprising the amino acid sequence of SEQ ID NO: 12 having 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 having up to 15 amino acid substitutions, wherein the amino acid substitutions are located at positions 3 to 25, 36 to 49, 97 to 82c, 84 to 93, or 103 to 113 of SEQ ID NO: 11; and (b) a variable light domain comprising the amino acid sequence of SEQ ID NO: 12 having up to 15 amino acid substitutions, wherein the amino acid substitutions are located at positions 1, 4, 6, 8 to 23, 35 to 48, 58 to 66, 70 to 88, or 98 to 107 of SEQ ID NO: 12, wherein the numbering of the VH domain and the VL domain is according to the Kabat numbering system.

[0025] On the one hand, the present invention provides an antibody that binds to human VEGF and to human IL-1β, wherein the antibody comprises a VH domain and a VL domain, the VH domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15, the VL domain comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8, the antibody comprising (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 11 having up to 15 amino acid substitutions; and (b) a variable light domain comprising the amino acid sequence of SEQ ID NO: 12 having up to 15 amino acid substitutions.

[0026] In one aspect, the present invention provides an antibody that binds to human VEGF and human IL-1β, wherein the antibody comprises a VH domain and a VL domain, the VH domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14, (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15, (d) a human heavy chain framework having (i) a FR1 comprising amino acid residues E2, G26, V28 and K30, (ii) a FR3 comprising amino acid residues R66, R83 and K94; the VL domain comprising: (e) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, (f) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17, (g) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 18 NO:8, and (h) a human light chain framework having (i) FR1 comprising amino acid residue I2, (ii) FR2 comprising amino acid residue Y49, and (iii) FR3 comprising amino acid residues G57, E67, D68 and Q69, wherein the numbering of the VH domain and the VL domain is according to the Kabat numbering system, 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 chain domain comprising the amino acid sequence of SEQ ID NO:12 with up to 15 amino acid substitutions.

[0027] In one aspect, the present invention provides an antibody that binds to human VEGF and human IL-1β, comprising a VH sequence of SEQ ID NO:11 and a VL sequence of SEQ ID NO:12.

[0028] In one aspect, the present invention provides an antibody that binds to human VEGF and human IL-1β, comprising a heavy chain amino acid sequence of SEQ ID NO:20 and a light chain amino acid sequence of SEQ ID NO:19.

[0029] In one aspect, the present invention provides an antibody that binds to human VEGF and human IL-1β, comprising a heavy chain amino acid sequence of SEQ ID NO:18 and a light chain amino acid sequence of SEQ ID NO:19.

[0030] In one aspect, the present invention provides an antibody that binds to human VEGF and human IL-1β, wherein the antibody comprises a VH domain and a VL domain, the VH domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14, (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15, (d) a human heavy chain framework having (i) a FR1 comprising amino acid residues E2, G26, V28 and K30, (ii) a FR3 comprising amino acid residues R66, R83 and K94; the VL domain comprising: (e) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, (f) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17, (g) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 18 NO:8, and (h) a human light chain framework having (i) FR1 comprising amino acid residue I2, (ii) FR2 comprising amino acid residue Y49, (iii) FR3 comprising amino acid residues G57, E67, D68 and Q69, wherein the numbering of the VH domain and the VL domain is according to the Kabat numbering system, wherein the antibody Fab fragment of the antibody binds to: (i) human VEGF121, wherein the K measured by surface plasmon resonance D less than 10 pM, and (ii) human IL-1β, where the K measured by surface plasmon resonance is D Less than 30pM.

[0031] In one aspect, the present invention provides an antibody that binds to human VEGF and to human IL-1β, wherein the antibody comprises a VH domain and a VL domain, the VH domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15; the VL domain comprising: (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8; 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: 12. NO:12 has an amino acid sequence with at least 90% sequence identity; wherein the antibody Fab fragment of the antibody binds to: (i) human VEGF121, wherein the K measured by surface plasmon resonance D less than 10 pM, and (ii) human IL-1β, where the K measured by surface plasmon resonance is D Less than 30pM.

[0032] In one aspect, the present invention provides an antibody that binds to human VEGF and to human IL-1β, wherein the antibody comprises a VH domain and a VL domain, the VH domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15, the VL domain comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8, 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: 12. The amino acid sequence of NO:12 has an amino acid sequence with at least 90% sequence identity; wherein the antibody Fab fragment of the antibody exhibits an aggregation onset temperature greater than 70°C.

[0033] In one aspect, the present invention provides an antibody that binds to human VEGF and to human IL-1β, wherein the antibody comprises a VH domain and a VL domain, the VH domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15, the VL domain comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8, 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: 12. The amino acid sequence of NO:12 has an amino acid sequence with at least 90% sequence identity; wherein the antibody Fab fragment of the antibody exhibits a melting temperature greater than 80°C as measured by dynamic light scattering.

[0034] In one aspect, the present invention provides an antibody that binds to human VEGF and to human IL-1β, wherein the antibody comprises a VH domain and a VL domain, the VH domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15, the VL domain comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8, 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: 12. The amino acid sequence of NO:12 has an amino acid sequence with at least 90% sequence identity; and wherein the binding of the antibody Fab fragment of the antibody to human IL-1β inhibits the binding of IL-1β to IL-1βR1, wherein the IC50 measured by surface plasmon resonance is less than 30nM.

[0035] One embodiment of the present invention relates to an antibody fragment that binds to human VEGF and human IL-1β. One embodiment of the present invention relates to a bispecific antibody fragment that binds to human VEGF and human IL-1β. In one embodiment, the antibody fragment is selected from Fv, Fab, Fab', Fab'-SH, F(ab')2 or a single-chain antibody derived therefrom. One embodiment of the present invention relates to a Fab fragment that binds to human VEGF and human IL-1β. One embodiment of the present invention relates to a Fv fragment that binds to human VEGF and human IL-1β.

[0036] One embodiment of the present invention relates to a full length IgG antibody that binds to human VEGF and human IL-1 β.

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

[0038] In one aspect, the invention provides a host cell comprising a nucleic acid of the invention.

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

[0040] In one aspect, the present invention provides a method for producing an antibody that binds to human VEGF and human IL-1β, comprising culturing the host cell of the present invention, thereby producing the antibody.

[0041] In one aspect, the invention provides antibodies produced by the methods of the invention.

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

[0043] 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 a vascular disease.

[0044] In one aspect, the present invention provides use of the antibody of the present invention or the pharmaceutical composition of the present invention in preparing a medicament. In one embodiment, it is used to prepare a medicament for treating vascular diseases.

[0045] In one aspect, the present invention provides a method of treating an individual suffering from a vascular disease, comprising administering to the individual an effective amount of an antibody of the present invention or a pharmaceutical composition of the present invention.

[0046] In one aspect, the present invention provides a method of inhibiting angiogenesis in an individual, comprising administering to the individual an effective amount of the antibody of the present invention or the pharmaceutical composition of the present invention to inhibit angiogenesis.

[0047] According to the present invention, therapeutic anti-VEGF / anti-IL-1β antibodies are provided, which are capable of simultaneously binding to their target antigens even when provided in the form of bispecific Fab fragments. In addition, the antibodies of the present invention provide several valuable properties that allow their therapeutic use, such as high affinity, hydrophilicity and high stability. The antibodies of the present invention can be provided in a high concentration liquid formulation, the viscosity of which is suitable for ocular application. The antibodies of the present invention are suitable for the treatment of ocular vascular diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 : Schematic diagram of the Fab fragment of the anti-VEGF / anti-IL-1β antibody of the present invention. Shown is a top view of the homologous VH / VL pair, including the arrangement of the CDR amino acids (upper figure). The VH domain is represented in gray and the VL domain is represented in white. In addition, the spatial arrangement of the CDR regions is indicated. The paratope region of the antibody of the present invention is highlighted (lower figure), wherein the VEGF paratope is arranged in the region of H-CDR2, L-CDR1 and L-CDR2, and the IL-1β paratope is arranged in the region of H-CDR1, H-CDR3 and L-CDR2.

[0049] Figure 2 : Amino acid sequence of the VH domain of an exemplary anti-VEGF / anti-IL-1β antibody of the invention. The Kabat numbering of the amino acid positions, as well as the CDR and FR regions are indicated. The amino acid positions contributing to the VEGF paratope, as well as the IL-1β paratope identified in Example 8 are highlighted.

[0050] Figure 3 : Amino acid sequence of the VL domain of an exemplary anti-VEGF / anti-IL-1β antibody of the invention. The Kabat numbering of the amino acid positions, as well as the CDR and FR regions are indicated. The amino acid positions contributing to the VEGF paratope, as well as the IL-1β paratope identified in Example 8 are highlighted.

[0051] Figure 4 : Simultaneous antigen binding of anti-VEGF / anti-IL-1β antibody 1HVL 12.85 to VEGF and IL-1β assessed via SPR according to Example 5.

[0052] Figure 5 : Simultaneous antigen binding of anti-VEGF / anti-IL-1β antibody RO7200394 to VEGF and IL-1β assessed via SPR according to Example 5.

[0053] Figure 6 : Prior art anti-VEGF / anti-IL-1β antibody 0032 evaluated via SPR according to Example 5 for simultaneous antigen binding to VEGF and IL-1β.

[0054] Fig. 7A : Inhibition of VEGF binding to hVEGFR2 in the presence of antibody RO7200394 (Fab fragment) as assessed in Example 6. Receptor binding inhibition was assessed in the presence and absence of other target antigens of the bispecific antibody IL-1 β.

[0055] Figure 7B : Inhibition of VEGF binding to hVEGFR2 in the presence of prior art antibody 0032 (full length IgG) as assessed in Example 6. Receptor binding inhibition was assessed in the presence and absence of other target antigens of the bispecific antibody IL-1 β.

[0056] Fig. 8A : Inhibition of IL-1β binding to IL-1β R1 in the presence of antibody RO7200394 (Fab fragment) as assessed in Example 6. Receptor binding inhibition was assessed in the presence and absence of other target antigens of the bispecific antibody VEGF.

[0057] Figure 8B : Inhibition of IL-1β binding to IL-1β R1 in the presence of prior art antibody 0032 (full length IgG) as assessed in Example 6. Receptor binding inhibition was assessed in the presence and absence of other target antigens of the bispecific antibody VEGF.

[0058] Fig. 9 : Competition ELISA evaluating VEGF121 binding to VEGF-R1 in the presence of the indicated antibodies as evaluated in Example 6.

[0059] Fig.10 : Competition ELISA evaluating VEGF165 binding to VEGF-R1 in the presence of the indicated antibodies as evaluated in Example 6.

[0060] Fig.11 : Results of hydrophobic interaction chromatography (HIC) of the antibodies of the invention as evaluated in Example 7

[0061] Fig.12 : Results of hydrophobic interaction chromatography (HIC) of prior art antibody 0032 (Example 7)

[0062] Fig.13 : Viscosity of antibody 1HVL12.85 as assessed in Example 8

[0063] Fig.14 : Viscosity of antibody RO7200394 as evaluated in Example 8 DETAILED DESCRIPTION

[0064] 1. definition

[0065] Unless otherwise defined herein, the scientific and technical terms used in conjunction with the present invention should have the meanings commonly understood by those of ordinary skill in the art. In addition, unless the context otherwise requires, singular terms should include plural terms, and plural terms should include singular terms. The methods and techniques disclosed herein are usually performed according to conventional methods well known in the art. Typically, the nomenclature and techniques associated with biochemistry, enzymology, molecule and cell biology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are well-known and commonly used in the art.

[0066] Unless otherwise defined herein, the term "comprising" shall include the term "consisting of."

[0067] As used herein, the term "about" in conjunction with a particular value (eg, temperature, concentration, time, etc.) shall mean a variation of + / - 1% of the particular value to which the term "about" refers.

[0068] The term "antibody" herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.

[0069] An "isolated" antibody is one that has been separated from a component of its natural environment. In some embodiments, the antibody is purified to a purity greater than 95% or 99% as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0070] The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous antibody population, that is, except for possible variant antibodies (e.g., containing naturally occurring mutations or produced during the production process of monoclonal antibody preparations, such variants are usually present in small amounts), each antibody comprising the population is identical and / or binds to the same epitope. Contrary to polyclonal antibody preparations that typically include different antibodies for different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed to a single determinant on an antigen. Therefore, the modifier "monoclonal" indicates that the characteristic of the antibody is obtained from a substantially homogeneous antibody population, and should not be interpreted as requiring antibodies to be produced by any particular method.

[0071] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein.

[0072] The "class" of an antibody refers to the type of constant domain or constant region possessed by the heavy chain of the antibody. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. In certain embodiments, the antibody is an IgG1 isotype. In certain embodiments, the antibody is an IgG1 isotype with P329G, L234A, and L235A mutations to reduce the effector function of the Fc region. In other embodiments, the antibody is an IgG2 isotype. In certain embodiments, the antibody is an IgG4 isotype with an S228P mutation in the hinge region to improve the stability of the IgG4 antibody. The heavy chain constant domains corresponding to different classes of immunoglobulins are referred to as α, δ, ε, γ, and μ, respectively. The light chain of an antibody can be classified into one of two types based on the amino acid sequence of its constant domain, which are called kappa (κ) and lambda (λ).

[0073] The term "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the 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, which is also referred to as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0074] "Effector functions" refer to those biological activities attributable to the Fc region of an antibody, which vary with 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.

[0075] The term "variable region" or "variable domain" refers to the domain of an antibody heavy chain or light chain that participates in the binding of an antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, wherein each domain comprises four conserved framework regions (FRs) and three hypervariable regions (HVRs) (see, e.g., Kindt et al., Kuby Immunology, 6th edition, WH Freeman and Co., p. 91 (2007)). In the antibodies of the present invention, a single VH domain and a VL domain pair, i.e. a homologous VH / VL pair, specifically bind to its two targets: VEGF and IL-1β.

[0076] "DutaFab" is a bispecific antibody as disclosed in WO2012 / 163520. In DutaFab, a single VH domain and VL domain pair specifically binds to two different epitopes, wherein one paratope comprises amino acid residues from CDR-H2, CDR-L1 and CDR-L3 and the other paratope comprises amino acid residues from CDR-H1, CDR-H3 and CDR-L2. DutaFab contains two non-overlapping paratopes within a cognate VH / VL pair and can bind to two different epitopes simultaneously. DutaFab and methods of producing same by screening libraries comprising monospecific Fab fragments are disclosed in WO2012 / 163520.

[0077] A "human antibody" is an antibody having an amino acid sequence corresponding to that of an antibody produced by a human or human cell, or derived from a non-human source utilizing a human antibody library or other human antibody encoding sequences. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues.

[0078] "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. In general, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. In general, the subgroup of sequences is a subgroup as described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), Volumes 1-3. In one embodiment, for VL, the subgroup is subgroup κI as in Kabat et al., supra. In one embodiment, for VH, the subgroup is subgroup III as in Kabat et al., supra.

[0079] "Antibody fragment" refers to a molecule other than an intact antibody, which comprises 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.

[0080] As used interchangeably herein, "paratope" or "antigen binding site" refers to the portion of an antibody that recognizes and binds an antigen. The paratope is formed by several single amino acid residues from the variable domains of the antibody heavy and light chains, which are arranged in spatial proximity in the tertiary structure of the Fv region. The antibodies of the present invention contain two "non-overlapping" paratopes in a homologous VH / VL pair. "Non-overlapping" means that the amino acids contained in one of the two paratopes are not contained in the other paratope.

[0081] 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.

[0082] As used herein, "IL-1β paratope" is the paratope or antigen binding site to which IL-1β binds. The IL-1β paratope of the antibodies of the invention comprises amino acid residues from CDR-H1, CDR-H3, and CDR-L2 of the antibody.

[0083] Unless otherwise indicated, the term "VEGF" as used herein refers to any native VEGF from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses "full-length" unprocessed VEGF, as well as any form of VEGF produced by processing in cells. The term also encompasses naturally occurring variants of VEGF, such as splice variants or allelic variants. The amino acid sequence of an exemplary human VEGF is shown in SEQ ID NO: 26.

[0084] The terms "anti-VEGF antibody" and "antibody that binds to VEGF" refer to an antibody that is capable of binding to VEGF with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent targeting VEGF. 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, the antibody that binds to VEGF has a dissociation constant (K) of 1 nM, ≤0.1 nM, or ≤0.01 nM. D ). When the antibody KD An antibody is said to "specifically bind" VEGF if the concentration of the antibody is 1 μM or less.

[0085] Unless otherwise indicated, the term "IL-1β" as used herein refers to any natural IL-1β from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term includes "full-length" unprocessed IL-1β, as well as any form of IL-1β produced by processing in cells. The term also encompasses naturally occurring variants of IL-1β, such as splice variants or allelic variants. The amino acid sequence of an exemplary human IL-1β is shown in SEQ ID NO: 27.

[0086] The terms "anti-IL-1β antibody" and "antibody that binds to anti-IL-1β" refer to an antibody that is capable of binding to IL-1β with sufficient affinity so that the antibody can be used as a diagnostic and / or therapeutic agent targeting IL-1β. In one embodiment, the extent of binding of the anti-IL-1β antibody to an unrelated, non-anti-IL-1β protein is less than about 10% of the binding of the antibody to anti-IL-1β, as measured, for example, by surface plasmon resonance (SPR). In certain embodiments, the antibody that binds to IL-1β has a dissociation constant (K) of 1 nM, ≤0.1 nM, or ≤0.03 nM. D ). When the antibody K D If the concentration of IL-1β is 1 μM or less, the antibody is said to "specifically bind" to anti-IL-1β.

[0087] The antibodies of the present invention "simultaneously bind to human VEGF and human IL-1β", which means that (a) the Fab fragments of the antibodies of the present invention that bind to human IL-1β (also) specifically bind to human VEGF, and (b) the Fab fragments of the antibodies of the present invention that bind to human VEGF (also) specifically bind to human IL-1β. Simultaneous binding can be assessed by methods known in the art (e.g., by surface plasmon resonance as described herein).

[0088] As used herein, the term "complementarity determining region" or "CDR" refers to each region of an antibody variable domain that is highly variable in sequence and contains antigen contact residues. Typically, an antibody contains 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 indicated, CDR residues and other residues (e.g., FR residues) in the variable domain are numbered herein 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).

[0089] As used herein, "framework" or "FR" refers to the variable domain amino acid residues other than the CDR residues. The framework of the variable domain is generally composed of the following four framework domains: FR1, FR2, FR3, and FR4. Thus, the CDR and FR amino acid sequences generally appear in the following sequence in (a) the VH domain: FR1—CDR-H1—FR2—CDR-H2—FR3—CDR-H3—FR4; in (b) the VL domain: FR1—CDR-L1—FR2—CDR-L2—FR3—CDR-L3—FR4.

[0090] According to the Kabat numbering system used herein, the framework and CDR regions are located in the following regions of the variable domain:

[0091] FR1 CDR-1 FR2 CDR2 FR3 CDR3 FR4 VH 1-30 31-35b* 36-49 50-65 66-94 95-102 103-113 V L 1-23 24-34 35-49 50-56 57-88 89-97 98-107

[0092] *like Figure 2 As shown, in CDR-H1, there may be additional amino acids between positions 35b and 36, referred to herein as positions "35c", "35d" and "35e".

[0093] The amino acid positions according to the Kabat numbering system referred to herein are shown in the table below in comparison with the amino acid sequences of the antibodies of the invention. Figure 2 Reference is made herein to an amino acid at a certain position within an amino acid sequence as is well known in the art by indicating the respective amino acid and amino acid position, e.g. an amino acid sequence, e.g. "E2" refers to the glutamic acid residue at Kabat position 2 of the amino acid sequence of the corresponding antibody domain.

[0094] "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 intrinsic binding affinity, which 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 can generally be expressed by the dissociation constant (K D ) represents. Affinity can be measured by conventional methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity will be described herein.

[0095] The term "epitope" refers to a site on a protein or non-protein antigen to which an antigen binds. An epitope can be formed by a continuous stretch of amino acids (linear epitope) or by the inclusion of non-continuous amino acids (conformational epitope), for example due to antigen folding, i.e., due to the tertiary folding of the protein antigen and in spatial proximity. After the protein antigen is exposed to a denaturant, a linear epitope is usually still bound by an antibody, while a conformational epitope is usually destroyed after treatment with a denaturant. 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 three-dimensional conformation.

[0096] Screening for antibody binding can be performed using methods routine in the art, such as, but not limited to, alanine scanning, peptide blotting (see Meth. Mol. Biol. 248 (2004) 443-463), peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of antigens (see Prot. Sci. 9 (2000) 487-496) and cross-blocking (see "Antibodies", Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY).

[0097] Antigen structure-based antibody profiling (ASAP), also known as modification-assisted profiling (MAP), allows the classification of a variety of monoclonal antibodies that specifically bind to VEGF or IL-1β based on the binding profile of each antibody from a large number of antibodies and chemically or enzymatically modified antigen surfaces (see, e.g., US2004 / 0101920). The antibodies in each group bind to the same epitope, which can be a unique epitope that is significantly different from or partially overlaps with the epitope represented by another group.

[0098] Competitive binding can also be used to readily determine whether an antibody binds to the same epitope of VEGF or IL-1β as a reference antibody of the present invention, or competes for binding to a reference antibody of the present invention. For example, an "antibody that binds to the same epitope on VEGF and IL-1β" as a reference antibody refers to an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in the respective competition assays, and conversely, the reference antibody blocks the binding of the antibody to its antigen by 50% or more in the respective competition assays. Also for example, to determine whether an antibody binds to the same epitope as a reference antibody, the reference antibody is allowed to bind to VEGF or IL-1β under saturating conditions. After removing excess reference antibody, the ability of the antibody in question to bind to VEGF or IL-1β is assessed. If the antibody in question is able to bind to VEGF or IL-1β after saturation binding of the reference antibody, it can be concluded that the antibody in question binds to a different epitope than the reference antibody. However, if the antibody in question is unable to bind to VEGF or IL-1β after saturation binding of the reference antibody, the antibody in question may bind to the same epitope as the reference antibody. To confirm whether the antibodies in question bind to the same epitope or are 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 any other quantitative or qualitative antibody binding assay available in the art). The assay should be performed in two settings, i.e., both antibodies are saturated antibodies. If in both settings, only the first (saturated) antibody is able to bind to VEGF or IL-1β, then it can be concluded that the antibody in question and the reference antibody compete for binding to VEGF or IL-1β.

[0099] In some embodiments, two antibodies are considered to bind to the same or overlapping epitope if one antibody inhibits the binding of the other antibody by at least 50%, at least 75%, at least 90%, or even 99% or more by 1, 5, 10, 20, or 100-fold excess as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 50 (1990) 1495-1502).

[0100] In some embodiments, two antibodies are considered to bind to the same epitope if substantially all 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 amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other antibody.

[0101] " Amino acid sequence identity percentage " about reference polypeptide sequence is defined as the percentage of amino acid residues in candidate sequence identical with the amino acid residues in reference polypeptide sequence after comparing sequences and introducing room (if necessary) to realize maximum sequence identity percentage, without considering any conservative substitution as a part of sequence identity for the purpose of comparison. Comparison for determining amino acid sequence identity percentage can be realized by various means within the technical scope of the art, for example, using publicly available computer software, such as BLAST, BLAST-2, ClustalW, Megalign (DNASTAR) software or FASTA program package. Those skilled in the art can determine the appropriate parameters for comparing sequences, including any algorithm required for realizing maximum comparison on the total length of the compared sequence. Alternatively, sequence comparison computer program ALIGN-2 can be used to generate identity percentage values. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc. and the source code has been filed with user documentation in the US Copyright Office, Washington D.C., 20559, where it is registered under US Copyright Registration No. TXU510087 and as described in WO 2000 / 005319.

[0102] However, unless otherwise stated, for the purposes of this article, the BLOSUM50 comparison matrix is ​​used to generate amino acid sequence identity % values ​​using the ggsearch program of the FASTA package version 36.3.8c or higher. The FASTA package is by W. R. Pearson and D. J. Lipman (1988), "Improved Tools for Biological Sequence Analysis", PNAS 85:2444-2448; W. R. Pearson (1996) "Effective protein sequence comparison" Meth. Enzymol. 266:227-258; and Pearson et al., (1997) Genomics 46:24-36 creation, and is publicly available from www.fasta.bioch.virginia.edu / fasta_www2 / fasta_down.shtml or www.ebi.ac.uk / Tools / sss / fasta. Alternatively, sequences can be compared using a 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) to ensure that a global rather than a local alignment is performed. The amino acid identity percentages are given in the alignment header of the output.

[0103] The term "nucleic acid molecule" or "polynucleotide" includes any compound and / or substance comprising a nucleotide polymer. Each nucleotide is composed of a base, particularly 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. Typically, nucleic acid molecules are described by base sequences, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. Base sequences are typically expressed as from 5' to 3'. In this article, the term nucleic acid molecule encompasses synthetic forms of deoxyribonucleic acid (DNA) (including, for example, complementary DNA (cDNA) and genomic DNA), ribonucleic acid (RNA) (particularly messenger RNA (mRNA)), DNA or RNA, and mixed polymers comprising two or more of these molecules. Nucleic acid molecules can be linear or cyclic. In addition, the term nucleic acid molecule includes sense strands and antisense strands, as well as single-stranded and double-stranded forms. In addition, nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases with derivatized sugar or phosphate backbone bonds or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules suitable as carriers for direct expression in vitro and / or in vivo (e.g., in a host or patient) of the antibodies of the present invention. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors may be unmodified or modified. For example, mRNA may be chemically modified to enhance the stability of the RNA vector and / or the expression of the coding molecule so that mRNA may be injected into a subject to produce antibodies in vivo (see, e.g., Stadler et al., Nature Medicine 2017, published online on June 12, 2017, doi:10.1038 / nm.4356 or EP 2 101 823 B1).

[0104] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that normally contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0105] "Isolated nucleic acid encoding an antibody" refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including such nucleic acid molecules in a single vector or different vectors, and such nucleic acid molecules present in one or more locations in a host cell.

[0106] As used herein, the term "vector" refers to a nucleic acid molecule capable of carrying another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures, as well as vectors that integrate into the genome of a host cell into which it has been 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."

[0107] 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 primary transformed cells and progeny derived therefrom, without regard to the number of passages. Progeny may not be identical to the nucleic acid content of the parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected in the original transformed cell are included herein.

[0108] The term "pharmaceutical composition" or "pharmaceutical formulation" refers to a preparation that is in a form that permits the biological activity of the active ingredient contained therein to be effective, and that contains no additional components that are unacceptably toxic to a subject to which the pharmaceutical composition would be administered.

[0109] "Pharmaceutically acceptable excipient" refers to an ingredient in a pharmaceutical composition or formulation other than the active ingredient, which is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0110] An "effective amount" of an agent (eg, a pharmaceutical composition) refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0111] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, 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, the individual or subject is a human.

[0112] As used herein, "treatment" (and grammatical variants thereof, such as "treat" or "treating") refers to an attempt to alter the natural course of a disease in the individual being treated, and may be performed for prevention or as a clinical intervention performed during clinical pathology. Desired effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating the disease state, and alleviating or improving prognosis. In some embodiments, the antibodies of the invention are used to delay the development of a disease or slow the progression of a disease.

[0113] As used herein, the term "eye disease" includes any eye disease associated with pathological angiogenesis and / or atrophy. Eye diseases may be characterized by new blood vessel changes or unregulated proliferation and / or invasion into structures of eye tissues such as the retina or cornea. Eye diseases may be characterized by atrophy of retinal tissues (photoreceptors and underlying retinal pigment epithelium (RPE) and choriocapillaris). Non-limiting eye 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., localized, non-central DME and diffuse, centrally involved DME), retinopathy, diabetic retinopathy (DR) (e.g., proliferative DR (PDR), non-proliferative DR (NPDR), and high altitude DR), other ischemia-related retinopathy, ROP, retinal vein occlusion (RVO) (e.g., central (CRVO) and branch (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, Hippel-Lindau syndrome, ocular histoplasmosis, FEVR, Coronary artery disease, Norrie disease, bone marrow disease, retinal vascular disease, vascular disease, myopia, optic disc disease, vascular disease ... Retinal abnormalities associated with OPPG, subconjunctival hemorrhage, erythema, ocular neovascular disease, neovascular glaucoma, retinitis pigmentosa (RP), hypertensive retinopathy, retinal angiomatous proliferation, macular telangiectasia, 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., allergic conjunctivitis)), Leber congenital amaurosis (also known as Reye's amaurosis or LCA), uveitis (including infectious and non-infectious uveitis), choroiditis (e.g., multifocal choroiditis), ocular histoplasmosis, blepharitis, dry eye, ocular trauma, sicca disease, and other ophthalmic diseases (wherein the disease is associated with ocular neovascularization, vascular leakage and / or retinal edema or retinal atrophy). Additional exemplary ocular diseases include retinoschisis (abnormal splitting of the neurosensory layer of the retina), diseases associated with rubeosis iridis (corner neovascularization), 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 overdose, atopic keratitis, superior limbal keratitis, keratitis sicca, Sjögren's syndrome, acne rosacea, bullous disease, syphilis, mycobacterial infection, lipid degeneration, chemical burns, bacterial ulcers, fungal ulcers, herpes simplex virus infection, herpes zoster infection, protozoan infection, Kaposi's sarcoma, Mooren's ulcer, Terrien's marginal degeneration, marginal keratolysis, rheumatoid arthritis, systemic lupus, multiple arteriosclerosis, trauma, Wegener's sarcoidosis, scleritis, Stevens-Johnson syndrome, radial keratotomy, and corneal rejection. Exemplary diseases associated with choroidal neovascularization and defects in the retinal vasculature, including increased vascular leakage, aneurysms, and capillary detachment, include, but are not limited to, diabetic retinopathy, macular degeneration, sickle cell anemia, sarcoidosis, syphilis, pseudoxanthoma elasticum, Paget's disease, venous occlusion, arterial occlusion, carotid occlusive disease, chronic uveitis / vitritis, mycobacterial infection, Lyme disease, systemic lupus erythematosus, retinopathy of prematurity, retinal edema (including macular edema), Eal's disease, Behcet's disease, infectious retinitis or choroiditis (e.g., multifocal choroiditis), presumed ocular histoplasmosis, Best's disease (vitreomacular degeneration), myopia, optic nerve pits, 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 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, fundus dystrophy, retinoschisis, and retinitis pigmentosa.

[0114] The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and / or warnings concerning the use of such therapeutic products. 2. DETAILED DESCRIPTION

[0116] In one aspect, the invention is based in part on providing bispecific antibodies for therapeutic applications. In certain aspects, an antibody is provided that binds to human VEGF and human IL-1β. The antibodies of the invention can be used, for example, to diagnose or treat vascular diseases, such as ocular vascular diseases.

[0117] A. Exemplary Antibodies that Bind to Human VEGF and Human IL-1β

[0118] In one aspect, the present invention provides an antibody that binds to human VEGF and human IL-1β. In one aspect, an isolated antibody that binds to human VEGF and human IL-1β is provided. In one aspect, the present invention provides an antibody that specifically binds to human VEGF and human IL-1β.

[0119] In certain aspects, an antibody that binds to human VEGF and to human IL-1β is provided, wherein the antibody comprises a VEGF paratope (i.e., an antigen binding site that binds to VEGF) and an IL-1β paratope (i.e., an antigen binding site that binds to IL-1β) within a cognate pair of a VL domain and a VH domain,

[0120] wherein the VEGF paratope comprises amino acid residues from CDR-H2, CDR-L1 and CDR-L3 of an antibody, wherein the IL-1β paratope comprises amino acid residues from CDR-H1, CDR-H3 and CDR-L2 of an antibody; and / or

[0121] The variable light chain domain and variable heavy chain domain pair bind to human VEGF and human IL-1β simultaneously; and / or

[0122] None of the amino acids contained in the VEGF paratope are contained in the IL-1β paratope; and / or

[0123] None of the amino acids contained in the IL-1β paratope are contained in the VEGF paratope; and / or

[0124] The antibody binds to the same epitope on human VEGF and to the same epitope on human IL-1β as an antibody having a variable heavy chain domain of SEQ ID NO: 11 and a variable light chain domain of SEQ ID NO: 12; and / or

[0125] The antibody Fab fragment of the antibody binds to: (i) human VEGF121, wherein the K measured by surface plasmon resonance is D less than 10 pM, and (ii) human IL-1β, where the K measured by surface plasmon resonance is D Less than 30 pM; and / or

[0126] The antibody Fab fragment of the antibody exhibits an aggregation onset temperature greater than 70°C; and / or

[0127] an antibody Fab fragment of said antibody exhibits a melting temperature greater than 80°C as measured by dynamic light scattering; and / or

[0128] · Binding of the antibody Fab fragment of the antibody to human VEGF inhibits the binding of VEGF to VEGFR2, wherein the IC50 measured by surface plasmon resonance is less than 50nM; and wherein binding of the antibody Fab fragment of the antibody to human IL-1β inhibits the binding of IL-1β to IL-1βR1, wherein the IC50 measured by surface plasmon resonance is less than 30nM.

[0129] On the other hand, the present invention provides an antibody comprising a VH domain and a VL domain, the VH domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15, and the VL domain comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17 and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8.

[0130] In another aspect, the present invention provides an antibody comprising a VH domain and a VL domain, wherein the VH domain comprises: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14, (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15, and (d) a human heavy chain framework having (i) a FR1 comprising amino acid residues E2, G26, V28 and K30, (ii) a FR3 comprising amino acid residues R66, R83 and K94; and the VL domain comprises (e) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, (f) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17, (g) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 18 NO:8, and (h) a human light chain framework having (i) a FR1 comprising amino acid residue I2, (ii) a FR2 comprising amino acid residue Y49: (iii) a FR3 comprising amino acid residues G57, E67, D68 and Q69, wherein the numbering of the VH domain and the VL domain is according to the Kabat numbering system.

[0131] In another aspect, the invention provides an antibody comprising a VH domain and a VL domain, wherein the VH domain comprises amino acid residues E2, G26, V28, K30, W31, N35b, D35c, K52a, D55, H56, Y58, T61, K62, F63, I64, R66, R83, K94, D95, V96, F98 and D101, and the VL domain comprises amino acid residues I2, Y27, W27a, S27c, S27d, L32, Y49, D50, Y53, K54, L56, G57, E67, D68, Q69, Y91, R92, Y93, H94 and Y96, wherein the numbering of the VH domain and the 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: D55, H56, Y58, T61, K62, F63, I64, R66, and R83, and the following amino acid residues in the VL domain: I2, Y27, W27a, S27c, S27d, E67, D68, Q69, R92, Y93, H94, and Y96; and an IL-1 β paratope comprising the following amino acid residues in the VH domain: E2, G26, V28, K30, W31, N35b, D35c, K52a, K94, D95, V96, F98, and D101, and the following amino acid residues in the VL domain: L32, Y49, D50, Y53, K54, L56, G57, Y91.

[0132] 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; 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:12.

[0133] 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, wherein the VH domain comprises amino acid residues E2, G26, V28, K30, W31, N35b, D35c, K52a, D55, H56, Y58, T61, K62, F63, I64, R66, R83, K94, D95, V96, F98 and D101; 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: 11, wherein the VH domain comprises amino acid residues E2, G26, V28, K30, W31, N35b, D35c, K52a, D55, H56, Y58, T61, K62, F63, I64, R66, R83, K94, D95, V96, F98 and D101. The amino acid sequence of NO:12 has an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, wherein the VL domain comprises amino acid residues I2, Y27, W27a, S27c, S27d, L32, Y49, D50, Y53, K54, L56, G57, E67, D68, Q69, Y91, R92, Y93, H94 and Y96, and the numbering of the VH domain and the VL domain is according to the Kabat numbering system.

[0134] On the other hand, the present invention provides an antibody comprising a VH domain and a VL domain, the VH domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15; the VL domain comprising: (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8, the antibody comprising (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 13, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15; IDNO:11 has 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; 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:12.

[0135] On the other hand, the present invention provides an antibody comprising a VH domain and a VL domain, wherein the VH domain comprises: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15; the VL domain comprises: (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8; the antibody comprises (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 13, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15; ID NO:11 has 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, wherein the VH domain comprises amino acid residues E2, G26, V28, K30, R66, R83 and K94; and (b) a VL domain comprising the amino acid residues SEQ ID NO:12. The amino acid sequence of NO:12 has an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, wherein the VL domain comprises amino acid residues I2, Y49, G57, E67, D68 and Q69, and wherein the numbering of the VH domain and the VL domain is according to the Kabat numbering system.

[0136] In another aspect, the present invention provides an antibody comprising a VH domain and a VL domain, wherein the VH domain comprises: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14, (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15, (d) a human heavy chain framework having (i) a FR1 comprising amino acid residues E2, G26, V28 and K30, (ii) a FR3 comprising amino acid residues R66, R83 and K94; and the VL domain comprises (e) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16; (f) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17, (g) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 18. NO:8, and (h) a human light chain framework having (i) FR1 comprising amino acid residue I2, (ii) FR2 comprising amino acid residue Y49, and (iii) FR3 comprising amino acid residues G57, E67, D68 and Q69, wherein the numbering of the VH domain and the VL domain is according to the Kabat numbering system, the 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; 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:11; The amino acid sequence of NO:12 has an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

[0137] 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: 12 having 1 to 15, 1 to 10, or 1 to 5 amino acid substitutions.

[0138] 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 25, 36 to 49, 97 to 82c, 84 to 93, or 103 to 113 of SEQ ID NO: 11; and (b) a variable light chain domain comprising the amino acid sequence of SEQ ID NO: 12 having 1 to 15, 1 to 10, or 1 to 5 amino acid substitutions, wherein the amino acid substitutions are located at positions 1, 4, 6, 8 to 23, 35 to 48, 58 to 66, 70 to 88, or 98 to 107 of SEQ ID NO: 12, wherein the numbering of the VH domain and the VL domain is according to the Kabat numbering system.

[0139] On the other hand, the present invention provides an antibody comprising a VH domain and a VL domain, the VH domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15, the VL domain comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8, the 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: 12 having 1 to 15, 1 to 10, or 1 to 5 amino acid substitutions.

[0140] In another aspect, the present invention provides an antibody comprising a VH domain and a VL domain, wherein the VH domain comprises: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14, (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15, (d) a human heavy chain framework having (i) a FR1 comprising amino acid residues E2, G26, V28 and K30, (ii) a FR3 comprising amino acid residues R66, R83 and K94; and the VL domain comprises (e) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, (f) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17, (g) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 18. NO:8, and (h) a human light chain framework having (i) FR1 comprising amino acid residue I2, (ii) FR2 comprising amino acid residue Y49, and (iii) FR3 comprising amino acid residues G57, E67, D68 and Q69, wherein the numbering of the VH domain and the VL domain is according to the Kabat numbering system, the 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:12 having 1 to 15, 1 to 10, or 1 to 5 amino acid substitutions.

[0141] In one aspect, the present invention provides an antibody comprising a VH domain, wherein the VH domain has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO: 11. In certain aspects, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity comprises a substitution (e.g., a conservative substitution), insertion or deletion relative to the reference sequence, but the antibody comprising the VH sequence that binds to human VEGF and human IL-1β retains the ability to bind to human VEGF and human IL-1β. In certain aspects, in SEQ ID NO: 11, a total of 1 to 10 amino acids have been substituted, inserted and / or deleted. In certain aspects, the substitution, insertion or deletion occurs in a region outside the CDR (i.e., in the FR). In a specific aspect, VH comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:13, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:14, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:15.

[0142] In one aspect, the present invention provides an antibody comprising a VL domain, wherein the VL domain has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO: 12. In certain aspects, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity comprises a substitution (e.g., a conservative substitution), insertion or deletion relative to the reference sequence, but the antibody comprising the VL sequence that binds to human VEGF and human IL-1β retains the ability to bind to human VEGF and human IL-1β. In certain aspects, in SEQ ID NO: 12, a total of 1 to 10 amino acids have been substituted, inserted and / or deleted. In certain aspects, the substitution, insertion or deletion occurs in a region outside the CDR (i.e., in the FR). In a specific aspect, VL comprises (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:16, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:17, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:8.

[0143] In another aspect, an antibody that binds to human VEGF and human IL-1β is provided, wherein the antibody comprises a VH sequence as in any aspect provided above and a VL sequence as in any aspect provided above. In one aspect, the antibody comprises the VH and VL sequences in SEQ ID NO: 11 and SEQ ID NO: 12, respectively, including post-translational modifications of those sequences.

[0144] In another aspect, an antibody that binds to human VEGF and human IL-1β is provided, wherein the antibody comprises a heavy chain amino acid sequence of SEQ ID NO:20 and a light chain amino acid sequence of SEQ ID NO:19.

[0145] In another aspect, an antibody that binds to human VEGF and human IL-1β is provided, wherein the antibody comprises a heavy chain amino acid sequence of SEQ ID NO:18 and a light chain amino acid sequence of SEQ ID NO:19.

[0146] In another aspect of the invention, the antibody that binds to human VEGF and human IL-1β according to any of the above aspects is a monoclonal antibody. In one aspect, the antibody that binds to human VEGF and human IL-1β is an antibody fragment, such as a Fv, Fab, Fab', scFv, diabody or F(ab')2 fragment. In another aspect, the antibody is a full-length antibody.

[0147] In another aspect, an antibody that binds to human VEGF and human IL-1 β according to any aspect above may incorporate any of the features, alone or in combination, as described in Sections 1-7 below:

[0148] 1. Antibody affinity

[0149] In certain embodiments, the antibodies provided herein are characterized by a dissociation constant (K D )≤1 nM, ≤0.1 nM or ≤0.01 nM binds to VEGF. In certain embodiments, the antibody that binds to IL-1β has a dissociation constant (K) of 1 nM, ≤0.1 nM or ≤0.03 nM. D ).

[0150] On the one hand, using Surface plasmon resonance assay to measure K D .

[0151] For example, at 25°C, VEGF121 immobilized on a C1 chip was detected at ∼10 response units (RU). or The K of antibody binding to VEGF was measured in an assay using BIAcore, Inc., Piscataway, NJ. D For kinetic measurements, two-fold serial dilutions of Fab (1.2–100 nM) were injected into 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. A simple one-to-one Langmuir binding model ( Evaluation Software version 3.2), and the association rate (k) was calculated by simultaneously fitting the association and dissociation sensorgrams. on ) and dissociation rate (k off ). Equilibrium dissociation constant (K D ) is calculated as the ratio k off / k on See, e.g., Chen, Y. et al., J. Mol. Biol. 293:865-881 (1999).

[0152] For example, at 25°C, using a bispecific antibody immobilized on a C1 chip, at ∼20 response units (RU), or The K of antibody binding to IL-1β was measured in an assay using BIAcore, Inc., Piscataway, NJ. DFor kinetic measurements, two-fold serial dilutions of human IL-1β (0.74 nM to 60 nM) were injected into 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. A simple one-to-one Langmuir binding model ( Evaluation Software version 3.2), and the association rate (k) was calculated by simultaneously fitting the association and dissociation sensorgrams. on ) and dissociation rate (k off ). Equilibrium dissociation constant (K D ) is calculated as the ratio k off / k on See, e.g., Chen, Y. et al., J. Mol. Biol. 293:865-881 (1999).

[0153] 2. Antibody fragments

[0154] In certain aspects, the antibodies provided herein are antibody fragments.

[0155] In one aspect, antibody fragments are Fab', Fab'-SH or F(ab')2 fragments, particularly Fab fragments. Papain digestion of whole antibodies produces two identical antigen-binding fragments called "Fab" fragments, each of which contains a heavy chain variable domain and a light chain variable domain (VH and VL, respectively) and a constant domain (CL) of a light chain and a first constant domain (CH1) of a heavy chain. Thus, the term "Fab fragment" refers to an antibody fragment comprising a light chain comprising a VL domain and a CL domain and a heavy chain fragment comprising a VH domain and a CH1 domain. The difference between Fab' fragments and Fab fragments is that Fab' fragments have added residues at the carboxyl terminus of the CH1 domain, which residues include one or more cysteines from the hinge region of the antibody. Fab'-SH is a Fab' fragment in which the cysteine ​​residues of the constant domains have free sulfhydryl groups. Pepsin treatment produces a F(ab')2 fragment having two antigen-binding sites (two Fab fragments) and a portion of the Fc region. For a discussion of Fab fragments and F(ab')2 fragments comprising salvage receptor binding epitope residues and having increased in vivo half-lives, see US Pat. No. 5,869,046.

[0156] Antibody fragments can be prepared by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and recombinant production by recombinant host cells (eg, E. coli, CHO), as described herein.

[0157] 3. Thermal stability

[0158] The antibodies provided herein exhibit excellent thermal stability. In certain embodiments, the Fab fragments of the antibodies provided herein exhibit an aggregation onset temperature greater than 70°C. In certain embodiments, the Fab fragments of the antibodies provided herein exhibit a melting temperature greater than 80°C as measured by dynamic light scattering.

[0159] 4. Library-derived Antibodies

[0160] In certain aspects, the antibodies provided herein are derived from libraries. Antibodies of the present invention can be isolated by screening combinatorial libraries for antibodies with one or more desired activities. Methods for screening combinatorial libraries are reviewed in, for example, Lerner et al., Nature Reviews 16: 498-508 (2016). For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies with desired binding characteristics. Such methods are reviewed, 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), as well as in Hoogenboom et al., Methods in Molecular Biology 178: 1-37 (O'Brien et al., eds., Human Press, Totowa, NJ, 2001) and in Marks and Bradbury, Methods in Molecular Biology 248: 161-175 (Lo, ed., Human Press, Totowa, NJ, 2003).

[0161] In certain phage display methods, libraries of VH and VL genes are cloned separately by polymerase chain reaction (PCR) and randomly recombined in phage libraries, which can then be screened for phage that bind to the antigen, as described by Winter et al. in Annual Review of Immunology 12:433-455 (1994). Phage typically display antibody fragments as single-chain Fv (scFv) fragments or Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, an initial library (e.g., from humans) can be cloned to provide a single source of antibodies for a variety of non-self antigens and self antigens without any immunization, as described by Griffiths et al. in EMBO Journal 12:725-734 (1993). In addition, natural libraries are synthesized by cloning unrearranged V gene segments from stem cells; and using PCR primers containing random sequences to encode highly variable CDR3 regions and complete in vitro rearrangement, 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 Publication Nos. 2005 / 0079574, 2007 / 0117126, 2007 / 0237764 and 2007 / 0292936.

[0162] Other examples of methods known in the art for screening combinatorial libraries of antibodies with one or more desired activities include ribosome and mRNA display, and methods for displaying antibodies and selecting bacteria, mammalian cells, insect cells or yeast cells. Methods for yeast surface display are summarized in, for example, Scholler et al., Methods in Molecular Biology 503:135-56 (2012) and Cherf et al., Methods in Molecular biology 1319:155-175 (2015) and Zhao et al., Methods in Molecular Biology 889:73-84 (2012). Methods for ribosome display are described in, for example, He et al., Nucleic Acids Research 25:5132-5134 (1997) and Hanes et al., PNAS 94:4937-4942 (1997).

[0163] Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments herein.

[0164] 5. Multispecific Antibodies

[0165] In some aspects, the antibodies provided herein are multispecific antibodies. A "multispecific antibody" is a monoclonal antibody that has binding specificity to at least two different sites (i.e., different epitopes on different antigens or different epitopes on the same antigen). In some aspects, a multispecific antibody has three or more binding specificities.

[0166] Multispecific antibodies having three or more binding specificities, comprising antibodies provided herein, can be provided in an asymmetric form, with domain crossover in one or more binding arms having 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, e.g., WO 2009 / 080253) or complete Fab arms (see, e.g., WO 2009 / 080251, WO 2016 / 016299, see also Schaefer et al., PNAS, 108 (2011) 1187-1191, and Klein et al., MAbs 8 (2016) 1010-20). Various other 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).

[0167] 6. Antibody variants

[0168] In some aspects, it is contemplated that the amino acid sequence variants of the antibodies provided herein. For example, it may be desirable to change the binding affinity and / or other biological properties of the antibody. The amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, the deletion of residues in the antibody amino acid sequence, and / or insertion and / or substitution. Any combination of deletion, insertion and substitution can be performed to achieve the final construct, provided that the final construct has the desired characteristics, such as antigen binding.

[0169] In some aspects, antibody variants with one or more amino acid substitutions are provided. The sites of interest for substitution mutagenesis include CDR and FR. Conservative substitutions are presented under the "preferred substitutions" heading in the following table. More substantial changes are provided under the "exemplary substitutions" heading in Table 1, as further described below with reference to the amino acid side chain categories. Amino acid substitutions can be introduced into the target antibody, and the product is screened for desired activity (e.g., retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC).

[0170] surface

[0171]

[0172]

[0173] Amino acids can be grouped according to common side chain properties:

[0174] (1) Hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile;

[0175] (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;

[0176] (3) Acidic: Asp, Glu;

[0177] (4) Basic: His, Lys, Arg;

[0178] (5) Residues that affect chain orientation: Gly, Pro;

[0179] (6) Aromatic: Trp, Tyr, Phe.

[0180] Non-conservative substitutions will entail exchanging a member of one of these classes for a member of another class.

[0181] One type of substitution variant involves replacing one or more hypervariable region residues of a parent antibody (e.g., humanized or human antibody). Typically, relative to the parent antibody, one or more resulting variants selected for further study will have changes (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) and / or will substantially retain certain biological properties of the parent antibody. Exemplary substitution variants are affinity matured antibodies, which can be conveniently generated, for example, using affinity maturation techniques based on phage display such as those described herein. Briefly, one or more CDR residues are mutated and the variant antibodies are displayed on phage and screened for specific biological activity (e.g., binding affinity).

[0182] Changes (e.g., substitutions) can be made in the CDR, for example, to improve antibody affinity. Such changes can be made in CDR "hot spots", i.e., residues encoded by codons that undergo high-frequency mutations during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008)) and / or residues in contact with the antigen, wherein the resulting variant VH or VL is tested for binding affinity. Affinity maturation achieved by construction and reselection from a secondary library has been described, for example, by Hoogenboom et al. in Methods in Molecular Biology 178: 1-37 (O'Brien et al., Human Press, Totowa, NJ, (2001)). In certain aspects of affinity maturation, diversity is introduced into 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 created. The library is then screened to identify any antibody variant with the desired affinity. Another method of introducing diversity involves a CDR-directed approach 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. CDR-H3 and CDR-L3 in particular are often targeted.

[0183] In some aspects, substitution, insertion or deletion may occur in one or more CDRs, as long as such changes do not substantially reduce the antigen binding ability of the antigen binding molecule. 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 changes may be, for example, outside of the antigen contact residues in the CDRs. In some variant VH and VL sequences provided above, each CDR either remains unchanged or contains no more than one, two or three amino acid substitutions.

[0184] The method that can be used to identify antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis", as described by Cunningham and Wells (1989) Science, 244: 1081-1085. In this method, a residue or a group of target residues (e.g., charged residues such as arg, asp, his, lys and glu) are identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Other substitutions can be introduced at amino acid positions that exhibit functional sensitivity to the initial substitution. Alternatively or additionally, the crystal structure of the antigen-antibody complex can be used to identify the contact points between the antibody and the antigen. Such contact residues and adjacent residues that are candidates for substitution can be targeted or eliminated. Variants can be screened to determine whether they have the desired properties.

[0185] Amino acid sequence insertions include amino and / or carboxyl terminal fusions ranging in length from one residue to a polypeptide containing one hundred or more residues, and intrasequence insertions of one or more amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertion variants of antibody molecules include fusing the N-terminus or C-terminus of the antibody to an enzyme (e.g., for ADEPT (antibody directed enzyme prodrug therapy)) or a polypeptide that increases the serum half-life of the antibody.

[0186] a) Glycosylation variants

[0187] In certain aspects, the antibodies provided herein are altered to increase or decrease the extent of antibody glycosylation. Glycosylation sites can be conveniently added or deleted to an antibody by altering the amino acid sequence to create or remove one or more glycosylation sites.

[0188] When antibody comprises Fc district, the oligosaccharide connected thereto can be changed.Natural antibodies produced by mammalian cells usually comprise biantennary oligosaccharides with side chains, and the biantennary oligosaccharides are usually connected to the Asn297 of the CH2 domain in the Fc district by N-connection.See, for example, Wright et al. TIBTECH 15:26-32 (1997).Oligosaccharides can include various carbohydrates, for example, mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, and the fucose of the GlcNAc in the " stem " of biantennary oligosaccharide structure.In some aspects, the oligosaccharides in the antibody of the present invention can be modified to produce antibody variants with some improved characteristics.

[0189] In one aspect, antibody variants are provided having non-fucosylated oligosaccharides, i.e., oligosaccharide structures lacking fucose attached (directly or indirectly) to the Fc region. Such non-fucosylated oligosaccharides (also referred to as "defucosylated" oligosaccharides) are particularly N-linked oligosaccharides lacking a fucose residue attached to the first GlcNAc in the stem of the biantennary oligosaccharide structure. In one aspect, antibody variants are provided having an increased proportion of non-fucosylated oligosaccharides in the Fc region compared to a native or parent antibody. For example, the proportion of non-fucosylated oligosaccharides can be at least about 20%, at least about 40%, at least about 60%, at least about 80%, or even about 100% (i.e., the absence of fucosylated oligosaccharides). The percentage of non-fucosylated oligosaccharides, as described, for example, in WO2006 / 082515, as measured by MALDI-TOF mass spectrometry, is the (average) amount of oligosaccharides lacking a fucose residue relative to the sum of all oligosaccharides (e.g., complex, hybrid and high mannose structures) linked to Asn 297. Asn297 refers to the asparagine residue located at approximately position 297 in the Fc region (EU numbering of residues in the Fc region); however, due to minor sequence variations in antibodies, Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such antibodies having an increased proportion of non-fucosylated oligosaccharides in the Fc region may have improved FcγRIIIa receptor binding and / or improved effector function, in particular improved ADCC function. See, for example, US 2003 / 0157108; US2004 / 0093621.

[0190] Examples of cell lines capable of producing antibodies with reduced fucosylation include Lec13 CHO cells lacking protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US 2003 / 0157108; and WO 2004 / 056312, particularly in Example 11), and knockout cell lines, such as α-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614-622 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO 2004 / 056312, particularly in Example 11). 2003 / 085107), or cells with reduced or abolished GDP-fucose synthesis or transporter activity (see, e.g., US2004259150, US2005031613, US2004132140, US2004110282).

[0191] On the other hand, antibody variants provide bisected oligosaccharides, for example, wherein the biantennary oligosaccharides attached to the Fc region of the antibody are bisected by GlcNAc. As described above, such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described in, for example, Umana et al., Nat Biotechnol 17, 176-180 (1999); Ferrara et al., Biotechn Bioeng 93, 851-861 (2006); WO 99 / 54342; WO 2004 / 065540, WO 2003 / 011878.

[0192] Also provided are antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region. Such antibody variants may have improved CDC function. Such antibody variants are described in, for example, WO 1997 / 30087; WO 1998 / 58964; and WO 1999 / 22764.

[0193] b) Fc region variants

[0194] In certain aspects, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. An Fc region variant can comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0195] In certain aspects, the present invention contemplates antibody variants with some but not all effector functions, making them ideal candidates for applications where the in vivo half-life of the antibody is important and certain effector functions (e.g., complement dependent cytotoxicity (CDC) and antibody dependent cell-mediated cytotoxicity (ADCC)) are unnecessary or harmful. In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / depletion of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and therefore may lack ADCC activity), but retains FcRn binding ability. Primary cells NK cells that mediate ADCC express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Kinet, Annu. Rev. Immunol. 9: 457-492 (1991). Other non-limiting examples of in vitro assays for evaluating ADCC activity of target molecules are described in U.S. Pat. Nos. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, 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 assays can be used (see, e.g., ACTI for flow cytometry). TM Nonradioactive cytotoxicity assay (Cell Technology, Inc. Mountain View, CA; and CytoTox Nonradioactive cytotoxicity assays (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, ADCC activity of the molecule of interest can be assessed in vivo, for example, in an animal model such 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 cannot bind to C1q and therefore lacks CDC activity. See, e.g., 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). FcRn binding and in vivo clearance / half-life assays 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); WO 2013 / 120929 Al).

[0196] Antibodies with reduced effector function include those with substitutions of one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include those with substitutions at two or more of amino acids 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant, in which residues 265 and 297 are substituted with alanine (U.S. Pat. No. 7,332,581).

[0197] Certain antibody variants with improved or decreased binding to FcRs are described (see, e.g., U.S. Patent No. 6,737,056; WO 2004 / 056312, and Shields et al., J. Biol. Chem 9(2):6591-6604 (2001)).

[0198] In certain aspects, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, for example, substitutions at positions 298, 333, and / or 334 of the Fc region (EU numbering of residues).

[0199] In some aspects, antibody variants include Fc regions with one or more amino acid substitutions that reduce FcγR binding, such as substitutions at positions 234 and 235 of the Fc region (EU numbering of residues). On the one hand, substitutions are L234A and L235A (LALA). In some aspects, antibody variants are further included in D265A and / or P329G in the Fc region derived from human IgG1 Fc region. On the one hand, in the Fc region derived from human IgG1 Fc region, substitutions are L234A, L235A and P329G (LALA-PG). (See, e.g., WO 2012 / 130831). On the other hand, in the Fc region derived from human IgG1 Fc region, substitutions are L234A, L235A and D265A (LALA-DA).

[0200] In some aspects, changes are made in the Fc region that result in altered (ie, improved or reduced) CIq 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).

[0201] Antibodies with extended half-life and improved neonatal Fc receptor (FcRn) binding, responsible for the transfer of maternal IgG to the fetus (Guyer, RL et al., J. Immunol. 117:587 (1976), and Kim, JK et al., J. Immunol. 24:249 (1994)) are described in US 2005 / 0014934 (Hinton et al.). Those antibodies comprise an Fc region having one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include those having a substitution at one or more of the following 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, such as a substitution at Fc region residue 434 (see, e.g., U.S. Pat. No. 7,371,826; Dall'Acqua, WF et al. J. Biol. Chem. 281 (2006) 23514-23524).

[0202] Fc region residues that are critical for mouse Fc-mouse FcRn interactions have been identified by site-directed mutagenesis (see, e.g., Dall'Acqua, WF et al. J. Immunol 169 (2002) 5171-5180). The interaction involves residues 1253, H310, H433, N434, and H435 (EU index numbering) (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 1253, 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 on the human Fc-human FcRn complex have shown that residues I253, S254, H435 and Y436 are essential for the interaction (Firan, M. et al., Int. Immunol. 13 (2001) 993; Shields, RL et al., J. Biol. Chem. 276 (2001) 6591-6604). Various mutants of residues 248 to 259, 301 to 317, 376 to 382 and 424 to 437 have been reported and examined in Yeung, YA et al. (J. Immunol. 182 (2009) 7667-7671).

[0203] In some aspects, the antibody variant comprises an Fc region with one or more amino acid substitutions that reduce FcRn binding, for example, substitutions at positions 253, and / or 310 and / or 435 of the Fc region (EU numbering of residues). In some aspects, the antibody variant comprises an Fc region with amino acid substitutions at positions 253, 310 and 435. On the one hand, in an Fc region derived from a human IgG1 Fc region, the substitutions are I253A, H310A and H435A. See, e.g., Grevys, A. et al., J. Immunol. 194 (2015) 5497-5508.

[0204] In some aspects, the antibody variant comprises an Fc region with one or more amino acid substitutions that reduce FcRn binding, for example, substitutions at 310, and / or 433 and / or 436 of the Fc region (EU numbering of residues). In some aspects, the antibody variant comprises an Fc region with amino acid substitutions at positions 310, 433 and 436. On the one hand, in an Fc region derived from a human IgG1 Fc region, the substitutions are H310A, H433A and Y436A. (See, e.g., WO 2014 / 177460 A1).

[0205] In some aspects, the antibody variant comprises an Fc region with one or more amino acid substitutions that increase FcRn binding, for example, substitutions at 252, and / or 254 and / or 256 in the Fc region (EU numbering of residues). In some aspects, the antibody variant comprises an Fc region with amino acid substitutions at positions 252, 254 and 256. On the one hand, in an Fc region derived from a human IgG1 Fc region, the substitutions are M252Y, S254T and T256E. 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.

[0206] The C-terminus of the heavy chain of the antibody as reported herein can be a complete C-terminus ending with amino acid residue PGK. The C-terminus of the heavy chain can be a shortened C-terminus, in which one or two C-terminal amino acid residues have been removed. In a preferred aspect, the C-terminus of the heavy chain is a shortened C-terminus ending with PG. In one aspect of all aspects reported herein, the antibody comprising a heavy chain including a C-terminal CH3 domain as specified herein, comprises a C-terminal glycine-lysine dipeptide (G446 and K447, the EU index numbering of amino acid position). In one aspect of all aspects reported herein, the antibody comprising a heavy chain including a C-terminal CH3 domain as specified herein, comprises a C-terminal glycine residue (G446, the EU index numbering of amino acid position).

[0207] c) Cysteine ​​engineered antibody variants

[0208] In certain aspects, it may be desirable to generate antibodies with engineered cysteine ​​residues, such as THIOMAB. TM Antibodies, wherein one or more residues of the antibody are replaced by cysteine ​​residues. In a specific embodiment, the substituted residue is present in an accessible site of the antibody. As further described herein, by replacing those residues with cysteine, reactive thiol groups are positioned in accessible sites of the antibody, and can be used to conjugate the antibody with other parts (such as drug moieties or linker-drug moieties) to produce immunoconjugates. Cysteine ​​engineered antibodies can be produced as described in, for example, U.S. Patent Nos. 7,521,541, 8,30,930, 7,855,275, 9,000,130 or WO 2016040856.

[0209] 7. Immunoconjugates

[0210] The present invention also provides immunoconjugates comprising an antibody that binds to human VEGF and human IL-1β disclosed herein, which is conjugated (chemically bonded) 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 a fragment thereof), or a radioactive isotope.

[0211] On the one hand, immunoconjugates are antibody-drug conjugates (ADCs), wherein the antibody is conjugated to one or more therapeutic agents described above. A linker is typically used to connect the antibody to one or more therapeutic agents. An overview of ADC technology is listed in Pharmacol Review 68:3-19 (2016), which includes examples of therapeutic agents, drugs, and linkers.

[0212] B. Recombinant Methods and Compositions

[0213] Antibodies can be produced using recombinant methods and compositions, for example, as described in US 4,816,567. For these methods, one or more isolated nucleic acids encoding the antibodies are provided.

[0214] In one aspect, an isolated nucleic acid encoding an antibody of the invention is provided.

[0215] In one aspect, a method for preparing an antibody that binds to human VEGF and human IL-1β is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody as provided above under conditions suitable for expressing the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0216] In order to recombinantly produce antibodies that bind to human VEGF and human IL-1β, nucleic acids encoding the antibodies, such as those described above, are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that specifically bind to genes encoding heavy and light chains of antibodies), or produced by recombinant methods or obtained by chemical synthesis.

[0217] Suitable host cells for cloning or expressing vectors encoding antibodies include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. Regarding expressing antibody fragments and polypeptides in bacteria, see, for example, US 5,648,237, US 5,789,199 and US 5,840,523, (see also Charlton, KA, in: Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2003), pp. 245-254, describing the expression of antibody fragments in Escherichia coli.) Antibodies can be separated from bacterial cell paste in a soluble fraction after expression, and can be further purified.

[0218] Vertebrate cells can 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 lines (COS-7) transformed by SV40; human embryonic kidney cell lines (such as 293 or 293T cells described in, for example, Graham, FL et al., J. Gen Virol. 36 (1977) 59-74); small hamster kidney cells (BHK); mouse Sertoli cells (such as TM4 cells described in Mather, JP, Biol. Reprod. 23 (1980) 243-252); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); Buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells (as described, for example, in Mather, JP et al., Annals N.Y. Acad. Sci. 383 (1982) 44-68); MRC 5 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 cell lines 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.

[0219] In one aspect, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell or a lymphocyte (eg, a Y0, NS0, Sp20 cell).

[0220] C. Pharmaceutical Compositions

[0221] In other aspects, a pharmaceutical composition comprising any of the antibodies provided herein is provided, for example, for use in any of the following methods of treatment. On the one hand, the pharmaceutical composition comprises any of the antibodies provided herein and at least one pharmaceutical excipient. On the other hand, the pharmaceutical composition comprises any of the antibodies provided herein and at least one additional therapeutic agent, for example, as described below.

[0222] Pharmaceutical compositions of antibodies that bind to human VEGF and human IL-1β as described herein are prepared by mixing the antibodies having the desired purity with one or more optional pharmaceutical carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)) in the form of lyophilized compositions or aqueous solutions. Pharmaceutical carriers are generally nontoxic to recipients at the doses and concentrations employed, and include, but are not limited to: buffers such as histidine, phosphates, citrates, acetates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl alcohol, or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; metacresol); low molecular weight (less than about 10 residues) polypeptides; 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 dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., zinc protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutical carriers herein also include interstitial drug dispersants such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 ( Halozyme, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[0223] Exemplary lyophilized antibody compositions are described in US Pat. No. 6,267,958. Aqueous antibody compositions include those described in US Pat. No. 6,171,586 and WO 2006 / 044908, the latter compositions comprising a histidine-acetate buffer.

[0224] The pharmaceutical compositions herein may also contain more than one active ingredient essential for the particular indication being treated, preferably active ingredients with complementary activities that do not adversely affect each other. Such active ingredients are suitably present in combination in amounts effective for the intended purpose.

[0225] The active ingredient can be embedded in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively); in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules); or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. ed. (1980).

[0226] 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.

[0227] Pharmaceutical compositions for in vivo administration are generally sterile. For example, sterility can be easily achieved by filtration through sterile filtration membranes.

[0228] D. Methods of treatment and routes of administration

[0229] Any of the antibodies provided herein that bind to human VEGF and human IL-1 β can be used in therapeutic methods.

[0230] On the one hand, an antibody for use as a drug that binds to human VEGF and human IL-1β is provided. In other aspects, an antibody for treating vascular diseases that binds to human VEGF and human IL-1β is provided. In certain aspects, an antibody that binds to human VEGF and human IL-1β for use in a method of treatment is provided. In certain aspects, the present invention provides an antibody that binds to human VEGF and human IL-1β, for use in a method of treating an individual with a vascular disease, the method comprising administering to the individual an effective amount of an antibody that binds to human VEGF and human IL-1β. In one such aspect, such as described below, 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). In other aspects, the present invention provides an antibody that binds to human VEGF and human IL-1β, for inhibiting angiogenesis. In certain aspects, the present invention provides an antibody that binds to human VEGF and human IL-1β, for use in inhibiting angiogenesis in an individual, the method comprising administering to the individual an effective amount of an antibody that binds to human VEGF and human IL-1β to inhibit angiogenesis. The "individual" according to any of the above aspects is preferably a human.

[0231] In other aspects, an antibody for treating an eye disease that binds to human VEGF and human IL-1β is provided. In one embodiment, the eye disease is selected from: AMD (e.g., wet AMD, dry AMD, moderate AMD, advanced AMD, and geographic atrophy (GA)), macular degeneration, macular edema, DME (in one embodiment, localized, 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 retinopathy associated with ischemia, ROP, retinal vein occlusion (RVO) (in one embodiment, central (CRVO) and branch (BRVO) forms), 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, Hippel-Lindau syndrome, ocular histoplasmosis, FEVR, crown Tz's disease, Norrie's disease, retinal abnormalities associated with osteoporosis-pseudoglioma syndrome (OPPG), subconjunctival hemorrhage, redness, ocular neovascular disease, neovascular glaucoma, retinitis pigmentosa (RP), hypertensive retinopathy, retinal angiomatous proliferation, macular telangiectasia, iris neovascularization, intraocular neovascularization, retinal degeneration, cystoid macular edema (CME), vasculitis, papilledema, retinitis (including but not limited to CMV retinitis), melanoma In one embodiment, the eye disease is selected from: AMD (in one embodiment, wet AMD, dry AMD, moderate AMD, advanced AMD, and geographic atrophy (GA)), macular degeneration, macular edema, DME (in one embodiment, localized, 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).

[0232] In other aspects, the present invention provides a use of an antibody that binds to human VEGF and human IL-1β in the production or preparation of a drug. In one aspect, the drug is used to treat vascular disease. In other aspects, the drug is used in a method for treating vascular disease, the method comprising administering an effective amount of the drug to an individual suffering from vascular disease. In one such aspect, such as described below, the method further comprises administering an effective amount of at least one additional therapeutic agent to the individual.

[0233] In one aspect, the medicament is used to treat an eye disease. In other aspects, the medicament is used in a method for treating an eye disease, the method comprising administering an effective amount of the medicament to an individual suffering from the eye disease. In one such aspect, such as described below, the method further comprises administering an effective amount of at least one additional therapeutic agent to the individual.

[0234] In other aspects, the invention provides methods for treating vascular diseases. In one aspect, the method comprises administering to an individual suffering from such vascular disease an effective amount of an antibody that binds to human VEGF and human IL-1β. In one such aspect, as described below, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent.

[0235] In another aspect, the invention provides a method for treating an eye disease. In one aspect, the method comprises administering to an individual suffering from such an eye disease an effective amount of an antibody that binds to human VEGF and human IL-1β. In one such aspect, as described below, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent.

[0236] According to any of the above embodiments, an "individual" may be a human being.

[0237] In other aspects, the present invention provides pharmaceutical compositions comprising any antibody that binds to human VEGF and human IL-1β as provided herein, for example, for use in any of the above methods of treatment. On the one hand, the pharmaceutical composition comprises any antibody that binds to human VEGF and human IL-1β as provided herein and a pharmaceutically acceptable carrier. On the other hand, the pharmaceutical composition comprises any antibody that binds to human VEGF and human IL-1β as provided herein and at least one additional therapeutic agent, for example, as described below.

[0238] The antibodies of the present invention can be administered alone or used in combination therapy. For example, the combination therapy includes administering an antibody of the present invention and administering at least one additional therapeutic agent (e.g., one, two, three, four, five or six additional therapeutic agents).

[0239] For example, in certain embodiments, any of the foregoing methods further comprises administering one or more additional compounds. In certain embodiments, the antibodies provided herein that bind to human VEGF and human IL-1β are administered simultaneously with the additional compounds. In certain embodiments, the antibodies that bind to human VEGF and human IL-1β are administered before or after the additional compounds. In certain embodiments, the additional 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, B factor, H factor, 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 additional compound is an antibody or antigen-binding fragment thereof.

[0240] In certain embodiments according to (or as applied to) any of the above embodiments, the eye 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 retinopathy, diabetic macular edema, pathological myopia, Hippel-Lindau syndrome, ocular histoplasmosis, retinal vein occlusion (RVO) including CRVO and BRVO, corneal neovascularization, retinal neovascularization and retinopathy of prematurity (ROP).

[0241] In some cases, the antibodies provided herein that bind to human VEGF and human IL-1β can be administered in combination with at least one other therapeutic agent for the treatment of an ocular disorder, such as an ocular disorder described herein (e.g., AMD (e.g., wet AMD), DME, DR, RVO, or GA). Exemplary other therapeutic agents for combination therapy for the treatment of ocular disorders include, but are not limited to, anti-angiogenic agents, such as VEGF antagonists, including, for example, anti-VEGF antibodies (e.g., anti-VEGF Fab (ranibizumab)), soluble receptor fusion proteins (e.g., recombinant soluble receptor fusion proteins (Aflibercept, also known as VEGF Trap Eye; Regeneron / Aventis), aptamers (e.g. anti-VEGF PEGylated aptamers (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), semasanib (SU5416; SUGEN), and (sunitinib); tryptophanyl-tRNA synthetase (TrpRS); squalamine; (Anecortave for long-acting suspension; Alcon); Complistatin A4 prodrug (CA4P); (mifepristone-ru486); subconjunctival triamcinolone acetonide; intravitreal crystalline triamcinolone acetonide; matrix metalloproteinase inhibitors (e.g., pumastat (AG3340; Pfizer)); fluocinolone acetonide (including fluocinolone acetonide intraocular implant; Bausch & Lom / controlled delivery system); tricarboxyaminoquinoline; inhibitors of integrin β3 function; angiostatin, and combinations thereof. These and other therapeutic agents that can be administered in combination with antibodies that bind to human VEGF and human IL-1β of the present invention are described, for example, in U.S. Patent Application No. 2014 / 0017244, which is incorporated herein by reference in its entirety.

[0242] Further examples of other therapeutic agents that can be used in combination with the antibodies that bind to human VEGF and human IL-1β provided herein to treat ocular disorders (e.g., AMD, DME, DR, RVO or GA) include, but are not limited to: (verteporfin; a light-activated drug often used in combination with nonthermal laser photodynamic therapy), PKC412, Endovion (NS3728; NeuroSearch A / S), neurotrophic factors (such as glial-derived neurotrophic factor (GDNF) and ciliary neurotrophic factor (CNTF)), diltiazem, dorzolamide, 9-cis-retinal, ophthalmic drugs (e.g., iodine phosphide, fosfothioline, or carbonic anhydrase inhibitors), veovastat (AE-941; AEterna Laboratories, Inc.), Sirna-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., used by EntreMed, Inc.), cardiotrophin-1 (Genentech), 2-methoxyestradiol (Allergan / Oculex), DL-8234 (Toray Industries, Inc.), 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., inhibitors from Allergan, SUGEN, or Pfizer), NX-278-L (NeXstar Pharmaceuticals / Gilead Sciences), Opt-24 (OPTISFrance SA), retinal ganglion neuroprotectants (Cogent Neurosciences), N-nitropyrazole derivatives (Texas A&M University System), KP-102 (Krenitsky Pharmaceuticals), cyclosporin A, therapeutic agents for photodynamic therapy (e.g., receptor-targeted PDT, Bristol-Myers Squibb; porfimer sodium with PDT for injection; verteporfin, QLT Inc; ropeporfin with PDT, Miravent Medical Technologies; talaporfin sodium with PDT, Nippon Oil Corporation; and motexafin lutetium, Pharmacyclics, Inc.), antisense oligonucleotides (including, for example, products tested by Novagali Pharma SA and ISIS-13650, Ionis Pharmaceuticals), and combinations thereof.

[0243] An antibody that binds to human VEGF and human IL-1β as provided herein can be administered in combination with a therapy or surgical procedure for treating an eye disorder (e.g., AMD, DME, DR, RVO or GA), including, for example, laser photocoagulation (e.g., panretinal photocoagulation (PRP)), drusen laser, macular hole surgery, macular shift surgery, implantable microtelescope, PHI sports angiography (also known as microlaser therapy and feeding vessel therapy), proton beam therapy, microstimulation therapy, retinal detachment and vitreous surgery, scleral buckle, macular surgery, transpupillary thermal therapy, light system The invention relates to methods of treating a patient's condition with a conventional method, including conventional therapy, use of RNA interference (RNAi), in vitro fertilization (also known as membrane differential filtration and rheotherapy), microchip implantation, stem cell therapy, gene replacement therapy, ribozyme gene therapy (including gene therapy for hypoxia response elements, Oxford Biomedical Products; Lentipak, Genetix; and PDEF gene therapy, GenVec), photoreceptor / retinal cell transplantation (including transplantable retinal epithelial cells, Diacrin, Inc.; retinal cell transplantation, such as Astras Pharmaceuticals USA, ReNeuron, CHA Biotech), acupuncture, and combinations thereof.

[0244] In some cases, antibodies that bind to human VEGF and human IL-1β can be administered in combination with anti-angiogenic agents to treat eye diseases (e.g., AMD, DME, DR, RVO, or GA). Any suitable anti-angiogenic agent can be used in combination with the antibodies that bind to human VEGF and human IL-1β of the present invention, including but not limited to those listed in Carmeliet et al. Nature 407:249-257, 2000. In some embodiments, the anti-angiogenic agent is a VEGF antagonist, including but not limited to: anti-VEGF antibodies (e.g., anti-VEGF Fab (ranibizumab), RTH-258 (formerly known as ESBA-1008, anti-VEGF single-chain antibody fragment; Novartis), or bispecific anti-VEGF antibodies (e.g., anti-VEGF / anti-angiogenic peptide 2 bispecific antibodies, such as faricimab; Roche)), soluble recombinant receptor fusion proteins (e.g. (aflibercept), VEGF variants, soluble VEGFR fragments, aptamers capable of blocking VEGF (e.g., pegaptanib sodium) or VEGFR, neutralizing anti-VEGFR antibodies, small molecule inhibitors of VEGFR tyrosine kinase, anti-VEGF (e.g., abicipar pegol, Molecular Partners AG / Allergan), small interfering RNA that inhibits VEGF or VEGFR expression, 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), semasanib (SU5416; SUGEN), and (sunitinib)) and combinations thereof.

[0245] Other suitable anti-angiogenic agents that can be administered in combination with an antibody that binds to human VEGF and human IL-1β as provided herein to treat an ocular disorder (e.g., AMD, DME, DR, RVO, or GA) include corticosteroids, antagonist steroids, anecortave acetate, angiostatin, endostatin, tyrosine kinase inhibitors, matrix metalloproteinase (MMP) inhibitors, insulin-like growth factor binding protein 3 (IGFBP3), 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 the localization of stem cells (e.g., endothelial progenitor cells) to sites of new blood vessel formation (e.g., anti-vascular endothelial cadherin (CD-144) antibodies and / or anti-SDF-1 antibodies), and combinations thereof.

[0246] In other examples, in some cases, antibodies and / or polymer formulations thereof that bind to human VEGF and human IL-1β can be administered in combination with an agent having anti-neovascular activity to treat eye diseases (e.g., AMD, DME, DR, RVO, or GA), such as anti-inflammatory drugs, rapamycin inhibitors (e.g., rapamycin, (everolimus) and (temsirolimus), cyclosporine, a tumor necrosis factor (TNF) antagonist (e.g., an anti-TNFα antibody or antigen-binding fragment thereof (e.g., infliximab, adalimumab, cefixime and golimumab) or a soluble receptor fusion protein (e.g., etanercept)), an anti-complement drug, a nonsteroidal anti-inflammatory agent (NSAID), or a combination thereof.

[0247] In yet further examples, antibodies that bind to human VEGF and human IL-1β can be administered in combination with agents that have neuroprotective effects and can potentially reduce the conversion of dry AMD to wet AMD, such as drugs known as "neurosteroids," which include, for example, dehydroepiandrosterone (DHEA) (brand name: PRASTERA TM and ), dehydroepiandrosterone sulfate, and pregnenolone sulfate.

[0248] Any suitable AMD therapeutic agent may be administered as an additional therapeutic agent in combination with the antibodies that bind to human VEGF and human IL-1β described herein for treating an ocular disorder (e.g., AMD, DME, DR, RVO, or GA), including but not limited to VEGF antagonists, such as anti-VEGF antibodies (e.g., (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-angiogenic peptide 2 bispecific antibody, such as faricimab; Roche)), a soluble VEGF receptor fusion protein (e.g., (Aflibercept), anti-VEGF (e.g. abicipar pegol); MolecularPartners AG / Allergan) or anti-VEGF aptamers (e.g. (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., 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); combined with photodynamic therapy (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, such as CLG-561; Alcon), or complement factor D antagonists (e.g., anti-complement factor D antibodies, such as lambalizumab; Roche)); C3 blocking peptides (e.g., APL-2, Appellis); visual cycle modulators (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, e.g., NT-501 (Renexus); PH-05206388 (Pfizer), huCNS-SC cell transplantation (StemCells), CNTO-2476 (umbilical cord stem cell line; Janssen), OpRegen (RPE cell suspension; 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, e.g., GSK-933776); S1P antagonists (e.g., anti-S1P antibodies, e.g., iSONEP TM ; Lpath Inc); ROB04 antagonists (e.g., anti-ROBO4 antibodies, such as DS-7080a; Daiichi Sankyo Co., Ltd.); lentiviral vectors expressing endostatin and angiostatin (e.g., RetinoStat); and any combination thereof. In some cases, AMD therapeutics (including any prior AMD therapeutics) can be co-formulated. For example, the anti-PDGFR antibody REGN2176-3 can be co-formulated with aflibercept Co-formulation. In some cases, such co-formulations may be administered in combination with antibodies of the invention that bind to human VEGF and human IL-1 β. In some cases, the eye disorder is AMD (eg, wet AMD).

[0249] The antibody of the present invention that binds to human VEGF and human IL-1β can be used together with (ranibizumab) is administered in combination to treat an eye disease (e.g., AMD, DME, DR, RVO, or GA). In some cases, the eye disease is AMD (e.g., wet AMD). In some cases, the eye disease is GA.

[0250] The antibody of the present invention that binds to human VEGF and human IL-1β can be used together with (aflibercept) is administered in combination to treat an eye condition (e.g., AMD, DME, DR, RVO, or GA). In some cases, the eye condition is AMD (e.g., wet AMD). In some cases, the eye condition is GA.

[0251] The antibody of the present invention that binds to human VEGF and human IL-1β can be used together with (pegaptanib sodium) is administered in combination to treat an eye disease (e.g., AMD, DME, DR, RVO, or GA). In some cases, the eye disease is AMD (e.g., wet AMD). In some cases, the eye disease is GA.

[0252] The antibody of the present invention that binds to human VEGF and human IL-1β can be combined with photodynamic therapy. (verteporfin) is administered in combination to treat an eye disease (e.g., AMD, DME, DR, RVO, or GA). In some cases, the eye disease is AMD (e.g., wet AMD). In some cases, the eye disease is GA.

[0253] The antibodies of the present invention that bind to human VEGF and human IL-1β can be administered in combination with a PDGF antagonist to treat eye disorders (e.g., AMD, DME, DR, RVO, or GA). Exemplary PDGF antagonists that can be used in combination with the antibodies of the present invention that bind to human VEGF and human IL-1β include anti-PDGF antibodies, anti-PDGFR antibodies, small molecule inhibitors (e.g., squalamine), anti-PDGF-B PEGylated aptamers, e.g., (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, It can be administered as an adjunct therapy to the antibodies of the present invention that bind to human VEGF and human IL-1β. OHR-102 can be administered in combination with VEGF antagonists (e.g. or In some embodiments, the antibodies of the present invention that bind to human VEGF and human IL-1β can be administered in combination with OHR-102, and / or In some cases, the eye condition is AMD (eg, wet AMD). In some cases, the eye condition is GA.

[0254] The antibodies of the invention that bind to human VEGF and human IL-1β can be administered in combination with RTH-258 to treat eye diseases (e.g., AMD, DME, DR, RVO, or GA). RTH-258 can be administered, for example, by intravitreal injection or intraocular infusion. In some cases, the eye disease is AMD (e.g., wet AMD). In some cases, the eye disease is GA.

[0255] Antibodies that bind to human VEGF and human IL-1β of the invention can be administered in combination with abicipar pegol to treat an eye disorder (e.g., AMD, DME, DR, RVO, or GA). In some cases, the eye disorder is AMD (e.g., wet AMD). In some cases, the eye disorder is GA.

[0256] Any suitable DME and / or DR therapeutic agent, including but not limited to VEGF antagonists (e.g., or ), corticosteroids (e.g., corticosteroid implants (e.g. (dexamethasone intravitreal implant) or (fluocinolone acetonide intravitreal implant), or a corticosteroid formulated for administration by intravitreal injection (e.g., triamcinolone acetonide), or a combination thereof. In certain instances, the ocular condition is DME and / or DR.

[0257] The antibody of the present invention that binds to human VEGF and human IL-1β can be used together with (ranibizumab) in combination to treat DME and / or DR (e.g., NPDR or PDR).

[0258] The antibody of the present invention that binds to human VEGF and human IL-1β can be used together with (aflibercept) in combination to treat DME and / or DR (eg, NPDR or PDR).

[0259] The antibody of the present invention that binds to human VEGF and human IL-1β can be used together with (dexamethasone intravitreal implant) in combination to treat DME and / or DR.

[0260] The antibody of the present invention that binds to human VEGF and human IL-1β can be used together with (dexamethasone intravitreal implant) in combination to treat DME and / or DR.

[0261] In some cases, the TAO / PRN treatment regimen or the TAE treatment regimen can be used to administer an AMD therapeutic agent (e.g., ranibizumab or aflibercept) in combination with an antibody that binds to human VEGF and human IL-1β of the present invention, and / or a polymer formulation thereof. In some cases, the eye disorder is AMD (e.g., wet AMD). In some cases, the eye disorder is GA.

[0262] Such combination therapies described above encompass combined administration (where two or more therapeutic agents are included in the same or different formulations) and separate administration, in which case the administration of the antibodies of the invention described herein that bind to human VEGF and human IL-1β can be performed before, simultaneously with, and / or after the administration of the additional therapeutic agent or agent. In one embodiment, the administration of the antibodies of the invention that bind to human VEGF and human IL-1β and the administration of the additional therapeutic agent each occur within about one, two, three, four, or five months, or within about one, two, three, four, five, or six days.

[0263] The antibodies of the present invention (and any additional therapeutic agents) can be administered by any suitable means, including parenteral, intrapulmonary and intranasal, and if necessary for local treatment, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration can be carried out by any suitable route, for example by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-lived or long-term. Various dosing schedules are contemplated herein, including but not limited to single or multiple administrations, push administrations, and pulse infusions at various time points.

[0264] The antibodies of the present invention will be formulated, dosed and applied in a manner consistent with good medical practice. Factors to be considered in this case include the specific illness being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the illness, the delivery site of the medicament, the method of administration, the time schedule of administration, and other factors known to practitioners. The antibody is not necessary, but is optionally co-formulated with one or more preparations currently used to prevent or treat the illness in question. The effective amount of these other preparations depends on the amount of the antibody present in the pharmaceutical composition, the type of illness or treatment, and other factors discussed above. These are generally used with the same dosages and routes of administration as described herein, or with about 1% to 99% of the dosages described herein, or with any dosage and any route determined to be suitable empirically / clinically.

[0265] For the prevention or treatment of disease, the appropriate dose of the antibody of the present invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the molecule is used for prevention or treatment purposes, the patient's medical history and the response to the antibody, and the discretion of the attending physician. The antibody is appropriately administered to the patient once or in a series of treatments. Depending on the type and severity of the disease, an antibody of about 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg-10 mg / kg) can be, for example, an initial candidate dose administered to the patient by one or more separate administrations or by continuous infusion. Depending on the above factors, a typical daily dose can range from about 1 μg / kg to 100 mg / kg or more. For repeated administration for several days or longer, depending on the condition, treatment will generally continue until the desired disease symptom suppression occurs. An exemplary dose of an antibody ranges from about 0.05 mg / kg to about 10 mg / kg. Therefore, 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, weekly or every three weeks (e.g., so that the patient receives about two to about twenty, or for example about six doses of the antibody). An initial higher loading dose may be administered, followed by one or more lower doses. The progress of the therapy is easily monitored by conventional techniques and assays.

[0266] E. Products

[0267] In another aspect of the present invention, a product is provided, which contains substances that can be used to treat, prevent and / or diagnose the above-mentioned diseases. The product includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The container can be formed of a variety of materials such as glass or plastic. The container holds a composition that is effective for treating, preventing and / or diagnosing the disease itself or in combination with another composition, and the container may have a sterile access port (for example, the container may be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). At least one active agent in the composition is an antibody of the present invention. The label or package insert indicates that the composition is used to treat the selected disease. In addition, the product may include (a) a first container containing a composition comprising an antibody of the present invention; and (b) a second container containing a composition comprising an additional cytotoxic agent or other therapeutic agent. The product in this aspect of the present invention may also include a package insert indicating that the composition can be used to treat a specific disease. Alternatively or additionally, the article of manufacture may further include a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. The article of manufacture may also include other substances required from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0268] 3. Specific embodiments of the present invention

[0269] Hereinafter, specific embodiments of the present invention are listed.

[0270] 1. An antibody that binds to human VEGF and human IL-1β, comprising a VEGF paratope and an IL-1β paratope within a cognate pair of a variable light chain domain (VL domain) and a variable heavy chain domain (VH domain), wherein the VEGF paratope comprises amino acid residues from CDR-H2, CDR-L1 and CDR-L3 of the antibody, wherein the IL-1β paratope comprises amino acid residues from CDR-H1, CDR-H3 and CDR-L2 of the antibody.

[0271] 2. An antibody that binds to human VEGF and human IL-1β, comprising a VEGF paratope and an IL-1β paratope within a cognate pair of a variable light chain domain (VL domain) and a variable heavy chain domain (VH domain), wherein the variable light chain domain and variable heavy chain domain pair simultaneously bind to human VEGF and human IL-1β.

[0272] 3. An antibody that binds to human VEGF and human IL-1β, which comprises a VEGF paratope and an IL-1β paratope within a homologous pair of a variable light chain domain (VL domain) and a variable heavy chain domain (VH domain), wherein none of the amino acids contained in the VEGF paratope are contained in the IL-1β paratope.

[0273] 4. An antibody that binds to human VEGF and to human IL-1β, comprising a VEGF paratope and an IL-1β paratope within one cognate pair of a variable light chain domain (VL domain) and a variable heavy chain domain (VH domain), wherein the antibody binds to the same epitope on human VEGF and to the same epitope on human IL-1β as an antibody having a variable heavy chain domain of SEQ ID NO: 11 and a variable light chain domain of SEQ ID NO: 12.

[0274] 5. An antibody that binds to human VEGF and to human IL-1β, comprising a VEGF paratope and an IL-1β paratope within a homologous pair of a variable light chain domain (VL domain) and a variable heavy chain domain (VH domain), wherein

[0275] wherein the VEGF paratope comprises amino acid residues from CDR-H2, CDR-L1 and CDR-L3 of an antibody, wherein the IL-1β paratope comprises amino acid residues from CDR-H1, CDR-H3 and CDR-L2 of an antibody; and / or

[0276] The variable light chain domain and variable heavy chain domain pair bind to human VEGF and human IL-1β simultaneously; and / or

[0277] None of the amino acids contained in the VEGF paratope are contained in the IL-1β paratope; and / or

[0278] The antibody binds to the same epitope on human VEGF and to the same epitope on human IL-1β as an antibody having a variable heavy chain domain of SEQ ID NO: 11 and a variable light chain domain of SEQ ID NO: 12; and / or

[0279] The antibody Fab fragment of the antibody binds to: (i) human VEGF121, wherein the K measured by surface plasmon resonance is D less than 10 pM, and (ii) human IL-1β, where the K measured by surface plasmon resonance is D Less than 30 pM; and / or

[0280] The antibody Fab fragment of the antibody exhibits an aggregation onset temperature greater than 70°C; and / or

[0281] an antibody Fab fragment of said antibody exhibits a melting temperature greater than 80°C as measured by dynamic light scattering; and / or

[0282] · Binding of the antibody Fab fragment of the antibody to human VEGF inhibits the binding of VEGF to VEGFR2, wherein the IC50 measured by surface plasmon resonance is less than 50nM; and wherein binding of the antibody Fab fragment of the antibody to human IL-1β inhibits the binding of IL-1β to IL-1βR1, wherein the IC50 measured by surface plasmon resonance is less than 30nM.

[0283] 6. An antibody according to any one of the preceding embodiments, wherein the antibody comprises a VH domain and a VL domain, the VH domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15, and the VL domain comprises (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8.

[0284] 7. An antibody that specifically binds to human VEGF and human IL-1β, wherein the antibody comprises a VH domain and a VL domain, the VH domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15, and the VL domain comprises (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8.

[0285] 8. The antibody of any one of the preceding embodiments, wherein the antibody comprises a VH domain and a VL domain, the VH domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14, (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15, and (d) a human heavy chain framework having (i) a FR1 comprising amino acid residues E2, G26, V28, and K30, (ii) a FR3 comprising amino acid residues R66, R83, and K94; the VL domain comprising (e) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, (f) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 17,

[0286] L2, (g) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:8, and (h) a human light chain framework having (i) a FR1 comprising amino acid residue I2, (ii) a FR2 comprising amino acid residue Y49, and (iii) a FR3 comprising amino acid residues G57, E67, D68 and Q69, wherein the numbering of the VH domain and the VL domain is according to the Kabat numbering system.

[0287] 9. An antibody that specifically binds to human VEGF and human IL-1β, wherein the antibody comprises a VH domain and a VL domain, the VH domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14, (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15, (d) a human heavy chain framework having (i) a FR1 comprising amino acid residues E2, G26, V28 and K30, (ii) a FR3 comprising amino acid residues R66, R83 and K94; the VL domain comprising (e) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, (f) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 17

[0288] L2, (g) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:8, and (h) a human light chain framework having (i) a FR1 comprising amino acid residue I2, (ii) a FR2 comprising amino acid residue Y49, and (iii) a FR3 comprising amino acid residues G57, E67, D68 and Q69, wherein the numbering of the VH domain and the VL domain is according to the Kabat numbering system.

[0289] 10. The antibody of any of the preceding embodiments, wherein the antibody comprises a VH domain comprising amino acid residues E2, G26, V28, K30, W31, N35b, D35c, K52a, D55, H56, Y58, T61, K62, F63, I64, R66, R83, K94, D95, V96, F98, and D101, and a VL domain comprising amino acid residues I2, Y27, W27a, S27c, S27d, L32, Y49, D50, Y53, K54, L56, G57, E67, D68, Q69, Y91, R92, Y93, H94, and Y96, wherein the numbering of the VH domain and the VL domain is according to the Kabat numbering system.

[0290] 11. The antibody of embodiment 10, comprising

[0291] - a VEGF paratope comprising the following amino acid residues in said VH domain:

[0292] D55, H56, Y58, T61, K62, F63, I64, R66 and R83; and the following amino acid residues in the VL domain: I2, Y27, W27a, S27c, S27d, E67, D68, Q69, R92, Y93, H94 and Y96; and

[0293] - an IL-1β paratope comprising the following amino acid residues in the VH domain: E2, G26, V28, K30, W31, N35b, D35c, K52a, K94, D95, V96, F98 and D101; and the following amino acid residues in the VL domain: L32, Y49, D50, Y53, K54, L56, G57, Y91.

[0294] 12. An antibody that specifically binds to human VEGF and human IL-1β, comprising in a pair of VL domains and VH domains: (i) a VH domain comprising amino acid residues E2, G26, V28, K30, W31, N35b, D35c, K52a, D55, H56, Y58, T61, K62, F63, I64, R66, R83, K94, D95, V96, 6, F98 and D101, (ii) a VL domain comprising amino acid residues I2, Y27, W27a, S27c, S27d, L32, Y49, D50, Y53, K54, L56, G57, E67, D68, Q69, Y91, R92, Y93, H94 and Y96, wherein the numbering of the VH domain and the VL domain is according to the Kabat numbering system.

[0295] 13. The antibody of embodiment 12, comprising

[0296] - a VEGF paratope comprising the following amino acid residues in the VH domain: D55, H56, Y58, T61, K62, F63, I64, R66, and R83, and the following amino acid residues in the VL domain: I2, Y27, W27a, S27c, S27d, E67, D68, Q69, R92, Y93, H94, and Y96; and

[0297] -IL-1β paratope comprising the following amino acid residues in the VH domain: E2, G26, V28, K30, W31, N35b, D35c, K52a, K94, D95, V96, F98 and D101, and the following amino acid residues in the VL domain: L32, Y49, D50, Y53, K54, L56, G57, Y91.

[0298] 14. An antibody according to any 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: 12.

[0299] 15. The antibody of any of the preceding embodiments, comprising (a) a VH domain comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 11, wherein the VH domain comprises amino acid residues E2, G26, V28, K30, W31, N35b, D35c, K52a, D55, H56, Y58, T61, K62, F63, I64, R66, R83, K94, D95, V96, F98, and D101; 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: 11. The amino acid sequence of NO:12 has an amino acid sequence with at least 90% sequence identity, wherein the VL domain comprises amino acid residues I2, Y27, W27a, S27c, S27d, L32, Y49, D50, Y53, K54, L56, G57, E67, D68, Q69, Y91, R92, Y93, H94 and Y96, and the numbering of the VH domain and the VL domain is according to the Kabat numbering system.

[0300] 16. An antibody that specifically binds to human VEGF and to human IL-1β, wherein the antibody comprises a VH domain and a VL domain, the VH domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15, the VL domain comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8, 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: 12.

[0301] 17. An antibody that specifically binds to human VEGF and to human IL-1β, wherein the antibody comprises a VH domain and a VL domain, the VH domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15, and (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8, 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, wherein the VH domain comprises amino acid residues E2, G26, V28, K30, R66, R83 and K94; and (b) a VL domain comprising the amino acid sequence of SEQ ID NO: 1 The amino acid sequence of NO:12 has an amino acid sequence with at least 90% sequence identity, wherein the VL domain comprises amino acid residues I2, Y49, G57, E67, D68 and Q69, and wherein the numbering of the VH domain and the VL domain is according to the Kabat numbering system.

[0302] 18. An antibody that specifically binds to human VEGF and human IL-1β, wherein the antibody comprises a VH domain and a VL domain, the VH domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14, (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15, (d) a human heavy chain framework having (i) a FR1 comprising amino acid residues E2, G26, V28 and K30, (ii) a FR3 comprising amino acid residues R66, R83 and K94; the VL domain comprising (e) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, (f) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17, (g) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 18. NO:8, and (h) a human light chain framework having (i) FR1 comprising amino acid residue I2, (ii) FR2 comprising amino acid residue Y49, and (iii) FR3 comprising amino acid residues G57, E67, D68 and Q69, wherein the numbering of the VH domain and the VL domain is according to the Kabat numbering system, the antibody comprising (a) a VH domain comprising an amino acid sequence having at least 90% sequence identity 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:12.

[0303] 19. An antibody according to any of the preceding embodiments, comprising: (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 11 having up to 15 amino acid substitutions; and (b) a variable light chain domain comprising the amino acid sequence of SEQ ID NO: 12 having up to 15 amino acid substitutions.

[0304] 20. The antibody of any of the preceding embodiments, comprising (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 11 having up to 15 amino acid substitutions, wherein the amino acid substitutions are located at positions 3 to 25, 36 to 49, 97 to 82c, 84 to 93, or 103 to 113 of SEQ ID NO: 11; and (b) a variable light domain comprising the amino acid sequence of SEQ ID NO: 12 having up to 15 amino acid substitutions, wherein the amino acid substitutions are located at positions 1, 4, 6, 8 to 23, 35 to 48, 58 to 66, 70 to 88, or 98 to 107 of SEQ ID NO: 12, wherein the numbering of the VH domain and the VL domain is according to the Kabat numbering system.

[0305] 21. An antibody that specifically binds to human VEGF and to human IL-1β, wherein the antibody comprises a VH domain and a VL domain, the VH domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15, the VL domain comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8, the antibody comprising (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 11 having up to 15 amino acid substitutions; and (b) a variable light domain comprising the amino acid sequence of SEQ ID NO: 12 having up to 15 amino acid substitutions.

[0306] 22. An antibody that specifically binds to human VEGF and human IL-1β, wherein the antibody comprises a VH domain and a VL domain, the VH domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14, (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15, (d) a human heavy chain framework having (i) a FR1 comprising amino acid residues E2, G26, V28 and K30, (ii) a FR3 comprising amino acid residues R66, R83 and K94; the VL domain comprising (e) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, (f) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17, (g) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 18 NO:8, and (h) a human light chain framework having (i) FR1 comprising amino acid residue I2, (ii) FR2 comprising amino acid residue Y49, and (iii) FR3 comprising amino acid residues G57, E67, D68 and Q69, wherein the numbering of the VH domain and the VL domain is according to the Kabat numbering system, 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 chain domain comprising the amino acid sequence of SEQ ID NO:12 with up to 15 amino acid substitutions.

[0307] 23. The antibody according to any one of the preceding embodiments, comprising the VH sequence of SEQ ID NO:11 and the VL sequence of SEQ ID NO:12.

[0308] 24. An antibody that specifically binds to human VEGF and human IL-1β, comprising the VH sequence of SEQ ID NO: 11 and the VL sequence of SEQ ID NO: 12.

[0309] 25. The antibody according to any one of the preceding embodiments, comprising the heavy chain amino acid sequence of SEQ ID NO:20 and the light chain amino acid sequence of SEQ ID NO:19.

[0310] 26. An antibody that specifically binds to human VEGF and human IL-1β, comprising the heavy chain amino acid sequence of SEQ ID NO: 20 and the light chain amino acid sequence of SEQ ID NO: 19.

[0311] 27. The antibody according to any one of the preceding embodiments, comprising the heavy chain amino acid sequence of SEQ ID NO:18 and the light chain amino acid sequence of SEQ ID NO:19.

[0312] 28. An antibody that specifically binds to human VEGF and human IL-1β, comprising the heavy chain amino acid sequence of SEQ ID NO: 18 and the light chain amino acid sequence of SEQ ID NO: 19.

[0313] 29. The antibody according to any one of the preceding embodiments, wherein the antibody Fab fragment of the antibody binds to (i) human VEGF121, wherein the K measured by surface plasmon resonance is D less than 10 pM, and (ii) binds to human IL-1β with a K measured by surface plasmon resonance D Less than 30pM.

[0314] 30. An antibody that specifically binds to human VEGF and to human IL-1β, wherein an antibody Fab fragment of the antibody binds to (i) human VEGF121, wherein the K measured by surface plasmon resonance is D less than 10 pM, and (ii) binds to human IL-1β with a K measured by surface plasmon resonance D Less than 30pM.

[0315] 31. An antibody that specifically binds to human VEGF and human IL-1β, wherein the antibody comprises a VH domain and a VL domain, the VH domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15, the VL domain comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8, wherein

[0316] The antibody Fab fragment of the antibody binds to (i) human VEGF121, wherein the K measured by surface plasmon resonance D less than 10 pM, and (ii) binds to human IL-1β with a K measured by surface plasmon resonance D Less than 30pM.

[0317] 32. An antibody that specifically binds to human VEGF and to human IL-1β, wherein the antibody comprises a VH domain and a VL domain, the VH domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14, (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15, (d) a human heavy chain framework having (i) a FR1 comprising amino acid residues E2, G26, V28 and K30, (ii) a FR3 comprising amino acid residues R66, R83 and K94; the VL domain comprising (e) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, (f) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 17

[0318] L2, (g) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:8, and (h) a human light chain framework having (i) a FR1 comprising amino acid residue I2, (ii) a FR2 comprising amino acid residue Y49, and (iii) a FR3 comprising amino acid residues G57, E67, D68, and Q69, wherein the numbering of the VH domain and the VL domain is according to the Kabat numbering system, wherein the antibody Fab fragment of the antibody binds to (i) human VEGF121, wherein the K measured by surface plasmon resonance D less than 10 pM, and (ii) binds to human IL-1β with a K measured by surface plasmon resonance D Less than 30pM.

[0319] 33. An antibody that specifically binds to human VEGF and human IL-1β, comprising in a pair of VH and VL domains: (i) a VH domain comprising amino acid residues E2, G26, V28, K30, W31, N35b, D35c, K52a, D55, H56, Y58, T61, K62, F63, I64, R66, R83, K94, D95, V96, F98 and D101, (ii) a VL domain comprising amino acid residues comprising amino acid residues I2, Y27, W27a, S27c, S27d, L32, Y49, D50, Y53, K54, L56, G57, E67, D68, Q69, Y91, R92, Y93, H94 and Y96, wherein the numbering of the VH domain and the VL domain is according to the Kabat numbering system, wherein the antibody Fab fragment of the antibody binds to (i) human VEGF121, wherein the K measured by surface plasmon resonance D less than 10 pM, and (ii) binds to human IL-1β with a K measured by surface plasmon resonance D Less than 30pM.

[0320] 34. An antibody that specifically binds to human VEGF and to human IL-1β, wherein the antibody comprises a VH domain and a VL domain, the VH domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15, the VL domain comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8, 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: 12. NO:12 has an amino acid sequence with at least 90% sequence identity; wherein the antibody Fab fragment of the antibody binds to (i) human VEGF121, wherein the K measured by surface plasmon resonance D less than 10 pM, and (ii) binds to human IL-1β with a K measured by surface plasmon resonance D Less than 30pM.

[0321] 35. The antibody of any preceding embodiment, wherein an antibody Fab fragment of said antibody exhibits an aggregation onset temperature greater than 70°C.

[0322] 36. An antibody that specifically binds to human VEGF and to human IL-1β, wherein an antibody Fab fragment of the antibody exhibits an aggregation onset temperature greater than 70°C.

[0323] 37. An antibody that specifically binds to human VEGF and human IL-1β, wherein the antibody comprises a VH domain and a VL domain, the VH domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15, the VL domain comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8, wherein

[0324] The antibody Fab fragment of the antibody exhibits an aggregation onset temperature greater than 70°C.

[0325] 38. An antibody that specifically binds to human VEGF and to human IL-1β, wherein the antibody comprises a VH domain and a VL domain, the VH domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14, (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15, (d) a human heavy chain framework having (i) a FR1 comprising amino acid residues E2, G26, V28 and K30, (ii) a FR3 comprising amino acid residues R66, R83 and K94; the VL domain comprising (e) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, (f) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 17

[0326] L2, (g) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:8, and (h) a human light chain framework having (i) a FR1 comprising amino acid residue I2, (ii) a FR2 comprising amino acid residue Y49, and (iii) a FR3 comprising amino acid residues G57, E67, D68 and Q69, wherein the numbering of the VH domain and the VL domain is according to the Kabat numbering system, wherein the antibody Fab fragment of the antibody exhibits an aggregation onset temperature greater than 70°C.

[0327] 39. An antibody that specifically binds to human VEGF and human IL-1β, comprising in a pair of VH domains and VL domains: (i) a VH domain comprising amino acid residues E2, G26, V28, K30, W31, N35b, D35c, K52a, D55, H56, Y58, T61, K62, F63, I64, R66, R83, K94, D95, V96, F98 and D101, (ii) A VL domain comprising amino acid residues I2, Y27, W27a, S27c, S27d, L32, Y49, D50, Y53, K54, L56, G57, E67, D68, Q69, Y91, R92, Y93, H94 and Y96, wherein the numbering of the VH domain and the VL domain is according to the Kabat numbering system, wherein the antibody Fab fragment of the antibody exhibits an aggregation onset temperature greater than 70°C.

[0328] 40. An antibody that specifically binds to human VEGF and to human IL-1β, wherein the antibody comprises a VH domain and a VL domain, the VH domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15, the VL domain comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8, 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: 12. The amino acid sequence of NO:12 has an amino acid sequence with at least 90% sequence identity; wherein the antibody Fab fragment of the antibody exhibits an aggregation onset temperature greater than 70°C.

[0329] 41. The antibody of any preceding embodiment, wherein an antibody Fab fragment of said antibody exhibits a melting temperature greater than 80°C as measured by dynamic light scattering.

[0330] 42. An antibody that specifically binds to human VEGF and to human IL-1 β, wherein an antibody Fab fragment of said antibody exhibits a melting temperature greater than 80°C as measured by dynamic light scattering.

[0331] 43. An antibody that specifically binds to human VEGF and to human IL-1β, wherein the antibody comprises a VH domain and a VL domain, the VH domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15, the VL domain comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8, wherein the antibody Fab fragment of the antibody exhibits a melting temperature greater than 80°C as measured by dynamic light scattering.

[0332] 44. An antibody that specifically binds to human VEGF and to human IL-1β, wherein the antibody comprises a VH domain and a VL domain, the VH domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14, (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15, (d) a human heavy chain framework having (i) a FR1 comprising amino acid residues E2, G26, V28 and K30, (ii) a FR3 comprising amino acid residues R66, R83 and K94; the VL domain comprising (e) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, (f) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 17

[0333] L2, (g) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:8, and (h) a human light chain framework having (i) FR1 comprising amino acid residue I2, (ii) FR2 comprising amino acid residue Y49, (iii) FR3 comprising amino acid residues G57, E67, D68 and Q69, wherein the numbering of the VH domain and the VL domain is according to the Kabat numbering system, wherein the antibody Fab fragment of the antibody exhibits a melting temperature greater than 80°C as measured by dynamic light scattering.

[0334] 45. An antibody that specifically binds to human VEGF and human IL-1β, comprising in a pair of VH domains and VL domains: (i) a VH domain comprising amino acid residues E2, G26, V28, K30, W31, N35b, D35c, K52a, D55, H56, Y58, T61, K62, F63, I64, R66, R83, K94, D95, V96, F98 and D101, (ii) a VL domain A domain comprising amino acid residues I2, Y27, W27a, S27c, S27d, L32, Y49, D50, Y53, K54, L56, G57, E67, D68, Q69, Y91, R92, Y93, H94 and Y96, wherein the numbering of the VH domain and the VL domain is according to the Kabat numbering system, wherein the antibody Fab fragment of the antibody exhibits a melting temperature greater than 80°C as measured by dynamic light scattering.

[0335] 46. ​​An antibody that specifically binds to human VEGF and to human IL-1β, wherein the antibody comprises a VH domain and a VL domain, the VH domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15, the VL domain comprising: (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8, 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: 12. The amino acid sequence of NO:12 has an amino acid sequence with at least 90% sequence identity; wherein the antibody Fab fragment of the antibody exhibits a melting temperature greater than 80°C as measured by dynamic light scattering.

[0336] 47. An antibody according to any of the preceding embodiments, wherein the binding of the antibody Fab fragment of the antibody to human VEGF inhibits the binding of VEGF to VEGFR2, wherein the IC50 measured by surface plasmon resonance is less than 50nM; and wherein the binding of the antibody Fab fragment of the antibody to human IL-1β inhibits the binding of IL-1β to IL-1βR1, wherein the IC50 measured by surface plasmon resonance is less than 30nM.

[0337] 48. An antibody that specifically binds to human VEGF and to human IL-1β, wherein the binding of the antibody Fab fragment of the antibody to human VEGF inhibits the binding of VEGF to VEGFR2, wherein the IC50 measured by surface plasmon resonance is less than 50nM; and wherein the binding of the antibody Fab fragment of the antibody to human IL-1β inhibits the binding of IL-1β to IL-1βR1, wherein the IC50 measured by surface plasmon resonance is less than 30nM.

[0338] 49. An antibody that specifically binds to human VEGF and to human IL-1β, wherein the antibody comprises a VH domain and a VL domain, the VH domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15, the VL domain comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8, and wherein binding of an antibody Fab fragment of the antibody to human IL-1β inhibits binding of IL-1β to IL-1βR1, wherein the IC50 measured by surface plasmon resonance is less than 30 nM.

[0339] 50. An antibody that specifically binds to human VEGF and to human IL-1β, wherein the antibody comprises a VH domain and a VL domain, the VH domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14, (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15, (d) a human heavy chain framework having (i) a FR1 comprising amino acid residues E2, G26, V28 and K30, (ii) a FR3 comprising amino acid residues R66, R83 and K94; the VL domain comprising (e) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, (f) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17, (g) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 18 NO:8, and (h) a human light chain framework having (i) FR1 comprising amino acid residue I2, (ii) FR2 comprising amino acid residue Y49, and (iii) FR3 comprising amino acid residues G57, E67, D68 and Q69, wherein the numbering of the VH domain and the VL domain is according to the Kabat numbering system, and wherein the binding of the antibody Fab fragment of the antibody to human IL-1β inhibits the binding of IL-1β to IL-1βR1, wherein the IC50 measured by surface plasmon resonance is less than 30nM.

[0340] 51. An antibody that specifically binds to human VEGF and human IL-1β, comprising in a pair of VH and VL domains: (i) a VH domain comprising amino acid residues E2, G26, V28, K30, W31, N35b, D35c, K52a, D55, H56, Y58, T61, K62, F63, I64, R66, R83, K94, D95, V96, F98 and D101, (ii) a VL domain comprising amino acid residues I2, Y27 , W27a, S27c, S27d, L32, Y49, D50, Y53, K54, L56, G57, E67, D68, Q69, Y91, R92, Y93, H94 and Y96, wherein the numbering of the VH domain and the VL domain is according to the Kabat numbering system; and wherein the binding of the antibody Fab fragment of the antibody to human IL-1β inhibits the binding of IL-1β to IL-1βR1, wherein the IC50 measured by surface plasmon resonance is less than 30nM.

[0341] 52. An antibody that specifically binds to human VEGF and to human IL-1β, wherein the antibody comprises a VH domain and a VL domain, the VH domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15, the VL domain comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8, 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: 12. The amino acid sequence of NO:12 has an amino acid sequence with at least 90% sequence identity; and wherein the binding of the antibody Fab fragment of the antibody to human IL-1β inhibits the binding of IL-1β to IL-1βR1, wherein the IC50 measured by surface plasmon resonance is less than 30nM.

[0342] 53. The antibody of any preceding embodiment, which is a monoclonal antibody.

[0343] 54. The antibody of any preceding embodiment, which is an antibody fragment that binds to human VEGF and human IL-1 β.

[0344] 55. The antibody of any one of the preceding embodiments, wherein the antibody is bispecific.

[0345] 56. The antibody of any preceding embodiment, wherein the antibody is a Fab fragment.

[0346] 57. The antibody of any one of the preceding embodiments, wherein the antibody is a bispecific antibody fragment.

[0347] 58. The antibody of any one of the preceding embodiments, wherein the antibody is a multispecific antibody.

[0348] 59. An isolated nucleic acid encoding the antibody of any one of embodiments 1 to 58.

[0349] 60. A host cell comprising the nucleic acid of embodiment 59.

[0350] 61. An expression vector comprising the nucleic acid of embodiment 61.

[0351] 62. A method for producing an antibody that binds to human VEGF and human IL-1β, comprising culturing the host cell according to embodiment 60, thereby producing the antibody.

[0352] 63. The method of embodiment 62, further comprising recovering the antibody from the host cell.

[0353] 64. An antibody produced by the method described in embodiment 62 or 63.

[0354] 65. A pharmaceutical formulation comprising the antibody of any one of embodiments 1 to 58 and a pharmaceutically acceptable carrier.

[0355] 66. The antibody according to any one of embodiments 1 to 58 for use as a medicament.

[0356] 67. The antibody of any one of embodiments 1 to 58 for use in treating a vascular disease.

[0357] 68. The antibody of any one of embodiments 1 to 58 for use in treating an ocular vascular disease.

[0358] 69. Use of the antibody of any one of embodiments 1 to 58 or the pharmaceutical composition of embodiment 65 in the preparation of a medicament.

[0359] 70. Use of a compound according to any one of embodiments 1 to 58 or a pharmaceutical composition according to embodiment 65 for the preparation of a medicament for inhibiting angiogenesis.

[0360] 71. A method of treating an individual suffering from a vascular disease, comprising administering to the individual an effective amount of the antibody of any one of embodiments 1 to 58 or the pharmaceutical composition of embodiment 65.

[0361] 72. A method of treating an individual suffering from an ocular vascular disease, comprising administering to the individual an effective amount of the antibody of any one of embodiments 1 to 58 or the pharmaceutical composition of embodiment 65.

[0362] 73. A method of inhibiting angiogenesis in an individual, comprising administering to the individual an effective amount of the antibody of any one of embodiments 1 to 58 or the pharmaceutical composition of embodiment 65 to inhibit angiogenesis.

[0363] Description of amino acid sequence

[0364]

[0365]

[0366]

[0367] Examples

[0368] The following examples are provided to aid the understanding of the present invention, the true scope of the invention being set forth in the appended claims. It should be understood that modifications may be made to the procedures set forth without departing from the spirit of the present invention.

[0369] Example 1:

[0370] Generation of bispecific anti-VEGF / anti-IL-1β Fab fragments

[0371] Bispecific anti-VEGF / anti-IL-1β Fab fragments are generated by independently screening monospecific antibodies with non-overlapping paratopes that bind to VEGF and IL-1β, and then merging the amino acid sequences into a biparatopic VH / VL pair that binds to VEGF and IL-1β as described previously, for example, in WO2012 / 163520.

[0372] Two different phage display libraries of synthetic Fab fragments were utilized, wherein in the first phage display library, the residues in the CDR-H1, CDR-H3 and CDR-L2 regions of the Fab fragments were diversified, and wherein in the second phage display library, the residues in the CDR-L1, CDR-L3 and CDR-H2 regions of the Fab fragments were diversified. In each library, the other three CDR regions remained unchanged as unchanged pseudosequences. In both libraries, the CH1 domain of the Fab fragments was fused to the truncated gene III protein via a linker to facilitate phage display.

[0373] The first library was enriched for binders to human IL-1β and the second library was enriched for binders to human VEGF-A by panning of phage libraries. After panning, plasmid minipreps were generated for the two 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 IL-1β binders or VEGF-A binders, respectively. These vector pools were transformed into TG1 E. coli cells and individual colonies were picked and cultured to solublely express individual Fab clones in microtiter plates. Supernatants containing soluble Fab fragments were screened for binding to IL-1β or VEGF-A using standard ELISA methods, and DNA plasmid preparation and sequencing were then performed on TG1 clones producing specific binders to obtain VH and VL sequence pairs that specifically bind to IL-1β or VEGF-A, respectively.

[0374] A pair of bispecific anti-VEGF / anti-IL-1β VH and VL sequences were designed in silico by (1) replacing unrelated VH residues 52b to 65 in the VH sequence of an IL-1β-specific Fab with VH residues 52b to 65 of a selected VEGF-A-specific Fab, thereby substituting CDR-H2 residues that are likely to be part of the VEGF-A-specific paratope into the IL-1β binder heavy chain, and (2) replacing unrelated VL residues 49 to 57 in the VL sequence of a VEGF-A-specific Fab with VL residues 49 to 57 of a selected IL-1β-specific Fab, thereby substituting CDR-L2 residues that are likely to be part of the IL-1β-specific paratope into the VEGF-A binder light chain.

[0375] Example 2:

[0376] Expression of the bispecific anti-VEGF / anti-IL-1β Fab fragment 1HVL2.3

[0377] The resulting designed bispecific anti-VEGF / anti-IL-1β VH and VL sequence pairs were synthesized and cloned into an E. coli expression vector in frame with the gene sequences encoding the CH1 and Cκ domains. The vector was transformed into TG1 E. coli cells, and single colonies were cultured for soluble expression of the bispecific antibody Fab fragment. The bispecific antibody was purified from the TG1 culture supernatant by affinity chromatography, and specific binding to both IL-1β and VEGF-A was confirmed.

[0378] The bispecific anti-VEGF / anti-IL-1β antibody "1HVL2.3" was selected, characterized in that the heavy chain is SEQ ID NO:9 and the light chain is SEQ ID NO:10.

[0379] For further analysis, the anti-VEGF / anti-IL-1β antibodies of the invention were transformed into and expressed from HEK293 cells by standard recombinant methods.

[0380] Example 3:

[0381] Characterization of the bispecific anti-VEGF / anti-IL-1β Fab fragment 1HVL2.3

[0382] The binding affinity, hydrophilicity and thermal stability of the bispecific antibody 1HVL2.3 were evaluated as follows:

[0383] VEGF binding kinetics assessed by surface plasmon resonance (SPR):

[0384] The anti-His capture antibody (GE Healthcare 28995056) was immobilized on an S-series sensor chip C1 (GE Healthcare 29104990) using standard amine coupling chemistry to obtain a surface density of approximately 500 resonance units (RU). HBS-P+ (10mM HEPES, 150mM NaCl pH 7.4, 0.05% surfactant P20) was used as running and dilution buffer. Human VEGF121-His was captured to the surface and the resulting ligand density was approximately 10RU and 20RU, respectively. A series of dilutions of the bispecific anti-VEGF / anti-IL-1βFab fragment (1.2-100nM, 1:3 dilution) were injected sequentially for 90s each, and the dissociation was monitored for 3600s at a flow rate of 30μl / min (single cycle kinetics). Surface regeneration was performed by injecting 10mM, pH 1.5 glycine for 60s. Large refractive index deviations were corrected by subtracting blank injections and by subtracting the responses obtained from a control flow cell without capturing human VEGF121. Curve fitting was performed using a 1:1 Langmuir binding model within the Biacore evaluation software. To provide a more robust fit, the "Multiple Rmax" option was selected to perform a global fit using both ligand densities simultaneously.

[0385] IL-1b binding kinetics assessed by surface plasmon resonance (SPR):

[0386] The anti-Fab capture antibody (GE Healthcare 28958325) was immobilized on an S-series sensor chip C1 (GE Healthcare 29104990) using standard amine coupling chemistry to obtain a surface density of approximately 500 resonance units (RU). The bispecific anti-VEGF / anti-IL-1β Fab fragment was captured to the surface to obtain a capture level of approximately 20 RU. Serial dilutions of human IL-1β (PeproTech 200-01B) or cynomolgus monkey IL-1β (SinoBiological90010-CNAE) ranging from 0.74nM to 60nM (1:3 dilution) were injected for 90s and dissociation was monitored for at least 600s at a flow rate of 30m rate / min. Surface regeneration was performed by two consecutive injections of 10mM, pH 2.1 glycine for 60s each. Large refractive index deviations were corrected by subtracting blank injections and by subtracting the response obtained from a control flow cell without captured bispecific anti-VEGF / anti-IL-1β Fab fragment. Curve fitting was performed using a 1:1 Langmuir binding model within the Biacore evaluation software.

[0387] Hydrophobic Interaction Chromatography (HIC):

[0388] The apparent hydrophobicity was determined by injecting 20 μg of the bispecific anti-VEGF / anti-IL-1β Fab fragment onto a HIC-Ether-5PW (Tosoh) column equilibrated with 25 mM sodium phosphate, 1.5 M ammonium sulfate, pH 7.0. Elution was performed using a linear gradient from 0 to 100% of buffer B (25 mM sodium phosphate, pH 7.0) over 60 minutes. Retention times were compared to protein standards with known hydrophobicity.

[0389] Thermal stability:

[0390] Samples of bispecific anti-VEGF / anti-IL-1βFab fragments were prepared at a concentration of 1 mg / mL in 20 mM histidine / histidine chloride, 140 mM NaCl, pH 6.0, transferred to 384-well optical plates by centrifugation through a 0.4 μm filter plate, and covered with paraffin oil. The hydrodynamic radius was measured repeatedly by dynamic light scattering on a DynaPro plate reader (Wyatt) while the samples were heated from 25°C to 80°C at a rate of 0.05°C / min. Alternatively, the samples were transferred to a 10 μL microcuvette array and static light scattering data and fluorescence data after 266 nm laser excitation were recorded with an Optim1000 instrument (Avacta Inc.) while they were heated from 25°C to 90°C at a rate of 0.1°C / min.

[0391] The aggregation onset temperature is defined as the temperature at which the hydrodynamic radius (DLS) or the temperature at which the scattered light intensity (Optim 1000) begins to increase. The melting temperature is defined as the inflection point in a graph of fluorescence intensity versus wavelength.

[0392] The results are shown in Tables 1 and 2.

[0393] Table 1: VEGF and IL-1β binding kinetics of 1HVL2.3 assessed by SPR

[0394]

[0395] Table 2: Thermal stability and hydrophobicity of 1HVL2.3

[0396] Tagg(℃) Tm(℃)DLS HIC (relative retention time) 75(+ / -1) 87(+ / -1) 0.58

[0397] Example 4:

[0398] Improvement of the bispecific anti-VEGF / anti-IL-1β Fab fragment 1HVL2.3

[0399] As described above, the bispecific anti-VEGF / anti-IL-1β Fab fragment 1HVL2.3, although highly stable, exhibits affinity for IL-1β in the nanomolar range and significant hydrophobicity. For the treatment of ocular vascular diseases that require injection of therapeutic agents into the eye, it is desirable to provide therapeutic agents with high affinity for the target antigen and very high concentrations to increase the durability of the therapeutic effect and minimize the inconvenience of treatment. Therefore, for the intended purpose, it is necessary to increase the affinity of the antibody and reduce the hydrophobicity to ensure solubility in high concentration isotonic buffers.

[0400] Therefore, for clinical application, antibodies need further improvement, for example, with respect to IL-1β binding (particularly by improving the dissociation rate) and reducing hydrophobicity. 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 1HVL2.3 were screened and selected based on their desired properties in terms of yield, affinity, simultaneous antigen binding, hydrophilicity, stability, viscosity and other parameters.

[0401] Improved candidate antibodies 1HVL5.15, 1HVL12.85 and RO7200394 were selected from a plurality of tested candidate antibody molecules. The amino acid sequences of those improved bispecific anti-VEGF / anti-IL-1β Fab fragments are identified in Table 3.

[0402] Table 3: Amino acid sequences of bispecific anti-VEGF / anti-IL-1β Fab fragments 1HVL2.3, 1HVL5.15, 1HVL12.85 and RO7200394 (numbers refer to SEQ ID NOs used herein)

[0403]

[0404]

[0405] Figure 2 and Figure 3 The alignment of the variable heavy chain domain and variable light chain domain of the generated bispecific anti-VEGF / anti-IL-1β Fab fragment is shown. The amino acid position numbering within the VH and VL domains is based on the Kabat numbering system. For simplicity, the numbering is included in the figure to further illustrate the position of the framework and CDR amino acids.

[0406] Example 5:

[0407] Improved antigen binding kinetics of bispecific anti-VEGF / anti-IL-1β Fab fragments

[0408] The binding kinetics of the candidate antibodies to VEGF and IL-1β were evaluated using the indicated bispecific anti-VEGF / anti-IL-1β Fab fragments (amino acid sequences are shown in Table 3) as described in Example 3. For comparison, the antigen binding kinetics of the prior art anti-VEGF / anti-IL-1β antibody 0032 (full-length IgG antibody) as disclosed in WO2016 / 075034 were described.

[0409] The results of IL-1β binding kinetics are shown in Tables 4 and 5.

[0410] Table 4: Human IL-1β binding kinetics of bispecific anti-VEGF / anti-IL-1β antibodies assessed by SPR (data from prior art antibody 0032 of WO2016 / 075034)

[0411]

[0412] *n=4

[0413] Table 5: Cynomolgus monkey IL-1β binding kinetics of bispecific anti-VEGF / anti-IL-1β antibodies assessed by SPR

[0414]

[0415] The binding kinetics of antibodies 1HVL5.15 and 1HVL12.85 to IL-1β of other species and related proteins were evaluated by SPR using the same experimental setup as described in Example 3. No binding was observed to rat IL-1β, porcine IL-1β, human IL-1α, and human IL-1RA. Weak binding was observed to mouse and rabbit IL-1β.

[0416] The results of VEGF binding kinetics are shown in Table 6.

[0417] Table 6: Human VEGF121 binding kinetics of bispecific anti-VEGF / anti-IL-1β antibodies assessed by SPR (data from prior art antibody 0032 of WO2016 / 075034)

[0418]

[0419] Simultaneous antigen binding of candidate antibodies to VEGF and IL-1β was evaluated as follows:

[0420] The anti-His capture antibody (GE Healthcare 28995056) was immobilized on an S-series sensor chip C1 (GE Healthcare 29104990) using standard amine coupling chemistry to obtain a surface density of approximately 500 resonance units (RU). HBS-P+ (10mM HEPES, 150mM NaCl pH 7.4, 0.05% surfactant P20) was used as a running and dilution buffer. Human VEGF121-His was captured on the surface, followed by continuous injection of candidate antibodies and IL-1β. Surface regeneration was performed by injecting 10mM, pH 1.5 glycine for 60s. Large refractive index deviations were corrected by subtracting blank injections and by subtracting the response obtained from a control flow cell without capturing human VEGF121.

[0421] For all improved bispecific anti-VEGF / anti-IL-1 β Fab fragments, ie, 1HVL5.15, 1HVL12.85 and RO7200394, simultaneous binding of the candidate antibodies to VEGF and IL-1 β was confirmed. Figure 4 Simultaneous binding of anti-VEGF / anti-IL-1β1 HVL12.85 is demonstrated. Figure 5 Simultaneous combination of anti-VEGF / anti-IL-1β RO7200394 is demonstrated.

[0422] The simultaneous antigen binding of the full-length IgG prior art antibody 0032 (WO2016 / 075034) was also evaluated using the same experimental setup. Figure 6 Shown in.

[0423] Example 6:

[0424] Inhibits the binding of VEGF and IL-1β to their respective receptors

[0425] Receptor inhibition assay

[0426] The inhibition of the binding of VEGF and IL-1β to their respective receptors hVEGFR2 and IL-1βR1 in the presence of candidate antibody RO7200394 (Fab fragment) was evaluated as described below. For comparison, the kinetics of the prior art anti-VEGF / anti-IL-1β antibody 0032 (full length IgG) disclosed in WO2016 / 075034 was also evaluated.

[0427] hVEGFR2 (R&D Systems 357-KD) and IL-1bR1 (SinoBiological Inc. 10126-H02H) were immobilized on different flow cells of S-series sensor chip CM5 (GE Healthcare 29104988) using standard amine coupling chemistry, resulting in surface densities of approximately 8000 and 20000 resonance units (RU), respectively. HBS-P+ (10 mM HEPES, 150 mM NaCl pH 7.4, 0.05% surfactant P20) was used as running and dilution buffer.

[0428] To evaluate VEGF receptor binding inhibition, RO7200394 at a final concentration of 200 nM or 400 nM Antibody 0032 was pre-incubated with 50 nM VEGF121. To evaluate IL-1β receptor binding inhibition, RO7200394 at a final concentration of 200 nM or 200 nM Antibody 0032 was pre-incubated with 50 nM IL-1β. Samples were diluted (1:2) in the corresponding 50 nM VEGF121 or IL-1β solution.

[0429] Antibody / ligand mixture is injected on VEGFR2 or IL-1R1 surface with the flow velocity of 5ml / min for 60s.After dissociation stage 60s, regenerate VEGFR2 surface by injecting 5mM NaOH 60s, and regenerate IL-1R1 surface by injecting 10mM glycine pH3.0, 5mM NaOH continues 60s respectively subsequently.By deducting blank injection and correcting large refractive index deviation by deducting the response obtained from blank control flow cell.In order to assess, take in conjunction with response after injection ends in 5 seconds.The derivatization response in RU is converted into the conjunction with response relative to the initial signal corresponding to the part without antibody.4 parameter logistic model (XLfit, ID Business Solutions Ltd.) is used to calculate IC50 value.

[0430] The results are shown in Tables 7 and 8; Fig. 7A (VEGFR2 inhibition in the presence of antibody RO7200394), Figure 7B (VEGFR2 inhibition in the presence of antibody 0032), Fig. 8A (IL-1R1 inhibition in the presence of antibody RO7200394) and Figure 8B (IL-1R1 inhibition in the presence of antibody 0032).

[0431] Table 7: Inhibition of VEGR2 binding by bispecific anti-VEGF / anti-IL-1β antibodies in the absence (-IL-1β) or presence of IL-1β (+IL-1β)

[0432]

[0433] Table 8: Inhibition of IL-1βR1 binding by bispecific anti-VEGF / anti-IL-1β antibodies in the absence (-VEGF) or presence of VEGF (+VEGF).

[0434]

[0435] It has been demonstrated that the binding of IL-1β to the bispecific anti-VEGF / anti-IL-1β Fab fragments of the invention does not interfere with the inhibition of the VEGF / VEGFR2 interaction. Furthermore, the binding of VEGF to the bispecific anti-VEGF / anti-IL-1β Fab fragments of the invention does not interfere with the inhibition of the IL-1β / IL-1βR1 interaction.

[0436] VEGF competition ELISA

[0437] The following buffers were used: PBS (1x PBS pH 7.4); PBST (1x PBS supplemented with 0.1% v / v Tween-20); PBST 1% BSA (PBST supplemented with 1% BSA (30% bovine serum albumin solution, obtained from Sigma-Aldrich, A0336)); NaHCO3 (NaHCO3 solution, pH 9.4, prepared from BupH Buffer Packs (ThermoScientific, 28382)); 2% MPBST (PBST supplemented with 2% (w / v) skim milk powder (Carl Roth, T145.3)).

[0438] 96-well plates (Maxisorp Nunc-Immoplates) were coated with 50 μL / well of rhVEGFR-1-Fc (R&D #321-FL-050) at a final concentration of 1 μg / ml in 200 mM NaHCO 3 , pH 9.4 at room temperature for 1 hour.

[0439] At the same time, different concentrations of antibody Fab fragments were pre-incubated with VEGF to form an antibody Fab-VEGF premix as shown below: A dilution series of antibody Fab fragments was prepared by adding 280 μl of antibody Fab fragment solution (409.6 nM antibody in PBST-1% BSA) to the first column of wells of a round-bottom 96-well PS plate. Each well in columns 2 to 12 of the same plate was filled with 140 μl PBST-1% BSA. Subsequently, a 1:2 dilution was performed by transferring 140 μl of the antibody solution to the next column, mixing thoroughly, and then transferring 140 μl of the diluted antibody solution to the next column. This operation was repeated until the 11th column. The excess 140 μl was discarded so that all wells contained 140 μl. The 12th column was used as a control (blank). For pre-incubation with VEGF, 50 μl / well of 2 nM VEGF121 (Humanzyme, HZ-1206, Lot0614-01) or 2 nM VEGF165 (Humanzyme, HZ-1153, Lot 0716-01) in PBST-1% BSA was pre-filled in a round-bottom 96-well PS plate. 50 μl of the dilution series of the respective antibody Fab fragments were added to VEGF121 or VEGF165, respectively; mixed thoroughly and incubated at room temperature for 1.5 hours.

[0440] The rhVEGFR-1-Fc coated plate was washed twice with PBST and blocked with 200 μl of 2% MPBST for 45 min. After rinsing the MPBST solution twice with PBST, 50 μl of antibody Fab-VEGF premix was added to the plate and incubated at room temperature for 1.5 hours. Subsequently, the plate was washed twice with PBST. Then, 50 μl of a solution containing a biotinylated anti-VEGF antibody (R&D, BAF293; diluted 1:2000 in PBST) and SA-HRP (KPL, 14-30-00; diluted 1:2000 in PBST) was added and incubated at room temperature for 30 min. After washing 6 times with PBST, 50 μl of TMB substrate solution (two-component HRP substrate (KPL, 34021); used at room temperature) was added and incubated at room temperature for 30 min. 50 μl of 1N H2SO4 was added and the absorbance was read at 450 nm.

[0441] The results are shown in Fig. 9 and Fig.10 and demonstrates improved inhibition of VEGF-R1 binding by 1HVL12.85 and RO7200394 compared to 1HVL2.3.

[0442] Example 7:

[0443] Improved biophysical properties (stability and hydrophobicity) of bispecific anti-VEGF / anti-IL-1β Fab fragments

[0444] As described in Example 3, the indicated biophysical properties of the candidate antibodies were evaluated using the indicated bispecific anti-VEGF / anti-IL-1β Fab fragments (amino acid sequences are shown in Table 3).

[0445] Table 9 illustrates the thermal stability and hydrophobicity of the analyzed antibodies. For comparison, the thermal stability of the prior art anti-VEGF / anti-IL-1β antibody 0032 (full-length IgG antibody) as disclosed in WO2016 / 075034 is included. The HIC chromatogram of the bispecific anti-VEGF / anti-IL-1beta Fab fragment is shown in Fig.11 As shown, the HIC chromatogram of the prior art anti-VEGF / anti-IL-1βIgG antibody 0032 is as shown in Fig.12 shown.

[0446] Table 9: Thermal stability and hydrophobicity of bispecific anti-VEGF / anti-IL-1β antibodies (data from prior art antibody 0032 of WO2016 / 075034)

[0447] Antibody Tagg(℃) Tm(℃)DLS HIC (relative retention time) 0032 IgG 55 62.5 0.81 1HVL2.3 Fab 75(+ / -1) 87(+ / -1) 0.58 1HVL12.85 Fab 73(+ / -1) 85(+ / -1) 0.03 RO7200394 Fab 73(+ / -1) 85(+ / -1) 0.04

[0448] Example 8:

[0449] Improved biophysical properties (dynamic viscosity) of bispecific anti-VEGF / anti-IL-1β Fab fragments

[0450] The viscosity of the candidate antibodies was assessed using the bispecific anti-VEGF / anti-IL-1β Fab fragments shown (amino acid sequences are shown in Table 3) as follows:

[0451] Viscosity was measured by latex bead DLS method as previously described (He F et al.; Anal Biochem. 2010 Apr 1; ​​399(1): 141-3). Briefly, samples were concentrated to >200 mg / mL (based on material availability) using a centrifugal concentration device, such as Amicon Ultra-0.5 mL centrifugal filter, Ultracel-10K, Cat.-No. UFC501096.

[0452] A dilution series from about 10 mg / mL to the maximum feasible concentration was prepared, and polysorbate 20 and beads (nanoscale size standard, Nom diameter: 300 nm, 1% solid content, ThermoFisher Cat-No. 3300A) were added to final concentrations of 0.02% (PS20) and 0.03% (w / w, beads), respectively.

[0453] Small aliquots of these samples were centrifuged at maximum speed for 1 minute and then the protein concentration was determined by UV280 absorbance.

[0454] The remaining sample was transferred to a 384-well optical plate, covered with a layer of paraffin oil to prevent evaporation, and DLS data were recorded at the indicated temperatures. From the DLS data of the apparent hydrodynamic radius of the latex beads, the viscosity of the solution was calculated as described by He F et al. above. The viscosity measured at the highest concentration is reported in Table 10. The results are also shown in Fig.13 (1HVL12.85) and Fig.14 (RO7200394).

[0455] Table 10: Viscosity of bispecific anti-VEGF / anti-IL-1β antibodies at 15°C

[0456] Antibody Viscosity 1HVL12.85 Fab 18.4cP@195mg / ml RO7200394 Fab 14.6cP@210mg / ml

[0457] The results show that the antibodies of the invention can be formulated at high concentrations comprising viscosities below the acceptable viscosity limit for injectability, which is as high as 30 cP. While both tested antibodies were shown to be highly concentrable, the effect was more prominent in the RO7200394 antibody.

[0458] As a result, the antibodies of the invention are very suitable for intraocular application, as they allow high molar doses to be provided in a limited injection volume, which, when combined with high potency, results in high durability and thus reduced dosing frequency, which is desirable to reduce difficulties on the part of patients.

[0459] Example 9:

[0460] Improved chemical stability of bispecific anti-VEGF / anti-IL-1β Fab fragments

[0461] Chemical degradation testing:

[0462] The antibody samples were prepared in 20 mM His / HisCl, 140 mM NaCl, pH 6.0 and divided into three aliquots: one aliquot was rebuffered in PBS and two aliquots were kept in the original formulation. The PBS aliquot and one His / HisCl aliquot were incubated at 40°C (His / NaCl) or 37°C (PBS) at 1 mg / ml for 2 weeks (2w), and the PBS sample was incubated for 4 weeks (4w). The third control aliquot sample was stored at -80°C. After the incubation, the samples were analyzed for relative activity concentration (Biacore; the activity concentration of the two pressurized aliquots of each conjugate was normalized to the unpressurized 4°C aliquot), aggregation (SEC) and fragmentation (capillary electrophoresis or SDS-PAGE) and compared with untreated controls.

[0463] Binding activity after stress was assessed as follows:

[0464] The anti-Fab capture antibody (GE Healthcare 28958325) was immobilized on an S-series sensor chip CM5 (GE Healthcare 29104988) using standard amine coupling chemistry to obtain a surface density of 4000–6000 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. The antibody anti-VEGF / anti-IL-1β antibody 1HVL12.85 was injected at a concentration of 2 μg / ml for 60 s at a flow rate of 5 μg / min. HuVEGF121 (prepared in-house) or huIL-1β (Peprotech 200-01B) was injected at a concentration of 2 μg / ml for 60 s, and dissociation was monitored at a flow rate of 5 μl / min for 60 s. Surface regeneration was performed by two consecutive injections of 10 mM, pH 2.1 glycine for 60 s each. Large refractive index deviations were corrected by subtracting blank injections and by subtracting the response obtained from a blank control flow cell. For evaluation, binding responses were taken 5 seconds after the end of the injection. To normalize the binding signal, the VEGF or IL-1β binding response was divided by the anti-Fab response. Relative activity concentrations were calculated by comparing each temperature stressed sample to the corresponding stressed sample.

[0465] The results are shown in Tables 11 and 12.

[0466] Table 11: Binding activity of anti-VEGF / anti-IL-1β antibody 1HVL12.85 (amino acid sequence see Table 3) after pressurization

[0467]

[0468] Table 12: Molecular integrity of anti-VEGF / anti-IL-1β antibody 1HVL12.85 (amino acid sequence see Table 3) after compression

[0469]

[0470] Example 10:

[0471] Structural analysis of the improved bispecific anti-VEGF / anti-IL-1β Fab fragment 1HVL5.15

[0472] The structural analysis of the anti-VEGF / anti-IL-1β Fab fragment 1HVL5.15 was performed by X-ray crystallography as shown below:

[0473] Complex formation and crystallization of the ternary complex IL1β-VEGF121-Fab 1HVL5.15

[0474] To form the complex, the antibody 1HVL5.15 Fab fragment and human IL1β (Peprotech) were mixed at a molar ratio of 1:1.1. After incubation at 4°C for 16 hours overnight, human VEGF121 (prepared in-house) was added to obtain a ternary complex, which was concentrated to 10 mg / ml. Initial crystallization experiments were performed at 21°C in a sitting drop evaporation diffusion setup. The first microcrystals appeared from 1.4M sodium malonate within 4 days. Subsequent inoculation experiments produced crystals from 0.1M sodium cacodylate, pH 5.5, 0.1M calcium acetate, 12% PEG8000. Crystals can be harvested directly from the screening plate without any further optimization steps.

[0475] Data collection and structure determination

[0476] To collect the data, the crystals were rapidly cooled at 100 K in a precipitant solution with 15% ethylene glycol added as a cryoprotectant. Diffraction data were collected at a wavelength of 1.0000 Å on beamline X10SA at the Swiss Light Source (Willingen, Switzerland) using a PILATUS 6M detector. The data have been processed using XDS (Kabsch, W. Acta Cryst. D66, 133-144 (2010)) and scaled using SADABS (BRUKER). The crystals belong to the unit cell axis α=β=γ=90° space group C2221, and diffracts to Resolution. The structure was determined by molecular replacement with PHASER (McCoy, AJ et al. J. Appl. Cryst. 40, 658-674 (2007)), using the relevant internal structure of the Fab fragment, the coordination of the pdb entry 1MKK of IL1β and VEGF as a search model. Programs from the CCP4 suite (Collaborative Computational Project, Number 4 Acta Cryst. D50, 760-763 (1994)) and Buster (Bricogne, G., et al. (2011) Buster version 2.9.5 Cambridge, UK: Global Phasing Ltd) have been used to refine the structure. Proteins were manually reconstructed using differential electron density with COOT (Emsley, P. et al. Acta Cryst D66, 486-501 (2010)). Table 13 summarizes data collection and optimization statistics. All figures were prepared using PYMOL (DeLano Scientific, Palo Alto, CA, 2002). Structures were analyzed using the CONTACT program in the CCP4 suite (Collaborative Computational Project, Number 4 Acta Cryst. D50, 760-763 (1994)) using the maximum The contact distances between paratope and epitope residues are identified.

[0477] Table 13: Data collection and structure refinement statistics (X-ray crystallography)

[0478]

[0479]

[0480] 1 The values ​​in brackets refer to the highest resolution group.

[0481] 2 R merge =Σ|I- | / ΣI, where I is the intensity.

[0482] 3 R work =Σ|F o - <F c >| / ΣF o , where F o is the observed structure factor amplitude, F c is the calculated structure factor amplitude.

[0483] 4 R free It is calculated based on 5% of the total data omitted during the refinement process.

[0484] The amino acid residues that contact the respective antigens VEGF and IL-1β were identified from the crystal structure of the bispecific anti-VEGF / anti-IL-1β Fab fragment 1HVL5.15 in complex. The positions of the amino acid residues in the paratope within the VH and VL domains are illustrated as follows Figure 2 and Figure 3 shown.

[0485] 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, heavy chain CDR1 and heavy chain CDR3. The IL-1β paratope does not include amino acids from the light chain CDR2.

[0486] The amino acid residues identified as contributing to antigen binding are identified in Table 14 (for variable heavy chain domain amino acid residues) and Table 15 (for variable light chain domain amino acid residues). Amino acid positions are numbered according to the Kabat numbering system (in Figure 2 and 3 The same numbering is used in ). The amino acid positions involved in antigen binding are determined by their Kabat positions in the VH or VL domain (see also Figure 2 and 3 ).

[0487] Table 14: Variable heavy chain amino acid residues involved in antigen binding as identified by crystal structure analysis of the bispecific anti-VEGF / anti-IL-1 β antibody 1HVL5.15.

[0488] VH VEGF IL-1β FR1 - 2、26、28、30 H-CDR1 - 31, 35b, 35c FR2 - - H-CDR2 55、56、58、61、62、63、64 52a FR3 66、83 94 H-CDR3 - 95、96、98、101 FR4 - -

[0489] Table 15: Variable light chain amino acid residues involved in antigen binding as identified by crystal structure analysis of the bispecific anti-VEGF / anti-IL-1 β antibody 1HVL5.15.

[0490] V L VEGF IL-1β FR1 2 - L-CDR1 27, 27a, 27c, 27d 32 FR2 - 49 L-CDR2 - 50、53、54、56 FR3 67、68、69 57 L-CDR3 92、93、94、96 91 FR4 - -

Claims

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

2. An antibody according to one of the preceding claims, wherein the antibody comprises a VH domain and a VL domain, the VH domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15; and the VL domain comprising: (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:

8.

3. An antibody according to one of the preceding claims, wherein the antibody comprises a VH domain and a VL domain, the VH domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14, (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15, (d) a human heavy chain framework having (i) a FR1 comprising amino acid residues E2, G26, V28 and K30, (ii) a FR3 comprising amino acid residues R66, R83 and K94; and the VL domain comprising: (e) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, (f) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17, (g) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 18 NO:8, and (h) a human light chain framework having (i) a FR1 comprising amino acid residue I2, (ii) a FR2 comprising amino acid residue Y49, and (iii) a FR3 comprising amino acid residues G57, E67, D68 and Q69, wherein the numbering of the VH domain and the VL domain is according to the Kabat numbering system.

4. The antibody of one of the preceding claims, wherein the antibody comprises a VH domain comprising amino acid residues E2, G26, V28, K30, W31, N35b, D35c, K52a, D55, H56, Y58, T61, K62, F63, I64, R66, R83, K94, D95, V96, F98 and D101, and a VL domain comprising amino acid residues I2, Y27, W27a, S27c, S27d, L32, Y49, D50, Y53, K54, L56, G57, E67, D68, Q69, Y91, R92, Y93, H94 and Y96, wherein the numbering of the VH domain and the VL domain is according to the Kabat numbering system.

5. The antibody according to claim 4, comprising A VEGF paratope comprising the following amino acid residues in the VH domain: D55, H56, Y58, T61, K62, F63, I64, R66 and R83; and the following amino acid residues in the VL domain: I2, Y27, W27a, S27c, S27d, E67, D68, Q69, R92, Y93, H94 and Y96; and An IL-1β paratope comprising the following amino acid residues in the VH domain: E2, G26, V28, K30, W31, N35b, D35c, K52a, K94, D95, V96, F98 and D101; and the following amino acid residues in the VL domain: L32, Y49, D50, Y53, K54, L56, G57, Y91.

6. An antibody according to any of the preceding claims, 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:

12.

7. An antibody according to any one of the preceding claims, 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: 12 with up to 15 amino acid substitutions.

8. The antibody according to any one of the preceding claims, comprising the VH sequence of SEQ ID NO: 11 and the VL sequence of SEQ ID NO:

12.

9. An antibody that specifically binds to human VEGF and human IL-1β, comprising the VH sequence of SEQ ID NO: 11 and the VL sequence of SEQ ID NO:

12.

10. The antibody according to any one of the preceding claims, wherein the antibody Fab fragment of the antibody binds to: (i) human VEGF121, wherein the K measured by surface plasmon resonance is D less than 10 pM, and (ii) human IL-1β, where the K measured by surface plasmon resonance is D Less than 30pM.

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