Anti-VEGF antibodies and methods of use thereof

Abstract

The present invention relates to an anti-VEGF antibody and a preparation method and use thereof.

Description

Field of the Invention

[0001] The present invention relates to antibodies that inhibit angiogenesis, more particularly to anti-VEGF antibodies, and methods of use thereof. Background Art

[0002] The present invention provides anti-VEGF antibodies that preferentially inhibit the binding of VEGF to VEGF-R2 rather than to VEGF-R1. While inhibiting angiogenesis, such antibodies have improved safety due to their specific blocking properties.

[0003] Angiogenesis is the development of new vasculature from pre-existing blood vessels and / or circulating endothelial stem cells (Asahara, M., et al., Science, 275(5302):964-967, 1997; Springer et al., Mol. Cell, 2(5):549-558, 1998; Folkman and Shing, J. Biol. Chem., 267:10931-10934, 1992). Although angiogenesis plays an important role in many physiological processes, it has also been implicated in the pathogenesis of a variety of conditions, including solid tumors, intraocular neovascular syndromes such as proliferative retinopathy or age-related macular degeneration (AMD), rheumatoid arthritis, and psoriasis (Folkman, J., et al., J. Biol. Chem. 267 (1992) 10931-10934; Klagsbrun, M., et al., Annu. Rev. Physiol. 53 (1991) 217-239; and Garner, A., Vascular diseases, in: Pathobiology of ocular disease, A dynamic approach, Garner, A., and Klintworth, GK (eds.), 2nd ed., Marcel Dekker, New York (1994), pp. 1625-1710).

[0004] Angiogenesis in normal and malignant tissues is regulated by a balance of angiogenic stimulants and angiogenic inhibitors, which are produced in target tissues and at distant sites (Fidler et al., Cancer J. Sci. Am., 4 Suppl 1: S58-66, 1998; McNamara et al., Br. J Surg., 85(8): 1044-1055, 1998). Vascular endothelial growth factor-A (VEGF, also known as vascular permeability factor, VPF) is a major stimulator of angiogenesis. Human VEGF (SEQ ID No: 29) is described, for example, in Leung, DW, et al., Science 246 (1989). VEGF is involved in the regulation of normal and abnormal angiogenesis and neovascularization associated with tumors and intraocular disorders (Ferrara, N., et al., Endocr. Rev. 18 (1997) 4-25; Berkman, RA, et al., J. Clin. Invest. 91 (1993) 153-159; Brown, LF, et al., Human Pathol. 26 (1995) 86-91; Brown, LF, et al., Cancer Res. 53 (1993) 4727-4735; Mattern, J., et al., Brit. J. Cancer. 73 (1996) 931-934; and Dvorak, HF, et al., Am. J. Pathol. 146 (1995) 1029-1039). VEGF is a homodimeric glycoprotein that has been isolated from several sources. VEGF exhibits highly specific mitogenic activity on endothelial cells. VEGF has important regulatory functions in the formation of new blood vessels during embryonic angiogenesis and in angiogenesis during adult life (Carmeliet, P., et al., Nature, 380 (1996) 435-439; Ferrara, N., et al., Nature, 380 (1996) 439-442; reviewed in Ferrara, N., et al., Endocr. Rev. 18 (1997) 4-25).

[0005] The identification of VEGF as a key stimulus for angiogenesis in the pathogenesis of a variety of disorders has led to various attempts to block VEGF activity, for example, by anti-VEGF receptor antibodies, soluble receptor constructs, antisense strategies, RNA aptamers directed against VEGF, and low molecular weight VEGF receptor tyrosine kinase (RTK) inhibitors (Siemeister et al. Cancer Metastasis Rev., 17(2):241-248., 1998). Anti-VEGF antibodies have been described as inhibiting the growth of various human tumor cell lines in mice (Kim, KJ, et al., Nature 362 (1993) 841-844; Warren, SR, et al., J. Clin. Invest. 95 (1995) 1789-1797; Borgstrom, P., et al., Cancer Res. 56 (1996) 4032-4039; and Melnyk, O., et al., Cancer Res. 56 (1996) 921-924). WO 94 / 10202, WO 98 / 45332, WO 2005 / 00900 and WO 00 / 35956 relate to antibodies against VEGF. (sold) is an anti-VEGF antibody for tumor treatment in WO98 / 45331. ) is with bevacizumab A monoclonal antibody fragment derived from the same parent murine antibody. It is much smaller than the parent molecule and has been affinity matured to provide stronger binding to VEGF-A (WO98 / 45331). It is an anti-angiogenic compound approved for the treatment of the "wet" type of age-related macular degeneration (wAMD), a common form of age-related vision loss.

[0006] Anti-VEGF antibodies approved for clinical use, e.g. and Inhibits VEGF binding to two receptors, VEGF-R1 (FLT-1, fms-like tyrosine kinase) and VEGF-R2 (KDR / FLK-1, fetal liver kinase). VEGF-R1 and VEGF-R2 are closely related receptor tyrosine kinases (RTKs). Although VEGF-R2 is hypothesized to be primarily responsible for VEGF-mediated angiogenesis (Holash, J., et al., Proc Natl Acad Sci USA. 2002 Aug 20;99(17):11393-8), VEGF-R1 is known to have other important biological roles unrelated to angiogenesis, such as in osteoclast differentiation (Aldridge, SE, et al., Biochem Biophys Res Commun. 2005 Sep 30;335(3):793-8).

[0007] Some anti-VEGF antibodies have been reported that preferentially inhibit the binding of VEGF to VEGF-R2 but do not significantly inhibit the binding of VEGF to VEGF-R1 (WO200064946 describes an antibody called "2C3", WO2009060198 describes an antibody called "r84", WO2012089176 describes an antibody called "L3H6", and EP3006465 describes antibodies called "HF2-1", "HF2-5", "HF2-9" and "HF2-11"). By blocking VEGF binding to VEGF-R2, but not to VEGF-R1, the antibodies are described to have an improved safety profile and do not display common toxicity-related side effects associated with anti-VEGF therapy (Brekken, RA, et al., Cancer Res. 2000 Sep 15;60(18):5117-24; Sullivan, LA, et al., PLoS One, 2010 Aug 6;5(8):e12031).

[0008] Since the development of antibodies that offer such advantageous properties while simultaneously exhibiting the desired characteristics and sufficient affinity to make them suitable for clinical use is not a straightforward approach, there remains a need for improved VEGF inhibitors. SUMMARY OF THE INVENTION

[0010] The present invention provides anti-VEGF antibodies and methods of using the same.

[0011] One aspect of the invention is an antibody that binds to VEGF, wherein the binding of the antibody to VEGF significantly inhibits the binding of VEGF to the VEGF receptor VEGF-R2, but does not significantly inhibit the binding of VEGF to the VEGF receptor VEGF-R1.

[0012] Another aspect of the invention is an antibody that binds to VEGF, wherein the binding of the antibody to VEGF selectively inhibits the binding of VEGF to VEGF R2.

[0013] Another aspect of the invention is an antibody that binds to VEGF, wherein binding of the antibody to VEGF completely inhibits binding of VEGF to VEGF-R2, and wherein binding of the antibody to VEGF does not completely inhibit binding of VEGF to VEGF-R1.

[0014] Another aspect of the invention is an antibody that binds to VEGF, wherein the antibody binds to VEGF with an affinity of ≤150 pM as measured by surface plasmon resonance at a temperature of 25°C, and wherein the antibody binds to VEGF with a greater or about the same affinity as measured by surface plasmon resonance at a temperature of 37°C.

[0015] Another aspect of the invention is an antibody that binds to VEGF that binds to the same epitope as an antibody comprising the VH sequence of SEQ ID NO: 01 and the VL sequence of SEQ ID NO: 02.

[0016] Another aspect of the invention is an antibody that binds to VEGF, wherein the antibody comprises a heavy chain variable domain (VH) comprising

[0017] (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 03,

[0018] (B) CDR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 04, SEQ ID NO: 10, and SEQ ID NO: 12, and

[0019] (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 05; and

[0020] wherein the antibody comprises a light chain variable domain (VL) comprising

[0021] (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 06,

[0022] (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 07; and

[0023] (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 08.

[0024] In one embodiment, the antibody comprises a VH sequence of SEQ ID NO: 01 and a VL sequence of SEQ ID NO: 02. In one embodiment, the antibody comprises a VH sequence of SEQ ID NO: 09 and a VL sequence of SEQ ID NO: 02. In one embodiment, the antibody comprises a VH sequence of SEQ ID NO: 11 and a VL sequence of SEQ ID NO: 02. In one embodiment, the antibody comprises a VH sequence of SEQ ID NO: 33 and a VL sequence of SEQ ID NO: 02. In one embodiment, the antibody comprises a VH sequence of SEQ ID NO: 42 and a VL sequence of SEQ ID NO: 02. In one embodiment, the antibody comprises a VH sequence of SEQ ID NO: 44 and a VL sequence of SEQ ID NO: 02.

[0025] Another aspect of the invention is an antibody that specifically binds to VEGF, comprising the VH sequence of SEQ ID NO: 01 and the VL sequence of SEQ ID NO: 02. Another aspect of the invention is an antibody that is an affinity matured variant of the antibody having the VH sequence of SEQ ID NO: 01 and the VL sequence of SEQ ID NO: 02. Another aspect of the invention is an antibody that binds to VEGF that binds to the same epitope as the antibody having the VH sequence of SEQ ID NO: 01 and the VL sequence of SEQ ID NO: 02.

[0026] Another aspect of the present invention is an antibody that specifically binds to VEGF, comprising the VH sequence of SEQ ID NO: 09 and the VL sequence of SEQ ID NO: 02.

[0027] Another aspect of the present invention is an antibody that specifically binds to VEGF, comprising the VH sequence of SEQ ID NO: 11 and the VL sequence of SEQ ID NO: 02.

[0028] Another aspect of the present invention is an antibody that specifically binds to VEGF, comprising the VH sequence of SEQ ID NO: 33 and the VL sequence of SEQ ID NO: 02.

[0029] Another aspect of the present invention is an antibody that specifically binds to VEGF, comprising the VH sequence of SEQ ID NO: 42 and the VL sequence of SEQ ID NO: 02.

[0030] Another aspect of the present invention is an antibody that specifically binds to VEGF, comprising the VH sequence of SEQ ID NO: 44 and the VL sequence of SEQ ID NO: 02.

[0031] Another aspect of the invention is an isolated nucleic acid encoding an antibody of the invention.

[0032] Another aspect of the invention is a host cell comprising a nucleic acid of the invention.

[0033] Another aspect of the invention is a method for producing an antibody that binds to VEGF, comprising culturing the host cell of the invention under conditions suitable for expression of the antibody. Another aspect of the invention is an antibody produced by the method.

[0034] Another aspect of the present invention is a pharmaceutical composition comprising an antibody of the present invention and a pharmaceutically acceptable carrier.

[0035] Another aspect of the invention is an antibody of the invention or a pharmaceutical composition of the invention for use as a medicament.

[0036] Another aspect of the present invention is an antibody of the present invention or a pharmaceutical composition of the present invention for use in treating a VEGF-related disease, such as cancer or an eye disease.

[0037] Another aspect of the present invention is the use of the antibody of the present invention or the pharmaceutical composition of the present invention in the preparation of a medicament.

[0038] Another aspect of the present invention is use of the antibody of the present invention or the pharmaceutical composition of the present invention in the preparation of a medicament for inhibiting angiogenesis.

[0039] Another aspect of the invention is a method of treating an individual suffering from a VEGF-related disease, such as cancer or an eye disease, comprising administering to the individual an effective amount of an antibody of the invention or a pharmaceutical composition of the invention.

[0040] Another aspect of the present invention is a method of inhibiting angiogenesis in an individual, comprising administering to the individual an effective amount of an antibody of the present invention or a pharmaceutical composition of the present invention to inhibit angiogenesis.

[0041] The present invention provides novel anti-VEGF antibodies that exhibit particularly valuable properties, such as high affinity, high stability, and an improved safety profile, for example by avoiding side effects caused by blocking VEGF signaling through VEGF-R1. The antibodies provided herein exhibit high affinity, allowing for therapeutic applications of the antibody fragments. The antibodies of the present invention exhibit valuable properties that benefit patients suffering from VEGF-related diseases, such as cancer, vascular diseases, or eye diseases (e.g., age-related macular degeneration). BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 : Crystal structure of the VEGF dimer (purple) in complex with VEGF-R1 domain 2 (red) and with VEGF-R2 domains 2 and 3 (blue).

[0043] Figure 2 : Inhibition of VEGF binding to VEGF-R1 and VEGF-R2 in the presence of antibody Fab fragments as described in Example 2 (VEGF:VEGF-R2 / R1 inhibition ELISA).

[0044] Figure 3 : VEGF binding of anti-VEGF antibodies determined by ELISA as described in Example 4.

[0045] Figure 4A : Inhibition of VEGF binding to VEGF-R2 in the presence of the anti-VEGF antibodies VEGF-0089, VEGF-0113, VEGF-0114 and ranibizumab and L3H6 Fab of the present invention as described in Example 6 (0.4 nM VEGF).

[0046] Figure 4B : Inhibition of VEGF binding to VEGF-R2 in the presence of the anti-VEGF antibody VEGF-0089 of the present invention and prior art antibodies 2C3, r84 and L3H6 as described in Example 6, (0.7 nM VEGF).

[0047] Figure 5 : Inhibition of VEGF binding to VEGF-R1 in the presence of anti-VEGF antibodies as described in Example 6 (0.7 nM VEGF).

[0048] Figure 6 : Inhibition of VEGF binding to VEGF-R1 in the presence of anti-VEGF antibodies as described in Example 6 (0.34 nM VEGF).

[0049] Figure 7 : Inhibition of VEGF binding to VEGF-R2 in the presence of anti-VEGF antibodies as described in Example 6 (0.34 nM VEGF).

[0050] Figure 8 : Inhibition of VEGF binding to VEGF-R1 in the presence of anti-VEGF antibodies as described in Example 11 (0.34 nM VEGF).

[0051] Figure 9 : Inhibition of VEGF binding to VEGF-R1 in the presence of anti-VEGF antibodies as described in Example 11 (0.7 nM VEGF).

[0052] Figure 10 : Inhibition of VEGF binding to VEGF-R2 in the presence of anti-VEGF antibodies as described in Example 11 (0.34 nM VEGF).

[0053] Figure 11 : Inhibition of VEGF binding to VEGF-R2 in the presence of anti-VEGF antibodies as described in Example 11 (0.7 nM VEGF).

[0054] Figure 12 : Crystal structure of VEGF dimer (purple) in complex with anti-VEGF antibody VEGF-0089 as determined by X-ray crystallography according to Example 13.

[0055] Figure 13 : Epitope amino acids bound by the VEGF-0089 Fab fragment in the dimer of VEGF-A121 (SEQ ID NO: 45) as determined by X-ray crystallography according to Example 13. The amino acid positions contained in each VEGF-A121 molecule that contact the VEGF-0089 Fab fragment within a distance of 100 μm are highlighted in black. DETAILED DESCRIPTION

[0056] 1 .definition

[0057] The term "antibody" herein is used in the broadest sense to encompass various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0058] "Natural antibodies" refer to naturally occurring immunoglobulin molecules with variable structures. For example, natural IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, consisting of two identical light chains and two identical heavy chains bound by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable domain (VH), also referred to as a variable heavy domain or heavy chain variable region, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable domain (VL), also referred to as a variable light domain or light chain variable region, followed by a constant light (CL) domain.

[0059] 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.

[0060] An "isolated" antibody is one that has been separated from the components 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).

[0061] A "human antibody" is an antibody that possesses an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human or human cell, or derived from a non-human source using a human antibody repertoire or other human antibody encoding sequences. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues.

[0062] For the purposes herein, an "acceptor human framework" is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below.

[0063] "Framework" or "FR" refers to the variable domain residues excluding the hypervariable region (HVR) residues. The FR of a variable domain is typically composed of four FR domains: FR1, FR2, FR3, and FR4. The HVR and FR sequences in VH (or VL) typically appear in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0064] As used herein, the term "hypervariable region" or "HVR" refers to each of the regions of an antibody variable domain that are highly variable in sequence ("complementarity determining regions" or "CDRs") and / or form structurally defined loops ("hypervariable loops") and / or contain antigen contact residues ("antigen contacts"). Generally, antibodies comprise six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Exemplary HVRs herein include:

[0065] (a) Hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987));

[0066] (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991));

[0067] (c) antigens occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262:732-745 (1996)); and

[0068] (d) A combination of (a), (b) and / or (c), comprising HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3) and 94-102 (H3).

[0069] Unless otherwise indicated, HVR (eg, CDR) residues and other residues (eg, FR residues) in the variable domain are numbered herein according to Kabat et al. (supra).

[0070] The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these 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. Based on the amino acid sequence of its constant domain, the light chain of an antibody can be assigned to one of two types called kappa (κ) and lambda (λ).

[0071] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the 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. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system (also known as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0072] "Effector functions" refer to those biological activities attributed 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.

[0073] As used herein, unless otherwise indicated, the term "VEGF" 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 resulting from 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: 29.

[0074] The terms "anti-VEGF antibody" and "antibody that binds to VEGF" refer to an antibody that binds to VEGF with sufficient affinity to allow the antibody to be used as a diagnostic and / or therapeutic agent in 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 (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10-8 M or less, e.g., 10-8 M to 10-13 M, e.g., 10-9 M to 10-13 M). An antibody is said to "specifically bind" to VEGF when it has a Kd of 1 μM or less.

[0075] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by a dissociation constant (Kd). Affinity can be measured by conventional methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

[0076] For the purposes herein, an "acceptor human framework" is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below.

[0077] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007).) A single VH or VL domain may be sufficient to confer antigen-binding specificity. In addition, antibodies that bind to a specific antigen can be isolated using a VH or VL domain from an antibody that binds to that antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150: 880-887 (1993); Clarkson et al., Nature 352: 624-628 (1991).

[0078] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a group of substantially homogeneous antibodies, i.e., the individual antibodies constituting the group are identical and / or bind to the same epitope, except for possible variant antibodies (e.g., variant antibodies containing naturally occurring mutations or arising during the production of monoclonal antibody preparations, such variants typically existing in small amounts). Unlike polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on the antigen. Thus, the modifier "monoclonal" indicates the characteristic of an antibody obtained from a substantially homogeneous group of antibodies and should not be interpreted as requiring the antibody to be produced by any particular method. For example, the monoclonal antibodies according to the present invention can be prepared by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of a human immunoglobulin locus. Such methods and other exemplary methods for preparing monoclonal antibodies are described herein.

[0079] "Antibody fragments" refer to molecules other than intact antibodies that comprise a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0080] "Percent (%) amino acid sequence identity" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the candidate sequence with the reference polypeptide sequence and introducing gaps, if necessary, to achieve maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity for the purposes of the comparison. Alignments for determining percent amino acid sequence identity can be achieved in various ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software, or the FASTA package. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required to achieve maximum alignment over the full length of the compared sequences. However, for purposes herein, % amino acid sequence identity values ​​are generated using the ggsearch program in the FASTA package, version 36.3.8c or higher, and the BLOSUM50 comparison matrix. The FASTA program package was written by W. R. Pearson and DJ 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, and is publicly available at www.fasta.bioch.virginia.edu / fasta_www2 / fasta_down.shtml or www.ebi.ac.uk / Tools / sss / fasta. Alternatively, sequences can be compared using the public server accessible at fasta.bioch.virginia.edu / fasta_www2 / index.cgi using the ggsearch (global protein:protein) program and default options (BLOSUM50; open: -10; ext: -2; Ktup = 2) to ensure a global rather than a local alignment. The percent amino acid sequence identity is given in the output alignment header.

[0081] An "affinity matured" antibody is one with one or more alterations in one or more hypervariable regions (HVRs) which result in improvement in the affinity of the antibody for antigen, compared to a parent antibody which does not possess these alterations.

[0082] The term "epitope" refers to a site on a proteinaceous or non-proteinaceous antigen to which an anti-VEGF antibody binds. An epitope can be formed by a continuous stretch of amino acids (linear epitope) or comprise non-contiguous amino acids (conformational epitope), for example, brought into spatial proximity due to folding of the antigen (i.e., by tertiary folding of the protein antigen). Linear epitopes generally remain bound by anti-VEGF antibodies after exposure of the protein antigen to a denaturant, whereas conformational epitopes are generally 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 spatial conformation.

[0083] Screening for antibodies that bind to a specific epitope (i.e., those that bind to the same epitope) can be performed using routine methods in the art, such as, for example, 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).

[0084] Antigen structure-based antibody repertoires (ASAPs), also known as modification-assisted repertoires (MAPs), allow for the classification of multiple monoclonal antibodies that specifically bind to VEGF based on the binding profile of each antibody from multiple chemically or enzymatically modified antigen surfaces (see, e.g., US 2004 / 0101920). The antibodies in each bin 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 bin.

[0085] 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.

[0086] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules, including but not limited to a cytotoxic agent.

[0087] The terms "nucleic acid," "nucleic acid molecule," or "polynucleotide" include any compound and / or substance comprising a polymer of nucleotides. 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, a nucleic acid molecule is described by a sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is typically expressed as 5' to 3'. As used herein, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and polymers comprising a mixture of two or more of these molecules. Nucleic acid molecules can be linear or circular. In addition, the term nucleic acid molecule includes both sense and antisense strands, as well as single-stranded and double-stranded forms. Moreover, the nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides. The example of non-naturally occurring nucleotides includes modified nucleotide bases with derived sugar or phosphate backbone linkages or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules, which are suitable as carriers for directly expressing the antibodies of the present invention in vitro and / or in vivo, for example, in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors can be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA vector and / or the expression of the encoded molecule, so that mRNA can be injected into a subject to produce antibodies in vivo (see, e.g., Stadler et al., Nature Medicine 2017, published online June 12, 2017, doi: 10.1038 / nm.4356 or EP 2 101 823 B1).

[0088] 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 ordinarily 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.

[0089] "Isolated nucleic acid encoding an anti-VEGF antibody" refers to one or more nucleic acid molecules encoding the antibody heavy and light chains (or fragments thereof), including such one or more nucleic acid molecules in a single vector or separate vectors, and such one or more nucleic acid molecules present at one or more locations in a host cell.

[0090] As used herein, the term "vector" refers to a nucleic acid molecule capable of amplifying 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 the vector has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0091] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid is introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, without regard to the number of passages. Progeny may not be completely identical to the parent cell in nucleic acid content but may contain mutations. Mutant progeny having the same function or biological activity as screened or selected for in the initially transformed cell are included herein.

[0092] The term "pharmaceutical composition" or "pharmaceutical formulation" refers to a preparation that is in form permitting the biological activity of the active ingredient contained therein to be effective, and contains no additional ingredients that are unacceptably toxic to a subject to which the pharmaceutical composition would be administered.

[0093] An "effective amount" of an agent, such as a pharmaceutical composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0094] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic 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.

[0095] "Pharmaceutically acceptable carrier" refers to an ingredient other than the active ingredient in a pharmaceutical composition or formulation that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0096] 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.

[0097] As used herein, "treatment" (and grammatical variations such as "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of the disease in the individual being treated, and can be performed for prevention or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the onset or recurrence of the disease, alleviating symptoms, alleviating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or palliating the disease state, and regression or improved prognosis. In some embodiments, the antibodies of the invention are used to delay the development of the disease or slow the progression of the disease.

[0098] 2. Detailed description of the embodiments of the present invention

[0099] In one aspect, the present invention is based in part on the use of anti-VEGF antibodies to inhibit angiogenesis. In certain embodiments, antibodies that bind to VEGF are provided. The antibodies of the present invention can be used, for example, to diagnose or treat VEGF-related diseases, such as, for example, cancer or eye diseases.

[0100] A. Exemplary Anti-VEGF Antibodies

[0101] In one aspect, the present invention provides antibodies that bind to VEGF. In one aspect, isolated antibodies that bind to VEGF are provided. In one aspect, the present invention provides antibodies that specifically bind to VEGF. In certain embodiments, the anti-VEGF antibody

[0102] a) wherein the binding of the antibody to VEGF significantly inhibits the binding of VEGF to the VEGF receptor VEGF-R2, but does not significantly inhibit the binding of VEGF to the VEGF receptor VEGF-R1, and / or

[0103] b) wherein the antibody binds to VEGF with an affinity of ≤ 150 pM as measured by surface plasmon resonance at a temperature of 25°C, and wherein the antibody binds to VEGF with a greater or about the same affinity as measured by surface plasmon resonance at a temperature of 37°C.

[0104] In one aspect, the present invention provides an anti-VEGF antibody comprising at least one, two, three, four, five, or six CDRs selected from the group consisting of: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 03; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 04; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 05; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 06; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 07; and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 08. An exemplary antibody comprising this set of CDR amino acid sequences is the antibody referred to herein as "VEGF-0089."

[0105] In one aspect, the present invention provides an antibody comprising at least one, at least two, or all three VH CDR sequences selected from the group consisting of: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 03; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 04; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 05. In one embodiment, the antibody comprises CDR-H3 comprising the amino acid sequence of SEQ ID NO: 05. In another embodiment, the antibody comprises CDR-H3 and CDR-L3, wherein the CDR-H3 comprises the amino acid sequence of SEQ ID NO: 05 and the CDR-L3 comprises the amino acid sequence of SEQ ID NO: 08. In additional embodiments, the antibody comprises CDR-H3, CDR-L3, and CDR-H2, wherein the CDR-H3 comprises the amino acid sequence of SEQ ID NO: 05, the CDR-L3 comprises the amino acid sequence of SEQ ID NO: 08, and the CDR-H2 comprises the amino acid sequence of SEQ ID NO: 04. In additional embodiments, the antibody comprises (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 03; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 04; and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 05.

[0106] In another aspect, the present invention provides an antibody comprising at least one, at least two, or all three VL CDR sequences selected from the group consisting of: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 06; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 07; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 08. In one embodiment, the antibody comprises (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 06; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 07; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 08.

[0107] In another aspect, an antibody of the invention comprises (a) a VH domain comprising at least one, at least two or all three VH CDR sequences selected from the group consisting of: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 03; (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 04; and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 05; and (b) a VL domain comprising at least one, at least two or all three VLCDR sequences selected from the group consisting of: (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 06; (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 07; and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 08.

[0108] In another aspect, an antibody of the invention comprises (a) a VH domain comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 03: (b) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 05; and (b) a VL domain comprising (c) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 06; (d) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 07; and (e) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 08.

[0109] In another aspect, the present invention provides an antibody comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 03; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 04; (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 05; (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 06; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 07; and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 08. An exemplary antibody comprising this set of CDR amino acid sequences is the antibody referred to herein as "VEGF-0089."

[0110] In certain embodiments, any one or more amino acids of an anti-VEGF antibody provided above are mutated at the following CDR positions: positions 4, 6, 7, and 8 of CDR-H2 (SEQ ID NO: 04).

[0111] In certain embodiments, the substitution is a conservative substitution or deletion as provided herein. In certain embodiments, any one or more of the following substitutions or deletions can be performed in any combination: in CDR-H2 (SEQ ID NO: 04): position N4S, G6P, the deletion of the amino acid G at position 7, and I8F.

[0112] In another aspect, an antibody of the invention comprises (a) a VH domain comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 03: (ii) a CDR-H2, 2 of which have at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 04; and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 05; and (b) a VL domain comprising (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 06; (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 07; and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 08.

[0113] In another aspect, an antibody of the invention comprises (a) a VH domain comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 03; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 30; and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 05; and (b) a VL domain comprising (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 06; (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 07; and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 08. In the antibody according to this aspect of the invention, CDR-H2 comprises the amino acids of SEQ ID NO: 30, which is the following consensus sequence: SIGX1GX2X3X4YTYYADSVKG (SEQ ID NO: 30), wherein X1, X2, and X4 are independently selected from any naturally occurring amino acid, and wherein X3 is selected from any naturally occurring amino acid or a gap (no amino acid).

[0114] In another aspect, an antibody of the invention comprises (a) a VH domain comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 03; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 31; and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 05; and (b) a VL domain comprising (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 06; (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 07; and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 08. In the antibody according to this aspect of the invention, CDR-H2 comprises the amino acids of SEQ ID NO: 31, which is the following consensus sequence: SIGX1GX2X3X4YTYYADSVKG (SEQ ID NO: 31), wherein X1 is selected from N or S, X2 is selected from G or P, X3 is a gap (no amino acid) or G; and X4 is selected from I or F.

[0115] In another aspect, the present invention provides an antibody comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 03; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10; (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 05; (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 06; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 07; and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 08. Exemplary antibodies comprising this set of CDR amino acid sequences are those referred to herein as "VEGF-0113" and "VEGF-P1AE3520."

[0116] In another aspect, the present invention provides an antibody comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 03; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 12; (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 05; (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 06; (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 07; and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 08. Exemplary antibodies comprising this set of CDR amino acid sequences are those referred to herein as "VEGF-0114" and "VEGF-P1AE3521."

[0117] In one embodiment, the anti-VEGF antibody further comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0118] In another aspect, the anti-VEGF antibody comprises a heavy chain variable domain (VH) sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 01. In certain embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-VEGF antibody comprising that sequence retains the ability to bind to VEGF. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 01. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside of the CDRs (i.e., within the FRs). Optionally, the anti-VEGF antibody comprises the VH sequence in SEQ ID NO: 01, including post-translational modifications of that sequence. In a specific embodiment, VH comprises one, two or three CDRs selected from: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 03, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 04, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 05.

[0119] In another aspect, an anti-VEGF antibody is provided, wherein the antibody comprises a light chain variable domain (VL) sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 02. In certain embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-VEGF antibody comprising the sequence retains the ability to bind to VEGF. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 02. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside of the CDRs (i.e., within the FRs). Optionally, the anti-VEGF antibody comprises the VL sequence in SEQ ID NO: 02, including post-translational modifications of that sequence. In a specific embodiment, VL comprises one, two or three CDRs selected from: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 06; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 07; and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 08.

[0120] In another aspect, an anti-VEGF antibody is provided, wherein the antibody comprises a VH sequence as in any of the embodiments provided above and a VL sequence as in any of the embodiments provided above. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 01 and SEQ ID NO: 02, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH domain of SEQ ID NO: 01 and the VL domain of SEQ ID NO: 02. An exemplary antibody comprising such VH and VL domains is the antibody referred to herein as "VEGF-0089."

[0121] In another aspect, an anti-VEGF antibody is provided, wherein the antibody comprises a VH sequence as in any of the embodiments provided above and a VL sequence as in any of the embodiments provided above. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 33 and SEQ ID NO: 02, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH domain of SEQ ID NO: 33 and the VL domain of SEQ ID NO: 02. An exemplary antibody comprising such VH and VL domains is the antibody referred to herein as "VEGF-P1AD8675."

[0122] In another aspect, the anti-VEGF antibody comprises a heavy chain variable domain (VH) sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 09. In certain embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-VEGF antibody comprising that sequence retains the ability to bind to VEGF. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 09. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside of the CDRs (i.e., within the FRs). Optionally, the anti-VEGF antibody comprises the VH sequence in SEQ ID NO: 09, including post-translational modifications of that sequence. In a specific embodiment, VH comprises one, two or three CDRs selected from: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 03, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 10, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 05.

[0123] In another aspect, an anti-VEGF antibody is provided, wherein the antibody comprises a VH sequence as in any of the embodiments provided above and a VL sequence as in any of the embodiments provided above. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 09 and SEQ ID NO: 02, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH domain of SEQ ID NO: 09 and the VL domain of SEQ ID NO: 02. An exemplary antibody comprising such VH and VL domains is the antibody referred to herein as "VEGF-0113."

[0124] In another aspect, an anti-VEGF antibody is provided, wherein the antibody comprises a VH sequence as in any of the embodiments provided above and a VL sequence as in any of the embodiments provided above. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 43 and SEQ ID NO: 02, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH domain of SEQ ID NO: 43 and the VL domain of SEQ ID NO: 02. An exemplary antibody comprising such VH and VL domains is the antibody referred to herein as "VEGF-P1AE3520." In another aspect, the anti-VEGF antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 11. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-VEGF antibody comprising the sequence retains the ability to bind to VEGF. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 11. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside of the CDRs (i.e., within the FRs). Optionally, the anti-VEGF antibody comprises the VH sequence in SEQ ID NO: 11, including post-translational modifications of that sequence. In specific embodiments, the VH comprises one, two, or three CDRs selected from: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 03, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 12, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 05.

[0125] In another aspect, an anti-VEGF antibody is provided, wherein the antibody comprises a VH sequence as in any of the embodiments provided above and a VL sequence as in any of the embodiments provided above. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 11 and SEQ ID NO: 02, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH domain of SEQ ID NO: 11 and the VL domain of SEQ ID NO: 02. An exemplary antibody comprising such VH and VL domains is the antibody referred to herein as "VEGF-0114."

[0126] In another aspect, an anti-VEGF antibody is provided, wherein the antibody comprises a VH sequence as in any of the embodiments provided above and a VL sequence as in any of the embodiments provided above. In one embodiment, the antibody comprises the VH and VL sequences in SEQ ID NO: 45 and SEQ ID NO: 02, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the VH domain of SEQ ID NO: 45 and the VL domain of SEQ ID NO: 02. An exemplary antibody comprising such VH and VL domains is the antibody referred to herein as "VEGF-P1AE3521."

[0127] In one embodiment of all aspects the antibody comprises a CDR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:04, SEQ ID NO:10 and SEQ ID NO:12.

[0128] In one embodiment of all aspects the antibody comprises H-FR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:46 and SEQ ID NO:47.

[0129] In further aspects, the present invention provides antibodies that bind to the same epitope as the anti-VEGF antibodies provided herein. For example, in certain embodiments, antibodies are provided that bind to the same epitope as an anti-VEGF antibody comprising the VH sequence of SEQ ID NO: 01 and the VL sequence of SEQ ID NO: 02. In one embodiment, the anti-VEGF antibody binds to the same epitope as the antibody VEGF-0089 described herein, as measured by X-ray crystallography. In one embodiment, the anti-VEGF antibody binds to the same epitope as the antibody VEGF-0089 described herein, as measured by X-ray crystallography as described in Example 13. In one embodiment, an antibody is provided that binds to a conformational epitope on a dimer of VEGF-A121, wherein VEGF-A121 comprises the amino acid sequence of SEQ ID NO: 45, wherein the epitope comprises amino acids F17, M18, D19, Y21, Q22, R23, Y25, H27, P28, I29, E30, M55, N62, L66, N100, K101, C102, E103, C104, R105, and P106 in one of the individual VEGF-A121 molecules within the VEGF dimer; and amino acids E30, K48, M81, and Q87 in the other individual VEGF-A121 molecule within the VEGF dimer. Amino acid position numbering is based on the amino acid sequence of VEGF-A121 set forth in SEQ ID NO: 45 (see also SEQ ID NO: 45). Figure 13 In one embodiment, the epitope is determined by X-ray crystallography.

[0130] In another aspect of the invention, the anti-VEGF antibody according to any of the above embodiments is a monoclonal antibody, including a human antibody. In one embodiment, the anti-VEGF antibody is an antibody fragment, such as an Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment. In one embodiment, the anti-VEGF antibody is a Fab fragment. In another embodiment, the antibody is a full-length antibody, such as an intact IgG1 antibody or other antibody class or isotype as defined herein.

[0131] In another aspect, the anti-VEGF antibody according to any of the above embodiments may incorporate any of the features described in Sections 1-6 below, alone or in combination:

[0132] 1. Antibody affinity

[0133] In certain embodiments, the antibodies provided herein have a % saturation of ≤ 1 μM, ≤ 100 nM, ≤ 10 nM, or ≤ 1 nM (e.g., 10 -8 M or lower, such as 10 -8 M to 10 -13 M, for example 10-9 M to 10 -13 The dissociation constant (Kd) of the

[0134] In one embodiment, an antibody provided herein binds VEGF with an affinity of ≤ 150 pM (ie, has a dissociation constant (Kd) of ≤ 150 pM).

[0135] In one embodiment, the Fab fragments of the antibodies provided herein bind to VEGF with an affinity of ≤150 pM (i.e., have a dissociation constant (Kd) of ≤150 pM). In one embodiment, the antibodies provided herein bind to VEGF with an affinity of ≤150 pM, as measured by surface plasmon resonance at a temperature of 25°C. In one embodiment, the antibodies provided herein bind to VEGF with an affinity of ≤150 pM, as measured by surface plasmon resonance at a temperature of 37°C. In one embodiment, the antibodies provided herein bind to VEGF with an affinity of ≤150 pM, as measured by surface plasmon resonance at temperatures of 25°C and 37°C.

[0136] In one embodiment, the antibodies provided herein bind to VEGF with an affinity of ≤ 150 pM as measured by surface plasmon resonance at a temperature of 25° C., and the antibodies bind to VEGF with a greater or about the same affinity as measured by surface plasmon resonance at a temperature of 37° C. By “about the same affinity” is meant that the affinity of the antibody at 37° C. (i.e., Kd) is within + / - 5% of the affinity of the antibody at 25° C. (i.e., Kd).

[0137] In one embodiment, Kd is calculated using Measured by Surface Plasmon Resonance Assay. In one embodiment, Kd is measured using a surface plasmon resonance assay as described in Example 2.

[0138] For example, using -T-200a BIACORE The assay was performed using a BIAcore (BIAcore, Inc., Uppsala) at 25°C using an immobilized antigen CM5 chip with a response rate of ~10 RU. In one embodiment, a carboxymethylated dextran biosensor chip (CM5, BIAcore, Inc.) was activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Antigen was diluted to 5 μg / ml (~0.2 μM) with 10 mM sodium acetate, pH 4.8, and then injected at a flow rate of 5 μl / min to obtain approximately 10 response units (RU) of coupled protein. Following antigen injection, 1 M ethanolamine was injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) were injected in PBS with 0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at 25°C at a flow rate of approximately 25 μl / min. A simple one-to-one Langmuir binding model ( Evaluation Software version 3.2) and calculated the association rate (k on ) and dissociation rate (k off The equilibrium dissociation constant (Kd) was calculated as the ratio k off / k on See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). If the on-rate exceeds 106 M-1 s-1 by the surface plasmon resonance assay described above, the on-rate can be determined using a fluorescence quenching technique, i.e., measuring the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) of 20 nM anti-antigen antibody (Fab form) in PBS pH 7.2 at 25°C in the presence of increasing concentrations of antigen, as measured in a spectrometer such as a flow-blocking spectrophotometer (Aviv Instruments) or a 8000 Series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) using a stirred cuvette.

[0139] 2. Antibody fragments

[0140] In certain embodiments, the antibodies provided herein are antibody fragments. An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that retains the ability to specifically bind to an antigen. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, single-chain Fab (scFab); single-chain variable fragment (scFv) and single-domain antibodies (dAbs). For a review of certain antibody fragments, see Holliger and Hudson, Nature Biotechnology 23: 1126-1136 (2005).

[0141] In one embodiment, the antibody fragment is a Fab, Fab', Fab'-SH or F(ab')2 fragment, in particular a Fab fragment. Papain digestion of an intact antibody produces two identical antigen-binding fragments, called "Fab" fragments, each containing a heavy chain and a light chain variable domain (VH and VL, respectively) and a light chain constant domain (CL) and a heavy chain first constant domain (CH1). Thus, the term "Fab fragment" refers to an antibody fragment comprising a light chain containing a VL domain and a CL domain and a heavy chain fragment containing a VH domain and a CH1 domain. Fab' fragments differ from Fab fragments in that residues are added to the carboxyl terminus of the CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is a Fab' fragment in which the cysteine ​​residues of the constant domains carry a free thiol group. 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 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.

[0142] In another embodiment, the antibody fragment is a diabody, a triabody, or a tetrabody. Diabodies are antibody fragments with two antigen-binding sites that can be bivalent or bispecific. See, for example, EP 404,097; WO 1993 / 01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0143] In another embodiment, the antibody fragment is a single-chain Fab fragment. A "single-chain Fab fragment" or "scFab" is a polypeptide consisting of an antibody heavy chain variable domain (VH), an antibody heavy chain constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a linker, wherein the antibody domain and the linker have one of the following orders in the N-terminal to C-terminal direction: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1 or d) VL-CH1-linker-VH-CL. In particular, the linker is a polypeptide of at least 30 amino acids, preferably 32 to 50 amino acids. The single-chain Fab fragment is stabilized by the natural disulfide bond between the CL domain and the CH1 domain. In addition, these single-chain Fab fragments can be further stabilized by inserting cysteine ​​residues (e.g., position 44 in the variable heavy chain and position 100 in the variable light chain according to Kabat numbering) to produce interchain disulfide bonds.

[0144] In another embodiment, the antibody fragment is a single-chain variable region fragment (scFv). A "single-chain variable fragment" or "scFv" is a fusion protein of the heavy chain variable domain (VH) and light chain variable domain (VL) of an antibody connected by a linker. In particular, the linker is a short polypeptide of 10-25 amino acids, generally rich in glycine to provide flexibility, and rich in serine or threonine to provide solubility, and can connect the N-terminus of VH to the C-terminus of VL, or vice versa. Despite the removal of the constant region and the introduction of the linker, the protein still retains the specificity of the original antibody. For review of scFv fragments, see, e.g., Pluückthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore, eds., (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93 / 16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458.

[0145] 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), as described herein.

[0146] 3. Human Antibodies

[0147] In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be prepared using a variety of techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20: 450-459 (2008).

[0148] 4. Multispecific Antibodies

[0149] In certain embodiments, the antibodies provided herein are multispecific antibodies, such as bispecific antibodies. Multispecific antibodies are monoclonal antibodies that have binding specificity to at least two different sites (i.e., different epitopes on different antigens or different epitopes on the same antigen). In certain embodiments, multispecific antibodies have three or more binding specificities. In certain embodiments, one of the binding specificities is to VEGF, while the other (two or more) specificities are to any other antigen. In certain embodiments, bispecific antibodies can bind to two (or more) different epitopes of VEGF. Multispecific (e.g., bispecific) antibodies can also be used to localize cytotoxic agents or cells to cells expressing VEGF. Multispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0150] Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature, 305:537 (1983)), and “knob-in-hole” engineering (see, e.g., U.S. Pat. No. 5,731,168 and Atwell et al., J. Mol. Biol., 270:26 (1997)). Multispecific antibodies can also be prepared by the following methods: engineering electrostatic manipulation effects for preparing antibody Fc-heterodimer molecules (WO 2009 / 089004); cross-linking two or more antibodies or fragments (see, for example, U.S. Patent No. 4,676,980, and Brennan et al., Science, 229: 81 (1985)); using leucine zippers to generate bispecific antibodies (see, for example, Kostelny et al., J. Immunol., 148(5): 1547-1553 (1992) and WO 2011 / 034605); using common light chain technology to circumvent the light chain mispairing problem (see, for example, WO 98 / 50431); using "diabody" technology to prepare bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)); and preparing trispecific antibodies as described, e.g., in Tutt et al., J. Immunol. 147:60 (1991).

[0151] Also included herein are engineered antibodies with three or more antigen binding sites, including, for example, "Octopus antibodies" or DVD-Igs (see, for example, WO 2001 / 77342 and WO 2008 / 024715). Other examples of multispecific antibodies with three or more antigen binding sites can be found in WO 2010 / 115589, WO 2010 / 112193, WO 2010 / 136172, WO 2010 / 145792, and WO 2013 / 026831.

[0152] Multispecific antibodies can also be provided in an asymmetric form with domain crossover (i.e., by exchanging VH / VL domains) in one or more binding arms of the same antigen specificity (see, for example, WO2009 / 080252 and WO2015 / 150447), CH1 / CL domains (see, for example, WO2009 / 080253), or complete Fab arms (see, for example, WO2009 / 080251, WO2016 / 016299, see also Schaefer et al., PNAS, 108 (2011) 1187-1191, and Klein et al., MAbs 8 (2016) 1010-20). In one embodiment, the multispecific antibody comprises a spanning Fab fragment. The term "spanning Fab fragment" or "crossing Fab fragment" refers to a Fab fragment in which the variable or constant regions of the heavy and light chains are exchanged. The spanning Fab fragment comprises a polypeptide chain consisting of a light chain variable region (VL) and a heavy chain constant region 1 (CH1), and a polypeptide chain consisting of a heavy chain variable region (VH) and a light chain constant region (CL). Asymmetric Fab arms can also be engineered by introducing charged or uncharged amino acid mutations into the domain interfaces to guide correct Fab pairing. See, for example, WO2016 / 172485.

[0153] Various other molecular formats of multispecific antibodies are known in the art and are encompassed herein (see, e.g., Spiess et al., Mol Immunol 67 (2015) 95-106).

[0154] 5. Antibody variants

[0155] In certain embodiments, it is envisioned that the amino acid sequence variants of the antibodies provided herein. For example, it may be necessary 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. These modifications include, for example, the deletion of residues in the antibody amino acid sequence and / or insertion and / or replacement residues therein. Any combination of deletion, insertion, and replacement can be performed to obtain the final construct, provided that the final construct has the desired characteristics, such as antigen binding.

[0156] a) Substitution, insertion and deletion variants

[0157] In certain embodiments, antibody variants with one or more amino acid replacements are provided. Sites of interest for substitutional mutagenesis include HVR (e.g., CDR) and FR. Conservative replacements are shown under the heading "preferred replacements" in Table 1. More substantial changes are provided under the heading "exemplary replacements" in Table 1, and are further described below regarding amino acid side chain classes. Amino acid replacements can be introduced into the antibody of interest, and the product can be screened to obtain desired activity, such as maintained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0158] Table 1

[0159]

[0160]

[0161] Amino acids can be grouped according to common side chain properties:

[0162] (1) Hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile;

[0163] (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;

[0164] (3) Acidic: Asp, Glu;

[0165] (4) basic His, Lys, Arg;

[0166] (5) Residues that affect chain orientation: Gly, Pro;

[0167] (6) Aromatic: Trp, Tyr, Phe.

[0168] Non-conservative substitutions will entail exchanging a member of one of these classes for a member of another class.

[0169] One type of substitutional variant involves replacing one or more hypervariable region residues of a parent antibody (e.g., a humanized antibody or a human antibody). Generally, the resulting variant selected for further study will have changes (e.g., improvements) in certain biological properties relative to the parent antibody (e.g., increased affinity, reduced immunogenicity), and / or will substantially retain certain biological properties of the parent antibody. Exemplary substitutional 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. In short, one or more HVR (e.g., CDR) residues are mutated, and the variant antibodies are displayed on phage and screened for specific biological activity (e.g., binding affinity).

[0170] Changes (e.g., substitutions) can be made in HVRs (e.g., CDRs), for example, to improve antibody affinity. Such changes can be made in HVR "hotspots" (i.e., residues encoded by codons that undergo mutations at high frequency during somatic maturation) (see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008)); and / or in residues that contact the antigen, and the resulting variant VH or VL tested for binding affinity. Affinity maturation by construction and reselection of secondary libraries has been described, for example, in Hoogenboom et al., Methods in Molecular Biology 178: 1-37 (O'Brien et al., eds., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then established. The library is then screened to identify any antibody variants with the desired affinity. Another approach to introducing diversity involves an HVR (e.g., CDR)-directed approach, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR (e.g., CDR) residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. In particular, CDR-H3 and CDR-L3 are often targeted.

[0171] In certain embodiments, substitutions, insertions, or deletions may be made within one or more HVRs (e.g., CDRs), as long as these changes do not substantially reduce the ability of the antibody to bind to the antigen. For example, conservative changes (e.g., conservative substitutions, as provided herein) may be made in HVRs (e.g., CDRs) that do not substantially reduce binding affinity. For example, these changes may be outside of the antigen contact residues in the HVRs (e.g., CDRs). In certain embodiments of the variant VH and VL sequences provided above, each HVR is unchanged or contains no more than one, two, or three amino acid substitutions.

[0172] A method that can be used to identify residues or regions of an antibody that can be targeted for mutagenesis is called "alanine scanning mutagenesis," as described in Cunningham and Wells (1989) Science, 244: 1081-1085. In this method, residues or groups of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and 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. Further substitutions can be introduced at amino acid positions that demonstrate functional sensitivity to the initial substitutions. Alternatively or additionally, a crystal structure of the antigen-antibody complex can be used to identify contact points between the antibody and the antigen. These contact residues and neighboring residues can be targeted or eliminated as substitution candidates. Variants can be screened to determine whether they contain the desired properties.

[0173] Amino acid sequence insertions include amino-terminal and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertion variants of the antibody molecule include fusions of the N-terminus or C-terminus of the antibody with an enzyme (e.g., for ADEPT (antibody-directed enzyme prodrug therapy)) or a polypeptide that increases the serum half-life of the antibody.

[0174] b) Glycosylation variants

[0175] In certain embodiments, the antibodies provided herein are altered to increase or decrease the extent to which the antibodies are glycosylated. Addition or deletion of glycosylation sites to an antibody can be conveniently achieved by altering the amino acid sequence to create or remove one or more glycosylation sites.

[0176] In the case where the antibody comprises an Fc region, the oligosaccharides attached to the Fc region can be changed. Natural antibodies produced by mammalian cells typically contain branched, biantennary oligosaccharides that are generally attached to Asn297 of the CH2 domain of the Fc region via an N-link. See, for example, Wright et al., TIBTECH 15: 26-32 (1997). Oligosaccharides can include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to the GlcNAc in the "backbone" of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharides in the antibodies of the present invention can be modified to create antibody variants with certain improved properties.

[0177] In one embodiment, antibody variants are provided having non-fucosylated oligosaccharides, i.e., oligosaccharide structures that lack fucose attached (directly or indirectly) to the Fc region. Such non-fucosylated oligosaccharides (also referred to as "oligosaccharides without fucosylation") are particularly N-linked oligosaccharides that lack the fucose residue attached to the first GlcNAc in the backbone of the biantennary oligosaccharide structure. In one embodiment, antibody variants are provided that have 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 is the (average) amount of oligosaccharides lacking a fucose residue relative to the sum of all oligosaccharides (e.g., complex, hybrid, and high mannose structures) attached to Asn297, as measured by MALDI-TOF mass spectrometry, for example as described in WO2006 / 082515. Asn297 refers to the asparagine residue located at approximately position 297 in the Fc region (EU numbering of Fc region residues); 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 with an increased proportion of non-fucosylated oligosaccharides in the Fc region may have improved FcγRIIIa receptor binding and / or improved effector function, particularly improved ADCC function. See, for example, US 2003 / 0157108; US 2004 / 0093621.

[0178] Examples of cell lines capable of producing antibodies with reduced defucosylation include Lec13 CHO cells deficient in protein fucosylation (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); US 2003 / 0157108; and WO 2004 / 056312, especially Example 11); and gene knockout cell lines, such as CHO cells in which the α-1,6-fucosyltransferase gene FUT8 is knocked out (see, for example, Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614-622 (2004); Kanda, Y., et al., Biotechnol. Bioeng., 94(4): 680-688 (2006); and WO2003 / 085107); or cells with reduced or abolished GDP-fucose synthesis or transporter activity (see, for example, US2004259150, US2005031613, US2004132140, US2004110282).

[0179] In another embodiment, antibody variants having bisected oligosaccharides are provided, for example, biantennary oligosaccharides attached to the Fc region of the antibody are bisected by GlcNAc. These antibody variants may have reduced fucosylation and / or improved ADCC function as described above. 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.

[0180] Also provided are antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region. These antibody variants may have improved CDC function. These antibody variants are described, for example, in WO 1997 / 30087; WO 1998 / 58964; and WO 1999 / 22764.

[0181] c) Fc region variants

[0182] In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein to thereby generate an Fc region variant. The 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.

[0183] In certain embodiments, the present invention encompasses antibody variants that possess some, but not all, effector functions that make them ideal candidates for applications where the in vivo half-life of the antibody is important but certain effector functions (e.g., complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC)) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm 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 likely lacks ADCC activity) but retains FcRn binding ability. The primary cells for mediating ADCC, NK cells, 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, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays for evaluating ADCC activity of a molecule of interest 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, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see Bruggemann, M., et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays can be used (see, e.g., ACTI for flow cytometry). TM Nonradioactive cytotoxicity assay (CellTechnology, Inc. Mountain View, CA); and CytoTox Non-radioactive cytotoxicity assays (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) 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 that disclosed in Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays can also be performed to confirm that the antibody cannot bind to C1q and therefore lacks CDC activity. See, for example, 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., 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 A1).

[0184] Antibodies with reduced effector function include those with one or more substitutions at residues 238, 265, 269, 270, 297, 327, and 329 of the Fc region ( U.S. Pat. No. 6,737,056 ). Such Fc mutants include those with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant with residues 265 and 297 substituted with alanine ( U.S. Pat. No. 7,332,581 ).

[0185] Certain antibody variants with improved or decreased binding to FcRs have been 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)).

[0186] In certain embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, such as substitutions at positions 298, 333, and / or 334 (EU numbering of residues) of the Fc region.

[0187] In certain embodiments, antibody variants include an Fc region with one or more amino acid replacements that reduce FcγR binding, such as replacements at positions 234 and 235 (EU numbering of residues) in the Fc region. In one embodiment, the replacements are L234A and L235A (LALA). In certain embodiments, antibody variants further include D265A and / or P329G in the Fc region derived from human IgG1 Fc region. In one embodiment, the replacements in the Fc region derived from human IgG1 Fc region are L234A, L235A, and P329G (LALA-PG). (See, e.g., WO 2012 / 130831). In another embodiment, the replacements in the Fc region derived from human IgG1 Fc region are L234A, L235A, and D265A (LALA-DA).

[0188] In some embodiments, alterations are made in the Fc region that result in altered (i.e., improved or decreased) C1q binding and / or complement-dependent cytotoxicity (CDC), e.g., as described in U.S. Patent No. 6,194,551, WO 99 / 51642, and Idusogie et al., J. Immunol. 164:4178-4184 (2000).

[0189] US 2005 / 0014934 (Hinton et al.) describes antibodies with increased half-life and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)). These antibodies comprise an Fc region having one or more substitutions therein that improve the binding of the Fc region to FcRn. Such Fc variants include those having a substitution at one or more of Fc region residues 238, 252, 254, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434 (e.g., substitution of Fc region residue 434) (see, e.g., U.S. Patent No. 7,371,826; Dall'Acqua, WF, et al. J. Biol. Chem. 281 (2006) 23514-23524).

[0190] Fc region residues 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). Residues 1253, H310, H433, N434, and H435 (EU numbering according to Kabat) are implicated in the interaction (Medesan, C., et al., Eur. J. Immunol. 26 (1996) 2533; Firan, M., et al., Int. Immunol. 13 (2001) 993; Kim, JK, et al., Eur. J. Immunol. 24 (1994) 542). Residues I253, H310, and H435 were found to be critical for the interaction of human Fc with murine FcRn (Kim, JK, et al., Eur. J. Immunol. 29 (1999) 2819). Studies of the human Fc-human FcRn complex have shown that residues I253, S254, H435, and Y436 are critical for the interaction (Firan, M., et al., Int. Immunol. 13 (2001) 993; Shields, RL, et al., J. Biol. Chem. 276 (2001) 6591-6604). In Yeung, YA, et al. (J. Immunol. 182 (2009) 7667-7671), various mutants of residues 248 to 259 and 301 to 317 and 376 to 382 and 424-437 have been reported and examined.

[0191] In certain embodiments, the antibody variant comprises an Fc region having one or more amino acid substitutions that reduce FcRn binding, such as substitutions at positions 253, and / or 310, and / or 435 (EU numbering of residues) of the Fc region. In certain embodiments, the antibody variant comprises an Fc region having amino acid substitutions at positions 253, 310, and 435. In one embodiment, the substitutions are I253A, H310A, and H435A in an Fc region derived from a human IgG1 Fc region. See, e.g., Grevys, A., et al., J. Immunol. 194 (2015) 5497-5508.

[0192] In certain embodiments, the antibody variants comprise an Fc region with one or more amino acid substitutions that reduce FcRn binding, such as substitutions at positions 310, and / or 433, and / or 436 (EU numbering of residues) of the Fc region. In certain embodiments, the antibody variants comprise an Fc region with amino acid substitutions at positions 310, 433, and 436. In one embodiment, the substitutions are H310A, H433A, and Y436A in an Fc region derived from a human IgG1 Fc region. (See, e.g., WO 2014 / 177460A1).

[0193] In certain embodiments, the antibody variants comprise an Fc region with one or more amino acid substitutions that increase FcRn binding, such as substitutions at positions 252, and / or 254, and / or 256 (EU numbering of residues) of the Fc region. In certain embodiments, the antibody variants comprise an Fc region with amino acid substitutions at positions 252, 254, and 256. In one embodiment, the substitutions are M252Y, S254T, and T256E in an Fc region derived from a human IgG1 Fc region.

[0194] See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351 for additional examples of Fc region variants.

[0195] d) Cysteine-engineered antibody variants

[0196] In certain embodiments, it may be desirable to create cysteine ​​engineered antibodies, such as THIOMAB TM Antibodies, wherein one or more residues of the antibody are replaced with cysteine ​​residues. In a specific embodiment, the replaced residues appear at accessible sites of the antibody. By replacing those residues with cysteine, reactive sulfhydryl groups are thereby positioned at accessible sites of the antibody and can be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create immunoconjugates, as further described herein. Cysteine ​​engineered antibodies can be generated as described, for example, in U.S. Patent Nos. 7,521,541, 8,30,930, 7,855,275, 9,000,130 or WO2016040856.

[0197] 6. Immunoconjugates

[0198] The invention also provides immunoconjugates comprising an anti-VEGF antibody herein 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.

[0199] In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC), in which the antibody is conjugated to one or more therapeutic agents as described above. The antibody is typically linked to one or more therapeutic agents using a linker. An overview of ADC technology, including examples of therapeutic agents and drugs and linkers, is listed in Pharmacol Review 68: 3-19 (2016).

[0200] In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to an enzymatically active toxin or fragment thereof, including but not limited to diphtheria A chain, a non-binding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotonin, sapaonaria officinalis inhibitor, gelonin, mitomycin, restrictocin, phenomycin, enomycin, and the tricothecenes.

[0201] In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioactive isotopes can be used to produce radioconjugates. Examples include At 211 , I 131 , I 125 、Y 90 、Re 186 、Re 188 、Sm 153 、Bi 212 、P 32 、pb 212 When the radioconjugate is used for detection, it may contain a radioactive atom for scintigraphic studies, such as Ttc-99m or I 123, or spin labels for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

[0202] Conjugates of the antibody and cytotoxic agent can be prepared using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to antibodies. See WO 94 / 11026. The linker can be a "cleavable linker" that facilitates release of the cytotoxic drug in the cell. For example, an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker can be used (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Patent No. 5,208,020).

[0203] The immunoconjugates or ADCs herein specifically contemplate, but are not limited to, such conjugates prepared with the following cross-linkers, including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, Sulfo-EMCS, Sulfo-GMBS, Sulfo-KMUS, Sulfo-MBS, Sulfo-SIAB, Sulfo-SMCC, and Sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate), which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL., USA).

[0204] B. Recombinant methods and compositions

[0205] 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 antibody are provided.

[0206] In the case of natural antibodies or natural antibody fragments, two nucleic acids are required, one for the light chain or its fragment and one for the heavy chain or its fragment. Such nucleic acid encoding comprises the amino acid sequence of antibody VL and / or comprises the amino acid sequence of antibody VH (e.g., the light chain and / or heavy chain of the antibody). These nucleic acids can be on the same expression vector or on different expression vectors.

[0207] In the case of a bispecific antibody with a heterodimeric heavy chain, four nucleic acids are required: one for the first light chain, one for the first heavy chain comprising a first heterologous monomeric Fc region polypeptide, one for the second light chain, and one for the second heavy chain comprising a second heterologous monomeric Fc region polypeptide. These four nucleic acids can be contained in one or more nucleic acid molecules or expression vectors. Such nucleic acids encode an amino acid sequence comprising a first VL and / or an amino acid sequence comprising a first VH comprising a first heterologous monomeric Fc region and / or an amino acid sequence comprising a second VL and / or an amino acid sequence comprising a second VH comprising a second heterologous monomeric Fc region of the antibody (e.g., the first and / or second light chain and / or the first and / or second heavy chain of the antibody). These nucleic acids can be on the same expression vector or on different expression vectors, and typically these nucleic acids are located on two or three expression vectors, i.e., one vector can contain more than one of these nucleic acids. Examples of these bispecific antibodies are CrossMabs (see, e.g., Schaefer, W., et al., PNAS, 108 (2011) 11187-1191). For example, one of the heterologous monomer heavy chains comprises the so-called "knob mutations" (T366W and optionally one of S354C or Y349C) and the other comprises the so-called "hole mutations" (T366S, L368A and Y407V and optionally Y349C or S354C) (see, e.g., Carter, P., et al., Immunotechnol. 2 (1996) 73), numbered according to Kabat EU.

[0208] In one embodiment an isolated nucleic acid encoding an antibody for use in a method as reported herein is provided.

[0209] In one embodiment, a method of preparing an anti-VEGF antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody, as provided above, under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0210] For recombinant production of an anti-VEGF antibody, nucleic acid encoding the antibody (e.g., the antibodies described above) is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody), or produced by recombinant methods or obtained by chemical synthesis.

[0211] Suitable host cells for cloning or expressing vectors encoding antibodies include prokaryotic or eukaryotic cells described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, for example, US 5,648,237, US 5,789,199, and US 5,840,523. (See also Charlton, KA, In: Methods in Molecular Biology, Vol. 248, Lo, BKC ed., Humana Press, Totowa, NJ (2003), pp. 245-254, which describes expression of antibody fragments in E. coli.) After expression, the antibody can be separated from the bacterial cell paste as a soluble fraction and can be further purified.

[0212] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains whose glycosylation pathways have been "humanized" to produce antibodies with partially or fully human glycosylation patterns. See Gerngross, TU, Nat. Biotech. 22 (2004) 1409-1414; and Li, H., et al., Nat. Biotech. 24 (2006) 210-215.

[0213] Suitable host cells for expressing (glycosylated) antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. A number of baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.

[0214] Plant cell cultures can also be used as hosts. See, for example, US 5,959,177, US 6,040,498, US 6,420,548, US 7,125,978, and US 6,417,429 (which describe plant cell cultures for producing antibodies in transgenic plants). TM technology).

[0215] 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 line transformed with SV40 (COS-7); human embryonic kidney line (293 or 293T cells, as described, for example, in Graham, FL, et al., J. Gen Virol. 36 (1977) 59-74); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells, as described, for example, in Mather, JP, Biol. Reprod. 23 (1980) 243-252); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells (BRL3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT 060562); TRI cells (as described, for example, in Mather, JP, et al., Annals of Chemistry). 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 producing antibodies, 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.

[0216] In one embodiment, the host cell is eukaryotic, such as a Chinese hamster ovary (CHO) cell or a lymphoid cell (eg, Y0, NS0, Sp20 cell).

[0217] C. Binding assay

[0218] The anti-VEGF antibodies provided herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activities by various assays known in the art.

[0219] In one aspect, the antigen binding activity of the antibodies of the present invention is tested, for example, by known methods such as ELISA, Western blotting, and the like.

[0220] In another aspect, competition assays can be used to identify antibodies that compete for VEGF binding with an antibody comprising the VH domain of SEQ ID NO: 01 and the VL domain of SEQ ID NO: 02.

[0221] In an exemplary competition assay, immobilized VEGF is incubated in a solution containing a first labeled antibody that binds to VEGF (e.g., anti-VEGF-0089) and a second unlabeled antibody that is tested for its ability to compete with the first antibody for binding to VEGF. The second antibody can be present in the hybridoma supernatant. As a control, immobilized VEGF is incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. Following incubation under conditions permissive for binding of the first antibody to VEGF, excess unbound antibody is removed, and the amount of label associated with the immobilized VEGF is measured. If the amount of label associated with the immobilized VEGF in the test sample is substantially reduced relative to the control sample, this indicates that the second antibody competes with the first antibody for binding to VEGF. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0222] D. Pharmaceutical composition

[0223] In another aspect, a pharmaceutical composition comprising any of the antibodies provided herein is provided, e.g., for use in any of the following methods of treatment. In one aspect, the pharmaceutical composition comprises any of the antibodies provided herein and a pharmaceutically acceptable carrier. In another aspect, the pharmaceutical composition comprises any of the antibodies provided herein and at least one additional therapeutic agent, e.g., as described below.

[0224] Pharmaceutical compositions of the anti-VEGF antibodies described herein are prepared by mixing such antibodies of the desired purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized compositions or aqueous solutions. Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages 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 parahydroxybenzoates (alkyl esters)). paraben, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); 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., Zn-protein complexes); and / or nonionic surfactants, such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further 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 chondroitinase.

[0225] Exemplary lyophilized antibody compositions are described in US Patent No. 6,267,958. Aqueous antibody compositions include those described in US Patent No. 6,171,586 and WO 2006 / 044908, the latter compositions including a histidine-acetate buffer.

[0226] The active ingredient can be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or macroemulsions. These techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Osol, A., ed. (1980).

[0227] 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, eg, films, or microcapsules.

[0228] Pharmaceutical compositions for in vivo administration are generally sterile. Sterility can be readily accomplished, for example, by filtration through sterile filtration membranes.

[0229] E. Methods of treatment and routes of administration

[0230] Any of the anti-VEGF antibodies provided herein can be used in therapeutic methods.

[0231] In one aspect, an anti-VEGF antibody is provided for use as a medicament. In another aspect, an anti-VEGF antibody is provided for use in treating a VEGF-related disease (e.g., cancer or an eye disease). In certain embodiments, an anti-VEGF antibody is provided for use in a method of treating a subject having a VEGF-related disease, such as cancer or an eye disease, the method comprising administering to the subject an effective amount of an anti-VEGF antibody. In one such embodiment, the method further comprises administering to the subject an effective amount of at least one additional therapeutic agent (e.g., one, two, three, four, five, or six additional therapeutic agents), e.g., as described below. In a further embodiment, the present invention provides an anti-VEGF antibody for use in inhibiting angiogenesis. In certain embodiments, the present invention provides an anti-VEGF antibody for use in a method of inhibiting angiogenesis in a subject, the method comprising administering to the subject an effective amount of an anti-VEGF antibody to inhibit angiogenesis. The "subject" according to any of the above embodiments is preferably a human.

[0232] In yet another aspect, the present invention provides the use of an anti-VEGF antibody in the manufacture or preparation of a medicament. In one embodiment, the medicament is for use in the treatment of a VEGF-related disease, such as cancer or an eye disease. In a further embodiment, the medicament is for use in a method of treating a VEGF-related disease, such as cancer or an eye disease, comprising administering an effective amount of the medicament to an individual having a VEGF-related disease, such as cancer or an eye disease. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, such as described below. In a further embodiment, the medicament is for use in inhibiting angiogenesis. In a further embodiment, the medicament is for use in a method of inhibiting angiogenesis in an individual, comprising administering to the individual an effective amount of the medicament to inhibit angiogenesis. The "individual" according to any of the above embodiments may be a human.

[0233] In yet another aspect, the present invention provides methods for treating a VEGF-related disease, such as cancer or an eye disease. In one embodiment, the method comprises administering an effective amount of an anti-VEGF antibody to an individual having such a VEGF-related disease, such as cancer or an eye disease. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, as described below. The "individual" according to any of the above embodiments may be a human.

[0234] In yet another aspect, the present invention provides a method for inhibiting angiogenesis in an individual. In one embodiment, the method comprises administering to the individual an effective amount of an anti-VEGF antibody to inhibit angiogenesis. In one embodiment, the "individual" is a human.

[0235] In yet another aspect, a pharmaceutical composition comprising any of the anti-VEGF antibodies provided herein is provided, e.g., for use in any of the above methods of treatment. In one embodiment, the pharmaceutical composition comprises any of the anti-VEGF antibodies provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical composition comprises any of the anti-VEGF antibodies provided herein and at least one additional therapeutic agent, e.g., as described below.

[0236] The antibodies of the present invention can be used alone or in combination with other agents for treatment. For example, the antibodies of the present invention can be co-administered with at least one additional therapeutic agent.

[0237] 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 local treatment is required, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration can be by any appropriate route, for example, by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-term or long-term. Various dosing schedules are contemplated herein, including, but not limited to, single or multiple administrations at multiple time points, bolus administration and pulse infusion.

[0238] The antibodies of the present invention will be formulated, dosed, and applied in a manner consistent with good medical practice. Factors considered in this context include the specific condition being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the condition, the delivery site of the reagent, the method of administration, the administration time arrangement, and other factors known to medical practitioners. The antibodies need not but are optionally formulated together with one or more reagents currently used to prevent or treat the condition. The effective amount of such other reagents depends on the amount of the antibody present in the pharmaceutical composition, the type of condition or treatment, and other factors discussed above. These are typically used with the same dosage and route of administration as described herein, or with about 1 to 99% of the dosage described herein, or with any dosage, and are used by any approach determined to be suitable empirically / clinically.

[0239] For the prevention or treatment of disease, the appropriate dosage of the antibodies 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 antibody is administered for prevention or treatment purposes, previous treatment, the patient's clinical history and response to the antibody, and the judgment of the attending physician. The antibody is appropriately administered to the patient at one time or over 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 an initial candidate dose for administration to the patient, whether, for example, by one or more separate administrations or by continuous infusion. A typical daily dose may be in the range of about 1 μg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administration over several days or longer, depending on the patient's condition, treatment will generally be maintained until the desired containment of disease symptoms occurs. An exemplary dosage of the antibody will be in the range of about 0.05 mg / kg to about 10 mg / kg. Thus, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg or 10 mg / kg (or any combination thereof) can be administered to the patient. Such doses can 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 can be administered, followed by one or more lower doses. The progress of such treatment is easily monitored by conventional techniques and assays.

[0240] F. Products

[0241] In another aspect of the present invention, a product is provided, comprising materials that can be used to treat, prevent and / or diagnose the above-mentioned conditions. The product comprises 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 from various materials such as glass or plastic. The container contains a composition that is effective for treating, preventing and / or diagnosing the condition, either alone or in combination with another composition, and may have a sterile access port (for example, the container may be an intravenous solution bag or vial with a stopper pierceable 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 a selected condition. In addition, the product may comprise: (a) a first container containing a composition, wherein the composition comprises an antibody of the present invention; and (b) a second container containing a composition, wherein the composition comprises another cytotoxic agent or other therapeutic agent. The product in this embodiment of the present invention may further comprise a package insert indicating that the composition can be used to treat a specific condition. Alternatively, or in addition, the article of manufacture may further comprise 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. From a commercial and user perspective, it may further include other materials as required, including other buffers, diluents, filters, needles, and syringes.

[0242] 3. Specific embodiments of the present invention

[0243] Specific embodiments of the present invention are listed below.

[0244] 1. An antibody that binds to VEGF, wherein the binding of the antibody to VEGF significantly inhibits the binding of VEGF to the VEGF receptor VEGF-R2, but does not significantly inhibit the binding of VEGF to the VEGF receptor VEGF-R1.

[0245] 2. An antibody that binds to VEGF, wherein the antibody binds to VEGF with an affinity of ≤150 pM as measured by surface plasmon resonance at a temperature of 25°C, and wherein the antibody binds to VEGF with a greater or about the same affinity as measured by surface plasmon resonance at a temperature of 37°C.

[0246] 3. Antibodies that bind to VEGF,

[0247] a) wherein the binding of the antibody to VEGF significantly inhibits the binding of VEGF to the VEGF receptor VEGF-R2, but does not significantly inhibit the binding of VEGF to the VEGF receptor VEGF-R1, and / or

[0248] b) wherein the antibody binds to VEGF with an affinity of ≤ 150 pM as measured by surface plasmon resonance at a temperature of 25°C, and wherein the antibody binds to VEGF with a greater or about the same affinity as measured by surface plasmon resonance at a temperature of 37°C.

[0249] 4. The antibody of any one of embodiments 1 to 3, wherein the binding of the Fab fragment of the antibody significantly inhibits the binding of VEGF to the VEGF receptor VEGF-R2, but does not significantly inhibit the binding of VEGF to the VEGF receptor VEGF-R1.

[0250] 5. The antibody of any one of embodiments 1 to 4, wherein the Fab fragment of the antibody binds VEGF with an affinity of ≤150 pM as measured by surface plasmon resonance at a temperature of 25°C and as measured by surface plasmon resonance at a temperature of 37°C.

[0251] 6. An antibody that binds to VEGF, wherein the antibody comprises a heavy chain variable domain (VH) comprising

[0252] (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 03,

[0253] (b) CDR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 04, SEQ ID NO: 10 and SEQ ID NO: 12, and

[0254] (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 05; and

[0255] wherein the antibody comprises a light chain variable domain (VL) comprising

[0256] (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 06,

[0257] (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 07; and

[0258] (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 08.

[0259] 7. The antibody of any one of embodiments 1 to 5, wherein the antibody comprises a heavy chain variable domain (VH) comprising

[0260] (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 03,

[0261] (b) CDR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 04, SEQ ID NO: 10 and SEQ ID NO: 12, and

[0262] (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 05; and

[0263] wherein the antibody comprises a light chain variable domain (VL) comprising

[0264] (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 06,

[0265] (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 07; and

[0266] (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 08.

[0267] 8. The antibody of any one of embodiments 1 to 7, which is a monoclonal antibody.

[0268] 9. The antibody of any one of embodiments 1 to 8, which is a human antibody.

[0269] 10. The antibody of any one of embodiments 1 to 9, which is an antibody fragment that binds VEGF.

[0270] 11. The antibody of any one of embodiments 1 to 10, comprising a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 01; and a VL sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 02.

[0271] 12. The antibody of any one of embodiments 1 to 11, comprising a VH sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 01; and a VL sequence of SEQ ID NO: 02.

[0272] 13. The antibody of any one of embodiments 1 to 12, comprising the VH sequence of SEQ ID NO: 01 and the VL sequence of SEQ ID NO: 02.

[0273] 14. The antibody of any one of embodiments 1 to 12, comprising the VH sequence of SEQ ID NO: 09 and the VL sequence of SEQ ID NO: 02.

[0274] 15. The antibody of any one of embodiments 1 to 12, comprising the VH sequence of SEQ ID NO: 11 and the VL sequence of SEQ ID NO: 02.

[0275] 16. The antibody of any one of embodiments 1 to 12, comprising the VH sequence of SEQ ID NO: 33 and the VL sequence of SEQ ID NO: 02.

[0276] 17. The antibody of any one of embodiments 1 to 12, comprising the VH sequence of SEQ ID NO: 42 and the VL sequence of SEQ ID NO: 02.

[0277] 18. The antibody of any one of embodiments 1 to 12, comprising the VH sequence of SEQ ID NO: 44 and the VL sequence of SEQ ID NO: 02.

[0278] 19. An antibody that specifically binds to VEGF, comprising the VH sequence of SEQ ID NO: 01 and the VL sequence of SEQ ID NO: 02.

[0279] 20. An antibody that specifically binds to VEGF, comprising the VH sequence of SEQ ID NO: 09 and the VL sequence of SEQ ID NO: 02.

[0280] 21. An antibody that specifically binds to VEGF, comprising the VH sequence of SEQ ID NO: 11 and the VL sequence of SEQ ID NO: 02.

[0281] 22. An antibody that specifically binds to VEGF, comprising the VH sequence of SEQ ID NO: 33 and the VL sequence of SEQ ID NO: 02.

[0282] 23. An antibody that specifically binds to VEGF, comprising the VH sequence of SEQ ID NO: 42 and the VL sequence of SEQ ID NO: 02.

[0283] 24. An antibody that specifically binds to VEGF, comprising the VH sequence of SEQ ID NO: 44 and the VL sequence of SEQ ID NO: 02.

[0284] 25. The antibody of any one of embodiments 1 to 24, which is a full-length IgG1 antibody.

[0285] 26. The antibody of any one of embodiments 1 to 24, which is a Fab fragment.

[0286] 27. The antibody of any one of embodiments 1 to 26, wherein the antibody binds VEGF with an affinity of ≤ 150 pM as measured by surface plasmon resonance at a temperature of 25°C.

[0287] 28. The antibody of any one of embodiments 1 to 27, wherein the antibody is a multispecific antibody.

[0288] 29. The antibody of any one of embodiments 1 to 28, comprising

[0289] (a) a heavy chain of an amino acid sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 32, SEQ ID NO: 41 and SEQ ID NO: 43; and

[0290] (b) Light chain of SEQ ID NO:14.

[0291] 30. The antibody of any one of embodiments 1 to 28, comprising the heavy chain of SEQ ID NO: 13 and the light chain of SEQ ID NO: 14.

[0292] 31. The antibody of any one of embodiments 1 to 28, comprising the heavy chain of SEQ ID NO: 15 and the light chain of SEQ ID NO: 14.

[0293] 32. The antibody of any one of embodiments 1 to 28, comprising the heavy chain of SEQ ID NO: 16 and the light chain of SEQ ID NO: 14.

[0294] 33. The antibody of any one of embodiments 1 to 28, comprising the heavy chain of SEQ ID NO: 32 and the light chain of SEQ ID NO: 14.

[0295] 34. The antibody of any one of embodiments 1 to 28, comprising (a) a VH domain comprising (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 03; (ii) a CDR-H2 having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 04; and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 05; and (b) a VL domain comprising (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 06; (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 07; and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 08.

[0296] 35. An antibody that binds to VEGF, which is an affinity matured variant of the antibody having the VH sequence of SEQ ID NO: 01 and the VL sequence of SEQ ID NO: 02.

[0297] 36. An antibody that binds to VEGF, which is a variant of an antibody having a VH sequence of SEQ ID NO: 01 and a VL sequence of SEQ ID NO: 02, wherein the antibody significantly inhibits the binding of VEGF to the VEGF receptor VEGF-R2 without significantly inhibiting the binding of VEGF to the VEGF receptor VEGF-R1.

[0298] 37. An antibody that binds to VEGF that binds to the same epitope as an antibody having the VH sequence of SEQ ID NO: 01 and the VL sequence of SEQ ID NO: 02.

[0299] 38. The antibody of any of the preceding embodiments, wherein the antibody comprises a CDR-H2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 04, SEQ ID NO: 10, and SEQ ID NO: 12.

[0300] 39. The antibody of any of the preceding embodiments, wherein the antibody comprises H-FR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 46 and SEQ ID NO: 47.

[0301] 40. An isolated nucleic acid encoding the antibody of any one of embodiments 1 to 37.

[0302] 41. A host cell comprising the nucleic acid of embodiment 36.

[0303] 42. A method for producing an antibody that binds to VEGF, comprising culturing the host cell of embodiment 37 under conditions suitable for expression of the antibody.

[0304] 43. The method of embodiment 38, further comprising recovering the antibody from the host cell.

[0305] 44. An antibody produced by the method of embodiment 38 or 39.

[0306] 45. A pharmaceutical composition comprising the antibody of any one of embodiments 1 to 35 and a pharmaceutically acceptable carrier.

[0307] 46. ​​The antibody of any one of embodiments 1 to 35 or the pharmaceutical composition of embodiment 41 for use as a medicament.

[0308] 47. The antibody of any one of embodiments 1 to 35 or the pharmaceutical composition of any one of embodiment 41, for use in treating a VEGF-related disease.

[0309] 48. The antibody of any one of embodiments 1 to 35 or the pharmaceutical composition of any one of embodiment 41, for use in treating cancer.

[0310] 49. The antibody of any one of embodiments 1 to 35 or the pharmaceutical composition of any one of embodiment 41 for use in treating an eye disease.

[0311] 50. Use of the antibody of any one of embodiments 1 to 35 or the pharmaceutical composition of embodiment 41 in the preparation of a medicament for treating a VEGF-related disease.

[0312] 51. Use of the antibody of any one of embodiments 1 to 35 or the pharmaceutical composition of embodiment 41 in the preparation of a medicament for treating cancer.

[0313] 52. Use of the antibody of any one of embodiments 1 to 35 or the pharmaceutical composition of embodiment 41 in the preparation of a medicament for treating an eye disease.

[0314] 53. Use of the antibody of any one of embodiments 1 to 35 or the pharmaceutical composition of embodiment 41 in the preparation of a medicament for inhibiting angiogenesis.

[0315] 54. A method of treating an individual suffering from a VEGF-related disease, comprising administering to the individual an effective amount of the antibody of any one of embodiments 1 to 35 or the pharmaceutical composition of embodiment 41.

[0316] 55. 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 35 or the pharmaceutical composition of embodiment 41 to inhibit angiogenesis.

[0317] Description of amino acid sequences

[0318]

[0319]

[0320]

[0321]

[0322]

[0323] Listed below are the amino acid sequences of the VH and VL domains, including the labeled HVRs (HVRs are indicated by bold and underlined letters) of the anti-VEGF antibodies VEGF-0089, VEGF-0113, and VEGF-0114:

[0324] Antibody VEGF-0089:

[0325] VH domain (SEQ ID NO: 01)

[0326]

[0327] VL domain (SEQ ID NO: 02)

[0328]

[0329] Antibody VEGF-0113:

[0330] VH domain (SEQ ID NO: 09)

[0331]

[0332] VL domain (SEQ ID NO: 02)

[0333]

[0334] Antibody VEGF-0114:

[0335] VH domain (SEQ ID NO: 11)

[0336]

[0337] VL domain (SEQ ID NO: 02)

[0338]

[0339] Antibody VEGF-P1 AD8675:

[0340] VH domain (SEQ ID NO: 33)

[0341]

[0342] VL domain (SEQ ID NO: 02)

[0343]

[0344] Antibody VEGF-P1AE3520:

[0345] VH domain (SEQ ID NO: 42)

[0346]

[0347] VL domain (SEQ ID NO: 02)

[0348]

[0349] Antibody VEGF-P1AE3521:

[0350] VH domain (SEQ ID NO: 44)

[0351]

[0352] VL domain (SEQ ID NO: 02)

[0353]

[0354] Example

[0355] The following are examples of methods and compositions of the present invention. It should be understood that various other embodiments may be practiced, given the general description provided above.

[0356] Example 1:

[0357] Generation of human anti-VEGF antibody (antibody VEGF-0089)

[0358] Figure 1 An overlay of crystal structures of a human VEGF dimer in complex with VEGF-R1 domain 2 and VEGF-R3 domains 2 and 3 is depicted. This superimposition indicates that both VEGF receptors bind to highly similar regions on the VEGF dimer, and therefore it would appear to be highly challenging to generate antibodies that bind to VEGF in the same manner without inhibiting VEGF binding to both receptors VEGF-R1 and VEGF-R2. Consistent with this, among the various anti-VEGF antibodies known in the art, only a few have been reported to selectively block VEGF binding to VEGF-R2 but not to VEGF binding to VEGF-R1.

[0359] The antibody VEGF-0089 described herein is derived from Roche's proprietary transgenic rabbits that express a humanized antibody repertoire after immunization with a VEGF-derived antigen. Transgenic rabbits containing human immunoglobulin loci are reported in WO2000 / 46251, WO2002 / 12437, WO2005 / 007696, WO2006 / 047367, US2007 / 0033661, and WO2008 / 027986. Animals were housed in an AAALAC-accredited animal facility according to Appendix A "Guidelines for Accommodation and Care of Animals." All animal immunization protocols and experiments were approved by the Government of Upper Bavaria (Permit No. 55.2-1-54-2532-90-14) and performed in accordance with German animal welfare regulations and Directive 2010 / 63 of the European Parliament and Council.

[0360] Immunization of transgenic rabbits

[0361] Briefly, 12-16 week old rabbits (n=3) were immunized with recombinant human VEGF-121 protein conjugated to keyhole limpet hemocyanin (KLH) (prepared in-house). All animals were immunized with 400 μg of protein emulsified in complete Freund's adjuvant (CFA) by intradermal administration on day 0, followed by 200 μg of protein emulsion by alternating intramuscular and subcutaneous injections on weeks 1, 2, 6, 11, and 14. Starting with the fourth immunization, blood was collected on days 4, 5, and 6 after immunization. Serum was prepared for determination of immunogen-specific rabbit and human-specific immunoglobulin titers by ELISA, and peripheral mononuclear cells were isolated and used as a source of antigen-specific B cells in the B cell cloning process.

[0362] Cloning of B cells from transgenic rabbits

[0363] Isolation of Rabbit Peripheral Blood Mononuclear Cells (PBMCs): Blood samples were collected from immunized rabbits. Whole blood containing EDTA was diluted two-fold with 1× PBS (PAA) and then subjected to density centrifugation using mammalian lymphocyte separation medium (Cedarlane Laboratories) according to the manufacturer's instructions. PBMCs were washed twice with 1× PBS.

[0364] EL-4 B5 medium: RPMI 1640 (Pan Biotech) supplemented with 10% FCS (Pan Biotech), 2 mM glutamine, 1% penicillin / streptomycin solution (Gibco), 2 mM sodium pyruvate, 10 mM HEPES (PAN Biotech), and 0.05 mM β-mercaptoethanol (Invitrogen) was used.

[0365] Plate coating: Sterile cell culture 6-well plates were coated with 2 μg / ml KLH in carbonate buffer (0.1 M sodium bicarbonate, 34 mM disodium bicarbonate, pH 9.55) overnight at 4° C. Before use, the plates were washed three times in sterile PBS.

[0366] Depletion of Macrophages / Monocytes or Human Fc Binders: PBMCs were seeded in sterile 6-well plates (cell culture grade) to deplete macrophages and monocytes by nonspecific adhesion. Each well was filled with up to 4 ml of culture medium and up to 6 × 10e6 PBMCs from immunized rabbits and allowed to bind for 1 hour in an incubator at 37°C. Cells in the supernatant (peripheral blood lymphocytes (PBLs)) were used for the antigen panning step.

[0367] Immunofluorescence staining and flow cytometry: Anti-IgG FITC (AbD Serotec) and anti-huCk PE (Dianova) antibodies were used for single cell sorting. For surface staining, cells from the depletion and enrichment steps were incubated with anti-IgG FITC and anti-huCk PE antibodies in PBS and incubated at 4°C in the dark for 45 minutes. After staining, PBMCs were washed twice with ice-cold PBS. Finally, PBMCs were resuspended in ice-cold PBS and immediately subjected to FACS analysis. Propidium iodide (BD Pharmingen) at a concentration of 5 μg / ml was added before FACS analysis to distinguish between dead and live cells. TM Becton Dickinson FACSAria software TM (BD Biosciences) was used for single-cell sorting.

[0368] B cell culture: Rabbit B cells were cultured by the method described by Seeber et al. (S Seeber et al. PLoS One 9(2), e86184. February 4, 2014). Briefly, single sorted rabbit B cells were plated in 96-well plates with Cells (1: 100000) (Calbiochem), 5% rabbit thymocyte supernatant (MicroCoat) and γ-irradiated mouse EL-4B5 thymoma cells (5×10e5 cells / well) in 200 μl / well EL-4B5 medium were incubated in an incubator at 37° C. for 7 days. The supernatant of the B cell culture was removed for screening, and the remaining cells were immediately harvested and frozen at −80° C. in 100 μl RLT buffer (Qiagen).

[0369] Isolation of RNA encoding the antibody V domain. For recombinant expression of the antibody, the PCR product encoding VH or VL was cloned as cDNA into an expression vector and transiently transformed into HEK-293 cells.

[0370] An antibody comprising the VH domain of SEQ ID NO: 01 and the VL domain of SEQ ID NO: 02 was selected from the screen. This antibody is also referred to herein as the antibody "VEGF-0089." For subsequent analysis, an antibody was generated as a Fab fragment of VEGF-0089 (referred to herein as the "VEGF-0089 Fab fragment" or simply "VEGF-0089 Fab") having human VH and VL domains and rabbit-derived light chain constant domain (CLκ) and heavy chain constant domain (CH1). The amino acid sequence of the heavy chain of the VEGF-0089 Fab fragment is SEQ ID NO: 13. The amino acid sequence of the light chain of the VEGF-0089 Fab fragment is SEQ ID NO: 14.

[0371] Example 2:

[0372] Characterization of the generated human anti-VEGF antibody (antibody VEGF-0089)

[0373] Binding of VEGF to the antibody VEGF-0089 Fab fragment was assessed by surface plasmon resonance (SPR) as described below.

[0374] Determining Antibody Binding Affinity by Surface Plasmon Resonance (SPR)

[0375] An anti-His capture antibody (GE Healthcare 28995056) was immobilized to a Series S sensor chip C1 (GE Healthcare 29104990) using standard amine coupling chemistry, resulting in a surface density of 500-1000 resonance units (RU). HBS-P+ (10 mM HEPES, 150 mM NaCl, pH 7.4, 0.05% surfactant P20) was used as running and dilution buffer, and the measurement temperature was set to 25°C and 37°C, respectively. hVEGF-A121 was captured to the surface, resulting in capture levels of 5 to 35 RU. A dilution series of anti-VEGF antibody (0.37-30 nM) was injected for 120 s, and dissociation was monitored for at least 600 s at a flow rate of 30 μl / min. The surface was regenerated by injecting 10 mM glycine (pH 1.5) for 60 s. The bulk refractive index differences were corrected by subtracting blank injections and by subtracting the response obtained from a control flow cell in which hVEGF-A121 was not captured. The rate constants were calculated using the Langmuir 1:1 binding model in Calculated within the evaluation software.

[0376] As a result, the KD of the VEGF-0089 Fab fragment was determined to be 134 pM (at a temperature of 25°C).

[0377] To further characterize the antibodies, inhibition of VEGF binding to its receptors VEGF-R1 and VEGF-R2 in the presence of VEGF-0089 Fab fragment was assessed as follows:

[0378] Inhibition of VEGF binding to VEGF-R1 and VEGF-R2 in the presence of antibody Fab fragments (VEGF:VEGF-R2 / R1 inhibition ELISA).

[0379] 384-well streptavidin plates (Nunc / Microcoat #11974998001) were coated with 0.25 μg / ml biotinylated VEGF-R1 or 0.5 μg / ml biotinylated VEGF-R2 (prepared in-house, 25 μl / well each in DPBS (1x) (PAN, #P04-36500). The plates were incubated at room temperature for 1 hour. In parallel, VEGF-121-His (prepared in-house) at a concentration of 0.7 nM was incubated with various dilutions of the antibody (12 x 1:2 dilution steps, starting at a concentration of 500 nM). This preincubation step was performed in 384-well PP plates (Weidmann Medical Technology, #23490-101) in 1x OSEP buffer (10x double-distilled water, Roche, #11 666 789 001 + 0.5% bovine serum albumin fraction V, fatty acid-free, Roche, #10 735086 001 + 0.05% Tween 20). The plates were incubated at room temperature for 1 hour. After washing the VEGF-R1 / VEGF-R2-coated streptavidin plate three times with 90 μl / well of PBST buffer (10x double-distilled water, Roche #11666789001 + 0.1% Tween 20), 25 μl of the sample from the VEGF antibody preincubation plate was transferred to the coated streptavidin plate and incubated at room temperature for 1 hour. After washing three times with 90 μl / well PBST buffer, 25 μl / well of detection antibody (anti-His POD, Bethyl, #A190-114P, 1:12000) in 1x OSEP was added. After incubation at room temperature for 1 hour, the plate was washed three times with 90 μl PBST buffer. Simultaneously, 25 μl of TMB (Roche, #11835033001) was added to all wells. After incubation at room temperature for 10 minutes, the signal was detected at 370 nm / 492 nm on a Tecan Safire 2 reader.

[0380] As a representative of the prior art anti-VEGF antibodies used in control and clinical settings, (ranibizumab, heavy chain amino acid sequence of SEQ ID NO: 23, light chain amino acid sequence of SEQ ID NO: 24) were evaluated under the same conditions. The results are shown in Figure 2 middle.

[0381] The results indicate that the VEGF-0089 Fab fragment is able to completely block the binding of VEGF to VEGF-R2. The VEGF-0089 Fab fragment does not completely block the binding of VEGF to VEGF-R1. Therefore, it is believed that the VEGF-0089 Fab fragment selectively blocks VEGF signaling through VEGF-R2 rather than through VEGF-R1. Figure 2 As shown in the prior art antibodies It can completely block the binding of VEGF to two receptors, VEGF-R2 and VEGF R1.

[0382] Example 3:

[0383] Improvements in human anti-VEGF antibody VEGF-0089

[0384] To further improve the properties of the antibody, antibody variants of the antibody VEGF-0089 were generated.

[0385] From the various antibody candidates, two antibodies were selected in which the glycine in the middle of a three-glycine stretch within the H-CDR2 loop was substituted or deleted in the H-CDR2 loop.

[0386] VEGF-0113 antibody

[0387] A first variant of the VEGF-0089 antibody is an antibody comprising the heavy chain variable domain of SEQ ID NO: 09 and the light chain variable domain of SEQ ID NO: 02. This antibody is referred to herein as the "VEGF-0113" antibody, and—when provided as a Fab fragment having rabbit-derived CLκ and CH1 domains—the antibody is referred to as the "VEGF-0113 Fab fragment" or simply "VEGF-0113 Fab." The VEGF-0113 Fab fragment used herein comprises the heavy chain of SEQ ID NO: 15 and the light chain of SEQ ID NO: 14. The VEGF-0113 antibody differs from the VEGF-0089 antibody only in the amino acid sequence of the H-CDR2, as the glycine residue at position 6 of the H-CDR2 of the VEGF-0089 antibody is replaced by a proline residue:

[0388] H-CDR2 SIGNGGGIYTYYADSVKG of VEGF-0089 (SEQ ID NO: 04)

[0389] H-CDR2 SIGNGPGIYTYYADSVKG of VEGF-0113 (SEQ ID NO: 10)

[0390] The amino acid motif "GPG" implemented in H-CDR2 of VEGF-0113 confers less conformational freedom to the H-CDR2 loop when compared to the three glycine stretch "GGG" in H-CDR2 of VEGF-0089.

[0391] VEGF-0114 antibody

[0392] A second variant of the VEGF-0089 antibody is an antibody comprising the heavy chain variable domain of SEQ ID NO: 11 and the light chain variable domain of SEQ ID NO: 02. This antibody is referred to herein as the "VEGF-0114" antibody, and when provided as a Fab fragment having rabbit-derived CLκ and CH1 domains, the antibody is referred to as the "VEGF-0114 Fab fragment" or simply "VEGF-0114 Fab." The VEGF-0114 Fab fragment used herein comprises the heavy chain of SEQ ID NO: 16 and the light chain of SEQ ID NO: 14. The VEGF-0114 antibody differs from the VEGF-0089 antibody only in the amino acid sequence of the H-CDR2, as the asparagine residue at position 4 of the H-CDR2 of the VEGF-0089 antibody is replaced with a serine residue, the glycine residue at position 7 of the H-CDR2 of the VEGF-0089 antibody is removed, and the isoleucine residue at position 8 is replaced with a phenylalanine residue:

[0393] H-CDR2 SIGNGGGIYTYYADSVKG of VEGF-0089 (SEQ ID NO: 04)

[0394] H-CDR2 of VEGF-0114 (SEQ ID NO: 12)

[0395] To generate antibody VEGF-0114, the third glycine residue in the three-glycine stretch "GGG" in H-CDR2 of VEGF-0089 was removed. In addition, a phenylalanine residue was introduced after the three-glycine stretch to replace the isoleucine residue.

[0396] Example 4:

[0397] Evaluation of anti-VEGF antibody binding to VEGF by direct ELISA

[0398] Binding of prior art antibodies 2C3 and r84 to VEGF was assessed by direct ELISA as follows:

[0399] VEGF binding ELISA

[0400] Will MaxiSorp TM 384-well μ-transparent plates (#464718) were coated with 1 μg / ml VEGF-121-His (homemade) and incubated at room temperature for 1 hour. 90 μl / well PBST-buffer (double-distilled water, 10x PBS Roche #11666789001 + 0.1% 20) After washing three times, add 90 μl / well of blocking buffer (double distilled water, 10xPBS Roche #11666789001 + 2% bovine serum albumin fraction V, without fatty acids, Roche, #10735078001 + 0.05% 20) and incubated at room temperature for 1 hour. After washing three times with 90 μl / well PBST buffer, 25 μl of a dilution series of antibody starting at 60 nM (16 x 1:2 dilution steps) was added to the VEGF-coated wells. After incubation at room temperature for 1 hour, the plate was again washed three times with 90 μl / well PBST buffer. 25 μl / well of detection antibodies (anti-c-myc POD (Bethyl, #A190-104P, 1:16000) or anti-huλ LC (Bethyl, #A80-116P, 1:15000) or F(ab')2 fragment goat anti-hu IgG Fcγ fragment specific (JIR, #109-036-098, 1:2000) or goat anti-hu Igκ LC (Millipore, #AP502P, 1:2000) in 1x PBS (10x, Roche, #11666789001) + 0.5% bovine serum albumin fraction V, fatty acid-free, Roche, #10 735 086 001 + 0.05% Tween 20) were added and the plate was incubated for 1 hour at room temperature. After washing three times with 90 μl PBST buffer, 25 μl TMB (Roche, #11 835 033 001) was added to each well. After incubation for 3 min at room temperature, the signal was detected at 370 nm / 492 nm on a Tecan Safire 2 reader.

[0401] The Fab fragments of the antibodies of the present invention were tested separately. The following antibodies were tested (see Table 1):

[0402] Table 1: Amino acid sequences of antibodies of the present invention

[0403]

[0404]

[0405] The same method as described in Example 1 (transient transformation of HEK-293 cells) was used to generate prior art antibodies. The following prior art antibody Fab fragments were analyzed in parallel: Ranibizumab Fab fragments of prior art antibodies 2C3 (disclosed in WO200064946), r84 (disclosed in WO2009060198) and L3H6 (disclosed in WO2012089176) In addition, antibodies 2C3 and r84 were analyzed as full-length IgG1 antibodies.

[0406] The amino acid sequences of the prior art antibodies used are listed in Table 2.

[0407] Table 2: Amino acid sequences of prior art antibodies:

[0408] Heavy chain light chain 2C3 Fab SEQ ID NO: 17 SEQ ID NO: 18 r84 Fab SEQ ID NO: 19 SEQ ID NO: 20 2C3 IgG1 SEQ ID NO: 25 SEQ ID NO: 26 r84 IgG1 SEQ ID NO: 27 SEQ ID NO: 28 L3H6 Fab SEQ ID NO: 21 SEQ ID NO: 22 Ranibizumab SEQ ID NO: 23 SEQ ID NO: 24

[0409] The results are shown in Figure 3 While no VEGF binding was observed under the assay conditions used for antibody 2C3, VEGF binding was confirmed for the prior art antibodies r84 and L3H6. Antigen binding for antibody r84 was demonstrated to be significantly reduced when used as a Fab fragment rather than a full-length IgG1 due to a lack of avidity. VEGF binding was confirmed for all antibodies of the invention tested.

[0410] Example 5:

[0411] Binding affinity of the antibodies of the present invention

[0412] The affinity of the antibodies was determined by SPR using the same method as described in Example 2. All Fab fragments listed in Tables 1 and 2 were tested.

[0413] As described above, the affinity of the antibodies of the present invention and the prior art antibodies was evaluated. The results are shown in Table 3.

[0414] Table 3: Affinity of anti-VEGF antibodies

[0415] KD at 25℃[pM] KD at 37℃ [pM] VEGF-0089 Fab 134 99 VEGF-0113 Fab 21 17 VEGF-0114 Fab 55 56 2C3 Fab tbd tbd r84 Fab tbd tbd L3H6 Fab 3356 4517 Ranibizumab 154+ / -47 507

[0416] Example 6:

[0417] Inhibition of VEGF binding to VEGF-R2 in the presence of anti-VEGF antibodies

[0418] Binding of VEGF to VEGF-R2 and VEGF-R1, respectively, in the presence of the antibody Fab fragments of the invention is tested as follows.

[0419] Inhibition of VEGF binding to VEGF-R1 and VEGF-R2 in the presence of antibody Fab fragments (VEGF:VEGF-R2 / R1 inhibition ELISA).

[0420] A 384-well streptavidin plate (Nunc / Microcoat #11974998001) was coated with 0.25 μg / ml biotinylated VEGF-R1 or 0.5 μg / ml biotinylated VEGF-R2 (prepared in-house, 25 μl / well of each in DPBS (1x) (PAN, #P04-36500). The plate was incubated at room temperature for 1 hour. In parallel, VEGF-121-His (prepared in-house) at a concentration of 0.7 nM was incubated with various dilutions of the antibody (12 x 1:2 dilution steps, starting at a concentration of 500 nM). This pre-incubation step was performed in 384-well PP plates (Weidmann medical technology, #23490-101) in 1x OSEP buffer (10x double-distilled water, Roche, #11 666 789 001 + 0.5% bovine serum albumin fraction V, fatty acid-free, Roche, #10 735 086 001 + 0.05% Tween 20). The plates were incubated at room temperature for 1 hour. 90 μl / well of PBST buffer (10x double-distilled water, Roche #11666789001 + 0.1% 20) After washing the VEGF-R1 / VEGF-R2-coated streptavidin plate three times, 25 μl of sample from the VEGF antibody pre-incubated plate was transferred to the coated streptavidin plate, which was then incubated at room temperature for 1 hour. After washing three times with 90 μl / well PBST buffer, 25 μl / well of detection antibody (anti-His POD, Bethyl, #A190-114P, 1:12000) in 1x OSEP was added. After incubation at room temperature for 1 hour, the plate was washed three times with 90 μl PBST buffer. Simultaneously, 25 μl of TMB (Roche, #11 835 033 001) was added to all wells. After incubation at room temperature for 10 minutes, the signal was detected at 370 nm / 492 nm on a Tecan Safire 2 reader.

[0421] In the first experiment, the antibodies of the invention, VEGF-0089, VEGF-0113 and VEGF-0114 (see Table 1), were tested, and the prior art antibodies, L3H6 Fab and ranibizumab (see Table 2), were analyzed in the presence of 0.4 nM VEGF. As a negative control, ranibizumab was also analyzed in the absence of VEGF (denoted as "ranibizumab w / o hisVEGF" in the figure). The results are shown in FIG. Figure 4A middle.

[0422] In a second experiment, all antibodies listed in Tables 1 and 2 were analyzed as described in Example 2 in the presence of 0.7 nM VEGF. The results are shown in Figure 4B middle.

[0423] In the experimental setup used to test the prior art antibodies, inhibition of VEGF binding to VEGF-R2 was not assessed. The experiment was repeated under the same conditions as above, but using 0.34 nM VEGF-121-His instead of 0.7 nM. The results are shown in Figure 7 middle.

[0424] In this experimental setting, inhibition of VEGF binding to VEGF-R2 was observed for high concentrations of the prior art antibodies r84 IgG1, r84 Fab and L3H6 Fab. The antibodies of the invention VEGF-0089 Fab, VEGF-0113 Fab and VEGR-0114 Fab inhibited VEGF binding to VEGF-R2 at all concentrations tested.

[0425] Example 7:

[0426] Inhibition of VEGF binding to VEGF-R1 in the presence of anti-VEGF antibodies

[0427] Binding of VEGF to VEGF-R1 was tested in the presence of antibody Fab fragments of the invention as described in Example 6. All antibodies listed in Tables 1 and 2 were tested. The results are shown in Table 1. Figure 5 middle.

[0428] In the experimental setup used for the tested prior art antibodies 2C3, R84 and L3H6, inhibition of VEGF binding to VEGF-R1 was not assessed.

[0429] The experiment was repeated under the same conditions as above, but using 0.34 nM VEGF-121-His instead of 0.7 nM. The results are shown in Figure 5 middle.

[0430] Example 8:

[0431] IC50 evaluation of anti-VEGF antibodies by reporter gene assay (RGA)

[0432] The anti-VEGF antibodies of the invention (see Table 1) were tested at nine concentrations ranging from 20 nM to 0.02 nM. The prior art antibody Ranibizumab (see Table 2) was tested as a control.

[0433] Briefly, 37.5 μl of antibody solution / well was mixed with 37.5 μl of VEGF-A121: 25 ng / ml / well per well of a white 96-well multititer plate and incubated at room temperature for 30 minutes. Subsequently, 75 μl / well of HEK293-NFAT-RE-Luc2P / KDR suspension was added per 40,000 cells / well and incubated at 37°C, 5% CO2 for 5 hours. Finally, 100 μl / well of Bio-Glo TM The results are shown in Table 4.

[0434] Table 4: IC50 of anti-VEGF antibodies

[0435] IC50[nM] VEGF-0089 0,59 VEGF-0113 0,63 VEGF-0114 1,34 Ranibizumab 0,81

[0436] The data demonstrated antagonistic binding to VEGF by all antibodies tested.

[0437] Example 9:

[0438] Providing improved variants of the human anti-VEGF antibody VEGF-0089

[0439] Additional improved antibody variants of antibody VEGF-0089 were generated, particularly to improve the preference for inhibiting VEGF binding to VEGF-R2 rather than VEGF binding to VEGF-R1. From the various antibody candidates, three candidates listed in Table 5 were selected. The antibody Fab fragments listed in Table 5 were generated using the same method as described in Example 1 (transient transformation of HEK-293 cells).

[0440] Table 5: Amino acid sequences of antibodies of the present invention

[0441]

[0442]

[0443] Example 10:

[0444] Binding affinity of the antibodies of the present invention

[0445] The affinity of the antibodies was determined by SPR using the same method as described in Example 2. All Fab fragments of the antibodies of the invention listed in Tables 1 and 5 were tested. In addition, additional prior art antibodies HF2-1, HF2-5, HF2-9 and HF2-11 (disclosed in EP3006465) were evaluated as described above. The amino acid sequences of those additional prior art antibodies are shown in Table 6.

[0446] Table 6: Amino acid sequences of prior art antibodies disclosed in EP3006465

[0447] Heavy chain light chain HF2-1 Fab SEQ ID NO: 34 SEQ ID NO: 35 HF2-5 Fab SEQ ID NO: 36 SEQ ID NO: 35 HF2-9 Fab SEQ ID NO: 37 SEQ ID NO: 38 HF2-11 Fab SEQ ID NO: 39 SEQ ID NO: 40

[0448] The results are shown in Table 7.

[0449] Table 7: Affinity of anti-VEGF antibodies

[0450] KD at 25℃[pM] KD at 37℃ [pM] VEGF-0089 Fab 143 110 VEGF-0113 Fab 22 29 VEGF-0114 Fab 50 54 VEGF-P1AD8675 88 65 VEGF-P1AE3520 31 39 VEGF-P1AE3521 32 61 HF2-1 Fab 279 574 HF2-5 Fab 52 109 HF2-9 Fab 24 51 HF2-11 Fab 32 62

[0451] Example 11:

[0452] Inhibition of VEGF binding to VEGF-R2 or VEGF-R1 in the presence of anti-VEGF antibodies

[0453] Binding of VEGF to VEGF-R2 and VEGF-R2 was tested as described in Example 2 in the presence of antibody Fab fragments of the invention.

[0454] In these experiments, the antibodies of the invention, VEGF-0089, VEGF-0113, VEGF-0114, VEGF-P1AD8675, VEGF-P1AE3520 and VEGF-P1AE3521 (see Tables 1 and 5), were tested, and the prior art antibodies HF2-1, HF2-5, HF2-9 and HF2-11 (see Table 6) and ranibizumab (see Table 2) were analyzed in the presence of VEGF. The results are shown in Figure 8 and Figure 10 (in the presence of 0.34 nM VEGF) and Figure 9 and Figure 11 (in the presence of 0.7 nM VEGF).

[0455] The prior art antibodies tested did not significantly inhibit the binding of VEGF to the VEGF receptor VEGF-Rl. All antibodies tested of the present invention preferentially inhibited the binding of VEGF to VEGF-R2 rather than the binding of VEGF to VEGF-Rl.

[0456] Example 12:

[0457] Chemical stability of exemplary antibodies of the invention

[0458] The chemical stability of exemplary antibody Fab fragments of the present invention was tested as follows:

[0459] Chemical degradation testing:

[0460] 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 (2 weeks). The PBS sample was further incubated for a total of 4 weeks (4 weeks). A third control aliquot was stored at -80°C. At the end of the incubation period, the samples were analyzed for relative activity concentration (Biacore; the activity concentration of two stressed aliquots of each conjugate was normalized to that of the unstressed 4°C aliquot), aggregation (SEC), and fragmentation (capillary electrophoresis or SDS-PAGE) and compared to untreated controls.

[0461] For size exclusion UHPLC (=SEC), a chromatography gel such as TSKgel UP-SW3000 is used to separate proteins according to their molecular size in solution. Using this method, protein solutions are analyzed for the relative amounts of monomers, high molecular weight species (e.g., aggregates, dimers, impurities), and low molecular weight species (e.g., degradation products, impurities). 0.2 M potassium phosphate, 0.25 M KCl, pH 6.2 is used as the mobile phase. The protein solution is diluted, for example, ~50 μl of protein is injected in a volume of 5 μl and analyzed at 25°C at a flow rate of 0.3 ml / min, with protein detection at 280 nm. Peak definition and peak integration are performed as shown in the typical chromatogram in the product specific information file.

[0462] The Fab fragments of antibodies VEGF-0089, VEGF-0113, VEGF-0114, VEGF-P1AD8675, VEGF-P1AE3520 and VEGF-P1AE3521 were analyzed (see Tables 1 and 5). The results are shown in Tables 8 and 9.

[0463] Table 8: Improved VEGF binding activity after stress of antibody Fab fragments

[0464]

[0465] Table 9: Improved molecular integrity of antibody Fab fragments after stress (4 weeks, pH 7.4, 37°C)

[0466]

[0467] Example 13:

[0468] X-ray crystallography and epitope determination of the generated antibody VEGF-0089 in complex with VEGF-dimer

[0469] The crystal structure of the VEGF-0089 Fab fragment described above was analyzed according to standard methods known in the art.

[0470] X-ray crystallography of the VEGF-0089 Fab fragment in complex with VEGF-A121 was performed as follows:

[0471] Complex Formation and Purification of the Dimeric Complex VEGF-A121-VEGF-0089 Fab. To form the complex, the VEGF-0089 Fab fragment and human VEGF-A121 (Peprotech) were mixed at a 1.1:1 molar ratio. After incubation overnight at 4°C for 16 hours, the complex was purified by gel filtration chromatography on a Superdex 200 (16 / 600) column in 20 mM MES, 150 mM NaCl, pH 6.5. Fractions containing the dimeric complex were pooled and concentrated to 1.44 mg / ml.

[0472] Crystallization of the dimeric VEGF-A121-VEGF-0089 Fab complex. Initial crystallization trials were performed at 21°C in a sitting-drop vapor diffusion apparatus at a protein concentration of 11.5 mg / ml. Crystals emerged from 0.1 M Tris pH 8.5, 0.2 M LiSO₄, 1.26 M (NH₄)₂SO₄ within 1 day. Plate-shaped crystals grew to a final size of 150 x 100 x 30 μm within a week. Crystals were harvested directly from the screening plate without any further optimization steps.

[0473] Data collection and structure determination. For data collection, crystals were rapidly cooled at 100 K in a precipitant solution with 15% ethylene glycol added as a cryoprotectant. The crystals were detected at beamline X10SA of the Swiss Light Source (Villigen, Switzerland) using a PILATUS 6M detector. The diffraction data were collected at a wavelength of . The data have been processed with XDS (Kabsch, W. Acta Cryst. D 66, 133-144 (2010)) and scaled with SADABS (BRUKER). The crystal belongs to space group C2, with the cell axes being β=104.54°, and diffracts to Resolution. The coordinates of the relevant internal structures of the Fab fragment and VEGF were used as a search model, and the structure was determined by molecular replacement using PHASER (McCoy, A.J, Grosse-Kunstleve, R.W., Adams, P.D., Storoni, L.C., and Read, R.J. J. Appl. Cryst. 40, 658-674 (2007)). Programs from the CCP4 suite (Collaborative Computational Project, Number 4 Acta Cryst. D50, 760-763 (1994)) and Buster (Bricogne, G., Blanc, E., Brandl, M., Flensburg, C., Keller, P., Paciorek, W., Roversi, P., Sharff, A., Smart, O.S., Vonrhein, C., Womack, T.O. (2011). Buster version 2.9.5 Cambridge, United Kingdom: Global Phasing Ltd) were used for subsequent refinement of the data. Manual rebuilding of the protein using difference electron density was done with COOT (Emsley, P., Lohkamp, B., Scott, W.G. and Cowtan, K. Acta Cryst D66, 486-501 (2010)). Data collection and refinement statistics for both structures are summarized in Table 10. All illustrations were prepared with PYMOL (DeLano Scientific, Palo Alto, CA, 2002).

[0474] Table 10: Data collection and structure modification statistics

[0475]

[0476]

[0477] 1 Values in parentheses refer to the highest resolution shell.

[0478] 2 R merge = ∑|I - | / ∑I, where I is the intensity.

[0479] 3 R work = Σ|F o - <F c >| / ΣF o , where Fo is the observed and Fc is the calculated structure factor amplitude.

[0480] 4 R free Calculations are based on 5% of the total data omitted during the correction process.

[0481] Schematic diagram of the crystal structure of two VEGF-0089 Fab fragments in complex with human VEGF-A121 dimer Figure 12 As shown. The amino acid residues in the VEGF-A121 dimer that contact the VEGF-A0089 Fab fragment form a conformational epitope on the VEGF-A121 dimer that is bound by the VEGF-A0089 Fab. The amino acid sequence of VEGF-A121 is shown in SEQ ID NO: 45. Figure 13 Schematic representation of the amino acids contained in the epitope on the two VEGF-A121 molecules in the VEGF dimer is highlighted in FIG.

[0482] Antibody VEGF-0089 Fab binds to the following epitopes on the VEGF-A121 dimer:

[0483] - amino acids F17, M18, D19, Y21, Q22, R23, Y25, H27, P28, 129, E30, M55, N62, L66, N100, K101, C102, E103, C104, R105, and P106 of one of the individual VEGF-A121 molecules within the VEGF dimer; and

[0484] - Amino acids E30, K48, M81 and Q87 in another individual VEGF-A121 molecule within the VEGF dimer.

[0485] Although the foregoing invention has been described in detail by way of illustration and example for purposes of clarity of understanding, the description and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entirety.

Claims

1. Antibodies that bind to VEGF, It contains (a) the VH sequence of SEQ ID NO: 01 and the VL sequence of SEQ ID NO: 02, (b) the VH sequence of SEQ ID NO: 09 and the VL sequence of SEQ ID NO: 02, (c) the VH sequence of SEQ ID NO: 11 and the VL sequence of SEQ ID NO: 02, (d) a VH sequence of SEQ ID NO: 33 and a VL sequence of SEQ ID NO: 02, (e) the VH sequence of SEQ ID NO: 42 and the VL sequence of SEQ ID NO: 02, or (f) the VH sequence of SEQ ID NO: 44 and the VL sequence of SEQ ID NO:

02. The antibody according to claim 1 , which is a Fab fragment.

3. An isolated nucleic acid encoding the antibody of any one of claims 1 to 2. A host cell comprising the nucleic acid of claim 3 .

5. A method for producing an antibody that binds to VEGF, comprising culturing the host cell of claim 4 under conditions suitable for expression of the antibody. 6 . A pharmaceutical composition comprising the antibody according to claim 1 and a pharmaceutically acceptable carrier.

7. The pharmaceutical composition of claim 6, further comprising an additional therapeutic agent.

8. The antibody of any one of claims 1 to 2 or the pharmaceutical composition of any one of claims 6 to 7 for use as a medicament.

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