Antibodies that bind VEGF-A and IL6 and methods of use

Abstract

The present invention relates to, for example, an anti-VEGF-A / anti-IL6 antibody in the form of a bispecific Fab fragment and methods of using the same.

Description

[Technical Field]

[0001] The present invention relates to anti-VEGF-A / anti-IL6 antibodies and methods of use thereof. [Background technology]

[0002] Antibodies that bind to VEGF, such as ranibizumab, are used as therapeutic agents for treating ocular vascular diseases such as age-related macular degeneration. Antibodies that bind to IL6, such as those disclosed in WO 2014 / 074905, have been suggested for the treatment of ocular diseases.

[0003] WO 2012 / 163520 discloses a bispecific antibody ("DutaFab") containing two paratopes in a pair of VH and VL domains. Each paratope of the bispecific antibody of WO 2012 / 163520 contains amino acids from the heavy and light chain CDRs, with the heavy chain CDR-H1 and CDR-H3 and the light chain CDR-L2 contributing to the first paratope, and the light chain CDR-L1 and CDR-L3 and the heavy chain CDR-H2 contributing to the second paratope. Monospecific antibodies containing individual paratopes are independently isolated from different Fab libraries in which either the first or second paratope is diversified. The amino acid sequences of the monospecific antibodies are identified and fused to biparatopic VH and VL pairs. An example of an exemplary Fab fragment designated "VH6L" having a VL sequence of SEQ ID NO: 01 and a VH sequence of SEQ ID NO: 02 that specifically binds VEGF and IL-6 is disclosed in WO 2012 / 163520 as a proof-of-concept example.

[0004] Indeed, improved therapeutic antibodies that bind VEGF and IL6 for clinical application in ocular diseases are needed, for example, by improving efficacy over standard care and by improving duration of action, and thus reducing the frequency of intravitreal injections and the dosage burden for patients. Summary of the Invention

[0005] The present invention relates to bispecific anti-VEGF-A / anti-IL6 antibodies and methods of use thereof.

[0006] In one aspect, the present invention relates to an antibody that binds to human VEGF-A and human IL6, comprising: a VH domain comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 18, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 19, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 20; and a VL domain comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 15, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17; and comprising a variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 22 with up to five amino acid substitutions; and a variable light chain domain comprising the amino acid sequence of SEQ ID NO: 21 with up to five amino acid substitutions.

[0007] One embodiment of the present invention relates to an antibody that binds to human VEGF-A and human IL6, comprising the VH sequence of SEQ ID NO:22 and the VL sequence of SEQ ID NO:21.

[0008] One embodiment of the present invention relates to an antibody comprising the heavy chain amino acid sequence of SEQ ID NO:24 and the light chain amino acid sequence of SEQ ID NO:23.

[0009] One embodiment of the present invention relates to an antibody Fab fragment that binds to human VEGF-A and human IL6.

[0010] One embodiment of the present invention relates to a bispecific antibody Fab fragment that binds to human VEGF-A and human IL6.

[0011] In another aspect, the present invention provides an antibody that binds to IL6, which binds to the same epitope on IL6 as the antibody according to the present invention.

[0012] In another aspect, the present invention provides an antibody that binds to human IL6, a) a VH domain based on a human VH3 framework (the IL6 paratope comprises amino acid residues Y1, I2, Q3, Y26, E27, F28, T29, H30, Q31, D32, P52a, R94, I96, D97, F98, D101, and T102), and a VL domain based on a human Vkappa1 framework (the IL6 paratope comprises amino acid residues Y49, D50, S53, N54, Y55, P56, S57, Y91, and Y96); or b) An antibody is provided, comprising a VH domain based on a human VH3 framework (the IL6 paratope comprises amino acid residues Y1, P2, Q3, V26, L27, F28, K29, H30, Q31, D32, P52a, R94, L96, D97, F98, D101, E102), and a VL domain based on a human Vkappa1 framework (the IL6 paratope comprises amino acid residues Y49, D50, D53, R54, Y55, P56, E57, Y91, Y96) (numbering according to Kabat).

[0013] In another aspect, the invention provides an antibody that binds to IL6, which binds to the same epitope on IL6 as an antibody having a VL domain of SEQ ID NO: 35 and a VH domain of SEQ ID NO: 36. In one embodiment, the antibody comprises a VH domain having a human VH3 framework (the IL6 paratope comprises amino acid residues 1, 2, 3, 26, 27, 28, 29, 30, 31, 32, 52a, 94, 96, 97, 98, 101, 102) and a VL domain having a human Vkappa1 framework (the IL6 paratope comprises amino acid residues 49, 50, 53, 54, 55, 56, 57, 91, 96).

[0014] In one aspect, the invention provides an isolated nucleic acid encoding an antibody of the invention.

[0015] In one aspect, the invention provides a host cell comprising a nucleic acid of the invention. In one embodiment, the host cell is a CHO cell. In one embodiment, the host cell is an E. coli cell.

[0016] In one aspect, the invention provides an expression vector comprising a nucleic acid of the invention.

[0017] In one aspect, the invention provides a method for producing an antibody that binds human VEGF-A and human IL6, the method comprising culturing a host cell of the invention so that the antibody is produced.

[0018] In one aspect, the invention provides an antibody produced by the method of the invention.

[0019] In one aspect, the invention provides a pharmaceutical formulation comprising an antibody of the invention and a pharmaceutically acceptable carrier.

[0020] In one aspect, the invention provides a pre-filled syringe comprising an antibody of the invention and a pharmaceutically acceptable carrier.

[0021] In one aspect, the invention provides an ocular implant comprising an antibody of the invention and a pharmaceutically acceptable carrier. In one embodiment, the invention includes a port delivery device comprising an antibody of the invention.

[0022] In one aspect of the invention, the port delivery device administers an antibody or pharmaceutical formulation.

[0023] In one aspect, the invention provides an antibody of the invention for use as a medicament, in one embodiment for use in the treatment of vascular disease.

[0024] In one aspect, the invention provides use of an antibody of the invention or a pharmaceutical composition of the invention in the manufacture of a medicament, in one embodiment a medicament for treating a vascular disease.

[0025] In one aspect, the invention provides a method of treating an individual having a vascular disease, comprising administering to the individual an effective amount of an antibody of the invention or a pharmaceutical composition of the invention.

[0026] In one aspect, the invention provides a method for inhibiting angiogenesis in an individual, the method comprising administering to the individual an antibody of the invention or a pharmaceutical composition of the invention in an amount effective to inhibit angiogenesis.

[0027] The present invention provides therapeutic anti-VEGF-A / anti-IL6 antibodies capable of independently binding to their target antigens, even when provided in the form of antibody Fab fragments. They exhibit excellent KD within the pharmacologically relevant range and species cross-reactivity with the cynomolgus monkey target. The antibodies of the present invention are suitable for the treatment of ocular vascular diseases. The antibodies of the present invention offer several beneficial properties, including good expressibility and developability (e.g., high binding potency, high biophysical and biochemical stability, and high-concentration formulations) that enable their therapeutic application, high affinity for both targets that support particularly low effective doses, and advantageously high stability over long periods of time. Compared to non-antibody approaches, the antibodies of the present invention are more likely to be tolerated due to their high human nature and lack of artificial domains and linkers. Furthermore, the antibodies of the present invention are advantageously provided in highly concentrated liquid formulations with a viscosity suitable for ocular application. Because they can be provided at high concentrations, treatment with the antibodies of the present invention may be better tolerated by patients, as higher doses of therapeutic agent can be applied in a single treatment, allowing for longer treatment cycles. Bispecific Fab fragments, such as those described in the present invention, have an additional advantage over bispecific full-length IgG antibodies due to their much lower molecular weight. Fabs have a molecular weight of approximately 50 kDa, while full-length antibodies weigh three times as much (approximately 150 kDa) while providing the same number of binding sites. Therefore, for a given amount of drug, bispecific Fab fragments contain three times more binding sites than full-length IgG antibodies. [Brief explanation of the drawings]

[0028] [Figure 1] Binding of parental bispecific antibodies 6HVL_1 and V6HL_1 to human and cynomolgus monkey IL-6 as determined by surface plasmon resonance [Figure 2]VEGF IC50 of parent bispecific antibodies 6HVL_1 and V6HL_1 using human VEGF-165 [Figure 3] Improved bispecific antibody binding to human and cynomolgus IL6 as determined by surface plasmon resonance. [Figure 4] VEGF IC50 of improved bispecific antibodies using human VEGF-121 and VEGF-165. [Figure 5] Crystal structure of the Fab0182-IL-6 complex. Overall structure of IL-6 bound to Fab 0182. IL-6 is colored salmon, and the light and heavy chains of Fab 0182 are colored cyan and blue, respectively. [Figure 6] Crystal structure of the Fab 6HVL4.1-IL-6 complex. Overall structure of IL-6 bound to Fab 6HVL4.1. IL-6 is colored salmon, and the light and heavy chains of Fab 6HVL4.1 are colored wheat and blue, respectively. [Figure 7] Simultaneous binding of anti-VEGF / anti-IL-6 Fab to its targets assessed by SPR using immobilized anti-Fab antibodies [Figure 8] Blockade of VEGF-R2 binding by anti-VEGF / anti-IL-6 Fab in the presence of IL-6 as assessed by SPR using immobilized VEGF-A [Figure 9] The effect of VEGF binding on IL6 activity was assessed by a cell-based IL-6-specific reporter gene assay (without preincubation) as follows. [Figure 10] The effect of VEGF binding on IL6 activity was assessed by a cell-based IL-6-specific reporter gene assay (with preincubation) as follows. [Figure 11] Inhibition of IL-6 signaling in HRMECs by 6HVL_4 [Figure 12] Dose-dependent changes in the percentage inhibition of VEGF-A-induced HUVEC proliferation by 6HVL_4 [Figure 13]6HVL_4 rescues IL6 / IL6R / VEGF-induced barrier disruption on HRMVECs [Figure 14] Aflibercept reverses IL6 / IL6R / VEGF-induced barrier disruption on HRMVECs [Figure 15] Amino acid sequences of the VH and VL domains of the indicated antibodies. Kabat numbering of amino acid positions is shown. Amino acid positions contributing to the IL6 paratope identified in Example 13 are highlighted with black boxes. [Figure 16] Image of the IL6 / IL6R / gp130 complex (top; pdb-acc.#1p9m) compared with a structural superposition of Fab 0182 bound to IL6 and the IL6 / IL6R complex from pdb-acc.1p9m (bottom). [Figure 17] Image of the IL6 / IL6R / gp130 complex (top; pdb-acc.#1p9m) compared with a structural superposition of IL6-bound Fab 6HVL4.1 and the IL6 / IL6R complex from pdb-acc.1p9m (bottom). [Figure 18] SPR cross-linking experiments examining the ability of IL6R to bind IL6 when engaged in a preformed complex with Fab P1AE2421. [Figure 19] SPR binding experiments investigating the ability of Fab P1AE2421 to bind to human "hyper-IL6" (a chimera of human IL6 and IL6R). [Figure 20] ELISA competition experiments to determine the ability of Fab P1AE2421 to bind IL6 in a manner that blocks the binding of IL6 to IL6R. [Figure 21] Binding of the IL6-binding antibody 6HdL2.05 to human and cynomolgus IL6 as determined by surface plasmon resonance. DETAILED DESCRIPTION OF THE INVENTION

[0029] 1.Definition Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those skilled in the art. Furthermore, unless otherwise required by context, singular terms shall include the plural, and plural terms shall include the singular. The methods and techniques of the present disclosure are generally carried out according to conventional methods well known in the art. Generally, the nomenclatures and techniques used in connection with biochemistry, enzymology, molecular and cell biology, microbiology, genetics, and protein and nucleic acid chemistry, and hybridization described herein are those well known and commonly used in the art.

[0030] Unless otherwise defined herein, the term "comprising of" is intended to include the term "consisting of."

[0031] The term "about" as used herein in connection with a particular value (e.g., temperature, concentration, time, etc.) is intended to refer to a + / -1% variation of the particular value to which the term "about" refers.

[0032] The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0033] An "isolated" antibody is one that has been separated from a component of its natural environment. In some embodiments, the antibody is purified to greater than 95% or greater than 99% purity as determined by electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse-phase HPLC) methods. For a review of methods for assessing antibody purity, see, e.g., Flatman et al., J. Chromatogr B 848:79-87 (2007).

[0034] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind to the same epitope, except for variant antibodies that contain, for example, naturally occurring mutations or that may arise during production of the monoclonal antibody preparation, in which such variants are generally present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method.

[0035] The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a heavy chain that has a structure substantially similar to a native antibody structure or that contains an Fc region as defined herein.

[0036] The "class" of an antibody refers to the type of constant domain or constant region carried by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into "subclasses" (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. In certain embodiments, the antibody is of the IgG1 isotype. In certain embodiments, the antibody is of the IgG1 isotype with P329G, L234A, and L235A mutations to reduce Fc region effector function. In other embodiments, the antibody is of the IgG2 isotype. In certain embodiments, the antibody is of the IgG4 isotype with an S228P mutation in the hinge region to improve the stability of the IgG4 antibody. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The light chain of an antibody can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

[0037] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl terminus of the heavy chain. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also referred to as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0038] A "variable region" or "variable domain" is a domain of an antibody's heavy or light chain that is involved in binding the antibody to an antigen. The heavy and light chain variable domains (VH and VL, respectively) of natural antibodies generally have similar structures, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR) (see, for example, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)). In the antibody of the present invention, a single pair of VH and VL domains, i.e., a cognate VH / VL pair, specifically binds to its two targets: VEGF-A and IL6.

[0039] "DutaFab" is a bispecific antibody disclosed in WO 2012 / 163520. In DutaFab, a single pair of VH and VL domains specifically binds to two different epitopes, with one paratope comprising amino acid residues from CDR-H2, CDR-L1, and CDR-L3, and the other paratope comprising amino acid residues from CDR-H1, CDR-H3, and CDR-L2. DutaFab contains two non-overlapping paratopes within the cognate VH / VL pair and can simultaneously bind to two different epitopes. DutaFab and methods for producing them by screening libraries containing monospecific Fab fragments are disclosed in WO 2012 / 163520.

[0040] A "human antibody" is an antibody having an amino acid sequence corresponding to an antibody produced by a human or human cell, or to an antibody of non-human origin that utilizes the human antibody repertoire or other human antibody-encoding sequences. This definition of a human antibody specifically excludes humanized antibodies, which contain non-human antigen-binding residues. Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments herein.

[0041] A "human consensus framework" is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda, MD (1991), vols. 1-3. In one embodiment, for VL, the subgroup is subgroup kappa I in Kabat et al. (see above). In one embodiment, for VH, the subgroup is subgroup III as in Kabat et al., supra.

[0042] An "antibody fragment" is a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0043] The terms "paratope" and "antigen-binding site" refer to the portion of an antibody that recognizes and binds to an antigen, and are used interchangeably herein. The paratope is formed by multiple individual amino acid residues from the heavy and light chain variable domains of an antibody that are spatially adjacent in the tertiary structure of the Fv region. The antibodies of the present invention contain two paratopes in one cognate VH / VL pair.

[0044] As used herein, a "VEGF-A paratope" is a paratope or antigen-binding site that binds to VEGF-A. The VEGF-A paratope of an antibody of the invention comprises amino acid residues from CDR-H2, CDR-L1, and CDR-L3 of the antibody.

[0045] As used herein, an "IL6 paratope" is a paratope or antigen-binding site that binds to IL6. The IL6 paratope of the antibody of the present invention comprises amino acid residues from CDR-H1, CDR-H3, and CDR-L2 of the antibody.

[0046] As used herein, the term "vascular endothelial growth factor," abbreviated "VEGF," refers to any naturally occurring VEGF from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses not only "full-length," unprocessed VEGF, but also any form of VEGF that results from processing within a cell. The term also encompasses naturally occurring variants of VEGF, such as splice variants or allelic variants. The amino acid sequence of an exemplary human VEGF is set forth in SEQ ID NO: 27.

[0047] The terms "anti-VEGF-A antibody" and "antibody that binds to VEGF-A" refer to an antibody that can bind to VEGF-A with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting VEGF-A. In one embodiment, the extent of binding of the anti-VEGF-A antibody to an unrelated, non-VEGF-A protein is less than about 10% of the binding of the antibody to VEGF-A, as measured, for example, by surface plasmon resonance (SPR). In certain embodiments, an antibody that binds to VEGF-A has a dissociation constant (KD) of 1 nM or less, 0.1 nM or less, or 0.01 nM or less. An antibody that binds to VEGF-A has a KD of 1 μM or less. D An antibody is said to "specifically bind" to VEGF-A if it has

[0048] As used herein, the term "interleukin 6," or "IL6" for short, refers to any naturally occurring form of IL6 derived from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses "full-length," unprocessed IL6 and any form of IL6 resulting from intracellular processing. The term also encompasses naturally occurring variants of IL6, such as splice variants or allelic variants. An exemplary amino acid sequence of human IL6 is set forth in SEQ ID NO:28.

[0049] The antibodies of the invention "bind simultaneously to human VEGF-A and human IL6," meaning that (a) an antibody Fab fragment of the invention that binds to human IL6 also specifically binds to human VEGF-A, and (b) an antibody Fab fragment of the invention that binds to human VEGF-A also specifically binds to human IL6. Simultaneous binding can be assessed by methods known in the art, for example, by surface plasmon resonance as described herein.

[0050] As used herein, the term "complementarity-determining region" or "CDR" refers to each region of an antibody variable domain, which is hypervariable in sequence and contains residues that contact antigen. Antibodies generally contain six CDRs: three in the VH domain (CDR-H1, CDR-H2, CDR-H3) and three in the VL domain (CDR-L1, CDR-L2, CDR-L3). Unless otherwise specified, CDR residues and other residues in the variable domain (e.g., FR residues) are numbered according to the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991).

[0051] As used herein, "framework" or "FR" refers to the amino acid residues of a variable domain other than the CDR residues. The framework of a variable domain generally consists of four framework domains: FR1, FR2, FR3, and FR4. Thus, the CDR and FR amino acid sequences generally appear in the following order: (a) in the VH domain: FR1-CDR-H1-FR2-CDR-H2-FR3-CDR-H3-FR4; and (b) in the VL domain: FR1-CDR-L1-FR2-CDR-L2-FR3-CDR-L3-FR4.

[0052] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y is generally determined by the dissociation constant (K D ) Affinity can be measured by methods common in the art, including those described herein. Specific illustrative exemplary embodiments for measuring binding affinity are described herein.

[0053] The term "epitope" refers to a site on an antigen, either proteinaceous or non-proteinaceous, to which an antibody binds. Epitopes can be formed from a contiguous stretch of amino acids (linear epitopes) or can include noncontiguous amino acids (conformational epitopes), formed in close spatial proximity, for example, due to antigen folding (i.e., tertiary folding of a proteinaceous antigen). Linear epitopes are typically still bound by antibodies after exposure of a proteinaceous antigen to denaturing agents, whereas conformational epitopes are typically destroyed by treatment with denaturing agents. Epitopes contain at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial structure.

[0054] Screening for antibodies that bind to a specific epitope (i.e., antibodies that bind to the same epitope) can be performed using methods routine in the art, such as, but not limited to, alanine scanning, peptide blotting (Meth. Mol. Biol. 248 (2004) 443-463), peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of antigens (see Prot. Sci. 9 (2000) 487-496), and cross-blocking (see "Antibodies," Harlow and Lane, Cold Spring Harbor Press, Cold Spring Harb., NY).

[0055] Antigen Structure-based Antibody Profiling (ASAP), also known as Modification-Assisted Profiling (MAP), allows multiple monoclonal antibodies that specifically bind to VEGF-A or IL6 to be classified based on their respective binding profiles to chemically or enzymatically modified antigen surfaces (see, e.g., US2004 / 0101920). Each classified antibody binds to the same epitope, which may be distinct from epitopes represented by other classifications or may be a unique epitope that overlaps with other classifications.

[0056] Furthermore, competitive binding can be used to easily determine whether an antibody binds to the same epitope on VEGF-A or IL6 as a reference antibody of the present invention, or competes for binding. For example, an "antigen that binds to the same epitope on VEGF-A and IL6" as a reference antibody refers to an antibody that blocks the binding of the reference antibody to that antigen by 50% or more in each competitive assay; conversely, the reference antibody blocks the binding of the antibody to that antigen by 50% or more in each competitive assay. For example, to determine whether an antibody binds to the same epitope as a reference antibody, the reference antibody can be bound to VEGF-A or IL6 under saturating conditions. After removing excess reference antibody, the ability of the antibody to bind to VEGF-A or IL6 is evaluated. If the antibody can bind to VEGF-A or IL6 after saturating binding of the reference antibody, it can be concluded that the antibody of interest binds to a different epitope than the reference antibody. However, if the antibody of interest cannot bind to VEGF-A or IL6 after saturation binding of the reference antibody, it is possible that the antibody of interest binds to the same epitope as the reference antibody. To determine whether the antibody of interest binds to the same epitope or whether binding is simply hindered for steric reasons, routine experiments can be used (e.g., peptide mutations and binding analysis using ELISA, RIA, surface plasmon resonance, flow cytometry, or other quantitative or qualitative antibody binding assays available in the art). This assay should be performed in two setups, i.e., with both antibodies as saturating antibodies. If only the first (saturating) antibody can bind to VEGF-A or IL6 in both setups, it can be concluded that the antibody of interest and the reference antibody compete for binding to VEGF-A or IL6.

[0057] In some embodiments, two antibodies are considered to bind to the same or overlapping epitope if a 1-, 5-, 10-, 20-, or 100-fold excess of one antibody inhibits binding of the other by at least 50%, at least 75%, at least 90%, or even 99% or more, as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 50 (1990) 1495-1502).

[0058] In some embodiments, two antibodies are considered to bind to the same epitope if substantially all of the amino acid mutations in the antigen that reduce or eliminate binding of one antibody also reduce or eliminate binding of the other antibody. Two antibodies are considered to have "overlapping epitopes" if only a subset of the amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other antibody.

[0059] "Percent identity (%) of amino acid sequence" to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, without considering any conservative substitutions as part of sequence identity, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity for the purpose of alignment.Alignment for determining percent identity of amino acid sequence can be achieved in various ways within the skill of the art, for example, using publicly available computer software, such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software or FASTA program package.Those skilled in the art can determine the appropriate parameters for sequence alignment, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared.Alternatively, percent identity values ​​can be generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and the source code is on file in the user documentation at the U.S. Copyright Office, Washington, DC, 20559, registered under U.S. Copyright Registration No. TXU510087, and described in WO 2000 / 005319.

[0060] However, unless otherwise specified, for purposes herein, percent amino acid sequence identity values ​​are generated using the ggsearch program of the FASTA package version 36.3.8c, followed by the BLOSUM50 comparison matrix. The FASTA program package is described by W.R. Pearson and D.J. Lipman (1988), "Improved Tools for Biological Sequence Analysis," PNAS 85:2444-2448; W.R. Pearson (1996), "Effective protein sequence comparison," Meth. Enzymol. 266:227-258; and Pearson et al. (1997) Genomics 46:24-36, 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(globalprotein:protein) program and default options (BLOSUM50; open: -10; ext: -2; Ktup=2), ensuring a global rather than local alignment. The percent amino acid identity is given in the output alignment header.

[0061] The terms "nucleic acid molecule" or "polynucleotide" include any compound and / or substance comprising a polymer of nucleotides. Each nucleotide is composed of a base, specifically a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Nucleic acid molecules are often described by their base sequence, where the bases represent the primary (linear) structure of the nucleic acid molecule. The sequence of bases is typically represented 5' to 3'. As used herein, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA), e.g., complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers containing two or more of these molecules. Nucleic acid molecules may be linear or circular. In addition, the term nucleic acid molecule includes both sense and antisense strands, and both single- and double-stranded forms. Furthermore, the nucleic acid molecules described herein can contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases with derivatized sugar or phosphate backbone linkages or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules suitable as vectors for direct expression of the antibodies of the present invention in vitro and / or in vivo, e.g., 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 expression of the encoded molecule, allowing the mRNA to be injected into a subject to produce antibodies in vivo. (See, for example, Stadler et al, Nature Medicine 2017, published online 12 June 2017, doi:10.1038 / nm.4356 or EP 2101823 B1).

[0062] An "isolated" nucleic acid is a nucleic acid molecule that is separated from a component of its natural environment. Isolated nucleic acid includes a nucleic acid molecule contained within a cell that normally contains the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0063] An "isolated nucleic acid encoding" an antibody refers to one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of the antibody, wherein such nucleic acid molecules are contained in a single vector or in separate vectors, and wherein such nucleic acid molecules are present in one or more locations in a host cell.

[0064] The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors as autonomously replicating nucleic acid structures as well as vectors that are integrated into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."

[0065] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and its progeny, regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0066] The term "pharmaceutical composition" or "pharmaceutical formulation" refers to a preparation that is in a form such that the biological activity of the active ingredients contained in the preparation is effective, and that does not contain additional ingredients that are unacceptably toxic to the subject to which the pharmaceutical composition will be administered.

[0067] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical composition or formulation, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0068] An "effective amount" of an agent, eg, a pharmaceutical composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic or prophylactic result.

[0069] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates, such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0070] As used herein, "treatment" (and grammatical variants thereof, e.g., "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of disease in the treated individual and can be carried out prophylactically or during the course of clinical pathology. Desired effects of treatment include preventing the onset or recurrence of disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, remission or palliation of disease symptoms, and ameliorating or improving prognosis. In some embodiments, the antibodies of the invention are used to delay the onset of disease or to slow the progression of disease.

[0071] The term "ocular disease" as used herein includes any ocular disease associated with pathological neovascularization and / or atrophy. Ocular diseases can be characterized by altered or disregulated proliferation and / or infiltration of new blood vessels into structures of ocular tissues, such as the retina or cornea. Ocular diseases can be characterized by atrophy of retinal tissues (photoreceptors and the underlying retinal pigment epithelium (RPE) and choriocapillaris). Non-limiting ocular diseases include, for example, AMD (e.g., wet AMD, dry AMD, intermediate AMD, advanced AMD, and geographic atrophy (GA)), macular degeneration, macular edema, DME (e.g., focal, non-central DME, and diffuse, centrally involved DME), retinopathies, diabetic retinopathy (DR) (e.g., proliferative DR (PDR), non-proliferative DR (NPDR), and high altitude DR), other ischemia-related retinopathies, ROP, retinal edema, and retinal edema. Related to venous occlusion (RVO) (e.g., central (CRVO) and branch (BRVO) forms), CNV (e.g., myopic CNV), corneal neovascularization, diseases associated with corneal neovascularization, retinal neovascularization, diseases associated with retinal / choroidal neovascularization, central serous retinopathy (CSR), pathological myopia, von Hippel-Lindau disease, ocular histoplasmosis, FEVR, Coats disease, Norrie disease, and osteoporotic pseudoglioma syndrome (OPPG) Related retinal abnormalities, subconjunctival hemorrhage, rubeosis, ocular neovascular disease, neovascular glaucoma, retinitis pigmentosa (RP), hypertensive retinopathy, retinal angiomatous proliferation, macular vascular ectasia, iris neovascularization, intraocular neovascularization, retinal degeneration, cystoid macular edema (CME), vasculitis, papilledema, retinitis including, but not limited to: CMV retinitis, ocular melanoma, retinoblastoma, conjunctivitis (e.g., infectious conjunctivitis) and non-infectious (e.g., allergic Eye diseases include ocular neovascularization, ocular vascular leakage, retinal edema, retinal atrophy, ocular ocular malformation ...Further exemplary ocular diseases include diseases associated with retinoschisis (abnormal division of the retinal neurosensory layer), rubeosis (neovascularization of the angle), and diseases caused by abnormal proliferation of fibrovascular or fibrous tissue (including all forms of proliferative vitreoretinopathy). Exemplary diseases associated with corneal neovascularization include, but are not limited to, epidemic keratoconjunctivitis, vitamin A deficiency, contact lens overwear, atopic keratitis, superior limbal keratitis, pterygium, keratitis sicca, Sjogren's syndrome, acne rosacea, phylectenulosis, syphilis, mycobacterial infections, steatosis, chemical burns, bacterial ulcers, fungal ulcers, herpes simplex infections, herpes zoster infections, protozoal infections, Kaposi's sarcoma, Mooren's ulcer, Therrien's peripheral corneal degeneration, peripheral keratolysis, rheumatoid arthritis, generalized erythema, polyarteritis nodosa, trauma, Wegener's sarcoidosis, scleritis, Steven-Johnson syndrome, pemphigoid, radial keratotomy, and post-corneal transplant rejection. Exemplary diseases associated with choroidal neovascularization and defects in the retinal vasculature, including increased vascular leakage, aneurysms, and capillary dropout, include, but are not limited to, diabetic retinopathy, macular degeneration, sickle cell anemia, sarcoid, syphilis, pseudoxanthoma elasticum, Paget's disease, venous occlusion, arterial occlusion, carotid occlusive disease, chronic uveitis / vitreous inflammation, mycobacterial infections, Lyme disease, systemic lupus erythematosus, retinopathy of prematurity, retinal edema (including macular edema), Eales' disease, Behcet's disease, infections causing retinitis or choroiditis (e.g., multifocal choroid), presumed ocular histoplasmosis, Best's disease (vitreomacular degeneration), myopia, optic disc, pars planitis, retinal detachment (e.g., chronic retinal detachment), hyperviscosity syndrome, toxoplasmosis, trauma, and post-laser complications. Exemplary diseases associated with atrophy of retinal tissue (photoreceptors and the underlying RPE) include, but are not limited to, atrophic or non-exudative AMD (e.g., geographic atrophy or advanced dry AMD), macular atrophy (e.g., atrophy associated with neovascularization and / or geographic atrophy), diabetic retinopathy, Stargardt's disease, Sorsby Fundus dystrophy, retinoschisis, and retinitis pigmentosa.

[0072] The term "package insert" is used to refer to instructions customarily included in commercial packaging of therapeutic products that contain information about the indications, uses, dosages, administration, concomitant therapies, contraindications, and / or warnings regarding the use of such therapeutic product.

[0073] 2. Detailed Description of the Embodiments of the Invention In one embodiment, the present invention is based in part on the provision of bispecific antibodies for therapeutic applications. In a specific embodiment, an antibody that binds to human VEGF-A and human IL6 is provided. The antibodies of the present invention are useful, for example, in the treatment of vascular diseases, such as ocular vascular diseases.

[0074] A. Exemplary antibodies that bind to human VEGF-A and human IL6 In one aspect, the present invention provides an antibody that binds to human VEGF-A and human IL6. In one aspect, an isolated antibody that binds to human VEGF-A and human IL6 is provided. In one aspect, the present invention provides an antibody that specifically binds to human VEGF-A and human IL6.

[0075] In a particular embodiment, an antibody that binds to human VEGF-A and human IL6, wherein the antibody comprises a VEGF-A paratope (i.e., an antigen-binding site that binds to VEGF-A) and an IL6 paratope (i.e., an antigen-binding site that binds to IL6) within one cognate pair of VL and VH domains; the VEGF-A paratope comprises amino acid residues derived from CDR-H2, CDR-L1, and CDR-L3 of the antibody, and the IL6 paratope comprises amino acid residues derived from CDR-H1, CDR-H3, and CDR-L2 of the antibody; and / or • The IL6 paratope comprises amino acid residues derived from CDR-H2, CDR-L1 and CDR-L3 of the antibody, and the VEGF-A paratope comprises amino acid residues derived from CDR-H1, CDR-H3 and CDR-L2 of the antibody; the pair of variable light chain domains and variable heavy chain domains simultaneously binds human VEGF-A and human IL6; and / or the antibody binds to the same epitope on human VEGF-A and the same epitope on human IL6 as an antibody having a variable heavy chain domain of SEQ ID NO: 22 and a variable light chain domain of SEQ ID NO: 21; and / or The antibody Fab fragment of the antibody has (i) a K of less than 50 pM as measured by surface plasmon resonance D and (ii) binds to human VEGF-A121 with a K of less than 50 pM as measured by surface plasmon resonance. D binds to human IL6 at the antibody Fab fragment of the antibody exhibits an aggregation onset temperature of 60°C or higher, and in one embodiment 70°C or higher; and / or • The antibody Fab fragment of the antibody exhibits a melting temperature above 80°C as measured by dynamic light scattering.

[0076] In another aspect, the present invention provides an antibody that binds to human VEGF-A and human IL6, comprising a VH domain comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 18, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 19, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 20, and a VL domain comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 15, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17; The present invention provides an antibody comprising: (a) a VH domain comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 22; and (b) a VL domain comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 21.

[0077] In another aspect, the present invention provides an antibody that binds to human VEGF-A and human IL6, comprising: (a) a VH domain comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 22; and (b) a VL domain comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 21.

[0078] In another aspect, the invention provides an antibody that binds to human VEGF-A and human IL6, comprising: (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 22 with up to 15, up to 10, or up to 5 amino acid substitutions; and (b) a variable light chain domain comprising the amino acid sequence of SEQ ID NO: 21 with up to 15, up to 10, or up to 5 amino acid substitutions.

[0079] In another aspect, the present invention provides an antibody that binds to human VEGF-A and human IL6, comprising a VH domain comprising (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 18, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 19, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 20, and a VL domain comprising (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 15, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17, wherein the antibody comprises: (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 22 with up to 15, up to 10, or up to 5 amino acid substitutions; and (b) a variable light chain domain comprising the amino acid sequence of SEQ ID NO: 21 with up to 15, up to 10, or up to 5 amino acid substitutions.

[0080] In one embodiment, the present invention provides an antibody that binds to human VEGF-A and human IL6, comprising a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 22. 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 compared to the reference sequence, but an antibody that binds to human VEGF-A and human IL6 comprising that sequence retains the ability to bind to human VEGF-A and human IL6. In certain embodiments, a total of up to 10 amino acids have been substituted, inserted, and / or deleted in SEQ ID NO: 22. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). In a particular embodiment, the VH comprises a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 18, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 19, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 20.

[0081] In one embodiment, the present invention provides an antibody that binds to human VEGF-A and human IL6, comprising a VL domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 21. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions compared to the reference sequence, but an antibody that binds to human VEGF-A and human IL6 comprising that sequence retains the ability to bind to human VEGF-A and human IL6. In certain embodiments, a total of up to 10 amino acids have been substituted, inserted, and / or deleted in SEQ ID NO: 21. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (i.e., in the FRs). In a particular aspect, the VL comprises (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 15, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17.

[0082] In another aspect, an antibody that binds to human VEGF-A and human IL6 is provided, the antibody comprising the VH sequence of any of the aspects provided above and the VL sequence of any of the aspects provided above. In one aspect, the antibody comprises the VH and VL sequences of SEQ ID NO: 22 and SEQ ID NO: 21, respectively, including post-translational modifications of these sequences.

[0083] In another aspect, an antibody that binds to human VEGF-A and human IL6 is provided, wherein the antibody comprises a heavy chain amino acid sequence of SEQ ID NO:24 and a light chain amino acid sequence of SEQ ID NO:23.

[0084] In a further embodiment of the present invention, the antibody that binds to human VEGF-A and human IL6 according to any of the above embodiments is a monoclonal antibody. In one embodiment, the antibody that binds to human VEGF-A and human IL6 is an antibody fragment, such as an Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment. In another embodiment, the antibody is a full-length antibody.

[0085] In another aspect, the present invention provides an antibody that binds to IL6, derived from the antibody of the present invention. The IL6 paratope disclosed for the antibody of the present invention can be used to provide additional antibodies, such as monospecific antibodies or bispecific antibodies that bind to IL6 and another antigen. The IL6 paratope of the antibody 6HVL4.1 disclosed herein was identified by X-ray crystallography (Example 13). Antibody 6HVL4.1 is based on a VH domain with a human VH3 framework and a VL domain with a human Vkappa1 framework. Antibodies containing the IL6 paratope of antibody 6HVL4.1 bind to the same epitope on IL6. All embodiments disclosed herein for the antibodies of the present invention that bind to human VEGF-A and human IL6 also apply to antibodies that bind to IL6.

[0086] Thus, in one embodiment, the present invention provides an antibody that binds to human IL6, c) a VH domain based on a human VH3 framework (the IL6 paratope comprises amino acid residues Y1, I2, Q3, Y26, E27, F28, T29, H30, Q31, D32, P52a, R94, I96, D97, F98, D101, and T102), and a VL domain based on a human Vkappa1 framework (the IL6 paratope comprises amino acid residues Y49, D50, S53, N54, Y55, P56, S57, Y91, and Y96); or d) An antibody is provided, comprising a VH domain based on a human VH3 framework (the IL6 paratope comprises amino acid residues Y1, P2, Q3, V26, L27, F28, K29, H30, Q31, D32, P52a, R94, L96, D97, F98, D101, E102), and a VL domain based on a human Vkappa1 framework (the IL6 paratope comprises amino acid residues Y49, D50, D53, R54, Y55, P56, E57, Y91, Y96) (numbering according to Kabat).

[0087] In another aspect, the invention provides an antibody that binds to IL6, which binds to the same epitope on IL6 as an antibody having a VL domain of SEQ ID NO: 35 and a VH domain of SEQ ID NO: 36. In one embodiment, the antibody comprises a VH domain having a human VH3 framework (the IL6 paratope comprises amino acid residues 1, 2, 3, 26, 27, 28, 29, 30, 31, 32, 52a, 94, 96, 97, 98, 101, 102 of the antibody of the invention that binds to human VEGF-A and IL6), and a VL domain having a human Vkappa1 framework (the IL6 paratope comprises amino acid residues 49, 50, 53, 54, 55, 56, 57, 91, 96 of the antibody of the invention that binds to human VEGF-A and IL6).

[0088] In one embodiment, the antibody that binds to IL6 is a multispecific antibody that binds to IL6 and another target.

[0089] In further aspects, an antibody that binds human VEGF-A and human IL6 according to any of the above aspects, or an antibody that binds human IL6 according to any of the above aspects, may incorporate any of the features, alone or in combination, as described in sections 1 to 5 below.

[0090] 1. Antibody affinity In certain embodiments, the antibodies provided herein bind to VEGF-A with a dissociation constant (KD) of ≦1 nM, ≦0.1 nM, or ≦0.01 nM. In preferred embodiments, the antibodies provided herein bind to human VEGF-A with a dissociation constant (KD) of ≦10 pM, and in preferred embodiments, ≦5 pM. In preferred embodiments, the antibodies provided herein bind to human VEGFA-121 with a dissociation constant (KD) of ≦10 pM, and in preferred embodiments, ≦5 pM. In preferred embodiments, the antibodies provided herein bind to human VEGFA-165 with a dissociation constant (KD) of ≦10 pM, and in preferred embodiments, ≦5 pM.

[0091] In certain embodiments, antibodies that bind to IL6 have a dissociation constant (K D) of ≦1 nM, ≦0.1 nM, or ≦0.03 nM. In preferred embodiments, the antibodies provided herein have a dissociation constant (K) of ≦10 pM, and in preferred embodiments, ≦5 pM. D ) binds to human IL6. D is measured using a surface plasmon resonance assay, in one embodiment using a BIACORE® surface plasmon resonance assay.

[0092] In another embodiment, K D is measured using a KinExA assay. D is the K of VEGF-A binding D Detection of IL6 binding or K D is measured using the KinExA assay under conditions as described below in the Materials and General Methods section for the detection of

[0093] For example, the K DThe assay is performed using a KinExA 3200 instrument from Sapidyne Instruments (Boise, ID), where 30 μg of anti-VEGF antibody MAB293 (R&D) in 1 ml of PBS (pH 7.4) is used to coat PMMA beads with antigen according to the KinExA handbook protocol (Adsorption coating, Sapidyne). KinExA equilibrium assays are performed at room temperature using PBS (pH 7.4) containing 0.01% BSA and 0.01% Tween 20 as the running buffer, with samples and beads prepared in LowCross buffer (Candor Bioscience). The flow rate is 0.25 ml / min. A constant amount of VEGFA-121-His (50 pM and 500 pM in a second experiment) was titrated with the test antibody, and the equilibrated mixture was aspirated onto a column of beads coupled to an anti-VEGF antibody (Mab293) in the KinExA system in a volume of 750 μl for 50 pM constant VEGF and in a volume of 125 μl for 500 pM constant VEGF. Detection of bound VEGFA-121 was performed using a secondary biotinylated anti-VEGF antibody (BAF293) at a concentration of 250 ng / ml, followed by injection of 250 ng / ml of streptavidin Alexa Fluor™ 647 conjugate in sample buffer. D is obtained from nonlinear regression analysis of the data using the one-site homogeneous binding model included in the KinExA software (version 4.0.11) using the "standard analysis" method. D Calculate the data points and calculate the theoretical K D Determine the 95% confidence interval by fitting the curve. The 95% confidence interval is D Low and K D is given as high.

[0094] For example, the K DThe assay is measured using surface plasmon resonance (SPR) on a Biacore 8K instrument (Cytiva) at 25°C using HBS-EP+ (1x; BR100669; Cytiva) as the running buffer. Human Fab binder (28958325, Cytiva) is diluted to a final concentration of 10 μg / ml in 10 mM sodium acetate buffer, pH 5.0, and immobilized to a CM5 sensor chip using standard amine coupling chemistry. Prior to protein measurement, five initial cycles are optionally performed for conditioning purposes, with HBS-EP+ buffer flowing for approximately 120 seconds, followed by the application of 10 mM glycine buffer, pH 2.0, for 60 seconds to regenerate the derivatized chip surface. Antibody Fab fragments at a concentration of 75 nM are captured on this surface in HBS-EP+ buffer at a flow rate of 10 μl / min for 60 seconds. No Fab fragments are applied to the reference channel. Subsequently, human or cynomolgus IL-6 is applied in an appropriate dilution series in HBS-EP+ buffer at a flow rate of 30 μl / min (preferably using a contact time of 180 seconds and a dissociation time of 720 seconds). Regeneration of the derivatized chip surface is achieved as described above. Data is evaluated using 8K evaluation software (Biacore Insight Evaluation 3.0).

[0095] 2. Antibody fragments In certain aspects, the antibodies provided herein are antibody fragments.

[0096] In one embodiment, the antibody fragment is a Fab, Fab', Fab'-SH, or F(ab')2 fragment, particularly a Fab fragment. Papain digestion of an intact antibody generates two identical antigen-binding fragments (so-called "Fab" fragments), each containing the heavy and light chain variable domains (VH and VL, respectively) as well as the light chain constant domain (CL) and the first heavy chain constant domain (CH1). Thus, the term "Fab fragment" refers to an antibody fragment containing a light chain containing the VL and CL domains and a heavy chain fragment containing the VH and CH1 domains. "Fab' fragments" differ from Fab fragments by the addition of residues at the carboxy 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 ​​residue(s) in the constant domains retain a free thiol group. Pepsin treatment yields a F(ab')2 fragment containing two antigen-binding sites (two Fab fragments) and part of the Fc region. See US Pat. No. 5,869,046 for a description of Fab and F(ab')2 fragments that contain salvage receptor binding epitope residues and have increased in vivo half-lives.

[0097] Antibody fragments can be produced by a variety of techniques, including but not limited to, proteolytic digestion of intact antibodies and recombinant production by recombinant host cells (e.g., E. coli, CHO), as described herein.

[0098] In a preferred embodiment, the antibodies provided herein are Fab fragments.

[0099] In one embodiment, the VH domain of an antibody provided herein comprises a human VH3 framework.

[0100] In one embodiment, the VL domain of an antibody provided herein comprises a human Vkappa1 framework.

[0101] In one embodiment, the CL domain of the antibodies provided herein is of the kappa isotype.

[0102] In one embodiment, the CH1 domain of the antibodies provided herein is of the human IgG1 isotype.

[0103] In a preferred embodiment, the antibodies provided herein are Fab fragments comprising a CL domain of the kappa isotype and a CH1 domain of the human IgG1 isotype.

[0104] 3.Thermal stability The antibodies provided herein exhibit excellent thermal stability. In certain embodiments, the Fab fragments of the antibodies provided herein exhibit an aggregation onset temperature of 60°C or higher, and in one embodiment, 70°C or higher. In certain embodiments, the Fab fragments of the antibodies provided herein exhibit a melting temperature of greater than 80°C as measured by dynamic light scattering.

[0105] 4. Multispecific antibodies In certain embodiments, the antibodies provided herein are multispecific antibodies. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites, i.e., different epitopes on different antigens or different epitopes on the same antigen. In certain embodiments, multispecific antibodies have three or more binding specificities.

[0106] Multispecific antibodies with three or more binding specificities, including the antibodies provided herein, can also be provided in an asymmetric manner with domain crossovers in one or more binding arms of the same antigen specificity, i.e., by exchanging VH / VL domains (see, e.g., WO 2009 / 080252 and WO 2015 / 150447), CH1 / CL domains (see WO 2009 / 080253) or complete Fab arms (see WO 2009 / 080251, WO 2016 / 016299; see also Schaefer et al., PNAS, 108 (2011) 1187-1191, and Klein at al., MAbs 8 (2016) 1010-20). A variety of additional molecular formats of multispecific antibodies are known in the art and are included herein (see, e.g., Spiess et al., Mol Immunol 67 (2015) 95-106).

[0107] 5. Antibody Variants In certain embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to change the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of antibodies may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of, residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, as long as the final construct possesses the desired characteristics (e.g., antigen binding).

[0108] In certain embodiments, antibody variants with one or more amino acid substitutions are provided. Sites of interest for substitution mutagenesis include CDRs and FRs. Conservative substitutions are shown in the table below under the heading of "preferred substitutions." More substantial changes are provided in Table 1 under the heading of "exemplary substitutions," and are further described below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into the antibody of interest, and the products are screened for the desired activity, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC. [Table 1] TIFF0007801491000002.tif167161

[0109] Amino acids can be classified according to general side chain properties. (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) Residues affecting chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0110] Non-conservative substitutions will involve exchanging a member of one of these classes for a member of another class.

[0111] One type of substitutional variant involves substituting one or more CDR residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further study have altered (e.g., improved) specific biological properties (e.g., increased affinity, reduced immunogenicity) compared to the parent antibody and / or have substantially retained specific biological properties of the parent antibody. An exemplary substitutional variant is an affinity-matured antibody, which can be conveniently generated using, for example, phage-display-based affinity maturation techniques as described herein. Briefly, one or more CDR residues are mutated, and the variant antibodies displayed on phage are screened for a specific biological activity (e.g., binding affinity).

[0112] In certain embodiments, substitutions, insertions, or deletions may occur within one or more CDRs, as long as such changes do not substantially reduce the antibody's ability to bind to the antigen. For example, conservative changes (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in the CDRs. Such changes may, for example, be outside the antigen-contacting residues in the CDRs. In the above-mentioned specific variant VH and VL sequences, each CDR is either unchanged or has one, two, or three or fewer amino acid substitutions.

[0113] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, target residues or groups (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and substituted with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the antibody-antigen interaction is affected. Further substitutions can be introduced at amino acid positions that demonstrate functional sensitivity to the initial substitution. Alternatively, or in addition, crystal structures of antigen-antibody complexes can be used to identify contact points between the antibody and antigen. Such contact and neighboring residues can be targeted as candidates for substitution or removed. Variants can then be screened to determine whether they possess desired properties.

[0114] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of one or more amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of antibody molecules include the N- or C-terminal fusion of the antibody to an enzyme (e.g., ADEPT (for antibody-directed enzyme prodrug therapy) or a polypeptide which increases the serum half-life of the antibody.

[0115] a) Glycosylation variants In certain embodiments, the antibodies provided herein are altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody can be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

[0116] If the antibody contains an Fc region, the oligosaccharides attached to the antibody may be modified. Natural antibodies produced by mammalian cells typically contain branched, biantennary oligosaccharides that are generally linked to Asn297 in the CH2 domain of the Fc region via an N-linkage. See, for example, Wright et al., TIBTECH 15:26-32 (1997). The oligosaccharides may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose linked to the GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharides in the antibodies of the present invention may be performed to generate antibody variants with specific improved properties.

[0117] In one embodiment, antibody variants are provided that have nonfucosylated oligosaccharides, i.e., oligosaccharide structures lacking fucose linkage (direct or indirect) to the Fc region. Such nonfucosylated oligosaccharides (also referred to as "afucosylated" oligosaccharides) are particularly N-linked oligosaccharides lacking the first GlcNAc-linked fucose residue at the stem of the biantennary oligosaccharide structure. In one embodiment, antibody variants are provided that have an increased proportion of nonfucosylated oligosaccharides in the Fc region compared to the native or parent antibody. For example, the proportion of nonfucosylated oligosaccharides may be at least about 20%, at least about 40%, at least about 60%, at least about 80%, or even about 100% (i.e., no fucosylated oligosaccharides are present). The percentage of nonfucosylated oligosaccharides is the (average) amount of oligosaccharides lacking a fucose residue relative to the sum of all oligosaccharides (e.g., complex, hybrid, and high-mannose structures) attached to Asn297, as measured, for example, by MALDI-TOF mass spectrometry as described in WO 2006 / 082515. Asn297 refers to the asparagine residue located at approximately position 297 of the Fc region (EU numbering of Fc region residues); however, due to minor antibody sequence variations, Asn297 may also be located upstream or downstream of position 297, i.e., approximately ±3 amino acids between positions 294 and 300. Such antibodies with an increased percentage of nonfucosylated oligosaccharides in the Fc region may have improved FcγRIIIa receptor binding and / or improved effector function, particularly improved ADCC function. See, for example, U.S. Patent Application Publication Nos. 2003 / 0157108; 2004 / 0093621.

[0118] Examples of cell lines capable of producing antibodies with reduced fucosylation include Lec13 CHO cells, which are deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application Publication No. 2003 / 0157108; and WO 2004 / 056312, especially Example 11), and knockout cell lines, such as FUT8, knockout CHO cells for the alpha-1,6-fucosyltransferase gene (e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614-622 (2004); Kanda, Y. et al. al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO 2003 / 085107), or cells in which the activity of GDP-fucose synthesis or transporter proteins is reduced or abolished (see, e.g., U.S. Patent Application Publication Nos. 2004259150, 2005031613, 2004132140, and 2004110282).

[0119] In a further embodiment, antibody variants are provided with bisected oligosaccharides, e.g., biantennary oligosaccharides attached to the Fc region of the antibody are bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function, as described above. Examples of such antibody variants are described, for example, in Umana et al., Nat Biotechnol 17, 176-180 (1999); Ferrara et al., Biotechn Bioeng 93, 851-861 (2006); WO 99 / 54342, WO 2004 / 065540, and WO 2003 / 011878.

[0120] Also provided are antibody variants that have at least one galactose residue in the oligosaccharide attached to the Fc region. Such antibody variants can have improved CDC function. Examples of such antibody variants are described in, for example, WO 1997 / 30087; WO 1998 / 58964; and WO 1999 / 22764.

[0121] b) Fc region variants In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody presented herein, thereby creating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) containing an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0122] In certain embodiments, the present invention contemplates antibody variants that retain some, but not all, effector functions, making them desirable candidates for applications where the in vivo half-life of the antibody is important, yet certain effector functions (e.g., complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC)) are unnecessary or deleterious. To confirm reduced / absent CDC and / or ADCC activity, in vitro and / or in vivo cytotoxicity assays can be performed. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and thus likely lacks ADCC activity) but retains FcRn binding ability. NK cells, the primary cells for mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. Expression of FcR on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362 (see, e.g., Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see, Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be used (e.g., ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc. Mountain View, CA) and CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI)).Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, or in addition, the desired ADCC activity can be assessed in vivo in an animal model, such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay can also be performed to confirm that the antibody is unable to bind C1q and lacks CDC activity. See, for example, the C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). Determination of FcRn binding and in vivo clearance / half-life can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006); WO2013 / 120929A1).

[0123] Antibodies with reduced effector function include those with one or more substitutions at residues 238, 265, 269, 270, 297, 327, and 329 in the Fc region (U.S. Patent No. 6,737,056). Such Fc variants include Fc variants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variant, which has substitutions of residues 265 and 297 to alanine (U.S. Patent No. 7,332,581).

[0124] Certain antibody variants have been described with improved or diminished binding to FcRs (see, e.g., U.S. Pat. No. 6,737,056; WO 2004 / 056312; and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001)).

[0125] In certain embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, e.g., substitutions at positions 298, 333, and / or 334 (EU numbering of residues) of the Fc region.

[0126] In certain embodiments, the antibody variant comprises an Fc region having one or more amino acid substitutions that reduce FcγR binding, e.g., at positions 234 and 235 (EU numbering) of the Fc region. In one embodiment, the substitutions are L234A and L235A (LALA). In certain embodiments, the antibody variant further comprises D265A and / or P329G in an Fc region derived from a human IgG1 Fc region. In one embodiment, the substitutions are L234A, L235A, and P329G (LALA-PG) in an Fc region derived from a human IgG1 Fc region. (See, e.g., WO 2012 / 130831.) In another embodiment, the substitutions are L234A, L235A, and D265A (LALA-DA) in an Fc region derived from a human IgG1 Fc region.

[0127] In some embodiments, alterations occur within the Fc region that result in altered (i.e., either improved or decreased) C1q binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0128] Antibodies with increased half-lives and improved binding to fetal Fc receptors (FcRn) that are responsible for transferring maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) are described in U.S. Patent Application Publication No. 2005 / 0014934 (Hinton et al.). These antibodies comprise an Fc region with one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include those having substitutions at one or more of Fc region residues: 238, 252, 254, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, e.g., a substitution at Fc region residue 434 (see, e.g., U.S. Patent No. 7,371,826; Dall'Acqua, WF, et al. J. Biol. Chem. 281 (2006) 23514-23524).

[0129] The Fc region residues critical for mouse Fc-mouse FcRn interaction have been identified by site-directed mutagenesis (see, e.g., Dall'Acqua, WF, et al. J. Immunol 169 (2002) 5171-5180). Residues I253, H310, H433, N434, and H435 (EU index numbering) are involved in the interaction (Medesan, C., et al., Eur. J. Immunol. 26 (1996) 2533; Firan, M., et al., Int. Immunol. 13 (2001) 993; Kim, JK, et al., Eur. J. Immunol. 24 (1994) 542). Residues I253, H310, and H435 were found to be critical for the interaction between human Fc and mouse FcRn (Kim, JK, et al., Eur. J. Immunol. 29 (1999) 2819). Studies of the human Fc-human FcRn complex have shown that residues I253, S254, H435, and Y436 are critical for the interaction (Firan, M., et al., Int. Immunol. 13 (2001) 993; Shields, RL, et al., J. Biol. Chem. 276 (2001) 6591-6604). Yeung, YA, et al. (J. Immunol 182 (2009) 7667-7671) reported and investigated various mutants of residues 248-259, 301-317, 376-382, and 424-437.

[0130] In certain embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that reduce FcRn binding, e.g., mutations at Fc region positions 253, and / or 310, and / or 435 (residues according to EU numbering). In certain embodiments, the antibody variant comprises an Fc region with amino acid substitutions at positions 253, 310, and 435. In one embodiment, the substitutions are I253A, H310A, and H435A in the Fc region derived from a human IgG1 Fc region. See, e.g., Grevys, A., et al., J. Immunol. 194 (2015) 5497-5508.

[0131] In certain embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that reduce FcRn binding, e.g., mutations at positions 310, and / or 433, and / or 436 (EU numbering residues) of the Fc region. In certain embodiments, the antibody variant comprises an Fc region with amino acid substitutions at positions 310, 433, and 436. In one embodiment, the substitutions are H310A, H433A, and Y436A in the Fc region derived from a human IgG1 Fc region. (See, e.g., WO 2014 / 177460.)

[0132] In certain embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that increase FcRn binding, e.g., mutations at Fc region positions 252, and / or 254, and / or 256 (EU numbering residues). In certain embodiments, the antibody variant comprises an Fc region with amino acid substitutions at positions 252, 254, and 256. In one embodiment, the substitutions are M252Y, S254T, and T256E in the Fc region derived from a human IgG1 Fc region. For other examples of Fc region variants, see also Duncan & Winter, Nature 322:738-40 (1988), U.S. Patent No. 5,648,260, U.S. Patent No. 5,624,821, and WO 94 / 29351.

[0133] The C-terminus of the heavy chain of an antibody as reported herein may be a complete C-terminus ending with amino acid residue PGK. The C-terminus of the heavy chain may also be a shortened C-terminus in which one or two of the C-terminal amino acid residues are removed. In a preferred embodiment, the C-terminus of the heavy chain is a shortened C-terminus ending with PG. In one embodiment of all embodiments reported herein, an antibody comprising a heavy chain comprising a C-terminal CH3 domain as specified herein comprises a C-terminal glycine-lysine dipeptide (G446 and K447, EU index numbering of amino acid positions). In one embodiment of all embodiments reported herein, an antibody comprising a heavy chain comprising a C-terminal CH3 domain as specified herein comprises a C-terminal glycine residue (G446, EU index numbering of amino acid positions).

[0134] c) Cysteine ​​Engineered Antibody Variants In certain embodiments, it may be desirable to generate cysteine ​​engineered antibodies, e.g., THIOMAB™, in which one or more residues of an antibody are substituted with cysteine ​​residues. In certain embodiments, the substituted residues occur at accessible sites on the antibody. By replacing these residues with cysteine, reactive thiol groups are thereby placed at accessible sites on the antibody, which can be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create immunoconjugates, as further described herein. Cysteine ​​engineered antibodies can be generated, for example, as described in U.S. Pat. No. 7,521,541, U.S. Pat. No. 8,30,930, U.S. Pat. No. 7,855,275, U.S. Pat. No. 9,000,130, or WO2016040856.

[0135] B. Recombinant Methods and Compositions Antibodies can be produced using recombinant methods and compositions, for example, as described in U.S. Patent No. 4,816, 567. For these methods, one or more isolated nucleic acids encoding the antibody are provided.

[0136] In one aspect, an isolated nucleic acid encoding an antibody of the invention is provided.

[0137] In one aspect, a method is provided for producing an antibody that binds to human VEGF-A and human IL6, the method comprising culturing a host cell containing nucleic acid encoding the antibody under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture).

[0138] For recombinant production of antibodies that bind to human VEGF-A and human IL6, for example, nucleic acids encoding the above-mentioned antibodies are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of the antibody), or can be produced by recombinant methods, or can be obtained by chemical synthesis.

[0139] Suitable host cells for cloning or expressing antibody-encoding vectors include the prokaryotic or eukaryotic cells described herein. For example, antibodies may be produced in bacteria, particularly if glycosylation and Fc effector functions are not required. For the expression of antibody fragments and polypeptides in bacteria, see, e.g., US Pat. Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, KA, In: Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2003), pp. 245-254, which describes the expression of antibody fragments in E. coli.) After expression, the antibody may be isolated from the bacterial cell paste in appropriate fractions and further purified. In one embodiment, the host cell is an E. coli cell.

[0140] Vertebrate cells may also be used as hosts. For example, mammalian cell lines adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed with SV40 (COS-7), human embryonic kidney lines (e.g., 293 cells or 293T cells as described in Graham, FL et al., J. Gen Virol. 36 (1977) 59-74, baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4 cells as described in Mather, JP, Biol. Reprod. 23 (1980) 243-252), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor (MMT 060562), TRI cells (e.g., Mather, JP et al., Annals NY Acad. Sci. 383 (1982) 44-68), MRC5 cells, and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77 (1980) 4216-4220), and myeloma cell lines, such as Y0, NS0, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki, P. and Wu, A. M., Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268.

[0141] In one aspect, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell or a lymphocyte cell (e.g., Y0, NS0, Sp20 cell). In one preferred embodiment, the host cell is a CHO cell. Production of the antibody of the present invention in a CHO cell can improve the injectability of the antibody.

[0142] C. Pharmaceutical Compositions In a further aspect, a pharmaceutical composition is provided comprising any of the antibodies provided herein, e.g., for use in any of the following therapeutic methods. In one aspect, the pharmaceutical composition comprises any of the antibodies provided herein and a 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., those described below.

[0143] Pharmaceutical compositions of the antibodies that bind to human VEGF-A and human IL6 described herein are prepared by mixing such antibodies having 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 a lyophilized composition or aqueous solution. Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed and may contain buffers such as histidine, phosphate, citrate, acetate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polysaccharides. Examples of suitable pharmaceutically acceptable carriers include, but are not limited to, peptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or 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 non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include intercalating drug dispersants, such as soluble neutral active hyaluronidase glycoproteins (sHASEGPs), e.g., human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Halozyme, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Application Publication Nos. 2005 / 0260186 and 2006 / 0104968.In one embodiment, the sHASEGP is combined with one or more additional glycosaminoglycanases (eg, chondroitinases).

[0144] Exemplary lyophilized antibody compositions are described in U.S. Patent No. 6,267,958. Aqueous antibody compositions include those described in U.S. Patent No. 6,171,586 and WO2006 / 044908, the latter compositions comprising a histidine-acetate buffer.

[0145] The pharmaceutical compositions herein may also contain multiple active ingredients as needed for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other, and such active ingredients are suitably present in combination in amounts that are effective for the purpose intended.

[0146] The active ingredient may be encapsulated in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively), colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed., (1980).

[0147] Sustained-release pharmaceutical compositions can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

[0148] Pharmaceutical compositions to be used for in vivo administration are generally sterile. Sterilization may be readily accomplished, for example, by filtration through sterile filtration membranes.

[0149] D. Treatment Methods and Routes of Administration Any of the antibodies provided herein that bind to human VEGF-A and human IL6 can be used in therapeutic methods.

[0150] In one embodiment, an antibody that binds human VEGF-A and human IL6 is provided for use as a pharmaceutical. In a further embodiment, an antibody that binds human VEGF-A and human IL6 is provided for use in treating vascular disease. In certain embodiments, an antibody that binds human VEGF-A and human IL6 is provided for use in a method of treatment. In certain embodiments, the present invention provides an antibody that binds human VEGF-A and human IL6 for use in a method of treating an individual having vascular disease, the method comprising administering to the individual an effective amount of an antibody that binds human VEGF-A and human IL6. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent (e.g., one, two, three, four, five, or six additional therapeutic agents), e.g., as described below. In a further embodiment, the present invention provides an antibody that binds human VEGF-A and human IL6 for use in inhibiting angiogenesis. In a particular embodiment, the present invention provides an antibody that binds human VEGF-A and human IL6 for use in a method of inhibiting angiogenesis in an individual, the method comprising administering to the individual an effective amount of an antibody that binds human VEGF-A and human IL6 to inhibit angiogenesis. An "individual" according to any of the above embodiments is preferably a human.

[0151] In a further aspect, an antibody that binds human VEGF-A and human IL6 is provided for use in treating an ocular disease. In one embodiment, the ocular disease is AMD (in one embodiment, wet AMD, dry AMD, intermediate AMD, advanced AMD, and geographic atrophy (GA)), macular degeneration, macular edema, DME (in one embodiment, focal, non-central DME, and diffuse, centrally involved DME), retinopathies, diabetic retinopathy (DR) (in one embodiment, proliferative DR (PDR), non-proliferative DR (NPDR), and high altitude DR), other ischemia-related retinopathies, ROP, retinopathies. Retinal vein occlusion (RVO) (in one embodiment, central (CRVO) and branched (BRVO) forms), CNV (in one embodiment, myopic CNV), corneal neovascularization, diseases associated with corneal neovascularization, retinal neovascularization, diseases associated with retinal / choroidal neovascularization, central serous retinopathy (CSR), pathological myopia, von Hippel-Lindau disease, ocular histoplasmosis, FEVR, Coats disease, Norrie disease, osteoporotic pseudoglioma syndrome (OPPG) Associated retinal abnormalities, subconjunctival hemorrhage, rubeosis, ocular neovascular disease, neovascular glaucoma, retinitis pigmentosa (RP), hypertensive retinopathy, retinal angiomatous proliferation, macular vascular ectasia, iris neovascularization, intraocular neovascularization, retinal degeneration, cystoid macular edema (CME), vasculitis, papilledema, retinal inflammation including, but not limited to: CMV retinitis, ocular melanoma, retinoblastoma, conjunctivitis (in one embodiment, infectious conjunctivitis and non-infectious (in one embodiment, allergic In one embodiment, the ocular disease is selected from: ocular conjunctivitis, Leber's congenital black circle (also known as Leber's congenital black circle or LCA), uveitis (including infectious and non-infectious uveitis), choroiditis (in one embodiment, multifocal choroiditis), ocular histoplasmosis, blepharitis, dry eye, traumatic eye injury, Sjogren's disease, and other ocular diseases in which the disease or disorders are associated with ocular neovascularization, vascular leakage, and / or retinal edema or retinal atrophy. In one embodiment, the ocular disease is selected from AMD (in one embodiment, wet AMD, dry AMD, intermediate AMD, advanced AMD, and geographic atrophy (GA)), macular degeneration, macular edema, DME (in one embodiment, focal, non-central DME, and diffuse, centrally involved DME), retinopathy, diabetic retinopathy (DR) (in one embodiment, proliferative DR (PDR), non-proliferative DR (NPDR), and high altitude DR).

[0152] In a further aspect, the present invention provides use of an antibody that binds to human VEGF-A and human IL6 in the manufacture or preparation of a medicament. In one aspect, the medicament is for treating vascular disease. In a further aspect, the medicament is for use in a method of treating vascular disease, comprising administering an effective amount of the medicament to an individual having vascular disease. In one such aspect, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, such as those described below.

[0153] In one embodiment, the medicament is for treating an ocular disease. In a further embodiment, the medicament is for use in a method for treating an ocular disease, comprising administering an effective amount of the medicament to an individual having the ocular disease. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, such as those described below.

[0154] In a further aspect, the present invention provides a method for treating a vascular disease. In one aspect, the method comprises administering to an individual having such a vascular disease an effective amount of an antibody that binds to human VEGF-A and human IL6. In one such aspect, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, as described below.

[0155] In a further aspect, the present invention provides a method for treating an ocular disease. In one aspect, the method comprises administering to an individual having such an ocular disease an effective amount of an antibody that binds to human VEGF-A and human IL6. In one such aspect, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, as described below.

[0156] An "individual" according to any of the above embodiments may be a human.

[0157] In a further aspect, the present invention provides pharmaceutical compositions comprising any of the antibodies that bind human VEGF-A and human IL6 provided herein, e.g., for use in any of the above-described methods of treatment. In one aspect, the pharmaceutical composition comprises any of the antibodies that bind human VEGF-A and human IL6 provided herein and a pharmaceutically acceptable carrier. In another aspect, the pharmaceutical composition comprises any of the antibodies that bind human VEGF-A and human IL6 provided herein and at least one additional therapeutic agent, e.g., as described below.

[0158] The antibodies of the invention can be administered by intravitreal administration (e.g., intravitreal injection) or using a port delivery device. In one embodiment, the antibodies of the invention are administered using a port delivery device for a period of 6 months or more, in one embodiment 8 months or more, in one embodiment 9 months or more, and in one embodiment 12 months or more before the port delivery device is refilled. In one embodiment, the antibodies of the invention are administered using a port delivery device, and the antibody is applied to the port delivery device at a concentration of 150 mg / ml or more, in one embodiment 200 mg / ml or more.

[0159] The antibodies of the invention can be administered alone or in combination therapy, for example, a combination therapy includes administering an antibody of the invention and at least one additional therapeutic agent (e.g., 1, 2, 3, 4, 5, or 6 additional therapeutic agents).

[0160] In certain embodiments according to (or applied to) any of the above embodiments, the ocular disease is an intraocular neovascular disease selected from the group consisting of proliferative retinopathy, choroidal neovascularization (CNV), age-related macular degeneration (AMD), diabetic and other ischemia-related retinopathies, diabetic macular edema, pathological myopia, von Hippel-Lindau disease, ocular histoplasmosis, retinal vein occlusion (RVO) including CRVO and BRVO, corneal neovascularization, retinal neovascularization, and retinopathy of prematurity (ROP).

[0161] In some examples, the antibodies that bind human VEGF-A and human IL6 provided herein may be administered in combination with at least one additional therapeutic agent for treating an ocular disorder, e.g., an ocular disorder described herein (e.g., AMD (e.g., wet AMD), DME, DR, RVO, or GA).

[0162] Any suitable AMD therapeutic agent may be a VEGF antagonist, such as an anti-VEGF antibody (e.g., LUCENTIS® (ranibizumab), RTH-258 (formerly ESBA-1008, an anti-VEGF single-chain antibody fragment; Novartis), or a bispecific anti-VEGF antibody (e.g., an anti-VEGF / anti-angiopoietin 2 bispecific antibody, e.g., faricimab; Roche)), a soluble VEGF receptor fusion protein (e.g., EYLEA® (aflibercept)), an anti-VEGF DARPin® (e.g., abicipar pegol; Molecular Partners), or a VEGF receptor fusion protein (e.g., EYLEA® (aflibercept)). AG / Allergan), or anti-VEGF aptamers (e.g., MACUGEN® (pegaptanib sodium); platelet-derived growth factor (PDGF) antagonists, such as anti-PDGF antibodies, anti-PDGFR antibodies (e.g., REGN2176-3), anti-PDGF-BB pegylated aptamers (e.g., FOVISTA®; Ophthotech / Novartis), soluble PDGFR receptor fusion proteins, or dual PDGF / VEGF antagonists (e.g., small molecule inhibitors (e.g., DE-120 (Santen) or X-82 (TyrogeneX)) or bispecific anti-PDGF / anti-VEGF antibodies)); VI in combination with photodynamic therapy SUDYNE® (verteporfin); antioxidants; complement system antagonists, such as complement factor C5 antagonists (e.g., small molecule inhibitors (e.g., ARC-1905; Opthotech) or anti-C5 antibodies (e.g., LFG-316; Novartis), properdin antagonists (e.g., anti-properdin antibodies, e.g., CLG-561; Alcon), or complement factor D antagonists (e.g., anti-complement factor D antibodies, e.g., lampalizumab; Roche)); C3 blocking peptides (e.g., APL-2, Appellis); visual cycle modifiers (e.g., emixustat hydrochloride); squalamine (e.g., OHR-102; Ohr Pharmaceutical); vitamin and mineral supplements (e.g., Age-Related Eye Disease Study 1 (AREDS1; zinc and / or antioxidants) and Study 2 (AREDS2;zinc, antioxidants, lutein, zeaxanthin, and / or omega-3 fatty acids); cell-based therapies, e.g., NT-501 (Renexus); PH-05206388 (Pfizer), huCNS-SC cell transplantation (StemCells), CNTO-2476 (umbilical cord stem cell line; Janssen), OpRegen (suspension of RPE cells; Cell Cure Neurosciences), or MA09-hRPE cell transplantation (Ocata Therapeutics); tissue factor antagonists (e.g., hI-con1; Iconic Therapeutics); α-adrenergic receptor agonists (e.g., brimonidine tartrate; Allergan); peptide vaccines (e.g., S-646240; Shionogi); amyloid-β antagonists (e.g., anti-β amyloid monoclonal antibodies, e.g., GSK-933776); S1P antagonists (e.g., anti-S1P antibodies, e.g., iSONEP™; Lpath Inc); ROBO4 antagonists (e.g., anti-ROBO4 antibodies, e.g., DS-7080a; Daiichi Sankyo); lentiviral vectors expressing endostatin and angiostatin (e.g., RetinoStat); and any combination thereof may be administered as an additional therapeutic agent in combination with the antibodies binding to human VEGF and human IL6 provided herein for the treatment of ocular diseases (e.g., AMD, DME, DR, RVO, or GA). In some examples, AMD therapeutic agents (including any of the aforementioned AMD therapeutic agents) can be co-formulated. For example, the anti-PDGFR antibody REGN2176-3 can be co-formulated with aflibercept (EYLEA®). In some examples, such co-formulations can be administered in combination with antibodies of the present invention that bind to human VEGF and human IL6. In some examples, the ocular disorder is AMD (e.g., wet AMD).

[0163] Any suitable DME and / or DR therapeutic agent, including but not limited to a VEGF antagonist (e.g., LUCENTIS® or EYLEA®), a corticosteroid (e.g., a corticosteroid implant (e.g., OZURDEX® (dexamethasone intravitreal implant) or ILUVIEN® (fluocinolone acetonide intravitreal implant)) or a corticosteroid formulated for administration by intravitreal injection (e.g., triamcinolone acetonide)), or a combination thereof, can be administered in combination with an antibody that binds human VEGF and human IL6 of the present invention for the treatment of an ocular disorder (e.g., AMD, DME, DR, RVO, or GA). In some examples, the ocular disorder is DME and / or DR.

[0164] The antibodies that bind human VEGF and human IL6 provided herein are useful in treating retinal diseases, including retinal detachment and vitreous detachment, such as retinal detachment, vitreous detachment, and vitreous detachment. The antibodies that bind human VEGF and human IL6 provided herein are useful in treating retinal detachment and vitreous detachment, including retinal detachment and vitreous detachment, such as vitreous detachment, vitreous detachment, and vitreous detachment. The antibodies that bind human VEGF and human IL6 provided herein are useful in treating retinal detachment and vitreous detachment, including retinal detachment and vitreous detachment, such as vitreous detachment, vitreous detachment, and vitreous detachment, including vitreous detachment and vitreous detachment, such as vitreous detachment, vitreous detachment, and ... The present invention may be administered in conjunction with therapeutic or surgical procedures for the treatment of ocular disorders (e.g., AMD, DME, DR, RVO, or GA), including ocular therapy (e.g., ophthalmic solution), paracentesis, and combinations thereof.

[0165] Such combination therapy as described above encompasses combined administration (wherein two or more therapeutic agents are contained in the same or separate formulations) and separate administration, where administration of an antibody of the present invention that binds human VEGF and human IL6 can occur prior to, simultaneously with, and / or following administration of the additional therapeutic agent or agent. In one embodiment, administration of an antibody of the present invention that binds human VEGF and human IL6 and administration of the additional therapeutic agent occur within about 1, 2, 3, 4, or 5 months, or within about 1, 2, or 3 weeks, or within about 1, 2, 3, 4, 5, or 6 days of each other.

[0166] 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 desired for localized treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, for example, injection, such as intravenous or subcutaneous injection, depending in part on whether administration is short-term or long-term. Various dosing schedules are contemplated herein, including, but not limited to, single or multiple doses over various time points, bolus administration, and pulse infusion.

[0167] The antibodies of the present invention will be formulated, dosed, and administered in a manner consistent with good medical practice. Factors to consider in this regard include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of drug delivery, the method of administration, the administration schedule, and other factors known to medical professionals. The antibodies are optionally, but need not be, formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents will depend on the amount of antibody present in the pharmaceutical composition, the type of disease or treatment, and other factors discussed above. These will generally be used in the same dosages and via any route of administration as described herein, or about 1-99% of the dosages described herein, or at any dosage and via any route empirically / clinically determined to be appropriate.

[0168] For disease prevention or treatment, the appropriate dosage of the antibody of the invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease being treated, the type of antibody, the severity and course of the disease, whether the antibody is being administered for prophylactic or therapeutic purposes, previous therapy, the patient's medical history and response to the antibody, and the discretion of the attending physician. The antibody of the invention is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg to 10 mg / kg) of antibody may be an initial candidate dosage for administration to the patient, whether by one or more separate administrations or by continuous infusion, for example. Typical daily dosages may range from about 1 μg / kg to 100 mg / kg, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on symptoms, treatment is usually continued until a desired suppression of disease symptoms occurs. One exemplary dosage of antibody would be in the range of about 0.05 mg / kg to about 10 mg / kg. Thus, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, or 10 mg / kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, for example, weekly or every three weeks (e.g., so that the patient receives about two to about 20, or, for example, about six, doses of antibody). An initial higher loading dose may be administered, followed by one or more lower doses. The progress of this therapy is easily monitored by conventional techniques and assays.

[0169] E.Manufactured products In another aspect of the present invention, an article of manufacture containing materials useful for the treatment, prevention, and / or diagnosis of the aforementioned disorders is provided. The article of manufacture includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, vials, syringes, and the like. The container may be formed from a variety of materials, such as glass or plastic. The container holds a composition to be used alone or in combination with another composition effective in treating, preventing, and / or diagnosing a condition and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection 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 for treating the condition of choice.

[0170] Additionally, the article of manufacture may comprise (a) a first container containing a composition comprising an antibody of the invention; and (b) a second container containing a composition comprising an additional cytotoxic or other therapeutic agent. The article of manufacture in this aspect of the invention may further comprise a package insert indicating that the composition can be used to treat a particular condition. Alternatively, or in addition, the article of manufacture may further comprise a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, such as other buffers, diluents, filters, needles, syringes, etc.

[0171] F. Equipment The antibodies of the invention may be administered to the eye using an ocular implant, in one embodiment using a port delivery device.

[0172] A port delivery device is an implantable, rechargeable device that can release a therapeutic agent (e.g., an antibody of the invention) over several months (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or more). Exemplary port delivery devices that may be used include those from ForSight Labs, LLC and / or ForSight VISION4, as described, for example, in International Patent Application Publication Nos. WO 2010 / 088548, WO 2015 / 085234, WO 2013 / 116061, WO 2012 / 019176, WO 2013 / 040247, and WO 2012 / 019047, which are incorporated by reference in their entireties.

[0173] For example, the present invention provides a port delivery device comprising a reservoir containing any of the antibodies described herein. The port delivery device may further comprise a proximal region, a tubular body coupled to the proximal region in fluid communication with the reservoir, and one or more outlets in fluid communication with the reservoir and configured to release the composition into the eye. The tubular body may have an outer diameter configured to be inserted through an incision or opening in the eye of about 0.5 mm or less. The device may be about 1 mm to about 15 mm in length (e.g., about 1 mm, about 2 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 9 mm, about 11 mm, about 13 mm, or about 15 mm in length). The reservoir may have any suitable volume. In some cases, the reservoir has a volume of about 1 μl to about 100 μl (e.g., about 1 μl, about 5 μl, about 10 μl, about 20 μl, about 50 μl, about 75 μl, or about 100 μl). The device or its components may be made of any suitable material, for example polyimide.

[0174] In some examples, the port delivery device comprises a reservoir containing any of the antibodies described herein and one or more additional compounds.

[0175] In some examples, the port delivery device comprises any of the antibodies or antibody conjugates described herein and an additional VEGF antagonist.

[0176] 3. Specific Embodiments of the Invention Specific embodiments of the present invention are listed below.

[0177] 1. An antibody that binds to human VEGF-A and human IL6, comprising a VH domain comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 18, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 19, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 20, and a VL domain comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 15, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17, wherein (a) an antibody comprising: (a) a VH domain comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 22; and (b) a VL domain comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 21.

[0178] 2. An antibody that binds to human VEGF-A and human IL6, comprising: a VH domain comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 18, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 19, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 20; and a VL domain comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 15, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17, and comprising: a variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 22 with up to five amino acid substitutions; and a variable light chain domain comprising the amino acid sequence of SEQ ID NO: 21 with up to five amino acid substitutions.

[0179] 3. An antibody that binds to human VEGF-A and human IL6, comprising: (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 22 with up to 15, up to 10, or up to 5 amino acid substitutions; and (b) a variable light chain domain comprising the amino acid sequence of SEQ ID NO: 21 with up to 15, up to 10, or up to 5 amino acid substitutions.

[0180] 4. An antibody that binds to human VEGF-A and human IL6, comprising the VH sequence of SEQ ID NO: 22 and the VL sequence of SEQ ID NO: 21.

[0181] 5. The antibody of one of the preceding embodiments, comprising a heavy chain amino acid sequence of SEQ ID NO: 24 and a light chain amino acid sequence of SEQ ID NO: 23.

[0182] 6. The antibody of any one of the preceding embodiments, wherein the VEGF-A paratope comprises amino acid residues from CDR-H2, CDR-L1, and CDR-L3 of the antibody, and the IL6 paratope comprises amino acid residues from CDR-H1, CDR-H3, and CDR-L2 of the antibody, or the IL6 paratope comprises amino acid residues from CDR-H2, CDR-L1, and CDR-L3 of the antibody, and the VEGF-A paratope comprises amino acid residues from CDR-H1, CDR-H3, and CDR-L2 of the antibody; and / or the pair of variable light and heavy chain domains simultaneously binds human VEGF-A and human IL6; and / or the antibody binds to the same epitope on human VEGF-A and the same epitope on human IL6 as an antibody having a variable heavy chain domain of SEQ ID NO: 22 and a variable light chain domain of SEQ ID NO: 21; and / or The antibody Fab fragment of the antibody has (i) a K of less than 50 pM as measured by surface plasmon resonance D and (ii) binds to human VEGF-A121 with a K of less than 50 pM as measured by surface plasmon resonance. D binds to human IL6 at the antibody Fab fragment of the antibody exhibits an aggregation onset temperature of 60°C or higher, and in one embodiment 70°C or higher; and / or -An antibody, the antibody Fab fragment of which exhibits a melting temperature of greater than 80°C as measured by dynamic light scattering.

[0183] 7. An antibody that specifically binds to human VEGF-A and human IL6, comprising the heavy chain amino acid sequence of SEQ ID NO: 24 and the light chain amino acid sequence of SEQ ID NO: 23.

[0184] 8. The antibody of any one of the preceding embodiments, which is a Fab fragment.

[0185] 9. The antibody of any one of the preceding embodiments, which is a bispecific antibody fragment.

[0186] 10. The antibody of any one of the preceding embodiments, which is a monoclonal antibody.

[0187] 11. The antibody of any one of the preceding embodiments, wherein the antibody Fab fragment exhibits an aggregation onset temperature of 70°C or greater.

[0188] 12. The antibody of any one of the preceding embodiments, wherein the antibody Fab fragment of the antibody exhibits a melting temperature of greater than 80°C as measured by dynamic light scattering.

[0189] 13. The antibody of any one of the preceding embodiments, which is a monoclonal antibody.

[0190] 14. The antibody of any one of the preceding embodiments, which is an antibody fragment that binds to human VEGF-A and human IL6.

[0191] 15. The antibody of any one of the preceding embodiments, which is bispecific.

[0192] 16. The antibody of any one of the preceding embodiments, which is a Fab fragment.

[0193] 17. The antibody of any one of the preceding embodiments, which is a bispecific antibody fragment.

[0194] 18. The antibody of any one of the preceding embodiments, which is a multispecific antibody.

[0195] 19. The antibody of any one of the preceding embodiments, which specifically binds to human VEGF-A.

[0196] 20. The antibody of any one of the preceding embodiments, which specifically binds to human IL6.

[0197] 21. An antibody that binds to human IL6 and binds to the same epitope on IL6 as an antibody having the VL domain of SEQ ID NO: 35 and the VH domain of SEQ ID NO: 36.

[0198] 22. An antibody that binds to human IL6, wherein the antibody comprises a VH domain having a human VH3 framework (the IL6 paratope comprises amino acid residues 1, 2, 3, 26, 27, 28, 29, 30, 31, 32, 52a, 94, 96, 97, 98, 101, 102 of the antibody that binds to human VEGF-A and IL6 described in any one of embodiments 1 to 20), and a VL domain having a human Vkappa1 framework (the IL6 paratope comprises amino acid residues 49, 50, 53, 54, 55, 56, 57, 91, 96 of the antibody that binds to human VEGF-A and IL6 described in any one of embodiments 1 to 20).

[0199] 23. An antibody that binds to human IL6, a) a VH domain based on a human VH3 framework (the IL6 paratope comprises amino acid residues Y1, I2, Q3, Y26, E27, F28, T29, H30, Q31, D32, P52a, R94, I96, D97, F98, D101, and T102), and a VL domain based on a human Vkappa1 framework (the IL6 paratope comprises amino acid residues Y49, D50, S53, N54, Y55, P56, S57, Y91, and Y96); or b) An antibody comprising a VH domain based on the human VH3 framework (the IL6 paratope comprises amino acid residues Y1, P2, Q3, V26, L27, F28, K29, H30, Q31, D32, P52a, R94, L96, D97, F98, D101, E102) and a VL domain based on the human Vkappa1 framework (the IL6 paratope comprises amino acid residues Y49, D50, D53, R54, Y55, P56, E57, Y91, Y96) (numbering according to Kabat).

[0200] 24. An isolated nucleic acid encoding an antibody according to any one of embodiments 1 to 23.

[0201] 25. A host cell comprising the nucleic acid of embodiment 24.

[0202] 26. A method for producing an antibody that binds to human VEGF-A and human IL6, comprising culturing the host cell of embodiment 25 so that the antibody is produced.

[0203] 27. The method of embodiment 26, wherein the host cell is a CHO cell.

[0204] 28. A pharmaceutical formulation comprising an antibody according to any one of embodiments 1 to 23 and a pharmaceutically acceptable carrier.

[0205] 29. A port delivery device comprising an antibody according to any one of embodiments 1 to 23.

[0206] 30. The antibody according to any one of embodiments 1 to 23 for use as a medicament.

[0207] 31. The method of embodiment 26, further comprising recovering the antibody from the host cell.

[0208] 32. An antibody produced by the method of embodiment 26 or 31.

[0209] 33. A pharmaceutical formulation comprising an antibody according to any one of embodiments 1 to 23 and a pharmaceutically acceptable carrier.

[0210] 34. The antibody according to any one of embodiments 1 to 23 for use as a medicament.

[0211] 35. The antibody of any one of embodiments 1 to 23 for use in the treatment of a vascular disease.

[0212] 36. The antibody of any one of embodiments 1 to 23 for use in the treatment of ocular vascular diseases.

[0213] 37. Use of an antibody according to any one of embodiments 1 to 23 or a pharmaceutical composition according to embodiment 65 in the manufacture of a medicament.

[0214] 38. Use of the antibody according to any one of embodiments 1 to 23 or the pharmaceutical composition according to embodiment 65 in the manufacture of a medicament for inhibiting angiogenesis.

[0215] 39. A method for treating an individual having a vascular disease, comprising administering to the individual an effective amount of an antibody according to any one of embodiments 1 to 23 or a pharmaceutical formulation according to embodiment 33.

[0216] 40. A method for treating an individual having an ocular vascular disease, comprising administering to the individual an effective amount of an antibody according to any one of embodiments 1 to 23 or a pharmaceutical formulation according to embodiment 33.

[0217] 41. A method for inhibiting angiogenesis in an individual, comprising administering to the individual an antibody according to any one of embodiments 1 to 23 or a pharmaceutical formulation according to embodiment 33 in an amount effective to inhibit angiogenesis.

[0218] 42. A port delivery device comprising the antibody of any of embodiments 1 to 23 or the pharmaceutical formulation of embodiment 33.

[0219] 43. The antibody of any of embodiments 1 to 23 or the pharmaceutical formulation of embodiment 33 for ocular administration by a port delivery device.

[0220] 44. The antibody of any of embodiments 1 to 23 or the pharmaceutical formulation of embodiment 33 for ocular administration by a port delivery device of embodiment 42, wherein administration is for a period of at least 6 months, in one embodiment at least 8 months, and in one embodiment at least 9 months, before the port delivery device is refilled.

[0221] 45. Any of embodiments 1-23 or the pharmaceutical formulation of embodiment 33 for use as a medicament by administering the antibody or pharmaceutical formulation using a port delivery device, wherein the antibody is applied to the port delivery device at a concentration of 150 mg / ml or more, and in one embodiment at a concentration of 200 mg / ml or more. [Table 2] TIFF0007801491000004.tif220161 TIFF0007801491000005.tif220161 TIFF0007801491000006.tif212161 TIFF0007801491000007.tif211161 TIFF0007801491000008.tif208161 TIFF0007801491000009.tif227161 TIFF0007801491000010.tif233161 TIFF0007801491000011.tif197161 TIFF0007801491000012.tif112161 [Example]

[0222] The following examples are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.

[0223] Example 1: Generation of bispecific anti-VEGF / anti-6 Fab fragments Bispecific anti-VEGF / anti-IL-6 Fab fragments were generated by providing antibodies with distinct, non-overlapping paratopes that bind VEGF and IL-6, using methods similar to those previously described, for example, in WO 2012 / 163520.

[0224] Here, two different phage display libraries of synthetic Fab fragments were utilized: in the first, residues within the CDR-H1, CDR-H3, and CDR-L2 regions of the Fab fragments were diversified; in the second, residues within the CDR-L1, CDR-L3, and CDR-H2 regions of the Fab fragments were diversified. In each library, the other three CDR regions were maintained undiversified to represent paratopes capable of binding to VEGF-A, in contrast to the method of WO 2012 / 163520, which used constant, non-binding germline-like ("dummy") sequences.

[0225] For the first library, the paratopes capable of binding VEGF-A were derived from the VEGF-A binding paratopes described in WO 2021 / 198034.

[0226] For the second library, VEGF-A binding paratopes were obtained as follows: For naive selection, phage library panning was performed using a library in which CDR-H1, CDR-H3, and CDR-L2 were diversified as described in WO 2012 / 163520. The remaining CDR sequences were kept constant using non-binding germline-like sequences. Four rounds were performed: the first round was performed using 100 nM biotinylated VEGF-121 or VEGF-165 pre-immobilized on Dynabeads M-280 streptavidin (Thermofisher catalog number 11206D). Rounds 2–4 of panning were performed using 75, 15, and 3 nM biotinylated target in solution, respectively, followed by capture of the Fab-on-phage / target complexes on Dynabeads M-280 streptavidin. The phage / target / bead complexes were washed multiple times with PBST and PBS buffer. Captured phage clones carrying target-specific Fabs were eluted from M-280 beads using 100 mM DTT according to standard protocols, used to infect log-phase TG1 E. coli cells, and rescued using M13 K07 helper phage.

[0227] For screening of selection outputs, polyclonal plasmid minipreps were prepared from infected TG1 E. coli cells from each selection round. The plasmids were reformatted to produce soluble Fabs in the E. coli supernatant with a T7 tag at the C-terminus of the Fab CH1 domain. The ligated polyclonal plasmids encoding the T7-tagged Fabs were transformed into TG1 E. coli cells (Zymo Research catalog number T3017), and single colonies were picked into microtiter plates. Soluble Fabs were expressed in microtiter plates, and the supernatants were clarified by centrifugation. Target binding was assessed by ELISA against VEGF and competitive ELISA against VEGF receptor 2. Candidate binders were selected based on high binding signals to VEGF and good inhibition of receptor binding.

[0228] The binders were then expressed and purified in larger volumes, and binding to VEGF was assessed using SPR measurements. One of the resulting clones was further optimized through an iterative protein engineering and testing strategy and incorporated into a phage display library as the invariant sequences for CDR-H1, CDR-H3, and CDR-L2. Briefly, the protein engineering workstream consisted of an initial series of scout mutations to identify relevant beneficial mutations, followed by two successive rounds of affinity maturation based on oligonucleotide-based generation of a mutant library and phage display-based selection, followed by screening and further testing.

[0229] In both libraries, the CH1 domains of the Fab fragments were fused to the truncated gene-III protein via a linker to facilitate phage display. Thus, one library was intended to screen for bispecific Fab fragments whose IL6 paratope comprised amino acid residues from CDR-H1, CDR-H3, and CDR-L2 (referred to herein as the "6HVL" library), and the other library was intended to screen for bispecific Fab fragments whose IL6 paratope comprised amino acid residues from CDR-H2, CDR-L1, and CDR-L3 (referred to herein as the "VH6L" library).

[0230] Each library was enriched for human IL-6 binders by phage library panning. After panning, plasmid minipreps were generated for both enriched pools of phagemid vectors. The minipreps were digested with restriction enzymes to excise the region encoding the truncated gene-III protein and recircularized by ligation to obtain pools of expression vectors encoding soluble Fab fragments enriched for IL-6 binders. These vector pools were transformed into TG1 E. coli cells, and individual colonies were picked and cultured in microtiter plates for soluble expression of individual Fab clones. The supernatants containing the soluble Fab fragments were screened for binding to IL-6 and VEGF-A using standard ELISA methods.

[0231] Based on the screening data, bispecific anti-VEGF / anti-IL-6 Fab fragments were selected and TG1 clones producing specific binders were subjected to DNA plasmid preparation and sequencing to obtain a pair of VH and VL sequences from each library, respectively, that together encode one bispecific Fab fragment that specifically binds both IL-6 and VEGF-A: The clones were 6HVL_1, characterized by a heavy chain of SEQ ID NO: 03 and a light chain of SEQ ID NO: 04, and VH6L_1, characterized by a heavy chain of SEQ ID NO: 09 and a light chain of SEQ ID NO: 10.

[0232] Example 2: Expression and characterization of bispecific anti-VEGF / anti-IL-6 Fab fragments 6HVL_1 and VH6L_1 The resulting bispecific anti-VEGF / anti-IL-6 Fab fragments were characterized. The resulting vectors were transformed into TG1 E. coli cells as described in Example 1, and individual colonies were cultured for soluble expression of the bispecific antibody Fab fragments for both 6HVL_1 and VH6L_1. The bispecific antibodies were purified from TG1 culture supernatants by affinity chromatography. The binding of the bispecific antibodies 6HVL_1 and VH6L_1 to IL-6 from human and cynomolgus monkey IL-6, human VEGF121, and human VEGF165 was evaluated.

[0233] Example 3: Characterization of bispecific anti-VEGF / anti-IL-6 Fab fragments 6HVL_1 and VH6L_1 IL-6 binding kinetics as assessed by surface plasmon resonance (SPR): Surface plasmon resonance (SPR) was used to measure the binding kinetics and affinity of representative VEGF-IL-6 Fab fragments to human and cynomolgus IL-6 disclosed herein.

[0234] SPR analysis of the binding of human and cynomolgus monkey IL-6 Fab fragments was performed on a Biacore 8K instrument (Cytiva) at 25°C using HBS-EP+ (1x; BR100669; Cytiva) as the running buffer. Human Fab binder (28958325, Cytiva) was diluted to a final concentration of 10 μg / ml in 10 mM sodium acetate buffer, pH 5.0, and immobilized onto a CM5 sensor chip using standard amine coupling chemistry. This immobilization procedure resulted in a ligand density of approximately 5000 resonance units (RU). The reference channel was treated accordingly.

[0235] Prior to protein measurement, five conditioning cycles were performed. In each cycle, the derivatized chip surface was regenerated by flowing HBS-EP+ buffer for 120 seconds, followed by application of 10 mM glycine buffer, pH 2.0, for 60 seconds. Fab fragments at a concentration of 75 nM were captured on this surface for 60 seconds at a flow rate of 10 μl / min in HBS-EP+ buffer. No Fab fragments were applied to the reference channel. Subsequently, human or cynomolgus IL-6 was applied in appropriate dilutions in HBS-EP+ buffer at a flow rate of 30 μl / min (contact time: 180 seconds, dissociation time: 720 seconds). Regeneration of the derivatized chip surface was achieved as described above. Data were evaluated using 8K evaluation software (Biacore Insight Evaluation 3.0). Double referencing was used, and raw data was fitted using a 1:1 binding model.

[0236] Figure 1 shows representative SPR traces and fitting curves determined for the tested Fab fragments, with the corresponding Fab names indicated on the graph. Data are shown for binding to human and cynomolgus IL-6, as well as IL-1α (IL-1a) as a negative control. The affinities shown on the graph correspond to the mean and standard deviation of three independent experiments.

[0237] We observe clear binding of 6HVL_1 and VH6L_1 to human IL-6. Only VH6L_1 shows significant affinity for cyIL-6, albeit with an apparently very fast off-rate. No binding to the negative control target IL-1a is observed. The results of fitting the SPR data are shown in Table 1. Data are averaged for three experiments, and standard deviations are provided for the dissociation constant KD. For 6HVL_1, an affinity of KD = 0.9 nM is observed, while for VH6L_1, the affinity is KD = 10.7 nM. [Table 3]

[0238] VEGF binding assessed by competitive ELISA: To test the antibody concentrations required to block the interaction of VEGF121 and VEGF165 with their receptors, a competitive ELISA experiment using 6HVL_1 and VH6L_1 was performed. A VEGF-binding Fab fragment (ranibizumab) was used as a positive control, and an experiment using buffer alone was used as a negative control. Briefly, a 1:3 dilution series of all samples, starting at 20 nM, was mixed with a fixed concentration of 10 pM VEGF121 (Humanzyme HZ-1206) or 10 pM VEGF165 (Humanzyme HZ-1153) and incubated for 90 min. The mixture was then transferred to a Maxisorp plate coated with VEGF receptor 1 (VEGF-R1, R&D Systems, 1 μg / ml in NaHCO3, pH 9.4) after blocking the plate surface with 2% MPBST. The contact time between the Fab-antigen mixture and the receptor-coated plate was limited to 10 min at room temperature to minimize interference with binding equilibrium. After incubation and two washing steps, detection of VEGF121 / VEGF165 on the VEGFR1-coated plate was performed using a biotinylated anti-VEGF mAb (BAF203, R&D Systems) and horseradish peroxidase-conjugated streptavidin (HRP-streptavidin). The latter was detected using the chromogenic conversion of the HRP substrate 3,3',5,5'-tetramethylbenzidine TMB to 3,3',5,5'-tetramethylbenzidinediamine, followed by a change in absorbance at 450 nm. TMB was preheated to room temperature and incubated on the plate for 5 min, after which it was quenched with 1N H2SO4.

[0239] The results with the target VEGF165 are shown in Figure 2 and Tables 5 and 6. Clearly, both VH6L_1 and 6HVL_1 show a much improved ability to compete with the binding of both VEGF165 and VEGF121 to VEGFR1 compared to ranibizumab, a clinically well-established VEGF-A antagonist.

[0240] Example 4: Improved bispecific anti-VEGF-A / anti-IL6 Fab fragment As mentioned above, both antibodies showed no or low cross-reactivity with cynomolgus IL-6, which is desirable for clinical development. Additionally, treatment of ocular vascular disease requires injection of therapeutic agents into the eye. Consequently, optimal therapeutic agents should exhibit high affinity for the target antigen and high concentration to maximize the duration of therapeutic effect and patient convenience. Therefore, further improvement of the initially identified molecules for their intended purpose is desirable.

[0241] Several rounds of maturation were performed by introducing different amino acid substitutions into the VH and VL domains. During maturation, candidate antibodies derived from both the "parent" antibodies 6HVL_1 and VH6L_1 were screened and selected based on their desired properties in terms of yield, affinity, simultaneous antigen binding, hydrophilicity, stability, viscosity, and other parameters.

[0242] Improved candidate antibodies 6HVL_2, 6HVL_3, and 6HVL_4, as well as VH6L_2 and VH6L_3, were selected from multiple tested candidate antibody molecules from each round of maturation. Candidate selection was based on improving desired properties, particularly human IL6 binding and cynomolgus IL6 cross-reactivity, while ensuring injectability at high concentrations and maintaining other advantageous properties, such as VEGF-A affinity and thermal stability.

[0243] The improved candidate antibody 6HVL_4 was selected as the preferred candidate from multiple tested candidate antibody molecules. [Table 4]

[0244] All Fab fragments contained the same constant regions as contained in the full-length light and heavy chain amino acid sequences of antibody VH6L_4, namely CL having SEQ ID NO:29 and CH1 having SEQ ID NO:30.

[0245] Candidate antibodies were expressed as described in Example 2.

[0246] Example 5: Improved antigen binding kinetics of anti-VEGF-A / anti-IL6 Fab fragments The binding kinetics to human and cynomolgus IL6 and the competitive IC50 for VEGF / VEGFR1 competition for candidate antibodies were evaluated as described above using the indicated Fab fragments (amino acid sequences shown in Table 2 and Example 2). To determine the potency of the antibodies of the present invention relative to the prior art, the following controls were used: the bispecific antibody VH6L (VH / VL sequence disclosed in WO 2012 / 163520, herein referred to as "VH6L-BM"), the anti-VEGF antibody ranibizumab (INN), and an anti-IL6 antibody cross-reactive between human and cynomolgus IL6 (positive control) as disclosed in WO 2014 / 074905. The aforementioned prior art antibodies were prepared by recombinant expression.

[0247] Figure 3 and Tables 3 and 4 show the results of the human and cynomolgus IL6 binding assessment. Variants of 6HVL_4 with three additional framework amino acid mutations, 6HVL_4 and 6HVL_4-YHE, show improved human IL6 binding and cynomolgus IL6 cross-reactivity over the originally selected parent molecule in a pharmacologically relevant range. [Table 5] [Table 6]

[0248] Figure 4 and Tables 5 and 6 show the results of the evaluation of VEGF binding assessed by competitive ELISA using human VEGF 121 and human VEGF 165. Figure 4 illustrates that the prior art molecule 6HVL_BM exhibits an affinity that is too low to be detected under the conditions of the assay, and is therefore clearly much lower than the affinity of the antibody of the present invention. [Table 7] [Table 8] TIFF0007801491000019.tif40170

[0249] Example 6: Simultaneous binding of anti-VEGF / anti-IL-6 Fab fragments Simultaneous binding of the antibodies of the invention to their targets was assessed as follows by surface plasmon resonance using immobilized anti-Fab antibodies to capture the anti-VEGF / anti-IL-6 Fab fragments of the invention.

[0250] Approximately 5000 resonance units (RU) of anti-Fab antibody (Cytiva 28958325) were immobilized on a Series S Sensor Chip CM5 (Cytiva BR100530) using standard amine coupling chemistry. HBS-P+ (10 mM HEPES, 150 mM NaCl pH 7.4, 0.05% surfactant P20) was used as the running and dilution buffer, and the flow cell temperature was set to 25°C.

[0251] Anti-VEGF / anti-IL-6 Fab fragments were captured via their kappa chains by injecting a 10 μg / mL solution at a flow rate of 5 μL / min for 30 seconds to form anti-Fab / anti-VEGF / anti-IL-6 Fab complexes. To allow the formation of complexes containing anti-Fab, anti-VEGF / anti-IL-6 Fab, human VEGFA, and human IL-6, both antigens, human VEGFA121 (in-house production, P1AA1779-010) and human IL-6 (commercial, Peprotech #200-06), were added sequentially or simultaneously. Corresponding SPR response unit curves (Biacore T200, Cytiva) were monitored. For sequential binding, human VEGFA was injected at a concentration of 300 nM for 180 seconds, followed by a further injection of human IL-6 at a concentration of 300 nM for 180 seconds. The same concentrations were also injected in the reverse order (first human IL-6, then human VEGFA). Similarly, a mixture of both antigens was injected at a concentration of 300 nM each for 180 seconds. After each experiment, the surface was regenerated by injecting 10 mM glycine (pH 2.1) at a flow rate of 5 μL / min for 60 seconds. Differences in bulk refractive index were corrected by subtracting a blank injection and the response obtained from a control flow cell without Fab capture.

[0252] The results are shown in Figure 7. Addition of human VEGF-A to the anti-Fab / anti-VEGF / anti-IL-6 Fab complex resulted in binding and anti-Fab / Fab / VEGF-A complex formation. Sequential addition of human IL-6 resulted in the formation of an anti-Fab / DutaFab / VEGF-A / IL-6 complex (dashed curve). This clearly demonstrated that simultaneous binding of human VEGF-A and human IL-6 to anti-VEGF / anti-IL-6 Fab is possible.

[0253] When the reverse order was used, adding human IL-6 first, followed by the sequential addition of human VEGF-A, there was a significant reduction in simultaneous binding (dotted line), indicating that the binding of human IL-6 initially sterically interfered with the binding of human VEGF-A, resulting in a reduced but still possible binding capacity between the anti-VEGF / anti-IL-6 Fab and human VEGF-A.

[0254] In the presence of both targets, human IL-6 binding appeared to be favored, with reduced binding to human VEGF-A (solid line), an effect that was plausible due to the inherently higher affinity of the anti-VEGF / anti-IL-6 Fab for human IL-6 compared to human VEGF-A.

[0255] In another assay, blockade of VEGF-R2 by anti-VEGF / anti-IL-6 Fab fragments in the presence of IL-6 was assessed by a surface plasmon resonance inhibition assay using immobilized VEGF-A.

[0256] To demonstrate simultaneous binding of human VEGF-A and human IL-6 to anti-VEGF / anti-IL-6 Fab fragments, human VEGF receptor 2 (VEGFR2, commercially available from R&D Systems 357-KD) was immobilized on a Series S Sensor Chip CM5 (Cytiva BR100530) using standard amine coupling chemistry, yielding a surface density of approximately 11,000 resonance units (RU). HBS-P+ (10 mM HEPES, 150 mM NaCl pH 7.4, 0.05% Surfactant P20) was used as the running and dilution buffer.

[0257] VEGFA and VEGFR2 inhibition was tested using a 1:2 dilution series of 0–200 nM anti-VEGF / anti-IL-6 Fab fragments in 50 nM human VEGFA solution as a reference. The anti-VEGF / anti-IL-6 Fab fragment / VEGFA mixture was injected over the immobilized VEGFR2 surface at a flow rate of 5 μL / min for 30 seconds. After a 60-second dissociation phase, the VEGFR2 surface was regenerated by injecting 5 mM NaOH at a flow rate of 5 μL / min for 30 seconds. Bulk refractive index differences were corrected by subtracting a blank injection and the response obtained from a blank control flow cell. For evaluation, the binding response was obtained 5 seconds after the end of the injection. The induced response in RU was converted to a binding response by comparing it to the initial signal corresponding to the ligand(s) without the bispecific Fab. IC50 values ​​were calculated using a four-parameter logistic model (XLfit, ID Business Solutions Ltd.).

[0258] In addition to the reference, dilutions of anti-VEGF / anti-IL-6 Fab fragments from 0 to 200 nM in the presence of 10 nM human IL-6 were preincubated for 15 min and tested to calculate IC50 values ​​(Figure 3).

[0259] The results are shown in Figure 8. The graph shows the concentration-dependent inhibition of VEGFR2 / VEGF-A interaction with competing anti-VEGF / anti-IL-6 Fab. In the absence of anti-VEGF / anti-IL-6 Fab, 100% VEGFR2 / VEGF-A binding (0% inhibition) was achieved, whereas increasing anti-VEGF / anti-IL-6 Fab concentrations increased inhibition (solid cross line). The addition of human IL-6, which mimics therapeutically relevant conditions, did not affect the degree of VEGFR2 / VEGF-A inhibition, resulting in very similar IC50 values ​​(IC50 = 33 nM without human IL-6 (dashed triangle line) and IC50 = 37 nM with additional human IL-6 (dashed triangle black line)).

[0260] In a third assay, the effect of VEGF binding on IL6 activity was assessed by a cell-based IL-6-specific reporter gene assay as follows.

[0261] To evaluate the simultaneous binding of human VEGFA and human IL-6 to the anti-VEGF / anti-IL-6 Fab, we used an IL-6-specific cell-based reporter gene assay using the reporter cell line HEK-Blue™ IL-6 cells (InvivoGen). Cells were incubated with the anti-VEGF / anti-IL-6 Fab and human IL-6 for 20 + / - 1 hour in the absence and presence of excess human VEGF-A, either by simultaneous addition of the bispecific Fab and human IL-6 (Figure 9) or after preincubation (Figure 10). Binding of human IL-6 to its receptor, IL-6R, on the surface of HEK-Blue™ IL-6 cells triggers a signaling cascade via Janus family tyrosine kinases (JAK1, JAK2, and Tyk2) that leads to activation of signal transducer and activator of transcription 3 (STAT3) and subsequent secretion of secreted embryonic alkaline phosphatase (SEAP). Upon binding of anti-VEGF / anti-IL-6 Fab to human IL-6, signal transduction is inhibited and SEAP is not produced. Subsequently, SEAP levels in cell culture supernatants are quantified by adding QUANTI-Blue SEAP substrate to an aliquot of the supernatant. SEAP converts the QUANTI-Blue substrate to a product that can be measured by absorbance at 650 nm using a plate reader. Simultaneous binding of human VEGFA and human IL-6 is then assessed by graphing the mean absorbance versus the concentration of anti-VEGF / anti-IL-6 Fab, and the data are fitted to a constrained four-parameter curve. The relative potency (inhibitory concentration) of the sample is calculated using a four-parameter logistic curve fit.

[0262] The results are shown in Figures 9 and 10.

[0263] Figure 9 shows the results without pre-incubation. Titration of increasing amounts of anti-VEGF / anti-IL-6 Fab resulted in a calculated IC of 1.134 ng / mL (approximately 22.5 pM). 50A clear dose-response curve with IC values ​​was demonstrated, demonstrating clear inhibition of the human IL-6 response with increasing amounts of anti-VEGF / anti-IL-6 Fab. To address simultaneous binding of human IL-6 and VEGFA to the bispecific Fab, both target molecules were incubated simultaneously and the effect of human IL-6 was measured. Regardless of the ratio chosen (human VEGFA:human IL-6, 1:1 / 2.5:1 / 5:1), effective IC 50 Only a slight decrease in values ​​was observed. Values ​​are the IC in the absence of human VEGFA. 50 = 1.134 ng / mL, IC when human VEGF-A was present at a 5-fold excess 50 =1.724 ng / mL, a situation that closely reflects in vivo relevant conditions.

[0264] FIG. 10 shows the results from preincubation, which shows that binding of IL6 does not affect IL6 binding.

[0265] Example 7: Binding of anti-VEGF / anti-IL-6 Fab fragments to IL-6 determined by X-ray crystallography and proposed mode of action IL-6 signaling is initiated by the formation of a hexameric complex between IL-6, its non-signaling co-receptor IL-6R, and the cytokine receptor gp130. Three epitopes (sites 1, 2, and 3) have been defined to identify the contact surfaces within the complex (Boulanger MJ et al., Science 2003, 27;300(5628):2101-4). IL-6 initially binds to IL-6R via an interaction surface called "site 1." "site 2" is an epitope formed by the binary complex of IL-6 and IL-6R, which interacts with domains 2 and 3 of gp130. Subsequent interaction between "site 3" of IL-6 and domain 1 of gp130 leads to the formation of a dimer of the IL-6 / IL-6R / gp130 trimer, thus forming a hexameric signaling complex.

[0266] To understand which epitopes of IL-6 are bound by our two series of Fabs (6HVL and VH6L), we performed structural analysis of the complexes between IL-6 and antibody Fabs, each of which is representative of the anti-VEGF / anti-IL-6 Fab fragments of the present invention. The utilized Fab, 6HVL4.1, is very closely related to Fab 6HVL_2, differing by only two mutations, while Fab 0182 is most closely related to Fab VH6L_1. Due to the fact that all 6HVL clones are derived from the same Fab (6HVL_1), and similarly, all VH6L clones are each derived from Fab VH6L_1, we can safely assume that the structural results obtained below are applicable across each of the individual series of Fabs. The formation of complexes between Fabs and IL-6 and the analysis of the complex structures were performed by X-ray crystallography as follows.

[0267] An IL6-Fab complex was prepared by mixing equimolar amounts of Fab fragment 0182 (light chain amino acid sequence: SEQ ID NO: 33; heavy chain amino acid sequence: SEQ ID NO: 34) or 6HVL4.1 (light chain amino acid sequence: SEQ ID NO: 37; heavy chain amino acid sequence: SEQ ID NO: 38) with IL-6 (PeproTech, Lot No. 031316-2).

[0268] After 90 min of incubation on ice, the protein complexes were concentrated to 23.1 mg / ml for Fab fragment 0182 and 21.3 mg / ml for 6HVL4.1. Initial crystallization trials were performed in a sitting drop vapor diffusion setup at 21°C.

[0269] For Fab fragment 0182, needle-like crystals appeared within 2 days in 0.1 M MgCl2, 0.1 M sodium citrate pH 5, 15% (w / v) PEG 4000. The crystals were then used in seeding experiments, yielding large tetragonal crystals in 0.1 M calcium acetate, 12% (w / v) PEG 8000, 0.1 M sodium cacodylate, pH 5.5.

[0270] In 6HVL4.1, rhombohedral crystals appeared within 1 day from 0.2 M ammonium sulfate, 0.1 M Tris, pH 7.5, and 20% (w / v) PEG MME 5000.

[0271] For data collection, collected crystals were quenched at 100 K in crystallization solution supplemented with 15% ethylene glycol. X-ray diffraction data were collected at a wavelength of 0.9999 Å for Fab fragment 0182 and 0.9982 Å for 6HVL4.1 using a PILATUS 6M detector at beamline X10SA at the Swiss Light Source (Villigen, Switzerland). Data were processed with XDS (Kabsch, W., XDS. Acta Cryst. D66, 125-132 (2010)), scaled with AIMLESS (PREvans and GN Murshudov, "How good are my data and what is the resolution?" Acta Cryst. (2013). D69, 1204-1214), and analyzed for anisotropy with STARANISO (Tickle, IJ, Flensburg, C., Keller, P., Paciorek, W., Sharff, A., Vonrhein, C., Bricogne, G. (2018). STARANISO (http: / / staraniso.globalphasing.org / cgi-bin / staraniso.cgi). Cambridge, United Kingdom: Global Phasing Ltd.).

[0272] Crystals of the complex containing Fab 0182 belong to space group P21 with cell axes a=65.93 Å, b=65.46 Å, c=159.30 Å, β=91.65° and diffract to 2.18 Å resolution.

[0273] Crystals of the complex containing 6HVL4.1 belong to the space group P212121 with cell axes a = 57.56 Å, b = 64.98 Å, c = 203.68 Å and diffract to a resolution of 1.94 Å.

[0274] The structure was determined by molecular replacement with PHASER (McCoy, AJ, Grosse-Kunstleve, RW, Adams, PD, Winn, MD, Stroni, LC, & Read, RJP, PHASER crystallographic software. J Appl Cryst. 40, 658-674 (2007)) using the coordinates of an in-house Fab and IL-6 (pdb entry 1alu) as search models. Amino acids were changed according to sequence differences using different electron densities. The structure was refined using programs from the CCP4 suite (Winn, MD et al. Overview of the CCP4 suite and current developments. Acta. Cryst. D67, 235-242 (2011)) and BUSTER (Bricogne, Blanc, GE, Brandl, M., Flensburg, C., Keller, P., Paciorek, W., Roversi, P., Sharff, A., Smart, OS, Vonrhein, C., Womack, TO. Buster version 2.9.5 Cambridge, United Kingdom: Global Phasing Ltd. (2011)). Manual reconstruction was performed using COOT (Emsley, P., Lohkamp, ​​B., Scott, WG, Cowtan, K. Features and Development of COOT. Acta. Cryst. D66, 486-501 (2010)).

[0275] Data collection and refinement statistics are summarized in Table 7. All graphical displays were prepared using PYMOL (The Pymol Molecular Graphics System, Version 1.7.4. Schrodinger, LLC.). [Table 9] TIFF0007801491000021.tif167170

[0276] Structure of the Fab 0182-IL-6 complex The crystal structure of Fab 0182 (representative of the VH6L series of Fabs) in complex with IL-6 was determined at 2.18 Å resolution (Figure 5). The structure shows Fab 0182 bound to IL-6 through contributions from CDR2 of the heavy chain and CDR1 and CDR3 of the light chain. Further interaction with IL-6 is maintained by N-terminal residues Val3 and Gln4 of the light chain of Fab 0182. The interface contributed by IL-6 is formed by residues in helix A and helix C.

[0277] FIG. 16 shows the binding mode of Fab 0182 to IL6. For illustrative purposes, we generated two structural superpositions: first, a superposition of the complex structure of Fab with IL6; and second, a superposition of the complex structure of IL6R with IL6 (obtained from the co-crystal structure of IL6, IL6R, and gp130 with pdb accession code 1p9m (see Boulanger MJ et al., Science 2003, 27;300(5628):2101-4). Comparing this to the trimeric complex of IL6 with IL6R and gp130, it is clear that Fab binds IL6 in a manner very similar to gp130, i.e., binding to site 2 of IL6. This binding mode is expected to allow simultaneous binding of both Fab and IL6R to IL6; i.e., interaction of IL6 with IL6R should still be possible, and such IL6 antagonists are expected a priori to act by inhibiting the interaction of the IL6 / IL6R complex with gp130 via binding to site 2 of IL6.

[0278] Crystal structure of the Fab 6HVL4.1-IL-6 complex. We have determined the crystal structure of Fab 6HVL4.1 in complex with IL-6 at 1.94 Å resolution (Figure 6). This structure shows Fab 6HVL4.1 bound to IL-6, primarily through the contributions of CDR1 and CDR3 of the heavy chain and CDR2 and CDR3 of the light chain. Further interaction with IL-6 is maintained by the first three N-terminal residues of the heavy chain of Fab 6HVL4.1. The interface contributed by IL-6 is formed by residues in helix A and helix C.

[0279] Similar to what was done for Fab 0182, we analyzed the binding mode of Fab 6HVL4.1 to IL6 by using structural superposition and analyzing the interacting residues as described above. Figure 17 demonstrates that 6HVL4.1 also binds to IL6 in a manner very similar to gp130 and therefore, from a structural point of view, must be considered a site 2 binder.

[0280] Experimental investigation of the binding mode to IL-6 The fact that clones such as 6HVL4.1 and its derivatives are IL6 site 2 binders could be functionally confirmed by an assay utilizing surface plasmon resonance.

[0281] In one such assay, a Fab fragment (antibody "P1AE2421") representing a clone from the 6HVL series, including 6HVL4.1, was captured onto the surface of an SPR chip via an anti-Fab antibody, and IL6 and then IL6R were flowed over the chip surface at three different concentrations (250, 500, and 1000 nM). Here, we expect a two-step sequential signal increase if IL6 can bind to both the Fab and IL6R. Indeed, this was observed for the Fab tested (Figure 18): in the SPR signal trace, the addition of IL6 leads to a strong increase in signal, which is further enhanced after the addition of IL6R. This clearly demonstrates that simultaneous binding of IL6R and Fab to IL6 is possible. This finding is further supported by the fact that a covalent chimera of IL6 and IL6R (termed "hyper-IL6"), in which site 1 of IL6 is completely blocked and inaccessible, still facilitates binding of each Fab molecule when coated onto an SPR chip and probed with Fab at a concentration of 26 nM (Figure 19).

[0282] However, when we used ELISA experiments to examine whether the captured Fab fragments competed with IL6 binding to IL6R, we obtained surprising results. The assay setup was as follows: First, a fixed concentration of IL6 was preincubated with a titration series of Fab fragment P1AE2421, which was also used in SPR experiments and is representative of the 6HVL series of clones. This was then incubated on an ELISA plate directly coated with IL6R. After washing, plate-bound IL6R was detected with a biotinylated anti-IL6 antibody utilizing horseradish peroxidase-labeled streptavidin (Strep-HRP). In this assay (Figure 20), we observed results that strongly suggest almost complete inhibition of the IL6 / IL6R interaction by the Fab.

[0283] Given that available crystal structures and SPR experiments show that site 1 of IL6 remains accessible for binding, we must interpret these results as suggesting that the IL6 antibodies described in this patent are not only able to sterically block the binding of the IL6 / IL6R complex to gp130, but also to strongly allosterically reduce the binding affinity of IL6 to IL6R, i.e., to further functionally act as IL6 site 1 antagonists.

[0284] Such a mode of action is expected to have optimal properties for the following reasons.

[0285] 1. As IL6 site 2 binders, IL6 antagonists can equally effectively block the formation of a signaling complex resulting from IL6 binding to membrane-bound IL6R and gp130 (cis-signaling) or the formation of a preformed complex of IL6 and IL6R (trans-signaling). In contrast, IL6 site 1 binders cannot bind to the preformed complex of IL6 and IL6R, but can antagonize it only when the complex dissociates.

[0286] 2. Due to allosterically reducing the affinity of IL6 for IL6R, the IL6 antagonists described herein are expected to exhibit increased potency compared to Site 2 binders that do not exhibit this effect by disfavoring the formation of the first step in signal transduction, i.e., the formation of the IL6 / IL6R complex. Site 2 binders that do not allosterically interfere with Site 1 binding are expected to be particularly disadvantageous for cis-signaling, i.e., when the relative effective concentrations of IL6 / IL6R and gp130 are expected to be very high, and the second step of IL6 / IL6R-gp130 complex formation on the surface of cells must be blocked.

[0287] 3. IL6 site 1 binders, when used systemically as antibodies, are known to result in strong accumulation of IL6-antibody complexes due to a significantly increased half-life of the complex compared to IL6 alone. In contrast, IL6 site 2 binders are expected to still allow removal of IL6 / antibody complexes by binding to membrane-bound IL6R followed by internalization and degradation by cells that take up the complex. In that regard, the IL6 antagonists described herein are expected to combine the desirable properties of both site 1 and site 2 binders: functionally, they can block the first step in the formation of the IL6 / IL6R / gp130 signaling complex, while still allowing degradation of the IL6 / mAb complex via IL6R binding on cells.

[0288] 4. In ophthalmic indications, and when utilized as a Fab molecule, the expected behavior of such binders may be even more beneficial: Similar to IL6 site 1 binders, Fab / IL6 complexes can exit the ocular cavity relatively unimpeded by IL6R binding and can be rapidly cleared systemically by renal filtration.

[0289] Example 8: Improved thermal stability of anti-VEGF / anti-IL6 Fab fragments Additional sequence variants of improved anti-VEGF / anti-IL-6 antibodies were generated, including the amino acid sequences identified in Table 8. [Table 10]

[0290] The thermal stability of the indicated bispecific antibodies was assessed as follows.

[0291] Thermal stability: Bispecific antibody Fab fragment samples were prepared at 1 mg / mL in 20 mM histidine / histidine chloride, 140 mM NaCl, pH 6.0, and transferred to a 10 μL microcuvette array. Static light scattering data and fluorescence data upon excitation with a 266 nm laser were recorded using an UNcle instrument (Unchained Labs) while the samples were heated from 30°C to 90°C at a rate of 0.1°C / min. Samples were measured in triplicate.

[0292] The onset temperature was evaluated using UNcle analysis software. The aggregation onset temperature was defined as the temperature at which the scattered light intensity began to increase. Protein denaturation was monitored by the shift in the centroid mean (BCM) of the fluorescence signal versus heat. The melting temperature was defined as the inflection point of the BCM (nm) versus temperature curve. [Table 11]

[0293] Example 9: Biophysical properties of improved bispecific anti-VEGF / anti-IL6 Fab fragments (viscosity assessed by dynamic light scattering (DLS)) The aforementioned antibody Fab fragments were expressed in CHO cells by standard methods.

[0294] Viscosity was measured using the latex-bead DLS method as previously described (He F et al.; Anal Biochem. 2010 Apr 1;399(1):141-3). Specifically, the following protocol was followed using the indicated materials.

[0295] Viscosity Rating: Equipment and Materials Wyatt DLS plate reader with Greiner Bio-One microplates 3000 Series Nanosphere™ Size Standard (Thermofisher Catalog Number 3300A) Tween 20 (Roche, Cat. No. 11332465001) and silicone oil, e.g. (Alfa Aesar Cat. No. A12728) · UV photometer for concentration determination (e.g. Nanodrop 8000).

[0296] Sample preparation The antibody samples were rebuffered and diluted with 20 mM His / HCl, pH 5.5 (buffer) and 0.02% Tween 20 (final concentration). A bead concentration of 0.03% solids was added. At least three different concentrations were prepared, with the highest possible concentration being approximately 200 mg / mL. Two blank samples were required as antibody-free controls: one containing nanosphere beads resuspended in water and the other containing nanosphere beads resuspended in buffer. The samples were transferred to a microplate, and each well was covered with silicone oil.

[0297] Measurements using a Wyatt DLS plate reader All samples and blanks were analyzed at different temperatures from 15 °C to 35 °C in 5 °C steps. The acquisition time was 30 s and the number of acquisitions was 40 per sample and temperature.

[0298] Data analysis The raw data Dapp (apparent radius) in nm was shown in the overview of the software template (Microsoft Dynamics 7.10 or higher). The viscosity was calculated using the formula (ηreal = Dapp * ηHO / Dreal). Dreal is the bead size measured in a blank sample, which is equal to the bead size (300 nm). The calculated viscosity was shown as an Excel curve. Using Mooney curve fit (Excel), it is possible to extrapolate the viscosity at a given concentration. Here, the maximum protein concentration at which the viscosity exceeds 20 cP was calculated.

[0299] The maximum concentrations of the indicated antibodies to achieve a viscosity of 20 cP at 20° C. are shown below. [Table 12]

[0300] The results show that the antibodies of the present invention can be formulated at high concentrations with viscosities below the acceptable viscosity limit for injectability. As a result, the antibodies of the present invention are well suited for ocular applications, as they allow for the delivery of high molar doses in limited injection volumes, which, when combined with high efficacy, leads to high durability, thereby reducing dosing frequency, which is desirable for patient convenience and increased treatment compliance.

[0301] Example 10: A primary cell-based assay to demonstrate IL-6 inhibition mediated by the VEGF / IL-6 bispecific antibody 6HVL_4 (HRMEC) To measure IL-6 signaling activity in HRMEC, we established an assay to quantify ICAM-1 surface expression on HRMEC. HRMEC were stimulated with an equimolar concentration (2 nM) of human IL-6 and human IL-6R for 72 hours. ICAM-1 surface expression was assessed by flow cytometry. To measure the inhibitory activity of 6HVL_4, the IL-6 / IL-6R mixture was preincubated with increasing concentrations of antibody before application to the cells.

[0302] Cell Culture: HRMECs (catalog no. PEL-PB-CH-160-8511; PELOBiotech GmbH; Bayern, Germany) were thawed and cultured in 175 cm flasks in endothelial basal medium (EBM) (catalog no. CC-3156; Lonza; Basel, Switzerland) and endothelial growth medium (EGM-MV) containing 5% fetal bovine serum (FBS), hydrocortisone, human fibroblast growth factor B, VEGF, R3-IGF-1 (a recombinant analog of insulin-like growth factor-I with Glu replaced by Arg at position 3), ascorbic acid, human epidermal growth factor, and GA-1000 (all included in the EGM-MV Microvascular Endothelial SingleQuots™ Kit; catalog no. CC-4147; Lonza) at the manufacturer's recommended concentrations. Twenty-four hours after plating, the medium was replaced with fresh EGM-MV, and cells were grown for an additional 3 days before assay. Assay conditions were optimized across different passages and concentrations of IL-6 / soluble IL-6R. The final assay was performed using HRMECs at passage 6 and an equimolar stimulation of IL-6 / soluble IL-6R at a concentration of 2 nM.

[0303] Flow cytometry assay: HRMECs were detached from flasks by washing twice with Ca2+- and Mg2+-free phosphate-buffered saline (PBS) (catalog no. 10010023; Life Technologies) and once with the cell dissociation reagent Accutase (catalog no. A1110501; Thermo Fisher Scientific; Waltham, MA). After washing, 5 mL of cell dissociation reagent was added to the cells, and the flask was incubated at 37°C in a 5% CO2 incubator for 3 minutes. Dissociated cells were collected from the flask and placed in a 50 mL conical centrifuge tube. The tube was filled to 50 mL with EBM containing 2% FBS and centrifuged at 300 g for 6 minutes. The supernatant was discarded, and the pellet was resuspended in 5 mL of starvation medium (EBM containing 2% FBS). Cell numbers were quantified using a TC20 automated cell counter (Bio-Rad; Hercules, CA) and adjusted to 300,000 cells / mL using starvation medium. Then, 100 μL of the cell suspension was added to each well of a Costar 96-well plate (catalog no. 3596; Corning; Corning, NY) to obtain 30,000 cells / well. The plate was then incubated at 37°C in a 5% CO2 incubator for an additional 24 hours.

[0304] Recombinant human IL-6 (catalog no. 206-IL / CF; R&R&D Systems; Minneapolis, MN) and recombinant human IL-6R (catalog no. 227-SR-025; R&R&D Systems) were mixed at equal molar concentrations in starvation medium and incubated at room temperature for 1 hour to allow the formation of IL-6-I / L-6R complexes. Next, 50 μL of a dilution series (3-fold, 7-point dilutions) of 6HVL_4 was added to the cells and incubated at 37°C and 5% CO2 for 1 hour. Finally, 50 μL of IL-6-I / L-6R complex was added to the cells to obtain final concentrations of 2 nM for IL-6 and IL-6R, respectively, and 200.009 nM for 6HVL_4. Unstimulated cells and cells stimulated with the IL-6-I / L-6R complex without 6HVL_4 were also included to determine background ICAM-1 surface expression and 100% response levels, respectively. The cells were incubated at 37°C in a 5% CO2 incubator for 72 hours.

[0305] For analysis of ICAM-1 surface expression, cells were soaked in PBS (Ca 2+ , Mg 2+The plates were washed twice with Accutase (Cat. No. A1110501; Thermo Fisher Scientific) and once with cell dissociation reagent. Cells were detached from the plate using 50 μL of cell dissociation reagent (3 min, 37°C) and transferred to a Falcon 96-well flow cytometry storage plate (Cat. No. 353263; Corning). The original wells were washed once with 100 μL of PBS containing 2% FBS and 2 mM EDTA, and the wash medium containing the remaining cells was added to the flow cytometry plate. Cells were pelleted by centrifugation at 300 g for 6 min, and the supernatant was discarded. The pellet was resuspended in 100 μL of PBS, 2% FBS, and 2 mM EDTA containing 10 μg / mL human IgG (Cat. No. I2511; MilliporeSigma; Burlington, MA) to block nonspecific binding sites and incubated at room temperature for 15 min. After blocking, 0.5 μg of fluorescein-labeled anti-ICAM-1 antibody (catalog no. BBA20; R&R&D Systems) was added to the cells, and the reaction was incubated at 28°C for 45 minutes. After staining, the cells were pelleted by centrifugation at 300 g for 6 minutes, and the pellet was resuspended in 150 μL PBS containing 2% FBS and 2 mM EDTA. Fluorescein fluorescence was measured using an Attune NxT flow cytometer (Thermo Fisher Scientific).

[0306] Data analysis: For all three assays, each condition in all independent experiments was performed in quadruplicate. For each experiment, the background signal of unstimulated cells was subtracted from the background signal of the experimental wells to calculate the average signal per condition. The 100% response level was calculated from cells stimulated with IL-6 / IL-6R without 6HVL_4, and the inhibitory potency of 6HVL_4 was then expressed as the percentage inhibition of the 100% response. The percentage inhibition of each concentration of 6HVL_4 was measured in three independent experiments, and the mean and SEM were calculated. The mean of the three independent experiments was used to calculate the average IC50 and SE using ExcelXLfit software version 5.5.0 (IDBS; Guildford, UK). Concentration-response curves were fitted by nonlinear regression analysis using a five-parameter logistic model (A + ((BA) / (1 + (((BE)*((C / x)^D)) / (EA))))) calculated for basal and maximum inhibitory activity. [Table 13]

[0307] 6HVL_4 produced a dose-dependent inhibition of IL-6 signaling in HRMEC with a 50% inhibitory concentration (IC50) of 1.52 + / - 0.04 nM (Figure 11).

[0308] Example 11: A primary cell-based assay to demonstrate VEGF inhibition mediated by the VEGF / IL-6 bispecific antibody 6HVL4 (HUVEC) Assay: HUVECs were obtained from Lonza (catalog no. 00191027; Basel, Switzerland). Endothelial Basal Medium (EBM-2; catalog no. CC-3156) and EGM-2 Endothelial SingleQuots Kit (catalog no. CC4176), which together comprise the endothelial growth medium (EGM-2) and assay medium (EBM-2 with 0.5% fetal bovine serum [FBS]), were also purchased from Lonza.

[0309] HUVECs were maintained in T175 cell culture flasks (Corning, NY, catalog number 353112) coated with attachment factor (AF) (catalog number S-006-100; Gibco, Thermo Fisher Scientific, Waltham, MA). Cells were detached using StemPro Accutase (catalog number A11105-01; Gibco).

[0310] Cell viability / proliferation assays were performed in 96-well fibronectin-coated plates (catalog no. 354409; Corning) using alamarBlue (catalog no. DAL1100; Invitrogen, Thermo Fisher Scientific).

[0311] Recombinant human VEGF-A was obtained from R&D (catalog no. 293-VE; Minneapolis, MN) and Ca 2+ and Mg 2+ The antibody was dissolved in phosphate-buffered saline (PBS) (catalog no. 14190-094; Gibco) without ATP at a stock concentration of 100 μg / mL.

[0312] AlamarBlue contains the cell-permeable compound resazurin, which changes color by reducing the environment within healthy cells. The resulting pink color is a proportional marker of viable cells and can be used to detect proliferation by measuring absorbance at 570 nm. VEGF-A induces proliferation of HUVECs grown under cell-starvation conditions. Therefore, VEGF-A-induced HUVEC proliferation can be inhibited by using a VEGF-A neutralizing antibody or Fab.

[0313] HUVECs were maintained in EGM-2 in AF-coated T175 flasks up to passage 5. For viability assays, HUVECs were detached using Accutase and diluted 1:1.66 with assay medium (EBM-2 0.5% FBS). The cells were then centrifuged and resuspended in EBM-2 containing 0.5% FBS to a cell density of 100,000 cells / mL. 100 μL of the cell suspension was then seeded into a fibronectin-coated 96-well plate to obtain a cell density of 10,000 cells / well. The outer wells were not seeded with cells and were then filled with assay medium alone. The cells were incubated overnight at 37°C in a 5% CO2 incubator.

[0314] The next day, VEGF-A stock solution (100,000 ng / mL in PBS (Ca 2+ , Mg 2+ )) was used to prepare a 10x working solution (750 ng / mL) in assay medium (EBM 0.5% FBS).

[0315] The 6HVL_4 stock solution was also diluted with assay medium to prepare a 10x working solution, which was used to prepare eight 3x serial dilutions starting at 30,000 ng / mL and ending at 14 ng / mL.

[0316] Next, 12.5 μL of 10% prediluted 6HVL_4 solution and 12.5 μL of 10% VEGF-A solution (750 ng / mL) were added sequentially to each plate of cells in quadruplicate. VEGF-A was used at a constant final concentration (75 ng / mL), and 6HVL_4 was used in a dose-response format ranging from 3000 ng / mL to 1.4 ng / mL. Cells were incubated at 37°C and 5% CO2 for 72 hours. For analysis, 12 μL of alamarBlue was added to each well, followed by 3 hours of incubation in a cell culture incubator. Absorbance was measured at 570 nm with a reference wavelength of 600 nm using a Molecular Devices FlexStation 3 plate reader.

[0317] Data Analysis: For each experiment, each condition was performed in quadruplicate. A total of four independent experiments were performed. Two separate plates were treated on the same day, and each experiment was considered independent. Therefore, eight separate plates were used for analysis. The background signal of unstimulated cells was subtracted from the background signal of the experimental wells to calculate the average signal per condition. The 100% response level was calculated from cells stimulated with VEGF-A (75 ng / mL) without additional compound exposure, and the signal from the 6HVL_4-exposed wells was expressed as a percentage inhibition of the 100% response.

[0318] IC50 values ​​were calculated from the average data for each antibody concentration using ExcelXLfit software version 5.5.0 (IDBS; Guildford, UK). Concentration-response curves were fitted by nonlinear regression analysis using a four-parameter logistic model (A + ((BA) / (1 + ((C / x)^D)))) calculated for basal and maximum inhibitory activity. Data are presented as the mean from four independent experiments with standard error of the mean (SEM). [Table 14]

[0319] 6HVL_4 reduced VEGF-A-induced HUVEC proliferation with a 50% inhibitory concentration (IC50) of 2.06 + / - 0.30 nM (Figure 12). Example 12: Restoration of barrier function in the presence of both VEGF-A and IL-6 and the VEGF / IL-6 bispecific antibody 6HVL_4 indicates the biological activity of the molecules. To assess the dual biological activity of the antibodies of the present invention, i.e., simultaneous blockade of both targeted cytokines—VEGF-A and IL6 (in complex with IL6R)—a transendothelial resistance (TER) assay was performed. In this assay, electrically dense endothelial cell layers respond to the addition of both VEGF-A and IL6 by a loss of barrier function. Restoration of barrier function in the presence of both cytokines VEGF and IL6 by the VEGF / IL6 bispecific antibody 6HVL_4 was assessed as follows.

[0320] Assay: Human retinal microvascular endothelial cells (PELOBiotech; catalog number PEL-PB-CH-160-8511), also referred to as HRMVECs, were maintained in complete MV endothelial cell growth medium (MV-EGM-2 Lonza, catalog number CC-3202) in T175 flasks (Falcon Cat#353112) coated with attachment factor (Gibco, catalog number S-006-100) for up to five passages. For the transendothelial cell resistance assay, cells were detached using StemPro® Accutase® (Gibco, catalog number A11105-01). Cells were then seeded in 100 μl of MV-EGM-2 growth medium onto the upper chamber of a fibronectin-coated (catalog number 354008, Corning) Transwell filter (24-well Corning, catalog number 3470) at a cell density of 120,000 cells / well. The lower chamber of the Transwell filter was filled with 600 μl of MV-EGM-2 medium. The cells were incubated at 37°C with 5% CO2 for 3 days. The medium was then changed to assay conditions (MV-EGM-2 containing 2% FBS without VEGF), and the Transwell filter was transferred to the cellZcope system using 280 μl of medium for the upper chamber and 810 μl for the lower compartment. The cells were then incubated at 37°C with 5% CO2 for 24 hours, during which time TER was measured by cellZcope. The next day, cells were treated with a final concentration of 10 ng / ml VEGF (R&D Systems, Catalog No. 293-VE / CF), 50 ng / ml IL6 (R&D Systems, Catalog No. 206-IL / CF) in combination with 100 ng / ml IL6R (R&D Systems, Catalog No. 227-SR-025 / CF) and 10 ng / ml VEGF, or an equal volume of assay medium (8x per condition), and TER was measured until the following day. 6HVL_4 or aflibercept at final concentrations of 1 μg / ml or 2.3 μg / ml, respectively, or assay medium was then added to the cells, and TER was then measured for the next 24 hours. Thus, each condition was represented in quadruplicate.

[0321] Data Analysis: Data sets generated for one well were normalized to the TER value obtained immediately before the addition of the cytokine mix. For each condition, the mean signal and standard deviation were calculated from the normalized data.

[0322] result: The results are shown in Figure 12 (6HVL_4) and Figure 13 (aflibercept).

[0323] The barrier function of HRMVECs was reduced by the cytokine VEGF alone and in combination with IL6 / IL6R. Antibody 6HVL_4 restored the disrupted barrier function to 100% after 24 hours.

[0324] Example 13: Identification of the IL6 paratope region The crystal structure of the 6HVL4.1 complex with IL6 identified amino acid residues that contact IL6. An illustration of the location of paratope amino acid residues within the VH and VL domains is shown in Figure 15. To this end, we used the "byres" function in PyMOL and a 5 Å cutoff distance to identify residues likely to interact with IL6 in the Fab / IL6 complex. Here, we limited our analysis to residues 48-215 of IL6 (as defined by Uniprot number P05231). These residues are known to be commonly resolved in the structure of IL6 alone (see pdb accession numbers 1alu and 1IL6). Figure 15 also shows an alignment between 6HVL4.1 and the monospecific anti-IL6 antibody 6HdL2.05, based on the antibody of the present invention, in which the VEGF-paratope is replaced by a non-binding region. 6HdL2.05 has a VH domain of SEQ ID NO: 48 and a VL domain of SEQ ID NO: 47. When expressed and purified as described in Example 2 and subjected to SPR assays with human IL6 or cynomolgus IL6 performed as in Example 3, the antibody exhibited SPR sensorgrams as shown in Figure 20. After fitting the experimental data, 6HdL2.05 exhibited affinities comparable to the highest affinities obtained for the corresponding 6HVL series VEGF / IL6 bispecific antibodies (see Tables 3 and 4), with a fitted KD of 22 pM with human IL6 and 1.3 nM with cynomolgus IL6.

[0325] The amino acid residues identified as contributing to antigen binding are identified in Table 13 (for variable heavy domain amino acid residues) and Table 14 (for variable light domain amino acid residues). Amino acid positions are numbered according to the Kabat numbering system (the same numbering is used in Figures 1+5). Amino acid positions involved in antigen binding are identified by their Kabat position in the VH or VL domain. [Table 15]

Claims

1. An antibody that binds to human VEGF-A and human IL6, comprising the VH sequence of SEQ ID NO:22 and the VL sequence of SEQ ID NO:

21.

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

3. The antibody of claim 1 , wherein the antibody is a bispecific antibody fragment.

4. The antibody described in claim 1, comprising a heavy chain amino acid sequence of SEQ ID NO: 24 and a light chain amino acid sequence of SEQ ID NO:

23.

5. An isolated nucleic acid encoding the antibody of any one of claims 1 to 4.

6. A host cell comprising the nucleic acid of claim 5.

7. A method for producing an antibody that binds to human VEGF-A and human IL6, comprising culturing the host cell of claim 6 so that said antibody is produced.

8. 8. The method of claim 7, wherein the host cell is a CHO cell.

9. A pharmaceutical formulation comprising the antibody of any one of claims 1 to 4 and a pharmaceutically acceptable carrier.

10. The antibody according to any one of claims 1 to 4 for use as a pharmaceutical.

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