Antibodies that bind to 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 using the same.

Background Art

[0002] Antibodies that bind to VEGF, such as ranibizumab, are used as therapeutic agents for the treatment of ocular vascular diseases such as age-related macular degeneration. Antibodies that bind to IL6, such as those disclosed in International Publication No. 2014 / 074905, have been suggested for the treatment of ocular diseases.

[0003] International Publication No. 2012 / 163520 discloses a bispecific antibody that includes two paratopes in a pair of VH and VL domains (the "DutaFab"). Each paratope of the bispecific antibody disclosed in International Publication No. 2012 / 163520 includes amino acids from the heavy and light chain CDRs, where heavy chain CDR-H1 and CDR-H3 and light chain CDR-L2 contribute to the first paratope, and light chain CDR-L1 and CDR-L3 and heavy chain CDR-H2 contribute to the second paratope. Monospecific antibodies that include the individual paratopes are isolated independently from different Fab libraries in which either the first paratope or the 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 called "VH6L" having a VL sequence of SEQ ID NO: 01 and a VH sequence of SEQ ID NO: 02 that specifically binds to VEGF and IL-6 is disclosed in International Publication No. 2012 / 163520 as a proof of concept.

[0004] Indeed, improved therapeutic antibodies that bind to VEGF and IL6 for clinical application in ocular diseases are needed, for example, by improving the effectiveness against standard care, and by improving the duration of action, and thus reducing the frequency of intravitreal injections and the dosing burden on the patient.

Summary of the Invention

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

[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 a variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 22 having up to 5 amino acid substitutions; and a variable light chain domain comprising the amino acid sequence of SEQ ID NO: 21 having up to 5 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 and binds to the same epitope on IL6 as the antibody according to the present invention.

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

[0013] In another aspect, the present invention provides an antibody that binds to IL6 and binds to the same epitope on IL6 as the antibody having the VL domain of SEQ ID NO: 35 and the VH domain of SEQ ID NO: 36. In one embodiment, the antibody comprises a VH domain having a human VH3 framework (the IL6 paratope includes 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 includes amino acid residues 49, 50, 53, 54, 55, 56, 57, 91, 96).

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

[0015] In one aspect, the present invention provides a host cell comprising the nucleic acid of the present 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 present invention provides an expression vector comprising the nucleic acid of the present invention.

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

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

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

[0020] In one aspect, the present invention provides a prefilled syringe comprising the antibody of the present invention and a pharmaceutically acceptable carrier.

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

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

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

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

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

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

[0027] According to the present invention, there is provided a therapeutic anti-VEGF-A / anti-IL6 antibody which can bind independently to its target antigen even when provided in the form of an antibody Fab fragment. This exhibits excellent KD and cross-species reactivity with cynomolgus targets within a pharmacologically relevant range. The antibodies of the present invention are suitable for the treatment of ocular vascular diseases. The antibodies of the present invention support both high affinity for both targets, which enables their therapeutic application, good expressibility and developability (e.g., high binding potency, high biophysical and biochemical stability, high-concentration formulations), particularly a low effective dose, as well as high stability advantageously over a long period. Compared to non-antibody approaches, the antibodies of the present invention have a higher tendency to be tolerated due to their high human nature and lack of artificial domains and linkers. Furthermore, it is advantageous that the antibodies of the present invention are provided in a high-concentration liquid formulation having a viscosity suitable for ocular application. Since it can be provided at a high concentration, a higher dose of the therapeutic agent can be applied in a single treatment, enabling a longer treatment cycle, so that treatment with the antibodies of the present invention can be more tolerable for the patient. Bispecific Fab fragments such as those described in the present invention have further advantages over bispecific full-length IgG antibodies due to their much lower molecular weight. Fab has a molecular weight of about 50 kDa, while the weight of a full-length antibody is three times higher (about 150 kDa) while providing the same number of binding sites. Thus, for a given amount of drug, a bispecific Fab fragment contains three times more binding sites compared to a full-length IgG antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0028]

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Mode for Carrying Out the Invention

[0029] 1. Definitions Unless otherwise specifically defined herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. Further, unless the context specifically requires otherwise, singular terms shall include pluralities and plural terms shall include singulars. The methods and techniques of the present disclosure are generally performed according to conventional methods well known in the art. Generally, the nomenclature and techniques used in connection with biochemistry, enzymology, molecular biology, and cell biology, microbiology, genetics, and the chemistry of proteins and nucleic acids, as well as hybridization, described herein are well known and commonly used in the art.

[0030] Unless otherwise specifically defined herein, the term "comprising of" shall include the term "consisting of".

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

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

[0033] An "isolated" antibody is an antibody that has been separated from the components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or greater than 99% purity as determined by methods such as electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC). For a review of methods for assessing antibody purity, see, for example, 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, for example, naturally occurring mutations or variant antibodies that may occur during the production of the monoclonal antibody preparation, such variants generally being 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 obtained from a substantially homogeneous collection 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 structure substantially similar to a native antibody structure or having a heavy chain 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 main classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into "subclasses" (isotypes), for example, 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 having the 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 having the 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 that contains at least a portion of the constant region. This term includes the native sequence Fc region and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system, also called the EU index, described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0038] The "variable region" or "variable domain" refers to the domain of the heavy or light chain of an antibody that is involved in the binding of the antibody to an antigen. The variable domains (VH and VL, respectively) of the heavy and light chains of natural antibodies generally have a similar structure, and each domain contains 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 antibodies of the present invention, a single pair of VH and VL domains, i.e., a cognate VH / VL pair, specifically binds to its two targets: VEGF-A and IL6.

[0039] "DutaFab" is a bispecific antibody disclosed in International Publication No. WO 2012 / 163520. In DutaFab, a single pair of VH and VL domains specifically binds to two different epitopes, one paratope containing amino acid residues derived from CDR-H2, CDR-L1, and CDR-L3, and the other paratope containing amino acid residues derived from CDR-H1, CDR-H3, and CDR-L2. DutaFab contains two non-overlapping paratopes within a cognate VH / VL pair and can simultaneously bind to two different epitopes. Methods for their production by screening libraries containing DutaFab and monospecific Fab fragments are disclosed in International Publication No. 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 a non-human-derived antibody using a human antibody repertoire or a sequence encoding another human antibody, which definition clearly excludes humanized antibodies containing non-human antigen-binding residues. Antibodies or antibody fragments isolated from a human antibody library are regarded herein as human antibodies or human antibody fragments.

[0041] The "human consensus framework" is a framework that represents the most commonly occurring amino acid residues in the 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 such as those 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. (supra). In one embodiment, for VH, the subgroup is subgroup III as in the above Kabat et al.

[0042] An "antibody fragment" is a molecule other than an intact antibody that includes a portion of an intact antibody that binds to an 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] "Paratope" or "antigen-binding site" refers to a portion of an antibody that recognizes and binds to an antigen and is used interchangeably herein. A paratope is formed by a plurality of individual amino acid residues from the heavy and light chain variable domains of an antibody that are spatially proximate in the tertiary structure of the Fv region. The antibodies of the present invention contain two paratopes per homologous 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 paratopes of the antibodies of the present invention include amino acid residues derived from CDR-H2, CDR-L1 and CDR-L3 of the antibody.

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

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

[0047] The terms "anti-VEGF-A antibody" and "antibody that binds to VEGF-A" refer to an antibody that is capable of binding to VEGF-A with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in the targeting of VEGF-A. In one embodiment, the degree of binding of the anti-VEGF-A antibody to an irrelevant 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, the 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. When the antibody has a K D of 1 μM or less, the antibody is said to "specifically bind" to VEGF-A.

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

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

[0050] As used herein, the term "complementary determining region" or "CDR" refers to each region of the variable domain of an antibody that has a hypervariable sequence and contains residues that contact an antigen. Generally, an antibody contains six CDRs: three in the VH domain (CDR-H1, CDR-H2, CDR-H3) and three in the VL domain (CDR-L1, CDR-L2, CDR-L3). Unless otherwise specified, herein, CDR residues and other residues of 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 the variable domain other than the CDR residues. The framework of the 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 total strength of the 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 the 1:1 interaction between the members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for its partner Y can generally be represented 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 the site on an antigen, either proteinaceous or non-proteinaceous, to which an antibody binds. Epitopes can be formed from either contiguous stretches of amino acid sites (linear epitopes) or contain non-contiguous amino acids (structural epitopes), e.g., formed by being spatially proximal due to the folding of the antigen (i.e., by the tertiary folding of a proteinaceous antigen). Linear epitopes typically remain bound by an antibody even after the proteinaceous antigen has been exposed to a denaturing agent, whereas conformational epitopes are typically disrupted by treatment with a denaturing agent. An epitope contains 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 conventional in the art, such as, but not limited to, alanine scanning, peptide blotting (Meth. Mol. Biol. 248 (2004) 443-463), peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of the antigen (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), can classify a number of monoclonal antibodies that specifically bind to VEGF-A or IL6 based on the respective binding profiles of a number of antibodies to a chemically or enzymatically modified antigen surface (see, for example, US2004 / 0101920). Each of the classified antibodies binds to the same epitope, which may be a distinct epitope that is clearly different from or partially overlapping with the epitopes represented by other classifications.

[0056] Furthermore, competitive binding can be used to readily determine whether an antibody binds to the same epitope of VEGF-A or IL6 as the 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 the 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. Also, for example, to determine whether an antibody binds to the same epitope as the reference antibody, the reference antibody can be bound to VEGF-A or IL6 under saturation conditions. After removing the excess reference antibody, the ability of the antibody in question to bind to VEGF-A or IL6 is evaluated. If the antibody in question can bind to VEGF-A or IL6 after the saturation binding of the reference antibody, it can be concluded that the antibody of interest binds to a different epitope than the reference antibody. However, if the antibody in question cannot bind to VEGF-A or IL6 after the saturation binding of the reference antibody, the antibody of interest may bind to the same epitope as the epitope to which the reference antibody binds. Conventional experiments can be used to confirm whether the antibody in question binds to the same epitope or is only sterically hindered from binding (e.g., peptide mutations, ELISA, RIA, surface plasmon resonance, flow cytometry, or binding analysis using other quantitative or qualitative antibody binding assays available in the art). This assay should be performed in two setups, i.e., with both antibodies being saturation antibodies. In both settings, if only the first (saturation) antibody can bind to VEGF-A or IL6, 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, when measured in a competitive binding assay, two antibodies are considered to bind to the same or overlapping epitopes if a 1-, 5-, 10-, 20- or 100-fold excess of one antibody inhibits the binding of the other by at least 50%, at least 75%, at least 90%, or even 99% or more. (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 of the antigen that reduce or eliminate the binding of one antibody also reduce or eliminate the 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 the binding of one antibody reduce or eliminate the binding of the other antibody.

[0059] The "percent identity of amino acid sequences" with respect to a reference polypeptide sequence is, for the purpose of alignment, the sequences are aligned, and after introducing gaps if necessary to achieve the maximum sequence identity rate, without considering any conservative substitutions as part of the sequence identity, the ratio of the amino acid residues in the candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence. The alignment for determining the percent amino acid sequence identity can be achieved in various ways within the scope of the art, for example, using publicly available computer software, such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software or the FASTA program package. One of ordinary skill in the art can determine appropriate parameters for the alignment of sequences, including any algorithms necessary to achieve the maximum alignment over the entire length of the sequences being compared. Alternatively, the value of the identity rate can be generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., the source code is filed in the user documentation of the U.S. Copyright Office (Washington D.C., 20559), registered under U.S. Copyright Registration No. TXU510087, and is described in International Publication No. WO 2000 / 005319.

[0060] However, unless otherwise specified, for the purposes of this specification, the value of the percent amino acid sequence identity is generated using the ggsearch program of the FASTA package version 36.3.8c or, thereafter, using 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, the ggsearch (global protein:protein) program and default options (BLOSUM50; open: -10; ext: -2; Ktup = 2) can be used to compare sequences using a public server accessible at fasta.bioch.virginia.edu / fasta_www2 / index.cgi to ensure a global rather than local alignment is performed. The percent amino acid identity is given in the output alignment header.

[0061] The term "nucleic acid molecule" or "polynucleotide" includes any compound and / or substance that comprises a polymer of nucleotides. Each nucleotide is composed of a base, specifically a purine base or a 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. Often, nucleic acid molecules are described by a base sequence, where the bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is typically represented from 5' to 3'. As used herein, the term nucleic acid molecule includes deoxyribonucleic acid (DNA), such as complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and hybrid polymers comprising 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, as well as both single-stranded and double-stranded forms. Further, the nucleic acid molecules described herein may include naturally occurring nucleotides or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases or chemically modified residues having derivatized sugar or phosphate backbone linkages. Nucleic acid molecules also include, for example, DNA molecules and RNA molecules suitable as vectors for the direct expression of the antibodies of the present invention in vitro and / or in vivo in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors may or may not be modified. For example, mRNA may be chemically modified to enhance the stability of the RNA vector and / or the expression of the encoded molecule so that the mRNA can be injected into a subject to produce an antibody in vivo. (See, for example, Stadler et al, Nature Medicine 2017, published online 12 June 2017, doi:10.1038 / nm.4356 or European Patent No. 2101823 B1).

[0062] An "isolated" nucleic acid is a nucleic acid molecule that has been separated from the components of a natural environment. Isolated nucleic acids include nucleic acid molecules that are normally contained within a cell that contains nucleic acid molecules, but the nucleic acid molecules are present extrachromosomally or at a chromosomal location different from their 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 an antibody, such nucleic acid molecules being contained in a single vector or separate vectors, and such nucleic acid molecules being present at one or more locations in a host cell.

[0064] The term "vector" as used herein refers to a nucleic acid molecule capable of replicating a linked different nucleic acid. This term includes not only vectors as self-replicating nucleic acid structures, but also vectors integrated into the genome of a host cell into which the vector has been introduced. A particular vector can direct the expression of an operably linked nucleic acid. Such a vector is referred to herein as an "expression vector".

[0065] The terms "host cell", "host cell line", and "host cell culture" are used interchangeably and refer to a cell into which an exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells", which include primary transformed cells and progeny derived therefrom regardless of the number of passages. The progeny may not have exactly the same nucleic acid content as the parental cell and may contain mutations. Mutant progeny having the same function or biological activity as screened or selected in the original transformed cell are included herein.

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

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

[0068] An "effective amount" of a drug, e.g., a pharmaceutical composition, refers to an amount effective at the dosage and for the period of time necessary to achieve the desired therapeutic or prophylactic result.

[0069] "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, e.g., 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 its grammatical variations, e.g., "treat" or "treating") refers to a clinical intervention in a trial to alter the natural course of a disease in an individual being treated, which can be performed for prophylaxis or during the course of clinical pathology. Desired effects of treatment include preventing the onset or recurrence of the disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, remission or palliation of the condition, and recovery or improved prognosis. In some embodiments, the antibodies of the invention are used to delay the onset of a disease or to slow the progression of a disease.

[0071] As used herein, the term "ocular disease" includes any ocular disease associated with pathological angiogenesis and / or atrophy. Ocular diseases can be characterized by changes or dysregulation in the growth and / or infiltration of new blood vessels into the structure of ocular tissues such as the retina or cornea. Ocular diseases can be characterized by atrophy of retinal tissue (photoreceptors and the underlying retinal pigment epithelium (RPE) and choroidal capillary lamina). Non-limiting ocular diseases include, for example, AMD (e.g., exudative AMD, dry AMD, intermediate AMD, progressive AMD, and geographic atrophy (GA)), macular degeneration, macular edema, DME (e.g., focal, non-central DME, and diffuse, centrally involved DME), retinopathy, diabetic retinopathy (DR) (e.g., proliferative DR (PDR), non-proliferative DR (NPDR), and high altitude DR), other ischemia-related retinopathies, ROP, retinal vein occlusion (RVO) (e.g., central (CRVO) and branch (BRVO) forms), CNV (e.g., myopic CNV), corneal angiogenesis, diseases associated with corneal angiogenesis, retinal angiogenesis, diseases associated with retinal / choroidal angiogenesis, central serous retinopathy (CSR), pathologic myopia, von Hippel-Lindau disease, ocular histoplasmosis, FEVR, Coats disease, Norrie disease, retinal abnormalities associated with osteopetrosis pseudoglioma syndrome (OPPG), subconjunctival hemorrhage, rubeosis, ocular angiogenesis diseases, neovascular glaucoma, retinitis pigmentosa (RP), hypertensive retinopathy, retinal angiomatous proliferation, macular telangiectasia, iris angiogenesis, intraocular angiogenesis, retinal degeneration, cystoid macular edema (CME), vasculitis, papilledema, retinitis including but not limited to: CMV retinitis, ocular melanoma, retinoblastoma, conjunctivitis (e.g., infectious conjunctivitis and non-infectious (e.g., allergic) conjunctivitis), Leber congenital amaurosis (also known as Leber congenital amaurosis or LCA), uveitis (including infectious and non-infectious uveitis), choroiditis (e.g., multifocal choroiditis), ocular histoplasmosis, blepharitis, dry eye, traumatic eye injury, Sjogren's disease, and other ocular diseases where the disease or condition is associated with ocular angiogenesis, vascular leakage, and / or retinal edema or retinal atrophy.Additional exemplary eye diseases include retinal detachment (abnormal splitting of the neurosensory layer of the retina), diseases associated with 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 limbic keratitis, pterygium, dry keratitis, 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 ulcers, Terrien's marginal corneal degeneration, peripheral keratolysis, rheumatoid arthritis, systemic lupus erythematosus, polyarteritis, trauma, Wegener's granulomatosis, scleritis, Stevens-Johnson syndrome, pemphigoid, radial keratotomy, and 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, sarcoidosis, syphilis, pseudoxanthoma elasticum, Paget's disease, venous occlusion, arterial occlusion, carotid occlusive disease, chronic uveitis / vitritis, 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 choroiditis), presumed ocular histoplasmosis, Best's disease (vitellomacular dystrophy), myopia, optic disc, pars planitis, retinal detachment (e.g., chronic retinal detachment), hyperviscosity syndromes, toxoplasmosis, trauma, and laser complications. Exemplary diseases associated with atrophy of the retinal tissue (photoreceptors and underlying RPE) include, but are not limited to, atrophic or non-exudative AMD (e.g., geographic atrophy or progressive dry-type AMD), macular atrophy (e.g., atrophy associated with neovascularization and / or geographic atrophy), diabetic retinopathy, Stargardt's disease, Sorsby Fundus dystrophy, retinal detachment, and retinitis pigmentosa.

[0072] The term "accompanying document" is used to refer to the instructions customarily included in the commercial package of a therapeutic product that contain information about the indications, usage, dosage, administration, combination therapies, contraindications, and / or warnings regarding the use of such therapeutic product.

[0073] 2. Detailed Description of Embodiments of the Invention In one aspect, the present invention is based, in part, on the provision of bispecific antibodies for therapeutic applications. In certain aspects, antibodies that bind to human VEGF-A and human IL6 are 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 antibodies that bind to human VEGF-A and human IL6. In one aspect, isolated antibodies that bind to human VEGF-A and human IL6 are provided. In one aspect, the present invention provides antibodies that specifically bind to human VEGF-A and human IL6.

[0075] In certain aspects, an antibody that binds to human VEGF-A and human IL6, comprising 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 of the homologous pairs of the VL domain and the VH domain, ● 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 the variable light chain domain and the variable heavy chain domain binds simultaneously to human VEGF-A and human IL6; and / or ● The antibody binds to the same epitopes on human VEGF-A and the same epitopes on human IL6 as an antibody having the variable heavy chain domain of SEQ ID NO: 22 and the variable light chain domain of SEQ ID NO: 21; and / or ● The antibody Fab fragment of the antibody binds to human VEGF-A121 with a K D less than 50 pM as measured by surface plasmon resonance, and (ii) binds to human IL6 with a K D less than 50 pM as measured by surface plasmon resonance; and / or ● The antibody Fab fragment of the antibody exhibits an aggregation onset temperature of 60 °C or higher, in one embodiment 70 °C or higher; and / or ● The antibody Fab fragment of the antibody exhibits a melting temperature exceeding 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) 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 in the VH domain, and (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 in the VL domain, and (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 with 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 with 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, the antibody comprising: (a) a VH domain comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with 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 with the amino acid sequence of SEQ ID NO: 21.

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

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

[0080] In one aspect, the invention provides an antibody that binds to human VEGF-A and human IL6 and comprises 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 aspects, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity includes substitutions (e.g., conservative substitutions), insertions or deletions as compared to the reference sequence, but an antibody that binds to human VEGF-A and human IL6 and comprises such a sequence retains the ability to bind to human VEGF-A and human IL6. In certain aspects, a total of up to 10 amino acids are substituted, inserted and / or deleted in SEQ ID NO: 22. In certain aspects, the substitutions, insertions, or deletions occur in regions outside of the CDRs (i.e., in the FRs). In certain aspects, the VH comprises (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.

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

[0084] In a further aspect of the invention, an antibody that binds to human VEGF-A and human IL6 according to any of the above aspects is a monoclonal antibody. In one aspect, 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 aspect, the antibody is a full-length antibody.

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

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

[0087] In another aspect, the present invention provides an antibody that binds to IL6 and binds to the same epitope on IL6 as the antibody having the VL domain of SEQ ID NO: 35 and the 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 antibodies of the present invention that bind 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 antibodies of the present invention that bind to human VEGF-A and IL6).

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

[0089] In a further aspect, an antibody that binds to human VEGF-A and human IL6 according to any of the above aspects, or an antibody that binds to human IL6 according to any of the above aspects, may incorporate any of the following features alone or in combination as described in Sections 1-5 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, the antibody that binds to IL6 has a dissociation constant (K D) is 1 nM or less, 0.1 nM or less, or 0.03 nM or less. In a preferred embodiment, the antibodies provided herein have a dissociation constant (K D ) that binds to human IL6 of ≦10 pM, and in a preferred embodiment ≦5 pM. In one aspect, K D is measured using a surface plasmon resonance assay, and in one embodiment using a BIACORE® surface plasmon resonance assay.

[0092] In another aspect, K D is measured using a KinExA assay. In one embodiment, K D is measured using a KinExA assay under the conditions described below in the section on materials and general methods for the detection of K D for VEGF-A binding or the detection of K D for IL6 binding.

[0093] For example, the K Dwas measured in an assay using a KinExA 3200 instrument manufactured by Sapidyne Instruments (Boise, ID). PMMA beads were coated with antigen according to the protocol in the KinExA handbook (Adsorption coating, Sapidyne) using 30 μg of anti-VEGF antibody MAB293 (R&D) in 1 ml of PBS (pH 7.4). The KinExA equilibrium assay was performed at room temperature using PBS (pH 7.4) containing 0.01% BSA and 0.01% Tween 20 as the running buffer, and samples and beads were prepared in LowCross buffer (Candor Bioscience). The flow rate was 0.25 ml / min. A fixed amount of VEGFA-121-His (50 pM and 500 pM in the second experiment) was titrated with the test antibody, and the equilibrated mixture was aspirated into a column of beads conjugated with anti-VEGF antibody (Mab293) in the KinExA system at a volume of 750 μl for a fixed VEGF of 50 pM and 125 μl for a fixed VEGF of 500 pM. Detection of the 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. K D was obtained from non-linear regression analysis of data using a one-site homogeneous binding model included in the KinExA software (version 4.0.11) using the "standard analysis" method. The software calculates K D and determines the 95% confidence interval by fitting the data points to a theoretical K D curve. The 95% confidence interval is given as K D low and K D high.

[0094] For example, the K DIt is measured in an assay using surface plasmon resonance (SPR) on a Biacore 8K instrument (Cytiva) at 25 °C using HBS-EP+ (1x; BR100669; Cytiva) as the running buffer. The 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 on a CM5 sensor chip using standard amine coupling chemistry. Prior to protein measurement, 5 start cycles are optionally performed for conditioning purposes, flowing HBS-EP+ buffer for approximately 120 seconds in each cycle, followed by applying 10 mM glycine buffer pH 2.0 for 60 seconds to regenerate the derivatized chip surface. An antibody Fab fragment at a concentration of 75 nM is captured on this surface for 60 seconds at a flow rate of 10 μl / min in HBS-EP+ buffer. The Fab fragment is not 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. The data is evaluated using 8K evaluation software (Biacore Insight Evaluation 3.0).

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

[0096] In one aspect, 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 light chain constant domain (CL) and the first heavy chain constant domain (CH1) in addition to the variable domains of the heavy and light chains (VH and VL, respectively). Thus, the term “Fab fragment” refers to an antibody fragment comprising a light chain containing the VL domain and the CL domain and a heavy chain fragment containing the VH domain and the CH1 domain. A “Fab’ fragment” differs from a Fab fragment by having residues added at the carboxy terminus of the CH1 domain that contains one or more cysteines from the antibody hinge region. Fab’-SH is a Fab’ fragment in which the cysteine residue(s) of the constant domain retain a free thiol group. Pepsin treatment yields an F(ab’)2 fragment having two antigen-binding sites (two Fab fragments) and a portion of the Fc region. See U.S. Patent No. 5,869,046 for a description of Fab and F(ab’)2 fragments that contain salvage receptor binding epitope residues and have an increased in vivo half-life.

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

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

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

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

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

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

[0103] In a preferred embodiment, the antibody provided herein is a Fab fragment comprising a CL domain of the κ 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 fragment of the antibody provided herein exhibits an onset temperature of aggregation of 60°C or higher, in one embodiment 70°C or higher. In certain embodiments, the Fab fragment of the antibody provided herein exhibits a melting temperature of greater than 80°C as measured by dynamic light scattering.

[0105] 4. Multispecific antibodies In certain aspects, the antibodies provided herein are multispecific antibodies. A multispecific antibody is a monoclonal antibody that has binding specificity for at least two different sites, i.e., different epitopes on different antigens or different epitopes on the same antigen. In certain aspects, the multispecific antibody has three or more binding specificities.

[0106] Multispecific antibodies having three or more binding specificities, including the antibodies provided herein, can also be provided in an asymmetric form having 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, Schaefer et al, PNAS, 108(2011)1187-1191, and Klein at al., MAbs 8(2016)1010-20). Various further 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 alter the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions and substitutions can be made to arrive at the final construct so long as the final construct possesses the desired characteristics (e.g., antigen binding).

[0108] In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitution mutagenesis include the CDRs and FRs. Conservative substitutions are shown under the heading "Preferred Substitutions" in the table below. More substantial changes are provided under the heading "Exemplary Substitutions" in Table 1 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 can be screened for the desired activity, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC. [Table 1] TIFF2025521085000003.tif167161

[0109] Amino acids can be classified according to their general side chain characteristics. (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 would involve exchanging one member of one of these classes for another member of a different class.

[0111] Certain types of substitution variants involve 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 a modification (e.g., improvement) of certain biological properties (e.g., increased affinity, reduced immunogenicity) compared to the parent antibody and / or have certain biological properties of the parent antibody that are substantially retained. Exemplary substituted variants are affinity matured antibodies and can be readily generated, for example, using phage display-based affinity maturation techniques as described herein. Briefly, one or more CDR residues are mutated and the variant antibodies displayed on the phage are screened for a particular biological activity (e.g., binding affinity).

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

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

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

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

[0116] If the antibody comprises 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 attached by N-linkage to Asn297 of the CH2 domain of the Fc region. See, for example, Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharides can include various carbohydrates such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to the GlcNAc of the "stem" of the biantennary oligosaccharide structure. In some embodiments, modification of the oligosaccharides in the antibodies of the invention may be performed to generate antibody variants having certain improved properties.

[0117] In one aspect, provided is an antibody variant having an oligosaccharide structure that is a non-fucosylated oligosaccharide, i.e., lacking fucose attachment (directly or indirectly) to the Fc region. Such non-fucosylated oligosaccharides (also referred to as "afucosylated" oligosaccharides) are in particular N-linked oligosaccharides lacking a fucose residue to which a first GlcNAc is attached to the trunk of a bi-branched oligosaccharide structure. In one aspect, provided is an antibody variant having an increased ratio of non-fucosylated oligosaccharides in the Fc region as compared to the native or parental antibody. For example, the proportion of non-fucosylated oligosaccharides may be at least about 20%, at least about 40%, at least about 60%, at least about 80%, or in some cases about 100% (i.e., no fucosylated oligosaccharides are present). The proportion of non-fucosylated oligosaccharides is, for example, the (average) amount of oligosaccharides lacking a fucose residue relative to the total of all oligosaccharides (e.g., complex, hybrid, and high-mannose structures) attached to Asn297, measured by the MALDI-TOF mass spectrometry method described in WO 2006 / 082515. Asn297 refers to the asparagine residue located at position approximately 297 of the Fc region (EU numbering of Fc region residues); however, due to minor sequence variations in the antibody, Asn297 may be located upstream or downstream of position 297, i.e., within about ±3 amino acids between positions 294 and 300. Such antibodies having an increased proportion of non-fucosylated oligosaccharides in the Fc region can have improved FcγRIIIa receptor binding and / or improved effector function, particularly improved ADCC function. See, for example, US Patent Application Publication Nos. 2003 / 0157108; 2004 / 0093621.

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

[0119] In a further aspect, the antibody variant is provided, for example, with a bisected oligosaccharide in which the bisected oligosaccharide bound to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants can 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); International Publication No. 99 / 54342, International Publication No. 2004 / 065540, International Publication No. 2003 / 011878.

[0120] Also provided are antibody variants having 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, for example, in WO 1997 / 30087; WO 1998 / 58964; and WO 1999 / 22764.

[0121] b) Fc region variant In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of the antibodies presented herein, thereby creating Fc region variants. The Fc region variants can include a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) that contains amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0122] In certain embodiments, the invention contemplates antibody variants that, by having some but not all effector functions, are desirable candidates for applications where the in vivo half-life of the antibody is important while certain effector functions (e.g., complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC)) are unnecessary or harmful. To confirm reduction / abrogation of CDC and / or ADCC activity, in vitro and / or in vivo cytotoxicity assays can be performed. For example, an Fc receptor (FcR) binding assay can be performed to ensure that the antibody lacks FcγR binding (and thus is likely to lack ADCC activity), but retains FcRn binding ability. NK cells, which are the major cells mediating ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. Expression of FcRs in hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays for assessing the 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., the ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA), and the CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI)).Effector cells useful for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, or in addition, the ADCC activity of interest can be evaluated in vivo in an animal model such as that disclosed in Clynes et al. Proc. Nat’l Acad. Sci. USA 95:652-656 (1998). Also, a C1q binding assay may 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 of International Publication Nos. WO 2006 / 029879 and WO 2005 / 100402. A CDC assay can be performed to evaluate complement activation (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M. S. et al., Blood 101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie, Blood 103:2738-2743 (2004)), and FcRn binding and in vivo clearance / half-life determination can also be performed using methods known in the art (see, for example, Petkova, S. B. et al., Int’l. Immunol. 18(12):1759-1769 (2006); WO2013 / 120929Al).

[0123] Antibodies with reduced effector function include antibodies having 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 mutants include Fc mutants having substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called “DANA” Fc mutant having substitutions of residues 265 and 297 to alanine (U.S. Patent No. 7,332,581).

[0124] Certain antibody variants with improved or reduced binding to FcR are described. (See, for example, U.S. Patent No. 6,737,056; International Publication No. 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 having one or more amino acid substitutions that improve ADCC, such as substitutions at positions 298, 333, and / or 334 (EU numbering of residues) of the Fc region.

[0126] In certain embodiments, the antibody variant comprises an Fc region having one or more amino acid substitutions that reduce FcγR binding, such as residues at positions 234 and 235 (EU numbering of residues) 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 the Fc region derived from the human IgG1 Fc region. In one embodiment, the substitutions are L234A, L235A, and P329G (LALA-PG) within the Fc region derived from the human IgG1 Fc region. (See, for example, International Publication No. 2012 / 130831.) In another embodiment, the substitutions are L234A, L235A, and D265A (LALA-DA) within the Fc region derived from the human IgG1 Fc region.

[0127] In some embodiments, alterations that result in a change (i.e., either an improvement or a reduction) in C1q binding and / or complement-dependent cytotoxicity (CDC), as described, for example, in U.S. Patent No. 6,194,551, International Publication No. 99 / 51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000), occur within the Fc region.

[0128] Antibodies that play a role in transferring maternal IgG to the fetus, with an increased half-life and improved binding to the neonatal Fc receptor (FcRn) (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 contain an Fc region having one or more substitutions therein that improve the binding of the Fc region to FcRn. Such Fc variants include those having a substitution at one or more of Fc region residues: 238, 252, 254, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, or 434, for example, those having a substitution at Fc region residue 434 (see, e.g., U.S. Patent No. 7,371,826; Dall’Acqua, W.F., 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, for example, Dall’Acqua, W.F., 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, J.K., et al., Eur. J. Immunol. 24 (1994) 542). Residues I253, H310, and H435 were found to be critical for the interaction of human Fc with mouse FcRn (Kim, J.K., 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, R.L., et al., J. Biol. Chem. 276 (2001) 6591-6604). Yeung, Y.A., et al. (J. Immunol 182 (2009) 7667-7671) reported and investigated various mutants of residues 248-259 and 301-317 and 376-382 and 424-437.

[0130] In certain embodiments, the antibody variant comprises an Fc region having one or more amino acid substitutions that reduce FcRn binding, for example, 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 having amino acid substitutions at positions 253, 310, and 435. In one embodiment, the substitutions are I253A, H310A and H435A within an Fc region derived from the human IgG1 Fc region. See, for example, Grevys, A., et al., J. Immunol. 194 (2015) 5497-5508.

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

[0132] In certain embodiments, the antibody variant comprises an Fc region having one or more amino acid substitutions that increase FcRn binding, such as mutations at positions 252, and / or 254, and / or 256 (residues of EU numbering) of the Fc region. In certain embodiments, the antibody variant comprises an Fc region having amino acid substitutions at positions 252, 254, and 256. In one embodiment, the substitutions are M252Y, S254T, and T256E in an Fc region derived from the 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 can be a complete C-terminus ending with the amino acid residue PGK. The C-terminus of the heavy chain may be a shortened C-terminus with one or two of the C-terminal amino acid residues removed. In a preferred embodiment, the C-terminus of the heavy chain ends with PG in a shortened C-terminus. In one embodiment of all the embodiments reported herein, an antibody comprising a heavy chain containing the 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 the embodiments reported herein, an antibody comprising a heavy chain containing the 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 the antibody are replaced with cysteine residues. In certain embodiments, the replaced residues occur at accessible sites of the antibody. By replacing these residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody and can be used to conjugate the antibody to other sites, such as a drug site or a linker-drug site, to create an immunoconjugate, as further described herein. Cysteine-engineered antibodies can be generated, for example, as described in U.S. Patent No. 7,521,541, U.S. Patent No. 8,309,30, U.S. Patent No. 7,855,275, U.S. Patent No. 9,000,130, or International Publication No. 2016 / 040856.

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

[0138] Regarding the recombinant production of an antibody that binds to human VEGF-A and human IL6, for example, a nucleic acid encoding the above-mentioned antibody is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids can be easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to the 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 the cloning or expression of an antibody-encoding vector include prokaryotic or eukaryotic cells described herein. For example, the antibody may be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. For the expression of antibody fragments and polypeptides in bacteria, see, for example, US 5,648,237, US 5,789,199, and US 5,840,523. (See also Charlton, K.A., In: Methods in Molecular Biology, Vol. 248, Lo, B.K.C. (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 an appropriate fraction and further purified. In one embodiment, the host cell is an E. coli cell.

[0140] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in suspension can be useful. Other examples of useful mammalian host cell lines include monkey kidney CV1 cells transformed by SV40 (COS-7), human embryonic kidney cell lines (e.g., 293 cells or 293T cells as described in Graham, F.L. 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, J.P., Biol. Reprod. 23 (1980) 243-252), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), dog 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., as described in Mather, J.P. et al., Annals N.Y. 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, B.K.C. (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268.

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

[0142] C. Pharmaceutical composition In a further aspect, for example, a pharmaceutical composition is provided that comprises any of the antibodies provided herein for use in any of the following methods of treatment. In one aspect, the pharmaceutical composition comprises any of the antibodies provided herein and a pharmaceutically acceptable carrier. In another aspect, the pharmaceutical composition comprises any of the antibodies provided herein and at least one additional therapeutic agent, such as those described below.

[0143] The pharmaceutical composition of an antibody that binds to human VEGF-A and human IL6 described herein is prepared by mixing such an antibody having a desired purity with one or more pharmaceutically acceptable carriers (Remington’s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)) that are present as needed, in the form of a lyophilized composition or an aqueous solution. Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the dosages and concentrations used, and include buffers such as histidine, phosphate, citrate, acetate and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (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, etc.); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes), and / or nonionic surfactants such as polyethylene glycol (PEG), but are not limited thereto. An exemplary pharmaceutically acceptable carrier herein further includes an intervening drug dispersant such as a soluble neutral active hyaluronidase glycoprotein (sHASEGP), such as a human soluble PH-20 hyaluronidase glycoprotein 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 aspect, sHASEGP is combined with one or more additional glycosaminoglycanases (e.g., chondroitinase).

[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 composition containing a histidine - acetate buffer.

[0145] The pharmaceutical compositions herein may also include a plurality of active ingredients required for a particular indication being treated, preferably those having complementary activities that do not adversely affect each other. Such active ingredients are suitably present in combination in an amount effective for the intended purpose.

[0146] The active ingredients may be encapsulated, for example, in microcapsules prepared by coacervation techniques or by interfacial polymerization (e.g., hydroxy methylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively), or in 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 semi - permeable matrices of solid hydrophobic polymers containing the antibody, and these matrices are in the form of shaped articles, e.g., films or microcapsules.

[0148] Pharmaceutical compositions for in vivo administration are generally sterile. Sterilization can be readily achieved, for example, by filtration through sterile filtration membranes.

[0149] D. Methods of Treatment and Routes of Administration Any antibody that binds to human VEGF-A and human IL6 provided herein can be used in a method of treatment.

[0150] In one aspect, antibodies that bind to human VEGF-A and human IL6 for use as a medicament are provided. In a further aspect, antibodies that bind to human VEGF-A and human IL6 for use in the treatment of vascular diseases are provided. In certain aspects, antibodies that bind to human VEGF-A and human IL6 for use in a method of treatment are provided. In a particular aspect, the invention provides an antibody that binds to human VEGF-A and human IL6 for use in a method of treating an individual having a vascular disease, the method comprising administering to the individual an effective amount of the antibody that binds to human VEGF-A and human IL6. In such an aspect, the method further comprises, for example as described below, administering to the individual an effective amount of at least one additional therapeutic agent (e.g., 1, 2, 3, 4, 5, or 6 additional therapeutic agents). In a further aspect, the invention provides an antibody that binds to human VEGF-A and human IL6 for use in the inhibition of angiogenesis. In a particular aspect, the invention provides an antibody that binds to 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 the antibody that binds to human VEGF-A and human IL6 to inhibit angiogenesis. An "individual" according to any of the above aspects is preferably human.

[0151] In a further aspect, antibodies that bind to human VEGF-A and human IL6 for use in the treatment of eye diseases are provided. In one embodiment, the eye disease is AMD (in one embodiment, exudative AMD, dry AMD, intermediate AMD, progressive AMD, and geographic atrophy (GA)), macular degeneration, macular edema, DME (in one embodiment, focal, non-central DME, and diffuse, centrally involved DME), retinopathy, diabetic retinopathy (DR) (in one embodiment, proliferative DR (PDR), non-proliferative DR (NPDR), and high altitude DR), other ischemia-related retinopathies, ROP, retinal vein occlusion (RVO) (in one embodiment, central (CRVO) and branch (BRVO) forms), CNV (in one embodiment, myopic CNV), corneal neovascularization, diseases associated with corneal neovascularization, retinal neovascularization, diseases associated with retinal / choroidal neovascularization, central serous retinopathy (CSR), pathologic myopia, von Hippel-Lindau disease, ocular histoplasmosis, FEVR, Coats disease, Norrie disease, retinal abnormalities associated with osteopetrosis pseudoglioma syndrome (OPPG), subconjunctival hemorrhage, rubeosis, ocular neovascularization diseases, neovascular glaucoma, retinitis pigmentosa (RP), hypertensive retinopathy, retinal angiomatous proliferation, macular telangiectasia, iris neovascularization, intraocular neovascularization, retinal degeneration, cystoid macular edema (CME), vasculitis, papilledema, retinitis including but not limited to: CMV retinitis, ocular melanoma, retinoblastoma, conjunctivitis (in one embodiment, infectious conjunctivitis and non-infectious (in one embodiment, allergic) conjunctivitis), Leber congenital amaurosis (also known as Leber congenital amaurosis 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 eye diseases selected from diseases or disorders that are associated with ocular neovascularization, vascular leakage, and / or retinal edema or retinal atrophy. In one embodiment, the eye disease is AMD (in one embodiment, exudative AMD, dry AMD, intermediate AMD, progressive 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) selected from (in one embodiment, proliferative DR (PDR), non-proliferative DR (NPDR), and high altitude DR).

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

[0156] The "individual" according to any of the above aspects can be a human.

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

[0158] The antibodies of the present invention can be administered by intravitreal administration (e.g., intravitreal injection) or using a port delivery device. In one embodiment, the antibodies of the present invention are administered using a port delivery device over a period of 6 months or more, in one embodiment 8 months or more, in one embodiment 9 months or more, in one embodiment 12 months or more before the port delivery device is refilled. In one embodiment, the antibodies of the present 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 present invention can be administered alone or used in combination therapy. For example, combination therapy includes administering the antibody of the present invention and administering 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 applicable to) any of the above embodiments, the eye disease is an intraocular angiogenesis 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, pathologic myopia, von Hippel-Lindau disease, ocular histoplasmosis, retinal vein occlusion (RVO) including CRVO and BRVO, corneal angiogenesis, retinal angiogenesis, and retinopathy of prematurity (ROP).

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

[0162] Any suitable AMD therapeutic agent includes VEGF antagonists such as anti-VEGF antibodies (e.g., LUCENTIS® (ranibizumab), RTH-258 (formerly ESBA-1008, anti-VEGF single-chain antibody fragment; Novartis), or bispecific anti-VEGF antibodies (e.g., anti-VEGF / anti-angiopoietin 2 bispecific antibody, e.g., faricimab; Roche)), soluble VEGF receptor fusion proteins (e.g., EYLEA® (aflibercept)), anti-VEGF DARPin® (e.g., abicipar pegol; Molecular Partners AG / Allergan), or anti-VEGF aptamers (e.g., MACUGEN® (pegaptanib sodium)); platelet-derived growth factor (PDGF) antagonists such as anti-PDGF antibodies, anti-PDGFR antibodies (e.g., REGN2176-3), anti-PDGF-BB pegylated aptamers (e.g., FOVISTA®; Ophthotech / Novartis), soluble PDGFR receptor fusion proteins, or dual PDGF / VEGF antagonists (e.g., small molecule inhibitors (e.g., DE-120 (Santen) or X-82 (TyrogeneX)) or bispecific anti-PDGF / anti-VEGF antibodies)); VISUDYNE® (verteporfin) combined with photodynamic therapy; 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); vitamins and mineral supplements (e.g., Age-Related Eye Disease Study 1 (AREDS1; zinc and / or antioxidants) and Study 2 (AREDS2;those described in zinc, antioxidant, lutein, zeaxanthin, and / or omega-3 fatty acids); cell-based therapies, such as NT-501 (Renexus); PH-05206388 (Pfizer), huCNS-SC cell transplantation (StemCells), CNTO-2476 (umbilical cord stem cell line; Janssen), OpRegen (suspension of RPE cells; Cell Cure Neurosciences), or MA09-hRPE cell transplantation (Ocata Therapeutics); tissue factor antagonists (e.g., hI-con1; Iconic Therapeutics); α-adrenergic receptor agonists (e.g., brimonidine tartrate; Allergan); peptide vaccines (e.g., S-646240; Shionogi); amyloid-β antagonists (e.g., anti-β amyloid monoclonal antibodies, such as GSK-933776); S1P antagonists (e.g., anti-S1P antibodies, such as iSONEP (trademark); Lpath Inc); ROBO4 antagonists (e.g., anti-ROBO4 antibodies, such as DS-7080a; Daiichi Sankyo); lentiviral vectors expressing endostatin and angiostatin (e.g., RetinoStat); and any combination thereof, but not limited thereto, can be administered as additional therapeutic agents in combination with the antibodies that bind human VEGF and human IL6 provided herein for the treatment of eye diseases (e.g., AMD, DME, DR, RVO, or GA). In some examples, an AMD therapeutic agent (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 (registered trademark)). In some examples, such co-formulations can be administered in combination with the antibodies that bind human VEGF and human IL6 of the present invention. In some examples, the eye disorder is AMD (e.g., exudative AMD).;

[0163] Although not limited thereto, any suitable DME and / or DR therapeutic agent comprising 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 to human VEGF and human IL6 of the present invention for the treatment of an eye disorder (e.g., AMD, DME, DR, RVO, or GA). In some examples, the eye disorder is DME and / or DR.

[0164] The antibodies that bind to human VEGF and human IL6 provided herein can be administered in combination with a treatment or surgical procedure for the treatment of an eye disorder (e.g., AMD, DME, DR, RVO, or GA) including, for example, laser photocoagulation (e.g., panretinal photocoagulation (PRP)), drusen laser photocoagulation, macular hole surgery, macular translocation surgery, implantable miniature telescope, PHI kinetic angiography (also known as micro laser treatment and feeder vessel treatment), proton beam therapy, microstimulation therapy, retinal detachment and vitreous surgery, scleral buckling, submacular surgery, transpapillary thermotherapy, photochemical system I therapy, use of RNA interference (RNAi), extracorporeal rheopheresis (also known as membrane fraction filtration and rheopheresis), microchip transplantation, stem cell therapy, gene replacement therapy, ribozyme gene therapy (including gene therapy of hypoxia response element, Oxford Biomedica; Lentipak, Genetix; and PDEF gene therapy, GenVec), photoreceptor / retinal cell transplantation (including transplantable retinal epithelial cells, Diacrin, Inc.; retinal cell transplantation, e.g., Astellas Pharma US, Inc., ReNeuron, CHA Biotech), puncture, and combinations thereof.

[0165] Such combination therapies as described above include co - administration (wherein two or more therapeutic agents are included in the same or separate formulations) and separate administrations, in which case the administration of the antibodies of the present invention that bind to human VEGF and human IL6 can be carried out prior to, simultaneously with, and / or subsequent to the administration of an additional therapeutic agent or agent. In one embodiment, the administration of the antibodies of the present invention that bind to human VEGF and human IL6 and the administration of an additional therapeutic agent are carried out within about 1, 2, 3, 4, or 5 months of each other, or within about 1, 2, or 3 weeks of each other, 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, intralung, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intra - arterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, e.g., by injection such as intravenous or subcutaneous injection, depending in part on whether the administration is short - term or long - term. Various dosing schedules are contemplated herein, including single or multiple administrations over various time points, bolus dosing, and pulse infusions, but are not limited thereto.

[0167] The antibodies of the present invention will be formulated, dosed, and administered in a manner consistent with good medical practice. Factors to be considered in this regard include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the dosing schedule, and other factors known to the medical practitioner. The antibodies can optionally, although not necessarily, be formulated with one or more agents currently being 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 the other factors described above. These are generally used at the same dosages and by the same routes of administration as described herein, or at about 1 - 99% of the dosages described herein, or at any dosage and by any route that is empirically / clinically determined to be appropriate.

[0168] For the prevention or treatment of a disease, the appropriate dosage of the antibody of the present invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease being treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for prophylactic or therapeutic purposes, previous therapies, the patient's medical history and response to the antibody, and the discretion of the attending physician. The antibodies of the present invention are preferably administered to a patient once or over a series of treatments. Depending on the type and severity of the disease, an antibody in the range of about 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg to 10 mg / kg) can be an initial candidate dosage for administration to a patient, whether by, for example, one or more separate administrations or by continuous infusion. A typical daily dosage may range from about 1 μg / kg to 100 mg / kg, depending on the factors described above. In repeated administrations over several days or more, the treatment is usually continued until the desired suppression of the disease symptoms occurs, depending on the symptoms. One exemplary dosage of the antibody would be in the range of about 0.05 mg / kg to about 10 mg / kg. Thus, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, or 10 mg / kg (or any combination thereof) can be administered to a patient. Such doses can be administered intermittently, for example, weekly or every three weeks (e.g., such that the patient is administered about 2 to about 20 times, or for example, about 6 doses of the antibody). An initial higher loading dose, followed by one or more lower doses, may be administered. The progress of this therapy is readily monitored by conventional techniques and assays.

[0169] E. Manufactured article In another aspect of the invention, a manufactured article is provided that includes a material useful for treating, preventing, and / or diagnosing the above-described disorders. The manufactured article 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 that is used alone or in combination with another composition effective to treat, prevent, and / or diagnose a condition and may have a sterile access port (e.g., the container may be a vial having a stopper pierceable by an intravenous solution bag or a hypodermic needle). At least one active agent in the composition is an antibody of the invention. The label or package insert indicates that the composition is used to treat a selected condition.

[0170] Additionally, the manufactured article may comprise (a) a first container containing a composition comprising an antibody of the invention; and (b) a second container containing a composition comprising a further cytotoxic or other therapeutic agent. The manufactured article in this aspect of the invention may further include a package insert indicating that the composition can be used to treat a particular condition. Alternatively, or in addition, the manufactured article 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. Other buffers, diluents, filters, needles, syringes, and other materials desirable from a commercial and user perspective may further be included.

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

[0172] The port delivery device is an implantable and refillable device that can release a therapeutic agent (e.g., an antibody of the present 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 can be used include, for example, those from ForSight Labs, LLC and / or ForSight VISION4 as described in International Patent Application Publication No. 2010 / 088548, International Publication No. 2015 / 085234, International Publication No. 2013 / 116061, International Publication No. 2012 / 019176, International Publication No. 2013 / 040247, and International Publication No. 2012 / 019047, which are incorporated herein by reference in their entirety.

[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 eye incision or opening of about 0.5 mm or less. The device may have a length of about 1 mm to about 15 mm (e.g., a length of 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). 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, such as 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 present invention The following lists specific embodiments of the present invention.

[0177] 1. An antibody that binds to human VEGF-A and human IL6, 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 VH domain; and (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 a VL domain, wherein the VH domain comprises 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 with the amino acid sequence of SEQ ID NO: 22; and the VL domain comprises 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 with the amino acid sequence of SEQ ID NO: 21.

[0178] 2. An antibody that binds to human VEGF-A and human IL6, 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 VH domain; and (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 a VL domain, wherein the variable heavy chain domain comprises the amino acid sequence of SEQ ID NO: 22 with a maximum of 5 amino acid substitutions; and the variable light chain domain comprises the amino acid sequence of SEQ ID NO: 21 with a maximum of 5 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 having a maximum of 15, a maximum of 10, or a maximum of 5 amino acid substitutions; and (b) a variable light chain domain comprising the amino acid sequence of SEQ ID NO: 21 having a maximum of 15, a maximum of 10, or a maximum of 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 according to one of the preceding embodiments, comprising the heavy chain amino acid sequence of SEQ ID NO: 24 and the light chain amino acid sequence of SEQ ID NO: 23.

[0182] 6. The antibody according to any one of the preceding embodiments, wherein 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, 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, and / or ● The pair of the variable light chain domain and the variable heavy chain domain binds simultaneously to 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 the antibody having the variable heavy chain domain of SEQ ID NO: 22 and the variable light chain domain of SEQ ID NO: 21; and / or ● The antibody Fab fragment of the antibody binds to human VEGF-A121 with a K D of less than 50 pM as measured by surface plasmon resonance and binds to human IL6 with a K D of less than 50 pM as measured by surface plasmon resonance; and / or ● The antibody Fab fragment of the antibody exhibits an aggregation onset temperature of 60 °C or higher, in one embodiment 70 °C or higher; and / or ● An antibody in which the antibody Fab fragment of the antibody exhibits a melting temperature exceeding 80 °C as measured by dynamic light scattering.

[0183] 7. An antibody that specifically binds to human VEGF-A and human IL6 and comprises 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 according to any one of the preceding embodiments, which is a Fab fragment.

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

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

[0187] 11. The antibody according to any one of the preceding embodiments, in which the antibody Fab fragment exhibits an aggregation onset temperature of 70 °C or higher.

[0188] 12. The antibody according to any one of the preceding embodiments, in which the antibody Fab fragment exhibits a melting temperature exceeding 80 °C as measured by dynamic light scattering.

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

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

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

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

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

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

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

[0196] 20. An antibody according to 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 has a VH domain having a human VH3 framework (the IL6 paratope includes amino acid residues 1, 2, 3, 26, 27, 28, 29, 30, 31, 32, 52a, 94, 96, 97, 98, 101, 102 of an antibody that binds to human VEGF-A and IL6 according to any one of Embodiments 1 to 20), and a VL domain having a human Vkappa1 framework (the IL6 paratope includes amino acid residues 49, 50, 53, 54, 55, 56, 57, 91, 96 of an antibody that binds to human VEGF-A and IL6 according to 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 includes amino acid residues Y1, I2, Q3, Y26, E27, F28, T29, H30, Q31, D32, P52a, R94, I96, D97, F98, D101, T102), and a VL domain based on a human Vkappa1 framework (the IL6 paratope includes amino acid residues Y49, D50, S53, N54, Y55, P56, S57, Y91, 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 follows Kabat).

[0200] 24. An isolated nucleic acid encoding the 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, the method comprising culturing the host cell according to embodiment 25 such that the antibody is produced.

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

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

[0205] 29. A port delivery device comprising the 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 according to 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 preparation comprising the 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 medicine.

[0211] 35. The antibody according to any one of Embodiments 1 to 23 for use in the treatment of vascular diseases.

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

[0213] 37. 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 medicine.

[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 medicine for inhibiting angiogenesis.

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

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

[0217] 41. A method for inhibiting angiogenesis in an individual, the method comprising administering to the individual an effective amount of the antibody according to any one of Embodiments 1 to 23 or the pharmaceutical preparation according to Embodiment 33 for inhibiting angiogenesis.

[0218] 42. A port delivery device comprising the antibody according to any one of Embodiments 1 to 23 or the pharmaceutical preparation according to Embodiment 33.

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

[0220] 44. For ocular administration by a port delivery device according to embodiment 42, where the administration is over a period of 6 months or more, in one embodiment 8 months or more, and in one embodiment 9 months or more, before the port delivery device is refilled, an antibody according to any one of embodiments 1 to 23 or a pharmaceutical formulation according to embodiment 33.

[0221] 45. For use as a medicine by administering an antibody or a pharmaceutical formulation according to any one of embodiments 1 to 23 or a pharmaceutical formulation according to embodiment 33 using a port delivery device, where 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. [Table 2] TIFF2025521085000005.tif220161 TIFF2025521085000006.tif220161 TIFF2025521085000007.tif212161 TIFF2025521085000008.tif211161 TIFF2025521085000009.tif208161 TIFF2025521085000010.tif227161 TIFF2025521085000011.tif233161 TIFF2025521085000012.tif197161 TIFF2025521085000013.tif112161 [Examples]

[0222] The following examples are provided to assist in understanding the present invention, and the true scope thereof is set forth in the claims. It is understood that modifications may be made to the procedures described without departing from the spirit of the present invention.

[0223] Example 1: Generation of Bispecific Anti-VEGF / anti-IL-6 Fab Fragment The bispecific anti-VEGF / anti-IL-6 Fab fragment was generated by providing antibodies having distinct non-overlapping paratopes that bind to VEGF and IL-6 using a method similar to that previously described, for example, in International Publication No. WO 2012 / 163520.

[0224] Here, two different phage display libraries of synthetic Fab fragments were utilized. In the first phage display library, the residues within the CDR-H1, CDR-H3, and CDR-L2 regions of the Fab fragment were diversified, and in the second phage display library, the residues within the CDR-L1, CDR-L3, and CDR-H2 regions of the Fab fragment were diversified. In each library, the other three CDR regions were maintained without diversification and represented a paratope capable of binding to VEGF-A, in contrast to the method of International Publication No. WO 2012 / 163520 that used invariant non-binding germline-like ("dummy") sequences.

[0225] For the first library, the paratope capable of binding to VEGF-A was derived from the VEGF-A binding paratope described in International Publication No. WO 2021 / 198034.

[0226] For the second library, the VEGF-A binding paratope was 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 International Publication No. 2012 / 163520. The remaining CDR sequences were kept constant using non-germline-like sequences. In 4 rounds, 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 capturing the Fab-on-phage / target complex on Dynabeads M-280 streptavidin. The phage / target / bead complex was washed multiple times with PBST and PBS buffer. The captured phage clones with target-specific Fab were eluted from the M-280 beads using 100 mM DTT according to a standard protocol, used for infection of log-phase TG1 E. coli cells, and rescued using M13 K07 helper phage.

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

[0228] Thereafter, the binder was expressed, purified at a larger volume, and binding to VEGF was evaluated using SPR measurements. One of the resulting clones was further optimized by iterative protein engineering and screening strategies and incorporated into a phage display library as the invariant sequences of CDR-H1, CDR-H3, and CDR-L2. Briefly, the protein engineering workflow consisted of two successive rounds of affinity maturation based on an initial set of scout mutations to identify relevant beneficial mutations, followed by oligonucleotide-based generation of mutant libraries and phage display-based selection, followed by screening and further testing.

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

[0230] By phage library panning, each library was enriched for binders to human IL-6. After panning, plasmid minipreps were made for both enriched pools of phagemid vectors. The minipreps were digested with restriction enzymes to excise the region encoding the truncated gene-III protein and religated to obtain a pool of expression vectors encoding soluble Fab fragments enriched for IL-6 binders. These vector pools were transformed into TG1 E. coli cells, individual colonies were picked, and cultured for soluble expression of individual Fab clones in microtiter plates. Supernatants containing soluble Fab fragments were screened for binding to 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 pairs of VH and VL sequences encoding one bispecific Fab fragment that specifically binds to both IL-6 and VEGF-A from each library: The clones were 6HVL_1, characterized by the heavy chain of SEQ ID NO: 03 and the light chain of SEQ ID NO: 04, and VH6L_1, characterized by the heavy chain of SEQ ID NO: 09 and the 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 obtained bispecific anti-VEGF / anti-IL-6 Fab fragments were characterized. The vectors obtained as described in Example 1 were transformed into TG1 E. coli cells, and for both 6HVL_1 and VH6L_1, individual colonies were cultured for soluble expression of the bispecific antibody Fab fragments. The bispecific antibody was purified from the TG1 culture supernatant by affinity chromatography. The binding of the bispecific antibodies 6HVL_1 and VH6L_1 to human and cynomolgus monkey IL6, human VEGF121, and human VEGF165 to IL-6 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 Evaluated by Surface Plasmon Resonance (SPR): Surface plasmon resonance (SPR) was used to measure the binding rate and affinity of representative VEGF-IL-6 Fab fragments to human and cynomolgus monkey IL-6 disclosed herein.

[0234] SPR analysis of the binding of each Fab fragment IL-6 derived from human and cynomolgus monkey was performed at 25 °C on a Biacore 8K instrument (Cytiva) using HBS-EP+ (1x; BR100669; Cytiva) as the running buffer. The 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 on a CM5 sensor chip using standard amine coupling chemistry. This immobilization procedure yielded a ligand density of approximately 5000 resonance units (RU). The reference channel was treated accordingly.

[0235] Prior to protein measurement, five start-up cycles were performed for conditioning purposes. In each cycle, the derivatized chip surface was regenerated by flowing HBS-EP+ buffer for 120 seconds, followed by applying 10 mM glycine buffer pH 2.0 for 60 seconds. A 75 nM concentration of Fab fragment was captured on this surface for 60 seconds at a flow rate of 10 μl / min in HBS-EP+ buffer. The Fab fragment was not applied to the reference channel. Subsequently, human or cynomolgus IL-6 was applied in appropriate dilution series 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 reference was used and raw data were fitted using a 1:1 binding model.

[0236] Figure 1 shows representative SPR traces and fitting curves determined for the Fab fragments tested, with the corresponding Fab names shown on the graph. Data are shown for binding to human and cynomolgus IL-6, and 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] The inventors observe clear binding of 6HVL_1 and VH6L_1 to human IL-6. Only VH6L_1 shows a significant affinity for cyIL-6, although with a clearly 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. The data are averaged for three experiments and the standard deviation is 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 evaluated by competitive ELISA: To test what antibody concentrations are required to block the interaction of VEGF121 and VEGF165 with their receptors, competitive ELISA experiments were performed using 6HVL_1 and VH6L_1. A VEGF-binding Fab fragment (ranibizumab) was used as a positive control, and an experiment using only buffer was used as a negative control. Briefly, 1:3 dilution series of all samples starting from 20 nM were mixed with a constant concentration of 10 pM VEGF121 (Humanzyme HZ-1206) or 10 pM VEGF165 (Humanzyme HZ-1153) and incubated for 90 minutes. This mixture was then transferred to a Maxisorp plate surface blocked with 2% MPBST and then coated with VEGF receptor 1 (VEGF-R1, R&D Systems, 1 μg / ml in NaHCO3, pH 9.4). The contact time between the Fab antigen mixture and the receptor-coated plate was limited to 10 minutes at room temperature to minimize interference with the binding equilibrium. After incubation and two washing steps, detection of VEGF121 / VEGF165 on the VEGFR1-coated plate was performed using biotinylated anti-VEGF mAb (BAF203, R&D Systems) and horseradish peroxidase-labeled 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’-tetramethylbenzidine diamine, and a change in absorbance at 450 nm could occur thereafter. TMB was preheated to room temperature, incubated on the plate for 5 minutes, and then quenched with 1N H2SO4.

[0239] The results for 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: Improvement of Bispecific Anti-VEGF-A / Anti-IL6 Fab Fragment As described above, both antibodies showed no or low cross-reactivity with cynomolgus IL6, which is desirable for clinical development. In addition, the treatment of ocular vascular diseases requires injection of the therapeutic agent into the eye, and as a result, the optimal therapeutic agent should exhibit high affinity and high concentration for the target antigen in order to maximize the persistence of the therapeutic effect and patient convenience. Therefore, for the intended purpose, it is desirable to further improve the initially identified molecule.

[0241] Several rounds of maturation were performed by introducing different amino acid substitutions into the VH domain and VL domain. During maturation, candidate antibodies derived from both the "parent" antibodies 6HVL_1 and VH6L_1 were screened and selected based on their desired properties regarding 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 among the multiple tested candidate antibody molecules from each round of maturation. Candidate selection was based on the desired properties, particularly the improvement of 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 among the multiple tested candidate antibody molecules. [Table 4]

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

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

[0246] Example 5: Kinetics of Antigen Binding of Improved Anti-VEGF-A / Anti-IL6 Fab Fragments The binding kinetics to human and cynomolgus monkey IL6 and the competition IC50 for VEGF / VEGFR1 competition for the candidate antibodies were evaluated as 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 against the prior art, the following controls were used: bispecific antibody VH6L (VH / VL sequences disclosed in WO 2012 / 163520, referred to herein as "VH6L-BM"), anti-VEGF antibody ranibizumab (INN), and an anti-IL6 antibody that is cross-reactive between human and cynomolgus monkey IL6 as disclosed in WO 2014 / 074905 (positive control). The aforementioned prior art antibodies were prepared by recombinant expression.

[0247] Figure 3 and Tables 3 and 4 show the results of the evaluation of human and cynomolgus monkey IL6 binding. 6HVL_4 and 6HVL_4-YHE, variants of 6HVL_4 with three additional framework amino acid mutations, show improved human IL6 binding and cynomolgus monkey IL6 cross-reactivity beyond the initially 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 as evaluated by competitive ELISA using human VEGF121 and human VEGF165. Figure 4 illustrates that the prior art molecule 6HVL_BM exhibits an affinity that is too low to be detected under the assay conditions and is thus clearly much lower than the affinity of the antibodies of the present invention.

Table 7

Table 8

[0249] Example 6: Simultaneous Binding of Anti-VEGF / Anti-IL-6 Fab Fragments The simultaneous binding of the antibodies of the present invention to their targets was evaluated as follows by surface plasmon resonance using an immobilized anti-Fab antibody to capture the anti-VEGF / anti-IL-6 Fab fragments of the present 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 temperature of the flow cell was set at 25°C.

[0251] The anti-VEGF / anti-IL-6 Fab fragment was captured via the kappa chain by injecting a 10 μg / mL solution at a flow rate of 5 μL / min for 30 seconds to form an anti-Fab antibody / anti-VEGF / anti-IL-6 Fab complex. To enable the formation of complexes containing anti-Fab antibody, anti-VEGF / anti-IL-6 Fab, human VEGFA, and human IL-6, both antigens of human VEGFA121 (self-produced, P1AA1779-010) and human IL-6 (commercial, Peprotech#200-06) were added either continuously or simultaneously. The corresponding SPR response unit curves (Biacore T200, Cytiva) were monitored. For continuous binding, human VEGFA at a concentration of 300 nM was injected for 180 seconds, followed by an additional 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, followed by 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. The difference in bulk refractive index was corrected by subtracting a blank injection and the response obtained from a control flow cell without captured Fab was subtracted.

[0252] The results are shown in Figure 7. The addition of human VEGF-A to the anti-Fab / anti-VEGF / anti-IL-6 Fab complex resulted in binding and the formation of anti-Fab / Fab / VEGF-A complex. The continuous addition of human IL-6 resulted in the formation of 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] In the reverse order, when human IL-6 was added first, followed by continuous addition of human VEGF-A, the reduction in simultaneous binding was significant (dotted line). This indicated that the binding of human IL-6 sterically interfered with the binding of human VEGF-A first, resulting in a reduction in the binding ability between anti-VEGF / anti-IL-6 Fab and human VEGF-A, but it was still possible.

[0254] In the presence of both targets, human IL-6 binding appears to be preferred and binding to human VEGF-A is reduced (solid line). This effect was reasonable due to the intrinsically 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 the anti-VEGF / anti-IL-6 Fab fragment in the presence of IL-6 was evaluated by an inhibition assay using surface plasmon resonance with immobilized VEGF-A.

[0256] To show simultaneous binding of human VEGF-A and human IL-6 to the anti-VEGF / anti-IL-6 Fab fragment, human vascular endothelial growth factor 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 to obtain 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] As a reference, a 1:2 dilution series of 0 - 200 nM anti-VEGF / anti-IL-6 Fab fragment in 50 nM human VEGFA solution was used to test VEGFA and VEGFR2 inhibition. The anti-VEGF / anti-IL-6 Fab fragment / VEGFA mixture was injected onto 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. The difference in bulk refractive index was corrected by subtracting the blank injection and by subtracting the response obtained from the blank control flow cell. For the evaluation, the binding response 5 seconds after the end of the injection was obtained. The induced response in RU was converted to a binding response compared to the initial signal corresponding to the ligand(s) without bispecific Fab. The IC50 value was calculated using a four-parameter logistic model (XLfit, ID Business Solutions Ltd.).

[0258] In addition to the reference, dilutions of 0 - 200 nM anti-VEGF / anti-IL-6 Fab fragment in solution with 10 nM human IL-6 were pre-incubated for 15 minutes and tested to calculate the IC50 value (Figure 3).

[0259] The results are shown in Figure 8. The graph shows the inhibition of the VEGFR2 / VEGF-A interaction depending on the concentration of 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, but an increase in anti-VEGF / anti-IL-6 Fab concentration increased the inhibition (crossed solid line). The addition of human IL-6 mimicking the treatment-related conditions did not affect the degree of VEGFR2 / VEGF-A inhibition and resulted in very similar IC50 values (IC50 = 33 nM without human IL-6 (triangular dashed line), IC50 = 37 nM with additional human IL-6 (triangular dashed black line)).

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

[0261] To evaluate the simultaneous binding of human VEGFA and human IL-6 to anti-VEGF / anti-IL-6 Fab, an IL-6-specific cell-based reporter gene assay using the reporter cell line HEK-Blue™ IL-6 cells (InvivoGen) was used. The cells were incubated with anti-VEGF / anti-IL-6 Fab and human IL-6 for 20 ± 1 hours in the absence and presence of excess human VEGF-A, either by simultaneously adding the bispecific Fab and human IL-6 (Figure 9) or after pre-incubation (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 the Janus family of tyrosine kinases (JAK1, JAK2, and Tyk2) that results in the activation of signal transducer and activator of transcription 3 (STAT3) and subsequent secretion of SEAP (secreted embryonic alkaline phosphatase). In the case of binding of anti-VEGF / anti-IL-6 Fab to human IL-6, signaling is inhibited and SEAP is not produced. Subsequently, SEAP levels in the cell culture supernatant are quantified by adding the QUANTI-Blue SEAP substrate to an aliquot of the supernatant. SEAP converts the QUANTI-Blue substrate into a product that can be measured at an absorbance of 650 nm using a plate reader. The simultaneous binding of human VEGFA and human IL-6 is then evaluated by graphing the average absorbance against the concentration of anti-VEGF / anti-IL-6 Fab, and the data are fit to a constrained 4-parameter curve. The relative potency (inhibitory concentration) of the samples is calculated using a 4-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 (approx. 22.5 pM) 50showed a distinct dose response curve with values and demonstrated a distinct inhibition of human IL-6 response by incremental amounts of anti-VEGF / anti-IL-6 Fab. To address the 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 selected ratios of human VEGFA:human IL-6 (1:1 / 2.5:1 / 5:1), only a slight reduction in the effective IC 50 value was observed. The values changed slightly from an IC 50 = 1.134 ng / mL in the absence of human VEGFA to an IC 50 = 1.724 ng / mL when human VEGF-A was present in 5-fold excess, and this situation closely reflects in vivo relevant conditions.

[0264] Figure 10 shows the results by pre-incubation indicating that the binding of IL6 does not affect the binding of IL6.

[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 of IL6 with its non-signaling co-receptor IL6R and the cytokine receptor gp130. Here, three epitopes (site 1, 2 and 3) have been defined to identify the contact surfaces formed in the complex (Boulanger MJ et al., Science 2003, 27; 300(5628):2101-4.). IL-6 first binds to IL-6R via an interaction surface called "site 1". "Site 2" is an epitope formed by a binary complex of IL-6 and IL-6R that interacts with domains 2 and 3 of gp130. The subsequent interaction between "site 3" of IL6 and domain 1 of gp130 results in the formation of a dimer of the IL6 / IL6R / gp130 trimer and thus the formation of a hexameric signaling complex.

[0266] To understand which epitopes of IL6 are bound by our two series of Fabs (6HVL and VH6L), we performed structural analysis of the complexes of IL-6 with antibody Fabs, which are representative of the anti-VEGF / anti-IL-6 Fab fragments of the invention. The Fab 6HVL4.1 utilized is very closely related to Fab 6HVL_2, differing by only two mutations, and 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 derived from Fab VH6L_1 respectively, it can be safely assumed that the structural results obtained below are applicable across each of the Fabs of the individual series. Formation of the complex of Fab and IL-6 and analysis of the complex structure were performed by X-ray crystallography as follows.

[0267] IL6-Fab complexes were 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 number 031316-2), respectively.

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

[0269] For Fab fragment 0182, needle-like crystals appeared within two days from 0.1 M MgCl2, 0.1 M sodium citrate pH 5, 15% (w / v) PEG 4000. Subsequently, the crystals were used in a seeding experiment, and large square crystals could be obtained from 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 one day from 0.2 M ammonium sulfate, 0.1 M Tris, pH 7.5, 20% (w / v) PEG MME 5000.

[0271] For data collection, the collected crystals were flash-cooled to 100 K in the crystallization solution supplemented with 15% ethylene glycol. X-ray diffraction data were collected for Fab fragment 0182 at a wavelength of 0.9999 Å and for 6HVL4.1 at a wavelength of 0.9982 Å using a PILATUS 6M detector at the beamline X10SA of the Swiss Light Source (Villigen, Switzerland). The data were processed with XDS (Kabsch, W., XDS. Acta Cryst. D66, 125 - 132 (2010)), scaled with AIMLESS (P.R. Evans and G.N. Murshudov “How good are my data and what is the resolution?” Acta Cryst. (2013). D69, 1204 - 1214), and analyzed for anisotropy with STARANISO (Tickle, I.J., 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 the space group P21 with cell axes a = 65.93 Å, b = 65.46 Å, c = 159.30 Å, β = 91.65° and diffract to a resolution of 2.18 Å.

[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, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Storoni, L. C., & Read, R. J. Phaser crystallographic software. J Appl Cryst. 40, 658-674 (2007)) using the coordinates of the in-house Fab and IL-6 (pdb entry 1alu) as the search models. Amino acids were changed according to the sequence differences using different electron densities. The structure was refined using programs from the CCP4 suite (Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta. Cryst. D67, 235-242 (2011)) and BUSTER (Bricogne, Blanc, G. E., Brandl, M., Flensburg, C., Keller, P., Paciorek, W., Roversi, P., Sharff, A., Smart, O. S., Vonrhein, C., Womack, T. O. Buster version 2.9.5 Cambridge, United Kingdom: Global Phasing Ltd. (2011)). Manual rebuilding was performed using COOT (Emsley, P., Lohkamp, B., Scott, W. G., 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 representations were prepared using PYMOL (The Pymol Molecular Graphics System, Version 1.7.4. Schrodinger, LLC.).

Table 9

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

[0277] Figure 16 shows the binding mode of Fab 0182 to IL6. For illustrative purposes, the inventors generated a superposition of two structures: first, a superposition of the complex structure of Fab and IL6, and second, a superposition of the complex structure of IL6R and 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 with the trimeric complex of IL6 with IL6R and gp130, it is clear that Fab binds to IL6 in a manner very similar to gp130, i.e., it binds to site 2 of IL6. This binding mode is expected to allow simultaneous binding of both Fab and IL6R to IL6, i.e., the interaction between IL6 and IL6R should still be possible, and such an IL6 antagonist is expected to act empirically by inhibiting the interaction between the IL6 / IL6R complex and gp130 via binding to site 2 of IL6.

[0278] Crystal structure of the Fab 6HVL4.1-IL-6 complex. The inventors determined the crystal structure of the complex of Fab 6HVL4.1 with IL-6 at a resolution of 1.94 Å (Figure 6). This structure shows Fab 6HVL4.1 bound to IL-6 by the major contributions of heavy-chain CDR1, CDR3 and light-chain CDR2, CDR3. 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 of helix A and helix C.

[0279] Similar to what was done for Fab 0182, the inventors 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 very similar manner to gp130 and thus must be considered a site 2 binder from a structural point of view.

[0280] Experimental investigation of the binding mode to IL6 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") representative of clones of the 6HVL series containing 6HVL4.1 was captured on the surface of the SPR chip via an anti-Fab antibody, and IL6 and then IL6R were flowed over the chip surface at three different concentrations (250, 500, 1000 nM). Here, the inventors expected a two-step continuous signal increase if IL6 could bind to both Fab and IL6R. Indeed, for the Fab tested, this was what was observed (Figure 18): in the SPR signal trace, the addition of IL6 resulted in a strong increase in the signal, which was 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 in which site 1 of IL6 is completely shielded and inaccessible ("hyper-IL6"), when coated on the SPR chip and probed with Fab at a concentration of 26 nM, still facilitates the binding of each Fab molecule (Figure 19).

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

[0283] Considering that it has been found from the available crystal structures and SPR experiments that site 1 of IL6 remains accessible for binding, the inventors have to interpret these results such that the IL6 antibodies described in this patent can not only sterically block the binding of the IL6 / IL6R complex to gp130, but also strongly and allosterically reduce the binding affinity of IL6 to IL6R, i.e., can also function further as an IL6 site 1 antagonist.

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

[0285] 1. As an IL6 site 2 binder, the IL6 antagonist can equally effectively block the formation of the signaling complex by the binding of IL6 to membrane-bound IL6R and gp130 (cis-signaling), or the formation of the pre-formed complex of IL6 and IL6R (trans-signaling). In contrast, an IL6 site 1 binder cannot bind to the pre-formed complex of IL6 and IL6R and can only antagonize it when the complex dissociates.

[0286] 2. Due to the allosteric reduction of 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 show this effect by disadvantaging the formation of the first step of signaling, i.e., the formation of the IL6 / IL6R complex. A site 2 binder that does not allosterically interfere with site 1 binding is expected to be disadvantageous when it has to block the second step of IL6 / IL6R-gp130 complex formation on the cell surface, especially for cis-signaling, i.e., when the relative effective concentrations of IL6 / IL6R and gp130 are expected to be very high.

[0287] 3. When the IL6 site 1 binder is used systemically as an antibody, it is known to result in a strong accumulation of the IL6 / antibody complex because the half-life of the complex is significantly increased compared to IL6 alone. In contrast, the IL6 site 2 binder is expected to still enable the removal of the IL6 / antibody complex by internalization and degradation of the cells that bind to the membrane-bound IL6R and subsequently take up the complex. In that regard, the IL6 antagonists described herein are expected to combine the desirable properties of both the site 1 and site 2 binders: functionally, they can block the first step in the formation of the IL6 / IL6R / gp130 signaling complex, but still allow for the degradation of the IL6 / mAb complex via IL6R binding on the cell surface.

[0288] 4. In ophthalmic indications and when utilized as Fab molecules, the expected behavior of such binders can still be more beneficial. Similar to the IL6 site 1 binder, the Fab / IL6 complex can exit the eye cavity relatively unhindered by IL6R binding and can be rapidly eliminated systemically by renal filtration.

[0289] Example 8: Thermal stability of an improved anti-VEGF / anti-IL6 Fab fragment Additional sequence variants of an improved anti-VEGF / anti-IL-6 antibody were generated that contain the amino acid sequences identified in Table 8. [Table 10]

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

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

[0292] The evaluation of the starting temperature was performed by the UNcle analysis software. The aggregation starting temperature is defined as the temperature at which the scattered light intensity begins to increase. The denaturation of the protein was monitored by the shift of the barycentric mean (BCM) of the fluorescence signal with respect to heat. The melting temperature is defined as the inflection point of the BCM (nm) with respect to the temperature curve.

Table 11

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

[0294] Viscosity was measured using the latex-bead DLS method as described above (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 evaluation: Equipment and materials · Wyatt DLS plate reader equipped with Greiner Bio-One microplates · 3000 Series Nanosphere (trademark) size standard (Thermofisher catalog-number 3300A) · Tween20 (Roche, catalog number 11332465001) and silicone oil, e.g., (Alfa Aesar catalog number A12728) · UV spectrophotometer for concentration determination (e.g., Nanodrop 8000).

[0296] Sample Preparation The antibody sample was re-buffered and diluted with 20 mM His / HCl, pH 5.5 (buffer) and 0.02% Tween20 (final concentration). A bead concentration of 0.03% solids was added. At least three different concentrations were prepared, and the highest concentration possible was 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 microplates and each well was covered with silicone oil.

[0297] Measurement with 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 seconds 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 summary of the software template (Microsoft Dynamics 7.10 or higher). Viscosity was calculated using the formula (ηreal = Dapp * ηH2O / Dreal). Dreal is the bead size measured in the blank sample, which is equal to the bead size (300 nm). The calculated viscosities were shown in an Excel curve. It is possible to extrapolate the viscosity at a given concentration using the Mooney curve fit (Excel). Here, the maximum protein concentration when the viscosity exceeds 20 cP was calculated.

[0299] The maximum concentration of the indicated antibody to achieve a viscosity of 20 cP at 20 °C is 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 limits for injectability. As a result, the antibodies of the present invention are well-suited for ocular use as they enable the delivery of high molar doses in limited injection volumes, which, when combined with high efficacy, results in high durability, thereby reducing the frequency of administration, which is desirable for enhancing patient convenience and treatment compliance.

[0301] Example 10: Primary cell-based assay to demonstrate IL6 inhibition mediated by VEGF / IL-6 bispecific antibody 6HVL_4(HRMEC) To measure IL-6 signaling activity in HRMEC, an assay was established to quantify ICAM-1 surface expression in HRMEC. HRMEC were stimulated for 72 hours with a combination of equimolar concentrations (2 nM) of human IL-6 and human IL-6R. Surface expression of ICAM-1 was evaluated by flow cytometry. To measure the inhibitory activity of 6HVL_4, the IL-6 / IL-6R mixture was pre-incubated with increasing concentrations of the antibody prior to application to the cells.

[0302] Cell culture: HRMEC (catalog number PEL-PB-CH-160-8511; PELOBiotech Gmbh; Bayern, Germany) was thawed and cultured in endothelial growth medium (EGM-MV) containing endothelial basal medium (EBM) (catalog number CC-3156; Lonza; Basel, Switzerland) and 5% fetal bovine serum (FBS), hydrocortisone, human fibroblast growth factor B, VEGF, R3-IGF-1 (recombinant analog of insulin-like growth factor-I with Glu at position 3 replaced by Arg), ascorbic acid, human epidermal growth factor, and GA-1000 (all included in the EGM-2 MV Microvascular Endothelial SingleQuots™ kit; catalog number CC-4147; Lonza) at the manufacturer's recommended concentrations. Twenty-four hours after plating, the medium was replaced with fresh EGM-MV, and the cells were grown for an additional 3 days before the assay. The assay conditions were optimized over different passage numbers and concentrations of IL-6 / soluble IL-6R. The final assay was performed using 6th passage HRMEC and equimolar stimulation with 2 nM concentration of IL-6 / soluble IL-6R.

[0303] Flow cytometry assay: HRMECs were removed from the flask by washing twice with calcium- and magnesium-free phosphate-buffered saline (PBS) (catalog number 10010023; Life Technologies) and once with the cell dissociation reagent Accutase (catalog number A1110501; Thermo Fisher Scientific; Waltham, MA). After washing, 5 mL of the cell dissociation reagent was added to the cells, and the flask was incubated at 37 °C for 3 minutes in a 5% CO2 incubator. The dissociated cells were collected from the flask and placed into 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). The cell number was 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 number 3596; Corning; Corning, NY) to obtain 30,000 cells / well. The plate was then incubated for an additional 24 hours at 37 °C in a 5% CO2 incubator.

[0304] Recombinant human IL-6 (Catalog number 206-IL / CF; R&R&D Systems; Minneapolis, MN) and recombinant human IL-6R (Catalog number 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 form the IL-6-I / L-6R complex. Next, 50 L of a dilution series (3-fold, 7-point dilution) of 6HVL_4 was added to the cells and incubated at 37 °C and 5% CO2 for 1 hour. Finally, 50 μL of the 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. Background ICAM-1 surface expression and 100% response levels were determined for unstimulated cells and cells stimulated with the IL-6-I / L-6R complex without 6HVL_4, respectively. The cells were incubated at 37 °C in a 5% CO2 incubator for 72 hours.

[0305] For analysis of ICAM-1 surface expression, the cells were washed with PBS (Ca 2+ , Mg 2+;washed twice with Dulbecco's phosphate-buffered saline (PBS) (catalog number 14190250; Life Technologies) and once with Accutase cell dissociation reagent (catalog number A1110501; Thermo Fisher Scientific). Cells were detached from the plate using 50 μL of cell dissociation reagent (3 minutes, 37 °C) and transferred to a flow cytometry Falcon 96-well Storage plate (catalog number 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 minutes, and the supernatant was discarded. The pellet was resuspended in 100 μL of PBS, 2% FBS, 2 mM EDTA containing 10 μg / mL human IgG (catalog number I2511; MilliporeSigma; Burlington; MA) to block non-specific binding sites and incubated at room temperature for 15 minutes. After blocking, 0.5 μg of fluorescein-labeled anti-ICAM-1 antibody (catalog number BBA20; R&R&D Systems) was added to the cells, and the reaction was incubated at 28 °C for 45 minutes. After staining, cells were pelleted by centrifugation at 300 g for 6 minutes, and the pellet was resuspended in 150 μL of 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 every independent experiment 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 for each condition. The 100% response level was calculated from cells stimulated with IL-6 / IL-6R without 6HVL_4, and then the inhibitory ability of 6HVL_4 was expressed as the inhibition rate of the 100% response. The inhibition rate for 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 mean IC50 and SE using Excel XLfit software version 5.5.0 (IDBS; Guildford, UK). The concentration-response curve was fitted by non-linear regression analysis using a five-parameter logistic model (A + ((B - A) / (1 + (((B - E)*((C / x)^D)) / (E - A))))) calculated for the basal inhibition activity and the maximum inhibition activity.

Table 13

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

[0308] Example 11: Primary cell-based assay to demonstrate VEGF inhibition mediated by the VEGF / IL-6 bispecific antibody 6HVL_4 (HUVEC) Assay: HUVECs were obtained from Lonza (catalog number 00191027; Basel, Switzerland). Endothelial basal medium (EBM-2; catalog number CC-3156) and the EGM-2 Endothelial SingleQuots Kit (catalog number CC4176), which together constitute endothelial growth medium (EGM-2) and assay medium (EBM-2 containing 0.5% fetal bovine serum [FBS]), were also purchased from Lonza.

[0309] A T175 cell culture flask (catalog number 353112; Corning; Corning, NY) coated with adhesion factor (AF) (catalog number S-006-100; Gibco, Thermo Fisher Scientific; Waltham, MA) was used to maintain HUVEC. 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 number 354409; Corning) using alamarBlue (catalog number DAL1100; Invitrogen, Thermo Fisher Scientific).

[0311] Recombinant human VEGF-A was obtained from R&D (catalog number 293-VE; Minneapolis, MN) and dissolved in calcium- and magnesium-free phosphate-buffered saline (PBS) (catalog number 14190-094; Gibco) at a stock concentration of 100 μg / mL. 2+ and Mg 2+ AlamarBlue contains the cell-permeable compound resazurin. This compound changes its color by reducing the environment inside healthy cells. The resulting pink color is a proportional marker of live cells and can be used to detect proliferation by measuring the absorbance at 570 nm. VEGF-A induces the proliferation of HUVEC grown under cell starvation conditions. Therefore, the proliferation of VEGF-A-induced HUVEC can be inhibited by using a VEGF-A neutralizing antibody or Fab.

[0312]

[0313] ​HUVECs were maintained up to passage 5 in EGM-2 in a T175 flask coated with AF. For the viability assay, 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. Subsequently, 100 μL of the cell suspension was 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 only. The cells were incubated overnight at 37 °C in a 5% CO2 incubator.

[0314] The next day, a 10-fold working solution (750 ng / mL) was prepared in assay medium (EBM 0.5% FBS) using a VEGF-A stock solution (100,000 ng / mL in PBS (Ca2+, Mg2+)).

[0315] The 6HVL_4 stock solution was also diluted with assay medium to prepare a 10-fold working solution. Using this, a three-fold eight-step dilution series starting at 30,000 ng / mL and ending at 14 ng / mL was prepared.

[0316] Next, 12.5 μL of the 10-pre-diluted 6HVL_4 solution and 12.5 μL of the 10 VEGF-A solution (750 ng / mL) were successively added in quadruplicate to the cells in each plate. VEGF-A was used at a constant final concentration (75 ng / mL), and 6HVL_4 was used in a dose-response format with final concentrations ranging from 3000 ng / mL to 1.4 ng / mL. The 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 incubation in a cell culture incubator for 3 hours. Absorbance was measured at 570 nm with a reference wavelength of 600 nm using a FlexStation 3 plate reader manufactured by Molecular Devices.

[0317] Data analysis: For each experiment, each condition was performed in quadruplicate. A total of 4 independent experiments were conducted. Considering independent experiments, 2 separate plates were processed on the same day. Thus, 8 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 for each 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 the inhibition rate of the 100% response.

[0318] The IC50 values were calculated from the average data for each antibody concentration using Excel XLfit software version 5.5.0 (IDBS; Guildford, UK). The concentration-response curves were fitted by non-linear regression analysis using a four-parameter logistic model (A + ((B - A) / (1 + ((C / x)^D)))) calculated for the basal inhibition activity and the maximum inhibition activity. The data are presented as mean values from 4 independent experiments using the standard error of the mean (SEM).

Table 14

[0319] 6HVL_4 reduced VEGF-A-induced HUVEC proliferation and had a 50% inhibitory concentration (IC50) of 2.06 + / - 0.30 nM (Figure 12). Example 12: The restoration of the barrier function in the presence of both VEGF-A and IL6 and the VEGF / IL6 bispecific antibody 6HVL_4 indicates the biological activity of the molecule To evaluate the dual biological activity of the antibody of the present invention, i.e., the simultaneous blockade of both targeted cytokines - VEGF-A and IL6 (complex with IL6R), a transendothelial electrical resistance (TER) assay was performed. In this assay, an electrically tight endothelial cell layer responds to the addition of both VEGF-A and IL6 by loss of barrier function. The restoration of the barrier function in the presence of both cytokines VEGF and IL6 by the VEGF / IL6 bispecific antibody 6HVL_4 was evaluated as follows.

[0320] Assay: Human retinal microvascular endothelial cells, further referred to as HRMVEC (PELOBiotech; catalog number PEL-PB-CH-160-8511), were maintained up to passage 5 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 (Ginco, catalog number S-006-100). For the transendothelial cell resistance assay, cells were detached using StemPro® Accutase® (Gibco, catalog number A11105-01). Cells were then seeded at a cell density of 120,000 cells / well in 100 μl of MV-EGM-2 growth medium into the upper chamber of a fibronectin-coated (catalog number 354008, Corning) Transwell filter (24-well Corning, catalog number 3470). The lower chamber of the Transwell filter was filled with 600 μl of MV-EGM-2 medium. Cells were incubated at 37 °C with 5% CO2 for 3 days. The medium was then changed to assay conditions (MV-EGM-2 without VEGF containing 2% FBS), and the Transwell filter was transferred to the cellZcope system using 280 μl of medium in the upper chamber and 810 μl in the lower compartment. Cells were then incubated at 37 °C with 5% CO2 for 24 h, during which TER was measured by cellZcope. The next day, cells were treated with VEGF at a final concentration of 10 ng / ml (R&D Systems, catalog number 293-VE / CF), IL6 at 50 ng / ml (R&D Systems, catalog number 206-IL / CF) and IL6R at 100 ng / ml (R&D Systems, catalog number 227-SR-025 / CF) in combination with 10 ng / ml of VEGF, or with an equal volume of assay medium (8-fold per condition), and TER was measured until the next day. Subsequently, 6HVL_4 or aflibercept at final concentrations of 1 μg / ml and 2.3 μg / ml respectively, or assay medium, were added to the cells, and TER was then measured over the next 24 h. Thus, each condition was present 4 times.

[0321] Data analysis: The dataset generated for one well was normalized against 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] Results: The results are shown in Figure 12 (6HVL_4) and Figure 13 (aflibercept).

[0323] The barrier function of HRMVECs is reduced by cytokine VEGF alone and in combination with IL6 / IL6R. With antibody 6HVL_4, the disrupted barrier recovers to 100% after 24 hours.

[0324] Example 13: Identification of the IL6 paratope region From the crystal structure of the complex of 6HVL4.1 and IL6, the amino acid residues in contact with IL6 were identified. An example of the positions of the paratope amino acid residues in the VH domain and the VL domain is shown in FIG. 15. For this purpose, the "byres" function of PyMOL and a cut-off distance of 5 angstroms were used to identify residues in the Fab / IL6 complex that are likely to interact with IL6. Here, the inventors limited the analysis to residues 48 to 215 of IL6 (defined by Uniprot number P05231). These residues are known to be residues that are normally resolved in the structure of IL6 alone (see pdb accession numbers 1alu and 1IL6). FIG. 15 also shows the alignment between 6HVL4.1 and the single-specific anti-IL6 antibody 6HdL2.05 based on the antibody of the present invention, where the VEGF-paratope has been 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, purified, and subjected to an SPR assay using human IL6 or cynomolgus IL6 as described in Example 2 and performed in the same manner as in Example 3, the antibody showed an SPR sensorgram as shown in FIG. 20. After fitting of the experimental data, 6HdL2.05 showed an affinity comparable to the highest affinity obtained for the corresponding 6HVL series of VEGF / IL6 bispecific antibodies (see Tables 3 and 4), with a fitted KD of 22 pM for human IL6 and 1.3 nM for cynomolgus IL6.

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