Antibodies that bind to VEGF-A and IL-6, and methods of use.

Abstract

This invention provides improved therapeutic antibodies that bind to VEGF and IL6 for clinical application in ocular diseases. [Solution] A bispecific anti-VEGF-A / anti-IL6 antibody in the form of a Fab fragment and a method for using the same are provided.

Description

[Technical Field]

[0001] The present invention relates to an anti-VEGF-A / anti-IL6 antibody and a method for using the same. [Background technology]

[0002] Antibodies that bind to VEGF, such as ranibizumab, are used as therapeutic agents for ophthalmic vascular diseases such as age-related macular degeneration. Antibodies that bind to IL-6, such as those disclosed in International Publication No. 2014 / 074905, have been suggested for the treatment of ophthalmic diseases.

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

[0004] In fact, improved therapeutic antibodies that bind to VEGF and IL6 are needed for clinical application in ocular diseases, for example, by improving efficacy over standard care, improving duration of action, and ultimately reducing the frequency of intravitreal injections and thus the burden on patients. [Overview of the project]

[0005] The present invention relates to a bispecific anti-VEGF-A / anti-IL6 antibody and a method for using the same.

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

[0007] One embodiment of the present invention relates to an antibody that binds to human VEGF-A and human IL-6, 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 IL-6.

[0010] One embodiment of the present invention relates to a bispecific antibody Fab fragment that binds to human VEGF-A and human IL-6.

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

[0012] In another embodiment, the present invention relates to an antibody that binds to human IL-6, a) A VH domain based on the human VH3 framework (IL6 paratope containing 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 (IL6 paratope containing amino acid residues Y49, D50, S53, N54, Y55, P56, S57, Y91, Y96); or b) We provide antibodies containing a VH domain based on the human VH3 framework (IL6 paratope containing 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 (IL6 paratope containing amino acid residues Y49, D50, D53, R54, Y55, P56, E57, Y91, Y96) (numbering follows Kabat).

[0013] In another embodiment, the present invention provides an antibody that 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. 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 embodiment, the present invention provides isolated nucleic acids encoding the antibody of the present invention.

[0015] In one embodiment, the present invention provides a host cell containing 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 Escherichia 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, and 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, and 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 embodiment, the present invention provides a method for inhibiting angiogenesis in an organism, comprising administering to the organism an effective amount of the antibody or pharmaceutical composition of the present invention for inhibiting angiogenesis.

[0027] The present invention provides a therapeutic anti-VEGF-A / anti-IL6 antibody that can independently bind to its target antigen, even when provided in the form of an antibody Fab fragment. It exhibits excellent KD and species cross-reactivity with cynomolgus monkey targets within a pharmacologically relevant range. The antibody of the present invention is suitable for the treatment of ocular vascular diseases. The antibody of the present invention offers several beneficial properties, including good expressibility and developmental potential (e.g., high binding efficacy, high biophysical and biochemical stability, high-concentration formulations), high affinity for both targets, particularly supporting low effective doses, and high stability advantageous over long periods. Compared to non-antibody approaches, the antibody of the present invention tends to be more tolerable due to its high humanity and lack of artificial domains and linkers. Furthermore, it is advantageous that the antibody of the present invention is provided in a high-concentration liquid formulation with viscosity suitable for ocular application. Because it can be provided in high concentrations, higher doses of the therapeutic agent can be applied in a single treatment, allowing for longer treatment cycles, thus making treatment with the antibody of the present invention more tolerable for patients. Bispecific Fab fragments, such as those described in this invention, have further advantages over bispecific full-length IgG antibodies due to their much lower molecular weight. While Fab has a molecular weight of approximately 50 kDa, the weight of a full-length antibody is three times higher (approximately 150 kDa) while providing the same number of binding sites. Therefore, for a given amount of drug, a bispecific Fab fragment contains three times more binding sites compared to a full-length IgG antibody. [Brief explanation of the drawing]

[0028] [Figure 1] The binding of parental bispecific antibodies 6HVL_1 and V6HL_1 to human and cynomolgus monkey IL6 is determined by surface plasmon resonance. [Figure 2]VEGF IC50 of parental bispecific antibodies 6HVL_1 and V6HL_1 using human VEGF-165 [Figure 3] Improved bispecificity of antibodies to human and cynomolgus monkey IL6, determined by surface plasmon resonance. [Figure 4] VEGF IC50 of improved bispecific antibodies using human VEGF-121 and VEGF-165. [Figure 5] Crystal structure of the Fab0182-IL-6 complex. Overall diagram of the structure of IL-6 bound to Fab 0182. IL-6 is colored salmon, and the light and heavy chains of Fab 0182 are colored cyan and blue, respectively. [Figure 6] Crystal structure of the Fab 6HVL4.1-IL-6 complex. Overall structure of IL-6 bound to Fab 6HVL4.1. IL-6 is colored salmon, and the light and heavy chains of Fab 6HVL4.1 are colored wheat and blue, respectively. [Figure 7] Simultaneous binding of anti-VEGF / anti-IL-6 Fab to its target is evaluated by SPR using immobilized anti-Fab antibodies. [Figure 8] Blocking of VEGF-R2 binding by anti-VEGF / anti-IL-6 Fab in the presence of IL-6, as evaluated by SPR using immobilized VEGF-A. [Figure 9] The effect of VEGF binding on IL-6 activity was evaluated as follows using a cell-based IL-6-specific reporter gene assay (without pre-incubation). [Figure 10] The effect of VEGF binding on IL-6 activity was evaluated as follows using a cell-based IL-6-specific reporter gene assay (with pre-incubation). [Figure 11] Inhibition of IL-6 signaling in HRMEC by 6HVL_4 [Figure 12] Dose-dependent changes in % inhibition of VEGF-A-induced HUVEC proliferation by 6HVL_4 [Figure 13]Recovery of IL6 / IL6R / VEGF-induced barrier destruction on HRMVEC by 6HVL_4 [Figure 14] Recovery of IL6 / IL6R / VEGF-induced barrier destruction on HRMVEC by Aflibercept [Figure 15] The amino acid sequences of the VH and VL domains of the antibody shown. Kabat numbering of amino acid positions is indicated. Amino acid positions contributing to the IL6 paratope identified in Example 13 are highlighted with black squares. [Figure 16] Image of the IL6 / IL6R / gp130 complex (top; pdb-acc.#1p9m) compared with a superposition of the structures of the IL6 / IL6R complex (bottom) bound to IL6 from Fab 0182 and pdb-acc.1p9m. [Figure 17] Image of the IL6 / IL6R / gp130 complex (top; pdb-acc.#1p9m) compared with a superposition of the structures of the IL6 / IL6R complex (bottom) bound to IL6 from Fab 6HVL4.1 and pdb-acc.1p9m. [Figure 18] SPR crosslinking experiments to investigate the ability of IL6R to bind to IL6 when engaged with a pre-formed complex with Fab P1AE2421. [Figure 19] SPR binding experiments to investigate the ability of Fab P1AE2421 to bind to human "hyper IL6" (a chimera of human IL6 and IL6R). [Figure 20] ELISA competitive experiment to determine the ability of Fab P1AE2421 to bind to IL6 in a manner that blocks the binding of IL6 to IL6R. [Figure 21] The binding of the IL6-conjugating antibody 6HdL2.05 to human and cynomolgus monkey IL6 is determined by surface plasmon resonance. [Modes for carrying out the invention]

[0029] 1.Definition Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have meanings generally understood by those skilled in the art. Furthermore, unless otherwise specifically required by context, singular terms shall include plural forms and plural terms shall include singular forms. The methods and techniques of this disclosure are generally carried out in accordance with 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 protein and nucleic acid chemistry, as well as hybridization, as described herein are well known and commonly used in the art.

[0030] Unless otherwise defined herein, the term “comprising of” includes the term “consisting of.”

[0031] As used herein in relation to a specific value (e.g., temperature, concentration, time, etc.), the term "approximately" refers to a variation of + / - 1% of the specific value to which the term "approximately" refers.

[0032] In this specification, the term "antibody" is used in its broadest sense and includes, but is not limited to, 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.

[0033] "Isolated" antibodies are those separated from components of their natural environment. In some embodiments, antibodies are purified to a purity of over 95% or over 99% as determined by 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 evaluating 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 substantially homogeneous population of antibodies, i.e., the individual antibodies constituting the population are identical and / or bound to the same epitope, except for variant antibodies that include, for example, naturally occurring mutations or variant antibodies that may arise during the production of a monoclonal antibody preparation, such variants generally present in trace amounts. In contrast to polyclonal antibody preparations, which typically contain different antibodies directed toward different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed toward a single determinant on one antigen. Therefore, the modifier “monoclonal” indicates the characteristic of an antibody obtained from a substantially homogeneous collection of antibodies and should not be interpreted as requiring antibody production by any particular method.

[0035] The terms “full-length antibody,” “intact antibody,” and “whole antibody” are used herein synonymously to refer to antibodies having a structure substantially similar to that of a natural antibody or having a heavy chain containing an Fc region as defined herein.

[0036] The “class” of an antibody refers to the type of constant domain or constant region held by its heavy chain. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into “subclasses” (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. In certain embodiments, the antibody is the IgG1 isotype. In certain embodiments, the antibody is the IgG1 isotype with P329G, L234A, and L235A mutations to reduce the effector function of the Fc region. In other embodiments, the antibody is the IgG2 isotype. In certain embodiments, the antibody is the IgG4 isotype with the S228P mutation in the hinge region to improve the stability of the IgG4 antibody. The heavy chain constant domains corresponding to different classes of immunoglobulins are 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) or 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 includes at least a portion of the constant region. This term includes both 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 known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

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

[0039] DutaFab is a bispecific antibody disclosed in International Publication No. 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 congeneral VH / VL pair and can bind to two different epitopes simultaneously. Methods for producing DutaFab and libraries containing single-specific Fab fragments by screening are disclosed in International Publication No. 2012 / 163520.

[0040] A "human antibody" is defined as an antibody produced by a human or human cell, or an antibody having an amino acid sequence corresponding to a non-human antibody that utilizes a sequence encoding a human antibody repertoire or other human antibodies. This definition of a human antibody explicitly excludes humanized antibodies containing non-human antigen-binding residues. Antibodies or antibody fragments isolated from a human antibody library are considered human antibodies or human antibody fragments in this specification.

[0041] The "Human Consensus Framework" is a framework representing 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 subgroups of variable domain sequences. Generally, the sequence subgroups are those described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In one embodiment, for VL, the subgroup is subgroup Kappa I in Kabat et al. (see above). In one embodiment, for VH, the subgroup is subgroup III as described in Kabat et al. above.

[0042] An "antibody fragment" is a molecule other than an intact antibody that contains 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] The terms "paratope" or "antigen-binding site" refer to a portion of an antibody that recognizes and binds to an antigen, and are used interchangeably herein. A paratope is formed by a plurality of distinct amino acid residues from the heavy and light chain variable domains of the antibody, which are spatially adjacent in the tertiary structure of the Fv region. The antibodies of the present invention contain two paratopes in one homologous VH / VL pair.

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

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

[0046] As used herein, the term “vascular endothelial growth factor,” abbreviated as “VEGF,” refers to any natural VEGF from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. 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 allele variants. An example 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 and has sufficient affinity to be useful as a diagnostic and / or therapeutic agent in targeting VEGF-A. In one embodiment, the degree of binding of the anti-VEGF-A antibody to unrelated non-VEGF-A proteins is less than approximately 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. D If present, 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 form of IL6 derived from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. The term encompasses “full-length” untreated IL6 and any form of IL6 resulting from intracellular processing. The term also encompasses naturally occurring variants of IL6, such as splice variants or allele variants. An exemplary human IL6 amino acid sequence is shown in SEQ ID NO: 28.

[0049] The antibody of the present invention "simultaneously binds to human VEGF-A and human IL-6," meaning that (a) the antibody Fab fragment of the present invention bound to human IL-6 specifically binds to human VEGF-A as well, and (b) the antibody Fab fragment of the present invention bound to human VEGF-A specifically binds to human IL-6 as well. Simultaneous binding can be evaluated by methods known in the art, for example, by surface plasmon resonance as described herein.

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

[0051] As used herein, "framework" or "FR" refers to amino acid residues in the variable domain other than the CDR residue. The framework of the variable domain generally consists of four framework domains: FR1, FR2, FR3, and FR4. Therefore, 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 sum of the strength of non-covalent interactions between a single binding site between 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 is generally expressed by the dissociation constant (K). D ) can be expressed by. Affinity can be measured by methods common in the art, including those described herein. Specific descriptive and exemplary embodiments for measuring binding affinity are described herein.

[0053] The term "epitope" refers to a site on an antigen, whether proteinaceous or nonproteinaceous, to which an antibody binds. Epitopes can be formed from a continuous amino acid stretch site (linear epitopes) or from discontinuous amino acids (structural epitopes), and are formed spatially close together, for example, due to the folding of the antigen (i.e., by the tertiary folding of a proteinaceous antigen). Linear epitopes are typically still bound to antibodies even after the proteinaceous antigen is exposed to a denaturing agent, while conformational epitopes are typically destroyed by treatment with a denaturing agent. Epitopes contain at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial structure.

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

[0055] Antigen structure-based antibody profiling (ASAP), also known as modification-assisted profiling (MAP), allows for the classification of numerous monoclonal antibodies that specifically bind to VEGF-A or IL-6 based on the binding profiles of each antibody to chemically or enzymatically modified antigen surfaces (see, e.g., US2004 / 0101920). Each classified antibody binds to the same epitope, which may be distinctly different from or partially overlapping epitopes represented by other classifications.

[0056] Furthermore, competitive binding can be used to easily determine whether an antibody binds to the same VEGF-A or IL6 epitope as the reference antibody of the present invention, or whether it competes for binding. For example, an "antigen that binds to the same VEGF-A and IL6 epitope 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, and conversely, the reference antibody blocks the binding of the antibody to that antigen by 50% or more in each competitive assay. Alternatively, 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 saturated conditions. After removing 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 saturated binding of the reference antibody, it can be concluded that the antibody in question binds to a different epitope than the reference antibody. However, if the antibody in question cannot bind to VEGF-A or IL-6 after saturated binding to the reference antibody, the antibody in question may bind to the same epitope to which the reference antibody binds. Conventional experiments can be used to determine whether the antibody in question is binding to the same epitope or whether binding is simply being hindered for steric reasons (e.g., peptide mutation analysis or binding analysis using ELISA, RIA, surface plasmon resonance, flow cytometry, or other quantitative or qualitative antibody binding assays available in the art). This assay should be performed in two setups, i.e., with both antibodies being saturated antibodies. If, in both setups, only the first (saturated) antibody can bind to VEGF-A or IL-6, then it can be concluded that the antibody in question and the reference antibody compete for binding to VEGF-A or IL-6.

[0057] In some embodiments, competitive binding assays measure that 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 more than 99%, then the two antibodies are considered to bind to the same or overlapping epitopes. (See, for example, 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 a “duplicate epitope” if only a subset of the amino acid mutations that reduce or eliminate the binding of one antibody also reduces or eliminates the binding of the other antibody.

[0059] The "amino acid sequence identity percentage (%)" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, after aligning the sequences for alignment purposes and introducing gaps if necessary to achieve the maximum sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for determining the amino acid sequence identity percentage 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 FASTA program packages. Those skilled in the art can determine appropriate parameters for sequence alignment, including any algorithm necessary to achieve the maximum alignment over the entire length of the sequences being compared. Alternatively, the identity percentage value can be generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and its source code is filed in the user documentation of the US Copyright Office (Washington DC, 20559), registered under US Copyright Registration No. TXU510087, and published in International Publication No. 2000 / 005319.

[0060] However, unless otherwise specified, for the purposes of this specification, amino acid sequence identity % values ​​are generated using the ggsearch program in FASTA package version 36.3.8c, or subsequently using the BLOSUM50 comparison matrix. The FASTA program package is described by WRPearson and DJLipman (1988), "Improved Tools for Biological Sequence Analysis" PNAS 85:2444-2448; WRPearson (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, you can use the ggsearch(global protein:protein) program with default options (BLOSUM50;open:-10;ext:-2;Ktup=2) to compare sequences using a public server accessible at fasta.bioch.virginia.edu / fasta_www2 / index.cgi, ensuring a global rather than local alignment. The amino acid identity percentage is given in the output alignment header.

[0061] The terms “nucleic acid molecule” or “polynucleotide” include any compound and / or substance containing a polymer of nucleotides. Each nucleotide is composed of a base, specifically a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. 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 base sequence is typically represented 5' to 3'. In this specification, the term nucleic acid molecule includes deoxyribonucleic acid (DNA), e.g., complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), in particular messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers containing two or more of these molecules. Nucleic acid molecules may be linear or cyclic. In addition, the term nucleic acid molecule includes both sense and antisense strands, as well as both single-stranded and double-stranded forms. Furthermore, nucleic acid molecules described herein may include naturally occurring 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 DNA 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, for example. 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 mRNA can be injected into a subject in vivo to produce an antibody. (See, for example, Stadler et al, Nature Medicine 2017, published online 12 June 2017, doi:10.1038 / nm.4356 or European Patent No. 2101823B1).

[0062] "Isolated" nucleic acids are nucleic acid molecules that have been separated from their natural environment. Isolated nucleic acids include nucleic acid molecules that are normally found inside cells, but these nucleic acid molecules exist outside of chromosomes or at chromosomal locations different from their natural chromosomal locations.

[0063] An “isolated nucleic acid encoding an antibody” refers to one or more nucleic acid molecules that encode the heavy and light chains (or fragments thereof) of an antibody, and such nucleic acid molecules are contained in a single vector or separate vectors, and such nucleic acid molecules are located at one or more positions in a host cell.

[0064] The term “vector” as used herein refers to a nucleic acid molecule capable of replicating another nucleic acid it is linked to. This term includes not only vectors as self-replicating nucleic acid structures, but also vectors that have been incorporated into the genome of a host cell into which they have been introduced. Certain vectors can direct the expression of a functionally linked nucleic acid. Such vectors are referred to herein as “expression vectors.”

[0065] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, and also include the offspring of such cells. Host cells include “transformers” and “transformed cells,” which include primary transformed cells and their offspring, regardless of passage number. Offspring may not have nucleic acid content that is exactly the same as that of the parent cells and may contain mutations. Mutant offspring having the same function or biological activity as those screened or selected in the original transformed cells are included herein.

[0066] The terms "pharmaceutical composition" or "pharmaceutical preparation" refer to a preparation in which the active ingredients contained therein are in a form that allows for effective biological activity, and which does not contain additional ingredients that are unacceptably toxic to the subject to which the pharmaceutical composition is to be administered.

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

[0068] The “effective amount” of a drug, such as a pharmaceutical composition, refers to the amount that is effective in the dosage and duration required to achieve the desired therapeutic or preventive outcome.

[0069] The “individual” or “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates, 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., “to treat” or “treating”) refers to a clinical intervention in an attempt to alter the original course of a disease in the treated individual, and may be performed for preventive purposes or during the course of a clinicopathological disease. Desired effects of treatment include preventing the onset or recurrence of the disease, reducing symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, achieving remission or mitigation of the condition, and achieving a restored or improved prognosis. In some embodiments, the antibodies of the present invention are used to delay the onset of the disease or to slow the progression of the disease.

[0071] As used herein, the term “ocular disease” includes any ocular disease associated with pathological neovascularization and / or atrophy. Ocular diseases can be characterized by altered or unregulated proliferation and / or infiltration of neovascularization into the structures 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 tubules). 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-associated retinopathy, ROP, and retina. This includes venous occlusion (RVO) (e.g., central (CRVO) and branched (BRVO) forms), cerebral vascular contractures (CNV) (e.g., myopic CNV), corneal neovascularization, diseases associated with corneal neovascularization, retinal neovascularization, diseases associated with retinal / choroidal neovascularization, central serous retinopathy (CSR), pathological myopia, von Hippel-Lindau disease, ocular histoplasmosis, FEVR, Coats' disease, Norley's disease, and osteoporotic pseudoglioma syndrome (OPPG). Related retinal abnormalities, subconjunctival hemorrhage, rubeosis, ocular neovascular disease, neovascular glaucoma, retinitis pigmentosa (RP), hypertensive retinopathy, retinal angiomatoid proliferation, macular telangiectasia, iris neovascularization, intraocular neovascularization, retinal degeneration, cystic macular edema (CME), vasculitis, papilledema, and retinitis including, but not limited to, CMV retinitis, ocular melanoma, retinoblastoma, conjunctivitis (e.g., infectious conjunctivitis and non-infectious conjunctivitis (e.g., allergic conjunctivitis) - Conjunctivitis, Leber congenital blepharitis (also known as Leber congenital blepharitis or LCA), uveitis (including infectious and non-infectious uveitis), choroiditis (e.g., multifocal choroiditis), ocular histoplasmosis, blepharitis, dry eye, traumatic eye injury, Sjögren's disease, and other eye diseases in which the disease or condition is associated with ocular neovascularization, vascular leakage, and / or retinal edema or retinal atrophy.Further exemplary eye diseases include retinoschisis (abnormal splitting of the retinal nerve sensory layer), diseases associated with rubeosis (neovascularization of the iridocorneal angle), and diseases caused by abnormal proliferation of fibrovascular tissue or fibrous tissue (including all forms of proliferative vitreoretinopathy). Exemplary conditions associated with corneal neovascularization include, but are not limited to, epidemic keratoconjunctivitis, vitamin A deficiency, contact lens overwear, atopic keratitis, limbal keratitis, pterygium, keratitis sicca, Sjögren's syndrome, acne rosacea, phylectenulosis, syphilis, microbacterial infections, steatosis, chemical burns, bacterial ulcers, fungal ulcers, herpes simplex infections, herpes zoster, protozoal infections, Kaposi's sarcoma, Mohren's ulcer, pericorneal degeneration of Terien, peripheral keratolysis, rheumatoid arthritis, generalized erythema, polyarteritis, trauma, Wegener's sarcoidosis, scleritis, Stevens-Johnson syndrome, bullous pemphigoid, radial corneal incision, and corneal transplant rejection. Exemplary diseases associated with choroidal neovascularization and defects of the retinal vascular system, including increased vascular leakage, aneurysms, and capillary detachment, include, but are not limited to, diabetic retinopathy, macular degeneration, sickle cell anemia, sarcoid, syphilis, pseudoxanthoma elastica, Paget's disease, venous occlusion, arterial occlusion, carotid artery occlusive disease, chronic uveitis / vitritis, mycobacterial infections, Lyme disease, systemic lupus erythematosus, retinopathy of prematurity, retinal edema (including macular edema), Eels' disease, Behçet's disease, infections causing retinitis or choroiditis (e.g., multifocal choroid), presumptive ocular histoplasmosis, Best's disease (vitreomacular degeneration), myopia, optic disc, squamous cellulitis, retinal detachment (e.g., chronic retinal detachment), hyperviscosity syndrome, toxoplasmosis, trauma, and post-laser complications. Exemplary diseases associated with atrophy of retinal tissue (photoreceptors and the underlying RPE) include, but are not limited to, atrophic or non-exudative AMD (e.g., geographic atrophy or progressive dry AMD), macular atrophy (e.g., atrophy associated with neovascularization and / or geographic atrophy), diabetic retinopathy, Stargardt disease, Sorsby Fundus dystrophy, retinoschisis, and retinitis pigmentosa.

[0072] The term “packaging insert” is used to refer to the instructions typically included in the commercial packaging of a therapeutic product, which contain information about the indications, use, dosage, administration, combination therapies, contraindications, and / or warnings regarding the use of such therapeutic product.

[0073] 2. Detailed description of embodiments of the present invention In one embodiment, the present invention is based in part on providing bispecific antibodies for therapeutic application. In a particular embodiment, antibodies that bind to human VEGF-A and human IL-6 are provided. The antibodies of the present invention are useful, for example, for the treatment of vascular diseases, such as ophthalmic diseases.

[0074] A. Exemplary antibodies that bind to human VEGF-A and human IL-6 In one embodiment, the present invention provides antibodies that bind to human VEGF-A and human IL-6. In one embodiment, isolated antibodies that bind to human VEGF-A and human IL-6 are provided. In one embodiment, the present invention provides antibodies that specifically bind to human VEGF-A and human IL-6.

[0075] In a particular embodiment, an antibody that binds to human VEGF-A and human IL-6 comprises a VEGF-A paratope (i.e., an antigen-binding site that binds to VEGF-A) and an IL-6 paratope (i.e., an antigen-binding site that binds to IL-6) within one congeneral pair of the VL domain and VH domain, ●VEGF-A paratopes contain amino acid residues derived from the antibody's CDR-H2, CDR-L1, and CDR-L3, and IL6 paratopes contain amino acid residues derived from the antibody's CDR-H1, CDR-H3, and CDR-L2; and / or ● The IL6 paratope contains amino acid residues derived from the antibody's CDR-H2, CDR-L1, and CDR-L3, and the VEGF-A paratope contains amino acid residues derived from the antibody's CDR-H1, CDR-H3, and CDR-L2; ●The pair of variable light chain domains and variable heavy chain domains simultaneously binds to human VEGF-A and human IL-6; and / or ● The antibody binds to the same human VEGF-A epitope and the same human IL-6 epitope 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 was (i) measured by surface plasmon resonance and had a K content of less than 50 pM. D (ii) It binds to human VEGF-A121, and is measured by surface plasmon resonance to a K of less than 50 pM. D Binds to human IL-6; and / or ● The antibody Fab fragment of the antibody exhibits an aggregation initiation temperature of 60°C or higher, and in one embodiment, 70°C or higher; and / or ● The antibody Fab fragment of the antibody exhibits a melting temperature exceeding 80°C, as measured by dynamic light scattering.

[0076] In another embodiment, the present invention relates to an antibody that binds to human VEGF-A and human IL-6, 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, ( (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) an antibody comprising 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 IL-6, comprising (a) a VH domain containing 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 containing 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 embodiment, the present invention provides an antibody that binds to human VEGF-A and human IL-6, comprising (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 22 having up to 15, up to 10, or up to 5 amino acid substitutions; and (b) a variable light chain domain comprising the amino acid sequence of SEQ ID NO: 21 having up to 15, up to 10, or up to 5 amino acid substitutions.

[0079] In another embodiment, the present invention provides an antibody that binds to human VEGF-A and human IL-6, comprising a VH domain comprising (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 18, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 19, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 20, and a VL domain comprising (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 15, (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17, wherein the antibody comprises (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 22 having up to 15, up to 10, or up to 5 amino acid substitutions; and (b) a variable light chain domain comprising the amino acid sequence of SEQ ID NO: 21 having up to 15, up to 10, or up to 5 amino acid substitutions.

[0080] In one embodiment, the present invention provides antibodies that bind to human VEGF-A and human IL-6, comprising a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 22. In a particular embodiment, the 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 compared to the reference sequence, but antibodies that bind to human VEGF-A and human IL-6, comprising that sequence, retain the ability to bind to human VEGF-A and human IL-6. In a particular embodiment, a total of up to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 22. In a particular embodiment, the substitutions, insertions, or deletions occur in the region outside the CDR (i.e., in the FR). In certain embodiments, VH includes a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 18, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 19, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 20.

[0081] In one embodiment, the present invention provides antibodies that bind to human VEGF-A and human IL-6, comprising a VL domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 21. In a particular embodiment, the 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 compared to the reference sequence, but antibodies that bind to human VEGF-A and human IL-6, comprising that sequence, retain the ability to bind to human VEGF-A and human IL-6. In a particular embodiment, a total of up to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 21. In a particular embodiment, the substitutions, insertions, or deletions occur in the region outside the CDR (i.e., in the FR). In certain embodiments, VL includes (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 15, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 16, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 17.

[0082] In another embodiment, an antibody is provided that binds to human VEGF-A and human IL-6, comprising a VH sequence from any of the embodiments provided above and a VL sequence from any of the embodiments provided above. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO: 22 and SEQ ID NO: 21, respectively, and includes post-translational modifications of these sequences.

[0083] In another embodiment, an antibody is provided that binds to human VEGF-A and human IL-6, 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 further embodiments of the present invention, the antibody that binds to human VEGF-A and human IL-6 according to any of the above embodiments is a monoclonal antibody. In one embodiment, the antibody that binds to human VEGF-A and human IL-6 is an antibody fragment, such as Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment. In another embodiment, the antibody is a full-length antibody.

[0085] In another embodiment, the present invention provides an antibody that binds to IL6 derived from the antibody of the present invention. The IL6 paratopes disclosed for the antibody of the present invention can be used to provide further antibodies, for example, monospecific antibodies or bispecific antibodies that bind to IL6 and another antigen. The IL6 paratopes of antibody 6HVL4.1 disclosed herein were 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. An antibody containing the IL6 paratopes of antibody 6HVL4.1 binds to the same epitope on IL6. All embodiments disclosed herein for the antibody of the present invention that binds to human VEGF-A and human IL6 also apply to antibodies that bind to IL6.

[0086] Therefore, in one embodiment, the present invention relates to an antibody that binds to human IL-6, c) A VH domain based on the human VH3 framework (IL6 paratope containing 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 (IL6 paratope containing amino acid residues Y49, D50, S53, N54, Y55, P56, S57, Y91, Y96); or d) We provide antibodies containing a VH domain based on the human VH3 framework (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 (IL6 paratope includes amino acid residues Y49, D50, D53, R54, Y55, P56, E57, Y91, Y96) (numbering follows Kabat).

[0087] In another embodiment, the present invention provides an antibody that 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 of the antibody that binds to human VEGF-A and IL6 of the present invention), 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 the antibody that binds to human VEGF-A and IL6 of the present invention).

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

[0089] In further embodiments, the antibodies that bind to human VEGF-A and human IL-6 according to any of the above embodiments, or the antibodies that bind to human IL-6 according to any of the above embodiments, may incorporate any of the features individually or in combination, as described in Sections 1 to 5 below.

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

[0091] In certain embodiments, 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 DThe antigen was measured using an assay with a KinExA 3200 instrument from Sapidyne Instruments (Boise, ID), and PMMA beads were coated with the antigen using 30 μg of anti-VEGF antibody MAB293 (R&D) in 1 ml of PBS (pH 7.4) according to the protocol (Adsorption coating, Sapidyne) in the KinExA handbook. 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 the sample 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 a KinExA system at a volume of 750 μl for 50 pM constant VEGF and at a volume of 125 μl for 500 pM constant VEGF. Detection of conjugated VEGFA-121 was performed using a 250 ng / ml secondary biotinylated anti-VEGF antibody (BAF293), followed by injection of a 250 ng / ml streptavidin Alexa Fluor® 647 conjugate in sample buffer. D This is obtained from a nonlinear regression analysis of the data using the one-site homogeneous coupling model included in the KinExA software (version 4.0.11) using the "Standard Analysis" method. D Calculate the data points and theoretically determine K D The 95% confidence interval is determined by fitting the curve. The 95% confidence interval is K D Low and K D It is given as high.

[0094] For example, the K of an antibody that binds to IL6 DThe protein is measured using a surface plasmon resonance (SPR) assay on a Biacore 8K instrument (Cytiva) at 25°C with HBS-EP+ (1x; BR100669; Cytiva) as the running buffer. Human Fab conjugate (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 tip using a standard amine coupling reaction. Before protein measurement, five initiation cycles are optionally performed for conditioning purposes, with each cycle regenerating the derivatized tip surface by flowing HBS-EP+ buffer for approximately 120 seconds, followed by application of 10 mM glycine buffer, pH 2.0, for 60 seconds. A 75 nM antibody Fab fragment is captured on this surface in HBS-EP+ buffer at a flow rate of 10 ul / min for 60 seconds. The Fab fragment is not applied to the reference channel. Next, human or cynomolgus monkey 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 fragments In certain embodiments, the antibodies provided herein are antibody fragments.

[0096] In one embodiment, the antibody fragment is a Fab, Fab', Fab'-SH, or F(ab')2 fragment, particularly a Fab fragment. Papain digestion of an intact antibody produces two identical antigen-binding fragments (so-called "Fab" fragments), each containing the variable domains of the heavy and light chains (VH and VL, respectively), as well as the constant domain (CL) of the light chain and the first constant domain (CH1) of the heavy chain. Therefore, the term "Fab fragment" refers to an antibody fragment containing a light chain with VL and CL domains, and a heavy chain fragment containing the VH and CH1 domains. A "Fab' fragment" is distinct from a Fab fragment by adding a residue at the carboxyl terminus of the CH1 domain containing one or more cysteines from the antibody hinge region. Fab'-SH is a Fab' fragment in which the cysteine ​​residue (multivalent) of the constant domain retains 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. For a description of the Fab and F(ab')2 fragments, which contain salvage receptor-binding epitope residues and have a longer in vivo half-life, see U.S. Patent No. 5,869,046.

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

[0098] In preferred embodiments, the antibody provided herein is a Fab fragment.

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

[0100] In one embodiment, the VL domain of the antibody 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 a human IgG1 isotype.

[0103] In preferred embodiments, the antibody provided herein is a Fab fragment comprising a κ isotype CL domain and a human IgG1 isotype CH1 domain.

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

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

[0106] Multispecific antibodies having three or more binding specificities, including the antibodies provided herein, may also be provided in an asymmetric form with domain crossovers in one or more binding arms of the same antigen specificity, i.e., by exchanging VH / VL domains (see, e.g., International Publication Nos. 2009 / 080252 and International Publication Nos. 2015 / 150447), CH1 / CL domains (see International Publication No. 2009 / 080253), or complete Fab arms (see International Publication Nos. 2009 / 080251, International Publication Nos. 2016 / 016299, Schaefer et al, PNAS, 108(2011)1187-1191, and Klein et al., MAbs 8(2016)1010-20). Various further molecular forms of multispecific antibodies are known in the art and are included herein (see, for example, Spiess et al., Mol Immunol 67(2015) 95-106).

[0107] 5. Antibody variants In certain embodiments, amino acid sequence variants of antibodies provided herein are intended. For example, it may be desirable to alter the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of antibodies may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from residues in the amino acid sequence of the antibody, and / or insertions into residues in the amino acid sequence of the antibody, and / or substitutions of residues in the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be carried out so as to reach the final construct, insofar as the final construct has 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 substitutional mutagenesis include CDRs and FRs. Conservative substitutions are shown in the following table under the heading "Preferred Substitutions." More substantial substitutions are provided in Table 1 under the heading "Exemplary Substitutions" and are further described below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into the antibody of interest, and the product can be screened for desired activity, e.g., retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC. [Table 1] TIFF2026062918000003.tif167161

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

[0110] Non-conservative substitution involves exchanging a member of one class with a member of another class.

[0111] Certain types of substitution variants involve substituting one or more CDR residues in a parent antibody (e.g., a humanized antibody or a human antibody). Generally, the resulting variant(s) selected for further study have altered (e.g., improved) certain biological properties (e.g., increased affinity, decreased immunogenicity) and / or substantially retain certain biological properties of the parent antibody compared to the parent antibody. Exemplary substitution variants are affinity-matured antibodies, which can be readily generated using, for example, phage display-based affinity maturation techniques as described herein. In short, one or more CDR residues are mutated, and the variant antibody displayed on a phage is screened for specific biological activity (e.g., binding affinity).

[0112] In certain embodiments, substitutions, insertions, or deletions may occur within one or more CDRs, provided that such alterations do not substantially reduce the antibody's ability to bind to the antigen. For example, conservative alterations that do not substantially reduce binding affinity (e.g., conservative substitutions as provided herein) may be made within a CDR. Such alterations may, for example, be outside the antigen-contact residue in the CDR. In the specific 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 antibody residues or regions that can be targeted for mutagenesis is called “alanine scanning mutagenesis,” as described by Cunningham and Wells (1989) Science, 244:1081-1085. This method identifies target residues or groups (e.g., charged residues such as arg, asp, his, lys, and glu) and substituted them with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the antibody-antigen interaction is affected. Further substitutions may be introduced at amino acid positions that exhibit functional sensitivity to the initial substitution. Alternatively, or additionally, the crystal structure of the antigen-antibody complex can be used to identify contact points between the antibody and antigen. Such contact residues and adjacent residues may be targeted as candidates for substitution or removed. Variants may be screened to determine whether they possess the desired properties.

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

[0115] a) Glycosylated 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 an antibody can be conveniently achieved by altering the amino acid sequence so that one or more glycosylation sites are created or removed.

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

[0117] In one embodiment, an antibody variant is provided having an oligosaccharide structure lacking a non-fucosylated oligosaccharide, i.e., fucose binding (direct or indirect) to the Fc region. Such a non-fucosylated oligosaccharide (also called "afucosylated" oligosaccharide) is an N-linked oligosaccharide that lacks a fucose residue to which a first GlcNAc is bound in the stem of a branched oligosaccharide structure. In one embodiment, an antibody variant is provided having an increased proportion of non-fucosylated oligosaccharides in the Fc region compared to the natural or parent 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 possibly about 100% (i.e., no fucosylated oligosaccharides are present). The proportion of non-fucosylated oligosaccharides is the (average) amount of fucose-less oligosaccharides relative to the total of all oligosaccharides bound to Asn297 (e.g., complex, hybrid, and high-mannose structures), as measured by MALDI-TOF mass spectrometry, for example, as described in International Publication No. 2006 / 082515. Asn297 refers to the asparagine residue located at approximately position 297 of the Fc region (EU numbering of Fc region residues); however, due to minor sequence changes in the antibody, Asn297 may be located upstream or downstream of position 297, i.e., approximately ±3 amino acids between positions 294 and 300. Such antibodies with an increased proportion of non-fucosylated oligosaccharides in the Fc region may have improved FcγRIIIa receptor binding and / or improved effector function, particularly improved ADCC function. See, for example, U.S. Patent Application Publication No. 2003 / 0157108; and No. 2004 / 0093621.

[0118] Examples of cell lines capable of producing antibodies with reduced fucosylation include Lec13CHO cells lacking 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, and knockout CHO cells (e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614-622 (2004); Kanda, Y. et al.). Examples include cells in which GDP-fucose synthesis or transporter protein activity is reduced or lost (see, for example, U.S. Patent Application Publications No. 2004259150, 2005031613, 2004132140, and 2004110282).

[0119] In a further embodiment, the antibody variant is provided with a bifurcated oligosaccharide, for example, in which a bifurcated oligosaccharide bound to the Fc region of the antibody is bifurcated by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function, as described above. Examples of such antibody variants are described, for example, in Umana et al., Nat Biotechnol 17, 176-180 (1999); Ferrara et al., Biotechn Bioeng 93, 851-861 (2006); International Publication No. 99 / 54342, International Publication No. 2004 / 065540, and International Publication No. 2003 / 011878.

[0120] Antibody variants are also provided that have at least one galactose residue in the oligosaccharide attached to the Fc region. Such antibody variants may have improved CDC function. Examples of such antibody variants are described, for example, in International Publications 1997 / 30087; 1998 / 58964; and 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 to create an Fc region variant. The Fc region variant may include a human Fc region sequence (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc region) containing amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0122] In certain embodiments, the present invention conceives antibody variants that, by possessing some, but not all, effector functions, are desirable candidates for applications where the in vivo half-life of the antibody is important, but certain effector functions (e.g., complement-dependent cell-mediated cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC)) are unnecessary or harmful. In vitro and / or in vivo cytotoxic assays can be performed to confirm the reduction / loss of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that the antibody lacks FcγR binding (and therefore is likely to lack ADCC activity) but retains FcRn binding ability. NK cells, the primary cells for mediating ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. The expression of FcR 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 evaluating the ADCC activity of the target molecule are described in U.S. Patent No. 5,500,362 (see, e.g., Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be used (e.g., ACTI® non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA), and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI)).Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, or in addition, the desired ADCC activity can be evaluated in vivo in animal models, such as those disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). Furthermore, a C1q binding assay may be performed to confirm that the antibody cannot bind to C1q and lacks CDC activity. See, for example, the C1q and C3c binding ELISAs in International Publication Nos. 2006 / 029879 and 2005 / 100402. To evaluate complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al., J.Immunol.Methods202:163(1996); Cragg, MS et al., Blood101:1045-1052(2003); and Cragg, MS and MJ Glennie, Blood103:2738-2743(2004)). Determination of FcRn binding and in vivo clearance / half-life can also be performed using methods known in the art (see, e.g., Petkova, S B et al., Int'l.Immunol.18(12):1759-1769(2006); WO2013 / 120929Al).

[0123] Antibodies with reduced effector function include those 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 variants include the so-called "DANA" Fc variant, which has substitutions at residues 265 and 297 of alanine, as well as Fc variants having substitutions at two or more amino acid positions 265, 269, 270, 297, and 327 (U.S. Patent No. 7,332,581).

[0124] Specific antibody variants exhibiting improved or decreased 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 includes an Fc region having one or more amino acid substitutions that improve ADCC, for example, substitutions at positions 298, 333, and / or 334 (EU numbering of residues) of the Fc region.

[0126] In certain embodiments, the antibody variant includes an Fc region having one or more amino acid substitutions that reduce FcγR binding, e.g., Fc region positions 234 and 235 (residues in EU numbering). In one embodiment, the substitutions are L234A and L235A (LALA). In certain embodiments, the antibody variant further includes 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) in 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) in the Fc region derived from the human IgG1 Fc region.

[0127] In some embodiments, 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), modifications occur within the Fc region that result in alterations (i.e., either enhancement or reduction) of C1q binding and / or complement-dependent cytotoxicity (CDC).

[0128] Antibodies that play a role in transferring maternal IgG to the fetus, with increased half-life and improved binding to the embryonic Fc receptor (FcRn), 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. Examples of such Fc variants include substitutions in one or more of the 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, substitutions in the Fc region residue 434 (see, for example, U.S. Patent No. 7,371,826; Dall'Acqua, WF, et al. J. Biol. Chem. 281 (2006) 23514-23524).

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

[0130] In certain embodiments, the antibody variant includes an Fc region having one or more amino acid substitutions that reduce FcRn binding, e.g., mutations at Fc region positions 253 and / or 310 and / or 435 (residues in EU numbering). In certain embodiments, the antibody variant includes an Fc region having amino acid substitutions at positions 253, 310, and 435. In one embodiment, the substitutions are I253A, H310A, and H435A within the 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 includes an Fc region having one or more amino acid substitutions that reduce FcRn binding, e.g., mutations at positions 310 and / or 433 and / or 436 (residues in EU numbering) of the Fc region. In certain embodiments, the antibody variant includes an Fc region having amino acid substitutions at positions 310, 433, and 436. In one embodiment, the substitutions are H310A, H433A, and Y436A in the Fc region derived from the human IgG1 Fc region. (See, for example, International Publication No. 2014 / 177460.)

[0132] In certain embodiments, the antibody variant includes an Fc region having one or more amino acid substitutions that increase FcRn binding, e.g., mutations at Fc region positions 252 and / or 254 and / or 256 (residues in EU numbering). In certain embodiments, the antibody variant includes an Fc region having amino acid substitutions at positions 252, 254, and 256. In one embodiment, the substitutions are M252Y, S254T, and T256E in the Fc region derived from 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 International Publication No. 94 / 29351.

[0133] The C-terminus of the heavy chain of an antibody as reported herein may 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 from which one or two C-terminal amino acid residues have been removed. In one preferred embodiment, the C-terminus of the heavy chain is PG ending with 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 contains a C-terminal glycine-lysine dipeptide (G446 and K447, EU index numbering of amino acid position). In one embodiment of all the embodiments reported herein, an antibody comprising a heavy chain containing the C-terminal CH3 domain as specified herein contains a C-terminal glycine residue (G446, EU index numbering of amino acid position).

[0134] c) Cysteine-modified antibody variant In certain embodiments, it may be desirable to produce cysteine-modified antibodies, such as THIOMAB®, in which one or more residues of the antibody are substituted with cysteine ​​residues. In certain embodiments, the substituted residues occur at easily accessible sites on the antibody. By substituting these residues with cysteine, the reactive thiol group is positioned at an accessible site on the antibody and can be used to conjugate the antibody to other sites, such as a drug site or a linker drug site, as further described herein. Cysteine-modified antibodies can be produced, for example, as described in U.S. Patents 7,521,541, 8,30,930, 7,855,275, 9,000,130, or International Publication No. 2016040856.

[0135] B. Recombination 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 antibodies are provided.

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

[0137] One embodiment provides a method for producing antibodies that bind to human VEGF-A and human IL-6, comprising culturing host cells containing nucleic acids encoding the antibodies under conditions suitable for antibody expression, and optionally recovering the antibodies from the host cells (or host cell culture).

[0138] With regard to the recombinant production of antibodies that bind to human VEGF-A and human IL-6, for example, the nucleic acids encoding the aforementioned antibodies are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to the genes encoding the heavy and light chains of the antibody), or they can be produced by recombinant methods or obtained by chemical synthesis.

[0139] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies may be produced in bacteria, particularly when glycosylation and Fc effector function are not required. For the expression of antibody fragments and polypeptides in bacteria, see, for example, US5,648,237, US5,789,199 and US5,840,523. (See also Charlton, KA, In: Methods in Molecular Biology, Vol.248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2003), pp.245-254, describing the expression of antibody fragments in Escherichia coli.) After expression, antibodies may be isolated from bacterial cell paste in appropriate fractions and further purified. In one embodiment, the host cell is an Escherichia coli cell.

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

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

[0142] C. Pharmaceutical Compositions In further embodiments, pharmaceutical compositions are provided, for example, for use in any of the following therapeutic methods, comprising one of the antibodies provided herein. In one embodiment, the pharmaceutical composition comprises one of the antibodies provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical composition comprises one of the antibodies provided herein and at least one additional therapeutic agent, for example, one of those described below.

[0143] The pharmaceutical compositions of antibodies conjugating to human VEGF-A and human IL-6 described herein are prepared by mixing such antibodies of the desired purity with one or more optionally available pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)) in the form of lyophilized compositions or aqueous solutions. Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the dosage and concentration used and include buffering agents such as histidine, phosphates, citrates, acetates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol, etc.); and low molecular weight (less than about 10 residues). Lipeptides; 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). Exemplary pharmaceutically acceptable carriers herein further include intervening drug dispersants such as soluble neutrally active hyaluronidase glycoproteins (sHASEGP), e.g., human soluble PH-20 hyaluronidase glycoproteins such as rHuPH20 (HYLENEX®, Halozyme, Inc.). Certain exemplary sHASEGP and methods of use, including rHuPH20, are described in U.S. Patent Application Publications 2005 / 0260186 and 2006 / 0104968.In one embodiment, sHASEGP is combined with one or more further glycosaminoglycans (e.g., chondroitinase).

[0144] An example of a lyophilized antibody composition is described in U.S. Patent No. 6,267,958. Examples of aqueous antibody compositions are described in U.S. Patent No. 6,171,586 and WO2006 / 044908, the latter of which comprises a histidine-acetate buffer.

[0145] The pharmaceutical compositions described herein may also include multiple active ingredients required for the specific symptom being treated, preferably those having complementary activities that do not adversely affect one another. Such active ingredients are preferably present in combination in amounts effective for the intended purpose.

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

[0147] Sustained-release pharmaceutical compositions can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing antibodies, where these matrices are in the form of molded articles, such as films or microcapsules.

[0148] Pharmaceutical compositions used for in vivo administration are generally sterile. Sterilization can be easily achieved, for example, by filtration using a sterile filtration membrane.

[0149] D. Treatment methods and routes of administration Any antibody provided herein that binds to human VEGF-A and human IL-6 may be used in therapeutic methods.

[0150] In one embodiment, antibodies conjugating to human VEGF-A and human IL-6 are provided for use as pharmaceuticals. In further embodiments, antibodies conjugating to human VEGF-A and human IL-6 are provided for use in the treatment of vascular disease. In certain embodiments, antibodies conjugating to human VEGF-A and human IL-6 are provided for use in a treatment method. In certain embodiments, the present invention provides antibodies conjugating to human VEGF-A and human IL-6 for use in a method of treating an individual having vascular disease, comprising administering an effective amount of the antibody conjugated to human VEGF-A and human IL-6 to the individual. In such one embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent (e.g., one, two, three, four, five, or six additional therapeutic agents) to the individual, as described below, for example. In further embodiments, the present invention provides antibodies conjugating to human VEGF-A and human IL-6 for use in inhibiting angiogenesis. In certain embodiments, the present invention provides an antibody that binds to human VEGF-A and human IL-6 for use in a method of inhibiting angiogenesis in an organism, comprising administering an effective amount of the antibody that binds to human VEGF-A and human IL-6 to inhibit angiogenesis to the organism. The "organism" in any of the above embodiments is preferably human.

[0151] In a further embodiment, antibodies conjugating to human VEGF-A and human IL-6 are provided for use in the treatment of eye diseases. In one embodiment, the eye diseases are 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-associated retinopathy, ROP, reticular retinopathy. In one embodiment, membranous vein occlusion (RVO) (central (CRVO) and branched (BRVO) forms), cerebral vascular contracture (CNV) (in one embodiment, myopic CNV), corneal neovascularization, diseases associated with corneal neovascularization, retinal neovascularization, diseases associated with retinal / choroidal neovascularization, central serous retinopathy (CSR), pathological myopia, von Hippel-Lindau disease, ocular histoplasmosis, FEVR, Coats' disease, Norley's disease, osteoporotic pseudoglioma syndrome (OPPG) Related retinal abnormalities, subconjunctival hemorrhage, rubeosis, ocular neovascular disease, neovascular glaucoma, retinitis pigmentosa (RP), hypertensive retinopathy, retinal angiomatoid proliferation, macular telangiectasia, iris neovascularization, intraocular neovascularization, retinal degeneration, cystic macular edema (CME), vasculitis, papilledema, and retinitis including, but not limited to, CMV retinitis, ocular melanoma, retinoblastoma, conjunctivitis (in one embodiment, infectious conjunctivitis and non-infectious conjunctivitis (in one embodiment, allergic conjunctivitis) - Conjunctivitis), Leber congenital necrosis (also known as Leber congenital necrosis or LCA), uveitis (including infectious and non-infectious uveitis), choroiditis (in one embodiment, multifocal choroiditis), ocular histoplasmosis, blepharitis, dry eye, traumatic eye injury, Sjögren's disease, and other ocular diseases in which the disease or condition is associated with ocular neovascularization, vascular leakage, and / or retinal edema or retinal atrophy. In one embodiment, the ocular disease is selected from AMD (in one embodiment, 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).

[0152] In further embodiments, the present invention provides the use of antibodies conjugated to human VEGF-A and human IL-6 in the manufacture or preparation of a pharmaceutical. In one embodiment, the pharmaceutical is for the treatment of a vascular disease. In further embodiments, the pharmaceutical is for use in a method of treating a vascular disease, which includes administering an effective amount of the pharmaceutical to an individual having a vascular disease. In such an embodiment, the method further includes administering an effective amount of at least one additional therapeutic agent, e.g., one of those described below, to the individual.

[0153] In one embodiment, the pharmacopoeia is for the treatment of an eye disease. In a further embodiment, the pharmacopoeia is for use in a method of treating an eye disease, which includes administering an effective amount of the pharmacopoeia to an individual having an eye disease. In such an embodiment, the method further includes administering an effective amount of at least one additional therapeutic agent, for example, one of those described below, to the individual.

[0154] In further embodiments, the present invention provides a method for treating vascular disease. In one embodiment, the method comprises administering to an individual having such vascular disease an effective amount of antibodies that bind to human VEGF-A and human IL-6. In such an embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, as described below.

[0155] In further embodiments, the present invention provides a method for treating an eye disease. In one embodiment, the method comprises administering to an individual having such an eye disease an effective amount of antibodies that bind to human VEGF-A and human IL-6. In such an embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, as described below.

[0156] An "individual" in any of the above-described manner may be a human being.

[0157] In further embodiments, the present invention provides a pharmaceutical composition comprising one of the antibodies conjugating to human VEGF-A and human IL-6 provided herein, for use, for example, in any of the therapeutic methods described above. In one embodiment, the pharmaceutical composition comprises one of the antibodies conjugating to human VEGF-A and human IL-6 provided herein, and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical composition comprises one of the antibodies conjugating to human VEGF-A and human IL-6 provided herein, and at least one additional therapeutic agent, for example, described below.

[0158] The antibody of the present invention may be administered intravitreously (e.g., by intravitreous injection) or using a port delivery device. In one embodiment, the antibody of the present invention is administered using a port delivery device for a period of 6 months or more, in one embodiment 8 months or more, in one embodiment 9 months or more, and in one embodiment 12 months or more, before the port delivery device is refilled. In one embodiment, the antibody of the present invention is 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 antibody of the present invention may be administered alone or used in combination therapy. For example, combination therapy may include administering the antibody of the present invention and administering at least one additional therapeutic agent (e.g., additional therapeutic agents 1, 2, 3, 4, 5, or 6).

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

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

[0162] Any suitable AMD treatment includes VEGF antagonists, e.g., 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., falisimab; Roche)), soluble VEGF receptor fusion proteins (e.g., EYLEA® (aflibercept)), anti-VEGF DARPin® (e.g., abiscipal pegol; Molecular Partners) AG / Allergan), or anti-VEGF aptamers (e.g., MACUGEN® (pegaptanib sodium); platelet-derived growth factor (PDGF) antagonists, e.g., anti-PDGF antibodies, anti-PDGFR antibodies (e.g., REGN2176-3), anti-PDGF-BB pegylated aptamers (e.g., FOVISTA®; Opthotech / Novartis), soluble PDGFR receptor fusion proteins, or dual PDGF / VEGF antagonists (e.g., small molecule inhibitors (e.g., DE-120 (Santen) or X-82 (TyrogeneX)) or bispecific anti-PDGF / anti-VEGF antibodies)); VI in combination with photodynamic therapy SUDYNE® (verteporfin); antioxidants; complement system antagonists, e.g., complement factor C5 antagonists (e.g., small molecule inhibitors (e.g., ARC-1905; Optotech) 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., lamparizumab; Roche)); C3 blocking peptides (e.g., APL-2, Appellis); visual cycle modifiers (e.g., emixustat hydrochloride); squalamine (e.g., OHR-102; Ohr Pharmaceutical); vitamin and mineral supplements (e.g., Age-Related Eye Disease Study 1 (AREDS1; zinc and / or antioxidants) and Study 2 (AREDS2;(as listed in the text) (zinc, antioxidants, lutein, zeaxanthin, and / or omega-3 fatty acids); cell-based therapies, e.g., NT-501 (Renexus); PH-05206388 (Pfizer), huCNS-SC cell transplantation (StemCells), CNTO-2476 (umbilical cord stem cell line; Janssen), OpRegen (RPE cell suspension; Cell Cure Neurosciences), or MA09-hRPE cell transplantation (Ocata Therapeutics); tissue factor antagonists (e.g., hI-con1; Iconic These can be administered as additional therapeutic agents in combination with antibodies that bind to human VEGF and human IL6, provided herein for the treatment of eye diseases (e.g., AMD, DME, DR, RVO, or GA), including but not limited to: Therapeutics; α-adrenergic receptor agonists (e.g., brimonidine tartrate; Allergan); peptide vaccines (e.g., S-646240; Shionogi); amyloid-β antagonists (e.g., anti-β-amyloid monoclonal antibodies, e.g., GSK-933776); S1P antagonists (e.g., anti-S1P antibodies, e.g., iSONEP®; Lpath Inc); ROBO4 antagonists (e.g., anti-ROBO4 antibodies, e.g., DS-7080a; Daiichi Sankyo); lentiviral vectors expressing endostatin and angiostatin (e.g., RetinoStat); and any combination thereof. In some cases, AMD therapeutic agents (including any of the aforementioned AMD therapeutic agents) can be co-formulated. For example, the anti-PDGFR antibody REGN2176-3 can be co-formulated with aflibercept (EYLEA®). In some cases, such co-formulations can be administered in combination with the antibodies conjugating to human VEGF and human IL-6 of the present invention. In some cases, the ocular disorder is AMD (e.g., exudative AMD).

[0163] Any suitable DME and / or DR therapeutic agent, including, but not limited to, VEGF antagonists (e.g., LUCENTIS® or EYLEA®), corticosteroids (e.g., corticosteroid implants (e.g., OZURDEX® (dexamethasone intravitreal implant) or ILUVIEN® (fluocinolone acetonide intravitreal implant)) or corticosteroids formulated for administration by intravitreal injection (e.g., triamcinolone acetonide)), or combinations thereof, may be administered in combination with the antibodies conjugating to human VEGF and human IL6 of the present invention for the treatment of ocular disorders (e.g., AMD, DME, DR, RVO, or GA). In some examples, the ocular disorders are DME and / or DR.

[0164] Antibodies that bind to human VEGF and human IL6 provided herein are, for example, used in laser photocoagulation (e.g., panretinal photocoagulation (PRP)), drusen laser oscillation, macular hole surgery, macular translocation surgery, implantable miniature telescopes, PHI motor angiography (also known as microlaser therapy and feeder vascular treatment), proton beam therapy, microstimulation therapy, retinal detachment and vitrectomy, scleral buckling, submacular surgery, transpapillary thermotherapy, photosystem I therapy, use of RNA interference (RNAi), extracorporeal reophagesis (also known as membrane fractionation filtration and reotherapy), microchip implantation, stem cell therapy, gene replacement therapy, ribozyme gene therapy (including hypoxia-responsive element gene therapy, Oxford Biomedica; Lentipak, Genetix; and PDEF gene therapy, GenVec), photoreceptor / retinal cell transplantation (including transplantable retinal epithelial cells, Diacrin, Inc.; retinal cell transplantation, e.g., Astellas Pharma US, Inc., ReNeuron, CHA) It may be administered in combination with therapeutic or surgical procedures for the treatment of eye disorders (e.g., AMD, DME, DR, RVO, or GA), including biotech, puncture, and combinations thereof.

[0165] Such combination therapies described above encompass combined administration (where two or more therapeutic agents are contained 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 may be performed simultaneously with and / or prior to the administration of additional therapeutic agents or drugs. In one embodiment, the administration of the antibodies of the present invention that bind to human VEGF and human IL6 and the administration of additional therapeutic agents are performed within about 1, 2, 3, 4, or 5 months from each other, or within about 1, 2, or 3 weeks, or within about 1, 2, 3, 4, 5, or 6 days.

[0166] The antibodies (and any additional therapeutic agents) of the present invention may be administered by any suitable means, including parenteral, intrapulmonary, intranasal, and, if desired for topical treatment, intralesional administration. Parenteral administration includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Dosage may be by any preferred route, e.g., injection, such as intravenous or subcutaneous injection, depending in part whether the administration is short-term or long-term. Various dosing schedules, including but not limited to single doses, multiple doses over various time points, bolus administration, and pulse infusion, are contemplated herein.

[0167] The antibodies of the present invention will be formulated, administered, and given in a manner consistent with good medical practice. Factors to be considered in this regard include the specific disorder being treated, the specific mammal being treated, the clinical symptoms of the individual patient, the cause of the disorder, the site of drug delivery, the method of administration, the administration schedule, and other factors known to the healthcare professional. The antibodies will be formulated, optionally but not necessarily, with one or more drugs currently used to prevent or treat the disorder in question. The effective amount of such other drugs will depend on the amount of antibody present in the pharmaceutical composition, the type of disease or treatment, and the other factors mentioned above. These will generally be used by the same dosages and routes of administration as described herein, or at about 1–99% of the dosages described herein, or by any dosage and route that is empirically / clinically deemed appropriate.

[0168] For the prevention or treatment of 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 preventive or therapeutic purposes, previous therapies, the patient's medical history and response to the antibody, and the discretion of the attending physician. The antibody of the present invention is preferably administered to the patient in a single dose or over a series of treatments. Depending on the type and severity of the disease, an antibody of about 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg to 10 mg / kg) may be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate doses or by continuous infusions. A typical daily dose may range from about 1 μg / kg to 100 mg / kg, depending on the factors mentioned above. In repeated administrations over several days or more, treatment is usually continued, depending on the symptoms, until the desired suppression of disease symptoms occurs. One exemplary dosage of antibody would range from approximately 0.05 mg / kg to approximately 10 mg / kg. Therefore, one or more doses of approximately 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, or 10 mg / kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, for example, weekly or every three weeks (for example, so that the patient receives approximately 2 to approximately 20 doses of antibody, or for example, approximately 6 doses). A higher loading dose may be administered first, followed by one or more lower doses. The progress of this therapy is readily monitored by conventional techniques and assays.

[0169] E.Manufactured products In another aspect of the present invention, a product is provided comprising materials useful for treating, preventing and / or diagnosing the aforementioned disorders. The product comprises a container and a label or accompanying document on or associated with the container. Suitable containers include, for example, vials, syringes, etc. The container may be formed from a variety of materials, such as glass or plastic. The container holds the composition, which is used alone or in combination with another composition effective for treating, preventing and / or diagnosing the symptoms, and may have a sterile access port (for example, the container may be a vial with a stopper that can be punctured by an intravenous solution bag or a subcutaneous injection needle). At least one activator in the composition is an antibody of the present invention. The label or accompanying document indicates that the composition is used to treat a selected symptom.

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

[0171] F. Equipment The antibody of the present 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 capable of releasing a therapeutic agent (e.g., the antibody of the present invention) over several months (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or longer). Exemplary port delivery devices that may be used include, for example, those from ForSight Labs, LLC and / or ForSight VISION4, as described in International Patent Application Publications 2010 / 088548, 2015 / 085234, 2013 / 116061, 2012 / 019176, 2013 / 040247 and 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 and in fluid communication with the reservoir, and one or more outlets configured to be in fluid communication with the reservoir and to release the composition into the eye. The tubular body may have an outer diameter configured to be inserted through an incision or opening of the eye of about 0.5 mm or less. The device may be about 1 mm to about 15 mm in length (e.g., about 1 mm, about 2 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 9 mm, about 11 mm, about 13 mm, or about 15 mm in length). The reservoir may have any suitable volume. In some cases, the reservoir may have 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 apparatus 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 one of the antibodies described herein and one or more additional compounds.

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

[0176] 3. Specific Embodiments of the Invention The following lists specific embodiments of the present invention.

[0177] 1. An antibody that binds to human VEGF-A and human IL-6, comprising a VH domain comprising (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 18, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 19, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 20, and a VL domain comprising (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 15, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 16, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 17, (a) (b) an antibody comprising a VH domain containing 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 sequence number 22; and a VL domain containing 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 sequence number 21.

[0178] 2. An antibody that binds to human VEGF-A and human IL-6, comprising a VH domain containing (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 18, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 19, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 20, and a VL domain containing (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 15, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 16, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 17, and a variable heavy chain domain containing the amino acid sequence of SEQ ID NO: 22 with up to 5 amino acid substitutions; and a variable light chain domain containing the amino acid sequence of SEQ ID NO: 21 with up to 5 amino acid substitutions.

[0179] 3. An antibody that binds to human VEGF-A and human IL-6, comprising (a) a VH domain containing the amino acid sequence of SEQ ID NO: 22 having up to 15, up to 10, or up to 5 amino acid substitutions; and (b) a variable light chain domain containing the amino acid sequence of SEQ ID NO: 21 having up to 15, up to 10, or up to 5 amino acid substitutions.

[0180] 4. An antibody that binds to human VEGF-A and human IL-6, containing the VH sequence of SEQ ID NO: 22 and the VL sequence of SEQ ID NO: 21.

[0181] 5. An antibody according to one of the prior 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. An antibody according to any one of the prior 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 ● A pair of variable light chain domains and variable heavy chain domains simultaneously bind to human VEGF-A and human IL-6; and / or ● The antibody binds to the same human VEGF-A epitope and the same human IL-6 epitope 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 (i) was measured by surface plasmon resonance and had a K content of less than 50 pM. D (ii) It binds to human VEGF-A121, and is measured by surface plasmon resonance to a K of less than 50 pM. D Binds to human IL-6; and / or ● The antibody Fab fragment of the antibody exhibits an aggregation initiation temperature of 60°C or higher, and in one embodiment, 70°C or higher; and / or ●Antibodies in which the antibody Fab fragment 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 IL-6, containing 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. An antibody described in any one of the prior embodiments, which is a Fab fragment.

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

[0186] 10. An antibody described in any one of the prior embodiments, which is a monoclonal antibody.

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

[0188] 12. An antibody according to any one of the prior embodiments, wherein the antibody Fab fragment of the antibody exhibits a melting temperature above 80°C as measured by dynamic light scattering.

[0189] 13. An antibody described in any one of the prior embodiments, which is a monoclonal antibody.

[0190] 14. An antibody according to any one of the prior embodiments, which is an antibody fragment that binds to human VEGF-A and human IL-6.

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

[0192] 16. An antibody described in any one of the prior embodiments, which is a Fab fragment.

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

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

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

[0196] 20. An antibody according to any one of the prior embodiments, which specifically binds to human IL-6.

[0197] 21. An antibody that binds to human IL6, which binds to the same epitopes on IL6 as the 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 IL-6, wherein the antibody comprises a VH domain having a human VH3 framework (the IL-6 paratope includes amino acid residues 1, 2, 3, 26, 27, 28, 29, 30, 31, 32, 52a, 94, 96, 97, 98, 101, 102 of the antibody that binds to human VEGF-A and IL-6 described in any one of Embodiments 1 to 20), and a VL domain having a human Vkappa1 framework (the IL-6 paratope includes amino acid residues 49, 50, 53, 54, 55, 56, 57, 91, 96 of the antibody that binds to human VEGF-A and IL-6 described in any one of Embodiments 1 to 20).

[0199] 23. An antibody that binds to human IL-6, a) A VH domain based on the human VH3 framework (IL6 paratope containing 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 (IL6 paratope containing amino acid residues Y49, D50, S53, N54, Y55, P56, S57, Y91, Y96); or b) Antibodies containing a VH domain based on the human VH3 framework (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 (IL6 paratope includes amino acid residues Y49, D50, D53, R54, Y55, P56, E57, Y91, Y96) (numbering follows Kabat).

[0200] 24. An isolated nucleic acid encoding an antibody as described in any of Embodiments 1 to 23.

[0201] 25. A host cell containing the nucleic acid of Embodiment 24.

[0202] 26. A method for producing antibodies that bind to human VEGF-A and human IL-6, comprising culturing host cells as described in Embodiment 25 so that antibodies are produced.

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

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

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

[0206] 30. Antibodies described in any one of Embodiments 1 to 23 for use as pharmaceuticals

[0207] 31. The method according to Embodiment 26, further comprising recovering antibodies from host cells.

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

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

[0210] 34. An antibody according to any one of Embodiments 1 to 23 for use as a pharmaceutical.

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

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

[0213] 37. Use of an antibody described in any one of Embodiments 1 to 23 or a pharmaceutical composition described in Embodiment 65 in the manufacture of a pharmaceutical product.

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

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

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

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

[0218] 42. A port delivery device comprising an antibody according to any of Embodiments 1 to 23 or a pharmaceutical formulation according to Embodiment 33.

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

[0220] 44. An antibody according to any of Embodiments 1 to 23 or a pharmaceutical formulation according to Embodiment 33 for ocular administration by a port delivery device according to Embodiment 42, for a period of 6 months or more, 8 months or more in one embodiment, and 9 months or more in one embodiment, prior to the port delivery device being replenished.

[0221] 45. A pharmaceutical preparation according to any of Embodiments 1 to 23 or Embodiment 33, for use as a pharmaceutical by administering an antibody or pharmaceutical preparation using a port delivery device, wherein the antibody is applied to the port delivery device at a concentration of 150 mg / ml or higher, and in one embodiment at a concentration of 200 mg / ml or higher. [Table 2] TIFF2026062918000005.tif220161 TIFF2026062918000006.tif220161 TIFF2026062918000007.tif212161 TIFF2026062918000008.tif211161 TIFF2026062918000009.tif208161 TIFF2026062918000010.tif227161 TIFF2026062918000011.tif233161 TIFF2026062918000012.tif197161 TIFF2026062918000013.tif112161 [Examples]

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

[0223] Example 1: Bispecific anti-VEGF / anti-6 Fab fragment generation 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. 2012 / 163520.

[0224] Here, two different phage display libraries of the synthetic Fab fragment were used. In the first phage display library, residues within the CDR-H1, CDR-H3, and CDR-L2 regions of the Fab fragment were diversified, while in the second phage display library, 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, representing a paratope capable of binding to VEGF-A, in contrast to the method of International Publication No. 2012 / 163520, which used an immutable, non-binding germ cell-like ("dummy") sequence.

[0225] In the case of the first library, the paratopes capable of binding to VEGF-A were derived from the VEGF-A-binding paratopes described in International Publication No. 2021 / 198034.

[0226] In the case of the second library, the VEGF-A bound paratope was obtained as follows: For naive selection, phage library panning was performed using a library diversified with CDR-H1, CDR-H3, and CDR-L2 as described in International Publication No. 2012 / 163520. The remaining CDR sequences were kept constant using unbound germ cell-like sequences. In four rounds, the first round was performed using 100 nM biotinylated VEGF-121 or VEGF-165 pre-immobilized on Dynabeads M-280 streptavidin (Thermofisher catalog no. 11206D). Rounds 2-4 of panning were performed using 75, 15, and 3 nM biotinylated targets in solution, respectively, followed by capture of Fab-on-phage / target complexes on Dynabeads M-280 streptavidin. The phage / target / bead complexes were washed multiple times with PBST and PBS buffer. Capture phage clones with target-specific Fab were eluted from M-280 beads using 100 mM DTT according to a standard protocol, used to infect log-phase TG1 E. coli cells, and rescued using M13 K07 helper phage.

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

[0228] Subsequently, the binding agent was expressed, purified in larger volumes, and its binding to VEGF was evaluated using SPR measurement. One of the resulting clones was further optimized by repeated protein manipulation and testing strategies and incorporated into a phage display library as the invariant sequences CDR-H1, CDR-H3, and CDR-L2. In short, the protein manipulation workflow consisted of an initial series of scout mutations to identify relevant beneficial mutations, followed by two consecutive rounds of affinity maturation based on oligonucleotide-based generation and phage display-based selection of the mutant library, and then subsequent screening and further testing.

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

[0230] Phage library panning was used to enrich each library for human IL-6 binding agents. After panning, plasmid minipreps were prepared for both enrichment pools of phagemid vectors. The minipreps were digested with restriction enzymes to excise the region encoding the cleaved gene-III protein, and re-circularized by ligation to obtain a pool of expression vectors encoding soluble Fab fragments enriched with IL-6 binding agents. These vector pools were transformed into TG1 E. coli cells, individual colonies were harvested, and individual Fab clones were cultured for soluble expression in microtiter plates. The supernatant containing the soluble Fab fragments was screened for binding to IL-6 and VEGF-A using a standard ELISA method.

[0231] Based on 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 from each library, respectively, that co-encode a single bispecific Fab fragment that specifically binds to both IL-6 and VEGF-A: 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 obtained vectors were transformed into TG1 E. coli cells as described in Example 1, and individual colonies were cultured for both 6HVL_1 and VH6L_1 for soluble expression of the bispecific antibody Fab fragments. The bispecific antibodies were purified from the TG1 culture supernatant by affinity chromatography. The binding of the bispecific antibodies 6HVL_1 and VH6L_1 to IL-6 from human and cynomolgus monkey IL-6, human VEGF121, and human VEGF165 was evaluated.

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

[0234] SPR analysis of the binding of IL-6 to human and cynomolgus monkey-derived Fab fragments was performed on a Biacore 8K instrument (Cytiva) at 25°C using HBS-EP+ (1x; BR100669; Cytiva) as the running buffer. Human Fab conjugate (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 tip using a standard amine coupling reaction. This immobilization procedure yielded a ligand density of approximately 5000 resonance units (RUs). The reference channel was then processed accordingly.

[0235] Before 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 application of 10 mM glycine buffer pH 2.0 for 60 seconds. A 75 nM Fab fragment was captured on this surface in HBS-EP+ buffer at a flow rate of 10 ul / min for 60 seconds. The Fab fragment was not applied to the reference channel. Subsequently, human or cynomolgus monkey IL-6 was applied in an appropriate dilution series in HBS-EP+ buffer at a flow rate of 30 ul / min (contact time 180 seconds, dissociation time 720 seconds). Regeneration of the derivatized chip surface was achieved as described above. The data were evaluated using 8K evaluation software (Biacore Insight Evaluation 3.0). Raw data were fitted using a 1:1 binding model with dual references.

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

[0237] The inventors observed clear binding of 6HVL_1 and VH6L_1 to human IL-6. Only VH6L_1 showed significant affinity to cyIL-6, albeit at a clearly very rapid offrate. No binding to the negative control target IL-1a was observed. The results of fitting the SPR data are shown in Table 1. The data were averaged across three experiments, and the standard deviation is provided for the dissociation constant KD. For 6HVL_1, an affinity with KD = 0.9 nM was observed, while for VH6L_1, the affinity with KD = 10.7 nM was observed. [Table 3]

[0238] VEGF binding evaluated by competitive ELISA: Competitive ELISA experiments using 6HVL_1 and VH6L_1 were performed to test the antibody concentrations required to block the interaction between VEGF121 and VEGF165 and their receptors. VEGF-binding Fab fragment (ranibizumab) was used as a positive control, and experiments using buffer alone were used as a negative control. Briefly, a 1:3 dilution series of all samples, starting at 20 nM, was 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 Maxisorp plates coated with VEGF receptor 1 (VEGF-R1, R&D Systems, 1 μg / ml in NaHCO3, pH 9.4) after blocking the Maxisorp plate surface with 2% MPBST. 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 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 by utilizing the chromogenic conversion of the HRP substrate 3,3',5,5'-tetramethylbenzidine TMB to 3,3',5,5'-tetramethylbenzidinediamine, followed by a change in absorbance at 450 nm. TMB was preheated to room temperature, incubated on the plate for 5 minutes, and then quenched with 1N H2SO4.

[0239] The results for targeted VEGF165 are shown in Figure 2 and Tables 5 and 6. Clearly, both VH6L_1 and 6HVL_1 exhibit a much improved ability to compete for 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-IL6Fab fragments As described above, both antibodies showed no or low cross-reactivity with cynomolgus monkey IL-6, which is desirable for clinical development. In addition, treatment of ocular vascular disease requires injection of therapeutic agents into the eye, and therefore, the optimal therapeutic agent should exhibit high affinity and high concentration for the target antigen to maximize the duration of therapeutic effect and patient convenience. Thus, it is desirable to further improve the initially identified molecules for the intended purpose.

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

[0242] Improved candidate antibodies 6HVL_2, 6HVL_3, and 6HVL_4, as well as VH6L_2 and VH6L_3, were selected from multiple tested candidate antibody molecules from maturation in each round. Candidate selection was based on improvements in desired properties, particularly human IL-6 binding and cynomolgus monkey IL-6 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 several tested candidate antibody molecules. [Table 4]

[0244] All Fab fragments contained the same constant regions as those found in the full-length light and heavy chain amino acid sequences of the antibody VH6L_4, namely CL with SEQ ID NO: 29 and CH1 with 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 for 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 relative to the prior art, the following controls were used: bispecific antibody VH6L (VH / VL sequences disclosed in International Publication No. 2012 / 163520, referred to herein as "VH6L-BM"), anti-VEGF antibody ranibizumab (INN), and an anti-IL6 antibody (positive control) that is cross-reactive between human and cynomolgus monkey IL6 as disclosed in International Publication No. 2014 / 074905. The aforementioned prior art antibodies were prepared by recombinant expression.

[0247] Figure 3 and Tables 3 and 4 show the results of the 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 evaluated by competitive ELISA using human VEGF121 and human VEGF165. Figure 4 exemplifies 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] TIFF2026062918000020.tif40170

[0249] Example 6: Simultaneous binding of anti-VEGF / anti-IL-6 Fab fragments The simultaneous binding of the antibodies of the present invention to these targets was evaluated by surface plasmon resonance using immobilized anti-Fab antibodies to capture the anti-VEGF / anti-IL-6 Fab fragments of the present invention, as follows:

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

[0251] Anti-VEGF / anti-IL-6 Fab fragments were captured via kappa chains by injecting a 10 μg / mL solution at a flow rate of 5 μL / min for 30 seconds to form anti-Fab antibody / anti-VEGF / anti-IL-6 Fab complexes. Both human VEGFA121 (in-house produced, P1AA1779-010) and human IL-6 (commercial, Peprotech#200-06) antigens were added sequentially or simultaneously to enable the formation of complexes containing anti-Fab antibody, anti-VEGF / anti-IL-6 Fab, human VEGFA, and human IL-6. The corresponding SPR response unit curves (Biacore T200, Cytiva) were monitored. For sequential binding, human VEGFA at a concentration of 300 nM was injected for 180 seconds, followed by a further injection of human IL-6 at a concentration of 300 nM for 180 seconds. The same concentrations were also injected in the reverse order (human IL-6 first, followed by human VEGFA). Similarly, mixtures of both antigens were injected at a concentration of 300 nM for 180 seconds. After each experiment, the surface was regenerated by injecting 10 mM glycine (pH 2.1) at a flow rate of 5 μL / min for 60 seconds. Differences in bulk refractive index were corrected by subtracting blank injections and responses obtained from control flow cells that did not capture Fab.

[0252] The results are shown in Figure 7. Addition of human VEGF-A to the anti-Fab / anti-VEGF / anti-IL-6 Fab complex resulted in binding and formation of the anti-Fab / Fab / VEGF-A complex. Sequential addition of human IL-6 resulted in the formation of the anti-Fab / DutaFab / VEGF-A / IL-6 complex (dashed curve). This clearly demonstrates 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 human VEGF-A, a significant reduction in co-binding was observed (dotted line). This indicates that the binding of human IL-6 initially sterically interferes with the binding of human VEGF-A, resulting in a reduction in the binding ability between anti-VEGF / anti-IL-6 Fab and human VEGF-A, although binding is still possible.

[0254] In the presence of both targets, human IL-6 binding appeared preferred, and binding to human VEGF-A was reduced (solid line). This effect was reasonable due to the inherently higher affinity of anti-VEGF / anti-IL-6 Fab for human IL-6 compared to human VEGF-A.

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

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

[0257] For reference, VEGFA and VEGFR2 inhibition were tested using a 1:2 dilution series of 0–200 nM anti-VEGF / anti-IL-6 Fab fragments in 50 nM human VEGFA solution. The anti-VEGF / anti-IL-6 Fab fragment / VEGFA mixture was injected at a flow rate of 5 μL / min for 30 seconds onto an immobilized VEGFR2 surface. 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. Differences in bulk refractive index were corrected by subtracting the blank injection and the response obtained from the blank control flow cell. For evaluation, the binding response was obtained 5 seconds after the end of injection. The induced response at RU was translated into the binding response compared to the initial signal corresponding to the ligand(s) without bispecific Fab. IC50 values ​​were calculated using a 4-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 fragments were pre-incubated for 15 minutes in a solution containing 10 nM human IL-6, tested, and their IC50 values ​​were calculated (Figure 3).

[0259] The results are shown in Figure 8. The graph shows the inhibition of 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 increasing the concentration of anti-VEGF / anti-IL-6 Fab increased the inhibition (solid crosshairs). The addition of human IL-6, which mimics 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 (dashed triangle), IC50 = 37 nM with additional human IL-6 (black dashed triangle)).

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

[0261] To evaluate the simultaneous binding of human VEGFA and human IL-6 to 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. 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 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 activates 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 gave a calculated IC of 1.134 ng / mL (approx. 22.5 pM). 50The study showed clear dose-response curves with values, demonstrating clear inhibition of the human IL-6 response by gradually increasing doses of anti-VEGF / anti-IL-6 Fab. To address the simultaneous binding of human IL-6 and VEGFA to bispecific Fab, both target molecules were incubated simultaneously, and the effect of human IL-6 was measured. Regardless of the selected ratio of human VEGFA:human IL-6 (1:1 / 2.5:1 / 5:1), effective IC50 was observed. 50 Only a slight decrease in the value was observed. The value was the IC in the absence of human VEGFA. 50 =1.134 ng / mL, IC when human VEGF-A is present in a 5-fold excess. 50 It changed slightly to 1.724 ng / mL, a situation that closely reflects in vivo-related conditions.

[0264] Figure 10 shows pre-incubation results demonstrating that IL6 binding does not affect IL6 binding.

[0265] Example 7: Binding of anti-VEGF / anti-IL-6 Fab fragments to IL-6 and proposed mode of action determined by X-ray crystallography. IL-6 signaling is initiated by the formation of a hexameric complex between IL-6 and its non-signaling co-receptor IL6R and cytokine receptor gp130. Here, three epitopes (sites 1, 2, and 3) are defined to identify the contact surfaces formed within the complex (Boulanger MJ et al., Science 2003, 27;300(5628):2101-4). IL-6 initially binds to IL-6R via an interaction surface called "site 1". "Site 2" is an epitope formed by the two-component complex of IL-6 and IL-6R, which interacts with domains 2 and 3 of gp130. Subsequent interaction between "site 3" of IL-6 and domain 1 of gp130 leads to the formation of a dimer of the IL6 / IL6R / gp130 trimer, and thus the formation of a hexameric signaling complex.

[0266] To understand which epitopes of IL-6 are bound by our two series of Fab(6HVL and VH6L), we performed structural analysis of the complexes of IL-6 and antibody Fab, representative of the anti-VEGF / anti-IL-6 Fab fragments of the present invention. The used Fab 6HVL4.1 is very closely related to Fab 6HVL_2, differing by only two mutations, and Fab 0182 is most closely related to Fab VH6L_1. Given that all 6HVL clones originate from the same Fab(6HVL_1), and similarly all VH6L clones originate from Fab VH6L_1, it can be safely assumed that the structural results obtained below are applicable across each of the individual series of Fab. The formation of the Fab-IL-6 complex and the 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).

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

[0269] For Fab fragment 0182, needle-shaped crystals appeared within two days when mixed with 0.1M MgCl2, 0.1M sodium citrate (pH 5), and 15% (w / v) PEG 4000. Subsequently, these crystals were used in seeding experiments, and large tetragonal crystals were obtained from 0.1M calcium acetate, 12% (w / v) PEG 8000, 0.1M sodium cacodylate (pH 5.5).

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

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

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

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

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

[0275] The data collection and refined statistics are summarized in Table 7. All graphs were prepared using PYMOL (The Pymol Molecular Graphics System, Version 1.7.4, Schrodinger, LLC). [Table 9] TIFF2026062918000022.tif167170

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

[0277] Figure 16 shows the configuration of Fab 0182 being connected to IL6. For explanatory purposes, the inventors generated superpositions of two structures: firstly, a superposition of the composite structure of Fab and IL6; and secondly, a superposition of the composite structure of IL6R and IL6 (obtained from the cocrystal structure of IL6, IL6R, and gp130 having pdb accession code 1p9m (see Boulanger MJ et al., Science 2003, 27;300(5628):2101-4)). Comparing this to the trimer complex of IL6, IL6R, and gp130, it is clear that Fab binds to IL6 in a manner very similar to gp130, i.e., to site 2 of IL6. This binding mode is expected to allow simultaneous binding of both Fab and IL6R to IL6, i.e., interaction between IL6 and IL6R should still be possible, and such an IL6 antagonist is expected to act a priori 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 Fab 6HVL4.1 complex with IL-6 at a resolution of 1.94 Å (Figure 6). This structure shows Fab 6HVL4.1 bound to IL-6 by the main contributions of CDR1 and CDR3 of the heavy chain and CDR2 and CDR3 of the light chain. Further interaction with IL-6 is maintained by the first three N-terminal residues of the heavy chain of Fab 6HVL4.1. The interface to which IL-6 contributes is formed by the residues of helix A and helix C.

[0279] Similar to what was done with 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 manner very similar to gp130, and therefore, from a structural point of view, it must be considered a site 2 binder.

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

[0281] In one such assay, a Fab fragment (antibody "P1AE2421") representing a clone of the 6HVL series, including 6HVL4.1, was captured on the surface of an SPR chip via an anti-Fab antibody, and IL6 and subsequently IL6R were flowed onto the chip surface at three different concentrations (250, 500, and 1000 nM). Here, we anticipate a two-step, sequential signal increase if IL6 can bind to both Fab and IL6R. Indeed, this was observed for the Fab tested (Figure 18): in the SPR signal trace, the addition of IL6 resulted in a strong signal increase, which was further enhanced after the addition of IL6R. This clearly demonstrates that simultaneous binding of IL6R and IL6 to Fab is possible. This finding is further supported by the fact that a covalent chimera of IL6 and IL6R (referred to as "hyperIL6"), inaccessible due to site 1 of IL6 being completely shielded, still facilitates binding to each Fab molecule when coated onto an SPR tip and probed with Fab at a concentration of 26 nM (Figure 19).

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

[0283] Given that available crystal structures and SPR experiments indicate that site 1 of IL6 is still accessible for binding, we must interpret these results to mean that the IL6 antibody described in this patent can not only sterically block the binding of the IL6 / IL6R complex to gp130, but can also allosterically reduce the binding affinity of IL6 to IL6R, i.e., act more functionally as an IL6 site 1 antagonist.

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

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

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

[0287] 3. When IL6 site 1 conjugates are used systemically as antibodies, they are known to result in a significant increase in the half-life of the complex compared to IL6 alone, leading to robust accumulation of the IL6-antibody complex. In contrast, IL6 site 2 conjugates are expected to still allow for the removal of the IL6 / antibody complex by binding to membrane-bound IL6R and subsequently internalizing and degrading the complex within cells. In this regard, the IL6 antagonists described herein are expected to combine the desirable properties of both site 1 and site 2 conjugates: functionally they can block the first step in the formation of the IL6 / IL6R / gp130 signaling complex, while still allowing for the degradation of the IL6 / mAb complex via IL6R binding on cells.

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

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

[0290] The thermal stability of the 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 when excited with a 266 nm laser were recorded using an UNcle (Unchained Labs) instrument. The sample was measured in a triple-cuvette array.

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

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

[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 materials shown.

[0295] Viscosity evaluation: Equipment and materials Wyatt DLS plate reader with Greiner Bio-One microplates • 3000 Series Nanosphere (trademark) size specifications (Thermofisher catalog number 3300A) • Tween20 (Roche, catalog number 11332465001) and silicone oil, e.g. (Alfa Aesar catalog number A12728) • A UV photometer (e.g., Nanodrop 8000) for determining the concentration.

[0296] Sample preparation The antibody sample was rebuffered and diluted with 20 mM His / HCl, pH 5.5 (buffer) and 0.02% Tween 20 (final concentration). A bead concentration of 0.03% solids was added. At least three different concentrations were prepared, with the highest concentration possible being approximately 200 mg / mL. Two blank samples were needed as controls without antibody: 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 using a Wyatt DLS plate reader. All samples and blanks were analyzed at different temperatures from 15°C to 35°C in 5°C steps. The acquisition time was 30 seconds, and the number of acquisitions was 40 per sample and temperature.

[0298] Data Analysis The raw Dapp (apparent radius) data in nm is shown in the overview of the software template (Microsoft Dynamics 7.10 or later). 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 viscosity is shown as an Excel curve. It is possible to extrapolate viscosity to a given concentration using Mooney curve fitting (Excel). Here, the maximum protein concentration when the viscosity exceeds 20 cP was calculated.

[0299] The maximum antibody concentration shown to achieve a viscosity of 20 cP at 20°C is as follows. [Table 12]

[0300] The results demonstrate that the antibody of the present invention can be formulated at high concentrations containing a viscosity below an acceptable viscosity limit for injectability. As a result, the antibody of the present invention is highly suitable for ophthalmic applications because it allows for the delivery of high molar doses in a limited injection volume, which, when combined with high potency, results in high durability and consequently reduces the frequency of administration, which is desirable for improving patient convenience and treatment compliance.

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

[0302] Cell culture: HRMEC (catalog number PEL-PB-CH-160-8511; PELOBiotech GmbH; Bayern, Germany) was thawed and cultured in a 175 cm² flask in endothelial basal medium (EBM) (catalog number CC-3156; Lonza; Basel, Switzerland) and endothelial growth medium (EGM-MV) containing 5% fetal bovine serum (FBS), hydrocortisone, human fibroblast growth factor B, VEGF, R3-IGF-1 (a recombinant analog of insulin-like growth factor-I with Glu substituted with Arg at position 3), 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. 24 hours after plating, the medium was replaced with fresh EGM-MV, and the cells were grown for an additional 3 days before the assay. Assay conditions were optimized across different passages and concentrations of IL-6 / soluble IL-6R. The final assay was performed using 6th passage HRMEC and equimolar stimulation with 2 nM IL-6 / soluble IL-6R.

[0303] Flow cytometry assay: HRMEC cells were removed from the flask by washing twice with phosphate-buffered saline (PBS) (catalog no. 10010023; Life Technologies) that does not contain Ca2+ and Mg2+, and once with the cell dissociation reagent Accutase (catalog no. A1110501; Thermo Fisher Scientific; Waltham, MA). After washing, 5 mL of cell dissociation reagent was added to the cells, and the flask was incubated in a 5% CO2 incubator at 37°C for 3 minutes. The separated cells were collected from the flask and placed in a 50 mL conical centrifuge tube. The tube was filled with 50 mL of EBM containing 2% FBS and centrifuged at 300 g for 6 minutes. The supernatant was discarded, and the pellet was resuspended in 5 mL of starvation medium (EBM containing 2% FBS). Cell counts were quantified using a TC20 automated cell counter (Bio-Rad; Hercules, CA) and adjusted to 300,000 cells / mL using starvation medium. Then, 100 μL of the cell suspension was added to each well of a Costar 96-well plate (catalog number 3596; Corning; Corning, NY) to obtain 30,000 cells / well. The plates were then incubated for a further 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 in 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 of 6HVL_4 (3-fold, 7 dilutions) 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 both unstimulated cells and cells stimulated with the IL-6-I / L-6R complex without 6HVL_4. The cells were incubated in a 5% CO2 incubator at 37°C for 72 hours.

[0305] For the analysis of ICAM-1 surface expression, cells were treated with PBS (Ca 2+ Mg 2+The cells were washed twice with Life Technologies and once with the cell dissociation reagent Accutase (catalog no. A1110501; Thermo Fisher Scientific). Cells were detached from the plate using 50 μL of cell dissociation reagent (3 min, 37°C) and transferred to a flow cytometry Falcon 96-well Storage plate (catalog no. 353263; Corning). The original wells were washed once with 100 μL of PBS containing 2% FBS and 2 mM EDTA, and the washing medium containing the remaining cells was added to the flow cytometry plate. The cells were pelletized by centrifugation at 300 g for 6 minutes, and the supernatant was discarded. The pellet was resuspended in 100 μL of PBS, 2% FBS, and 2 mM EDTA containing 10 μg / mL human IgG (catalog no. I2511; MilliporeSigma; Burlington; MA) to block nonspecific 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 mixture was incubated at 28°C for 45 minutes. After staining, the cells were pelletized 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 was performed in a quadruple sequence in all independent experiments. For each experiment, the mean signal for each condition was calculated by subtracting the background signal of unstimulated cells from the background signal of the experimental well. The 100% response level was calculated from cells stimulated with IL-6 / IL-6R without 6HVL_4, and then the inhibitory activity of 6HVL_4 was expressed as the inhibition rate of the 100% response. The inhibition rates for each concentration of 6HVL_4 were measured in three independent experiments, and the mean and SEM were calculated. Using the mean of the three independent experiments, the mean IC50 and SE were calculated using ExcelXLfit software version 5.5.0 (IDBS; Guildford, UK). The concentration-response curves were fitted by nonlinear regression analysis using a 5-parameter logistic model (A + ((BA) / (1 + (((BE)*((C / x)^D)) / (EA))))) calculated for basal and maximal inhibitory activity. [Table 13]

[0307] 6HVL_4 produced dose-dependent inhibition of IL-6 signaling in HRMEC 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: HUVEC was obtained from Lonza (catalog number 00191027; Basel, Switzerland). Endothelial basal medium (EBM-2; catalog number CC-3156) and 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] T175 cell culture flasks (catalog number 353112; Corning; Corning, NY) coated with adhesion factor (AF) (catalog number S-006-100; Gibco, Thermo Fisher Scientific; Waltham, MA) were used to maintain HUVEC. Cells were detached using StemProAccutase (catalog number A11105-01; Gibco).

[0310] Cell viability / proliferation assays were performed using alamarBlue (catalog number DAL1100; Invitrogen, Thermo Fisher Scientific) on 96-well fibronectin-coated plates (catalog number 354409; Corning).

[0311] Recombinant human VEGF-A was obtained from R&D (catalog number 293-VE; Minneapolis, MN), Ca 2+ and Mg 2+ It was dissolved in phosphate-buffered saline (PBS) (catalog number 14190-094; Gibco) without the phosphate buffer at a stock concentration of 100 μg / mL.

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

[0313] HUVEC cells were maintained for up to 5 passages in EBM-2 in AF-coated T175 flasks. For viability assays, HUVEC cells 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 fibronectin-coated 96-well plates to obtain a cell density of 10,000 cells / well. Cells were not seeded in the outer wells, which were then filled only with assay medium. The cells were incubated overnight at 37°C in a 5% CO2 incubator.

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

[0315] The 6HVL_4 stock solution was also diluted in assay medium to prepare a 10-fold working solution. This was used to prepare an 8-step 3-fold dilution series starting at 30,000 ng / mL and ending at 14 ng / mL.

[0316] Next, 12.5 μL of 10-diluted 6HVL_4 solution and 12.5 μL of 10-VEGF-A solution (750 ng / mL) were added sequentially in four wells 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 a final concentration range of 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 Molecular Devices FlexStation 3 plate reader.

[0317] Data analysis: For each experiment, each condition was performed in a series of four. A total of four independent experiments were conducted. Independent experiments were considered by processing two separate plates on the same day. Therefore, eight separate plates were used for analysis. The average signal for each condition was calculated by subtracting the background signal of unstimulated cells from the background signal of the experimental well. The 100% response level was calculated from cells stimulated with VEGF-A (75 ng / mL) without additional compound exposure, and the signal from the 6 HVL_4 exposed wells was expressed as the inhibition rate of the 100% response.

[0318] IC50 values ​​were calculated from the average data for each antibody concentration using ExcelXLfit software version 5.5.0 (IDBS; Guildford, UK). Concentration-response curves were fitted by nonlinear regression analysis using a 4-parameter logistic model (A + ((BA) / (1 + ((C / x)^D)))) calculated for basal and maximal inhibitory activity. Data are presented as mean values ​​from four independent experiments using standard error of the mean (SEM). [Table 14]

[0319] 6HVL_4 reduced VEGF-A-induced HUVEC proliferation, with a 50% inhibitory concentration (IC50) of 2.06+ / -0.30 nM (Figure 12). Example 12: The restoration of barrier function in the presence of both VEGF-A and IL-6, as well as the VEGF / IL-6 bispecific antibody 6HVL_4, demonstrates the biological activity of the molecules. To evaluate the dual biological activity of the antibody of the present invention, namely the simultaneous blockade of both targeted cytokines—VEGF-A and IL6 (in complex with IL6R)—a transendothelial cell resistance (TER) assay was performed. In this assay, an electrically dense endothelial cell layer responds to the addition of both VEGF-A and IL6 by loss of barrier function. The restoration of 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 (PELOBiotech; catalog number PEL-PB-CH-160-8511), also known as HRMVECs, were maintained for up to 5 passages in complete MV endothelial cell growth medium (MV-EGM-2 Lonza, catalog number CC-3202) in T175 flasks (Falcon Cat#353112) coated with adhesion factor (Ginco, catalog number S-006-100). For transendothelial cell resistance assays, cells were detached using StemPro® Accutase® (Gibco, catalog number A11105-01). Subsequently, cells were seeded at a cell density of 120,000 cells / well in 100 μl of MV-EGM-2 growth medium in the upper chamber of a fibronectin-coated (Corning catalog number 354008) 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. Subsequently, the medium was 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 hours, during which time TER was measured by cellZcope. The following day, cells were treated with a combination of VEGF (R&D Systems, catalog number 293-VE / CF) at a final concentration of 10 ng / ml, IL6 (R&D Systems, catalog number 206-IL / CF) at 50 ng / ml, IL6R (R&D Systems, catalog number 227-SR-025 / CF) at 100 ng / ml, and VEGF at 10 ng / ml, or with an equal volume of assay medium (8 times per condition), and TER was measured until the next day. Subsequently, 6HVL_4 or aflibercept or assay medium at final concentrations of 1 μg / ml and 2.3 μg / ml, respectively, were added to the cells, and TER was measured for the next 24 hours. Thus, each condition was performed four times.

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

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

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

[0324] Example 13: Identification of the IL6 paratope region From the crystal structure of the 6HVL4.1-IL6 complex, amino acid residues in contact with IL6 were identified. An example of the positions of paratope amino acid residues within the VH and VL domains is shown in Figure 15. For this purpose, the PyMOL "byres" function and a 5-angstrom cutoff distance 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 48-215 of IL6 (defined by Uniprot number P05231). These residues are known to be normally degraded in the structure of IL6 alone (see pdb accession numbers 1alu and 1IL6). Figure 15 also shows the alignment between 6HVL4.1 and the monospecific anti-IL6 antibody 6HdL2.05 based on the antibody of the present invention, where the VEGF-paratope is replaced by an unbound region. 6HdL2.05 has the VH domain of SEQ ID NO: 48 and the VL domain of SEQ ID NO: 47. When the antibody was expressed and purified as described in Example 2 and subjected to an SPR assay using human IL6 or cynomolgus monkey IL6 as in Example 3, the antibody showed an SPR sensorgram as shown in Figure 20. After fitting the experimental data, 6HdL2.05 showed affinity comparable to the highest affinity obtained for the corresponding 6HVL series VEGF / IL6 bispecific antibody (see Tables 3 and 4), with a fitting KD of 22 pM for human IL6 and 1.3 nM for cynomolgus monkey IL6.

[0325] The amino acid residues identified as contributing to antigen binding are shown 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 Figures 1+5). The amino acid positions involved in antigen binding are identified by their Kabat positions within the VH or VL domains. [Table 15]

Claims

1. An antibody that binds to human VEGF-A and human IL-6, comprising a VH domain including (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 18, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 19, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 20, and a VL domain including (d) CDR-L1 containing the amino acid sequence of SEQ ID NO: 15, (e) CDR-L2 containing the amino acid sequence of SEQ ID NO: 16, and (f) CDR-L3 containing the amino acid sequence of SEQ ID NO: 17, and a variable heavy chain domain including the amino acid sequence of SEQ ID NO: 22 having up to five amino acid substitutions; and a variable light chain domain including the amino acid sequence of SEQ ID NO: 21 having up to five amino acid substitutions.

2. The antibody according to claim 1, wherein the aforementioned up to five amino acid substitutions occur in the FR region of each variable domain.

3. The antibody according to claim 1 or 2, comprising the VH sequence of SEQ ID NO: 22 and the VL sequence of SEQ ID NO:

21.

4. The antibody according to one of claims 1 to 3, comprising the heavy chain amino acid sequence of SEQ ID NO: 24 and the light chain amino acid sequence of SEQ ID NO:

23.

5. An antibody that binds to human VEGF-A and human IL-6, containing the VH sequence of SEQ ID NO: 22 and the VL sequence of SEQ ID NO:

21.

6. An antibody that binds to human IL-6, which binds to the same epitopes on IL-6 as the antibody having the VL domain of SEQ ID NO: 35 and the VH domain of SEQ ID NO:

36.

7. The antibody according to any one of claims 1 to 6, wherein the antibody is a Fab fragment.

8. The antibody according to any one of claims 1 to 7, wherein the antibody is a bispecific antibody fragment.

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

10. A host cell containing the nucleic acid described in claim 9.

11. A method for producing antibodies that bind to human VEGF-A and human IL-6, comprising culturing the host cells described in claim 10 so as to produce the antibodies.

12. The method according to claim 11, wherein the host cell is a CHO cell.

13. A pharmaceutical preparation comprising an antibody according to any one of claims 1 to 8 and a pharmaceutically acceptable carrier.

14. A port delivery device comprising the antibody according to any one of claims 1 to 8.

15. An antibody according to any one of claims 1 to 8, for use as a pharmaceutical.

16. A port delivery device comprising an antibody according to any one of claims 1 to 8 or a pharmaceutical preparation according to claim 13.

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