Antibodies binding to VEGF-a and il6 and methods of use

Abstract

The present invention relates to anti-VEGF-A / anti-IL6 antibodies, for example in the form of bispecific Fab fragments, and methods of use thereof.

Description

Technical Field

[0001] The present invention relates to anti-VEGF-A / anti-IL6 antibodies and methods of using the same. Background Art

[0002] Antibodies that bind to VEGF (e.g., ranibizumab) are used as therapeutic agents for treating ocular vascular diseases such as age-related macular degeneration. Antibodies that bind to IL6 (e.g., as disclosed in WO2014 / 074905) have been suggested for treating ocular diseases.

[0003] WO2012 / 163520 discloses bispecific antibodies comprising two paratopes in a pair of VH and VL domains ("DutaFab"). Each paratope of the bispecific antibody of WO2012 / 163520 comprises amino acids from the heavy chain and from the light chain CDR, wherein the heavy chain CDR-H1 and CDR-H3 and the light chain CDR-L2 contribute to the first paratope, while the light chain CDR-L1 and CDR-L3 and the heavy chain CDR-H2 contribute to the second paratope. Monospecific antibodies comprising each paratope are independently isolated from different Fab libraries, and the Fab libraries are diversified in the first or second paratope. The amino acid sequences of the monospecific antibodies are identified and merged into VH and VL pairs of biparatopes. An exemplary Fab fragment specifically binding to VEGF and IL-6 is disclosed in WO2012 / 163520 as a proof of concept example, the fragment is called "VH6L" and has a VL sequence of SEQ ID NO:01 and a VH sequence of SEQ ID NO:02.

[0004] There is a clear need for improved therapeutic antibodies that bind to VEGF and to IL6 for clinical use in ocular diseases, for example, by improving efficacy compared to standard of care and by improving duration of action and subsequently reducing the frequency of intravitreal injections so that the administration burden on patients is reduced. Summary of the invention

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

[0006] In one aspect, the present invention relates to an antibody that binds to human VEGF-A and to human IL6, the antibody comprising: a VH domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 18, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 19, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 20; and a VL domain comprising: (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 15, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17; the antibody comprises: a variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 22 having up to 5 amino acid substitutions; and a variable light chain domain comprising the amino acid sequence of SEQ ID NO: 21 having up to 5 amino acid substitutions.

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

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

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

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

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

[0012] In another aspect, the present invention provides an antibody that binds to human IL6, the antibody comprising:

[0013] a) A VH domain based on the human VH3 framework, wherein the IL6 paratope comprises the amino acid residues

[0014] Y1, I2, Q3, Y26, E27, F28, T29, H30, Q31, D32, P52a, R94, I96, D97, F98, D101, T102, and a VL domain based on a human Vκ1 framework wherein the IL6 paratope comprises amino acid residues Y49, D50, S53, N54, Y55, P56, S57, Y91, Y96; or

[0015] b) a VH domain based on a human VH3 framework, wherein the IL6 paratope comprises amino acid residues Y1, P2 、Q3、 V26 , L27 、F28、 K29 , H30, Q31, D32, P52a, R94, L96 、D97、F98、D101、 E102 , and a VL domain based on the human Vκ1 framework, wherein the IL6 paratope comprises amino acid residues Y49, D50, D53 , R54 、Y55、P56、 E57 , Y91, Y96 (according to Kabat numbering).

[0016] In another aspect, the invention provides an antibody that binds to IL6, and the antibody binds to the same epitope on IL6 as an antibody having a VL domain of SEQ ID NO: 35 and a VH domain of SEQ ID NO: 36. In one embodiment, the antibody comprises: a VH domain having a human VH3 framework, wherein the IL6 paratope comprises amino acid residues 1, 2, 3, 26, 27, 28, 29, 30, 31, 32, 52a, 94, 96, 97, 98, 101, 102; and a VL domain having a human Vκ1 framework, wherein the IL6 paratope comprises amino acid residues 49, 50, 53, 54, 55, 56, 57, 91, 96.

[0017] In one aspect, the invention provides an isolated nucleic acid encoding an antibody of the invention.

[0018] In one aspect, the invention provides a host cell comprising a nucleic acid of the invention. In one embodiment, the host cell is a CHO cell. In one embodiment, the host cell is an E. coli cell.

[0019] In one aspect, the invention provides an expression vector comprising a nucleic acid of the invention.

[0020] 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, thereby producing the antibody.

[0021] In one aspect, the invention provides antibodies produced by the methods of the invention.

[0022] In one aspect, the invention provides a pharmaceutical formulation comprising an antibody of the invention and a pharmaceutically acceptable carrier.

[0023] In one aspect, the invention provides a pre-filled syringe comprising an antibody of the invention and a pharmaceutically acceptable carrier.

[0024] In one aspect, the invention provides an ocular implant comprising an antibody of the invention and a pharmaceutically acceptable carrier. In one embodiment, the invention includes a port delivery device comprising an antibody of the invention.

[0025] In one aspect of the invention, a port delivery device administers an antibody or a pharmaceutical formulation.

[0026] In one aspect, the invention provides an antibody of the invention for use as a medicament, in one embodiment for use in the treatment of vascular disease.

[0027] In one aspect, the invention provides use of an antibody of the invention or a pharmaceutical composition of the invention in the preparation of a medicament, in one embodiment a medicament for treating a vascular disease.

[0028] In one aspect, the invention provides a method of treating an individual suffering from a vascular disease, the method comprising administering to the individual an effective amount of an antibody of the invention or a pharmaceutical composition of the invention.

[0029] In one aspect, the present invention provides a method of inhibiting angiogenesis in a subject, the method comprising administering to the subject an effective amount of the antibody of the present invention or the pharmaceutical composition of the present invention to inhibit angiogenesis.

[0030] According to the present invention, a therapeutic anti-VEGF-A / anti-IL6 antibody is provided, which is able to 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 antibodies of the present invention are suitable for the treatment of ocular vascular diseases. The antibodies of the present invention provide several valuable properties, including good expressibility and developability (e.g., high binding efficacy, high biophysical and biochemical stability, high concentration formulations) that allow their therapeutic applications, in particular, high affinity for both targets that support low effective doses, and high stability that is conducive to long durations. Compared with non-antibody methods, the antibodies of the present invention tend to be more acceptable due to their high humanization and lack of artificial domains and linkers. In addition, the antibodies of the present invention are advantageously provided in high-concentration liquid formulations, the viscosity of which is suitable for ocular applications. Because it can be provided in high concentrations, treatment with the antibodies of the present invention is more acceptable to patients because higher doses of therapeutic agents can be applied in one treatment, allowing longer treatment cycles. Bispecific Fab fragments such as those described in the present invention have the additional advantage over bispecific full-length IgG antibodies due to their much lower molecular weight. Whereas the molecular weight of a Fab is about 50 kDa, a full-length antibody weighs three times the molecular weight of a Fab (about 150 kDa) when providing the same number of binding sites. Thus, for a given amount of drug, a bispecific Fab fragment will contain three times as many binding sites as a full-length IgG antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 : Binding of parental bispecific antibodies 6HVL_1 and V6HL_1 to human and cynomolgus monkey IL6 determined by surface plasmon resonance

[0032] Figure 2 : VEGF IC50 of parental bispecific antibodies 6HVL_1 and V6HL_1 obtained using human VEGF-165

[0033] Figure 3 : Binding of improved bispecific antibodies to human and cynomolgus monkey IL6 determined by surface plasmon resonance.

[0034] Figure 4 : VEGF IC50 of improved bispecific antibodies using human VEGF-121 and VEGF-165.

[0035] Figure 5 : Crystal structure of the Fab0182–IL-6 complex. Overall view of the structure of IL-6 bound to Fab 0182. IL-6 is in light orange, and the light and heavy chains of Fab 0182 are in cyan and blue, respectively.

[0036] Figure 6 : Crystal structure of the Fab 6HVL4.1-IL-6 complex. Overall view of the structure of IL-6 bound to Fab 6HVL4.1. IL-6 is in light orange, and the light and heavy chains of Fab 6HVL4.1 are in wheat and blue, respectively.

[0037] Figure 7 : Simultaneous binding of anti-VEGF / anti-IL-6 Fab to their targets assessed by SPR using immobilized anti-Fab antibodies

[0038] Figure 8 : Blockade of VEGF-R2 binding by anti-VEGF / anti-IL-6 Fab in the presence of IL-6 assessed by SPR using immobilized VEGF-A

[0039] Fig. 9 The effect of VEGF binding on IL6 activity was assessed by a cell-based IL-6-specific reporter gene assay (without preincubation) as follows

[0040] Fig.10 The effect of VEGF binding on IL6 activity was assessed by a cell-based IL-6-specific reporter gene assay (with preincubation) as follows

[0041] Fig.11 : Inhibition of IL-6 signaling in HRMEC by 6HVL_4

[0042] Fig.12 : Dose-dependent changes in the inhibition percentage of 6HVL_4 on VEGF-A-induced HUVEC proliferation

[0043] Fig.13 :6HVL_4 restores IL6 / IL6R / VEGF-induced HRMVEC barrier disruption

[0044] Fig.14 : Aflibercept restores IL6 / IL6R / VEGF-induced HRMVEC barrier disruption

[0045] Fig.15 : Amino acid sequences of the VH and VL domains of the indicated antibodies. The Kabat numbering of the amino acid positions is shown. The amino acid positions contributing to the IL6 paratope identified in Example 13 are highlighted by black boxes.

[0046] Fig.16 : Comparison of the image of the IL6 / IL6R / gp130 complex (top; pdb-acc.#1p9m) with the superposition of the structure of Fab0182 bound to IL6 and the structure of the IL6 / IL6R complex from pdb-acc.1p9m (bottom).

[0047] Fig.17 : Comparison of the image of the IL6 / IL6R / gp130 complex (top; pdb-acc.#1p9m) with the superposition of the structure of Fab6HVL4.1 bound to IL6 and the structure of the IL6 / IL6R complex from pdb-acc.1p9m (bottom).

[0048] Fig.18 : SPR bridging experiments investigating the ability of IL6R to bind IL6 when involved in a preformed complex with Fab P1AE2421.

[0049] Fig.19 : SPR binding experiments investigating the ability of Fab P1AE2421 to bind to human "hyper-IL6" (a chimera of human IL6 and IL6R).

[0050] Fig. 20 : ELISA competition assay to determine the ability of Fab P1AE2421 to bind to IL6 in a manner that blocks the binding of IL6 to IL6R.

[0051] Fig.21 : Binding of the IL6-binding antibody 6HdL2.05 to human and cynomolgus monkey IL6 determined by surface plasmon resonance DETAILED DESCRIPTION

[0052] 1. definition

[0053] Unless otherwise defined herein, scientific and technical terms related to the present invention shall have the meanings commonly understood by those of ordinary skill in the art. In addition, unless the context otherwise requires, singular terms shall include plural terms, and plural terms shall include singular terms. The methods and techniques disclosed herein are generally carried out according to conventional methods well known in the art. Generally, the terms and techniques related to biochemistry, enzymology, molecular and cell biology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization as described herein are well known and commonly used in the art.

[0054] Unless otherwise defined herein, the term "comprising" shall include the term "consisting of."

[0055] As used herein, the term "about" in conjunction with a particular value (eg, temperature, concentration, time, etc.) shall mean a variation of + / - 1% of the particular value to which the term "about" refers.

[0056] The term "antibody" herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.

[0057] An "isolated" antibody is one that has been separated from a component of its natural environment. In some embodiments, the antibody is purified to a purity greater than 95% or 99% as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0058] The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous antibody population, that is, except for possible variant antibodies (e.g., containing naturally occurring mutations or produced during the production of monoclonal antibody preparations, such variants are generally present in small amounts), the individual antibodies comprising the population have identity and / or bind to the same epitope. Contrary to polyclonal antibody preparations that typically include different antibodies for different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed to a single determinant on an antigen. Therefore, the modifier "monoclonal" indicates that the characteristic of the antibody is obtained from a substantially homogeneous antibody population, and should not be interpreted as requiring antibodies to be produced by any particular method.

[0059] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein.

[0060] The "class" of an antibody refers to the type of constant domain or constant region possessed by the heavy chain of the antibody. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of them can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. In certain embodiments, the antibody is an IgG1 isotype. In certain embodiments, the antibody is an IgG1 isotype with P329G, L234A, and L235A mutations to reduce the effector function of the Fc region. In other embodiments, the antibody is an IgG2 isotype. In certain embodiments, the antibody is an IgG4 isotype with an S228P mutation in the hinge region to improve the stability of the IgG4 antibody. The heavy chain constant domains corresponding to different classes of immunoglobulins are referred to as α, δ, ε, γ, and μ, respectively. The light chain of an antibody can be classified into one of two types based on the amino acid sequence of its constant domain, which are called kappa (κ) and lambda (λ).

[0061] The term "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain, which comprises at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, which is also referred to as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0062] The term "variable region" or "variable domain" refers to the domain of an antibody heavy chain or light chain that participates in binding of an antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, wherein each domain comprises four conserved framework regions (FRs) and three hypervariable regions (HVRs) (see, e.g., Kindt et al., Kuby Immunology, 6th edition, WH Freeman and Co., p. 91 (2007)). In the antibodies of the present invention, a single VH domain and a VL domain pair, i.e. a homologous VH / VL pair, specifically bind to its two targets: VEGF-A and IL6.

[0063] "DutaFab" is a bispecific antibody as disclosed in WO2012 / 163520. In DutaFab, a single VH domain and VL domain pair specifically binds to two different epitopes, wherein one paratope comprises amino acid residues from CDR-H2, CDR-L1 and CDR-L3 and the other paratope comprises amino acid residues from CDR-H1, CDR-H3 and CDR-L2. DutaFab contains two non-overlapping paratopes within a cognate VH / VL pair and can bind to two different epitopes simultaneously. DutaFab and methods of producing same by screening libraries comprising monospecific Fab fragments are disclosed in WO2012 / 163520.

[0064] A "human antibody" is an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell, or derived from an amino acid sequence of an antibody of non-human origin utilizing a full library of human antibodies or other human antibody coding sequences. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen binding residues. Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments herein.

[0065] "People's common framework" is such a framework, which represents the most commonly present amino acid residues in the selection of human immunoglobulin VL or VH framework sequences. In general, the selection of human immunoglobulin VL or VH sequences comes from a subgroup of variable domain sequences. In general, the subgroup of sequences is such as Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), Volumes 1 to 3. In one embodiment, for VL, the subgroup is such as subgroup κI in Kabat et al. (supra). In one embodiment, for VH, the subgroup is such as subgroup III in Kabat et al. (supra).

[0066] "Antibody fragment" refers to a molecule other than an intact antibody, which comprises a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0067] As used interchangeably herein, "paratope" or "antigen binding site" refers to the portion of an antibody that recognizes and binds an antigen. The paratope is formed by several single amino acid residues from the variable domains of the antibody heavy and light chains, which are arranged in spatial proximity in the tertiary structure of the Fv region. The antibodies of the present invention contain two paratopes in one homologous VH / VL pair.

[0068] As used herein, a "VEGF-A paratope" is a paratope or antigen binding site that binds to VEGF-A. The VEGF-A paratope of an antibody of the invention comprises amino acid residues from CDR-H2, CDR-L1, and CDR-L3 of the antibody.

[0069] As used herein, "IL6 paratope" is the paratope or antigen binding site that binds to IL6. The IL6 paratope of the antibodies of the present invention comprises amino acid residues from CDR-H1, CDR-H3, and CDR-L2 of the antibody.

[0070] Unless otherwise indicated, the term "vascular endothelial growth factor" (abbreviated as "VEGF") as used herein refers to any native VEGF from any vertebrate source, including mammals, such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses "full-length" unprocessed VEGF, as well as any form of VEGF produced by processing in cells. The term also encompasses naturally occurring variants of VEGF, such as splice variants or allelic variants. The amino acid sequence of an exemplary human VEGF is shown in SEQ ID NO: 27.

[0071] The terms "anti-VEGF-A antibody" and "antibody that binds to VEGF-A" refer to an antibody that is capable of binding to anti-VEGF-A with sufficient affinity such that the antibody can be used as a diagnostic and / or therapeutic agent targeting VEGF-A. In one embodiment, the extent of binding of the anti-VEGF-A antibody to unrelated, non-VEGF-A proteins is less than about 10% of the binding of the antibody to VEGF-A, as measured, for example, by surface plasmon resonance (SPR). In certain embodiments, the antibody that binds to VEGF-A has a dissociation constant (KD) of 1 nM, ≤0.1 nM, or ≤0.01 nM. When the KD of the antibody is D An antibody is said to "specifically bind" VEGF-A if the concentration of the antibody is 1 μM or less.

[0072] Unless otherwise indicated, the term "interleukin-6" (abbreviated as "IL6") as used herein refers to any native IL6 from any vertebrate source, including mammals, such as primates (e.g., humans) and rodents (e.g., mice and rats). The term includes "full-length" unprocessed IL6, as well as any form of IL6 produced by processing in cells. The term also encompasses naturally occurring variants of IL6, such as splice variants or allelic variants. The amino acid sequence of an exemplary human IL6 is shown in SEQ ID NO: 28.

[0073] The antibodies of the present invention "simultaneously bind to human VEGF-A and human IL6", which means that (a) the Fab fragments of the antibodies of the present invention that bind to human IL6 (also) specifically bind to human VEGF-A, and (b) the Fab fragments of the antibodies of the present invention that bind to human VEGF-A (also) specifically bind to human IL6. Simultaneous binding can be assessed by methods known in the art (e.g., by surface plasmon resonance as described herein).

[0074] As used herein, the term "complementarity determining region" or "CDR" refers to each region of an antibody variable domain that is highly variable in sequence and contains antigen contact residues. Typically, an antibody contains six CDRs; three in the VH domain (CDR-H1, CDR-H2, CDR-H3), and three in the VL domain (CDR-L1, CDR-L2, CDR-L3). Unless otherwise indicated, CDR residues and other residues (e.g., FR residues) in the variable domain are numbered herein according to the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition Public Health Service, National Institutes of Health, Bethesda, MD, 1991).

[0075] As used herein, "framework" or "FR" refers to the variable domain amino acid residues other than the CDR residues. The framework of the variable domain is generally composed of the following four framework domains: FR1, FR2, FR3, and FR4. Thus, the CDR and FR amino acid sequences generally appear in the following sequence in (a) the VH domain: FR1—CDR-H1—FR2—CDR-H2—FR3—CDR-H3—FR4; in (b) the VL domain: FR1—CDR-L1—FR2—CDR-L2—FR3—CDR-L3—FR4.

[0076] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed by the dissociation constant (K D ) represents. Affinity can be measured by conventional methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity will be described herein.

[0077] The term "epitope" refers to a site on a protein or non-protein antigen to which an antibody binds. An epitope can be formed by a continuous stretch of amino acids (linear epitope) or by the inclusion of non-continuous amino acids (conformational epitope), for example due to antigen folding, i.e., due to the tertiary folding of the protein antigen and in spatial proximity. After the protein antigen is exposed to a denaturant, a linear epitope is usually still bound by the antibody, while a conformational epitope is usually destroyed after treatment with a denaturant. An epitope comprises at least 3, at least 4, at least 5, at least 6, at least 7, or 8 to 10 amino acids in a unique three-dimensional conformation.

[0078] Screening for antibody binding can be performed using methods routine in the art, such as, but not limited to, alanine scanning, peptide blotting (see Meth. Mol. Biol. 248 (2004) 443-463), peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of antigens (see Prot. Sci. 9 (2000) 487-496) and cross-blocking (see "Antibodies", Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY).

[0079] Antigen structure-based antibody profiling (ASAP), also known as modification-assisted profiling (MAP), allows classification of a variety of monoclonal antibodies that specifically bind to VEGF-A or IL6 based on the binding profile of each antibody from a large number of antibodies and chemically or enzymatically modified antigen surfaces (see, e.g., US2004 / 0101920). The antibodies in each group bind to the same epitope, which may be a unique epitope that is significantly different from or partially overlaps with the epitope represented by another group.

[0080] In addition, competitive binding can be used to easily determine whether an antibody binds to the same VEGF-A or IL6 epitope as a reference antibody of the present invention, or competes for binding to a reference antibody of the present invention. For example, an "antibody that binds to the same epitope on VEGF-A and IL6" as a reference antibody refers to an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in the respective competition assays, and conversely, the reference antibody blocks the binding of the antibody to its antigen by 50% or more in the respective competition assays. Also for example, to determine whether an antibody binds to the same epitope as a reference antibody, the reference antibody is allowed to bind to VEGF-A or IL6 under saturation conditions. After removing excess reference antibody, the ability of the antibody in question to bind to VEGF-A or IL6 is assessed. If the antibody in question is able to bind to VEGF-A or IL6 after saturation binding of the reference antibody, it can be concluded that the antibody in question binds to a different epitope than the reference antibody. However, if the antibody in question is unable to bind to VEGF-A or IL6 after saturation binding of the reference antibody, the antibody in question may bind to the same epitope as the reference antibody. To confirm whether the antibody in question binds to the same epitope or is hindered for steric reasons, routine experiments can be used (e.g., peptide mutation and binding analysis using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art). The assay should be performed in two settings, i.e., both antibodies are saturated antibodies. If in both settings, only the first (saturated) antibody is able to bind to VEGF-A or IL6, it can be concluded that the antibody in question and the reference antibody compete for binding to VEGF-A or IL6.

[0081] In some embodiments, two antibodies are considered to bind to the same or overlapping epitope if one antibody inhibits the binding of the other antibody by at least 50%, at least 75%, at least 90%, or even 99% or more by 1, 5, 10, 20, or 100-fold excess as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 50 (1990) 1495-1502).

[0082] In some embodiments, two antibodies are considered to bind to the same epitope if substantially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody also reduce or eliminate binding of the other antibody. Two antibodies are considered to have "overlapping epitopes" if only a subset of amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other antibody.

[0083] "Amino acid sequence identity percentage (%)" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence after aligning the candidate sequence with the reference polypeptide sequence and introducing spaces (if necessary) to achieve maximum sequence identity percentage, and for the purpose of alignment without considering any conservative substitutions as components of sequence identity. Alignment for determining amino acid sequence identity percentage can be achieved in various ways within the technical scope of the art, for example, using publicly available computer software, such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software or FASTA program package. Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithm required for achieving maximum alignment over the full length of the compared sequence. Alternatively, the sequence comparison computer program ALIGN-2 can be used to generate identity percentage values. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc. and the source code has been filed with user documentation in the US Copyright Office, Washington DC, 20559, where it is registered under US Copyright Registration No. TXU510087 and is described in WO 2000 / 005319.

[0084] However, unless otherwise stated, for the purposes of this article, the BLOSUM50 comparison matrix is ​​used to generate amino acid sequence identity % values ​​using the ggsearch program of the FASTA package version 36.3.8c or higher. The FASTA package is by W. R. Pearson and D. J. Lipman (1988), "Improved Tools for Biological Sequence Analysis", PNAS 85:2444-2448; W. R. Pearson (1996) "Effective protein sequence comparison" Meth. Enzymol. 266:227-258; and Pearson et al. (1997) Genomics 46:24-36 creation and can be obtained from www.fasta.bioch.virginia.edu / fasta_www2 / fasta_down.shtml or www.ebi.ac.uk / Tools / sss / fasta publicly. Alternatively, sequences can be compared using a public server accessible at fasta.bioch.virginia.edu / fasta_www2 / index.cgi, using the ggsearch (global protein:protein) program and default options (BLOSUM50; open: -10; ext: -2; Ktup = 2) to ensure that a global rather than a local alignment is performed. The amino acid identity percentage is given in the output alignment header.

[0085] The term "nucleic acid molecule" or "polynucleotide" includes any compound and / or substance comprising a nucleotide polymer. Each nucleotide is composed of a base, particularly a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e., deoxyribose or ribose) and a phosphate group. Typically, nucleic acid molecules are described by base sequences, wherein the bases represent the primary structure (linear structure) of the nucleic acid molecule. The base sequence is generally expressed as from 5' to 3'. In this article, the term nucleic acid molecule encompasses synthetic forms of deoxyribonucleic acid (DNA) (including, for example, complementary DNA (cDNA) and genomic DNA), ribonucleic acid (RNA) (particularly messenger RNA (mRNA)), DNA or RNA, and mixed polymers comprising two or more of these molecules. Nucleic acid molecules can be linear or cyclic. In addition, the term nucleic acid molecule includes sense strands and antisense strands, as well as single-stranded and double-stranded forms. In addition, the nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases with derivatized sugar or phosphate backbone bonds or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules suitable as carriers for direct expression in vitro and / or in vivo (e.g., in a host or patient) of the antibodies of the present invention. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) carriers may be unmodified or modified. For example, mRNA may be chemically modified to enhance the stability of the RNA carrier and / or the expression of the encoding molecule, so that mRNA may be injected into a subject to produce antibodies in vivo (see, e.g., Stadler et al., Nature Medicine 2017, published online on June 12, 2017, doi: 10.1038 / nm.4356 or EP 2 101 823 B1).

[0086] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that normally contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0087] "Isolated nucleic acid encoding an antibody" refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including such nucleic acid molecules in a single vector or different vectors, and such nucleic acid molecules present in one or more locations in a host cell.

[0088] The term "vector" as used herein refers to a nucleic acid molecule capable of carrying another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures, as well as vectors that are incorporated into the genome of a host cell into which they have been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."

[0089] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived from the primary transformed cell, regardless of the number of passages. Progeny may not be identical to the nucleic acid content of the parent cell, but may contain mutations. Mutant progeny as screened or selected for the same function or biological activity as in the original transformed cell are included herein.

[0090] The term "pharmaceutical composition" or "pharmaceutical formulation" refers to a preparation that is in effective form permitting the biological activity of the active ingredient contained therein, and that contains no additional components that are unacceptably toxic to a subject to which the pharmaceutical composition would be administered.

[0091] "Pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical composition or formulation other than the active ingredient, which is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0092] An "effective amount" of a pharmaceutical agent (eg, a pharmaceutical composition) refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0093] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0094] As used herein, "treatment" (and grammatical variants thereof such as treat or treat) refers to an attempt to alter the natural course of a disease in the individual being treated, and may be performed for prevention or clinical interventions that may be performed during clinical pathology. Desired effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating the disease state, and alleviating or improving prognosis. In some embodiments, the antibodies of the invention are used to delay the development of a disease or slow the progression of a disease.

[0095] As used herein, the term "eye disease" includes any eye disease associated with pathological angiogenesis and / or atrophy. Eye diseases may be characterized by new blood vessel changes or unregulated proliferation and / or invasion into structures of eye tissues such as the retina or cornea. Eye diseases may be characterized by atrophy of retinal tissues (photoreceptors and underlying retinal pigment epithelium (RPE) and choriocapillaris). Non-limiting eye diseases include, for example, AMD (e.g., wet AMD, dry AMD, intermediate AMD, advanced AMD, and geographic atrophy (GA)), macular degeneration, macular edema, DME (e.g., localized, non-central DME and diffuse, centrally involved DME), retinopathy, diabetic retinopathy (DR) (e.g., proliferative DR (PDR), non-proliferative DR (NPDR), and high altitude DR), other ischemia-related retinopathy, ROP, retinal vein occlusion (RVO) (e.g., central (CRVO) and branch (BRVO) forms), CNV (e.g., myopic CNV), corneal neovascularization, diseases associated with corneal neovascularization, retinal neovascularization, diseases associated with retinal / choroidal neovascularization, central serous retinopathy (CSR), pathological myopia, Hippel-Lindau syndrome, ocular histoplasmosis, FEVR, Coronary artery disease, Norrie disease, bone marrow disease, retinal vascular disease, vascular disease, myopia, optic disc disease, vascular disease ... Retinal abnormalities associated with OPPG, subconjunctival hemorrhage, erythema, ocular neovascular disease, neovascular glaucoma, retinitis pigmentosa (RP), hypertensive retinopathy, retinal angiomatous proliferation, macular telangiectasia, iris neovascularization, intraocular neovascularization, retinal degeneration, cystoid macular edema (CME), vasculitis, papilledema, retinitis (including but not limited to CMV retinitis), ocular melanoma, retinoblastoma, conjunctivitis (e.g., infectious conjunctivitis and non-infectious (e.g., allergic conjunctivitis)), Leber congenital amaurosis (also known as Reye's amaurosis or LCA), uveitis (including infectious and non-infectious uveitis), choroiditis (e.g., multifocal choroiditis), ocular histoplasmosis, blepharitis, dry eye, ocular trauma, sicca disease, and other ophthalmic diseases (wherein the disease is associated with ocular neovascularization, vascular leakage and / or retinal edema or retinal atrophy). Additional exemplary ocular diseases include retinoschisis (abnormal splitting of the neurosensory layer of the retina), diseases associated with rubeosis iridis (corner neovascularization), and diseases caused by abnormal proliferation of fibrovascular or fibrous tissue, including all forms of proliferative vitreoretinopathy.Exemplary diseases associated with corneal neovascularization include, but are not limited to, epidemic keratoconjunctivitis, vitamin A deficiency, contact lens overdose, atopic keratitis, superior limbal keratitis, pterygium keratitis sicca, Sjögren's syndrome, acne rosacea, bullous disease, syphilis, mycobacterial infection, lipid degeneration, chemical burns, bacterial ulcers, fungal ulcers, herpes simplex virus infection, herpes zoster infection, protozoan infection, Kaposi's sarcoma, Mooren's ulcer, Terrien's marginal degeneration, marginal keratolysis, rheumatoid arthritis, systemic lupus, multiple arteriosclerosis, trauma, Wegener's sarcoidosis, scleritis, Stevens-Johnson syndrome, radial keratotomy, and corneal transplant rejection. Exemplary diseases associated with choroidal neovascularization and defects in the retinal vasculature, including increased vascular leakage, aneurysms, and capillary detachment, include, but are not limited to, diabetic retinopathy, macular degeneration, sickle cell anemia, sarcoidosis, syphilis, pseudoxanthoma elasticum, Paget's disease, venous occlusion, arterial occlusion, carotid occlusive disease, chronic uveitis / vitritis, mycobacterial infection, Lyme disease, systemic lupus erythematosus, retinopathy of prematurity, retinal edema (including macular edema), Eal's disease, Behcet's disease, infectious retinitis or choroiditis (e.g., multifocal choroiditis), presumed ocular histoplasmosis, Best's disease (vitreomacular degeneration), myopia, optic nerve pits, pars planitis, retinal detachment (e.g., chronic retinal detachment), hyperviscosity syndrome, toxoplasmosis, trauma, and post-laser complications. Exemplary diseases associated with atrophy of retinal tissue (photoreceptors and underlying RPE) include, but are not limited to, atrophic or non-exudative AMD (e.g., geographic atrophy or advanced dry AMD), macular atrophy (e.g., atrophy associated with neovascularization and / or geographic atrophy), diabetic retinopathy, Stargardt's disease, fundus dystrophy, retinoschisis, and retinitis pigmentosa.

[0096] The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and / or warnings concerning the use of such therapeutic products. 2. DETAILED DESCRIPTION

[0098] In one aspect, the invention is based in part on providing bispecific antibodies for therapeutic applications. In certain aspects, antibodies that bind to human VEGF-A and human IL6 are provided. The antibodies of the invention can be used, for example, to treat vascular diseases, such as ocular vascular diseases.

[0099] A. Exemplary Antibodies that Bind to Human VEGF-A and Human IL6

[0100] In one aspect, the present invention provides antibodies that bind to human VEGF-A and human IL6. In one aspect, isolated antibodies that bind to human VEGF-A and human IL6 are provided. In one aspect, the present invention provides antibodies that specifically bind to human VEGF-A and human IL6.

[0101] In certain aspects, an antibody that binds to human VEGF-A and to human IL6 is provided, wherein the antibody comprises a VEGF-A paratope (i.e., an antigen binding site that binds to VEGF-A) and an IL6 paratope (i.e., an antigen binding site that binds to IL6) within a cognate pair of a VL domain and a VH domain, wherein

[0102] ● wherein the VEGF-A paratope comprises amino acid residues from CDR-H2, CDR-L1 and CDR-L3 of the antibody, wherein the IL6 paratope comprises amino acid residues from CDR-H1, CDR-H3 and CDR-L2 of the antibody; and / or

[0103] ● the IL6 paratope comprises amino acid residues from CDR-H2, CDR-L1, and CDR-L3 of the antibody, wherein the VEGF-A paratope comprises amino acid residues from CDR-H1, CDR-H3, and CDR-L2 of the antibody;

[0104] The variable light chain domain and variable heavy chain domain pair bind to human VEGF-A and human IL6 simultaneously; and / or

[0105] ● The antibody has a variable heavy chain domain having SEQ ID NO: 22 and a variable heavy chain domain having SEQ ID NO:

[0106] 21, binds to the same epitope on human VEGF-A and to the same epitope on human IL6 as compared to an antibody that contains a variable light chain domain of

[0107] ●The antibody Fab fragment of the antibody binds to: (i) human VEGF-A121, wherein

[0108] K measured by surface plasmon resonance D less than 50 pM, and (ii) human IL6, wherein the K measured by surface plasmon resonance is D Less than 50 pM; and / or

[0109] ● an antibody Fab fragment of the antibody exhibits an aggregation onset temperature of 60°C or higher, in some embodiments 70°C or higher; and / or

[0110] • The antibody Fab fragment of this antibody exhibits a melting temperature greater than 80°C as measured by dynamic light scattering.

[0111] On the other hand, the present invention provides an antibody that binds to human VEGF-A and human IL6, the antibody comprising: a VH domain, the VH domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 18, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 19, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 20; and a VL domain, the VL domain comprising: (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 15, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17; the antibody comprising: (a) a VH domain, the VH domain comprising the amino acid sequence of SEQ ID NO: 18, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 19, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 20; NO:22 has an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity; and (b) a VL domain comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:21.

[0112] In another aspect, the present invention provides an antibody that binds to human VEGF-A and to human IL6, the antibody comprising: (a) a VH domain comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence of SEQ ID NO: 22; and (b) a VL domain comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence of SEQ ID NO: 21.

[0113] On the other hand, the present invention provides an antibody that binds to human VEGF-A and to human IL6, 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.

[0114] In another aspect, the present invention provides an antibody that binds to human VEGF-A and to human IL6, the antibody comprising: a VH domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 18, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 19, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 20; and a VL domain comprising: (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 15, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17, the antibody comprising: (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 18 with at most 15, at most 10, or at most 5 amino acid substitutions. NO:22; 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.

[0115] In one aspect, the present invention provides an antibody that binds to human VEGF-A and human IL6, the antibody comprising: a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO: 22. In certain aspects, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity contains substitutions (e.g., conservative substitutions), insertions or deletions relative to the reference sequence, but the antibody that binds to human VEGF-A and human IL6 comprising the VH sequence retains the ability to bind to human VEGF-A and human IL6. In certain aspects, in SEQ ID NO: 22, a total of up to 10 amino acids have been substituted, inserted and / or deleted. In certain aspects, the substitutions, insertions or deletions occur in regions outside the CDRs (i.e., in the FRs). In specific aspects, the VH comprises: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 18, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 19, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 20.

[0116] In one aspect, the present invention provides an antibody that binds to human VEGF-A and human IL6, the antibody comprising: a VL domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO: 21. In certain aspects, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity contains substitutions (e.g., conservative substitutions), insertions or deletions relative to the reference sequence, but the antibody that binds to human VEGF-A and human IL6 comprising the VL sequence retains the ability to bind to human VEGF-A and human IL6. In certain aspects, in SEQ ID NO: 21, a total of up to 10 amino acids have been substituted, inserted and / or deleted. In certain aspects, the substitutions, insertions or deletions occur in regions outside the CDRs (i.e., in the FRs). In a specific aspect, the VL comprises: (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.

[0117] In another aspect, an antibody that binds to human VEGF-A and human IL6 is provided, wherein the antibody comprises a VH sequence as in any aspect provided above and a VL sequence as in any aspect provided above. In one aspect, the antibody comprises the VH and VL sequences in SEQ ID NO: 22 and SEQ ID NO: 21, respectively, including post-translational modifications of those sequences.

[0118] In another aspect, an antibody that binds to human VEGF-A and human IL6 is provided, wherein the antibody comprises a heavy chain amino acid sequence of SEQ ID NO:24 and a light chain amino acid sequence of SEQ ID NO:23.

[0119] In other aspects of the invention, the antibody that binds to human VEGF-A and human IL6 according to any of the above aspects is a monoclonal antibody. In one aspect, the antibody that binds to human VEGF-A and human IL6 is an antibody fragment, e.g., Fv, Fab, Fab', scFv, diabody or F(ab')2 fragment. In another aspect, the antibody is a full-length antibody.

[0120] On the other hand, the present invention provides an antibody that is bound to IL6, and the antibody is derived from the antibody of the present invention. The IL6 paratope disclosed for the antibody of the present invention can be used to provide other antibodies, for example, a monospecific antibody or a bispecific antibody that binds to IL6 and another antigen. The IL6 paratope of antibody 6HVL4.1 disclosed herein was identified via x-ray crystallography (Example 13). Antibody 6HVL4.1 is based on a VH domain with a human VH3 framework and a VL domain with a human Vκ1 framework. The antibody comprising the IL6 paratope of antibody 6HVL4.1 binds to the same epitope on IL6. All embodiments disclosed herein for the antibody of the present invention that is bound to human VEGF-A and human IL6 are also applicable to antibodies that are bound to IL6.

[0121] Therefore, in one embodiment, the present invention provides an antibody that binds to human IL6, the antibody comprising:

[0122] c) a VH domain based on a human VH3 framework, wherein the IL6 paratope comprises amino acid residues Y1, I2, Q3, Y26, E27, F28, T29, H30, Q31, D32, P52a, R94, I96, D97, F98, D101, T102, and a VL domain based on a human Vκ1 framework, wherein the IL6 paratope comprises amino acid residues Y49, D50, S53, N54, Y55, P56, S57, Y91, Y96; or

[0123] d) a VH domain based on a human VH3 framework, wherein the IL6 paratope comprises amino acid residues Y1, P2 、Q3、 V26 , L27 、F28、 K29 , H30, Q31, D32, P52a, R94, L96 、D97、F98、D101、 E102 , and a VL domain based on the human Vκ1 framework, wherein the IL6 paratope comprises amino acid residues Y49, D50, D53 , R54 、Y55、P56、 E57 , Y91, Y96 (according to Kabat numbering).

[0124] In another aspect, the invention provides an antibody that binds to IL6, and the antibody binds to the same epitope on IL6 as an antibody having a VL domain of SEQ ID NO: 35 and a VH domain of SEQ ID NO: 36. In one embodiment, the antibody comprises a VH domain having a human VH3 framework, wherein the IL6 paratope comprises amino acid residues 1, 2, 3, 26, 27, 28, 29, 30, 31, 32, 52a, 94, 96, 97, 98, 101, 102 of an antibody of the invention that binds to human VEGF-A and IL6; and a VL domain having a human Vκ1 framework, wherein the IL6 paratope comprises amino acid residues 49, 50, 53, 54, 55, 56, 57, 91, 96 of an antibody of the invention that binds to human VEGF-A and IL6.

[0125] In one embodiment, the antibody that binds to IL6 as described above is a multispecific antibody that binds to IL6 and another target.

[0126] In other aspects, an antibody that binds to human VEGF-A and human IL6 according to any of the above aspects or an antibody that binds to human IL6 according to any of the above aspects may combine any of the features, alone or in combination, as described in Sections 1 to 5 below:

[0127] 1. Antibody affinity

[0128] 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, 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, 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, in preferred embodiments, ≤5 pM.

[0129] In certain embodiments, the antibody that binds to IL6 has a dissociation constant (K) of ≤1 nM, ≤0.1 nM, or ≤0.03 nM. D In a preferred embodiment, the antibodies provided herein are characterized by a dissociation constant (K D )≤10pM, in a preferred embodiment ≤5pM binds to human IL6. In one aspect, K D Using surface plasmon resonance, in one embodiment Surface plasmon resonance measurement.

[0130] On the other hand, the KinExA assay was used to measure K D In one embodiment, the KinExA assay is used to detect the K of VEGF-A binding as described in the Materials and General Methods section below. D Or for detecting IL6 binding K D K is measured under the conditions D .

[0131] For example, the K of an antibody binding to VEGF-A D In the assay using the KinExA 3200 instrument from Sapidyne Instruments (Boise, ID), PMMA beads were coated with antigen using 30 μg of anti-VEGF antibody MAB293 (R&D) in 1 ml PBS (pH 7.4) according to the KinExA manual protocol (adsorption coating, Sapidyne). The KinExA equilibrium assay was performed at room temperature using PBS pH 7.4 containing 0.01% BSA and 0.01% Tween20 as the running buffer, and samples and beads were prepared in LowCross buffer (Candor Bioscience). A flow rate of 0.25 ml / min was used. A constant amount of VEGFA-121-His (50 pM and in a second experiment 500 pM) was titrated with the test antibody and the equilibrated mixture was passed through a column of anti-VEGF antibody (Mab293) coupled beads in the KinExA system in a volume of 750 μl for 50 pM constant VEGF and 125 μl for 500 pM constant VEGF. Bound VEGFA-121 was detected by using a second biotinylated anti-VEGF antibody (BAF293) at a concentration of 250 ng / ml followed by injection of 250 ng / ml streptavidin Alexa Fluor in sample buffer. TM 647 conjugate. K D The K values ​​were obtained by nonlinear regression analysis of the data using the “standard analysis” method using the single-site homogeneous binding model included in KinExA software (version 4.0.11). The software fits the data points to the theoretical K D Curve to calculate K D And determine the 95% confidence interval. The 95% confidence interval is expressed in K D Low and K D High given.

[0132] For example, the K of the antibody binding to IL6 DIn the determination using surface plasmon resonance (SPR), HBS-EP+ (1x; BR100669; Cytiva) was used as running buffer on a Biacore 8K instrument (Cytiva) at 25°C. 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 fixed on a CM5 sensor chip using standard amine coupling chemistry. Before protein measurement, five startup cycles were optionally performed for adjustment purposes, wherein in each cycle, HBS-EP+ buffer was flowed for approximately 120 seconds, followed by regeneration of the derivatized chip surface by applying 10 mM glycine buffer pH 2.0 for 60 seconds. Antibody Fab fragments at a concentration of 75 nM were captured on the surface at a flow rate of 10 ul / min in HBS-EP+ buffer for 60 seconds. Fab fragments were not applied to the reference channel. Subsequently, human or cynomolgus IL-6 was 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 was achieved as described above. Data were evaluated using 8K evaluation software (Biacore Insight Evaluation 3.0).

[0133] 2. Antibody fragments

[0134] In certain aspects, the antibodies provided herein are antibody fragments.

[0135] In one aspect, antibody fragments are Fab', Fab'-SH or F(ab')2 fragments, particularly Fab fragments. Papain digestion of whole antibodies produces two identical antigen-binding fragments called "Fab" fragments, each of which contains a heavy chain variable domain and a light chain variable domain (VH and VL, respectively) and a constant domain (CL) of a light chain and a first constant domain (CH1) of a heavy chain. Thus, the term "Fab fragment" refers to an antibody fragment comprising a light chain comprising a VL domain and a CL domain and a heavy chain fragment comprising a VH domain and a CH1 domain. The difference between Fab' fragments and Fab fragments is that Fab' fragments have added residues at the carboxyl terminus of the CH1 domain, which residues include one or more cysteines from the hinge region of the antibody. Fab'-SH is a Fab' fragment in which the cysteine ​​residues of the constant domains have free sulfhydryl groups. Pepsin treatment produces a F(ab')2 fragment having two antigen-binding sites (two Fab fragments) and a portion of the Fc region. For a discussion of Fab fragments and F(ab')2 fragments comprising salvage receptor binding epitope residues and having increased in vivo half-lives, see US Pat. No. 5,869,046.

[0136] Antibody fragments can be prepared by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and recombinant production by recombinant host cells (eg, E. coli, CHO), as described herein.

[0137] In preferred embodiments, the antibodies provided herein are Fab fragments.

[0138] In one embodiment, the VH domain of an antibody provided herein comprises a human VH3 framework.

[0139] In one embodiment, the VL domain of an antibody provided herein comprises a human Vkappa1 framework.

[0140] In one embodiment, the CL domain of an antibody provided herein is of the kappa isotype.

[0141] In one embodiment, the CH1 domain of an antibody provided herein is of human IgG1 isotype.

[0142] In a preferred embodiment, the antibody provided herein is a Fab fragment comprising a CL domain of the kappa isotype and a CH1 domain of the human IgG1 isotype.

[0143] 3. Thermal stability

[0144] The antibodies provided herein exhibit excellent thermal stability. In certain embodiments, the Fab fragments of the antibodies provided herein exhibit an aggregation onset temperature of 60°C or higher, in one embodiment 70°C or higher. In certain embodiments, the Fab fragments of the antibodies provided herein exhibit a melting temperature greater than 80°C measured by dynamic light scattering.

[0145] 4. Multispecific Antibodies

[0146] In some aspects, the antibodies provided herein are multispecific antibodies. A "multispecific antibody" is a monoclonal antibody that has binding specificity to at least two different sites (i.e., different epitopes on different antigens or different epitopes on the same antigen). In some aspects, a multispecific antibody has three or more binding specificities.

[0147] Multispecific antibodies with three or more binding specificities comprising antibodies provided herein can be provided in an asymmetric form with domain crossover in one or more binding arms with the same antigen specificity, i.e., by exchanging VH / VL domains (see, e.g., WO 2009 / 080252 and WO 2015 / 150447), CH1 / CL domains (see, e.g., WO 2009 / 080253) or complete Fab arms (see, e.g., WO 2009 / 080251, WO 2016 / 016299, see also Schaefer et al., PNAS, 108 (2011) 1187-1191, and Klein et al., MAbs 8 (2016) 1010-20). Various other molecular formats of multispecific antibodies are known in the art and are included herein (see, e.g., Spiess et al., Mol Immunol 67 (2015) 95-106).

[0148] 5. Antibody variants

[0149] In some aspects, it is contemplated that the amino acid sequence variants of the antibodies provided herein. For example, it may be desirable to change the binding affinity and / or other biological properties of the antibody. The amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, the disappearance and / or insertion and / or substitution of residues in the antibody amino acid sequence. Any combination of disappearance, insertion and substitution can be performed to achieve the final construct, provided that the final construct has the desired characteristics, such as antigen binding.

[0150] In certain aspects, antibody variants with one or more amino acid substitutions are provided. The sites of interest for substitution mutagenesis include CDR and FR. Conservative substitutions are shown under the "preferred substitutions" heading in the following table. More substantial changes are provided under the "exemplary substitutions" heading in Table 1, and will be further described below with reference to the amino acid side chain categories. Amino acid substitutions can be introduced into the target antibody, and the product can be screened for desired activity (e.g., retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC).

[0151] surface

[0152]

[0153]

[0154] Amino acids can be grouped according to common side chain properties:

[0155] (1) Hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile;

[0156] (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;

[0157] (3) Acidic: Asp, Glu;

[0158] (4) Basic: His, Lys, Arg;

[0159] (5) Residues that affect chain orientation: Gly, Pro;

[0160] (6) Aromatic: Trp, Tyr, Phe.

[0161] Non-conservative substitutions will entail exchanging a member of one of these classes for a member of another class.

[0162] One type of substitution variant involves replacing one or more CDR residues of a parent antibody (e.g., a humanized antibody or a human antibody). Typically, relative to the parent antibody, one or more resulting variants selected for further study will have changes (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) and / or will substantially retain certain biological properties of the parent antibody. Exemplary substitution variants are affinity matured antibodies, which can be conveniently generated, for example, using affinity maturation techniques based on phage display such as those described herein. In short, one or more CDR residues are mutated and the variant antibody is displayed on a phage and screened for a specific biological activity (e.g., binding affinity).

[0163] In certain aspects, substitutions, insertions or deletions may occur within one or more CDRs, as long as such changes do not substantially reduce the ability of the antibody to bind to antigen. For example, conservative changes (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in the CDRs. Such changes may be, for example, outside of the antigen contact residues in the CDRs. In certain variant VH and VL sequences provided above, each CDR either remains unchanged or contains no more than one, two or three amino acid substitutions.

[0164] The method that can be used to identify antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis", as described in Cunningham and Wells (1989) Science, 244: 1081-1085. In this method, a residue or a group of target residues (e.g., charged residues such as arg, asp, his, lys and glu) are identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Other substitutions can be introduced at amino acid positions that exhibit functional sensitivity to the initial substitution. Alternatively or additionally, the crystal structure of the antigen-antibody complex can be used to identify the contact points between the antibody and the antigen. Such contact residues and adjacent residues that are candidates for substitution can be targeted or eliminated. Variants can be screened to determine whether they have the desired properties.

[0165] Amino acid sequence insertions include amino and / or carboxyl terminal fusions ranging in length from one residue to a polypeptide containing one hundred or more residues, and intrasequence insertions of one or more amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertion variants of antibody molecules include fusing the N-terminus or C-terminus of the antibody to an enzyme (e.g., for ADEPT (antibody directed enzyme prodrug therapy)) or a polypeptide that increases the serum half-life of the antibody.

[0166] a) Glycosylation variants

[0167] In certain aspects, the antibodies provided herein are altered to increase or decrease the extent of antibody glycosylation. Glycosylation sites can be conveniently added or deleted to an antibody by altering the amino acid sequence to create or remove one or more glycosylation sites.

[0168] When antibody comprises Fc district, the oligosaccharide connected thereto can be changed.Native antibodies produced by mammalian cells usually comprise the biantennary oligosaccharide of side chain, and this biantennary oligosaccharide is usually connected to the Asn297 of the CH2 domain of Fc district by N-bonding.See, for example, Wright et al. TIBTECH 15:26-32 (1997).Oligosaccharide can include various carbohydrates, for example, mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, and the fucose of GlcNAc in the " backbone " of biantennary oligosaccharide structure.In some aspects, the oligosaccharide in the antibody of the present invention can be modified to produce antibody variants with some improved characteristics.

[0169] In one aspect, antibody variants are provided having non-fucosylated oligosaccharides, i.e., oligosaccharide structures lacking fucose attached (directly or indirectly) to the Fc region. Such non-fucosylated oligosaccharides (also referred to as "defucosylated" oligosaccharides) are particularly N-linked oligosaccharides lacking a fucose residue attached to the first GlcNAc in the stem of the biantennary oligosaccharide structure. In one aspect, antibody variants are provided having an increased proportion of non-fucosylated oligosaccharides in the Fc region compared to a native or parent antibody. For example, the proportion of non-fucosylated oligosaccharides can be at least about 20%, at least about 40%, at least about 60%, at least about 80%, or even about 100% (i.e., the absence of fucosylated oligosaccharides). The percentage of non-fucosylated oligosaccharides, as described, for example, in WO2006 / 082515, as measured by MALDI-TOF mass spectrometry, is the (average) amount of oligosaccharides lacking a fucose residue relative to the sum of all oligosaccharides (e.g., complex, hybrid and high mannose structures) linked to Asn 297. Asn297 refers to the asparagine residue located at approximately position 297 in the Fc region (EU numbering of residues in the Fc region); however, due to minor sequence variations in antibodies, Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such antibodies having an increased proportion of non-fucosylated oligosaccharides in the Fc region may have improved FcγRIIIa receptor binding and / or improved effector function, in particular improved ADCC function. See, for example, US2003 / 0157108 and US2004 / 0093621.

[0170] Examples of cell lines capable of producing antibodies with reduced fucosylation include Lec13 CHO cells lacking protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US 2003 / 0157108; and WO 2004 / 056312, particularly in Example 11), and knockout cell lines, such as α-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614-622 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO 2004 / 056312, particularly in Example 11). 2003 / 085107), or cells with reduced or abolished GDP-fucose synthesis or transporter activity (see, e.g., US2004259150, US2005031613, US2004132140, US2004110282).

[0171] On the other hand, antibody variants provide bisected oligosaccharides, for example, wherein the biantennary oligosaccharides attached to the Fc region of the antibody are bisected by GlcNAc. As described above, such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described in, for example, Umana et al., Nat Biotechnol 17, 176-180 (1999); Ferrara et al., Biotechn Bioeng 93, 851-861 (2006); WO 99 / 54342; WO 2004 / 065540, WO 2003 / 011878.

[0172] Also provided are antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region. Such antibody variants may have improved CDC function. Such antibody variants are described in, for example, WO 1997 / 30087, WO 1998 / 58964, and WO 1999 / 22764.

[0173] b) Fc region variants

[0174] In certain aspects, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. An Fc region variant can comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0175] In certain aspects, the present invention contemplates antibody variants with some but not all effector functions, making them ideal candidates for applications where the in vivo half-life of the antibody is important and certain effector functions (such as complement dependent cytotoxicity (CDC) and antibody dependent cell-mediated cytotoxicity (ADCC)) are unnecessary or harmful. In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / depletion of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and therefore may lack ADCC activity), but retains FcRn binding ability. NK cells, the main cells that mediate ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-492 (1991). Non-limiting examples of in vitro assays for evaluating ADCC activity of target molecules are described in U.S. Pat. Nos. 5,500,362 (see, e.g., Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays can be used (see, e.g., ACTI for flow cytometry). TM Nonradioactive cytotoxicity assay (Cell Technology, Inc. Mountain View, CA); and CytoTox Non-radioactive cytotoxicity assay (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, ADCC activity of the molecule of interest can be assessed in vivo, for example, in an animal model such as disclosed in Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays can also be performed to confirm that the antibody cannot bind to C1q and therefore lacks CDC activity. See, for example, C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996); Cragg, MS et al., Blood 101: 1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103: 2738-2743 (2004)). FcRn binding and in vivo clearance / half-life assays can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18 (12): 1759-1769 (2006); WO 2013 / 120929A1).

[0176] Antibodies with reduced effector function include those with substitutions of one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include those with substitutions at two or more of amino acids 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant, in which residues 265 and 297 are substituted with alanine (U.S. Pat. No. 7,332,581).

[0177] Certain antibody variants with improved or decreased binding to FcRs are described. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004 / 056312; and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001).)

[0178] In certain aspects, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, for example, substitutions at positions 298, 333, and / or 334 of the Fc region (EU numbering of residues).

[0179] In some aspects, antibody variants include Fc regions with one or more amino acid substitutions that reduce FcγR binding, such as substitutions at positions 234 and 235 in the Fc region (EU numbering of residues). On the one hand, substitutions are L234A and L235A (LALA). In some aspects, antibody variants are further included in D265A and / or P329G in the Fc region derived from human IgG1 Fc region. On the one hand, in the Fc region derived from human IgG1 Fc region, substitutions are L234A, L235A and P329G (LALA-PG). (See, for example, WO2012 / 130831). On the other hand, in the Fc region derived from human IgG1 Fc region, substitutions are L234A, L235A and D265A (LALA-DA).

[0180] In some aspects, changes are made in the Fc region that result in altered (ie, improved or reduced) CIq binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0181] Antibodies with extended half-life and improved neonatal Fc receptor (FcRn) binding, responsible for the transfer of maternal IgG to the fetus (Guyer, RL et al., J. Immunol. 117:587 (1976), and Kim, JK et al., J. Immunol. 24:249 (1994)) are described in US 2005 / 0014934 (Hinton et al.). Those antibodies comprise an Fc region having one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include those having a substitution at one or more of the following Fc region residues: 238, 252, 254, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424 or 434, such as a substitution at Fc region residue 434 (see, e.g., U.S. Pat. No. 7,371,826; Dall'Acqua, WF et al. J. Biol. Chem. 281 (2006) 23514-23524).

[0182] Fc region residues that are critical for mouse Fc-mouse FcRn interactions have been identified by site-directed mutagenesis (see, e.g., Dall'Acqua, WF et al. J. Immunol 169 (2002) 5171-5180). The interaction involves residues 1253, H310, H433, N434, and H435 (EU index numbering) (Medesan, C. et al., Eur. J. Immunol. 26 (1996) 2533; Firan, M. et al., Int. Immunol. 13 (2001) 993; Kim, JK et al., Eur. J. Immunol. 24 (1994) 542). Residues 1253, H310, and H435 were found to be critical for the interaction of human Fc with mouse FcRn (Kim, JK et al., Eur. J. Immunol. 29 (1999) 2819). Studies on the human Fc-human FcRn complex have shown that residues I253, S254, H435 and Y436 are essential for the interaction (Firan, M. et al., Int. Immunol. 13 (2001) 993; Shields, RL et al., J. Biol. Chem. 276 (2001) 6591-6604). Various mutants of residues 248 to 259, 301 to 317, 376 to 382 and 424 to 437 have been reported and examined in Yeung, YA et al. (J. Immunol. 182 (2009) 7667-7671).

[0183] In some aspects, the antibody variant comprises an Fc region with one or more amino acid substitutions that reduce FcRn binding, for example, substitutions at positions 253, and / or 310 and / or 435 of the Fc region (EU numbering of residues). In some aspects, the antibody variant comprises an Fc region with amino acid substitutions at positions 253, 310 and 435. On the one hand, in an Fc region derived from a human IgG1 Fc region, the substitutions are I253A, H310A and H435A. See, e.g., Grevys, A. et al., J. Immunol. 194 (2015) 5497-5508.

[0184] In some aspects, the antibody variant comprises an Fc region with one or more amino acid substitutions that reduce FcRn binding, for example, substitutions at 310, and / or 433 and / or 436 of the Fc region (EU numbering of residues). In some aspects, the antibody variant comprises an Fc region with amino acid substitutions at positions 310, 433 and 436. On the one hand, in an Fc region derived from a human IgG1 Fc region, the substitutions are H310A, H433A and Y436A. (See, e.g., WO 2014 / 177460 A1).

[0185] In certain aspects, the antibody variant comprises an Fc region having one or more amino acid substitutions that increase FcRn binding, for example, substitutions at positions 252, and / or 254 and / or 256 of the Fc region (EU numbering of residues). In certain aspects, the antibody variant comprises an Fc region having amino acid substitutions at positions 252, 254 and 256. On the one hand, in an Fc region derived from a human IgG1 Fc region, the substitutions are M252Y, S254T and T256E. For other examples of Fc region variants, see also: Duncan and Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351.

[0186] The C-terminus of the heavy chain of the antibody as reported herein can be a complete C-terminus ending with amino acid residue PGK. The C-terminus of the heavy chain can be a shortened C-terminus, in which one or two C-terminal amino acid residues have been removed. In a preferred aspect, the C-terminus of the heavy chain is a shortened C-terminus ending with PG. In one aspect of all aspects reported herein, the antibody comprising a heavy chain including a C-terminal CH3 domain as specified herein, comprises a C-terminal glycine-lysine dipeptide (G446 and K447, EU index numbering of amino acid position). In one aspect of all aspects reported herein, the antibody comprising a heavy chain including a C-terminal CH3 domain as specified herein, comprises a C-terminal glycine residue (G446, EU index numbering of amino acid position).

[0187] c) Cysteine ​​engineered antibody variants

[0188] In certain aspects, it may be desirable to generate antibodies with engineered cysteine ​​residues, such as THIOMAB. TM Antibodies, wherein one or more residues of the antibody are replaced by cysteine ​​residues. In a specific embodiment, the substituted residue is present in an accessible site of the antibody. As further described herein, by replacing those residues with cysteine, reactive thiol groups are positioned in accessible sites of the antibody, and can be used to conjugate the antibody with other parts (such as drug moieties or linker-drug moieties) to produce immunoconjugates. Cysteine ​​engineered antibodies can be produced as described in, for example, U.S. Patent Nos. 7,521,541, 8,30,930, 7,855,275, 9,000,130 or WO 2016040856.

[0189] B. Recombinant Methods and Compositions

[0190] Antibodies can be produced using recombinant methods and compositions, for example, as described in US 4,816,567. For these methods, one or more isolated nucleic acids encoding the antibodies are provided.

[0191] In one aspect, an isolated nucleic acid encoding an antibody of the invention is provided.

[0192] In one aspect, a method for preparing an antibody that binds to human VEGF-A and human IL6 is provided, wherein the method comprises: culturing a host cell containing a nucleic acid encoding the antibody as provided above under conditions suitable for expressing the antibody; and optionally recovering the antibody from the host cell (or host cell culture medium).

[0193] In order to recombinantly produce antibodies that bind to human VEGF-A and human IL6, nucleic acids encoding antibodies, such as those described above, are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that specifically bind to genes encoding heavy and light chains of antibodies), or produced by recombinant methods or obtained by chemical synthesis.

[0194] Suitable host cells for cloning or expressing vectors encoding antibodies include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. Regarding expressing antibody fragments and polypeptides in bacteria, see, for example, US 5,648,237, US 5,789,199, and US 5,840,523. (See also Charlton, KA, in: Methods in Molecular Biology, Vol. 248, Lo, BKC, ed., Humana Press, Totowa, NJ (2003), pp. 245-254, describing the expression of antibody fragments in E. coli.) Antibodies can be separated from bacterial cell paste in a soluble fraction after expression, and can be further purified. In certain embodiments, the host cell is an E. coli cell.

[0195] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 lines (COS-7) transformed by SV40; human embryonic kidney cell lines (such as 293 or 293T cells described in, for example, Graham, FL et al., J. Gen Virol. 36 (1977) 59-74); small hamster kidney cells (BHK); mouse Sertoli cells (such as TM4 cells described in Mather, JP, Biol. Reprod. 23 (1980) 243-252); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); Buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells (as described, for example, in Mather, JP et al., Annals N.Y. Acad. Sci. 383 (1982) 44-68); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub, G et al., Proc. Natl. Acad. Sci. USA 77 (1980) 4216-4220); and myeloma cell lines, such as Y0, NS0 and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki, P. and Wu, AM, Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268.

[0196] On the one hand, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell or a lymphocyte (such as a Y0, NS0, Sp20 cell). In a preferred embodiment, the host cell is a CHO cell. Producing the antibody of the present invention in a CHO cell can improve the injectability of the antibody.

[0197] C. Pharmaceutical Compositions

[0198] In other aspects, a pharmaceutical composition comprising any of the antibodies provided herein is provided, for example, for use in any of the following methods of treatment. In one aspect, the pharmaceutical composition comprises any of the antibodies provided herein and a pharmaceutically acceptable carrier. In another aspect, the pharmaceutical composition comprises any of the antibodies provided herein and at least one additional therapeutic agent, for example, as described below.

[0199] Pharmaceutical compositions of antibodies that bind to human VEGF-A and human IL6 as described herein (Remington's Pharmaceutical Sciences 16th edition, Osol, A. ed. (1980)) are prepared by mixing the antibodies having the desired purity with one or more optional pharmaceutical carriers, in the form of lyophilized compositions or aqueous solutions. Pharmaceutical carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as histidine, phosphates, citrates, acetates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl alcohol, or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; metacresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., zinc protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutical carriers herein also include interstitial drug dispersants such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 ( Halozyme, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[0200] Exemplary lyophilized antibody compositions are described in US Pat. No. 6,267,958. Aqueous antibody compositions include those described in US Pat. No. 6,171,586 and WO 2006 / 044908, the latter compositions comprising a histidine-acetate buffer.

[0201] The pharmaceutical compositions herein may also contain more than one active ingredient essential for the particular indication being treated, preferably active ingredients with complementary activities that do not adversely affect each other. Such active ingredients are suitably present in combination in amounts effective for the intended purpose.

[0202] The active ingredient can be embedded in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively); embedded in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules); or embedded in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. ed. (1980).

[0203] Pharmaceutical compositions can be prepared for sustained release. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, eg, films or microcapsules.

[0204] Pharmaceutical compositions for in vivo administration are generally sterile. For example, sterility can be easily achieved by filtration through sterile filtration membranes.

[0205] D. Methods of treatment and routes of administration

[0206] Any of the antibodies provided herein that bind to human VEGF-A and human IL6 can be used in therapeutic methods.

[0207] In one aspect, an antibody that binds to human VEGF-A and human IL6 is provided for use as a medicament. In other aspects, an antibody that binds to human VEGF-A and human IL6 is provided for the treatment of vascular diseases. In certain aspects, an antibody that binds to human VEGF-A and human IL6 is provided for a method of treating a subject suffering from a vascular disease. In certain aspects, the present invention provides an antibody that binds to human VEGF-A and human IL6 for use in treating a subject suffering from a vascular disease, the method comprising administering to the subject an effective amount of an antibody that binds to human VEGF-A and human IL6. In one such aspect, such as described below, the method further comprises administering to the subject an effective amount of at least one additional therapeutic agent (e.g., one, two, three, four, five or six additional therapeutic agents). In other aspects, the present invention provides an antibody that binds to human VEGF-A and human IL6 for inhibiting angiogenesis. In certain aspects, the present invention provides an antibody that binds to human VEGF-A and human IL6 for inhibiting angiogenesis in a subject, the method comprising administering to the subject an effective amount of an antibody that binds to human VEGF-A and human IL6 to inhibit angiogenesis. An "individual" according to any of the above aspects is preferably a human.

[0208] In other aspects, an antibody that binds to human VEGF-A and human IL6 is provided for treating an eye disease. In one embodiment, the eye disease is selected from: AMD (e.g., wet AMD, dry AMD, moderate AMD, advanced AMD, and geographic atrophy (GA)), macular degeneration, macular edema, DME (in one embodiment, localized, non-central DME and diffuse, centrally involved DME), retinopathy, diabetic retinopathy (DR) (in one embodiment, proliferative DR (PDR), non-proliferative DR (NPDR), and high altitude DR), other retinopathy associated with ischemia, ROP, retinal vein occlusion (RVO) (in one embodiment, central (CRVO) and branch (BRVO) forms), CNV (in one embodiment, myopic CNV), corneal neovascularization, diseases associated with corneal neovascularization, retinal neovascularization, diseases associated with retinal / choroidal neovascularization, central serous retinopathy (CSR), pathological myopia, Hippel-Lindau syndrome, ocular histoplasmosis, FEVR, crown Tz's disease, Norrie's disease, retinal abnormalities associated with osteoporosis-pseudoglioma syndrome (OPPG), subconjunctival hemorrhage, redness, ocular neovascular disease, neovascular glaucoma, retinitis pigmentosa (RP), hypertensive retinopathy, retinal angiomatous proliferation, macular telangiectasia, iris neovascularization, intraocular neovascularization, retinal degeneration, cystoid macular edema (CME), vasculitis, papilledema, retinitis (including but not limited to CMV retinitis), melanoma In one embodiment, the eye disease is selected from: AMD (in one embodiment, wet AMD, dry AMD, moderate AMD, advanced AMD, and geographic atrophy (GA)), macular degeneration, macular edema, DME (in one embodiment, localized, non-central DME and diffuse, centrally involved DME), retinopathy, diabetic retinopathy (DR) (in one embodiment, proliferative DR (PDR), non-proliferative DR (NPDR), and high altitude DR).

[0209] In other aspects, the present invention provides a use of an antibody that binds to human VEGF-A and human IL6 in the production or preparation of a drug. In one aspect, the drug is used to treat vascular disease. In other aspects, the drug is used in a method for treating vascular disease, the method comprising administering an effective amount of the drug to an individual suffering from vascular disease. In one such aspect, such as described below, the method further comprises administering an effective amount of at least one additional therapeutic agent to the individual.

[0210] In one aspect, the medicament is used to treat an eye disease. In other aspects, the medicament is used to treat a method for an eye disease, the method comprising administering an effective amount of the medicament to an individual suffering from the eye disease. In one such aspect, such as described below, the method further comprises administering an effective amount of at least one additional therapeutic agent to the individual.

[0211] In other aspects, the invention provides a method for treating a vascular disease. In one aspect, the method comprises administering an effective amount of an antibody that binds to human VEGF-A and human IL6 to an individual suffering from such a vascular disease. In one such aspect, as described below, the method further comprises administering an effective amount of at least one additional therapeutic agent to the individual.

[0212] In another aspect, the invention provides a method for treating an eye disease. In one aspect, the method comprises administering an effective amount of an antibody that binds to human VEGF-A and human IL6 to an individual suffering from the eye disease. In one such aspect, as described below, the method further comprises administering an effective amount of at least one additional therapeutic agent to the individual.

[0213] An "individual" according to any of the above aspects may be a human.

[0214] In other aspects, the present invention provides pharmaceutical compositions comprising any antibody that binds to human VEGF-A and human IL6 as provided herein, for example, for use in any of the above methods of treatment. In one aspect, the pharmaceutical composition comprises any antibody that binds to human VEGF-A and human IL6 as provided herein and a pharmaceutically acceptable carrier. On the other hand, the pharmaceutical composition comprises any antibody that binds to human VEGF-A and human IL6 as provided herein and at least one additional therapeutic agent, for example, as described below.

[0215] The antibodies of the invention can be administered intravitreally (e.g., intravitreal injection) or using a port delivery device. In one embodiment, the antibodies of the invention are administered using the port delivery device for a period of six months or longer, in one embodiment, 8 months or longer, in one embodiment, 9 months or longer, in one embodiment, 12 months or longer, prior to refilling the port delivery device. In one embodiment, the antibodies of the invention are administered using a port delivery device, wherein the antibodies are applied to the port delivery device at a concentration of 150 mg / ml or more, in one embodiment, at a concentration of 200 mg / ml or more.

[0216] The antibodies of the present invention can be administered alone or used in combination therapy. For example, the combination therapy includes administering an antibody of the present invention and administering at least one additional therapeutic agent (e.g., one, two, three, four, five or six additional therapeutic agents).

[0217] In certain embodiments according to (or as applied to) any of the above embodiments, the eye disease is an intraocular neovascular disease selected from the group consisting of: proliferative retinopathy, choroidal neovascularization (CNV), age-related macular degeneration (AMD), diabetic and other ischemia-related retinopathy, diabetic macular edema, pathological myopia, Hippel-Lindau syndrome, ocular histoplasmosis, retinal vein occlusion (RVO) including CRVO and BRVO, corneal neovascularization, retinal neovascularization and retinopathy of prematurity (ROP).

[0218] In some cases, an antibody that binds to human VEGF-A and human IL6 as provided herein can be administered in combination with at least one other therapeutic agent for the treatment of an ocular disorder, e.g., an ocular disorder described herein (e.g., AMD (e.g., wet AMD), DME, DR, RVO, or GA).

[0219] Any suitable AMD therapeutic agent can be administered as an additional therapeutic agent in combination with the antibodies that bind to human VEGF and human IL6 provided herein for treating an ocular disorder (e.g., AMD, DME, DR, RVO or GA), including but not limited to VEGF antagonists, such as anti-VEGF antibodies (e.g., (ranibizumab), RTH-258 (formerly ESBA-1008, an anti-VEGF single-chain antibody fragment; Novartis) or a bispecific anti-VEGF antibody (e.g., an anti-VEGF / anti-angiogenic peptide 2 bispecific antibody, such as faricimab; Roche)), a soluble VEGF receptor fusion protein (e.g., (Aflibercept), anti-VEGF (e.g. abicipar pegol); Molecular PartnersAG / Allergan) or anti-VEGF aptamers (e.g. (pegaptanib sodium)); platelet-derived growth factor (PDGF) antagonists, such as anti-PDGF antibodies, anti-PDGFR antibodies (e.g., REGN2176-3), anti-PDGF-BB PEGylated aptamers (e.g., Ophthotech / Novartis), soluble PDGFR receptor fusion proteins or dual PDGF / VEGF antagonists (e.g., small molecule inhibitors (e.g., DE-120 (Santen) or X-82 (TyrogeneX)) or bispecific anti-PDGF / anti-VEGF antibodies); combined with photodynamic therapy (verteporfin); antioxidants; complement system antagonists, such as complement factor C5 antagonists (e.g., small molecule inhibitors (e.g., ARC-1905; Opthotech) or anti-C5 antibodies (e.g., LFG-316; Novartis), properdin antagonists (e.g., anti-properdin antibodies, such as CLG-561; Alcon), or complement factor D antagonists (e.g., anti-complement factor D antibodies, such as lambalizumab; Roche)); C3 blocking peptides (e.g., APL-2, Appellis); visual cycle modulators (e.g., emixustat hydrochloride); squalamine (e.g., OHR-102; Ohr Pharmaceutical); vitamin and mineral supplements (e.g., those described in Age-Related Eye Disease Study 1 (AREDS1; zinc and / or antioxidants) and Study 2 (AREDS2; zinc, antioxidants, lutein, zeaxanthin, and / or omega-3 fatty acids); cell-based therapies, e.g., NT-501 (Renexus); PH-05206388 (Pfizer), huCNS-SC cell transplantation (StemCells), CNTO-2476 (umbilical cord stem cell line; Janssen), OpRegen (RPE cell suspension; Cell Cure Neurosciences), or MA09-hRPE cell transplantation (Ocata Therapeutics); tissue factor antagonists (e.g., hI-con1; Iconic Therapeutics); α-adrenergic receptor agonists (e.g., brimonidine tartrate; Allergan); peptide vaccines (e.g., S-646240; Shionogi); amyloid β antagonists (e.g., anti-β amyloid monoclonal antibodies, e.g., GSK-933776); S1P antagonists (e.g., anti-S1P antibodies, e.g., iSONEP TM; LpathInc); ROB04 antagonists (e.g., anti-ROBO4 antibodies, such as DS-7080a; Daiichi Sankyo Co., Ltd.); lentiviral vectors expressing endostatin and angiostatin (e.g., RetinoStat); and any combination thereof. In some cases, AMD therapeutics (including any prior AMD therapeutics) can be co-formulated. For example, the anti-PDGFR antibody REGN2176-3 can be co-formulated with aflibercept. Co-formulation. In some cases, such co-formulations can be administered in combination with antibodies of the invention that bind to human VEGF and human IL6. In some cases, the eye disorder is AMD (eg, wet AMD).

[0220] Any suitable DME and / or DR therapeutic agent, including but not limited to VEGF antagonists (e.g., or ), corticosteroids (e.g., corticosteroid implants (e.g. (dexamethasone intravitreal implant) or (fluocinolone acetonide intravitreal implant), or a corticosteroid formulated for administration by intravitreal injection (e.g., triamcinolone acetonide), or a combination thereof. In certain instances, the ocular condition is DME and / or DR.

[0221] An antibody that binds to human VEGF and human IL6 as provided herein can be administered in combination with a therapy or surgical procedure for treating an eye disorder (e.g., AMD, DME, DR, RVO or GA), including, for example, laser photocoagulation (e.g., panretinal photocoagulation (PRP)), drusen laser, macular hole surgery, macular shift surgery, implantable microtelescope, PHI sports angiography (also known as microlaser therapy and feeding vessel therapy), proton beam therapy, microstimulation therapy, retinal detachment and vitreous surgery, scleral buckle, macular surgery, transpupillary thermal therapy, light system The invention relates to methods of treating a patient's condition with a conventional method, including conventional therapy, use of RNA interference (RNAi), in vitro fertilization (also known as membrane differential filtration and rheotherapy), microchip implantation, stem cell therapy, gene replacement therapy, ribozyme gene therapy (including gene therapy for hypoxia response elements, Oxford Biomedical Products; Lentipak, Genetix; and PDEF gene therapy, GenVec), photoreceptor / retinal cell transplantation (including transplantable retinal epithelial cells, Diacrin, Inc.; retinal cell transplantation, such as Astras Pharmaceuticals USA, ReNeuron, CHA Biotech), acupuncture, and combinations thereof.

[0222] Such combination therapies described above encompass combined administration (wherein two or more therapeutic agents are included in the same or different formulations) and separate administration, in which case the administration of the antibody of the present invention that binds to human VEGF and human IL6 can be performed before, simultaneously with, and / or after the administration of the additional therapeutic agent or agent. In one embodiment, the administration of the antibody of the present invention that binds to human VEGF and human IL6 and the administration of the additional therapeutic agent each occur within about one, two, three, four, or five months, or within about one, two, three, four, five, or six days.

[0223] The antibodies of the present invention (and any additional therapeutic agents) can be administered by any suitable means, including parenteral, intrapulmonary and intranasal, and if necessary for local treatment, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration can be carried out by any suitable route, for example by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-lived or long-term. Various dosing schedules are contemplated herein, including but not limited to single or multiple administrations, push administrations, and pulse infusions at various time points.

[0224] The antibodies of the present invention will be formulated, dosed and applied in a manner consistent with good medical practice. Factors to be considered in this case include the specific illness being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the illness, the delivery site of the medicament, the method of administration, the time schedule of administration, and other factors known to practitioners. The antibody is not necessary, but is optionally co-formulated with one or more preparations currently used to prevent or treat the illness in question. The effective amount of these other preparations depends on the amount of the antibody present in the pharmaceutical composition, the type of illness or treatment, and other factors discussed above. These are generally used with the same dosage and route of administration as described herein, or with about 1% to 99% of the dosage described herein, or with any dosage and any route determined to be suitable empirically / clinically.

[0225] For the prevention or treatment of disease, the appropriate dose of the antibody of the present invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the molecule is used for prevention or treatment purposes, the patient's medical history and the response to the antibody, and the discretion of the attending physician. The antibody is appropriately administered to the patient once or in a series of treatments. Depending on the type and severity of the disease, an antibody of about 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg-10 mg / kg) can be, for example, an initial candidate dose administered to the patient by one or more separate administrations or by continuous infusion. Depending on the above factors, a typical daily dose can range from about 1 μg / kg to 100 mg / kg or more. For repeated administration for several days or longer, depending on the condition, treatment will generally continue until the desired disease symptom suppression occurs. An exemplary dose of an antibody ranges from about 0.05 mg / kg to about 10 mg / kg. Therefore, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg or 10 mg / kg (or any combination thereof) can be administered to the patient. Such doses can be administered intermittently, for example, weekly or every three weeks (for example, so that the patient receives about twice to about twenty times, or for example, about six doses of the antibody). An initial higher loading dose can be administered, followed by one or more lower doses. The progress of the therapy can be easily monitored by conventional techniques and determinations.

[0226] E. Products

[0227] In another aspect of the invention, a product is provided, which contains a substance that can be used to treat, prevent and / or diagnose the above-mentioned illness. The product includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, vials, syringes, etc. The container can be formed of a variety of materials such as glass or plastic. The container holds a composition that can effectively treat, prevent and / or diagnose the illness by itself or in combination with another composition, and the container can have a sterile access port (for example, the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic injection needle). At least one active agent in the composition is an antibody of the present invention. The label or package insert indicates that the composition is used to treat the selected illness.

[0228] In addition, the article may include (a) a first container, wherein the first container contains a composition comprising an antibody of the present invention; and (b) a second container, wherein the second container contains a composition comprising an additional cytotoxic agent or other therapeutic agent. The article in this aspect of the invention may also include a package insert indicating that the composition can be used to treat a specific condition. Alternatively or additionally, the article may further include a second (or third) container, which contains a pharmaceutical buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. The article may also include other substances required from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0229] F. Device

[0230] Ocular implants may be used, and in one embodiment, an infusion port delivery device is used to administer the antibodies of the invention to the eye.

[0231] A port delivery device is an implantable, refillable device that can release a therapeutic agent (e.g., an antibody of the invention) over a period of several months (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more months). Exemplary port delivery devices that can be used include those from ForSight Labs, LLC and / or ForSight VISION 4, e.g., as described in International Patent Application Publication Nos. WO 2010 / 088548, WO 2015 / 085234, WO 2013 / 116061, WO 2012 / 019176, WO 2013 / 040247, and WO 2012 / 019047, the entireties of which are incorporated herein by reference.

[0232] For example, the present invention provides an infusion port delivery device, which includes a reservoir containing any antibody described herein. The infusion port delivery device can also include a proximal region, a tubular body coupled to the proximal region that is in fluid communication with the reservoir, and one or more outlets that are in fluid communication with the reservoir and are configured to release the composition into the eye. The outer diameter of the tubular body can be configured to be inserted through an incision or opening of about 0.5mm or less in the eye. The length of the device can be about 1mm to about 15mm (for example, about 1mm, about 2mm, about 4mm, about 5mm, about 6mm, about 7mm, about 9mm, about 11mm, about 13mm or 15mm). The reservoir can have any suitable volume. In some cases, the reservoir has a volume of about 1μI to about 100μI (for example, about 1μI, about 5μI, about 10μI, about 20μI, about 50μI, about 75μI or about 100μI). The device or its components may be made of any suitable material, for example, polyimide.

[0233] In some cases, a port delivery device includes a reservoir containing any of the antibodies described herein and one or more additional compounds.

[0234] In some cases, a port delivery device includes any of the antibodies or antibody conjugates described herein and an additional VEGF antagonist.

[0235] 3. Specific embodiments of the present invention

[0236] Specific embodiments of the present invention are listed below.

[0237] 1. An antibody that binds to human VEGF-A and human IL6, the antibody comprising: a VH domain, the VH domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 18, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 19, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 20; and a VL domain, the VL domain comprising: (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 15, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17; the antibody comprising: (a) a VH domain, the VH domain comprising a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 19, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 20. NO:22 has an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity; and (b) a VL domain comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:21.

[0238] 2. An antibody that binds to human VEGF-A and human IL6, the antibody comprising: a VH domain, the VH domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 18, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 19, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 20; and a VL domain, the VL domain comprising: (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 15, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17; the antibody comprising: a variable heavy chain domain, the variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 22 having up to 5 amino acid substitutions; and a variable light chain domain, the variable light chain domain comprising the amino acid sequence of SEQ ID NO: 21 having up to 5 amino acid substitutions.

[0239] 3. An antibody that binds to human VEGF-A and to human IL6, the antibody comprising: (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 22 having at most 15, at most 10 or at most 5 amino acid substitutions; and (b) a variable light chain domain comprising the amino acid sequence of SEQ ID NO: 21 having at most 15, at most 10 or at most 5 amino acid substitutions.

[0240] 4. An antibody that binds to human VEGF-A and human IL6, comprising the VH sequence of SEQ ID NO: 22 and the VL sequence of SEQ ID NO: 21.

[0241] 5. The antibody according to one of the preceding embodiments, comprising the heavy chain amino acid sequence of SEQ ID NO:24 and the light chain amino acid sequence of SEQ ID NO:23.

[0242] 6. The antibody of any of the preceding embodiments, wherein the VEGF-A paratope comprises amino acid residues from CDR-H2, CDR-L1, and CDR-L3 of said antibody, and wherein the IL6 paratope comprises amino acid residues from CDR-H1, CDR-H3, and CDR-L2 of said antibody, or wherein the IL6 paratope comprises amino acid residues from CDR-H2, CDR-L1, and CDR-L3 of said antibody, and wherein the VEGF-A paratope comprises amino acid residues from CDR-H1, CDR-H3, and CDR-L2 of said antibody; and / or

[0243] The variable light chain domain and variable heavy chain domain pair bind to human VEGF-A and human IL6 simultaneously; and / or

[0244] ● the antibody binds to the same epitope on human VEGF-A and to the same epitope on human IL6 as an antibody having a variable heavy chain domain of SEQ ID NO: 22 and a variable light chain domain of SEQ ID NO: 21; and / or

[0245] The antibody Fab fragment of the antibody binds to: (i) human VEGF-A121, wherein the K measured by surface plasmon resonance D less than 50 pM, and (ii) human IL6, wherein the K measured by surface plasmon resonance is D Less than 50 pM; and / or

[0246] ● an antibody Fab fragment of the antibody exhibits an aggregation onset temperature of 60°C or higher, in one embodiment 70°C or higher; and / or

[0247] - The antibody Fab fragment of this antibody exhibits a melting temperature greater than 80°C as measured by dynamic light scattering.

[0248] 7. An antibody that specifically binds to human VEGF-A and human IL6, comprising the heavy chain amino acid sequence of SEQ ID NO: 24 and the light chain amino acid sequence of SEQ ID NO: 23.

[0249] 8. The antibody according to any one of the preceding embodiments, wherein the antibody is a Fab fragment.

[0250] 9. The antibody according to any one of the preceding embodiments, wherein the antibody is a bispecific antibody fragment.

[0251] 10. The antibody of any preceding embodiment, which is a monoclonal antibody.

[0252] 11. The antibody according to any one of the preceding embodiments, wherein the antibody Fab fragment of said antibody exhibits an aggregation onset temperature of 70°C and above.

[0253] 12. The antibody of any preceding embodiment, wherein an antibody Fab fragment of said antibody exhibits a melting temperature greater than 80°C as measured by dynamic light scattering.

[0254] 13. The antibody of any preceding embodiment, which is a monoclonal antibody.

[0255] 14. The antibody according to any one of the preceding embodiments, which is an antibody fragment that binds to human VEGF-A and to human IL6.

[0256] 15. The antibody of any preceding embodiment, wherein the antibody is bispecific.

[0257] 16. The antibody according to any one of the preceding embodiments, wherein the antibody is a Fab fragment.

[0258] 17. The antibody according to any one of the preceding embodiments, wherein the antibody is a bispecific antibody fragment.

[0259] 18. The antibody of any preceding embodiment, wherein the antibody is a multispecific antibody.

[0260] 19. The antibody of any one of the preceding embodiments, wherein the antibody specifically binds to human VEGF-A.

[0261] 20. The antibody of any preceding embodiment, wherein the antibody specifically binds to human IL6.

[0262] 21. An antibody that binds to human IL6, which binds to the same epitope on IL6 as an antibody having a VL domain of SEQ ID NO: 35 and a VH domain of SEQ ID NO: 36.

[0263] 22. An antibody that binds to human IL6, wherein the antibody comprises: a VH domain having a human VH3 framework, wherein the IL6 paratope comprises amino acid residues 1, 2, 3, 26, 27, 28, 29, 30, 31, 32, 52a, 94, 96, 97, 98, 101, 102 of the antibody that binds to human VEGF-A and IL6 according to any one of Examples 1 to 20; and a VL domain having a human Vκ1 framework, wherein the IL6 paratope comprises amino acid residues 49, 50, 53, 54, 55, 56, 57, 91, 96 of the antibody that binds to human VEGF-A and IL6 according to any one of Examples 1 to 20.

[0264] 23. An antibody that binds to human IL6, the antibody comprising:

[0265] a) a VH domain based on a human VH3 framework, wherein the IL6 paratope comprises amino acid residues Y1, I2, Q3, Y26, E27, F28, T29, H30, Q31, D32, P52a, R94, I96, D97, F98, D101, T102, and a VL domain based on a human Vκ1 framework, wherein the IL6 paratope comprises amino acid residues Y49, D50, S53, N54, Y55, P56, S57, Y91, Y96; or

[0266] b) a VH domain based on a human VH3 framework, wherein the IL6 paratope comprises amino acid residues Y1, P2 、Q3、 V26 , L27 、F28、 K29 , H30, Q31, D32, P52a, R94, L96 、D97、F98、D101、 E102 , and a VL domain based on the human Vκ1 framework, wherein the IL6 paratope comprises amino acid residues Y49, D50, D53 , R54 、Y55、P56、 E57 , Y91, Y96 (according to Kabat numbering).

[0267] 24. An isolated nucleic acid encoding the antibody of any one of embodiments 1 to 23.

[0268] 25. A host cell comprising the nucleic acid of embodiment 24.

[0269] 26. A method for producing an antibody that binds to human VEGF-A and to human IL6, comprising culturing the host cell according to embodiment 25, thereby producing the antibody.

[0270] 27. The method of embodiment 26, wherein the host cell is a CHO cell.

[0271] 28. A pharmaceutical formulation comprising the antibody of any one of embodiments 1 to 23 and a pharmaceutically acceptable carrier.

[0272] 29. A port delivery device comprising the antibody of any one of embodiments 1 to 23.

[0273] 30. The antibody according to any one of embodiments 1 to 23 for use as a medicament.

[0274] 31. The method of embodiment 26, further comprising recovering the antibody from the host cell.

[0275] 32. An antibody produced by the method of embodiment 26 or 31.

[0276] 33. A pharmaceutical formulation comprising the antibody of any one of embodiments 1 to 23 and a pharmaceutically acceptable carrier.

[0277] 34. The antibody according to any one of embodiments 1 to 23 for use as a medicament.

[0278] 35. The antibody of any one of embodiments 1 to 23 for use in treating a vascular disease.

[0279] 36. The antibody of any one of embodiments 1 to 23 for use in treating an ocular vascular disease.

[0280] 37. Use of the antibody according to any one of embodiments 1 to 23 or the pharmaceutical composition according to embodiment 65 in the preparation of a medicament.

[0281] 38. Use of the antibody according to any one of embodiments 1 to 23 or the pharmaceutical composition according to embodiment 65 in the preparation of a medicament for inhibiting angiogenesis.

[0282] 39. A method of treating an individual suffering from a vascular disease, comprising administering to the individual an effective amount of the antibody of any one of embodiments 1 to 23 or the pharmaceutical formulation of embodiment 33.

[0283] 40. A method of treating a subject having an ocular vascular disease, comprising administering to the subject an effective amount of the antibody of one of embodiments 1 to 23 or the pharmaceutical formulation of embodiment 33.

[0284] 41. A method of inhibiting angiogenesis in an individual, comprising administering to the individual an effective amount of the antibody of any one of embodiments 1 to 23 or the pharmaceutical formulation of embodiment 33 to inhibit angiogenesis.

[0285] 42. A port delivery device comprising the antibody of any one of embodiments 1 to 23 or the pharmaceutical formulation of embodiment 33.

[0286] 43. The antibody according to any one of embodiments 1 to 23 or the pharmaceutical formulation of embodiment 33 for ocular administration via a port delivery device.

[0287] 44. The antibody of any one of embodiments 1 to 23 or the pharmaceutical formulation of embodiment 33, for ocular administration via the port delivery device of embodiment 42, wherein administration is within a period of six months or more; in one embodiment, 8 months or more; in one embodiment, 9 months or more prior to refilling the port delivery device.

[0288] 45. The antibody of any one of embodiments 1 to 23 or the drug formulation of embodiment 33, for use as a medicament for administration of said antibody or said drug formulation by use of an infusion port delivery device, wherein said antibody is applied to said infusion port delivery device at a concentration of 150 mg / ml or more; in one embodiment, at a concentration of 200 mg / ml or more.

[0289] Description of amino acid sequence

[0290]

[0291]

[0292]

[0293]

[0294]

[0295]

[0296]

[0297] Examples

[0298] The following examples are provided to aid the understanding of the present invention, the true scope of the invention being set forth in the appended claims.It should be understood that modifications may be made to the procedures set forth without departing from the spirit of the present invention.

[0299] Example 1:

[0300] Generation of bispecific anti-VEGF / anti-6 Fab fragments

[0301] The bispecific anti-VEGF / anti-IL-6 Fab fragments were generated by providing antibodies with separate, non-overlapping paratopes binding to VEGF and IL-6 using methods similar to those described previously (e.g., methods described in WO2012 / 163520).

[0302] Here, two different phage display libraries of synthetic Fab fragments were utilized, wherein in the first phage display library, residues within the CDR-H1, CDR-H3, and CDR-L2 regions of the Fab fragments were diversified, and wherein in the second phage display library, residues within the CDR-L1, CDR-L3, and CDR-H2 regions of the Fab fragments were diversified. In each library, the corresponding other three CDR regions remained non-diversified and—in contrast to the method of WO2012 / 163520, which used invariant non-binding, germline-like ("virtual") sequences—represented a paratope capable of binding to VEGF-A.

[0303] In the case of the first library, the paratope capable of binding to VEGF-A is derived from the VEGF-A binding paratope described in WO 2021 / 198034.

[0304] In the case of the second library, the VEGF-A binding paratope was obtained as follows:

[0305] For initial selection, phage library panning was performed with a library in which CDR-H1, CDR-H3 and CDR-L2 were diversified, as described in WO2012 / 163520. The remaining CDR sequence was kept constant using non-bound germline sequences. Four rounds were performed, wherein the first round was performed using 100nM biotinylated VEGF-121 or VEGF-165 pre-fixed on Dynabeads M-280 streptavidin (Thermofisher catalog number 11206D). The 75nM, 15nM and 3nM biotinylated targets in the solution were used to perform the 2nd to 4th round of panning, and then the Fab / target complex on the phage was captured on Dynabeads M-280 streptavidin. The phage / target / bead complex was repeatedly washed with PBST and PBS buffer. Captured phage clones bearing target-specific Fab were eluted from M-280 beads using 100 mM DTT according to standard protocols, used to infect log-phase TG1 E. coli cells, and rescued using M13 K07 helper phage.

[0306] For the screening of selection output, the polyclonal plasmid small preparation of the corresponding selection round is prepared by the TG1 E. coli cells of infection. The plasmid is reformatted to produce soluble Fab in the E. coli supernatant, and the soluble Fab has a T7 tag at the C-terminal end of the Fab CH1 domain. The connection polyclonal plasmid encoding T7-tagged Fab is transformed into TG1 E. coli cells (Zymo Research catalog number T3017), and single colonies are picked into microtiter plates. Soluble Fab is expressed in microtiter plates, and the supernatant is clarified by centrifugation. Target binding is evaluated by ELISA measurement for VEGF and competitive ELISA for VEGF receptor 2. The selection of candidate binding substances is based on the high binding signal to VEGF and the good inhibition of receptor binding.

[0307] The binder was subsequently expressed and purified in a larger volume and the binding to VEGF was assessed using SPR measurements. One of the clones obtained was further optimized by an iterative protein engineering and testing strategy and integrated into a phage display library as an invariant sequence for CDR-H1, CDR-H3 and CDR-L2. In brief, the protein engineering workflow consisted of initial rounds of scout mutagenesis to identify relevant beneficial mutations, followed by two consecutive rounds of affinity maturation based on oligonucleotide-based mutation library generation and phage display-based selection, followed by screening and further testing.

[0308] In both libraries, the CH1 domain of the Fab fragments was fused to the truncated gene III protein via a linker to facilitate phage display. Thus, one library was designed to screen for bispecific Fab fragments in which the IL6 paratope comprised amino acid residues from CDR-H1, CDR-H3, and CDR-L2 (referred to herein as the "6HVL" library), while the other library was designed to screen for bispecific Fab fragments in which the IL6 paratope comprised amino acid residues from CDR-H2, CDR-L1, and CDR-L3 (referred to herein as the "VH6L" library).

[0309] By panning the phage libraries, binders to human IL-6 were enriched in each library. After panning, plasmid minipreps were generated for the two enrichment pools of phagemid vectors. The minipreps were digested with restriction enzymes to excise the region encoding the truncated gene III protein and recircularized by ligation to obtain a pool of expression vectors encoding soluble Fab fragments that were enriched for IL-6 binders. These vector pools were transformed into TG1 E. coli cells and single colonies were picked and cultured to solublely express single Fab clones in microtiter plates. Supernatants containing soluble Fab fragments were screened for binding to IL-6 and VEGF-A using standard ELISA methods.

[0310] Based on the screening data, bispecific anti-VEGF / anti-IL-6 Fab fragments were selected and DNA plasmid preparation and sequencing of TG1 clones producing specific binders were performed to obtain VH and VL sequence pairs that together encode a bispecific Fab fragment that specifically binds to both IL-6 and VEGF-A from each library:

[0311] These clones were 6HVL_1, characterized by a heavy chain of SEQ ID NO:03 and a light chain of SEQ ID NO:04, and VH6L_1, characterized by a heavy chain of SEQ ID NO:09 and a light chain of SEQ ID NO:10.

[0312] Example 2:

[0313] Expression and characterization of bispecific anti-VEGF / anti-IL-6 Fab fragments 6HVL_1 and VH6L_1

[0314] The resulting bispecific anti-VEGF / anti-IL-6 Fab fragments were characterized. The vectors obtained as described in Example 1 were transformed into TG1 E. coli cells, and separate colonies were cultured for soluble expression of the bispecific antibody Fab fragments for both 6HVL_1 and VH6L_1. The bispecific antibodies were purified from 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 IL6, human VEGF121, and human VEGF165 was assessed.

[0315] Example 3:

[0316] Characterization of the bispecific anti-VEGF / anti-IL-6 Fab fragments 6HVL_1 and VH6L_1 IL-6 binding kinetics assessed by surface plasmon resonance (SPR):

[0317] 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 disclosed herein.

[0318] The binding of the corresponding Fab fragment IL-6 from human and cynomolgus monkey was analyzed by SPR at 25°C on a Biacore 8K instrument (Cytiva) using HBS-EP+ (1x; BR100669; Cytiva) as running buffer. The 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 chip using standard amine coupling chemistry. This immobilization procedure yielded a ligand density of approximately 5000 resonance units (RU). The reference channel was treated accordingly.

[0319] Before protein measurement, five startup cycles were carried out for adjustment purposes. In each cycle, HBS-EP+ buffer was made to flow for about 120 seconds, then by applying 10mM glycine buffer pH2.0 for 60 seconds to regenerate the derivatization chip surface. The antibody Fab fragment with a concentration of 75nM was captured on this surface at a flow velocity of 10ul / min for 60 seconds in HBS-EP+ buffer. Fab fragment was not applied to the reference channel. Subsequently, people or cynomolgus monkey IL-6 (contact time 180 seconds, dissociation time 720 seconds) were applied in HBS-EP+ buffer with a flow velocity of 30ul / min. The regeneration of the derivatization chip surface was achieved as described above. 8K evaluation software (Biacore Insight Evaluation 3.0) was used to evaluate data. Dual reference was used and 1:1 binding model was used to fit raw data.

[0320] Figure 1 Representative SPR traces and fitting curves determined for the tested Fab fragments are shown, and the corresponding Fab names are provided in the figures. Data are depicted for binding to human and cynomolgus IL-6 and to IL-1α (IL-1a) as a negative control. The affinities provided in the figures correspond to the mean and standard deviation of three independent experiments.

[0321] We observed significant binding of 6HVL_1 and VH6L_1 to human IL-6. Only VH6L_1 showed significant affinity for cyIL-6, although the dissociation rate was apparently very fast. No binding to the negative control target IL-1a was observed. The SPR data fitting results are shown in Table 1. The data are the average of three experiments and the standard deviation of the dissociation constant KD is provided. For 6HVL_1, we observed an affinity of KD = 0.9 nM, while for VH6L_1, the affinity was KD = 10.7 nM.

[0322] Table 1: Affinity of the indicated antibodies for human and cynomolgus monkey IL6

[0323]

[0324] VEGF binding assessed by competition ELISA:

[0325] To test which antibody concentration is needed to block the interaction of VEGF121 and VEGF165 with their receptors, competition ELISA experiments were performed using 6HVL_1 and VH6L_1. The VEGF-binding Fab fragment ranibizumab was used as a positive control, and experiments using only buffer were used as negative controls. In brief, a 1:3 dilution series of all samples (starting from 20nM) was mixed with a constant concentration of 10pM VEGF121 (Humanzyme HZ-1206) or 10pM VEGF165 (Humanzyme HZ-1153) and incubated for 90 minutes. Then, after blocking the Maxisorp plate surface with 2% MPBST, the mixture was transferred to a Maxisorp plate coated with VEGF receptor 1 (VEGF-R1, R&Dsystems, 1μg / ml, in NaHCO3, pH 9.4). The contact time between the Fab-antigen mixture and the receptor-coated plate was limited to 10 minutes at room temperature to minimize interference with the binding equilibrium. After incubation and 2 washing steps, VEGF121 / VEGF165 on VEGFR1-coated plates were detected using biotinylated anti-VEGF mAb (BAF203, R&D systems) and horseradish peroxidase-labeled streptavidin (HRP-streptavidin). The latter was detected by color conversion to 3,3',5,5'-tetramethylbenzidine diamine using the HRP substrate 3,3',5,5'-tetramethylbenzidine TMB, followed by an absorbance change at 450 nm. TMB was pre-warmed to room temperature and incubated on the plate for 5 minutes, then quenched by the addition of 1N H2SO4.

[0326] The results of the target VEGF165 are as follows Figure 2 As shown in Tables 5 and 6. Obviously, compared with the clinically mature VEGF-A antagonist ranibizumab, both VH6L_1 and 6HVL_1 showed greatly improved ability to compete with both VEGF165 and VEGF121 for binding to VEGFR1.

[0327] Example 4:

[0328] Improvement of bispecific anti-VEGF-A / anti-IL6 Fab fragment

[0329] As described above, both antibodies showed no or low cross-reactivity with cynomolgus monkey IL6, but this cross-reactivity is desirable for clinical development. In addition, treatment of ocular vascular diseases 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 durability of the therapeutic effect and patient convenience. Therefore, further improvement of the initially identified molecules is required for the intended purpose.

[0330] Several rounds of maturation were performed by introducing different amino acid substitutions in the VH and VL domains. During maturation, candidate antibodies derived from the two "parental" antibodies 6HVL_1 and VH6L_1 were screened and selected based on their desired properties in terms of yield, affinity, simultaneous antigen binding, hydrophilicity, stability, viscosity and other parameters.

[0331] Improved candidate antibodies 6HVL_2, 6HVL_3 and 6HVL_4 as well as VH6L_2 and VH6L_3 were selected from candidate antibody molecules from multiple tests in each round of maturation. Candidate selection was based on the desired properties, in particular, improved human IL6 binding and cynomolgus IL6 cross-reactivity, while ensuring injectability at high concentrations and maintaining other favorable properties, such as VEGF-A affinity and thermal stability.

[0332] The improved candidate antibody 6HVL_4 was selected as the preferred candidate from multiple tested candidate antibody molecules.

[0333] Table 2: Amino acid sequences of the indicated bispecific Fab fragments (numbers refer to SEQ ID NOs used herein)

[0334] V L VH Light chain Heavy chain 6HVL_2 5 6 6HVL_3 7 8 VH6L_2 11 12 VH6L_3 13 14 6HVL_4 21 22 23 24 6HVL_4_YHE 25 26

[0335] All Fab fragments include the same constant regions as those contained in the full-length light chain and heavy chain amino acid sequences of antibody VH6L_4 (ie, CL having SEQ ID NO: 29 and CH1 having SEQ ID NO: 30).

[0336] Candidate antibodies were expressed as described in Example 2.

[0337] Example 5:

[0338] Improved antigen binding kinetics of anti-VEGF-A / anti-IL6 Fab fragments

[0339] As described above, the binding kinetics of the candidate antibodies to human and cynomolgus monkey IL6 and the competition IC50 for VEGF / VEGFR1 competition were assessed using the Fab fragments shown (amino acid sequences as shown in Table 2 and Example 2). In order to determine the efficacy 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 WO2012 / 163520, referred to herein as "VH6L-BM"), anti-VEGF antibody ranibizumab (INN), and anti-IL6 antibodies that cross-react between human and cynomolgus monkey IL6, as disclosed in WO2014 / 074905 (positive control). The above-mentioned prior art antibodies were prepared by recombinant expression.

[0340] Figure 3 The results of the evaluation of human and cynomolgus IL6 binding are shown in Tables 3 and 4. 6HVL_4 and 6HVL_4-YHE (a variant of 6HVL_4 with three additional framework amino acid mutations) showed improved human IL6 binding and cynomolgus IL6 cross-reactivity within the pharmacologically relevant range compared to the initially selected parental molecule.

[0341] Table 3: SPR human IL6

[0342] <![CDATA[K D [nM]]]> SD[nM] 6HVL_1 0.927 0.042 6HVL_2 0.036 0.003 6HVL_3 0.043 0.013 6HVL_4 0.069 0.002 6HVL_4_YHE 0.065 0.002 VH6L_1 10.7 0.3 VH6L_2 0.157 0.003 VH6L_3 0.191 0.027 VH6L_BM 3.5 0.6 Positive Control 0.035 0.007

[0343] Table 4: SPR cynomolgus monkey IL6

[0344]

[0345]

[0346] *No signal available for evaluation

[0347] Figure 4 And Tables 5 and 6 show the results of the assessment of VEGF binding using human VEGF121 and human VEGF165 by competition ELISA. Figure 4 It is shown that the prior art molecule 6HVL_BM exhibits an affinity that is too low to be detected under the assay conditions and is therefore certainly much lower than the affinity of the antibodies of the invention.

[0348] Table 5: IC50 VEGF121

[0349] IC50[pM] SEM[nM] 6HVL_1 173 12 6HVL_2 194 13 6HVL_3 171 11 6HVL_4 458 30 6HVL_4_YHE 528 35 VH6L_1 26 2 VH6L_2 38 3 VH6L_3 20 1 Ranibizumab 503 25

[0350] Table 6: IC50 VEGF165

[0351]

[0352]

[0353] Example 6:

[0354] Simultaneous binding of anti-VEGF / anti-IL-6 Fab fragments

[0355] Using immobilized anti-Fab antibodies to capture the anti-VEGF / anti-IL-6 Fab fragments of the invention, simultaneous binding of the antibodies of the invention to their targets was assessed by surface plasmon resonance as follows:

[0356] Approximately 5000 resonance units (RU) of anti-Fab antibody (Cytiva 28958325) were immobilized to S-series 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 running and dilution buffer, and the temperature of the flow cell was set to 25°C.

[0357] By injecting 10 μg / mL solution at a flow rate of 5 μL / min for 30 seconds, anti-VEGF / anti-IL-6Fab fragments are captured via κ chains to form anti-Fab antibody / anti-VEGF / anti-IL-6Fab complexes. Two antigens are added sequentially or simultaneously: human VEGFA121 (in-house production, P1AA1779-010) and human IL-6 (commercial, Peprotech#200-06), to allow the formation of a complex comprising anti-Fab antibody, anti-VEGF / anti-IL-6Fab, human VEGFA and human IL-6. The corresponding SPR response unit curve (BiacoreT200, Cytiva) is monitored. For sequential binding, a concentration of 300nM human VEGFA is injected for 180 seconds, and then a concentration of 300nM human IL-6 is injected for 180 seconds. The same concentration is also injected in reverse order (first injecting human IL-6, then injecting human VEGFA). Similarly, a mixture of two antigens is injected for 180 seconds at a concentration of each 300nM. After each experiment, the surface was regenerated by injecting 10 mM glycine pH 2.1 for 60 sec at a flow rate of 5 μL / min. Large refractive index deviations were corrected by subtracting blank injections and by subtracting the response obtained from a control flow cell without captured Fab.

[0358] The results are Figure 7. Addition of human VEGF-A to the anti-Fab / anti-VEGF / anti-IL-6Fab complex causes binding and formation of the anti-Fab / Fab / VEGF-A complex. Continuous addition of human IL-6 causes the formation of the anti-Fab / DutaFab / VEGF-A / IL-6 complex (dashed line). This clearly demonstrates that simultaneous binding of human VEGF-A and human IL-6 to anti-VEGF / anti-IL-6Fab is possible.

[0359] In the reverse order, human IL-6 was added first, followed by human VEGF-A, and a decrease in binding was evident (dashed line). This suggests that the binding of human IL-6 first sterically interferes with the binding of human VEGF-A, thereby reducing the binding capacity between anti-VEGF / anti-IL-6 Fab and human VEGF-A, but it is still possible.

[0360] In the presence of both targets, binding of human IL-6 appears to be preferred and binding to human VEGF-A is reduced (solid line). This effect is plausible because the anti-VEGF / anti-IL-6 Fab has a higher intrinsic affinity for human IL-6 than for human VEGF-A.

[0361] In another assay, blocking of VEGF-R2 by anti-VEGF / anti-IL-6 Fab fragments in the presence of IL-6 was assessed by an inhibition assay using surface plasmon resonance using immobilized VEGF-A:

[0362] To show the simultaneous binding of human VEGF-A and human IL-6 to anti-VEGF / anti-IL-6 Fab fragments, human VEGF receptor 2 (VEGFR2, commercial R&D Systems 357-KD) was immobilized to an S-series 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.

[0363] As a reference, a 1:2 dilution series of 0-200nM anti-VEGF / anti-IL-6Fab fragment in a 50nM human VEGFA solution was used, and VEGFA and VEGFR2 were tested for inhibition. Anti-VEGF / anti-IL-6Fab fragment / VEGFA mixture was injected onto the immobilized VEGFR2 surface at a flow rate of 5 μL / min for 30 seconds. After a dissociation period lasting 60 seconds, VEGFR2 surface regeneration was performed by injecting 5mM NaOH at a flow rate of 5 μL / min for 30 seconds. Large refractive index deviations were corrected by subtracting blank injections and by subtracting the response obtained from the blank control flow cell. For evaluation, the binding response was taken five seconds after the injection ended. The derivative response in RU was converted into a binding response relative to the initial signal corresponding to the ligand without bispecific Fab. IC50 values ​​were calculated using a 4-parameter logistic model (XLfit, ID Business Solutions Ltd.).

[0364] In addition to the reference, dilutions of 0-200 nM anti-VEGF / anti-IL-6 Fab fragments in solution in the presence of 10 nM human IL-6 were pre-incubated for 15 min and tested to calculate IC50 values ​​( Figure 3 ).

[0365] The results are Figure 8 . The figure shows that the inhibition of VEGFR2 / VEGF-A interaction depends on the concentration of competing anti-VEGF / anti-IL-6Fab. In the absence of anti-VEGF / anti-IL-6Fab, 100% VEGFR2 / VEGF-A binding was achieved (0% inhibition), while increasing the concentration of anti-VEGF / anti-IL-6Fab increased the inhibition (solid line with a cross). The addition of human IL-6 simulating treatment-related conditions did not affect the degree of inhibition of VEGFR2 / VEGF-A and produced very similar IC50 values ​​(IC50 = 33nM, without human IL-6 (dashed line with triangles); IC50 = 37nM, containing additional human IL-6 (black dashed line with triangles)).

[0366] In a third assay, the effect of VEGF binding on IL6 activity was assessed by a cell-based IL-6-specific reporter gene assay as follows:

[0367] To assess simultaneous binding of human VEGFA and human IL-6 to anti-VEGF / anti-IL-6 Fab, the reporter cell line HEK-Blue TM Cell-based IL-6-specific reporter gene assay using IL-6 cells (InvivoGen). In the absence and presence of excess human VEGF-A, the cells were simultaneously added with ( Fig. 9) or after preincubation of bispecific Fab and human IL-6 ( Fig.10 ), cells were incubated with anti-VEGF / anti-IL-6 Fab and human IL-6 for 20 + / - 1 hours. TM The binding of the receptor IL-6R on the surface of IL-6 cells triggers a signal transduction cascade through the tyrosine kinase of the Janus family (JAK1, JAK2 and Tyk2), thereby causing activation of signal transducers and activators of transcription 3 (STAT3) and subsequent secretion of SEAP (secreted embryonic alkaline phosphatase). In the case where anti-VEGF / anti-IL-6Fab is combined with human IL-6, signal transduction is inhibited and SEAP is not produced. Subsequently, the SEAP level in the cell culture supernatant is quantified by adding QUANTI-Blue SEAP substrate to the supernatant aliquot. SEAP converts QUANTI-Blue substrate into a product that can be measured at 650nm absorbance using an ELISA reader. Then the simultaneous binding of human VEGFA and human IL-6 is assessed by drawing a graph of average absorbance and anti-VEGF / anti-IL-6Fab concentration, and the data is fitted to a constrained 4-parameter curve. The relative efficacy (inhibitory concentration) of the sample is calculated using a 4-parameter logistic curve fitting.

[0368] The results are Fig. 9 and Fig.10 Shown in.

[0369] Fig. 9 Results without pre-incubation are shown. Titration of increasing amounts of anti-VEGF / anti-IL-6 Fab showed a clear dose response curve with the calculated IC 50 The value was 1.134 ng / mL (about 22.5 pM), indicating that the human IL-6 response was significantly inhibited by increasing the amount of anti-VEGF / anti-IL-6 Fab. To address the simultaneous binding of human IL-6 and VEGFA to the bispecific Fab, both target molecules were incubated simultaneously and the human IL-6 effect was measured. Regardless of the selected ratio of human VEGFA:human IL-6 (1:1 / 2.5:1 / 5:1), only effective IC 50 The value was slightly lower than the IC value in the absence of human VEGFA. 50 =1.134 ng / mL to IC when human VEGF-A is present in 5-fold excess 50 =1.724 ng / mL, which varies slightly and closely reflects the relevant conditions in vivo.

[0370] Fig.10 Results with pre-incubation are shown, indicating that binding of IL6 did not affect IL6 binding.

[0371] Example 7:

[0372] Binding of anti-VEGF / anti-IL-6 Fab fragments to IL-6 determined by X-ray crystallography and proposed mode of action

[0373] IL-6 signaling is initiated by forming a hexameric complex of IL6 with its non-signaling co-receptor IL6R and cytokine receptor gp130. Here, three epitopes (sites 1, 2 and 3) have been defined to identify the contact surface formed in the complex (Boulanger MJ et al., Science 2003, 27; 300 (5628): 2101-4.). IL-6 first binds to IL-6R through an interaction surface called "site 1". "Site 2" is an epitope that interacts with domains 2 and 3 of gp130 formed by a binary complex of IL-6 and IL-6R. Subsequently, the interaction between "site 3" of IL6 and domain 1 of gp130 causes the formation of a dimer of IL6 / IL6R / gp130 trimers, and thus forms a hexameric signaling complex.

[0374] In order to understand which epitopes of IL6 bind to our two Fab series (6HVL and VH6L), we performed structural analysis on the complex between IL-6 and the antibody Fabs representing the anti-VEGF / anti-IL-6 Fab fragments of the present invention, respectively. The Fab 6HVL4.1 used is very closely related to Fab 6HVL_2, and the difference is only two mutations, and Fab 0182 is most closely related to Fab VH6L_1. Due to the fact that all 6HVL clones are derived from the same Fab (6HVL_1), and all VH6L clones are also derived from Fab VH6L_1, respectively, it can be safely assumed that the structural results obtained below are applicable to each of the individual series of Fab. A complex of Fab and IL-6 was formed, and the analysis of the complex structure was performed by X-ray crystallography as follows:

[0375] The IL6-Fab complex was prepared by mixing equimolar amounts of Fab fragment 0182 (light chain amino acid sequence SEQ ID NO: 33, heavy chain amino acid sequence SEQ ID NO: 34) or 6HVL4.1 (light chain amino acid sequence SEQ ID NO: 37, heavy chain amino acid sequence SEQ ID NO: 38) with IL-6 (PeproTech, batch number 031316-2), respectively.

[0376] After incubation on ice for 90 minutes, the protein complexes were concentrated to 23.1 mg / ml for Fab fragment 0182 and 21.3 mg / ml for 6HVL4.1. Initial crystallization trials were performed at 21°C in a sitting drop vapor diffusion apparatus.

[0377] For Fab fragment 0182, needle-shaped crystals appeared within two days from 0.1M MgCl2, 0.1M sodium citrate pH 5, 15% (w / v) PEG 4000. The crystals were subsequently used for seeding experiments, and large tetragonal crystals could be obtained from 0.1M calcium acetate, 12% (w / v) PEG 8000, 0.1M sodium cacodylate pH 5.5.

[0378] For 6HVL4.1, rhombic crystals emerged from 0.2M ammonium sulfate, 0.1M Tris pH 7.5 pH and 20% (w / v) PEG MME5000 within one day.

[0379] For data collection, the collected crystals were rapidly cooled in a crystallization solution supplemented with 15% ethylene glycol at 100 K. The data were acquired using a PILATUS 6M detector at beamline X10SA of the Swiss Light Source (Villigen, Switzerland). (for Fab fragment 0182) and X-ray diffraction data were collected at a wavelength of (6HVL4.1). The data have been processed using XDS (Kabsch, W., XDS. Acta Cryst. D66, 125-132 (2010)), scaled using AIMLESS (PREvans and G.N. Murshudov "How good are my data and what is the resolution?" Acta Cryst. (2013). D69, 1204–1214) and analyzed using STARANISO (Tickle, I.J., Flensburg, C., Keller, P., Paciorek, W., Sharff, A., Vonrhein, C., Bricogne, G. (2018). http: / / staraniso.globalphasing.org / cgi-bin / staraniso.cgi ).Cambridge,United Kingdom:Global Phasing Ltd.)Analysis of anisotropy.

[0380] The crystals of the complex including Fab 0182 belong to space group P21, in which the unit cell axes are β=91.65°, and diffracts to resolution.

[0381] The crystals of the complex including 6HVL4.1 belong to the space group P212121, in which the unit cell axes are And diffracted to resolution.

[0382] The structure was determined by molecular replacement using PHASER (McCoy, AJ, Grosse-Kunstleve, RW, Adams, PD, Winn, MD, Storoni, LC, & Read, RJ Phaser crystallographic software. J Appl Cryst. 40, 658-674 (2007)) using the coordinates of the internal Fab and IL-6 (pdb entry 1 alu) as a search model. Depending on the sequence differences, the difference electron density was used to change the amino acids. The structure was refined using programs from the CCP4 suite (Winn, MD et al. Overview of the CCP4 suite and current developments. Acta. Cryst. D67, 235-242 (2011)) and BUSTER (Bricogne, Blanc, GE, Brandl, M., Flensburg, C., Keller, P., Paciorek, W., Roversi, P., Sharff, A., Smart, O.S., Vonrhein, C., Womack, T.O. Buster version 2.9.5 Cambridge, United Kingdom: Global Phasing Ltd. (2011)). Manual reconstruction was done using COOT (Emsley, P., Lohkamp, ​​B., Scott, WG, Cowtan, K. Features and Development of Coot. Acta Cryst. D66, 486-501 (2010)).

[0383] Data collection and optimization statistics are summarized in Table 7. All graphical presentations were performed using PyMOL (Pymol molecular graphics system, version 1.7.4. LLC.)

[0384] Table 7 Data collection and refinement statistics

[0385]

[0386]

[0387] *Values ​​in parentheses are for the highest resolution shell.

[0388] Structure of Fab 0182-IL-6 complex

[0389] by The crystal structure of Fab 0182 (a representative of the VH6L series of Fabs) in complex with IL-6 was determined at a resolution of Figure 5 ). The structure shows that Fab 0182 binds to IL-6 through the contribution of CDR2 of the heavy chain and CDR1 and CDR3 of 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 contributed by IL-6 is formed by the residues of helix A and helix C.

[0390] Fig.16 The binding mode of Fab 0182 to IL6 is shown. For the purpose of illustration, we generated a superposition of two structures: first, the superposition of the complex structure of Fab and IL6, and second, the superposition of the complex structure of IL6R and IL6 (taken from the co-crystal structure of IL6, IL6R and gp130, pdb accession number is 1p9m, see Boulanger MJ et al., Science 2003, 27; 300 (5628): 2101-4). Comparing it with the trimeric complex of IL6, IL6R and gp130, it is obvious that Fab binds to IL6 in a very similar way to gp130, i.e., it binds to site 2 of IL6. It is expected that this binding mode allows both Fab and IL6R to bind to IL6 at the same time, i.e., the interaction of IL6 with IL6R should still be possible, and it is a priori expected that this IL6 antagonist will play a role by inhibiting the interaction of IL6 / IL6R complex with gp130 via the binding to site 2 of IL6.

[0391] Structure of Fab 6HVL4.1-IL-6 complex

[0392] We The crystal structure of Fab 6HVL4.1 in complex with IL-6 was determined at a resolution of Figure 6 ). The structure shows that Fab 6HVL4.1 binds to IL-6 through the major contribution of CDR1, CDR3 of the heavy chain and CDR2, CDR3 of the light chain. Further interaction with IL-6 is maintained by the first three N-terminal residues of the heavy chain of Fab 6HVL4.1. The interface contributed by IL-6 is formed by the residues of helix A and helix C.

[0393] Similar to what was done for Fab 0182, we analyzed the binding mode of Fab 6HVL4.1 to IL6 using structural superposition and by analyzing the interacting residues as described above. Fig.17showed that 6HVL4.1 also binds to IL6 in a very similar manner to gp130 and must therefore be considered a site 2 binder from a structural point of view.

[0394] Experimental study on the binding mode of IL6

[0395] The fact that clones like 6HVL4.1 and their derivatives are IL6 site 2 binders could be functionally confirmed by assays using surface plasmon resonance.

[0396] In one such assay, Fab fragments representing the cloned 6HVL series, including 6HVL4.1 (antibody "P1AE2421"), were captured on the SPR chip surface via anti-Fab antibodies and IL6, and then three different concentrations (250nM, 500nM, 1000nM) of IL6R were flowed over the chip surface. Here, if IL6 could bind to both Fab and IL6R, we would expect a two-step sequential signal increase. And indeed, for the tested Fabs, the following was observed ( Fig.18 ): In the SPR signal trace, the addition of IL6 caused a strong increase in the signal, and the signal was further enhanced after the addition of IL6R. This clearly shows that simultaneous binding of IL6R and Fab to IL6 is possible. This finding is further confirmed by the fact that a covalent chimera of IL6 and IL6R (called "hyper-IL6", in which site 1 of IL6 is completely shielded and inaccessible) still promotes the binding of the corresponding Fab molecules when coated onto the SPR chip and probed with Fab at a concentration of 26nM ( Fig.19 ).

[0397] However, when an ELISA experiment was used to investigate whether the captured Fab fragments competed with IL6 for binding to IL6R, we obtained surprising results. The assay was set up as follows: First, a constant concentration of IL6 was pre-incubated with a titration series of the Fab fragment P1AE2421, which was also used for the SPR experiment and represents the cloned 6HVL series. It was then incubated on an ELISA plate directly coated with IL6R. After washing, IL6 bound to the plate-bound IL6R was detected with a biotinylated anti-IL6 antibody using horseradish peroxidase-labeled streptavidin (Strep-HRP). In this assay ( Fig. 20 ), the results we observed strongly suggest that the Fab almost completely inhibits the IL6 / IL6R interaction.

[0398] Given that we know from available crystal structures and SPR experiments that site 1 of IL6 is still available for binding, we must interpret these results so that the IL6 antibodies described in this patent not only sterically block the binding of the IL6 / IL6R complex to gp130, but also Allosteric mode Strongly reduces the binding affinity of IL6 to IL6R, ie additionally functionally acts as an IL6 site 1 antagonist.

[0399] This mode of action is expected to have optimal properties due to the following reasons:

[0400] 1. As IL6 site 2 binders, IL6 antagonists are equally effective in blocking the formation of a signaling complex formed by the binding of IL6 to membrane-bound IL6R and gp130 (cis signaling) or a preformed complex of IL6 and IL6R (trans signaling). In contrast, IL6 site 1 binders are unable to bind to the preformed complex of IL6 and IL6R but can antagonize it only when the complex is dissociated.

[0401] 2. Due to the allosterically reduced affinity of IL6 for IL6R, the IL6 antagonists described herein are expected to exhibit increased potency by disfavoring the formation of the first step of signaling (i.e., formation of the IL6 / IL6R complex) compared to site 2 binders that do not exhibit this effect. Site 2 binders that do not allosterically interfere with site 1 binding are expected to have disadvantages, particularly for cis signaling, i.e., when the second step of IL6 / IL6R-gp130 complex formation on the cell surface must be blocked, relatively effective concentrations of IL6 / IL6R and gp130 are expected to be very high.

[0402] 3. It is known that IL6 site 1 binders will lead to a strong accumulation of IL6 complexes with the antibody when used systemically, as the half-life of the complex is greatly increased compared to IL6 alone. In contrast, IL6 site 2 binders are expected to still eliminate the IL6 / antibody complex by binding to membrane-bound IL6R and subsequent internalization and degradation by cells that take up the complex. In this regard, it is expected that the IL6 antagonists described herein will combine the desired properties of both site 1 and site 2 binders: while functionally capable of blocking the first step of IL6 / IL6R / gp130 signaling complex formation, they still allow degradation of the IL6 / mAb complex via IL6R binding on cells.

[0403] 4. In ophthalmic indications and as Fab molecules, the expected behavior of such binders may still be more beneficial: similar to IL6 site 1 binders, the Fab / IL6 complex can leave the ocular space relatively unhindered by IL6R binding and can be rapidly eliminated systemically via renal filtration.

[0404] Example 8:

[0405] Improved thermal stability of anti-VEGF / anti-IL6 Fab fragments

[0406] Further sequence variants of improved anti-VEGF / anti-IL-6 antibodies were generated, including the amino acid sequences identified in Table 8.

[0407] Table 8: Amino acid sequences of the indicated bispecific Fab fragments (numbers refer to SEQ ID NOs used herein)

[0408] V L VH 6HVL_5 39 40 6HVL_6 41 42 VH6L_4 43 44 VH6L_5 45 46

[0409] The thermal stability of the indicated bispecific antibodies was assessed as follows.

[0410] Thermal stability:

[0411] Samples of bispecific antibody Fab fragments at a concentration of 1 mg / mL were prepared in 20 mM histidine / histidine chloride, 140 mM NaCl (pH 6.0) and transferred to a 10 μL microcuvette array. Static light scattering data and fluorescence data were recorded after 266 nm laser excitation using a UNcle instrument (UnchainedLabs) while the sample was heated from 30°C to 90°C at a rate of 0.1°C / min. Samples were measured three times.

[0412] The evaluation of the onset temperature was done by UNcle analysis software. The aggregation onset temperature was defined as the temperature at which the scattering intensity began to increase. The denaturation of the protein was monitored by the shift of the center of gravity mean (BCM) of the fluorescence signal with heat. The melting temperature was defined as the inflection point in the plot of BCM (nm) versus temperature.

[0413] Table 9: Thermal stability

[0414]

[0415]

[0416] Example 9:

[0417] Improved biophysical properties of bispecific anti-VEGF / anti-IL6 Fab fragments (viscosity assessment by dynamic light scattering (DLS))

[0418] Antibody Fab fragments as described above were expressed in CHO cells by standard methods.

[0419] Viscosity was measured by latex bead DLS method as previously described (He F et al.; Anal Biochem. 2010 Apr 1; ​​399(1): 141-3). Specifically, the following protocol was followed using the indicated materials.

[0420] Viscosity Assessment:

[0421] Instruments and Materials

[0422] Wyatt DLS plate reader with Greiner Bio-One microplate

[0423] ·3000 Series Nanosphere TM Size Standards (Thermofisher Cat. No. 3300A)

[0424] Tween 20 (Roche, catalog number 11332465001) and silicone oil, for example (Alfa Aesar catalog number A12728)

[0425] UV photometer for concentration determination (e.g. Nanodrop 8000).

[0426] Sample preparation

[0427] The antibody samples were rebuffered and diluted with 20 mM His / HCl (pH 5.5) (buffer) and 0.02% Tween 20 (final concentration). Beads were added at a solid concentration of 0.03%. At least three different concentrations were prepared, with the highest concentration being about 200 mg / mL where possible. Two blank samples were required as no antibody controls: one containing nanosphere beads resuspended in water and the other containing nanosphere beads resuspended in buffer. The samples were transferred to a microplate and each well was covered with silicone oil.

[0428] Measurements were performed using a Wyatt DLS microplate reader

[0429] All samples and blanks were analyzed at different temperatures from 15° C. to 35° C. in steps of 5° C. The acquisition time was 30 seconds and the number of acquisitions for each sample and temperature was 40.

[0430] Data analysis

[0431] The raw data Dapp (apparent radius) in nm is shown in the overview of the software template (Microsoft Dynamics 7.10 or higher). The viscosity is 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 (300nm). The calculated viscosity is shown in the Excel curve. Using Mooney curve fitting (in Excel), the viscosity at a given concentration can be inferred. Here, the maximum protein concentration at which the viscosity exceeds 20cP is calculated.

[0432] The maximum concentrations of the indicated antibodies that achieved a viscosity of 20 cP at 20°C are shown below.

[0433] Table 10: Viscosity measured by DLS beads method. The maximum feasible concentration of the indicated antibodies to achieve 20 cP at 20°C is shown.

[0434] Concentration [mg / ml] 6HVL_4 195.4 6HVL_5 192.4 VH6L_3 220.0 VH6L_4 226.8 VH6L_5 240.4

[0435] The results show that the antibodies of the invention can be formulated at high concentrations comprising viscosities below the acceptable viscosity limit for injectability. As a result, the antibodies of the invention are very suitable for intraocular applications because they allow high molar doses to be provided in a limited injection volume, which, when combined with high potency, results in high durability and thus reduced dosing frequency, which is desirable for increased patient convenience and treatment compliance.

[0436] Example 10:

[0437] Primary cell-based assay (HRMEC) to demonstrate VEGF / IL6 bispecific antibody 6HVL_4-mediated IL6 inhibition

[0438] In order to measure the IL-6 signaling activity in HRMEC, the determination of ICAM-1 surface expression in quantified HRMEC was established. The combination of human IL-6 and human IL-6R with equimolar concentration (2nM) was used to stimulate HRMEC for 72 hours. ICAM-1 surface expression was assessed by flow cytometry. In order to measure the inhibitory activity of 6HVL_4, before being applied to cells, the IL-6 / IL-6R mixture was pre-incubated with antibodies whose concentrations gradually increased.

[0439] Cell culture: HRMECs (Catalog No. PEL-PB-CH-160-8511; PELOBiotech Gmbh; Bayern, Germany) were thawed and cultured in 175 cm2 flasks in endothelial cell growth medium (EGM-MV), which contained endothelial cell basal medium (EBM) (Catalog No. CC-3156; Lonza; Basel, Switzerland) and 5% fetal bovine serum (FBS), hydrocortisone, human fibroblast growth factor B, VEGF, R3-IGF-1 (a recombinant analog of insulin-like growth factor-I in which Arg at position 3 is replaced by Glu), ascorbic acid, human epidermal growth factor, and GA-1000 (all included in the EGM-2MV Microvascular Endothelial Cell SingleQuotsTM Kit; Catalog No. CC-4147; Lonza) at concentrations recommended by the manufacturer. Twenty-four hours after plating, the medium was replaced with fresh EGM-MVs and the cells were grown for an additional 3 days before the assay. Assay conditions were optimized for different passage numbers and IL-6 / soluble IL-6R concentrations. The final assay was performed using HRMEC at passage 6 and equimolar stimuli of IL-6 / soluble IL-6R at a concentration of 2 nM.

[0440] Flow cytometry assay: HRMECs were separated from the flask by washing twice with phosphate buffered saline (PBS) without Ca2+ and Mg2+ (Cat. No. 10010023; Life Technologies) and once with cell dissociation reagent Accutase (Cat. No. A1110501; Thermo Fisher Scientific; Waltham, MA). After washing, 5 mL of cell dissociation reagent was added to the cells, and the flask was incubated at 37°C in a 5% CO2 incubator for 3 minutes. The separated cells were collected from the flask and placed in a 50 mL conical centrifuge tube. The tube was filled to 50 mL with EBM containing 2% FBS and centrifuged at 300g for 6 minutes. The supernatant was discarded and the pellet was resuspended in 5 mL of starvation medium (EBM containing 2% FBS). The number of cells was quantified using a TC20 automated cell counter (Bio-Rad; Hercules, CA) and adjusted to 300,000 cells / mL using starvation medium. Then, 100 μL of the cell suspension was added to each well of a Costar 96-well plate (Cat. No. 3596; Corning; Corning, NY) to yield 30,000 cells / well. The plate was then incubated for another 24 hours at 37° C. in a 5% CO 2 incubator.

[0441] Recombinant human IL-6 (Catalog No. 206-IL / CF; R&D Systems; Minneapolis, MN) and recombinant human IL-6R (Catalog No. 227-SR-025; R&D Systems) were mixed in starvation medium at equimolar concentrations and incubated at room temperature for 1 hour to allow the formation of IL-6-I / L-6R complexes. Next, 50 L of a 6HVL_4 dilution series (3-fold 7-point dilution) was added to the cells and incubated at 37° C., 5% CO2 for 1 hour. Finally, 50 μL of the IL-6-I / L-6R complex was added to the cells, resulting in a final concentration of 2 nM each of IL-6 and IL-6R, and a final concentration of 200.009 nM of 6HVL_4. Unstimulated cells and cells stimulated with IL-6-I / L-6R complexes without 6HVL_4 were also included to determine background ICAM-1 surface expression and 100% response levels, respectively. The cells were incubated at 37°C in a 5% CO2 incubator for 72 hours.

[0442] To analyze ICAM-1 surface expression, PBS (Ca 2+ Mg 2+ ; Life Technologies) washed cells twice and washed once with cell dissociation reagent Accutase (Cat. No. A1110501; Thermo Fisher Scientific). Cells were separated from the plate using 50 μL cell dissociation reagent (3 minutes, 37°C) and transferred to a flow cytometry Falcon 96-well storage plate (Cat. No. 353263; Corning). The original wells were washed once with 100 μL PBS containing 2% FBS and 2mM EDTA, and the wash medium containing the remaining cells was added to the flow cytometry plate. Cells were precipitated by centrifugation at 300 g for 6 minutes, and the supernatant was discarded. The precipitate was resuspended in 100 μL PBS, 2% FBS, 2mM EDTA containing 10 μg / mL human IgG (Cat. No. I2511; MilliporeSigma; Burlington; MA) to block nonspecific binding sites and incubated at room temperature for 15 minutes. After blocking, 0.5 μg of fluorescein-labeled anti-ICAM-1 antibody (Catalog No. BBA20; R&D Systems) was added to the cells and the reaction was incubated at 28°C for 45 minutes. After staining, the cells were pelleted by centrifugation at 300 g for 6 minutes and the pellet was resuspended in 150 μL PBS containing 2% FBS and 2 mM EDTA. Fluorescence was measured using an Attune NxT flow cytometer (ThermoFisher Scientific).

[0443] Data analysis: For all 3 determinations, each condition in each independent experiment was performed in quadruplicate. For each experiment, the background signal of unstimulated cells was subtracted from the experimental wells and the average signal of each condition was calculated. 100% response level was calculated from cells stimulated with IL-6 / IL-6R without 6HVL_4, and then the inhibition potential of 6HVL_4 was expressed as the inhibition percentage of 100% response. The inhibition percentage of 6HVL_4 for each concentration was measured in 3 independent experiments, and the mean value and SEM were calculated. The mean value of 3 independent experiments was used to calculate the average IC50 and SE using ExcelXLfit software version 5.5.0 (IDBS; Guildford, UK). Concentration response curves were fitted by nonlinear regression analysis using a 5-parameter logistic model (A+((BA) / (1+(((BE)*((C / x)^D)) / (EA))))).

[0444] Table 11: Calculated % inhibition of IL-6-induced ICAM-1 expression on the surface of HRMEC by 6HVL_4

[0445]

[0446]

[0447] 6HVL_4 caused a dose-dependent inhibition of IL-6 signaling in HRMECs, with a 50% inhibitory concentration (IC50) of 1.52 + / - 0.04 nM ( Fig.11 ).

[0448] Example 11:

[0449] Primary cell-based assay (HUVEC) to demonstrate VEGF / IL6 bispecific antibody 6HVL_4-mediated VEGF inhibition

[0450] Determination:

[0451] HUVECs were obtained from Lonza (Cat. No. 00191027; Basel, Switzerland). Endothelial cell basal medium (EBM-2; Cat. No. CC-3156), which together comprised endothelial cell growth medium (EGM-2) and assay medium (EBM-2, containing 0.5% fetal bovine serum [FBS]), and the EGM-2 Endothelial Cell SingleQuots Kit (Cat. No. CC4176) were also purchased from Lonza.

[0452] T175 cell culture flasks (Catalog No. 353112; Corning; Corning, NY) coated with attachment factor (AF) (Catalog No. S-006-100; Gibco, Thermo Fisher Scientific; Waltham, MA) were used to maintain HUVECs. StemProAccutase (Catalog No. A11105-01; Gibco) was used to detach the cells.

[0453] Cell viability / proliferation assays were performed using alamarBlue (Cat. No. DAL1100; Invitrogen, Thermo Fisher Scientific) in 96-well fibronectin-coated plates (Cat. No. 354409; Corning).

[0454] Recombinant human VEGF-A was obtained from R&D (Catalog No. 293-VE; Minneapolis, MN) and dissolved in Ca-free medium at a stock concentration of 100 μg / mL. 2+ and Mg 2+ The cells were then added to phosphate buffered saline (PBS) (Cat. No. 14190-094; Gibco).

[0455] Alamarblue contains the cell permeable compound resazurin. This compound changes color in response to the reducing environment within healthy cells. The resulting pink color is a ratiometric marker of viable cells and can be used to detect proliferation by measuring absorbance at 570 nm. VEGF-A induces proliferation of HUVECs grown under conditions of cell starvation. Therefore, VEGF-A-induced HUVEC proliferation can be inhibited by using VEGF-A neutralizing antibodies or Fabs.

[0456] HUVECs were maintained in EGM-2 in T175 flasks coated with AF until the 5th generation. For viability assays, HUVECs were separated using Accutase and diluted 1:1.66 in assay medium (EBM-2 0.5% FBS). The cells were then centrifuged and resuspended in EBM-2 containing 0.5% FBS to a cell density of 100,000 cells / mL. 100 μL of the cell suspension was then inoculated onto a fibronectin-coated 96-well plate to produce a cell density of 10,000 cells / well. The outer wells were not inoculated with cells and were then filled with assay medium only. The cells were incubated overnight at 37°C in a 5% CO2 incubator.

[0457] The next day, VEGF-A stock solution (100,000 ng / mL in PBS (Ca2+, Mg2+)) was used to prepare a 10x working solution (750 ng / mL) in assay medium (EBM 0.5% FBS).

[0458] The 6HVL_4 stock solution was also diluted in assay medium to prepare a 10-fold working solution. This was used to prepare a 3-fold 8-point dilution series starting at 30,000 ng / mL and ending at 14 ng / mL.

[0459] Next, 12.5 μ L 10 pre-diluted 6HVL_4 solution and 12.5 μ L 10VEGF-A solution (750 ng / mL) are added to the cells in each plate in turn, in quadruplicate. VEGF-A is used with a constant final concentration (75 ng / mL), and 6HVL_4 is used in a dose response form, with a final concentration range of 3000 ng / mL to 1.4 ng / mL. Cells are incubated for 72 hours at 37 ° C, 5% CO2. For analysis, 12 μ L alamarBlue is added to each well, and then incubated for 3 hours in a cell culture box. FlexStation 3 microplate readers from Molecular Devices are used to measure absorbance at 570 nm, with a reference wavelength of 600 nm.

[0460] Data Analysis:

[0461] For each experiment, each condition was carried out in quadruplicate. A total of 4 independent experiments were performed. Independent experiments were considered to be 2 separate plates processed on the same day. Therefore, 8 separate plates were used for analysis. The background signal of unstimulated cells was subtracted from the experimental wells and the average signal of each condition was calculated. 100% response level was calculated from cells stimulated by VEGF-A (75 ng / mL) without additional compound exposure, and the signal from 6HVL_4 exposure wells was expressed as the inhibition percentage of 100% response.

[0462] IC50 values ​​were calculated from the mean data of 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 relative to basal and maximum inhibitory activity. Data are presented as the mean of 4 independent experiments with standard error of the mean (SEM).

[0463] Table 12: Calculated % inhibition of VEGF-A-induced HUVEC proliferation by 6HVL_4

[0464] concentration inhibition[%] SEM [%] 62.4 87.8 5.3 20.8 87.8 6.5 6.9 90.9 5.3 2.3 58.4 8.5 0.77 6.6 6.3 0.26 7.1 3.1 0.09 7.0 2.0

[0465] 6HVL_4 reduced VEGF-A-induced HUVEC proliferation with a 50% inhibitory concentration (IC50) of 2.06+ / -0.30 nM ( Fig.12).

[0466] Example 12:

[0467] Restoration of barrier function in the presence of both VEGF-A and IL6 as well as the VEGF / IL6 bispecific antibody 6HVL_4 demonstrates the biological activity of this molecule

[0468] In order to assess the dual biological activity of the antibodies of the invention, i.e., simultaneous blocking of two targeted cytokines (VEGF-A and IL6 (complexed with IL6R)), a transendothelial electrical resistance (TER) assay was performed. In this assay, the electrically dense endothelial cell layer responds to the addition of both VEGF-A and IL6 by losing barrier function. The restoration of barrier function by the VEGF / IL6 bispecific antibody 6HVL-4 in the presence of two cytokines, VEGF and IL6, was assessed as follows:

[0469] Determination:

[0470] Human retinal microvascular endothelial cells (PELO Biotech; catalog number PEL-PB-CH-160-8511), further designated as HRMVECs, were maintained in complete MV endothelial cell growth medium (MV-EGM-2 Lonza, catalog number CC-3202) in T175 flasks (Falcon catalog number 353112) coated with attachment factor (Ginco, catalog number S-006-100) until passage 5. For transendothelial electrical resistance assays, 10 μg / mL of 10 μg / mL of 2% endothelial cells were used. (Gibco, catalog number A11105-01) to separate cells. Thereafter, the cells were seeded into the upper chamber of a fibronectin-coated (catalog number 354008, Corning) Transwell filter (24-well Corning, catalog number 3470) in 100 μl MV-EGM-2 growth medium at a cell density of 120.000 cells / well. The lower chamber of the Transwell filter was filled with 600 μl MV-EGM-2 culture medium. The cells were incubated at 37°C and 5% CO2 for 3 days. The culture medium was then changed to the assay conditions (MV-EGM-2 without VEGF, containing 2% FBS), and the Transwell filter was transferred to the cellZcope system using 280 μl culture medium (upper chamber) and 810 μl culture medium (lower chamber). The cells were then incubated at 37°C and 5% CO2 for 24 hours while measuring TER by cellZcope. The next 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 amount of assay medium (eight times per condition), and TER was measured until the next day. Thereafter, 6HVL-4 or aflibercept at a final concentration of 1 μg / ml, 2.3 μg / ml, or assay medium were added to the cells, and TER was subsequently measured for the next 24 hours. Each condition therefore occurred four times.

[0471] Data Analysis:

[0472] The data set generated from one well was normalized to the TER value obtained shortly before the addition of the cytokine mix. For each condition, the mean signal and standard deviation were calculated from the normalized data.

[0473] result:

[0474] The results are Fig.12 (6HVL_4) and 13 (Aflibercept) are shown.

[0475] The barrier function of HRMVEC was reduced by using the cytokine VEGF alone and in combination with IL6 / IL6R. When the antibody 6HVL_4 was used, the disrupted barrier was restored to 100% after 24 hours.

[0476] Example 13:

[0477] Identification of the IL6 complementary region

[0478] The amino acid residues that contact IL6 were identified from the crystal structure of the complex between 6HVL4.1 and IL6. The positions of the amino acid residues in the paratope within the VH and VL domains are illustrated as follows Fig.15 As shown. To this end, residues in the Fab / IL6 complex that could potentially interact with IL6 were identified using the “byres” function of PyMOL and a cutoff distance of 5 Å. Here, we restricted the analysis to residues 48-215 of IL6 (as defined in Uniprot ID P05231), which are residues found to be commonly resolved in the structure of IL6 alone (see pdb accession numbers 1alu and 1IL6). Fig.15 Also shown is an alignment between 6HVL4.1 and a monospecific anti-IL6 antibody 6HdL2.05 based on an antibody of the invention, wherein the VEGF paratope is replaced by a non-binding region. 6HdL2.05 has a VH domain of SEQ ID NO: 48 and a VL domain of SEQ ID NO: 47. When expressed and purified as described in Example 2 and subjected to an SPR assay using human IL6 or cynomolgus monkey IL6 similar to that performed in Example 3, the antibody exhibited Fig. 20 SPR sensorgrams depicted. After fitting the experimental data, 6HdL2.05 exhibited an affinity comparable to the highest affinity obtained for the corresponding 6HVL series of VEGF / IL6 bispecific antibodies (see Tables 3 and 4), with a fitted KD of 22 pM for human IL6 and 1.3 nM for cynomolgus monkey IL6.

[0479] The amino acid residues identified as contributing to antigen binding are identified in Table 13 (for variable heavy chain domain amino acid residues) and Table 14 (for variable light chain domain amino acid residues). Amino acid positions are numbered according to the Kabat numbering system (in Figure 1 +5). Amino acid positions involved in antigen binding are identified by their Kabat position in the VH or VL domain.

[0480] Table 13: Shows the amino acid residues at the same Kabat positions for 6HdL2.05, i.e. the variable domain amino acid residues involved in IL6 binding identified by crystal structure analysis

[0481]

Claims

1. An antibody that binds to human VEGF-A and human IL6, the antibody comprising: a VH domain, the VH domain comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 18, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 19, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 20; and a VL domain, the VL domain comprising: (d) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 15, (e) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, and (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17; the antibody comprising: a variable heavy chain domain, the variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 22 with up to 5 amino acid substitutions; and a variable light chain domain, the variable light chain domain comprising the amino acid sequence of SEQ ID NO: 21 with up to 5 amino acid substitutions.

2. The antibody according to claim 1, wherein the at most 5 amino acid substitutions occur in the FR region of the corresponding 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 the preceding claims, 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 IL6, comprising the VH sequence of SEQ ID NO: 22 and the VL sequence of SEQ ID NO:

21.

6. An antibody that binds to human IL6, said antibody binding to the same epitope on IL6 as an antibody having a VL domain of SEQ ID NO: 35 and a VH domain of SEQ ID NO:

36.

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

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

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

10. A host cell comprising the nucleic acid according to claim 9.

11. A method for producing an antibody that binds to human VEGF-A and human IL6, the method comprising culturing the host cell according to claim 10, thereby producing the antibody. The method according to claim 11 , wherein the host cell is a CHO cell.

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

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

15. The antibody according to any one of claims 1 to 8 for use as a medicament.

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

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