HK40114363A - Antibody that binds to vegf-a and il6 and methods of use - Google Patents

Description

Antibodies that bind to VEGF-A and IL6 and their usage

Technical Field

This invention relates to anti-VEGF-A/anti-IL6 antibodies and their methods of use.

Background Technology

Antibodies that bind to VEGF (e.g., ranibizumab) are used as therapeutic agents for the treatment of ocular vascular diseases such as age-related macular degeneration. Antibodies that bind to IL6 (e.g., as disclosed in WO2014/074905) have been proposed for the treatment of ocular diseases.

WO2012/163520 discloses a bispecific antibody containing two complementary sites within a pair of VH and VL domains (“DutaFab”). Each complementary site of the bispecific antibody of WO2012/163520 contains amino acids from the heavy chain and from the light chain CDR, wherein heavy chain CDR-H1 and CDR-H3 and light chain CDR-L2 constitute the first complementary site, while light chain CDR-L1 and CDR-L3 and heavy chain CDR-H2 constitute the second complementary site. Monospecific antibodies containing each complementary site are independently isolated from different Fab libraries, which are diverse in both the first and second complementary sites. The amino acid sequences of the monospecific antibodies are identified and merged into VH and VL pairs with dual complementary sites. WO2012/163520 discloses an exemplary Fab fragment, referred to as “VH6L”, which binds specifically to VEGF and IL-6 as a proof-of-concept example and has the VL sequence of SEQ ID NO:01 and the VH sequence of SEQ ID NO:02.

There is indeed a need for improved therapeutic antibodies that bind to VEGF and IL6 for clinical application in ocular diseases, for example, by improving efficacy compared to standard care and by improving the duration of action and consequently reducing the frequency of intravitreal injections to reduce the burden of administration to patients.

Summary of the Invention

This invention relates to a bispecific anti-VEGF-A/anti-IL6 antibody and its method of use.

In one aspect, the present invention relates to an antibody that binds to human VEGF-A and 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-H3 comprising the amino acid sequence of SEQ ID NO:15. The antibody comprises: (e) CDR-L1 containing the amino acid sequence of SEQ ID NO:16, (f) CDR-L3 containing 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.

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

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

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

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

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

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

a) VH domain based on the human VH3 framework, where the IL6 complementary site contains amino acid residues.

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

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

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

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

In one aspect, the present invention provides a host cell comprising the nucleic acid of the present invention. In one embodiment, the host cell is a CHO cell. In another embodiment, the host cell is an Escherichia coli cell.

In one aspect, the present invention provides an expression vector comprising the nucleic acid of the present invention.

In one aspect, the present invention provides a method for generating antibodies that bind to human VEGF-A and human IL6, the method comprising culturing the host cells of the present invention to generate antibodies.

In one aspect, the present invention provides antibodies generated by the method of the present invention.

In one aspect, the present invention provides a pharmaceutical formulation comprising the antibody and pharmaceutical carrier of the present invention.

In one aspect, the present invention provides a pre-filled syringe comprising the antibody and pharmaceutical carrier of the present invention.

In one aspect, the present invention provides an ocular implant comprising the antibody of the present invention and a pharmaceutical carrier. In one embodiment, the present invention includes a port delivery device comprising the antibody of the present invention.

In one aspect of the invention, the infusion port delivery device administers an antibody or pharmaceutical preparation.

In one aspect, the present invention provides an antibody for use as a medicine, and in one embodiment, for use in the treatment of vascular diseases.

In one aspect, the present invention provides the use of the antibody or pharmaceutical composition of the present invention in the preparation of a medicament, in one embodiment of which is a medicament for treating vascular diseases.

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

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

According to the present invention, a therapeutic anti-VEGF-A/anti-IL6 antibody is provided, which is capable of independently binding to its target antigen even when provided in the form of an antibody Fab fragment. It exhibits excellent KD and species cross-reactivity with the cynomolgus monkey target within the pharmacologically relevant range. The antibody of the present invention is suitable for the treatment of ocular vascular diseases. The antibody of the present invention provides several valuable properties, including good expressibility and developability for its therapeutic applications (e.g., high binding potency, high biophysical and biochemical stability, high-concentration formulations), particularly supporting high affinity for both targets at low effective doses, and high stability favorable for long durations. Compared to non-antibody methods, the antibody of the present invention tends to be more acceptable due to its high humanization and lack of artificial domains and linkers. Furthermore, the antibody of the present invention is advantageously provided in a high-concentration liquid formulation with a viscosity suitable for ocular application. Because it can be provided in high concentrations, treatment using the antibody of the present invention is more acceptable to patients, as a higher dose of therapeutic agent can be applied in a single treatment, thus allowing for longer treatment cycles. Bispecific Fab fragments, such as those described in this invention, offer additional advantages over full-length bispecific IgG antibodies due to their significantly lower molecular weight. While providing the same number of binding sites, the molecular weight of Fab is approximately 50 kDa, whereas the full-length antibody weighs three times as much (approximately 150 kDa). Therefore, for a given amount of drug, a bispecific Fab fragment will contain three times as many binding sites as a full-length IgG antibody.

Attached Figure Description

Figure 1: Binding of parental bispecific antibodies 6HVL_1 and V6HL_1 to human and cynomolgus monkey IL6, as determined by surface plasmon resonance.

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

Figure 3: Binding of the improved bispecific antibody to human and cynomolgus monkey IL6 as determined by surface plasmon resonance.

Figure 4: VEGF IC50 of the improved bispecific antibody obtained using human VEGF-121 and VEGF-165.

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

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

Figure 7: Simultaneous binding of anti-VEGF/anti-IL-6Fab to its target as assessed by SPR using immobilized anti-Fab antibodies

Figure 8: Blocking effect of anti-VEGF/anti-IL-6Fab on VEGF-R2 binding in the presence of IL-6, as assessed by SPR using immobilized VEGF-A.

Figure 9: Evaluation of the effect of VEGF binding on IL-6 activity by cell-based IL-6 specific reporter gene assay (without pre-incubation)

Figure 10: Evaluation of the effect of VEGF binding on IL-6 activity by cell-based IL-6-specific reporter gene assay (with pre-incubation) as follows.

Figure 11: Suppression of IL-6 signaling in HRMEC by 6HVL_4

Figure 12: Dose-dependent changes in the inhibition rate (%) of 6HVL_4 on VEGF-A-induced HUVEC proliferation.

Figure 13: Recovery of IL6/IL6R/VEGF-induced HRMVEC barrier disruption by 6HVL_4

Figure 14: Recovery of IL6/IL6R/VEGF-induced HRMVEC barrier disruption by aflibercept

Figure 15: Amino acid sequences of the VH and VL domains of the antibody shown. Kabat numbers of the amino acid positions are displayed. The amino acid positions that contribute to the IL6 complementation site identified in Example 13 are highlighted with black boxes.

Figure 16: Image of the IL6/IL6R/gp130 complex (top; pdb-acc.#1p9m) compared with the superimposed structure of Fab0182 bound to IL6 and the structure of the IL6/IL6R complex from pdb-acc.1p9m (bottom).

Figure 17: Image of the IL6/IL6R/gp130 complex (top; pdb-acc.#1p9m) compared with the superimposed structure of Fab6HVL4.1 bound to IL6 and the structure of the IL6/IL6R complex from pdb-acc.1p9m (bottom).

Figure 18: SPR bridging experiments investigating the ability of IL6R to bind with IL6 when involved in a pre-formed complex with Fab P1AE2421.

Figure 19: SPR binding experiment to study the ability of Fab P1AE2421 to bind to human "hyper-IL6" (a chimera of human IL6 and IL6R).

Figure 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.

Figure 21: Binding of IL6-binding antibody 6HdL2.05 to human and cynomolgus monkey IL6, as determined by surface plasmon resonance.

Detailed Implementation

1. Definition

Unless otherwise defined herein, scientific and technical terms related to this invention shall have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms shall include plural terms, and plural terms shall include singular terms. The methods and techniques of this disclosure are generally performed according to conventional methods known in the art. Typically, 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.

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

When the term “about” is used in conjunction with specific values (e.g., temperature, concentration, time, etc.) in this article, it should refer to a change of +/-1% of the specific value referred to by the term “about”.

The term “antibody” is used in the broadest sense and covers a wide range of antibody structures, including but not limited to monoclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity.

"Isolated" antibodies are antibodies that have been separated from components in their natural environment. In some embodiments, antibodies are purified to a purity greater than 95% or 99%, as determined by methods such as 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, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

As used herein, the term "monoclonal antibody" refers to an antibody derived from a substantially homogeneous group of antibodies; that is, individual antibodies comprising this group are identical and/or bind to the same epitopes, except for possible variant antibodies (e.g., those containing naturally occurring mutations or generated during the production of the monoclonal antibody formulation, which are typically present in small quantities). In contrast to polyclonal antibody formulations, which typically comprise different antibodies targeting different determinants (epitaxes), each monoclonal antibody in a monoclonal antibody formulation targets a single determinant on the antigen. Therefore, the modifier "monoclonal" indicates that the antibody is characterized by being derived from a substantially homogeneous group of antibodies and should not be interpreted as requiring the antibody to be produced by any particular method.

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

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

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

The term "variable region" or "variable domain" refers to a domain of the antibody heavy or light chain involved in antibody-antigen binding. The variable domains (VH and VL, respectively) of the heavy and light chains of natural antibodies typically have similar structures, with each domain containing four conserved frame regions (FRs) and three hypervariable regions (HVRs) (see, for example, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., p. 91 (2007)). In the antibodies of this invention, a single VH and VL domain pair, i.e., a homologous VH/VL pair, specifically binds to its two targets: VEGF-A and IL6.

"DutaFab" is a bispecific antibody disclosed in WO2012/163520. In DutaFab, a single VH and VL domain pair specifically binds to two distinct epitopes, one complementary site containing amino acid residues from CDR-H2, CDR-L1, and CDR-L3, and the other complementary site containing amino acid residues from CDR-H1, CDR-H3, and CDR-L2. DutaFab contains two non-overlapping complementary sites within homologous VH/VL pairs and can bind to two different epitopes simultaneously. WO2012/163520 discloses DutaFab and a method for generating it by screening libraries containing a single-specific Fab fragment.

"Human antibody" is an antibody whose amino acid sequence corresponds to that of antibodies produced by humans or human cells, or to non-human antibodies derived from a complete human antibody library or other human antibody-coding sequences. This definition of human antibody specifically excludes humanized antibodies containing non-human antigen-binding residues. Antibodies or antibody fragments isolated from human antibody libraries in this paper are considered human antibodies or human antibody fragments.

The “human common framework” is a framework that represents the most frequently present amino acid residues in the selection of the human immunoglobulin VL or VH framework sequence. Generally, the selection of the human immunoglobulin VL or VH sequence is derived from a subgroup of variable domain sequences. Typically, the sequence subgroups are those listed in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, NIH Publication 91-3242, Bethesda MD (1991), Volumes 1-3. In one embodiment, for VL, this subgroup is subgroup κI as listed in Kabat et al. (ibid.). In one embodiment, for VH, this subgroup is subgroup III as listed in Kabat et al. (ibid.).

An "antibody fragment" refers to a molecule other than a complete antibody that contains a portion of the complete antibody that binds to the antigen bound by the complete antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; bisomatic antibodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

As used interchangeably herein, "complementary site" or "antigen-binding site" refers to a portion of an antibody that recognizes and binds to an antigen. A complementary site is formed by several single amino acid residues arranged spatially adjacently in the tertiary structure of the Fv region, derived from the variable domains of the antibody heavy and light chains. The antibody of this invention contains two complementary sites in a homologous VH/VL pair.

As used herein, “VEGF-A complementary site” is a complementary site or antigen-binding site that binds to VEGF-A. The VEGF-A complementary site of the antibody of the present invention comprises amino acid residues from the antibody’s CDR-H2, CDR-L1, and CDR-L3.

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

Unless otherwise specified, as used herein, the term "vascular endothelial growth factor" (abbreviated as "VEGF") means any naturally occurring VEGF from any vertebrate source, including mammals (such as primates (e.g., humans)) and rodents (e.g., mice and rats). The term covers "full-length" unprocessed VEGF, as well as any form of VEGF produced through cellular processing. The term also covers naturally occurring variants of VEGF, such as splice variants or allelic variants. An exemplary amino acid sequence of human VEGF is shown in SEQ ID NO:27.

The terms "anti-VEGF-A antibody" and "VEGF-A-binding antibody" refer to antibodies that bind to anti-VEGF-A with sufficient affinity, making them suitable for use as diagnostic and/or therapeutic agents targeting VEGF-A. In one embodiment, for example, as measured by surface plasmon resonance (SPR), the binding degree of the anti-VEGF-A antibody to unrelated, non-VEGF-A proteins is less than about 10% of the antibody's binding to VEGF-A. In some embodiments, the VEGF-A-binding antibody has a dissociation constant (KD) of 1 nM, ≤0.1 nM, or ≤0.01 nM. When the _KD of an antibody is 1 μM or less, the antibody is said to "specifically bind" to VEGF-A.

Unless otherwise specified, as used herein, the term "interleukin-6" (abbreviated as "IL6") means any naturally occurring 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 through cellular processing. The term also covers naturally occurring variants of IL6, such as splice variants or allelic variants. An exemplary amino acid sequence of human IL6 is shown in SEQ ID NO:28.

The antibody of the present invention "binds simultaneously to human VEGF-A and human IL6," meaning that (a) the antibody Fab fragment of the present invention, which binds to human IL6, also specifically binds to human VEGF-A, and (b) the antibody Fab fragment of the present invention, which binds to human VEGF-A, also specifically binds to human IL6. Simultaneous binding can be assessed using methods known in the art, such as surface plasmon resonance as described herein.

As used herein, the term “complementarity-determining region” or “CDR” refers to each region of the antibody variable domain that is sequence-hypervariant 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 stated, CDR residues and other residues (e.g., FR residues) in the variable domain are numbered in this document 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).

As used herein, “framework” or “FR” refers to the variable domain amino acid residues other than the CDR residues. A variable domain framework typically consists of four frame domains: FR1, FR2, FR3, and FR4. Therefore, the CDR and FR amino acid sequences typically appear in (a) the VH domain as: FR1—CDR-H1—FR2—CDR-H2—FR3—CDR-H3—FR4; and in (b) the VL domain: FR1—CDR-L1—FR2—CDR-L2—FR3—CDR-L3—FR4.

"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 stated, as used herein, "binding affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its partner Y can generally be represented by the dissociation constant (K_{_D} ). Affinity can be measured by conventional methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity will be described herein.

The term "epitope" refers to a site on a protein or non-protein antigen that an antibody binds to. Epitopes can be formed from continuous amino acid elongations (linear epitopes) or from discontinuous amino acid sequences (conformal epitopes), for example, due to antigen folding, i.e., spatial proximity due to the tertiary folding of protein antigens. Linear epitopes typically remain bound to antibodies after exposure to a denaturing agent, while conformational epitopes are usually destroyed upon treatment with the denaturing agent. Epitopes contain at least 3, at least 4, at least 5, at least 6, at least 7, or 8 to 10 amino acids in a unique stereoconformation.

Screening for antibody binding can be performed using methods conventional in the art, such as, but not limited to, alanine scanning, Western 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).

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

Furthermore, competitive binding can be used to readily determine whether an antibody binds to the same VEGF-A or IL6 epitope as the reference antibody of the present invention, or to compete for binding to the reference antibody of the present invention. For example, "antibody binding to the same epitopes 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 its respective competitive assay, and conversely, the reference antibody blocks the binding of the antibody to its antigen by 50% or more in its respective competitive assay. Similarly, for example, to determine whether an antibody binds to the same epitope as a reference antibody, the reference antibody is bound 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 evaluated. If the antibody in question is able to bind to VEGF-A or IL6 after saturation binding with the reference antibody, it can be concluded that the antibody in question binds to a different epitope than the reference antibody. However, if the antibody in question cannot bind to VEGF-A or IL6 after saturation binding with the reference antibody, then 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 blocked due to steric hindrance, standard experiments can be used (e.g., peptide mutation and binding assays using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art). This 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.

In some embodiments, such as as measured in a competitive binding assay, if one antibody inhibits the binding of another antibody by at least 50%, at least 75%, at least 90%, or even 99% or higher by 1, 5, 10, 20, or 100-fold excess, the two antibodies are considered to bind to the same or overlapping epitopes (see, for example, Junghans et al., Cancer Res. 50 (1990) 1495-1502).

In some embodiments, if substantially all amino acid mutations in the antigen that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of another antibody, then the two antibodies are considered to bind to the same epitope. If only a subset of amino acid mutations that reduce or eliminate the binding of one antibody reduces or eliminates the binding of another antibody, then the two antibodies are considered to have “overlapping epitopes.”

The "percentage of amino acid sequence identity (%)" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to those in the reference polypeptide sequence after aligning the candidate sequence with the reference polypeptide sequence and introducing vacancies (if necessary) to achieve the maximum percentage of sequence identity, and for alignment purposes without considering any conserved substitutions as part of sequence identity. Alignment used to determine the percentage of amino acid sequence identity can be performed in various ways within the scope of the art, such as using publicly available computer software, such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software, or the FASTA package. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared. Alternatively, the sequence comparison computer program ALIGN-2 can be used to generate the percentage of identity values. The ALIGN-2 sequence comparison computer program was written by Genentech, and the source code has been submitted with the user documentation to the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registry No. TXU510087 and described in WO 2000/005319.

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

The terms "nucleic acid molecule" or "polynucleotide" include any compound and/or substance comprising a polymer of nucleotides. Each nucleotide consists of bases, particularly purine or pyrimidine bases (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate ester group. Typically, nucleic acid molecules are described by a base sequence, where the bases represent the primary structure (linear structure) of the nucleic acid molecule. Base sequences are typically represented from 5' to 3'. In this document, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA) (including, for example, complementary DNA (cDNA) and genomic DNA), ribonucleic acid (RNA) (particularly messenger RNA (mRNA)), synthetic forms of DNA or RNA, and mixed polymers containing two or more of these molecules. Nucleic acid molecules can be linear or circular. Furthermore, the term nucleic acid molecule includes sense and antisense strands, as well as single-stranded and double-stranded forms. Additionally, 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 having derivatized sugar or phosphate backbone bonds or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules suitable as vectors for direct in vitro and/or in vivo (e.g., in a host or patient) expression of antibodies used in this invention. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors can be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA vector and/or the expression of the encoding molecule, enabling the mRNA to be injected into a subject to generate antibodies in vivo (see, for example, Stadler et al., Nature Medicine 2017, published online June 12, 2017, doi:10.1038/nm.4356 or EP 2 101 823 B1).

"Isolated" nucleic acids refer to nucleic acid molecules that have been separated from components of their natural environment. Isolated nucleic acids include nucleic acid molecules that are contained in cells that normally contain nucleic acid molecules, but which are located outside the chromosome or at a chromosomal location different from their natural chromosomal location.

"Isolated nucleic acid encoding antibody" refers to one or more nucleic acid molecules that encode the heavy and light chains (or fragments thereof) of an antibody, including such nucleic acid molecules in a single or different vectors, and such nucleic acid molecules present at one or more locations in a host cell.

As used herein, the term "vector" refers to a nucleic acid molecule capable of carrying another nucleic acid linked to it. This term includes vectors that function as self-replicating nucleic acid structures, as well as vectors incorporated into the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of the nucleic acid to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells in which exogenous nucleic acids have been introduced, including progeny cells. Host cells include “transformations” and “transformed cells,” which include primary transformed cells and progeny derived from those primary transformed cells, regardless of passage number. Progeny cells may not have completely identical nucleic acid contents to the parent cells and may contain mutations. This article includes mutant progeny with the same function or biological activity as those screened or selected from the original transformed cells.

The term "pharmaceutical composition" or "pharmaceutical formulation" refers to a formulation in which the active ingredient contained therein is in a biologically effective form and does not contain any additional components that would have unacceptable toxicity to a subject to be administered the pharmaceutical composition.

"Pharmaceutical carrier" refers to a component in a pharmaceutical composition or formulation other than the active ingredient, which is non-toxic to the subject. Pharmaceutical carriers include, but are not limited to, buffer solutions, excipients, stabilizers, or preservatives.

The “effective amount” of a pharmaceutical agent (such as a pharmaceutical composition) refers to the amount that is sufficient to effectively achieve the desired therapeutic or preventative outcome at the necessary dose for the necessary period of time.

"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., human and non-human primates, such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

As used herein, “treatment” (and its grammatical variations such as treat or treating) refers to an attempt to alter the natural course of a disease in the treated individual and is a clinical intervention that can be performed for prevention or may be performed during a clinicopathological process. The desired effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the disease state, and mitigating or improving prognosis. In some embodiments, the antibodies of the present invention are used to delay the development of disease or slow its progression.

As used herein, the term "eye disease" includes any eye condition associated with pathological angiogenesis and/or atrophy. Eye diseases can be characterized by altered or unregulated proliferation and/or invasion of ocular tissues such as the retina or cornea. A characteristic feature of eye diseases may be atrophy of retinal tissues (photoreceptors and the underlying retinal pigment epithelium (RPE) and choroidal capillaries). Non-restrictive eye diseases include, for example, AMD (e.g., wet AMD, dry AMD, moderate AMD, advanced AMD, and geographic atrophy (GA)), macular degeneration, macular edema, DME (e.g., focal, non-central DME, and diffuse, central DME), retinopathy, diabetic retinopathy (DR) (e.g., proliferative DR (PDR), non-proliferative DR (NPDR), and high-altitude DR), other ischemic 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's disease, and bone disease. Osteoporosis-pseudoglioma syndrome (OPPG)-related retinal abnormalities, subconjunctival hemorrhage, redness, ocular neovascularization, neovascular glaucoma, retinitis pigmentosa (RP), hypertensive retinopathy, retinal angioma proliferation, macular telangiectasia, iris neovascularization, intraocular neovascularization, retinal degeneration, cystic macular edema (CME), vasculitis, papilledema, retinitis (including but not limited to CMV retinitis), ocular melanoma, retinoblastoma, conjunctivitis (e.g., infectious conjunctivitis and non-infectious conjunctivitis (e.g., allergic conjunctivitis)), Lieber's 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, dry eye disease, and other ophthalmic diseases (wherein the disease is associated with ocular neovascularization, vascular leakage, and/or retinal edema or retinal atrophy). Other exemplary eye diseases include retinal schizophrenia (abnormal splitting of the neurosensory layer of the retina), diseases associated with iris redness (corneal 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 overuse, atopic keratitis, superior limbal keratitis, pterygium keratoconjunctivitis, Sjögren's syndrome, rosacea, bullous keratosis, syphilis, mycobacterial infection, lipid degeneration, chemical burns, bacterial ulcers, fungal ulcers, herpes simplex virus infection, herpes zoster infection, protozoan infection, Kaposi's sarcoma, keratoconjunctivitis, Terrien's limbic degeneration, marginal keratolysis, rheumatoid arthritis, systemic lupus erythematosus, multiple arteriosclerosis, trauma, Wegener's sarcoidosis, scleritis, Stephen Johnson's syndrome, radial keratotomy, and corneal transplant rejection. Examples of diseases associated with choroidal neovascularization and defects in the retinal vascular system (including increased vascular leakage, aneurysms, and capillary detachment) include, but are not limited to, diabetic retinopathy, macular degeneration, sickle cell anemia, sarcoidosis, syphilis, elastic fiber pseudoxanthoma, Paget's disease, venous occlusion, arterial occlusion, carotid artery occlusive disease, chronic uveitis/hyalitis, mycobacterial infection, Lyme disease, systemic lupus erythematosus, retinopathy of prematurity, retinal edema (including macular edema), Ilse's disease, Behcet's disease, infectious retinitis or choroiditis (e.g., multifocal choroiditis), suspected ocular histoplasmosis, Best's disease (vitrectomyces macular degeneration), myopia, optic cupping, pars plana inflammation of the ciliary body, retinal detachment (e.g., chronic retinal detachment), hyperviscosity syndrome, toxoplasmosis, and complications following trauma and laser treatment. Exemplary diseases associated with atrophy of retinal tissues (photoreceptors and underlying RPE) include, but are not limited to, atrophic or non-exudative AMD (e.g., geographic atrophy or late dry AMD), macular atrophy (e.g., atrophy associated with neovascularization and/or geographic atrophy), geographic atrophy, diabetic retinopathy, Sturges disease, fundus dystrophy, retinal schizophrenia, and retinitis pigmentosa.

The term "packaging insert" is used to refer to the instruction leaflet typically included in the commercial packaging of a therapeutic product, which contains information concerning the indications, usage, dosage, administration, combination therapy, contraindications, and/or warnings related to the use of such therapeutic products.

2. Detailed Implementation

On one hand, the present invention is based in part on providing bispecific antibodies for therapeutic applications. In some aspects, antibodies binding to human VEGF-A and human IL6 are provided. The antibodies of the present invention can be used, for example, to treat vascular diseases, such as ocular vascular diseases.

A. Exemplary antibodies that bind to human VEGF-A and human IL6

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

In some respects, an antibody is provided that binds to human VEGF-A and human IL6, wherein the antibody contains a VEGF-A complementary site (i.e., an antigen-binding site for VEGF-A) and an IL6 complementary site (i.e., an antigen-binding site for IL6) within a homologous pair of the VL and VH domains, wherein

●The VEGF-A complementary site contains amino acid residues from CDR-H2, CDR-L1, and CDR-L3 of the antibody, and the IL6 complementary site contains amino acid residues from CDR-H1, CDR-H3, and CDR-L2 of the antibody; and/or

●The IL6 complementary site contains amino acid residues from the antibody's CDR-H2, CDR-L1, and CDR-L3, while the VEGF-A complementary site contains amino acid residues from the antibody's CDR-H1, CDR-H3, and CDR-L2.

●The variable light chain domain and the variable heavy chain domain bind simultaneously to human VEGF-A and human IL6; and/or

●The antibody contains a variable heavy chain domain having SEQ ID NO:22 and SEQ ID NO:

Compared to antibodies with a variable light chain domain of 21, it binds to the same epitope on human VEGF-A and the same epitope on human IL6; and/or

●The antibody Fab fragment binds to the following: (i) human VEGF-A121, wherein

(ii) _IL6 , wherein _KD measured by surface plasmon resonance is less than 50 pM; and/or

● The antibody Fab fragment exhibits an aggregation initiation temperature of 60°C or higher, and in some embodiments 70°C or higher; and/or

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

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

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

In another aspect, the present invention provides an antibody that binds to human VEGF-A and human IL6, the antibody comprising: (a) a VH domain comprising the amino acid sequence of SEQ ID NO:22 having 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.

In another aspect, the present invention provides an antibody that binds to human VEGF-A and 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) CDR-L2 comprising the amino acid sequence of SEQ ID NO:16 and (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:17, the 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.

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 some aspects, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conserved substitutions), insertions, or deletions relative to a reference sequence, but the antibody containing this VH sequence retains its ability to bind to human VEGF-A and human IL6. In some aspects, in SEQ ID NO:22, a total of up to 10 amino acids have been substituted, inserted, and/or deleted. In some respects, substitutions, insertions, or deletions occur in regions outside the CDR (i.e., in the FR). In particular, VH comprises: (a) CDR-H1, which comprises the amino acid sequence of SEQ ID NO:18, (b) CDR-H2, which comprises the amino acid sequence of SEQ ID NO:19, and (c) CDR-H3, which comprises the amino acid sequence of SEQ ID NO:20.

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 some aspects, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conserved substitutions), insertions, or deletions relative to a reference sequence, but the antibody containing this VL sequence retains its ability to bind to human VEGF-A and human IL6. In some aspects, in SEQ ID NO:21, a total of up to 10 amino acids have been substituted, inserted, and/or deleted. In some respects, substitutions, insertions, or deletions occur in regions outside the CDR (i.e., in the FR). In particular, the VL comprises: (d) CDR-L1, which comprises the amino acid sequence of SEQ ID NO:15; (e) CDR-L2, which comprises the amino acid sequence of SEQ ID NO:16; and (f) CDR-L3, which comprises the amino acid sequence of SEQ ID NO:17.

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

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

In other aspects of the invention, the antibody binding to human VEGF-A and human IL6 according to any of the foregoing aspects is a monoclonal antibody. In one aspect, the antibody binding to human VEGF-A and human IL6 is an antibody fragment, such as Fv, Fab, Fab', scFv, a biantibody, or an F(ab') ₂ fragment. In another aspect, the antibody is a full-length antibody.

In another aspect, the present invention provides an antibody that binds to IL6, derived from the antibody of the present invention. The IL6 complementary site disclosed herein for the antibody of the present invention can be used to provide other antibodies, such as monospecific or bispecific antibodies that bind to IL6 and another antigen. The IL6 complementary site of the antibody 6HVL4.1 disclosed herein was identified by X-ray crystallography (Example 13). Antibody 6HVL4.1 is based on a VH domain having a human VH3 framework and a VL domain having a human Vκ1 framework. Antibodies containing the IL6 complementary site of antibody 6HVL4.1 bind to the same epitope on IL6. All embodiments disclosed herein for the antibody of the present invention binding to human VEGF-A and human IL6 are also applicable to antibodies binding to IL6.

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

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

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

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

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

In other respects, antibodies binding to human VEGF-A and human IL6 according to any of the foregoing aspects, or antibodies binding to human IL6 according to any of the foregoing aspects, may bind any of the features alone or in combination, as described in Sections 1 to 5 below:

1. Antibody affinity

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

In some embodiments, the antibody binding to IL6 has a dissociation constant (K_{_D}) of ≤1 nM, ≤0.1 nM, or ≤0.03 nM. In a preferred embodiment, the antibody provided herein binds to human IL6 with a dissociation constant (K _{_D} ) ≤10 pM, and in a preferred embodiment ≤5 pM. In one aspect, K _{_D} is measured using surface plasmon resonance assay, and in one embodiment, surface plasmon resonance assay.

In another aspect, _KD is measured using the KinExA assay. In one embodiment, KD is measured using the _KinExA assay under the conditions described below for detecting _KD of VEGF-A binding or for detecting _KD of IL6 binding.

For example, the _KD of antibody-VEGF-A binding was measured using a KinExA 3200 instrument from SAPidayne 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, SAPidayne). KinExA equilibration assays were performed at room temperature using PBS pH 7.4 containing 0.01% BSA and 0.01% Tween 20 as the run buffer. 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 500 pM in the second experiment) was titrated with the antibody to be tested, and the equilibrated mixture was passed through a column of anti-VEGF antibody (Mab293) conjugated beads in a KinExA system, with a volume of 750 μl for 50 pM constant VEGF and 125 μl for 500 pM constant VEGF. VEGFA-121 binding was detected by injecting a second biotinylated anti-VEGF antibody (BAF293) at a concentration of 250 ng/ml followed by injection of a 250 ng/ml streptavidin Alexa Fluor ^™ 647 conjugate into the sample buffer. _{K_D} was obtained by performing nonlinear regression analysis on the data using the "Standard Analysis" method, based on the unit point homogeneous binding model included in KinExA software (version 4.0.11). The software calculates K_{_D} by fitting the data points to the theoretical K_{_D} curve and determining the 95% confidence interval. The 95% confidence intervals are given as _{KD_low} and _{KD_high} .

For example, _KD of antibody-IL6 binding was measured on a Biacore 8K instrument (Cytiva) at 25°C using HBS-EP+ (1x; BR100669; Cytiva) as the run buffer in a surface plasmon resonance (SPR) assay. 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 chemically immobilized on a CM5 sensor chip using standard amine coupling. Optionally, five start-up cycles were performed for conditioning purposes prior to protein measurements, in which, in each cycle, the HBS-EP+ buffer was flowed for approximately 120 seconds, followed by regeneration of the derivatized chip surface by applying 10 mM glycine buffer at pH 2.0 for 60 seconds. A 75 nM antibody Fab fragment was captured on this surface in HBS-EP+ buffer at a flow rate of 10 μL/min for 60 seconds. The Fab fragment was not applied to the reference channel. Subsequently, human or cynomolgus monkey IL-6 was appropriately diluted 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).

2. Antibody fragments

In some respects, the antibodies presented in this article are antibody fragments.

On one hand, antibody fragments are Fab', Fab'-SH, or F(ab') ₂ fragments, especially Fab fragments. Papain digestion of an intact antibody produces two identical antigen-binding fragments called "Fab" fragments. Each "Fab" fragment contains a heavy chain variable domain and a light chain variable domain (VH and VL, respectively), as well as a light chain constant domain (CL) and a heavy chain first constant domain (CH1). Therefore, the term "Fab fragment" refers to an antibody fragment comprising a light chain containing the VL and CL domains and a heavy chain containing the VH and CH1 domains. Fab' fragments differ from Fab fragments in that Fab' fragments have residues added to the carboxyl terminus of the CH1 domain, including one or more cysteine residues from the antibody hinge region. Fab'-SH is a Fab' fragment in which the cysteine residues in the constant domain have a free thiol group. Pepsin treatment produces an F(ab') ₂ fragment, which has two antigen-binding sites (two Fab fragments) and a portion of the Fc region. For a discussion of the Fab and F(ab') ₂ fragments, which contain rescue receptor-binding epitope residues and have extended in vivo half-lives, see U.S. Patent No. 5,869,046.

Antibody fragments can be prepared using various techniques, including but not limited to the proteolytic digestion of intact antibodies and recombinant production from recombinant host cells (e.g., Escherichia coli, CHO), as described herein.

In a preferred embodiment, the antibody provided herein is a Fab fragment.

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

In one embodiment, the VL domain of the antibody provided herein contains the human Vkappa1 framework.

In one embodiment, the CL domain of the antibody provided herein is a kappa isotype.

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

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.

3. Thermal stability

The antibodies provided herein exhibit excellent thermal stability. In some embodiments, the Fab fragments of the antibodies provided herein exhibit aggregation initiation temperatures of 60°C or higher, and in one embodiment, 70°C or higher. In some embodiments, the Fab fragments of the antibodies provided herein exhibit melting temperatures greater than 80°C as measured by dynamic light scattering.

4. Multispecific antibodies

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

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

5. Antibody variants

In some respects, amino acid sequence variants of the antibodies presented herein are envisioned. For example, it may be desirable to alter the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of antibodies can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletion, and/or insertion and/or substitution of residues within the antibody amino acid sequence. Any combination of deletions, insertions, and substitutions can be performed to achieve the final construct, provided that the final construct possesses the desired characteristics, such as antigen binding.

In some respects, antibody variants with one or more amino acid substitutions are provided. Sites of interest for substitution mutagenesis include CDR and FR. Conserved substitutions are shown under the heading “Preferred Substitutions” in the table below. Further substantial variations are provided under the heading “Exemplary Substitutions” in Table 1, and are described further below with reference to the amino acid side chain categories. Amino acid substitutions can be introduced into target antibodies, and the products can be screened for desired activities (e.g., retained/improved antigen binding, reduced immunogenicity, or improved ADCC or CDC).

surface

Amino acids can be grouped based on their common side-chain characteristics:

(1) Hydrophobicity: Leucine, Met, Ala, Val, Leu, Ile;

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

(3) Acidity: Asp, Glu;

(4) Alkaline: His, Lys, Arg;

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

(6) Fang tribe: Trp, Tyr, Phe.

Non-conservative substitution would require swapping members of one of these categories for members of another category.

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, one or more resulting variants selected for further research will alter (e.g., improve) certain biological properties (e.g., increased affinity, decreased immunogenicity) and/or will substantially retain certain biological properties of the parent antibody relative to the parent antibody. An exemplary substitution variant is an affinity-matured antibody, which can be conveniently generated, for example, using phage display-based affinity maturation techniques 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 specific biological activities (e.g., binding affinity).

In some respects, substitution, insertion, or deletion can occur within one or more CDRs, as long as such changes do not substantially reduce the antibody's ability to bind to the antigen. For example, conserved changes that do not substantially reduce binding affinity (e.g., conserved substitutions as provided herein) can be made in the CDRs. Such changes can, for example, be external to the antigen-contacting residues in the CDR. In some variant VH and VL sequences provided above, each CDR either remains unchanged or contains no more than one, two, or three amino acid substitutions.

A method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scan mutagenesis," as described by 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) is identified and replaced with a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the antibody-antigen interaction is affected. Additional substitutions can be introduced at amino acid positions that exhibit functional sensitivity to the initial substitution. Alternatively or additionally, the contact points between the antibody and antigen can be identified using the crystal structure of the antigen-antibody complex. Such contact residues and adjacent residues can be targeted or eliminated as candidates for substitution. Variants can be screened to determine if they possess the desired properties.

Amino acid sequence insertions include the fusion of the amino and/or carboxyl termini of peptides ranging in length from one residue to one hundred or more residues, as well as intra-sequence insertions of one or more amino acid residues. Examples of terminal insertions include antibodies having an N-terminal methionine residue. Other insertion variants of antibody molecules include the fusion of the N-terminus or C-terminus of the antibody with an enzyme (e.g., for ADEPT (antibody-directed enzyme prodrug therapy)) or peptide that increases the serum half-life of the antibody.

a) Glycosylation variants

In some respects, the antibodies presented herein can be modified to increase or decrease the degree of antibody glycosylation. The addition or deletion of glycosylation sites to antibodies can be conveniently achieved by altering the amino acid sequence to create or remove one or more glycosylation sites.

When an antibody contains an Fc region, the oligosaccharide associated with it can be modified. Natural antibodies produced by mammalian cells typically contain branched biantennary oligosaccharides, which are usually linked to Asn297 of the CH2 domain of the Fc region via N-bonding. See, for example, Wright et al., TIBTECH 15:26-32 (1997). Oligosaccharides can include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose of GlcNAc attached to the “backbone” of the biantennary oligosaccharide structure. In some aspects, the oligosaccharides in the antibodies of the present invention can be modified to produce antibody variants with certain improved properties.

On the one hand, antibody variants with unfucosylated oligosaccharides are provided, i.e., oligosaccharide structures lacking (directly or indirectly) fucose linked to the Fc region. Such unfucosylated oligosaccharides (also known as "defucosylated" oligosaccharides) are particularly N-linked oligosaccharides that lack the fucose residues linking the first GlcNAc in the stem of the biantennary oligosaccharide structure. On the other hand, antibody variants with an increased proportion of unfucosylated oligosaccharides in the Fc region compared to natural or parental antibodies are provided. For example, the proportion of unfucosylated 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., no fucose-linked oligosaccharides present). The percentage of non-fucosylated oligosaccharides, as described, for example, in WO2006/082515, and as measured by MALDI-TOF mass spectrometry, is the (average) amount of oligosaccharides lacking fucosylated residues relative to the sum of all oligosaccharides (e.g., complex, heterozygous, and high-mannose structures) linked to Asn 297. Asn297 refers to the asparagine residue (EU number of Fc region residues) located at approximately position 297 in the Fc region; however, due to minor sequence variations in antibodies, Asn297 can also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such antibodies with an increased proportion of non-fucosylated oligosaccharides in the Fc region may exhibit improved FcγRIIIa receptor binding and/or improved effector function, particularly improved ADCC function. See, for example, US2003/0157108 and US2004/0093621.

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); US2003/0157108; and WO 2004/056312, especially in Example 11), and knockout cell lines, such as those with the α-1,6-fucosylation gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnu). Ki et al. Biotech. Bioeng. 87:614-622 (2004); Kanda, Y. et al. Biotechnol. Bioeng., 94(4):680-688 (2006); and WO 2003/085107), or cells with reduced or cancelled GDP-fucose synthesis or transporter activity (see, for example, US2004259150, US2005031613, US2004132140, US2004110282).

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

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

b) Fc region variants

In some respects, one or more amino acid modifications may be introduced into the Fc region of the antibody provided herein, thereby generating an Fc region variant. The Fc region variant may contain a human Fc region sequence (e.g., human _IgG1 , _IgG2 , _IgG3 , or _IgG4 Fc region) containing an amino acid modification (e.g., substitution) at one or more amino acid positions.

In some respects, the present invention considers antibody variants possessing some, but not all, effector functions, making them ideal candidates for applications where the in vivo half-life of the antibody is important, but certain effector functions (such as complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC)) are unnecessary or detrimental. In vitro and/or in vivo cytotoxicity assays can be performed to confirm a 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 capacity. The primary cells mediating ADCC, NK cells, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays for evaluating ADCC activity of target molecules are described in U.S. Patent Nos. 5,500,362 (see, for example, 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, for example, the ACTI ^™ non-radioactive cytotoxicity assay for flow cytometry (Cell Technology, Inc. Mountain View, CA); and the CytoTox non-radioactive cytotoxicity assay (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, ADCC activity of the molecule of interest can be assessed in vivo, for example, in animal models such as those 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 does not bind to C1q and therefore lacks CDC activity. See, for example, WO 2006/029879 and WO C1q and C3c binding ELISA in 2005/100402. To assess complement activation, CDC assays can be performed (see, for example, 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, for example, Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006); WO 2013/120929A1).

Antibodies with reduced effector function include those with substitutions of one or more of the following Fc region residues: 238, 265, 269, 270, 297, 327, and 329 (US Patent No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of the following amino acid positions: 265, 269, 270, 297, and 327, including the so-called “DANA” Fc mutant, in which residues 265 and 297 are substituted with alanine (US Patent No. 7,332,581).

Certain antibody variants with improved or reduced binding to FcR are described. (See, for example, U.S. Patent No. 6,737,056; WO 2004/056312; and Shields et al., J. Biol. Chem. 9(2):6591-6604(2001).)

In some respects, antibody variants contain an Fc region with one or more amino acid substitutions that improve ADCC, such as substitutions at positions 298, 333, and/or 334 of the Fc region (EU numbers of the residues).

In some aspects, the antibody variant includes an Fc region with one or more amino acid substitutions that reduce FcγR binding, such as substitutions at positions 234 and 235 of the Fc region (EU numbers of the residues). In one aspect, the substitutions are L234A and L235A (LALA). In some aspects, the antibody variant further includes D265A and/or P329G in an Fc region derived from the human IgG1 _Fc region. In one aspect, in the Fc region derived from the human _IgG1 Fc region, the substitutions are L234A, L235A, and P329G (LALA-PG). (See, for example, WO2012/130831). In another aspect, in the Fc region derived from the human _IgG1 Fc region, the substitutions are L234A, L235A, and D265A (LALA-DA).

In some respects, alterations are made in the Fc region that result in changes (i.e., improvements or reductions) in C1q binding and/or complement-dependent cytotoxicity (CDC), for example, as described in U.S. Patent Nos. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

Antibodies with extended half-lives and improved neonatal Fc receptor (FcRn) binding responsible for transferring maternal IgG to the fetus (Guyer, R.L. et al., J. Immunol. 117:587 (1976), and Kim, J.K. et al., J. Immunol. 24:249 (1994)) are described in US2005/0014934 (Hinton et al.). These antibodies contain an Fc region with one or more substitutions that improve the binding of the Fc region to the FcRn. Such Fc variants include Fc variants with substitutions 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, for example, a substitution of Fc region residue 434 (see, for example, U.S. Patent No. 7,371,826; Dall'Acqua, W.F. et al. J. Biol. Chem. 281 (2006) 23514-23524).

Fc region residues crucial for the mouse Fc-mouse FcRn interaction have been identified through site-directed mutagenesis (see, for example, Dall’Acqua, W.F. et al. J. Immunol 169 (2002) 5171-5180). The interaction involves residues I253, H310, H433, N434, and H435 (EU index numbers) (Medesan, C. et al. Eur. J. Immunol. 26 (1996) 2533; Firan, M. et al. Int. Immunol. 13 (2001) 993; Kim, J.K. et al. Eur. J. Immunol. 24 (1994) 542). Residues I253, H310, and H435 were found to be crucial for the interaction between human Fc and mouse FcRn (Kim, J.K. 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 crucial for the interaction (Firan, M. et al., Int. Immunol. 13(2001) 993; Shields, R.L. 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, Y.A. et al. (J. Immunol. 182(2009) 7667-7671).

In some respects, antibody variants comprise Fc regions 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 numbers of the residues). In some respects, antibody variants comprise Fc regions with amino acid substitutions at positions 253, 310, and 435. In one instance, in the Fc region derived from the human IgG1 Fc region, the substitutions are I253A, H310A, and H435A. See, for example, Grevys, A. et al., J. Immunol. 194 (2015) 5497-5508.

In some respects, antibody variants comprise Fc regions with one or more amino acid substitutions that reduce FcRn binding, for example, substitutions at positions 310, and/or 433, and/or 436 of the Fc region (EU numbers of the residues). In some respects, antibody variants comprise Fc regions with amino acid substitutions at positions 310, 433, and 436. In one instance, in the Fc region derived from the human IgG1 Fc region, the substitutions are H310A, H433A, and Y436A. (See, for example, WO 2014/177460 A1).

In some respects, antibody variants comprise 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 numbers of the residues). In some respects, antibody variants comprise an Fc region having amino acid substitutions at positions 252, 254, and 256. In one respect, in the Fc region derived from the 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.

The C-terminus of the heavy chain of the antibody reported herein may be a full C-terminus ending with the amino acid residue PGK. The C-terminus of the heavy chain may 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, as specified herein, an antibody comprising a heavy chain including a C-terminal CH3 domain comprises a C-terminal glycine-lysine dipeptide (G446 and K447, EU index numbers of amino acid positions). In one aspect of all aspects reported herein, as specified herein, an antibody comprising a heavy chain including a C-terminal CH3 domain comprises a C-terminal glycine residue (G446, EU index number of amino acid position).

c) Cysteine-engineered antibody variants

In some respects, it may be necessary to produce cysteine-engineered antibodies, such as THIOMAB ^™ antibodies, in which one or more residues of the antibody are replaced by cysteine residues. In certain embodiments, the substituted residues are located at accessible sites on the antibody. As further described herein, by replacing those residues with cysteine, a reactive thiol group is positioned at an accessible site on the antibody and can be used to conjugate the antibody with other parts, such as a pharmaceutical part or a linker-pharmaceutical part, to produce an immunoconjugate. Cysteine-engineered antibodies can be produced, for example, as described in U.S. Patent Nos. 7,521,541, 8,30,930, 7,855,275, 9,000,130, or WO 2016040856.

B. Recombination methods and compositions

Antibodies can be generated using recombinant methods and compositions, such as those described in US 4,816,567. For these methods, one or more isolated nucleic acids encoding the antibody are provided.

On the one hand, isolated nucleic acids encoding the antibodies of the present invention are provided.

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 antibody expression; and optionally recovering the antibody from the host cell (or host cell culture medium).

To recombinantly generate antibodies that bind to human VEGF-A and human IL6, nucleic acids encoding the 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 readily isolated and sequenced using routine procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibodies), or obtained through recombinant methods or chemical synthesis.

Suitable host cells for cloning or expressing vectors encoding antibodies include prokaryotic or eukaryotic cells as described herein. Antibodies can be generated in bacteria, for example, particularly when glycosylation and Fc effector function are not required. For information on the expression of antibody fragments and peptides in bacteria, see, for example, US 5,648,237, US 5,789,199, and US 5,840,523. (See also Charlton, K.A., in Methods in Molecular Biology, Vol. 248, edited by Lo, B.K.C., Humana Press, Totowa, NJ (2003), pp. 245-254, describing the expression of antibody fragments in *E. coli*.) Antibodies can be separated from the bacterial cell paste in a soluble fraction after expression and can be further purified. In some embodiments, the host cell is *E. coli* cells.

Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for growth in suspension may be useful. Other examples of useful mammalian host cell lines are the monkey kidney CV1 line (COS-7) transformed with SV40; human embryonic kidney cell lines (such as 293 or 293T cells as described, for example, in Graham, F.L. et al., J. Gen. Virol. 36 (1977) 59-74); hamster kidney cells (BHK); mouse Sertoli cells (such as TM4 cells as described, for example, in Mather, J.P., Biol. Reprod. 23 (1980) 243-252); and monkey kidney cells. (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); Buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumors (MMT 060562); TRI cells (as described, for example, in Mather, J.P. 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, NSO, and Sp2/0. For reviews of certain mammalian host cell lines applicable to antibody production, see, for example, Yazaki, P. and Wu, A.M., Methods in Molecular Biology, Vol. 248, Lo, B.K.C. (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268.

On the one hand, the host cell is a eukaryotic cell, such as Chinese hamster ovary (CHO) cells or lymphocytes (e.g., Y0, NSO, Sp20 cells). In a preferred embodiment, the host cell is a CHO cell. Generating the antibody of the present invention in CHO cells can improve the injectability of the antibody.

C. Pharmaceutical Composition

In other aspects, pharmaceutical compositions comprising any of the antibodies provided herein are provided, for example, in any of the following treatment methods. In one aspect, the pharmaceutical composition comprises any of the antibodies provided herein and a pharmaceutical carrier. In another aspect, the pharmaceutical composition comprises any of the antibodies provided herein and at least one additional therapeutic agent, such as those described below.

Pharmaceutical compositions of antibodies binding to human VEGF-A and human IL6, as described herein, are prepared by mixing antibodies of the desired purity with one or more optional pharmaceutical carriers (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)), in the form of lyophilized compositions or aqueous solutions. Pharmaceutical carriers are generally non-toxic to recipients at the doses and concentrations used 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 octadecyl dimethyl benzyl ammonium chloride; hexamethyl diammonium chloride; benzalkonium chloride; benzyl chloride; phenol, butanol, or benzyl alcohol; alkyl esters of parabens, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; m-cresol); low molecular weight (less than about 10). (10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., zinc protein complexes); and/or nonionic surfactants, such as polyethylene glycol (PEG). Exemplary pharmaceutical carriers described herein also include interstitial drug dispersants, such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), such as human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (Halozyme, Inc.). Certain exemplary sHASEGP and methods of use (including rHuPH20) are described in U.S. Patent Publications 2005/0260186 and 2006/0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycans (such as chondroitinase).

Exemplary lyophilized antibody compositions are described in U.S. Patent No. 6,267,958. Aqueous antibody compositions include those described in U.S. Patent Nos. 6,171,586 and WO 2006/044908, the latter of which contain histidine-acetate buffer.

The pharmaceutical compositions described herein may also contain more than one active ingredient essential for the specific indication being treated, preferably active ingredients having complementary activities that do not adversely affect each other. Such active ingredients are appropriately combined in amounts effective for the intended purpose.

The active ingredient can be encapsulated in microcapsules (e.g., hydroxymethyl cellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules) prepared by, for example, cohesive techniques or interfacial polymerization; encapsulated in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules); or encapsulated in crude emulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, ed. Osol, A. (1980).

Pharmaceutical compositions for sustained release can be prepared. Suitable examples of sustained-release formulations include semi-permeable matrices of solid hydrophobic polymers containing antibodies, which are in the form of molded articles such as membranes or microcapsules.

Pharmaceutical compositions intended for internal administration are typically sterile. For example, sterility can be readily achieved through filtration using a sterile filter membrane.

D. Treatment methods and routes of administration

Any antibody that binds to human VEGF-A and human IL6, as described in this article, can be used for therapeutic purposes.

In one aspect, an antibody binding to human VEGF-A and human IL6 is provided for use as a medicament. In other aspects, an antibody binding to human VEGF-A and human IL6 is provided for treating vascular diseases. In some aspects, an antibody binding to human VEGF-A and human IL6 is provided for a treatment method. In some aspects, the present invention provides a method for treating an individual with vascular disease using an antibody binding to human VEGF-A and human IL6, the method comprising administering to the individual an effective amount of the antibody binding to human VEGF-A and human IL6. In one such aspect, as described below, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent (e.g., one, two, three, four, five, or six additional therapeutic agents). In other aspects, the present invention provides an antibody binding to human VEGF-A and human IL6 for inhibiting angiogenesis. In some aspects, the present invention provides a method for inhibiting angiogenesis in an individual using an antibody binding to human VEGF-A and human IL6, the method comprising administering to the individual an effective amount of the antibody binding to human VEGF-A and human IL6 to inhibit angiogenesis. The “individual” in any of the above aspects is preferably a person.

In other aspects, an antibody binding to human VEGF-A and human IL6 is provided for the treatment of ocular diseases. In one embodiment, the ocular 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, central DME), retinopathy, diabetic retinopathy (DR) (in one embodiment, proliferative DR (PDR), non-proliferative DR (NPDR), and high-altitude DR), other ischemic retinopathy, ROP, retinal vein occlusion (RVO) (in one embodiment, central (CRVO) and branched (BRVO) forms), CNV (in one embodiment, myopic CNV), corneal neovascularization, diseases associated with corneal neovascularization, retinal neovascularization, diseases associated with retinal/choroidal neovascularization, central serous retinopathy (CSR), pathological myopia, Hippel-Lindau syndrome, ocular histoplasmosis, FEVR, and coronary artery disease. Norie's disease, retinal abnormalities associated with osteoporosis-pseudoglioma syndrome (OPPG), subconjunctival hemorrhage, redness and swelling, ocular neovascularization, neovascular glaucoma, retinitis pigmentosa (RP), hypertensive retinopathy, retinal angioma proliferation, macular telangiectasia, iris neovascularization, intraocular neovascularization, retinal degeneration, cystic macular edema (CME), vasculitis, papilledema, retinitis (including but not limited to CMV retinitis), and ocular melanosis. Retinoblastoma, conjunctivitis (e.g., infectious conjunctivitis and non-infectious conjunctivitis (in one embodiment, allergic conjunctivitis)), Lieber's 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 syndrome, ocular trauma, dry eye disease, and other ophthalmic diseases (wherein the disease is associated with ocular neovascularization, vascular leakage, and/or retinal edema or retinal atrophy). In one embodiment, the ocular disease is selected from: AMD (in one embodiment, wet AMD, dry AMD, moderate AMD, advanced AMD, and geographic atrophy (GA)), macular degeneration, macular edema, DME (in one embodiment, focal, non-central DME, and diffuse, centrally involved DME), retinopathy, diabetic retinopathy (DR) (in one embodiment, proliferative DR (PDR), non-proliferative DR (NPDR), and high-altitude DR).

In other aspects, the present invention provides the use of an antibody binding to human VEGF-A and human IL6 in the production or preparation of a medicament. In one aspect, the medicament is used to treat vascular diseases. In other aspects, a method of using the medicament to treat vascular diseases includes administering an effective amount of the medicament to an individual suffering from a vascular disease. In one such aspect, as described below, the method further includes administering an effective amount of at least one additional therapeutic agent to the individual.

In one aspect, the drug is used to treat an eye disease. In another aspect, the drug is used as a method for treating an eye disease, the method comprising administering an effective amount of the drug to an individual suffering from an eye disease. In one such aspect, as described below, the method further comprises administering an effective amount of at least one other therapeutic agent to the individual.

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

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

An "individual" can be a person according to any of the above aspects.

In other aspects, the present invention provides pharmaceutical compositions comprising any antibody provided herein that binds to human VEGF-A and human IL6, for example, for any of the above-described treatment methods. In one aspect, the pharmaceutical composition comprises any antibody provided herein that binds to human VEGF-A and human IL6 and a pharmaceutical carrier. In another aspect, the pharmaceutical composition comprises any antibody provided herein that binds to human VEGF-A and human IL6 and at least one additional therapeutic agent, such as those described below.

The antibodies of the present invention can be administered via intravitreal administration (e.g., intravitreal injection) or using an infusion port delivery device. In one embodiment, the antibodies of the present invention are administered over a period of six months or longer before refilling the infusion port delivery device; in another embodiment, over a period of eight months or longer; in yet another embodiment, over a period of nine months or longer; and in yet another embodiment, over a period of 12 months or longer. In one embodiment, the antibodies of the present invention are administered using an infusion port delivery device, wherein the antibodies are applied to the infusion port delivery device at a concentration of 150 mg/ml or higher; and in yet another embodiment, at a concentration of 200 mg/ml or higher.

The antibody of the present invention can be administered alone or in combination therapy. For example, such combination therapy includes administering the 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).

In some embodiments according to (or applied to) any of the above embodiments, the ocular disease is an intraocular neovascular disease selected from the group consisting of: proliferative retinopathy, choroidal neovascularization (CNV), age-related macular degeneration (AMD), diabetic and other ischemic 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).

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

Any suitable AMD therapeutic agent can be administered as an additional treatment in combination with the antibodies described herein that bind to human VEGF and human IL6 for the treatment of ocular diseases (e.g., AMD, DME, DR, RVO, or GA). The suitable AMD therapeutic agents include, but are 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 bispecific anti-VEGF antibodies (e.g., anti-VEGF/anti-angiogenic peptide 2 bispecific antibodies, such as faricimab; Roche)), soluble VEGF receptor fusion proteins (e.g., aflibercept), and anti-VEGF (e.g., abicipar pegol); Molecular Partners AG/Allergan) or anti-VEGF aptamers (e.g., (pigatanib 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); with photodynamic therapy Therapeutic combinations include: (verteporfin); antioxidants; complement system antagonists, e.g., complement factor C5 antagonists (e.g., small molecule inhibitors (e.g., ARC-1905; Opthotech) or anti-C5 antibodies (e.g., LFG-316; Novartis), properdin antagonists (e.g., anti-properdin antibodies, e.g. CLG-561; Alcon), or complement factor D antagonists (e.g., anti-complement factor D antibodies, e.g., lanpalizumab; Roche)); C3 blocking peptides (e.g., APL-2, Appellis); visual cycle modulators (e.g., emixustat hydrochloride); squalene (e.g., OHR-102; Or...). Pharmaceuticals; vitamin and mineral supplements (e.g., those described in Study 1 (AREDS1; zinc and/or antioxidants) and Study 2 (AREDS2; zinc, antioxidants, lutein, zeaxanthin, and/or omega-3 fatty acids) of age-related eye diseases); cell-based therapies, such as NT-501 (Renexus); PH-05206388 (Pfizer), huCNS-SC cell transplantation (StemCells), CNTO-2476 (umbilical cord stem cell line; Janssen), OpRegen (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 ^™ ; Lpath Inc.); ROBO4 antagonists (e.g., anti-ROBO4 antibodies, e.g., DS-7080a; Daiichi Sankyo Co., Ltd.); lentiviral vectors expressing endostatin and angiostatin (e.g., RetinoStat); and any combination thereof. In some cases, AMD treatments (including any prior AMD treatments) may be co-formulated. For example, the anti-PDGFR antibody REGN2176-3 may be co-formulated with aflibercept. In some cases, such co-formulations may be administered in combination with the antibodies of the present invention that bind to human VEGF and human IL6. In some cases, eye conditions are AMD (e.g., wet AMD).

Any suitable DME and/or DR therapeutic agent can be administered in combination with the antibody of the present invention that binds to human VEGF and human IL6 for the treatment of ocular diseases (e.g., AMD, DME, DR, RVO, or GA). The suitable DME and/or DR therapeutic agents include, but are not limited to, VEGF antagonists (e.g., or), corticosteroids (e.g., corticosteroid implants (e.g., dexamethasone intravitreal implants or fluocinolone acetonide intravitreal implants), or corticosteroids formulated for intravitreal injection (e.g., triamcinolone acetonide) or combinations thereof. In some cases, the ocular disease is DME and/or DR.

An antibody that binds to human VEGF and human IL6, as described herein, can be used in combination with therapies or surgical procedures for treating ocular diseases (e.g., AMD, DME, DR, RVO, or GA), including: for example, laser photocoagulation (e.g., panretinal photocoagulation (PRP)), drusen laser, macular hole surgery, macular displacement surgery, implantable microtelescopes, PHI motor angiography (also known as microlaser therapy and feeder vascular therapy), proton beam therapy, microstimulation therapy, retinal detachment and vitrectomy, scleral buckling, macular surgery, transpupillary thermotherapy, photosystem I therapy, and the use of RN. RNA interference (RNAi), in vitro evacuation (also known as membrane differential filtration and rheology therapy), microchip implantation, stem cell therapy, gene replacement therapy, ribozyme gene therapy (including gene therapy for hypoxia-responsive elements, Oxford Biopharmaceuticals; Lentipak, Genetix; and PDEF gene therapy, GenVec), photoreceptor/retinal cell transplantation (including transplantable retinal epithelial cells, Diacrin, Inc.; retinal cell transplantation, such as AstraZeneca Pharmaceuticals USA, ReNeuron, CHA Biotech), acupuncture and combinations thereof.

The aforementioned combination therapies encompass both combined administration (where two or more therapeutic agents are included in the same or different formulations) and single administration. In the case of single administration, 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 other therapeutic agents or pharmaceuticals. In one embodiment, the administration of the antibody of the present invention that binds to human VEGF and human IL6 and the administration of other therapeutic agents each occur within approximately one, two, three, four, or five months, or within approximately one, two, or three weeks, or within approximately one, two, three, four, five, or six days.

The antibodies (and any other therapeutic agents) of this invention can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal administration, and, if desired, for local treatment or intralesional application. Parenteral infusion includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Administration can be carried out by any suitable route, such as by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is transient or long-term. Various dosing schedules are considered herein, including but not limited to single or multiple administrations at various time points, bolus administration, and pulsatile infusion.

The antibodies of this invention will be formulated, administered, and applied in accordance with good medical practice. Factors to be considered in this context include the specific disease being treated, the specific mammal being treated, the individual patient's clinical condition, the cause of the disease, the site of delivery of the drug, the method of administration, the timing of administration, and other factors known to a practicing physician. The antibody is not mandatory, but may optionally be formulated in conjunction with one or more formulations currently used for the prevention or treatment of the disease in question. The effective amount of these other formulations depends on the amount of antibody present in the pharmaceutical composition, the type of disease or treatment, and other factors discussed above. These are generally used at the same dosage and route of administration as described herein, or at about 1% to 99% of the dosage described herein, or at any dosage and via any route determined empirically/clinically as appropriate.

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 administered for preventive or therapeutic purposes, the patient's medical history and 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) may be an initial candidate dose administered to the patient, for example, by a single or multiple administrations alone or by continuous infusion. Depending on the above factors, a typical daily dose range may be from about 1 μg/kg to 100 mg/kg or more. For repeated administration over several days or longer, depending on the condition, treatment typically continues until the desired suppression of disease symptoms occurs. An exemplary dose range for the antibody is from about 0.05 mg/kg to about 10 mg/kg. Therefore, patients can be administered 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). Such doses can be administered intermittently, for example weekly or every three weeks (e.g., so that the patient receives about two to about twenty doses, or, for example, about six doses of the antibody). An initial higher loading dose can be administered, followed by one or more lower doses. Progression of the therapy can be easily monitored using routine techniques and assays.

E. Products

In another aspect of the invention, an article is provided containing a substance that can be used to treat, prevent, and/or diagnose the aforementioned conditions. The article 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 from a variety of materials such as glass or plastic. The container contains a composition that, alone or in combination with another composition, is effective in treating, preventing, and/or diagnosing the condition, and the container may have a sterile inlet (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be punctured by a hypodermic needle). At least one active agent in the composition is an antibody of the present invention. The label or package insert indicates that the composition is intended to treat the selected condition.

Furthermore, the article may include (a) a first container containing a composition comprising the antibody of the present invention; and (b) a second container containing a composition comprising additional cytotoxic agents or other therapeutic agents. The article in this aspect of the invention may also include a packaging 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 containing pharmaceutical buffers, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and glucose solution. The article may also include other substances required from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

F. Device

An ocular implant can be used; in one embodiment, an infusion port delivery device is used to administer the antibody of the present invention into the eye.

An infusion port delivery device is an implantable, refillable device that can release a therapeutic agent (e.g., the antibody of the present 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 infusion port delivery devices that may be used include those from ForSight Labs, LLC and/or ForSightVISION4, such as those described in International Patent Application Publications WO 2010/088548, WO2015/085234, WO 2013/116061, WO 2012/019176, WO 2013/040247 and WO 2012/019047, the entire contents of which are incorporated herein by reference.

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

In some cases, infusion port delivery devices include a reservoir containing any of the antibodies described herein and one or more additional compounds.

In some cases, infusion port delivery devices include any antibody or antibody conjugate described herein and additional VEGF antagonists.

3. Specific embodiments of the present invention

Specific embodiments of the present invention are listed below.

1. An antibody that binds to human VEGF-A and 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) an amino acid sequence of SEQ ID NO:17. The antibody comprises: (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.

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) an amino acid sequence comprising the amino acid sequence of SEQ ID NO:15. The antibody comprises: (e) CDR-L1 containing the amino acid sequence of SEQ ID NO:16, (f) CDR-L3 containing 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.

3. An antibody that binds to human VEGF-A and human IL6, the antibody comprising: (a) a VH domain comprising the amino acid sequence of SEQ ID NO:22 having 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.

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.

5. The antibody according to any one of the foregoing 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.

6. The antibody according to any one of the foregoing embodiments, wherein the VEGF-A complementary site comprises amino acid residues from CDR-H2, CDR-L1, and CDR-L3 of the antibody, wherein the IL6 complementary site comprises amino acid residues from CDR-H1, CDR-H3, and CDR-L2 of the antibody, or wherein the IL6 complementary site comprises amino acid residues from CDR-H2, CDR-L1, and CDR-L3 of the antibody, wherein the VEGF-A complementary site comprises amino acid residues from CDR-H1, CDR-H3, and CDR-L2 of the antibody; and/or

●The variable light chain domain and the variable heavy chain domain bind simultaneously to human VEGF-A and human IL6; and/or

●The antibody, compared to antibodies having the variable heavy chain domain of SEQ ID NO:22 and the variable light chain domain of SEQ ID NO:21, binds to the same epitope on human VEGF-A and the same epitope on human IL6; and/or

● The antibody Fab fragment binds to the following: (i) human VEGF-A121, wherein the _KD measured by surface plasmon resonance is less than 50 pM, and (ii) human IL6, wherein the _KD measured by surface plasmon resonance is less than 50 pM; and/or

● The antibody Fab fragment of the antibody exhibits an aggregation initiation temperature of 60°C or higher, and in one embodiment 70°C or higher; and/or

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

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.

8. The antibody according to any of the foregoing embodiments, wherein the antibody is a Fab fragment.

9. The antibody according to any of the foregoing embodiments, wherein the antibody is a bispecific antibody fragment.

10. The antibody according to any one of the foregoing embodiments is a monoclonal antibody.

11. The antibody according to any one of the foregoing embodiments, wherein the antibody Fab fragment of said antibody exhibits an aggregation initiation temperature of 70°C and higher.

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

13. The antibody according to any one of the foregoing embodiments is a monoclonal antibody.

14. The antibody according to any one of the foregoing embodiments is an antibody fragment that binds to human VEGF-A and human IL6.

15. The antibody according to any of the foregoing embodiments, wherein the antibody is bispecific.

16. The antibody according to any of the foregoing embodiments, wherein the antibody is a Fab fragment.

17. The antibody according to any of the foregoing embodiments, wherein the antibody is a bispecific antibody fragment.

18. The antibody according to any one of the foregoing embodiments, wherein the antibody is a multispecific antibody.

19. The antibody according to any of the foregoing embodiments, wherein the antibody specifically binds to human VEGF-A.

20. The antibody according to any of the foregoing embodiments, wherein the antibody specifically binds to human IL6.

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

22. An antibody that binds to human IL6, wherein the antibody comprises: a VH domain having a human VH3 framework, wherein the IL6 complementary site 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 complementary site 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.

23. An antibody that binds to human IL6, said antibody comprising:

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

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

24. An isolated nucleic acid encoding an antibody according to any one of Examples 1 to 23.

25. A host cell comprising the nucleic acid as described in Example 24.

26. A method for producing antibodies that bind to human VEGF-A and human IL6, comprising culturing host cells according to Example 25 to produce said antibodies.

27. The method according to Example 26, wherein the host cell is a CHO cell.

28. A pharmaceutical preparation comprising an antibody according to any one of Examples 1 to 23 and a pharmaceutical carrier.

29. An infusion port delivery device comprising an antibody according to any one of Examples 1 to 23.

30. The antibody according to any one of Examples 1 to 23, which is used as a drug.

31. The method according to Example 26, further comprising recovering antibodies from host cells.

32. Antibodies produced by the method described in Example 26 or 31.

33. A pharmaceutical preparation comprising an antibody according to any one of Examples 1 to 23 and a pharmaceutical carrier.

34. The antibody according to any one of Examples 1 to 23, which is used as a drug.

35. The antibody according to any one of Examples 1 to 23, used for the treatment of vascular diseases.

36. The antibody according to any one of Examples 1 to 23, used for the treatment of ocular vascular diseases.

37. Use of the antibody according to any one of Examples 1 to 23 or the pharmaceutical composition according to Example 65 in the preparation of a medicament.

38. Use of the antibody according to any one of Examples 1 to 23 or the pharmaceutical composition according to Example 65 in the preparation of a medicament for inhibiting angiogenesis.

39. A method of treating an individual suffering from vascular disease, comprising administering to the individual an effective amount of an antibody according to any one of Examples 1 to 23 or a pharmaceutical preparation according to Example 33.

40. A method of treating an individual suffering from an ocular vascular disease, comprising administering to the individual an effective amount of an antibody according to any one of Examples 1 to 23 or a pharmaceutical preparation according to Example 33.

41. A method for inhibiting angiogenesis in an individual, comprising administering to the individual an effective amount of an antibody according to any one of Examples 1 to 23 or a pharmaceutical preparation according to Example 33 to inhibit angiogenesis.

42. An infusion port delivery device comprising an antibody according to any one of Examples 1 to 23 or a pharmaceutical preparation according to Example 33.

43. An antibody according to any one of Examples 1 to 23 or a pharmaceutical preparation of Example 33 is used for ocular application via a port-of-care delivery device.

44. An antibody according to any one of Examples 1 to 23 or a pharmaceutical preparation according to Example 33, for ocular administration via an infusion port delivery device according to Example 42, wherein the administration is performed over a period of six months or longer before refilling the infusion port delivery device; in one example, over a period of eight months or longer; in one example, over a period of nine months or longer.

45. An antibody according to any one of Examples 1 to 23 or a pharmaceutical preparation according to Example 33, for use as a medicine by administering the antibody or the pharmaceutical preparation using an infusion port delivery device, wherein the antibody is administered at a concentration of 150 mg/ml or higher; in one embodiment, at a concentration of 200 mg/ml or higher to the infusion port delivery device.

Description of amino acid sequence

Example

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

Example 1:

Generation of bispecific anti-VEGF/anti-6Fab fragments

The bispecific anti-VEGF/anti-IL-6 Fab fragment is generated by providing an antibody with separate, non-overlapping complementary sites that bind to VEGF and IL-6 using a method similar to that previously described (e.g., the method described in WO2012/163520).

Here, two distinct phage display libraries of the synthesized Fab fragment are used, wherein the residues within the CDR-H1, CDR-H3, and CDR-L2 regions of the Fab fragment are diverse in the first phage display library, and the residues within the CDR-L1, CDR-L3, and CDR-H2 regions of the Fab fragment are diverse in the second phage display library. In each library, the corresponding other three CDR regions remain non-diversified and—in contrast to the method of WO2012/163520 using invariant non-binding, germline-like (“virtual”) sequences—represent complementary sites capable of binding to VEGF-A.

In the case of the first library, the complementary bit that can bind to VEGF-A originates from the VEGF-A binding complementary bit described in WO 2021/198034.

In the case of the second library, the VEGF-A binding complementary bit is obtained as follows:

For initial selection, phage library panning was performed using diversified libraries of CDR-H1, CDR-H3, and CDR-L2, as described in WO2012/163520. The remaining CDR sequences were kept constant using non-binding germline-like sequences. The process was performed in four rounds, with the first round using 100 nM biotinylated VEGF-121 or VEGF-165 pre-immobilized on Dynabeads M-280 streptavidin (Thermofisher catalog number 11206D). Rounds 2 through 4 were performed using 75 nM, 15 nM, and 3 nM biotinylated targets in solution, respectively, to capture the phage Fab/target complex on Dynabeads M-280 streptavidin. The phage/target/bead complex was washed multiple times with PBST and PBS buffer. According to the standard protocol, phage clones with target-specific Fab captured from M-280 beads were eluted using 100mM DTT. These phage clones were then used to infect logarithmic TG1 Escherichia coli (E. coli) cells and rescued using M13 K07 helper phages.

For the selection of outputs, small-scale preparations of polyclonal plasmids for the corresponding selection rounds were prepared from infected TG1 *E. coli* cells. The plasmids were reformatted to generate soluble Fab in *E. coli* supernatant, which was tagged with a T7 at the C-terminus of the Fab CH1 domain. The ligation polyclonal plasmid encoding the T7-tagged Fab was transformed into TG1 *E. coli* cells (Zymo Research catalog number T3017), and single colonies were picked into microtiter plates. The soluble Fab was expressed in the microtiter plates, and the supernatant was clarified by centrifugation. Target binding was assessed by ELISA against VEGF and a competitive ELISA against VEGF receptor 2. Candidate binders were selected based on high binding signal for VEGF and good inhibition of receptor binding.

The conjugates were subsequently expressed and purified in larger volumes, and binding to VEGF was assessed using SPR measurements. One of the resulting clones was further optimized through iterative protein engineering and testing strategies and integrated into a phage display library as an invariant sequence against CDR-H1, CDR-H3, and CDR-L2. In summary, the protein engineering workflow consists of: initial rounds of reconnaissance mutations to identify relevant beneficial mutations, followed by two consecutive rounds of affinity maturation based on oligonucleotide-based mutant library generation and phage display selection, followed by screening and further testing.

In both libraries, the CH1 domain of the Fab fragments was fused to a truncated gene III protein via a linker to facilitate phage display. Therefore, one library was designed to screen for bispecific Fab fragments, where the IL6 complement contained 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, where the IL6 complement contained amino acid residues from CDR-H2, CDR-L1, and CDR-L3 (referred to herein as the "VH6L" library).

Phage library panning enriched the bindings against human IL-6 in each library. Following panning, microparticle preparations of plasmids were generated for two enrichment pools of phage vectors. These microparticle preparations were digested with restriction enzymes to remove the region encoding a truncated gene III protein, and then recirculated by ligation to obtain expression vector pools encoding soluble Fab fragments enriched with IL-6 bindings. These vector pools were transformed into TG1 *E. coli* cells, and single colonies were selected and cultured for soluble expression of single Fab clones in microtiter plates. Supernatants containing the soluble Fab fragments were screened using standard ELISA methods for binding to IL-6 and VEGF-A.

Based on the screening data, a bispecific anti-VEGF/anti-IL-6 Fab fragment was selected, and DNA plasmids were prepared and sequenced from the TG1 clones that produced specific bindings to obtain VH and VL sequence pairs that co-encode a bispecific Fab fragment that specifically binds to both IL-6 and VEGF-A from each library.

These clones are 6HVL_1, characterized by the heavy chain of SEQ ID NO:03 and the light chain of SEQ ID NO:04, and VH6L_1, characterized by the heavy chain of SEQ ID NO:09 and the light chain of SEQ ID NO:10.

Example 2:

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

The obtained bispecific anti-VEGF/anti-IL-6 Fab fragments were characterized. The vector obtained as described in Example 1 was 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 evaluated.

Example 3:

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

Surface plasmon resonance (SPR) was used to measure the binding kinetics and affinity of representative VEGF-IL-6 Fab fragments with human and cynomolgus monkey IL-6 disclosed herein.

SPR analysis of the binding of the corresponding Fab fragments IL-6 from humans and cynomolgus monkeys was performed at 25 °C on a Biacore 8K instrument (Cytiva) using HBS-EP+ (1x; BR100669; Cytiva) as the run 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 then processed accordingly.

Five start-up cycles were performed for conditioning purposes prior to protein measurements. In each cycle, the HBS-EP+ buffer was flown for approximately 120 seconds, followed by regeneration of the derivatized chip surface by applying 10 mM glycine buffer at pH 2.0 for 60 seconds. A 75 nM antibody Fab fragment was captured on the surface in HBS-EP+ buffer at a flow rate of 10 μL/min for 60 seconds. No Fab fragment was applied to the reference channel. Subsequently, human or cynomolgus monkey IL-6 was serially applied in HBS-EP+ buffer at an appropriate dilution at a flow rate of 30 μL/min (contact time 180 s, dissociation time 720 s). Regeneration of the derivatized chip surface was achieved as described above. Data were evaluated using 8K evaluation software (Biacore Insight Evaluation 3.0). A dual reference was used, and a 1:1 binding model was used to fit the raw data.

Figure 1 shows representative SPR traces and fitted curves determined for the tested Fab fragments, with the corresponding Fab names provided in the figure. Data are depicted for binding with human and cynomolgus monkey IL-6 and with IL-1α (IL-1a) as a negative control. The affinities provided in the figure correspond to the mean and standard deviation of the three independent experiments.

We observed significant binding of 6HVL_1 and VH6L_1 to human IL-6. Only VH6L_1 showed a significant affinity for cyIL-6, although the dissociation rate was significantly very rapid. No binding with the negative control target IL-1a was observed. The SPR data fitting results are shown in Table 1. Data are the averages of the 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.

Table 1: Affinity of the antibodies shown to human and cynomolgus monkey IL6

VEGF binding assessed by competitive ELISA:

To test which antibody concentration was needed to block the interaction of VEGF121 and VEGF165 with their receptors, competitive ELISA assays were performed using 6HVL_1 and VH6L_1. VEGF-binding Fab fragment ranibizumab was used as a positive control, and assays using only buffer were used as negative controls. Briefly, a 1:3 series of all samples (starting from 20 nM) was mixed with a constant concentration of 10 pM VEGF121 (Humanzyme HZ-1206) or 10 pM VEGF165 (Humanzyme HZ-1153) and incubated for 90 min. The mixture was then transferred to Maxisorp plates coated with VEGF receptor 1 (VEGF-R1, R&Dsystems, 1 μg/ml, in NaHCO3, pH 9.4) after blocking the Maxisorp plate surface with 2% MPBST. The contact time between the Fab-antigen mixture and the receptor-coated plate was limited to 10 min at room temperature to minimize interference with binding equilibrium. Following incubation and two 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 colorimetric conversion of the HRP substrate 3,3',5,5'-tetramethylbenzidine TMB to 3,3',5,5'-tetramethylbenzidine diamine, which was subsequently detected by an absorbance change at 450 nm. The TMB was preheated to room temperature and incubated on the plates for 5 minutes, followed by _quenching with 1 N _H₂SO₄ .

The results for the target VEGF165 are shown in Figure 2 and Tables 5 and 6. Clearly, compared to the clinically established VEGF-A antagonist ranibizumab, both VH6L_1 and 6HVL_1 demonstrated significantly enhanced ability to compete with both VEGF165 and VEGF121 for binding to VEGFR1.

Example 4:

Improved bispecific anti-VEGF-A/anti-IL6 Fab fragment

As mentioned above, neither antibody showed cross-reactivity with cynomolgus monkey IL6 or showed low cross-reactivity, but such cross-reactivity is required for clinical development. Furthermore, the 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 to the target antigen to maximize the durability of therapeutic effect and patient convenience. Therefore, further refinement of the initially identified molecule is needed for its intended purpose.

Several rounds of maturation were conducted by introducing different amino acid substitutions into the VH and VL domains. During maturation, candidate antibodies derived from the two “parent” antibodies, 6HVL_1 and VH6L_1, were screened and selected based on their expected properties in terms of yield, affinity, simultaneous antigen binding, hydrophilicity, stability, viscosity, and other parameters.

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 mature tests in each round. Candidate selection was based on desired properties, particularly improved human IL6 binding and cynomolgus monkey IL6 cross-reactivity, while ensuring injectability at high concentrations and maintaining other favorable properties such as VEGF-A affinity and thermostability.

The improved candidate antibody 6HVL_4 was selected as the preferred candidate from among multiple candidate antibody molecules to be tested.

Table 2: Amino acid sequences of the bispecific Fab fragments shown (numbers refer to SEQ ID NO as used in this paper).

VL VH Light chain Heavy chain 6HVL_2 5 6 6HVL_3 7 8 VH6L_2 11 12 VH6L_3 13 14 6HVL_4 twenty one twenty two twenty three twenty four 6HVL_4_YHE 25 26  

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

Express candidate antibodies as described in Example 2.

Example 5:

Improved antigen-binding kinetics of anti-VEGF-A/anti-IL6 Fab fragment

As described above, the binding kinetics of the candidate antibody against human and cynomolgus monkey IL6 and the competitive IC50 against VEGF/VEGFR1 were evaluated using the Fab fragments shown (amino acid sequences as shown in Table 2 and Example 2). To determine the potency of the antibody of the present invention relative to the prior art, the following controls were used: the bispecific antibody VH6L (the VH/VL sequence disclosed in WO2012/163520, referred to herein as “VH6L-BM”), the anti-VEGF antibody ranibizumab (INN), and an anti-IL6 antibody that cross-reacts between human and cynomolgus monkey IL6, as disclosed in WO2014/074905 (positive control). The aforementioned prior art antibodies were prepared via recombinant expression.

Figure 3 and Tables 3 and 4 show the evaluation results of human and cynomolgus monkey IL6 binding. 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 monkey IL6 cross-reactivity within the pharmacologically relevant range compared to the originally selected parental molecule.

Table 3: SPR Human IL6

<![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

Table 4: SPR cynomolgus monkey IL6

*No signals available for evaluation

Figure 4 and Tables 5 and 6 show the results of VEGF binding assessment using human VEGF121 and human VEGF165 via competitive ELISA. Figure 4 shows that the affinity exhibited by the prior art molecule 6HVL_BM was too low to be detected under the assay conditions, and therefore certainly much lower than that of the antibody of the present invention.

Table 5: IC50 VEGF121

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 Lanidomide 503 25

Table 6: IC50 VEGF165

Example 6:

Simultaneous binding of anti-VEGF/anti-IL-6Fab fragments

The anti-VEGF/anti-IL-6Fab fragment of the present invention was captured using an immobilized anti-Fab antibody, and the simultaneous binding of the antibody to its target was evaluated by surface plasmon resonance as follows:

Approximately 5000 resonance units (RUs) of the anti-Fab antibody (Cytiva 28958325) were immobilized onto the 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 the run and dilution buffer, and the flow cell temperature was set to 25°C.

An anti-Fab antibody/anti-VEGF/anti-IL-6Fab complex was formed by capturing the anti-VEGF/anti-IL-6Fab fragment via the κ chain through injection of a 10 μg/mL solution at a flow rate of 5 μL/min for 30 seconds. Two antigens, human VEGFA121 (internally generated, P1AA1779-010) and human IL-6 (commercial, Peprotech #200-06), were added sequentially or simultaneously 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 (Biacore T200, Cytiva) was monitored. For sequential binding, human VEGFA at a concentration of 300 nM was injected for 180 seconds, followed by human IL-6 at a concentration of 300 nM for 180 seconds. The same concentrations were also injected in reverse order (human IL-6 first, then human VEGFA). Similarly, a mixture of the two antigens was injected at a concentration of 300 nM each for 180 seconds. After each experiment, surface regeneration was performed by injecting 10 mM glycine at a flow rate of 5 μL/min for 60 seconds at pH 2.1. Large refractive index bias was corrected by subtracting the blank injection and by subtracting the response obtained from the control flow cell without the capture Fab.

The results are shown in Figure 7. Addition of human VEGF-A to the anti-Fab/anti-VEGF/anti-IL-6Fab complex induces binding and formation of the anti-Fab/Fab/VEGF-A complex. The sequential addition of human IL-6 induces 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.

In reverse order, human IL-6 was added first, followed by human VEGF-A, and the reduction in binding was significant (dashed line). This indicates that the binding of human IL-6 first spatially interferes with the binding of human VEGF-A, thereby reducing the binding ability between anti-VEGF/anti-IL-6Fab and human VEGF-A, but this is still possible.

In the presence of both targets, binding to human IL-6 appears to be preferred, with reduced binding to human VEGF-A (solid line). This effect is reasonable given that anti-VEGF/anti-IL-6Fab has a higher intrinsic affinity for human IL-6 compared to human VEGF-A.

In another assay, immobilized VEGF-A was used to assess the blocking effect of anti-VEGF/anti-IL-6Fab fragments on VEGF-R2 in the presence of IL-6 by surface plasmon resonance inhibition assays.

To demonstrate the simultaneous binding of human VEGF-A and human IL-6 to the anti-VEGF/anti-IL-6Fab fragment, human VEGF receptor 2 (VEGFR2, commercial R&D Systems 357-KD) was immobilized onto the S-Series sensor chip CM5 (Cytiva BR100530) using standard amine coupling chemistry, resulting in 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.

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

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

The results are shown in Figure 8. This figure shows that inhibition of the VEGFR2/VEGF-A interaction depends on the concentration of competitive 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 inhibition (solid line with crosses). Adding human IL-6, mimicking the treatment-related condition, did not affect the degree of VEGFR2/VEGF-A inhibition and produced very similar IC50 values (IC50 = 33 nM, no human IL-6 (dashed line with triangles); IC50 = 37 nM, containing additional human IL-6 (black dashed line with triangles)).

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

To assess the simultaneous binding of human VEGFA and human IL-6 to anti-VEGF/anti-IL-6Fab, a cell-based IL-6-specific reporter gene assay was performed using the reporter cell line HEK-Blue ^™ IL-6 cells (InvivoGen). Cells were incubated with anti-VEGF/anti-IL-6Fab and human IL-6 for 20 ± 1 h in the absence and presence of human VEGF-A, either simultaneously (Fig. 9) or after pre-incubation with bispecific Fab and human IL-6 (Fig. 10). Binding of human IL-6 to its receptor IL-6R on the surface of HEK-Blue ^™ IL-6 cells triggers a signaling cascade via Janus family (JAK1, JAK2, and Tyk2) tyrosine kinases, leading to activation of signal transducers and activator of transcription 3 (STAT3) and subsequent secretion of SEAP (secretory embryonic alkaline phosphatase). In the presence of anti-VEGF/anti-IL-6Fab binding to human IL-6, signaling was inhibited and no SEAP was produced. The SEAP level in the cell culture supernatant was then quantified by adding QUANTI-Blue SEAP substrate to aliquots of the supernatant. SEAP converts the QUANTI-Blue substrate into a product measurable at 650 nm using a microplate reader. The simultaneous binding of human VEGFA and human IL-6 was then assessed by plotting average absorbance against anti-VEGF/anti-IL-6Fab concentrations, and the data were fitted to a constrained 4-parameter curve. The relative potency (inhibitory concentration) of the samples was calculated using 4-parameter logistic curve fitting.

The results are shown in Figures 9 and 10.

Figure 9 shows the results without pre-incubation. Titration with increasing amounts of anti-VEGF/anti-IL-6Fab showed a clear dose-response curve, with a calculated _IC50 of 1.134 ng/mL (approximately 22.5 pM), indicating that increasing the amount of anti-VEGF/anti-IL-6Fab significantly inhibited the human IL-6 response. 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 chosen human VEGFA:human IL-6 ratio (1:1/2.5:1/5:1), only a slight decrease in the effective _IC50 was observed. This value changed slightly from _IC50 = 1.134 ng/mL in the absence of human VEGFA to _IC50 = 1.724 ng/mL when human VEGF-A was present in a 5-fold excess, closely reflecting the relevant in vivo conditions.

Figure 10 shows the results with pre-incubation, indicating that IL6 binding does not affect IL6 binding.

Example 7:

The binding of the anti-VEGF/anti-IL-6 Fab fragment to IL-6 and the proposed mode of action, as determined by X-ray crystallography.

IL-6 signaling is initiated by the formation of a hexameric complex of IL-6 with its non-signaling co-receptor IL-6R and the cytokine receptor gp130. Three epitopes (sites 1, 2, and 3) have been defined to recognize the contact surfaces formed in the complex (Boulanger MJ et al., Science 2003, 27; 300(5628):2101-4.). IL-6 first binds to IL-6R via an interacting surface called “site 1”. “Site 2” is an epitope formed by the IL-6 and IL-6R binary complex that interacts with domains 2 and 3 of gp130. Subsequently, the interaction between “site 3” of IL-6 and domain 1 of gp130 leads to the formation of a dimer of the IL-6/IL-6R/gp130 trimer, thus forming the hexameric signaling complex.

To understand which epitopes of IL6 bind to our two Fab series (6HVL and VH6L), we performed structural analysis on the complexes between IL-6 and the antibody Fabs representing the anti-VEGF/anti-IL-6 Fab fragments of this invention, respectively. The Fab 6HVL4.1 used is very closely related to Fab 6HVL_2, differing only in two mutations, and Fab 0182 is most closely related to Fab VH6L_1. Since all 6HVL clones originate from the same Fab (6HVL_1), and similarly all VH6L clones originate from Fab VH6L_1, it is safe to assume that the structural results obtained below apply to each of the individual Fab series. Complexes of Fab and IL-6 were formed, and the structural analysis of the complexes was performed by X-ray crystallography as follows:

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).

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 assays were performed at 21 °C in a sitting-drop vapor diffusion apparatus.

For Fab fragment 0182, needle-like crystals appeared within two days in 0.1M _MgCl₂ , 0.1M sodium citrate at pH 5, and 15% (w/v) PEG4000. The crystals were subsequently used in inoculation experiments, and large tetragonal crystals could be obtained from 0.1M calcium acetate, 12% (w/v) PEG8000, and 0.1M sodium dimethylarsinate at pH 5.5.

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

For data collection, the collected crystals were rapidly cooled at 100 K in a crystallization solution supplemented with 15% ethylene glycol. X-ray diffraction data were collected using a PILATUS 6M detector at beamline X10SA from a Swiss source (Villigen, Switzerland) at wavelengths (for Fab fragment 0182) and (for 6HVL 4.1). The data were 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 anisotropy was analyzed using STARANISO (Tickle, IJ, Flensburg, C., Keller, P., Paciorek, W., Sharff, A., Vonnhein, C., Bricogne, G. (2018). STARANISO ( http://staraniso.globalphasing.org/cgi-bin/staraniso.cgi ). Cambridge, United Kingdom: Global Phasing Ltd.).

The crystals of the complex including Fab 0182 belong to space group P2 ₁ , with cell axes of β = 91.65°, and are diffracted to a resolution of [resolution missing].

The crystals of the complex including 6HVL4.1 belong to space group P2 ₁ 2 ₁ 2 ₁ , where the unit cell axis is and the diffraction resolution is .

The structure was determined using PHASER (McCoy, A.J., Grosse-Kunstleve, R.W., Adams, P.D., Winn, M.D., Storoni, L.C., & Read, R.J. Phaser crystallographic software. J Appl Cryst. 40, 658-674 (2007)), employing the coordinates of the internal Fab and IL-6 (pdb entry 1alu) as a search model via molecular substitution. Based on sequence differences, differential electron density was used to modify amino acids. The architecture was refined using procedures from the CCP4 suite (Winn, M.D. et al. Overview of the CCP4 suite and current developments. Acta. Cryst. D67, 235-242 (2011)) and BUSTER (Bricogne, Blanc, G.E., Brandl, M., Flensburg, C., Keller, P., Paciorek, W., Roversi, P., Sharff, A., Smart, O.S., Vornhein, C., Womack, T.O. Buster version 2.9.5 Cambridge, United Kingdom: Global Phasing Ltd. (2011)). Manual reconstruction was performed using COOT (Emsley, P., Lohkamp, B., Scott, W.G., Cowtan, K. Features and Development of Coot. Acta Cryst. D66, 486-501 (2010)).

Table 7 summarizes the data collection and optimization statistics. All graphical representations were prepared using PYMOL (Pymol Molecular Graphics System, version 1.7.4.LLC.).

Table 7. Data Collection and Refinement Statistics

The value in parentheses refers to the highest resolution shell.

Structure of the Fab 0182-IL-6 complex

The crystal structure of the complex of Fab 0182 (representative of the VH6L series of Fab) and IL-6 was determined at 1000 ppm (Figure 5). The structure shows that Fab 0182 binds to IL-6 through contributions from the CDR2 of the heavy chain and the CDR1 and CDR3 of the light chain. Further interaction with IL-6 is maintained by the N-terminal residues Val3 and Gln4 of the Fab 0182 light chain. The interface contributed by IL-6 is formed by residues of helical A and helical C.

Figure 16 illustrates the binding mode of Fab 0182 with IL6. For illustrative purposes, we generated a superposition of two structures: first, a superposition of the Fab-IL6 complex structure, and second, a superposition of the IL6R-IL6 complex structure (derived from the eutectic structure of IL6, IL6R, and gp130, PDB accession number 1p9m, see Boulanger MJ et al., Science 2003, 27; 300(5628):2101-4). Comparing this to the trimeric complex of IL6 with IL6R and gp130, it is clear that Fab binds to IL6 in a very similar manner to gp130, i.e., it binds to site 2 of IL6. This binding mode is expected to allow both Fab and IL6R to bind to IL6 simultaneously, meaning that the interaction between IL6 and IL6R should still be possible, and it is a priori expected that this IL6 antagonist functions by inhibiting the interaction between the IL6/IL6R complex and gp130 via binding to site 2 of IL6.

Structure of the Fab 6HVL4.1-IL-6 complex

We determined the crystal structure of the Fab 6HVL4.1-IL-6 complex at a high resolution (Figure 6). The structure shows that Fab 6HVL4.1 binds to IL-6 through major contributions from CDR1 and CDR3 of the heavy chain and CDR2 and CDR3 of the light chain. Further interaction with IL-6 is maintained by the first three N-terminal residues of the heavy chain of Fab 6HVL4.1. The interface contributed by IL-6 is formed by residues of helical A and C.

Similar to the approach used for Fab 0182, we employed structural superposition and analyzed the interacting residues as described above to determine the binding mode of Fab 6HVL4.1 to IL6. Figure 17 shows that 6HVL4.1 also binds to IL6 in a manner very similar to gp130, and therefore, from a structural perspective, it must be considered a site 2 binding compound.

Experimental study on the binding mode of IL6

The fact that clones like 6HVL4.1 and their derivatives are IL6 site 2 binding compounds can be functionally confirmed by measurements using surface plasmon resonance.

In one such assay, Fab fragments representing the 6HVL family (including 6HVL4.1 (antibody “P1AE2421”)) were captured on the surface of an SPR chip via an anti-Fab antibody and IL6, and then three different concentrations (250 nM, 500 nM, 1000 nM) of IL6R were passed through the chip surface. Here, if IL6 can bind to both Fab and IL6R, we expect a two-step sequential signal increase. And indeed, for the Fab tested, the following was observed (Figure 18): in the SPR signal trace, the addition of IL6 caused a strong signal increase, and the signal was further enhanced after the addition of IL6R. This clearly demonstrates that simultaneous binding of IL6R and Fab to IL6 is possible. This finding is further corroborated by the fact that covalent chimeras of IL6 and IL6R (referred to as “hyper-IL6”, in which site 1 of IL6 is completely shielded and inaccessible) still promote the binding of the corresponding Fab molecules when coated onto an SPR chip, and this was detected using Fab at a concentration of 26 nM (Figure 19).

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

Given that we know from the available crystal structure and SPR experiments that site 1 of IL6 is still available for binding, we must interpret these results so that the IL6 antibody described in this patent can not only spatially block the binding of the IL6/IL6R complex to gp130, but also strongly reduce the binding affinity of IL6 to IL6R in an allosteric manner , i.e., additionally act as an IL6 site 1 antagonist.

This mode of action is expected to have optimal properties for the following reasons:

1. As IL6 site 2 binders, IL6 antagonists can equally effectively block the formation of signal transduction complexes (cis signal transduction) formed by the binding of IL6 to membrane-bound IL6R and gp130, or the pre-formed complex of IL6 and IL6R (trans signal transduction). Conversely, IL6 site 1 binders cannot bind to the pre-formed complex of IL6 and IL6R, but can only antagonize it upon dissociation of the complex.

2. Because they allosterically reduce the affinity of IL6 for IL6R, the IL6 antagonists described herein are expected to exhibit increased potency by inhibiting the formation of the first step of signaling (i.e., the formation of the IL6/IL6R complex) compared to site 2 conjugates that do not exhibit this effect. Site 2 conjugates 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, the relatively effective concentrations of IL6/IL6R and gp130 are expected to be very high.

3. It is known that when used systemically as an antibody, IL6 site 1 bindings lead to a strong accumulation of the IL6/antibody complex due to the significantly increased half-life of the complex compared to IL6 alone. Conversely, IL6 site 2 bindings are expected to still eliminate the IL6/antibody complex via binding to membrane-bound IL6R and subsequent internalization and degradation by cells that take up the complex. In this regard, the IL6 antagonists described herein are expected to combine the desired properties of both site 1 and site 2 bindings: while functionally blocking the first step in the formation of the IL6/IL6R/gp130 signaling complex, they still allow for the degradation of the IL6/mAb complex via cellular IL6R binding.

4. In ophthalmic indications and as Fab molecules, the expected behavior of such conjugates may still be more beneficial: similar to IL6 site 1 conjugates, Fab/IL6 complexes can make the ocular space relatively unimpeded by IL6R binding and can be rapidly eliminated systemically by renal filtration.

Example 8:

Improved thermal stability of VEGF/IL6 Fab fragments

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

Table 8: Amino acid sequences of the bispecific Fab fragments shown (numbers refer to SEQ ID NO as used in this paper).

VL VH 6HVL_5 39 40 6HVL_6 41 42 VH6L_4 43 44 VH6L_5 45 46

The thermal stability of the bispecific antibodies is evaluated as follows.

Thermal stability:

A sample containing a 1 mg/mL bispecific antibody Fab fragment was prepared in 20 mM histidine/histidine chloride and 140 mM NaCl (pH 6.0) and transferred to a 10 μL microcuvette array. Static light scattering and fluorescence data were recorded using an Uncle instrument (UnchainedLabs) after excitation with a 266 nm laser, while the sample was heated from 30 °C to 90 °C at a rate of 0.1 °C/min. Measurements were repeated three times.

The assessment of the onset temperature was performed using UNcle analysis software. The aggregation onset temperature was defined as the temperature at which the scattering intensity begins to increase. Protein denaturation was monitored by the shift of the centroid mean (BCM) of the fluorescence signal with heat. The melting temperature was defined as the inflection point in the curve of BCM (nm) versus temperature.

Table 9: Thermal Stability

Example 9:

Biophysical properties of the improved bispecific anti-VEGF/anti-IL6 Fab fragment (viscosity assessment by dynamic light scattering (DLS))

The antibody Fab fragment, as described above, was expressed in CHO cells using standard methods.

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

Viscosity assessment:

Instruments and materials

• Wyatt DLS microplate reader with Greiner Bio-One microplate

• 3000 Series Nanosphere ^™ Standard Size (Thermofisher Catalog No. 3300A)

• Twain 20 (Roche, catalog number 11332465001) and silicone oils, such as (Alfa Aesar catalog number A12728).

• Ultraviolet spectrophotometers used for concentration determination (e.g., Nanodrop 8000).

Sample preparation

Rebuffer the antibody sample and dilute it with 20 mM His/HCl (pH 5.5) (buffer) and 0.02% Tween 20 (final concentration). Add beads to a solid concentration of 0.03%. Prepare at least three different concentrations, with the highest concentration being approximately 200 mg/mL if possible. Two blank samples are required as antibody-free controls: one containing nanobeads resuspended in water and the other containing nanobeads resuspended in buffer. Transfer the samples to microplates and cover each well with silicone oil.

Measurements were performed using a Wyatt DLS microplate reader.

All samples and blanks were analyzed at different temperatures ranging from 15°C to 35°C in 5°C increments. The acquisition time was 30 seconds, and each sample and temperature was acquired 40 times.

Data Analysis

The overview in the software template (Microsoft Dynamics 7.10 or later) shows the raw data Dapp (apparent radius) in nm. 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 (300 nm). The calculated viscosity is displayed as a curve in Excel. Viscosity at a given concentration can be inferred using Mooney curve fitting (in Excel). Here, the maximum protein concentration with a viscosity exceeding 20 cP is calculated.

The maximum concentration of the antibody shown is as follows, which reaches a viscosity of 20 cP at 20°C.

Table 10: Viscosity measured by DLS bead method. The maximum feasible concentration of the indicated antibody at 20 cP is shown at 20°C.

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

The results show that the antibodies of the present invention can be formulated at high concentrations, containing viscosities below the acceptable viscosity limit for injectability. Consequently, the antibodies of the present invention are well-suited for intraocular application because they allow for the delivery of high molar doses in limited injection volumes, which, when combined with potent drugs, results in high durability and thus reduces the frequency of administration, which is desirable for increased patient convenience and treatment adherence.

Example 10:

Primary cell-based assay (HRMEC) used to demonstrate VEGF/IL6 bispecific antibody 6HVL_4-mediated IL6 inhibition.

To measure IL-6 signaling activity in HRMECs, a assay was established to quantify ICAM-1 surface expression in HRMECs. HRMECs were stimulated for 72 hours with an equimolar concentration (2 nM) of a combination of human IL-6 and human IL-6R. ICAM-1 surface expression was assessed by flow cytometry. To measure 6HVL4 inhibitory activity, the IL-6/IL-6R mixture was pre-incubated with gradually increasing concentrations of antibody before application to cells.

Cell Culture: HRMEC (catalog number PEL-PB-CH-160-8511; PELOBiotech GmbH; Bayern, Germany) was thawed and cultured in 175 cm² flasks in endothelial cell growth medium (EGM-MV) containing endothelial cell basal medium (EBM) (catalog number CC-3156; Lonza; Basel, Switzerland) and 5% fetal bovine serum (FBS), hydrocortisone, human fibroblast growth factor B, VEGF, R3-IGF-1 (a recombinant analog of insulin-like growth factor-I, where Arg at position 3 is replaced with Glu), ascorbic acid, human epidermal growth factor, and GA-1000 (all included in the EGM-2MV Microvascular Endothelial Cell SingleQuots™ Kit; catalog number CC-4147; Lonza) at the manufacturer's recommended concentrations. Twenty-four hours after plating, the culture medium was replaced with fresh EGM-MV, and the cells were regenerated for 3 days before assay. Assay conditions were optimized for different passage numbers and IL-6/soluble IL-6R concentrations. The final assay was performed using 6th-generation HRMECs and an equimolar concentration of 2 nM IL-6/soluble IL-6R.

Flow cytometry assay: HRMECs were separated from the flask by washing twice with Ca2+ and Mg2+-free phosphate-buffered saline (PBS) (catalog number 10010023; Life Technologies) and once with the cell dissociation reagent Accutase (catalog number A1110501; Thermo Fisher Scientific; Waltham, MA). After washing, 5 mL of cell dissociation reagent was added to the cells, and the flask was incubated in a 5% CO2 incubator at 37°C for 3 minutes. The separated cells were collected from the flask and transferred to a 50 mL conical centrifuge tube. The tube was filled to 50 mL with EBM containing 2% FBS and centrifuged at 300 g for 6 minutes. The supernatant was discarded, and the pellet was resuspended in 5 mL of starvation medium (EBM containing 2% FBS). Cell counts were quantified using a TC20 automated cell counter (Bio-Rad; Hercules, CA) and adjusted to 300,000 cells/mL using starved medium. Then, 100 μL of cell suspension was added to each well of a Costar 96-well plate (catalog number 3596; Corning; Corning, NY) to produce 30,000 cells/well. The plates were then incubated at 37°C for 24 hours in a 5% CO2 incubator.

Recombinant human IL-6 (catalog number 206-IL/CF; R&D Systems; Minneapolis, MN) and recombinant human IL-6R (catalog number 227-SR-025; R&D Systems) were mixed at equimolar concentrations in starved medium and incubated at room temperature for 1 hour to allow the formation of the IL-6-I/L-6R complex. Next, 50 μL of a serial 6HVL_4 dilution (3-fold 7-spot dilution) was added to the cells and incubated at 37°C and 5% CO2 for 1 hour. Finally, 50 μL of the IL-6-I/L-6R complex was added to the cells, resulting in final concentrations 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 the IL-6-I/L-6R complex without 6HVL_4 were also included to determine background ICAM-1 surface expression and 100% response levels, respectively. The cells were incubated at 37°C in a 5% CO2 incubator for 72 hours.

To analyze ICAM-1 surface expression, cells were washed twice with PBS ( ^Ca²⁺ , ^Mg²⁺ ; Life Technologies) and once with Accutase (catalog number A1110501; Thermo Fisher Scientific). Cells were separated from the plate using 50 μL of Accutase (3 min, 37°C) and transferred to a flow cytometry Falcon 96-well storage plate (catalog number 353263; Corning). The original wells were washed once with 100 μL PBS containing 2% FBS and 2 mM EDTA, and wash medium containing the remaining cells was added to the flow cytometry plate. Cells were pelleted by centrifugation at 300 g for 6 min, and the supernatant was discarded. The pellet was resuspended in 100 μL PBS, 2% FBS, and 2 mM EDTA containing 10 μg/mL human IgG (catalog number I2511; MilliporeSigma; Burlington; MA) to block non-specific binding sites and incubated at room temperature for 15 min. After inhibition, 0.5 μg of fluorescein-labeled anti-ICAM-1 antibody (catalog number BBA20; R&D Systems) was added to the cells, and the reaction was incubated at 28°C for 45 min. Following staining, cells were pelleted by centrifugation at 300g for 6 min, and the pellet was resuspended in 150 μL PBS containing 2% FBS and 2 mM EDTA. Fluorescein fluorescence was measured using an Attune NxT flow cytometer (ThermoFisher Scientific).

Data Analysis: For all three assays, 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 mean signal for each condition was calculated. The 100% response level was calculated from cells stimulated with IL-6/IL-6R without 6HVL_4, and the inhibitory potential of 6HVL_4 was then expressed as the percentage inhibition at 100% response. The percentage inhibition of 6HVL_4 for each concentration was measured in the three independent experiments, and the mean and SEM were calculated. The mean IC50 and SE were calculated using the mean from the three independent experiments using ExcelXLfit software version 5.5.0 (IDBS; Guildford, UK). The concentration-response curves were fitted using a 5-parameter logistic model (A+((B-A)/(1+(((B-E)*((C/x)^D))/(E-A)))))) by nonlinear regression analysis.

Table 11: Calculated % inhibition of 6HVL_4 on IL-6-induced ICAM-1 expression on HRMEC surfaces

6HVL4 induces dose-dependent inhibition of IL-6 signaling in HRMEC, with a 50% inhibitory concentration (IC50) of 1.52 +/- 0.04 nM (Figure 11).

Example 11:

Primary cell-based assay (HUVEC) used to demonstrate VEGF/IL6 bispecific antibody 6HVL_4-mediated VEGF inhibition.

Measurement:

HUVECs were obtained from Lonza (catalog number 00191027; Basel, Switzerland). The endothelial cell basal medium (EBM-2; catalog number CC-3156), which together form the endothelial cell growth medium (EGM-2) and the assay medium (EBM-2, containing 0.5% fetal bovine serum [FBS]), and the EGM-2 endothelial cell SingleQuots kit (catalog number CC4176) were also purchased from Lonza.

T175 cell culture flasks coated with adhesion factor (AF) (catalog number S-006-100; Gibco, Thermo Fisher Scientific; Waltham, MA) (catalog number 353112; Corning; Corning, NY) were used to maintain HUVECs. StemProAccutase (catalog number A11105-01; Gibco) was used for cell isolation.

Cell viability/proliferation was measured using alamarBlue (catalog number DAL1100; Invitrogen, Thermo Fisher Scientific) in 96-well fibronectin-coated plates (catalog number 354409; Corning).

Recombinant human VEGF-A was obtained from R&D (catalog number 293-VE; Minneapolis, MN) and dissolved at a stock concentration of 100 μg/mL in phosphate-buffered saline (PBS) (catalog number 14190-094; Gibco) free of Ca2 ⁺ and Mg2 ⁺ .

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

HUVECs were maintained in AF-coated T175 flasks in EGM-2 until passage 5. For viability assays, HUVECs were isolated using Accutase and diluted 1:1.66 in assay medium (EBM-2 0.5% FBS). Cells were then centrifuged and resuspended in EGM-2 containing 0.5% FBS to a cell density of 100,000 cells/mL. 100 μL of the cell suspension was then seeded into fibronectin-coated 96-well plates to achieve a cell density of 10,000 cells/well. The outer wells were left unseeded and then filled only with assay medium. Cells were incubated overnight at 37°C in a 5% CO2 incubator.

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

The 6HVL_4 stock solution was also diluted in the 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.

Next, 12.5 μL of 10⁻¹⁰ prediluted 6HVL₄ solution and 12.5 μL of 10⁻¹⁰ VEGF-A solution (750 ng/mL) were added sequentially to the cells in each plate, in quadruplicate. VEGF-A was used at a constant final concentration (75 ng/mL), and 6HVL₄ was used in a dose-responsive manner, with a final concentration range of 3000 ng/mL to 1.4 ng/mL. Cells were incubated at 37 °C and 5% CO₂ for 72 h. For analysis, 12 μL of alamarBlue was added to each well, and the cells were then incubated in a cell culture incubator for 3 h. Absorbance was measured at 570 nm using a FlexStation 3 microplate reader from Molecular Devices, with a reference wavelength of 600 nm.

Data Analysis:

For each experiment, each condition was performed in quadruplicate. A total of four independent experiments were conducted. An independent experiment was considered to be two separate plates processed on the same day. Therefore, eight separate plates were used for analysis. The background signal of unstimulated cells was subtracted from the experimental wells, and the mean signal for each condition was calculated. The 100% response level was calculated from cells stimulated with VEGF-A (75 ng/mL) without additional compound exposure, and the signal from the 6HVL_4 exposure well is expressed as the percentage of inhibition of 100% response.

IC50 values were calculated from mean data for each antibody concentration using ExcelXLfit software version 5.5.0 (IDBS; Guildford, UK). Concentration-response curves were fitted using a 4-parameter logistic model (A + ((B-A)/(1 + ((C/x)^D))))) calculated relative to baseline and maximum inhibitory activity via nonlinear regression analysis. Data are presented as means from four independent experiments, with standard error (SEM) of the means.

Table 12: Calculated % inhibition of 6HVL_4 on VEGF-A-induced HUVEC proliferation

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

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

Example 12:

The restoration of barrier function in the presence of both VEGF-A and IL6, as well as the VEGF/IL6 bispecific antibody 6HVL_4, demonstrates the bioactivity of this molecule.

To evaluate the dual biological activity of the antibody of this invention, namely the simultaneous blocking of two targeting cytokines (VEGF-A and IL6 (complexed with IL6R)), transendothelial cell electrical resistance (TER) measurements were performed. In this measurement, the electrically dense endothelial cell layer responds to the addition of both VEGF-A and IL6 by losing its barrier function. The restoration of barrier function by the VEGF/IL6 bispecific antibody 6HVL_4 in the presence of both cytokines VEGF and IL6 was evaluated as follows:

Measurement:

Human retinal microvascular endothelial cells, further named HRMVECH (PELOBiotech; catalog number PEL-PB-CH-160-8511), 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 adhesion factor (Ginco, catalog number S-006-100) until passage 5. For transendothelial cell resistance measurements, cells were isolated using (Gibco, catalog number A11105-01). Subsequently, cells were seeded at a density of 120,000 cells/well in 100 μl of MV-EGM-2 growth medium into the upper chamber of a fibronectin-coated (Catalog number 354008, Corning) Transwell filter (24-well Corning, catalog number 3470). The lower chamber of the Transwell filter contained 600 μl of MV-EGM-2 medium. Cells were incubated at 37°C and 5% CO2 for 3 days. The medium was then changed to assay conditions (MV-EGM-2 without VEGF, containing 2% FBS), and the Transwell filter was transferred to the cellZcope system using 280 μl of medium (upper chamber) and 810 μl of medium (lower chamber). Cells were then incubated at 37°C and 5% _CO2 for 24 hours, while TER was measured using cellZcope. On the second day, cells were treated with either a final concentration of 10 ng/ml VEGF (R&D Systems, catalog 293-VE/CF), 50 ng/ml IL6 (R&D Systems, catalog 206-IL/CF) combined with 100 ng/ml IL6R (R&D Systems, catalog 227-SR-025/CF) and 10 ng/ml VEGF, or with an equal volume of assay medium (eightfold for each condition), and TER was measured until the next day. Subsequently, 6HVL4 or aflibercept or assay medium at final concentrations of 1 μg/ml and 2.3 μg/ml were added to the cells, and the TER was measured over the next 24 hours. Thus, each condition was performed four times.

Data Analysis:

The dataset generated from one well was normalized to TER values obtained shortly before the addition of the cytokine mixture. For each condition, the mean signal and standard deviation were calculated from the normalized data.

result:

The results are shown in Figures 12 (6HVL_4) and 13 (Abercept).

The barrier function of HRMVECs was reduced using the cytokine VEGF alone, as well as in combination with IL6/IL6R. When the antibody 6HVL_4 was used, the damaged barrier recovered to 100% after 24 hours.

Example 13:

Identification of IL6 complementary regions

The amino acid residues in contact with IL6 were identified from the crystal structure of the complex between 6HVL4.1 and IL6. The positions of complementary amino acid residues within the VH and VL domains are illustrated in Figure 15. For this purpose, the "byres" function of PyMOL and a cutoff distance of 5 Å were used to identify residues in the Fab/IL6 complex that may interact with IL6. Here, we analyze residues 48-215 confined to IL6 (as defined in Uniprot ID P05231), which are residues typically resolved in the structure of IL6 alone (see PDB accessions 1alu and 1IL6). Figure 15 also shows an alignment between 6HVL4.1 and the monospecific anti-IL6 antibody 6HdL2.05 based on the antibody of the present invention, where the VEGF complementary site is replaced by a non-binding region. 6HdL2.05 has the VH domain of SEQ ID NO:48 and the VL domain of SEQ ID NO:47. When expressed and purified as described in Example 2 and subjected to SPR assays using human IL6 or cynomolgus monkey IL6 similar to those in Example 3, the antibody exhibited the SPR sensing pattern depicted in Figure 20. After fitting the experimental data, 6HdL2.05 demonstrated an affinity comparable to the highest affinity obtained with 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 a fitted KD of 1.3 nM for cynomolgus monkey IL6.

Amino acid residues identified as contributing to antigen binding were identified in Tables 13 (for amino acid residues in the variable heavy chain domain) and 14 (for amino acid residues in the variable light chain domain). Amino acid positions were numbered according to the Kabat numbering system (the same numbering was used in Figures 1+5). The positions of amino acids involved in antigen binding were identified by their Kabat positions within the VH or VL domains.

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

Claims (16)

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) an amino acid sequence of SEQ ID NO:15. The antibody comprises: (e) CDR-L1 containing the amino acid sequence of SEQ ID NO:16, (f) CDR-L3 containing 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. 2. The antibody according to claim 1, wherein the up to 5 amino acid substitutions occur in the FR region of the corresponding variable structural 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 any 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 the VL domain of SEQ ID NO:35 and the VH domain of SEQ ID NO:36. 7. The antibody according to any one of 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 an 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 generating antibodies that bind to human VEGF-A and human IL6, the method comprising culturing host cells according to claim 10 to generate the antibodies. 12. The method according to claim 11, wherein the host cell is a CHO cell. 13. A pharmaceutical preparation comprising an antibody according to any one of claims 1 to 8 and a pharmaceutical carrier. 14. An infusion port delivery device comprising an antibody according to any one of claims 1 to 8. 15. The antibody according to any one of claims 1 to 8, for use as a medicine. 16. An infusion port delivery device comprising an antibody according to any one of claims 1 to 8 or a pharmaceutical preparation according to claim 13.