Modified antibodies and methods of making the same

Abstract

The present invention relates to an improved modified antibody, which has increased solubility and can be concentrated to have a high concentration, and a method for preparing the same; and to a modified antibody in which an amino acid located in a framework region of the antibody is substituted, and a method for preparing the same.

Description

TECHNICAL FIELD

[0001] The present application relates to improved modified antibodies having increased solubility and capable of being highly concentrated, and a method for producing the same, and more particularly, to improved modified antibodies in which amino acids in the backbone thereof are substituted, and a method for producing the same. BACKGROUND

[0002] Antibodies are very effective as therapeutic agents in treating diseases such as cancer and autoimmune diseases. The developability of antibodies is evaluated in consideration of high production, stable formulation, high physicochemical and in vivo stability, high solubility, and PK characteristics (Next generation antibody drugs: pursuit of the 'High-hanging fruit'. Nat. Rev. Drug Discovery., 2018, 17: 197-223.)

[0003] Antibodies as protein drugs can be chemically unstable due to changes in covalent bonds, or can be physically unstable due to deformation of the three-dimensional spatial structure. Antibodies can be chemically unstable due to hydrolysis, oxidation, deamidation, disulfide bond modification, or racemization, and can be physically unstable due to aggregation, adsorption, or dissolution.

[0004] The instability of antibodies can affect solubility, efficacy, etc., and reduce the stability and solubility of antibodies, thereby causing a decrease in productivity and production yield, and an increase in the aggregation tendency, which increases the risk of immunogenicity.

[0005] In this technical background, the present inventors have endeavored to develop modified antibodies that can be highly concentrated by improving the solubility of antibodies. As a result, the present inventors have found that highly concentrated can be achieved based on the improvement of solubility by amino acid substitution at a specific position, and completed the present application based on the same. SUMMARY

[0006] Accordingly, it is an object of the present application to provide improved modified antibodies having increased solubility and fragments thereof.

[0007] It is another object of the present application to provide a method of producing the modified antibodies and fragments thereof.

[0008] According to one aspect of the present application, the above and other objects can be achieved by providing modified antibodies and fragments thereof in which at least one amino acid selected from the group consisting of amino acids at positions 13 and 42 of the heavy chain variable region (based on the AHO numbering system) and an amino acid at position 94 of the light chain variable region (based on the AHO numbering system) is substituted.

[0009] According to another aspect of the present application, there is provided a method of producing a modified antibody and fragments thereof in which at least one amino acid selected from the group consisting of amino acids at positions 13 and 42 of the heavy chain variable region (based on the AHO numbering system) and amino acid at position 94 of the light chain variable region (based on the AHO numbering system) is substituted. BRIEF DESCRIPTION OF DRAWINGS

[0010] Figure 1 Results of antibody structure modeling and hotspot analysis are shown.

[0011] Figure 2 Results of expression testing of L11T, V13S and V103S (based on the AHO numbering system, hereinafter "L11T", "V12S" and "V93S") in antibody hotspots are shown.

[0012] Figure 3 Results of expression testing of N42T, Q77R and N94S (based on the AHO numbering system, hereinafter "N35T", "Q61R" and "N76S") in antibody hotspots are shown.

[0013] Figure 4 Results of expression testing of V13S, N42T and N94S (based on the AHO numbering system, hereinafter "V12S", "N35T" and "N76S") in antibody hotspots are shown.

[0014] FIGS. 5A to 5G show results of purification of antibodies by SDS-PAGE.

[0015] Figure 6 Results of thermal stability analysis of anti-Ang2 antibodies according to the present application are shown.

[0016] FIGS. 7A to 7C show results of evaluation of anti-angiogenic efficacy of anti-Ang2 antibodies according to the present application using a mouse CNV (choroidal neovascularization) model. DETAILED DESCRIPTION

[0017] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this application pertains. Generally, the nomenclature used herein is well known and commonly used in the art.

[0018] The present inventors have found that modified antibodies and fragments thereof exhibit improved solubility, can be concentrated to high levels, and thus exhibit improved productivity, in which at least one amino acid selected from the group consisting of amino acids at positions 13 (V12) and 42 (N35) in the heavy chain variable region (based on the AHO numbering system) and amino acid at position 94 (N76) in the light chain variable region (based on the AHO numbering system) is substituted.

[0019] In particular, the present inventors have found that modified antibodies and fragments thereof exhibit improved solubility, can be concentrated to high levels, and thus exhibit improved productivity, in which the amino acid at position 13 (V12) in the heavy chain variable region is substituted with serine (S), the amino acid at position 42 (N35) in the heavy chain variable region is substituted with threonine (T), or the amino acid at position 94 (N76) in the light chain variable region is substituted with serine (S), wherein the numbering is based on the AHO numbering system.

[0020] Based on this, in one aspect, the present application relates to modified antibodies and fragments thereof, in which at least one amino acid selected from the group consisting of amino acids at positions 13 and 42 in the heavy chain variable region (based on the AHO numbering system) and amino acid at position 94 in the light chain variable region (based on the AHO numbering system) is substituted.

[0021] In another aspect, the present application relates to a method for producing modified antibodies and fragments thereof, in which at least one amino acid selected from the group consisting of amino acids at positions 13 and 42 in the heavy chain variable region (based on the AHO numbering system) and amino acid at position 94 in the light chain variable region (based on the AHO numbering system) is substituted.

[0022] The positions of the amino acids of the antibodies or fragments thereof disclosed herein can be defined or identified based on the AHO numbering system. In some cases, the positions of the amino acids of the antibodies or fragments thereof disclosed herein can mean positions numbered in order starting from No. 1 of the amino acid from the N-terminus of the amino acid sequence of the heavy chain variable region of SEQ ID NO: 34 and the light chain variable region of SEQ ID NO: 32.

[0023] In one embodiment, the amino acids at positions 13 and 42 in the heavy chain variable region (based on the AHO numbering system) and the amino acid at position 94 in the light chain variable region (based on the AHO numbering system) can be substituted with serine (S) or threonine (T).

[0024] In a specific embodiment, the amino acid substitution can include substitution of the amino acid at one or more positions (based on the AHO numbering system) selected from the group consisting of:

[0025] substitution of valine (V) at position 13 in the heavy chain variable region with serine (S);

[0026] substitution of asparagine (N) at position 42 in the heavy chain variable region with threonine (T); and

[0027] substitution of asparagine (N) at position 94 in the light chain variable region with serine (S).

[0028] In particular embodiments of the application, the amino acid substitutions can include amino acid substitutions at the following positions (based on the AHO numbering system):

[0029] substitution of valine (V) at position 13 in the heavy chain variable region with serine (S);

[0030] substitution of asparagine (N) at position 42 in the heavy chain variable region with threonine (T);

[0031] substitution of asparagine (N) at position 94 in the light chain variable region with serine (S).

[0032] substitution of valine (V) at position 13 in the heavy chain variable region with serine (S), and substitution of asparagine (N) at position 42 in the heavy chain variable region with threonine (T);

[0033] substitution of valine (V) at position 13 in the heavy chain variable region with serine (S), and substitution of asparagine (N) at position 94 in the light chain variable region with serine (S);

[0034] substitution of asparagine (N) at position 42 in the heavy chain variable region with threonine (T), and substitution of asparagine (N) at position 94 in the light chain variable region with serine (S); or

[0035] substitution of valine (V) at position 13 in the heavy chain variable region with serine (S), substitution of asparagine (N) at position 42 in the heavy chain variable region with threonine (T), and substitution of asparagine (N) at position 94 in the light chain variable region with serine (S).

[0036] The above amino acid substitutions result in an increase in the solubility of the antibody, an increase in the ability to concentrate to high levels, and an increase in productivity.

[0037] Unlike the above amino acid substitutions, it was found that substitution of leucine at position 11 in the heavy chain variable region (based on the AHO numbering system) with threonine, substitution of valine at position 103 in the heavy chain variable region (based on the AHO numbering system) with serine, or substitution of glutamine at position 77 in the light chain variable region (based on the AHO numbering system) with arginine did not exhibit the desired effect of increasing solubility.

[0038] In particular embodiments, the modified antibody or fragment thereof according to the present application can comprise one or more amino acid substitutions in the framework regions selected from the group consisting of:

[0039] QVQLVESGGGLX1KPGGSLRLSCAAS (wherein X1 is S);

[0040] MX2WVRQAPGKGLEWVSS (wherein X2 is T); and

[0041] NLQSGVSSQFSGSGSGTDFTLTIX3SLQPEDSATYYC (wherein X3 is S).

[0042] In particular, the anti-Ang2 antibody clone O4 comprising the heavy chain variable region of SEQ ID NO: 34 and the light chain variable region of SEQ ID NO: 32 can comprise substitution of one or more amino acids selected from the group consisting of amino acids at positions 12 and 35 in the heavy chain variable region and amino acid at position 76 in the light chain variable region with serine (S) or threonine (T).

[0043] In one embodiment, the anti-Ang2 antibody clone O4 comprising the heavy chain variable region of SEQ ID NO: 34 and the light chain variable region of SEQ ID NO: 32 can comprise one or more amino acid substitutions selected from the group consisting of:

[0044] substitution of valine (V) at position 12 in the heavy chain variable region with serine (S);

[0045] substitution of asparagine (N) at position 35 in the heavy chain variable region with threonine (T); and

[0046] substitution of asparagine (N) at position 76 in the light chain variable region with serine (S).

[0047] In particular, according to the present application, the anti-Ang2 antibody clone O4 can comprise the following amino acid substitutions:

[0048] substitution of valine (V) at position 12 in the heavy chain variable region with serine (S);

[0049] substitution of asparagine (N) at position 35 in the heavy chain variable region with threonine (T);

[0050] substitution of asparagine (N) at position 76 in the light chain variable region with serine (S).

[0051] substitution of valine (V) at position 12 in the heavy chain variable region with serine (S), and substitution of asparagine (N) at position 35 in the heavy chain variable region with threonine (T);

[0052] substitution of valine (V) at position 12 in the heavy chain variable region with serine (S), and substitution of asparagine (N) at position 76 in the light chain variable region with serine (S);

[0053] substitution of asparagine (N) at position 35 in the heavy chain variable region with threonine (T), and substitution of asparagine (N) at position 76 in the light chain variable region with serine (S); or

[0054] substitution of valine (V) at position 12 in the heavy chain variable region with serine (S), substitution of asparagine (N) at position 35 in the heavy chain variable region with threonine (T), and substitution of asparagine (N) at position 76 in the light chain variable region with serine (S).

[0055] An attempt was made to improve the physical properties of O4, which is a clone of an anti-Ang2 antibody comprising a heavy chain variable region of SEQ ID NO: 34 and a light chain variable region of SEQ ID NO: 32. As a result, it was shown that when O4 has a framework region of the sequence QVQLVESGGGLX1KPGGSLRLSCAAS (wherein X1 is S) in FR1 of the heavy chain variable region, or a framework region of the sequence MX2WVRQAPGKGLEWVSS (wherein X2 is T) in FR3, or a framework region of the sequence QVQLVESGGGLX1KPGGSLRLSCAAS (wherein X1 is S) in FR1 and a framework region of the sequence MX2WVRQAPGKGLEWVSS (wherein X2 is T) in FR3, it can be highly concentrated due to an increase in the solubility of the antibody, thereby exhibiting improved productivity.

[0056] As a result, it was shown that when O4 has a framework region of the sequence NLQSGVSSQFSGSGSGTDFTLTIX3SLQPEDSATYYC (wherein X3 is S) in FR3 of the light chain variable region, it can be highly concentrated due to an increase in the solubility of the antibody, thereby exhibiting improved productivity.

[0057] As a result, it was shown that when O4 has a framework region of the sequence QVQLVESGGGLX1KPGGSLRLSCAAS (wherein X1 is S) in FR1 of the heavy chain variable region and a framework region of the sequence NLQSGVSSQFSGSGSGTDFTLTIX3SLQPEDSATYYC (wherein X3 is S) in FR3 of the light chain variable region, or

[0058] O4 has a framework region in FR3 of the heavy chain variable region with the sequence MX2WVRQAPGKGLEWVSS (wherein X2 is T) and a framework region in FR3 of the light chain variable region with the sequence NLQSGVSSQFSGSGSGTDFTLTI X3SLQPEDSATYYC (wherein X3 is S), or

[0059] O4 has a framework region in FR1 of the heavy chain variable region with the sequence QVQLVESGGGLX1KPGGSLRLSCAAS (wherein X1 is S), a framework region in FR3 of the heavy chain variable region with the sequence MX2WVRQAPGKGLEWVSS (wherein X2 is T) and a framework region in FR3 of the light chain variable region with the sequence NLQSGVSSQFSGSGSGTDFTLTI X3SLQPEDSATYYC (wherein X3 is S), it can be highly concentrated due to the improved solubility of the antibody, thereby exhibiting improved productivity.

[0060] In some cases, the modified antibody or fragment thereof according to the present application can be an antibody or antigen-binding fragment thereof that binds to Ang2 (angiopoietin-2).

[0061] In this case, the antibody or antigen-binding fragment thereof can include the following:

[0062] a heavy chain variable region comprising a heavy chain CDR1 selected from the group consisting of SEQ ID NOs: 1, 7, 13, 19, and 25, a heavy chain CDR2 selected from the group consisting of SEQ ID NOs: 2, 8, 14, 20, and 26, and

[0063] a heavy chain CDR3 selected from the group consisting of SEQ ID NOs: 3, 9, 15, 21, 27, 43, 44, and 45; and

[0064] a light chain variable region comprising a light chain CDR1 selected from the group consisting of SEQ ID NOs: 4, 10, 16, 22, and 28, a light chain CDR2 selected from the group consisting of SEQ ID NOs: 5, 11, 17, 23, and 29, and

[0065] a light chain CDR3 selected from the group consisting of SEQ ID NOs: 6, 12, 18, 24, and 30.

[0066]

[0067]

[0068]

[0069] As used herein, the term "antibody" refers to an anti-Ang2 antibody that specifically binds to Ang2. Complete antibodies and antigen-binding fragments of antibody molecules are within the scope of the present application.

[0070] A whole antibody has a structure having two full-length light chains and two full-length heavy chains, and each light chain is bonded to a heavy chain by a disulfide bond. The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types, and is subdivided into gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1), and alpha 2 (α2). The light chain constant region has kappa (κ) and lambda (λ) types.

[0071] An antigen-binding fragment or antibody fragment of an antibody is a fragment having an antigen-binding ability, and includes Fab, F(ab'), F(ab')2, Fv, etc. In the antibody fragment, Fab refers to a structure including the variable region of each of the heavy chain and the light chain, the constant region of the light chain, and the first constant domain (CH1) of the heavy chain, each of which has one antigen-binding site. Fab' differs from Fab in that it further includes a hinge region including at least one cysteine residue at the C-terminus of the CH1 domain of the heavy chain. F(ab')2 is produced by a disulfide bond between the cysteine residues in the hinge region of Fab'. Fv is the smallest antibody fragment having only the variable region of the heavy chain and the variable region of the light chain. Double-chain Fv is a fragment in which the heavy chain variable region and the light chain variable region are connected by a non-covalent bond; and single-chain Fv (scFv) is a fragment in which the heavy chain variable region and the light chain variable region are connected by a peptide linker therebetween, or are directly connected at the C-terminus, typically by a covalent bond, thereby forming a dimer-like structure like double-chain Fv. Such antibody fragments can be obtained using a protease (for example, Fab can be obtained by limited cleavage of an intact antibody with papain, and F(ab')2 fragments can be obtained by cleavage of an intact antibody with pepsin), and can also be prepared using a genetic recombination technique.

[0072] In one embodiment, the antibody of the present application is in the form of Fv (e.g., scFv) or a whole antibody. In addition, the heavy chain constant region can be selected from the group consisting of gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) isotypes. For example, the constant region can be γ1 (IgG1), γ3 (IgG3), or γ4 (IgG4). The light chain constant region can be κ or λ.

[0073] The term "heavy chain" as used herein encompasses both full-length heavy chains, which include a variable domain (VH) containing an amino acid sequence with sufficient variability to confer specificity to an antigen, and three constant domains (CHI, CH2, and CH3), and fragments thereof. The term "light chain" as used herein encompasses both full-length light chains, which include a variable domain (VL) containing an amino acid sequence with sufficient variability to confer specificity to an antigen, and a constant domain (CL), and fragments thereof.

[0074] Antibodies of the present application include, but are not limited to, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single-chain Fvs (scFV), single-chain antibodies, Fab fragments, F(ab') fragments, disulfide-bond Fvs (sdFV), anti-idiotypic (anti-Id) antibodies, epitope-binding fragments of such antibodies, and the like.

[0075] The term "monoclonal antibody" refers to a homogeneous antibody population (i.e., the individual antibodies that make up the population are identical to one another, except for possible naturally occurring mutations that can be present in minor amounts) obtained from the proliferation and differentiation of a single cell, all of which express the same antibodies. Monoclonal antibodies are highly specific, being induced by a single antigenic site and reacting with the exact determinant.

[0076] The term "epitope" refers to a protein determinant capable of specific binding to an antibody. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually are formed by at least one conformational epitope and optionally at least one linear epitope. Conformational epitopes are distinguished from non-conformational epitopes in that the bonds of the sugar chain of the former are broken by the denaturing solvent used to elute the antibody from the solid phase.

[0077] A "humanized" form of a non-human (e.g., murine) antibody is a chimeric antibody that includes one or more amino acid sequences derived from a non-human antibody (donor or source antibody), such as a CDR sequence, containing minimal sequence derived from a non-human immunoglobulin. In most instances, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate that have the desired specificity, affinity, and capacity.

[0078] The term "human antibody", as used herein, refers to a molecule derived from human immunoglobulin, in which all of the amino acid sequences comprising the complementarity determining regions and the structural regions that make up the antibody are composed of human immunoglobulin.

[0079] A portion of a heavy and / or light chain is identical with or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) includes "imported" sequences from another species or belonging to another antibody class or subclass, as well as such antibody as a fragment thereof, which exhibits the desired biological activity.

[0080] As used herein, the term "antibody variable domain" refers to the light and heavy chain regions of an antibody molecule, which include the amino acid sequences of the complementarity determining regions (CDRs; i.e., CDR1, CDR2, and CDR3) and framework regions (FRs). VH refers to the variable domain of the heavy chain. VL refers to the variable domain of the light chain.

[0081] The term "complementarity determining region" (CDR, i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues in the variable domain of an antibody that are necessary for antigen binding. Each variable domain typically has three CDR regions identified as CDR1, CDR2, and CDR3. The complementarity determining regions of the present invention include a heavy chain variable region comprising a heavy chain CDR3 of SEQ ID NO: 1; and a light chain variable region comprising a light chain CDR3 of SEQ ID NO: 2.

[0082] Specifically, the antibody or antigen-binding fragment thereof that binds to Ang2 includes a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 1, a heavy chain CDR2 of SEQ ID NO: 2, and a heavy chain CDR3 of SEQ ID NO: 3; and a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 4, a light chain CDR2 of SEQ ID NO: 5, and a light chain CDR3 of SEQ ID NO: 6,

[0083] a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 7, a heavy chain CDR2 of SEQ ID NO: 8, and a heavy chain CDR3 of SEQ ID NO: 9; and a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 10, a light chain CDR2 of SEQ ID NO: 11, and a light chain CDR3 of SEQ ID NO: 12,

[0084] a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 13, a heavy chain CDR2 of SEQ ID NO: 14, and a heavy chain CDR3 of SEQ ID NO: 15; and a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 16, a light chain CDR2 of SEQ ID NO: 17, and a light chain CDR3 of SEQ ID NO: 18,

[0085] a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 19, a heavy chain CDR2 of SEQ ID NO: 20, and a heavy chain CDR3 of SEQ ID NO: 21; and a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 22, a light chain CDR2 of SEQ ID NO: 23, and a light chain CDR3 of SEQ ID NO: 24,

[0086] a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 25, a heavy chain CDR2 of SEQ ID NO: 26, and a heavy chain CDR3 of SEQ ID NO: 27; and a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 28, a light chain CDR2 of SEQ ID NO: 29, and a light chain CDR3 of SEQ ID NO: 30,

[0087] a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 13, a heavy chain CDR2 of SEQ ID NO: 14, and a heavy chain CDR3 of SEQ ID NO: 43; and a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 16, a light chain CDR2 of SEQ ID NO: 17, and a light chain CDR3 of SEQ ID NO: 18,

[0088] a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 13, a heavy chain CDR2 of SEQ ID NO: 14, and a heavy chain CDR3 of SEQ ID NO: 44; and a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 16, a light chain CDR2 of SEQ ID NO: 17, and a light chain CDR3 of SEQ ID NO: 18, or

[0089] a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 13, a heavy chain CDR2 of SEQ ID NO: 14, and a heavy chain CDR3 of SEQ ID NO: 45; and a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 16, a light chain CDR2 of SEQ ID NO: 17, and a light chain CDR3 of SEQ ID NO: 18.

[0090] The term "framework region" (FR) refers to variable domain residues other than CDR residues. Each variable domain generally has four FRs, identified as FR1, FR2, FR3, and FR4. The inventors of the present application induced mutations in the framework region with the aim of improving productivity and solubility in order to develop a high-concentration formulation with improved productivity.

[0091] An "Fv" fragment is an antibody fragment that contains a complete antibody recognition and binding site. Such regions include dimers that are composed of one heavy chain variable domain and one light chain variable domain connected in very close proximity to each other, for example by scFv, covalently.

[0092] A "Fab" fragment contains the variable and constant domains of the light chain and the variable domain of the heavy chain and the first constant domain (CH1). F(ab')2 antibody fragments typically include a pair of Fab fragments which are covalently linked near their carboxy termini by disulfide bonds between their hinge cysteines.

[0093] A "single-chain Fv" or "scFv" antibody fragment includes the VHand VLdomains of an antibody, wherein these domains are present in a single polypeptide chain. The Fv polypeptide can further include a polypeptide linker between the VHand VLdomains to enable the scFv to form the desired structure for antigen binding.

[0094] Functionally, the binding affinity of the antibody according to the present application ranges from 10 -5 M to 10 -12 M. The amino acid substitution according to the present application neither reduces the affinity for the antigen nor adversely affects the affinity.

[0095] For example, the binding affinity of the modified antibody according to the present application for the antigen is 10 -6 M to 10 -12 M, 10 -7 M to 10 -12 M, 10 -8 M to 10 -12 M, 10 -9 M to 10 -12 M, 10 -5 M to 10 -11 M, 10 -6M to 10 -11 M, 10 -7 M to 10 -11 M, 10 -8 M to 10 - 11 M, 10 -9 M to 10 -11 M, 10 -10 M to 10 -11 M, 10 -5 M to 10 -10 M, 10 -6 M to 10 -10 M, 10 -7 M to 10 -10 M, 10 -8 M to 10 - 10 M, 10 -9 M to 10 -10 M, 10 -5 M to 10 -9 M, 10 -6 M to 10 -9 M, 10 -7 M to 10 -9 M, 10 -8 M to 10 -9 M, 10 -5 M to 10 -8 M, 10 - 6 M to 10 -8 M, 10 -7 M to 10 -8 M, 10 -5 M to 10 -7 M, 10 -6 M to 10 -7 M or 10 -5 M to 10 -6 M.

[0096] The modified antibody according to the application can comprise a heavy chain variable region selected from the group consisting of SEQ ID NO: 31, 33 and 36 and / or a light chain variable region of SEQ ID NO: 35.

[0097] In a particular embodiment according to the application, the modified antibody according to the application can comprise a heavy chain variable region of SEQ ID NO: 31 and a light chain variable region of SEQ ID NO: 32;

[0098] a heavy chain variable region of SEQ ID NO: 33 and a light chain variable region of SEQ ID NO: 32;

[0099] a heavy chain variable region of SEQ ID NO: 34 and a light chain variable region of SEQ ID NO: 35;

[0100] a heavy chain variable region of SEQ ID NO: 36 and a light chain variable region of SEQ ID NO: 32;

[0101] a heavy chain variable region of SEQ ID NO: 31 and a light chain variable region of SEQ ID NO: 35;

[0102] a heavy chain variable region of SEQ ID NO: 33 and a light chain variable region of SEQ ID NO: 35; or

[0103] a heavy chain variable region of SEQ ID NO: 36 and a light chain variable region of SEQ ID NO: 35.

[0104] The modified antibody or fragment thereof according to the present application can include not only the sequence of the antibody, but also a biological equivalent thereof, as long as it can maintain the desired function. For example, additional variations can be made to the amino acid sequence of the antibody to further improve the binding affinity and / or other biological properties of the antibody. Such variations include, for example, deletion, insertion, and / or substitution of residues of the amino acid sequence of the antibody. Such amino acid mutations are based on the relative similarity of the amino acid side chain substituents, such as their hydrophobicity, hydrophilicity, charge, and size. It can be seen through analysis of the size, shape, and type of the amino acid side chain substituents that arginine, lysine, and histidine are all positively charged residues; alanine, glycine, and serine have similar sizes; and phenylalanine, tryptophan, and tyrosine have similar shapes. Therefore, based on these considerations, arginine, lysine, and histidine; alanine, glycine, and serine; and phenylalanine, tryptophan, and tyrosine are considered to be biological functional equivalents.

[0105] When considering mutations having biologically equivalent activity, the antibody according to the application or the encoding nucleic acid molecule thereof is interpreted to include sequences having substantial identity to the sequences shown in the sequence listing. The term "substantial identity" means that, when comparing the sequences of the application with any other sequences to correspond with each other as much as possible and analyzing the aligned sequences using an algorithm commonly used in the art, the sequences have at least 90% homology, preferably at least 90% homology, most preferably at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% homology. Alignment methods for sequence comparison are well known in the art. NCBI Basic Local Alignment Search Tool (BLAST) is available through NCBI and can be used in conjunction with sequence analysis programs on the Internet (such as BLASTP, BLASTM, BLASTX, TBLASTN, and TBLASTX). BLAST is available at www.ncbi.nlm.nih.gov / BLAST / . Methods for comparing sequence homology using this program can be found at www.ncbi.nlm.nih.gov / BLAST / blast_help.html.

[0106] Based on this, the antibody according to the application or the antigen-binding fragment thereof can have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology compared to the sequence disclosed herein or the entirety thereof. The homology can be determined by sequence comparison and / or alignment by methods known in the art. For example, the percentage of sequence homology of the nucleic acid or protein according to the application can be determined using a sequence comparison algorithm (i.e., BLAST or BLAST 2.0), manual alignment, or visual inspection.

[0107] In another aspect, the present application relates to a nucleic acid encoding the modified antibody or fragment thereof.

[0108] The modified antibody or fragment thereof can be produced recombinantly by isolating a nucleic acid encoding the antibody or antigen-binding fragment thereof of the present application. The nucleic acid is isolated and inserted into a replicable vector, after which further cloning (DNA amplification) or further expression is carried out. Based on this, in another aspect, the present application relates to a vector comprising the nucleic acid.

[0109] The term "nucleic acid" is intended to encompass both DNA (gDNA and cDNA) and RNA molecules, and the nucleotides, which are the basic building blocks of nucleic acids, include naturally occurring nucleotides as well as analogs thereof having modified sugar or base moieties. The sequences of the nucleic acids encoding the heavy chain variable region and the light chain variable region of the present application can vary. Such variations include additions, deletions, or non-conservative or conservative substitutions of nucleotides.

[0110] In one specific embodiment according to the present application, the nucleic acid can comprise the following sequence:

[0111] a sequence encoding the heavy chain variable region of SEQ ID NO: 37 and a sequence encoding the light chain variable region of SEQ ID NO: 38;

[0112] a sequence encoding the heavy chain variable region of SEQ ID NO: 39 and a sequence encoding the light chain variable region of SEQ ID NO: 38;

[0113] a sequence encoding the heavy chain variable region of SEQ ID NO: 40 and a sequence encoding the light chain variable region of SEQ ID NO: 41;

[0114] a sequence encoding the heavy chain variable region of SEQ ID NO: 42 and a sequence encoding the light chain variable region of SEQ ID NO: 38;

[0115] a sequence encoding the heavy chain variable region of SEQ ID NO: 37 and a sequence encoding the light chain variable region of SEQ ID NO: 41;

[0116] a sequence encoding the heavy chain variable region of SEQ ID NO: 39 and a sequence encoding the light chain variable region of SEQ ID NO: 41; or

[0117] a sequence encoding the heavy chain variable region of SEQ ID NO: 42 and a sequence encoding the light chain variable region of SEQ ID NO: 41.

[0118] DNA encoding the antibodies can be readily isolated or synthesized using conventional procedures (e.g., using oligonucleotide probes that are specific for DNA encoding the heavy and light chains of the antibodies). A variety of vectors are available. Vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0119] As used herein, the term “vector” refers to a tool for expressing a target gene in a host cell and includes plasmid vectors, cosmid vectors, and viral vectors (such as phage vectors, adenoviral vectors, retroviral vectors, and adeno-associated viral vectors). The nucleic acid encoding the antibody in the vector is operably linked to a promoter.

[0120] The term “operably linked” means functional linkage between a nucleic acid expression regulatory sequence, such as a promoter, a signal sequence, or an array of binding sites for a transcriptional modulator, and another nucleic acid sequence, and enables the regulation of the transcription and / or translation of the other nucleic acid sequence.

[0121] When a prokaryotic cell is used as a host, it generally includes an efficient promoter capable of transcription (e.g., a tac promoter, a lac promoter, a lacUV5 promoter, a lpp promoter, a pL lambda promoter, a pR lambda promoter, a rac5 promoter, an amp promoter, a recA promoter, a SP6 promoter, a trp promoter, or a T7 promoter), a ribosome binding site for initiation of translation, and a transcription / translation termination sequence. In addition, for example, when a eukaryotic cell is used as a host, it includes a promoter derived from a mammalian cell genome (e.g., a metallothionein promoter, a beta-actin promoter, a human hemoglobin promoter, or a human muscle creatine promoter) or a promoter derived from a mammalian virus (e.g., an adenovirus late promoter, a vaccinia virus 7.5K promoter, an SV40 promoter, a cytomegalovirus (CMV) promoter, an HSV tk promoter, a mouse mammary tumor virus (MMTV) promoter, an HIV LTR promoter, a Moloney virus promoter, an Epstein-Barr virus (EBV) promoter, or a Rous sarcoma virus (RSV) promoter), and generally has a polyadenylation sequence as a transcription termination sequence.

[0122] Optionally, the vector can be fused with another sequence to facilitate purification of the antibody expressed therefrom. The sequence to be fused therewith can include, for example, glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA), 6x His (six histidines; Qiagen, USA), etc.

[0123] The vector includes an antibiotic resistance gene commonly used as a selective marker in the art, and examples thereof include genes conferring resistance to ampicillin, gentamycin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline.

[0124] In another aspect, the present application relates to a cell transformed with the above-mentioned vector. The cell for producing the antibody of the present application can be a prokaryote, a yeast, or a higher eukaryote, but is not limited thereto.

[0125] Prokaryotic host cells, such as Escherichia coli; Bacillus strains, such as Bacillus subtilis and Bacillus thuringiensis; Streptomyces spp.; Pseudomonas spp. (e.g., Pseudomonas putida); Proteus mirabilis; and Staphylococcus spp. (e.g., Staphylococcus carnosus) can be used.

[0126] Animal cells are of greatest interest, and examples of useful host cell lines include, but are not limited to, COS-7, BHK, CHO, CHO / K1, DXB-11, DG-44, CHO / -DHFR, CV1, COS-7, HEK293, BHK, TM4, VERO, HELA, MDCK, BRL 3A, W138, Hep G2, SK-Hep, MMT, TRI, MRC 5, FS4, 3T3, RIN, A549, PC12, K562, PER.C6, SP2 / 0, NS-0, U20S, and HT1080.

[0127] In another aspect, the present application relates to a method of producing a modified antibody or fragment thereof, in which at least one amino acid at position 12 or 35 of the heavy chain variable region (based on the AHO numbering system) and / or one amino acid at position 76 of the light chain variable region (based on the AHO numbering system) is substituted with serine (S) or threonine (T).

[0128] The nucleic acid encoding the modified antibody or fragment thereof can be introduced into a cell. The cell can be cultured in various media. Any commercially available medium can be used without limitation as the culture medium. All other necessary supplements well known to those skilled in the art can be included at appropriate concentrations. The culture conditions (e.g., temperature and pH) are those conventionally used to select a host cell for expression, as will be apparent to those skilled in the art.

[0129] Recovery of the antibody or fragment thereof can be performed, for example, by centrifugation or ultrafiltration to remove impurities from the resulting product, and purifying the resulting product using, for example, affinity chromatography. Other additional purification techniques can be used, such as anion or cation exchange chromatography, hydrophobic interaction chromatography, and hydroxyapatite (HA) chromatography.

[0130] In another aspect, the present application relates to a composition for preventing or treating a tumor comprising the antibody or a fragment thereof as an active ingredient. The antibody can be IgG or a fragment including a variable region (i.e., ScFv or Fab). In addition, the variable region of the heavy chain can be IgG1, IgG2, IgG3, or IgG4.

[0131] In another aspect, the present application relates to a pharmaceutical composition for preventing or treating an ocular disease, comprising (a) a pharmaceutically effective amount of the antibody or a fragment thereof according to the present application; and (b) a pharmaceutically acceptable carrier. In another aspect, the present application relates to a method for preventing or treating an ocular disease, comprising administering the antibody or a fragment thereof according to the present application to a patient with an ocular disease. In addition, the present application relates to the use of the antibody or an antigen-binding fragment thereof for inhibiting the mechanism of Ang2, and its use based thereon for preventing or treating an ocular disease.

[0132] With respect to an ocular disease, the cornea is an avascular tissue and must always maintain transparency to protect vision. However, it is known that angiogenesis also occurs in the eye, thereby causing ocular angiogenesis-related diseases. In other words, neovascularization in the cornea impairs the transparency of the eyeball, thereby causing vision loss, and neovascularization in the retina induces the formation of abnormal blood vessels and the exudation of blood, thereby causing blindness through the degeneration of retinal cells.

[0133] Based on this, the present application can be used for preventing or treating an ocular disease, such as retinopathy of prematurity, corneal neovascularization, diabetic retinopathy, choroidal neovascular disease, macular degeneration (e.g., age-related macular degeneration), etc.

[0134] In another aspect, the present application relates to a pharmaceutical composition for preventing or treating a tumor, comprising (a) a pharmaceutically effective amount of the antibody or a fragment thereof according to the present application; and (b) a pharmaceutically acceptable carrier. In another aspect, the present application relates to a method for preventing or treating a tumor, comprising administering the antibody or a fragment thereof according to the present application to a patient with a tumor. In addition, the present application relates to the use of the antibody or an antigen-binding fragment thereof for inhibiting the mechanism of Ang2, and its use based thereon for preventing or treating a tumor.

[0135] Tumors or cancers as therapeutic targets include, but are not limited to, melanoma (e.g., metastatic malignant melanoma), renal cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone-refractory prostate adenocarcinoma), pancreatic adenocarcinoma, breast cancer (in some cases, triple-negative breast cancer), colon cancer, lung cancer (e.g., non-small cell lung cancer), esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma, and other neoplastic cancers. In addition, tumors or cancers according to the present application include refractory cancers or recurrent cancers that can be treated using the antibodies of the present application.

[0136] In another aspect, the present application relates to a composition for inhibiting angiogenesis comprising the antibody or fragment thereof as an active ingredient. In another aspect, the present application relates to a pharmaceutical composition for preventing and / or treating a disease associated with Ang2 activation and / or overproduction comprising the antibody or fragment thereof as an active ingredient.

[0137] The present application provides, for example, a method for inhibiting angiogenesis comprising administering to a patient in need thereof a therapeutically effective amount of the antibody or fragment thereof. The method for inhibiting angiogenesis can further comprise identifying a patient in need of angiogenesis inhibition prior to administration. In another aspect, the present application relates to a method for preventing and / or treating a disease associated with Ang2 activation and / or overproduction comprising administering to a patient in need thereof a therapeutically effective amount of the antibody or fragment thereof. The method for preventing and / or treating a disease associated with Ang2 activation and / or overproduction can further comprise identifying a patient in need of prevention and / or treatment of a disease associated with Ang2 activation and / or overproduction prior to administration.

[0138] The pharmaceutical composition can further comprise a pharmaceutically acceptable carrier, and can include, but is not limited to, at least one selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, benzoic acid, propyl gallate, talc, magnesium stearate, mineral oil, and the like, which are commonly used in formulating pharmaceuticals. The pharmaceutical composition can further comprise at least one selected from the group consisting of diluents, excipients, lubricants, wetting agents, sweetening agents, flavoring agents, emulsifiers, suspending agents, and preservatives, which are commonly used in preparing pharmaceutical compositions.

[0139] The pharmaceutical composition or the antibody or antigen-binding fragment thereof can be administered orally or parenterally in an effective amount. The parenteral administration includes intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intradermal administration, topical administration, intranasal administration, intrapulmonary administration, intrarectal administration, etc. After oral administration, since a protein or a peptide is digested, an active drug-coated or formulated oral composition should be used to avoid degradation of the protein or the peptide in the stomach. In addition, the pharmaceutical composition can be administered using any device capable of delivering the active substance to target cells.

[0140] The content or dosage of the antibody or fragment thereof in the pharmaceutical composition can vary depending on factors such as the formulation method, the administration method, and age, body weight, sex, pathological condition, diet, administration time, administration interval, administration route, excretion rate, and patient responsiveness. For example, the daily dosage of the antibody or fragment thereof can range from 0.001 mg / kg to 1,000 mg / kg, specifically 0.01 mg / kg to 100 mg / kg, more specifically 0.1 mg / kg to 50 mg / kg, even more specifically 0.1 mg / kg to 20 mg / kg, but the present application is not limited thereto. The daily dosage can be prepared by formulating into a single dosage having a unit dosage, formulating or packaging in a multi-dose container with an appropriate dosage.

[0141] The pharmaceutical composition can be administered in combination with other drugs, such as other angiogenesis inhibitors or therapeutic agents for diseases associated with Ang2 activation and / or overproduction, and the dosage, administration method, and type of the other drugs are appropriately selected depending on the patient's condition.

[0142] The pharmaceutical composition can be in the form of a solution, a suspension, a syrup, or an emulsion in an oily or aqueous medium, or can be formulated in the form of an extract, a powder, a granule, a tablet, or a capsule. The pharmaceutical composition can further include a dispersant or a stabilizer.

[0143] In particular, the pharmaceutical composition containing the antibody or fragment thereof can be formulated into an immunoliposome since it includes the antibody and antigen-binding fragments thereof. The liposome containing the antibody can be prepared according to methods well known in the art. The immunoliposome is a lipid composition containing phosphatidylcholine, cholesterol, and polyethylene glycol-derivatized phosphatidylethanolamine, and can be prepared by reverse-phase evaporation (KR Patent No. 10-2015-0089329). For example, the Fab' fragment of the antibody can be conjugated with the liposome via a disulfide bond exchange reaction.

[0144] Meanwhile, the antibody or fragment thereof specifically binds to Ang2, and thus can be used to determine whether Ang2 is activated and / or overproduced. Accordingly, in another aspect, the present application relates to a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof for diagnosing Ang2 activation and / or overproduction and / or a disease associated with Ang2 activation and / or overproduction. In another aspect, the present application relates to a diagnostic method or providing information for diagnosis, which comprises treating a biological sample obtained from a patient with the antibody or fragment thereof, identifying an antigen-antibody reaction, and determining that the patient has a symptom of Ang2 activation and / or overproduction or a disease associated with Ang2 activation and / or overproduction when the antigen-antibody reaction is detected. The biological sample can be selected from cells, tissues, and body fluids derived from the patient.

[0145] Whether the antigen-antibody reaction occurs can be identified by various methods known in the art, for example, conventional enzyme reaction, fluorescence, luminescence, and / or radio detection, particularly by a method selected from the group consisting of immunochromatography, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescence immunoassay (FIA), luminescence immunoassay (LIA), Western blotting, etc., but the present application is not limited thereto.

[0146] The patient to whom the pharmaceutical composition is administered or the patient diagnosed can be a mammal, including a primate (including a human, a monkey, etc.) or a rodent (including a mouse, a rat, etc.).

[0147] The disease associated with Ang2 activation and / or overproduction includes cancer; cancer metastasis; an ocular disease such as retinopathy of prematurity, corneal neovascularization, diabetic retinopathy, choroidal neovascular disease, and macular degeneration (e.g., age-related macular degeneration); asthma; rheumatoid arthritis; psoriasis; an inflammatory disease such as pneumonia and chronic inflammation; and a cardiovascular disease such as hypertension or arteriosclerosis or sepsis. The cancer can overexpress Ang2, and can be a solid cancer or a blood cancer, but is not limited thereto, and can include at least one selected from the group consisting of squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, skin cancer, cutaneous or intraocular melanoma, rectal cancer, perianal cancer, esophageal cancer, small bowel cancer, endocrine gland cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, chronic or acute leukemia, lymphocytic lymphoma, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver tumor, breast cancer (in some cases, triple-negative breast cancer), colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, head and neck cancer, brain cancer, osteosarcoma, etc. The cancer can be a primary cancer or a metastatic cancer.

[0148] Example

[0149] Hereinafter, the present application will be described in more detail with reference to Examples. However, it will be apparent to those skilled in the art that these Examples are provided merely to illustrate the present application and should not be construed to limit the scope of the present application.

[0150] It is expected that the application of a large amount of anti-Ang2 antibody to an ocular disease through injection will provide a better effect. Since the volume suitable for injection into the eye is limited, a formulation that is stable at a higher concentration than a conventional substance is required. In order to prepare a formulation at a higher concentration than a conventional anti-Ang2 antibody concentration, a high-concentration formulation was successfully prepared based on hot spot analysis of antibody structure and formulation buffer.

[0151] Example 1. Production of variants to improve physical properties

[0152] Efforts were made to improve the physical properties of anti-Ang2 antibody clone O4 (heavy chain variable region of SEQ ID NO: 34 and light chain variable region of SEQ ID NO: 32).

[0153] [Table 1]

[0154]

[0155] Amino acids that can cause aggregation of the anti-Ang2 antibody were analyzed using Discovery Studio (BIOBIA) software. Amino acids Leu 11, Val 12, Asn 35, Val 93 in VH and amino acids Gln 61 and Asn 76 in VL were analyzed as hot spots that affect physical properties (Table 2). Figure 1 The amino acids analyzed as hot spots were mutated, expressed in variants with improved physical properties, and selected.

[0156] [Table 2]

[0157]

[0158] Example 2. Production of variants to improve physical properties

[0159] To improve the physical properties of the anti-Ang2 antibody (04), substitutions were made to the amino acids selected by hot spot analysis. Expression tests were performed on the constructs after the amino acid changes. The clones with point mutations in the anti-Ang2 antibody (04) gene were amplified using PCR. The cloning was completed by inserting the amplified gene into an expression vector (Merck Millipore, pET 22b(+)). The transformation was performed by inserting the cloned plasmid into an expression host cell (Merck Millipore, BL21(DE3)). To perform the expression test, 100 pg / ml ampicillin was added to the 2XYT medium, and the transformed cells were inoculated and cultured at 37°C for 8 hours. After reducing the temperature to 25°C, 0.5 mM IPTG was added to induce expression, and incubation was performed for 18 hours. The cells were harvested after centrifugation at 8,000 rpm for 10 minutes. The precipitated cells were suspended in a 50 mM Tris, 150 mM NaCl pH 7.4 buffer, and were disrupted by ultrasonication. The disrupted cells were separated from the supernatant by centrifugation at 1,3000 rpm for 1 hour, and expression was detected by SDS-PAGE Figure 2 , 3 and 4).

[0160] Example 3. ScFv production of the optimized anti-Ang2 antibody

[0161] The anti-Ang2 antibody with improved physical properties was cloned into a pET-22b vector (Novagen) in order to express the antibody in E. coli. Selection was performed on the colonies produced by transformation into BL21(DE3), and 1% of E. coli pre-cultured in LB medium containing 100 pg / ml ampicillin at 37°C, 200 rpm was inoculated in LB (lysozyme broth) medium containing 100 pg / ml ampicillin. The colonies were cultured at 37°C and 200 rpm, and when the OD 600 reached 0.6 to 0.8, the temperature of the incubator was reduced to 20°C, and 0.5 mM IPTG was added thereto, after which culture was performed for 16 hours.

[0162] The E. coli after culture was collected by centrifugation at 8,000 rpm for 10 minutes. After removing the culture medium, the cells were resuspended with 10 ml of lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM PMSF (phenylmethylsulfonyl fluoride)) per g of cell weight. The cells were disrupted using an ultrasonicator under the conditions (power: 20W, rest: 3 sec, work: 3 sec, time: 10 min). The disrupted cells were centrifuged at 13,000 rpm for 1 hour to separate the supernatant from the precipitate from each other.

[0163] ScFv were expressed in insoluble form and the pellets were washed for refolding. After homogenization with a homogenizer using 50 mM Tris-HCl pH 7.4 and 150 mM NaCl buffer, the pellets were washed twice by centrifugation at 13,000 rpm for 1 hour. The remaining E. coli-derived material in the pellets was removed using a buffer containing 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 2 M urea and 0.5% Triton X-100 and washed repeatedly three times with a pH 7.4 buffer containing 50 mM Tris-HCl and 150 mM NaCl. After resuspension of the inclusion bodies with a buffer containing 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 8 M urea and 10 mM DTT, the reaction was allowed to proceed for about 30 minutes to obtain unfolded ScFv and centrifuged at 13,000 rpm for 1 hour to separate the supernatant from the precipitate.

[0164] ScFv antibodies were refolded by removing urea via step dialysis. Dialysis buffers were prepared by reducing the concentration of urea by 1 / 2 based on 50 mM Tris-HCl pH 7.4 and 150 mM NaCl. Refolding was performed with the addition of 0.1 M L-arginine and 10% glycerol to inhibit aggregation in the 4-2-1 M urea concentration range where most structures form. Refolded scFv antibodies were isolated and purified using HisTrap and Capo L columns.

[0165] The separation and purification were performed as follows. Purification was performed using AKTA purifier (GE healthcare) system, and 5 ml HisTrap packed column. After equilibration with a buffer containing 50 mM Tris, 500 mM NaCl and 10 mM imidazole, pH 7.4, in a column volume corresponding to 10 times the volume of HisTrap column, the ScFv antibody sample was allowed to flow through the HisTrap column at a flow rate of 3 ml / min to bind to the resin in the column. In order to remove non-specifically bound substances present in the resin, a buffer containing 50 mM Tris, 150 mM NaCl and 400 mM imidazole, pH 7.4 was allowed to flow in an amount corresponding to about 10 times the column volume, and then a buffer containing 50 mM Tris, 150 mM NaCl and 400 mM imidazole, pH 7.4 was allowed to flow in a concentration gradient to elute the scFv antibody. The antibody sample eluted was purified using a 5 ml Capto L column as follows. After equilibrating the Capto L column with a buffer containing 50 mM Tris and 150 mM NaCl, pH 7.4, the sample was allowed to flow through the Capto L column at a flow rate of 3 ml / min and bind to the resin in the column. PBS buffer was allowed to pass through the column in an amount of about 10 times the column volume to remove non-specifically bound substances. Non-specifically bound substances that could not be removed with the PBS buffer were removed by flowing a 10 CV pH 5.5 buffer containing 50 mM MES, 400 mM NaCl and 0.2% tween 20. A buffer containing 0.1 M glycine and 50 mM NaCl, pH 2.5 was allowed to flow through the column in an amount of about 10 times the column volume to elute the scFv antibody. The pH of the eluted sample was neutralized with a 1 M Tris buffer, pH 7.4. FIGS. 5A to 5G show the results of SDS-PAGE of the protein obtained after the final purification. As a result, a high-purity ScFv antibody was obtained.

[0166] Specific sequences of anti-Ang2 antibodies having improved physical properties are as follows.

[0167] [Table 3]

[0168]

[0169]

[0170] Example 4. ScFv concentration of optimized anti-Ang2 antibody

[0171] To analyze the highest concentration that does not cause aggregation, concentration was performed using a centrifugation method. The purified anti-Ang2 antibody was dialyzed against PBS and a formulation buffer (10 mM sodium phosphate, 40 mM NaCl, 0.03% polysorbate, 5% sucrose, pH 6.2) containing the formulation composition used in the commercial product to perform a buffer exchange. 10 ml of PBS or formulation buffer was added to a Viva Spin Turbo 5,000 MWCO PEG tube (Sartorius) and centrifuged at 3,400 g for 10 minutes to equilibrate, and 10 ml of the sample after buffer exchange was injected into the tube and concentrated by centrifugation at 3,400 g and 4°C for 10 minutes. The absorbance was measured with a NanoDrop (Thermo) to measure the concentration (Table 4).

[0172] [Table 4]

[0173]

[0174] Example 5. Analysis of binding specificity of optimized anti-Ang2 ScFv antibody

[0175] To perform the analysis of binding specificity, the binding constant was measured using the Octet system (Pall Fortebio LLC., USA).

[0176] The binding ability of the purified ScFv antibody to human Ang2 and mouse Ang2 was measured using the Octet system (Fortebio Inc., USA). The AR2G biosensor was hydrated for 20 minutes. The AR2G biosensor was activated using a solution of 5% s-NHS and 5% EDC in 90% tri-distilled water. The anti-Ang2 antibody was immobilized on the AR2G sensor at a concentration of 1 µg / ml. The antibody was quenched with 1 M ethanolamine. The antibody was equilibrated with PBS, and the association kinetics were measured at human Ang2 concentrations of 50 nM, 40 nM, 30 nM, and 20 nM to calculate the association rate constant (k a ), the dissociation rate constant (k dis ), and the association constant (K D ) (Table 5).

[0177] [Table 5]

[0178]

[0179] Example 6: Analysis of thermal stability of anti-Ang2 scFv antibody

[0180] To analyze the thermal stability of the anti-Ang2 scFv antibody, Tm was measured using RT-PCR. The analysis samples were prepared to have a composition of 5 μΐ of protein thermal shift buffer (Thermo Fisher), 12.5 μΐ of anti-Ang2 scFv antibody (1 mg / ml in PBS pH 7.4 buffer), and 2.5 μΐ of 8X protein thermal shift dye, and the experiments were enabled to be repeated twice. The Tm analysis was performed by using the melting curve experiment method in a QuantStudio 5 thermal cycler (Thermo Fisher) to increase the temperature from 25 °C to 99 °C at a rate of 1.6 °C / s, measuring the fluorescence intensity (absorbance a (580 ± 10 nm) - absorbance b (623 ± 14 nm)), and calculating the Tm value using the protein thermal shift software (Thermo Fisher). The Tm B value of the anti-Ang2 scFv antibody calculated according to the Boltzmann equation was 77.31 °C, which was increased by more than 7 °C compared to before the technical modification, which indicates that the thermal stability was greatly improved due to the technical modification. Figure 6

[0181] Example 7: Evaluation of anti-angiogenic effect of anti-Ang2 scFv antibody using a mouse CNV model

[0182] To determine whether the anti-Ang2 scFv antibody has an effect of inhibiting angiogenesis, a drug efficacy test was performed in a mouse model of laser-induced choroidal neovascularization. The commercial drug aflibercept was used as a control to test efficacy. After general anesthesia of the mice with ketamine, additional local anesthesia was performed by dropping anesthetic eye drops on the eyes, and further mydriatics were added to the eyes to induce mydriasis. The mice were placed on an execution table, and a Micron-IV laser burn was used to damage the Bruch's membrane under CNV induction conditions (wavelength 532 nm, diameter 50 μιη, duration 80 mS, power level 200 mW). No lesions with bubbling were observed during the laser burn induction process were classified as unsuccessful laser burns, and were excluded from the analysis and statistical processing based on the exclusion criteria modified based on the criteria proposed by Gong Y. et al.

[0183] ​To determine the role of drugs in inhibiting CNV-induced angiogenesis, mice were anesthetized with ketamine on day 10 after CNV induction, and a fluorescent contrast agent was injected intraperitoneally. Anesthetic eye drops were instilled into the eyes to induce additional local anesthesia, and a mydriatic agent was further added to induce pupillary dilation. Mice were placed on a sacrifice table, and an imaging camera using a Micron-IV was focused on the fundus. A lubricating gel was applied to the eyeball, and an OCT lens was brought into contact with the cornea. After FFA / OCT imaging, a drop of antibiotic ophthalmic solution was applied to the mouse's eye. The FFA and OCT images were analyzed using the Image-J program.

[0184] Electroretinography (ERG) was performed to determine the optic nerve recovery effect. Twelve hours prior to ERG evaluation, mice underwent dark acclimatization in a dark room. On the day of evaluation (11 days after CNV induction), mice were... and After general anesthesia, add [medication] to the eyes. Additional local anesthesia was induced, and a mydriatic agent was further added to induce pupil dilation. Mice were placed on an ERG euthanasia table, and the ERG probe was brought into contact with the tail, head, and cornea, respectively. ERG was measured as a single flash of light stimulation (0.9 log cds / m²). 2 Electroretinogram (ERG) changes (10 responses / intensities). One drop of Tobradex was applied to the mice's eyes upon completion of the ERG evaluation. ERG analysis was performed using the LabScribeERG (iWorx data acquisition software) program.

[0185] Aflibercept was diluted in sterile PBS at a dose of 20 μg / μl / eye, and the diluted solution was administered to anti-Ang2 scFv antibody via intravitreal injection at doses of 5 μg / μl / eye, 10 μg / μl / eye, and 20 μg / μl / eye on the second day after CNV induction. On day 10 after CNV induction, retinal images were evaluated using FFA (fundus fluorescein angiography) and OCT (optical coherence tomography), and the size and cross-sectional area of ​​the CNV lesion were measured and compared. Additionally, on day 11 after CNV induction, monophtosis ERG (electroretinography) was performed to detect single flashes (0.9 log cds / m²). 2) The stimulated retinal electrography changes were summarized as the amplitude from the A wave to the B wave, and then compared and evaluated. As a result, in terms of the size of the CNV lesion measured on the FFA, the lesion size in the aflibercept administration group of 10 μg / μl / eye and 20 μg / μl / eye (P < 0.01) and the anti-Ang2 scFv antibody administration group was statistically significantly smaller than that in the vehicle administration group (P < 0.05, respectively) (Fig. 7A). In addition, the results of measuring the CNV lesion volume measured on the OCT indicated that the CNV lesion volume in the group administered with 10 μg / μl / eye and 20 μg / μl / eye of the anti-Ang2 scFv antibody was statistically significantly decreased (P < 0.01 and P < 0.001, respectively) (Fig. 7B). When the retinal electrography on the ERG was compared with the vehicle administration group, the aflibercept administration group and the anti-Ang2 scFv antibody administration group (G3, G4, and G5) showed a significantly increased retinal electrography compared to the CNV control group G1 (P < 0.0001, P < 0.0001, P < 0.0001, and P < 0.01, respectively) (Fig. 7C).

[0186] Industrial applicability

[0187] The antibody or fragment thereof according to the present application includes a mutation of a framework region, thereby improving solubility, enabling high concentration, thereby improving productivity of the antibody.

[0188] While particular configurations of the application have been described in detail herein, it will be appreciated by those skilled in the art that the foregoing detailed description is provided by way of preferred embodiments and is not to be construed in a limiting sense. Thus, the spirit and scope of the application are defined by the appended claims and their equivalents.

[0189] SEQUENCE LIST FREE TEXT

[0190] Electronic filing attached. <110> Drug Antibody Company <120> Modified Antibodies and Methods for Making the Same <130> PP-B2632 <160> 51 <170> PatentIn version 3.5 <210> 1 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> HCDR1 <400> 1 Gly Phe Ser Phe Asp Asp Tyr Ala <400> 1 <210> 2 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> HCDR2 <400> 2 Ile Lys Asp Asp Gly Ser Gln Thr <400> 2 <210> 3 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> HCDR3 <400> 3 Thr Thr Glu Gly Leu Met Asn Gly Leu His Phe Asp Met <400> 3 <210> 4 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> LCDR1 <400> 4 Ser Ser Asn Ile Gly Ala Gly Tyr Asp <400> 4 <210> 5 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> LCDR2 <400> 5 Gly Asn Asn 1 <210> 6 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> LCDR3 <400> 6 Gln Ser Tyr Asp Ser Arg Leu Gly Val Val 1 5 10 <210> 7 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> HCDR1 <400> 7 Gly Tyr Ser Phe Thr Ser Tyr Trp 1 5 <210> 8 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> HCDR2 <400> 8 Ile Tyr Pro Gly Asn Ser Asp Thr 1 5 <210> 9 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> HCDR3 <400> 9 Thr Thr Glu Gly Leu Met Asn Gly Leu His Phe Asp Met 1 5 10 <210> 10 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> LCDR1 <400> 10 Gln Ser Leu Leu His Ser Leu Gly Asp Asn Tyr 1 5 10 <210> 11 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> LCDR2 <400> 11 Leu Gly Ser 1 <210> 12 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> LCDR3 <400> 12 Met Gln Ser Leu Gln Thr Pro Pro Tyr Thr 1 5 10 <210> 13 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> HCDR1 <400> 13 Gly Phe Thr Phe Ser Ser Tyr Ser 1 5 <210> 14 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> HCDR2 <400> 14 Ile Ser Ala Ser Asp Gly Ala Thr 1 5 <210> 15 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> HCDR3 <400> 15 Ala Lys Ile Leu Ala Gly Tyr Ser Gly Pro Met Gly Gly Met Asp Val 1 5 10 15 <210> 16 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> LCDR1 <400> 16 Arg Asp Ile Ser Asn Tyr 1 5 <210> 17 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> LCDR2 <400> 17 Gly Ala Ser 1 <210> 18 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> LCDR3 <400> 18 Gln Gln Tyr Tyr Ser Tyr Pro Leu Thr 1 5 <210> 19 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> HCDR1 <400> 19 Gly Phe Ala Phe Gly Arg Tyr Glu 1 5 <210> 20 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> HCDR2 <400> 20 Ile Asp Thr Gly Gly Gly Ala Lys 1 5 <210> 21 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> HCDR3 <400> 21 Thr Thr Glu Gly Leu Met Asn Gly Leu His Phe Asp Met 1 5 10 <210> 22 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> LCDR1 <400> 22 Gln Ala Ile Ser Thr Trp 1 5 <210> 23 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> LCDR2 <400> 23 Thr Ala Ser 1 <210> 24 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> LCDR3 <400> 24 Gln Gln Leu Asn Ser Tyr Pro Tyr Thr 1 5 <210> 25 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> HCDR1 <400> 25 Gly Phe Thr Phe Asp Asp Cys Ala 1 5 <210> 26 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> HCDR2 <400> 26 Ile Ser Gly Asn Ser Lys Asn Val 1 5 <210> 27 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> HCDR3 <400> 27 Ala Arg Asp Pro Ala Tyr Ser Gln Phe Asp Tyr 1 5 10 <210> 28 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> LCDR1 <400> 28 Ser Ser Asn Val Gly Gly Tyr Pro 1 5 <210> 29 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> LCDR2 <400> 29 Thr Asp Tyr 1 <210> 30 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> LCDR3 <400> 30 Ala Thr Trp Asp Asp Asn Leu Asn Gly Tyr Val 1 5 10 <210> 31 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> V12S VH <400> 31 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Ser Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Ala Ser Asp Gly Ala Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Arg Ser Thr Leu Tyr 65 70 75 80 Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Thr Leu Ala Gly Tyr Ser Gly Pro Met Gly Gly Met Asp Val 100 105 110 Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 32 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> V12S VL <400> 32 Asp Ile Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Arg Asp Ile Ser Asn Tyr 20 25 30 Leu Ala Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Ser Leu Ile 35 40 45 Tyr Gly Ala Ser Asn Leu Gin Ser Gly Val Ser Ser Gin Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Gin Pro 65 70 75 80 Glu Asp Ser Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Asp Ile Lys Arg 100 105 <210> 33 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> N35T VH <400> 33 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ser Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Ala Ser Asp Gly Ala Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Arg Ser Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Thr Leu Ala Gly Tyr Ser Gly Pro Met Gly Gly Met Asp Val 100 105 110 Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 34 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> N76S VH <400> 34 Gin Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ser Met Asn Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser lie Ser Ala Ser Asp Gly Ala Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Arg Gly Arg Phe Thr lie Ser Arg Asp Asn Ser Arg Ser Thr Leu Tyr 65 70 75 80 Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Thr Leu Ala Gly Tyr Ser Gly Pro Met Gly Gly Met Asp Val 100 105 110 Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 35 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> N76S VL <400> 35 Asp lie Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr lie Thr Cys Arg Ala Ser Arg Asp lie Ser Asn Tyr 20 25 30 Leu Ala Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Ser Leu lie 35 40 45 Tyr Gly Ala Ser Asn Leu Gin Ser Gly Val Ser Ser Gin Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Ser Leu Gin Pro 65 70 75 80 Glu Asp Ser Ala Thr Tyr Tyr Cys Gin Gin Tyr Tyr Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Asp Ile Lys Arg 100 105 <210> 36 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> V12S-N35T VH <400> 36 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Ser Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ser Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Ala Ser Asp Gly Ala Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Arg Ser Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Thr Leu Ala Gly Tyr Ser Gly Pro Met Gly Gly Met Asp Val 100 105 110 Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 37 <211> 369 <212> DNA <213> Artificial Sequence <220> <223> V12S <400> 37 caggtgcagc tggtagagtc tgggggaggc ctgtccaagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt caccttcagt agctatagca tgaactgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcatcc attagtgcta gtgatggtgc cacatactac 180 gcagactccg tgaggggccg gttcaccatc tccagagaca attccaggag cacactgtat 240 ctgcaaatga acagtctgag agccgaggac acggccgtgt attactgtgc gaaaactctc 300 gcgggatata gtggcccaat gggtggcatg gacgtctggg gccaagggac cacggtcacc 360 gtctcctca 369 <210> 38 <211> 324 <212> DNA <213> Artificial Sequence <220> <223> VL‑O4 <400> 38 gacatccaga tgacccagtc tccatcctca ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgtc gggcgagtcg ggacattagc aactatttag cctggtatca gcagaaacca 120 gggaaagccc ctaagtccct gatctatgga gcatccaatt tacaaagtgg ggtctcatca 180 cagttcagcg gcagtggatc cgggacagat ttcaccctca ccatcaacag cctgcagcct 240 gaagattctg caacttatta ctgtcaacag tactatagtt acccgctcac ttttggcgga 300 gggaccaagg tggatatcaa acgt 324 <210> 39 <211> 369 <212> DNA <213> Artificial Sequence <220> <223> N35T <400> 39 caggtgcagc tggtagagtc tgggggaggc ctggtcaagc ctggggggc cctgagactc 60 tcctgtgcag cctctggatt caccttcagt agctatagca tgacctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcatcc attagtgcta gtgatggtgc cacatactac 180 gcagactccg tgaggggccg gttcaccatc tccagagaca attccaggag cacactgtat 240 ctgcaaatga acagtctgag agccgaggac acggccgtgt attactgtgc gaaaactctc 300 gcgggatata gtggcccaat gggtggcatg gacgtctggg gccaagggac cacggtcacc 360 gtctcctca 369 <210> 40 <211> 369 <212> DNA <213> Artificial Sequence <220> <223> VH‑O4 <400> 40 caggtgcagc tggtagagtc tgggggaggc ctggtcaagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt caccttcagt agctatagca tgaactgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcatcc attagtgcta gtgatggtgc cacatactac 180 gcagactccg tgaggggccg gttcaccatc tccagagaca attccaggag cacactgtat 240 ctgcaaatga acagtctgag agccgaggac acggccgtgt attactgtgc gaaaactctc 300 gcgggatata gtggcccaat gggtggcatg gacgtctggg gccaagggac cacggtcacc 360 gtctcctca 369 <210> 41 <211> 324 <212> DNA <213> Artificial Sequence <220> <223> N76S <400> 41 gacatccaga tgacccagtc tccatcctca ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgtc gggcgagtcg ggacattagc aactatttag cctggtatca gcagaaacca 120 gggaaagccc ctaagtccct gatctatgga gcatccaatt tacaaagtgg ggtctcatca 180 cagttcagcg gcagtggatc cgggacagat ttcaccctca ccatcagcag cctgcagcct 240 gaagattctg caacttatta ctgtcaacag tactatagtt acccgctcac ttttggcgga 300 gggaccaagg tggatatcaa acgt 324 <210> 42 <211> 369 <212> DNA <213> Artificial Sequence <220> <223> V12S / N35T <400> 42 caggtgcagc tggtagagtc tgggggaggc ctgtccaagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt caccttcagt agctatagca tgacctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcatcc attagtgcta gtgatggtgc cacatactac 180 gcagactccg tgaggggccg gttcaccatc tccagagaca attccaggag cacactgtat 240 ctgcaaatga acagtctgag agccgaggac acggccgtgt attactgtgc gaaaactctc 300 gcgggatata gtggcccaat gggtggcatg gacgtctggg gccaagggac cacggtcacc 360 gtctcctca 369 <210> 43 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic CDR3 <400> 43 Ala Lys Thr Leu Ala Gly Tyr Ser Gly Pro Met Gly Gly Met Asp Val 1 5 10 15 <210> 44 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic CDR3 <400> 44 Ala Lys Ile Leu Val Gly Tyr Ser Gly Pro Met Gly Gly Met Asp Val 1 5 10 15 <210> 45 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic CDR3 <400> 45 Ala Lys Ser Leu Ala Ser Tyr Ser Gly Pro Met Gly Gly Met Asp Val 1 5 10 15 <210> 46 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> L11T <400> 46 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Thr Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Ala Ser Asp Gly Ala Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Arg Ser Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Thr Leu Ala Gly Tyr Ser Gly Pro Met Gly Gly Met Asp Val 100 105 110 Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 47 <211> 369 <212> DNA <213> Artificial Sequence <220> <223> L11T <400> 47 caggtgcagc tggtagagtc tgggggaggc acggtcaagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt caccttcagt agctatagca tgaactgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcatcc attagtgcta gtgatggtgc cacatactac 180 gcagactccg tgaggggccg gttcaccatc tccagagaca attccaggag cacactgtat 240 ctgcaaatga acagtctgag agccgaggac acggccgtgt attactgtgc gaaaactctc 300 gcgggatata gtggcccaat gggtggcatg gacgtctggg gccaagggac cacggtcacc 360 gtctcctca 369 <210> 48 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> V93S <400> 48 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Ala Ser Asp Gly Ala Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Arg Ser Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ser Tyr Tyr Cys 85 90 95 Ala Lys Thr Leu Ala Gly Tyr Ser Gly Pro Met Gly Gly Met Asp Val 100 105 110 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 49 <211> 369 <212> DNA <213> Artificial Sequence <220> <223> V93S <400> 49 caggtgcagc tggtagagtc tgggggaggc ctggtcaagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt caccttcagt agctatagca tgaactgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcatcc attagtgcta gtgatggtgc cacatactac 180 gcagactccg tgaggggccg gttcaccatc tccagagaca attccaggag cacactgtat 240 ctgcaaatga acagtctgag agccgaggac acggcctcgt attactgtgc gaaaactctc 300 gcgggatata gtggcccaat gggtggcatg gacgtctggg gccaagggac cacggtcacc 360 gtctcctca 369 <210> 50 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Q61R <400> 50 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Arg Asp Ile Ser Asn Tyr 20 25 30 Leu Ala Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Ser Leu Ile 35 40 45 Tyr Gly Ala Ser Asn Leu Gin Ser Gly Val Ser Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Gin Pro 65 70 75 80 Glu Asp Ser Ala Thr Tyr Tyr Cys Gin Gin Tyr Tyr Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Asp Ile Lys Arg 100 105 <210> 51 <211> 324 <212> DNA <213> Artificial Sequence <220> <223> Q61R <400> 51 gacatccaga tgacccagtc tccatcctca ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgtc gggcgagtcg ggacattagc aactatttag cctggtatca gcagaaacca 120 gggaaagccc ctaagtccct gatctatgga gcatccaatt tacaaagtgg ggtctcatca 180 cggttcagcg gcagtggatc cgggacagat ttcaccctca ccatcaacag cctgcagcct 240 GAGAATTCTG CAACCTGATT ACTGTCAACG GAGTAGTAGT ACCCGCTCAC TTTTGGCGGA 300 GGGACCAAGG TGGATATCAA ACCT 324

Claims

1. A modified antibody or antigen-binding fragment thereof that binds to Ang2 (angiopoietin-2) comprising an amino acid substitution, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the sequence of SEQ ID No: 34 and a light chain variable region having the sequence of SEQ ID No: 32, wherein the substitution is selected from the group consisting of: substitution of a serine (S) for an asparagine (N) at position 42 in the heavy chain variable region; and substitution of a serine (S) for an asparagine (N) at position 94 in the light chain variable region.

2. The modified antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain variable region having the amino acid substitution comprises framework regions having the sequences QVQLVESGGGLX1KPGGSLRLSCAAS and MX2WVRQAPGKGLEWVSS, wherein X1 is S, and wherein X2 is T.

3. The modified antibody or antigen-binding fragment thereof of claim 1, wherein the light chain variable region having the amino acid substitution comprises framework regions having the sequence NLQSGVSSQFSGSGSGTDFTLTIX3SLQPEDSATYYC, wherein X3 is S.

4. The modified antibody or antigen-binding fragment thereof of claim 1, wherein the modified antibody or antigen-binding fragment thereof comprises a plurality of framework regions selected from the group consisting of: QVQLVESGGGLX1KPGGSLRLSCAAS, wherein X1 is S; MX2WVRQAPGKGLEWVSS, wherein X2 is T; and NLQSGVSSQFSGSGSGTDFTLTIX3SLQPEDSATYYC, wherein X3 is S.

5. The modified antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising heavy chain CDR1, CDR2, and CDR3 of SEQ ID NOs: 13, 14, and 43; and a light chain variable region comprising light chain CDR1, CDR2, and CDR3 of SEQ ID NOs: 16, 17, and 18.

6. The modified antibody or antigen-binding fragment thereof of claim 1, wherein the modified antibody or antigen-binding fragment thereof comprises a heavy chain variable region selected from the group consisting of SEQ ID NOs: 33 and 36.

7. The modified antibody or antigen-binding fragment thereof of claim 1, wherein the modified antibody or antigen-binding fragment thereof comprises a light chain variable region of SEQ ID NO:

35.

8. The modified antibody or antigen-binding fragment thereof of claim 1, wherein the modified antibody or antigen-binding fragment thereof comprises: a heavy chain variable region of SEQ ID NO: 33 and a light chain variable region of SEQ ID NO: 32; a heavy chain variable region of SEQ ID NO: 34 and a light chain variable region of SEQ ID NO: 35; ​ ​ ​ ​ ​ ​ ​ ​ ​ the heavy chain variable region of SEQ ID NO: 36 and the light chain variable region of SEQ ID NO: 32; the heavy chain variable region of SEQ ID NO: 31 and the light chain variable region of SEQ ID NO: 35; the heavy chain variable region of SEQ ID NO: 33 and the light chain variable region of SEQ ID NO: 35; or the heavy chain variable region of SEQ ID NO: 36 and the light chain variable region of SEQ ID NO:

35.

9. A nucleic acid encoding the modified antibody or antigen-binding fragment thereof of any one of claims 1 to 8.

10. An expression vector comprising the nucleic acid of claim 9.

11. A cell transformed with the expression vector of claim 10.

12. A method for producing a modified antibody or antigen-binding fragment thereof, the method comprising: (a) culturing the cell of claim 11 ; and (b) collecting the modified antibody or antigen-binding fragment thereof from the cultured cell.

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