CN117999283A - TIE2 agonistic antibodies and uses thereof - Google Patents

Abstract

The present invention relates to an agonistic antibody of Tie2 or an antigen binding fragment thereof that binds to the Ig3Fn3 domain of human Tie2, wherein homodimeric Tie2 can form polygonal assemblies by binding of the antibody, thereby obtaining aggregation and activation.

Description

TIE2 agonistic antibodies and uses thereof

Technical Field

The present invention relates to Tie2 agonistic antibodies or antigen binding fragments thereof which bind to the Ig3 Fn3 (membrane proximal fibronectin type III) domain of human Tie2, wherein homodimeric Tie2 can form polygonal assemblies by binding of antibodies, and thus aggregate and activate.

Background

Angiogenesis occurs dynamically through various regulatory factors during the development, growth, preservation, and homeostasis of an organism. Thus, the newly formed blood vessels act as transport channels for various biological materials such as nutrients, oxygen, hormones, etc. to surrounding cells. Blood vessels that are abnormal in function and structure are the direct or indirect cause of the occurrence and progression of various diseases. Tumor blood vessels exacerbate hypoxia due to functional and structural defects, leading to tumor progression and metastasis to other tissues, and preventing anticancer drugs from being well delivered to the center of tumor tissue. In addition to cancer, defective blood vessels may also be found in various other diseases or conditions. Examples include various eye diseases (e.g., diabetic macular edema, age-related macular degeneration), viral infections, and acute inflammatory reactions such as sepsis, etc. Thus, if a therapeutic agent capable of normalizing a pathological blood vessel is present, it is viable for use in treating various patients with vascular abnormalities.

In order to inhibit abnormal angiogenesis and reduce vascular permeability, methods of directly activating Tie2 are being considered. Recombinant proteins that bind directly to the Tie2 receptor and induce Tie2 phosphorylation and activation are also being developed and tested for therapeutic effects in many preclinical cancers and eye models. COMP-Ang1 and angiopeptide are representative examples. Although these agents exhibit anti-angiogenic and anti-invasive activity, they have the disadvantage of having a very short half-life and of being unstable in physicochemical properties. In addition, a small molecule compound (AKB-9778) has been developed as an inhibitor of the dephosphorylating enzyme VE-PTP. VE-PTP inactivates Tie2 by removing the phosphate group on phosphorylated Tie 2. The disadvantage of these compounds is that they activate other receptors non-specifically, but they indirectly increase Tie2 activity by inhibiting VE-PTP. In addition, tie2 activating antibodies have been developed (US 6365154B1, US20170174789 A1). These antibodies inhibit vascular leakage by increasing the survival rate of vascular endothelial cells. Interestingly, a plant extract is said to induce Tie2 activity and to be useful as a skin care cosmetic (e.g., JP2011102273A, JP2018043949A, JP2015168656 a).

Tie2 is a receptor tyrosine kinase specific to endothelial cells, promotes vascular growth and stabilization, and may be an attractive therapeutic target for ischemic and inflammatory vascular diseases. Tie2 agonistic antibodies and oligomeric angiopoietin (Angpt 1) variants have been developed as potential therapeutic agents. However, the underlying mechanisms of its role in Tie2 aggregation and activation have not been clearly determined. Furthermore, the Angpt variant has difficulties in terms of production and storage.

Against this background, the inventors of the present application have made an effort to develop Tie2 agonistic antibodies, and thus human Tie2 agonistic antibodies, and determined that such antibodies specifically bind to Fn3 domain of Tie2, so that homodimeric Tie2 can form polygonal assemblies, and thus be aggregated and activated, thereby completing the present application.

Disclosure of Invention

It is an object of the present invention to provide a Tie2 agonistic antibody or antigen binding fragment thereof.

It is another object of the present invention to provide a nucleic acid encoding the antibody or antigen binding fragment thereof.

It is still another object of the present invention to provide a vector comprising a nucleic acid, a cell transformed with the vector, and a method of producing the same.

It is a further object of the present invention to provide a composition for preventing or treating an angiogenic disease, which comprises the antibody or antigen binding fragment thereof.

It is a further object of the present invention to provide a composition for co-administration with other therapeutic agents for angiogenic diseases, comprising the antibody or antigen binding fragment thereof.

In order to achieve the above object, the present invention provides a Tie2 agonistic antibody or an antigen binding fragment thereof, wherein the antibody binds to an Ig3 Fn3 (membrane proximal fibronectin type III) domain of human Tie2, and homodimer Tie2 is formed into a polygonal assembly by the binding of the antibody, and is thus aggregated and activated.

In particular, the present invention provides a Fab comprising: a heavy chain variable region (VH) comprising the sequence of SEQ ID NO.1, a heavy chain constant region (CH) comprising the sequence of SEQ ID NO. 3, a light chain variable region (VL) comprising the sequence of SEQ ID NO. 2, and a light chain constant region (CL) comprising the sequence of SEQ ID NO. 4.

In particular, the invention provides humanized antibodies comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 5 or SEQ ID NO. 7 and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 6 or SEQ ID NO. 8.

Furthermore, the present invention provides a nucleic acid encoding the antibody or antigen binding fragment thereof.

Furthermore, the present invention provides a vector comprising the nucleic acid.

In addition, the present invention provides cells transformed with the vector.

In addition, the present invention provides a method of producing the antibody or antigen-binding fragment thereof, comprising (a) culturing the cells and (b) recovering the antibody or antigen-binding fragment thereof from the cultured cells.

Furthermore, the present invention provides a composition for preventing or treating angiogenesis-related diseases, which comprises the antibody or antigen-binding fragment thereof as an active ingredient.

Furthermore, the present invention provides a composition for co-administration with other therapeutic agents for angiogenic diseases, comprising the antibody or antigen binding fragment thereof. The present invention provides a method for preventing or treating angiogenic diseases comprising administering the antibody or antigen binding fragment thereof to a patient. The invention provides the use of the antibody or antigen binding fragment thereof in the manufacture of a medicament for the prevention or treatment of angiogenic diseases.

Furthermore, the present invention provides an antibody polygonal assembly comprising a Tie2 agonistic antibody or antigen binding fragment thereof and forming with homodimer Tie2 by binding of the antibody to the Ig3 Fn3 (membrane proximal fibronectin type III) domain of human Tie 2.

Drawings

Figures 1a and 1b show the generation of Tie2 activated mouse monoclonal antibodies,

A shows schematic domain structure of Tie2 receptor and strategy for generation of monoclonal antibodies by hybridoma technique, ig: immunoglobulin-like domain, EGF: epidermal growth factor-like domain, fn: fibronectin type III domain, and

B shows the binding kinetics of hTAAB to human (left) or mouse (right) Tie2 as determined by SPR and BLI analysis, where the equilibrium dissociation constant (K _D, M) is calculated as the ratio of dissociation rate to binding rate (K _off/k_on) and the kinetic parameters are determined by the global fitting function of the Biacore Insight assessment software using a 1:1 binding model.

Figures 2a to 2d show a structural comparison of Tie2 Fn2-3,

A shows the results of size exclusion chromatography of chimeric hTAAB Fab alone (black line) and in complex with Tie2 ECD variants (Ig 3-Fn3, red line; fn1-3, blue line; and Fn2-3, yellow line) (left), and analysis of eluted fractions by SDS-PAGE and Coomassie brilliant blue staining under reducing and non-reducing conditions (right),

B shows a comparison of the Tie2 Fn2-3 monomer (black) combined with the chimeric hTAAB Fab with previously reported monomer structures of Tie2 Fn2-3 (red, PDB:5MYB chain A) and Fn1-3 (green, PDB:5UTK chain A), wherein the structures are represented as a band diagram,

C shows a comparison of Tie2 Fn2-3 dimer (black) bound to chimeric hTAAB Fab with previously reported Tie2 Fn2-3 dimer structure (red, PDB:5 MYB), and

D shows a comparison of Tie2 Fn2-3 dimer (black) bound to chimera hTAAB Fab with previously reported Tie2 Fn1-3 dimer structure (PDB: 5 UTK), wherein Tie2 Fn1-3 dimer in two asymmetric units in the lattice are colored green and cyan, respectively;

Figures 3a to 3d show the general structure of hTAAB Fab composited with Tie2 Fn2-3,

A shows the overall structure of a 2:2 hTie2 Fn 2-3/chimeric hTAAB Fab complex, wherein the 2-heavy axis (2-fold axis) of the dimer complex is represented as a black ellipse, the heavy and light chains of chimeric hTAAB Fab are colored dark orange and light orange, respectively, and the hTie2 Fn2-3 dimer is colored blue-green and cyan,

B shows a sequence comparison of human and mouse Tie2 Fn2-3 domains, wherein the dots in the mouse Tie2 sequence represent the same residues as human Tie2,

C shows an open-book view showing the interaction interface of Tie2 Fn2-3 (top) and chimera hTAAB Fab (bottom) as surface representations, wherein the color schemes of hTie2 Fn2-3 and chimera hTAAB Fab are the same as in fig. 3a, but are reversed for each binding surface, the six CDR loops of chimera hTAAB Fab are represented by different colored lines (bottom), and

D shows the sequence comparison of hTAAB heavy chain variable region (top) and light chain variable region (bottom) with the closest human and mouse variable region germline genes, where the points in the mouse and human germline sequences represent the same residues as hTAAB, and in (b-d) the hTie2 residues interacting with the heavy and light chains of chimeric hTAAB Fab are colored dark orange and light orange, respectively, V730 interacting with the heavy and light chains are highlighted in red (b and c, top), and the chimeric hTAAB Fab residues interacting with hTie2 Fn3 are colored cyan (c, bottom, and d);

FIGS. 4a to 4d show the results of analysis of the binding interface between the chimeric hTAAB Fab and the hTie2 Fn3,

A shows a close-up view of the key molecular interactions (A, B and C region), where residues involved in the hTie2Fn 3/chimeric hTAAB Fab interactions (red, green and blue boxes) and hTie2 dimerization (black boxes) are represented as rods and labeled, hydrogen bonds and electrostatic interactions are represented as dashed lines,

B shows the key amino acid residues of A, B and C regions involved in hTAAB binding, with the interaction residue of hTAAB listed on the left and the corresponding interaction residue of Tie2 on the right, and interactions between the residue partners are represented in red (ionic interactions), black (hydrogen bonds) and blue (hydrophobic interactions).

C shows the electrostatic potential of the hTie2 Fn 2-3/chimeric hTAAB Fab complex calculated according to the Poisson-Boltzmann equation in PyMOL, where the structure is characterized as an open book style view and a surface view, and the color map reflects the electrostatic properties (blue: positively charged, red: negatively charged), where the interaction region is highlighted with yellow lines, and

D shows hydrophobic residues in hTie2 Fn2-3 and chimerism hTAAB Fab present on the surface, where the interaction interface is represented by a black line;

FIGS. 5a to 5d show the sequence alignment of Tie2, tie1 Fn2-Fn3 domains and chimeric hTAAB Fab,

A shows the sequence alignment of human (homo sapiens (H.sapiens), uniProt: Q02763), mouse (M.musculus), uniProt: Q02858), rat (brown rat (R.norvegicus), uniProt: D3ZCD 0), monkey (cynomolgus monkey (M.fascicularis), uniProt: A0A2K5VRI 3), cow (cattle (B.taurus), uniProt: Q06807) and dog (domestic dog (C.fasmiaria), uniProt: F1P8U 6) Tie2 Fn2-3,

B shows the alignment of the sequences of human (Chile, uniProt: Q02763) and mouse (mouse, uniProt: Q02858) Tie2 Fn2-3, and of human (Chile, uniProt: P35590) and mouse (mouse, uniProt Q06806) Tie1 Fn2-3,

C shows the alignment of the heavy chain variable region of chimeric hTAAB and the closest mouse and human germline gene (top) and the alignment of the heavy chain gamma 1 constant region of chimeric hTAAB (hIGHV x 01) and the closest mouse germline gene (bottom), and d shows the alignment of the light chain variable region of chimeric hTAAB and the closest mouse and human germline gene (top) and the alignment of the light chain kappa constant region of chimeric hTAAB (hIGKC x 01) and the closest mouse germline gene (bottom), and

In (a-d), blue boxes represent interacting residues in the interface of chimeric hTAAB Fab heavy and hTie2 Fn3, white boxes represent interacting residues between chimeric hTAAB Fab light and hTie2 Fn3, red circles represent residue V730 of hTie2, which interacts with both heavy and light chains, grey circles represent residues involved in interactions between chimeric hTAAB Fab heavy and light chains, residues involved in dimer interactions between Fn3 and Fn3 domains of Tie2 are represented as orange triangles, red boxes represent perfect sequence conservation, yellow boxes represent residues with more than 70% similarity based on physicochemical properties, secondary structural elements are marked above the alignment with arrows (β -chain) and helices (α -helices), and sequence alignment is created using T-Coffee (http:// tcoffe. Crg. Cat) and ESPript servers (http:///esppript. Freb);

The results shown in figure 6 show hTAAB IgG binding of polygonal assemblies mediating Tie2 dimer,

A shows the results of immunoblot analysis of the relative phosphorylation ratios of Tie2 after treatment of HUVEC with chimeric hTAAB Fab or hTAAB mouse IgG1 (1 and 10 μg/ml) (left), and optical density analysis of the p-Tie2/Tie2 ratios (right; mean ± SD n=3; p <0.001vs control),

B shows the results of immunoblot analysis of the relative phosphorylation ratios of Akt after treatment of HUVEC with chimeric hTAAB Fab or hTAAB IgG1 (1 and 10 μg/ml) (left), and of the optical density analysis of the p-Akt/Akt ratio (right; mean ± SD n=3; p < 0.05; p <0.001vs control),

C shows the results of size exclusion chromatography-multi angle light scattering (SEC-MALS) analyses of hTie2 Fn2-3, hTie2 ECD, hTAAB/hTie2 Fn2-3 complexes and hTAAB/hTie2 ECD complexes, wherein the samples were run in 0.5ml/min of PBS at a protein concentration of 3mg/ml (hTie 2 Fn 2-3) or 1mg/ml (all others) on a Superdex 200INCREASE 10/300GL column and the absorbance at molecular weights (kDa) and 280nm were plotted against elution volume (ml),

D shows the design of Tie2 dimerization mutants in which residues D682 and N691 (yellow) on both ends of the antiparallel beta-sheet interaction interface between Fn3 domains are mutated to cysteines to allow the formation of two disulfide bonds, resulting in a constitutive Tie2 Ig3-Fn3 dimer,

E shows the results of analysis of purified hTie2 Ig3-Fn 3D 682C/N691C by SDS-PAGE and Coomassie brilliant blue staining under reducing and non-reducing conditions,

F shows the results of size exclusion chromatography with hTAAB IgG a composited hTie2 Ig3-Fn 3D 682C/N691C, wherein purified hTie2 Ig3-Fn 3D 682C/N691C was incubated with hTAAB IgG1 for 2 hours at a molar ratio of 2:1 (Tie 2 monomer: hTAAB IgG 1) and size exclusion chromatography was performed, the fractions for negative staining EM and class 2D averages were expressed as EM analysis,

G shows representative class 2D average (top) of Tie2 dimerization mutant/hTAAB IgG1 complexes, where the cyclic higher order structure of Tie2 dimer/hTAAB IgG1 complexes in 4 to 4, 5 to 5 and 6 to 6 assemblies was modeled based on class 2D average of corresponding tetragonal, pentagonal and hexagonal closed loop structures using Tie2 Fn 2-3/chimeric hTAAB Fab complexes, tie2 Ig1-Fn1 (PDB: 4K0V 11) and Fc fragment of human IgG1 (PDB: 5 VGP) (lower graph), where the scale is 20nm, and

H shows a polygonal 5 to 5 assembly 3D model of Tie2 dimer/hTAAB IgG1 complex observed on negative staining EM carrier, where 2:2tie 2 Fn2-3/chimeric hTAAB Fab complex in asymmetric units of crystal is represented by red line;

FIG. 7 shows the results of negative staining EM analysis of Tie2 dimerisation mutant/hTAAB IgG1 complex,

A shows representative photomicrographs of negative staining EM analysis of purified hTie2 Ig3-Fn 3N 691C/D682C complexed with hTAAB IgG1 (left; scale bar, 100 nm), and representative particle images of tetragonal, pentagonal and hexagonal closed-loop structures (right; scale bar, 20 nm), and

B shows representative photomicrographs of negative staining EM analysis of aggregation peaks of size exclusion chromatography of hTie2 Ig3-Fn 3N 691C/D682C and hTAAB IgG1 complexes, with scale bar 100nm;

The results shown in figure 8 indicate that ligand independent Tie2 dimerization is critical for hTAAB-mediated Tie2 aggregation and activation,

A shows the design of Tie2 monomer mutants in which residues V685, V687 and K700 (yellow) at the Fn3-Fn3 'dimer interface are mutated to negatively charged residues (V685D/V687D/K700E) for disrupting the Fn3-Fn3' dimer interface by charge repulsion, resulting in a constitutive Tie2 monomer (left), and full length Tie2-GFP WT in HEK293T cells for live cell imaging and Tie2 activation assays, and schematic diagrams of constitutive Tie2 dimerization mutants and monomer mutants (right).

B shows the copolymerization Jiao Yanshi image of Tie2 (full length Tie 2WT, tie2 dimerisation mutant (D682C/N691C), or monomer mutant (V685D/V687D/K700E)) in HEK293T cells after treatment with hTAAB and COMP-Ang1, where the image is presented at 60 Xmagnification and scale of 10 μm,

C and D show the results of immunoblot analysis of the relative phosphorylation ratios of Tie2 and Akt in HEK293T cells transiently expressing WT Tie2, a constitutive dimeric Tie2 mutant (D682C/N691C) or a constitutive monomeric Tie2 mutant (V685D/V687D/K700E) after treatment with COMP-Angpt1 (1 μg/ml) or hTAAB IgG1 (10 μg/ml) for 1 hour (C), and optical density analysis of the p-Tie2/Tie2 and p-Akt/Akt ratios (D) (mean ± SD n=3; p <0.05, < p <0.01, < p <0.001vs. control), and

E shows a hTAAB-mediated model of Tie2 activation on cell membranes, wherein hTAAB IgG binds to the Fn3 domain of homodimeric Tie2 and induces ligand-independent Tie2 dimer aggregation into higher order cyclic assemblies, and Tie2 aggregation organizes inactive kinase dimers in a manner optimal for autophosphorylation between adjacent dimers;

figure 9 shows hTAAB structure-based humanization,

A shows a schematic representation of chimeric hTAAB antibodies in subclasses IgG1, igG2 and IgG4, in which the interchain disulfide bonds are represented by black lines, the heavy and light chain variable regions are colored dark orange and light orange, respectively, the heavy and light chain constant regions are colored green and blue, respectively,

B and c show the results of immunoblot analysis of the relative phosphorylation ratios of Tie2 and Akt after HUVEC treatment with hTAAB in the form of mouse IgG1 or human chimeric IgG1, igG2 or IgG4 (1 and 10 μg/ml), and of the optical density analysis of the ratios of p-Tie2/Tie2 and p-Akt/Akt (c) (mean ± SD n=3; p < 0.05; p < 0.01; p <0.001vs control),

D shows alignment of hTAAB and humanized TAAB (hzTAAB) heavy and light chain variable region sequences with the closest human germline gene, where the points in the human germline and hzTAAB sequences represent the same residues as parent hTAAB, the hTAAB CDRs and hzTAAB grafted CDRs are colored blue, the back mutated residues of parent hTAAB are colored green and represented as green triangles, and the secondary structural elements (beta-strands) are represented by arrows,

E-H shows a homologous model structure of hzTAAB-H1L1 Fv superimposed on a chimeric hTAAB Fv structure, which chimeric hTAAB Fv structure was adapted from the crystal structure of the Tie2 Fn2-Fn 3/chimeric hTAAB Fab complex, wherein the heavy and light chains of chimeric hTAAB are colored dark orange and light orange, respectively, and the heavy and light chains of hzTAAB-H1L1 are colored purple and pink,

F-h shows the principle of structure-based humanization of Tie 2-activated antibodies, in which residues in the light chain that are critical for maintaining VH-VL pairing and CDR conformation (f) and affinity for Tie2 Fn3 (g) are back mutated to the mouse hTAAB sequence, residues in the heavy chain that are critical for maintaining CDR conformation are back mutated to the mouse hTAAB sequence (h), and the mutation is represented by an arrow, and the staining of the residues is the same as in (e), and

I shows the binding kinetics of hTie2 ECD measured by SPR analysis to humanizations TAAB (hzTAAB-H1L 1, hzTAAB-H2L1, hzTAAB-H1L2 and hzTAAB-H2L2, and the previously reported humanized construct 3H7H12G 4), where the equilibrium dissociation constant (K _D, M) is calculated as the ratio of dissociation rate to binding rate (K _off/k_on), the kinetic parameters are determined by the global fitting function of the Biacore Insight assessment software using a 1:1 binding model, and the vertical dashed line represents the start of the dissociation phase;

figures 10a to 10c show the results of an analysis of humanized Tie2 activating antibodies,

A shows the results of size exclusion chromatography of humanized Tie2 activating antibodies based on IgG1 (hzTAAB H L1, H1L2, H2L1 and H2L 2),

B shows the analysis of the concentrated eluted fractions of hzTAAB by SDS-PAGE and Coomassie blue staining under reducing (left) and non-reducing (right) conditions, and

C shows the binding kinetics of hzTAAB to mTie2Ig3-Fn3 measured by BLI (biological layer interferometry) analysis, wherein the binding (a) and dissociation (D) of mTie2Ig3-Fn3 are depicted as a sensorgram obtained using the Octet RED96 system, wherein the indicated antibodies are immobilized on an anti-human IgG Fc Capture (AHC) biosensor, followed by measuring binding by immersing the biosensor in a well containing 1600nM of mouse Tie2Ig3-Fn3 solution, and then measuring dissociation by washing with kinetic buffer;

FIG. 11 shows the results of humanization TAAB activating Tie2 and its downstream signaling in HUVECs,

A and b show immunoblot analysis results of the relative phosphorylation ratios of Tie2 after HUVEC treatment with different concentrations (0.02, 0.1, 0.5, 2.5, 12.5 and 50 μg/ml) of ptaab (a) or 3H7H12G4 (b), with ABTAA (2.5 μg/ml) + Angpt2 serving as positive control, and densitometric analysis of the p-Tie2/Tie2 ratio (right),

C and d show immunoblot analysis results (c) of the relative phosphorylation ratios of Tie2 and Akt after HUVEC treatment with different concentrations (1 and 10 μg/ml) of humanised TAAB (hzTAAB-H1L 1, H1L2, H2L1 and H2L 2) or hTAAB (c), and optical density analysis (d) of the p-Tie2/Tie2 and p-Akt/Akt ratios (mean ± SD n = 3; p < 0.05; p < 0.01; p <0.001vs control),

E and f show immunoblot analysis results of the relative phosphorylation ratios of Tie2 and Akt after HUVEC treatment with different concentrations (0.1, 0.5, 2.5 and 12.5. Mu.g/ml) of 3H7H12G4 or hzTAAB-H2L2, and optical density analysis of the p-Tie2/Tie2 and p-Akt/Akt ratios (f),

G and H show biological effects of hzTAAB-H2L2 in HUVEC and effects of hzTAAB-H2L2 on serum deprivation induced apoptosis of HUVEC (top), wherein HUVEC was incubated in serum-free medium containing 10 μg/ml of hzTAA-H2L2, hTAAB or 1 μg/ml of COMP-Angpt1, migration activity was measured using a modified Boyden chamber assay (middle), HUVEC was inoculated in the upper layer of 8- μm pore membrane, serum-free medium containing 10 μg/ml of hzTAA-H2L2, hTAAB or 1 μg/ml of COMP-Angpt1 was added to the bottom chamber, migrated cells were fixed and stained after 9 hours of incubation, HUVEC was plated on matrigel coated wells and subjected to a serum-free treatment of COMP-Angpt1 containing 10 μg/ml of hzTAA-H2L2, hTAAB or 1 μg/ml of COMP-Angpt (average value of 7) was obtained and the average value of < p-p =0.0.0.0, and the average value of p-p (p=0.0.0.0.0.v.m), and the positive values of p.0.v.p.m were obtained as controls

I shows confocal images of HUVEC representing hzTAAB-H2L 2-induced translocation of Tie2 to cell-cell contact and hzTAAB-H2L 2-induced nuclear clearance of FOXO1, where serum starved HUVEC was treated with hTAAB-H2L2 (10 μg/ml) or COMP-Angpt1 (1 μg/ml) for 30 min, goat anti-human Tie2 and donkey anti-goat antibody conjugated to Alexa Fluor488 were used for Tie2 visualization without untreated group as control, and rabbit anti-FOXO 1 and donkey anti-rabbit antibody conjugated to Alexa Fluor 594 were used for FOXO1 visualization, with the scale of 20 μm, DAPI:4', 6-diamidino-2-phenylindole; and

Figures 12a to 12d show a model of full length Tie2 aggregation,

A shows the crystal stacking interaction between the chimeric hTAAB Fab/Tie2 Fn2-3 complex, wherein the color scheme of the chimeric hTAAB Fab and Tie2 Fn2-3 is depicted in fig. 3a, and the lateral interaction between Fn2 domains introduced by crystal stacking is represented as a black box,

B shows a close-up of the crystal stacking interactions shown in black boxes in (a), where critical binding residues mediated by crystal stacking are shown and hydrogen bonding and electrostatic interactions are indicated by dashed lines.

C shows a recommended model of hTAAB-mediated Tie2 aggregation, where the circulating higher order structure of the full length Tie2 dimer/hTAAB IgG1 complex in the 5 to 5 assembly was modeled using the crystal structure of Tie2 Fn 2-3/chimeric hTAAB Fab, the Fc fragment of human IgG1 (PDB: 5 VGP) and the previously reported Tie2 construct Tie2 Ig1-Fn1 (PDB: 4K 0V), tie2 Fn1-3 (PDB: 5 MYA) and Tie2 tyrosine kinase domain (PDB: 6 MWE) based on the class 2D average of pentagonal closed loop structures, and the transmembrane domain was adapted from the NMR structure of the EGFR dimerization motif previously reported (PDB: 5LV 6), and

D shows a recommended model of COMP-Ang1 mediated Tie2 aggregation, where the circulating complex of full length Tie2 dimer with COMP-Ang1 complex in a5 to 1 assembly is modeled using the model structure of Tie2 (C) and Ang1 FLD/Tie2 Ig1-Fn1 complex (PDB: 4K 0V) and COMP coiled-coil domain (PDB: 1 VDF) (C and D) based on a model of hTAAB mediated full length Tie2 aggregation (C), and Tie2 Ig2 domain for Angpt FLD binding is colored yellow.

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the terms used herein are well known in the art and are typical.

The inventors of the present application developed a human Tie2 agonistic antibody hTAAB that targets the Tie2 Fn (membrane proximal fibronectin type III) domain. The Tie2/hTAAB composite structure functions in a new mode of Tie2 aggregation.

HTAAB is a human Tie2 agonistic antibody that acts in a novel pattern of Tie2 aggregation by forming a Tie2/hTAAB complex structure and specifically binds to the Tie2 Fn3 domain, linking the Tie2 homodimers into polygonal assemblies. This structure is in contrast to the transverse Tie2 arrays observed in previous lattices. In addition, disruption of the Fn3-Fn3' dimer interface inactivates Tie2 aggregation signals induced by hTAAB. These results underscore the importance of Fn 3-mediated Tie2 homodimerization for hTAAB-induced Tie2 polygonal assemblies and provide insight into how Tie2 agonists induce Tie2 aggregation and activation. Furthermore, the success of constructing humanized antibodies based on hTAAB structures creates potential clinical possibilities.

Accordingly, one aspect of the present invention relates to a Tie2 agonistic antibody or antigen binding fragment thereof, wherein the antibody binds to the Ig3 Fn3 (membrane proximal fibronectin type III) domain of human Tie2, and by binding of the antibody, homodimer Tie2 is formed into a polygonal assembly and is thus aggregated and activated.

Furthermore, the present invention relates to an antibody polygonal assembly comprising a Tie2 agonistic antibody or antigen binding fragment thereof and forming with homodimer Tie2 by binding of the antibody to the Ig3 Fn3 (membrane proximal fibronectin type III) domain of human Tie 2.

As used herein, the term "antibody" refers to an antibody that specifically binds Tie 2. The scope of the invention includes not only intact antibody forms that specifically bind to Tie2, but also antigen-binding fragments of antibody molecules.

The structure of an intact antibody has two full length light chains and two full length heavy chains, and each light chain is linked to a heavy chain by disulfide bonds. The heavy chain constant regions are gamma (gamma), mu (mu), alpha (alpha), delta (delta), and epsilon (epsilon) types and are subdivided into gamma 1 (gamma 1), gamma 2 (gamma 2), gamma 3 (gamma 3), gamma4 (gamma 4), alpha 1 (alpha 1), and alpha 2 (alpha 2) subclasses. The constant regions of the light chain are of the kappa (kappa) and lambda (lambda) types.

An antigen binding fragment or antibody fragment of an antibody refers to a fragment having antigen binding capacity and includes Fab, F (ab') 2, and Fv. Among the numerous antibody fragments, fab has a structure with light and heavy chain variable regions, a light chain constant region, and a first heavy chain constant region (CH 1), and has one antigen binding site. Fab 'differs from Fab in that Fab' has a hinge region comprising at least one cysteine residue at the C-terminus of the CH1 domain of the heavy chain. F (ab ') 2 antibodies are produced by disulfide bonds between cysteine residues in the hinge region of Fab'. Fv is the smallest antibody fragment with only the heavy and light chain variable regions. Double-chain Fv is configured such that the heavy and light chain variable regions are joined by a non-covalent bond, while single-chain Fv (scFv) is configured such that the heavy and light chain variable regions are joined by a covalent bond, typically via a peptide linker therebetween, or directly at the C-terminus, forming a dimeric structure, such as a double-chain Fv. Such antibody fragments may be obtained using proteolytic enzymes (e.g., fab may be obtained by restriction cleavage of the whole antibody with papain, and F (ab') 2 may be obtained by restriction cleavage of the whole antibody with pepsin), or may be constructed by genetic recombination techniques.

In embodiments, an antibody according to the invention is in Fv form (e.g., scFv) or in whole antibody form. Furthermore, the heavy chain constant region may be any one selected from the group consisting of numerous isoforms such as gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε). For example, the constant region may be γ1 (IgG 1), γ3 (IgG 3), or γ4 (IgG 4). The light chain constant region may be kappa or lambda.

As used herein, the term "heavy chain" is understood to include full length heavy chains including a variable region domain VH comprising an amino acid sequence having a variable region sequence sufficient to confer antigen specificity, and three constant region domains CH1, CH2 and CH3, as well as fragments thereof. Furthermore, the term "light chain" as used herein is understood to include full length light chains, including a variable region domain VL comprising an amino acid sequence having a variable region sequence sufficient to confer antigen specificity, and constant region domain CL, as well as fragments thereof.

Examples of antibodies of the invention include, but are not limited to, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single chain Fv (scFV), single chain antibodies, fab fragments, F (ab') fragments, disulfide-like Fv (sdFV), anti-idiotype (anti-Id) antibodies, epitope-binding fragments of these antibodies, and the like.

Monoclonal antibodies are antibodies obtained from a population of substantially homogeneous antibodies, wherein the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may occur at a low frequency. Monoclonal antibodies are highly specific and are induced against a single antigenic site. In contrast to typical (polyclonal) antibodies, which typically comprise different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.

The term "epitope" refers to a protein determinant to which an antibody is capable of specific binding. The epitope typically consists of a set of chemically active surface molecules, such as amino acids or sugar side chains, and typically has specific three-dimensional structural features and specific charge properties. Conformational epitopes differ from non-conformational epitopes in that binding is lost in the presence of denaturing solvents, whereas binding is not lost.

A "humanized" form of a non-human (e.g., murine) antibody is a chimeric antibody that includes at least one amino acid sequence (e.g., CDR sequence) derived from at least one non-human antibody (donor or source antibody) that comprises a minimal sequence derived from a non-human immunoglobulin. In most cases, the humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the hypervariable region of the recipient are replaced with residues from the hypervariable region of a non-human species (donor antibody) having the desired specificity, affinity and capacity, e.g., mouse, rat, rabbit or non-human primate. For humanization, residues within at least one framework domain (FR) of the variable region of the recipient human antibody may be substituted with corresponding residues from a non-human species donor antibody. This helps maintain the correct three-dimensional configuration of the grafted CDR or CDRs, thereby improving affinity and antibody stability. Humanized antibodies may include novel residues that are not present in additional acceptor or donor antibodies, e.g., to further improve antibody performance.

"Human antibody" is a molecule derived from a human immunoglobulin and refers to an antibody comprising all amino acid sequences of the complementarity determining regions and framework regions that are comprised of the human immunoglobulin.

A portion of the heavy and/or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remaining chain(s) include "chimeric" antibodies (immunoglobulins) identical or homologous to corresponding sequences in antibodies derived from other species or belonging to other antibody classes or subclasses, as well as fragments of such antibodies which exhibit the desired biological activity.

As used herein, the "variable region" of an antibody refers to the light or heavy chain portion of an antibody molecule that includes the amino acid sequences of complementarity determining regions (CDRs; i.e., CDR1, CDR2, and CDR 3) and Framework Regions (FR). VH refers to the variable region of the heavy chain, while VL refers to the variable region of the light chain.

"Complementarity determining regions" (CDRs; i.e., CDR1, CDR2 and CDR 3) are amino acid residues of an antibody variable domain that are essential for antigen binding. Each variable domain typically has three CDRs, identified as CDR1, CDR2, and CDR3.

"Framework region" (FR) is a variable domain residue other than a CDR residue. Each variable domain typically has four FR, identified as FR1, FR2, FR3, and FR4.

Tie2 antibodies are monovalent or bivalent and comprise single or double chains. Functionally, tie2 antibodies have a binding affinity in the range of 10 ^-5 M to 10 ^-12 M. For example, the binding affinity of Tie2 antibodies may be 10 ^-6 M to 10 ^-12M、10^-7 M to 10 ^-12M、10^-8 M to 10 ^-12M、10^-9 M to 10 ^-12M、10^-5 M to 10 ^-11M、10^-6 M to 10 ^-11M、10^-7 M to 10 ^-11M、10^-8 M to 10 ^{- 11}M、10^-9 M to 10 ^-11M、10^-10 M to 10 ^-11M、10^-5 M to 10 ^-10M、10^-6 M to 10 ^-10M、10^-7 M to 10 ^-10M、10^-8 M to 10 ^{- 10}M、10^-9 M to 10 ^-10M、10^-5 M to 10 ^-9M、10^-6 M to 10 ^-9M、10^-7 M to 10 ^-9M、10^-8 M to 10 ^-9M、10^-5 M to 10 ^-8M、10^{- 6} M to 10 ^-8M、10^-7 M to 10 ^-8M、10^-5 M to 10 ^-7M、10^-6 M to 10 ^-7 M or 10 ^-5 M to 10 ^-6 M.

According to a specific embodiment of the application, the antibody may be a Fab comprising a heavy chain variable region (VH) comprising the sequence of SEQ ID NO. 1, a heavy chain constant region (CH) comprising the sequence of SEQ ID NO. 3, a light chain variable region (VL) comprising the sequence of SEQ ID NO. 2 and a light chain constant region (CL) comprising the sequence of SEQ ID NO. 4.

In a specific embodiment according to the invention, a humanized antibody hzTAAB, such as hzTAAB-H1 or hzTAAB-L1, is constructed. Humanized antibodies hzTAAB may comprise a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 5 or SEQ ID NO. 7 and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 6 or SEQ ID NO. 8.

In particular, in claim 3, the humanized antibody may comprise:

A heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 5 and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 6;

A heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 7 and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 6;

A heavy chain variable region comprising the amino acid sequence of SEQ ID NO.5 and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 8; or (b)

A heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 7 and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 8.

Within the scope of being able to specifically recognize Tie2, an antibody or antibody fragment according to the invention may include not only the sequences of the anti-Tie 2 antibodies listed herein, but also biological equivalents thereof. For example, additional modifications may be made to the amino acid sequence of the antibody to further increase the binding affinity and/or other biological properties of the antibody. Such modifications include, for example, deletions, insertions, and/or substitutions of amino acid sequence residues of antibodies. Amino acid variations are based on the relative similarity of amino acid side chain substituents in terms of, for example, hydrophobicity, hydrophilicity, charge, size, and the like. Arginine, lysine and histidine are all positively charged residues, with alanine, glycine and serine being similar in size and phenylalanine, tryptophan and tyrosine being similar in shape based on analysis of the size, shape and type of amino acid side chain substituents. Thus, based on these considerations, arginine, lysine, and histidine may be considered as biofunctionally equivalent, alanine, glycine, and serine may be considered as biofunctionally equivalent, and phenylalanine, tryptophan, and tyrosine may be considered as biofunctionally equivalent.

In view of the above-described variations having equivalent biological activities, the amino acid sequence of the antibody of the present invention or the nucleic acid molecule encoding the same should be understood to include sequences having substantial identity to the sequences listed in the sequence numbers. When sequences of the invention and any other sequences are aligned to correspond as much as possible to each other and the aligned sequences are analyzed using algorithms commonly used in the art, "substantially identical" means that the sequences exhibit at least 90% homology, most preferably at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology or at least 99% homology. Alignment methods for sequence comparison are well known in the art. NCBI Basic Local Alignment Search Tools (BLAST) are accessible through NCBI and the like and can be used over the Internet in conjunction with sequence analysis programs such as blastp, blasm, blastx, tblastn and tblastx. BLAST can be obtained at www.ncbi.nlm.nih.gov/BLAST. Methods for comparing sequence homology using this program can be found in www.ncbi.nlm.nih.gov/BLAST/blast_hellp.html.

Based on this, an antibody or antigen binding fragment thereof according to the present invention may have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology to the specific sequences or all sequences listed herein. Such homology can be determined by sequence comparison and/or alignment using methods known in the art. For example, the percent sequence homology of a nucleic acid or protein of the invention can be determined using a sequence comparison algorithm (i.e., BLAST or BLAST 2.0), manual alignment, or visual inspection.

Another aspect of the invention relates to nucleic acids encoding the antibodies or antigen binding fragments thereof.

The nucleic acid may comprise a sequence selected from the group consisting of SEQ ID NOS 9 to 14.

According to the invention, an antibody or antigen-binding fragment thereof may be recombinantly produced by isolating nucleic acids encoding the antibody or antigen-binding fragment thereof. The nucleic acid may be isolated and inserted into a replicable vector for further cloning (DNA amplification) or for further expression. Based thereon, a further aspect of the invention relates to a vector comprising the nucleic acid.

Herein, "nucleic acid" has a meaning that comprehensively encompasses DNA (gDNA and cDNA) and RNA molecules, and nucleotides as essential components of nucleic acids include not only natural nucleotides but also analogues in which sugar or base regions are modified. The sequences of the nucleic acids encoding the heavy chain variable region and the light chain variable region of the present invention may be modified. Such modifications include additions, deletions or non-conservative or conservative substitutions of nucleotides.

DNA encoding an antibody can be readily isolated or synthesized using conventional techniques (e.g., using oligonucleotide probes that are capable of specifically binding to DNA encoding the heavy and light chains of the antibody). Many vectors are available. The vector component generally includes, but is not limited to, at least one selected from the group consisting of a signal sequence, an origin of replication, at least one marker gene, an enhancer element, a promoter, and a transcription termination sequence.

As used herein, the term "vector" refers to a means for expressing a target gene in a host cell, and includes plasmid vectors, cosmid vectors, viral vectors such as phage vectors, adenovirus vectors, retrovirus vectors, adeno-associated virus vectors, and the like. In the vector, the nucleic acid encoding the antibody is operably linked to a promoter.

Here, the term "operably linked" means a functional linkage between a nucleic acid expression control sequence (e.g., a promoter, a signal sequence, or an array of transcription regulatory binding sites) and a different nucleic acid sequence whereby the control sequence is used to control transcription and/or translation of the different nucleic acid sequence.

When a prokaryotic cell is used as a host, it typically includes a strong promoter capable of promoting transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLlambda promoter, pRlambda promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter or T7 promoter), a ribosome binding site for initiating translation, and a transcription/translation termination sequence. In addition, for example, when eukaryotic cells are used as hosts, promoters derived from the genome of mammalian cells (e.g., metallothionein promoter, β -actin promoter, human hemoglobin promoter, or human muscle creatine promoter), or promoters derived from mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, tk promoter of HSV, mouse Mammary Tumor Virus (MMTV) promoter, LTR promoter of HIV, promoter of moloney virus, epstein-Barr virus (EBV), or promoter of Rous Sarcoma Virus (RSV)) may be used, and polyadenylation sequences are generally used as transcription termination sequences.

In some cases, the vector may be fused to another sequence to facilitate purification of the antibody expressed therefrom. Examples of sequences fused thereto include glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA) and 6 XHis (hexahistidine; qiagen, USA).

The vector comprises an antibiotic resistance gene commonly used in the art as a selectable marker, such as a gene conferring resistance to ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin or tetracycline.

A further aspect of the invention relates to a cell transformed with the vector described above. Examples of cells for producing antibodies of the invention may include, but are not limited to, prokaryotic cells, yeast cells, and higher eukaryotic cells.

Prokaryotic host cells such as E.coli (ESCHERICHIA COLI), B.subtilis (Bacillus subtilis) and B.thuringiensis (Bacillus thuringiensis) can be used as strains belonging to the genus Bacillus (Bacillus), streptomyces (Streptomyces), pseudomonas (e.g., pseudomonas putida (Pseudomonas putida)), proteus mirabilis (Proteus mirabilis) and Staphylococcus (e.g., staphylococcus botulinum (Staphylococcus carnosus)).

Here, interest in animal cells is greatest, and examples of useful host cell lines may include, but are not limited to COS-7、BHK、CHO、CHOK1、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.

A further aspect of the invention relates to a method of producing the antibody or antigen-binding fragment thereof, comprising (a) culturing the above-described cells and (b) recovering the antibody or antigen-binding fragment thereof from the cultured cells.

Cells can be cultured in a variety of media. Any commercially available medium may be used as the medium without limitation. All other necessary supplements known to those skilled in the art may be included in suitable concentrations. Culture conditions such as temperature and pH have been used for the host cell selected for expression, as will be apparent to those skilled in the art.

For recovering the antibody or antigen-binding fragment thereof, impurities may be removed by, for example, centrifugation or ultrafiltration, and the resulting product may be purified using, for example, affinity chromatography or the like. Other additional purification techniques may be used, such as anion or cation exchange chromatography, hydrophobic interaction chromatography, and hydroxyapatite chromatography.

Still another aspect of the present invention relates to a composition for preventing or treating angiogenic diseases, which comprises the antibody or antigen binding fragment thereof as an active ingredient.

Here, "angiogenesis" refers to the formation or growth of new blood vessels from preexisting blood vessels, and "angiogenesis-related diseases" refers to diseases related to the occurrence or progression of angiogenesis. The disease may fall within the scope of angiogenesis-related diseases without limitation, as long as it can be treated with an antibody. Examples of angiogenesis-related diseases may include, but are not limited to, cancer, tumor metastasis, diabetic retinopathy, retinopathy of prematurity, corneal graft rejection, macular degeneration, neovascular glaucoma, polycythemia, proliferative retinopathy, psoriasis, hemophilia arthritis, capillary formation of atherosclerotic plaques, keloids, wound granulations, vascular adhesions, rheumatoid arthritis, osteoarthritis, autoimmune diseases, crohn's disease, post-operative restenosis, atherosclerosis, intestinal adhesions, cat scratch disease, ulcers, liver cirrhosis, nephritis, diabetic nephropathy, diabetes, inflammatory diseases, and neurodegenerative diseases. Furthermore, the cancer may be selected from the group consisting of: esophageal cancer, gastric cancer, colorectal cancer, rectal cancer, oral cancer, pharyngeal cancer, laryngeal cancer, lung cancer, colon cancer, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, prostate cancer, testicular cancer, bladder cancer, renal cancer, liver cancer, pancreatic cancer, bone cancer, connective tissue cancer, skin cancer, brain cancer, thyroid cancer, leukemia, hodgkin's lymphoma, and multiple bone marrow blood cancers, but are not limited thereto.

As used herein, the term "prevention" or "prophylaxis" refers to any effect of inhibiting or delaying the onset of a disease of interest by administering an antibody or composition according to the invention. The term "treatment or therapy" refers to any action that ameliorates or reduces the symptoms of a disease of interest by the administration of an antibody or composition according to the invention.

The composition comprising the antibody of the invention may be a pharmaceutical composition and may further comprise suitable carriers, excipients or diluents commonly used in the art.

Pharmaceutical compositions comprising a pharmaceutically acceptable carrier may be provided in a variety of oral or parenteral dosage forms such as tablets, pills, powders, granules, capsules, suspensions, oral solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solutions, suspensions, lyophilisates and suppositories. In this regard, the pharmaceutical compositions of the present invention may be formulated in combination with diluents or excipients such as fillers, thickeners, binders, wetting agents, disintegrants, surfactants, and the like. Solid formulations for oral administration may be in the form of tablets, pills, powders, granules, capsules and the like. With respect to these solids, the compounds of the present invention may be formulated in combination with at least one excipient, such as starch, calcium carbonate, sucrose, lactose or gelatin. Lubricants such as simple excipients, magnesium stearate, talc, and the like may be additionally used. Liquid formulations for oral administration may be suspensions, oral solutions, emulsions, syrups and the like. Various excipients such as simple diluents such as water or liquid paraffin, humectants, sweeteners, fragrances, preservatives and the like may be included in the liquid formulation. Furthermore, the pharmaceutical compositions of the present invention may be in parenteral dosage forms such as sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilisates, suppositories and the like. Injectable propylene glycol, polyethylene glycol, vegetable oils such as olive oil and esters such as ethyl oleate may be suitable for use in non-aqueous solvents and suspensions. The basic materials of the suppository include semisynthetic fatty acid esters (Witepsol), polyethylene glycol, tween 61, cocoa butter, laurel oil and glycerogelatin.

The compositions of the present invention are administered in a pharmaceutically effective amount. As used herein, the term "pharmaceutically effective amount" refers to an amount of a pharmaceutical composition sufficient to treat a disease at a reasonable benefit/risk ratio applicable to any drug treatment. The effective amount depends on various factors including the severity of the disease to be treated, the age and sex of the patient, the type of disease, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration, the rate of secretion, the period of treatment, the co-administration of the drugs and other parameters known in the art. The compositions of the present invention may be administered alone or in combination with other therapeutic agents. Thus, the composition may be administered sequentially or concurrently with conventional therapies. Furthermore, the composition may be administered in a single dose or in multiple doses. Taking these factors into account, it is important that the minimum amount sufficient to obtain maximum effect without side effects is administered, and the dosage can be readily determined by an expert in the art. Although the dosage of the pharmaceutical composition of the present invention is not particularly limited, it may vary depending on various factors including the health and weight of the patient, the severity of the disease, the type of the drug, the administration route and the administration time. The composition may be administered to a mammal (including rats, mice, livestock, humans, etc.) by a generally acceptable route (e.g., orally, intrarectally, intravenously, subcutaneously, intrauterine or intracranially) in single or multiple doses per day.

A further aspect of the invention relates to a method of inhibiting angiogenesis or a method of preventing or treating an angiogenesis-related disease comprising administering the antibody or the composition to a subject in need thereof.

The methods of the invention comprise administering to a subject in need of inhibition of angiogenesis a pharmaceutically effective amount of a pharmaceutical composition. The subject may be a mammal such as, but not limited to, a dog, cow, horse, rabbit, mouse, rat, chicken, or human. The pharmaceutical composition may be administered parenterally, subcutaneously, intraperitoneally, intrapulmonary, or intranasally, and, as desired, by a suitable method, including intralesional administration for topical treatment. The preferred dosage of the pharmaceutical composition of the present invention depends on various factors including the health and weight of the subject, the severity of the disease, the type of drug, the route of administration and the time of administration, and can be readily determined by one skilled in the art.

Still further aspects of the invention relate to a pharmaceutical composition comprising the antibody for preventing or treating cancer, or a method of preventing or treating cancer, comprising administering the antibody or composition to a subject in need thereof. Here, the terms "antibody", "prophylaxis" and "treatment" are as described above.

The type of cancer is not limited as long as it can be treated with the antibody of the present invention. In particular, the antibodies of the invention are capable of preventing the onset or progression of cancer by inhibiting angiogenesis. Examples of cancers include, but are not limited to, esophageal cancer, gastric cancer, colorectal cancer, rectal cancer, oral cancer, pharyngeal cancer, laryngeal cancer, lung cancer, colon cancer, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, prostate cancer, testicular cancer, bladder cancer, renal cancer, liver cancer, pancreatic cancer, bone cancer, connective tissue cancer, skin cancer, brain cancer, thyroid cancer, leukemia, hodgkin's lymphoma, and multiple myeloma.

Furthermore, the antibodies of the invention may be used in combination with other antibodies or bioactive agents or materials for various purposes. Still a further aspect of the invention relates to a composition for co-administration with an additional therapeutic agent for an angiogenic disease comprising the antibody or antigen binding fragment thereof.

Examples of additional therapeutic agents for angiogenic diseases may include anti-angiogenic, anti-inflammatory, and/or anti-cancer drugs. Thus, these can overcome each other's resistance and improve efficacy.

When the composition according to the present invention is co-administered with an additional therapeutic agent for angiogenic diseases, the Tie2 antibody and the additional therapeutic agent for angiogenic diseases may be administered sequentially or simultaneously. For example, an anti-angiogenic, anti-inflammatory and/or anti-cancer drug may be administered to a subject, and then a composition comprising as an active ingredient a Tie2 antibody or antigen-binding fragment thereof may be administered thereto, or the composition may be administered to a subject, and then an anti-angiogenic, anti-inflammatory and/or anti-cancer drug is administered thereto. In some cases, the composition may be administered to the subject concurrently with the anti-angiogenic, anti-inflammatory, and/or anti-cancer drug.

Examples

The invention will be better understood by the following examples. It will be apparent to those skilled in the art that these examples are for illustration of the invention only and are not intended to limit the scope of the invention.

EXAMPLE 1 preparation of Tie2 activated mouse monoclonal antibodies

1.1 Expression and purification of recombinant proteins

Recombinant Tie2 proteins for mouse immunization were prepared by cloning the gene encoding hTie2 Ig3-Fn3 (human Tie2 residue 349-738, genbank accession No. AAH 35514.2) into a pFuse-hIgG1-Fc vector (pFuse-hg 1Fc1, invivoGen) and then transiently expressing in an Expi293F cell.

Human Tie2 Ig3-Fn3 (349T-738P)

Amino acid sequence (SEQ ID NO: 19)

TPKIVDLPDHIEVNSGKFNPICKASGWPLPTNEEMTLVKPDGTVLHPKDFNHTDHFSVAIFTIHRILPPDSGVWVCSVNTVAGMVEKPFNISVKVLPKPLNAPNVIDTGHNFAVINISSEPYFGDGPIKSKKLLYKPVNHYEAWQHIQVTNEIVTLNYLEPRTEYELCVQLVRRGEGGEGHPGPVRRFTTASIGLPPPRGLNLLPKSQTTLNLTWQPIFPSSEDDFYVEVERRSVQKSDQQNIKVPGNLTSVLLNNLHPREQYVVRARVNTKAQGEWSEDLTAWTLSDILPPQPENIKISNITHSSAVISWTILDGYSISSITIRYKVQGKNEDQHVDVKIKNATITQYQLKGLEPETAYQVDIFAENNIGSSNPAFSHELVTLPESQAP

DNA sequence (SEQ ID NO: 20)

ACCCCAAAGATAGTGGATTTGCCAGATCATATAGAAGTAAACAGTGGTAAATTTAATCCCATTTGCAAAGCTTCTGGCTGGCCGCTACCTACTAATGAAGAAATGACCCTGGTGAAGCCGGATGGGACAGTGCTCCATCCAAAAGACTTTAACCATACGGATCATTTCTCAGTAGCCATATTCACCATCCACCGGATCCTCCCCCCTGACTCAGGAGTTTGGGTCTGCAGTGTGAACACAGTGGCTGGGATGGTGGAAAAGCCCTTCAACATTTCTGTTAAAGTTCTTCCAAAGCCCCTGAATGCCCCAAACGTGATTGACACTGGACATAACTTTGCTGTCATCAACATCAGCTCTGAGCCTTACTTTGGGGATGGACCAATCAAATCCAAGAAGCTTCTATACAAACCCGTTAATCACTATGAGGCTTGGCAACATATTCAAGTGACAAATGAGATTGTTACACTCAACTATTTGGAACCTCGGACAGAATATGAACTCTGTGTGCAACTGGTCCGTCGTGGAGAGGGTGGGGAAGGGCATCCTGGACCTGTGAGACGCTTCACAACAGCTTCTATCGGACTCCCTCCTCCAAGAGGTCTAAATCTCCTGCCTAAAAGTCAGACCACTCTAAATTTGACCTGGCAACCAATATTTCCAAGCTCGGAAGATGACTTTTATGTTGAAGTGGAGAGAAGGTCTGTGCAAAAAAGTGATCAGCAGAATATTAAAGTTCCAGGCAACTTGACTTCGGTGCTACTTAACAACTTACATCCCAGGGAGCAGTACGTGGTCCGAGCTAGAGTCAACACCAAGGCCCAGGGGGAATGGAGTGAAGATCTCACTGCTTGGACCCTTAGTGACATTCTTCCTCCTCAACCAGAAAACATCAAGATTTCCAAC ATTACACACTCCTCGGCTGTGATTTCTTGGACAATATTGGATGGCTATTCTATTTCTTCTATTACTATCCGTTACAAGGTTCAAGGCAAGAATGAAGACCAGCACGTTGATGTGAAGATAAAGAATGCCACCATCACTCAGTATCAGCTCAAGGGCCTAGAGCCTGAAACAGCATACCAGGTGGACATTTTTGCAGAGAACAACATAGGGTCAAGCAACCCAGCCTTTTCTCATGAACTGGTGACCCTCCCAGAATCTCAAGCACCA

Specifically, the Expi293F cells (2 x10 ⁶ cells/ml) were cultured in an Expi293 expression medium (a 1435103, sameifeier) and then transfected with a plasmid encoding hTie2Ig3-Fn3 using a ExpiFectamine293 transfection kit (a 14524, sameifeier). Cells were incubated in a shaker incubator (orbital shaker, 125 rpm) at 37℃and 8% CO ₂ for 5 days. After removal of the cells by centrifugation, the culture supernatant comprising the secreted hTie2Ig3-Fn3-Fc fusion protein was purified on an AKTA purification system (GE healthcare) equipped with HiTrap MabSelect SuRe affinity column (11003494, GE healthcare (GE HEALTHCARE)). Purified hTie2Ig3-Fn3-Fc fusion protein was concentrated using an Amicon ultracentrifuge filter (UFC 8030, millipore) and buffer replaced with PBS. The Fc tag of the fusion protein was removed by thrombin cleavage (27-0846-01, GE medical) at 22℃for 18 hours. The hTie2Ig3-Fn3 protein was further purified by removing the cleaved Fc tag on HiTrap MabSelect SuRe affinity columns.

To prepare human Tie2 for crystallization, the gene encoding hTie2 Fn2-3 (residues 541-735) (described below) was cloned into pET-28a (69864, novagen), and expressed in E.coli (E.coli) BL21 (DE 3) RIL (230240, agilent technologies (Agilent Technologies)).

Human Tie2 Fn2-3 (541I-735S)

Amino acid sequence (SEQ ID NO: 21)

IGLPPPRGLNLLPKSQTTLNLTWQPIFPSSEDDFYVEVERRSVQKSDQQNIKVPGNLTSVLLNNLHPREQYVVRARVNTKAQGEWSEDLTAWTLSDILPPQPENIKISNITHSSAVISWTILDGYSISSITIRYKVQGKNEDQHVDVKIKNATITQYQLKGLEPETAYQVDIFAENNIGSSNPAFSHELVTLPES

DNA sequence (SEQ ID NO: 22)

ATCGGACTCCCTCCTCCAAGAGGTCTAAATCTCCTGCCTAAAAGTCAGACCACTCTAAATTTGACCTGGCAACCAATATTTCCAAGCTCGGAAGATGACTTTTATGTTGAAGTGGAGAGAAGGTCTGTGCAAAAAAGTGATCAGCAGAATATTAAAGTTCCAGGCAACTTGACTTCGGTGCTACTTAACAACTTACATCCCAGGGAGCAGTACGTGGTCCGAGCTAGAGTCAACACCAAGGCCCAGGGGGAATGGAGTGAAGATCTCACTGCTTGGACCCTTAGTGACATTCTTCCTCCTCAACCAGAAAACATCAAGATTTCCAACATTACACACTCCTCGGCTGTGATTTCTTGGACAATATTGGATGGCTATTCTATTTCTTCTATTACTATCCGTTACAAGGTTCAAGGCAAGAATGAAGACCAGCACGTTGATGTGAAGATAAAGAATGCCACCATCACTCAGTATCAGCTCAAGGGCCTAGAGCCTGAAACAGCATACCAGGTGGACATTTTTGCAGAGAACAACATAGGGTCAAGCAACCCAGCCTTTTCTCATGAACTGGTGACCCTCCCAGAATCT

Cells were grown at 37℃in LB medium supplemented with 50. Mu.g/ml kanamycin until OD600 was 0.4. Protein expression was induced in 0.05mM IPTG (isopropyl. Beta. -d-1-thiogalactoside) and incubated at 18℃for 15 hours. Cells were recovered by centrifugation, resuspended in lysis buffer (20mM HEPES,pH 7.5 and 200mM NaCl), and lysed by sonication on ice. After removal of cell debris by centrifugation (0.5 hours at 13,000Xg at 4 ℃), the supernatant was loaded onto a Ni-NTA agarose affinity column (30210, kaiji). After washing with a washing buffer (20mM HEPES,pH 7.5, 200mM NaCl and 50mM imidazole) corresponding to 5 column volumes, the proteins were eluted with an elution buffer (20mM HEPES,pH 7.5, 200mM NaCl and 400mM imidazole) and further purified by size exclusion chromatography using a HiLoad16/600Superdex 200pg column (28-9893-35, GE medical) equilibrated with 20mM HEPES,pH 7.5 and 200mM NaCl. To prepare the chimeric hTAAB Fab for crystallization, the heavy and light chains of the synthesized chimeric hTAAB Fab were cloned into a modified pBAD expression vector for periplasmic secretion. Coli Top10F (Invitrogen) cells were transformed with plasmid pBAD-Fab and then grown at 37℃in LB medium supplemented with 100. Mu.g/ml ampicillin. Protein expression was induced with 0.2% arabinose at an OD600 of 1.0 and cells were grown at 30 ℃ for 15 hours. Cells were recovered by centrifugation, resuspended in lysis buffer (20mM HEPES,pH 7.5 and 200mM NaCl), and lysed by sonication on ice. After removal of cell debris by centrifugation (at 13,000Xg for 30min at 4 ℃) the supernatant comprising the soluble chimeric hTAAB Fab was loaded onto a Ni-NTA agarose affinity chromatography column (QIAGEN) and washed with a washing buffer (20mM HEPES,pH 7.5, 200mM NaCl and 50mM imidazole) corresponding to 5 column volumes. Proteins were eluted with elution buffer (20mM HEPES,pH 7.5, 200mM NaCl and 400mM imidazole) and further purified by size exclusion chromatography using a HiLoad16/600Superdex 200pg column equilibrated with 20mM HEPES,pH 7.5 and 200mM NaCl. Purified proteins and antibodies were split and stored at-80 ℃.

1.2 Immunization and Generation of B cell hybridomas

Five week old BALB/c mice were vaccinated twice weekly for 6 weeks with purified hTie2-Ig3-Fn3 (100 μg/injection) mixed with adjuvant. anti-Tie 2 antibodies in serum of immunized mice were evaluated by hTie2 ELISA (enzyme linked immunosorbent assay). When antibody titer (diluted 1:5000) was sufficiently increased (OD > 1.0), B lymphocytes were isolated from the spleen of immunized mice and fused with cultured myeloma cells (SP 2/0). The fused cells were cultured in HAT (hypoxanthine, aminopterin, and thymidine) medium, and hybridoma cells were selected and cultured only with the fused myeloma cells and B lymphocytes.

B cell hybridomas were maintained in DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% Fetal Bovine Serum (FBS), penicillin (100U/ml) and streptomycin (100 mg/ml). To produce anti-Tie 2 antibodies in B cell hybridomas, cells were washed with PBS and cultured in serum-free medium (SFM, 12045-076, gibco) for 3 days.

Viable hybridoma cells were dispersed in 96-well plates and culture supernatants were tested by hTie2 ELISA. For clone selection by limiting dilution, hybridoma pools showing a +signal were selected.

1.3 Sequencing of DNA encoding monoclonal antibodies

Hybridoma cells (2×10 ⁶ cells/ml) were cultured in DMEM containing 10% FBS and total RNA was obtained using RNEASY MINI kit (qiagen). RNA concentration was measured and cDNA was synthesized by Reverse Transcription (RT). PCR was performed on heavy and light chain variable region gene sequences using a Mouse Ig-primer set (Mouse Ig-PRIMER SET) (Novagen) and synthesized cDNA as templates, i.e., at 94℃for 5 minutes, followed by 35 cycles: at 94℃for 1 minute, at 50℃for 1 minute, at 72℃for 2 minutes, and then gradually cooled from 72℃to 4℃over 6 minutes. The PCR products obtained from each reaction were cloned into TA vectors and DNA sequenced, producing nucleotide sequences encoding the heavy and light chain variable regions of each antibody.

1.4 Preparation of Tie 2-activated mouse monoclonal antibody

The ECD of Tie2 forms dimers through the membrane proximal Fn3, independent of ligand binding. Multimers Angpt1 that bind to the Ligand Binding Domain (LBD) crosslink these preformed Tie2 dimers into higher order oligomers or "Tie2 clusters" for Tie2 activation and downstream signaling. However, the LBD of the preformed Tie2 homodimer is too far apart for a single antibody to target LBD So that Tie2 aggregation cannot be induced. Based on this concept, it is assumed that anti-Tie 2 antibodies that bind to the membrane proximal Tie2Fn domain induce Tie2 oligomerization and activation, such as multimer Angpt1.

An Ig3-Fn3 that targets hTie2, but not mTie2, was generated as hTie 2-activated mouse monoclonal antibody hTAAB.

Example 2 demonstration of binding of two hTAAB Fab to the V-Tie 2 dimer by Crystal Structure

Human-mouse chimeric IgG1, igG2, and IgG4 antibodies of Tie2 activating antibody hTAAB were generated by cloning the heavy or light chain variable region (VH or VL) of mouse hTAAB into a backbone vector expressing a human heavy or light chain constant region (CH or CL). The DNA fragment encoding the mouse hTAAB heavy chain variable region was synthesized as the sequence "EcoRV-signal sequence-VH-NheI" (Bioneer, inc.). The synthesized DNA fragment was digested with EcoRV and NheI and then cloned into pFUSE-CHIg-hG1 (IgG 1 isotype) and pFUSE-CHIg-hG2 (IgG 2 isotype) vectors (InvivoGen). To construct human IgG4 chimeric antibodies, DNA encoding the mouse hTAAB heavy chain variable region (VH) was amplified by PCR using primers that included EcoRI and NheI sites for cloning. The PCR product was then subcloned into a modified pOptiVEC-TOPO vector expressing the human IgG4 antibody heavy chain constant region (CH). The chimeric light chain expression vector was constructed by amplifying a DNA fragment encoding the mouse hTAAB light chain variable region by PCR using primers comprising EcoRI and BsiWI restriction sites.

Human IgG1 heavy chain constant region (CH 1-CH 3)

Amino acid sequence (SEQ ID NO: 23)

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

DNA sequence (SEQ ID NO: 24)

GCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGA GCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA

Human IgG2 heavy chain constant region (CH 1-CH 3)

Amino acid sequence (SEQ ID NO: 25)

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

DNA sequence (SEQ ID NO: 26)

GCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAA GGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA

Human IgG4 heavy chain constant region (CH 1-CH 3)

Amino acid sequence (SEQ ID NO: 27)

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

DNA sequence (SEQ ID NO: 28)

GCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCATCATGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAA AGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTCACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAA

Next, the PCR product was subcloned into a modified pcDNA3.3-TOPO vector expressing human kappa light chain constant regions.

Human kappa light chain constant region (GenBank accession number: AAA 58989.1)

Amino acid sequence (SEQ ID NO: 29)

TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

DNA sequence (SEQ ID NO: 30)

ACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGC

Chimeric antibodies were produced using the Expi293 expression system (zemoeifeil). The Expi293F cells were co-transfected with heavy and light chain expression vectors using ExpiFectamine293 transfection kit (a 14524, zemoeimer), after which the transfected cells were cultured in Expi293 expression medium for 5 days. The culture supernatant was filtered through a 0.45- μm filter and the antibody was purified using an AKTA purification device (GE medical) equipped with HiTrap MabSelect SuRe column (11003494, GE medical).

To map the hTAAB-bound epitope, the minimal domain of Tie2 for hTAAB binding was identified. Since hTie2 Ig3-Fn3 was used to immunize mice to produce antibodies, three recombinant proteins were produced, including Ig3-Fn3, fn1-3, or Fn2-3 of hTie2 (fig. 1, tie2 structure). Size Exclusion Chromatography (SEC) was used to test binding to recombinant chimeric hTAAB Fab, the recombinant chimeric hTAAB Fab was constructed from a fusion of the hTAAB heavy chain variable region to the human γ1 constant region and the fusion of the light chain variable region to the human kappa constant region. When chimeric hTAAB Fab was mixed with hTie2 Ig3-Fn3, fn1-3, or Fn2-3, the chromatograms of chimeric hTAAB Fab alone showed a single peak shifted forward (fig. 2 a). In addition, the chimeric hTAAB Fab and hTie2 domains were co-eluted. These results indicate that hTie2 Fn2-3 is sufficient for hTAAB binding and that hTie2 Fn2-3 and chimeric hTAAB Fab are capable of forming stable complexes (fig. 2a, yellow).

Determination by molecular substitution using the DOWN-Li Youshan antibody (durvalumab) Fab (PDB: 5X 8M) and Tie2 Fn2-3 structure (PDB: 5MYA chain B) as search modelsThe crystal structure of the hTie2 Fn 2-3/chimeric hTAAB Fab complex (Table 1). The crystal asymmetry unit comprises a 2-fold symmetrical heterotetramer hTie2 Fn 2-3/chimeric hTAAB Fab complex (2:2 stoichiometry).

TABLE 1

Two chimeric hTAAB Fab are bound to the side of each hTie 2Fn 3 domain at an inclination of about 15 ° relative to the plane perpendicular to the 2-fold axis (fig. 3a, right). This crystal structure reveals two major epitopes for the hTAAB-bound Fn3 domain, the heavy chain being bound to dark orange and the light chain to light orange (fig. 3b and c, top). The two hTie 2Fn2-3 monomers in the heterotetrameric hTie2 Fn2-3/chimeric hTAAB Fab complex were found to have the same overall structure and to be similar to the previously reported crystal structures of Tie2Fn2-3 (PDB: 5MYB; red) and Fn1-3 (PDB: 5UTK; green), indicating the presence of a rigid linkage between Fn2 and Fn3 (FIG. 2b; the root mean square deviation of C.alpha.between the hTie 2Fn2-3 monomers is about5MYB is/>5UTK is/>). Notably, the configuration and interface between hTie2 dimers in the hTie2 Fn 2-3/chimeric hTAAB Fab complex was also consistent with that found in previous studies (dimer 2 model of 5MYB and 5 UTK) (fig. 2c and d).

These results indicate that hTAAB binding did not cause a change in the conformation of the hTie2 homodimer. The four domains of chimeric hTAAB Fab (heavy chain variable region (VH), heavy chain constant region (CH), light chain variable region (VL) and light chain constant region (CL)) are folded with a typical Ig domain consisting of a pair of β -sheets. The heavy chain CDRs (HCDR 1, HCDR2 and HCDR 3) and the light chain CDRs (LCDR 1, LCDR2 and LCDR 3) are involved in contact with the hTie2 Fn3 domain (fig. 3c, bottom). In addition to the six CDRs, light chain Framework Region (FR) residues R46, Y49, S53 and R66 also interact with the hTie2 Fn3 domain (fig. 3 d). Thus, the discovery of the crystal structure revealed that two hTAAB Fab bound to the sides of the V-shaped Tie2 dimer, particularly the Fn3 domain.

Example 3 interaction of Tie2 FN3 with hTAAB and interaction with homoFn 3

Chimeric hTAAB Fab provides a total embedding surface area ofIs bound to hTie2 Fn3, the total entrapment surface area is contacted by about 57% of the heavy chain (/ >)Dark orange) and 43% light chain contactLight orange) composition (fig. 3 c). The binding interface between the chimera hTAAB Fab and the hTie2Fn3 can be divided into three regions: A. regions B and C (fig. 4a, B and 5).

The interaction of the a region is mediated primarily by ionic interactions and hydrogen bonding between hTAAB heavy chains (HCDR 1 and HCDR 2) and the hTie2 Fn3 domain. The residues on hTAAB HCDR1 (S28, T30, S31 and W33) and HCDR2 (H52, D55 and E57) form multiple interactions with residues on hTie2 Fn3βA [ E643, N644, I645 (backbone), K646, I647 (backbone) ] and βG (H727) (FIG. 4a, top left and bottom left, and FIG. 4 c).

In zone B, hTAAB of HCDR3, LCDR1, LCDR2 and LCDR3 cover a large surface area of hTie2 Fn3 by hydrophobic interactions with a total embedded surface area ofA. Residues I647, I650, a707, V730 and L732 of hTie2Fn3 form a network of hydrophobic core interactions with residues L100 and Y101 on HCDR3 and I31, Y49, a50, Y91 and a92 on LCDR (fig. 4a, bottom left, and fig. 4 d). These hydrophobic interactions are stabilized by adjacent C-region interactions that are involved in hydrogen bonding and electrostatic interactions between hTie2Fn3 residues (Q677, E705, and E728) and hTAAB light chain residues (S53, R66, and R46) (fig. 4a, middle bottom, and fig. 4C).

Interestingly, the hTAAB light chain residues involved in the C-region interaction are located on the β chain of FR (R46 on βc ', S53 on βc', R66 on βd), rather than the CDR loop. Most of the hTie2 Fn3 residues involved in hTAAB interactions are highly conserved in numerous species (fig. 5 a), but the hydrophobic valine (V730) of hTie2 in the center of the hTAAB binding interface in mice and rats will be replaced by a long charged side chain (arginine) (fig. 3b and 5 a), which explains why hTAAB cannot bind to mouse Tie2 (fig. 1e, right). However, all epitopes of hTAAB were conserved between human and monkey Tie2, indicating that hTAAB cross-reacted with monkey Tie2 (fig. 5 a). Furthermore, hTAAB is less likely to bind to Tie1 due to the low sequence similarity between Tie2 and Fn2-3 domains of Tie1, especially residues involved in hTAAB interactions (fig. 5 b).

Consistent with the previous Tie2 apo-structure (dimer 2 model of 5MYB and 5 UTK), the Fn3-Fn3 'interface consists of hydrogen bonds between backbone atoms of homotype Fn3 βc' (D682-K690), forming a continuous antiparallel β -sheet (fig. 4a, right). The dimer interface is further stabilized by hydrophobic interactions involving V685 and V687, and by mutual electrostatic interactions, especially D682-N691', D682' -N691, Y697-Q683', Y697' -Q683, K700-E703 'and K700' -E703 (FIG. 4a, bottom right). Wide embedding interface between Fn3-Fn3The low solvation free energy of-4.7 kcal/mol (Pisa server analysis) and the consistent V-configuration of Tie2 homodimers, whether or not hTAAB was present, suggest that specific interactions between homomembranous proximal Fn3 domains contribute to the formation of ligand-independent Tie2 homodimers.

Crystal structure and functional analysis using Fn2 mutants showed that Fn2 interactions are essential for lateral aggregation of preformed Tie2 dimers. Surprisingly, even when the structure comprises Tie2 Fn2-3/hTAAB Fab complex and the spatial population of protein crystals differs between the two structures, the same interaction between Fn2 domains of adjacent homodimers is observed in the crystal packing (fig. 12a and b). However, fab and Fab that bind to Tie2 Fn3 in the crystal stacking lattice are nearly parallel to each other (used to represent domains or residues in the symmetrical alignment of Tie2 Fn2-3/hTAAB Fab complex), thus deviating from the possible angle (115-148 °) between the two Fab arms of one IgG 1. Therefore hTAAB IgG1 is unlikely to be involved in Fn2 mediated lateral aggregation of preformed Tie2 dimers, but this type of Tie2 aggregation may occur in the higher order Angpt 1.

Example 4 Induction of polygonal Assembly of Tie2 dimer by hTAAB IgG binding

Human umbilical vein endothelial cells (HUVEC; C2519A, longza) were maintained in EGM-2 endothelial cell proliferation medium (CC-3162; longza) and incubated at 37℃and 5% CO ₂ in a humid incubator. The Expi293F cells (a 14527, zemoeimerter) were maintained in Expi293 expression medium (a 1435102, zemoeimerter) and incubated at 37 ℃ and 8% CO ₂ in a humidified shaker incubator.

Multimers Angpt 1or COMP-Angpt1 are able to induce higher order aggregation of Tie2, which is critical for activating Tie2 and its downstream signaling in EC to stabilize blood vessels. The phosphorylation level of Akt, the major downstream signaling protein of Tie2 receptor, was measured using immunoblotting techniques. Interestingly, not only hTAAB Fab but also hTAAB IgG1 induced concentration-dependent activation of Tie2 and Akt in primary cultured HUVECs (fig. 6a and 6 b). This result means that hTAAB Fab binds to Tie2 itself sufficiently to induce Tie2 aggregation and activation. Briefly, hTAAB IgG1 was able to promote Tie2 aggregation by a bivalent Fab arm. However, in the heterotetrameric hTie2 Fn 2-3/chimeric hTAAB Fab complex, the distance between the two C-termini of the Fab heavy chain is greater than(FIG. 3 a), which shows that the two Fab's bound to the homoTie 2 dimer are from two different IgG molecules. In contrast to the crystal structure of the hetero-tetrameric hTie2Fn 2-3/chimeric hTAAB Fab complex, SEC-multi-angle light scattering (MALS) analysis showed that hTie2Fn2-3 and hTie2 ECD were monomeric in solution. Furthermore, hTAAB bound to hTie2Fn2-3 or hTie2 ECD could not induce oligomerization (fig. 6 c). Current data and previous reports are contradictory regarding the multimeric state of the recombinant Tie2 ectodomain in solution. However, ligand-independent Tie2 dimerization can occur on cell membranes under physiological conditions through the steric constraints imposed by full-length Tie 2. This dimerization may occur particularly through the YIA sequence between the catalytic and activating loops of the intracellular kinase domain and shows homotype Fn3-Fn3' interactions in the crystal structure.

To mimic ligand-independent Tie2 dimers in physiological cell membranes, constitutive Tie2 dimers were artificially generated. Two residues D682 and N691 involved in homotypic Fn3-Fn3' interactions were mutated to cysteines (D682C and N691C) to introduce disulfide bonds (fig. 6D and e). The purified hTie2 Ig3-Fn3D682C/N691C dimer was then incubated with hTAAB IgG1 in a 1:1 ratio to investigate how full length hTAAB IgG1 induced aggregation of the preformed Tie2 dimer. After aggregate removal by SEC, the structural arrangement of the resulting complex (shoulder peak) was examined using a negative staining Electron Microscope (EM) (fig. 6f and 7). Surprisingly, the 2D classification of negatively stained EM particles showed higher order assembly cross-linking of full length hTAAB IgG with quadrangles, pentagons and hexagons of hTie2 Ig3-Fn3D682C/N691C dimer (fig. 6 g). These polygonal assemblies are characterized by the Fc domain of IgG1 at each vertex and one Tie2 dimer that links the two IgG1 by binding to one of the Fab arms. Although the Ig3 domain appears diffuse in the 2D average of negatively stained EM due to the flexibility of the loop between Ig3 and Fn1 domains, the rigid Fn1-3 domain of the hTie2 Ig3-Fn3D682C/N691C dimer is evident on both sides of the polygon. Based on observations of negative staining EM and crystal structure of hTie2 Fn 2-3/chimeric hTAAB Fab complex, a 3D model of pentagonal assembly (5 to 5hTAAB IgGs and hTie2 Ig3-Fn3D682C/N691C dimer) could be generated (fig. 6 h).

Example 5 ligand independent Tie2 dimerization is critical for hTAAB-mediated Tie2 aggregation and activation.

The biological relevance and significance of Tie2/hTAAB polygonal assemblies in cell membranes was evaluated. Tie2 dimer (D682C/N691C) was used to construct Tie2 monomers (V685D/V687D/K700E) with full-length Tie2-GFP constructs by disrupting the Fn3-Fn3' dimer interface (FIG. 8 a). This construct was used to transiently transfect HEK293T cells that were unable to endogenously express Tie 2. After confirming adequate plasma membrane expression of full length Tie2-GFP wild-type (WT) and constitutive dimeric Tie2 mutants and monomeric Tie2 mutants in cells (fig. 8 b), aggregation of GFP-tagged Tie2 WTs and mutants was monitored. Live cell imaging showed that after hTAAB or COMP-Angpt1 addition, clusters of WT Tie2 and Tie2 dimer formed on the cell surface, whereas Tie2 monomer failed to form clusters (fig. 8 b). Next, the effect of hTAAB IgG1 or COMP-Angpt1 on Tie2 activation was measured by monitoring phosphorylation of Tie2 and Akt under the same conditions. Although disruption of the Fn3-Fn3 dimer interface (constitutive Tie2 monomer) abrogates Tie2 and Akt phosphorylation induced by hTAAB and COMP-Angpt1, WT Tie2 and dimer Tie2 are similarly activated by both Tie2 agonists (fig. 8c and d). These results indicate that WT Tie2 exists as a homodimer in the plasma membrane and that ligand-independent homodimerization (due to intermolecular interactions between Fn3 domains) is essential for hTAAB-mediated Tie2 aggregation and activation.

Example 6 activation of Tie2 and its downstream Signaling in HUVEC by humanization TAAB

6.1 Production of humanized antibodies

The Fv region of mouse hTAAB was humanised based on the Tie 2Fn 2-3/chimeric hTAAB Fab complex structure and model structures of IGHV1-46 x 01 and IGKV1-17 x 01 complexes.

The sequence of mouse hTAAB Fv was compared to the sequence of human germline genes using the IMGT/domain pairing on-line tool of the International immunogenetics information System (International ImmunoGeneTics Information System, IMGT) (http:// www.imgt.org). IGHV1-46 x 01 and IGKV1-17 x 01, which show the highest sequence identity with the heavy and light chain variable regions of mouse hTAAB, respectively, were selected as human Framework (FR) donors. The CDRs (defined by IMGT numbers) of IGHV1-46 x 01 and IGKV1-17 x 01 are replaced by corresponding portions in mouse hTAAB, resulting in hzTAAB-H1 and hzTAAB-L1. Selected residues critical for maintaining VH-VL pairing, CDR conformation and Tie2 Fn3 affinity are further replaced at hzTAAB-H1 and hzTAAB-L1, resulting in hzTAAB-H2 and hzTAAB-L2. The resulting humanized Fv genes (hzTAAB-H1, hzTAAB-H2, hzTAAB-L1 and hzTAAB-L2) were synthesized (Baiori) and their expression in mammalian cells was optimized. First, the human immunoglobulin kappa light chain constant region (GenBank accession number: AAA 58989.1) and the human immunoglobulin gamma 1 heavy chain constant region (GenBank accession number: AWK 57454.1) were cloned into pOptiVEC-TOPO and pcDNA3.3-TOPO vectors, and then the variable regions of the synthetic humanized light and heavy chains (hzTAAB-H1, hzTAAB-H2, hzTAAB-L1 or hzTAAB-L2) were cloned into the vectors.

Human kappa light chain constant region (GenBank accession number: AAA 58989.1)

Amino acid sequence (SEQ ID NO: 31)

TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

DNA sequence (SEQ ID NO: 32)

ACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGAC AGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGC

Human IgG1 heavy chain constant region (CH 1-CH 3) (GenBank accession number: AWK 57454.1) amino acid sequence (SEQ ID NO: 33)

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

DNA sequence (SEQ ID NO: 34)

GCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTG GACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA

The use of ExpiFectamine 293 transfection kit (A14524, siemens) transfected with a plasmid containing humanized light and heavy chains. Cells were incubated in a shaker incubator (orbital shaker, 125 rpm) at 37℃and 8% CO ₂ for 5 days. The resulting culture supernatant was centrifuged to remove cells and the antibodies were isolated by affinity chromatography on a ProA column (Amicogen) equilibrated with PBS. Bound antibody was eluted with 0.1M glycine-HCl, pH 2.7, and neutralized with 1M Tris-Cl, pH 9.0. The eluted antibodies were further purified by size exclusion chromatography using a Superdex 200Gain 10/300GL column (28-9909-44, GE medical) equilibrated with PBS. Antibody purity was assessed using reduced and non-reduced SDS-PAGE, and purified antibodies were aliquoted and stored at-80 ℃.

6.2 Homology modeling of humanized Tie2 activating antibodies

SWISS-MODEL homology modeling was performed with sequences of IGHV1-46 x 01 and IGKV1-17 x 01 transplanted with hTAAB CDR human germline using the structure of hTAAB Fv in the Tie2 Fn 2-3/chimeric hTAAB Fab complex structure as a template. The resulting model with the highest QMEAN-Z (qualitative model energy analysis-Z) score (-0.37) was aligned with the hTAAB Fv structure for further structure-based humanization.

Potent hTAAB Tie2 agonistic activity is promising in clinical applications in vascular diseases. Thus, attempts were made to humanize mice hTAAB while maintaining the ability to bind and activate Tie2, but minimizing immunogenicity due to their mouse origin. The hinge flexibility of the IgG subclass varies slightly due to the variation in length, sequence and number of disulfide bonds at the hinge between Fab and Fc domains, and the Fab-Fab angle and Fab-to-Fc domain orientation of each subclass also varies.

Considering that the Fab-Fab angle and Fab orientation of hTAAB may be key determinants of Tie2 polygonal assemblies, it was investigated whether such hinge flexibility influences Tie2 activation. To this end, chimeric IgG1, igG2 or IgG4 comprising the hTAAB variable region was constructed (fig. 9 a) and its Tie2 agonistic activity was compared to that of the parent mouse hTAAB. Phosphorylation of Tie2 and Akt is related to hinge flexibility of the IgG subclass (IgG 1> IgG4> IgG 2). The IgG2 isotype with the most rigid hinge showed the lowest Tie2 agonistic activity (fig. 9b and c). These results indicate that polygonal Tie2 aggregation induced by hTAAB is critical for Tie2 activation. Based on these observations, the IgG1 format used for hTAAB humanization was selected.

CDR boundaries were defined using IMGT/domain gap alignment tools (http:// www.imgt.org) from IMGT (international immunogenetic information system) and the human germline genes IGHV1-46 x 01 and IGKV1-17 x 01 were selected as human FR donors, as they showed the highest sequence identity with the heavy chain variable region (66%) and the light chain variable region (68%) of mouse hTAAB (fig. 9D and 5C-D).

Binding kinetics of mouse hTAAB and humanized IgG1 to human Tie2 were measured by Surface Plasmon Resonance (SPR) using a Biacore T200 system equipped with an authentication-grade CM5 series S sensor chip (BR 100399, GE healthcare). HEPES buffered saline (0.01M HEPES and 0.15M NaCl) containing 3mM EDTA (ethylenediamine tetraacetic acid) and 0.05% (v/v) P20 detergent (HBS-EP+) was used as a reaction and running buffer (BR 100669, GE medical treatment). Human Tie2 ECD (residues 23-735 of human Tie 2; genBank accession number: AAH 35514.2) was immobilized on the surface of a CM5 sensor chip by amine coupling using 10mM acetate at pH 5.5. Thereafter, mice hTAAB and humanized IgG1 diluted in HBS-EP+ buffer were applied at 7 different concentrations (0, 2,4, 8, 16, 32 and 64 nM) at a flow rate of 30 μl/min for 300 seconds on antigen-immobilized sensor chips. The analyte bound to the sensor chip was dissociated by washing with HBS-ep+ running buffer for 300 seconds. The association (k _on,M^-1s^-1) and dissociation (k _off,,s^-1) constants were measured every 300 seconds. The equilibrium dissociation constant (K _D, M) was calculated as the ratio of dissociation rate to association rate (K _off/k_on). The global fitting function of the software was evaluated using Biacore weight, and kinetic parameters were determined using a 1:1 binding model.

The binding kinetics of mouse hTAAB and humanized IgG1 to mouse Tie2 were measured by BLI (biofilm interference technique) using the Octet system (arey (ForteBio)). The analysis was carried out at 30 ℃. After 10 minutes of hydration in kinetic buffer (0.01% endotoxin free BSA, 0.002% Tween-20 and 0.005% NaN ₃ in PBS), mice hTAAB (10. Mu.g/ml each) were loaded onto an anti-mouse IgG Fc capture (AMC) biosensor (forteBio). Ab-coated sensors were incubated with 1600nM solutions of mouse Tie2Ig3-Fn3 (residues 349-737) and thus correlation curves were recorded for 600 seconds. Dissociation was measured in kinetic buffer for 600 seconds.

Conventional CDR grafting is performed by simply replacing the CDR portions in the selected human germline FR donors with the corresponding CDRs of the hybridoma mouse antibody hTAAB. To maintain the hTAAB HCDR2 conformation, S59 was substituted with the R59 residues flanking hTAAB heavy chain HCDR2 in the humanized antibody. The CDR-grafted variable regions (hzTAAB-H1 and hzTAAB-L1) of the heavy and light chains of the humanized Tie 2-activated antibody (hzTAAB) were cloned into the pOptiVEC-TOPO vector (GenBank accession number: AWK 57454.1) comprising the human Ig gamma 1 heavy chain constant region and the pcDNA3.3-TOPO vector (GenBank accession number: AAA 58989.1) comprising the human Ig kappa light chain constant region, respectively. After hzTAAB-H1 and hzTAAB-L1 were co-expressed in HEK293F cells, the antibodies were purified to homogeneity as full length IgG1 by protein A affinity chromatography and SEC (FIGS. 10a and b) and the binding kinetics of hzTAAB-H1L1 to hTie2 ECD was assessed using SPR. hzTAAB-H1L1 binding to hTie2 showed much lower affinity and faster off-rate (k_on＝2.5x10⁵M^-1s^-1;k_off＝5.6x10^-3 s^-1;K_D＝2.2x10^-8 M)( FIG. 9 f), indicating that conventional CDR grafting was insufficient to maintain the binding affinity of hTAAB to hTie 2.

Regarding hTAAB structure-based humanization, homology modeling was performed on hzTAAB-H1 and hzTAAB-L1 using Fv (variable fragment) structures of parent hTAAB in the crystal structure as templates. The parental hTAAB Fv structure was placed on the resulting hzTAAB-H1L1Fv model to identify FR residues that are critical to maintaining VH-VL pairing, CDR conformation, and binding affinity to hTie2 (fig. 9 e). Comparative analysis between hzTAAB-H1L1 and the VH-VL interface of parent hTAAB showed that the hTAAB light chain residues N34 and L36 on LFR2 (G34 and Y36 in hzTAAB-L1) stabilize Y101 on HCDR3 and reduce the steric hindrance of W105 on HFR 4. Furthermore, hTAAB light chain residue D55 on LFR3 (Q55 in hzTAAB-L1) is critical to maintaining the conformation of the LCDR2 hairpin loop by interacting with R46 on LFR2 (f in fig. 9 e). hTAAB light chain residue R66 on LFR3 (G66 in hzTAAB-L1) is also critical for binding Tie2 through charge interactions with E705 of hTie2 (G in fig. 9E). Based on this, hzTAAB-L2 was generated by back-mutating selected hzTAAB-L1 residues to the corresponding mouse hTAAB residues (G34N, Y36L, Q D and G66R) (FIG. 9D). For the heavy chain (hzTAAB-H2), R72 and T74 on HFR3 of hzTAAB-H1 were mutated to parent hTAAB residues V72 and K74, which potentially stabilized the HCDR2 conformation by interaction with P53 and S54 of HCDR2 (H in FIG. 9 e).

After hzTAAB-H1L1, three other hzTAAB (hzTAAB-H1L 2, hzTAAB-H2L1 and hzTAAB-H2L 2) were produced, all full length IgG1 in HEK293F cells, based on different combinations of heavy and light chains (FIGS. 10a and b). After purification, the binding kinetics of the obtained antibodies to hTie2 ECD were assessed using SPR. hzTAAB-H2L1 has a binding affinity (k_on＝2.8x10⁵ M^-1s^-1;k_off＝3.3x10^-3 s^-1;K_D＝1.1x10^-8 M) slightly higher than the binding affinity (k_on＝2.5x10⁵ M^-1s^-1;k_off＝5.6x10^-3 s^-1;K_D＝2.2x10^-8 M), of hzTAAB-H1L1 but a much higher dissociation rate than that of parent hTAAB, (k_on＝1.4x10⁵ M^-1s^-1;k_off＝6.0x10^-4 s^-1;K_D＝4.2x10^-9 M)( FIG. 9 i). However, back mutations in the light chain significantly improved the affinities of hzTAAB-H1L2(k_on＝1.1x10⁵ M^-1s^-1;k_off＝9.6x10^-4 s^-1;K_D＝8.5x10^-9 M) and hzTAAB H2L2(k_on＝1.4x10⁵ M^-1s^-1;k_off＝7.3x10^-4 s^-1;K_D＝5.2x10^-9 M) for hTie2 ECD (fig. 9 i). The affinity of the obtained humanized antibodies (hzTAAB-H1L 2 and hzTAAB-H2L 2) was comparable to that of the parent hTAAB and was achieved mainly by a substantial reduction of the off-rate. Furthermore, hzTAAB-H2L2 exhibited 4,700-fold higher affinity than 3H7H12G4 when comparing Tie2 binding affinity to the previously reported humanized antibody 3H7 known as 3H7H12G4, indicating an improvement in the structure-based hTAAB humanization process (fig. 9 i). Similar to the parent hTAAB, no humanized antibodies were bound to mTie2 (fig. 10 c).

Subsequently, phosphorylation of Tie2 and Akt after hzTAAB treatment was detected and compared to phosphorylation after hTAAB or 3H7H12G4 treatment. Consistent with Tie2 binding affinity, the previously developed 3H7H12G4 showed very low Tie2 agonistic activity compared to hTAAB (fig. 11a and b). Of the four hzTAAB, hzTAAB-H2L2 was most effective in inducing phosphorylation of Tie2 and Akt in HUVECs (FIGS. 11c and d). This is comparable to the Tie2 agonistic activity of hTAAB. In addition, hzTAAB-H2L2 exhibited excellent Tie2 agonistic activity when hzTAAB-H2L2 was compared to 3H7H12G4 (FIGS. 11e and f).

Thus hzTAAB-H2L2 effectively induced survival, migration and tube formation of EC, and translocation of Tie2 to the cell-cell contact site and translocation of FOXO1 from the nucleus to the cytosol to a similar extent as COMP-Angpt1 and parent hTAAB (fig. 11 g-i). Taken together, the structure-based humanization strategy successfully retained the binding affinity and Tie2 agonistic activity of the parent hTAAB.

Industrial applicability

The inventors of the present application developed a human Tie2 agonistic antibody hTAAB targeting the Tie2 Fn (membrane proximal fibronectin type III) domain and a humanized antibody thereof. The Tie2/hTAAB composite structure functions in a new mode of Tie2 aggregation. It was determined that hTAAB acted in a novel fashion by Tie2 aggregation by forming a Tie2/hTAAB composite structure and specifically bound the Tie2 Fn3 domain, linking the Tie2 homodimers into polygonal assemblies. The importance of Fn 3-mediated Tie2 homodimerization for hTAAB-induced Tie2 polygonal assemblies can be demonstrated, and it can also be understood how Tie2 agonists induce Tie2 aggregation and activation. Furthermore, potential clinical applicability can be demonstrated by constructing humanized antibodies based on hTAAB structures.

While certain portions of the present invention have been described in detail above, it will be apparent to those skilled in the art that these are merely preferred embodiments, and the scope of the present invention is not limited thereby. Accordingly, the substantial scope of the present invention is defined by the appended claims and equivalents thereof.

Free text of sequence Listing

An electronic file is attached.

<110> Basic science institute (Institute for Basic Science)

Korean science and technology institute (korea advanced institute of SCIENCE AND technology)

<120> TIE2 agonistic antibodies and uses thereof

(Tie2 Agonistic Antibodies and Uses Thereof)

<130> PP-B2798

<160> 36

<170> PatentIn version 3.2

<210> 1

<211> 115

<212> PRT

<213> Artificial sequence

<220>

<223> hTAAB VH

<400> 1

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr

20 25 30

Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Met Ile His Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe

50 55 60

Met Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Leu Tyr Gly Asn Ser Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ala

115

<210> 2

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> hTAAB VL

<400> 2

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Glu Arg Val Ser Leu Thr Cys Arg Ala Ser Gln Asp Ile Gly Ile Ser

20 25 30

Leu Asn Trp Leu Gln Gln Glu Pro Asp Gly Thr Ile Lys Arg Leu Ile

35 40 45

Tyr Ala Thr Ser Ser Leu Asp Ser Gly Val Pro Lys Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser

65 70 75 80

Glu Asp Phe Val Asp Tyr Tyr Cys Leu Gln Tyr Ala Ser Ser Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 3

<211> 103

<212> PRT

<213> Artificial sequence

<220>

<223> HTAAB heavy chain constant region

<400> 3

Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala

1 5 10 15

Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu

50 55 60

Ser Ser Ser Val Thr Val Pro Ser Ser Pro Arg Pro Ser Glu Thr Val

65 70 75 80

Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys

85 90 95

Ile Val Pro Arg Asp Cys Gly

100

<210> 4

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> HTAAB light chain constant region

<400> 4

Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu

1 5 10 15

Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe

20 25 30

Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg

35 40 45

Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu

65 70 75 80

Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser

85 90 95

Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

100 105

<210> 5

<211> 115

<212> PRT

<213> Artificial sequence

<220>

<223> hzTAAB-H1L1 VH

<400> 5

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile His Pro Ser Asp Ser Glu Thr Arg Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Leu Tyr Gly Asn Ser Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 6

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> hzTAAB-H1L1 VL

<400> 6

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 Gln Asp Ile Gly Ile Ser

20 25 30

Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile

35 40 45

Tyr Ala Thr Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Ala Ser Ser Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 7

<211> 115

<212> PRT

<213> Artificial sequence

<220>

<223> hzTAAB-H2L1 VH

<400> 7

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile His Pro Ser Asp Ser Glu Thr Arg Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Val Asp Lys Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Leu Tyr Gly Asn Ser Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 8

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> hzTAAB-H1L2 VL

<400> 8

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 Gln Asp Ile Gly Ile Ser

20 25 30

Leu Asn Trp Leu Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile

35 40 45

Tyr Ala Thr Ser Ser Leu Asp Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Ala Ser Ser Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 9

<211> 345

<212> DNA

<213> Artificial sequence

<220>

<223> hTAAB VH

<400> 9

caggtccaac tgcagcagcc tggggctgag ctggtgaggc ctggagcttc agtgaagctg 60

tcctgcaagg cttctggcta ctccttcacc agctactgga tgaactgggt gaagcagagg 120

cctggacaag gccttgagtg gattggcatg attcatcctt ccgatagtga aactaggtta 180

aatcagaagt tcatggacaa ggccacattg actgtagaca aatcctccag cacagcctac 240

atgcagctca gcagcccgac atctgaggac tctgcggtct attactgtgc tcgtggcctc 300

tatggtaact cttggggcca agggactctg gtcactgtct ctgca 345

<210> 10

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> hTAAB VL

<400> 10

gacatccaga tgacccagtc tccatcctcc ttatctgcct ctctgggaga aagagtcagt 60

ctcacttgtc gggcaagtca ggacattggt attagcttaa actggcttca gcaggaacca 120

gatggaacta ttaaacgcct gatctacgcc acatccagtt tagattctgg tgtccccaaa 180

aggttcagtg gcagtaggtc tgggtcagat tattctctca ccatcagcag ccttgagtct 240

gaagattttg tagactatta ctgtctacaa tatgctagtt ctccgtacac gttcggaggg 300

gggaccaagc tggaaataaa a 321

<210> 11

<211> 345

<212> DNA

<213> Artificial sequence

<220>

<223> hzTAAB-H1L1 VH

<400> 11

caggttcagc tggtccagag cggggctgag gtcaagaaac caggtgcatc cgttaaggtg 60

tcatgtaaag cctcggggta cagtttcacc agctactgga tgcactgggt gagacaggcc 120

cctggtcaag gccttgaatg gatgggaatt atccatccct ccgattccga aactagatat 180

gcacaaaagt ttcagggcag agtgactatg accagagaca catctacgtc taccgtctat 240

atggagttgt caagtctgcg cagcgaggac acagctgtat actattgcgc caggggactc 300

tatggcaata gctggggaca gggcactctg gtgacagtgt cttca 345

<210> 12

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> hzTAAB-H1L1 VL

<400> 12

gatatccaga tgactcaatc tcctagctcc ctcagcgctt cagtgggcga cagagtcacc 60

attacctgtc gcgcttctca ggatatcggg attagcctgg gatggtacca gcagaagcct 120

ggtaaggcac ccaaacggct gatttacgcc acatcgagtc tgcagtctgg agtgccaagc 180

aggttttcag gaagcgggtc cggcaccgag tttactctga caatatctag tctgcagcca 240

gaagacttcg ccacatacta ttgcttgcag tatgcatcct caccctatac gttcggccaa 300

ggaaccaaac ttgagatcaa g 321

<210> 13

<211> 345

<212> DNA

<213> Artificial sequence

<220>

<223> hzTAAB-H2L1 VH

<400> 13

caggtccagc tggtgcagag tggcgccgag gtcaagaagc ctggagcatc agttaaagtg 60

tcttgtaaag ctagtggcta ctcgtttact tcttactgga tgcactgggt gagacaagcc 120

ccaggtcaag gattggagtg gatgggcatt atccatccca gcgactccga aacgagatat 180

gctcagaagt tccaggggcg ggtgacaatg accgtcgata aatctacaag caccgtgtac 240

atggagcttt ccagtctgag atccgaagac actgccgtat actattgcgc aaggggcctc 300

tatggtaatt catggggaca ggggaccctg gtgacagtta gctcc 345

<210> 14

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> hzTAAB-H1L2 VL

<400> 14

gacatccaaa tgactcagag cccctccagt ctgtcggcat ctgtgggaga tcgcgtcacc 60

attacatgcc gggccagcca ggacatcggc attagtttga actggcttca gcagaagcct 120

gggaaagccc caaaaagact catatacgct acttcttccc tcgactctgg cgtgccctca 180

agatttagcg gaagcaggtc aggcactgag ttcaccctga caattagctc cctgcaacca 240

gaagatttcg ctacatacta ttgtctgcag tacgcctcat ccccttatac ctttggtcag 300

gggactaagc tggagatcaa g 321

<210> 15

<211> 115

<212> PRT

<213> Artificial sequence

<220>

<223> 3H7H12G4 VH

<400> 15

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Met Ile His Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe

50 55 60

Met Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Leu Tyr Gly Asn Ser Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 16

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> 3H7H12G4 VL

<400> 16

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 Gln Asp Ile Gly Ile Ser

20 25 30

Leu Asn Trp Leu Gln Gln Glu Pro Gly Lys Ala Pro Lys Arg Leu Ile

35 40 45

Tyr Ala Thr Ser Ser Leu Asp Ser Gly Val Pro Lys Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Ala Ser Ser Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 17

<211> 345

<212> DNA

<213> Artificial sequence

<220>

<223> 3H7H12G4 VH

<400> 17

caggtgcagc tggtccaatc cggggctgag gtgaagaagc ctggagcatc agtgaaagtt 60

tcatgcaaag ctagtggtta caccttcacc agctattgga tgaactgggt gcggcaggcc 120

cccggtcagg ggcttgagtg gatgggcatg atccacccat ccgactctga gactaggctg 180

aaccagaagt ttatggatag agtgaccatg acaagagata cgtccacttc tactgtctat 240

atggaactga gcagtctgag atctgaagac acagccgttt actactgtgc tcgcggactc 300

tatggcaata gctggggcca aggaacattg gtaaccgtct cttct 345

<210> 18

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> 3H7H12G4 VL

<400> 18

gacatccaga tgactcagtc cccctcgagc ctctcagctt ctgttggaga cagagtgaca 60

attacatgcc gggcctcaca ggatattggg atctccctga actggctgca acaggaacca 120

ggaaaggccc ctaagcgcct gatatatgcc acatcctctc ttgactcagg ggtcccaaag 180

aggtttagcg gcagtggatc aggtactgag ttcactctca ccatctctag cctgcagcct 240

gaggattttg caacctacta ttgtttgcaa tacgctagtt ccccctacac gttcggccag 300

ggcaccaaag tggaaatcaa a 321

<210> 19

<211> 390

<212> PRT

<213> Artificial sequence

<220>

<223> Human Tie2 Ig3-Fn3 349T-738P

<400> 19

Thr Pro Lys Ile Val Asp Leu Pro Asp His Ile Glu Val Asn Ser Gly

1 5 10 15

Lys Phe Asn Pro Ile Cys Lys Ala Ser Gly Trp Pro Leu Pro Thr Asn

20 25 30

Glu Glu Met Thr Leu Val Lys Pro Asp Gly Thr Val Leu His Pro Lys

35 40 45

Asp Phe Asn His Thr Asp His Phe Ser Val Ala Ile Phe Thr Ile His

50 55 60

Arg Ile Leu Pro Pro Asp Ser Gly Val Trp Val Cys Ser Val Asn Thr

65 70 75 80

Val Ala Gly Met Val Glu Lys Pro Phe Asn Ile Ser Val Lys Val Leu

85 90 95

Pro Lys Pro Leu Asn Ala Pro Asn Val Ile Asp Thr Gly His Asn Phe

100 105 110

Ala Val Ile Asn Ile Ser Ser Glu Pro Tyr Phe Gly Asp Gly Pro Ile

115 120 125

Lys Ser Lys Lys Leu Leu Tyr Lys Pro Val Asn His Tyr Glu Ala Trp

130 135 140

Gln His Ile Gln Val Thr Asn Glu Ile Val Thr Leu Asn Tyr Leu Glu

145 150 155 160

Pro Arg Thr Glu Tyr Glu Leu Cys Val Gln Leu Val Arg Arg Gly Glu

165 170 175

Gly Gly Glu Gly His Pro Gly Pro Val Arg Arg Phe Thr Thr Ala Ser

180 185 190

Ile Gly Leu Pro Pro Pro Arg Gly Leu Asn Leu Leu Pro Lys Ser Gln

195 200 205

Thr Thr Leu Asn Leu Thr Trp Gln Pro Ile Phe Pro Ser Ser Glu Asp

210 215 220

Asp Phe Tyr Val Glu Val Glu Arg Arg Ser Val Gln Lys Ser Asp Gln

225 230 235 240

Gln Asn Ile Lys Val Pro Gly Asn Leu Thr Ser Val Leu Leu Asn Asn

245 250 255

Leu His Pro Arg Glu Gln Tyr Val Val Arg Ala Arg Val Asn Thr Lys

260 265 270

Ala Gln Gly Glu Trp Ser Glu Asp Leu Thr Ala Trp Thr Leu Ser Asp

275 280 285

Ile Leu Pro Pro Gln Pro Glu Asn Ile Lys Ile Ser Asn Ile Thr His

290 295 300

Ser Ser Ala Val Ile Ser Trp Thr Ile Leu Asp Gly Tyr Ser Ile Ser

305 310 315 320

Ser Ile Thr Ile Arg Tyr Lys Val Gln Gly Lys Asn Glu Asp Gln His

325 330 335

Val Asp Val Lys Ile Lys Asn Ala Thr Ile Thr Gln Tyr Gln Leu Lys

340 345 350

Gly Leu Glu Pro Glu Thr Ala Tyr Gln Val Asp Ile Phe Ala Glu Asn

355 360 365

Asn Ile Gly Ser Ser Asn Pro Ala Phe Ser His Glu Leu Val Thr Leu

370 375 380

Pro Glu Ser Gln Ala Pro

385 390

<210> 20

<211> 1170

<212> DNA

<213> Artificial sequence

<220>

<223> Human Tie2 Ig3-Fn3 349T-738P

<400> 20

accccaaaga tagtggattt gccagatcat atagaagtaa acagtggtaa atttaatccc 60

atttgcaaag cttctggctg gccgctacct actaatgaag aaatgaccct ggtgaagccg 120

gatgggacag tgctccatcc aaaagacttt aaccatacgg atcatttctc agtagccata 180

ttcaccatcc accggatcct cccccctgac tcaggagttt gggtctgcag tgtgaacaca 240

gtggctggga tggtggaaaa gcccttcaac atttctgtta aagttcttcc aaagcccctg 300

aatgccccaa acgtgattga cactggacat aactttgctg tcatcaacat cagctctgag 360

ccttactttg gggatggacc aatcaaatcc aagaagcttc tatacaaacc cgttaatcac 420

tatgaggctt ggcaacatat tcaagtgaca aatgagattg ttacactcaa ctatttggaa 480

cctcggacag aatatgaact ctgtgtgcaa ctggtccgtc gtggagaggg tggggaaggg 540

catcctggac ctgtgagacg cttcacaaca gcttctatcg gactccctcc tccaagaggt 600

ctaaatctcc tgcctaaaag tcagaccact ctaaatttga cctggcaacc aatatttcca 660

agctcggaag atgactttta tgttgaagtg gagagaaggt ctgtgcaaaa aagtgatcag 720

cagaatatta aagttccagg caacttgact tcggtgctac ttaacaactt acatcccagg 780

gagcagtacg tggtccgagc tagagtcaac accaaggccc agggggaatg gagtgaagat 840

ctcactgctt ggacccttag tgacattctt cctcctcaac cagaaaacat caagatttcc 900

aacattacac actcctcggc tgtgatttct tggacaatat tggatggcta ttctatttct 960

tctattacta tccgttacaa ggttcaaggc aagaatgaag accagcacgt tgatgtgaag 1020

ataaagaatg ccaccatcac tcagtatcag ctcaagggcc tagagcctga aacagcatac 1080

caggtggaca tttttgcaga gaacaacata gggtcaagca acccagcctt ttctcatgaa 1140

ctggtgaccc tcccagaatc tcaagcacca 1170

<210> 21

<211> 195

<212> PRT

<213> Artificial sequence

<220>

<223> Human Tie2 Fn2-3 541I-735S

<400> 21

Ile Gly Leu Pro Pro Pro Arg Gly Leu Asn Leu Leu Pro Lys Ser Gln

1 5 10 15

Thr Thr Leu Asn Leu Thr Trp Gln Pro Ile Phe Pro Ser Ser Glu Asp

20 25 30

Asp Phe Tyr Val Glu Val Glu Arg Arg Ser Val Gln Lys Ser Asp Gln

35 40 45

Gln Asn Ile Lys Val Pro Gly Asn Leu Thr Ser Val Leu Leu Asn Asn

50 55 60

Leu His Pro Arg Glu Gln Tyr Val Val Arg Ala Arg Val Asn Thr Lys

65 70 75 80

Ala Gln Gly Glu Trp Ser Glu Asp Leu Thr Ala Trp Thr Leu Ser Asp

85 90 95

Ile Leu Pro Pro Gln Pro Glu Asn Ile Lys Ile Ser Asn Ile Thr His

100 105 110

Ser Ser Ala Val Ile Ser Trp Thr Ile Leu Asp Gly Tyr Ser Ile Ser

115 120 125

Ser Ile Thr Ile Arg Tyr Lys Val Gln Gly Lys Asn Glu Asp Gln His

130 135 140

Val Asp Val Lys Ile Lys Asn Ala Thr Ile Thr Gln Tyr Gln Leu Lys

145 150 155 160

Gly Leu Glu Pro Glu Thr Ala Tyr Gln Val Asp Ile Phe Ala Glu Asn

165 170 175

Asn Ile Gly Ser Ser Asn Pro Ala Phe Ser His Glu Leu Val Thr Leu

180 185 190

Pro Glu Ser

195

<210> 22

<211> 585

<212> DNA

<213> Artificial sequence

<220>

<223> Human Tie2 Fn2-3 541I-735S

<400> 22

atcggactcc ctcctccaag aggtctaaat ctcctgccta aaagtcagac cactctaaat 60

ttgacctggc aaccaatatt tccaagctcg gaagatgact tttatgttga agtggagaga 120

aggtctgtgc aaaaaagtga tcagcagaat attaaagttc caggcaactt gacttcggtg 180

ctacttaaca acttacatcc cagggagcag tacgtggtcc gagctagagt caacaccaag 240

gcccaggggg aatggagtga agatctcact gcttggaccc ttagtgacat tcttcctcct 300

caaccagaaa acatcaagat ttccaacatt acacactcct cggctgtgat ttcttggaca 360

atattggatg gctattctat ttcttctatt actatccgtt acaaggttca aggcaagaat 420

gaagaccagc acgttgatgt gaagataaag aatgccacca tcactcagta tcagctcaag 480

ggcctagagc ctgaaacagc ataccaggtg gacatttttg cagagaacaa catagggtca 540

agcaacccag ccttttctca tgaactggtg accctcccag aatct 585

<210> 23

<211> 330

<212> PRT

<213> Artificial sequence

<220>

<223> Human IgG1 heavy chain constant region

<400> 23

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 24

<211> 990

<212> DNA

<213> Artificial sequence

<220>

<223> Human IgG1 heavy chain constant region

<400> 24

gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60

ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120

tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180

ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240

tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300

aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360

ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420

gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480

tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 540

agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600

gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660

aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag 720

atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840

ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 900

cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960

cagaagagcc tctccctgtc tccgggtaaa 990

<210> 25

<211> 326

<212> PRT

<213> Artificial sequence

<220>

<223> Human IgG2 heavy chain constant region

<400> 25

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro

100 105 110

Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

115 120 125

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

130 135 140

Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly

145 150 155 160

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn

165 170 175

Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp

180 185 190

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro

195 200 205

Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu

210 215 220

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn

225 230 235 240

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

245 250 255

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

260 265 270

Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

275 280 285

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

290 295 300

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

305 310 315 320

Ser Leu Ser Pro Gly Lys

325

<210> 26

<211> 978

<212> DNA

<213> Artificial sequence

<220>

<223> Human IgG2 heavy chain constant region

<400> 26

gctagcacca agggcccatc ggtcttcccc ctggcgccct gctccaggag cacctccgag 60

agcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120

tggaactcag gcgctctgac cagcggcgtg cacaccttcc cagctgtcct acagtcctca 180

ggactctact ccctcagcag cgtggtgacc gtgccctcca gcaacttcgg cacccagacc 240

tacacctgca acgtagatca caagcccagc aacaccaagg tggacaagac agttgagcgc 300

aaatgttgtg tcgagtgccc accgtgccca gcaccacctg tggcaggacc gtcagtcttc 360

ctcttccccc caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacgtgc 420

gtggtggtgg acgtgagcca cgaagacccc gaggtccagt tcaactggta cgtggacggc 480

gtggaggtgc ataatgccaa gacaaagcca cgggaggagc agttcaacag cacgttccgt 540

gtggtcagcg tcctcaccgt tgtgcaccag gactggctga acggcaagga gtacaagtgc 600

aaggtctcca acaaaggcct cccagccccc atcgagaaaa ccatctccaa aaccaaaggg 660

cagccccgag aaccacaggt gtacaccctg cccccatccc gggaggagat gaccaagaac 720

caggtcagcc tgacctgcct ggtcaaaggc ttctacccca gcgacatcgc cgtggagtgg 780

gagagcaatg ggcagccgga gaacaactac aagaccacgc ctcccatgct ggactccgac 840

ggctccttct tcctctacag caagctcacc gtggacaaga gcaggtggca gcaggggaac 900

gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacacgca gaagagcctc 960

tccctgtctc cgggtaaa 978

<210> 27

<211> 327

<212> PRT

<213> Artificial sequence

<220>

<223> Human IgG4 heavy chain constant region

<400> 27

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

130 135 140

Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

210 215 220

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210> 28

<211> 981

<212> DNA

<213> Artificial sequence

<220>

<223> Human IgG4 heavy chain constant region

<400> 28

gctagcacca agggcccatc ggtcttcccc ctggcgccct gctccaggag cacctccgag 60

agcacagccg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120

tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180

ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacgaagacc 240

tacacctgca acgtagatca caagcccagc aacaccaagg tggacaagag agttgagtcc 300

aaatatggtc ccccatgccc atcatgccca gcacctgagt tcctgggggg accatcagtc 360

ttcctgttcc ccccaaaacc caaggacact ctcatgatct cccggacccc tgaggtcacg 420

tgcgtggtgg tggacgtgag ccaggaagac cccgaggtcc agttcaactg gtacgtggat 480

ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagttcaa cagcacgtac 540

cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaacggcaa ggagtacaag 600

tgcaaggtct ccaacaaagg cctcccgtcc tccatcgaga aaaccatctc caaagccaaa 660

gggcagcccc gagagccaca ggtgtacacc ctgcccccat cccaggagga gatgaccaag 720

aaccaggtca gcctgacctg cctggtcaaa ggcttctacc ccagcgacat cgccgtggag 780

tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 840

gacggctcct tcttcctcta cagcaggctc accgtggaca agagcaggtg gcaggagggg 900

aatgtcttct catgctccgt gatgcatgag gctctgcaca accactacac acagaagagc 960

ctctccctgt ctctgggtaa a 981

<210> 29

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> Human kappa light chain constant region

<400> 29

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

1 5 10 15

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

20 25 30

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

35 40 45

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

50 55 60

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

65 70 75 80

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

85 90 95

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 30

<211> 318

<212> DNA

<213> Artificial sequence

<220>

<223> Human kappa light chain constant region

<400> 30

acggtggctg caccatctgt cttcatcttc ccgccatctg atgagcagtt gaaatctgga 60

actgcctctg ttgtgtgcct gctgaataac ttctatccca gagaggccaa agtacagtgg 120

aaggtggata acgccctcca atcgggtaac tcccaggaga gtgtcacaga gcaggacagc 180

aaggacagca cctacagcct cagcagcacc ctgacgctga gcaaagcaga ctacgagaaa 240

cacaaagtct acgcctgcga agtcacccat cagggcctga gctcgcccgt cacaaagagc 300

ttcaacaggg gagagtgc 318

<210> 31

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> Human kappa light chain constant region

<400> 31

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

1 5 10 15

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

20 25 30

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

35 40 45

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

50 55 60

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

65 70 75 80

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

85 90 95

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 32

<211> 318

<212> DNA

<213> Artificial sequence

<220>

<223> Human kappa light chain constant region

<400> 32

acggtggctg caccatctgt cttcatcttc ccgccatctg atgagcagtt gaaatctgga 60

actgcctctg ttgtgtgcct gctgaataac ttctatccca gagaggccaa agtacagtgg 120

aaggtggata acgccctcca atcgggtaac tcccaggaga gtgtcacaga gcaggacagc 180

aaggacagca cctacagcct cagcagcacc ctgacgctga gcaaagcaga ctacgagaaa 240

cacaaagtct acgcctgcga agtcacccat cagggcctga gctcgcccgt cacaaagagc 300

ttcaacaggg gagagtgc 318

<210> 33

<211> 330

<212> PRT

<213> Artificial sequence

<220>

<223> Human IgG1 heavy chain constant region

<400> 33

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 34

<211> 990

<212> DNA

<213> Artificial sequence

<220>

<223> Human IgG1 heavy chain constant region

<400> 34

gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60

ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120

tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180

ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240

tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300

aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360

ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420

gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480

tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 540

agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600

gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660

aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag 720

atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840

ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 900

cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960

cagaagagcc tctccctgtc tccgggtaaa 990

<210> 35

<211> 309

<212> PRT

<213> Artificial sequence

<220>

<223> HTAAB heavy chain constant region

<400> 35

Gly Cys Thr Ala Ala Ala Ala Cys Cys Ala Cys Ala Cys Cys Thr Cys

1 5 10 15

Cys Cys Ala Gly Cys Gly Thr Gly Thr Ala Cys Cys Cys Thr Cys Thr

20 25 30

Gly Gly Cys Cys Cys Cys Ala Gly Gly Ala Thr Cys Ala Gly Cys Ala

35 40 45

Gly Cys Cys Cys Ala Gly Ala Cys Cys Ala Ala Thr Ala Gly Cys Ala

50 55 60

Thr Gly Gly Thr Gly Ala Cys Ala Cys Thr Cys Gly Gly Ala Thr Gly

65 70 75 80

Cys Cys Thr Gly Gly Thr Ala Ala Ala Gly Gly Gly Cys Thr Ala Thr

85 90 95

Thr Thr Cys Cys Cys Thr Gly Ala Gly Cys Cys Thr Gly Thr Gly Ala

100 105 110

Cys Gly Gly Thr Cys Ala Cys Thr Thr Gly Gly Ala Ala Thr Ala Gly

115 120 125

Thr Gly Gly Gly Ala Gly Thr Thr Thr Gly Thr Cys Thr Ala Gly Thr

130 135 140

Gly Gly Thr Gly Thr Thr Cys Ala Cys Ala Cys Ala Thr Thr Thr Cys

145 150 155 160

Cys Cys Gly Cys Thr Gly Thr Cys Cys Thr Gly Cys Ala Gly Ala Gly

165 170 175

Cys Gly Ala Thr Thr Thr Ala Thr Ala Cys Ala Cys Ala Cys Thr Thr

180 185 190

Ala Gly Cys Thr Cys Cys Thr Cys Thr Gly Thr Gly Ala Cys Cys Gly

195 200 205

Thr Gly Cys Cys Ala Thr Cys Cys Thr Cys Thr Cys Cys Thr Ala Gly

210 215 220

Ala Cys Cys Gly Thr Cys Ala Gly Ala Ala Ala Cys Thr Gly Thr Gly

225 230 235 240

Ala Cys Thr Thr Gly Thr Ala Ala Cys Gly Thr Cys Gly Cys Cys Cys

245 250 255

Ala Thr Cys Cys Ala Gly Cys Ala Thr Cys Ala Thr Cys Cys Ala Cys

260 265 270

Cys Ala Ala Ala Gly Thr Thr Gly Ala Cys Ala Ala Gly Ala Ala Gly

275 280 285

Ala Thr Cys Gly Thr Thr Cys Cys Cys Ala Gly Ala Gly Ala Cys Thr

290 295 300

Gly Thr Gly Gly Cys

305

<210> 36

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> HTAAB light chain constant region

<400> 36

cgcgctgatg ccgctcctac cgtctccata tttccgccct cctcagagca gctcacatca 60

ggtggggcgt ccgtggtgtg ctttctgaat aatttctatc cgaaggatat taacgtcaaa 120

tggaaaattg acggctcgga acgtcaaaac ggtgttctga actcctggac cgaccaggat 180

tctaaggata gtacttatag tatgagtagc acattgacac ttactaaaga cgaatatgag 240

cgtcacaata gctacacctg tgaggccacc cataaaacga gcacctcgcc gatcgttaag 300

agctttaatc gcaatgaatg c 321

Claims (14)

1. A Tie2 agonistic antibody or antigen binding fragment thereof, wherein the antibody binds to the Ig3 Fn3 (membrane proximal fibronectin type III) domain of human Tie2, and by binding of the antibody, homodimer Tie2 is formed into a polygonal assembly, and is thus aggregated and activated.

2. The antibody or antigen binding fragment thereof of claim 1, wherein the antibody is a Fab comprising: a heavy chain variable region (VH) comprising the sequence of SEQ ID NO. 1, a heavy chain constant region (CH) comprising the sequence of SEQ ID NO. 3, a light chain variable region (VL) comprising the sequence of SEQ ID NO.2, and a light chain constant region (CL) comprising the sequence of SEQ ID NO. 4.

3. The antibody or antigen-binding fragment thereof of claim 1, comprising:

a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 5 or SEQ ID NO. 7; and

A light chain variable region comprising the amino acid sequence of SEQ ID NO. 6 or SEQ ID NO. 8.

4. The antibody or antigen-binding fragment thereof of claim 3, comprising:

A heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 5 and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 6;

A heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 7 and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 6;

A heavy chain variable region comprising the amino acid sequence of SEQ ID NO.5 and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 8; or (b)

A heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 7 and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 8.

5. A nucleic acid encoding the antibody or antigen-binding fragment thereof of any one of claims 1 to 4.

6. The nucleic acid of claim 5, comprising a sequence selected from the group consisting of SEQ ID NOs 9 to 14.

7. An expression vector comprising the nucleic acid of claim 5.

8. A cell transfected with the expression vector of claim 7.

9. A method of producing a Tie2 agonistic antibody or antigen binding fragment thereof, comprising:

(a) Culturing the cell of claim 8; and

(B) Recovering the antibody or antigen binding fragment thereof from the cultured cells.

10. A composition for preventing or treating an angiogenic disease, comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 as an active ingredient.

11. The composition of claim 10, wherein the angiogenesis-related disease is selected from the group consisting of: cancer, tumor metastasis, diabetic retinopathy, retinopathy of prematurity, corneal graft rejection, macular degeneration, neovascular glaucoma, polycythemia, proliferative retinopathy, psoriasis, hemophilia arthritis, capillary blood vessel formation of atherosclerotic plaques, keloids, wound granulations, vascular adhesions, rheumatoid arthritis, osteoarthritis, autoimmune diseases, crohn's disease, post-operative restenosis, atherosclerosis, intestinal adhesions, cat scratch, ulcers, liver cirrhosis, nephritis, diabetic nephropathy, diabetes mellitus, inflammatory diseases and neurodegenerative diseases.

12. The composition of claim 11, wherein the cancer is selected from the group consisting of: esophageal cancer, gastric cancer, colorectal cancer, rectal cancer, oral cancer, pharyngeal cancer, laryngeal cancer, lung cancer, colon cancer, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, prostate cancer, testicular cancer, bladder cancer, renal cancer, liver cancer, pancreatic cancer, bone cancer, connective tissue cancer, skin cancer, brain cancer, thyroid cancer, leukemia, hodgkin's lymphoma, and multiple bone marrow-blood cancers.

13. A composition for co-administration with an additional therapeutic agent for angiogenic disease comprising an antibody or antigen binding fragment thereof according to any one of claims 1 to 4.

14. An antibody polygonal assembly comprising a Tie2 agonistic antibody or antigen binding fragment thereof and forming a homodimeric Tie2 by binding of said antibody to the Ig3 Fn3 (membrane proximal fibronectin type III) domain of human Tie 2.