# Patent

## Title
Antibody specifically binding to ANG2 and use thereof

## Abstract

An Ang2 specific antibody, a method of preventing and/or treating a disease related to the activation and/or overproduction (overexpression) of Ang2 by administering the antibody to a subject, and a method of screening a candidate substance for diagnosing, preventing, or treating a disease related to activation or overproduction of Ang2.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of Korean Patent Application No. 10-2014-0009815 filed on Jan. 27, 2014 in the Korean Intellectual Property Office, the entire disclosure of which is hereby incorporated by reference.
INCORPORATION BY REFERENCE OF ELECTRONICALLY SUBMITTED MATERIALS
Incorporated by reference in its entirety herein is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: One 25,877 Byte ASCII (Text) file named “719241 ST25.TXT-Revised” created on Jan. 26, 2015.
BACKGROUND OF THE INVENTION
1. Field
Provided is an antibody specifically binding to an angiogenesis-inducing factor angiopoietin-2 (Ang2) to inhibit the function thereof, and a use thereof. In particular, provided are an Ang2 specific antibody, a method of preventing and/or treating a disease related to the activation and/or overproduction (overexpression) of Ang2, including administering the antibody to a subject in need thereof, and a method of screening a candidate substance for diagnosing, preventing, or treating a disease related to activation or overproduction of Ang2.
2. Description of the Related Art
Angiogenesis refers to a mechanism through which a new blood vessel is formed from a pre-existing blood vessel, and has been known to play an essential role in the formation of organs, normal physiological growth, wound healing and so on. Also, abnormal angiogenesis has been known to play a crucial role in diseases or symptoms, such as tumor growth and metastasis, age-related macular degeneration, diabetic retinopathy, psoriasis, rheumatoid arthritis, chronic inflammation, and the like.
Angiogenesis has been known to play an important role in tumor growth and metastasis, and various intensive research into angiogenesis mechanisms for developing new cancer therapies has been going on by developed countries and multinational pharmaceutical companies. One of the proteins that has been a target of such research is Angiopoietin, which has been known to be involved in blood vessel development and angiogenesis after birth. Known are Ang-1, 2, 3 and 4.
Among them, angiogenesis process related to Angiopoietin-2 (Ang2) in a cancer tissue is thought to be as follows. First, for angiogenesis in the cancer tissue, cooption occurs, wherein cancer cells select pre-existing blood vessels to form new blood vessels in a cancer tissue. Thereafter, blood vessel regression occurs, during which the functions of the pre-existing blood vessels are destroyed by Ang2 pathway. The regression of the pre-existing vessels causes hypoxic environment within the cancer tissue, which is an environment where the formation of new blood vessels is possible. Under such conditions, the expression of vascular endothelial cell growth factor (VEGF) is increased, and new blood vessels are thus formed. For such a reason, Ang2 is one of the important targets in the development of angiogenesis inhibitors, and various kinds of angiogenesis inhibitors are currently being developed and actively undergoing preclinical or clinical trials.
As Ang2 is of increasing importance as a target for developing an angiogenesis inhibitor, there is a need of developing an effective and strong Ang2 targeting substance.
BRIEF SUMMARY OF THE INVENTION
One embodiment provides an anti-Ang2 antibody or an antigen-binding fragment thereof, which specifically binds to Ang2.
Another embodiment provides a pharmaceutical composition including the anti-Ang2 antibody or an antigen-binding fragment thereof as an active ingredient.
Another embodiment provides a method of inhibiting angiogenesis, including administering the anti-Ang2 antibody or an antigen-binding fragment thereof to a subject.
Another embodiment provides a method of preventing and/or treating a disease related to activation or overproduction (overexpression) of Ang2, including administering the anti-Ang2 antibody or an antigen-binding fragment thereof to a subject.
Another embodiment provides a method of screening a substance for utility in diagnosing, preventing, or treating a disease related to activation or overproduction of Ang2.
Still another embodiment provides a novel polypeptide comprising an antigen binding site for an anti-Ang2 antibody.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic diagram showing an Ang2 antibody binding region (epitope) on Ang2.
FIG. 2 is an image of an immunoblot showing the degree of Tie2 phosphorylation by administering an anti-Ang2 antibody.
FIG. 3 is a graph showing the degree of vascular endothelial cell migration during administration of an anti-Ang2 antibody.
FIG. 4 is a graph showing the total length of vascular endothelial cell tube formation during administration of an anti-Ang2 antibody.
FIG. 5A is a graph showing a volume change of colorectal cancer cells by administering an anti-Ang2 antibody according to the days of administration (x axis: day after grouping).
FIG. 5B is a graph showing a volume change of colorectal cancer cells at 18th day of administering an anti-Ang2 antibody according to the kind of antibodies administered.



DETAILED DESCRIPTION OF THE INVENTION
Described is an antibody inhibiting the functions of an angiogenesis-inducing factor angiopoietin-2 (Ang2), and particularly, an antibody useful for diagnosing and/or treating a disease associated with the activation and/or overproduction (overexpression) of Ang2 and suppressing angiogenesis in a cancer tissue, by hindering an angiogenesis inducing factor, Ang2, which is essential for the growth of cancer cells in the cancer tissue, from binding to its intracellular receptor, Tie2 receptor.
Ang2 protein is closely related to angiogenesis. It is a soluble ligand present in the blood, and it is a therapeutic target protein which attracts attention for new drug development. Provided is an anti-Ang2 antibody or an antigen-binding fragment thereof, capable of recognizing and binding to Ang2 (e.g., a human Ang2) protein that is widely involved in angiogenesis, metastasis, cancer cell invasion, and the like, thereby preventing Ang2 from binding to its receptor, Tie2 receptor. Therefore, the anti-Ang2 antibody capable of suppressing angiogenesis, cancer incidence, and cancer cell metastasis, and can be used for diagnosis and/or treatment of a disease associated with the activation and/or overproduction (overexpression) of Ang2.
The term “antibody” as used herein refers to a substance generated by the stimulus of an antigen in immune system, and is not particularly limited to specific kinds. The antibody in the invention includes any animal antibodies, chimeric antibodies, humanized antibodies or fully human antibodies. Furthermore, the antibody in the invention also includes antigen-binding fragments of an antibody which possess an antigen binding potential. The term “antibody” is used broadly to encompass antigen-binding antibody fragments unless otherwise noted. Meanwhile, complementarity-determining regions (CDR) as used herein refer to regions which contribute specificity for antigen binding, as part of the variable regions of an antibody. The antigen-binding fragments of an antibody may be antibody fragments including at least one complementarity-determining region.
In connection with angiogenesis process in a cancer tissue, cooption occurs, wherein cancer cells select pre-existing blood vessels to form new blood vessels in a cancer tissue. Thereafter, blood vessel regression occurs, during which the functions of the pre-existing blood vessels are destroyed by Ang2 pathway. The regression of the pre-existing vessels causes hypoxic environment within the cancer tissue, which is an environment where the formation of new blood vessels is possible. Under such conditions, the expression of vascular endothelial cell growth factor (VEGF) is increased, and new blood vessels are thus formed. As angiopoietin proteins, Ang1, Ang2, Ang3, and Ang4, are known and of them, Ang2 is also known as ANGPT2 and is expressed in blood vessel remodeling areas.
Ang2, which becomes a target of an antibody to be provided in one embodiment, is closely related to angiogenesis, is a soluble ligand present in blood, and it is widely involved in angiogenesis, metastasis, and cancer cell invasion. The Ang2 may be derived (originated) from mammals including primates such as humans and monkeys and rodents such as rats and mice and for example, it may be selected from the group consisting of a human Ang2 (e.g., NCBI Accession #015123), a monkey Ang2 (e.g., NCBI Accession No. Q8MIK6 etc.), a mouse Ang2 (NCBI Accession # NP_031452, Accession #O35608, etc.), a rat Ang2 (e.g., NCBI Accession No. O35462, etc.), and any combination thereof.
Presently, antibodies are widely used for treating diseases. As antibodies are very stable in vivo as well as in vitro and have a long half-life, they are favorable for mass expression and production. Also, since an antibody has intrinsically a dimer structure, it has a fairly high avidity.
In one embodiment, the antibody has an activity of binding to Ang2 and an effect of suppressing angiogenesis in a cancer tissue by hindering Ang2 from binding to its intracellular receptor, Tie2 receptor. Specifically, the antibody may inhibit binding between Ang2 and Tie2 receptor by competing with Tie2 in binding to Ang2 and for example, it may inhibit binding between Ang2 and Tie2 receptor by recognizing and/or binding to a binding site of Ang2 for binding to Tie2 receptor.
An embodiment provides an anti-Ang2 antibody or an antigen-binding fragment thereof. The anti-Ang2 antibody may recognize all or a part (for example, at least one amino acid selected from the group consisting of the amino acid residue regions exposed to the outside of each loop) of the regions consisting of loop 3 (a region covering from 460th to 468th amino acids of SEQ ID NO: 27) of human Ang2 (hAng2; SEQ ID NO: 27; Accession #O15123), or an amino acid sequence region including about 2 to about 20, about 2 to about 15, about 2 to about 10, or about 2 to about 5 consecutive amino acids including at least one exposed amino acid residue, which is exposed to the outside of loop 3 of SEQ ID NO: 27, as an epitope, or specifically bind to the region. An “exposed” amino acid refers to an amino acid that is exposed to solution and available for binding when a protein (e.g., Ang2) is in its native conformation in a biological medium or other solution under physiological conditions (e.g., physiological pH, isotonicity, temperature, etc.).
For example, the amino acid residue exposed to the outside of loop 3 of SEQ ID NO: 27 may be at least one selected from the group consisting of Y460 and K468 (which are marked in bold and italic letters in the following sequence).









Ang2



(SEQ ID NO: 27)



MWQIVFFTLS CDLVLAAAYN NFRKSMDSIG KKQYQVQHGS



 



CSYTFLLPEM



 



DNCRSSSSPY VSNAVQRDAP LEYDDSVQRL QVLENIMENN



 



TQWLMKLENY



 



IQDNMKKEMV EIQQNAVQNQ TAVMIEIGTN LLNQTAEQTR



 



KLTDVEAQVL



 



NQTTRLELQL LEHSLSTNKL EKQILDQTSE INKLQDKNSF



 



LEKKVLAMED KHIIQLQSIK EEKDQLQVLV SKQNSIIEEL



 



EKKIVTATVN NSVLQKQQHD LMETVNNLLT MMSTSNSAKD



 



PTVAKEEQIS FRDCAEVFKS GHTTNGIYTL TFPNSTEEIK



 



AYCDMEAGGG GWTIIQRRED GSVDFQRTWK EYKVGFGNPS



 



GEYWLGNEFV SQLTNQQRYV LKIHLKDWEG NEAYSLYEHF



 



YLSSEELNYR



 



IHLKGLTGTA GKISSISQPG NDFSTKDGDN DKCICKCSQM



 



LTGGWWFDAC GPSNLNGMY FN GIKWYYWKGS



 



GYSLKATTMM IRPADF






For example, the anti-Ang2 antibody may recognize at least one amino acid residue selected from the group consisting of Y460 and K468 positioned at loop 3 of human Ang2 of SEQ ID NO: 27, or an amino acid sequence region including about 2 to about 20, about 2 to about 15, about 2 to about 10, or about 2 to about 5 consecutive amino acids including the above at least one amino acid residue, as an epitope, and/or specifically bind thereto.
The above epitope sites are exposed amino acid residues positioned at loop 3 of the three dimensional structure of Ang2, and they directly participate in binding with a Tie2 receptor or they are positioned within the binding site with the Tie2 receptor or neighboring thereupon (see FIG. 1). Accordingly, the anti-Ang2 antibody or an antigen-binding fragment thereof recognizing and binding to the above at least one epitope competes with the Tie2 receptor to bind to Ang2 and thus, inhibits binding between Ang2 and the Tie2 receptor.
The term “consecutive amino acid” may refer to amino acids which are adjacent to one another on the secondary or tertiary structure of a protein as well as amino acids which are continuous on their primary amino acid sequences. Accordingly, the “consecutive amino acid residues” as used herein may refer to contiguous amino acid residues on the primary, secondary, or tertiary structure of a protein.
In addition, not only an anti-Ang2 antibody recognizing and/or specifically binding to the epitope sites, but also an antibody or an antigen-binding fragment thereof which competes with the anti-Ang2 antibody for binding to Ang2 can compete with the Tie2 receptor to bind to Ang2. Therefore, the antibody (competitively-binding antibody) or an antigen-binding fragment thereof which competes with the anti-Ang2 antibody for binding to Ang2 can inhibit binding between Ang2 and the Tie2 receptor. This competitively-binding antibody may be an antibody recognizing a site adjacent to the aforementioned epitopes on its three dimensional structure as an epitope. The competitively-binding antibody may have an Ang2 binding affinity (Kd) of about 10 nM or less, for example, about 1 pM to about 10 nM, about 10 pM to 10 nM, or about 100 pM to about 10 nM.
Therefore, the anti-Ang2 antibody or an antigen-binding fragment thereof may be at least one selected from the group consisting of an antibody or an antigen-binding fragment thereof recognizing and/or specifically binding to the aforementioned epitope, and an antibody competing therewith for binding to Ang2 or an antigen-binding fragment thereof.
In a particular embodiment, the anti-Ang2 antibody or an antigen-binding fragment thereof may comprise, as a heavy chain complementarity determining region (CDR), at least one selected from the group consisting of a polypeptide (CDR-H1) comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, a polypeptide (CDR-H2) comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, and a polypeptide (CDR-H3) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12.
The anti-Ang2 antibody or an antigen-binding fragment thereof may comprise, as a light chain complementarity determining region (CDR), at least one selected from the group consisting of a polypeptide (CDR-L1) comprising the amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16, a polypeptide (CDR-L2) comprising the amino acid sequence of General Formula 1 (SEQ ID NO: 25), and a polypeptide (CDR-L3) comprising an amino acid sequence of General Formula 2 (SEQ ID NO: 26):







[General Formula 1]









(SEQ ID NO: 25)



X1-X2-S-X3-X4-X5-X6






wherein X1 is arginine(R) or tyrosine(Y),
X2 is alanine(A) or threonine(T) (for example, alanine(A)),
X3 is asparagine(N), arginine(R), or serine(S) (for example, asparagine(N)),
X4 is leucine(L) or arginine(R),
X5 is aspartic acid(D), histidine(H), or tyrosine(Y) (for example, aspartic acid(D), or tyrosine(Y)), and
X6 is serine(S) or proline(P);







[General Formula 2]









(SEQ ID NO: 26)



Q-Q-X7-X8-X9-X10-P-X11-T






wherein X7 is serine(S), glycine(G), aspartic acid(D), or tyrosine(Y) (for example, serine(S) or aspartic acid(D)),
X8 is asparagine(N), tyrosine(Y), or serine(S) (for example, asparagine(N) or tyrosine(Y)),
X9 is glutamic acid(E), threonine(T), or lysine(K) (for example, glutamic acid(E) or threonine(T)),
X10 is aspartic acid(D), serine(S), or leucine(L) (for example, aspartic acid(D) or serine(S)), and
X11 is leucine(L), tryptophan(W), or tyrosine(Y) (for example, leucine(L) or tryptophan(W)).
In a specific embodiment, the anti-Ang2 antibody or an antigen-binding fragment thereof may comprise or consist essentially of the above heavy chain complementarity determining region, light chain complementarity determining region, or combination thereof.
In particular, the anti-Ang2 antibody or an antigen-binding fragment thereof may comprise or consist essentially of:
at least one heavy chain complementarity determining region selected from the group consisting of a polypeptide (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, a polypeptide (CDR-H2) comprising the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6, and a polypeptide (CDR-H3) comprising an amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10, or a heavy chain variable region comprising the at least one heavy chain complementarity determining region;
at least one light chain complementarity determining region selected from the group consisting of a polypeptide (CDR-L1) comprising the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14, a polypeptide (CDR-L2) comprising the amino acid sequence of SEQ ID NO: 25, and a polypeptide (CDR-L3) comprising the amino acid sequence of SEQ ID NO: 26, or a light chain variable region comprising the at least one light chain complementarity determining region;
a combination of the at least one heavy chain complementarity determining region and the at least one light chain complementarity determining region; or
a combination of the heavy chain variable region and the light chain variable region.
More particularly, the anti-Ang2 antibody or an antigen-binding fragment thereof may comprise or consist essentially of:
at least one heavy chain complementarity determining region selected from the group consisting of a polypeptide (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, a polypeptide (CDR-H2) comprising the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6, and a polypeptide (CDR-H3) comprising an amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10, or a heavy chain variable region comprising the at least one heavy chain complementarity determining region;
at least one light chain complementarity determining region selected from the group consisting of a polypeptide (CDR-L1) comprising the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14, a polypeptide (CDR-L2) comprising the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 18, and a polypeptide (CDR-L3) comprising the amino acid sequence of SEQ ID NO: 21 or SEQ ID NO: 22, or a light chain variable region comprising the at least one light chain complementarity determining region;
a combination of the at least one heavy chain complementarity determining region and the at least one light chain complementarity determining region; or
a combination of the heavy chain variable region and the light chain variable region.
In one embodiment, the heavy chain variable region may comprise or consist essentially of the amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 29, and the light chain variable region may comprise or consist essentially of the amino acid sequence of SEQ ID NO: 36 or SEQ ID NO: 37.
The anti-Ang2 antibody or an antigen-binding fragment thereof may not be one consisting of at least one selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 18, and SEQ ID NO: 22, or may not be one consisting of the amino acid sequence of SEQ ID NO: 37 only.
In another embodiment, the anti-Ang2 antibody or an antigen-binding fragment thereof may comprise or consist essentially of:
at least one heavy chain complementarity determining region selected from the group consisting of a polypeptide (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 3, a polypeptide (CDR-H2) comprising the amino acid sequence of SEQ ID NO: 7, and a polypeptide (CDR-H3) comprising an amino acid sequence of SEQ ID NO: 11, or a heavy chain variable region comprising the at least one heavy chain complementarity determining region;
at least one light chain complementarity determining region selected from the group consisting of a polypeptide (CDR-L1) comprising the amino acid sequence of SEQ ID NO: 15, a polypeptide (CDR-L2) comprising the amino acid sequence of SEQ ID NO: 19, and a polypeptide (CDR-L3) comprising the amino acid sequence of SEQ ID NO: 23, or a light chain variable region comprising the at least one light chain complementarity determining region;
a combination of the at least one heavy chain complementarity determining region and the at least one light chain complementarity determining region; or
a combination of the heavy chain variable region and the light chain variable region.
In one embodiment, the heavy chain variable region may comprise or consist essentially of the amino acid sequence of SEQ ID NO: 30, and the light chain variable region may comprise or consist essentially of the amino acid sequence of SEQ ID NO: 38.
In another embodiment, the anti-Ang2 antibody or an antigen-binding fragment thereof may comprise or consist essentially of:
at least one heavy chain complementarity determining region selected from the group consisting of a polypeptide (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 4, a polypeptide (CDR-H2) comprising the amino acid sequence of SEQ ID NO: 8, and a polypeptide (CDR-H3) comprising an amino acid sequence of SEQ ID NO: 12, or a heavy chain variable region comprising the at least one heavy chain complementarity determining region;
at least one light chain complementarity determining region selected from the group consisting of a polypeptide (CDR-L1) comprising the amino acid sequence of SEQ ID NO: 16, a polypeptide (CDR-L2) comprising the amino acid sequence of SEQ ID NO: 20, and a polypeptide (CDR-L3) comprising the amino acid sequence of SEQ ID NO: 24, or a light chain variable region comprising the at least one light chain complementarity determining region;
a combination of the at least one heavy chain complementarity determining region and the at least one light chain complementarity determining region; or
a combination of the heavy chain variable region and the light chain variable region.
In one embodiment, the heavy chain variable region may comprise or consist essentially of the amino acid sequence of SEQ ID NO: 31, and the light chain variable region may comprise or consist essentially of the amino acid sequence of SEQ ID NO: 39.
The heavy chain CDR of the anti-Ang2 antibody may comprise amino acid sequences, for example, as set forth in the following Table 1.







TABLE 1







Amino acid sequence of heavy chain CDR









CDRH1-KABAT
CDRH2-KABAT
CDRH3-KABAT





SYWLE
EILPGSGSTNYNEKFRG
GNHNSYYYAMDY


(SEQ ID NO: 1)
(SEQ ID NO: 5)
(SEQ ID NO: 9)


 


DYYMK
EINPKNGDTFYNQIFKG
ENDYDVGFFDY


(SEQ ID NO: 2)
(SEQ ID NO: 6)
(SEQ ID NO: 10)


 


NYGMN
WINTYTGEPTYADDFKG
DHDGYLMDY


(SEQ ID NO: 3)
(SEQ ID NO: 7)
(SEQ ID NO: 11)


 


DPYIH
RIDPANGNTKYDPKFQG
RWDGGGFDY


(SEQ ID NO: 4)
(SEQ ID NO: 8)
(SEQ ID NO: 12)









The light chain CDR of the anti-Ang2 antibody may comprise amino acid sequences, for example, as set forth in the following Table 2.







TABLE 2







Amino acid sequence of light chain CDR









CDRL1-KABAT
CDRL2-KABAT
CDRL3-KABAT





RASESVDSYGNSFMH
RASNLDS (SEQ ID
QQSNEDPLT (SEQ ID


(SEQ ID NO: 13)
NO: 17)
NO: 21)


 


KASQSVSNDVA (SEQ
YASNRYP (SEQ
QQDYTSPWT


ID NO: 14)
ID NO: 18)
(SEQ ID NO: 22)


 


STSQGISNYLN
YTSSLHS
QQYSKLPYT


(SEQ ID NO: 15)
(SEQ ID NO: 19)
(SEQ ID NO: 23)


 


RASQDISNYLN
YTSRLHS
QQGNTLPWT (SEQ


(SEQ ID NO: 16)
(SEQ ID NO: 20)
ID NO: 24)









An animal-derived antibody which is produced by immunizing an animal with a desired antigen may generally trigger an immune rejection response when administered to humans for treatment purpose, and a chimeric antibody has been developed to suppress such immune rejection response. A chimeric antibody is formed by replacing the constant region of an animal-derived antibody, which is a cause of anti-isotype response, with the constant region of a human antibody using genetic engineering methods. The chimeric antibody has considerably improved anti-isotype response in comparison with animal-derived antibodies, but animal-derived amino acids are still present in its variable regions and thus it still contains potential side effects resulting from an anti-idiotypic response. It is a humanized antibody that has been thus developed to improve such side effects. This is manufactured by grafting CDR (complementarity determining regions) which, of the variable regions of a chimeric antibody, has an important role in antigen binding into a human antibody framework.
An important consideration in CDR grafting technology for manufacturing a humanized antibody is to select an optimized human antibody which can optimally incorporate the CDR of an animal-derived antibody and for this, utilization of antibody database, analysis of crystal structure, molecule modeling technology, etc. are employed. However, although the CDR of an animal-derived antibody is grafted into an optimized human antibody framework, there are a considerable number of cases where antigen binding affinity is not preserved because there are amino acids which affect antigen binding while being positioned at the framework of the animal-derived antibody. In this regard, it may be essential to apply an additional antibody engineering technology for restoring antigen binding affinity.
According to one embodiment, the antibody may be a mouse-derived antibody, a mouse-human chimeric antibody, a humanized antibody, or a human antibody. The antibody or antigen-binding fragment thereof may be isolated from a living body or non-naturally occurring. The antibody or antigen-binding fragment thereof may be recombinant or synthetic. The antibody or antigen-binding fragment thereof may be monoclonal.
An intact antibody has a structure with two full-length light chains and two full-length heavy chains, and each light chain is linked to each heavy chain via a disulfide bond. The constant region of an antibody is divided into a heavy chain constant region and a light chain constant region, and the heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ) and epsilon (ε) types, and has gamma1 (γ1), gamma2 (γ2), gamma3 (γ3), gamma4 (γ4), alpha1 (α1) and alpha2 (α2) as its subclass. The light chain constant region has kappa (κ) and lambda (λ) types.
The term “heavy chain” is understood to include a full-length heavy chain and fragments thereof, the full-length heavy chain including a variable region domain VH including an amino acid sequence having sufficient variable region sequences that contribute the specificity for antigen binding and three constant region domains CH1, CH2 and CH3 domains and a hinge. The term “light chain” is understood to include a full-length light chain and fragments thereof, the full-length light chain including a variable region domain VL including an amino acid sequence having sufficient variable region sequences that contribute to the specificity for antigen binding and a constant region domain CL.
The term “CDR (complementarity determining region)” refers to an amino acid sequence found in the hypervariable region of a heavy chain and a light chain of an immunoglobulin. The heavy and light chain may each include three CDRs (CDRH1, CDRH2, CDRH3, and CDRL1, CDRL2, CDRL3). The CDRs of an antibody can provide an essential contact residue for binding to an antigen or an epitope. Throughout the specification, the terms “specifically binding” or “specifically recognizing” has the same meaning as generally known to an ordinary person in the art, indicating that an antigen and an antibody specifically interact with each other to lead to an immunological response.
The light chain constant region and the heavy chain constant region except the aforementioned CDR regions or light chain variable regions and heavy chain variable regions of the anti-Ang2 antibody may be light chain constant regions and heavy chain constant regions of any subtypes of an immunoglobulin, for example, light chain constant regions and heavy chain constant regions of IgA, IgD, IgE, IgG (e.g., IgG1, IgG2, IgG 3, and IgG4), IgM, etc.
In an embodiment, the antigen-binding fragment may at least one be selected from the group consisting of an scFv, an (scFv)2, an scFv-Fc, an Fab, an Fab′ and an F(ab′)2.
The term “antigen-binding fragment,” which is a fragment of the full structure of an immunoglobulin, refers to some of a polypeptide including a portion to which an antigen can bind. For example, it may be an scFv, an (scFv)2, an Fab, an Fab′, or an F(ab′)2, but is not limited thereto. Among the antigen-binding fragments, an Fab, which is a structure having the light chain and heavy chain variable regions, the light chain constant region, and the heavy chain first constant region (CH1), has one antigen binding site.
An Fab′ differs from the Fab in that the Fab′ has a hinge region including at least one cysteine residue at the C-terminal of the heavy chain CH1 domain.
An F(ab′)2 antibody is produced when cysteine residues at the hinge region of the Fab′ are joined by a disulfide bond. An Fv is a minimal antibody fragment, having only heavy chain variable regions and light chain variable regions, and a recombinant technique for producing the Fv fragment is well known in the art.
A two-chain Fv may have a structure in which heavy chain variable regions are linked to light chain variable regions by a non-covalent bond, and a single-chain Fv may generally form a dimer structure as in the two-chain Fv, wherein heavy chain variable regions are covalently bound to light chain variable regions via a peptide linker or the heavy and light chain variable regions are directly linked to each other at the C-terminals thereof. The peptide linker may include 1 to 100 or 2 to 50 amino acids, and proper sequences thereof have been known in the art.
The antigen-binding fragment may be obtained using a protease (for example, a whole antibody can be digested with papain to obtain Fab fragments, or can be digested with pepsin to obtain F(ab′)2 fragments), or may be prepared by a genetic recombinant technique.
The term “hinge region” refers to a region included in the heavy chains of an antibody, which is present between the CH1 and CH2 regions, and provides flexibility to the antigen binding site in the antibody.
When an animal-derived antibody goes through a chimerization process, an animal-derived IgG1 hinge is replaced with a human IgG1 hinge, but a length of the animal-derived IgG1 hinge is shorter than the human IgG1 hinge, and disulfide bonds between two heavy chains are reduced from 3 to 2. Thus, rigidity of the hinges may have different effects. Therefore, modification of a hinge region can increase an antigen binding efficiency of a humanized antibody. Methods of deleting, inserting, or substituting an amino acid for modifying amino acid sequences of the hinge region are well known in the art.
The anti-Ang2 antibody may be a monoclonal antibody. The monoclonal antibody may be prepared by methods well known in the art. For example, it may be prepared using a phage display technique. Alternatively, the anti-Ang2 antibody may also be prepared as a mouse-derived monoclonal antibody using the method of Schwaber, et al. (Schwaber, J and Cohen, E. P., “Human×Mouse Somatic Cell Hybrid Clones Secreting Immunoglobulins of Both Parental Types,” Nature, 244 (1973), 444-447).
Meanwhile, individual monoclonal antibodies may be screened using a typical ELISA (Enzyme-Linked ImmunoSorbent Assay) format, based on the binding potential with Ang2 Inhibitory activities can be verified through functional analysis such as competitive ELISA for verifying the molecular interaction of binding assemblies or functional analysis such as a cell-based assay. Then, with regard to monoclonal antibody members selected on the basis of their strong inhibitory activities, their affinities (Kd values) to Ang2 may each be verified.
Finally selected antibodies may be subjected to humanization as well as prepared as an antibody where the portions not including the antigen-binding regions are derived from a human immunoglobulin antibody. The method of humanization is generally known to the relevant art (Almagro, J. C. and Fransson, J., “Humanization of antibodies,” Frontiers in Bioscience, 13(2008), 1619-1633).
Another embodiment provides a hybridoma which produces the antibody. The hybridoma may be at least one selected from the group consisting of deposit numbers KCLRF-BP-00305, KCLRF-BP-00306, KCLRF-BP-00307, and KCLRF-BP-00308.
Another embodiment provides a pharmaceutical composition comprising the anti-Ang2 antibody or an antigen-binding fragment thereof. In one embodiment, the pharmaceutical composition may be used for inhibiting angiogenesis. In another embodiment, the pharmaceutical composition may be used for preventing and/or treating a disease associated with activation and/or overexpression of Ang2.
Another embodiment provides a method of inhibiting angiogenesis comprising administering the anti-Ang2 antibody or an antigen-binding fragment thereof to a subject in need of inhibiting angiogenesis. The method of inhibiting angiogenesis may further comprise a step of identifying the subject who is in need of angiogenesis inhibition, before the step of administering. Another embodiment provides a method of preventing and/or treating a disease associated with Ang2 activation and/or overexpression, comprising administering the anti-Ang2 antibody or an antigen-binding fragment thereof to a subject in need of preventing and/or treating the disease. The method of preventing and/or treating a disease associated with Ang2 activation and/or overexpression may further comprise a step of identifying the subject who is in need of preventing and/or treating a disease associated with Ang2 activation and/or overexpression, before the step of administering. In the above methods, the anti-Ang2 antibody or an antigen-binding fragment thereof may be administered in a pharmaceutically effective amount.
The pharmaceutical composition may further include a pharmaceutically acceptable carrier, and the carrier may be those commonly used in the formulation of drugs, which may be one or more selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginates, gelatin, calcium silicate, micro-crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, and mineral oil, but is not limited thereto. The pharmaceutical composition may further include one or more selected from the group consisting of a diluent, an excipient, a lubricant, a wetting agent, a sweetener, a flavor enhancer, an emulsifying agent, a suspension agent, and a preservative.
The pharmaceutical composition may be administered orally or parenterally. The parenteral administration may include intravenous injection, subcutaneous injection, muscular injection, intraperitoneal injection, endothelial administration, local administration, intranasal administration, intrapulmonary administration, and rectal administration. Since oral administration leads to digestion of proteins or peptides, an active ingredient in the compositions for oral administration must be coated or formulated to prevent digestion in stomach. In addition, the composition may be administered using an optional device that enables an active substance to be delivered to target cells.
The content of the anti-Ang2 antibody or an antigen-binding fragment thereof in the pharmaceutical composition may be prescribed in a variety of ways, depending on factors such as formulation methods, administration methods, age of subjects, body weight, gender, pathologic conditions, diets, administration time, administration interval, administration route, excretion speed, and reaction sensitivity. For example, a daily dosage of the anti-Ang2 antibody or an antigen-binding fragment thereof may be within the range of 0.001 to 1000 mg/kg, 0.01 to 100 mg/kg, 0.1 to 50 mg/kg, or 0.1 to 20 mg/kg, but is not limited thereto. The daily dosage may be formulated into a single formulation in a unit dosage form or formulated in suitably divided dosage forms, or it may be manufactured to be contained in a multiple dosage container. The term “pharmaceutically effective amount” as used herein refers to a content or dose of an active ingredient capable of showing desirable pharmacological effects and it may be determined in a variety of ways, depending on factors such as formulation methods, administration methods, age of subjects, body weight, gender, pathologic conditions, diets, administration time, administration interval, administration route, excretion speed, and reaction sensitivity.
The pharmaceutical composition may be formulated into a form of a solution in oil or an aqueous medium, a suspension, syrup, an emulsifying solution, an extract, powder, granules, a tablet, or a capsule, and may further include a dispersing or a stabilizing agent for the formulation.
In particular, the pharmaceutical composition comprising the anti-Ang2 antibody or an antigen-binding fragment thereof may be formulated into an immunoliposome since it contains an antibody or an antigen-binding fragment. A liposome containing an antibody may be prepared using any methods widely known in the art. The immunoliposome may be a lipid composition including phosphatidylcholine, cholesterol, and polyethyleneglycol-derivatized phosphatidylethanolamine, and may be prepared by a reverse phase evaporation method. For example, Fab′ fragments of an antibody may be conjugated to the liposome through a disulfide-exchange reaction.
Meanwhile, as the anti-Ang2 antibody or an antigen-binding fragment thereof specifically binds to Ang2, this can be used for detecting Ang2, or the activation and/or overproduction (overexpression) of Ang2.
Accordingly, another embodiment provides a composition for detecting Ang2, wherein the composition comprises the anti-Ang2 antibody or the antigen-binding fragment thereof. Another embodiment provides a method for detecting Ang2 comprising contacting the anti-Ang2 antibody or the antigen-binding fragment thereof with a biological sample; and measuring an antigen-antibody reaction. In this detection method, when the antigen-antibody reaction is detected, it can be determined that Ang2 is present in the biological sample. Another embodiment provides a use of the anti-Ang2 antibody or an antigen-binding fragment thereof for detecting Ang2.
Another embodiment provides a composition for detecting the activation and/or overproduction (overexpression) of Ang2, or a pharmaceutical composition for diagnosing a disease related to the activation and/or overproduction (overexpression) of Ang2, wherein the composition comprises the anti-Ang2 antibody or the antigen-binding fragment thereof. Another embodiment provides a method for detecting the activation and/or overproduction (overexpression) of Ang2, or a method for diagnosing a disease related to the activation and/or overproduction (overexpression) of Ang2, or a method of providing information for diagnosis thereof, wherein the method comprises contacting the anti-Ang2 antibody or the antigen-binding fragment thereof with a biological sample obtained from a subject; and measuring an antigen-antibody reaction. The detection method, the diagnose method, or the method of providing information for diagnosis may further comprise a step of determining the subject to have Ang2 activation and/or overproduction (overexpression) symptoms or to have a disease related to Ang2 activation and/or overproduction (overexpression) in case that the antigen-antibody reaction is detected, subsequently to the step of measuring an antigen-antibody reaction. The biological sample may be at least one selected from the group consisting of cells, tissues, and body fluid (e.g., blood, serum, blood plasma, etc.), which are obtained (isolated) from a subject, and culture thereof.
The step of measuring the antigen-antibody reaction may be performed using various methods known in the art. For example, it may be measured through an ordinary enzyme reaction, fluorescence, luminescence, and/or radioactivity detection and particularly, it may be measured by a method selected from the group consisting of immunochromatography, immunohistochemistry, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescence immunoassay (FIA), luminescence immunoassay (LIA), western blotting, microarray, etc., but is not limited thereto.
The subjects which the anti-Ang2 antibody or an antigen binding fragment is administered to or is aimed to diagnose may be selected from mammals including primates such as humans and monkeys, or rodents such as rats and mice.
The diseases related to the activation and/or overproduction (overexpression) of Ang2 may be cancer; cancer metastasis; cancer invasion/penetration; eye diseases such as retinopathy of prematurity, macular degeneration (e.g., age-related macular degeneration), diabetic retinopathy, neovascular glaucoma, etc.; asthma; rheumatoid arthritis; inflammatory diseases such as psoriasis, chronic inflammation, pneumonia, septicemia, etc.; cardiovascular disease such as hypertension, arteriosclerosis, etc.; sepsis, etc. The cancer may be associated with overexpression of Ang2, it may be a solid cancer or a blood cancer, and it may be, but is not limited to, selected from the group consisting of squamous cell carcinoma, small-cell lung cancer, non-small-cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, peritoneal carcinoma, skin cancer, melanoma in the skin or eyeball, rectal cancer, cancer near the anus, esophagus cancer, small intestinal tumor, endocrine gland cancer, parathyroid cancer, adrenal cancer, soft-tissue sarcoma, urethral cancer, chronic or acute leukemia, lymphocytic lymphoma, hepatocellular cancer, gastric cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, large intestine cancer, endometrial carcinoma or uterine carcinoma, salivary gland tumor, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, head or neck cancer, brain cancer, osteosarcoma, and the like. The cancer may be a primary cancer or metastatic cancer.
In another embodiment, provided is a method for screening a candidate substance (compound) for utility in diagnosing, preventing, and/or treating a disease related to the activation and/or overproduction (overexpression) of Ang2 using the above epitope. The screening method includes

    
    
        (a) contacting a candidate compound to the aforementioned epitope of three dimensional structure of Ang2; and
        (b) measuring binding between the epitope and the candidate compound.
    
    


In the step of measuring binding, when the epitope and the candidate compound show binding affinity of 10 nM or less, for example, 1 pM to 10 nM, 10 pM to 10 nM, or 100 pM to 10 nM, the candidate compound can be determined to be a candidate substance for diagnosing, preventing, and/or treating a disease related to the activation and/or overproduction (overexpression) of Ang2.
The step of measuring binding may be carried out using various methods known in the art. For example, it may be measured through an ordinary enzyme reaction, fluorescence, luminescence, and/or radioactivity detection and particularly, it may be measured by a method selected from the group consisting of surface plasmon resonance (SPR), immunochromatography, immunohistochemistry, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescence immunoassay (FIA), luminescence immunoassay (LIA), western blotting, microarray, etc., but is not limited thereto.
The epitope may be all or a part (for example, at least one selected from the group consisting of the amino acid residue regions exposed to the outside of loop 3) of loop 3 (a region covering from 460th to 468th amino acids of SEQ ID NO: 27) of human Ang2 (hAng2; SEQ ID NO: 27), or an amino acid sequence region including 2 to 20, 2 to 15, 2 to 10, or 2 to 5 contiguous amino acids including at least one amino acid residue exposed to the outside of loop 3 of SEQ ID NO: 27 and for example, it may be at least one amino acid residue selected from the group consisting of Y460 and K468, which are positioned at loop 3, or an amino acid sequence region including 2 to 20, 2 to 15, 2 to 10, or 2 to 5 contiguous amino acids within Ang2, which comprises the above at least one amino acid residue (e.g., Y460 and/or K468).
The candidate compounds may be one or more selected from the group consisting of various artificially-synthesized or natural compounds (chemical), polypeptides, oligopeptides, polynucleotides, oligonucleotides, antisense-RNA, shRNA (short hairpin RNA), siRNA (small interference RNA), aptamers, natural product extracts and so on.
The step of measuring the binding affinity between the epitope and the candidate compound may be carried out using various methods known in the art. For example, the binding affinity may be measured using Biacore machine. In general, the range within which the binding affinity is considered as a drug for treatment may be defined to have a binding constant KD value of 10 mM or less. For instance, in case that the binding affinity between the epitope of Ang2 and a candidate compound (for example, antibody) to be analyzed is 1 pM to 10 nM, 10 pM to 10 nM, or 100 pM to 10 nM when measured using surface plasmon resonance methods such as Biacore machine, the candidate compound (for example, antibody) can be determined to be a candidate substance for diagnosing, preventing, and/or treating a disease related to the activation and/or overproduction (overexpression) of Ang2.
In another embodiment, provided is polypeptide molecule comprising the heavy chain complementarity determining region, the light chain complementarity determining region or the combination thereof or the heavy chain variable region, the light chain variable region or the combination thereof, of the anti-Ang2 antibody as described above. The polypeptide molecule may function as a precursor or a component of an antagonist against Ang2 as well as an antibody or an antigen-binding fragment thereof. For example, the polypeptide molecule may function as an Ang2 antigen binding site, and can be included as a component of a protein scaffold (e.g., peptibody, nanobody, etc.), a bispecific antibody, and a multi-specific antibody having a similar structure to an antibody. The polypeptide molecule may be non-naturally occurring. For example, the polypeptide molecule may be synthetic or recombinant.
The term “antagonist” as used herein is interpreted to encompass all molecules that partially or entirely block, suppress or neutralize at least one biological activity of its target (e.g., Ang2).
The term “peptibody (peptide+antibody)” used herein refers to a fusion protein including a peptide and all or part of the constant region of an antibody such as an Fc portion wherein the peptide serves as an antigen binding site (heavy chain and/or light chain CDR or variable regions) thereby to render a protein having similar framework and functions to an antibody
The term “nanobody” used herein is called a single-domain antibody, refers to an antibody fragment including a single variable domain of an antibody as a monomer form, and has characteristics of selectively binding to a specific antigen similarly to an antibody having an intact structure. The molecular weight of the nanobody is generally about 12 kDa to about 15 kDa, which is very little when compared to the normal molecular weight (about 150 kDa or about 160 kDa) of an intact antibody (including two heavy chains and two light chains) and in some cases it is smaller than an Fab fragment or scFv fragment.
The term “bispecific antibody” or “multi-specific antibody” used herein refers to an antibody recognizing and/or binding to two (bispecific antibody) or more (multi-specific antibody) different antigens, or recognizing and/or binding to different sites of the same antigen, and one antigen binding site of the bispecific antibody or multi-specific antibody may include the polypeptide described above.
The polypeptide molecule may comprise or consist essentially of at least one of SEQ ID NOs: 1-24.
In an embodiment, the polypeptide molecule may comprise or consist essentially of:
at least one selected from the group consisting of a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, and a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12;
at least one selected from the group consisting of a polypeptide comprising the amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16, a polypeptide comprising the amino acid sequence of SEQ ID NO: 25 (for example, an amino acid sequence selected from the group consisting of SEQ ID NOS: 17 to 20), and a polypeptide comprising the amino acid sequence of SEQ ID NO: 26 (for example, an amino acid sequence selected from the group consisting of SEQ ID NOS: 21 to 24); or
any combination thereof.
In an embodiment, the polypeptide molecule may comprise or consist essentially of at least one selected from SEQ ID NOS: 28 to 31 and 36 to 39. Specifically, the polypeptide molecule may comprise or consist essentially of the amino acid sequence selected from the group consisting of SEQ ID NOS: 28 to 31; the amino acid sequence selected from the group consisting of SEQ ID NOS: 36 to 39; or any combination thereof.
In an embodiment, the polypeptide molecule may not be one consisting only of at least one selected from the group consisting of amino acid sequences of SEQ ID NO: SEQ ID NO: 14, SEQ ID NO: 18, and SEQ ID NO: 22, or may not be one consisting only of the amino acid sequence of SEQ ID NO: 37.
The above polypeptide molecule may be non-naturally occurring, and for example, may be synthetic or recombinant.
The above bispecific antibody or multi-specific antibody may refer to an antibody including each antigen binding site to different two or more kinds of antigens and recognizing the two or more kinds of antigens at the same time, wherein one of the antigen binding sites may include the aforementioned polypeptide molecule. In particular, the polypeptide molecule serving as Ang2 antigen binding site may form a dimer or multimer together with an antigen binding site to another antigen to constitute a bi-specific antibody or a multi-specific antibody. Accordingly, in one embodiment, there is provided a bi-specific antibody or a multi-specific antibody including the polypeptide molecule as an Ang2 antigen binding site.
In another embodiment, provided is a protein scaffold comprising at least one (e.g., 1 to 5, particularly 2 to 4) peptide complex, which comprises one or more of the aforementioned polypeptide molecules or a repeat where the polypeptide molecules are repeatedly linked by a linker (hereafter, ‘first peptide’) and a polypeptide having a structural function (hereafter, ‘second peptide’; e.g., a heavy chain or light chain constant region of an antibody, or an Fc fragment of an antibody), wherein the at least one peptide complex is bound to each other at the second peptide (e.g., Fc fragment) to form a multimer structure.
In another embodiment, provided is a polynucleotide molecule encoding the polypeptide molecule or a recombinant vector comprising the polynucleotide molecule. Particularly, the polynucleotide molecule may comprise or consist essentially of a nucleotide sequence encoding the amino acid sequence selected from the group consisting of SEQ ID NOS: 28 to 31, or a nucleotide sequence encoding the amino acid sequence selected from the group consisting of SEQ ID NOS: 36 to 39. For example, the polynucleotide molecule may comprise or consist essentially of the nucleotide sequence selected from the group consisting of SEQ ID NOS: 32 to 35, or the nucleotide sequence selected from the group consisting of SEQ ID NOS: 40 to 43.
The term “vector” used herein refers to a means for expressing a target gene in a host cell. For example, the vector may include a plasmid vector, a cosmid vector, and a virus vector, such as a bacteriophage vector, an adenovirus vector, a retrovirus vector, and an adeno-associated virus vector. The recombinant vector may be prepared by manipulating a plasmid (for example, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, and pUC19), a phage (for example, λgt4λB, λ-Charon, λΔz1, and M13), or a virus (for example, SV40) often used in the art, but is not limited thereto.
In the recombinant vector, the polynucleotide may be operatively linked to a promoter. The term “operatively linked” used herein means a functional linkage between a nucleotide expression regulating sequence (for example, a promoter sequence) and other nucleotide sequences. Thus, the regulating sequence may regulate the transcription and/or translation of the other nucleotide sequences by being “operatively linked”.
The recombinant vector may be generally constructed as a cloning vector or an expression vector. The expression vector may be a vector commonly used in the art for expressing a foreign protein in a plant, animal or microorganism. The recombinant vector may be constructed using various methods known in the art.
The recombinant vector may be constructed for use in prokaryotic or eukaryotic host cells. For example, when a prokaryotic cell is used as a host cell, the expression vector used generally includes a strong promoter capable of initiating transcription (for example, pLλ promoter, CMV promoter, trp promoter, lac promoter, tac promoter, T7 promoter), a ribosome binding site for initiating translation, and a transcription/translation termination sequence. When a eukaryotic cell is used as a host cell, an origin of replication acting in the eukaryotic cell to be included in the vector may include fl origin of replication, SV40 origin of replication, pMB1 origin of replication, adeno origin of replication, AAV origin of replication, or BBV origin of replication, but is not limited thereto. The promoter in an expression vector for a eukaryotic host cell may be a promoter derived from genomes of mammalian cells (for example, a metallothionein promoter) or a promoter derived from mammalian viruses (for example, an adenovirus late promoter, a vaccinia virus 7.5K promoter, a SV40 promoter, a cytomegalovirus promoter, and a tk promoter of HSV), and the transcription termination sequence may have, in general, a polyadenylation sequence.
Another embodiment provides a recombinant cell comprising the recombinant vector.
The recombinant cell may be obtained by introducing the recombinant vector into a suitable host cell. The host cell, which is capable of stably and consecutively cloning or expressing the recombinant vector, may be any host cells known in the art. The prokaryotic cell may be E. coli JM109, E. coli BL21, E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, a Bacillus genus bacterial cell such as Bacillus subtilis or Bacillus thuringiensis, intestinal bacteria such as Salmonella typhimurium, Serratia marcescens, or various Pseudomonas species. A eukaryotic host cell may be a yeast (Saccharomyce cerevisiae), an insect cell, a plant cell, or an animal cell, for example, Sp2/0, CHO (Chinese hamster ovary) K1, CHO DG44, PER.C6, W138, BHK, COS-7, 293, HepG2, Huh7, 3T3, RIN, MDCK cell line and so on, but is not limited thereto.
The polynucleotide or the recombinant vector including the same may be transferred (introduced) into the host cell by using a method widely known in the art. For example, when a prokaryotic cell is used as the host cell, the transfer may be performed using a CaCl2 method or an electroporation method, and when a eukaryotic cell is used as the host cell, the transfer may be performed by microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, or gene bombardment, but is not limited thereto.
The transformed host cell may be selected using a phenotype expressed by a selectable marker by known methods in the art. For example, when the selectable marker is a specific antibiotic resistance gene, a transformant is cultured in a medium containing the antibiotic, and thus the transformant may easily be selected.
Provided is an anti-Ang2 antibody for target-treating to Ang2, which, among the factors involved in neovascular formation and growth in a cancer tissue, has been drawing a new attention lately by the elucidation of its molecular mechanism, and it can be usefully developed as a new innovative antibody drug having excellent effects compared to the pre-existing antibody drugs. In addition, it is expected that the use of the anti-Ang2 antibody may be expanded to development of multi-specific antibody and the like.
Examples
Hereafter, the present disclosure will be described in detail by examples.
The following examples are intended merely to illustrate the invention and are not construed to restrict or limit the invention in any way.
Example 1
Preparation of an Anti-Ang2 Antibody
An anti-Ang2 antibody was prepared by inducing immune response through administering human Ang2 protein (R&D systems) together with and an adjuvant to 5-week old BALB/c mice, and then preparing hybridomas each of which produces each antibody according to a known method described in the paper written by Schwaber et al. (Schwaber, J and Cohen, E. P., “Human×Mouse Somatic Cell Hybrid Clones Secreting Immunoglobulins of Both Parental Types,” Nature, 244 (1973), 444-447).
In particular, to obtain immunized mice necessary for preparing hybridoma cell lines, 100 μg of human Ang2 protein (R&D Systems) mixed with the same amount of a complete Freund's adjuvant was administered via an intraperitoneal injection to each of five 4-6-week-old BALB/c mice (Japan SLC, Inc.). After two weeks, the antigen (half of the previously injected amount) mixed with an incomplete Freund's adjuvant using the same method as described above was administered to each mouse via an intraperitoneal injection. After one additional week, a final boosting was performed and three days later, blood was collected from the tail of each mouse to obtain serum, which was then diluted at 1/1000 with PBS and subjected to an ELISA to verify that the titer of an antibody recognizing Ang2 was increased. From the results, mice in which a sufficient amount of the antibody was obtained were selected, and a cell fusion process was performed on the selected mice.
Three days before the cell fusion experiment, a mixture of 50 μg of PBS and 100 μg of human Ang2 protein (R&D systems) was administered via an intraperitoneal injection to BALB/c mice (Japan SLC, Inc.), and after each immunized mouse was anesthetized, its spleen located on the left side of the body was extracted. The extracted spleen was ground with a mesh to isolate cells, which were mixed with a culture medium (DMEM, Hyclon) to prepare a spleen cell suspension. The suspension was centrifuged to collect a cell layer. The obtained 1×108 spleen cells were mixed with 1×107 myeloma cells (Sp2/0), and the mixture was centrifuged to precipitate the cells. The centrifuged precipitate was slowly dispersed, treated with 1 ml of 45% polyethylene glycol (PEG 1500) contained in a culture medium (DMEM), and maintained at 37° C. for one minute before adding 1 ml of a culture medium (DMEM). Subsequently, 10 ml of the culture medium (DMEM) was added for 1 minute to the resultant, which was incubated in a water bath at 37° C. for 5 minutes and then re-centrifuged after the total volume was adjusted to 50 ml. The resulting cell precipitate was re-suspended in an isolation medium (HAT medium) at a concentration of 1 ̃2×105/ml, and the resultant suspension was distributed at 0.1 ml to the each well of a 96-well plate, which was then incubated in a carbon dioxide incubator at 37° C. to prepare the hybridoma cell groups.
Example 2
Selection of Anti-Ang2 Antibody Producing Clone and Purification of Antibody
The above obtained individual antibody producing hybridomas were screened using a typical ELISA format to select hybridomas which produce 95 anti-Ang2 monoclonal antibodies among the hybridomas differentiated from their mother hybridomas, based on their binding potential with Ang2.
More specifically, to select the hybridoma cells that specifically react only to Ang2 protein among the hybridoma cell groups prepared in Example 1 above, an ELISA assay method using a human Ang2 protein as an antigen was used for screening.
Human Ang-2 protein was added at 100 ng per well to a microtiter plate to be adhered to the surface of the plate, and unreacted antigens were removed by washing. 50 μl of the hybridoma cell culture obtained in Example 1 above was added to each well to react for 1 hour and then, the wells were sufficiently washed with phosphate buffered saline-TWEEN 20 (PBST) solution to remove unreacted culture solution. Goat anti-mouse IgG-horseradish peroxidase (goat anti-mouse IgG-HRP) was added thereto, a reaction was allowed to occur at a room temperature for 1 hour and then, washing was sufficiently performed with the TBST solution. Subsequently, substrate solution (OPD) of peroxidase was added to each well to react, and the reaction degree was measured by the absorption at 450 nm using an ELISA reader to repeatedly select hybridoma cell lines that secret antibodies having specifically high binding affinity only to human Ang2 protein. A limiting dilution was performed on the hybridoma cell lines obtained through repetitive selection to obtain final 58 clones of hybridoma cell lines producing monoclonal antibodies.
Each hybridoma obtained above was cultured in DMEM (Dulbeco's Modified Eagle's Medium) and then, the culture solutions were collected and subjected to Protein G-affinity chromatography method to purify anti-Ang2 monoclonal antibodies produced from each hybridoma.
First, the hybridoma cells cultured in 50 ml of culture medium (DMEM) containing 10% (v/v) FBS were centrifuged to obtain a cell precipitate, which was washed at least twice with 20 ml of PBS to remove the FBS. The cell precipitate was re-suspended in 50 ml of the culture medium (DMEM) and then incubated in a carbon dioxide incubator at 37° C. for 3 days. Subsequently, the cell culture was centrifuged to remove the antibody-producing cells, and the culture medium including the secreted antibodies was isolated and then, stored at 4° C. or used directly. Antibodies were purified from 50 to 300 ml of the culture medium using an AKTA purification device (GE Healthcare) equipped with an affinity column (protein G agarose column; Pharmacia, USA). The purified antibodies were stored for subsequent use after replacing the supernatant with PBS using a filter for protein aggregation (Amicon).
Example 3
Examination of Ang2:Tie-2 Binding Inhibition
In order to confirm whether the antibody binding to Ang2 prepared in Example 2 is a neutralizing antibody which actually inhibits the binding of Ang2 and Tie-2, Ang2-Tie2 binding competition ELISA was conducted using the antibody.
More specifically, MaxiSorpTM flat-bottom plate (Nunc) of 96-well was coated with hTie2-Fc (R&D Systems) which is a protein bound with 4 μg/ml of Fc of human IgG1. After coating, the plate was washed five times with 0.05% (v/v) Tween-20-containing PBS (phosphate buffer saline) and then blocked with 1% (v/v) BSA (bovine serum albumin; Sigma)-containing PBS at a room temperature for 2 hour.
For Ang2:Tie2 competition ELISA, each anti-Ang2 antibody obtained in Example 2 was placed at various concentrations of 400 nM-0.001 nM into each well coated with the hTie-2/Fc fusion protein along with 1% (v/v) BSA and 400 ng/ml of a FLAG-tagged hAng-2 and then, the plate was allowed to react at a room temperature for 2 hours and washed five times with PBST. After that, a HRP-conjugated anti-FLAG antibody (Sigma) diluted in 1% (v/v) BSA-containing PBS at a ratio of 1:5,000 (v/v) was added in an amount of 1000 to each well to react at a room temperature for 1 hour and then, the plate was washed five times with PBST. Lastly, 100 μl of TMB substrate (Cell Signaling) was added to each well of the plate to induce color development for 3 min. and then, the reaction was ceased by the addition of 100 μl of Stop solution (Cell Signaling) and OD450 values were measured on a plate reader (Molecular Devices).
From the obtained results, the concentration (50% inhibition concentration, IC50) where 50% of Ang2:Tie-2 binding is inhibited was obtained, which indicates that the anti-Ang2 antibody can neutralize the binding intensity between Ang2 and Tie-2. The obtained results are shown in Table 3:










TABLE 3








50% inhibition concentration to



Antibody
Ang2:Tie-2 binding (IC50, nM)









SAIT-ANG2-AB-m1A10
1.34



SAIT-ANG2-AB-m1B6
2.89



SAIT-ANG2-AB-m3E2
0.55



SAIT-ANG2-AB-m8D3
0.99










Example 4
Examination of Binding Affinity of an Anti-Ang2 Antibody to hAng2 and hAng1
To more exactly measure the binding affinity of the anti-Ang2 antibodies to antigen Ang2, the binding affinity of the above antibodies to the antigen was measured by an surface plasmon resonance (SPR) method using BIAcore T100 (GE Healthcare). The SPR method uses refractive index change of light which passes a sensor chip according to the state of materials coated onto the sensor chip, and if an antigen or an antibody is flowed onto a chip coated with the antigen or antibody, it causes changes in refractive index due to their binding and Kd values are thus calculated from the measured values.
First, anti-His antibody was immobilized on a CM5 sensor chip (GE healthcare) up to 8,000 RU levels using a pH 5.0 acetate solution and an amine coupling kit (GE Healthcare). 6 μg/ml of a recombinant hAng-2 (C-His, R&D Systems) protein was flowed onto the chip to be captured at 100 to 200 RU levels. The antibody obtained in Example 2 above was diluted serially twice each time starting from 100 nM concentration and it was each flowed onto the chip to allow it to be bound to (on), dissociated from (off), and regenerated (using 10 mM NaOH solution) from the antigen captured on the sensor chip, thereby measuring antigen-antibody affinity. With regard to hAng1, such experiments were conducted, and the results are as shown in the following Table 4.









TABLE 4





Antibody
hAng2 (Kd, nM)
hAng1 (Kd, nM)







SAIT-ANG2-AB-m1A10
4.2
No binding


SAIT-ANG2-AB-m1B6
2.3
No binding


SAIT-ANG2-AB-m3E2
1.3
No binding


SAIT-ANG2-AB-m8D3
4.0
No binding









The hybridomas producing the 4 antibodies were deposited in the Korean Cell Line Bank located at Yongon-dong, Chongno-gu, Seoul, South Korea, as of Dec. 20, 2013 and received accession numbers KCLRF-BP-00305 (SAIT-ANG2-AB-m3E2), KCLRF-BP-00306 (SAIT-ANG2-AB-m1A10), KCLRF-BP-00307 (SAIT-ANG2-AB-m1B6), and KCLRF-BP-00308 (SAIT-ANG2-AB-m8D3), respectively.
Example 5
Examination of Binding Affinity of an Anti-Ang2 Antibody to mAng2
An ELISA was conducted to measure the binding affinity of the antibodies prepared in Example 2 to mouse Ang2(mAng2). MaxiSorpTM flat-bottom plate (Nunc) of 96-well was coated with 5-20 μg/ml of mouse Ang2 (Mouse Ang2; Accession # NP_031452) (R&D Systems). Thereafter, the plate was washed five times with 0.05% (v/v) Tween-20-containing PBS (PBST) and then blocked with 1% (v/v) BSA (bovine serum albumin; Sigma)-containing PBS at a room temperature for 2 hour. Each of the anti-Ang2 antibodies was added to each well of the plate at various concentrations, and the plate was allowed to react at a room temperature for 2 hours.
Thereafter, the plate was washed five times with PBST. Then, a HRP-conjugated anti-mouse IgG antibody (Santacruz) diluted in 1% (v/v) BSA-containing PBS at a ratio of 1:1,000 (v/v) was added in an amount of 50 μl to each well to react at a room temperature for 1 hour and then, the plate was washed five times with PBST. Lastly, 100 μl of TMB substrate (Cell Signaling) was added to each well of the plate to induce color development for 3 min. and then, the reaction was ceased by the addition of 100 μl of Stop solution (Cell Signaling) and OD450 values were measured on a plate reader (Molecular Devices).
From the obtained results, 50% binding concentration (Kd) of the anti-Ang2 antibody to Ang2 protein is obtained, thereby measuring the binding intensity of each of the anti-Ang2 antibodies to Ang2. The obtained results are shown in Table 5:










TABLE 5







Antibody
mAng2 (Kd, nM)









SAIT-ANG2-AB-m1A10
No binding



SAIT-ANG2-AB-m1B6
2.1



SAIT-ANG2-AB-m3E2
130



SAIT-ANG2-AB-m8D3
141










Example 6
Identification of Antigen Recognition Region (Epitope) of the Anti-Ang2 Antibodies
To identify the epitope for each of the anti-Ang2 antibodies obtained in Example 2, ELISA was performed using a recombinant protein wherein receptor binding site of Ang2 protein is tagged with Flag or an artificial mutation is introduced into full-length Ang2.
A 96-well MaxiSorpTM flat-bottom plate (Nunc) was coated with 50 μl of each anti-Ang2 antibody (400 nM). After that, the plate was washed five times with 0.05% (v/v) Tween-20-containing PBS (PBST) and then blocked with 1% (v/v) BSA-containing PBS at a room temperature for 2 hours. S417, P419, N421, I434, D448, A449, P452, Y460, N467, K468, or F469 residue of Ang2 (SEQ ID NO: 27) was mutated (substituted) with alanine and tagged with FLAG (N-DYKDDDDK-C; 1012 Da) and then 250 ng of them was each added to each well to the plate, which was allowed to react at a room temperature for 2 hours.
Thereafter, the plate was washed five times with 0.05% (v/v) Tween-20-containing PBS and then, an HRP-conjugated anti-FLAG antibody (SIGMA) diluted in 1% (v/v) BSA-containing PBS at 1:5,000 ratio (v/v) was added in the amount of 50 μl to each well to react at a room temperature for 1 hour, followed by washing five times with 0.1% (v/v) Tween-20-containing PBS.
Finally, 100 μl of TMB substrate (cell signal) was added to each well of the plate to induce color development for 3 minutes and then, the reaction was ceased by the addition of 100 μl of stop solution (Cell signaling) and OD450 values were measured on a plate reader (Molecular Devices). By comparing binding intensity to mutated Ang2 to binding intensity to non-mutated (native) Ang2, each epitope for the Ang2 antibodies was identified. The obtained binding intensity (%) (binding intensity to mutant Ang2/binding intensity to native Ang2) was shown in Tables 6 and FIG. 1.








Table 6








Binding (%)












Loop 1
Loop 2
Loop 3
Loop4






















RBD
S417
Q418
P419
N421
I434
D448
A449
P452
Y460
N467
K468
F469


mutant














1A10
99.96
97.2
101.6
89.3
93.2
95.3
95.6
104.7
8.3
79.4
27.3
77.9


Ang2
—
—
—
—
I434
—
—
—
—
—
K468
F469


mutant














8D3




137.3





20.5
41.9









(In Table 6, “RBD mutant” refers to a mutant prepared using the receptor binding domain (amino acid residues from 276th to 496th positions) of Ang2;
“1A10” and “8D3” refer to “SAIT-ANG2-AB-m1A10” and “SAIT-ANG2-AB-m8D3”, respectively)
As shown in Table 6, the binding intensities to mutant Ang2, Y460 and K468, are decreased as 30% or less of that to native Ang2.
Example 7
Examination of Ang2 Receptor Activity Inhibition by Ang2 Antibodies
As Ang2 induces a change in vascular endothelial cells by binding to a Tie2 receptor expressed in the vascular endothelial cells to induce the phosphorylation of the receptor and activate it, the Ang2 inhibitory activities of the anti-Ang2 antibodies were verified through the functional analysis of the antibodies using a cell-based assay.
For this, 1×106 of Tie2-overexpressed CHO (Chinese hamster ovary) cell (Kim et al., Biochim Bioohys Acta., 2009) were cultured in a 60 mm culture dish using 5% (v/v) FBS (Gibco)-containing IMDM media (Gibco) at 37° C. and when they reached 80 ̃90% confluency, the media were replaced with serum-free IMDM media and further cultured at 37° C. for 16 hours. The dish was washed once with PBS and after the replacement with 0.1 nM sodium orthovanadate-mixed IMDM media, they were further cultured for 10 min. After washed once with PBS, the cultured cells were treated with a mixture prepared by mixing the anti-Ang2 antibodies prepared in Example 2 at various concentrations with 20 nM human Ang2 protein (R&D systems) and letting them stand for 20 min, and further cultured for 10 min.
The cells were washed using a cold PBS, treated with 300 μl of lysis buffer (Roche), collected to a tube to allow them to be dissolved at 4° C. for 30 minutes, and then, centrifuged at 13,000 rpm for 15 minutes to quantify a supernatant. 2 μg of anti Tie2 antibody (R&D system) was added to 0.5 mg of a cell lysate, which was then overnight reacted at 4° C. and then subjected to immunoprecipitation by the addition of protein A bead (GE Healthcare) thereto.
The reactant obtained above was centrifuged at 13,000 rpm for 15 minutes to obtain a pellet, which was washed two to three times with lysis buffer (Roche). A sample buffer (Invitrogen) mixed with a reducing agent was added thereto. The obtained mixture was boiled at 95° C. for 5 minutes, and then, applied to NuPAGE Novex 4-12% Bis-Tris gel (Invitrogen) and transferred onto Nitrocellulose membrane (Invitrogen).
To verify the presence of the phosphorylation of Tie2, the above blots were blocked with PBST mixed with 3% (v/v) skim milk (Sigma) for 30 minutes and identified using an HRP-conjugated anti-phospho tyrosine antibody (Millipore). For Tie2 identification, the blots were reacted in a stripping buffer (Thermo) for 15 minutes and then blocked again and identified using an anti Tie2 antibody (Santa cruz). After band intensities were measured using Image J software (http://rsb.info.nih.gov/ij/index.html), the inhibitory degrees of Tie2 phosphorylation after the treatment of the anti-Ang2 antibodies were calculated in relative % against the Ang2 single treatment group, and the results are shown in the following Table 7 and FIG. 2.










TABLE 7







Antibody
Tie2 phosphorylation



(treated amount: 50 nM)
inhibition (%)









SAIT-ANG2-AB-m1A10
82.01%



SAIT-ANG2-AB-m3E2
76.89%



SAIT-ANG2-AB-m8D3
76.69%










As shown in Table 7 and FIG. 2, the anti-Ang2 antibodies can effectively inhibit the phosphorylation of Tie2.
Example 8
Inhibition of Cell Migration by Anti-Ang2 Antibodies
To confirm whether the above prepared anti-Ang2 antibody can inhibit the migration of vascular endothelial cells, an assay using Oris cell migration kit (Platypus technology) was performed.
Vascular endothelial cells (Human umbilical vein endothelial cells (HVUEC), ATCC) were sub-confluently cultured in EGM-2 medium (Lonza). 2.5×104 cells of the cultured HVUEC were seeded onto each well of a migration plate equipped with a stopper, and cultured for 24 hours. The stopper was removed from the confluently cultured cells, to make cell free zone, and then, the plate was washed once with PBS.
Various concentrations of each of the anti-Ang2 antibodies prepared in Example 2 were mixed with 40 nM Ang2 protein (R&D systems) in serum-free medium, and allowed to be reacted for 20 minutes. The cultured cells were treated with the reactant and further cultured for 24 hours. A mixture where Calcein (BD) was mixed with serum-free medium to the concentration of 8 μg/ml was added to each well at the amount of 5 μl, and left for reaction for 30 minutes, to stain the cells. Thereafter, a blocker was equipped in the back of the plate, and fluorescence images were obtained using InCell analyzer6000 (GE Healthcare). Then, the fluorescence intensity in migration zone was quantified, and shown in FIG. 3 as migration index. As shown in FIG. 3, the anti-Ang2 antibody effectively inhibits the cell migration.
Example 9
Inhibition of In Vitro Vasculogenesis by Anti-Ang2 Antibodies
To confirm whether the prepared anti-Ang2 antibodies can inhibit tube formation of vascular endothelial cells by Ang2, HUVEC tube formation assay was performed.
Vascular endothelial cells (Human umbilical vein endothelial cells (HVUEC), ATCC) were sub-confluently cultured in EGM-2 medium (Lonza). After replacement with serum-free medium, the cells were further cultured for 6 hours. 200 nM of each of the anti-Ang2 antibodies prepared in Example 2 was mixed with 80 nM Ang2 protein (R&D systems) in serum-free medium, and the obtained mixture was mixed with 8×105 cells/ml of HVUEC suspension at the ratio of 1:1. 50 μl of the obtained mixture was seeded into each well of Matrigel-coated 96-well plate (BD BioCoat Angiogenesis System-Endothelial Cell Tube Formation) and cultured for 18 hours. Thereafter, and 50 μl of a mixture, where calcein (BD) was mixed with serum-free medium to the concentration of 8 m/ml, was added to each well and left for reaction for 30 minutes, to stain the cells. Fluorescence images were obtained using a fluorescence microscope (Zeiss). From the images, the length of the formed tube was measured using AxioVision (Zeiss) and the sum thereof is shown in FIG. 4.
Example 10
Inhibition of Colo205 Tumor Cell Growth by Anti-Ang2 Antibodies
To verify the tumor cell growth inhibition effect of the anti-Ang2 antibodies, an experiment was performed for a colorectal cancer cell xenograft model using human colorectal cancer cell line Colo205 (ATCC). Colo205 cells were cultured in 10% (v/v) FBS (Gibco)-containing RPMI-1640 medium (Gibco). 5×105 cells of the cultured Colo205 cells were re-suspended in 100 μl of serum-free medium, and administered via subcutaneous injection using 1 ̃2% (v/v) isoflurane to anesthetized BALB/c nude mice (4 ̃5 weeks old; Shanghai SLAC Laboratory Animal Co. Ltd.). When the size of the tumor reaches 100 ̃200 mm3, each of the anti-Ang2 antibodies prepared in Example 2 was administered via intraperitoneal injection at the concentration of 10 mg/kg twice a week, and then the tumor size was measured. The tumor size (V) was calculated by the following formula: V=(length×width2)/2. The obtained results are shown in FIG. 5A (change in the tumor size according to administration days) and FIG. 5B (the tumor size at day 18 according to the kind of the antibodies). X axis in FIG. 5A shows days after grouping, which refers to the number of antibody administration days.
Example 11
Gene Cloning of Anti-Ang2 Antibodies
Total RNA was obtained from each of the prepared antibody producing hybridomas (2×106 cells) using RNeasy mini kit (Qiagen). Then, using the RNA as a template, gene sequences of a heavy chain variable region and light chain variable region were amplified from a monoclonal antibody produced from each of the hybridomas using OneStep RT-PCR kit (Qiagen), Mouse Ig-Primer Set (Novagen) and thermocycler (GeneAmp PCR System 9700, Applied Biosystem): at 94° C. for 5 minutes; [at 50° C. for 30 minutes and at 95° C. for 15 minutes], [at 94° C. for 1 minute, at 50° C. for 1 minute, and 72° C. for 2 minutes]×35 cycles; at 72° C. for 6 minutes; cooled to 4° C.
The PCR product obtained from each of the above reactions was subjected to direct DNA sequencing, to obtain sequences of CDRs, heavy chain variable region and light chain variable region of each antibody. The obtained sequences of CDRs, heavy chain variable region and light chain variable region of each antibody are shown in Table 8 (heavy chain CDRs), Table 9 (light chain CDRs), and Table 10 (heavy chain variable region and light chain variable region).







TABLE 8







Amino acid sequence of heavy chain CDRs










Antibody
CDRH1-KABAT
CDRH2-KABAT
CDRH3-KABAT





SAIT-ANG-2-AB-m1A10
SYWLE
EILPGSGSTNYNEKFRG
GNHNSYYYAMDY



(SEQ ID NO: 1)
(SEQ ID NO: 5)
(SEQ ID NO: 9)


 


SAIT-ANG-2-AB-m8D3
DYYMK
EINPKNGDTFYNQIFKG
ENDYDVGFFDY



(SEQ ID NO: 2)
(SEQ ID NO: 6)
(SEQ ID NO: 10)


 


SAIT-ANG-2-AB-m1B6
NYGMN
WINTYTGEPTYADDFKG
DHDGYLMDY



(SEQ ID NO: 3)
(SEQ ID NO: 7)
(SEQ ID NO: 11)


 


SAIT-ANG-2-AB-m3E2
DPYIH
RIDPANGNTKYDPKFQG
RWDGGGFDY



(SEQ ID NO: 4)
(SEQ ID NO: 8)
(SEQ ID NO: 12)
















TABLE 9







Amino acid sequence of light chain CDRs










Antibody
CDRL1-KABAT
CDRL2-KABAT
CDRL3-KABAT





SAIT-ANG-2-AB-m1A10
RASESVDSYGNSFMH
RASNLDS (SEQ ID
QQSNEDPLT (SEQ ID



(SEQ ID NO: 13)
NO: 17)
NO: 21)


 


SAIT-ANG-2-AB-m8D3
KASQSVSNDVA (SEQ
YASNRYP (SEQ
QQDYTSPWT (SEQ



ID NO: 14)
ID NO: 18)
ID NO: 22)


 


SAIT-ANG-2-AB-m1B6
STSQGISNYLN
YTSSLHS
QQYSKLPYT



(SEQ ID NO: 15)
(SEQ ID NO: 19)
(SEQ ID NO: 23)


 


SAIT-ANG-2-AB-m3E2
RASQDISNYLN (SEQ ID
YTSRLHS
QQGNTLPWT (SEQ



NO: 16)
(SEQ ID NO: 20)
ID NO: 24)


















TABLE 10






Heavy chain variable region
Light chain variable region







SAIT-
QVQLQQSGAELMKPGASVKISCKATDY
DIVLTQSPASLAVSLGQRATISCRASES


ANG-
TFSSYWLEWLIQRPGHGLEWIGEILPGS
VDSYGNSFMHWYQQKPGQPPKLLIYR


2-AB-
GSTNYNEKFRGKATFTEDTSSNTAYMQ
ASNLDSGIPARFSGSGSRTDFTLTINPVE


m1A10
LSSLTSEDSAVYYCARGNHNSYYYAMD
ADDVATYYCQQSNEDPLTFGAGTKLEL



YWGQGTSVTVSS (SEQ ID NO: 28)
K (SEQ ID NO: 36)



caggttcagctgcagcagtctggagctgagctgatgaagcct
gacattgtgctgacccaatctccagcttctctggctgtgtctcta



ggggcctcagtgaagatatcctgcaaggctactgactacacat
ggtcagagggccaccatatcctgcagagccagtgaaagtgtt



tcagtagctactggctagagtggttaatacagaggcctggaca
gatagttatggcaatagttttatgcactggtaccagcagaaac



tggccttgagtggattggagagatatacctggaagtggtagta
caggacagccacccaaactcctcatctatcgtgcatccaacct



ctaactacaatgagaagttcaggggcaaggccacattcactg
agattctgggatccctgccaggttcagtggcagtgggtctag



aagatacatcctccaacacagcctacatgcaactcagcagcct
gacagacttcaccctcaccattaatcctgtggaggctgatgat



gacatctgaggactctgccgtctattactgtgcaagaggtaac
gttgcaacctattactgtcagcaaagtaatgaggatcctctcac



cacaactcctattactatgctatggactactggGGTCAAG
gttcggtgctgggaccaagctggagctgaaa (SEQ ID



GAACCTCAGTCACCGTCTCCTCA (SEQ
NO: 40)



ID NO: 32)



 


SAIT
EVQLQQSGPELVKPGDSVKMSCKASGY
TIVMTQTPKFLLVSAGDRITITCKASQS


ANG-
TFTDYYMKWVRQSHGKSLQWVGEINP
VSNDVAWYQQKPGQSPKLLIYYASNR


2-AB-
KNGDTFYNQIFKGKATLTVDKSSSTAY
YPGVPDRFTGSGYGTDFTFTISTVQAED


m8D3
MQLTSLTSEDSAVYYCTRENDYDVGFF
LAVYFCQQDYTSPWTFGGGTELEIK



DYWGQGTSVTVSS (SEQ ID NO: 29)
(SEQ ID NO: 37)



Gaggtccagctgcaacagtctggacctgagctggtgaagcc
ActattgtgatgacccagactcccaaattcctgCttgtatcag



tggggattcagtgaagatgtcctgcaaggcttctggatacacct
caggagacaggattaccataacctgcaaggccagtcagagt



tcactgactactacatgaagtgggtgaggcagagccatggaa
gtgagtaatgatgtagcctggtatcaacagaagccagggca



agagccttcagtgggttggagaaattaatcctaagaatggtgat
gtctcctaaactgctgatatactatgcatccaatcgctaccctg



actttctacaaccagatattcaagggcaaggccacattgactgt
gagtccctgatcgcttcactggcagtggatatgggacggattt



agacaaatcctccagcacagcctacatgcaactcaccagcct
cactttcaccatcagcactgtgcaggctgaagacctggcagtt



gacatctgaggactctgcagtctattactgtacacgtgagaatg
tatttctgtcagcaggattatacctctccgtggacgttcggtgg



attacgacgtgggattctttgactactggGGTCAAGGA
aggcaccgagctggaaatcaaa (SEQ ID NO: 41)



ACCTCAGTCACCGTCTCCTCA (SEQ ID




NO: 33)



 


SAIT-
QIQLVQSGPELKKPGETVKISCKASGYT
DIQMTQTTSSLSASLGDRVTISCSTSQGI


ANG-
FTNYGMNWVKQAPGKGLKWMGWINT
SNYLNWYQQKPDGTVKLLIFYTSSLHS


2-AB-
YTGEPTYADDFKGRFAFSLETSASTAYL
GVPSRFSGSGSGTDYSLTISNLEPEDIAT


m1B6
QINNLKNEDTATYFCARDHDGYLMDY
YYCQQYSKLPYTFGGGTKLEIK (SEQ



WGQGTSVTVSS (SEQ ID NO: 30)
ID NO: 38)



cagatccagttggtgcagtctggacctgagctgaagaagcct
Gatatccagatgacacagactacatcctccctgtctgcctctc



ggagagacagtcaagatctcctgcaaggcttctggatatacct
tgggagacagagtcaccatcagttgtagtacaagtcagggca



tcacaaactatggaatgaactgggtgaagcaggctccaggaa
ttagcaattatttgaactggtatcagcagaaaccagatggaact



agggtttaaagtggatgggctggataaacacctacactggag
gttaaactcctgatcttttacacatcaagtttacactcaggagtc



agccaacatatgctgatgacttcaagggacgttttgccttctcttt
ccatcaaggttcagtggcagtgggtctgggactgattattctct



ggaaacctctgccagcactgcctatttgcagatcaacaacctc
caccatcagcaacctggaacctgaagatattgccacttactatt



aaaaatgaggacacggctacatatttctgtgcaagagatcatg
gtcagcagtatagtaagcttccgtacacgttcggggggggga



atggttaccttatggactactggGGTCAAGGAACCT
ccaagctggaaataaaa (SEQ ID NO: 42)



CAGTCACCGTCTCCTCA (SEQ ID NO: 




34)



 


SAIT-
EVQLQQSGAELVKPGASVKLSCTASGF
DIQMTQTTSSLSASLGDRVTISCRASQD


ANG-
NIKDPYIHWVKQRPEQGLEWIGRIDPAN
ISNYLNWYQQKPDGTVKLLIYYTSRLH


2-AB-
GNTKYDPKFQGKATITADTSSNTAYLQ
SGVPSRFSGSGSGTDYSLTITNLEQEDIA


m3E2
LSSLTSEDTAVYYCVRRWDGGGFDYW
TYFCQQGNTLPWTFGGGTKLEIK (SEQ



GQGTSVTVSS (SEQ ID NO: 31)
ID NO: 39)



gaggttcagctgcagcagtctggggcagagcttgtgaagcca
gatatccagatgacacagactacatcctccctgtctgcctctct



ggggcctcagtcaagttgtcctgcacagcttctggcttcaacat
gggagacagagtcaccatcagttgcagggcaagtcaggac



taaagacccctatatacactgggtgaaacagaggcctgaaca
attagcaattatttaaactggtatcagcagaaaccagatggaa



gggcctggagtggattggaaggattgatcctgcgaatggtaat
ctgttaaactcctgatctactacacatcaagattacactcagga



actaaatatgacccgaagttccagggcaaggccactataacg
gtcccatcaaggttcagtggcagtgggtctggaacagattatt



gcagacacatcctccaacacagcctacctccagctcagcagc
ctctcaccattaccaacctggagcaagaagatattgccactta



ctgacatctgaggacactgccgtctattactgtgttagaaggtg
cttttgccaacagggtaatacgcttccgtggacgttcggtgga



ggacggggggggattgactactggGGTCAAGGAA
ggcaccaagctggaaatcaaa (SEQ ID NO: 43)



CCTCAGTCACCGTCTCCTCA (SEQ ID




NO: 35)









All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.
The use of the terms “a” and “an” and “the” and “at least one” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

## Claims

1. An anti-Ang2 antibody or an antigen-binding fragment thereof, comprising
a heavy chain variable region comprising a polypeptide (CDR-H1) comprising SEQ ID NO: 4, a polypeptide (CDR-H2) comprising SEQ ID NO: 8, and a polypeptide (CDR-H3) comprising SEQ ID NO: 12; and
a light chain variable region comprising a polypeptide (CDR-L1) comprising SEQ ID NO: 16, a polypeptide (CDR-L2) comprising SEQ ID NO: 20, and a polypeptide (CDR-L3) comprising SEQ ID NO: 24.

2. The anti-Ang2 antibody or an antigen-binding fragment thereof of claim 1, wherein the antibody or antigen binding fragment thereof comprises a heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 31, and a light chain variable region comprises the amino acid sequence of SEQ ID NO: 39.

3. A hybridoma of KCLRF-BP-00305.

4. A method of treating a colorectal cancer in a subject, comprising administering the anti-Ang2 antibody or an antigen-binding fragment thereof of claim 1 to the subject.