WO2015083978A1

WO2015083978A1 - Bispecific anti-vegf-c/anti-ang2 antibody

Info

Publication number: WO2015083978A1
Application number: PCT/KR2014/011420
Authority: WO
Inventors: 이정준; 한상열; 김석균; 이광훈; 김형찬; 김경은; 이윤숙; 김수진
Original assignee: 삼성전자 주식회사
Priority date: 2013-12-02
Filing date: 2014-11-26
Publication date: 2015-06-11
Also published as: KR20150063728A

Abstract

Provided are a bispecific anti-VEGF-C/anti-Ang2 antibody, a pharmaceutical composition for preventing and/or treating diseases associated with Ang2 and/or VEGF-C, comprising the same, and a method for preventing and/or treating diseases associated with Ang2 and/or VEGF-C using the same.

Description

【Specification】

[Name of invention]

Anti VEGF-C / Anti Ang2 Bispecific Antibodies

Technical Field

Anti-VEGF-C / anti Ang2 bispecific antibodies, including Ang2 and / or

Pharmaceutical compositions for preventing and / or treating VEGF-C related diseases, and methods for preventing and / or treating ANGGF and / or VEGF-C related diseases using the same are provided. Background Art

Angiogenesis (angiogenes i s) refers to the mechanism by which new blood vessels are formed from existing blood vessels. ¾ phase angiogenesis plays an important role in organ formation, normal physiological growth, and wound healing, whereas abnormal angiogenesis is associated with tumor growth and metastasis, and / or age-related macular degeneration, diabetic retinopathy, psoriasis, and rheumatism. It plays a decisive role in the development or exacerbation of diseases such as arthritis and chronic inflammation. Thus, factors involved in angiogenesis are important targets for the development of new therapeutics for diseases such as cancer. Factors known to be involved in angiogenesis include angiopoietins such as Angl, Ang2, Ang3, Ang4, VEGF-A, VEGF-B, and VEGF-C, which are known to be involved in angiogenesis and postnatal angiogenesis. , VEGF-D and other vascular endothelial cell growth factors.

Among these, the angiogenesis process of cancer tissue related to Ang2 is as follows. First, public vessels (coopt ion) that cancer cells are selected existing vessel for angiogenesis in cancer tissues occurs ^■. Thereafter, vascular degeneration occurs by the Ang2 pathway, which destroys the function of existing blood vessels. Due to the degeneration of existing blood vessels, the environment within the cancerous tissue becomes a hypoxic environment, providing conditions for the formation of new blood vessels. Under these conditions, the expression of the vascular endothelial cell growth factor (VEGF) is increased to generate new blood vessels. In addition, VEGF-C, a member of the Vascul ar endothelial growth f actor (VEGF) family, promotes lymphatic neovascularization, lymphatic endothelial cell growth and migration upon binding to VEGFR3, a receptor. In addition, VEGF-C has been shown to be involved in lymphat i c- medi ated metastasis, breast cancer metastasis and human nodules through induction of tumor-associated lymphangiogenesis in a number of solid cancers such as gastric cancer, prostate cancer, human colorectal cancer and invasive cervical cancer. Promotes melanoma metastasis In addition, VEGF-C overexpression in cancer cells increases metastasis to regional lymph nodes by increasing tumor-associated lymphangiogenesis.

Therefore, by inhibiting the two proteins at the same time is expected to further enhance the therapeutic effect of related diseases such as cancer, the development of a dual inhibitor having such activity is required.

[Detailed Description of the Invention]

[Technical problem]

One example provides an anti VEGF-C / anti Ang2 bispecific antibody comprising an anti VEGF-C antibody or antigen binding fragment thereof, and an anti Ang2 antibody or antigen binding fragment thereof.

Another example provides a composition for preventing and / or treating Ang2 and / or VEGF-C related diseases comprising the anti-VEGF-C / anti Ang2 bispecific antibody as an active ingredient.

Another example provides a method of preventing and / or treating Ang2 and / or VEGF-C related diseases comprising administering the anti-VEGF-C / anti Ang2 bispecific antibody.

Technical Solution

The present invention relates to an antibody which simultaneously inhibits the function of Ang2 of VEGF-C, an angiogenesis inducer, and more specifically, VEGF-C, which is an inducer of angiogenesis, is essential for cancer cell growth in cancer tissues. Lesions by interfering with binding of its intracellular receptor VEGF-R3 and at the same time preventing Ang2 from binding its intracellular receptor Ti e2 It provides antibodies useful for inhibiting neovascularization and metastasis in tissue (eg cancer tissue) and for diagnosing and / or treating diseases associated with the activity and / or overproduction of Ang2 and / or VEGF-C.

One example provides an anti VEGF-C / anti Ang2 bispecific antibody comprising an anti VEGF-C antibody or antigen binding fragment thereof, and an anti Ang2 antibody or antigen binding fragment thereof. The antigen-binding fragment may be selected from the group consisting of scFv, (scFv) 2, scFvFc, Fab, Fab ¹ and F (ab ') 2.

VEGF-C, one of the targets of the bispecific antibody, is a ligand of VEGFR-3 and is related to angiogenesis, lymphangiogenesis, tumor formation, tumor metastasis, and the like. The VEGF-C may be derived from mammals including primates such as humans and monkeys, rodents such as rats and mice. For example, Human VEGF-C (eg, NCBI Accession No. P49767) derived from humans, originated from Monkey VEGF-C (eg, NCBI Accession No. B6EBK4), mouse-derived VEGF-C (NCBI Accession No. P97953), rat-derived VEGF-C (eg, NCBI Accession No. 035757), and the like, It is not limited to this.

Ang2, one of the targets of the bispecific antibody, is a soluble ligand present in the blood, and is an eye disease accompanied by angiogenesis, metastasis, cancer cell invasion, abnormal angiogenesis, and rheumatoid arthritis. It works extensively on a variety of pathological symptoms, including The Ang2 may be derived from a mammal, including a human, a primate such as a rodent, a rodent such as a rat or a mouse. For example, a human-derived Human Ang2 (Accession # 015123), a monkey derived from an Ang2 (eg, NCBI) Accession No. Q8MIK6, etc.), mouse Ang2 derived from a mouse (Accession # NP — 031452, Accession # 035608, etc.), rat Ang2 derived from a rat (eg, NCBI Accession No. 035462, etc.), and the like, but are not limited thereto.

The anti-VEGF-C / anti Ang2 bispecific antibody may comprise an anti VEGF-C antibody and an anti Ang2 antibody in the form of a complete immunoglobulin (eg, IgG). In this case, the anti-VEGF-C antibody and anti-Ang2 antibody may be in the form of being connected to each other at the N terminal or C terminal. In another embodiment, the anti-VEGF-C / anti Ang2 bispecific antibody has an anti-VEGF-C antibody in the form of a complete immunoglobulin (eg, IgG) and an antibinding ability to Ang2 linked to the N or C terminus of the antibody. It may be one comprising an antigen binding fragment (eg scFV) of an Ang2 antibody. In another example, the anti-VEGF—C / anti Ang2 bispecific antibody is an anti VEGF that retains the ability to bind an anti Ang2 antibody in the form of a complete immunoglobulin (eg, IgG) to VEGF-C linked to the N or C terminus of the antibody. And may comprise an antigen binding fragment (eg scFv) of an -C antibody. More specifically, the anti-VEGF-C / anti Ang2 bispecific antibody is the N terminus or C terminus of the antibody in the form of a complete immunoglobulin (eg, IgG) and the immunoglobulin form, specifically the C terminus of the heavy chain of each monomer. It may be an up-down asymmetric bispecific antibody including antigen-binding fragments (eg, fragments in scFv form) each bound thereto. In this case, the immunoglobulin type antibody has a binding site for VEGF-C and the antigen binding fragment (eg, a scFv form fragment) has a binding site for Ang2 (see FIG. 1), or the immune An antibody in globulin form may have a binding site for Ang2 and the antigen binding fragment (eg, a fragment in scFv form) may have a binding site for VEGF-C.

In the anti-VEGF-C / anti Ang2 bispecific antibody, the anti VEGF-C antibody or antigen binding fragment thereof and the anti Ang2 antibody or antigen binding fragment thereof may be linked via or without a linker such as a peptide linker. In addition, the heavy and light chain portions in the antigen binding fragment, such as the heavy and light chain variable regions in the scFV fragment, can also be linked via or without a peptide linker. The peptide linker linking the anti-VEGF-C antibody or antigen-binding fragment thereof and the anti Ang2 antibody or antigen-binding fragment thereof and the peptide linker linking the heavy and light chain portions in the antigen-binding fragment may be the same or different. The peptide linker is 1 to 100 or 2 It may be a polypeptide consisting of from 50 to 50 amino acids, the amino acid type included there is no limitation. The peptide linker is, for example, _{G 1} y,

Asn and / or Ser residues may be included, and neutral amino acids such as Thr and / or Al a may also be included. Suitable amino acid sequences for peptide linkers are known in the art. On the other hand, the linker may determine the length of the linker, as long as it does not affect the function of the bispecific antibody. For example, the peptide linker may be made of one or more selected from the group consisting of Gly, Asn, Ser, Thr, and Al a, including 1 to 100, 2 to 50, or 5 to 25. In one example, the peptide linker is (G4S) n (n is a repeating number of (G4S)), it may be represented by an integer of 1 to 10, for example, an integer of 2 to 5).

Specifically, the anti Ang2 antibody or antigen-binding fragment thereof may be one that inhibits binding of Ang2 and Ti e2 receptor by competing with Ti e2 receptor in binding to Ang2, for example, with Ti e2 receptor of An _g 2 It may be to inhibit the binding of Ang2 and Tie2 receptor by recognizing and / or binding the binding site of.

In addition, the anti Ang2 antibody or antigen-binding fragment thereof may further have an activity of inhibiting binding between Ang2 and integrin. The integr in is a representative protein that mediates cell adhesion, and is a heterodimer structure comprising an alpha (α) subunit and a beta (β) subunit. In mammals, 18 alpha subunits and 8 beta subunits have been identified. The integrin may be derived from mammals such as humans, primates such as monkeys, rodents such as mice, rats, and the like, and may be, for example, human integrins, native integrins, mouse integrins, rats integrins, and the like. Twenty-four integrins are known for each species, and their amino acid sequences are well identified and are obvious to those of ordinary skill in the art. For example, the integrin may be human integrin, representative integrin Alpha5betal (α5β1) (α5: NCBI Accession No. P08648, βΐ: NCBI Accession No. P05556), alphaVbetaK aV131) (aV: NCBI Accession No. P06756, βΐ: NCBI Accession No. P05556), and alphaVbeta3 (ανβ3 (aV: NCBI Accession No. P06756, β3: NCBI Accession No. P05106), and the like, but are not limited thereto.

In one embodiment, the anti Ang2 antibody or antigen-binding fragment thereof is loop 1 (sites from 417 to 434 of SEQ ID NO: 70) of human Ang2 (hAng2; SEQ ID NO: 70; Accession # 015123), loop 2 (Sites from 447 amino acids to 454 amino acids of SEQ ID NO: 70), and all or part of a portion consisting of loop 3 (sites from 460 amino acids to 468 amino acids of SEQ ID NO: 70) (eg, the outer portion of each loop) At least one selected from the group consisting of amino acid residues exposed to C), or at least 2 to 20 contiguous one or more amino acid residues exposed outside of Loop 1, Loop 2, or Loop 3 in SEQ ID NO: 70 Recognize as an epitope an amino acid sequence region comprising from 15 to 15, from 2 to 10, or from 2 to 5 amino acids, or specifically at this site It may be joined to. .

For example, the anti Ang2 antibody or antigen-binding fragment thereof is at least one amino acid residue selected from the group consisting of 1434 located in loop 1 of human Ang2, A449 and P452 located in loop 2, and N467 located in loop 3, or Recognize and / or specifically bind an amino acid sequence region comprising two to twenty, two to fifteen two to ten, or two to five amino acids comprising one or more amino acid residues as an epitope It may be. In one embodiment, the anti Ang2 antibody or antigen-binding fragment thereof comprises one or more amino acid residues selected from the group consisting of 1434 located in loop 1 of human Ang2, A449 and P452 located in loop 2, and N467 located in loop 3 It may be recognized as an epitope or specifically bound to this part. [Ang2 (SEQ ID NO: 70; Loop 1, Loop 2, and Loop 3 are each underlined in order, and each epitope is bold)]

MWQIVFFTLS CDLVLAAAYN NFRKSMDSIG KQYQVQHGS CSYTFLLPEM DNCRSSSSPY VSNAVQRDAP LEYDDSVQRL QVLENIMENN TQWLMKLENY IQD 顺 KKEMV EIQQNAVQNQ TAVMIEIGTN LLNQTAEVTR LTDVEA

NQTTRLELQL LEHSLSTNKL EKQILDQTSE INKLQDKNSF LEKKVLAMED KHI IQLQSIK EEKDQLQVLV SKQNSI IEEL EKKIVTATVN NSVLQKQQHD LMETVNNLLT 画 STSNSAKD PTTAKEEQIS FRDCAEVGITL

TFPNSTEEIK AYCDMEAGGG GWTI IQRRED GSVDFQRTWK EYKVGFGNPS GEYWLGNEFV SQLTNQQRYV LKIHLKDWEG NEAYSLYEHF YLSSEELNYR

IHL GLTGTA GKISSI SQPG NDFSTKDGDN DKC I CKCSQM LTGGWWFDAC GPSNLNGMYY PQRQNTNKFN GIKWYYWKGS GYSLKAT 1M IRPADF

The epitope region is an exposed amino acid residue located at loops 1, 2, or 3 of the three-dimensional structure of Ang2, and directly participates in binding to the Ti e2 receptor, or is located at or adjacent to the binding site with the Ti e2 receptor. do. Therefore, the anti Ang2 antibody or antigen-binding fragment thereof that recognizes and binds one or more of the epitopes competes with the Tie2 receptor in binding to Ang2, thereby inhibiting the binding of Ang2 and Ti e2 receptor.

The "adjacent amino acids" may refer to amino acids located adjacent to each other on the secondary structure or the tertiary structure, as well as the primary structure of the protein. Accordingly, the term "adjacent amino acid" in the present specification may mean an amino acid residue that is contiguous on the primary, secondary or tertiary structure of the protein, and also recognizes and / or specifically binds the epitope region. In addition to the anti-Ang2 antibody, the antibody or antigen-binding fragment thereof that competitively binds the anti-Ang2 antibody can also compete with the Ti e2 receptor in binding to Ang2, thereby inhibiting the binding of Ang2 and Tie2 receptor. May be an antibody that recognizes an epitope as described above and an adjacent site on a three-dimensional structure as an epitope. The binding antibody may have a binding affinity (Kd) of Ang2 of 0.001 to ΙΟη ^ specifically 0.01 to 1 nM, more specifically 0.1 to 0.8 nM.

Thus, the anti-Ang2 antibody or antigen-binding fragment thereof may be selected from the group consisting of an antibody or antigen-binding fragment thereof that recognizes and / or specifically binds the epitope described above, and an antibody or antigen-binding fragment thereof that competitively binds Ang2. It may be one or more selected.

In embodiments, the anti-An _g 2 antibody or antigen-binding fragment thereof is a polypeptide having the amino acid sequence of SEQ ID NO: 49 as the complementarity determining region (CDR) of the heavy chain (CDR-HI) , A polypeptide having an amino acid sequence of SEQ ID NO: 50 (CDR-H2), and a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 17-24, such as SEQ ID NOS: 17-22 from the group consisting of -H3) may be one containing one or more selected: ^■

Xi-YX ₂ -MS (SEQ ID NO: 49)

X ₃ -IX ₄ — X ₅ -X ₆ -X Figure X ₈ -X ₉ -X ₁₀ -YYADSVKG (SEQ ID NO: 50)

In SEQ ID NO: 49,

¾ is aspartic acid (D), serine (S), or asparagine (N), such as aspartic acid (D) or asparagine (N),

¾ is alanine (A), aspartic acid (D), or tyrosine (Y),

In SEQ ID NO: 50,

¾ is alanine (A), glycine (G), leucine (L), or serine (S), for example alanine (A), glycine (G), or serine (S),

X ₄ is tyrosine (Y) or serine (S),

¾ is proline (P), histidine (H), or serine (S),

¾ is aspartic acid (D), glycine (G), or serine (S),

X? Is serine (S), glycine (G), or aspartic acid (D),

¾ is glycine (G) or serine (S),- ¾ is asparagine (N) or serine (S),

X ₁₀ is lysine 00, isoleucine (I), or threonine (T).

In addition, the anti Ang2 antibody or antigen-binding fragment thereof is a light chain complementarity determining site, the polypeptide having the amino acid sequence of SEQ ID NO: 51 (CDR-L1), a poly having the amino acid sequence of SEQ ID NO: 52 It may comprise one or more selected from the group consisting of a peptide (CDR-L2) and a polypeptide having an amino acid sequence of SEQ ID NO: 53 (CDR 'L3):

X _u -GS— S— SN— IGX ₁₂ -N-¾— VX ₁₄ (SEQ ID NO: 51)

Xi5- i6-Xi7-Xi8-R-P-S (SEQ ID NO: 52)

Xi9-X2o-WDX ₂ iSLX ₂₂ -X ₂₃ (SEQ ID NO: 53)

In SEQ ID NO: 51,

乂 ^ is serine (S) or threonine (T),

2 is asparagine (N) or serine (S),

( ₁₃ is alanine (A), tyrosine (Y), or aspartic acid (D),

4 is asparagine (N), serine (S), threonine (T), or tyrosine (Y), and in SEQ ID NO: 52,

Χι ₅ is alanine (A) or serine (S),

6 is aspartic acid (D) or asparagine (N),

7 is serine (S) or asparagine (N), such as serine (S),

} ₁₈ is asparagine (N), lysine (K), histidine 00, or glutamine (Q),

In SEQ ID NO: 53,

9 is glycine (G) or alanine (A),

X ₂₀ is serine (S), alanine (A), or threonine (T), such as serine (S) or threonine (T),

( ₂₁ is tyrosine (Y), or aspartic acid (D), such as tyrosine (Y), ¾ ₂ is serine (S) or asparagine (N), such as serine (S), ¾ ₃ is glycine (G) or alanine (A).

In an embodiment, the anti Ang2 antibody or antigen-binding fragment thereof may be one comprising the heavy chain complementarity determining site, light chain complementarity determining site, or a combination thereof described above.

More specifically, the anti Ang2 antibody or antigen-binding fragment thereof includes a polypeptide having an amino acid sequence of SEQ ID NO: 49 (CDR-H1), a polypeptide having an amino acid sequence of SEQ ID NO: 50 (CDR-H2), and SEQ ID NOs: 17 to 24 At least one heavy chain complementarity determining site selected from the group consisting of an amino acid sequence selected from the group consisting of a polypeptide (CDR-H3) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 17 to 22, or the at least one heavy chain complementarity determining site A heavy chain variable region comprising a;

A group consisting of a polypeptide having an amino acid sequence of SEQ ID NO: 51 (CDR-L1), a polypeptide having an amino acid sequence of SEQ ID NO: 52 (CDR-L2), and a polypeptide having an amino acid sequence of SEQ ID NO: 53 (CDR ′ L3) One or more light chain complementarity determining sites selected from or a light chain variable region comprising the one or more light chain complementarity determining sites;

A combination of said at least one heavy chain complementarity determining site and at least one light chain complementarity determining site; or

Combination of the heavy chain variable region and light chain variable region

It may be to include.

Specifically, the reprint CDR of an anti Ang2 antibody may be, for example, having the amino acid sequence of Table ^' 1 below.

Table 1

NYAMS (SEQ ID NO: 3) AISSGGGNIYYADSVKG (SEQ ID NO: 11) AKSGIQPSPPSMSSAYAMDV (SEQ ID NO: 19)

DYAMS (SEQ ID NO: 4) SIYPDDGNTYYADSVKG (SEQ ID NO: 12) ARHTSHHTSIDGYYYYGMDG (SEQ ID NO: 20)

DYDMS (SEQ ID NO: 5) SISHGDSNKYYADSVKG (SEQ ID NO: 13) AKSSGIQESPPTYYYYGMDV (SEQ ID NO: 21)

DYAMS (SEQ ID NO: 6) SIYPDDGNTYYADSVKG (SEQ ID NO: ⁴ ) AKHPVRLNLHPMYYYYGMDV (SEQ ID NO: 22)

SYDMS (SEQ ID NO: 7) USPDSSSIYYADSVKG (SEQ ID NO: 15) MDLISFWRGGFDY (SEQ ID NO: 23)

DYDMS (SEQ ID NO: 8) GISSDDGNTYYADSVKG (SEQ ID NO: 16) ARPTIDKYTLRGYYSYGMDV (SEQ ID NO: 24) The light chain CDRs of the anti Ang2 antibody may also be, for example, those having the amino acid sequence of Table 2 below.

Table 2

An amino acid sequence selected from SEQ ID NOs: 1-8, such as SEQ ID NOs: 1-8. A polypeptide having an amino acid sequence selected from 6 (CDR-H1), an amino acid sequence selected from SEQ ID NOs: 9-16, such as a polypeptide (CDR-H2) having an amino acid sequence selected from SEQ ID NOs: 9-14, and SEQ ID NO: 17 At least one heavy chain complementarity determining site selected from the group consisting of a polypeptide having an amino acid sequence selected from: 24 to 24, for example, SEQ ID NOs: 17 to 22 (CDR—H3), or a heavy chain variable region comprising the same;

A polypeptide having an amino acid sequence selected from SEQ ID NOs: 25 to 32, such as an amino acid sequence selected from SEQ ID NOs: 25 to 30, (CDR-L1), A polypeptide having an amino acid sequence selected from SEQ ID NOs: 33 to 40, such as a polypeptide having an amino acid sequence selected from SEQ ID NOs: 33 to 38, and an amino acid sequence selected from SEQ ID NOs: 41 to 48, such as amino acids selected from SEQ ID NOs: 41 to 46 One or more light chain complementarity determining sites selected from the group consisting of a polypeptide having a sequence (CDR-L3) or a light chain variable region comprising the same;

Combination of the heavy chain variable region and light chain variable region

It may be to come.

In one example, the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 54 to 61, such as an amino acid sequence selected from the group consisting of SEQ ID NO: 54 to 59, the light chain variable region consists of SEQ ID NOs: 62 to 69 It may include an amino acid sequence selected from the group consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 62 to 67. Accordingly, the anti Ang2 antibody or antigen-binding fragment thereof may comprise a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 54 to 61, such as an amino acid sequence selected from the group consisting of SEQ ID NOs: 54 to 59; A light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 62 to 69, such as an amino acid sequence selected from the group consisting of SEQ ID NOs: 62 to 67; Or a combination of the heavy chain variable region and the light chain variable region.

In another example, an anti Ang2 antibody or antigen-binding fragment thereof may be a polypeptide having an amino acid sequence of SEQ ID NO: 7 or 8 (CDR-H1), a polypeptide having an amino acid sequence of SEQ ID NO: 15 or 16 (CDR-H2) And one or more heavy chain complementarity determining sites selected from the group consisting of a polypeptide having an amino acid sequence of SEQ ID NO: 23 or 24 (CDR-H3) or a heavy chain variable region comprising the same; A polypeptide having an amino acid sequence of SEQ ID NO: 31 or 32 (CDR-L1), a polypeptide having an amino acid sequence of SEQ ID NO: 39 or 40 (CDR-L2), and a polypeptide having an amino acid sequence of SEQ ID NO: 47 or 48 ( CDR ′ L3) at least one light chain complementarity determining site selected from the group consisting of or a light chain variable region comprising the same;

Combination of the heavy chain variable region and light chain variable region

It may be to include.

In one example, the heavy chain variable region may comprise an amino acid sequence of SEQ ID NO: 60 or 61, the light chain variable region may comprise an amino acid sequence of SEQ ID NO: 68 or 69.

Thus, the anti Ang2 antibody or antigen-binding fragment thereof may comprise a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 60 or 61; Light chain variable region comprising the amino acid sequence of SEQ ID NO: 68 or 69; Or it may be one comprising the combination of the heavy ^chain, variable region and light chain variable region.

In one embodiment, the anti-VEGF-C antibody or antigen-binding fragment thereof, CDR-H1 comprising the amino acid sequence of SEQ ID NO: 89, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 90, and the amino acid sequence of SEQ ID NO: 91 One or more heavy chain complementarity determining regions selected from the group consisting of CDR-H3 comprising; or a heavy chain variable comprising the one or more heavy chain complementarity determining regions

-ᄇ Τ "τ1 · ，

One or more light chain complementarity determining regions selected from the group consisting of: CDR—L1 comprising the amino acid sequence of SEQ ID NO: 92, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 93, and CDR ′ L3 comprising the amino acid sequence of SEQ ID NO: 94 Or a light chain variable region comprising said at least one light chain complementarity determining region;

Said at least one heavy chain complementarity determining region and said at least one light chain Combination of complementarity determining regions; or

Combination of the heavy chain variable region and the light chain variable region

It may be to include.

The amino acid sequence of each CDR of said anti-VEGF-C is summarized in Table 3 below.

Table 3

For example, the anti-VEGF-C antibody or antigen-binding fragment thereof may comprise a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 95, a light chain variable region comprising the amino acid sequence of SEQ ID NO: 96, or a combination thereof. It may be to include.

SEQ ID NO: 95 Heavy chain variable region of anti-VEGF-C antibody>

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMS VRQAPGKGLEWVSAISYDNGSTYYADSV KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDPYLARLNTFDYWGQGTLVTVSS

(The parts shown in bold in the sequence are CDR sites, in order CDR-Hl, CDR-H2, and CDR—H3)

SEQ ID NO: 96 Light chain variable region of anti-VEGF-C antibody>

QSVLTQPPSASGTPGQ VTISCSGSSSNIGSNNVSWYQQLPGTAPKLL I YYNSHRPSGVPDRFS GSKSGTSASLA I SGLRSEDEADYYCATWDSSLNGYVFGGGTKLT

(The parts shown in bold in the sequence are CDR sites, and in turn are CDR-L1, CDR-L2, and CDR-L3)

As used herein, the term "antibody" refers to a substance produced by stimulation of an antigen in the immune system, and includes not only those produced in vivo but also those produced synthetically or recombinantly. It doesn't work. In the present invention, the antibody refers to an antibody having an antigen binding ability. Antigen binding fragments are also included. Meanwhile, "Complementarity Determining Area

(Complementarity-determining regions, CDR) "means a site that confers binding specificity with the antigen among the variable regions of the antibody. The antigen-binding fragment of the antibody described above is an antibody fragment comprising at least one of the complementarity determining regions. For example, it may be selected from the group consisting of scFv, (scFv) 2, scFv-Fc, Fab, Fab ¹ and F (ab ') 2.

In the anti-Ang2 antibody or antigen-binding fragment thereof and the anti-VEGF-C antibody or antigen-binding fragment thereof, the remaining regions except for the heavy chain CDRs and light chain CDR regions defined above, or the heavy chain variable region and the light chain variable region are all subtypes of immunoglobulins ( For example, it may be derived from IgA, IgD, IgE, IgG (IgGl, IgG2, IgG3, IgG4), IgM, etc.), for example from the light chain constant region and / or heavy chain constant region of all the subtypes of immunoglobulins. It may be derived.

Animal-derived antibodies produced by immunizing a desired antigen with an immunized animal are generally capable of immunorejection when administered to humans for therapeutic purposes, and chimeric antibodies have been developed to suppress such rejection. Chimeric antibodies are those in which the unchanged regions of animal-derived antibodies that cause anti-isotype reactions are replaced with the constant regions of human antibodies using genetic engineering methods. Chimeric antibodies have significantly improved anti-isotype reactions compared to animal-derived antibodies, but still contain side effects for potential anti-idiotypic reactions, as animal-derived amino acids are present in the variable region. Doing. Humanized antibodies have been developed to improve these side effects. It is prepared by implanting a region of the antibody region, which plays an important role in the binding of antigens in the variable regions of chimeric antibodies, to the human antibody framework.

The most important aspect of CDR grafting technology for the production of humanized antibodies is that it is best able to accept the CDR regions of animal-derived antibodies. In order to select an optimized human antibody, the use of an antibody database, analysis of crystal structures, molecular modeling techniques, and the like are utilized. However, even when the CDR region of an animal-derived antibody is implanted into an optimized human antibody skeleton, the amino acid located in the skeleton of an animal-derived antibody may exist and affect antigen binding. As such, the application of additional antibody engineering techniques to restore antigen binding capacity is essential.

In the present invention, the antibody includes all animal antibodies, chimeric antibodies, humanized antibodies, or human antibodies. Specifically, 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. For example, the antibody or antigen-binding fragment thereof may be produced synthetically or recombinantly. The antibody may be a monoclonal antibody.

A complete antibody is a structure having two full length light chains and two full length heavy chains, each of which is linked by heavy and disulfide bonds. The constant region of the antibody is divided into a heavy chain constant region and a light chain constant region, and the heavy chain constant region has a gamma (γ), mu (μ), alpha (α), delta (δ) and epsilon (ε) type, subclass Gamma 1 (γ1), gamma 2 ( _Υ2 ), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1) and alpha 2 (α2). The constant region of the light chain has kappa ( _κ ) and lambda (λ) types.

The term "heavy chain" refers to the variable region domain V _H and the three constant region domains C _H1 , C _H2 and C _H3 and the hinge, comprising an amino acid sequence having sufficient variable region sequence to confer specificity to the antigen ( It is interpreted to include both the full length heavy chain including the hinge and fragments thereof. The term “light chain” also refers to both the full length light chain and fragment thereof comprising the variable region domain V _L and the variable region domain C _L comprising an amino acid sequence having sufficient variable region sequence to confer specificity to the antigen. In the sense to include Interpreted

The term "complement ar i ty determining region" (CDR) refers to the amino acid sequence of the hypervariable regions of the heavy and light chains of immunoglobulins. The heavy and light chains may each comprise three CDRols (CDRH1, CDRH2 CDRH3 and CDRLl, CDRL2, CDRL3). The CDRs can provide key contact residues for the antibody to bind antigen or epitope. On the other hand, in the present specification, the term "specifically binds" ¹ or "specifically recognized"'is the same as that commonly known to those skilled in the art, antigen and antibody specifically interacts with immunological reactions It means to do.

The "antigen binding fragment"'is a fragment thereof for the entire structure of the immunoglobulin, and refers to a portion of a polypeptide including a portion to which an antigen can bind, for example, scFv, (scFv) _2l scFv-Fc, Fab, Fab 'or F (ab') ₂ , but is not limited to such.

The antigen-binding fragment of the antibody in the present invention is an antibody fragment comprising at least one of the complementarity determining regions, for example, in the group consisting of scFv, (scFv) 2, scFv-Fc, Fab, Fab ¹ and F (ab ') 2. It may be selected.

Fab in the antigen-binding fragment has one antigen binding site in a structure having variable regions of the light and heavy chains, a constant region of the light chain and the first constant region of the heavy chain (C _H1 ).

Fab ¹ differs from Fab in that it has a hinge region comprising at least one cysteine residue at the C-terminus of the heavy chain C _H1 domain. F (ab ') ₂ antibodies are produced when the cysteine residues of the hinge region of Fab' form disulfide bonds.

Recombinant techniques for generating Fv fragments with minimal antibody fragments in which F has only heavy and variable chain regions are well known in the art. Double-chain Fv (two-chain Fv) is a non-covalent bond in which the heavy chain variable region and the light chain variable region are linked, and the single chain FV (single chain Fv) is generally shared by the variable region of the heavy chain and the single chain variable region through a peptide linker. It may be linked by a bond or directly at the C-terminus to lead to a dimer-like structure, such as a double chain Fv. The peptide linker may be as described above, and may be, for example, 1 to 100, for example, 2 to 50 or 5 to 25 amino acids in length, and the amino acid type included therein is not limited.

The antigen binding fragments can be obtained using proteolytic enzymes (e.g., by limiting cleavage of the entire antibody to papain, a Fab can be obtained and pepsin cleavage can yield an F (ab ') ₂ fragment). It can be produced through genetic recombination technology.

The term “hunge region” refers to a region included in the heavy chain of an antibody, which exists between the CH1 and CH2 regions and functions to provide flexibility of the antigen binding site in the antibody.

When an animal-derived antibody undergoes chimerization, an IgGl hinge derived from an animal is replaced by a human IgGl hinge, but an animal-derived IgGl hinge is shorter than a human IgGl hinge and has a disulfide bond between two heavy chains ( Disulfide bonds are reduced from three to two, resulting in different rigidity of the hinges. Thus, modification of the hinge region can increase the antigen binding efficiency of the humanized antibody. Deletion, addition or substitution of amino acids for modifying the amino acid sequence of the hinge region is well known to those skilled in the art.

The anti Ang2 antibody and / or anti VEGF-C antibody may be a monoclonal antibody. Monoclonal antibodies can be prepared according to methods well known in the art. For example, it can be manufactured using a phage display technique.

On the other hand, individual monoclonal antibodies can be screened based on their ability to bind Ang2 using a typical EL ISA (Enzyme—Linked I unoSorbent Assay) format. Assays can be assayed for inhibitory activity through functional assays such as competitive ELISA or cell-based assays for assaying molecular interactions on conjugates. Then for the selected monoclonal antibody members based on strong inhibitory activity

Test each affinity (Kd values) for Ang2.

In an embodiment, the anti VEGF-C / anti An _g 2 bispecific antibody is an antigen binding fragment of an anti VEGF-C antibody and an anti Ang2 antibody linked to the C terminus of the anti VEGF-C antibody, ie scFv, (scFv ) ₂ , Fab, Fab ¹ or F (ab ') ₂ , such as scFv. As long as the antigen-binding fragment of the anti-Ang2 antibody maintains the binding ability to Ang2, any fragment including at least one of the CDRs for Ang2 shown above may be used. For example, scFv, (scFv) 2, Fab, Fab 'or F (ab') ₂ of the anti Ang2 antibody is a heavy chain variable region and SEQ ID NO: 62 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 54 to SEQ ID NO: 61 To light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 69.

Thus, in one embodiment, the anti-VEGF-C / anti Ang2 bispecific antibody is an amino acid selected from the group consisting of anti-VEGF-C antibodies and SEQ ID NO: 54 to SEQ ID NO: 61 linked to the C terminus of the anti VEGF-C antibody ScFv, (scFV) ₂ , Fab, Fab 'or F (ab) of an anti Ang2 antibody comprising a heavy chain variable region comprising a sequence and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 62 to 69 ') ₂ may be included.

In another embodiment, the anti VEGF-C / anti Ang2 bispecific antibody is an anti Ang2 antibody, and an scFv, (scFv) ₂ , Fab, Fab 'or F of an anti VEGF— C antibody linked to the C terminus of the anti Ang2 antibody. (ab ') ₂ , such as including scFv. For example, the scFv, (scFv) ₂ , Fab, Fab 'or F (ab') ₂ of the anti-VEGF-C antibody is at least one of the CDRs for VEGF-C presented above as long as it maintains avidity for VEGF ᅳ C. All fragments including can be used.

The anti-VEGF-C / anti Ang2 bispecific antibody has superior anti-cancer effects such as inhibiting tumor cell migration and inhibiting tumor cell proliferation, as compared to antibodies that inhibit Ang2 or VEGF-C alone, It is also remarkable compared with the case of co-administration with antibodies that independently inhibit VEGF-C. Has a synergistic effect. In addition, the anti-VEGF-C / anti Ang2 bispecific antibody can reduce the dose and / or intervals between administrations compared to the single or combined administration of the antibody that inhibits Ang2 or VEGF-C alone, side effects of the drug There is an advantage that can be reduced. Anti-VEGF-C / anti Ang2 bispecific antibodies are also desired for cancer patients or individual patients that are resistant to existing Ang2 inhibitors (eg anti Ang2 antibodies) or VEGF-C inhibitors (eg anti VEGF-C antibodies). Can be effective.

Another example provides a pharmaceutical composition comprising the anti-VEGF-C / anti Ang2 bispecific antibody.

Specifically, one embodiment provides a pharmaceutical composition for preventing and / or treating diseases related to Ang2 and / or VEGF-C comprising the anti-VEGF-C / anti Ang2 bispecific antibody as an active ingredient.

Another example provides a method of preventing and / or treating a disease associated with Ang2 and / or VEGF-C comprising administering to a patient a pharmaceutically effective amount of said anti-VEGF-C / anti Ang2 bispecific antibody. The patient may be a patient in need of prevention and / or treatment of diseases associated with Ang2 and / or VEGF-C. The method of preventing and / or treating cancer may further comprise identifying a patient in need of prevention and / or treatment of cancer prior to the administering step.

Another example is provided for the use of the anti-VEGF-C / anti Ang2 bispecific antibody for the prevention and / or treatment of cancer.

The disease associated with Ang2 and / or VEGF-C is a disease involving the Ang2 / Ti e2 signaling system and the VEGF-C / VEGFR signaling system, and overactivation or overproduction of Ang2 and / or VEGF—C (overexpression) Or a disease caused by abnormal action. For example, diseases associated with Ang2 and / or VEGF-C include cancer; Cancer metastasis; Dark infiltration / invasion; Eye diseases such as macular degeneration (eg, age-related macular degeneration), diabetic retinopathy, inflammatory diseases such as psoriasis, rheumatoid arthritis, chronic inflammation, and sepsis; Malaria; The cancer may be a solid cancer or a hematologic cancer and may be a cancer secreting Ang2 and / or VEGF ᅳ C. The cancer may include, but is not limited to, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, skin cancer, skin or intraocular myeloma, rectal cancer, anal muscle cancer, Esophageal cancer, small bowel cancer, endocrine cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, chronic or acute leukemia ᅳ lymphocyte lymphoma, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, neck cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, Colon cancer, colon cancer, endometrium or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, head and neck cancer, brain cancer, osteosarcoma and the like. Such cancers include primary cancer as well as metastatic cancer.

In the pharmaceutical composition or method, the pharmaceutically effective amount of the anti-VEGF-C / anti Ang2 bispecific antibody is provided with at least one additive selected from the group consisting of pharmaceutically acceptable carriers, diluents, excipients, and the like. Can be.

The pharmaceutically acceptable carrier is commonly used in the preparation of antibodies, lactose, dextrose, sucrose, sorbbi, manny, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, fine 1 type selected from the group consisting of crystalline cellulose, polyvinylpyridone, cellulose, water, syrup, methyl cellulose, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate, mineral oil, etc. It may be more than, but is not limited thereto. In addition to the above components, the pharmaceutical composition may include at least one member selected from the group consisting of diluents, excipients, lubricants, wetting agents, sweetener flavoring agents, emulsifiers, suspending agents, preservatives, and the like, which are commonly used in preparing pharmaceutical compositions. It can be included as.

The pharmaceutical composition, the anti-VEGF-C / anti Ang2 bispecific antibody can be administered orally or parenterally. In the case of parenteral administration, it can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, pulmonary administration and rectal administration. When taken orally, protein or Since the peptide is digested, the oral composition must be formulated to coat the active agent or to protect it from degradation in the stomach. In addition, the composition may be administered by any device in which the active agent may migrate to the target cell.

Appropriate dosages of the pharmaceutical composition or the anti-VEGF-C / anti Ang2 bispecific antibody may be determined by the method of formulation, mode of administration, age, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion and response of the patient. It can be prescribed in various ways by factors such as sensibility. Preferred dosages of the compositions are in the range of 0.001 to 100 mg / kg on an adult basis. For example, the daily dosage of the pharmaceutical composition or the anti-VEGF-C / anti Ang2 dual specific antibody may range from 0.001 to 1000 mg / kg, specifically from 0.01 to 100 mg / kg, more specifically from 0.1 to 50 _mg / kg May be, but is not limited thereto. The daily dosage may be formulated into one formulation in unit dosage form, may be formulated in appropriate quantities or may be prepared by incorporation into a multi-dose container.

The term “pharmaceutically effective amount” means an amount in which the active ingredient (ie, the anti VEGF-C / anti Ang2 bispecific antibody) can exhibit the desired effect, i.e., the effect of preventing and / or treating cancer, and formulating It can be prescribed in various ways, such as by method, mode of administration, age, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion and response.

The pharmaceutical composition may be prepared in unit dose form or formulated into a multi-dose container by formulating with a pharmaceutically acceptable carrier and / or excipient, according to methods readily available to those skilled in the art. . The formulation may be in the form of solutions, suspensions, syrups or emulsions in oil or aqueous media, or may be in the form of extracts, powders, powder granules, tablets or accelerators, and may further comprise dispersants or stabilizers.

In addition, the pharmaceutical composition may be administered as a separate therapeutic agent or other It may be administered in combination with a therapeutic agent, and may be administered sequentially or simultaneously with a conventional therapeutic agent.

On the other hand, since the pharmaceutical composition comprises an antibody or antigen-binding fragment, it can be formulated with an immune liposome. Liposomes comprising the antibody can be prepared according to methods well known in the art. The immune liposomes can be prepared by reverse phase evaporation as a lipid composition comprising phosphatidylcholine, cholesterol and polyethyleneglycol-derivatized phosphatidylethane amines. For example, Fab 'fragments of antibodies can be conjugated to liposomes via disulfide replacement reactions. Chemotherapeutic agents such as doxorubicin may further be included in the liposomes.

Subjects to which the pharmaceutical composition or the anti-VEGF-C / anti Ang2 bispecific antibody is administered or subject to the prophylactic and / or therapeutic method are mammals including primates such as humans and monkeys, or rodents such as rats and mice, or the like. It may be selected from the group consisting of cells, tissues and the like derived from these, but is not limited thereto.

Another example provides a composition for detecting Ang2 and / or VEGF-C comprising the anti-VEGF-C / anti Ang2 bispecific antibody and a pharmaceutical composition for diagnosing Ang2 and / or VEGF-C related diseases. In another example, treating a biological sample with the anti-VEGF-C / anti Ang2 bispecific antibody; And it provides a method for detecting Ang2 and / or VEGF-C comprising the step of identifying the antigen-antibody reaction, and a method for providing information on the diagnosis or diagnosis of An _g 2 and / or VEGF-C-related diseases. The detecting method, the diagnostic method or the method of providing information to the diagnosis may include Ang2 and / or VEGF-C present in the biological sample when the antigen-antibody reaction is detected after the step of confirming whether the antigen-antibody reaction is detected. the steps ^you, or a patient in which the biological sample is derived to determine that may further comprise a step of determining to have Ang2 and / or VEGF-C-related diseases. The biological sample is selected from the group consisting of (isolated) cells, tissues, body fluids (e.g. serum), or cultures thereof obtained from the patient. It may be. The patient to be detected or diagnosed may be a mammal including a human, a primate including a roundabout, a rodent including a mouse, a rat, and the like.

The step of determining whether the antigen-antibody reaction can be carried out through a variety of methods known in the art. For example, it can be measured by conventional enzymatic reaction, fluorescence, luminescence and / or radiation detection. Specifically, immunochromatography (I 腿 unochromatography),

Immunohistochemical staining (I 隱 unohi stochemi stry), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescence immunoassay ( Floresence immunoassay (FIA), luminescence immunoassay (LIA), Western blotting (Western blot ting) can be measured by a method selected from the group consisting of, but is not limited thereto.

Advantageous Effects

The anti-VEGF-C / anti Ang2 dual antibody of the present invention is a therapeutic agent having both characteristics of an inhibitor of Ang2 / Ti e2 signaling and an inhibitor of VEGF-C / HGF signaling, and has the following effects:

1. Improved efficacy and decreased dose level than Ang2 / Tie2 inhibitor and HGF / VEGF-C inhibitor alone.

2. Decreased dose concentrations when combined with Ang2 / Ti e2 inhibitors and HGF / VEGF-C inhibitors.

3. Used as a treatment for metastatic cancer as well as primary cancer

4. Application to other diseases involving Ang2 / Ti e2 signaling system and VEGF-C / VEGFR signaling system other than cancer

[Brief Description of Drawings]

1 is a schematic diagram showing the structure of an anti-VEGF-C / anti Ang2 bispecific antibody according to one embodiment.

2 shows anti-VEGF-C / anti Ang2 bispecific antibodies according to one embodiment A graph showing the degree of cell proliferation at the time of treatment.

3 is a graph showing the degree of cell migration when the anti-VEGF-C / anti Ang2 bispecific antibody treatment according to one embodiment.

[Form for implementation of invention]

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples. Example 1 Preparation of Anti-Ang2 Antibodies

1.1. Preparation of Ang2-binding scFv Clones

Fully human Ang2 antibodies were constructed using phage display scFv libraries (obtained from Ewha Womans University), for human Ang2 polypeptides (R & D systems; Human Ang2; Accession # 015123 (hAng2); SEQ ID NO: 70). The specific protocol is as follows.

Antibodies that react with ANG2 through primary, secondary and tertiary panning by angling the Ang2 polypeptide in maxifum immunotubes in amounts of about 10 ug (microgram) / ml, 1 ug / ml and 0.1 ug / ml, respectively The process of enriching was performed. After blocking the surface of the immunotubes using about 3? ¾ (v / v) milk dissolved in PBS, approximately 0.5 ml of 3% (v / v) milk derived from the same phage display scFv library described above ¹² phage particles were added and blocked together with incubation ^at 37 ^° C for 1 hour. After that, the milk-blocked phages were placed in an immunotube plated with Ang2, and incubated at room temperature for 1 hour to bind An _g 2 and phages.

After incubation of the phage, the phage surface was washed 3 to 5 times with PBS and about 0.1% (v / v) 20, and the bound phage was eluted with 100 mM triethanolamine. Eluted phages were infected and amplified into E. coli ER2537 cells (New England Biolabs, USA) and prepared for use in the next step of screening. This process maxithorpe the Ang2 polypeptide Immunotubes were plated in amounts of about 10 ug / ml, 1 ug / ml and 0.1 ug / ml, respectively, and repeated three times, followed by ELISA (Enzyme-L inked Immunosorbent Assay) affinity assay (Example 1.2). About 600 specific Ang2-binding scFv clones were recognized that recognize human Ang2 (Accession # 015123) and mouse Ang2 (Accession # NP_031452Ang2Ang2), as measured using reference).

1.2. Anti-Ang2 Antibody Production Clone Selection and Antibody Purification

Seventy clones producing anti-Ang2 antibodies were selected from the about 600 Ang2-binding scFv clones obtained in Example 1.1 above based on their ability to bind Ang2 using the ELISA format. Specifically, clones with high ELISA 0D were selected from the clones that can bind Ang2 and inhibit Tie2 binding. Each clone was then cultured in SB Media with ampicillin added up to 0D 600 = 1.0, injected with 1 mM IPTGdsopropyl-β-D- Thiogalactopyranoside, and then collected periplasm fractions. Anti-Ang2 monoclonal antibody was partially purified.

1.3. Ang2: Tie-2 neutralization ELISA (Competitive ELISA)

Competitive ELISAs were performed to assay molecular interactions on the conjugates. 96-well MaxiSorp ™ flat-bottom plates (Nunc) were coated with hTie2-Fc (R & D Systems), a protein that binds 4 ugyl il Fc of human IgGl. Then, the plate was washed five times with PBS (Phosphate Buffer Saline) containing 0.05¾ (v / v) Tween-20, followed by PBS containing l% (v / v) BSA (Bovine serum albumin; Sigma). It was blocked for 2 hours at room temperature.

In order to perform Ang2: Tie-2 neutralization ELISA, each scfv-type anti-Ang2 antibody (1, 10, 100, 1000 nM) purified in Example 2 was treated with 1% (ν / ν) of BSA and 400 After putting ng / ml FLAG ᅳ Tagging hAng2 into each well of the hTie2-Fc coated plate, the plate was allowed to react at room temperature for 2 hours. The plate is then 0.05 ¾ ^ Tween- After 5 washings with PBS containing 20, HRP-conjugated anti-FLAG antibody (SIGMA) was diluted with 1% (ν / ν) of BSA at 1: 5,000 ratio (v / v). Each well was added in an amount of 100 ul (microliter) and reacted at room temperature for 1 hour, followed by washing 5 times with PBS containing 0.1% (v / v) of Tween-20. Finally, by the addition of TMB substrate (cell signal) of 100 ul (microliter) to each well of the plate was derived the color development reaction was performed for 3 minutes at room temperature, ^° C. The reaction was then stopped with 50 ul of 5N H ₂ SO ₄ solution and the OD450 values were measured on a plate reader (Molecular Devices). Through this, 50% inhibition concentration (IC50) of the binding force of Ang2: Tie-2 was obtained and shown in Table 4 below.

Table 4

As shown in Table 4, it was confirmed that the anti-Ang2 antibody can increase the binding force between Ang2 and Tie_2 receptor (neutralization).

1.4. Binding ELISA of hAng2, mAng2,

For measuring the binding force to the respective antigens of the produced antibody

ELISA was performed. 96-well MaxiSorp ™ flat-bottom plates (Nunc) were coated with 5-20 ug / ml of human Ang2, mouse Ang2 (Mouse Ang2; Accession # NP_031452) (above R & D Systems). Then, the plate was washed five times with PBS containing 0.05% (v / v) Tween-20, and then blocked with PBS containing l% (v / v) BSA for 2 hours at room temperature. Of each of the prepared scFV forms After the anti-Ang2 antibody was placed in each well of the plate, the plate was reacted in silver for 2 hours.

Then, after washing five times with PBS containing 0.05% (v / v) of Tween-20, H-conjugated HA-probe Antibody (F-7) HRP conjugated antibody (Santacruz) was used. Dilute to 1: 1000 ratio (v / v) with PBS containing (v / v) BSA and add 50 ul to each well of the plate, and react for 1 hour at silver, 0.1¾) ( v / v) and washed 5 times with PBS containing Tween-20. Finally, 100 ul TMB substrate (ceil signal) was added to each well of the plate to induce color reaction for 3 minutes at room temperature, after which the reaction was stopped with 50ul of 5N H ₂ S0 ₄ solution and the value 0D450. Was measured on a plate reader (Molecular Devices). Through this, 50% binding concentration (Kd) for human Ang2 and mouse Ang2 proteins was obtained to determine the extent of anti-Ang2 antibody binding to each protein. The results obtained are shown in Table 5 below:

Table 5

A heavy chain of monoclonal antibody produced in each clone using a thermocycler (GeneAmp PCR System 9700, Applied Biosystem) from the respective antibody-producing E. coli glycerol stock obtained from the upper body selection result; The variable region gene sequence of the light chain was amplified:

PCR condition

5 minutes at 94 ^° C .;

[1 minute, 1 minute at 55 ^° C, 2 min at 72 ^° C at 94 ^° C] x 30 cycles;

6 minutes at 72 ^° C .;

Cooled to 4 ^° C

Primer: pC3X-f: 3'-GCACGACAGG1TTCCCGAC-5 '(SEQ ID NO: 87), pC3X-b: 3 ¹ -MCCATCGATAGCAGCACCG-5 ¹ (SEQ ID NO: 88).

PCR products obtained from each reaction were washed with a QIAquick Mult iwell PCR Purification kit (Qiagen) according to the manufacturer's protocol.

After cloning the obtained PCR results, DNA sequencing was performed by the disclosed method. As a result, the CDR sequences and variable regions shown in Tables 6 to 8 below could be obtained.

Table 6

Table 7

SAIT-ANG2-AB-8-A5 SGSSSNIGNNYVT ADSHRPS ATWDYSLSG (SEQ ID NO 26) (SEQ ID NO 34) (SEQ ID NO 42)

SAIT— ANG2-AB— 7-C9 SGSSSNIGNNDVY ANSHRPS GTWDYSLSG

(SEQ ID NO 27) (SEQ ID NO 35) (SEQ ID NO 43)

SAIT-ANG2-AB-4-C11 TGSSSNIGNNDVS SDSKRPS GSWDYSLSG

(SEQ ID NO 28) (SEQ ID NO 36) (SEQ ID NO 44)

SAIT-ANG2-AB-4-F5 SGSSSNIGSNAVN ADSNRPS GSWDYSLSG

(SEQ ID NO 29) (SEQ ID NO 37) (SEQ ID NO 45)

SAIT-ANG2-AB-4-F11 TGSSSNIGNNAVS SDSQRPS ATWDYSLSA

(SEQ ID NO: 30) (SEQ ID NO: 38) (SEQ ID NO: 46)

SAIT-A G2-AB-4-H10 SGSSSNIGSNYVN SDSHRPS GAWDDSLSG

(SEQ ID NO 31) (SEQ ID NO 39) (SEQ ID NO 47)

SAIT-ANG2-AB-3-D3 TGSSSNIGS YVS SDNKRPS GTWDDSLNG

(SEQ ID NO 32) (SEQ ID NO 40) (SEQ ID NO 48)

[Table 8]

IYYADSVKGRFTI SRDNS NTLYLQMNSL VPDRFSGSKSGTSASLA I SGLRSEDEAD

AEDTAVYYCAKDLISFWRGGFDYWGQGT YYCGSWDYSLSGYVFGGGTKLTVLG

LVTVSS (SEQ ID NO: 60) (SEQ ID NO: 68)

SAIT-ANG-2-AB-3-D3 EVQLLESGGGLVQPGGSLRLSCAASGFT QSVLTQPPSASGTPGQRVTISC GSSSNI

FSDYDMSWVRQAPG GLEWVSGISSDDGN GSNYVSWYQQLPGTAP LL IYSDNKRPSG TYYADSVKGRFT I SRDNSKNTLYLQMNSL VPDRFSGSKSGTSASLA I SGLRSEDEAD RAEDTAVYYCARPTIDKYTLRGYYSYGMD YYCGTWDDSLKLTVGGVV

.6. Preparation of Anti Ang2 scFV

Example 1 above. Anti-Hang2 scFv was prepared using the CDR of 4-H10 among the CDR sequences obtained in step 5. That is, the heavy chain variable region (SEQ ID NO: 60) comprising the amino acid sequence of SEQ ID NO: 7 (CDR— HI), the amino acid sequence of SEQ ID NO: 15 (CDR-H2), and the amino acid sequence of SEQ ID NO: 23 (CDR-H3), A light chain variable region (SEQ ID NO: 68) comprising SEQ ID NO: 31 (CDR-L1), SEQ ID NO: 39 (CDR-L2), and the amino acid sequence of SEQ ID NO: 47 (CDR— L3), and a peptide linker (GGGGSGGGGSGSGSGGS) Anti Ang2 scFv (SEQ ID NO: 99) was synthesized (Bi oneer).

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMS VRLAPGKGLEWVSLISPDSSSIYYADSV KGRFT I SRDNSKNTLYLQMNSLMEDTA YCAKDLISFWRGGFDYWGQGTLVTVSSGGGGSGGGGSGSGG SQSVLTQPPSASGTPGQRVT I SCSGSSSNIGSNYVNWYQQLPGTAPKLL I YSDSHRPSGVPDRFSGSKSGT SASLA I SGLRSEDEADYYCGAWDDSLSGYVFGGGTKLTVLGQ

Example 2. Anti-VEGF-C Antibody Preparation

2.2 VEGF-C-binding scFv clones

For human VEGF-C polypeptides (R & D systems; Human VEGF-C; Access i on # P49767 (hVEGF-C)), a full human VEGF-C antibody was prepared using a phage display scFV library (from Ewha Womans University). Produced. The specific protocol is as follows.

The VEGF-C polypeptides were plated in maxifum immunotubes in amounts of about 10 ug (mi crogram) / ml, 1 ug / ml and 0.1 ug / ml, respectively, through first, second and third panning. To VEGF-C _. The process of enr i ch Was performed. After blocking the surface of the immunotube with about 3% (v / v) milk dissolved in PBS ((Phosphate Buffer Saline), the same phage display scFv described above in about 0.5% of 3% (v / v) milk. About ¹² xl phage particles derived from the library were added and blocked together with incubation for 1 hour at 37 ^° C. Then, milk-blocked phages were placed in an immunotube plated with VEGF-C and incubated for 1 hour at room temperature. Treatment was performed to bind VEGF-C and phage.

The phage terms were washed three to five times with PBS and about 0.1% (v / v) 20 after treatment, and the bound phages were eluted using 100 mM triethanolamine. Eluted phages were infected and amplified into E. coli ER2537 cells (New England Biolabs., USA) and obtained for use in the next step of screening. This process was carried out three times after the VEGF-C polypeptide was plated on maxifum immunotubes in an amount of about 10 ug / ml, 1 Lig / ml and 0.1 ug / ml, respectively, followed by EL ISA ( About 600 specific VEGF-O binding scFv clones that recognize human VEGF_C (Accession # P49767) measured using the Enzyme-Linked I 瞧 unoSorbent Assay affinity assay (see Example 2.2 below) were identified.

2.2. Anti-VEGF-C Antibody Production Clone Selection and Antibody Purification

Twenty clones producing anti-VEGF-C antibodies were selected from the about 600 VEGF-C-binding scFv clones obtained in Example 1, based on their ability to bind VEGF-C using the ELISA format. Specifically, clones with high ELISA 0D were selected from the clones that bind VEGF-C and inhibit the binding of VEGF-C and VEGR-R3. Each clone was then cultured in SB Media (BD Bioscience) with ampicillin added to 0D 600 = 1.0 and injected with 1 mM IPTG (I sopropy 1-β -D-T o ogal act opyr anos i de) (periplasm) fractions were collected and partially purified by scFv-type anti-VEGF-C monoclonal antibody using NI-NTA column (QIAGEN).

2.3. VEGF-C: VEGR- using anti-VEGF-C antibody in scFv form R3 neutralization ELISA (Competitive ELISA)

In order to determine the inhibitory effect of VEGF-C: VEGR-R3 binding of the scFv-type anti—VEGF-C antibody purified in Example 2.2, a competitive ELISA was performed to assay molecular interactions with the conjugates. 96 μlwell MaxiSorp ™ flat-bottom plates (Nunc) were coated with hVEGR—R3-Fc (R & D Systems. Accession No. P35916), a protein that binds 2 ug / ul of Fc of human IgGl. Then, the plate was washed five times with Phosphate Buffer Saline (PBS) containing 0.05% (v / v) Tween-20, followed by PBS containing l% (v / v) Bovine serum albumin (Sigma). It was blocked for 2 hours at room temperature.

In order to perform VEGF-C: VEGR-R3 neutralization ELISA, the scFv-type anti-VEGF-C antibody (1, 10, 100, 1000 nM) purified in Example 2 was added with BSA of (v / v) and 200. ng / ml FLAG-Tagging hVEGF-CXAccession # P49767) into each well of the hVEGR_R3-Fc coated plate, and then the plate was allowed to react at room temperature for 2 hours. Then, the plate was washed five times with PBS containing 0.05% Tween-20, and then HRP-conjugated anti-FLAG antibody (SIGMA) was added to PBS containing 1% (ν / ν) of BSA. Dilute to a ratio of: 5,000 (v / v), add 100 ul (microliter) to each well, and react for 1 hour at phase silver, followed by PBS containing 0 · 1% (ν / ν) of Tween-20. Washed five times. Due to addition of the last all-TMB substrate (cell signal) of 100 ul (microliter) to each well of the plate was banung induce color development for 3 minutes at room temperature, ^° C. The reaction was then stopped with 50 ul of 5N H ₂ SO ₄ solution and the 0D450 value was measured on a plate reader (Molecular Devices). Through this, 50% inhibition concentration (IC50) of the binding force of VEGF-C: VEGR-R3 was obtained and shown in Table 9 below.

Table 9

As shown in Table 9, the anti-VEGF-C antibody between the VEGF-C and VEGR-R3 It was confirmed that the binding force can be neutralized.

2.4. Binding ELISA with hVEGF-C

An ELISA was performed to determine the binding strength of the antibody purified in Example 2 to the antigen. 96-well MaxiSorp ™ flat-bottom plates (Nunc) were coated with 1 ug / ml human VEGF-C (R & D Systems). Then, the plate was washed five times with PBS containing 0.05 (v / v) Tween-20, and then blocked with PBS containing 1% (ν / ν) BSACBovine Serum Albumin) for 2 hours at room temperature. After the anti-VEGF-C antibody of the scFV form purified in Example 2 was put in each well of the plate, the plate was reacted at room temperature for 2 hours.

After washing 5 times with PBS containing 0.05¾) (v / v) of Tween-20, HRP-conjugated anti-HA (HA—probe Antibody (F H 7) HRP conjugated) antibody (Santacruz) To 1% (ν / ν) BSA containing PSA 1: 1000 ratio (v / v) diluted in 50 ul of each well of the plate, the reaction at room temperature for 1 hour, 0.1 Washed 5 times with PBS containing% (v / v) of Tween-20. Finally, 100 ul TMB substrate (cell signal) was added to each well of the plate to induce color reaction for 3 minutes at room temperature. Then, the reaction was stopped with 50ul of 5N H ₂ S0 ₄ solution, and the 0D450 value was plated. Measurements were taken on a reader (Molecular Devices). The degree of binding of the anti-VEGF-C antibody to the protein was determined by obtaining 50% binding concentration (Kd) of the human VEGF-C protein. The results obtained are shown in Table 9 below:

Table 10

2. 5. Anti-VEGF-C Human Antibody Gene Cloning

The variable region gene sequences of the heavy and light chains of the monoclonal antibody of Example 2.2 were amplified (PC3X Reverse primer: 5′—AAC CAT CGA TAG CAG CAC CG ′ 3 ′, PC3X Forward primer: 5 ′ — GCA CGA CAG GTT TCC CGA 03 '):

PCR condition

5 minutes at 94 ^° C .;

[1 minute, 1 minute at 55 ^° C, 2 min at 72 ^° C at 94 ^° C] x 30 cycles;

6 minutes at 72 ^° C .;

Cool to 4 ^° C.

PCR products obtained from each reaction were washed with a QIAquick Multiwell PCR Purification kit (Qiagen) according to the manufacturer's protocol.

After cloning the secured PCR results, DNA sequencing was performed by the disclosed method. As a result, the amino acid sequences of CDR and SEQ ID NO: 95 (heavy chain variable region) and SEQ ID NO: 96 (light chain variable region) shown in Table 11 were obtained.

Table 11

Cloning the base sequence (SEQ ID NO: 97) encoding the heavy chain variable region (SEQ ID NO: 95) and the base sequence encoding the light chain variable region (SEQ ID NO: 96) obtained in Example 2.5 to different vectors, respectively. I was.

The heavy chain variable region was cloned into a vector pOPTI-VAC (Invitrogen) containing a CMV promoter (cytomegalovirus promoter) and including a constant region and an Fc region of human IgGl. The light chain variable region has a CMV promoter and has been cloned into a vector pFUSE2-CLIg-hl2 (Invivogen) containing a blunt region of human IgGl.

Specifically, the heavy chain variable region and the vector comprising the same are treated with restriction enzymes of ecorl (neb) and Nhel (neb), and the light chain The variable region and the vector comprising the same are treated with restriction enzymes of ecorl (neb) and avrll (neb), and then ligated with T4 DNA Ligase (New England Biolab) to express the antibody containing the desired variable region. Heavy and light chain vectors were prepared.

The heavy and light chain vectors thus obtained were transfected into 293-F cells (invitrogen) as well. The cells were cultured in 293_f expression medium (Invitrogen) medium without serum, and the culture solution was recovered on the fifth day. The culture solution obtained through the culture contained an antibody consisting of a heavy chain and a light chain having a variable region of SEQ ID NO: 55 or SEQ ID NO: 56. After centrifuging the culture medium containing the expressed antibody for 10 min at lOOOxg to remove residual cells and impurities, affinity chromatography using Protein A (GE-Healthcare) having a strong affinity with the antibody Fc region. IgG anti-VEGF-C antibody was purified by low PH e hit ion.

SEQ ID NO: 95 Heavy chain variable region of anti-VEGF-C antibody>

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVSAISYDNGSTYYADSV KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDPYLARLNTFDYWGQGTLVTVSS

(The parts shown in bold in the sequence are CDR sites, and in turn are CDR-Hl, CDR-H2, and CDR-H3)

SEQ ID NO: 96 Light chain variable region of anti-VEGF-C antibody>

QSVLTQPPSASGTPGQRVT I SCSGSSSNIGSNNVSWYQQLPGTAPKLL I YYNSHRPSGVPDRFS GSKSGTSASLA I SGLRSEDEADYYCATWDSSLNGYVFGGGTKLT

2.7. VEGF-C: Tie-2 neutralization ELISA using anti-VEGF-C antibody in IgG form (competitive ELISA)

In order to determine the inhibitory effect of VEGF-C: VEGR—R3 binding of the anti-VEGF ᅳ C antibody in the IgG form purified in Example 2.6 above, Competitive EUSA was performed to test the interaction.

96-well MaxiSorp ™ flat-bottom plates (Nunc) were coated with hVEGFR3—Fc (R & D Systems), a protein that binds the Fc of 2 ugAil of human IgGl. Then, the plate was washed five times with PBSCPhosphate Buffer Saline (0.05% (v / v) Tween—20), followed by room temperature with PBS containing l% (v / v) BSA (Bovine serum albumin; Sigma). Blocking for 2 hours.

In order to perform VEGF-R3: VEGF-C neutralization EUSA, the IgG form of the anti-VEGF-C antibody (1, 10, 100, 1000 nM) purified in Example 6 was l¾) (v / v) of BSA. And 200 ng / ml FLAG-Tagging VEGF-C were added to each well of the hVEGFR3-Fc coated plate, and the plates were allowed to react for 2 hours at silver. Then, the plate was washed five times with PBS containing 0.05% (v / v) Tween-20, and then HRP-conjugated anti—FLAG antibody (SIGMA) contained l¾ (v / v) of BSA. Diluted with PBS at a ratio of 1: 5,000 (v / v) and added to each well in an amount of 100 ul and reacted at room temperature for 1 hour, followed by PBS containing 0.1% (v / v) of Tween-20. Washed once. Finally, 100 wells of TMB substrate (cell signal) was added to each well of the plate to induce color reaction for 3 minutes at room temperature. The reaction was then stopped with 50ul of 5N H ₂ SO ₄ solution and the 0D450 value was measured on a plate reader (Molecular Devices). Through this, 50% inhibition concentration (IC50) of the binding force of VEGF-C: VEGF-R3 was obtained and shown in Table 12 below.

Table 12

As shown in Table 12, it was confirmed that the anti-VEGF-C antibody can neutralize the binding capacity between VEGF ᅳ C and VEGF-R3.

Example 3. Construction of Anti-VEGF-C / Anti Ang2 Bispecific Antibodies

The anti Ang2 scFv prepared in Example 1.6 (SEQ ID NO: 99) An anti-VEGF-C / anti Ang2. Double specific antibody (tetra complex: heavy chain of an anti Ang2 antibody) is linked to the C terminus of each monomer of the anti-VEGF-C antibody (including SEQ ID NO: 95 and SEQ ID NO: 96) obtained in Example 2.6, respectively. Variable region + light chain variable region of anti Ang2 antibody + heavy chain of anti VEGF-C antibody (including heavy chain variable region of SEQ ID NO: 95) + light chain of anti VEGF-C antibody (including light chain variable region of SEQ ID NO: 96) Was made (see FIG. 1).

To this end, a heavy chain coding DNA fragment (SEQ ID NO: 97) and a light chain (light chain of the anti-VEGF-C antibody) for encoding the heavy chain (consisting of an anti-VEGF-C antibody plus an anti Ang2 scFv) of the above-specific antibody DNA fragment (SEQ ID NO: 98) is expressed in human embryonic kidney 293F cells (invi trogen) in a ratio of 2: 2 and affinity chromatography using Protein A (GE-Heal thcare) The bispecific antibody was purified by low PH elution.

To this end, the following process was carried out.

The amino acid sequence of the VEGF-C recognition site is sequence number as described above.

The amino acid sequence of 95 and SEQ ID NO: 96 was used. The base sequence (SEQ ID NO: 100) of the coding DNA fragment of the heavy chain variable region (SEQ ID NO: 95) and the base sequence (SEQ ID NO: 101) of the coding DNA fragment of the light chain variable region (SEQ ID NO: 96) are as follows:

GACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGGACTCT

TCTACTCCCCTCAGCAGCGTGGTGACCGTGCCCTCCAG

GGC

The heavy chain variable region coding DNA fragment using EcoRI / Nhel restriction enzyme (NEB), and the light chain variable region coding DNA fragment using EcoRI / BsiWI restriction enzyme (NEB), contained in the pCDNA3.3 vector (Invi trogen). Anti-VEGF-C antibodies were prepared by subcloning the IgG2 backbone and kappa light chain, respectively.

An Ang2 recognition site (labeled 4H10) was used as the anti Ang2 scFv (SEQ ID NO: 99) prepared in Example 1.6. The nucleotide sequence of the DNA fragment encoding it (SEQ ID NO: 102) is as follows:

GAGGTACAACTGCTGGAGAGTGGAGGAGGTCTCGTCCAGCCGGGAGGGAGCCTAAGAnAAGn

TCATCACGGCGCCTACCCTTATGATGTGCCCGATTATGCCTCT

The anti Ang2 scFv coding DNA fragment was synthesized by optimization for production in human cells (Bioneer).

The anti Ang2 scFv coding DNA fragment thus prepared was subcloned to the 3 'end of the heavy chain coding DNA fragment of the anti-VEGF-C antibody prepared previously using BamHI / XhoI restriction enzyme (NEB). At this time, the coding DNA fragment of the peptide linker (GGGGSGGGGS) was used to connect them.

The prepared DNA fragments were transfected into Exp i 293 cells (Invitrogen) to express the dual antibody protein. The double antibodies were selectively isolated from the culture using MabSelectSuRe column (GE Healthcare), and then elution with IgG elution buffer (Thermo Scientific) solution and neutralized with 1/20 (v / v) 1M Tris (pH 8.0) solution. After that, it was replaced with PBS solution.

Example 4. Affinity of VEGF-C and Ang2 of Anti-VEGF-C / Anti Ang2 Bispecific Antibodies

Example above _. Each affinity for VEGF-C of the bispecific antibody prepared in 3 was confirmed using Biacore T100 (GE). Human Fab conjugates (# 28-9583-25, GE Healthcare) were immobilized on the surface of CM5 chips (# BR-1005-30, GE) according to the manufacturer's instructions. About 90 120 RU of bispecific antibodies were captured and various concentrations of VEGF-C-Fc (# 358-MT / CF, R & D Systems) were injected into the captured antibodies. The surface was then regenerated by injecting a 10 mM Glycine-HCKpH 1.5) solution therein. In order to measure the affinity, the data obtained in the above experiments were fitted using BIAevaluation software (GE Healthcare, Biacore T100 evaluation software).

In addition, for the An _g 2 of the bispecific antibody prepared in Example 3 ELISA was performed to determine the binding force. 96-well MaxiSorp ™ flat-bottom plates (Nunc) were coated at 50 ul / well with 1 ug / ul of human Ang2 (R & D Systems). Then, the plate was washed five times with PBS (Phosphate Buffer Saline) containing 0.05% Tween-20 and blocked with PBS containing \ BSA for 2 hours at silver. After putting the prepared bispecific antibody into each well, the plate was reacted at room temperature for 1 hour. Then, after washing 5 times with PBS containing 0.05 ¾ ^ Tween ᅳ 20, HRP-conjugated anti-Human Fc antibody (Sigma) was diluted with PBS containing 1% of BSA at a ratio of 1: 5,000 at room temperature. After reacting for 1 hour at, it was washed 5 times with PBS containing 0.1% Tween-20. Finally, lOOul of TMB substrate (SIGMA) was added to each plate to induce color reaction. Then, the reaction was stopped with 50ul of 5N H2S04 solution, and the 0D450 value was measured by a plate reader (Molecular Devices). Through this, the binding force was measured by obtaining 50% binding concentration (Kd) of the human _An g2 protein.

The results obtained are shown in Table 13 below:

Table 13

Example 5: anti-VEGF-C / anti-Ang2 inhibition of cell proliferation, wherein the bispecific antibody of the effect produced VEGF-C / anti-Ang2 to test the cytostatic effects of the bispecific antibody, Cell Counting Kit ^~ 8 (CCK- 8, supplied by Dojinho Molecular Technologies, Inc. Cat. # CK04-01) was used to determine the extent of cell proliferation.

Lymphatic endothelial cells (LEC; Lonza, CC-2811) were incubated in EGM-2MV medium (Lonza) under conditions of 5% CO ₂ and 37 ^° C. For cell proliferation assay, the cultured cells were passaged in a 96 well plate at a concentration of 5000 cels / wel 1 (1¾> FBS in EBM2) in Example 3 The prepared anti-VEGF-C / anti Ang2 bispecific antibody 60 nM was treated and incubated for 72 hours. At this time, human Ang2 (R & D systems; Accession # 015123 (hAng2)) 2ug (microgram) / ml (40nM) and VEGF- (XP49767) lug / mK30nM) were treated together. Anti-ang2 antibody 4H10 60nM alone, anti-VEGF-C antibody 4A2 (Example 2.6) 60nl alone treatment, and anti Ang2 antibody 4H10 60nM And a combination treatment group of anti-VEGF-C antibody 60nM was used.

The anti-Ang2 antibody 4H10 is a vector comprising a heavy chain variable region (SEQ ID NO: 60) shown in Table 8 of Example 1.6, a constant region of the CMV promoter (cytomegalovirus promoter) and human IgGl, and an Fc region; Cloned to VAC (Invitrogen) (using ecorl (neb) and Nhel (neb) restriction enzymes and T4 DNA Ligase (New Engl nd Biolab)), the light chain variable region of SEQ ID NO: 8 (SEQ ID NO: 68) was obtained from CMV promoter and human IgGl. After cloning (using ecorl (neb) and avrll (neb) restriction enzymes and T4 DNA Ligase (New England Biolab)) in a vector pFUSE2- CLIg-hl2 (Invivogen) containing a constant region of the obtained heavy chain vector and light chain The vector was transfected with 293-F cells (invitrogen) and cultured on day 5 at 37 ^° C in 293—f expression medium (Invitrogen) medium without serum. Recovered and centrifuged at 1000 xg for 10 minutes After removing the cells and impurities, was purified by low PH elution by affinity chromatography method using Protein A (GE-Healthcare).

After incubation, cell counting kit-8 assay (Dojindo Molecular Technologies, Gaithersburg, MD) was used to analyze the degree of cell proliferation according to the manufacturer's instructions. In brief, after 48 hours and 72 hours of incubation, CCK8 solution was added to each well by 10 ul (microl iter) for 2.5 hours, and then the absorbance at 450 nm was read with a microplate reader.

The obtained result is shown in FIG. As shown in FIG. 2 Likewise, when the anti-VEGF-C / anti Ang2 bispecific antibody treatment, the anti-ang2 antibody or anti-VEGF-C antibody alone showed a significant cell proliferation inhibitory effect, anti Ang2 antibody or anti-VEGF ᅳ C antibody In comparison with the combination treatment of can be seen to show an enhanced cell growth inhibitory effect.

Example 6 Inhibition of Cell Migration of Anti-VEGF-C / Anti Ang2 Prophylactic Antibodies In order to confirm the cell migration inhibitory effect of the anti-VEGF-C / anti Ang2 bispecific antibody obtained in Example 3, The effect on LEC migration induced by Ang2 and VEGF-C was tested.

The mobility of vascular endothelial cells was measured using GE Healthcare's xCelligence RTCA (Realtime cell analyzer). RTCA is a non-invasive cell monitoring system that can identify cell changes by measuring impedance in real time. CIM-platel6 (GE Healthcare) consisting of a lower chamber and an upper chamber was used for cell migration assay. A microelectrode for measuring impedance is arranged in the upper chamber, and when the cells seeded in the chamber move through the micropores, they are attached to the microelectrode. It was.

Lymphatic endothelial cells (Lonza, CC-2811; P5-7) cultured in EGM2-MV medium were grown for 6 hours in EBM medium with 1% FBS. 1 ug / ml VEGF-C and 1 ug / ml Ang2 (R & D systems) and ΙΟΟηΜ bispecific antibody were added to EBM medium containing 1% FBS in each well of the lower chamber of the CIM-platel6, followed by fibronectin-coated upper chamber. assembly. After 30 ul of serum free EBM medium was added to the upper chamber, the plate was placed in the incubator for 1 hour for equi librat ion between the medium and the CIM-plate at the device station in the incubator. The vascular endothelial cells resuspended in serum-free media were seeded at 40,000 / well, left to settle down for 15 minutes, and mounted on the device to measure cell movement in real time. The degree of cell migration is represented by the cell index value. The obtained result is shown in FIG. As shown in Figure 3, when the anti-VEGF-C / anti Ang2 bispecific antibody treatment, it can be seen that a significant cell migration inhibitory effect than the anti-Ang2 antibody or anti-VEGF-C antibody alone treatment, the anti-Ang2 antibody Or it can be seen that shows an enhanced cell migration inhibitory effect compared with the combination treatment of anti-VEGF-C antibody.

Claims

【Scope of Claim】

【Claim 1】

Includes an anti-VEGF-C antibody or antigen-binding fragment thereof, and an anti-Ang2 antibody or antigen-binding fragment thereof,

The anti-VEGF-C antibody or antigen-binding fragment thereof is

One or more heavy chain complementarity determining regions selected from the group consisting of CDR-H1 comprising the amino acid sequence of SEQ ID NO: 89, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 90, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 91 , or a heavy chain variable region comprising one or more of the heavy chain complementarity-determining regions;

One or more light chain complementarity determining regions selected from the group consisting of CDR-L1 comprising the amino acid sequence of SEQ ID NO: 92, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 93, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 94 , or a light chain variable region comprising the one or more light chain complementarity-determining regions;

a combination of said one or more heavy chain complementarity determining regions and said one or more light chain complementarity determining regions; or

It includes all,

The anti-Ang2 antibody or antigen-binding fragment thereof is

A polypeptide having the amino acid sequence of SEQ ID NO: 49 (CDR-H1), a polypeptide having the amino acid sequence of SEQ ID NO: 50 (CDR-H2), and an amino acid sequence selected from the group consisting of SEQ ID NO: 17 to 24, such as SEQ ID NO: 17 to At least one heavy chain complementarity-determining region selected from the group consisting of a polypeptide (CDR-H3) having an amino acid sequence selected from the group consisting of 22, or a heavy chain variable region comprising the at least one heavy chain complementarity-determining region;

A group consisting of a polypeptide having the amino acid sequence of SEQ ID NO: 51 (CDR-L1), a polypeptide having the amino acid sequence of SEQ ID NO: 52 (CDR-L2), and a polypeptide having the amino acid sequence of SEQ ID NO: 53 (CDR-L3) One or more light chain complementarity-determining regions selected from or the one or more light chain complementarity-determining regions A light chain variable region comprising;

A combination of said at least one heavy chain complementarity determining region and at least one light chain complementarity determining region; or

Combination of the heavy chain variable region and light chain variable region

which includes,

Anti-VEGF-C/anti-Ang2 bispecific antibody.

[Claim 2]

The method of claim 1, wherein the anti-Ang2 antibody or antigen-binding fragment thereof is a polypeptide having an amino acid sequence selected from SEQ ID NOs: 1 to 8 (CDR-HI), a polypeptide having an amino acid sequence selected from SEQ ID NOs: 9 to 16 ( CDR-H2), and a polypeptide having an amino acid sequence selected from SEQ ID NOs: 17 to 24 (CDR-H3); at least one heavy chain complementarity determining region or a heavy chain variable region comprising the same;

A polypeptide having an amino acid sequence selected from SEQ ID NOs: 25 to 32 (CDR-L1), a polypeptide having an amino acid sequence selected from SEQ ID NOs: 33 to 40 (CDR-L2), and a polypeptide having an amino acid sequence selected from SEQ ID NOs: 41 to 48. At least one light chain complementarity determining region selected from the group consisting of polypeptide (CDR-L3) or a light chain variable region comprising the same; A combination of said at least one heavy chain complementarity determining region and at least one light chain complementarity determining region; or

Combination of the heavy chain variable region and light chain variable region

which includes,

Anti-VEGF-C/anti-Ang2 bispecific antibody.

[Claim 3]

The method of claim 1, wherein the anti-Ang2 antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 54 to 61; A light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 62 to 69; or the heavy chain variable region and the light chain variable region Containing a combination of parts,

Anti-VEGF-C/anti-Ang2 bispecific antibody.

【Claim 4】

The method of claim 1, wherein the anti-VEGF-C antibody or antigen-binding fragment thereof has a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 95,

A light chain variable region containing the amino acid sequence of SEQ ID NO: 96, or

Their combination _β

Containing, anti-VEGF-C/anti-Ang2 bispecific antibody.

【Claim 5】

The method of claim 1, wherein the anti-VEGF-C/anti-Ang2 bispecific antibody is

A form comprising an anti-VEGF-C antibody and an antigen-binding fragment of an anti-Ang2 antibody linked to the C terminus of the anti-VEGF-C antibody, or

A form comprising an anti-Ang2 antibody and an antigen-binding fragment of an anti-VEGF-C antibody linked to the C terminus of the anti-Ang2 antibody,

Anti-VEGF-C/anti-Ang2 bispecific antibody.

【Claim 6】

The method of any one of claims 1 to 5, wherein the antigen-binding fragment is selected from the group consisting of scFv, (scFv)2, scFvFc, Fab, Fab ¹ , and F(ab ')2,

Anti-VEGF-C/anti-Ang2 bispecific antibody.

【Claim 7】

A pharmaceutical composition for preventing or treating diseases related to Ang2 or VEGF-C, comprising the anti-VEGF-C/anti-Ang2 bispecific antibody of any one of claims 1 to 5 as an active ingredient.

【Claim 8】

The disease related to Ang2 or VEGF-C according to claim 17, wherein the disease related to Ang2 or VEGF-C is selected from the group consisting of cancer, cancer metastasis, cancer infiltration or invasion, eye disease, inflammatory disease, and malaria. For prevention or treatment of Pharmaceutical composition.

【Claim 9】

A composition for detecting Ang2 or VEGF-C comprising the anti-VEGF-C/anti-Ang2 bispecific antibody of any one of claims 1 to 5.

【Claim 10】

A composition for diagnosing diseases related to _Ang 2 or VEGF-C, comprising the anti-VEGF-C/anti-Ang2 bispecific antibody of any one of claims U to 5.

【Claim 11】

The diagnostic composition of claim 10, wherein the disease related to Ang2 or VEGF-C is selected from the group consisting of cancer, cancer metastasis, cancer infiltration or invasion, eye disease, inflammatory disease, and malaria.

【Claim 12】

A method for preventing or treating diseases related to Ang2 or VEGF-C, comprising administering the anti-VEGF—C/anti-Ang2 bispecific antibody of paragraph 1 to a patient.

【Claim 13】

The method of claim 12, wherein the disease related to Ang2 or VEGF-C is selected from the group consisting of cancer, cancer metastasis, cancer infiltration or invasion, eye disease, inflammatory disease, and malaria. Prevention or treatment method.

【Claim 14】

A method for detecting Ang2 or VEGF-C comprising contacting a biological sample with the anti-VEGF-C/anti-Ang2 bispecific antibody of claim 1.

【Claim 15】

A method for diagnosing diseases related to Ang2 or VEGF-C, comprising contacting a biological sample with the anti-VEGF-C/anti-Ang2 antibody of claim 1.

【Claim 16】

The method of claim 15, wherein the disease related to Ang2 or VEGF-C consists of cancer, cancer metastasis, cancer infiltration or invasion, eye disease, inflammatory disease, and malaria. A diagnostic method selected from the group.

【Claim 17】

The anti-VEGF-C/anti-Ang2 bispecific antibody according to any one of items 1 to 6 for use in the prevention or treatment of diseases related to Ang2 or VEGF-C.

【Claim 18】

The method of claim 17, wherein the disease related to Ang2 or VEGF-C is selected from the group consisting of cancer, cancer metastasis, cancer infiltration or invasion, eye disease, inflammatory disease, and malaria, anti-VEGF-C/anti-Ang2 disease. Specific antibodies.

【Claim 19】

The anti-VEGF-C/anti-Ang2 bispecific antibody of any one of claims 1 to 6 for use in the detection of Ang2 or VEGF-C.

【Claim 20】

The anti-VEGF-C/anti-Ang2 bispecific antibody of any one of claims 1 to 6 for use in the diagnosis of diseases related to Ang2 or VEGF-C.

【Claim 21】

The method of claim 20, wherein the disease related to Ang2 or VEGF-C is selected from the group consisting of cancer, cancer metastasis, cancer infiltration or invasion, eye disease, inflammatory disease, and malaria. Anti-VEGF-C/anti-Ang2 dual Specific antibodies.