KR102089766B1 - Pharmaceutical composition for a combination therapy containing an angiogenesis inhibitor and anti-c-Met antibody - Google Patents

Abstract

Provided is a pharmaceutical composition for combined administration for the prevention and / or treatment of c-Met and angiogenesis-inducing disease diseases comprising an angiogenesis inhibitor and an anti-c-Met antibody or antigen-binding fragment thereof as an active ingredient. The pharmaceutical composition for combined administration exhibits an excellent synergistic effect, and more effective treatment is possible.

Description

Pharmaceutical composition for a combination therapy containing an angiogenesis inhibitor and anti-c-Met antibody

The present invention relates to a pharmaceutical composition for combined administration for the prevention and / or treatment of c-Met and VEGF-induced diseases comprising an angiogenesis inhibitor and an anti-c-Met antibody or antigen-binding fragment thereof as an active ingredient.

c-Met is a representative receptor tyrosine kinase (RTK) that is present on the cell surface. It binds to its ligand, Hepatocyte Growth Factor (HGF), promotes intracellular signaling and promotes cell growth. In addition, it is overexpressed in cancer cells and is widely involved in cancer development, cancer metastasis, cancer cell migration, cancer cell penetration, and new blood vessel formation. c-Met is overexpressed in many types of cancer, and especially, patients with c-Met overexpression have a poor prognosis of cancer.

Angiogenesis inhibitors collectively refer to drugs designed to inhibit the growth of cancer by blocking blood supply to cancer cells, and examples thereof include vascular endothelial growth factor (VEGF) antagonists (inhibitors). Vascular Endothelial Cell Growth Factor (VEGF) is also present in normal cells, and is secreted from cancer cells, and binds to its receptor, VEGFR (VEGF Receptor), which causes angiogenesis, and cancer cells through the new blood vessels Nutrients necessary for growth are provided.

Therefore, both c-Met and angiogenesis-related VEGF are of great importance as targets in the development of anticancer agents.

However, a technique for treating cancer by administering a drug targeting c-Met and a drug targeting angiogenesis-related factors (eg, VEGF) has not been proposed.

The present inventors developed a cancer treatment technology with better anti-cancer effect and good prognosis, while administering a drug targeting c-Met and an angiogenesis inhibitor (for example, a drug targeting VEGF), a remarkable synergistic effect. The present invention was completed by developing a combination treatment method to obtain.

Therefore, an example of the present invention is an angiogenesis inhibitor (eg, VEGF antagonist) and an anti-c-Met antibody or a combination thereof for the prevention and / or treatment of c-Met and VEGF-induced diseases comprising an antigen-binding fragment thereof as an active ingredient Provided is a pharmaceutical composition for administration.

Another example is a first pharmaceutical composition comprising an effective amount of an angiogenesis inhibitor (eg, VEGF antagonist) as an active ingredient, a second pharmaceutical composition comprising an effective amount of an anti-c-Met antibody or antigen-binding fragment thereof as an active ingredient, and packaging Kits for the prevention and / or treatment of c-Met and VEGF-induced diseases, including containers.

In another example, a pharmaceutically effective amount of an angiogenesis inhibitor (e.g., a VEGF antagonist) and a pharmaceutically effective amount of an anti-c-Met antibody or antigen-binding fragment thereof require prevention and / or treatment of c-Met and VEGF-induced diseases Provided is a method for preventing and / or treating c-Met and VEGF-induced diseases, comprising administering to a patient in combination.

The present inventors completed the present invention by confirming that if both the c-Met and the angiogenesis-related factor (eg, VEGF) are targeted during the study for the development of an anticancer agent having a better effect, cancer cell growth can be inhibited more effectively. Accordingly, the present invention inhibits the growth of cancer cells by targeting c-Met, and simultaneously inhibits angiogenesis by targeting VEGF to block the supply of nutrients essential for cancer cell growth, thereby inhibiting cancer cell growth, thereby inhibiting these two factors. It is characterized by being able to obtain all of the enhanced synergistic effect in inhibiting the growth of cancer cells by targeting them all. Moreover, since the production of new blood vessels is an essential element of primary metastatic cancer by providing new pathways for cancer cells to travel at short or long distances, it inhibits VEGF to block the VEGF / VEGFR signaling system, thereby inhibiting the formation of new blood vessels, thereby metastasis of cancer. It is possible to obtain an advantageous effect in suppressing.

Therefore, by simultaneously targeting two factors, c-Met and angiogenesis-related factor (VEGF), it is possible to simultaneously inhibit growth of cancer tissue and growth and metastasis due to angiogenesis.

Thus, an example of the present invention is an angiogenesis inhibitor and an anti-c-Met antibody, or an antigen-binding fragment thereof, as an active ingredient, c-Met and / or angiogenesis-inducing diseases for the prevention and / or treatment of a combination for pharmaceutical administration Provided is a composition.

In another example, a pharmaceutically effective amount of an angiogenesis inhibitor and a pharmaceutically effective amount of an anti-c-Met antibody or antigen-binding fragment thereof are used in combination with a patient in need of prevention and / or treatment of c-Met and / or VEGF-induced disease Provided is a method for preventing and / or treating c-Met and VEGF-induced diseases, comprising administering. The method may further include identifying a patient in need of prevention and / or treatment of c-Met and / or VEGF-induced disease prior to the combination administration step.

In one embodiment, the pharmaceutical composition for co-administration may be for administration by mixing an effective amount of an angiogenesis inhibitor and an effective amount of an anti-c-Met antibody or an antigen-binding fragment thereof, to prepare a mixture, and to administer simultaneously.

In another embodiment, the pharmaceutical composition for co-administration may be formulated such that an effective amount of an angiogenesis inhibitor and an effective amount of an anti-c-Met antibody or antigen-binding fragment thereof are formulated and administered simultaneously or sequentially. In this case, the pharmaceutical composition for co-administration includes a first pharmaceutical composition comprising an effective amount of an angiogenesis inhibitor as an active ingredient and a second pharmaceutical composition comprising an effective amount of an anti-c-Met antibody or an antigen-binding fragment thereof as an active ingredient. It may be a pharmaceutical composition for pharmaceutical combination administration for simultaneous or sequential administration. In the case of sequential administration, the order may be changed.

Another example is a first pharmaceutical composition comprising an effective amount of an angiogenesis inhibitor as an active ingredient, a second pharmaceutical composition comprising an effective amount of an anti-c-Met antibody or antigen-binding fragment thereof as an active ingredient, and a packaging container, c -Provides a kit for preventing and / or treating Met and angiogenic diseases.

In the present invention, an angiogenesis inhibitor, such as a drug targeting VEGF (VEGF antagonist), and an anti-c-Met antibody or an antigen-binding fragment thereof, may be administered in combination, thereby achieving excellent synergistic effects compared to using a single drug. .

In addition, excellent effects can be obtained even in patients resistant to angiogenesis inhibitors (eg, VEGF antagonists) by co-administration of angiogenesis inhibitors with anti-c-Met antibodies or antigen-binding fragments thereof. Therefore, in another example, for combined administration to overcome resistance to angiogenesis inhibitors (e.g., VEGF antagonists) and angiogenesis inhibitors (e.g., VEGF antagonists) comprising an anti-c-Met antibody or antigen-binding fragment thereof as an active ingredient. Pharmaceutical compositions are provided.

In addition, the anti-c-Met antibody or antigen-binding fragment thereof exhibits c-Met decomposition activity by the lysosomal pathway, not the proteasome pathway mediated by Cbl, independent of Cbl, which is a representative RTK negative regulator, and thus Cbl It mediates LRIG1, another negative regulator that acts independently of Cbl, even when administered to patients who do not function normally due to factors such as mutation, small expression of Cbl, or mutation of c-Met Cbl binding site. Since c-Met can be inhibited, the combination administration of an anti-c-Met antibody or an antigen-binding fragment thereof and a VEGF antagonist can be particularly useful for patients in which Cbl does not function normally.

In the present invention, by administering an anti-c-Met antibody and an angiogenesis inhibitor in combination, it is possible to obtain not only an excellent synergistic effect compared to the case of using an anti-c-Met antibody or angiogenesis inhibitor alone, but also anti-c- When Met antibodies or angiogenesis inhibitors are used respectively, excellent therapeutic effects can be obtained even in diseases that have no effect on diseases (eg cancer) or anti-c-Met antibodies or angiogenesis inhibitors.

The angiogenesis inhibitor may be any substance that is known to inhibit angiogenesis, and examples thereof include a drug that targets vascular endothelial cell growth factor (VEGF) and inhibits action (VEGF antagonist).

The "Vascular Endothelial Cell Growth Factor (VEGF)" is also present in normal cells, and is secreted from cancer cells, in particular, binds to its receptor VEGFR (VEGF Receptor) to cause angiogenesis, and the cancer cells are used to develop the new blood vessels. Through this, they receive the nutrients necessary for growth. Overexpression of VEGF causes various diseases, and is particularly involved in the prognosis of cancer as well as invasion and metastasis. For this reason, VEGF is an important target in anticancer therapy.

The VEGF may be derived from VEGF selected from the group consisting of human, monkey, mouse, and rat. For example, the protein is GenBank Accession Number NM_001025366.2. NM_001025367.2. NM_001025368.2. NM_001025369.2. NM_001025370.2. NM_001033756.2. NM_001171622.1. NM_001171623.1. NM_001171624.1. NM_001171625.1. NM_001171626.1. NM_001171627.1. NM_001171628.1. NM_001171629.1. NM_001171630.1. NM_001204384.1. NM_001204385.1. Or it may be a polypeptide encoded by a nucleotide sequence (mRNA) provided in NM_003376.5 or the like.

), 수니티닙 말레이트, AEE-788 (Novartis; AE-788 또는 NVP-AEE-788), 악시티닙 (axitinib; Pfizer; AG-13736 또는 AG-013736), AG-028262 (Pfizer), 컴브레타스타틴 A4 아날로그 (combretastatin A4; AVE-8062; Ajinomoto Co. and Sanofi-aventis; AC-7700), 세디라닙(cediranib; AZD-2171; AstraZeneca; AZ-2171), 소라페닙(sorafenib; NEXAVAR

; Bayer AG and Onyx; CAS Registry Number 284461-73-0, BAY-43-9006, raf kinase inhibitor, IDDBCP150446), BMS-387032 (Sunesis and Bristol-Myers Squibb; SNS-032 또는 CAS Registry Number 345627-80-7), CEP-7055 (Cephalon and Sanofi-aventis; CEP-11981 또는 SSR-106462), CHIR-258 (Chiron; CAS Registry Number 405169-16-6, GFKI, 또는 GFKI-258), CP-547632 (OSI Pharmaceuticals and Pfizer; CAS Registry Number 252003-65-9) 또는 이의 유사 억제제인 CP-564959, E-7080 (Eisai Co.; CAS Registry Number 417716-92-8, ER-203492-00), 파조파닙(Pazopanib; GlaxoSmithKline), GW-654652 (GlaxoSmithKline) 또는 이와 관련된 인다졸릴피리미딘 Kdr 억제제 (indazolylpyrimidine Kdr inhibitors), KRN-951 (Kirin Brewery Co.) 또는 이와 관련된 퀴놀린-우레아 VEGF 억제제, 미도스타우린(midostaurin; PKC-412; Novartis; CAS Registry Number 120685-11-2, benzoylstaurosporine, CGP-41251, STI-412), 바탈라닙(vatalanib; PTK-787; Novartis and Schering; CAS Registry Numbers 212141-54-3 및 212142-18-2; PTK/ZK, PTK-787/ZK-222584, ZK-22584, VEGF-TKI, VEGF-RKI, PTK-787A, DE-00268, CGP-79787, CGP-79787D, ZK-222584) 또는 이와 관련된 아닐리노프탈라진 유도체 VEGF 억제제(anilinophthalazine derivative VEGF inhibitors), 셈마사닙(semaxanib; SU-5416; Sugen and Pfizer/Pharmacia; CAS Registry Number 194413-58-6, 204005-46-9), SU-6668 (Sugen and Taiho; CAS Registry Number 252916-29-3, SU-006668, TSU-68), 탈리도미드(thalidomide; Celgene; CAS Registry Number 50-35-1, Synovir, Thalidomide Pharmion, Thalomid), XL-647 (Exelixis; EXEL-7647), XL-999 (Exelixis; EXEL-0999), 반데타닙(vandetanib; ZD-6474 AstraZeneca; CAS Registry Number 443913-73-3, Zactima, AZD-6474) 또는 이와 관련된 아닐리노퀴나졸린 VEGF 억제제(anilinoquinazoline VEGF inhibitors), ZK-304709 (Schering; CDK inhibitors (indirubin derivatives), ZK-CDK, MTGI, multi-target tumor growth inhibitor) 또는 인디루빈 유도체 VEGF 억제제(indirubin derivative VEGF inhibitors; WO 00/234717, WO 02/074742, WO 02/100401, WO 00/244148, WO 02/096888, WO 03/029223, WO 02/092079, WO 02/094814 참조), CDP791, 엔자스타우린(Enzastaurin), BIBF 1120(Boehringer Ingelheim), BAY 573952, BAY 734506, XL 184, IMC-1121B, CEP 701, SU 014813, SU 10944, SU 12662, OSI-930, BMS 582664, 등으로 이루어진 군에서 선택된 1종 이상의 것일 수 있다.The drug targeting the VEGF (VEGF antagonist) may be one or more selected from the group consisting of VEGF antibodies and small molecule drugs, specifically, avastin, VEGF-trap (VEGF-trap), sunitinib (sunitinib; Sugen and Pfizer; SU-11248, SU-011248, SU-11248J, SUTENT

), Sunitinib Malate, AEE-788 (Novartis; AE-788 or NVP-AEE-788), Axitinib (Pitizer; AG-13736 or AG-013736), AG-028262 (Pfizer), Com Bretastatin A4 analog (combretastatin A4; AVE-8062; Ajinomoto Co. and Sanofi-aventis; AC-7700), cediranib (cediranib; AZD-2171; AstraZeneca; AZ-2171), sorafenib (sorafenib; NEXAVAR

; Bayer AG and Onyx; CAS Registry Number 284461-73-0, BAY-43-9006, raf kinase inhibitor, IDDBCP150446), BMS-387032 (Sunesis and Bristol-Myers Squibb; SNS-032 or CAS Registry Number 345627-80-7), CEP-7055 (Cephalon and Sanofi-aventis; CEP-11981 or SSR-106462), CHIR-258 (Chiron; CAS Registry Number 405169-16-6, GFKI, or GFKI-258), CP-547632 (OSI Pharmaceuticals and Pfizer; CAS Registry Number 252003-65-9) or similar inhibitors CP-564959, E-7080 (Eisai Co .; CAS Registry Number 417716-92-8, ER-203492-00), Pazopanib (GlaxoSmithKline), GW -654652 (GlaxoSmithKline) or related indazolylpyrimidine Kdr inhibitors, KRN-951 (Kirin Brewery Co.) or a related quinoline-urea VEGF inhibitor, midostaurin (PKC-412; Novartis; CAS) Registry Number 120685-11-2, benzoylstaurosporine, CGP-41251, STI-412), vatalanib; PTK-787; Novartis and Schering; CAS Registry Numbers 212141-54-3 and 212142-18-2 ; PTK / ZK, PTK-787 / ZK-222584, ZK-22584, VEGF-TKI, VEGF-RKI, PTK-787A, DE-00268, CGP-79787, CGP-79787D, ZK-222584) or related anilinos Phthalazine derivative VEGF inhibitors, semasanib (semaxanib; SU-5416; Sugen and Pfizer / Pharmacia; CAS Registry Number 194413-58-6, 204005-46-9), SU-6668 (Sugen and Taiho; CAS Registry Number 252916-29-3, SU-006668, TSU-68), thalidomide (Celgene; CAS Registry Number 50-35-1, Synovir, Thalidomide Pharmion, Thalomid), XL-647 (Exelixis; EXEL-7647), XL-999 (Exelixis; EXEL-0999), vandetanib (ZD-6474 AstraZeneca; CAS Registry Number 443913-73-3, Zactima, AZD-6474) or related anilinoquinazoline VEGF inhibitors, ZK-304709 (Schering; CDK inhibitors (indirubin derivatives), ZK-CDK, MTGI, multi- target tumor growth inhibitor or indirubin derivative VEGF inhibitors; WO 00/234717, WO 02/074742, WO 02/100401, WO 00/244148, WO 02/096888, WO 03/029223, WO 02 / 092079, see WO 02/094814), CDP791, Enzastaurin, BIBF 1120 (Boehringer Ingelheim), BAY 573952, BAY 734506, XL 184, IMC-1121B, CEP 701, SU 014813, SU 10944, SU 12662, OSI-930, BMS 582664, etc. It may be one or more selected from the group.

N-acetylcolchinol phosphate (ZD-6126; AstraZeneca and Angiogene; CAS Registry Number 219923-05-4, ANG-453, as an angiogenesis inhibitor having an action of indirectly inhibiting VEGF other than a VEGF antagonist AZD-6126, ZD-6126 derivative, ZM-445526) or ANG-400 series drugs, Imatinib; Novartis; CAS Registry Numbers 152459-95-5, 220127-57-1, Glivec, Gleevec, STI- 571, CGP-57148), everolimus (RAD-001; Novartis; CAS Registry Number 159351-69-6, RAD-001, SDZ-RAD, Certican), dasatinib (BMS-354825; Bristol -Myers Squibb; CAS Registry Number 302962-49-8, Src / Abl kinase inhibitor).

 “C-Met” or “c-Met protein” means a receptor tyrosine kinase that binds to hepatocyte growth factor. The c-Met protein may be derived from all species, for example, may be derived from mammals including primates such as humans and monkeys, and rodents such as mice and rats. More specifically, the c-Met protein is derived from a primate such as human c-Met (eg NP_000236), monkey c-Met (eg Macaca mulatta, NP_001162100), or mouse c-Met (eg NP_032617.2 ), Rat c-Met (eg, NP_113705.1), and the like. The protein includes, for example, a polypeptide encoded by the nucleotide sequence provided in GenBank Aceession Number NM_000245, or a protein encoded by the polypeptide sequence provided in GenBank Aceession Number NM_000236, or an extracellular domain thereof. Receptor tyrosine kinase c-Met is involved in various mechanisms, such as cancer development, cancer metastasis, cancer cell migration, cancer cell penetration, and angiogenesis.

Fragment of the anti-c-Met antibody refers to a fragment containing the antigen-binding site of the antibody, complementarity determining region (CDR), CDR and Fc region of the anti-c-Met antibody, scFv, (scFv) ₂ , scFv-Fc, Fab, Fab 'or F (ab') ₂ , and the like.

In the present invention, an anti-c-Met antibody is used in the sense to include a variant of the antibody. The antibody variants may include isotypes of human and animal antibodies present in nature, and / or Fc of all human and animal antibodies in which amino acids constituting the hinge have been modified. Unless otherwise stated in the present invention, c-Met antibodies are understood to include variants of the antibody.

The anti-c-Met antibody may be a specific region of c-Met, for example, a specific region in the SEMA domain as an epitope, and all antibodies that act on c-Met to induce internalization and degradation. Or an antigen-binding fragment thereof.

C-Met, a receptor for HGF (Hepatocyte growth factor), is divided into three parts: an extracellular site, a membrane permeation site, and an intracellular site. In the extracellular site, α-subunits and β-subunits are formed by disulfide bonds. In the connected form, the HGF binding domain is composed of a SEMA domain, a PSI domain (plexin-semaphorins-integrin homology domain) and an IPT domain (immunoglobulin-like fold shared by plexins and transcriptional factors domain). The SEMA domain of the c-Met protein may include the amino acid sequence of SEQ ID NO: 79, and is a domain present in the extracellular region of c-Met, which corresponds to a region to which HGF binds. A region of the SEMA domain comprising an amino acid sequence of SEQ ID NO: 71 corresponding to a specific region, for example, 106 to 124, is a loop between the 2 and 3 propeller domains of the epitope in the SEMA domain of the c-Met protein. ), And may act as an epitope of the anti-c-Met antibody proposed in the present invention.

The term "epitope" is an antigenic determinant and is interpreted to mean a portion of the antigen recognized by the antibody. According to one embodiment, the epitope is a region comprising 5 or more consecutive amino acids in the SEMA domain (SEQ ID NO: 79) of the c-Met protein, eg, the 106 th in the SEMA domain (SEQ ID NO: 79) of the c-Met protein. It may be one containing 5 to 19 consecutive amino acids located in SEQ ID NO: 71 corresponding to the 124th. For example, the epitope may be composed of 5 to 19 consecutive amino acids, including SEQ ID 73 (EEPSQ) among the amino acid sequences of SEQ ID NO: 71, for example, the amino acid sequence of SEQ ID NO: 71, SEQ ID NO: 72 or SEQ ID NO: 73 It may be a polypeptide comprising a.

The epitope having the amino acid sequence of SEQ ID NO: 72 corresponds to the outermost portion of the loop region between domains 2 and 3 of the propeller structure in the SEMA domain of the c-Met protein, and the amino acid sequence of SEQ ID NO: 73. The epitope to have is a site to which the antibody or antigen-binding fragment according to one embodiment binds most specifically.

Thus, the anti-c-Met antibody may be one that specifically binds to an epitope comprising 5 to 19 consecutive amino acids comprising SEQ ID NO: 73 (EEPSQ) among the amino acid sequences of SEQ ID NO: 71, such as the sequence It may be an antibody or antigen-binding fragment that specifically binds an epitope comprising the amino acid sequence of SEQ ID NO: 71, SEQ ID NO: 72, or SEQ ID NO: 73.

According to one embodiment, the anti-c-Met antibody,

CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, amino acid sequence of SEQ ID NO: 5, amino acid sequence of SEQ ID NO: 2, or consecutive 8 to 9 amino acids in the amino acid sequence of SEQ ID NO: 2 CDR-H2 comprising the amino acid sequence consisting of 19 amino acids, and the amino acid sequence of SEQ ID NO: 6, the amino acid sequence of SEQ ID NO: 85, or the sequence of amino acids 1 to 6 in the amino acid sequence of SEQ ID NO: 85 At least one heavy chain complementarity determining region (CDR) selected from the group consisting of CDR-H3 comprising an amino acid sequence consisting of 6 to 13 amino acids, or a heavy chain variable region comprising said at least one heavy chain complementarity determining region;

CDR-L1 comprising the amino acid sequence of SEQ ID NO: 7, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 8, and the amino acid sequence of SEQ ID NO: 9, the amino acid sequence of SEQ ID NO: 86, or the amino acid of SEQ ID NO: 89 One or more light chain complementarity determining regions selected from the group consisting of CDR-L3 comprising an amino acid sequence consisting of 9 to 17 amino acids comprising the first to ninth amino acids in the sequence, or the one or more light chain complementarity determining regions Light chain variable region;

A combination of the one or more heavy chain complementarity determining regions and the one or more light chain complementarity determining regions; or

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

Including,

The SEQ ID NO: 4 to SEQ ID NO: 9 may be an antibody or an antigen-binding fragment that is an amino acid sequence represented by the following general formulas I to VI:

Formula Ⅰ

Xaa ₁ -Xaa ₂ -Tyr-Tyr-Met-Ser (SEQ ID NO: 4),

Formula Ⅱ

Arg-Asn-Xaa ₃ -Xaa ₄ -Asn-Gly-Xaa ₅ -Thr (SEQ ID NO: 5),

Formula Ⅲ

Asp-Asn-Trp-Leu-Xaa ₆ -Tyr (SEQ ID NO: 6),

Formula Ⅳ

Lys-Ser-Ser-Xaa ₇ -Ser-Leu-Leu-Ala-Xaa ₈ -Gly-Asn-Xaa ₉ -Xaa ₁₀ -Asn-Tyr-Leu-Ala (SEQ ID NO: 7)

General formula Ⅴ

Trp-Xaa ₁₁ -Ser-Xaa ₁₂ -Arg-Val-Xaa ₁₃ (SEQ ID NO: 8)

General formula Ⅵ

Xaa ₁₄ -Gln-Ser-Tyr-Ser-Xaa ₁₅ -Pro-Xaa ₁₆ -Thr (SEQ ID NO: 9)

In the general formula (I), Xaa ₁ is absent or is Pro or Ser, Xaa ₂ is Glu or Asp,

In the general formula II, Xaa ₃ is Asn or Lys, Xaa ₄ is Ala or Val, Xaa ₅ is Asn or Thr,

In the general formula III, Xaa ₆ is Ser or Thr,

In the general formula IV, Xaa ₇ is His, Arg, Gln or Lys, Xaa ₈ is Ser or Trp, Xaa ₉ is His or Gln, Xaa ₁₀ is Lys or Asn,

In the general formula V, Xaa ₁₁ is Ala or Gly, Xaa ₁₂ is Thr or Lys, Xaa ₁₃ is Ser or Pro,

In the general formula VI, Xaa ₁₄ is Gly, Ala or Gln, Xaa ₁₅ is Arg, His, Ser, Ala, Gly or Lys, and Xaa ₁₆ is Leu, Tyr, Phe or Met.

In one embodiment, the CDR-H1 may include an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24. The CDR-H2 may include an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 25, and SEQ ID NO: 26. The CDR-H3 may include an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 85.

The CDR-L1 may include an amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 106. The CDR-L2 may include an amino acid sequence selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36. The CDR-L3 may include an amino acid sequence selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 37, SEQ ID NO: 86, and SEQ ID NO: 89. .

In one embodiment, the antibody or antigen-binding fragment is

Polypeptide (CDR-H1) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 25, and SEQ ID NO: 26 A polypeptide comprising an amino acid sequence (CDR-H2), and a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 85 (CDR-H3) Heavy chain variable region;

Polypeptide (CDR-L1) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 106, SEQ ID NO: 11, SEQ ID NO: Polypeptide (CDR-L2) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36, and SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, A light chain variable region comprising a polypeptide (CDR-L3) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 86, and SEQ ID NO: 89; or

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

It may be to include.

According to one embodiment, the anti-c-Met antibody or antigen-binding fragment has the amino acid sequence of SEQ ID NO: 17, SEQ ID NO: 74, SEQ ID NO: 87, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93 or SEQ ID NO: 94. The heavy chain variable region comprising, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 75, SEQ ID NO: 88, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: The light chain variable region comprising the amino acid sequence of 99 or SEQ ID NO: 107, or may include a combination of the heavy chain variable region and the light chain variable region.

In one embodiment, the anti-c-Met antibody may be a monoclonal antibody that specifically binds to the extracellular region of the c-Met protein produced in hybridoma cells with accession number KCLRF-BP-00220. (See Republic of Korea Patent Publication No. 2011-0047698; the above documents are incorporated herein by reference).

The anti-c-Met antibody may include all of the antibodies defined in Korean Patent Publication No. 2011-0047698.

According to one embodiment, the anti-c-Met antibody,

Amino acid sequence of SEQ ID NO: 62 (of which the 1st to 17th amino acid sequences are signal peptides), 18th to 462th amino acid sequences of SEQ ID NO: 62, and amino acid sequence of SEQ ID NO: 64 (from 1st among them) 17th amino acid sequence is a signal peptide) or 18th to 461th amino acid sequence of SEQ ID NO: 64, amino acid sequence of SEQ ID NO: 66 (of which the 1st to 17th amino acid sequence is a signal peptide), And A heavy chain comprising an amino acid sequence selected from the group consisting of the 18th to 460th amino acid sequence of SEQ ID NO: 66; And

Amino acid sequence of SEQ ID NO: 68 (the amino acid sequence from 1 to 20 of which is a signal peptide), amino acid sequence of 21 to 240 of SEQ ID NO: 68, amino acid sequence of SEQ ID NO: 70 (from 1 of 1) 20th amino acid sequence is a signal peptide), a light chain comprising an amino acid sequence selected from the group consisting of the amino acid sequence of the 21st to 240th SEQ ID NO: 70, and the amino acid sequence of SEQ ID NO: 108

It may be to include.

For example, the anti-c-Met antibody,

The heavy chain comprising the amino acid sequence of SEQ ID NO: 62 or the 18 to 462 amino acid sequence of SEQ ID NO: 62 and the light chain comprising the amino acid sequence of SEQ ID NO: 68 or the 21 to 240 amino acid sequence of SEQ ID NO: 68 Containing antibody;

The heavy chain comprising the amino acid sequence of SEQ ID NO: 64 or the 18th to 461th amino acid sequence of SEQ ID NO: 64 and the light chain comprising the amino acid sequence of SEQ ID NO: 68 or the 21st to 240th amino acid sequence of SEQ ID NO: 68 Containing antibody;

The heavy chain comprising the amino acid sequence of SEQ ID NO: 66 or the 18 to 460 amino acid sequence of SEQ ID NO: 66 and the light chain comprising the amino acid sequence of SEQ ID NO: 68 or the 21 to 240 amino acid sequence of SEQ ID NO: 68 Containing antibody;

The heavy chain comprising the amino acid sequence of SEQ ID NO: 62 or the 18th to 462th amino acid sequence of SEQ ID NO: 62 and the light chain comprising the amino acid sequence of SEQ ID NO: 70 or the 21st to 240th amino acid sequence of SEQ ID NO: 70. Containing antibody;

The heavy chain comprising the amino acid sequence of SEQ ID NO: 64 or the 18 to 461 amino acid sequence of SEQ ID NO: 64 and the light chain comprising the amino acid sequence of SEQ ID NO: 70 or the 21 to 240 amino acid sequence of SEQ ID NO: 70 Containing antibody; or

A heavy chain comprising the amino acid sequence of SEQ ID NO: 66 or the 18th to 460th amino acid sequence of SEQ ID NO: 66 and a light chain comprising the amino acid sequence of the amino acid sequence of SEQ ID NO: 70 or 21 to 240 of SEQ ID NO: 70. Containing antibody

An antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 62 or the amino acid sequence from 18 to 462 of SEQ ID NO: 62 and a light chain comprising the amino acid sequence of SEQ ID NO: 108;

An antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 64 or the 18 to 461 amino acid sequence of SEQ ID NO: 64 and a light chain comprising the amino acid sequence of SEQ ID NO: 108; And

Antibodies comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 66 or the amino acid sequence of 18 to 460 of SEQ ID NO: 66 and a light chain comprising the amino acid sequence of SEQ ID NO: 108

It may be selected from the group consisting of.

On the other hand, the polypeptide having the amino acid sequence of SEQ ID NO: 70 is a light chain consisting of a human kappa constant region, and the polypeptide having the amino acid sequence of SEQ ID NO: 68 is 36 (from kabat numbering) of the polypeptide having the amino acid sequence of SEQ ID NO: 70. Accordingly, histidine is a polypeptide in the form of tyrosine substituted. Due to the substitution, the production amount of the antibody according to one embodiment may be increased. In addition, the polypeptide having the amino acid sequence of SEQ ID NO: 108 is located at the 27e position by kabat numbering in the polypeptide having the amino acid sequence of the 21st to 240th excluding the signal peptide from 1st to 20th of the amino acid sequence of SEQ ID NO: 68. Serin (Ser) of (in accordance with kabat numbering, position 32 in SEQ ID NO: 108; CDR-L1) is substituted with tryptophan (Trp), and due to the substitution, activity of the antibody according to one embodiment (eg, binding affinity to c-Met, c-Met decomposition activity and Akt phosphorylation inhibitory activity, etc.) may be further enhanced.

Animal-derived antibodies produced by immunizing an immunized animal with a desired antigen may generally undergo an immune rejection reaction when administered to a human for therapeutic purposes, and chimeric antibodies have been developed to suppress such immune rejection. Chimeric antibodies are those that replace the constant regions of animal-derived antibodies that cause anti-isotype reactions with the constant regions of human antibodies using genetic engineering methods. Chimeric antibodies have improved significantly in anti-isotype reactions compared to animal-derived antibodies, but still have animal-derived amino acids present in the variable region, thus implicating side effects on potential anti-idiotypic responses. Doing. Humanized antibodies have been developed to improve these side effects. It is produced by transplanting a region of complementary determining regions (CDRs) that play an important role in antigen binding among variable regions of a chimeric antibody into a human antibody framework.

The most important thing in the CDR grafting technology for the production of humanized antibodies is to select an optimized human antibody that can best accept the CDR regions of animal-derived antibodies. structure), molecular modeling technology, etc. are utilized. However, even when the CDR regions of animal-derived antibodies are grafted onto the optimized human antibody skeleton, there are cases where amino acids that affect the antigen binding while being located on the skeleton of the animal-derived antibody are often not preserved. Since it exists, it can be said that the application of additional antibody engineering techniques to restore antigen binding ability is essential.

According to one embodiment, the antibody may be a mouse-derived antibody, a mouse-human chimeric antibody, a humanized antibody, or a human antibody.

A complete antibody has a structure having two full length light chains and two full length heavy chains, and each light chain is connected by a heavy chain and a disulfide bond. 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 gamma (γ), mu (μ), alpha (α), delta (δ) and epsilon (ε) types, and subclasses. Gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1) and alpha 2 (α2). The constant regions of the light chain have 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 (A) comprising an amino acid sequence with sufficient variable region sequences to confer specificity to the antigen ( It is interpreted as including both the full-length heavy chain and the fragments thereof. In addition, the term “light chain” refers to both the full-length light chain comprising the variable region domain V _L and the constant region domain C _L and fragments thereof comprising an amino acid sequence having sufficient variable region sequences to confer specificity to the antigen. It is interpreted as including meaning.

The term "complementarity determining region (CDR)" refers to the amino acid sequence of the hypervariable regions of the heavy and light chains of immunoglobulins. The heavy chain and light chain may each include three CDRs (CDRH1, CDRH2, CDRH3 and CDRL1, CDRL2, CDRL3). The CDRs can provide a major contact residue for the antibody to bind to an antigen or epitope. On the other hand, in the present specification, the term, "specifically binding" or "specifically recognized" has the same meaning as commonly known to those skilled in the art, and antigens and antibodies specifically interact to perform an immunological reaction. Means

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

When the animal-derived antibody undergoes a chimerization process, the animal-derived IgG1 hinge is replaced with a human IgG1 hinge, but the animal-derived IgG1 hinge is shorter in length than the human IgG1 hinge and disulfide bond between the two heavy chains ( The disulfide bond is reduced from 3 to 2, and the rigidity of the hinge shows different effects. Thus, modification of the hinge region can increase the antigen binding efficiency of the humanized antibody. Methods of deletion, addition or substitution of amino acids to modify the amino acid sequence of the hinge region are well known to those skilled in the art.

Thus, in one embodiment of the present invention, in order to enhance antigen binding efficiency, the anti-c-Met antibody or antigen-binding fragment includes a hinge region in which one or more amino acids are deleted, added or substituted to modify the amino acid sequence. You can. For example, the antibody may include a hinge region comprising the amino acid sequence of SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, or SEQ ID NO: 105. More specifically, the hinge region may include the amino acid sequence of SEQ ID NO: 100 or SEQ ID NO: 101.

The light chain constant region and the heavy chain constant region except the previously defined CDR regions or light chain variable regions and heavy chain variable regions of the anti-c-Met antibody or anti-EGFR antibody are immunoglobulins of all subtypes (eg, IgA, IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), IgM, etc.) may be a light chain constant region and a heavy chain constant region.

As used herein, the term “antigen-binding fragment” is a fragment of the entire structure of an immunoglobulin and refers to a portion of a polypeptide that includes a portion to which an antigen can bind, for example, scFv, (scFv) ₂ , scFv-Fc, Fab, Fab ', or F (ab') ₂ , but is not limited thereto. Antigen-binding fragments of an antibody in the present invention include an antibody fragment comprising one or more of the above-described complementarity determining regions, such as scFv, (scFv) 2, scFv-Fc, Fab, Fab 'and F (ab') 2 It may be selected from.

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

Fab 'differs from Fab in that it has a hinge region containing one or more cysteine residues at the C-terminus of the heavy chain C _H1 domain. The F (ab ') ₂ antibody is produced by cysteine residues in the hinge region of Fab' forming disulfide bonds.

Fv is a minimal antibody fragment having only a heavy chain variable region and a light chain variable region. Recombination techniques for generating Fv fragments are well known in the art. The double-chain Fv (two-chain Fv) is a non-covalently linked heavy chain variable region and a light chain variable region, and the single-chain Fv (single-chain Fv) is generally shared between the variable region of the heavy chain and the variable region of the single chain through a peptide linker. It can be linked by a bond or directly linked at the C-terminus to form 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 long, and the types of amino acids included are not limited.

The antigen-binding fragment can be obtained using proteolytic enzymes (e.g., digestion of the whole antibody with papain to obtain Fab, and digestion with pepsin to obtain F (ab ') ₂ fragment), It can be produced through genetic recombination technology.

On the other hand, since the mixture or pharmaceutical composition contains an antibody or an antigen-binding fragment, it can be formulated as an immune liposome. Liposomes comprising antibodies can be prepared according to methods well known in the art. The immune liposome is a lipid composition comprising phosphatidylcholine, cholesterol and polyethylene glycol-derivatized phosphatidylethanolamine, and may be prepared by reverse phase evaporation. For example, Fab 'fragments of an antibody can be conjugated to liposomes through a disulfide-replacement reaction. Chemotherapeutic agents, such as doxorubicin, may additionally be included in the liposome.

According to one embodiment, the antibody may be to act as an antagonist of the c-Met protein.

The term “antagonist” is interpreted as the concept encompassing all molecules that partially, completely block, inhibit, or neutralize one or more of the biological activities of a target (eg, c-Met). For example, “antagonist” antibody refers to an antibody that inhibits or reduces the biological activity of the antigen (eg c-Met) to which the antibody binds. The antagonist may act to reduce receptor phosphorylation, disable cells, or kill cells that have been activated by the ligand by binding the receptor to the ligand. In addition, the antagonist may completely disrupt the interaction between the receptor and the ligand, or substantially reduce the interaction by a change in the tertiary structure of the receptor, or down regulation.

A first pharmacy comprising an effective amount of an angiogenesis inhibitor (e.g., VEGF antagonist) and an effective amount of an anti-c-Met antibody or antigen-binding fragment thereof as a mixture, an effective amount of an angiogenesis inhibitor (e.g., VEGF antagonist) The second pharmaceutical composition comprising an effective amount of an anti-c-Met antibody or antigen-binding fragment thereof as a composition or an active ingredient may be provided together with a pharmaceutically acceptable carrier, diluent, and / or excipient.

The mixture, or a pharmaceutically acceptable carrier included in the pharmaceutical composition, is commonly used for the formulation of drugs, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, In the group consisting of gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc. It may be one or more selected, but is not limited thereto. The blending agent or pharmaceutical composition is one or more selected from the group consisting of diluents, excipients, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc., which are commonly used in the manufacture of pharmaceutical compositions in addition to the above components. It can contain.

The mixture or pharmaceutical composition may be administered orally or parenterally. In the case of parenteral administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, intrapulmonary administration, and rectal administration may be administered. When administered orally, proteins or peptides are digested, so oral compositions 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 capable of transporting the active substance to target cells.

The effective amount of the angiogenesis inhibitor (e.g., VEGF antagonist) and the effective amount of the anti-c-Met antibody or antigen-binding fragment thereof for a single administration are the formulation method, mode of administration, age, weight, sex, morbidity, food of the patient, It can be variously prescribed by factors such as administration time, administration interval, administration route, excretion rate and response sensitivity. For example, an effective amount of the angiogenesis inhibitor (eg, VEGF antagonist) for one administration may be 0.001 to 1000 mg / kg, specifically 0.01 to 100 mg / kg, more specifically 0.1 to 50 mg / kg, The effective amount of the anti-c-Met antibody or antigen-binding fragment thereof for single administration is 0.001 to 1000 mg / kg, specifically 0.01 to 100 mg / kg, more specifically 0.1 to 50 mg / kg It may be a range, but is not limited thereto. The effective amount for the single administration may be formulated as a single unit in the form of a unit dose, or may be prepared by appropriately dispensing, or incorporated into a multi-dose container. In the kit, an effective amount of an angiogenesis inhibitor (eg, VEGF antagonist) for single administration and an effective amount of an anti-c-Met antibody or antigen-binding fragment thereof may be included in a packaging container as a basic unit.

The administration interval between the combination administrations, which is defined as a period from the combination administration to the next combination administration, is not limited thereto, but may be 5 hours to 30 days, specifically 5 to 14 days. In the combination administration, the first pharmaceutical composition comprising an effective amount of an angiogenesis inhibitor (eg, VEGF antagonist) as an active ingredient and a second pharmaceutical composition comprising an effective amount of an anti-c-Met antibody or an antigen-binding fragment thereof as an active ingredient May be administered in combination at predetermined time intervals (eg, minutes, hours, or days, or weeks) depending on the type of disease, the condition of the patient, and the like. For example, the first pharmaceutical composition and the second pharmaceutical composition may be administered simultaneously (within 1 minute of the administration interval) or sequentially (more than 1 minute of administration interval), and when administered sequentially, the first pharmaceutical composition and The interval between administrations of the second pharmaceutical composition may be 1 minute to 30 days, specifically 1 minute to 7 days, 1 minute to 24 hours, or 1 minute to 60 minutes, more specifically 1 minute to 10 minutes, and the order of administration is as follows. It is okay to change.

The mixture or pharmaceutical composition may be in the form of a solution, suspension, syrup or emulsion in an oil or aqueous medium, or may be formulated in the form of ex-agent, powder, granule, tablet or capsule, and dispersing agent for formulation. Or it may further include a stabilizer.

In particular, since the pharmaceutical composition comprising the anti-c-Met antibody or an effective amount thereof contains an antibody or antigen-binding fragment, it can be formulated as an immune liposome. Liposomes comprising antibodies can be prepared according to methods well known in the art. The immune liposome is a lipid composition comprising phosphatidylcholine, cholesterol and polyethylene glycol-derivatized phosphatidylethanolamine, and may be prepared by reverse phase evaporation. For example, Fab 'fragments of an antibody can be conjugated to liposomes through a disulfide-replacement reaction. Chemotherapeutic agents, such as doxorubicin, may additionally be included in the liposome.

Another example induces c-Met and angiogenesis factors (such as VEGF) comprising administering to a patient an effective amount of an angiogenesis inhibitor (e.g., VEGF antagonist) and an effective amount of an anti-c-Met antibody or antigen-binding fragment thereof. It provides a method of preventing and / or treating disease. The method of prevention and / or treatment may further include identifying a patient in need of prevention and / or treatment of c-Met and angiogenic factors (such as VEGF) -induced diseases prior to the administration step.

In one embodiment, the combination administration may be performed by administering a mixture of an effective amount of an angiogenesis inhibitor (eg, VEGF antagonist) and an effective amount of an anti-c-Met antibody or antigen-binding fragment thereof. In another embodiment, the combination administration is a first step of administering an effective amount of an angiogenesis inhibitor (eg, VEGF antagonist) as an active ingredient and a second step of administering an effective amount of an anti-c-Met antibody or antigen-binding fragment thereof as an active ingredient The steps may be performed simultaneously or sequentially. When sequentially administered, the order may be changed.

The patient may be a mammal including primates including humans, monkeys, etc., rodents including mice, rats, and the like. In addition, the patient may be a cancer patient or a patient resistant to angiogenesis inhibitors (eg, VEGF antagonists). Thus, the method of prevention and / or treatment may further include identifying a patient resistant to angiogenesis inhibitors (eg, VEGF antagonists) prior to the administration step.

In one embodiment, the patient does not have Cbl or is present at low concentration (eg, '+1' or '-when Cbl is immunohistochemically stained using an anti-Cbl antibody available for immunohistochemical staining) 'Exists at a concentration corresponding to'), a functional mutation of Cbl is induced, or an interaction site of c-Met with Cbl is mutated, such that the existing c-Met antibody may not exhibit c-Met decomposition activity. In addition, the patient may be a patient capable of decomposing c-Met by LRIG1 mediated by the intrinsic c-Met antibody as a pathway independent of Cbl due to the high expression level of LRIG1.

Thus, the method of prevention and / or treatment may further include identifying a patient in which Cbl is inactivated and / or has a high expression level of LRIG1 prior to the administration step.

The step of identifying the patient

(1) determining the Cbl concentration, the Cbl mutation, and / or the c-Met's interaction with Cbl in the cell sample isolated from the patient; And

(2) when the concentration of Cbl corresponds to '+1' or '-' when immunohistochemical staining using an anti-Cbl antibody that can be used for immunohistochemical staining, when a Cbl mutation exists, and / Or if there is a variation in the site of interaction of c-Met with Cbl, determining the cell or the patient from which the cell is derived as a target for administration of the pharmaceutical composition for co-administration

It may be to include.

In embodiments, the step of identifying the patient,

Determining the LRIG1 concentration of the cell sample isolated from the patient, and / or the LRIG1 concentration corresponds to +2 or +3 when immunohistochemical staining using an anti-LRIG1 antibody available for immunohistochemical staining In the case, it may further include the step of determining the administration target of the pharmaceutical composition for combined administration.

"Cbl", "Cbl protein", or "Cbl enzyme" is also referred to as E3 ligase (E3 ligase), and is a protein involved in cellular signaling and protein ubiquitination, and c-Met protein located in the cell membrane of cancer cells It is a substance that serves to decompose by internalizing inside. The protein may be a polypeptide encoded by the nucleotide sequence provided in GenBank Accession Number (NM_005188, NM_007619, NM_170662 or NM_001033238), or a polypeptide comprising the amino acid sequence of GenBank Accession Number (NP_005199, NP_031645, NP_733762 or NP_001028410) .

"LRIG1 (Leucine-rich repeats and immunoglobulin-like domains protein 1)" is a transmembrane protein that interacts with receptor tyrosine kinase, such as the EGFR-line, MET and RET proteins. The LRIG1 may be derived from a mammal including humans, monkeys, etc., a mammal including rats, mice, etc., and may be specifically human LRIG1 (Accession No. NM_015541 or NP_056356).

Confirmation of the Cbl concentration or LRIG1 concentration can be performed by measuring by a conventional protein quantitative analysis means and / or evaluating the measured results. For example, the concentration of Cbl or LRIG1 can be measured through conventional enzyme reaction, fluorescence, luminescence, and / or radiation detection using antibodies, aptamers, etc. that specifically bind to Cbl or LRIG1, respectively. Chromatography (Immunochromatography), immunohistochemical staining, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescence immunoassay : FIA), luminescence immunoassay (LIA), Western blotting, etc., may be measured by a method selected from the group consisting of, but is not limited to. The detection material for measuring the concentration of Cbl or LRIG1 may be at least one selected from the group consisting of antibodies, aptamers, etc. that specifically bind to Cbl or LRIG1.

The Cbl mutation is a mutation and / or Cbl of all Cbl genes that results in loss of function associated with interaction with c-Met (eg, binding) and / or cell internalization of c-Met and / or degradation of c-Met. It may be a sequence or structural variation of the protein. In an embodiment, the Cbl variant is 51 or more contiguous, such as 51 to 200 contiguous nucleotides in the region from 1169 to 1414 in the nucleotide sequence provided in GenBank Accession Number NM_005188 deleted, or substituted with a different nucleotide, or In the amino acid sequence of GenBank Accession Number NP_005179, 3 or more consecutive 17 or more amino acids in the region from 343 to 424 may be deleted or substituted with different amino acids. This mutation induces the modification of Cbl's RING Finger Motif, thereby losing its function as an E3 ligase enzyme. In other words, the ability to break down other proteins is lost due to mutations in nucleotides or amino acids.

The determination of whether or not such a Cbl mutation can be performed by a method such as direct analysis of a nucleotide sequence or amino acid sequence, RT-PCR or DNA sequence sequencing, and / or by evaluating the measured result, but is not limited thereto. no.

In addition, the Cbl mutation detection material may be one or more selected from the group consisting of primers capable of detecting such mutations, and anti-Cbl antibodies specifically binding to the mutated Cbl, aptamers, and the like. It is not. The primer capable of detecting the Cbl mutation is a sequence of 20 to 50 sequences and / or complementary sequences containing the mutation site among the nucleotide sequences of the mutated Cbl gene, or 80% or more of these sequences to enable hybridization with them , It may be a sequence having a sequence homology of 90% or more, and more specifically 95% or more.

The c-Met mutation refers to a mutation occurring at a site where Cbl of c-Met recognizes and / or binds, and even when Cbl is quantitatively present or a loss of function mutation does not occur, Cbl and c-Met It refers to a variation that prevents interaction (eg binding).

"C-Met interaction site with Cbl" is a site where Cbl recognizes and acts in the structure of c-Met, and is a site that enables intracellular movement and degradation of c-Met by Cbl. Typical c-Met interaction sites with Cbl include a region encoded by tyrosine (Y1003), a 1003 amino acid residue that binds to Cbl, or exon 14 of the c-Met gene. The exon 14 region of the c-Met gene may be a region from 3075 to 3215 in the full-length nucleotide sequence of NM_000245, or a region from 964 to 1009 in the full-length amino acid sequence of NP_000236. The c-Met mutation is a tyrosine (Y1003), the 1003th amino acid residue of c-Met, is deleted or another amino acid (e.g., alanine, isoleucine, leucine, ketionine, phenylalanine, proline, tryptophan, valine, asparagine, cysteine, An amino acid selected from the group consisting of glutamine, glycine, serine, threonine, aspartic acid, glutamic acid, arginine, histidine, and lysine, specifically phenylalanine), or 141 consecutive exon 14 segments of the c-Met gene Or more, such as 141 to 300 consecutive nucleotides deleted or substituted with other nucleotides, and / or 46 or more consecutive 46 to 100 sequential polypeptide sites encoded by the exon 14 region, such as 46 to 100 consecutive Dog amino acids may be deleted or substituted with other amino acids. In an embodiment, the variation of c-Met is a deletion of tyrosine (Y1003), a 1003th amino acid residue of c-Met, or substitution with phenylalanine (ie, Y1003F, etc.), or deletion of the exon 14 region of c-Met gene, or It may be a polypeptide deletion encoded by the exon 14 region of c-Met protein.

The c-Met may be confirmed by mutation, nucleotide sequence or amino acid sequence analysis, RT-PCR experiment, or DNA sequence sequencing, or the like, and / or evaluation of the measured results. It is not limited thereto. In addition, primers capable of detecting such mutations with a c-Met mutation detection agent (20 to 50 consecutive sequences including a mutation site in the nucleotide sequence of the mutated c-Met gene and / or complementary thereto) Sequences and / or sequences having sequence homology of at least 80%, specifically at least 90%, more specifically at least 95%) with the sequence to enable hybridization with the sequences), antibodies specifically binding to the modified c-Met, Aptamers and the like, but are not limited thereto.

The pharmaceutical composition for combination administration proposed in the present invention is a disease caused by c-Met and angiogenic factors (eg, VEGF), such as an increase in the number of copies and / or an increase in expression and / or angiogenesis factors of c-Met. (E.g., VEGF) can be used for the prevention and / or treatment of diseases caused by overexpression, typically cancer. The cancer may be an overexpression of c-Met and angiogenic factors (eg, VEGF), and may be solid cancer, but is not limited to squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung Squamous carcinoma, peritoneal cancer, skin cancer, melanoma in the skin or eye, rectal cancer, anus congenital cancer, esophageal cancer, small intestine cancer, endocrine adenocarcinoma, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, chronic or acute leukemia, lymphocyte lymphoma, hepatocyte Cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver tumor, breast cancer, colon cancer, colon cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulva cancer, thyroid cancer, It may be one or more selected from the group consisting of liver cancer, head and neck cancer, brain cancer, osteosarcoma, and soft sarcoma. In another example, the disease caused by the c-Met and angiogenic factors may be gestational diabetes, diabetic retinopathy, macular degeneration (eg, wet senile macular degeneration (wet AMD), etc.).

The preventive and / or therapeutic effect of the cancer includes not only the effect of inhibiting the growth of cancer cells, but also the effect of suppressing the exacerbation of cancer due to migration, invasion, metastasis, and the like. Accordingly, cancers treatable by the combination treatment of the present invention include both primary and metastatic cancers.

In another aspect, a combination treatment partner is provided that can overcome each other's resistance and enhance efficacy. Specifically, an angiogenesis inhibitor is proposed as a co-administration administration partner that allows the anti-c-Met antibody to exert its effects on diseases that have had no efficacy and exertion alone or resistance to it. In addition, an anti-c-Met antibody is proposed as a combination administration partner that allows angiogenesis inhibitors to exert their effects on diseases that have not been effective or exerted alone, or diseases with resistance to them. That is, it provides an effect of enhancing the efficacy of each anti-c-Met antibody and angiogenesis inhibitor relative to the inhibitor.

Accordingly, another example provides a pharmaceutical composition for enhancing the efficacy of an anti-c-Met antibody comprising an angiogenesis inhibitor. Another example provides a method of enhancing the efficacy of an anti-c-Met antibody, comprising administering an angiogenesis inhibitor in combination with an anti-c-Met antibody. Another example provides use for enhancing the efficacy of anti-c-Met antibodies of angiogenesis inhibitors.

Another example provides a pharmaceutical composition for enhancing the efficacy of an angiogenesis inhibitor comprising an anti-c-Met antibody. Another example provides a method of enhancing the efficacy of an angiogenesis inhibitor comprising administering an anti-c-Met antibody with an angiogenesis inhibitor. Another example provides the use of anti-c-Met antibody agents to enhance the efficacy of angiogenesis inhibitors.

Enhancement of the efficacy of the anti-c-Met antibody agent or angiogenesis inhibitor is resistant to diseases (e.g., cancer) and / or each inhibitor that each inhibitor has no effect when administered alone or has a minor effect. It means to have an effective effect on a disease (eg, cancer).

By the co-administration of the anti-c-Met antibody and an angiogenesis inhibitor (eg, VEGF antagonist), not only a remarkably enhanced synergistic effect is exhibited as compared to their administration alone, but also reduces the dose of each drug. Can be, Cbl and / or c-Met is mutated to obtain an excellent effect even in patients who have not had an effect with the existing anti-c-Met antibody, and is not only effective in primary cancer but also metastatic cancer, as well as its treatment The target disease can be extended to other diseases in which c-Met / HGF signaling systems other than cancer and VEGF / VEGFR signaling systems are involved.

1 is a graph showing the results of performing the BrdU assay after treating an anti-c-Met antibody to an NCI-H441 lung cancer cell line, and shows the degree of agonism of the anti-c-Met antibody.
2 is a graph showing the results of measuring the phosphorylation level of Akt by ELISA method after treatment with anti-c-Met antibody on Caki-1 cell line.
Figure 3 is a graph showing the results of measuring the total amount of c-Met remaining after treatment with the anti-c-Met antibody to the MKN45 cell line, and shows the degree of c-Met degradation.
Figure 4 is a graph showing the results of analyzing the cell growth degree when the combination administration of L3-1Y and avastin (avastin) by real time cell analysis (red: VEGF + HGF treatment, blue: avastin alone Treatment, light green: antibody L3-1Y alone treatment, purple: antibody L3-1Y and avastin combination treatment, light blue: no treatment (media only).
5 is a graph showing the results of analyzing 92 hours, 18 minutes, and 9 seconds data as an end point in real-time cell analysis.
Figure 6 is a graph showing the cell growth inhibitory effect according to the concentration of L3-1Y and avastin (avastin) in combination administration.
FIG. 7 is a photograph showing calcein AM staining in Oris 96-well plates of antibodies and / or avastin-treated cells (HuVEC, Human Umbilical Vein Endothelial Cells), and shows cell metastasis evaluation results.
8 is a graph showing the effect of inhibiting cell metastasis measured using an Oris 96-well plate according to the concentration of the treated drug.
9 is a graph showing the results of comparing the effect of inhibiting cell metastasis measured using the Oris 96-well plate at the highest concentration of the treated drug.
10 is a photograph showing the penetration ability of HUVEC cells according to the treatment drug.
11 is a graph showing the penetration area of HUVEC cells according to the treatment drug.
12 is a graph showing the cell growth level when L3-1Y / IgG2 and avastin are administered in combination (pink: VEGF + HGF treatment, sky blue: avastin alone treatment with VEGF + HGF, yellow: L3- with VEGF + HGF) 1Y / IgG2 treatment alone, purple: L3-1Y / IgG2 combined with VEGF + HGF and avastin treatment, blue: no treatment (media only).
13 is a graph showing the relative cell index (%) after L3-1Y / IgG2 administration alone, avastin alone administration, or L3-1Y / IgG2 and avastin combination administration (for real time cell analysis, 71 hours 54 minutes 17 seconds) Is the end point).
14 shows amino acid sequences of the heavy and light chains of the L3-1Y / IgG2 antibody according to an embodiment.

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

However, the following examples are only to illustrate the present invention, the content of the present invention is not limited to the following examples.

Reference example : c- Met  Production of inhibitors

1.1. c- Met Mouse Antibodies '' AbF46 ' Production of

1.1.1. Immunization of mice

To obtain an immunized mouse required for the development of the hybridoma cell line, 100 μg of human c-Met / Fc fusion protein (R & D Systems) per 5 mice and the same amount of complete Freund's adjuvant Were mixed and injected into the intraperitoneal cavity of 4-6 week-old BALB / c mice (Japan SLC, Inc.). After 2 weeks, 50 µg, which is half of the amount injected with the human c-Met / Fc fusion protein used as the antigen, was mixed with the same amount of incomplete Freund's adjuvant in the same manner as described above. It was injected intraperitoneally. After one week, the last boosting was performed, and after 3 days, blood was collected from the tail of the mouse to obtain serum, and it was confirmed that the titer of the antibody recognizing c-Met was increased by ELISA by dilution in PBS with 1/1000. As a result of the above, a mouse having a sufficient amount of antibody was selected and the following cell fusion process was performed.

1.1.2. Cell fusion and Hybridoma  Produce

Three days before the cell fusion experiment, a human c-Met / Fc fusion protein mixture was injected into the intraperitoneal cavity of a BALB / c mouse (Japan SLC, Inc.) in PBS of 50 µg, and the immunized mouse was anesthetized, and then on the left side of the body. The located spleen was removed. The extracted spleen was ground with a mesh to separate the cells, and mixed with a culture medium (DMEM, GIBCO, Invitrogen) to make a spleen cell suspension. The suspension was centrifuged to recover the cell layer. A mixture of the resulting spleen cells, 1 x 10 ⁸ dogs and myeloma cells (Sp2 / 0) 1 x 10 8 gae precipitate the following, the cells by centrifugation. The centrifuged precipitate was slowly dispersed, treated with 45% polyethylene glycol (PEG) (1 mL) in culture medium (DMEM), maintained at 37 ° C. for 1 minute, and then 1 mL of culture medium (DMEM). Was added. After that, 10 ml of culture medium (DMEM) was added for 1 minute, left in water at 37 ° C. for 5 minutes, and then adjusted to 50 ml and centrifuged again. The cell precipitate was re-suspended in a separation medium (HAT medium) to about 1 to 2 × 10 ⁵ / ml, and 0.1 ml each was dispensed into a 96-well plate, and then cultured in a 37 ° C carbon dioxide incubator to prepare a hybridoma cell population. Did.

1.1.3. c- Met  Producing monoclonal antibodies against proteins Hybridoma  Cell sorting

Human c-Met / Fc fusion protein and human Fc protein were used as antigens to select hybridoma cells that specifically respond to c-Met protein among the hybridoma cell groups prepared in Reference Example 1.1.2. Screening was performed through an ELISA analysis method.

To the microtiter plate, 50 μl (2 ug (microgram) / ml) of human c-Met / Fc fusion protein was added to each well and attached to the plate surface, and unreacted antigen was washed and removed. In order to select and exclude antibodies that bind to Fc other than c-Met, human Fc proteins were attached to the plate surface in the same manner as above.

50 µl of each of the prepared hybridoma cell cultures obtained in Reference Example 1.1.2 was added to each well prepared for reaction for 1 hour, followed by sufficient washing with phosphate buffer solution-Tween 20 (TBST) solution to remove unreacted culture. Did. Goat anti-mouse IgG-horseradish peroxidase (goat anti-mouse IgG-HRP) was added thereto, reacted at room temperature for 1 hour, and then thoroughly washed with TBST solution. Subsequently, a peroxidase substrate solution (OPD) was added to react, and the degree of reaction was confirmed by measuring absorbance at 450 nm with an ELISA Reader.

By confirming the degree of reaction as described above, hybridoma cell lines that do not bind to human Fc and secrete antibodies with high binding power specifically for human c-Met protein were selected repeatedly. By limiting dilution of the hybridoma cell line obtained through repeated screening, one clone of a hybridoma cell line producing a monoclonal antibody was finally obtained. The final selected monoclonal antibody-producing hybridoma was deposited on October 6, 2009 to the Korea Cell Line Research Foundation located in Yeongeon-dong, Jongno-gu, Seoul, which is an international depository organization under the Budapest Treaty, and was granted the accession number KCLRF-BP-00220 (Korea See Patent Publication No. 2011-0047698).

1.1.4. Production and purification of monoclonal antibodies

The hybridoma cells obtained in Reference Example 1.1.3 were cultured in a serum-free medium, and monoclonal antibodies were produced and purified from the culture medium.

First, by centrifuging the hybridoma cells cultured in 50 ml of culture medium (DMEM) medium containing 10% (v / v) FBS, the cell precipitate was washed twice or more with 20 ml PBS to remove FBS. , The cell precipitate was resuspended in 50 ml of culture medium (DMEM) medium, and then cultured in a 37 ° C. carbon dioxide incubator for 3 days.

Subsequently, by centrifugation, the cells producing the antibody were removed, and the culture medium from which the antibodies were secreted was isolated and stored at 4 ° C. or collected immediately and used for purification of the antibody. After purification of the antibody from 50 ml to 300 ml of the prepared culture medium using an AKTA purification device (GE Healthcare) equipped with an affinity column (Protein G agarose column; Pharmacia, USA), a filter for protein aggregation (Amicon) was used. The purified antibody was stored by substituting the supernatant with PBS, and used in the following examples.

1.2. c- Met About Chimeric  Antibody chAbF46 Production of

In general, since a mouse antibody is likely to show an immunogenicity when injected into a human for therapeutic purposes, in order to solve this, from the mouse antibody AbF46 produced in Example 1 above, a mutation region related to antigen binding ( A chimeric antibody chAbF46 was constructed to replace the constant region except the variable region with the sequence of a human IgG1 antibody.

The nucleotide sequence corresponding to the heavy chain is' EcoRI-signal sequence-VH-NheI-CH-TGA-XhoI '(SEQ ID NO: 38), and the nucleotide sequence corresponding to the light chain is' EcoRI-signal sequence-VL- BsiWI-CL-TGA Genes were synthesized by designing each with -XhoI '(SEQ ID NO: 39). Subsequently, the pOptiVEC ^TM -TOPO TA Cloning Kit included in the OptiCHO ^TM Antibody Express Kit (Cat no. 12762-019) from Invitrogen was used to prepare a DNA fragment having the nucleotide sequence corresponding to the heavy chain (SEQ ID NO: 38), pcDNA ^TM 3.3 -TOPO DNA fragments having the nucleotide sequence corresponding to the light chain (SEQ ID NO: 39) in the TA Cloning Kit (Cat no. 8300-01) were cloned using EcoRI (NEB, R0101S) and XhoI (NEB, R0146S) restriction enzymes, respectively. , A vector containing a heavy chain and a vector containing a light chain for expression of a chimeric antibody were constructed, respectively.

Each of the constructed vectors was amplified using Qiagen Maxiprep kit (Cat no. 12662), and temporary expression is Freestyle ^TM It was performed using the MAX 293 Expression System (invitrogen). The cell line used was 293 F cell, and was cultured in a floating culture method using FreeStyle ™ 293 Expression Medium as a medium. One day before the temporary expression, cells were prepared at a concentration of 5x10 ⁵ cells / ml, and after 24 hours, temporary expression was performed when the number of cells reached 1x10 ⁶ cells / ml. Transfection was performed by liposomal reagent method using Freestyle ^TM MAX reagent (invitrogen), and DNA was prepared in a ratio of heavy chain DNA: light chain DNA = 1: 1 in a 15 ml tube and mixed with 2 ml of OptiPro ™ SFM (invtrogen) (A), 100ml of Freestyle ^TM MAX reagent and 2ml of OptiPro ™ SFM in another 15ml tube, mix (B), mix (A) and (B) for 15 minutes, incubate for 15 minutes, The mixture was slowly mixed. After completion of transduction, the cells were cultured for 5 days in a 37 ° C., 80% humidity, 8% CO _{2 ,} 130 rpm incubator.

The cultured cells were centrifuged to take 100 ml of the supernatant, respectively, and purified using AKTA Prime (GE healthcare). After installing Protein A column (GE healthcare, 17-0405-03) in AKTA Prime and flowing the culture solution at a flow rate of 5 ml / min, it was eluted with an IgG elution buffer (Thermo Scientific, 21004). The obtained eluate was exchanged with PBS buffer, and finally the chimeric antibody AbF46 (hereinafter referred to as chAbF46) was purified.

1.3. Chimeric  Antibody chAbF46 From humanized antibodies huAbF46 Production of

1.3.1. Heavy chain  Humanization ( Heavy chain humanization )

For the design of H1-heavy and H3-heavy, the VH gene of mouse antibody AbF46 purified in Reference Example 1.2 above through Ig Blast (http://www.ncbi.nlm.nih.gov/igblast/) The human germline gene with the highest homology was analyzed. As a result, it was confirmed that VH3-71 has 83% homology at the amino acid level, and CDR-H1, CDR-H2, and CDR-H3 of the mouse antibody AbF46 are defined by Kabat numbering, and the CDR portion of the mouse antibody AbF46 is It was designed to be introduced into the framework of VH3-71. At this time, amino acids 30 (S → T), 48 (V → L), 73 (D → N), and 78 (T → L) were back-mutated with the amino acid sequence of the original mouse AbF46 antibody. Thereafter, H1 was additionally mutated to amino acids 83 (R → K) and 84 (A → T) to construct H1-heavy (SEQ ID NO: 40) and H3-heavy (SEQ ID NO: 41).

As a result of finding the framework sequence of human antibody for the design of H4-heavy, the sequence is very similar to the mouse skeleton sequence of AbF46 antibody, and it is defined as Kabat numbering using the VH3 subtype known to be the most stable. CDR-H1, CDR-H2 and CDR-H3 of the mouse antibody AbF46 were introduced. Through this, H4-heavy (SEQ ID NO: 42) was constructed.

1.3.2. Light chain  Humanization ( Light chain humanization )

For the design of two H1-light (SEQ ID NO: 43) and H2-light (SEQ ID NO: 44), mouse antibody AbF46 via Ig Blast (http://www.ncbi.nlm.nih.gov/igblast/) Human germline genes with the highest homology to VL gene were analyzed. As a result, it was confirmed that VK4-1 has 75% homology at the amino acid level, and CDR-L1, CDR-L2, and CDR-L3 of the mouse antibody AbF46 are defined by Kabat numbering, and the CDR portion of the mouse antibody AbF46 is It was designed to be introduced into the framework of VK4-1. At this time, H1-light back-mutated 3 amino acids 36 (Y → H), 46 (L → M), and 49 (Y → I), and H2-light was amino acid 49 (Y → I). It was constructed by back-mutation of only one.

For the design of H3-light (SEQ ID NO: 45), the human germline gene having the highest homology with the VL gene of the mouse antibody AbF46 was obtained through Blast (http://www.ncbi.nlm.nih.gov/igblast/). Among the analyzed results, VK2-40 was selected in addition to VK4-1. It was confirmed that the mouse antibodies AbF46 VL and VK2-40 had 61% homology at the amino acid level, and CDR-L1, CDR-L2, and CDR-L3 of the mouse antibody AbF46 were defined by Kabat numbering, and CDRs of the mouse antibody AbF46 The part was designed to be introduced into the framework of VK4-1. At this time, H3-light was constructed by back-mutating the 3 amino acids 36 (Y → H), 46 (L → M), and 49 (Y → I).

For the design of H4-light (SEQ ID NO: 46), as a result of finding the framework sequence of the human antibody, CDR-L1 of the mouse antibody AbF46 defined by Kabat numbering using the Vk1 subtype known to be the most stable, CDR-L2 and CDR-L3 were introduced. At this time, H4-light was constructed by additionally back-mutating 3 amino acids 36 (Y → H), 46 (L → M), and 49 (Y → I).

Subsequently, the DNA fragment having the nucleotide sequence corresponding to the heavy chain in pOptiVEC ^TM -TOPO TA Cloning Kit included in the OptiCHO ^TM Antibody Express Kit (Cat no. 12762-019) from Invitrogen (H1-heavy; SEQ ID NO: 47, H3) -heavy; SEQ ID NO: 48, H4-heavy; SEQ ID NO: 49) pcDNA ^TM 3.3-TOPO DNA fragments (H1-light; SEQ ID NO: 50, H2-light; SEQ ID NO: 51, H3-light; SEQ ID NO: 52, H4-light; SEQ ID NO: 53) each having a nucleotide sequence corresponding to the light chain in the TA Cloning Kit A vector for expression of humanized antibodies was constructed by cloning using EcoRI (NEB, R0101S) and XhoI (NEB, R0146S) restriction enzymes.

Each of the constructed vectors was amplified using Qiagen Maxiprep kit (Cat no. 12662), and temporary expression is Freestyle ^TM It was performed using the MAX 293 Expression System (invitrogen). The cell line used was 293 F cell, and was cultured in a floating culture method using FreeStyle ™ 293 Expression Medium as a medium. One day before the temporary expression, cells were prepared at a concentration of 5x10 ⁵ cells / ml, and after 24 hours, temporary expression was performed when the number of cells reached 1x10 ⁶ cells / ml. Transfection was performed by liposomal reagent method using Freestyle ^TM MAX reagent (invitrogen), and DNA was prepared in a ratio of heavy chain DNA: light chain DNA = 1: 1 in a 15 ml tube and mixed with 2 ml of OptiPro ™ SFM (invtrogen) (A), 100ml of Freestyle ^TM MAX reagent and 2ml of OptiPro ™ SFM in another 15ml tube, mix (B), mix (A) and (B) for 15 minutes, incubate for 15 minutes, The mixture was slowly mixed. After completion of transduction, the cells were cultured for 5 days in a 37 ° C., 80% humidity, 8% CO _{2 ,} 130 rpm incubator.

The cultured cells were centrifuged to take 100 ml of each supernatant and purified using AKTA Prime (GE healthcare). After installing Protein A column (GE healthcare, 17-0405-03) in AKTA Prime and flowing the culture solution at a flow rate of 5 ml / min, it was eluted with an IgG elution buffer (Thermo Scientific, 21004). This was exchanged with PBS buffer to finally purify the humanized antibody AbF46 (hereinafter referred to as huAbF46). On the other hand, the heavy chain and light chain combinations of the humanized antibody huAbF46 used in Examples are H4-heavy (SEQ ID NO: 42) and H4-light (SEQ ID NO: 46).

1.4. huAbF46  Antibody scFv  Library production

The gene for constructing the scFv of the huAbF46 antibody was designed using the heavy chain variable region and the light chain variable region of the huAbF46 antibody. Each heavy chain variable region and light chain variable region were in the form of 'VH-linker-VL', and the linker was designed to have the amino acid sequence of 'GLGGLGGGGSGGGGSGGSSGVGS' (SEQ ID NO: 54). A polynucleotide (SEQ ID NO: 55) encoding the scFv of the huAbF46 antibody thus designed was synthesized by requesting from Bonia, and a vector for expressing it is shown in SEQ ID NO: 56.

Then, expressed from the vector By analyzing the result, it was confirmed that it showed a specific binding force to c-Met.

1.5. Affinity maturation ( affinity maturation Of library genes for)

1.5.1. Target CDR Selection of and primer  making

For affinity maturation of the huAbF46 antibody, six complementary determining regions (CDRs) were defined by 'Kabat numbering' from the mouse antibody AbF46 prepared above, and each CDR is shown in Table 1 below. .

CDR Amino acid sequence CDR-H1 DYYMS (SEQ ID NO: 1) CDR-H2 FIRNKANGYTTEYSASVKG (SEQ ID NO: 2) CDR-H3 DNWFAY (SEQ ID NO: 3) CDR-L1 KSSQSLLASGNQNNYLA (SEQ ID NO: 10) CDR-L2 WASTRVS (SEQ ID NO: 11) CDR-L3 QQSYSAPLT (SEQ ID NO: 12)

Primers were prepared as follows for random sequence introduction of antibody CDRs. In the conventional random sequence introduction method, N codons were used to introduce the same ratio of bases (25% A, 25% G, 25% C, 25% T) to the site to be mutated, but in this example, the huAbF46 antibody In order to introduce a random base into the CDR of the three, encoding the amino acid of each CDR Among the wild-type nucleotides, 85% of the first and second nucleotides were preserved, and 5% of the remaining three bases were introduced, respectively. In addition, primers were designed such that the third nucleotide was introduced the same (33% G, 33% C, 33% T).

1.5.2. huAbF46  Antibody library construction and c- Met Check the binding force for

Construction of the antibody library gene through the random sequence introduction of CDR was performed using primers prepared in the same manner as in Reference Example 1.5.1 above. Using the polynucleotide containing the scFv of the huAbF46 antibody as a template, two PCR fragments were prepared as in the method shown in FIG. 1, and through the overlap extension PCR method, only the desired CDRs were respectively obtained. The scFv library gene of the mutated huAbF46 antibody was secured to build libraries targeting each of the 6 CDRs produced.

The library produced in this way confirmed the binding ability of the wild-type and each library to c-Met, and each library tended to have a lower binding force to c-Met than the wild-type, but some of the binding strength to c-Met The retained mutations were identified.

1.6. From the produced library Affinity  Improved antibody screening

After improving the binding ability of the library to c-Met from the constructed library, the gene sequence of scFv from each individual clone was analyzed. The secured gene sequences are shown in Table 2, respectively, and these were converted into IgG forms. Among the following clones, four antibodies produced from L3-1, L3-2, L3-3, and L3-5 were selected to perform subsequent experiments .

Clone name Derived library CDR sequences H11-4 CDR-H1 PEYYMS (SEQ ID NO: 22) YC151 CDR-H1 PDYYMS (SEQ ID NO: 23) YC193 CDR-H1 SDYYMS (SEQ ID NO: 24) YC244 CDR-H2 RNNANGNT (SEQ ID NO: 25) YC321 CDR-H2 RNKVNGYT (SEQ ID NO: 26) YC354 CDR-H3 DNWLSY (SEQ ID NO: 27) YC374 CDR-H3 DNWLTY (SEQ ID NO: 28) L1-1 CDR-L1 KSSHSLLASGNQNNYLA (SEQ ID NO: 29) L1-3 CDR-L1 KSSRSLLSSGNHKNYLA (SEQ ID NO: 30) L1-4 CDR-L1 KSSKSLLASGNQNNYLA (SEQ ID NO: 31) L1-12 CDR-L1 KSSRSLLASGNQNNYLA (SEQ ID NO: 32) L1-22 CDR-L1 KSSHSLLASGNQNNYLA (SEQ ID NO: 33) L2-9 CDR-L2 WASKRVS (SEQ ID NO: 34) L2-12 CDR-L2 WGSTRVS (SEQ ID NO: 35) L2-16 CDR-L2 WGSTRVP (SEQ ID NO: 36) L3-1 CDR-L3 QQSYSRPYT (SEQ ID NO: 13) L3-2 CDR-L3 GQSYSRPLT (SEQ ID NO: 14) L3-3 CDR-L3 AQSYSHPFS (SEQ ID NO: 15) L3-5 CDR-L3 QQSYSRPFT (SEQ ID NO: 16) L3-32 CDR-L3 QQSYSKPFT (SEQ ID NO: 37)

1.7. Of selected antibodies IgG Convert to

The polynucleotide encoding the heavy chain of the selected four antibodies consists of 'EcoRI-signal sequence-VH-NheI-CH-XhoI' (SEQ ID NO: 38), and in the case of heavy chain, the amino acid of the antibody is not changed after affinity maturation As it was not, the heavy chain of huAbF46 antibody was used as it was. However, the hinge region (hinge region) was replaced with a U6-HC7 hinge (SEQ ID NO: 57) rather than the hinge of human IgG1. The light chain was designed to consist of 'EcoRI-signal sequence-VL-BsiWI-CL-XhoI', respectively, to synthesize genes, and after the affinity maturation, polynucleotides encoding the light chain variable regions of the four antibodies selected (sequences) No. 58 to SEQ ID No. 61) were synthesized by requesting Bioneer. Subsequently, the pOptiVEC ^TM -TOPO TA Cloning Kit included in the OptiCHO ^TM Antibody Express Kit (Cat no. 12762-019) from Invitrogen was used to prepare a DNA fragment having the nucleotide sequence corresponding to the heavy chain (SEQ ID NO: 38), pcDNA ^TM 3.3 -TOPO DNA fragment having a nucleotide sequence corresponding to the light chain in the TA Cloning Kit (Cat no. 8300-01) (a DNA fragment comprising CDR-L3 derived from L3-1: SEQ ID NO: 58, including CDR-L3 derived from L3-2) DNA fragments comprising: SEQ ID NO: 59, DNA fragments comprising CDR-L3 derived from L3-3: SEQ ID NO: 60, DNA fragments comprising CDR-L3 derived from L3-5: SEQ ID NO: 61, respectively, EcoRI (NEB, R0101S) And cloning using XhoI (NEB, R0146S) restriction enzyme to construct a vector for expression of affinity matured antibodies.

Each of the constructed vectors was amplified using Qiagen Maxiprep kit (Cat no. 12662), and temporary expression is Freestyle ^TM It was performed using the MAX 293 Expression System (invitrogen). The cell line used was 293 F cell, and was cultured in a floating culture method using FreeStyle ™ 293 Expression Medium as a medium. One day before the temporary expression, cells were prepared at a concentration of 5x10 ⁵ cells / ml, and after 24 hours, temporary expression was performed when the number of cells reached 1x10 ⁶ cells / ml. Transfection was performed by liposomal reagent method using Freestyle ^TM MAX reagent (invitrogen), and DNA was prepared in a ratio of heavy chain DNA: light chain DNA = 1: 1 in a 15 ml tube and mixed with 2 ml of OptiPro ™ SFM (invtrogen) (A), 100ml of Freestyle ^TM MAX reagent and 2ml of OptiPro ™ SFM in another 15ml tube, mix (B), mix (A) and (B) for 15 minutes, incubate for 15 minutes, The mixture was slowly mixed. After completion of transduction, the cells were cultured for 5 days in a 37 ° C., 80% humidity, 8% CO _{2 ,} 130 rpm incubator.

The cultured cells were centrifuged to take 100 ml of each supernatant and purified using AKTA Prime (GE healthcare). After installing Protein A column (GE healthcare, 17-0405-03) in AKTA Prime and flowing the culture solution at a flow rate of 5 ml / min, it was eluted with an IgG elution buffer (Thermo Scientific, 21004). These were exchanged with PBS buffer, and finally, four mature antibodies (hereinafter, huAbF46-H4-A1 (derived from L3-1), huAbF46-H4-A2 (derived from L3-2), and huAbF46-H4-A3 (L3- 3), and huAbF46-H4-A5 (named L3-5)) were purified.

1.8. Constant region and / or Hinge area  Substituted huAbF46 - H4 -Preparation of A1

Of the four antibodies selected in Reference Example 1.7 above, the binding affinity with c-Met was the highest, and the huAbF46-H4-A1, which was measured to have the lowest Akt phosphorylation and c-Met differentiation, was hinged. Alternatively, an antibody in which the constant region and the hinge region were substituted was prepared.

huAbF46 antibody consisting of heavy chain variable region of huAbF46-H4-A1, heavy chain consisting of U6-HC7 hinge and human IgG1 constant region, and light chain composed of light chain variable region of huAbF46-H4-A1 and human kappa constant region -H4-A1 (U6-HC7); huAbF46-H4-An antibody consisting of a heavy chain variable region of huAbF46-H4-A1, a heavy chain consisting of a human IgG2 hinge and a human IgG1 constant region, and a light chain consisting of a light chain variable region of huAbF46-H4-A1 and a human kappa constant region, huAbF46-H4- With A1 (IgG2 hinge); huAbF46-H4-An antibody consisting of a heavy chain variable region of huAbF46-H4-A1, a heavy chain consisting of a human IgG2 hinge and a human IgG2 constant region, and a light chain consisting of a light chain variable region of huAbF46-H4-A1 and a human kappa constant region, huAbF46-H4- A1 (IgG2 Fc). In addition, on the other hand, in order to increase the production of the three antibodies, all of histidine 36 of the light chain consisting of the human kappa constant region was replaced with tyrosine.

To construct the three antibodies, a polynucleotide (SEQ ID NO: 63), huAbF46- encoding a polypeptide (SEQ ID NO: 62) consisting of a heavy chain variable region of huAbF46-H4-A1, a U6-HC7 hinge and a human IgG1 constant region. Polynucleotide (SEQ ID NO: 65) encoding a polypeptide consisting of H4-A1 heavy chain variable region, human IgG2 hinge and human IgG1 constant region (SEQ ID NO: 64), heavy chain variable region of huAbF46-H4-A1, human IgG2 Polynucleotide (SEQ ID NO: 66) encoding a polypeptide consisting of the hinge and human IgG2 constant region (SEQ ID NO: 66), light chain variable region of huAbF46-H4-A1 in which histitin 36 is substituted with tyrosine and human kappa constant region Polynucleotides encoding SEQ ID NO: 68 (SEQ ID NO: 68) (SEQ ID NO: 69) were synthesized by requesting Bioneer. Then, pOptiVEC ^TM -TOPO TA Cloning Kit included in Invitrogen's OptiCHO ^TM Antibody Express Kit (Cat no. 12762-019) was subjected to DNA fragments having a base sequence corresponding to the heavy chain, pcDNA ^TM 3.3-TOPO A DNA fragment having a base sequence corresponding to the light chain was inserted into a TA Cloning Kit (Cat no. 8300-01) to construct a vector for expression of the antibody.

Each of the constructed vectors was amplified using Qiagen Maxiprep kit (Cat no. 12662), and temporary expression is Freestyle ^TM It was performed using the MAX 293 Expression System (invitrogen). The cell line used was 293 F cell, and was cultured in a floating culture method using FreeStyle ™ 293 Expression Medium as a medium. One day before the temporary expression, cells were prepared at a concentration of 5x10 ⁵ cells / ml, and after 24 hours, temporary expression was performed when the number of cells reached 1x10 ⁶ cells / ml. Transfection was performed by liposomal reagent method using Freestyle ^TM MAX reagent (invitrogen), and DNA was prepared in a ratio of heavy chain DNA: light chain DNA = 1: 1 in a 15 ml tube and mixed with 2 ml of OptiPro ™ SFM (invtrogen) (A), 100ml of Freestyle ^TM MAX reagent and 2ml of OptiPro ™ SFM in another 15ml tube, mix (B), mix (A) and (B) for 15 minutes, incubate for 15 minutes, The mixture was slowly mixed. After completion of transduction, the cells were cultured for 5 days in a 37 ° C., 80% humidity, 8% CO _{2 ,} 130 rpm incubator.

The cultured cells were centrifuged to take 100 ml of each supernatant and purified using AKTA Prime (GE healthcare). After installing Protein A column (GE healthcare, 17-0405-03) in AKTA Prime and flowing the culture solution at a flow rate of 5 ml / min, it was eluted with an IgG elution buffer (Thermo Scientific, 21004). This was exchanged with PBS buffer, and finally, three antibodies (huAbF46-H4-A1 (U6-HC7), huAbF46-H4-A1 (IgG2 hinge), and huAbF46-H4-A1 (IgG2 Fc)) were purified. In the present specification, the three purified antibodies were named as follows and used in the following examples:

huAbF46-H4-A1 (U6-HC76): L3-1Y / U6-HC7 / IgG1 or L3-1Y

huAbF46-H4-A1 (IgG2 hinge): L3-1Y / U3-HC9 / IgG1 or L3-1Y / U3HC9

huAbF46-H4-A1 (IgG2 Fc): L3-1Y / U3-HC9 / IgG2 or L3-1Y / IgG2.

Example 1: Side-effect confirmation test of c-Met antibody

C-Met antibody L3-1Y produced in the above reference example, its modification L3-1Y / U3HC9 / IgG1 (the hinge region of L3-1Y is modified to U3HC9), and L3-1Y / U3HC9 / IgG2 (L3-1Y The degree of agonism of the hinge and Fc modification) was measured, and the results were compared with the previously developed c-Met antibody 5D5 (American Type Culture Collection (ATCC, Manassas, VA)) as a positive control.

BrdU assay was performed to view side effects (agonism; vulnerability to antibody safety).

First, the BrdU method is as follows. Human lung cancer cells, NCI-H441 cells (ATCC Cat. # HTB-174), were suspended in serum-free RPMI 1640 medium (Gibco) at 2 x 10 ⁵ cells / ml, and the suspension was 96-well tissue culture plate with 100 ul per well. (Corning, Lowell, MA). After incubation for 24 hours at 37? With 5% CO ₂ , the medium was completely removed, and then the antibody (L3-1Y, L3-1Y / U3HC9, L3-1Y / IgG2, 5D5, IgG (eBioscience)) was 10 ug / It was replaced with RPMI 1640 medium serially diluted from ml to 0.001 ug / ml. After incubation at 37? For 5 hours under conditions of 5% CO ₂ for 21 hours, 5-bromo-2'-deoxyuridine (5-bromo-2'-deoxyuridine, BrdU) was added and cultured for 3 hours. After that, BrdU assay (Roche, Indianapolis, IN) was performed. After denaturation / fixation of the cells on the plate, the anti-BrdU antibody was added, and after 1 hour, the substrate was added and the color development reaction was measured with an ELISA spectraMax reader (Molecular Devices, Sunnyvale, CA) at 370 nm. The comparison was based on the functionality of the mouse AbF46 antibody. As a negative control, mouse IgG was used, and a 5D5 antibody (ATCC Cat. # HB11895 hybridoma cells isolated and purified), known as an agonist, was used as a positive control.

Finally, the value of cells not containing BrdU was used as a background control, and after restriction, it was calculated as a relative value to a value (100%) of a control group not treated with an anti-c-Met antibody, and is shown in FIG. 1.

As shown in Figure 1, it was confirmed that the antibody L3-1Y and its variants showed significantly lower side effects compared to the positive control 5D5. In Figure 1, L3-1Y / U6-HC7 / IgG1, L3-1Y / U3-HC9 / IgG1, and L3-1Y / U3-HC9 / IgG2 are L3-1Y, L3-1Y U3HC9, and L3-1Y IgG2, respectively. it means.

DNA synthesis is essential for cells to grow and divide. DNA replication occurs in the S phase, and at this time, AT and GC bonds occur, and bromide is required. Therefore, the growth of cells can be measured by the amount of bromide, and the above BrdU assay can be used as an agonism assay because it is judged that there is agoonism when the cells are grown at the same time. If the BrdU value is small, the amount of DNA synthesized within the same time is small, and the growth of cells is slow, so it can be considered as a decrease in agonism.

Example 2: AKT phosphorylation inhibition test of c-Met antibody

The low side effects of the anti-c-Met antibody presented in the present invention were verified once again by safety and efficacy as a mechanism-based experiment.

In order to see the safety of the antibody, AKT phosphorylation was quantitatively measured by ELISA. Cellular functions regulated by AKT include cell proliferation, cell survival, cell size control, responsiveness to available nutrients, intermediate metabolic processes, angiogenesis, and tissue invasion. All of these processes are representative cancer cell characteristics, and numerous oncoproteins and tumor suppressors interact with each other on the AKT pathway, and the action of cells at the junction of signal transduction and classical metabolic regulation. It performs the function of finely adjusting. Therefore, the higher the degree of AKT phosphorylation and activation, the higher the tumor-forming ability. Therefore, the anti-cancer effect of the antibody was tested by measuring the degree of phosphorylation inhibition of AKT according to antibody treatment.

AKT phosphorylation site was Ser473, and AKT phosphorylation was measured using PathScan phospho-AKT1 (Ser473) chemiluminescent Sandwich ELISA kit (Cell signaling).

The day before the test, the Caki-1 kidney cancer cell line (HTB-46; American Type Culture Collection (ATCC), Manassas, VA) cultured at 2X10 ⁵ cells / ml was mixed with serum-free DMEM medium for 5 minutes / ml and treated for 30 minutes. After that, the experiment was conducted using the ELISA kit.

The obtained result was measured with a Perkins Elmer machine. The degree of phosphorylation of AKT of the antibody was determined as a relative value based on the degree of phosphorylation by 5D5 used as a positive control (100%).

The obtained results are shown in FIG. 2. As shown in Figure 2, it can be seen that the antibody L3-1Y and its variants have a significantly higher inhibitory effect on AKT phosphorylation compared to the positive control 5D5.

Example 3: c-Met antibody c-Met degradation induction test

It was verified by measuring the total amount of c-Met that the antibody L3-1Y and its variants had low side effects and excellent induction of c-Met degradation. Since it is already known that the binding of c-Met and HGF promotes the growth of cancer cells, it is proved that the growth of cancer cells decreases when the total amount of c-Met decreases by antibody treatment, and thereby the anti-cancer activity of the antibody can be demonstrated. .

The c-Met decomposition inducing activity of the c-Met antibody L3-1Y and L3-1Y / U3HC9 (hinge modification) and L3-1Y / IgG2 (hinge and Fc modification) produced in the reference example were measured. The degree of cM-Met degradation in this example was expressed through the total amount of c-Met measured using ELISA.

After mixing MKN45 gastric cancer cell line 2X10 ⁵ cells / ml with 5ug / ml of each antibody for 24 hours (RPMI medium, GIBCO), ELISA experiment using human total HGF R / c-Met ELISA kit (R & D systems) Was performed. Finally, Super Aquablue (eBiosciences) was added for reaction, and colorimetric signals were measured with an OD value at a wavelength of 450 nm. The measured values for each antibody are shown in FIG. 3 in terms of relative values to the control group (media only, 100%) that was not treated with the antibody.

As shown in FIG. 3, it can be seen that c-Met antibody L3-1Y and its variants are superior in c-Met decomposition efficacy compared to the control group without antibody.

Example 4: Inhibition effect of cell growth by coadministration of anti-c-Met antibody and VEGF antagonist

Anti-c-Met antibody L3-1Y or L3-1Y / IgG2 and a real-time cell assay using xCelligence system (Roche) to confirm the degree of cell growth inhibition by co-administration of antagonist Avastin (Roche) with VRGF antagonist ( Real time cell analysis) was performed.

Cells (HuVEC, ATCC) treated with 100 ug cells / well c-Met and VEGFR ligands HGF (R & D systems) 0.4 ug / ml and VEGF (R & D systems) 0.4 ug / ml to improve the growth of cells -Met antibody L3-1Y or L3-1Y / IgG2, Avastin, or how to inhibit their combination administration was investigated.

The instrument used in this analysis is the xCelligence-RTCA (Real time cell analyzer) DP system, which measures the number of cells by measuring the impedance generated when cells are attached on a gold microelectrode array in real time. Device.

Using E-plate 16, generated after adding 10000 cells / well of cells (HuVEC, ATCC) and 10 ug / ml of each antibody, 10 ug / ml of Roche, or a mixture thereof to each well of the plate The impedance value was measured in real time. Figures 4 (L3-1Y) and 12 (L3-1Y / IgG2) show relative values (Cell Index) of the impedance values generated when cells are attached to the bottom.

In FIG. 4, red is the result of cells treated with VEGF + HGF, blue is the result of cells treated with Avastin alone, light green is the result of cells treated with antibody L3-1Y alone, purple is antibody L3-1Y, and Results in cells treated with avastin combination, sky blue indicates results in untreated (media only) cells. As can be seen in Figure 4, it can be seen that cell growth is significantly inhibited when these combinations are treated rather than when the antibodies L3-1Y and Avastin are treated alone.

In FIG. 12, red is the result of cells treated with VEGF + HGF, blue is the result of cells treated with Avastin alone, light green is the result of cells treated with antibody L3-1Y / IgG2 alone, purple is the antibody L3- Results in cells treated with 1Y / IgG2 and Avastin combined, sky blue indicates results in cells that were untreated (media only). As can be seen in Figure 4, it can be seen that cell growth is significantly inhibited when these combinations are treated rather than when the antibodies L3-1Y / IgG2 and Avastin are treated alone.

As a result of the real-time cell analysis of FIG. 4, when the positive control (VEGF + HGF treated group) reached the highest growth (92 hours 18 minutes 9 seconds), inhibition of the antibody L3-1Y, avastin, and combination treatment thereof The degree is calculated as a relative value to the positive control (100%) and is shown in FIG. 5. In addition, as a result of real-time cell analysis of FIG. 12, when the positive control (VEGF + HGF treated group) reached the best growth (71 hours 54 minutes 17 seconds), the antibody L3-1 L3-1Y / IgG2Y, avastin, and The degree of inhibition at the time of these combination treatments is calculated as a relative value to the positive control (100%) and is shown in FIG. 13.

5 and 13, when the antibody L3-1Y or L3-1Y / IgG2 and avastin were administered in combination, it was confirmed that a significantly superior cell growth inhibitory effect was obtained compared to the case where they were treated alone.

As described above, after determining the degree of inhibition of cell growth by the combination administration of anti-c-Met antibody and Avastin using the xCelligence system (Roche), each administration using CCK-8 assay in 96-well The patterns of water concentrations were examined.

First, HuVEC cells (ATCC) were seeded in complete medium (lonza, cc-3162) in an amount of 10000 per well in a 96-well plate. After incubation for 24 hours, the medium was replaced with serum-free medium (lonza, cc-3121) mixed with various concentrations (0.05 to 5 ug / ml) of anti-c-Met antibody L3-1Y, avastin, or a mixture thereof. After 72 hours, CCK-8 analysis (Dojindo, CK-04) was performed to read and analyze the generated signal.

The obtained results are shown in Fig. 6. As shown in Figure 6, when combined administration of the anti-c-Met antibody L3-1Y and avastin shows a higher cell growth inhibitory effect than their respective administration, it was observed that this effect shows a tendency to increase in proportion to the concentration .

Example  5: anti-c- Met  With antibodies VEGF  Cells by combined administration of antagonists Metastatic ability  Inhibitory effect

Both c-Met and VEGF are factors involved in cell growth as well as cancer metastasis. When each inhibitor was used in combination, it was tested whether it showed a synergistic effect on cell motility related to cancer cell metastasis.

Oris 96-well plate (Platypus, OrisTM Cell migration assay) method was performed. Each well of a 96-well plate with a built-in stopper was seeded with 10000 HuVEC cells (ATCC) and treated with HGF 0.4 ug / ml and VEGF 0.4 ug / ml in serum-free medium (EBM, Lonza) for 24 hours. After incubation, the stopper was removed. As the stopper is a circular rubber material, cells cannot grow in the place where the stopper was located, so if the stopper is removed after 24 hours, only the place creates a circular space.

After the stopper was removed, the anti-c-Met antibody L3-1Y, avastin, and mixtures thereof were treated with an amount of 10 ug / ml, and after 24 hours, when stained with the fluorescent substance calcein AM (BD), only the cells were stained, and the cells were stained. The missing part remains a space. Therefore, as the cell migration is inhibited, the size of the unstained space is large, and the degree of cell migration can be measured by observing the unstained space.

Since cell migration is directly involved in the metastasis of cancer, this assay is a general-purpose method for evaluating cell metastasis. In particular, this assay is a method used to determine whether cells move to adjacent regions.

The results of the cell staining and unstained space after the test were photographed using a fluorescent microscope, and are shown in FIG. 7. As shown in FIG. 7, it can be seen that treatment of growth factors HGF and VEGF with serum-free medium enhances cell migration. However, when the anti-c-Met antibody L3-1Y, avastin, or a mixture thereof is treated together with the growth factors, enhanced cell migration is inhibited again by the growth factor, resulting in a large unstained empty space, particularly anti c. It can be seen that the effect of the -Met antibody L3-1Y and Avastin in combination is marked.

Anti-c-Met antibody L3-1Y and / or anti-c-Met antibody L3 at various concentrations (0.05 to 5 ug / ml) in the same manner as the method obtained in FIG. 7 in order to see the effect according to the concentration of Avastin The fluorescence signal intensity obtained by treating -1Y, Avastin, or a mixture thereof is converted into a signal intensity of 100% of the VEGF + HGF treatment group and converted to a value read by a multilabel reader (Perkin-Elmer, Envision). . As shown in Figure 8, the anti-c-Met antibody L3-1Y and avastin show a cell growth inhibitory effect in a concentration-dependent manner, and this effect is significantly increased when the anti-c-Met antibody L3-1Y and avastin are treated in combination. You can see that

The effect at the highest concentration (5 ug / ml) among the results of FIG. 8 is summarized in a bar graph and is shown in FIG. 9. As shown in FIG. 9, when avastin alone was administered, there was almost no cell migration inhibitory effect, but when the anti-c-Met antibody L3-1Y and avastin were treated in combination, it was confirmed that the efficacy of cell migration inhibition was significantly enhanced.

Example  6: Anti-c- Met  With antibodies VEGF  Cells by combined administration of antagonists Penetration  Inhibitory effect

In order to confirm whether the cell penetration ability is inhibited by the combination administration of the anti-c-Met antibody L3-1Y and Avastin, it is divided into an upper chamber and a lower chamber, and a collagen-encoded cell penetration ability assay plate (BioCoat Growth Factor Reduced) Cell infiltration capacity was tested using a MATRIGEL Invasion Chamber; BD science, Cat #. 354483).

In the upper chamber, HUVEC cells (ATCC; 5 × 10 ⁴ cells) were dispensed, and in the lower chamber, anti-c-Met antibody L3-1Y, Avastin, or a mixture thereof was mixed with the medium (EBM) in an amount of 10 ug / ml, respectively. The mixture was added and cultured at 37 ° C (HGF + VEGF was added to the lower chmaber, 0.4 ug / ml each). After incubation for 24 hours, the collagen layer of the upper chamber was pierced, and cells descending to the lower chamber were stained with calcein and observed with a fluorescence microscope. The ability of such cells to penetrate the collagen layer is often used to determine the ability of cells to transfer to distant tissues or organs. The obtained fluorescence image is shown in FIG. 10. As shown in FIG. 10, after treating both VEGF and HGF, when the anti-c-Met antibody L3-1Y and Avastin were used in combination, it was confirmed that the cell penetration ability was significantly inhibited compared to the case where they were treated individually. You can.

Meanwhile, the fluorescence area is calculated from the fluorescence image obtained above and is shown in FIG. 11. As shown in FIG. 11, it can be confirmed that when the anti-c-Met antibody L3-1Y and avastin were used in combination, the area occupied by the cells was significantly reduced.

Korea Cell Line Research Foundation KCLRFBP00220 20091006

<110> Samsung Electronics Co. Ltd <120> Pharmaceutical composition for a combination therapy containing          an angiogenesis inhibitor and anti-c-Met antibody <130> DPP20134677KR <150> KR 10-2012-0101177 <151> 2012-09-12 <160> 108 <170> KopatentIn 1.71 <210> 1 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> heavy chain CDR1 of AbF46 <400> 1 Asp Tyr Tyr Met Ser   1 5 <210> 2 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> heavy chain CDR2 of AbF46 <400> 2 Phe Ile Arg Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ser Ala Ser   1 5 10 15 Val Lys Gly             <210> 3 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> heavy chain CDR3 of AbF46 <400> 3 Asp Asn Trp Phe Ala Tyr   1 5 <210> 4 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> heavy chain CDR1 of c-Met antibody <220> <221> UNSURE <222> (1) <223> X is Pro or Ser or absent <220> <221> UNSURE <222> (2) <223> X is Glu or Asp <400> 4 Xaa Xaa Tyr Tyr Met Ser   1 5 <210> 5 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> heavy chain CDR2 of c-Met antibody <220> <221> UNSURE <222> (3) <223> X is Asn or Lys <220> <221> UNSURE <222> (4) <223> X is Ala or Val <220> <221> UNSURE <222> (7) <223> X is Asn or Thr <400> 5 Arg Asn Xaa Xaa Asn Gly Xaa Thr   1 5 <210> 6 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> heavy chain CDR3 of c-Met antibody <220> <221> UNSURE <222> (5) <223> X is Ser or Thr <400> 6 Asp Asn Trp Leu Xaa Tyr   1 5 <210> 7 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> light chain CDR1 of c-Met antibody <220> <221> UNSURE <222> (4) <223> X is His, Arg, Gln or Lys <220> <221> UNSURE <222> (12) <223> X is His or Gln <220> <221> UNSURE <222> (13) <223> X is Lys or Asn <220> <221> UNSURE <222> (9) <223> X is Ser or Trp <400> 7 Lys Ser Ser Xaa Ser Leu Leu Ala Xaa Gly Asn Xaa Xaa Asn Tyr Leu   1 5 10 15 Ala     <210> 8 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> light chain CDR2 of c-Met antibody <220> <221> UNSURE <222> (2) <223> X is Ala or Gly <220> <221> UNSURE <222> (4) <223> X is Thr or Lys <220> <221> UNSURE <222> (7) <223> X is Ser or Pro <400> 8 Trp Xaa Ser Xaa Arg Val Xaa   1 5 <210> 9 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> light chain CDR3 of c-Met antibody <220> <221> UNSURE <222> (1) <223> X is Gly, Ala or Gln <220> <221> UNSURE <222> (6) <223> X is Arg, His, Ser, Ala, Gly or Lys <220> <221> UNSURE <222> (8) <223> X is Leu, Tyr, Phe or Met <400> 9 Xaa Gln Ser Tyr Ser Xaa Pro Xaa Thr   1 5 <210> 10 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> light chain CDR1 of AbF46 <400> 10 Lys Ser Ser Gln Ser Leu Leu Ala Ser Gly Asn Gln Asn Asn Tyr Leu   1 5 10 15 Ala     <210> 11 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> light chain CDR2 of AbF46 <400> 11 Trp Ala Ser Thr Arg Val Ser   1 5 <210> 12 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> light chain CDR3 of AbF46 <400> 12 Gln Gln Ser Tyr Ser Ala Pro Leu Thr   1 5 <210> 13 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR-L3 derived from L3-1 clone <400> 13 Gln Gln Ser Tyr Ser Arg Pro Tyr Thr   1 5 <210> 14 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR-L3 derived from L3-2 clone <400> 14 Gly Gln Ser Tyr Ser Arg Pro Leu Thr   1 5 <210> 15 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR-L3 derived from L3-3 clone <400> 15 Ala Gln Ser Tyr Ser His Pro Phe Ser   1 5 <210> 16 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR-L3 derived from L3-5 clone <400> 16 Gln Gln Ser Tyr Ser Arg Pro Phe Thr   1 5 <210> 17 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> heavy chain variable region of anti c-Met humanized          antibody (huAbF46-H4) <400> 17 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr              20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu          35 40 45 Gly Phe Ile Arg Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ser Ala      50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr  65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr                  85 90 95 Tyr Cys Ala Arg Asp Asn Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu             100 105 110 Val Thr Val Ser Ser         115 <210> 18 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> light chain variable region of anti c-Met humanized          antibody (huAbF46-H4) <400> 18 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Leu Ala Ser              20 25 30 Gly Asn Gln Asn Asn Tyr Leu Ala Trp His Gln Gln Lys Pro Gly Lys          35 40 45 Ala Pro Lys Met Leu Ile Ile Trp Ala Ser Thr Arg Val Ser Gly Val      50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln                  85 90 95 Ser Tyr Ser Arg Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile             100 105 110 Lys Arg         <210> 19 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> light chain variable region of anti c-Met humanized          antibody (huAbF46-H4) <400> 19 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Leu Ala Ser              20 25 30 Gly Asn Gln Asn Asn Tyr Leu Ala Trp His Gln Gln Lys Pro Gly Lys          35 40 45 Ala Pro Lys Met Leu Ile Ile Trp Ala Ser Thr Arg Val Ser Gly Val      50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gly Gln                  85 90 95 Ser Tyr Ser Arg Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile             100 105 110 Lys Arg         <210> 20 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> light chain variable region of anti c-Met humanized          antibody (huAbF46-H4) <400> 20 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Leu Ala Ser              20 25 30 Gly Asn Gln Asn Asn Tyr Leu Ala Trp His Gln Gln Lys Pro Gly Lys          35 40 45 Ala Pro Lys Met Leu Ile Ile Trp Ala Ser Thr Arg Val Ser Gly Val      50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Ala Gln                  85 90 95 Ser Tyr Ser His Pro Phe Ser Phe Gly Gln Gly Thr Lys Val Glu Ile             100 105 110 Lys Arg         <210> 21 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> light chain variable region of anti c-Met humanized          antibody (huAbF46-H4) <400> 21 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Leu Ala Ser              20 25 30 Gly Asn Gln Asn Asn Tyr Leu Ala Trp His Gln Gln Lys Pro Gly Lys          35 40 45 Ala Pro Lys Met Leu Ile Ile Trp Ala Ser Thr Arg Val Ser Gly Val      50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln                  85 90 95 Ser Tyr Ser Arg Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile             100 105 110 Lys Arg         <210> 22 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> CDR-H1 derived from H11-4 clone <400> 22 Pro Glu Tyr Tyr Met Ser   1 5 <210> 23 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> CDR-H1 derived from YC151 clone <400> 23 Pro Asp Tyr Tyr Met Ser   1 5 <210> 24 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> CDR-H1 derived from YC193 clone <400> 24 Ser Asp Tyr Tyr Met Ser   1 5 <210> 25 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR-H2 derived from YC244 clone <400> 25 Arg Asn Asn Ala Asn Gly Asn Thr   1 5 <210> 26 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> CDR-H2 derived from YC321 clone <400> 26 Arg Asn Lys Val Asn Gly Tyr Thr   1 5 <210> 27 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> CDR-H3 derived from YC354 clone <400> 27 Asp Asn Trp Leu Ser Tyr   1 5 <210> 28 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> CDR-H3 derived from YC374 clone <400> 28 Asp Asn Trp Leu Thr Tyr   1 5 <210> 29 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR-L1 derived from L1-1 clone <400> 29 Lys Ser Ser His Ser Leu Leu Ala Ser Gly Asn Gln Asn Asn Tyr Leu   1 5 10 15 Ala     <210> 30 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR-L1 derived from L1-3 clone <400> 30 Lys Ser Ser Arg Ser Leu Leu Ser Ser Gly Asn His Lys Asn Tyr Leu   1 5 10 15 Ala     <210> 31 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR-L1 derived from L1-4 clone <400> 31 Lys Ser Ser Lys Ser Leu Leu Ala Ser Gly Asn Gln Asn Asn Tyr Leu   1 5 10 15 Ala     <210> 32 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR-L1 derived from L1-12 clone <400> 32 Lys Ser Ser Arg Ser Leu Leu Ala Ser Gly Asn Gln Asn Asn Tyr Leu   1 5 10 15 Ala     <210> 33 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR-L1 derived from L1-22 clone <400> 33 Lys Ser Ser His Ser Leu Leu Ala Ser Gly Asn Gln Asn Asn Tyr Leu   1 5 10 15 Ala     <210> 34 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR-L2 derived from L2-9 clone <400> 34 Trp Ala Ser Lys Arg Val Ser   1 5 <210> 35 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR-L2 derived from L2-12 clone <400> 35 Trp Gly Ser Thr Arg Val Ser   1 5 <210> 36 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR-L2 derived from L2-16 clone <400> 36 Trp Gly Ser Thr Arg Val Pro   1 5 <210> 37 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR-L3 derived from L3-32 clone <400> 37 Gln Gln Ser Tyr Ser Lys Pro Phe Thr   1 5 <210> 38 <211> 1416 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence of heavy chain of chAbF46 <220> <221> misc_feature <222> (1) .. (6) <223> EcoRI restriction site <220> <221> misc_feature <222> (7) .. (66) <223> signal sequence <220> <221> misc_feature <222> (67) .. (417) <223> VH-heavy chain variable region <220> <221> misc_feature <222> (418) .. (423) <223> NdeI restriction site <220> <221> misc_feature <222> (418) .. (1407) <223> CH-heavy chain constant region <220> <221> misc_feature <222> (1408) .. (1410) <223> TGA-stop sodon <220> <221> misc_feature <222> (1411) .. (1416) <223> XhoI restriction site <400> 38 gaattcgccg ccaccatgga atggagctgg gtttttctcg taacactttt aaatggtatc 60 cagtgtgagg tgaagctggt ggagtctgga ggaggcttgg tacagcctgg gggttctctg 120 agactctcct gtgcaacttc tgggttcacc ttcactgatt actacatgag ctgggtccgc 180 cagcctccag gaaaggcact tgagtggttg ggttttatta gaaacaaagc taatggttac 240 acaacagagt acagtgcatc tgtgaagggt cggttcacca tctccagaga taattcccaa 300 agcatcctct atcttcaaat ggacaccctg agagctgagg acagtgccac ttattactgt 360 gcaagagata actggtttgc ttactggggc caagggactc tggtcactgt ctctgcagct 420 agcaccaagg gcccatcggt cttccccctg gcaccctcct ccaagagcac ctctgggggc 480 acagcggccc tgggctgcct ggtcaaggac tacttccccg aaccggtgac ggtgtcgtgg 540 aactcaggcg ccctgaccag cggcgtgcac accttcccgg ctgtcctaca gtcctcagga 600 ctctactccc tcagcagcgt ggtgaccgtg ccctccagca gcttgggcac ccagacctac 660 atctgcaacg tgaatcacaa gcccagcaac accaaggtgg acaagaaagt tgagcccaaa 720 tcttgtgaca aaactcacac atgcccaccg tgcccagcac ctgaactcct ggggggaccg 780 tcagtcttcc tcttcccccc aaaacccaag gacaccctca tgatctcccg gacccctgag 840 gtcacatgcg tggtggtgga cgtgagccac gaagaccctg aggtcaagtt caactggtac 900 gtggacggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gtacaacagc 960 acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa tggcaaggag 1020 tacaagtgca aggtctccaa caaagccctc ccagccccca tcgagaaaac catctccaaa 1080 gccaaagggc agccccgaga accacaggtg tacaccctgc ccccatcccg ggaggagatg 1140 accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctatcccag cgacatcgcc 1200 gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg 1260 gactccgacg gctccttctt cctctacagc aagctcaccg tggacaagag caggtggcag 1320 caggggaacg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacgcag 1380 aagagcctct ccctgtctcc gggtaaatga ctcgag 1416 <210> 39 <211> 759 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence of light chain of chAbF46 <220> <221> misc_difference <222> (1) .. (6) <223> EcoRI restriction site <220> <221> misc_difference <222> (7) .. (90) <223> signal sequence <220> <221> misc_difference <222> (91) .. (432) <223> VL-light chain variable region <220> <221> misc_difference <222> (430) .. (435) <223> BsiWI restriction site <220> <221> misc_difference <222> (433) .. (750) <223> CL-light chain constant region <220> <221> misc_difference <222> (751) .. (753) <223> stop codon <220> <221> misc_difference <222> (754) .. (759) <223> XhoI restriction site <400> 39 gaattcacta gtgattaatt cgccgccacc atggattcac aggcccaggt cctcatgttg 60 ctgctgctat cggtatctgg tacctgtgga gacattttga tgacccagtc tccatcctcc 120 ctgactgtgt cagcaggaga gaaggtcact atgagctgca agtccagtca gagtctttta 180 gctagtggca accaaaataa ctacttggcc tggcaccagc agaaaccagg acgatctcct 240 aaaatgctga taatttgggc atccactagg gtatctggag tccctgatcg cttcataggc 300 agtggatctg ggacggattt cactctgacc atcaacagtg tgcaggctga agatctggct 360 gtttattact gtcagcagtc ctacagcgct ccgctcacgt tcggtgctgg gaccaagctg 420 gagctgaaac gtacggtggc tgcaccatct gtcttcatct tcccgccatc tgatgagcag 480 ttgaaatctg gaactgcctc tgttgtgtgc ctgctgaata acttctatcc cagagaggcc 540 aaagtacagt ggaaggtgga taacgccctc caatcgggta actcccagga gagtgtcaca 600 gagcaggaca gcaaggacag cacctacagc ctcagcagca ccctgacgct gagcaaagca 660 gactacgaga aacacaaagt ctacgcctgc gaagtcaccc atcagggcct gagctcgccc 720 gtcacaaaga gcttcaacag gggagagtgt tgactcgag 759 <210> 40 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of H1-heavy <400> 40 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr              20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu          35 40 45 Gly Phe Ile Arg Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ser Ala      50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Ser  65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr                  85 90 95 Tyr Cys Ala Arg Asp Asn Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu             100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu         115 120 125 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys     130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser                 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser             180 185 190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn         195 200 205 Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His     210 215 220 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr                 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu             260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys         275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser     290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile                 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro             340 345 350 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu         355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn     370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg                 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu             420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys         435 440 445 <210> 41 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of H3-heavy <400> 41 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr              20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu          35 40 45 Gly Phe Ile Arg Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ser Ala      50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Ser  65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr                  85 90 95 Tyr Cys Ala Arg Asp Asn Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu             100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu         115 120 125 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys     130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser                 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser             180 185 190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn         195 200 205 Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His     210 215 220 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr                 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu             260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys         275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser     290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile                 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro             340 345 350 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu         355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn     370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg                 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu             420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys         435 440 445 <210> 42 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of H4-heavy <400> 42 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr              20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu          35 40 45 Gly Phe Ile Arg Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ser Ala      50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr  65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr                  85 90 95 Tyr Cys Ala Arg Asp Asn Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu             100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu         115 120 125 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys     130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser                 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser             180 185 190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn         195 200 205 Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His     210 215 220 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr                 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu             260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys         275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser     290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile                 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro             340 345 350 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu         355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn     370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg                 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu             420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys         435 440 445 <210> 43 <211> 220 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of H1-light <400> 43 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly   1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Ala Ser              20 25 30 Gly Asn Gln Asn Asn Tyr Leu Ala Trp His Gln Gln Lys Pro Gly Gln          35 40 45 Pro Pro Lys Met Leu Ile Ile Trp Ala Ser Thr Arg Val Ser Gly Val      50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln                  85 90 95 Ser Tyr Ser Ala Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile             100 105 110 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp         115 120 125 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn     130 135 140 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 145 150 155 160 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp                 165 170 175 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr             180 185 190 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser         195 200 205 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys     210 215 220 <210> 44 <211> 220 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of H2-light <400> 44 Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly   1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Ala Ser              20 25 30 Gly Asn Gln Asn Asn Tyr Leu Ala Trp His Leu Gln Lys Pro Gly Gln          35 40 45 Ser Pro Gln Met Leu Ile Ile Trp Ala Ser Thr Arg Val Ser Gly Val      50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys  65 70 75 80 Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln                  85 90 95 Ser Tyr Ser Ala Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Leu             100 105 110 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp         115 120 125 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn     130 135 140 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 145 150 155 160 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp                 165 170 175 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr             180 185 190 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser         195 200 205 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys     210 215 220 <210> 45 <211> 220 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of H3-light <400> 45 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly   1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Ala Ser              20 25 30 Gly Asn Gln Asn Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln          35 40 45 Pro Pro Lys Leu Leu Ile Ile Trp Ala Ser Thr Arg Val Ser Gly Val      50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln                  85 90 95 Ser Tyr Ser Ala Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile             100 105 110 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp         115 120 125 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn     130 135 140 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 145 150 155 160 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp                 165 170 175 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr             180 185 190 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser         195 200 205 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys     210 215 220 <210> 46 <211> 219 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of H4-light <400> 46 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Leu Ala Ser              20 25 30 Gly Asn Gln Asn Asn Tyr Leu Ala Trp His Gln Gln Lys Pro Gly Lys          35 40 45 Ala Pro Lys Met Leu Ile Ile Trp Ala Ser Thr Arg Val Ser Gly Val      50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln                  85 90 95 Ser Tyr Ser Ala Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile             100 105 110 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp         115 120 125 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn     130 135 140 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 145 150 155 160 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp                 165 170 175 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr             180 185 190 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser         195 200 205 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu     210 215 <210> 47 <211> 1350 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence of H1-heavy <400> 47 gaggtgcagc tggtggagtc tgggggaggc ttggtccagc ctggagggtc cctgagactc 60 tcctgtgcag cctctggatt caccttcact gactactaca tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg gttgggcttt attagaaaca aagctaacgg ttacaccaca 180 gaatacagtg cgtctgtgaa aggcagattc accatctcaa gagataattc aaagaactca 240 ctgtatctgc aaatgaacag cctgaaaacc gaggacacgg ccgtgtatta ctgtgctaga 300 gataactggt ttgcttactg gggtcaagga accctggtca ccgtctcctc ggctagcacc 360 aagggcccat cggtcttccc cctggcaccc tcctccaaga gcacctctgg gggcacagcg 420 gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca 480 ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac 540 tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc 600 aacgtgaatc acaagcccag caacaccaag gtggacaaga aagttgagcc caaatcttgt 660 gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 720 ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 780 tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 840 ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 900 cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 960 tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 1020 gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggagga gatgaccaag 1080 aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 1140 tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 1200 gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 1260 aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 1320 ctctccctgt ctccgggtaa atgactcgag 1350 <210> 48 <211> 1350 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence of H3-heavy <400> 48 gaggtgcagc tggtggagtc tgggggaggc ttggtccagc ctggagggtc cctgagactc 60 tcctgtgcag cctctggatt caccttcact gactactaca tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg gttgggcttt attagaaaca aagctaacgg ttacaccaca 180 gaatacagtg cgtctgtgaa aggcagattc accatctcaa gagataattc aaagaactca 240 ctgtatctgc aaatgaacag cctgcgtgct gaggacacgg ccgtgtatta ctgtgctaga 300 gataactggt ttgcttactg gggtcaagga accctggtca ccgtctcctc ggctagcacc 360 aagggcccat cggtcttccc cctggcaccc tcctccaaga gcacctctgg gggcacagcg 420 gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca 480 ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac 540 tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc 600 aacgtgaatc acaagcccag caacaccaag gtggacaaga aagttgagcc caaatcttgt 660 gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 720 ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 780 tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 840 ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 900 cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 960 tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 1020 gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggagga gatgaccaag 1080 aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 1140 tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 1200 gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 1260 aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 1320 ctctccctgt ctccgggtaa atgactcgag 1350 <210> 49 <211> 1350 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence of H4-heavy <400> 49 gaggttcagc tggtggagtc tggcggtggc ctggtgcagc cagggggctc actccgtttg 60 tcctgtgcag cttctggctt caccttcact gattactaca tgagctgggt gcgtcaggcc 120 ccgggtaagg gcctggaatg gttgggtttt attagaaaca aagctaatgg ttacacaaca 180 gagtacagtg catctgtgaa gggtcgtttc actataagca gagataattc caaaaacaca 240 ctgtacctgc agatgaacag cctgcgtgct gaggacactg ccgtctatta ttgtgctaga 300 gataactggt ttgcttactg gggccaaggg actctggtca ccgtctcctc ggctagcacc 360 aagggcccat cggtcttccc cctggcaccc tcctccaaga gcacctctgg gggcacagcg 420 gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca 480 ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac 540 tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc 600 aacgtgaatc acaagcccag caacaccaag gtggacaaga aagttgagcc caaatcttgt 660 gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 720 ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 780 tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 840 ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 900 cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 960 tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 1020 gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggagga gatgaccaag 1080 aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 1140 tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 1200 gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 1260 aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 1320 ctctccctgt ctccgggtaa atgactcgag 1350 <210> 50 <211> 669 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence of H1-light <400> 50 gacatcgtga tgacccagtc tccagactcc ctggctgtgt ctctgggcga gagggccacc 60 atcaactgca agtccagcca gagtctttta gctagcggca accaaaataa ctacttagct 120 tggcaccagc agaaaccagg acagcctcct aagatgctca ttatttgggc atctacccgg 180 gtatccgggg tccctgaccg attcagtggc agcgggtctg ggacagattt cactctcacc 240 atcagcagcc tgcaggctga agatgtggca gtttattact gtcagcaatc ctatagtgct 300 cctctcacgt tcggaggcgg taccaaggtg gagatcaaac gtacggtggc tgcaccatct 360 gtcttcatct tcccgccatc tgatgagcag ttgaaatctg gaactgcctc tgttgtgtgc 420 ctgctgaata acttctatcc cagagaggcc aaagtacagt ggaaggtgga taacgccctc 480 caatcgggta actcccagga gagtgtcaca gagcaggaca gcaaggacag cacctacagc 540 ctcagcagca ccctgacgct gagcaaagca gactacgaga aacacaaagt ctacgcctgc 600 gaagtcaccc atcagggcct gagctcgccc gtcacaaaga gcttcaacag gggagagtgt 660 tgactcgag 669 <210> 51 <211> 669 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence of H2-light <400> 51 gatattgtga tgacccagac tccactctcc ctgcccgtca cccctggaga gccggcctcc 60 atctcctgca agtccagtca gagtctttta gctagtggca accaaaataa ctacttggcc 120 tggcacctgc agaagccagg gcagtctcca cagatgctga tcatttgggc atccactagg 180 gtatctggag tcccagacag gttcagtggc agtgggtcag gcactgattt cacactgaaa 240 atcagcaggg tggaggctga ggatgttgga gtttattact gccagcagtc ctacagcgct 300 ccgctcacgt tcggacaggg taccaagctg gagctcaaac gtacggtggc tgcaccatct 360 gtcttcatct tcccgccatc tgatgagcag ttgaaatctg gaactgcctc tgttgtgtgc 420 ctgctgaata acttctatcc cagagaggcc aaagtacagt ggaaggtgga taacgccctc 480 caatcgggta actcccagga gagtgtcaca gagcaggaca gcaaggacag cacctacagc 540 ctcagcagca ccctgacgct gagcaaagca gactacgaga aacacaaagt ctacgcctgc 600 gaagtcaccc atcagggcct gagctcgccc gtcacaaaga gcttcaacag gggagagtgt 660 tgactcgag 669 <210> 52 <211> 669 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence of H3-light <400> 52 gacatcgtga tgacccagtc tccagactcc ctggctgtgt ctctgggcga gagggccacc 60 atcaactgca agtccagcca gagtctttta gctagcggca accaaaataa ctacttagct 120 tggtaccagc agaaaccagg acagcctcct aagctgctca ttatttgggc atctacccgg 180 gtatccgggg tccctgaccg attcagtggc agcgggtctg ggacagattt cactctcacc 240 atcagcagcc tgcaggctga agatgtggca gtttattact gtcagcaatc ctatagtgct 300 cctctcacgt tcggaggcgg taccaaggtg gagatcaaac gtacggtggc tgcaccatct 360 gtcttcatct tcccgccatc tgatgagcag ttgaaatctg gaactgcctc tgttgtgtgc 420 ctgctgaata acttctatcc cagagaggcc aaagtacagt ggaaggtgga taacgccctc 480 caatcgggta actcccagga gagtgtcaca gagcaggaca gcaaggacag cacctacagc 540 ctcagcagca ccctgacgct gagcaaagca gactacgaga aacacaaagt ctacgcctgc 600 gaagtcaccc atcagggcct gagctcgccc gtcacaaaga gcttcaacag gggagagtgt 660 tgactcgag 669 <210> 53 <211> 669 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence of H4-light <400> 53 gatatccaga tgacccagtc cccgagctcc ctgtccgcct ctgtgggcga tagggtcacc 60 atcacctgca agtccagtca gagtctttta gctagtggca accaaaataa ctacttggcc 120 tggcaccaac agaaaccagg aaaagctccg aaaatgctga ttatttgggc atccactagg 180 gtatctggag tcccttctcg cttctctgga tccgggtctg ggacggattt cactctgacc 240 atcagcagtc tgcagccgga agacttcgca acttattact gtcagcagtc ctacagcgct 300 ccgctcacgt tcggacaggg taccaaggtg gagatcaaac gtacggtggc tgcaccatct 360 gtcttcatct tcccgccatc tgatgagcag ttgaaatctg gaactgcctc tgttgtgtgc 420 ctgctgaata acttctatcc cagagaggcc aaagtacagt ggaaggtgga taacgccctc 480 caatcgggta actcccagga gagtgtcaca gagcaggaca gcaaggacag cacctacagc 540 ctcagcagca ccctgacgct gagcaaagca gactacgaga aacacaaagt ctacgcctgc 600 gaagtcaccc atcagggcct gagctcgccc gtcacaaaga gcttcaacag gggagagtgt 660 tgactcgag 669 <210> 54 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> linker between VH and VL <400> 54 Gly Leu Gly Gly Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly   1 5 10 15 Gly Ser Ser Gly Val Gly Ser              20 <210> 55 <211> 1088 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding scFv of huAbF46 antibody <400> 55 gctagcgttt tagcagaagt tcaattggtt gaatctggtg gtggtttggt tcaaccaggt 60 ggttctttga gattgtcttg tgctgcttct ggttttactt tcaccgatta ttacatgtcc 120 tgggttagac aagctccagg taaaggtttg gaatggttgg gtttcattag aaacaaggct 180 aacggttaca ctaccgaata ttctgcttct gttaagggta gattcaccat ttctagagac 240 aactctaaga acaccttgta cttgcaaatg aactccttga gagctgaaga tactgctgtt 300 tattactgcg ctagagataa ttggtttgct tattggggtc aaggtacttt ggttactgtt 360 tcttctggcc tcgggggcct cggaggagga ggtagtggcg gaggaggctc cggtggatcc 420 agcggtgtgg gttccgatat tcaaatgacc caatctccat cttctttgtc tgcttcagtt 480 ggtgatagag ttaccattac ttgtaagtcc tcccaatctt tgttggcttc tggtaatcag 540 aacaattact tggcttggca tcaacaaaaa ccaggtaaag ctccaaagat gttgattatt 600 tgggcttcta ccagagtttc tggtgttcca tctagatttt ctggttctgg ttccggtact 660 gattttactt tgaccatttc atccttgcaa ccagaagatt tcgctactta ctactgtcaa 720 caatcttact ctgctccatt gacttttggt caaggtacaa aggtcgaaat caagagagaa 780 ttcggtaagc ctatccctaa ccctctcctc ggtctcgatt ctacgggtgg tggtggatct 840 ggtggtggtg gttctggtgg tggtggttct caggaactga caactatatg cgagcaaatc 900 ccctcaccaa ctttagaatc gacgccgtac tctttgtcaa cgactactat tttggccaac 960 gggaaggcaa tgcaaggagt ttttgaatat tacaaatcag taacgtttgt cagtaattgc 1020 ggttctcacc cctcaacaac tagcaaaggc agccccataa acacacagta tgttttttga 1080 gtttaaac 1088 <210> 56 <211> 5597 <212> DNA <213> Artificial Sequence <220> <223> expression vector including polynucleotide encoding scFv of          huAbF46 antibody <220> <221> misc_difference <222> (573) .. (578) <223> NheI restriction site <220> <221> misc_difference <222> (588) .. (938) <223> huAbF46 VH <220> <221> misc_difference <222> (939) .. (1007) <223> linker <220> <221> misc_difference <222> (1008) .. (1349) <223> huAbF46 VL <220> <221> misc_difference <222> (1350) .. (1355) <223> EcoRI restriction site <220> <221> misc_difference <222> (1356) .. (1397) <223> V5 epitope <220> <221> misc_difference <222> (1398) .. (1442) <223> (G4S) 3 linker <220> <221> misc_difference <222> (1443) .. (1649) <223> Aga2 <220> <221> misc_difference <222> (1650) .. (1652) <223> TGA (stop codon) <220> <221> misc_difference <222> (1653) .. (1660) <223> PmeI restriction site <400> 56 acggattaga agccgccgag cgggtgacag ccctccgaag gaagactctc ctccgtgcgt 60 cctcgtcttc accggtcgcg ttcctgaaac gcagatgtgc ctcgcgccgc actgctccga 120 acaataaaga ttctacaata ctagctttta tggttatgaa gaggaaaaat tggcagtaac 180 ctggccccac aaaccttcaa atgaacgaat caaattaaca accataggat gataatgcga 240 ttagtttttt agccttattt ctggggtaat taatcagcga agcgatgatt tttgatctat 300 taacagatat ataaatgcaa aaactgcata accactttaa ctaatacttt caacattttc 360 ggtttgtatt acttcttatt caaatgtaat aaaagtatca acaaaaaatt gttaatatac 420 ctctatactt taacgtcaag gagaaaaaac cccggatcgg actactagca gctgtaatac 480 gactcactat agggaatatt aagctaattc tacttcatac attttcaatt aagatgcagt 540 tacttcgctg tttttcaata ttttctgtta ttgctagcgt tttagcagaa gttcaattgg 600 ttgaatctgg tggtggtttg gttcaaccag gtggttcttt gagattgtct tgtgctgctt 660 ctggttttac tttcaccgat tattacatgt cctgggttag acaagctcca ggtaaaggtt 720 tggaatggtt gggtttcatt agaaacaagg ctaacggtta cactaccgaa tattctgctt 780 ctgttaaggg tagattcacc atttctagag acaactctaa gaacaccttg tacttgcaaa 840 tgaactcctt gagagctgaa gatactgctg tttattactg cgctagagat aattggtttg 900 cttattgggg tcaaggtact ttggttactg tttcttctgg cctcgggggc ctcggaggag 960 gaggtagtgg cggaggaggc tccggtggat ccagcggtgt gggttccgat attcaaatga 1020 cccaatctcc atcttctttg tctgcttcag ttggtgatag agttaccatt acttgtaagt 1080 cctcccaatc tttgttggct tctggtaatc agaacaatta cttggcttgg catcaacaaa 1140 aaccaggtaa agctccaaag atgttgatta tttgggcttc taccagagtt tctggtgttc 1200 catctagatt ttctggttct ggttccggta ctgattttac tttgaccatt tcatccttgc 1260 aaccagaaga tttcgctact tactactgtc aacaatctta ctctgctcca ttgacttttg 1320 gtcaaggtac aaaggtcgaa atcaagagag aattcggtaa gcctatccct aaccctctcc 1380 tcggtctcga ttctacgggt ggtggtggat ctggtggtgg tggttctggt ggtggtggtt 1440 ctcaggaact gacaactata tgcgagcaaa tcccctcacc aactttagaa tcgacgccgt 1500 actctttgtc aacgactact attttggcca acgggaaggc aatgcaagga gtttttgaat 1560 attacaaatc agtaacgttt gtcagtaatt gcggttctca cccctcaaca actagcaaag 1620 gcagccccat aaacacacag tatgtttttt gagtttaaac ccgctgatct gataacaaca 1680 gtgtagatgt aacaaaatcg actttgttcc cactgtactt ttagctcgta caaaatacaa 1740 tatacttttc atttctccgt aaacaacatg ttttcccatg taatatcctt ttctattttt 1800 cgttccgtta ccaactttac acatacttta tatagctatt cacttctata cactaaaaaa 1860 ctaagacaat tttaattttg ctgcctgcca tatttcaatt tgttataaat tcctataatt 1920 tatcctatta gtagctaaaa aaagatgaat gtgaatcgaa tcctaagaga attgggcaag 1980 tgcacaaaca atacttaaat aaatactact cagtaataac ctatttctta gcatttttga 2040 cgaaatttgc tattttgtta gagtctttta caccatttgt ctccacacct ccgcttacat 2100 caacaccaat aacgccattt aatctaagcg catcaccaac attttctggc gtcagtccac 2160 cagctaacat aaaatgtaag ctctcggggc tctcttgcct tccaacccag tcagaaatcg 2220 agttccaatc caaaagttca cctgtcccac ctgcttctga atcaaacaag ggaataaacg 2280 aatgaggttt ctgtgaagct gcactgagta gtatgttgca gtcttttgga aatacgagtc 2340 ttttaataac tggcaaaccg aggaactctt ggtattcttg ccacgactca tctccgtgca 2400 gttggacgat atcaatgccg taatcattga ccagagccaa aacatcctcc ttaggttgat 2460 tacgaaacac gccaaccaag tatttcggag tgcctgaact atttttatat gcttttacaa 2520 gacttgaaat tttccttgca ataaccgggt caattgttct ctttctattg ggcacacata 2580 taatacccag caagtcagca tcggaatcta gagcacattc tgcggcctct gtgctctgca 2640 agccgcaaac tttcaccaat ggaccagaac tacctgtgaa attaataaca gacatactcc 2700 aagctgcctt tgtgtgctta atcacgtata ctcacgtgct caatagtcac caatgccctc 2760 cctcttggcc ctctcctttt cttttttcga ccgaatttct tgaagacgaa agggcctcgt 2820 gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttagg acggatcgct 2880 tgcctgtaac ttacacgcgc ctcgtatctt ttaatgatgg aataatttgg gaatttactc 2940 tgtgtttatt tatttttatg ttttgtattt ggattttaga aagtaaataa agaaggtaga 3000 agagttacgg aatgaagaaa aaaaaataaa caaaggttta aaaaatttca acaaaaagcg 3060 tactttacat atatatttat tagacaagaa aagcagatta aatagatata cattcgatta 3120 acgataagta aaatgtaaaa tcacaggatt ttcgtgtgtg gtcttctaca cagacaagat 3180 gaaacaattc ggcattaata cctgagagca ggaagagcaa gataaaaggt agtatttgtt 3240 ggcgatcccc ctagagtctt ttacatcttc ggaaaacaaa aactattttt tctttaattt 3300 ctttttttac tttctatttt taatttatat atttatatta aaaaatttaa attataatta 3360 tttttatagc acgtgatgaa aaggacccag gtggcacttt tcggggaaat gtgcgcggaa 3420 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac 3480 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg 3540 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc 3600 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg 3660 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga 3720 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc 3780 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag 3840 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga 3900 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg 3960 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga 4020 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt 4080 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact 4140 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt 4200 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg 4260 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacgggcagt caggcaacta 4320 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac 4380 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta 4440 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt 4500 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt 4560 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt 4620 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc 4680 agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg 4740 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg 4800 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt 4860 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac 4920 tgagatacct acagcgtgag cattgagaaa gcgccacgct tcccgaaggg agaaaggcgg 4980 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg 5040 ggaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat 5100 ttttgtgatg ctcgtcaggg gggccgagcc tatggaaaaa cgccagcaac gcggcctttt 5160 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg 5220 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa 5280 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc 5340 ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga 5400 aagcgggcag tgagcgcaac gcaattaatg tgagttacct cactcattag gcaccccagg 5460 ctttacactt tatgcttccg gctcctatgt tgtgtggaat tgtgagcgga taacaatttc 5520 acacaggaaa cagctatgac catgattacg ccaagctcgg aattaaccct cactaaaggg 5580 aacaaaagct ggctagt 5597 <210> 57 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> U6-HC7 hinge <400> 57 Glu Pro Lys Ser Cys Asp Cys His Cys Pro Pro Cys Pro   1 5 10 <210> 58 <211> 435 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding CDR-L3 derived from L3-1 clone <400> 58 gaattcacta gtgattaatt cgccgccacc atggattcac aggcccaggt cctcatgttg 60 ctgctgctat cggtatctgg tacctgtgga gatatccaga tgacccagtc cccgagctcc 120 ctgtccgcct ctgtgggcga tagggtcacc atcacctgca agtccagtca gagtctttta 180 gctagtggca accaaaataa ctacttggcc tggcaccaac agaaaccagg aaaagctccg 240 aaaatgctga ttatttgggc atccactagg gtatctggag tcccttctcg cttctctgga 300 tccgggtctg ggacggattt cactctgacc atcagcagtc tgcagccgga agacttcgca 360 acttattact gtcagcagtc ctacagccgc ccgtacacgt tcggacaggg taccaaggtg 420 gagatcaaac gtacg 435 <210> 59 <211> 435 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding CDR-L3 derived from L3-2 clone <400> 59 gaattcacta gtgattaatt cgccgccacc atggattcac aggcccaggt cctcatgttg 60 ctgctgctat cggtatctgg tacctgtgga gatatccaga tgacccagtc cccgagctcc 120 ctgtccgcct ctgtgggcga tagggtcacc atcacctgca agtccagtca gagtctttta 180 gctagtggca accaaaataa ctacttggcc tggcaccaac agaaaccagg aaaagctccg 240 aaaatgctga ttatttgggc atccactagg gtatctggag tcccttctcg cttctctgga 300 tccgggtctg ggacggattt cactctgacc atcagcagtc tgcagccgga agacttcgca 360 acttattact gtgggcagtc ctacagccgt ccgctcacgt tcggacaggg taccaaggtg 420 gagatcaaac gtacg 435 <210> 60 <211> 435 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding CDR-L3 derived from L3-3 clone <400> 60 gaattcacta gtgattaatt cgccgccacc atggattcac aggcccaggt cctcatgttg 60 ctgctgctat cggtatctgg tacctgtgga gatatccaga tgacccagtc cccgagctcc 120 ctgtccgcct ctgtgggcga tagggtcacc atcacctgca agtccagtca gagtctttta 180 gctagtggca accaaaataa ctacttggcc tggcaccaac agaaaccagg aaaagctccg 240 aaaatgctga ttatttgggc atccactagg gtatctggag tcccttctcg cttctctgga 300 tccgggtctg ggacggattt cactctgacc atcagcagtc tgcagccgga agacttcgca 360 acttattact gtgcacagtc ctacagccat ccgttctctt tcggacaggg taccaaggtg 420 gagatcaaac gtacg 435 <210> 61 <211> 435 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding CDR-L3 derived from L3-5 clone <400> 61 gaattcacta gtgattaatt cgccgccacc atggattcac aggcccaggt cctcatgttg 60 ctgctgctat cggtatctgg tacctgtgga gatatccaga tgacccagtc cccgagctcc 120 ctgtccgcct ctgtgggcga tagggtcacc atcacctgca agtccagtca gagtctttta 180 gctagtggca accaaaataa ctacttggcc tggcaccaac agaaaccagg aaaagctccg 240 aaaatgctga ttatttgggc atccactagg gtatctggag tcccttctcg cttctctgga 300 tccgggtctg ggacggattt cactctgacc atcagcagtc tgcagccgga agacttcgca 360 acttattact gtcagcagtc ctacagccgc ccgtttacgt tcggacaggg taccaaggtg 420 gagatcaaac gtacg 435 <210> 62 <211> 462 <212> PRT <213> Artificial Sequence <220> <223> polypeptide consisting of heavy chain of huAbF46-H4-A1, U6-HC7          hinge and constant region of human IgG1 <400> 62 Met Glu Trp Ser Trp Val Phe Leu Val Thr Leu Leu Asn Gly Ile Gln   1 5 10 15 Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly              20 25 30 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp          35 40 45 Tyr Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp      50 55 60 Leu Gly Phe Ile Arg Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ser  65 70 75 80 Ala Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn                  85 90 95 Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val             100 105 110 Tyr Tyr Cys Ala Arg Asp Asn Trp Phe Ala Tyr Trp Gly Gln Gly Thr         115 120 125 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro     130 135 140 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 145 150 155 160 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn                 165 170 175 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln             180 185 190 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser         195 200 205 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser     210 215 220 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Cys His 225 230 235 240 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe                 245 250 255 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro             260 265 270 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val         275 280 285 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr     290 295 300 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 305 310 315 320 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys                 325 330 335 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser             340 345 350 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro         355 360 365 Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val     370 375 380 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 385 390 395 400 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp                 405 410 415 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp             420 425 430 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His         435 440 445 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys     450 455 460 <210> 63 <211> 1410 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding polypeptide consisting of heavy chain of          huAbF46-H4-A1, U6-HC7 hinge and constant region of human IgG1 <400> 63 gaattcgccg ccaccatgga atggagctgg gtttttctcg taacactttt aaatggtatc 60 cagtgtgagg ttcagctggt ggagtctggc ggtggcctgg tgcagccagg gggctcactc 120 cgtttgtcct gtgcagcttc tggcttcacc ttcactgatt actacatgag ctgggtgcgt 180 caggccccgg gtaagggcct ggaatggttg ggttttatta gaaacaaagc taatggttac 240 acaacagagt acagtgcatc tgtgaagggt cgtttcacta taagcagaga taattccaaa 300 aacacactgt acctgcagat gaacagcctg cgtgctgagg acactgccgt ctattattgt 360 gctagagata actggtttgc ttactggggc caagggactc tggtcaccgt ctcctcggct 420 agcaccaagg gcccatcggt cttccccctg gcaccctcct ccaagagcac ctctgggggc 480 acagcggccc tgggctgcct ggtcaaggac tacttccccg aaccggtgac ggtgtcgtgg 540 aactcaggcg ccctgaccag cggcgtgcac accttcccgg ctgtcctaca gtcctcagga 600 ctctactccc tcagcagcgt ggtgaccgtg ccctccagca gcttgggcac ccagacctac 660 atctgcaacg tgaatcacaa gcccagcaac accaaggtgg acaagaaagt tgagcccaaa 720 agctgcgatt gccactgtcc tccatgtcca gcacctgaac tcctgggggg accgtcagtc 780 ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 840 tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 900 ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 960 cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 1020 tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 1080 gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggagga gatgaccaag 1140 aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 1200 tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 1260 gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 1320 aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 1380 ctctccctgt ctccgggtaa atgactcgag 1410 <210> 64 <211> 461 <212> PRT <213> Artificial Sequence <220> <223> polypeptide consisting of heavy chain of huAbF46-H4-A1, human          IgG2 hinge and constant region of human IgG1 <400> 64 Met Glu Trp Ser Trp Val Phe Leu Val Thr Leu Leu Asn Gly Ile Gln   1 5 10 15 Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly              20 25 30 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp          35 40 45 Tyr Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp      50 55 60 Leu Gly Phe Ile Arg Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ser  65 70 75 80 Ala Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn                  85 90 95 Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val             100 105 110 Tyr Tyr Cys Ala Arg Asp Asn Trp Phe Ala Tyr Trp Gly Gln Gly Thr         115 120 125 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro     130 135 140 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 145 150 155 160 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn                 165 170 175 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln             180 185 190 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser         195 200 205 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser     210 215 220 Asn Thr Lys Val Asp Lys Lys Val Glu Arg Lys Cys Cys Val Glu Cys 225 230 235 240 Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu                 245 250 255 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu             260 265 270 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys         275 280 285 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys     290 295 300 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 305 310 315 320 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys                 325 330 335 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys             340 345 350 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser         355 360 365 Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys     370 375 380 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 385 390 395 400 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly                 405 410 415 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln             420 425 430 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn         435 440 445 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys     450 455 460 <210> 65 <211> 1407 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding polypeptide consisting of heavy chain of          huAbF46-H4-A1, human IgG2 hinge and constant region of human IgG1 <400> 65 gaattcgccg ccaccatgga atggagctgg gtttttctcg taacactttt aaatggtatc 60 cagtgtgagg ttcagctggt ggagtctggc ggtggcctgg tgcagccagg gggctcactc 120 cgtttgtcct gtgcagcttc tggcttcacc ttcactgatt actacatgag ctgggtgcgt 180 caggccccgg gtaagggcct ggaatggttg ggttttatta gaaacaaagc taatggttac 240 acaacagagt acagtgcatc tgtgaagggt cgtttcacta taagcagaga taattccaaa 300 aacacactgt acctgcagat gaacagcctg cgtgctgagg acactgccgt ctattattgt 360 gctagagata actggtttgc ttactggggc caagggactc tggtcaccgt ctcctcggct 420 agcaccaagg gcccatcggt cttccccctg gcaccctcct ccaagagcac ctctgggggc 480 acagcggccc tgggctgcct ggtcaaggac tacttccccg aaccggtgac ggtgtcgtgg 540 aactcaggcg ccctgaccag cggcgtgcac accttcccgg ctgtcctaca gtcctcagga 600 ctctactccc tcagcagcgt ggtgaccgtg ccctccagca gcttgggcac ccagacctac 660 atctgcaacg tgaatcacaa gcccagcaac accaaggtgg acaagaaagt tgagaggaag 720 tgctgtgtgg agtgcccccc ctgcccagca cctgaactcc tggggggacc gtcagtcttc 780 ctcttccccc caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacatgc 840 gtggtggtgg acgtgagcca cgaagaccct gaggtcaagt tcaactggta cgtggacggc 900 gtggaggtgc ataatgccaa gacaaagccg cgggaggagc agtacaacag cacgtaccgt 960 gtggtcagcg tcctcaccgt cctgcaccag gactggctga atggcaagga gtacaagtgc 1020 aaggtctcca acaaagccct cccagccccc atcgagaaaa ccatctccaa agccaaaggg 1080 cagccccgag aaccacaggt gtacaccctg cccccatccc gggaggagat gaccaagaac 1140 caggtcagcc tgacctgcct ggtcaaaggc ttctatccca gcgacatcgc cgtggagtgg 1200 gagagcaatg ggcagccgga gaacaactac aagaccacgc ctcccgtgct ggactccgac 1260 ggctccttct tcctctacag caagctcacc gtggacaaga gcaggtggca gcaggggaac 1320 gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacacgca gaagagcctc 1380 tccctgtctc cgggtaaatg actcgag 1407 <210> 66 <211> 460 <212> PRT <213> Artificial Sequence <220> <223> polypeptide consisting of heavy chain of huAbF46-H4-A1, human          IgG2 hinge and constant region of human IgG2 <400> 66 Met Glu Trp Ser Trp Val Phe Leu Val Thr Leu Leu Asn Gly Ile Gln   1 5 10 15 Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly              20 25 30 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp          35 40 45 Tyr Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp      50 55 60 Leu Gly Phe Ile Arg Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ser  65 70 75 80 Ala Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn                  85 90 95 Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val             100 105 110 Tyr Tyr Cys Ala Arg Asp Asn Trp Phe Ala Tyr Trp Gly Gln Gly Thr         115 120 125 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro     130 135 140 Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly 145 150 155 160 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn                 165 170 175 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln             180 185 190 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser         195 200 205 Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser     210 215 220 Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu Cys 225 230 235 240 Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe                 245 250 255 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val             260 265 270 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe         275 280 285 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro     290 295 300 Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr 305 310 315 320 Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val                 325 330 335 Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr             340 345 350 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg         355 360 365 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly     370 375 380 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 385 390 395 400 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser                 405 410 415 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln             420 425 430 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His         435 440 445 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys     450 455 460 <210> 67 <211> 1404 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding polypeptide consisting of heavy chain of          huAbF46-H4-A1, human IgG2 hinge and constant region of human IgG2 <400> 67 gaattcgccg ccaccatgga atggagctgg gtttttctcg taacactttt aaatggtatc 60 cagtgtgagg ttcagctggt ggagtctggc ggtggcctgg tgcagccagg gggctcactc 120 cgtttgtcct gtgcagcttc tggcttcacc ttcactgatt actacatgag ctgggtgcgt 180 caggccccgg gtaagggcct ggaatggttg ggttttatta gaaacaaagc taatggttac 240 acaacagagt acagtgcatc tgtgaagggt cgtttcacta taagcagaga taattccaaa 300 aacacactgt acctgcagat gaacagcctg cgtgctgagg acactgccgt ctattattgt 360 gctagagata actggtttgc ttactggggc caagggactc tggtcaccgt ctcctcggct 420 agcaccaagg gcccatcggt cttccccctg gcgccctgct ccaggagcac ctccgagagc 480 acagcggccc tgggctgcct ggtcaaggac tacttccccg aaccggtgac ggtgtcgtgg 540 aactcaggcg ctctgaccag cggcgtgcac accttcccag ctgtcctaca gtcctcagga 600 ctctactccc tcagcagcgt ggtgaccgtg ccctccagca acttcggcac ccagacctac 660 acctgcaacg tagatcacaa gcccagcaac accaaggtgg acaagacagt tgagcgcaaa 720 tgttgtgtcg agtgcccacc gtgcccagca ccacctgtgg caggaccgtc agtcttcctc 780 ttccccccaa aacccaagga caccctcatg atctcccgga cccctgaggt cacgtgcgtg 840 gtggtggacg tgagccacga agaccccgag gtccagttca actggtacgt ggacggcgtg 900 gaggtgcata atgccaagac aaagccacgg gaggagcagt tcaacagcac gttccgtgtg 960 gtcagcgtcc tcaccgttgt gcaccaggac tggctgaacg gcaaggagta caagtgcaag 1020 gtctccaaca aaggcctccc agcccccatc gagaaaacca tctccaaaac caaagggcag 1080 ccccgagaac cacaggtgta caccctgccc ccatcccggg aggagatgac caagaaccag 1140 gtcagcctga cctgcctggt caaaggcttc taccccagcg acatcgccgt ggagtgggag 1200 agcaatgggc agccggagaa caactacaag accacgcctc ccatgctgga ctccgacggc 1260 tccttcttcc tctacagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc 1320 ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc 1380 ctgtctccgg gtaaatgact cgag 1404 <210> 68 <211> 240 <212> PRT <213> Artificial Sequence <220> <223> polypeptide consisting of light chain of huAbF46-H4-A1 (H36Y) and          human kappa constant region <400> 68 Met Asp Ser Gln Ala Gln Val Leu Met Leu Leu Leu Leu Seru Val Ser   1 5 10 15 Gly Thr Cys Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser              20 25 30 Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser          35 40 45 Leu Leu Ala Ser Gly Asn Gln Asn Asn Tyr Leu Ala Trp Tyr Gln Gln      50 55 60 Lys Pro Gly Lys Ala Pro Lys Met Leu Ile Ile Trp Ala Ser Thr Arg  65 70 75 80 Val Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp                  85 90 95 Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr             100 105 110 Tyr Cys Gln Gln Ser Tyr Ser Arg Pro Tyr Thr Phe Gly Gln Gly Thr         115 120 125 Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe     130 135 140 Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys 145 150 155 160 Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val                 165 170 175 Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln             180 185 190 Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser         195 200 205 Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His     210 215 220 Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 240 <210> 69 <211> 758 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding polypeptide consisting of light chain of          huAbF46-H4-A1 (H36Y) and human kappa constant region <400> 69 aattcactag tgattaattc gccgccacca tggattcaca ggcccaggtc ctcatgttgc 60 tgctgctatc ggtatctggt acctgtggag atatccagat gacccagtcc ccgagctccc 120 tgtccgcctc tgtgggcgat agggtcacca tcacctgcaa gtccagtcag agtcttttag 180 ctagtggcaa ccaaaataac tacttggcct ggtaccaaca gaaaccagga aaagctccga 240 aaatgctgat tatttgggca tccactaggg tatctggagt cccttctcgc ttctctggat 300 ccgggtctgg gacggatttc actctgacca tcagcagtct gcagccggaa gacttcgcaa 360 cttattactg tcagcagtcc tacagccgcc cgtacacgtt cggacagggt accaaggtgg 420 agatcaaacg tacggtggct gcaccatctg tcttcatctt cccgccatct gatgagcagt 480 tgaaatctgg aactgcctct gttgtgtgcc tgctgaataa cttctatccc agagaggcca 540 aagtacagtg gaaggtggat aacgccctcc aatcgggtaa ctcccaggag agtgtcacag 600 agcaggacag caaggacagc acctacagcc tcagcagcac cctgacgctg agcaaagcag 660 actacgagaa acacaaagtc tacgcctgcg aagtcaccca tcagggcctg agctcgcccg 720 tcacaaagag cttcaacagg ggagagtgtt gactcgag 758 <210> 70 <211> 240 <212> PRT <213> Artificial Sequence <220> <223> polypeptide consisting of light chain of huAbF46-H4-A1 and human          kappa constant region <400> 70 Met Asp Ser Gln Ala Gln Val Leu Met Leu Leu Leu Leu Seru Val Ser   1 5 10 15 Gly Thr Cys Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser              20 25 30 Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser          35 40 45 Leu Leu Ala Ser Gly Asn Gln Asn Asn Tyr Leu Ala Trp His Gln Gln      50 55 60 Lys Pro Gly Lys Ala Pro Lys Met Leu Ile Ile Trp Ala Ser Thr Arg  65 70 75 80 Val Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp                  85 90 95 Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr             100 105 110 Tyr Cys Gln Gln Ser Tyr Ser Arg Pro Tyr Thr Phe Gly Gln Gly Thr         115 120 125 Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe     130 135 140 Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys 145 150 155 160 Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val                 165 170 175 Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln             180 185 190 Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser         195 200 205 Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His     210 215 220 Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 240 <210> 71 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> epitope in SEMA domain of c-Met <400> 71 Phe Ser Pro Gln Ile Glu Glu Pro Ser Gln Cys Pro Asp Cys Val Val   1 5 10 15 Ser Ala Leu             <210> 72 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> epitope in SEMA domain of c-Met <400> 72 Pro Gln Ile Glu Glu Pro Ser Gln Cys Pro   1 5 10 <210> 73 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> epitope in SEMA domain of c-Met <400> 73 Glu Glu Pro Ser Gln   1 5 <210> 74 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> heavy chain variable region of anti-c-Met antibody (AbF46 or          huAbF46-H1) <400> 74 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr              20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu          35 40 45 Gly Phe Ile Arg Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ser Ala      50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Ser  65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr                  85 90 95 Tyr Cys Ala Arg Asp Asn Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu             100 105 110 Val Thr Val Ser Ser         115 <210> 75 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> light chain variable region of anti-c-Met antibody (AbF46 or          huAbF46-H1) <400> 75 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly   1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Ala Ser              20 25 30 Gly Asn Gln Asn Asn Tyr Leu Ala Trp His Gln Gln Lys Pro Gly Gln          35 40 45 Pro Pro Lys Met Leu Ile Ile Trp Ala Ser Thr Arg Val Ser Gly Val      50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln                  85 90 95 Ser Tyr Ser Ala Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile             100 105 110 Lys Arg         <210> 76 <211> 1416 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence of heavy chain of nti-c-Met antibody (AbF46          or huAbF46-H1) <220> <221> misc_feature <222> (1) .. (6) <223> EcoRI restriction site <220> <221> misc_feature <222> (7) .. (66) <223> signal sequence <220> <221> misc_feature <222> (67) .. (417) <223> VH-heavy chain variable region <220> <221> misc_feature <222> (418) .. (423) <223> NdeI restriction site <220> <221> misc_feature <222> (418) .. (1407) <223> CH-heavy chain constant region <220> <221> misc_feature <222> (1408) .. (1410) <223> TGA-stop sodon <220> <221> misc_feature <222> (1411) .. (1416) <223> XhoI restriction site <400> 76 gaattcgccg ccaccatgga atggagctgg gtttttctcg taacactttt aaatggtatc 60 cagtgtgagg tgaagctggt ggagtctgga ggaggcttgg tacagcctgg gggttctctg 120 agactctcct gtgcaacttc tgggttcacc ttcactgatt actacatgag ctgggtccgc 180 cagcctccag gaaaggcact tgagtggttg ggttttatta gaaacaaagc taatggttac 240 acaacagagt acagtgcatc tgtgaagggt cggttcacca tctccagaga taattcccaa 300 agcatcctct atcttcaaat ggacaccctg agagctgagg acagtgccac ttattactgt 360 gcaagagata actggtttgc ttactggggc caagggactc tggtcactgt ctctgcagct 420 agcaccaagg gcccatcggt cttccccctg gcaccctcct ccaagagcac ctctgggggc 480 acagcggccc tgggctgcct ggtcaaggac tacttccccg aaccggtgac ggtgtcgtgg 540 aactcaggcg ccctgaccag cggcgtgcac accttcccgg ctgtcctaca gtcctcagga 600 ctctactccc tcagcagcgt ggtgaccgtg ccctccagca gcttgggcac ccagacctac 660 atctgcaacg tgaatcacaa gcccagcaac accaaggtgg acaagaaagt tgagcccaaa 720 tcttgtgaca aaactcacac atgcccaccg tgcccagcac ctgaactcct ggggggaccg 780 tcagtcttcc tcttcccccc aaaacccaag gacaccctca tgatctcccg gacccctgag 840 gtcacatgcg tggtggtgga cgtgagccac gaagaccctg aggtcaagtt caactggtac 900 gtggacggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gtacaacagc 960 acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa tggcaaggag 1020 tacaagtgca aggtctccaa caaagccctc ccagccccca tcgagaaaac catctccaaa 1080 gccaaagggc agccccgaga accacaggtg tacaccctgc ccccatcccg ggaggagatg 1140 accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctatcccag cgacatcgcc 1200 gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg 1260 gactccgacg gctccttctt cctctacagc aagctcaccg tggacaagag caggtggcag 1320 caggggaacg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacgcag 1380 aagagcctct ccctgtctcc gggtaaatga ctcgag 1416 <210> 77 <211> 759 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence of light chain of anti-c-Met antibody (AbF46          or huAbF46-H1) <220> <221> misc_difference <222> (1) .. (6) <223> EcoRI restriction site <220> <221> misc_difference <222> (7) .. (90) <223> signal sequence <220> <221> misc_difference <222> (91) .. (432) <223> VL-light chain variable region <220> <221> misc_difference <222> (430) .. (435) <223> BsiWI restriction site <220> <221> misc_difference <222> (433) .. (750) <223> CL-light chain constant region <220> <221> misc_difference <222> (751) .. (753) <223> stop codon <220> <221> misc_difference <222> (754) .. (759) <223> XhoI restriction site <400> 77 gaattcacta gtgattaatt cgccgccacc atggattcac aggcccaggt cctcatgttg 60 ctgctgctat cggtatctgg tacctgtgga gacattttga tgacccagtc tccatcctcc 120 ctgactgtgt cagcaggaga gaaggtcact atgagctgca agtccagtca gagtctttta 180 gctagtggca accaaaataa ctacttggcc tggcaccagc agaaaccagg acgatctcct 240 aaaatgctga taatttgggc atccactagg gtatctggag tccctgatcg cttcataggc 300 agtggatctg ggacggattt cactctgacc atcaacagtg tgcaggctga agatctggct 360 gtttattact gtcagcagtc ctacagcgct ccgctcacgt tcggtgctgg gaccaagctg 420 gagctgaaac gtacggtggc tgcaccatct gtcttcatct tcccgccatc tgatgagcag 480 ttgaaatctg gaactgcctc tgttgtgtgc ctgctgaata acttctatcc cagagaggcc 540 aaagtacagt ggaaggtgga taacgccctc caatcgggta actcccagga gagtgtcaca 600 gagcaggaca gcaaggacag cacctacagc ctcagcagca ccctgacgct gagcaaagca 660 gactacgaga aacacaaagt ctacgcctgc gaagtcaccc atcagggcct gagctcgccc 720 gtcacaaaga gcttcaacag gggagagtgt tgactcgag 759 <210> 78 <211> 4170 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding c-Met protein <400> 78 atgaaggccc ccgctgtgct tgcacctggc atcctcgtgc tcctgtttac cttggtgcag 60 aggagcaatg gggagtgtaa agaggcacta gcaaagtccg agatgaatgt gaatatgaag 120 tatcagcttc ccaacttcac cgcggaaaca cccatccaga atgtcattct acatgagcat 180 cacattttcc ttggtgccac taactacatt tatgttttaa atgaggaaga ccttcagaag 240 gttgctgagt acaagactgg gcctgtgctg gaacacccag attgtttccc atgtcaggac 300 tgcagcagca aagccaattt atcaggaggt gtttggaaag ataacatcaa catggctcta 360 gttgtcgaca cctactatga tgatcaactc attagctgtg gcagcgtcaa cagagggacc 420 tgccagcgac atgtctttcc ccacaatcat actgctgaca tacagtcgga ggttcactgc 480 atattctccc cacagataga agagcccagc cagtgtcctg actgtgtggt gagcgccctg 540 ggagccaaag tcctttcatc tgtaaaggac cggttcatca acttctttgt aggcaatacc 600 ataaattctt cttatttccc agatcatcca ttgcattcga tatcagtgag aaggctaaag 660 gaaacgaaag atggttttat gtttttgacg gaccagtcct acattgatgt tttacctgag 720 ttcagagatt cttaccccat taagtatgtc catgcctttg aaagcaacaa ttttatttac 780 ttcttgacgg tccaaaggga aactctagat gctcagactt ttcacacaag aataatcagg 840 ttctgttcca taaactctgg attgcattcc tacatggaaa tgcctctgga gtgtattctc 900 acagaaaaga gaaaaaagag atccacaaag aaggaagtgt ttaatatact tcaggctgcg 960 tatgtcagca agcctggggc ccagcttgct agacaaatag gagccagcct gaatgatgac 1020 attcttttcg gggtgttcgc acaaagcaag ccagattctg ccgaaccaat ggatcgatct 1080 gccatgtgtg cattccctat caaatatgtc aacgacttct tcaacaagat cgtcaacaaa 1140 aacaatgtga gatgtctcca gcatttttac ggacccaatc atgagcactg ctttaatagg 1200 acacttctga gaaattcatc aggctgtgaa gcgcgccgtg atgaatatcg aacagagttt 1260 accacagctt tgcagcgcgt tgacttattc atgggtcaat tcagcgaagt cctcttaaca 1320 tctatatcca ccttcattaa aggagacctc accatagcta atcttgggac atcagagggt 1380 cgcttcatgc aggttgtggt ttctcgatca ggaccatcaa cccctcatgt gaattttctc 1440 ctggactccc atccagtgtc tccagaagtg attgtggagc atacattaaa ccaaaatggc 1500 tacacactgg ttatcactgg gaagaagatc acgaagatcc cattgaatgg cttgggctgc 1560 agacatttcc agtcctgcag tcaatgcctc tctgccccac cctttgttca gtgtggctgg 1620 tgccacgaca aatgtgtgcg atcggaggaa tgcctgagcg ggacatggac tcaacagatc 1680 tgtctgcctg caatctacaa ggttttccca aatagtgcac cccttgaagg agggacaagg 1740 ctgaccatat gtggctggga ctttggattt cggaggaata ataaatttga tttaaagaaa 1800 actagagttc tccttggaaa tgagagctgc accttgactt taagtgagag cacgatgaat 1860 acattgaaat gcacagttgg tcctgccatg aataagcatt tcaatatgtc cataattatt 1920 tcaaatggcc acgggacaac acaatacagt acattctcct atgtggatcc tgtaataaca 1980 agtatttcgc cgaaatacgg tcctatggct ggtggcactt tacttacttt aactggaaat 2040 tacctaaaca gtgggaattc tagacacatt tcaattggtg gaaaaacatg tactttaaaa 2100 agtgtgtcaa acagtattct tgaatgttat accccagccc aaaccatttc aactgagttt 2160 gctgttaaat tgaaaattga cttagccaac cgagagacaa gcatcttcag ttaccgtgaa 2220 gatcccattg tctatgaaat tcatccaacc aaatctttta ttagtggtgg gagcacaata 2280 acaggtgttg ggaaaaacct gaattcagtt agtgtcccga gaatggtcat aaatgtgcat 2340 gaagcaggaa ggaactttac agtggcatgt caacatcgct ctaattcaga gataatctgt 2400 tgtaccactc cttccctgca acagctgaat ctgcaactcc ccctgaaaac caaagccttt 2460 ttcatgttag atgggatcct ttccaaatac tttgatctca tttatgtaca taatcctgtg 2520 tttaagcctt ttgaaaagcc agtgatgatc tcaatgggca atgaaaatgt actggaaatt 2580 aagggaaatg atattgaccc tgaagcagtt aaaggtgaag tgttaaaagt tggaaataag 2640 agctgtgaga atatacactt acattctgaa gccgttttat gcacggtccc caatgacctg 2700 ctgaaattga acagcgagct aaatatagag tggaagcaag caatttcttc aaccgtcctt 2760 ggaaaagtaa tagttcaacc agatcagaat ttcacaggat tgattgctgg tgttgtctca 2820 atatcaacag cactgttatt actacttggg tttttcctgt ggctgaaaaa gagaaagcaa 2880 attaaagatc tgggcagtga attagttcgc tacgatgcaa gagtacacac tcctcatttg 2940 gataggcttg taagtgcccg aagtgtaagc ccaactacag aaatggtttc aaatgaatct 3000 gtagactacc gagctacttt tccagaagat cagtttccta attcatctca gaacggttca 3060 tgccgacaag tgcagtatcc tctgacagac atgtccccca tcctaactag tggggactct 3120 gatatatcca gtccattact gcaaaatact gtccacattg acctcagtgc tctaaatcca 3180 gagctggtcc aggcagtgca gcatgtagtg attgggccca gtagcctgat tgtgcatttc 3240 aatgaagtca taggaagagg gcattttggt tgtgtatatc atgggacttt gttggacaat 3300 gatggcaaga aaattcactg tgctgtgaaa tccttgaaca gaatcactga cataggagaa 3360 gtttcccaat ttctgaccga gggaatcatc atgaaagatt ttagtcatcc caatgtcctc 3420 tcgctcctgg gaatctgcct gcgaagtgaa gggtctccgc tggtggtcct accatacatg 3480 aaacatggag atcttcgaaa tttcattcga aatgagactc ataatccaac tgtaaaagat 3540 cttattggct ttggtcttca agtagccaaa ggcatgaaat atcttgcaag caaaaagttt 3600 gtccacagag acttggctgc aagaaactgt atgctggatg aaaaattcac agtcaaggtt 3660 gctgattttg gtcttgccag agacatgtat gataaagaat actatagtgt acacaacaaa 3720 acaggtgcaa agctgccagt gaagtggatg gctttggaaa gtctgcaaac tcaaaagttt 3780 accaccaagt cagatgtgtg gtcctttggc gtgctcctct gggagctgat gacaagagga 3840 gccccacctt atcctgacgt aaacaccttt gatataactg tttacttgtt gcaagggaga 3900 agactcctac aacccgaata ctgcccagac cccttatatg aagtaatgct aaaatgctgg 3960 caccctaaag ccgaaatgcg cccatccttt tctgaactgg tgtcccggat atcagcgatc 4020 ttctctactt tcattgggga gcactatgtc catgtgaacg ctacttatgt gaacgtaaaa 4080 tgtgtcgctc cgtatccttc tctgttgtca tcagaagata acgctgatga tgaggtggac 4140 acacgaccag cctccttctg ggagacatca 4170 <210> 79 <211> 444 <212> PRT <213> Artificial Sequence <220> <223> SEMA domain of c-Met <400> 79 Leu His Glu His His Ile Phe Leu Gly Ala Thr Asn Tyr Ile Tyr Val   1 5 10 15 Leu Asn Glu Glu Asp Leu Gln Lys Val Ala Glu Tyr Lys Thr Gly Pro              20 25 30 Val Leu Glu His Pro Asp Cys Phe Pro Cys Gln Asp Cys Ser Ser Lys          35 40 45 Ala Asn Leu Ser Gly Gly Val Trp Lys Asp Asn Ile Asn Met Ala Leu      50 55 60 Val Val Asp Thr Tyr Tyr Asp Asp Gln Leu Ile Ser Cys Gly Ser Val  65 70 75 80 Asn Arg Gly Thr Cys Gln Arg His Val Phe Pro His Asn His Thr Ala                  85 90 95 Asp Ile Gln Ser Glu Val His Cys Ile Phe Ser Pro Gln Ile Glu Glu             100 105 110 Pro Ser Gln Cys Pro Asp Cys Val Val Ser Ala Leu Gly Ala Lys Val         115 120 125 Leu Ser Ser Val Lys Asp Arg Phe Ile Asn Phe Phe Val Gly Asn Thr     130 135 140 Ile Asn Ser Ser Tyr Phe Pro Asp His Pro Leu His Ser Ile Ser Val 145 150 155 160 Arg Arg Leu Lys Glu Thr Lys Asp Gly Phe Met Phe Leu Thr Asp Gln                 165 170 175 Ser Tyr Ile Asp Val Leu Pro Glu Phe Arg Asp Ser Tyr Pro Ile Lys             180 185 190 Tyr Val His Ala Phe Glu Ser Asn Asn Phe Ile Tyr Phe Leu Thr Val         195 200 205 Gln Arg Glu Thr Leu Asp Ala Gln Thr Phe His Thr Arg Ile Ile Arg     210 215 220 Phe Cys Ser Ile Asn Ser Gly Leu His Ser Tyr Met Glu Met Pro Leu 225 230 235 240 Glu Cys Ile Leu Thr Glu Lys Arg Lys Lys Arg Ser Thr Lys Lys Glu                 245 250 255 Val Phe Asn Ile Leu Gln Ala Ala Tyr Val Ser Lys Pro Gly Ala Gln             260 265 270 Leu Ala Arg Gln Ile Gly Ala Ser Leu Asn Asp Asp Ile Leu Phe Gly         275 280 285 Val Phe Ala Gln Ser Lys Pro Asp Ser Ala Glu Pro Met Asp Arg Ser     290 295 300 Ala Met Cys Ala Phe Pro Ile Lys Tyr Val Asn Asp Phe Phe Asn Lys 305 310 315 320 Ile Val Asn Lys Asn Asn Val Arg Cys Leu Gln His Phe Tyr Gly Pro                 325 330 335 Asn His Glu His Cys Phe Asn Arg Thr Leu Leu Arg Asn Ser Ser Gly             340 345 350 Cys Glu Ala Arg Arg Asp Glu Tyr Arg Thr Glu Phe Thr Thr Ala Leu         355 360 365 Gln Arg Val Asp Leu Phe Met Gly Gln Phe Ser Glu Val Leu Leu Thr     370 375 380 Ser Ile Ser Thr Phe Ile Lys Gly Asp Leu Thr Ile Ala Asn Leu Gly 385 390 395 400 Thr Ser Glu Gly Arg Phe Met Gln Val Val Val Ser Arg Ser Gly Pro                 405 410 415 Ser Thr Pro His Val Asn Phe Leu Leu Asp Ser His Pro Val Ser Pro             420 425 430 Glu Val Ile Val Glu His Thr Leu Asn Gln Asn Gly         435 440 <210> 80 <211> 451 <212> PRT <213> Artificial Sequence <220> <223> PSI-IPT domain of c-Met <400> 80 Tyr Thr Leu Val Ile Thr Gly Lys Lys Ile Thr Lys Ile Pro Leu Asn   1 5 10 15 Gly Leu Gly Cys Arg His Phe Gln Ser Cys Ser Gln Cys Leu Ser Ala              20 25 30 Pro Pro Phe Val Gln Cys Gly Trp Cys His Asp Lys Cys Val Arg Ser          35 40 45 Glu Glu Cys Leu Ser Gly Thr Trp Thr Gln Gln Ile Cys Leu Pro Ala      50 55 60 Ile Tyr Lys Val Phe Pro Asn Ser Ala Pro Leu Glu Gly Gly Thr Arg  65 70 75 80 Leu Thr Ile Cys Gly Trp Asp Phe Gly Phe Arg Arg Asn Asn Lys Phe                  85 90 95 Asp Leu Lys Lys Thr Arg Val Leu Leu Gly Asn Glu Ser Cys Thr Leu             100 105 110 Thr Leu Ser Glu Ser Thr Met Asn Thr Leu Lys Cys Thr Val Gly Pro         115 120 125 Ala Met Asn Lys His Phe Asn Met Ser Ile Ile Ile Ser Asn Gly His     130 135 140 Gly Thr Thr Gln Tyr Ser Thr Phe Ser Tyr Val Asp Pro Val Ile Thr 145 150 155 160 Ser Ile Ser Pro Lys Tyr Gly Pro Met Ala Gly Gly Thr Leu Leu Thr                 165 170 175 Leu Thr Gly Asn Tyr Leu Asn Ser Gly Asn Ser Arg His Ile Ser Ile             180 185 190 Gly Gly Lys Thr Cys Thr Leu Lys Ser Val Ser Asn Ser Ile Leu Glu         195 200 205 Cys Tyr Thr Pro Ala Gln Thr Ile Ser Thr Glu Phe Ala Val Lys Leu     210 215 220 Lys Ile Asp Leu Ala Asn Arg Glu Thr Ser Ile Phe Ser Tyr Arg Glu 225 230 235 240 Asp Pro Ile Val Tyr Glu Ile His Pro Thr Lys Ser Phe Ile Ser Thr                 245 250 255 Trp Trp Lys Glu Pro Leu Asn Ile Val Ser Phe Leu Phe Cys Phe Ala             260 265 270 Ser Gly Gly Ser Thr Ile Thr Gly Val Gly Lys Asn Leu Asn Ser Val         275 280 285 Ser Val Pro Arg Met Val Ile Asn Val His Glu Ala Gly Arg Asn Phe     290 295 300 Thr Val Ala Cys Gln His Arg Ser Asn Ser Glu Ile Ile Cys Cys Thr 305 310 315 320 Thr Pro Ser Leu Gln Gln Leu Asn Leu Gln Leu Pro Leu Lys Thr Lys                 325 330 335 Ala Phe Phe Met Leu Asp Gly Ile Leu Ser Lys Tyr Phe Asp Leu Ile             340 345 350 Tyr Val His Asn Pro Val Phe Lys Pro Phe Glu Lys Pro Val Met Ile         355 360 365 Ser Met Gly Asn Glu Asn Val Leu Glu Ile Lys Gly Asn Asp Ile Asp     370 375 380 Pro Glu Ala Val Lys Gly Glu Val Leu Lys Val Gly Asn Lys Ser Cys 385 390 395 400 Glu Asn Ile His Leu His Ser Glu Ala Val Leu Cys Thr Val Pro Asn                 405 410 415 Asp Leu Leu Lys Leu Asn Ser Glu Leu Asn Ile Glu Trp Lys Gln Ala             420 425 430 Ile Ser Ser Thr Val Leu Gly Lys Val Ile Val Gln Pro Asp Gln Asn         435 440 445 Phe Thr Gly     450 <210> 81 <211> 313 <212> PRT <213> Artificial Sequence <220> <223> TyrKc domain of c-Met <400> 81 Val His Phe Asn Glu Val Ile Gly Arg Gly His Phe Gly Cys Val Tyr   1 5 10 15 His Gly Thr Leu Leu Asp Asn Asp Gly Lys Lys Ile His Cys Ala Val              20 25 30 Lys Ser Leu Asn Arg Ile Thr Asp Ile Gly Glu Val Ser Gln Phe Leu          35 40 45 Thr Glu Gly Ile Ile Met Lys Asp Phe Ser His Pro Asn Val Leu Ser      50 55 60 Leu Leu Gly Ile Cys Leu Arg Ser Glu Gly Ser Pro Leu Val Val Leu  65 70 75 80 Pro Tyr Met Lys His Gly Asp Leu Arg Asn Phe Ile Arg Asn Glu Thr                  85 90 95 His Asn Pro Thr Val Lys Asp Leu Ile Gly Phe Gly Leu Gln Val Ala             100 105 110 Lys Gly Met Lys Tyr Leu Ala Ser Lys Lys Phe Val His Arg Asp Leu         115 120 125 Ala Ala Arg Asn Cys Met Leu Asp Glu Lys Phe Thr Val Lys Val Ala     130 135 140 Asp Phe Gly Leu Ala Arg Asp Met Tyr Asp Lys Glu Tyr Tyr Ser Val 145 150 155 160 His Asn Lys Thr Gly Ala Lys Leu Pro Val Lys Trp Met Ala Leu Glu                 165 170 175 Ser Leu Gln Thr Gln Lys Phe Thr Thr Lys Ser Asp Val Trp Ser Phe             180 185 190 Gly Val Leu Leu Trp Glu Leu Met Thr Arg Gly Ala Pro Pro Tyr Pro         195 200 205 Asp Val Asn Thr Phe Asp Ile Thr Val Tyr Leu Leu Gln Gly Arg Arg     210 215 220 Leu Leu Gln Pro Glu Tyr Cys Pro Asp Pro Leu Tyr Glu Val Met Leu 225 230 235 240 Lys Cys Trp His Pro Lys Ala Glu Met Arg Pro Ser Phe Ser Glu Leu                 245 250 255 Val Ser Arg Ile Ser Ala Ile Phe Ser Thr Phe Ile Gly Glu His Tyr             260 265 270 Val His Val Asn Ala Thr Tyr Val Asn Val Lys Cys Val Ala Pro Tyr         275 280 285 Pro Ser Leu Leu Ser Ser Glu Asp Asn Ala Asp Asp Glu Val Asp Thr     290 295 300 Arg Pro Ala Ser Phe Trp Glu Thr Ser 305 310 <210> 82 <211> 1332 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding SEMA domain of c-Met <400> 82 ctacatgagc atcacatttt ccttggtgcc actaactaca tttatgtttt aaatgaggaa 60 gaccttcaga aggttgctga gtacaagact gggcctgtgc tggaacaccc agattgtttc 120 ccatgtcagg actgcagcag caaagccaat ttatcaggag gtgtttggaa agataacatc 180 aacatggctc tagttgtcga cacctactat gatgatcaac tcattagctg tggcagcgtc 240 aacagaggga cctgccagcg acatgtcttt ccccacaatc atactgctga catacagtcg 300 gaggttcact gcatattctc cccacagata gaagagccca gccagtgtcc tgactgtgtg 360 gtgagcgccc tgggagccaa agtcctttca tctgtaaagg accggttcat caacttcttt 420 gtaggcaata ccataaattc ttcttatttc ccagatcatc cattgcattc gatatcagtg 480 agaaggctaa aggaaacgaa agatggtttt atgtttttga cggaccagtc ctacattgat 540 gttttacctg agttcagaga ttcttacccc attaagtatg tccatgcctt tgaaagcaac 600 aattttattt acttcttgac ggtccaaagg gaaactctag atgctcagac ttttcacaca 660 agaataatca ggttctgttc cataaactct ggattgcatt cctacatgga aatgcctctg 720 gagtgtattc tcacagaaaa gagaaaaaag agatccacaa agaaggaagt gtttaatata 780 cttcaggctg cgtatgtcag caagcctggg gcccagcttg ctagacaaat aggagccagc 840 ctgaatgatg acattctttt cggggtgttc gcacaaagca agccagattc tgccgaacca 900 atggatcgat ctgccatgtg tgcattccct atcaaatatg tcaacgactt cttcaacaag 960 atcgtcaaca aaaacaatgt gagatgtctc cagcattttt acggacccaa tcatgagcac 1020 tgctttaata ggacacttct gagaaattca tcaggctgtg aagcgcgccg tgatgaatat 1080 cgaacagagt ttaccacagc tttgcagcgc gttgacttat tcatgggtca attcagcgaa 1140 gtcctcttaa catctatatc caccttcatt aaaggagacc tcaccatagc taatcttggg 1200 acatcagagg gtcgcttcat gcaggttgtg gtttctcgat caggaccatc aacccctcat 1260 gtgaattttc tcctggactc ccatccagtg tctccagaag tgattgtgga gcatacatta 1320 aaccaaaatg gc 1332 <210> 83 <211> 1299 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding PSI-IPT domain of c-Met <400> 83 tacacactgg ttatcactgg gaagaagatc acgaagatcc cattgaatgg cttgggctgc 60 agacatttcc agtcctgcag tcaatgcctc tctgccccac cctttgttca gtgtggctgg 120 tgccacgaca aatgtgtgcg atcggaggaa tgcctgagcg ggacatggac tcaacagatc 180 tgtctgcctg caatctacaa ggttttccca aatagtgcac cccttgaagg agggacaagg 240 ctgaccatat gtggctggga ctttggattt cggaggaata ataaatttga tttaaagaaa 300 actagagttc tccttggaaa tgagagctgc accttgactt taagtgagag cacgatgaat 360 acattgaaat gcacagttgg tcctgccatg aataagcatt tcaatatgtc cataattatt 420 tcaaatggcc acgggacaac acaatacagt acattctcct atgtggatcc tgtaataaca 480 agtatttcgc cgaaatacgg tcctatggct ggtggcactt tacttacttt aactggaaat 540 tacctaaaca gtgggaattc tagacacatt tcaattggtg gaaaaacatg tactttaaaa 600 agtgtgtcaa acagtattct tgaatgttat accccagccc aaaccatttc aactgagttt 660 gctgttaaat tgaaaattga cttagccaac cgagagacaa gcatcttcag ttaccgtgaa 720 gatcccattg tctatgaaat tcatccaacc aaatctttta ttagtggtgg gagcacaata 780 acaggtgttg ggaaaaacct gaattcagtt agtgtcccga gaatggtcat aaatgtgcat 840 gaagcaggaa ggaactttac agtggcatgt caacatcgct ctaattcaga gataatctgt 900 tgtaccactc cttccctgca acagctgaat ctgcaactcc ccctgaaaac caaagccttt 960 ttcatgttag atgggatcct ttccaaatac tttgatctca tttatgtaca taatcctgtg 1020 tttaagcctt ttgaaaagcc agtgatgatc tcaatgggca atgaaaatgt actggaaatt 1080 aagggaaatg atattgaccc tgaagcagtt aaaggtgaag tgttaaaagt tggaaataag 1140 agctgtgaga atatacactt acattctgaa gccgttttat gcacggtccc caatgacctg 1200 ctgaaattga acagcgagct aaatatagag tggaagcaag caatttcttc aaccgtcctt 1260 ggaaaagtaa tagttcaacc agatcagaat ttcacagga 1299 <210> 84 <211> 939 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding TyrKc domain of c-Met <400> 84 gtgcatttca atgaagtcat aggaagaggg cattttggtt gtgtatatca tgggactttg 60 ttggacaatg atggcaagaa aattcactgt gctgtgaaat ccttgaacag aatcactgac 120 ataggagaag tttcccaatt tctgaccgag ggaatcatca tgaaagattt tagtcatccc 180 aatgtcctct cgctcctggg aatctgcctg cgaagtgaag ggtctccgct ggtggtccta 240 ccatacatga aacatggaga tcttcgaaat ttcattcgaa atgagactca taatccaact 300 gtaaaagatc ttattggctt tggtcttcaa gtagccaaag gcatgaaata tcttgcaagc 360 aaaaagtttg tccacagaga cttggctgca agaaactgta tgctggatga aaaattcaca 420 gtcaaggttg ctgattttgg tcttgccaga gacatgtatg ataaagaata ctatagtgta 480 cacaacaaaa caggtgcaaa gctgccagtg aagtggatgg ctttggaaag tctgcaaact 540 caaaagttta ccaccaagtc agatgtgtgg tcctttggcg tgctcctctg ggagctgatg 600 acaagaggag ccccacctta tcctgacgta aacacctttg atataactgt ttacttgttg 660 caagggagaa gactcctaca acccgaatac tgcccagacc ccttatatga agtaatgcta 720 aaatgctggc accctaaagc cgaaatgcgc ccatcctttt ctgaactggt gtcccggata 780 tcagcgatct tctctacttt cattggggag cactatgtcc atgtgaacgc tacttatgtg 840 aacgtaaaat gtgtcgctcc gtatccttct ctgttgtcat cagaagataa cgctgatgat 900 gaggtggaca cacgaccagc ctccttctgg gagacatca 939 <210> 85 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> heavy chain CDR3 of anti-c-Met antibody <400> 85 Asp Asn Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val   1 5 10 <210> 86 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> light chain CDR3 of anti-c-Met antibody <400> 86 Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu   1 5 10 <210> 87 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> heavy chain variable region of monoclonal antibody AbF46 <400> 87 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Thr Asp Tyr              20 25 30 Tyr Met Ser Trp Val Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu          35 40 45 Gly Phe Ile Arg Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ser Ala      50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Gln Ser Ile  65 70 75 80 Leu Tyr Leu Gln Met Asp Thr Leu Arg Ala Glu Asp Ser Ala Thr Tyr                  85 90 95 Tyr Cys Ala Arg Asp Asn Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu             100 105 110 Val Thr Val Ser Ala         115 <210> 88 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> light chain variable region of anti-c-Met antibody <400> 88 Asp Ile Leu Met Thr Gln Ser Pro Ser Ser Leu Thr Val Ser Ala Gly   1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Ala Ser              20 25 30 Gly Asn Gln Asn Asn Tyr Leu Ala Trp His Gln Gln Lys Pro Gly Arg          35 40 45 Ser Pro Lys Met Leu Ile Ile Trp Ala Ser Thr Arg Val Ser Gly Val      50 55 60 Pro Asp Arg Phe Ile Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr  65 70 75 80 Ile Asn Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln                  85 90 95 Ser Tyr Ser Ala Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu             100 105 110 Lys Arg         <210> 89 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> light chain CDR3 of anti-c-Met antibody <400> 89 Gln Gln Ser Tyr Ser Ala Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu   1 5 10 15 Glu     <210> 90 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> heavy chain variable region of AT-VH1 <400> 90 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Thr Asp Tyr              20 25 30 Tyr Met Ser Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu          35 40 45 Gly Phe Ile Arg Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ser Ala      50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Ser Thr  65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Ser Ala Thr Tyr                  85 90 95 Tyr Cys Ala Arg Asp Asn Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu             100 105 110 Val Thr Val Ser Ser         115 <210> 91 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> heavy chain variable region of AT-VH2 <400> 91 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Thr Asp Tyr              20 25 30 Tyr Met Ser Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu          35 40 45 Gly Phe Ile Arg Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ser Ala      50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Ser Thr  65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr                  85 90 95 Tyr Cys Ala Arg Asp Asn Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu             100 105 110 Val Thr Val Ser Ser         115 <210> 92 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> heavy chain variable region of AT-VH3 <400> 92 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Thr Asp Tyr              20 25 30 Tyr Met Ser Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu          35 40 45 Gly Phe Ile Arg Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ser Ala      50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Ser Thr  65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr                  85 90 95 Tyr Cys Ala Arg Asp Asn Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu             100 105 110 Val Thr Val Ser Ser         115 <210> 93 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> heavy chain variable region of AT-VH4 <400> 93 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Thr Asp Tyr              20 25 30 Tyr Met Ser Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu          35 40 45 Gly Phe Ile Arg Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ser Ala      50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr  65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr                  85 90 95 Tyr Cys Ala Arg Asp Asn Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu             100 105 110 Val Thr Val Ser Ser         115 <210> 94 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> heavy chain variable region of AT-VH5 <400> 94 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Thr Asp Tyr              20 25 30 Tyr Met Ser Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu          35 40 45 Gly Phe Ile Arg Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ser Ala      50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr  65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr                  85 90 95 Tyr Cys Ala Arg Asp Asn Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu             100 105 110 Val Thr Val Ser Ser         115 <210> 95 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> light chain variable region of anti c-Met humanized          antibody (huAbF46-H4) <400> 95 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Leu Ala Ser              20 25 30 Gly Asn Gln Asn Asn Tyr Leu Ala Trp His Gln Gln Lys Pro Gly Lys          35 40 45 Ala Pro Lys Met Leu Ile Ile Trp Ala Ser Thr Arg Val Ser Gly Val      50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln                  85 90 95 Ser Tyr Ser Ala Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile             100 105 110 Lys Arg         <210> 96 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> light chain variable region of AT-Vk1 <400> 96 Asp Ile Leu Met Thr Gln Ser Pro Ser Ser Leu Thr Ala Ser Val Gly   1 5 10 15 Asp Arg Val Thr Met Thr Cys Lys Ser Ser Gln Ser Leu Leu Ala Ser              20 25 30 Gly Asn Gln Asn Asn Tyr Leu Ala Trp His Gln Gln Lys Pro Gly Lys          35 40 45 Ala Pro Lys Met Leu Ile Ile Trp Ala Ser Thr Arg Val Ser Gly Val      50 55 60 Pro Asp Arg Phe Ile Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln                  85 90 95 Ser Tyr Ser Ala Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile             100 105 110 Lys     <210> 97 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> light chain variable region of AT-Vk2 <400> 97 Asp Ile Leu Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Leu Ala Ser              20 25 30 Gly Asn Gln Asn Asn Tyr Leu Ala Trp His Gln Gln Lys Pro Gly Lys          35 40 45 Ala Pro Lys Met Leu Ile Ile Trp Ala Ser Thr Arg Val Ser Gly Val      50 55 60 Pro Asp Arg Phe Ile Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln                  85 90 95 Ser Tyr Ser Ala Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile             100 105 110 Lys     <210> 98 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> light chain variable region of AT-Vk3 <400> 98 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Leu Ala Ser              20 25 30 Gly Asn Gln Asn Asn Tyr Leu Ala Trp His Gln Gln Lys Pro Gly Lys          35 40 45 Ala Pro Lys Met Leu Ile Ile Trp Ala Ser Thr Arg Val Ser Gly Val      50 55 60 Pro Asp Arg Phe Ile Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln                  85 90 95 Ser Tyr Ser Ala Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile             100 105 110 Lys     <210> 99 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> light chain variable region of AT-Vk4 <400> 99 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Leu Ala Ser              20 25 30 Gly Asn Gln Asn Asn Tyr Leu Ala Trp His Gln Gln Lys Pro Gly Lys          35 40 45 Ala Pro Lys Met Leu Ile Ile Trp Ala Ser Thr Arg Val Ser Gly Val      50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln                  85 90 95 Ser Tyr Ser Ala Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile             100 105 110 Lys     <210> 100 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> modified hinge region (U7-HC6) <400> 100 Glu Pro Ser Cys Asp Lys His Cys Cys Pro Pro Cys Pro   1 5 10 <210> 101 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> modified hinge region (U6-HC7) <400> 101 Glu Pro Lys Ser Cys Asp Cys His Cys Pro Pro Cys Pro   1 5 10 <210> 102 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> modified hinge region (U3-HC9) <400> 102 Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro   1 5 10 <210> 103 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> modified hinge region (U6-HC8) <400> 103 Glu Pro Arg Asp Cys Gly Cys Lys Pro Cys Pro Pro Cys Pro   1 5 10 <210> 104 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> modified hinge region (U8-HC5) <400> 104 Glu Lys Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro   1 5 10 <210> 105 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> human hinge region <400> 105 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro   1 5 10 15 <210> 106 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR-L1 of antibody L3-11Y <400> 106 Lys Ser Ser Gln Ser Leu Leu Ala Trp Gly Asn Gln Asn Asn Tyr Leu   1 5 10 15 Ala     <210> 107 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of light chain variable region of antibody          L3-11Y <400> 107 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Leu Ala Trp              20 25 30 Gly Asn Gln Asn Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys          35 40 45 Ala Pro Lys Met Leu Ile Ile Trp Ala Ser Thr Arg Val Ser Gly Val      50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln                  85 90 95 Ser Tyr Ser Arg Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile             100 105 110 Lys Arg         <210> 108 <211> 220 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of light chain of antibody L3-11Y <400> 108 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Leu Ala Trp              20 25 30 Gly Asn Gln Asn Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys          35 40 45 Ala Pro Lys Met Leu Ile Ile Trp Ala Ser Thr Arg Val Ser Gly Val      50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln                  85 90 95 Ser Tyr Ser Arg Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile             100 105 110 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp         115 120 125 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn     130 135 140 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 145 150 155 160 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp                 165 170 175 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr             180 185 190 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser         195 200 205 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys     210 215 220

Claims (17)

VEGF inhibitor and anti-c-Met antibody or antigen-binding fragment thereof, as an active ingredient,
The anti-c-Met antibody,
CDR-H1 consisting of the amino acid sequence of SEQ ID NO: 1, CDR-H2 consisting of the amino acid sequence of SEQ ID NO: 2, CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 3, CDR-L1 consisting of the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: CDR-L2 consisting of the amino acid sequence of 11, and CDR-L3 consisting of the amino acid sequence of SEQ ID NO: 13, 14, 15, or 16, or
CDR-H1 consisting of the amino acid sequence of SEQ ID NO: 1, CDR-H2 consisting of the amino acid sequence of SEQ ID NO: 2, CDR-H3 consisting of the amino acid sequence of SEQ ID NO: 3, CDR-L1 consisting of the amino acid sequence of SEQ ID NO: 106, SEQ ID NO: An antibody or antigen-binding fragment comprising CDR-L2 consisting of the amino acid sequence of 11 and CDR-L3 consisting of the amino acid sequence of SEQ ID NO: 13,
A pharmaceutical composition for combined administration for the prevention or treatment of c-Met and angiogenic factors induced diseases selected from cancer, gestational diabetes, diabetic retinopathy, and macular degeneration. According to claim 1,
The pharmaceutical composition for co-administration is in the form of a mixture of angiogenesis inhibitors and anti-c-Met antibodies, or angiogenesis inhibitors and anti-c-Met antibodies are formulated and administered simultaneously or sequentially.
A pharmaceutical composition for use in combination for the prevention or treatment of c-Met and angiogenesis-induced diseases. delete The method according to claim 1, wherein the anti-c-Met antibody or antigen-binding fragment thereof is specific to an epitope consisting of 5 to 19 consecutive amino acids comprising the amino acid sequence of SEQ ID NO: 73 (EEPSQ) in the amino acid sequence of SEQ ID NO: 71. To combine,
A pharmaceutical composition for use in combination for the prevention or treatment of c-Met and angiogenesis-induced diseases. delete delete According to claim 1, wherein the anti-c-Met antibody or antigen-binding fragment thereof,
A heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 17; And
Light chain variable region consisting of the amino acid sequence of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 107
That includes,
A pharmaceutical composition for use in combination for the prevention or treatment of c-Met and angiogenesis-induced diseases. According to claim 1, wherein the anti-c-Met antibody,
The heavy chain comprising the amino acid sequence of SEQ ID NO: 62 or the 18 to 462 amino acid sequence of SEQ ID NO: 62 and the light chain comprising the amino acid sequence of SEQ ID NO: 68 or the 21 to 240 amino acid sequence of SEQ ID NO: 68 Containing antibody;
The heavy chain comprising the amino acid sequence of SEQ ID NO: 64 or the 18th to 461th amino acid sequence of SEQ ID NO: 64 and the light chain comprising the amino acid sequence of SEQ ID NO: 68 or the 21st to 240th amino acid sequence of SEQ ID NO: 68 Containing antibody;
The heavy chain comprising the amino acid sequence of SEQ ID NO: 66 or the 18 to 460 amino acid sequence of SEQ ID NO: 66 and the light chain comprising the amino acid sequence of SEQ ID NO: 68 or the 21 to 240 amino acid sequence of SEQ ID NO: 68 Containing antibody;
The heavy chain comprising the amino acid sequence of SEQ ID NO: 62 or the 18th to 462th amino acid sequence of SEQ ID NO: 62 and the light chain comprising the amino acid sequence of SEQ ID NO: 70 or the 21st to 240th amino acid sequence of SEQ ID NO: 70. Containing antibody;
The heavy chain comprising the amino acid sequence of SEQ ID NO: 64 or the 18 to 461 amino acid sequence of SEQ ID NO: 64 and the light chain comprising the amino acid sequence of SEQ ID NO: 70 or the 21 to 240 amino acid sequence of SEQ ID NO: 70 Containing antibody; or
A heavy chain comprising the amino acid sequence of SEQ ID NO: 66 or the 18th to 460th amino acid sequences of SEQ ID NO: 66 and a light chain comprising the amino acid sequence of the amino acid sequences of SEQ ID NO: 70 or 21 to 240 of SEQ ID NO: 70. Containing antibody
An antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 62 or the amino acid sequence from 18 to 462 of SEQ ID NO: 62 and a light chain comprising the amino acid sequence of SEQ ID NO: 108;
An antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 64 or the amino acid sequence from 18 to 461 of SEQ ID NO: 64 and a light chain comprising the amino acid sequence of SEQ ID NO: 108; And
Antibodies comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 66 or the amino acid sequence of 18 to 460 of SEQ ID NO: 66 and a light chain comprising the amino acid sequence of SEQ ID NO: 108
Is selected from the group consisting of,
A pharmaceutical composition for use in combination for the prevention or treatment of c-Met and angiogenesis-induced diseases. The method of claim 8, wherein the anti-c-Met antibody is the amino acid sequence of SEQ ID NO: 66 or the heavy chain comprising the amino acid sequence of 18 to 460 of SEQ ID NO: 66 and the amino acid sequence of SEQ ID NO: 68 or 21 of SEQ ID NO: 68 An antibody comprising a light chain comprising an amino acid sequence from th to 240 th,
A pharmaceutical composition for use in combination for the prevention or treatment of c-Met and angiogenesis-induced diseases. delete According to claim 1,
The anti-c-Met antibody is a monoclonal antibody,
A pharmaceutical composition for use in combination for the prevention or treatment of c-Met and angiogenesis-induced diseases. According to claim 1,
The anti-c-Met antibody is a mouse-derived antibody, a mouse-human chimeric antibody or a humanized antibody,
A pharmaceutical composition for use in combination for the prevention or treatment of c-Met and angiogenesis-induced diseases. According to claim 1,
The antigen-binding fragment is selected from the group consisting of scFv, (scFv) 2, Fab, Fab 'and F (ab') 2,
A pharmaceutical composition for use in combination for the prevention or treatment of c-Met and angiogenesis-induced diseases. delete According to claim 1,
The VEGF inhibitor is avastin, VEGF-trap, sunitinib, sunitinib malate, AEE-788, axitinib, AG-028262, combretastatin A4 analog (combretastatin A4 analog), cediranib (cediranib), sorafenib (sorafenib), BMS-387032 (CAS Registry Number 345627-80-7), CEP-7055, CHIR-258 (CAS Registry Number 405169-16-6 ), CP-547632 (CAS Registry Number 252003-65-9), CP-564959, E-7080 (CAS Registry Number 417716-92-8), Pazopanib, GW-654652, Indazolylpyrimidine Kdr Inhibitors (indazolylpyrimidine Kdr inhibitors), KRN-951, quinoline-urea VEGF inhibitors, midostaurin, vatalanib, anilinophthalazine derivatives VEGF inhibitors (anilinophthalazine derivative VEGF inhibitors), semasanib (semaxanib) ), SU-6668 (CAS Registry Number 252916-29-3), thalidomide, XL-647, XL-999, vandetanib, anilinoquinazoline VEGF inhibitor (anilinoquinazoline VEGF) inhibitors), ZK-304709, indirubin derivative VEGF inhibitors, CDP791, Enzastaurin, BIBF 1120 (Boehringer Ingelheim), BAY 573952, BAY 734506, XL 184, IMC-1121B, CEP 701 , SU 014813, SU 10944, SU 12662, OSI-930, BMS 582664, N-acetylcolchinol phosphate, ANG-400 series drugs, Imatinib, everolimus, and all At least one selected from the group consisting of satinib,
A pharmaceutical composition for use in combination for the prevention or treatment of c-Met and angiogenesis-induced diseases. delete According to claim 1,
The cancer is squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, peritoneal cancer, skin cancer, melanoma of the skin or eye, rectal cancer, anal muscle cancer, esophageal cancer, small intestine cancer, endocrine gland cancer, Parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, chronic or acute leukemia, lymphocyte lymphoma, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver tumor, breast cancer, colon cancer, colon cancer, For the prevention or treatment of endometrial or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulva cancer, thyroid cancer, liver cancer, head and neck cancer, brain cancer, osteosarcoma, or soft sarcoma, c-Met and angiogenic factors Pharmaceutical composition for combined administration.

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