KR101985299B1 - Anti-c-Met/anti-Nrp1 bispecific antibody - Google Patents

Abstract

An anti-c-Met / anti-Nrp1 bispecific antibody comprising an anti-c-Met antibody or antigen binding fragment thereof and an anti-Nrp1 antibody or antigen binding fragment thereof, and a pharmaceutical composition comprising the same are provided.

Description

Anti-c-Met / anti-Nrp1 bispecific antibody

An anti-c-Met / anti-Nrp1 bispecific antibody comprising an anti-c-Met antibody or antigen binding fragment thereof and an anti-Nrp1 antibody or antigen binding fragment thereof, and a pharmaceutical composition comprising the same are provided.

Neuropilin (Nrp) is a protein receptor that is active in neurons, and there are two types of neurons, Nrp1 and Nrp2. Nrp1 consists of about 923 amino acids, Nrp2 consists of about 926 amino acids, has a common domain structure in common, and is known to have 44% amino acid homology overall.

Among these, Nrp1 is a receptor that binds to the semaphorin 3A ligand, which initially interacts with the flexin co-receptor to regulate axon induction. Nrp1 mediates vascular development by binding to vascular endothelial growth factor (VEGF). Nrp1 is expressed in various types of human tumor cell lines and human tumors (glioblastoma, astrocytoma, glioma, neuroblastoma, testicular cancer, colorectal cancer, melanoma, pancreatic cancer, lung cancer, breast cancer, esophageal cancer and prostate cancer, etc.), VEGF and Semaphorin is involved in the proliferation, survival and migration of cancer cells. It is also known that the progression of cancer increases or the prognosis of cancer patients is poor depending on the expression level of Nrp1.

On the other hand, c-Met is a representative RTK (Receptor Tyrosine Kinase) present on the cell surface and binds to its ligand, HGF / SF (Hepatocyte Growth Factor / Scattering Factor), which promotes intracellular signaling and promotes cell growth. In addition, it is overexpressed in many types of cancer cells and is widely involved in cancer development, cancer metastasis, cancer cell migration, cancer cell infiltration, and neovascularization. In addition, c-Met signaling through HGF / SF is a representative early cancer metastasis protein that causes scattering by weakening cell-cell contact of almost all kinds of epithelial tumors.

Therefore, if Nrp1 and c-Met are simultaneously targeted, the growth and metastasis of cancer cells will be more effectively suppressed. For this reason, development of a drug targeting both Nrp1 and c-Met is required.

Republic of Korea Patent Publication No. 10-2008-0068105

One example provides an anti-c-Met / anti-Nrp1 bispecific antibody comprising an anti-c-Met antibody or antigen binding fragment thereof and an anti-Nrp1 antibody or antigen binding fragment thereof.

The anti-c-Met antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof that specifically binds to an epitope consisting of five or more contiguous amino acids including “EEPSQ” in the SEMA domain of the c-Met protein.

Another example provides a pharmaceutical composition comprising the anti-c-Met / anti-Nrp1 bispecific antibody as an active ingredient.

Another example includes overproduction (eg, overexpression) and / or abnormal activation of c-Met and / or Nrp1, comprising administering to the patient a pharmaceutically effective amount of an anti-c-Met / anti-Nrp1 bispecific antibody. Methods of preventing and / or treating related diseases are provided.

The pharmaceutical compositions or methods may be used for the prevention and / or treatment of diseases associated with overproduction (eg overexpression) and / or abnormal activation of c-Met and / or Nrp1, such as cancer or cancer metastasis. The cancer may be cancer having resistance to anticancer agents.

Another example provides a pharmaceutical composition for overcoming anticancer drug resistance comprising the anti-c-Met / anti-Nrp1 bispecific antibody as an active ingredient.

Another example provides a method for overcoming anticancer drug resistance comprising administering to a patient a pharmaceutically effective amount of an anti-c-Met / anti-Nrp1 bispecific antibody.

c-Met is a representative RTK (Receptor Tyrosine Kinase) present on the cell surface and is known to be involved in cell growth, cancer development, cancer metastasis and neovascularization. Nrp1 is also known to play a role in cell growth, cancer metastasis, and neovascularization.

Therefore, the present specification proposes that by simultaneously targeting c-Met and Nrp1, anti-cancer and / or anti-tumor effect of cancer is enhanced.

In addition, by simultaneously targeting c-Met and Nrp1 involved in cancer cell growth, it is possible to overcome resistance in cancers that exhibit innate and / or acquired resistance to existing anticancer agents. Suggest.

First, one example provides an anti-c-Met / anti-Nrp1 bispecific antibody comprising an anti-c-Met antibody or antigen binding fragment thereof and an anti-Nrp1 antibody or antigen binding fragment thereof. The bispecific antibody may exert an anticancer effect and / or a cancer metastasis inhibiting effect by simultaneously recognizing and binding c-Met and Nrp1 to inhibit function.

In the present invention, "antibody" means a substance produced by stimulation of an antigen in the immune system, and may be produced in vivo, recombinantly generated, or artificially synthesized. The kind is not particularly limited. Antibodies in the present invention include both animal antibodies, chimeric antibodies, humanized antibodies, or human antibodies. In the present invention, the antibody also includes an antigen-binding fragment of an antibody having antigen-binding ability. On the other hand, the term "Complementarity-determining regions (CDRs)" means a site that provides binding specificity with the antigen in the variable region of the antibody. "Antigen binding fragment" may be one selected from the group consisting of an antibody fragment comprising at least one of said complementarity determining regions, such as scFv, (scFv) 2, scFv-Fc, Fab, Fab 'and F (ab') 2 have.

In the following description of the anti-c-Met antibody or antigen-binding fragment thereof and the anti-Nrp1 antibody or antigen-binding fragment thereof, all subtypes except for the heavy chain and light chain CDR regions, or the heavy chain variable region and the light chain variable region are all subtypes. Immunoglobulins (eg, IgA, IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), IgM, etc.), for example light chain constant regions and / or immunoglobulins of all these subtypes It may be derived from a heavy chain constant region.

One target of the bispecific antibodies provided herein "c-Met" refers to receptor tyrosine kinases that bind to hepatocyte growth factor. The c-Met protein may be from any species, eg, from a primate such as human c-Met (eg NP_000236.2), monkey c-Met (eg Macaca mulatta, NP_001162100), or mouse rodents such as 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 a nucleotide sequence provided in GenBank Aceession Number NM_000245.2, or a protein having an amino acid sequence provided in GenBank Aceession Number NM_000236.2, or an extracellular domain thereof. Receptor tyrosine kinase c-Met is involved in various mechanisms, for example, cancer development, cancer metastasis, cancer cell migration, cancer cell infiltration, neovascularization process.

Another target of the bispecific antibodies provided herein is "Nrp1", which binds to vascular endothelial growth factor (VEGF) and mediates angiogenesis, and is characterized by various types of human tumor cell lines and human tumors (glioblastoma, astrocytoma, neurons). Glioma, neuroblastoma, testicular cancer, colorectal cancer, melanoma, pancreatic cancer, lung cancer, breast cancer, esophageal cancer and prostate cancer, etc.) and is involved in the effects of VEGF and semaphorin on the proliferation, survival and migration of cancer cells. Nrp1 may be derived from mammals including humans, primates such as monkeys, rodents such as rats, mice, and the like, for example, Human Nrp1 derived from humans (amino acid sequence: NCBI Accession # NP_001019799.1; coding polynucleotide sequence: NCBI Accession) # NM_001024628.2; etc.), Mouse Nrp1 from the mouse (amino acid sequence: Accession # NP_032763.2; coding polynucleotide sequence: NCBI Accession # NM_008737.2; etc.), and the like.

Anti-Nrp1 antibodies or antigen binding fragments thereof included in the bispecific antibodies provided herein have the property of internalizing after binding to Nrp1 expressed on the surface of a cell (eg, cancer cell). In the following examples it was confirmed that the anti-Nrp1 antibody of the present invention is bound to Nrp1 expressed on the surface of cancer cells, and then internalized into cells. Unlike conventional antibodies that bind to Nrp1 on the cell surface and block downstream signaling at that site, the antibody provided herein binds to Nrp1 on the cell surface and enters the cell, thus providing an absolute amount of Nrp1 present on the cell surface. Can be reduced and block all signaling associated with Nrp1, it can expect a greater therapeutic effect than conventional antibodies.

The anti-Nrp1 antibody or antigen-binding fragment thereof,

A polypeptide comprising the amino acid sequence of SEQ ID NO: 139 (CDR-H1), a polypeptide comprising the amino acid sequence of SEQ ID NO: 140 (CDR-H2), and a polypeptide comprising the amino acid sequence of SEQ ID NO: 141 (CDR-H3) A heavy chain complementarity determining region (CDR) comprising one or more selected from the group consisting of: or a heavy chain variable region comprising the heavy chain complementarity determining region:

A polypeptide comprising the amino acid sequence of SEQ ID NO: 142 (CDR-L1), a polypeptide comprising the amino acid sequence of SEQ ID NO: 143 (CDR-L2), and a polypeptide comprising the amino acid sequence of SEQ ID NO: 144 (CDR-L3) A light chain complementarity determining region comprising at least one selected from the group consisting of or a light chain variable region comprising the light chain complementarity determining regions;

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

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

It may be to include:

[SEQ ID NO: 139]

Xaa1 Tyr Xaa2 Met Ser (Xaa1 is Ser or Gly, Xaa2 is Tyr or Ala)

[SEQ ID NO 140]

Xaa3 Ile Ser Pro Gly Ser Xaa4 Xaa5 Xaa6 Tyr Tyr Ala Asp Ser Val Xaa7 Gly (Xaa3 is Ala or Gly, Xaa4 is Gly or Ser, Xaa5 is Ser or Asn, Xaa6 is Thr or Lys, Xaa7 is Gln or Lys)

[SEQ ID NO: 141]

Arg Lys Xaa8 Xaa9 Phe Asp Tyr (Xaa8 is Thr, Lys, or Tyr, Xaa9 is Arg, Ser, or Met)

[SEQ ID NO: 142]

Xaa10 Gly Xaa11 Ser Ser Asn Ile Gly Asn Asn Xaa12 Val Xaa13 (Xaa10 is Ser or Thr, Xaa11 is Pro or Ser, Xaa12 is Ser or Asp, Xaa13 is Ser or Tyr)

[SEQ ID NO: 143]

Xaa14 Xaa15 Xaa16 Xaa17 Arg Pro Ser (Xaa14 is Ser or Ala, Xaa15 is Asp or Asn, Xaa16 is Asn or Ser, Xaa17 is Asn or Lys)

[SEQ ID NO: 144]

Xaa18 Xaa19 Trp Xaa20 Xaa21 Ser Leu Xaa22 Xaa23 Tyr Val (Xaa18 is Ala or Gly, Xaa19 is Ala or Ser, Xaa20 is Asp or Val, Xaa21 is Ser or Ala, Xaa22 is Asn or Ser, Xaa23 is Gly or Ala).

More specifically, the anti-Nrp1 antibody or antigen-binding fragment thereof

A polypeptide comprising the amino acid sequence of SEQ ID NO: 110 or 121 (CDR-H1), a polypeptide comprising the amino acid sequence of SEQ ID NO: 111, 116, or 122 (CDR-H2), and SEQ ID NO: 112, 117 or 123 A heavy chain complementarity determining region comprising one or more selected from the group consisting of a polypeptide comprising an amino acid sequence (CDR-H3), or a heavy chain variable region comprising the heavy chain complementarity determining region; And / or

A polypeptide comprising the amino acid sequence of SEQ ID NO: 113, 118, or 124 (CDR-L1), a polypeptide comprising the amino acid sequence of SEQ ID NO: 114, 119, or 125 (CDR-L2), and SEQ ID NOs: 115, 120 Or a light chain complementarity determining region comprising one or more selected from the group consisting of a polypeptide (CDR-L3) comprising an amino acid sequence of 126, or a light chain variable region comprising the light chain complementarity determining regions

It may be to include.

Specifically, the complementarity determining site of the anti-Nrp1 antibody or antigen-binding fragment thereof may be, for example, having the amino acid sequence of Table 1 below.

Heavy chain CDR amino acid sequence of anti-Nrp1 antibody CDRH1-KABAT CDRH2-KABAT CDRH3-KABAT SYYMS (SEQ ID NO: 110)
GYAMS (SEQ ID NO: 121) AISPGSSNKYYADSVQG (SEQ ID NO: 111)
AISPGSSNKYYADSVKG (SEQ ID NO: 116)
GISPGSGSTYYADSVKG (SEQ ID NO: 122) RKKSFDY (SEQ ID NO: 112)
RKYMFDY (SEQ ID NO: 117)
RKTRFDY (SEQ ID NO: 123) Light chain CDR amino acid sequence of anti-Nrp1 antibody CDRL1-KABAT CDRL2-KABAT CDRL3-KABAT SGPSSNIGNNDVS (SEQ ID NO: 113)
TGSSSNIGNNDVY (SEQ ID NO: 118)
SGSSSNIGNNSVY (SEQ ID NO: 124) SDNNRPS (SEQ ID NO: 114)
SDSNRPS (SEQ ID NO: 119)
ANNKRPS (SEQ ID NO: 125) GAWVASLSAYV (SEQ ID NO: 115)
ASWDSSLSGYV (SEQ ID NO: 120)
AAWDSSLNGYV (SEQ ID NO: 126

In one embodiment, the anti-Nrp1 antibody or antigen binding fragment thereof

A heavy chain variable region comprising the amino acid sequence of SEQ ID 127, 129, or 131;

A light chain variable region comprising the amino acid sequence of SEQ ID 128, 130, or 132; or

Combination of these

It may be to include.

In one embodiment, the anti-Nrp1 antibody or antigen binding fragment thereof,

Or comprise the amino acid sequences of SEQ ID NOs: 110 (CDR-H1), 111 (CDR-H2), 112 (CDR-H3), 113 (CDR-L1), 114 (CDR-L2), and 115 (CDR-L3) ,

Or comprise the amino acid sequences of SEQ ID NOs: 110 (CDR-H1), 116 (CDR-H2), 117 (CDR-H3), 118 (CDR-L1), 119 (CDR-L2), and 120 (CDR-L3) ,

Comprising amino acid sequences of SEQ ID NOs: 121 (CDR-H1), 122 (CDR-H2), 123 (CDR-H3), 124 (CDR-L1), 125 (CDR-L2), and 126 (CDR-L3) It may be.

In another embodiment, the anti-Nrp1 antibody or antigen binding fragment thereof,

A heavy chain variable region of SEQ ID NO: 127 and a light chain variable region of SEQ ID NO: 128, or

A heavy chain variable region of SEQ ID NO: 129 and a light chain variable region of SEQ ID NO: 130, or

The heavy chain variable region of SEQ ID NO: 131 and the light chain variable region of SEQ ID NO: 132 may be included.

The anti-c-Met antibody may be one that recognizes a specific site of c-Met, such as a specific site within the SEMA domain, as an epitope, and all acts on c-Met to induce intracellular migration and degradation. Antibody or antigen binding fragment thereof.

C-Met, a receptor for hepatocyte growth factor, is divided into three parts: extracellular site, transmembrane site, and intracellular site. In the extracellular site, the α- and β-subunits are separated by disulfide bonds. In linked form, it consists of the HGF binding domain, the SEMA domain, the PSI domain (plexin-semaphorins-integrin homology domain), and the 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 existing at the extracellular site of c-Met and corresponds to a site to which HGF binds. A region comprising the amino acid sequence of SEQ ID NO: 71 corresponding to a specific site of the SEMA domain, eg, 106 to 124, is a loop between propeller domains 2 and 3 of the epitopes in the SEMA domain of the c-Met protein. ), And can act as an epitope of the anti-c-Met antibody proposed in the present invention.

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

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

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

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

CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4, an amino acid sequence of SEQ ID NO: 5, an amino acid sequence of SEQ ID NO: 2, or a contiguous 8 to 3rd amino acid including the amino acids 3 to 10 in the amino acid sequence of SEQ ID NO: 2; CDR-H2 comprising an 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 first to sixth amino acids within 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, amino acid sequence of SEQ ID NO: 15, amino acid sequence of SEQ ID NO: 86, or sequence 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 amino acids 1-9 in the amino acid sequence of No. 89, or said one or more light chain complementarities A light chain variable region comprising a crystal 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,

SEQ ID NO: 4 to SEQ ID NO: 9 may be an antibody or antigen-binding fragment which is an amino acid sequence represented by the general formula I to formula VI, respectively:

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 Formula I, Xaa ₁ is absent or Pro or Ser, Xaa ₂ is Glu or Asp,

In Formula II, Xaa ₃ is Asn or Lys, Xaa ₄ is Ala or Val, Xaa ₅ is Asn or Thr,

In Formula III, Xaa ₆ is Ser or Thr,

In 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 Formula V, Xaa ₁₁ is Ala or Gly, Xaa ₁₂ is Thr or Lys, Xaa ₁₃ is Ser or Pro,

In 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 comprise 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 a polypeptide 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 (CDR-H1), SEQ ID NO: 2, sequence A polypeptide (CDR-H2) comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 25, and SEQ ID NO: 26, and 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 A heavy chain variable region comprising a polypeptide comprising (CDR-H3); And a polypeptide (CDR-L1), SEQ ID NO: 11, 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; A 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 , SEQ ID NO: 37, SEQ ID NO: 86, and SEQ ID NO: 89 may include a light chain variable region comprising a polypeptide (CDR-L3) comprising an amino acid sequence selected from the group consisting of.

Thus, in one embodiment of the present invention, to enhance antigen binding efficiency, the anti-c-Met antibody or antigen binding fragment comprises a hinge region in which the amino acid sequence is modified by deletion, addition or substitution of one or more amino acids. It may be. For example, the antibody may include a hinge region including 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.

According to one embodiment, the anti-c-Met antibody or antigen-binding fragment is an amino acid 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 Said heavy chain variable region comprising a sequence; SEQ ID NO: 109, 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: 99, or SEQ ID NO: It may include the light chain variable region comprising the amino acid sequence of 107, or a combination of the heavy chain variable region and the light chain variable region.

In one embodiment, the anti-c-Met antibody is a monoclonal antibody that specifically binds to an extracellular region of the c-Met protein produced in hybridoma cells with accession number KCLRF-BP-00220. (See Korean Patent Publication No. 2011-0047698; which document is incorporated herein by reference).

The anti-c-Met antibody may include all of the antibodies defined in Korean Patent Publication Nos. 2011-0047698 and 2013-0037189.

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

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

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

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

It may be to include.

For example, the anti-c-Met antibody,

A heavy chain comprising the amino acid sequence of SEQ ID NO: 62 or the amino acid sequence of the 18th to 462th sequence of SEQ ID NO: 62 and a light chain comprising the amino acid sequence of SEQ ID NO: 68 or the amino acid sequence of the 21st to 240th amino acids of SEQ ID NO: 68; Antibody comprising;

A heavy chain comprising the amino acid sequence of SEQ ID NO: 64 or the amino acid sequence of the 18th to 461th sequences of SEQ ID NO: 64 and a light chain comprising the amino acid sequence of SEQ ID NO: 68 or the amino acid sequence of the 21st to 240th amino acids of SEQ ID NO: 68; Antibody comprising;

A heavy chain comprising the amino acid sequence of SEQ ID NO: 66 or the amino acid sequence of the 18th to 460th sequence of SEQ ID NO: 66 and a light chain comprising the amino acid sequence of SEQ ID NO: 68 or the amino acid sequence of the 21st to 240th amino acids of SEQ ID NO: 68; Antibody comprising;

A heavy chain comprising the amino acid sequence of SEQ ID NO: 62 or the amino acid sequence of the 18th to 462th sequence of SEQ ID NO: 62 and a light chain comprising the amino acid sequence of SEQ ID NO: 70 or the amino acid sequence of the 21st to 240th amino acids of SEQ ID NO: 70; Antibody comprising;

A heavy chain comprising the amino acid sequence of SEQ ID NO: 64 or the amino acid sequence of the 18th to 461th sequence of SEQ ID NO: 64 and a light chain comprising the amino acid sequence of SEQ ID NO: 70 or the amino acid sequence of the 21st to 240th amino acids of SEQ ID NO: 70; Antibody comprising; or

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

An antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 62 or the amino acid sequence of the 18th to 462th sequence 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 of the 18th to 461th sequences of SEQ ID NO: 64 and a light chain comprising the amino acid sequence of SEQ ID NO: 108; And

An antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 66 or the amino acid sequence of the 18th to 460th sequence 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 comprising the amino acid sequence of SEQ ID NO: 70 is a light chain consisting of a human kappa constant region, the polypeptide comprising the amino acid sequence of SEQ ID NO: 68 in the polypeptide comprising the amino acid sequence of SEQ ID NO: 70 36 (according to kabat numbering, 62nd amino acid position in SEQ ID NO: 68) histidine is a polypeptide in the form of tyrosine substituted. Due to the substitution, the yield of the antibody according to one embodiment may be increased. In addition, the polypeptide comprising the amino acid sequence of SEQ ID NO: 108 is kabat numbering in the polypeptide comprising the amino acid sequence of the 21st to 240th except for the 1st to 20th signal peptide of the amino acid sequence of SEQ ID NO: 68 Serine of position 27e (depending on kabat numbering, position 32 in SEQ ID NO: 108; inside CDR-L1) was substituted with tryptophan (Trp), and due to the substitution, Activity (eg, binding affinity for c-Met, c-Met degrading activity and Akt phosphorylation inhibitory activity, etc.) can be further enhanced.

Hereinafter, description common to the anti-c-Met antibody and anti-Nrp1 antibody will be described.

Animal-derived antibodies that are produced by immunizing a desired antigen with an immunized animal are generally capable of immunorejection upon administration to humans for therapeutic purposes, and chimeric antibodies have been developed to suppress such rejection. Chimeric antibodies are obtained by replacing the constant region of an animal-derived antibody causing an anti-isotype reaction with the constant region of a human antibody using genetic engineering methods. Chimeric antibodies have been significantly improved in anti-isotype responses compared to animal derived antibodies, but still contain adverse effects on potential anti-idiotypic responses due to the presence of animal derived amino acids in the variable region. Doing. Humanized antibodies have been developed to ameliorate these side effects. It is produced by implanting a region of complementarity determining regions (CDRs) that play an important role in binding of antigens in the variable regions of chimeric antibodies to the human antibody framework.

The most important aspect of CDR grafting technology for producing humanized antibody is to select an optimized human antibody that can best accept the CDR region of an animal-derived antibody. structure analysis and molecular modeling techniques. However, even when the CDR region of an animal-derived antibody is implanted into an optimized human antibody skeleton, there are many amino acids that are located in the skeleton of an animal-derived antibody and affect antigen binding. As such, the application of additional antibody engineering techniques to restore antigen binding capacity is essential.

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

A complete antibody is a structure having two full length light chains and two full length heavy chains, each of which is linked by heavy and disulfide bonds. The constant region of the antibody is divided into a heavy chain constant region and a light chain constant region, and the heavy chain constant region has a gamma (γ), mu (μ), alpha (α), delta (δ) and epsilon (ε) type, subclass Gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1) and alpha 2 (α2). The constant regions of the light chains have kappa (κ) and lambda (λ) types.

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

The term “complementarity determining region” refers to the amino acid sequences of the hypervariable regions of the heavy and light chains of immunoglobulins. The heavy and light chains may each comprise three CDRs (CDRH1, CDRH2, CDRH3 and CDRL1, CDRL2, CDRL3). The CDRs can provide key contact residues for the antibody to bind antigen or epitope. Meanwhile, in the present specification, the term "specifically binds" or "specifically recognized" is the same as commonly known to those skilled in the art, and the antigen and the antibody specifically interact with each other to perform an immunological response. Means that.

The term “antigen binding fragment” refers to a portion of a polypeptide that includes a portion to which an antigen can bind, as a fragment thereof for the entire structure of an immunoglobulin. For example, it may be, but is not limited to, scFv, (scFv) ₂ , scFv-Fc, Fab, Fab ', or F (ab') ₂ .

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

Fab 'differs from Fab in that it has a hinge region comprising one or more cysteine residues at the C-terminus of the heavy chain C _H1 domain.

F (ab ') ₂ antibodies are produced when the cysteine residues of the hinge region of Fab' form disulfide bonds. Recombinant techniques for generating Fv fragments with minimal antibody fragments in which Fv has only heavy chain variable regions and light chain variable regions are well known in the art.

Double-chain Fv is a non-covalent bond in which the heavy chain variable region and the light chain variable region are linked, and the single-chain Fv is generally shared by the variable region of the heavy chain and the short chain variable region through a peptide linker. It may be linked by bond or directly at the C-terminus to form a dimer-like structure such as a double chain Fv. scFv-Fc is a structure in which a single chain Fv and a constant region are linked with or without a peptide linker. The peptide linker may be composed of 1 to 100 or 2 to 50 arbitrary amino acids, and those skilled in the art may appropriately select the amino acid length and type.

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

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

When an animal-derived antibody undergoes chimerization, the animal-derived IgG1 hinge is replaced with a human IgG1 hinge, but the animal-derived IgG1 hinge is shorter than the human IgG1 hinge and has a disulfide bond between two heavy chains. Disulfide bonds are reduced from three to two, resulting in different rigidity of the hinges. Thus, modification of the hinge region can increase the antigen binding efficiency of the humanized antibody. Deletion, addition or substitution of amino acids for modifying the amino acid sequence of the hinge region is well known to those skilled in the art.

The antibody may be a monoclonal antibody. Monoclonal antibodies can be prepared according to methods well known in the art. For example, it can be manufactured using a phage display technique. In addition, the antibody may be a disulfide-binding Fvs (sdFV) or anti-idiotype (anti-Id) antibody.

The antibodies or antibody fragments described herein can include not only the sequences of the antibodies described herein, but also their biological equivalents within the scope of specifically recognizing each antigen, ie, Nrp1 or c-Met. . For example, further changes can be made to the amino acid sequence of the antibody to further improve the binding affinity and / or other biological properties of the antibody. Such modifications include, for example, deletions, insertions and / or substitutions of amino acid sequence residues of the antibody. Such amino acid variations can be made based on the relative similarity of amino acid side chain substituents such as hydrophobicity, hydrophilicity, charge, size, and the like. By analysis of the size, shape and type of amino acid side chain substituents, arginine, lysine and histidine are all positively charged residues; Alanine, glycine and serine have similar sizes; It can be seen that phenylalanine, tryptophan and tyrosine have a similar shape. Thus, based on these considerations, arginine, lysine and histidine; Alanine, glycine and serine; Phenylalanine, tryptophan and tyrosine are biologically equivalent functions.

In introducing mutations, the hydropathic index of amino acids can be considered. Each amino acid is assigned a hydrophobicity index according to its hydrophobicity and charge: isoleucine (+4.5); Valine (+4.2); Leucine (+3.8); Phenylalanine (+2.8); Cysteine / cysteine (+2.5); Methionine (+1.9); Alanine (+1.8); Glycine (-0.4); Threonine (-0.7); Serine (-0.8); Tryptophan (-0.9); Tyrosine (-1.3); Proline (-1.6); Histidine (-3.2); Glutamate (-3.5); Glutamine (-3.5); Aspartate (-3.5); Asparagine (-3.5); Lysine (-3.9); And arginine (-4.5).

The hydrophobic amino acid index is very important in conferring the interactive biological function of proteins. It is known that substitution with amino acids having similar hydrophobicity indexes can retain similar biological activity. When introducing mutations with reference to the hydrophobic index, substitutions between amino acids which exhibit a hydrophobic index difference of preferably within ± 2, more preferably within ± 1 and even more preferably within ± 0.5 are performed.

On the other hand, it is also well known that substitutions between amino acids having similar hydrophilicity values result in proteins with equivalent biological activity. For example, the following hydrophilicity values are assigned to each amino acid residue: arginine (+3.0); Lysine (+3.0); Asphaltate (+ 3.0 ± 1); Glutamate (+ 3.0 ± 1); Serine (+0.3); Asparagine (+0.2); Glutamine (+0.2); Glycine (0); Threonine (-0.4); Proline (-0.5 ± 1); Alanine (-0.5); Histidine (-0.5); Cysteine (-1.0); Methionine (-1.3); Valine (-1.5); Leucine (-1.8); Isoleucine (-1.8); Tyrosine (-2.3); Phenylalanine (-2.5); Tryptophan (-3.4).

Amino acid substitutions in proteins that do not alter the activity of the molecule as a whole are well known in the art. For example, the most commonly occurring substitutions are amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thr / Phe, Substitutions between Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu and / or Asp / Gly.

Given the above-described variations with biologically equivalent activity, an antibody described herein or a nucleic acid molecule encoding the same is also interpreted to include sequences that exhibit substantial identity with the sequences listed in the Sequence Listing. This substantial identity is at least 61% homology when the sequences of the invention described above are aligned as closely as possible with any other sequence and the aligned sequences are analyzed using algorithms commonly used in the art. , At least 70% homology, at least 80% homology, at least 90%, at least 95%, or at least 99% homology. Alignment methods for sequence comparison are known in the art. For example, the NCBI Basic Local Alignment Search Tool (BLAST) is accessible from NBCI and the like, and can be used in conjunction with sequencing programs such as blastp, blasm, blastx, tblastn and tblastx on the Internet. BLSAT is accessible at www.ncbi.nlm.nih.gov/BLAST/. Sequence homology comparisons using this program can be found at www.ncbi.nlm.nih.gov/BLAST/blast_help.html.

In one embodiment, the anti-c-Met / anti-Nrp1 bispecific antibody is an anti-c-Met antibody or antigen binding fragment thereof, and the C terminus or N terminus of the anti-c-Met antibody or antigen binding fragment thereof, For example, an anti-Nrp1 antibody or antigen binding fragment thereof linked to the C terminus.

In this case, in the anti-c-Met / anti-Nrp1 bispecific antibody, the anti-c-Met antibody plays a role in mediating the movement and degradation of the c-Met protein into the cell, thereby fully performing this role. It may be advantageous to have a complete antibody structure (eg, IgG type antibody). Anti-Nrp1 antibodies are important for their specific recognition and binding to Nrp1, and do not have a complete antibody structure and are capable of internalization even in the form of antigen-binding fragments such as, for example, scFv, and thus included in the form of antigen-binding fragments that recognize Nrp1. It may be okay. Thus, in one embodiment, the anti-c-Met / anti-Nrp1 bispecific antibody is a complete form of an anti-c-Met antibody (eg IgG type antibody) against c-Met and an anti-c-Met antibody. It may include, but is not limited to, an antigen binding fragment (eg scFv or scFv-Fc) of an anti-Nrp1 antibody linked to the C terminus (eg, the C terminus of the heavy chain of an anti-c-Met antibody).

In the anti-c-Met / anti-Nrp1 bispecific antibody, the anti-c-Met antibody or antigen-binding fragment thereof and the anti-Nrp1 antibody or antigen-binding fragment thereof may be linked with or without a linker, such as a peptide linker. Can be. In addition, heavy and light chain portions in antigen binding fragments, such as heavy and light chain variable regions in scFv fragments, can also be linked with or without a peptide linker. The peptide linker linking the anti-c-Met antibody or antigen-binding fragment thereof and the anti-Nrp1 antibody or antigen-binding fragment thereof and the peptide linker linking the heavy and light chain portions in the antigen-binding fragment may be the same or different. The peptide linker may be a polypeptide consisting of 1 to 100, 2 to 50, 2 to 30, 2 to 20, or 2 to 15 arbitrary amino acids, and the amino acid type included therein is not limited. The peptide linker may comprise, for example, Gly, Asn and / or Ser residues, and may also include neutral amino acids such as Thr and / or Ala. Suitable amino acid sequences for peptide linkers are known in the art. On the other hand, the linker can be variously determined the length, as long as it does not affect the function of the bispecific antibody. For example, the peptide linker may include one to at least one selected from the group consisting of Gly, Asn, Ser, Thr, and Ala, including 1 to 100, 2 to 50, or 5 to 25. In one example, the peptide linker is (G4S) n (n is the number of repetitions of (G4S)), it may be represented by an integer of 1 to 10, for example, an integer of 2 to 5).

In an embodiment, the anti-c-Met / anti-Nrp1 bispecific antibody is an anti-c-Met antibody (eg, an antibody in IgG form), and an anti-Nrp1 antibody linked to the C terminus of the anti-c-Met antibody. ScFv, (scFv) ₂ , scFv-Fc, Fab, Fab 'or F (ab') ₂ , such as scFv.

In one embodiment, the heavy chain of the anti-c-Met / anti-Nrp1 bispecific antibody comprises the heavy chain of the anti-c-Met antibody described above on the N-terminal side, and the scFv, (scFv) ₂ of the anti-Nrp1 antibody on the C-terminal side. , scFv-Fc, Fab, Fab 'or F (ab') ₂ may be included, with or without a linker, for example, may include an amino acid sequence selected from SEQ ID NO: 145 to 147.

The light chain of the anti-c-Met / anti-Nrp1 bispecific antibody may be the same as the light chain of the anti-c-Met antibody described above, for example, the amino acid sequence of SEQ ID NO: 68 (the amino acids 1st to 20th of these) The sequence is a signal peptide), the 21st to 240th amino acid sequence of SEQ ID NO: 68, the amino acid sequence of SEQ ID NO: 70 (the 1st to 20th amino acid sequence is the signal peptide), 21 of SEQ ID NO: 70 It may include an amino acid sequence selected from the group consisting of the amino acid sequence of the second to 240, and the amino acid sequence of SEQ ID NO: 108.

The anti-c-Met / anti-Nrp1 bispecific antibodies may maintain binding affinity for c-Met or Nrp1 similar to that of monoclonal antibodies, respectively. For example, the affinity (Kd) of the anti-c-Met / anti-Nrp1 bispecific antibody for c-Met, as measured by surface plasmon resonance (SPR; e.g. Biacore), is about 0.001 to 50 nM, 0.001 to 30 nM , 0.001 to 10 nM, 0.001 to 1 nM, or 0.001 to 0.5 nM, and the affinity (Kd) for Nrp1 may be about 0.001 to 100 nM, 0.001 to 70 nM, 0.001 to 50 nM, 0.001 to 40 nM, 0.001 to 30 nM, 0.001 to 10 nM, or 0.001 to 1 nM.

The anti-c-Met / anti-Nrp1 bispecific antibody is not only inhibited c-Met and Nrp1 activity but also c-Met and Nrp1 by cell internalization and degradation activity of the anti-c-Met antibody. Decomposition can reduce the total level to allow more fundamental blocking. Thus, the anti-c-Met / anti-Nrp1 bispecific antibody may be an anticancer agent (eg gemcitabine or the like) and / or a c-Met target therapeutic (eg anti-c-Met antibody) and / or Nrp1 target therapeutic (eg , Anti-Nrp1 antibody) can also be effective when applied to patients with resistance.

In addition, the anti-c-Met / anti-Nrp1 bispecific antibody, therefore, the anti-c-Met / anti-Nrp1 bispecific antibody, as well as when administered in combination with the c-Met inhibitor and Nrp1 inhibitor alone Compared with, it has a significantly elevated anti-cancer and / or cancer metastasis inhibiting effect. Due to such an elevated effect, the anti-c-Met / anti-Nrp1 bispecific antibody can exert an excellent effect even at a low dose as compared to the single or combined administration of c-Met inhibitor and Nrp1 inhibitor. In addition, the side effects of high doses can be reduced.

Thus, another example of the present invention is a disease associated with overproduction (eg, overexpression) and / or abnormal activation of c-Met and / or Nrp1 comprising the anti-c-Met / anti-Nrp1 bispecific antibody as an active ingredient. It provides a pharmaceutical composition for the prophylaxis and / or treatment of. The pharmaceutical composition may further include a pharmaceutically acceptable carrier in addition to the bispecific antibody.

Another example is the prevention and / or treatment of diseases associated with overproduction (eg, overexpression) and / or abnormal activation of c-Met and / or Nrp1 with a pharmaceutically effective amount of the anti-c-Met / anti-Nrp1 bispecific antibody. A method of preventing and / or treating a disease associated with overproduction (eg, overexpression) and / or abnormal activation of c-Met and / or Nrp1 comprising administering to a patient in need thereof. The method of preventing and / or treating a disease associated with overproduction (eg, overexpression) and / or abnormal activation of c-Met and / or Nrp1 may include overproduction of c-Met and / or Nrp1 prior to the administering step. , Overexpression) and / or abnormal activation may further comprise identifying a patient in need of prevention and / or treatment of a disease associated with abnormal activation.

As used herein, the term "prevention" means any action that inhibits or delays the occurrence of a disease by administration of a composition of the invention, and "treatment" refers to the inhibition of the development of the disease, alleviation of symptoms, amelioration, And / or removal of the etiology (eg, removal of tumor cells or tumor tissue in the case of tumors).

Diseases associated with overproduction (eg overexpression) and / or abnormal activation of c-Met and / or Nrp1 may be, for example, cancer or cancer metastasis.

Accordingly, there is provided a pharmaceutical composition for preventing and / or treating cancer or cancer metastasis comprising the anti-c-Met / anti-Nrp1 bispecific antibody as an active ingredient.

In addition, a method of preventing and / or treating cancer, comprising administering a pharmaceutically effective amount of the anti-c-Met / anti-Nrp1 bispecific antibody to a patient in need of the prevention and / or treatment of cancer or cancer metastasis. To provide. The method of preventing and / or treating cancer or cancer metastasis may further comprise identifying a patient in need of prevention and / or treatment of cancer or cancer metastasis prior to said administering.

The cancer may be associated with overexpression and / or abnormal activation of Nrp1 and / or c-Met. The cancer may be solid or hematological cancer, for example, but not limited to, lung cancer (eg, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, etc.), peritoneal cancer, skin cancer, black Species (eg, skin or intraocular melanoma, etc.), rectal cancer, anal muscle cancer, esophageal cancer, gastric cancer, small intestine cancer, endocrine cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, chronic or acute leukemia, lymphocyte lymphoma, hepatocytes Cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma (or glioblastoma), astrocytoma, glioma, neuroblastoma, testicular cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colon cancer, endometrial or uterine cancer, salivary gland cancer, It may be one or more selected from the group consisting of kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, head and neck cancer, brain cancer, osteosarcoma and the like. The cancer may be primary or metastatic cancer.

The anti-c-Met / anti-Nrp1 bispecific antibody provided herein simultaneously inhibits c-Met and Nrp1, thereby at least one anticancer agent selected from the group consisting of anticancer agents such as gemcitabine and / or the like. Anticancer effects may also be exerted against cancers that are resistant or resistant to c-Met targeted therapeutics (eg anti-c-Met antibodies) and / or Nrp1 targeted therapeutics (eg anti-Nrp1 antibodies).

Thus, the cancer may be one or more anticancer agents selected from the group consisting of existing anticancer agents such as gemcitabine and / or c-Met targeted therapeutics (eg anti-c-Met antibodies) and / or Nrp1 targeted therapeutics. Cancers that have innate or acquired resistance to (eg, anti-Nrp1 antibodies).

In addition, another example provides a pharmaceutical composition for overcoming anticancer drug resistance comprising the anti-c-Met / anti-Nrp1 bispecific antibody. Another example provides a method of overcoming anticancer resistance, comprising administering a pharmaceutically effective amount of the anti-c-Met / anti-Nrp1 bispecific antibody to a patient in need of overcoming anticancer resistance.

As described above, the anticancer agent resistance is at least one anticancer agent selected from the group consisting of gemcitabine and / or c-Met targeted therapeutics (e.g., anti-c-Met antibodies) and / or Nrp1 targeted therapeutics (e.g., , Anti-Nrp1 antibody) may be innate resistance or acquired resistance.

In the pharmaceutical composition or method, the anti-c-Met / anti-Nrp1 bispecific antibody may be provided with one or more additives selected from the group consisting of pharmaceutically acceptable carriers, diluents, excipients, and the like. .

The pharmaceutically acceptable carrier is conventionally used for the preparation of antibodies, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline Cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc. It is not limited. The pharmaceutical composition further includes at least one selected from the group consisting of diluents, excipients, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, and the like, which are conventionally used for preparing pharmaceutical compositions, in addition to the above components. can do.

The pharmaceutical composition, the anti-c-Met / anti-Nrp1 bispecific antibody may be administered orally or parenterally. In the case of parenteral administration, it can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, pulmonary administration and rectal administration. In oral administration, because proteins or peptides are digested, oral compositions should be formulated to coat the active agent or to protect it from degradation in the stomach. In addition, the composition may be administered by any device in which the active substance may migrate to the target cell.

Appropriate doses of the pharmaceutical composition, the anti-Nrp1 antibody or antigen-binding fragment thereof, or the anti-c-Met / anti-Nrp1 bispecific antibody may be formulated by the method of administration, mode of administration, age, weight, sex, pathology of the patient. Various prescriptions can be made by factors such as condition, food, time of administration, route of administration, rate of excretion, and response sensitivity. Preferred dosages of the compositions may range from 0.0001 to 100 mg / kg on an adult basis. The daily dosage may be formulated into one formulation in unit dosage form, may be formulated in appropriate quantities or may be prepared within a multi-dose container. The term "pharmaceutically effective amount" refers to an amount in which the active ingredient (ie, the anti-c-Met / anti-Nrp1 bispecific antibody) may exhibit a desired effect, i.e., prevent and / or treat cancer. It can be prescribed in various ways such as, formulating method, mode of administration, age, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion and response to the patient.

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

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

A patient to be administered the pharmaceutical composition or a subject to be administered the prophylactic and / or therapeutic method may be a mammal, including a primate or rat such as a human or a monkey, or a rodent such as a mouse, or a cell or tissue separated from the mammal, or a subject thereof. It may be a culture, but is not limited thereto, and may be a cancer patient having resistance to an existing anticancer agent.

The anti-c-Met / anti-Nrp1 antibodies provided herein have the following advantages:

1. Increased efficacy and decreased dose concentrations when combined with c-Met and Nrp1 inhibitors alone or in combination.

2. Efficacy in inhibiting cancer growth as well as cancer metastasis.

3. It also shows anticancer effect even against cancer that has resistance to existing anticancer drugs.

4. Application to other diseases involving overexpression or abnormal activation of Nrp1 and / or c-Met other than cancer.

1 is a schematic diagram showing the structure of an anti-c-Met / anti-Nrp1 bispecific antibody.
2 is a schematic diagram of phagemid vector for production of anti-Nrp1 scFv antibody fragment.
3 shows Coomassie staining results of each purified anti-Nrp1 scFv antibody fragment.
4 is a graph of the binding capacity of three anti-Nrp1 scFv antibody fragments to Nrp1.
FIG. 5 is a graph showing the binding force to Nrp1 according to the concentration of three anti-Nrp1 scFv antibody fragments (y-axis: OD value (46 nm); x-axis: scFv concentration (ng / ml)).
6 is a graph showing the effect of inhibiting cell proliferation against gastric cancer cell line MKN45 of anti-c-Met / anti-Nrp1 bispecific antibody.
FIG. 7 is a fluorescence image showing the effect of cell migration inhibition on kidney cancer cell line 786-O of anti-c-Met / anti-Nrp1 bispecific antibody.
FIG. 8 is a graph showing quantitative fluorescence intensity of FIG. 7.
9 is a fluorescence image showing the invasion inhibitory effect of anti-c-Met / anti-Nrp1 bispecific antibody on kidney cancer cell line 786-O.
FIG. 10 is a graph showing quantitative fluorescence intensity of FIG. 9.
FIG. 11 is a graph showing the effect of inhibiting cell proliferation on c-Met antibody resistant kidney cancer cell line 786-O of anti-c-Met / anti-Nrp1 bispecific antibody.
12 is an immunoblotting result showing Nrp1 expression level in gemcitabine resistant MKN45 cell line.
FIG. 13 is a graph showing the effect of inhibiting cell proliferation on MKN45 cell line (left) and gemcitabine resistant MKN45 cell line (right) of anti-c-Met / anti-Nrp1 bispecific antibody.
14 shows immunoblotting results showing changes in c-Met and Nrp1 levels in gemcitabine resistant MKN45 cell line following anti-c-Met / anti-Nrp1 bispecific antibody treatment.

Hereinafter, the following examples will be described in more detail with reference to the present invention. However, these examples are merely illustrative of one or more embodiments, and the scope of the invention is not limited to these examples.

Reference Example  1: Construction of Anti-c-Met Antibody

1.1. mouse antibody against c-Met '' AbF46 Production

1.1.1. Immunization of Mice

To obtain the immunized mice required for the development of hybridoma cell lines, 5 mice each had 100 μg of human c-Met / Fc fusion protein (R & D Systems) equivalent to a complete Freund's adjuvant. Were mixed and injected intraperitoneally of 4-6 week old BALB / c mice (Japan SLC, Inc.). Two weeks later, 50 μg of the previous injection of 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 above. Intraperitoneal injection. One week later, the last boosting (boosting) was performed 3 days after the blood was collected from the tail of the mouse to obtain a serum was diluted in PBS to 1/1000 to confirm that the titer of the antibody that recognizes c-Met by ELISA. As a result, mice obtained with sufficient amounts of antibodies were selected, and the following cell fusion process was performed.

1.1.2. Cell fusion and Hybridoma  Produce

Three days before the cell fusion experiment, 50 μg of PBS was injected with a mixture of human c-Met / Fc fusion protein intraperitoneally of BALB / c mice (Japan SLC, Inc.), and the anesthetized immunized mice were placed on the left side of the trunk. Located spleen was removed. Cells were isolated by grinding the spleens with a mesh and mixed with culture medium (DMEM, GIBCO, Invitrogen) to make a splenocyte 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) contained in the culture medium (DMEM), held at 37 ° C. for 1 minute, and then 1 mL of the culture medium (DMEM). Was added. Thereafter, 10 ml of culture medium (DMEM) was added for 1 minute, left for 5 minutes in water at 37 ° C., and centrifuged again at 50 ml. The cell precipitate was resuspended in a separation medium (HAT medium) at about 1 × 2 × 10 ⁵ / mL, and 0.1 mL was dispensed in a 96-well plate, followed by culturing in a 37 ° C. carbon dioxide incubator to prepare a hybridoma cell group. It was.

1.1.3. to produce monoclonal antibodies against c-Met protein Hybridoma  Selection of cells

Human c-Met / Fc fusion protein and human Fc protein were used as antigens to select hybridoma cells that specifically react with c-Met protein among the hybridoma cell groups prepared in Reference Example 1.1.2. Screened through ELISA assay method.

50 μl (2 ug / ml) of each human c-Met / Fc fusion protein was added to the microtiter plate and attached to the plate surface, and unreacted antigens were washed out. Human Fc protein was attached to the plate surface in the same manner as above to select and exclude antibodies that bind to Fc but not c-Met.

50 μl of each of the hybridoma cells obtained in Reference Example 1.1.2 was added to each of the prepared wells, followed by reaction for 1 hour, followed by sufficient washing with phosphate buffer-twin 20 (TBST) solution to remove unreacted culture. It was. To this was added goat anti-mouse IgG-horseradish peroxidase (goat anti-mouse IgG-HRP), reacted at room temperature for 1 hour, and then sufficiently washed with TBST solution. Subsequently, the substrate solution (OPD) of peroxidase was added to react, and the reaction degree 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 but secrete antibodies with high specificity only to human c-Met protein were repeatedly selected. Limiting dilution of the hybridoma cell line obtained through repeated screening yielded a final clone of one hybridoma cell line producing monoclonal antibody. The final screened monoclonal antibody-producing hybridomas were deposited on October 6, 2009 with the Korea Cell Line Research Foundation, located in Yeongun-dong, Jongno-gu, Seoul, under the Treaty of Budapest, and received accession number KCLRF-BP-00220 (Korea See Publication 2011-0047698).

1.1.4. Production and Purification of Monoclonal Antibodies

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

First, the hybridoma cells cultured in 50 ml of culture medium (DMEM) medium containing 10% (v / v) FBS were centrifuged, and the cell precipitates were washed two or more times 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, the cells producing the antibody were removed by centrifugation, and the culture medium in which the antibodies were secreted was separated and stored at 4 ° C. or collected immediately and used for separation and purification of the antibody. After purely purifying the antibody from 50 ml to 300 ml of the prepared culture medium using an AKTA purification apparatus (GE Healthcare) equipped with a Protein G agarose column (Pharmacia, USA), a protein aggregation filter (Amicon) was used. Purified antibody was stored by substituting the supernatant with PBS for use in subsequent examples.

1.2. for c-Met Chimeric  Antibodies of chAbF46  making

In general, mouse antibodies are highly likely to show immunogenicity when injected into humans for therapeutic purposes. To address this, mutations related to antigen binding from the mouse antibody AbF46 produced in Reference Example 1.1.4 above may be solved. The chimeric antibody chAbF46 was constructed in which the constant region except the variable region was replaced with the sequence of the 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' Each gene was designed to be composed of -XhoI '(SEQ ID NO: 39). Subsequently, the DNA fragment (SEQ ID NO: 38) containing the nucleotide sequence corresponding to the heavy chain was added to the pOptiVEC ^™ -TOPO TA Cloning Kit included in the OptiCHO ^™ Antibody Express Kit (Cat no. 12762-019) of Invitrogen, pcDNA ^™ 3.3-TOPO Cloning the DNA fragment (SEQ ID NO: 39) containing the nucleotide sequence corresponding to the light chain in TA Cloning Kit (Cat no. 8300-01) using EcoRI (NEB, R0101S) and XhoI (NEB, R0146S) restriction enzymes, respectively. As a result, a vector containing a heavy chain and a vector containing a light chain were constructed, respectively.

The constructed vectors were amplified using Qiagen Maxiprep kit (Cat no. 12662), respectively, and the temporary expression was Freestyle ^TM. This was done using the MAX 293 Expression System (invitrogen). The cell line used was 293 F cells, and cultured by suspension culture using FreeStyle ™ 293 Expression Medium as a medium. The cells were prepared at a concentration of 5x10 ⁵ cells / ml one day before the temporary expression, and then after 24 hours, the cells were temporarily expressed when the number of cells reached 1x10 ⁶ cells / ml. Transfection was performed by liposomal reagent method using Freestyle ^TM MAX reagent (invitrogen), and the DNA was prepared in a ratio of heavy chain DNA to light chain DNA = 1: 1 in a 15 ml tube and mixed with 2 ml of OptiPro ™ SFM (invtrogen). and (a), also after the other Freestyle ^TM MAX reagent 100㎕ with OptiPro ™ SFM 2ml mix (B) to the 15ml tube, then incubation 15 minutes to mix the (a) and (B), the cells prepared the day before The mixture was mixed slowly. After completion of the transduction, incubation for 5 days at 37 ℃, 80% humidity, 8% CO _2, 130 rpm incubator.

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

1.3. Chimeric  Antibodies from chAbF46  Humanized antibodies of huAbF46  making

1.3.1. Heavy chain  Heavy chain humanization

For the design of H1-heavy and H3-heavy, first, the VH gene of the mouse antibody AbF46 purified in Reference Example 1.2 above through Ig Blast (http://www.ncbi.nlm.nih.gov/igblast/) The germline genes of humans with the highest homology with were analyzed. As a result, it was confirmed that VH3-71 had 83% homology at the amino acid level, CDR-H1, CDR-H2, CDR-H3 of mouse antibody AbF46 were defined as Kabat numbering, and the CDR portion of mouse antibody AbF46 was defined as 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. Afterwards, H1 mutated the amino acids 83 (R → K) and 84 (A → T) to finally construct H1-heavy (SEQ ID NO: 40) and H3-heavy (SEQ ID NO: 41).

For the design of H4-heavy, the framework sequence of human antibody was found. As a result, Kabat numbering was defined using the most stable VH3 subtype known to be similar to that of AbF46 antibody. CDR-H1, CDR-H2, CDR-H3 of the mouse antibody AbF46 were introduced. This established H4-heavy (SEQ ID NO: 42).

1.3.2. Light chain  Light chain humanization

For the design of 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 having the highest homology with the VL gene were analyzed. As a result, it was confirmed that VK4-1 had 75% homology at the amino acid level, CDR-L1, CDR-L2, CDR-L3 of mouse antibody AbF46 were defined as Kabat numbering, and the CDR portion of mouse antibody AbF46 was defined as It was designed to be introduced into the backbone of VK4-1. At this time, H1-light back-mutated three amino acids 36 (Y → H), 46 (L → M), 49 (Y → I), and H2-light 49 amino acid (Y → I) Only one was constructed by back-mutation.

For the design of H3-light (SEQ ID NO: 45), Blast (http://www.ncbi.nlm.nih.gov/igblast/) was used to identify a human germline gene with the highest homology with the VL gene of the mouse antibody AbF46. 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, CDR-L1, CDR-L2, CDR-L3 of the mouse antibody AbF46 was defined as Kabat numbering, and the CDR of the mouse antibody AbF46 was determined. The part was designed to be introduced into the backbone of VK4-1. In this case, H3-light was constructed by back-mutating three amino acids 36 (Y → H), 46 (L → M), and 49 (Y → I).

For the design of H4-light (SEQ ID NO: 46), we found the framework sequences of human antibodies and found that CDR-L1 of mouse antibody AbF46, defined by Kabat numbering, using the most known Vk1 subtype, CDR-L2, CDR-L3 were introduced. At this time, H4-light was constructed by further back-mutation of three amino acids 36 (Y → H), 46 (L → M), 49 (Y → I).

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

The constructed vectors were amplified using Qiagen Maxiprep kit (Cat no. 12662), respectively, and the temporary expression was Freestyle ^TM. This was done using the MAX 293 Expression System (invitrogen). The cell line used was 293 F cells, and cultured by suspension culture using FreeStyle ™ 293 Expression Medium as a medium. The cells were prepared at a concentration of 5x10 ⁵ cells / ml one day before the temporary expression, and then after 24 hours, the cells were temporarily expressed when the number of cells reached 1x10 ⁶ cells / ml. Transfection was performed by liposomal reagent method using Freestyle ^TM MAX reagent (invitrogen), and the DNA was prepared in a ratio of heavy chain DNA to light chain DNA = 1: 1 in a 15 ml tube and mixed with 2 ml of OptiPro ™ SFM (invtrogen). and (a), also after the other Freestyle ^TM MAX reagent 100㎕ with OptiPro ™ SFM 2ml mix (B) to the 15ml tube, then incubation 15 minutes to mix the (a) and (B), the cells prepared the day before The mixture was mixed slowly. After completion of the transduction, incubation for 5 days at 37 ℃, 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). Protein A column (GE healthcare, 17-0405-03) was installed in AKTA Prime, and the culture solution was flowed at a flow rate of 5 ml / min, and then eluted with IgG elution buffer (Thermo Scientific, 21004). This was exchanged with PBS buffer to finally purify the humanized antibody AbF46 (hereinafter referred to as huAbF46). Meanwhile, the heavy and light chain combinations of the humanized antibody huAbF46 used in the following examples are H4-heavy (SEQ ID NO: 42) and H4-light (SEQ ID NO: 46).

1.4. huAbF46  Antibody scFv  Library Authoring

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

Then, expressed from the vector Analysis of the results, it was confirmed that showing a specific binding force to c-Met.

1.5. Construction of Library Genes for Affinity Maturation

1.5.1. Target CDR  Selection and primer  making

Six complementarity determining regions (CDRs) were defined by 'Kabat numbering' from the mouse antibody AbF46 prepared above for affinity maturation of the huAbF46 antibody, and each CDR is shown in Table 2 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 to introduce random sequences of antibody CDRs. Conventional random sequence introduction method using N codon 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 huAbF46 antibody To introduce a random base into the CDRs of the 85% of the first and second nucleotides of the wild-type nucleotides were preserved and 5% of the remaining three bases were introduced. In addition, primers were designed such that the third nucleotide was introduced equally (33% G, 33% C, 33% T).

1.5.2. huAbF46  Library Preparation of Antibodies and Confirmation of Avidity for c-Met

Construction of the antibody library gene by random sequence introduction of CDRs was performed using primers prepared in the same manner as in Reference Example 1.5.1. Using the polynucleotide containing the scFv of the huAbF46 antibody as a template, two PCR fragments were prepared as shown in FIG. 1, and each of the desired CDRs was subjected to overlap extension PCR. The scFv library genes of the mutated huAbF46 antibody were obtained to construct libraries targeting each of the six CDRs.

The prepared library was found to bind the wild type and c-Met of each library, each library showed a tendency to lower the binding capacity to c-Met than the wild type, but the binding capacity to some c-Met Remaining mutations were identified.

1.6. From the authored library Affinity  Improved Screening of Antibodies

After enhancing the binding capacity of the library to c-Met from the constructed library, the gene sequence of scFv was analyzed from each individual clone. The obtained gene sequences are shown in Table 7, respectively, and these were converted into IgG forms. Of the following clones, four antibodies produced from L3-1, L3-2, L3-3, L3-5 were selected for subsequent experiments .

Clone name Derived library CDR sequence 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 To IgG  conversion

The polynucleotides encoding the heavy chains of the four selected antibodies consist of 'EcoRI-signal sequence-VH-NheI-CH-XhoI' (SEQ ID NO: 38), and the heavy chains do not alter the amino acid of the antibody after affinity maturation. The heavy chain of huAbF46 antibody was used as it was. However, the hinge region was replaced with the U6-HC7 hinge (SEQ ID NO: 57), not the hinge of human IgG1. The light chains were designed to be composed of 'EcoRI-signal sequence-VL-BsiWI-CL-XhoI', and the genes were synthesized. No. 58 to SEQ ID NO: 61) were synthesized by applying to Bioneer. Subsequently, the DNA fragment (SEQ ID NO: 38) containing the nucleotide sequence corresponding to the heavy chain was added to the pOptiVEC ^™ -TOPO TA Cloning Kit included in the OptiCHO ^™ Antibody Express Kit (Cat no. 12762-019) of Invitrogen, pcDNA ^™ 3.3-TOPO DNA fragments comprising the nucleotide sequence corresponding to the light chain (TA fragment containing L3-1 derived CDR-L3: SEQ ID NO: 58, L3-2 derived CDR-L3) in TA Cloning Kit (Cat no. 8300-01) DNA fragment comprising: SEQ ID NO: 59, DNA fragment comprising L3-3-derived CDR-L3: SEQ ID NO: 60, DNA fragment comprising L3-5-derived CDR-L3: SEQ ID NO: 61) to EcoRI (NEB, R0101S) ) And XhoI (NEB, R0146S) restriction enzymes were used to construct vectors for expression of affinity matured antibodies.

The constructed vectors were amplified using Qiagen Maxiprep kit (Cat no. 12662), respectively, and the temporary expression was Freestyle ^TM. This was done using the MAX 293 Expression System (invitrogen). The cell line used was 293 F cells, and cultured by suspension culture using FreeStyle ™ 293 Expression Medium as a medium. The cells were prepared at a concentration of 5x10 ⁵ cells / ml one day before the temporary expression, and then after 24 hours, the cells were temporarily expressed when the number of cells reached 1x10 ⁶ cells / ml. Transfection was performed by liposomal reagent method using Freestyle ^TM MAX reagent (invitrogen), and the DNA was prepared in a ratio of heavy chain DNA to light chain DNA = 1: 1 in a 15 ml tube and mixed with 2 ml of OptiPro ™ SFM (invtrogen). and (a), also after the other Freestyle ^TM MAX reagent 100㎕ with OptiPro ™ SFM 2ml mix (B) to the 15ml tube, then incubation 15 minutes to mix the (a) and (B), the cells prepared the day before The mixture was mixed slowly. After completion of the transduction, incubation for 5 days at 37 ℃, 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). Protein A column (GE healthcare, 17-0405-03) was installed in AKTA Prime, and the culture solution was flowed at a flow rate of 5 ml / min, and then eluted with IgG elution buffer (Thermo Scientific, 21004). It was exchanged with PBS buffer to finally produce four types of antibodies (hereinafter, huAbF46-H4-A1 (from L3-1), huAbF46-H4-A2 (from L3-2), huAbF46-H4-A3 (L3-). 3), and huAbF46-H4-A5 (named from L3-5)).

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

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

An antibody consisting of a heavy chain consisting of a heavy chain variable region of huAbF46-H4-A1, a U6-HC7 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 was selected from huAbF46 -H4-A1 (U6-HC7); An antibody consisting of a heavy chain consisting of a heavy chain variable region of huAbF46-H4-A1, 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 was selected from huAbF46-H4- With Al (IgG2 hinge); An antibody consisting of a heavy chain consisting of a heavy chain variable region of huAbF46-H4-A1, 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 was selected from huAbF46-H4- Each named A1 (IgG2 Fc). On the other hand, the three antibodies were all substituted histidine No. 36 of the light chain consisting of the human kappa constant region in order to increase the production of tyrosine (tyrosine).

To prepare the three antibodies, a polynucleotide encoding the polypeptide consisting of the heavy chain variable region of huAbF46-H4-A1, the U6-HC7 hinge and the IgG1 constant region of human (SEQ ID NO: 62) (SEQ ID NO: 63), huAbF46 A polynucleotide encoding the polypeptide consisting of the heavy chain variable region of -H4-A1, the human IgG2 hinge and the human IgG1 constant region (SEQ ID NO: 64) (SEQ ID NO: 65), the heavy chain variable region of huAbF46-H4-A1, human Polynucleotide encoding the polypeptide consisting of the IgG2 hinge of human and IgG2 constant region of human (SEQ ID NO: 66) (SEQ ID NO: 67), light chain variable region of huAbF46-H4-A1 where histidine 36 is substituted with tyrosine A polynucleotide encoding the polypeptide consisting of the kappa constant region (SEQ ID NO: 68) (SEQ ID NO: 69) was synthesized by applying to Bioneer. Subsequently, the DNA fragment containing the nucleotide sequence corresponding to the heavy chain in the pOptiVEC ^™ -TOPO TA Cloning Kit included in the OptiCHO ^™ Antibody Express Kit (Cat no. 12762-019) manufactured by Invitrogen, pcDNA ^™ 3.3-TOPO A DNA fragment containing a nucleotide 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.

The constructed vectors were amplified using Qiagen Maxiprep kit (Cat no. 12662), respectively, and the temporary expression was Freestyle ^TM. This was done using the MAX 293 Expression System (invitrogen). The cell line used was 293 F cells, and cultured by suspension culture using FreeStyle ™ 293 Expression Medium as a medium. The cells were prepared at a concentration of 5x10 ⁵ cells / ml one day before the temporary expression, and then after 24 hours, the cells were temporarily expressed when the number of cells reached 1x10 ⁶ cells / ml. Transfection was performed by liposomal reagent method using Freestyle ^TM MAX reagent (invitrogen), and the DNA was prepared in a ratio of heavy chain DNA to light chain DNA = 1: 1 in a 15 ml tube and mixed with 2 ml of OptiPro ™ SFM (invtrogen). and (a), also after the other Freestyle ^TM MAX reagent 100㎕ with OptiPro ™ SFM 2ml mix (B) to the 15ml tube, then incubation 15 minutes to mix the (a) and (B), the cells prepared the day before The mixture was mixed slowly. After completion of the transduction, incubation for 5 days at 37 ℃, 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). Protein A column (GE healthcare, 17-0405-03) was installed in AKTA Prime, and the culture solution was flowed at a flow rate of 5 ml / min, and then eluted with IgG elution buffer (Thermo Scientific, 21004). Finally, three antibodies (huAbF46-H4-A1 (U6-HC7), huAbF46-H4-A1 (IgG2 hinge), huAbF46-H4-A1 (IgG2 Fc)) were purified. Among them, huAbF46-H4-A1 (IgG2 Fc) was selected for the anti-c-Met antibody according to the present invention and used in the following examples, and the antibody was named as anti-c-Met antibody SAIT301.

Reference Example  2. Anti- Nrp1  Antibody manufacturing

2.1. Identification of Internalizing Antibodies Using Patient-derived Cells

Among the patient-derived cells possessed by the project group (Samsung Hospital Intractor Cancer Research Group) to select the cells needed for cell panning to identify anti-Nrp1 antibody fragments, cells with high expression level of Nrp1 were selected from FACS ( Fluorescence Activated Cell Sorting) was selected. Of these, 131 cells expressing Nrp1 on the surface (patient-derived cells obtained from the National Cancer Institute (NCI) institution) were used for cell panning.

Using a synthetic scFv antibody fragment phage library (Yang et al., Mol. Cells, 27: 225-235, 2009) previously prepared, scFv antibody fragments binding to human Nrp1 were identified through phage display screening. Four sub-library samples were collected in 400 ml of each culture medium (SB / ampicillin / 2% glucose) to recover phagemid vectors introduced into E. coli host ER2537 (New England Biolab) in phage form. Incubated for 2 hours. When the absorbance at OD 600 was about 0.5-0.7, the supernatant was removed by centrifugation at 5000 g for 20 minutes and then suspended in 400 ml of secondary culture medium (SB / ampicillin), followed by 10 ¹² pfu (plaque forming unit). Helper phage (VCSM13; Stratagene) was added and incubated for 1 hour. Next, 70 μg / ml of kanamycin antibiotic (antibiotic gene introduced into helper phage) was added and cultured overnight at 30 ° C. to allow phage library to be secreted out of the host cell. Then, the culture obtained by centrifugation was precipitated only phage form using polyethylene glycol (PEG) solution to obtain a phage library.

The phage library thus obtained was mixed with 131 cells (4 X 10 ⁶ cells), which are patient-derived cells with high Nrp1 expression, and placed in a total of 5 ml NBA (neurobasal medium), fixed at 4 ° C, and then rotated 360 degrees for 1-2 hours. Rotated. Then, cells were separated by centrifugation at 300 g for 5 minutes to remove phage particles that did not bind to 131 cells, followed by washing with 5 ml NBA. This process was repeated four times, and in the final process, 131 cells and phages were placed in a T flask using 5 ml of NBA previously placed in an incubator at 37 ° C and incubated at 37 ° C for 30 minutes to internalize phage particles on the cell surface. ) Into the cells.

 Then, the cells were transferred to a 15 ml conical tube and centrifuged at 300 g for 5 minutes to separate the cells. This process was repeated 6 times with 5 ml of cold PBS (Phosphate buffered saline). Repeated cell panning cycles increased the frequency of this process. Subsequently, 5 ml of 0.1 M glycine (pH 2.2) was added, and allowed to stand at room temperature for 5 minutes to separate phage particles on the cell surface from the cell surface. Thereafter, centrifuged at 300 g for 5 minutes to separate the cells, 0.5 mL 100 mM TEA was added thereto, and the resulting mixture was transferred to an e-tube and placed at room temperature for 15 minutes. Next, centrifuge for 5 minutes at 12,000 rpm to separate the cell debris, and the supernatant containing the phage particles in the cells, neutralized by mixing with 1 ml 2M Tris (pH 8), 8.5 ml containing pre-grown ER2537 Phage particles were infected with E. coli host ER2537 by culturing at 120 rpm at 37 ° C. Thereafter, centrifuged at 3,000 rpm for 15 minutes, the ER2537, which was settled, was mixed with 500 µl of culture medium (SB), smeared on 15 cm culture medium, and cultured by adding 5 ml of SB culture medium (50% glycerol) to the colonies. Recovered and stored (-80 ° C). Phage particle amplification was then performed by taking 1 ml of the previous round of phage solution stored for the repeated cell panning round. Phage particles recovered by incorporating helper phage after incubation in host cell ER2537 were separated by PEG precipitation, and the same was used for the next round panning. Three rounds of panning were performed. As the repetition was repeated, it was confirmed that the ratio of phage particles before and after panning increased, which means that internalized phage particles were amplified through cell panning, and the results are shown in Table 4.

2.2. term- Nrp1 scFv  ELISA and Sequence Analysis for Candidate Selection

Phage particles recovered in the third round of cell panning were identified as colonies in culture medium via host cell (ER2537) infection. These colonies were taken and inoculated in 96-well plates containing 200 μl SB / ampicillin culture medium and then incubated at 37 ° C. for 2-3 hours.

 Then, to induce scFv-pIII protein expression, each well was treated with IPTG (Isopropyl β-D-1-thiogalactopyranoside) at a final concentration of 1 mM and incubated overnight at 30 ° C. The cultured plate was centrifuged at 3,000 rpm for 15 minutes to remove supernatant and 40 μl of TES solution (20% w / v sucrose, 50 mM) per well to recover phage particles in cultured periplasm. Tris, 1 mM EDTA, pH 8.0) was added and the cells were lysed by standing at 4 ° C for 30 minutes. Thereafter, 60 μl of 0.2X TES solution was treated and placed at 4 ° C. for 30 minutes to decompose the cells by osmotic pressure. Then, the plate was centrifuged at 3,000 rpm for 15 minutes to obtain supernatant scFv-pIII protein.

25 μl of the supernatant obtained in a 96-well plate coated with human Nrp1 protein prepared in advance was added to each corresponding well, and then bound at room temperature for 1 hour, and then washed six times using TBST and distilled water. Then, after binding for 1 hour at room temperature using an HRP-bound anti-HA antibody that can bind to the HA tag of scFv-pIII, and washed again 6 times with TBST (0.1% Tween20) and distilled water. After inducing color reaction using TMB solution, the color reaction was stopped with H ₂ SO ₄ solution, and the value was measured at OD 450 nm.

The total number of clones analyzed was 384, of which 41 clones (binding capacity> 2) showed high binding capacity to human Nrp1. As a control, a BSA solution was used, and among these 41 clones, 10 clones having high binding capacity were selected through reconfirmation ELISA. Thereafter, phagemids were recovered from 10 clones, followed by DNA sequencing, and clones having a total of six different sequences were selected. Except for 3H10, the same sequence as 1C08, clones with different sequences were selected and finally 3H10, 1A03 and 4F12 clones were selected as anti-Nrp1 scFv candidates. The CDR amino acid sequences, variable region amino acid sequences and DNA sequences encoding them of the selected 3H10, 1A03 and 4F12 clones are shown in Tables 5 and 6.

CDR amino acid sequence of anti-Nrp1 scFv Heavy chain CDR amino acid sequence of anti-Nrp1 antibody Anti-Nrp1 Clone CDRH1-KABAT CDRH2-KABAT CDRH3-KABAT 1A03 SYYMS (SEQ ID NO: 110) AISPGSSNKYYADSVQG (SEQ ID NO: 111) RKKSFDY (SEQ ID NO: 112) 3H10 SYYMS (SEQ ID NO: 110) AISPGSSNKYYADSVKG (SEQ ID NO: 116) RKYMFDY (SEQ ID NO: 117) 4F12 GYAMS (SEQ ID NO: 121) GISPGSGSTYYADSVKG (SEQ ID NO: 122) RKTRFDY (SEQ ID NO: 123) Light chain CDR amino acid sequence of anti-Nrp1 antibody Anti-Nrp1 Clone CDRL1-KABAT CDRL2-KABAT CDRL3-KABAT 1A03 SGPSSNIGNNDVS (SEQ ID NO: 113) SDNNRPS (SEQ ID NO: 114) GAWVASLSAYV (SEQ ID NO: 115) 3H10 TGSSSNIGNNDVY (SEQ ID NO: 118) SDSNRPS (SEQ ID NO: 119) ASWDSSLSGYV (SEQ ID NO: 120) 4F12 SGSSSNIGNNSVY (SEQ ID NO: 124) ANNKRPS (SEQ ID NO: 125) AAWDSSLNGYV (SEQ ID NO: 126)

Heavy and light chain variable regions of anti-Nrp1 scFv Heavy chain variable region of anti-Nrp1 antibody Anti-Nrp1 Clone Amino acid sequence Coding DNA Sequence 1A03 SEQ ID NO: 127 SEQ ID NO: 133 3H10 SEQ ID NO: 129 SEQ ID NO: 135 4F12 SEQ ID NO: 131 SEQ ID NO: 137 Light chain variable region of anti-Nrp1 antibody Anti-Nrp1 Clone Amino acid sequence Coding DNA Sequence 1A03 SEQ ID NO: 128 SEQ ID NO: 134 3H10 SEQ ID NO: 130 SEQ ID NO: 136 4F12 SEQ ID NO: 132 SEQ ID NO: 138

2.3. term- Nrp1 scFv  Production and Nrp1 Binding capacity  Confirm

The basic composition of phagemid can be confirmed in FIG. 2, and in the case of the host cell ER2537 used in the above example, scFv expression alone is not possible because it inhibits the transcriptional codon (amber codon (UAG)) located in front of phage pIII. Therefore, phagemid was transduced into the expression strain using an expression strain (TOP10F '; Thermo Fisher Scientific), which is a non-suppressor strain. Thereafter, each phagemid was identified through the DNA sequencing, and the expression strains introduced without mutation were taken as colonies, and then inoculated in 3 ml of LB / ampicillin culture medium and then cultured overnight at 37 ° C. Then, 3 ml of overnight culture was transferred to 400 ml culture medium (SB / Ampicillin) and cultured until 0.5-0.7 in O.D 600, and the final concentration of 1 mM IPTG was added and cultured overnight at 30 ° C. After centrifugation, the expression host was dissolved using 40 ml of TES solution, and 60 ml of O.2X TES was added to recover phage particles in the periplasm. The recovered supernatant was filtered through a 0.45 μm filter. The scFv protein present in the filtered solution was combined with 1 ml of Ni-NTA bead (Qiagen) for 1 hour at room temperature for His-tag purification, and then packed in a gravity column (Bio-rad) to 200 mM. Recovered through imidazole solution. After expression and purification of each clone, SDS-PAGE and coomassie blue staining (coomassie blue staining) confirmed the corresponding scFv size of about 28 kDa (FIG. 3).

ELISA was performed using the purified scFv to determine whether binding ability to the target Nrp1 exists. 96 wells coated with 200 ng Nrp1 protein and 96 wells coated with 200 ng BSA as a control were bound to 5 μg / ml level for each clone for 1 hour at room temperature by ELISA (3 repetitions). Thereafter, washing with 0.1% TBST three times, followed by treatment with HRP-bound HA antibody for 1 hour, washing again, and then standing for 5 minutes with TMB solution. The OD value was measured after stopping the color reaction with 2M sulfuric acid solution. The obtained result is shown in FIG. As shown in FIG. 4, 1A03, 3H10, and 4F12 scFv showed specific binding ability to Nrp1 compared to 12B scFv (comparative antibody: amino acid sequence: SEQ ID NO: 151; coding DNA sequence: SEQ ID NO: 152) that does not bind Nrp1. .

Next, in order to measure the binding ability according to the concentration of each antibody fragment to human Nrp1, each scFv in 200 ng Nrp1 or BSA-coated 96 well, 2,000 ng / ㎖, 1,000 ng / ㎖, 500 ng / ㎖ OD value changes were analyzed by treatment at concentrations of 250 ng / ml, 125 ng / ml, 62.5 ng / ml, 31.25 ng / ml, or 15.62 ng / ml.

The obtained results are shown in Fig. 5 (y-axis: OD value (46 nm); x-axis: scFv concentration (ng / ml). As shown in Fig. 5, the concentration in the case of 12B scFv in terms of binding capacity to Nrp1. While there was no change in OD value, the 3H10 and 4F12 scFv showed higher scFv bound to Nrp1 compared to BSA at higher concentrations.

To quantify the strength of the three scFv antibody fragments to Nrp1 protein by quantification, the final KD values through the ka and kd values were determined using biacore T100, a surface plasmon resonance (SPR) instrument. Got it. The KD value is a value obtained by dividing the kd value by the ka value. The lower the KD value, the higher the binding ability to the substance. As a result, 4F12 scFv had the lowest KD (M) value of 73.60 X 10 ^-9 , followed by 1A03 scFv with a KD (M) value of 89.40 X 10 ^-9 and 3H10 scFv of 295.4 X 10 ^-9 KD (M) values are shown.

Example  1: anti-c-Met / anti- Nrp1  Construction of Bispecific Antibodies

A bispecific antibody fused with the scFv (3H10, 1A03 or 4F12) of the three anti-Nrp1 antibodies prepared in Reference Example 2.2 to the c-terminus of the Fc of the anti-c-Met antibody SAIT301 prepared in Reference Example 1 Produced. The production process is as follows.

First, the heavy chain portion of the SAIT301 antibody used for the bispecific antibody cloning was prepared as follows. The heavy chain coding sequence of the anti-c-Met antibody was synthesized by deleting the C-terminal sequence from position 1396 of SEQ ID NO: 67 and inserting ggcggtggtggttccggaggcggcggatcc instead (Bionia).

Thereafter, the DNA fragment was ligated to the pOptiVEC ^™ -TOPO TA Cloning vector included in the OptiCHOTM Antibody Express Kit (Cat no. 12762-019) manufactured by Invitrogen.

On the other hand, anti-Nrp1 scFv of the heavy and light chain variable regions of the three anti-Nrp1 scFv was linked to the peptide linker (G ₄ S) ₂ was produced (synthesis by Bionic; N-terminal BamHI restriction enzyme recognition site And forms containing C-terminal XhoI restriction enzyme recognition sites).

Thereafter, the anti-Nrp1 scFv prepared above was cloned using a BamHI restriction enzyme and a XhoI restriction enzyme into a vector containing a previously prepared SAIT301 portion to construct an expression vector including the heavy chain of the bispecific antibody.

The amino acid sequence of the heavy chain of the constructed bispecific antibody (the C terminus of the heavy chain of the anti-c-Met antibody and the N terminus of the anti-Nrp1 scFv (3H10, 1A03 or 4F12) are connected by (G ₄ S) ₂ linker) SEQ ID NOs: 142 to 144, respectively, and the DNA sequences encoding them are shown in SEQ ID NOs: 145 to 147, respectively.

In addition, the light chain coding nucleotide sequence of the anti-c-Met antibody was synthesized to have the nucleotide sequence of SEQ ID NO: 69 (Bionia). PcDNA ^TM 3.3-TOPO included in Invitrogen's OptiCHO ^TM Antibody Express Kit (Cat no. 12762-019) A DNA fragment containing a nucleotide sequence corresponding to the light chain was inserted into a TA cloning vector (Cat no. 8300-01) to construct a vector expressing the light chain (SEQ ID NO: 68) of the bispecific antibody.

The constructed heavy chain expression vector and light chain expression vector were amplified using Qiagen Maxiprep kit (Cat no. 12662), respectively, and temporary expression was performed using FreestyleTM MAX 293 Expression System (invitrogen). The cell line used was 293F cells, and cultured by suspension culture using FreeStyle ™ 293 Expression Medium as a medium. The cells were prepared at a concentration of 5x10 ⁵ cells / ml one day before the temporary expression, and then after 24 hours, the cells were temporarily expressed when the number of cells reached 1x10 ⁶ cells / ml. Transfection was performed using a liposomal reagent method using FreestyleTM MAX reagent (invitrogen), and the 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), mix (B) 100µl of FreestyleTM MAX reagent and 2ml of OptiPro ™ SFM in another 15ml tube, mix (A) and (B), incubate for 15 minutes, and mix the cells into the cells prepared one day before. Mix slowly. After completion of the transduction, the cells were cultured in 37 ° C., 80% humidity, 8% CO ₂ , and 130 rpm incubator for 4 days.

The cultured cells were centrifuged to obtain 100 ml of supernatant, respectively, and purified using AKTA Prime (GE healthcare). A HiTrap MabSelect SuRe column (GE healthcare, 11-0034-95) was installed in AKTA Prime, the culture was allowed to flow at a flow rate of 5 ml / min, eluted with IgG elution buffer (Thermo Scientific, 21004), and then the eluate obtained. Was exchanged with PBS buffer.

The anti-c-Met / anti-Nrp1 bispecific antibodies in which three anti-Nrp1 scFvs were fused to the c-terminus of the anti-c-Met antibody SAIT301 prepared above are summarized in Table 7 below.

Double antibodies Anti-c-Met antibody Anti-Nrp1 scFv Heavy chain Light chain MN02 SAIT301 1A03 SEQ ID NO: 145
(DNA: SEQ ID NO: 148) SEQ ID NO: 68 MN03 3H10 SEQ ID NO: 146
(DNA: SEQ ID NO: 149) MN04 4F12 SEQ ID NO: 147
(DNA: SEQ ID NO: 150)

Example  2: anti-c-Met / anti- Nrp1  C-Met of bispecific antibodies and On Nrp1  For binding affinity

The affinity for c-Met of the three anti-c-Met / anti-Nrp1 bispecific antibodies prepared in Example 1 was confirmed using Biacore T100 (GE), respectively. Human Fab binders (# 28-9583-25, GE Healthcare) were immobilized on the surface of CM5 chips (# BR-1005-30, GE) according to the manufacturer's instructions. About 90-120 RU of the bispecific antibody MA01 was captured and various concentrations of c-Met-Fc (# 358-MT / CF, R & D Systems) were injected into the captured antibody. The surface was regenerated by injecting a 10 mM Glycine-HCl (pH 1.5) solution. In order to measure the affinity, the data obtained in the above experiments were fitted using BIAevaluation software (GE Healthcare, Biocore T100 evaluation software).

In addition, the affinity for Nrp1 of the three anti-c-Met / anti-Nrp1 bispecific antibodies prepared in Example 1 was confirmed using Biacore T100 (GE), respectively. Anti-histidine antibody (R & D Systems) was immobilized on the surface of the CM5 chip (# BR-1005-30, GE) according to the manufacturer's instructions. C-terminal histidine-tagged human Nrp1 (R & D Systems) was captured and various concentrations of bispecific antibody were injected into the captured antigen (Nrp1). The surface was regenerated by injecting a 10 mM Glycine-HCl (pH 1.5) solution. In order to measure the affinity, the data obtained in the above experiments were fitted using BIAevaluation software (GE Healthcare, Biocore T100 evaluation software).

The results obtained are shown in Table 8 below:

Double antibodies KD against c-Met (nM) KD against Nrp1 (nM) KD against Nrp1, scFv, (nM) MN02 0.08 22.31 138.5 MN03 0.09 11.43 67.21 MN04 0.05 0.38 0.62

In Table 8, "KD against c-Met" is the binding affinity for c-Met of the double antibody, "KD against Nrp1" is the binding affinity for Nrp1 of the double antibody, "KD against Nrp1, scFv," The binding affinity of -Nrp1 scFv to Nrp1 is shown, respectively.

As confirmed in Table 8 above, it can be seen that the anti-c-Met / anti-Nrp1 bispecific antibody maintains avidity for c-Met and Nrp1 at a similar level as in a single antibody.

Example  3: anti-c-Met / anti- Nrp1  Inhibitory Effects of Bispecific Antibodies on Cancer Cell Growth

The cancer cell killing effect of the anti-c-Met / anti-Nrp1 bispecific antibody prepared in Example 1 was tested in gastric cancer cell line MKN45.

Specifically, 5000 MKN45 cells (JCRB0254) were dispensed into each well containing a medium (GIBCO RPMI 1640, 100 ul / well), and the three bispecific antibodies prepared in Example 1 were each 60 nM (1). treatment for 5 days). Cell number change was measured by CellTiter Glo (CTG) assay. Specifically, after incubating for 5 days, 100 microliters of CTG solution (Promega) was added to each well, and then incubated at room temperature for 30 minutes. The luminescence signal obtained was recorded using an Envision 2104 Multi-label Reader (Perkin Elmer, Waltham, Massachusetts, USA).

For comparison, the antibody control group (control), the anti-c-Met antibody SAIT301 alone treatment group (60nM) prepared in Reference Example 1, the anti-Nrp1 antibody alone treatment group (1A: 1A03 clone of Reference Example 2.2 IgG antibody comprising 6 CDRs; 3H: IgG antibody comprising 6 CDRs of 3H10 clone of Reference Example 2.2; 4F: IgG antibody comprising 6 CDRs of 4F12 clone of Reference Example 2.2; 60 nM each)), And the combination of the anti-c-Met antibody SAIT301 and anti-Nrp1 antibody (1A, 3H, 4F) (60 nM, respectively) produced in Reference Example 1 was also performed.

The obtained result is shown in FIG. As shown in FIG. 6, anti-c-Met / anti-Nrp1 bispecific antibodies have a superior anti-cancer cell growth inhibitory effect than anti-c-Met antibody alone and anti-Nrp1 antibody alone as well as combination thereof. You can see that.

Example  4: anti-c-Met / anti- Nrp1  Inhibition of cancer cell metastasis by bispecific antibodies

The effect of the anti-c-Met / anti-Nrp1 bispecific antibody prepared in Example 1 on cancer cell motility was observed. The inhibitory effect on cancer cell migration by VEGF + HGF treatment of the bispecific antibody was tested. For the test, kidney cancer cell line 786-O (ATCC) resistant to the anti-c-Met antibody SAIT301 prepared in Reference Example 1 was used (no anticancer effect upon treatment with SAIT301 alone).

To determine whether anti-c-Met / anti-Nrp1 bispecific antibodies inhibit cell migration induced by VEGF and HGF, a cell migration assay using the Oris Migration System (Platypus Thechnologies) was performed. Was performed.

Specifically, kidney cancer cell line 786-O (ATCC) was inoculated in a 96-well plate equipped with a stopper in an amount of 50,000 per well. After incubation for 24 hours, the stopper was removed and each of the duplicates prepared in Example 1 above in RPMI medium (containing 10% FBS; GIBCO) with 0.1 ug (microgram) / ml HGF and 0.1 ug / ml VEGF added. Treatment with specific antibody (60 nM each) for 24 hours. The cells were then visualized by treatment with Hoechst 33342 (bisbenzimide; Life Technologies). Cell migration was determined by counting the number of cells with nuclei stained with Hoechst 33342. Cell number measurement was performed using InCell Analyzer 6000 (GE Healthcares) and the results obtained were converted to relative values for the group treated with VEGF + HGF only (100%).

For comparison, the antibody-free group (vehicle only, HGF only, VEGF only, and VEGF + HGF only), the anti-c-Met antibody SAIT301 alone treatment group (60nM) prepared in Reference Example 1, The same test was also performed on the single treatment group (60 nM) of each of the anti-Nrp1 antibodies comprising the six CDR3s of the clone prepared in Reference Example 2.2.

The obtained visualization result (fluorescence image) is shown in FIG. 7, and a graph quantifying the fluorescence intensity of FIG. 7 is shown in FIG. 8. As shown in FIG. 7 and FIG. 8, the anti-c-Met / anti-Nrp1 bispecific antibody had a cell migration by VEGF + HGF as compared to the case where SAIT301 and anti-Nrp1 antibodies were treated alone. It showed an excellent synergistic effect on inhibition.

In addition, in order to test the cancer metastasis inhibitory effect of the double antibody, the degree of Invasion inhibition of the antibody against the kidney cancer cell line 786-O was tested.

In order to confirm the invasion inhibition effect by the antibody treatment, BioCoat Growth Factor Reduced MATRIGEL Invasion Chamber (BD science, Cat #. 354483) was used. The chamber was divided into an upper chamber and a lower chamber, and the upper chamber was coated with collagen according to the manufacturer's manual.

Kidney cancer cell line 786-O (ATCC; 10 ⁵ cells / well) was inoculated into the upper chamber, and the lower chamber was treated with each bispecific antibody (60 nM each) prepared in Example 1 and then incubated at 37 ° C. . At this time, VEGF or HGF was added to the lower chamber in an amount of 0.1 ug / ml, respectively. After 24 hours, the cells that penetrated the collagen layer of the upper chamber and migrated to the lower chamber were stained with calcein AM (BD) and observed with a fluorescence microscope (ZEISS), and the fluorescence intensity was measured using an Envision 2104 Multi-label Reader (Perkin Elmer). Quantification.

For comparison, the antibody-free group (treated only with VEGF + HGF), the anti-c-Met antibody SAIT301 alone treated group (60 nM) prepared in Reference Example 1 above, and the six CDR3 clones of the clone prepared in Reference Example 2.2 above The same test was performed for the single treatment group (60 nM) of each of the anti-Nrp1 antibodies comprising.

The obtained fluorescence image is shown in FIG. 9, and a graph quantifying fluorescence intensity is shown in FIG. 10. 9 and 10, anti-c-Met / anti-Nrp1 bispecific antibodies have better cancer cell invasion than anti-c-Met antibody alone and anti-Nrp1 antibody alone as well as combinations thereof. It can be seen that the inhibitory effect.

Example  5: anti-c-Met / anti- Nrp1  Anti-c-Met Antibody Resistance Overcoming Test by Bispecific Antibody

In order to confirm the possibility of overcoming the resistance of the anti-c-Met / anti-Nrp1 bispecific antibody to the c-Met target therapeutic agent, kidney cancer cell line 786- having resistance to the anti-c-Met antibody SAIT301 prepared in Reference Example 1 O was used (no anticancer effect when treated with SAIT301 alone). Referring to Example 3, the bispecific antibody prepared in Example 1 to 5000 cells were each treated at a concentration of 60 nM, and after 3 days, cell number change was measured by CellTiter Glo assay.

For comparison, six of the 4F12 clones obtained from the antibody-free group (denoted as Cont), the anti-c-Met antibody SAIT301-treated group prepared in Reference Example 1 alone (60nM; denoted S301), and Reference Example 2.2 above The same test was also performed for the anti-Nrp1 antibody alone treatment group containing the CDRs (60 nM; denoted 4F) and the combination administration group of SAIT301 and anti-Nrp1 antibody (60 nM; denoted S + 4F, respectively).

The obtained result is shown in FIG. As shown in FIG. 11, the anti-c-Met / anti-Nrp1 bispecific antibody is a c-Met therapeutic agent in renal cancer cells compared to the case of treatment with SAIT301 and 4F12 alone and in combination. It can be seen that the effect of overcoming the resistance to.

Example  5: anti-c-Met / anti- Nrp1  Anticancer Drug Overcoming Test by Bispecific Antibody

To determine the possibility of overcoming the resistance of the anti-c-Met / anti-Nrp1 bispecific antibody to the anticancer drug (gemcitabine), gemcitabine was repeatedly administered to gastric cancer cell line MKN45 to gemcitabine resistant MKN45 ('MKN45-Gem Re'). Indication) was prepared.

Specifically, the MKN45-Gem Re cells were prepared by the following procedure. MKN45 cells (ATCC) were exposed to gemcitabine (Eli Lilly) for 3 months. At this time, gemcitabine was treated with increasing concentration in the concentration range of 10 to 100 uM ( in vitro ). In order to determine whether the treated cells have a continuous resistance to gemcitabine, by measuring the cell viability in the group cultured by treating the cells with gemcitabine for two weeks and the group cultured for two weeks without treatment, It was confirmed that the cells showed an equivalent degree of cell survival regardless of gemcitabine treatment.

MKN45 cells (ATCC; no resistance; indicated as 'parental'), and the gemcitabine resistant cell line MKN45-Gem Re prepared above were incubated in 60 mm plates at a concentration of 2x10 ⁵ cells / mL, respectively, and after 24 hours, lysis buffer (Roche Cell proteins were extracted. 20 ug of the extracted cell protein was loaded onto a western blot gel, followed by western blot, followed by anti-c-Met antibody (abcam), anti-Nrp1 antibody (Cell signaling), anti-p-Akt antibody (Cell signaling), and anti- Each protein was detected using a -GAPDH antibody (Cell signaling), respectively.

The obtained result is shown in FIG.

As shown in Figure 12, when gemcitabine resistance is induced, it can be seen that the Nrp1 expression level is increased.

Referring to Example 3, 5000 MKN45 and MKN45-Gem Re cells were prepared each, and the bispecific antibodies prepared in Example 1 were treated at concentrations of 0.2, 1, or 5 ug / mL, respectively, for 3 days. Thereafter, cell number change was measured by CellTiter Glo assay.

For comparison, the same test was performed on the anti-c-Met antibody SAIT301 alone treatment group prepared in Reference Example 1.

The obtained result is shown in FIG. As shown in FIG. 13, anti-c-Met / anti-Nrp1 bispecific antibody showed better cell proliferation inhibitory effect in both MKN45 and MKN45-Gem Re cells than SAIT301 alone, which is anti-c It can be said that the -Met / anti-Nrp1 bispecific antibody is excellent in overcoming the anticancer drug resistance.

Changes in total c-Met and Nrp1 amounts were tested upon anti-c-Met / anti-Nrp1 bispecific antibody treatment in gemcitabine resistant MKN45 cell line.

The prepared MKN45-Gem Re cells were incubated in 60 mm plates at a concentration of 2x10 ⁵ cells / mL, and after 24 hours, the bispecific antibodies prepared in Example 1 were treated at a concentration of 5 ug / mL for 24 hours, respectively. Cell cells were extracted by treating the cells with lysis buffer (Roche). 20 ug of the extracted protein was loaded onto a western blot gel, followed by western blot, followed by anti-c-Met antibody (abcam), anti-Nrp1 antibody (Cell signaling), and anti-GAPDH antibody (Cell signaling). Protein was detected.

For comparison, instead of the bispecific antibody, each anti-Nrp1 antibody comprising six CDRs of the PBS treated group, the SAIT301 antibody prepared in Reference Example 1 and the clone made in Reference Example 2.2 The same test was performed for the group treated with.

The obtained Western blot results are shown in FIG. 14. As shown in FIG. 14, c-Met and Npr1 were treated with anti-c-Met / anti-Nrp1 bispecific antibodies in MKN45-Gem Re cells compared with the anti-c-Met and anti-Nrp1 antibodies. It can be seen that the total amount of is significantly reduced, which shows that the double antibody has the internalization and degradation effects of the antigen.

Korea Cell Line Research Foundation KCLRFBP00220 20091006

<110> Samsung Electronics Co. Ltd          SAMSUNG LIFE PUBLIC WELFARE FOUNDATION <120> Anti-c-Met / anti-Nrp1 bispecific antibody <130> DPP20154809KR <160> 152 <170> KopatentIn 2.0 <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> MOD_RES <222> (1) <223> X is Pro or Ser or absent <220> <221> MOD_RES <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> MOD_RES <222> (3) <223> X is Asn or Lys <220> <221> MOD_RES <222> (4) <223> X is Ala or Val <220> <221> MOD_RES <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> MOD_RES <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> MOD_RES <222> (4) <223> X is His, Arg, Gln or Lys <220> <221> MOD_RES <222> (12) <223> X is His or Gln <220> <221> MOD_RES <222> (13) <223> X is Lys or Asn <220> <221> MOD_RES <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> MOD_RES <222> (2) <223> X is Ala or Gly <220> <221> MOD_RES <222> (4) <223> X is Thr or Lys <220> <221> MOD_RES <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> MOD_RES <222> (1) <223> X is Gly, Ala or Gln <220> <221> MOD_RES <222> (6) <223> X is Arg, His, Ser, Ala, Gly or Lys <220> <221> MOD_RES <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 (418) .. (1407) CH-heavy chain constant region <220> <221> misc_feature (1408) .. (1410) <223> TGA-stop codon <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) V223-light chain variable region <220> <221> misc_difference <222> (430) .. (435) <223> BsiWI restriction site <220> <221> misc_difference (222) (433) .. (750) CL-light chain constant region <220> <221> misc_difference (222) (751) .. (753) <223> stop codon <220> <221> misc_difference 754 (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 (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> 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 Ser 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 Ser 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 anti-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 (418) .. (1407) CH-heavy chain constant region <220> <221> misc_feature (1408) .. (1410) <223> TGA-stop codon <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) V223-light chain variable region <220> <221> misc_difference <222> (430) .. (435) <223> BsiWI restriction site <220> <221> misc_difference (222) (433) .. (750) CL-light chain constant region <220> <221> misc_difference (222) (751) .. (753) <223> stop codon <220> <221> misc_difference 754 (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 <210> 109 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> light chain variable region of anti c-Met antibody <400> 109 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 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> 110 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDRH1 of 1A03 or 3H10 <400> 110 Ser Tyr Tyr Met Ser   1 5 <210> 111 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDRH2 of 1A03 <400> 111 Ala Ile Ser Pro Gly Ser Ser Asn Lys Tyr Tyr Ala Asp Ser Val Gln   1 5 10 15 Gly <210> 112 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDRH3 of 1A03 <400> 112 Arg Lys Lys Ser Phe Asp Tyr   1 5 <210> 113 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> CDRL1 of 1A03 <400> 113 Ser Gly Pro Ser Ser Asn Ile Gly Asn Asn Asp Val Ser   1 5 10 <210> 114 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDRL2 of 1A03 <400> 114 Ser Asp Asn Asn Arg Pro Ser   1 5 <210> 115 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDRL3 of 1A03 <400> 115 Gly Ala Trp Val Ala Ser Leu Ser Ala Tyr Val   1 5 10 <210> 116 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDRH2 of 3H10 <400> 116 Ala Ile Ser Pro Gly Ser Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys   1 5 10 15 Gly <210> 117 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDRH3 of 3H10 <400> 117 Arg Lys Tyr Met Phe Asp Tyr   1 5 <210> 118 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> CDRL1 of 3H10 <400> 118 Thr Gly Ser Ser Ser Asn Ile Gly Asn Asn Asp Val Tyr   1 5 10          <210> 119 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDRL2 of 3H10 <400> 119 Ser Asp Ser Asn Arg Pro Ser   1 5 <210> 120 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDRL3 of 3H10 <400> 120 Ala Ser Trp Asp Ser Ser Leu Ser Gly Tyr Val   1 5 10 <210> 121 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDRH1 of 4F12 <400> 121 Gly Tyr Ala Met Ser   1 5 <210> 122 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDRH2 of 4F12 <400> 122 Gly Ile Ser Pro Gly Ser Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys   1 5 10 15 Gly <210> 123 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDRH3 of 4F12 <400> 123 Arg Lys Thr Arg Phe Asp Tyr   1 5 <210> 124 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> CDRL1 of 4F12 <400> 124 Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn Ser Val Tyr   1 5 10 <210> 125 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDRL2 of 4F12 <400> 125 Ala Asn Asn Lys Arg Pro Ser   1 5 <210> 126 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDRL3 of 4F12 <400> 126 Ala Ala Trp Asp Ser Ser Leu Asn Gly Tyr Val   1 5 10 <210> 127 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> VH of 1A03 <400> 127 Glu Val Gln Leu Leu 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 Ser Ser Tyr              20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Ala Ile Ser Pro Gly Ser Ser Asn Lys Tyr Tyr Ala Asp Ser Val      50 55 60 Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Arg Lys Lys Ser Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val             100 105 110 Thr Val Ser Ser         115 <210> 128 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> VL of 1A03 <400> 128 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln   1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Pro Ser Ser Asn Ile Gly Asn Asn              20 25 30 Asp Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu          35 40 45 Ile Tyr Ser Asp Asn Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser      50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg  65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Ala Trp Val Ala Ser Leu                  85 90 95 Ser Ala Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu             100 105 110 <210> 129 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> VH of 3H10 <400> 129 Glu Val Gln Leu Leu 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 Ser Ser Tyr              20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Ala Ile Ser Pro Gly Ser Ser Asn Lys Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Arg Lys Tyr Met Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val             100 105 110 Thr Val Ser Ser         115 <210> 130 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> VL of 3H10 <400> 130 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln   1 5 10 15 Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Asn Asn              20 25 30 Asp Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu          35 40 45 Ile Tyr Ser Asp Ser Asn Arg Pro Ser Gly Val Pro Asp Arg Phe Ser      50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg  65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ser Trp Asp Ser Ser Leu                  85 90 95 Ser Gly Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu             100 105 110 <210> 131 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> VH of 4F12 <400> 131 Glu Val Gln Leu Leu 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 Ser Gly Tyr              20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Gly Ile Ser Pro Gly Ser Gly Ser Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Lys Arg Lys Thr Arg Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val             100 105 110 Thr Val Ser Ser         115 <210> 132 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> VL of 4F12 <400> 132 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Arg   1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn              20 25 30 Ser Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu          35 40 45 Ile Tyr Ala Asn Asn Lys Arg Pro Ser Gly Val Pro Asp Arg Phe Ser      50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg  65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Ser Ser Leu                  85 90 95 Asn Gly Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu             100 105 110 <210> 133 <211> 348 <212> DNA <213> Artificial Sequence <220> <223> VH of 1A03 <133> 133 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc agttattata tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagcg atctctcctg gtagtagtaa taaatattac 180 gctgattctg tacaaggtcg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gagaaggaag 300 aagtcgttcg actactgggg ccagggtaca ctggtcaccg tgagctca 348 <210> 134 <211> 330 <212> DNA <213> Artificial Sequence <220> <223> VL of 1A03 <400> 134 cagtctgtgc tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc 60 tcttgtagtg gcccttcatc taatattggc aataatgatg tctcctggta ccagcagctc 120 ccaggaacgg ctcccaaact cctcatctat tctgataata atcggccaag cggggtccct 180 gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccgg 240 tccgaggatg aggctgatta ttactgtggt gcttgggttg ctagcctgag tgcttatgtc 300 ttcggcggag gcaccaagct gacggtccta 330 <210> 135 <211> 348 <212> DNA <213> Artificial Sequence <220> <223> VH of 3H10 <400> 135 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc agttattata tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcagcg atctctcctg gtagtagtaa taaatattac 180 gctgattctg taaaaggtcg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gagaaggaag 300 tatatgttcg actactgggg ccagggtaca ctggtcaccg tgagctca 348 <210> 136 <211> 330 <212> DNA <213> Artificial Sequence <220> <223> VL of 3H10 <400> 136 cagtctgtgc tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc 60 tcttgtactg gctcttcatc taatattggc aataatgatg tctactggta ccagcagctc 120 ccaggaacgg cacccaaact cctcatctat tctgatagta atcggccaag cggggtccct 180 gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccgg 240 tccgaggatg aggctgatta ttactgtgct tcttgggatt ctagcctgag tggttatgtc 300 ttcggcggag gcaccaagct gacggtccta 330 <210> 137 <211> 348 <212> DNA <213> Artificial Sequence <220> <223> VH of 4F12 <400> 137 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc ggttatgcta tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcaggg atctctcctg gtagtggtag tacatattac 180 gctgattctg taaaaggtcg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gaaacgtaag 300 actaggttcg actactgggg ccagggtaca ctggtcaccg tgagctca 348 <210> 138 <211> 330 <212> DNA <213> Artificial Sequence <220> <223> VL of 4F12 <400> 138 cagtctgtgc tgactcagcc accctcagcg tctgggaccc ccgggcggag ggtcaccatc 60 tcttgtagtg gctcttcatc taatattggc aataattctg tctactggta ccagcagctc 120 ccaggaacgg cccccaaact cctcatctat gctaataata agcggccaag cggggtccct 180 gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccgg 240 tccgaggatg aggctgatta ttactgtgct gcttgggatt ctagcctgaa tggttatgtc 300 ttcggcggag gcaccaagct gacggtccta 330 <139> <211> 5 <212> PRT <213> Artificial Sequence <220> <221> MOD_RES <222> (1) <223> Xaa is Gly or Ser <220> <221> MOD_RES <222> (3) <223> Xaa is Ala or Tyr <220> <223> CDRH1 of anti Nrp1 antibody <400> 139 Xaa Tyr Xaa Met Ser   1 5 <210> 140 <211> 17 <212> PRT <213> Artificial Sequence <220> <221> MOD_RES <222> (1) <223> Xaa is Ala or Gly <220> <221> MOD_RES <222> (7) <223> Xaa is Gly or Ser <220> <221> MOD_RES <222> (8) <223> Xaa is Ser or Asn <220> <221> MOD_RES <222> (9) <223> Xaa is Thr or Lys <220> <221> MOD_RES <222> (16) <223> Xaa is Gln or Lys <220> <223> CDRH2 of anti Nrp1 antibody <400> 140 Xaa Ile Ser Pro Gly Ser Xaa Xaa Xaa Tyr Tyr Ala Asp Ser Val   1 5 10 15 Xaa Gly <210> 141 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> MOD_RES <222> (3) <223> Xaa is Thr, Lys, or Tyr <220> <221> MOD_RES <222> (4) <223> Xaa is Arg, Ser, or Met <220> <223> CDRH3 of anti Nrp1 antibody <400> 141 Arg Lys Xaa Xaa Phe Asp Tyr   1 5 <210> 142 <211> 13 <212> PRT <213> Artificial Sequence <220> <221> MOD_RES <222> (1) <223> Xaa is Ser or Thr <220> <221> MOD_RES <222> (3) <223> Xaa is Pro or Ser <220> <221> MOD_RES <222> (11) <223> Xaa is Ser or Asp <220> <221> MOD_RES <222> (13) <223> Xaa is Ser or Tyr <220> <223> CDRL1 of anti Nrp1 antibody <400> 142 Xaa Gly Xaa Ser Ser Asn Ile Gly Asn Asn Xaa Val Xaa   1 5 10 <210> 143 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> MOD_RES <222> (1) <223> Xaa is Ser or Ala <220> <221> MOD_RES <222> (2) <223> Xaa is Asp or Asn <220> <221> MOD_RES <222> (3) <223> Xaa is Asn or Ser <220> <221> MOD_RES <222> (4) <223> Xaa is Asn or Lys <220> <223> CDRL2 of anti-Nrp1 antibody <400> 143 Xaa Xaa Xaa Xaa Arg Pro Ser   1 5 <210> 144 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> MOD_RES <222> (1) <223> Xaa is Ala or Gly <220> <221> MOD_RES <222> (2) <223> Xaa is Ala or Ser <220> <221> MOD_RES <222> (4) <223> Xaa is Asp or Val <220> <221> MOD_RES <222> (5) <223> Xaa is Ser or Ala <220> <221> MOD_RES <222> (8) <223> Xaa is Asn or Ser <220> <221> MOD_RES <222> (9) <223> Xaa is Gly or Ala <220> <223> CDRL3 of anti-Nrp1 antibody <400> 144 Xaa Xaa Trp Xaa Xaa Ser Leu Xaa Xaa Tyr Val   1 5 10 <210> 145 <211> 694 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain of bispecific antibody MN02 <400> 145 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 Cys Ser Arg Ser Thr Ser Glu Ser 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 Asn             180 185 190 Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn         195 200 205 Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro     210 215 220 Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro 225 230 235 240 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr                 245 250 255 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn             260 265 270 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg         275 280 285 Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val     290 295 300 Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 305 310 315 320 Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys                 325 330 335 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu             340 345 350 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe         355 360 365 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu     370 375 380 Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe 385 390 395 400 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly                 405 410 415 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr             420 425 430 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser         435 440 445 Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu     450 455 460 Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe 465 470 475 480 Thr Phe Ser Ser Tyr Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys                 485 490 495 Gly Leu Glu Trp Val Ser Ala Ile Ser Pro Gly Ser Ser Asn Lys Tyr             500 505 510 Tyr Ala Asp Ser Val Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser         515 520 525 Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr     530 535 540 Ala Val Tyr Tyr Cys Ala Arg Arg Lys Lys Ser Phe Asp Tyr Trp Gly 545 550 555 560 Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly                 565 570 575 Gly Gly Ser Gly Gly Gly Gly Ser Gln Ser Val Leu Thr Gln Pro Pro             580 585 590 Ser Ala Ser Gly Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Ser Gly         595 600 605 Pro Ser Ser Asn Ile Gly Asn Asn Asp Val Ser Trp Tyr Gln Gln Leu     610 615 620 Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr Ser Asp Asn Asn Arg Pro 625 630 635 640 Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala                 645 650 655 Ser Leu Ala Ile Ser Gly Leu Arg Ser Glu Asp Glu Ala Asp Tyr Tyr             660 665 670 Cys Gly Ala Trp Val Ala Ser Leu Ser Ala Tyr Val Phe Gly Gly Gly         675 680 685 Thr Lys Leu Thr Val Leu     690 <210> 146 <211> 694 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain of bispecific antibody MN03 <400> 146 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 Cys Ser Arg Ser Thr Ser Glu Ser 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 Asn             180 185 190 Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn         195 200 205 Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro     210 215 220 Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro 225 230 235 240 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr                 245 250 255 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn             260 265 270 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg         275 280 285 Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val     290 295 300 Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 305 310 315 320 Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys                 325 330 335 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu             340 345 350 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe         355 360 365 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu     370 375 380 Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe 385 390 395 400 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly                 405 410 415 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr             420 425 430 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser         435 440 445 Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu     450 455 460 Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe 465 470 475 480 Thr Phe Ser Ser Tyr Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys                 485 490 495 Gly Leu Glu Trp Val Ser Ala Ile Ser Pro Gly Ser Ser Asn Lys Tyr             500 505 510 Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser         515 520 525 Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr     530 535 540 Ala Val Tyr Tyr Cys Ala Arg Arg Lys Tyr Met Phe Asp Tyr Trp Gly 545 550 555 560 Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly                 565 570 575 Gly Gly Ser Gly Gly Gly Gly Ser Gln Ser Val Leu Thr Gln Pro Pro             580 585 590 Ser Ala Ser Gly Thr Pro Gly Gln Arg Val Thr Ile Ser Cys Thr Gly         595 600 605 Ser Ser Ser Asn Ile Gly Asn Asn Asp Val Tyr Trp Tyr Gln Gln Leu     610 615 620 Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr Ser Asp Ser Asn Arg Pro 625 630 635 640 Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala                 645 650 655 Ser Leu Ala Ile Ser Gly Leu Arg Ser Glu Asp Glu Ala Asp Tyr Tyr             660 665 670 Cys Ala Ser Trp Asp Ser Ser Leu Ser Gly Tyr Val Phe Gly Gly Gly         675 680 685 Thr Lys Leu Thr Val Leu     690 <210> 147 <211> 694 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain of bispecific antibody MN04 <400> 147 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 Cys Ser Arg Ser Thr Ser Glu Ser 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 Asn             180 185 190 Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn         195 200 205 Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro     210 215 220 Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro 225 230 235 240 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr                 245 250 255 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn             260 265 270 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg         275 280 285 Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val     290 295 300 Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 305 310 315 320 Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys                 325 330 335 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu             340 345 350 Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe         355 360 365 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu     370 375 380 Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe 385 390 395 400 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly                 405 410 415 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr             420 425 430 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser         435 440 445 Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu     450 455 460 Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe 465 470 475 480 Thr Phe Ser Gly Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys                 485 490 495 Gly Leu Glu Trp Val Ser Gly Ile Ser Pro Gly Ser Gly Ser Thr Tyr             500 505 510 Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser         515 520 525 Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr     530 535 540 Ala Val Tyr Tyr Cys Ala Lys Arg Lys Thr Arg Phe Asp Tyr Trp Gly 545 550 555 560 Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly                 565 570 575 Gly Gly Ser Gly Gly Gly Gly Ser Gln Ser Val Leu Thr Gln Pro Pro             580 585 590 Ser Ala Ser Gly Thr Pro Gly Arg Arg Val Thr Ile Ser Cys Ser Gly         595 600 605 Ser Ser Ser Asn Ile Gly Asn Asn Ser Val Tyr Trp Tyr Gln Gln Leu     610 615 620 Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr Ala Asn Asn Lys Arg Pro 625 630 635 640 Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala                 645 650 655 Ser Leu Ala Ile Ser Gly Leu Arg Ser Glu Asp Glu Ala Asp Tyr Tyr             660 665 670 Cys Ala Ala Trp Asp Ser Ser Leu Asn Gly Tyr Val Phe Gly Gly Gly         675 680 685 Thr Lys Leu Thr Val Leu     690 <210> 148 <211> 2082 <212> DNA <213> Artificial Sequence <220> <223> DNA encoding heavy chain of bispecific antibody MN02 <400> 148 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 cctggcgccc tgctccagga gcacctccga gagcacagcg 420 gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca 480 ggcgctctga ccagcggcgt gcacaccttc ccagctgtcc tacagtcctc aggactctac 540 tccctcagca gcgtggtgac cgtgccctcc agcaacttcg gcacccagac ctacacctgc 600 aacgtagatc acaagcccag caacaccaag gtggacaaga cagttgagcg caaatgttgt 660 gtcgagtgcc caccgtgccc agcaccacct gtggcaggac cgtcagtctt cctcttcccc 720 ccaaaaccca aggacaccct catgatctcc cggacccctg aggtcacgtg cgtggtggtg 780 gacgtgagcc acgaagaccc cgaggtccag ttcaactggt acgtggacgg cgtggaggtg 840 cataatgcca agacaaagcc acgggaggag cagttcaaca gcacgttccg tgtggtcagc 900 gtcctcaccg ttgtgcacca ggactggctg aacggcaagg agtacaagtg caaggtctcc 960 aacaaaggcc tcccagcccc catcgagaaa accatctcca aaaccaaagg gcagccccga 1020 gaaccacagg tgtacaccct gcccccatcc cgggaggaga tgaccaagaa ccaggtcagc 1080 ctgacctgcc tggtcaaagg cttctacccc agcgacatcg ccgtggagtg ggagagcaat 1140 gggcagccgg agaacaacta caagaccacg cctcccatgc tggactccga cggctccttc 1200 ttcctctaca gcaagctcac cgtggacaag agcaggtggc agcaggggaa cgtcttctca 1260 tgctccgtga tgcatgaggc tctgcacaac cactacacgc agaagagcct ctccctgtct 1320 ccgggtaaag gcggtggtgg ttccggaggc ggcggatccg aggtgcagct gttggagtct 1380 gggggaggct tggtacagcc tggggggtcc ctgagactct cctgtgcagc ctctggattc 1440 acctttagca gttattatat gagctgggtc cgccaggctc cagggaaggg gctggagtgg 1500 gtctcagcga tctctcctgg tagtagtaat aaatattacg ctgattctgt acaaggtcgg 1560 ttcaccatct ccagagacaa ttccaagaac acgctgtatc tgcaaatgaa cagcctgaga 1620 gccgaggaca cggccgtgta ttactgtgcg agaaggaaga agtcgttcga ctactggggc 1680 cagggtacac tggtcaccgt gagctcaggt ggaggcggtt caggcggagg tggatccggc 1740 ggtggcggat cgcagtctgt gctgactcag ccaccctcag cgtctgggac ccccgggcag 1800 agggtcacca tctcttgtag tggcccttca tctaatattg gcaataatga tgtctcctgg 1860 taccagcagc tcccaggaac ggctcccaaa ctcctcatct attctgataa taatcggcca 1920 agcggggtcc ctgaccgatt ctctggctcc aagtctggca cctcagcctc cctggccatc 1980 agtgggctcc ggtccgagga tgaggctgat tattactgtg gtgcttgggt tgctagcctg 2040 agtgcttatg tcttcggcgg aggcaccaag ctgacggtcc ta 2082 <210> 149 <211> 2082 <212> DNA <213> Artificial Sequence <220> <223> DNA encoding heavy chain of bispecific antibody MN03 <400> 149 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 cctggcgccc tgctccagga gcacctccga gagcacagcg 420 gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca 480 ggcgctctga ccagcggcgt gcacaccttc ccagctgtcc tacagtcctc aggactctac 540 tccctcagca gcgtggtgac cgtgccctcc agcaacttcg gcacccagac ctacacctgc 600 aacgtagatc acaagcccag caacaccaag gtggacaaga cagttgagcg caaatgttgt 660 gtcgagtgcc caccgtgccc agcaccacct gtggcaggac cgtcagtctt cctcttcccc 720 ccaaaaccca aggacaccct catgatctcc cggacccctg aggtcacgtg cgtggtggtg 780 gacgtgagcc acgaagaccc cgaggtccag ttcaactggt acgtggacgg cgtggaggtg 840 cataatgcca agacaaagcc acgggaggag cagttcaaca gcacgttccg tgtggtcagc 900 gtcctcaccg ttgtgcacca ggactggctg aacggcaagg agtacaagtg caaggtctcc 960 aacaaaggcc tcccagcccc catcgagaaa accatctcca aaaccaaagg gcagccccga 1020 gaaccacagg tgtacaccct gcccccatcc cgggaggaga tgaccaagaa ccaggtcagc 1080 ctgacctgcc tggtcaaagg cttctacccc agcgacatcg ccgtggagtg ggagagcaat 1140 gggcagccgg agaacaacta caagaccacg cctcccatgc tggactccga cggctccttc 1200 ttcctctaca gcaagctcac cgtggacaag agcaggtggc agcaggggaa cgtcttctca 1260 tgctccgtga tgcatgaggc tctgcacaac cactacacgc agaagagcct ctccctgtct 1320 ccgggtaaag gcggtggtgg ttccggaggc ggcggatccg aggtgcagct gttggagtct 1380 gggggaggct tggtacagcc tggggggtcc ctgagactct cctgtgcagc ctctggattc 1440 acctttagca gttattatat gagctgggtc cgccaggctc cagggaaggg gctggagtgg 1500 gtctcagcga tctctcctgg tagtagtaat aaatattacg ctgattctgt aaaaggtcgg 1560 ttcaccatct ccagagacaa ttccaagaac acgctgtatc tgcaaatgaa cagcctgaga 1620 gccgaggaca cggccgtgta ttactgtgcg agaaggaagt atatgttcga ctactggggc 1680 cagggtacac tggtcaccgt gagctcaggt ggaggcggtt caggcggagg tggatccggc 1740 ggtggcggat cgcagtctgt gctgactcag ccaccctcag cgtctgggac ccccgggcag 1800 agggtcacca tctcttgtac tggctcttca tctaatattg gcaataatga tgtctactgg 1860 taccagcagc tcccaggaac ggcacccaaa ctcctcatct attctgatag taatcggcca 1920 agcggggtcc ctgaccgatt ctctggctcc aagtctggca cctcagcctc cctggccatc 1980 agtgggctcc ggtccgagga tgaggctgat tattactgtg cttcttggga ttctagcctg 2040 agtggttatg tcttcggcgg aggcaccaag ctgacggtcc ta 2082 <210> 150 <211> 2082 <212> DNA <213> Artificial Sequence <220> <223> DNA encoding heavy chain of bispecific antibody MN04 <400> 150 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 cctggcgccc tgctccagga gcacctccga gagcacagcg 420 gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca 480 ggcgctctga ccagcggcgt gcacaccttc ccagctgtcc tacagtcctc aggactctac 540 tccctcagca gcgtggtgac cgtgccctcc agcaacttcg gcacccagac ctacacctgc 600 aacgtagatc acaagcccag caacaccaag gtggacaaga cagttgagcg caaatgttgt 660 gtcgagtgcc caccgtgccc agcaccacct gtggcaggac cgtcagtctt cctcttcccc 720 ccaaaaccca aggacaccct catgatctcc cggacccctg aggtcacgtg cgtggtggtg 780 gacgtgagcc acgaagaccc cgaggtccag ttcaactggt acgtggacgg cgtggaggtg 840 cataatgcca agacaaagcc acgggaggag cagttcaaca gcacgttccg tgtggtcagc 900 gtcctcaccg ttgtgcacca ggactggctg aacggcaagg agtacaagtg caaggtctcc 960 aacaaaggcc tcccagcccc catcgagaaa accatctcca aaaccaaagg gcagccccga 1020 gaaccacagg tgtacaccct gcccccatcc cgggaggaga tgaccaagaa ccaggtcagc 1080 ctgacctgcc tggtcaaagg cttctacccc agcgacatcg ccgtggagtg ggagagcaat 1140 gggcagccgg agaacaacta caagaccacg cctcccatgc tggactccga cggctccttc 1200 ttcctctaca gcaagctcac cgtggacaag agcaggtggc agcaggggaa cgtcttctca 1260 tgctccgtga tgcatgaggc tctgcacaac cactacacgc agaagagcct ctccctgtct 1320 ccgggtaaag gcggtggtgg ttccggaggc ggcggatccg aggtgcagct gttggagtct 1380 gggggaggct tggtacagcc tggggggtcc ctgagactct cctgtgcagc ctctggattc 1440 acctttagcg gttatgctat gagctgggtc cgccaggctc cagggaaggg gctggagtgg 1500 gtctcaggga tctctcctgg tagtggtagt acatattacg ctgattctgt aaaaggtcgg 1560 ttcaccatct ccagagacaa ttccaagaac acgctgtatc tgcaaatgaa cagcctgaga 1620 gccgaggaca cggccgtgta ttactgtgcg aaacgtaaga ctaggttcga ctactggggc 1680 cagggtacac tggtcaccgt gagctcaggt ggaggcggtt caggcggagg tggatccggc 1740 ggtggcggat cgcagtctgt gctgactcag ccaccctcag cgtctgggac ccccgggcgg 1800 agggtcacca tctcttgtag tggctcttca tctaatattg gcaataattc tgtctactgg 1860 taccagcagc tcccaggaac ggcccccaaa ctcctcatct atgctaataa taagcggcca 1920 agcggggtcc ctgaccgatt ctctggctcc aagtctggca cctcagcctc cctggccatc 1980 agtgggctcc ggtccgagga tgaggctgat tattactgtg ctgcttggga ttctagcctg 2040 aatggttatg tcttcggcgg aggcaccaag ctgacggtcc ta 2082 <210> 151 <211> 252 <212> PRT <213> Artificial Sequence <220> <223> anti-Nrp scFv 12B <400> 151 Glu Val Gln Leu Leu 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 Ser Asn Tyr              20 25 30 Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Trp Ile Ser Pro Gly Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Lys Arg Ile Gly Ile Lys Leu His Leu Leu Ile Tyr Tyr Ser Tyr             100 105 110 Ala Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly         115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Thr Gln Ser     130 135 140 Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln Arg Val 145 150 155 160 Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Asn Asn Asp Val                 165 170 175 Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu Ile Tyr             180 185 190 Ser Asp Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly Ser         195 200 205 Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg Ser Glu     210 215 220 Asp Glu Ala Asp Tyr Tyr Cys Gly Ala Trp Asp Ser Ser Leu Asn Ala 225 230 235 240 Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu                 245 250 <210> 152 <211> 756 <212> DNA <213> Artificial Sequence <220> <223> DNA encoding anti-Nrp scFv 12B <400> 152 gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttagc aattattcta tgagctgggt ccgccaggct 120 ccagggaagg ggctggagtg ggtctcatgg atctctcctg gtgatggtag tacatattac 180 gctgattctg taaaaggtcg gttcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gaaacgtatt 300 ggtattaagc ttcatttgct gatttattat tcttatgcta tggacgtctg gggccagggt 360 acactggtca ccgtgagctc aggtggaggc ggttcaggcg gaggtggatc cggcggtggc 420 ggaacgcagt ctgtgctgac tcagccaccc tcagcgtctg ggacccccgg gcagagggtc 480 accatctctt gtactggctc ttcatctaat attggcaata atgatgtctc ctggtaccag 540 cagctcccag gaacggctcc caaactcctc atctattctg ataatcagcg gccaagcggg 600 gtccctgacc gattctctgg ctccaagtct ggcacctcag cctccctggc catcagtggg 660 ctccggtccg aggatgaggc tgattattac tgtggtgctt gggattctag cctgaatgct 720 tatgtcttcg gcggaggcac caagctgacg gtccta 756

Claims (13)

Anti-c-Met antibodies or antigen binding fragments thereof; And an anti-Nrp1 antibody or antigen binding fragment thereof,
The anti-c-Met antibody or antigen-binding fragment thereof
CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1,
CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2,
CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3,
CDR-L1 comprising the amino acid sequence of SEQ ID NO: 10,
CDR-L2 comprising the amino acid sequence of SEQ ID NO: 11, and
CDR-L3 comprising 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, and SEQ ID NO: 16,
The anti-Nrp1 antibody or antigen-binding fragment thereof
Or comprise the amino acid sequences of SEQ ID NOs: 110 (CDR-H1), 111 (CDR-H2), 112 (CDR-H3), 113 (CDR-L1), 114 (CDR-L2), and 115 (CDR-L3) ,
Or comprise the amino acid sequences of SEQ ID NOs: 110 (CDR-H1), 116 (CDR-H2), 117 (CDR-H3), 118 (CDR-L1), 119 (CDR-L2), and 120 (CDR-L3) , or
Comprising amino acid sequences of SEQ ID NOs: 121 (CDR-H1), 122 (CDR-H2), 123 (CDR-H3), 124 (CDR-L1), 125 (CDR-L2), and 126 (CDR-L3) That,
Anti-c-Met / anti-Nrp1 bispecific antibody. delete delete delete The method of claim 1, wherein the anti-Nrp1 antibody or antigen-binding fragment thereof,
A heavy chain variable region of SEQ ID NO: 127 and a light chain variable region of SEQ ID NO: 128, or
A heavy chain variable region of SEQ ID NO: 129 and a light chain variable region of SEQ ID NO: 130, or
It comprises a heavy chain variable region of SEQ ID NO: 131 and a light chain variable region of SEQ ID NO: 132,
Anti-c-Met / anti-Nrp1 bispecific antibody. delete delete The method of claim 1, wherein the anti-c-Met antibody or antigen-binding fragment thereof,
A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 17, and
Light chain variable region comprising the amino acid sequence of SEQ ID 109, SEQ ID 18, SEQ ID 19, SEQ ID 20, or SEQ ID 21
It comprises, anti-c-Met / anti-Nrp1 bispecific antibody. The method according to any one of claims 1, 5, and 8,
The antigen binding fragment is selected from the group consisting of scFv, (scFv) 2, scFv-Fc, Fab, Fab 'and F (ab') 2,
Anti-c-Met / anti-Nrp1 bispecific antibody. The method according to any one of claims 1, 5, and 8,
Wherein the anti-c-Met antibody and anti-Nrp1 antibody are mouse derived antibodies, mouse-human chimeric antibodies, humanized antibodies, or human antibodies, respectively
Anti-c-Met / anti-Nrp1 bispecific antibody. The method according to any one of claims 1, 5, and 8,
An antigen-binding fragment of an anti-c-Met antibody in IgG form and an anti-Nrp1 antibody linked to the C terminus of the Fc of the anti-c-Met antibody,
Anti-c-Met / anti-Nrp1 bispecific antibody. A pharmaceutical composition for preventing or treating cancer or cancer metastasis, comprising the anti-c-Met / anti-Nrp1 bispecific antibody of any one of claims 1, 5, and 8 as an active ingredient. The pharmaceutical composition of claim 12, wherein the cancer is an anticancer drug resistant cancer.

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