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Patent Analysis of

Anti-cMet/anti-EGFR/anti-HER3 multispecific antibodies and use thereof

Updated Time 12 June 2019

Patent Registration Data

Publication Number

US10000569

Application Number

US14/692934

Application Date

22 April 2015

Publication Date

19 June 2018

Current Assignee

SAMSUNG ELECTRONICS CO., LTD.

Original Assignee (Applicant)

SAMSUNG ELECTRONICS CO., LTD.

International Classification

C07K16/40,C07K16/28,C07K16/30,C07K16/32,C12N15/09

Cooperative Classification

C07K16/2863,C07K16/32,C07K16/40,A61K39/395,A61K39/39558

Inventor

CHEONG, KWANG HO,HWANG, JAE WOONG,LEE, SEUNG HYUN,LIN, POWEI,CHO, MI YOUNG

Patent Images

This patent contains figures and images illustrating the invention and its embodiment.

US10000569 Anti-cMet/anti-EGFR/anti-HER3 multispecific antibodies 1 US10000569 Anti-cMet/anti-EGFR/anti-HER3 multispecific antibodies 2 US10000569 Anti-cMet/anti-EGFR/anti-HER3 multispecific antibodies 3
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Abstract

An anti-EGFR/anti-HER3 antibody or antigen binding fragment thereof, and a method of preventing and/or treating a cancer using the same.

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Claims

1. An anti-c-Met/anti-EGFR/anti-HER3 multispecific antibody comprising a complete form of an anti-c-Met antibody and an anti-EGFR/anti-HER3 bispecific scFv conjugated to the C-terminus of the anti-c-Met antibody, wherein the anti-c-Met antibody comprises a heavy chain comprising the 18th to 460th positions of SEQ ID NO: 66, and a light chain comprising the 21st to 240th positions of SEQ ID NO: 68; and the anti-EGFR/anti-HER3 bispecific scFv comprises SEQ ID NO: 119.

2. A method of treating a cancer comprising administering the anti-c-Met/anti-EGFR/anti-HER3 multispecific antibody of claim 1 to a subject in need of cancer treatment.

3. A pharmaceutical composition comprising the anti-c-Met/anti-EGFR/anti-HER3 multispecific antibody of claim 1 and a pharmaceutically acceptable carrier.

4. A method of preparing the anti-c-Met/anti-EGFR/anti-HER3 multispecific antibody of claim 1, the method comprising expressing a nucleic acid encoding the anti-c-Met/anti-EGFR/anti-HER3 multispecific antibody of claim 1 in an isolated host cell.

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Claim Tree

  • 1
    1. An anti-c-Met/anti-EGFR/anti-HER3 multispecific antibody comprising
    • a complete form of an anti-c-Met antibody and an anti-EGFR/anti-HER3 bispecific scFv conjugated to the C-terminus of the anti-c-Met antibody, wherein the anti-c-Met antibody comprises a heavy chain comprising the 18th to 460th positions of SEQ ID NO: 66, and a light chain comprising the 21st to 240th positions of SEQ ID NO: 68
    • and the anti-EGFR/anti-HER3 bispecific scFv comprises SEQ ID NO: 119.
  • 2
    2. A method of treating a cancer comprising
    • administering the anti-c-Met/anti-EGFR/anti-HER3 multispecific antibody of claim 1 to a subject in need of cancer treatment.
  • 3
    3. A pharmaceutical composition comprising
    • the anti-c-Met/anti-EGFR/anti-HER3 multispecific antibody of claim 1 and a pharmaceutically acceptable carrier.
  • 4
    4. A method of preparing the anti-c-Met/anti-EGFR/anti-HER3 multispecific antibody of claim 1, the method comprising
    • expressing a nucleic acid encoding the anti-c-Met/anti-EGFR/anti-HER3 multispecific antibody of claim 1 in an isolated host cell.
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Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of Korean Patent Application No. 10-2014-0055664 on May 9, 2014 with the Korean Intellectual Property Office, the disclosure of which is herein incorporated by reference in its entirety.

INCORPORATION-BY-REFERENCE OF MATERIAL ELECTRONICALLY SUBMITTED

Incorporated by reference in its entirety herein is a computer-readable nucleotide/amino acid sequence listing submitted herewith and identified as follows: One 141,477 byte ASCII (Text) file named “718579_ST25_revised-2.TXT,”-created on Sep. 7, 2016.

BACKGROUND

1. Field

Provided are an anti-c-Met/anti-EGFR/anti-HER3 multispecific antibody, and a method of preventing and/or treating a cancer using the same.

2. Description of the Related Art

c-Met and EGFR proteins interact with each other and are involved in various mechanisms related to tumors. These proteins (targets) are typical receptor tyrosine kinases (RTKs) present at the surface of cells, capable of inducing the proliferation of cancer cells, the penetration of the cancer cells, angiogenesis, etc. Also, these proteins participate in each other's signal transduction systems by interacting with each other, thereby inducing resistance against each other's therapeutic agents.

The EGFR family comprises EGFR (HER1), HER2 (Erbb2), HER3 (Erbb3) and HER4 (Erbb4), and forms a homodimer or a heterodimer to mediate signal transduction. Inhibiting the signal is important to cancer development and growth. If resistance to anti-EGFR family or anti-c-Met therapy is developed, HER3 may be activated to exert signal transduction, thus, it is hardly expected to yield a satisfactory therapeutic result.

Multispecific antibodies targeting two or more antigens have been developed in various kinds and forms and are expected as a new drug antibody having excellent therapeutic effects compared to a monoclonal antibody. Most of multispecific antibodies have been developed so that their therapeutic effects on cancers can be increased by recognizing an antigen of cytotoxic cells (killer cells) and other antigen of cancer cells at the same time thus allowing the cancer cells to be killed by the cytotoxic cells. However, research in the field indicates that cancer cells themselves can be mutated to proliferate and penetrate even by intracellular ligands or various antigens of the same cancer cells other than the targeted antigen, it is expected that a multispecific antibody capable of recognizing another antigen of the cancer cells as well as an antigen of the killer cells will be also useful in treating cancers.

In addition, various multispecific antibodies have been developed, but their efficiency was not proved in clinical tests or several side effects were observed. For these reasons, there were many cases which were not approved by FDA and were not marketed as therapeutic antibodies. One of the biggest reasons for which, in spite of the fact that multispecific antibodies having various forms and mechanisms have been developed, the multispecific antibodies were not marketed, is a problem in the stability and productivity of the antibodies. In the production of early multispecific antibodies having an IgG form, random combination of light chains and heavy chains of the antibodies, made it very difficult to separate and purify the desired kind of multispecific antibodies, providing an obstacle to mass production. Also, in case of multispecific antibodies with other than IgG forms, their stabilities as a drug were not verified in fields such as protein folding, pharmacokinetics, and the like.

Accordingly, there is a need for the development of a multispecific antibody which is predicted to achieve effective cancer treatment effects by recognizing two or more kinds of antigens in cancer cells at the same time. Furthermore, there is a need for the development of a bispecific antibody which can enhance cancer treatment as well as solve the side-effect and resistance problems of existing cancer treatment regimens.

SUMMARY

One embodiment provides a polypeptide including one amino acid sequence or a combination of two or more amino acid sequences selected from the group consisting of SEQ ID NO: 109 to SEQ ID NO: 114.

Another embodiment provides a polynucleotide encoding the polypeptide, and method of preparing the polypeptide by expressing the polynucleotide in a cell.

Another embodiment provides an anti-EGFR/anti-HER3 bispecific antibody or an antigen-binding fragment thereof including at least one heavy chain complementarity determining region selected from the group consisting of a CDR-H1 including the amino acid sequence of SEQ ID NO: 109, a CDR-H2 including the amino acid sequence of SEQ ID NO: 110, and a CDR-H3 including the amino acid sequence of SEQ ID NO: 111; at least one light chain complementarity determining region selected from the group consisting of a CDR-L1 including the amino acid sequence of SEQ ID NO: 112, a CDR-L2 including the amino acid sequence of SEQ ID NO: 113, and a CDR-L3 including the amino acid sequence of SEQ ID NO: 114; or a combination of the at least one heavy chain complementarity determining region and the at least one light chain complementarity determining region.

Another embodiment provides a polynucleotide encoding the bispecific antibody or an antigen-binding fragment thereof, and method of preparing the bispecific antibody or an antigen-binding fragment thereof by expressing the polynucleotide in a cell.

Another embodiment provides an anti-c-Met/anti-EGFR/anti-HER3 multispecific antibody including an anti-c-Met antibody or an antigen-binding fragment thereof and an anti-EGFR/anti-HER3 bispecific antibody or an antigen-binding fragment thereof, wherein the anti-c-Met antibody or an antigen-binding fragment thereof specifically binds to an epitope including 5 or more contiguous amino acids within SEMA domain (SEQ ID NO: 79) of c-Met protein, and the anti-EGFR/anti-HER3 bispecific antibody or an antigen-binding fragment thereof includes at least one heavy chain complementarity determining region selected from the group consisting of a CDR-H1 including the amino acid sequence of SEQ ID NO: 109, a CDR-H2 including the amino acid sequence of SEQ ID NO: 110, and a CDR-H3 including the amino acid sequence of SEQ ID NO: 111; at least one light chain complementarity determining region selected from the group consisting of a CDR-L1 including the amino acid sequence of SEQ ID NO: 112, a CDR-L2 including the amino acid sequence of SEQ ID NO: 113, and a CDR-L3 including the amino acid sequence of SEQ ID NO: 114; or a combination of the at least one heavy chain complementarity determining region and the at least one light chain complementarity determining region.

Another embodiment provides a polynucleotide encoding the anti-c-Met/anti-EGFR/anti-HER3 multispecific antibody including an anti-c-Met antibody or an antigen-binding fragment thereof and an anti-EGFR/anti-HER3 bispecific antibody or an antigen-binding fragment thereof, and method of preparing the anti-c-Met/anti-EGFR/anti-HER3 multispecific antibody including an anti-c-Met antibody or an antigen-binding fragment thereof and an anti-EGFR/anti-HER3 bispecific antibody or an antigen-binding fragment thereof by expressing the polynucleotide in a cell.

Another embodiment provides a pharmaceutical composition including the anti-EGFR/anti-HER3 bispecific antibody or the antigen-binding fragment thereof, and a pharmaceutically acceptable carrier.

Another embodiment provides a method of preventing and/or treating a cancer including administering a pharmaceutically effective amount of the anti-EGFR/anti-HER3 bispecific antibody or an antigen-binding fragment thereof to a subject in need thereof.

Another embodiment provides a pharmaceutical composition including the anti-c-Met/anti-EGFR/anti-HER3 multispecific antibody or the antigen-binding fragment thereof, and a pharmaceutically acceptable carrier.

Another embodiment provides a method of preventing and/or treating a cancer including administering a pharmaceutically effective amount of the anti-c-Met/anti-EGFR/anti-HER3 multispecific antibody or the antigen-binding fragment thereof to a subject in need thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of an anti-c-Met/anti-EGFR/anti-HER3 multispecific antibody according to an embodiment.

FIG. 2 is the result of size exclusion chromatography indicating purification of an anti-c-Met/anti-EGFR/anti-HER3 multispecific antibody according to an embodiment.

FIG. 3 is the result of western blot assay indicating target degradation activity of an anti-c-Met/anti-EGFR/anti-HER3 multispecific antibody according to an embodiment.

FIG. 4 is a graph displaying the activity of anti-c-Met/anti-EGFR/anti-HER3 multispecific antibody according to an embodiment for inhibiting growth of human epidermoid carcinoma cell line A431.

FIG. 5 is a graph displaying the activity of anti-c-Met/anti-EGFR/anti-HER3 multispecific antibody according to an embodiment for inhibiting growth of human gastric cancer cell line SNU5.

FIG. 6 is a graph displaying the activity of anti-c-Met/anti-EGFR/anti-HER3 multispecific antibody according to an embodiment for inhibiting growth of human pancreas cancer cell line BxPC3.

FIG. 7 is a graph displaying the of anti-c-Met/anti-EGFR/anti-HER3 multispecific antibody according to an embodiment for inhibiting growth of human lung cancer cell line NCI-H1993.

FIG. 8 is a graph displaying the activity of anti-c-Met/anti-EGFR/anti-HER3 multispecific antibody according to an embodiment for inhibiting growth of human breast cancer cell line HCC1954.

FIG. 9 is a graph displaying the activity of anti-c-Met/anti-EGFR/anti-HER3 multispecific antibody according to an embodiment for inhibiting growth of human liver cancer cell line Huh7.

FIG. 10 is a graph displaying the activity of anti-c-Met/anti-EGFR/anti-HER3 multispecific antibody according to an embodiment for inhibiting growth of human colon cancer cell line HT29.

DETAILED DESCRIPTION

It is verified that a multispecific antibody recognizing c-Met, EGFR and HER3 at the same time prevents the development of cancer resistance and shows excellent cancer cell inhibitory effects, even in cancer cells having resistance, by previously blocking c-Met-implicated signal transduction which causes resistance against drugs.

A multispecific antibody in which an anti-c-Met antibody is fused to an antibody recognizing a secondary target, EGFR and HER3, or an antigen binding fragment thereof (e.g., scFv) can improve the stability of the multispecific antibodies.

One embodiment provides a polypeptide including a novel amino acid sequence. The polypeptide may have a function as a CDR of an anti-EGFR and/or anti-HER3 antibody. In particular, the polypeptide may include one amino acid sequence or a combination of two or more amino acid sequences selected from the group consisting of SEQ ID NO: 109 to SEQ ID NO: 114.

The polypeptide including the amino acid sequence of SEQ ID NO: 109 to SEQ ID NO: 114 as a CDR of an anti-EGFR/anti-HER3 antibody is summarized in Table 1, as follows:


TABLE 1
amino acid sequence
SEQ ID NO.
Heavy
CDR-H1
GDWIH
109
chain CDR
CDR-H2
WVGEISAAGGYTDYADSVKG
110
CDR-H3
ESRVSFEAAMDY
111
Light
CDR-L1
RASQNIATDVA
112
chain
CDR-L2
SASFLYS
113
CDR
CDR-L3
QQSEPEPY
114

In one particular embodiment, the polypeptide may be a polypeptide including a polypeptide of SEQ ID NO: 115, a polypeptide of SEQ ID NO: 116 or a combination thereof, or a polypeptide of SEQ ID NO: 119. The polypeptide of SEQ ID NO: 115 includes the amino acid sequences of SEQ ID NOS: 109 to 111, and may have a function as a heavy chain variable region of an anti-EGFR/anti-HER3 antibody. In addition, the polypeptide including the amino acid sequence of SEQ ID NO: 116 includes the amino acid sequences of SEQ ID NOS: 112 to 114, and may have a function as a light chain variable region of an anti-EGFR/anti-HER3 antibody. The polypeptide of SEQ ID NO: 119 includes the polypeptides of SEQ ID NOs: 115 and 116 that are connected via a linker.

<SEQ ID NO: 115, Capable of Acting as a Heavy Chain Variable Region of an Anti-EGFR/Anti-HER3 Antibody>


EVQLVESGGGLVQPGGSLRLSCAASGFTLSGDWIHWVRQAPGKCLEWVGE
ISAAGGYTDYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARES
RVSFEAAMDYWGQGTLVTVSS

(wherein the CDRs are marked in bold type, indicating CDR-H1, CDR-H2, and CDR-H3 in sequence.)

<SEQ ID NO: 116, Capable of Acting as a Light Chain Variable Region of an Anti-EGFR/Anti-HER3 Antibody>


DIQMTQSPSSLSASVGDRVTITCRASQNIATDVAWYQQKPGKAPKLLIYS
ASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSEPEPYTFGC
GTKVEIK

(wherein the CDRs are marked in bold type, indicating CDR-H1, CDR-H2, and CDR-H3 in sequence.)

<SEQ ID NO: 117, a Nucleic Acid Sequence Encoding a Heavy Chain Variable Region of an Anti-EGFR/Anti-HER3 Antibody>


gaggttcagctggtggagtctggcggtggcctggtgcagccagggggctc
actccgtttgtcctgtgcagcttctggcttcaccctttctggcgactgga
tacactgggtgcgtcaggccccgggtaagtgcctggaatgggttggagag
atttctgctgcgggtggttatactgactatgccgatagcgtcaagggccg
tttcactataagcgcagacacatccaaaaacacagcctacctgcagatga
acagcctgcgtgctgaggacactgccgtctattattgtgctagagagagt
agggtcagcttcgaggctgcgatggactactggggtcaaggaaccctggt
caccgtctcctcg

<SEQ ID NO: 118, a Nucleic Acid Sequence Encoding a Light Chain Variable Region of an Anti-EGFR/Anti-HER3 Antibody>


gatatccagatgacccagtccccgagctccctgtccgcctctgtgggcga
tagggtcaccatcacctgccgtgccagtcagaatatcgctactgatgtag
cctggtatcaacagaaaccaggaaaagctccgaaactactgatttactcg
gcatccttcctctactctggagtcccttctcgcttctctggttccggatc
tgggacggatttcactctgaccatcagcagtctgcagccggaagacttcg
caacttattactgtcagcaatctgagcctgaaccttatacgttcggatgc
ggtaccaaggtggagatcaaa

The polypeptide may act as a precursor or a component of EGFR and HER3 antagonist, such as an anti-EGFR/anti-HER3 antibody, an antigen-binding fragment thereof, or an anti-EGFR/anti-HER3 antibody analogue (e.g., a peptibody, nanobody, etc.).

Therefore, another embodiment provides an anti-EGFR/anti-HER3 antagonist including the polypeptide. The antagonist inhibits the EGFR and HER3 activity, and may be one or more selected from the group consisting of an anti-EGFR/anti-HER3 antibody, an antigen-binding fragment thereof, an anti-EGFR/anti-HER3 antibody analogue (e.g., a peptibody, nanobody, etc.), and the like.

The term “antagonist” may include any molecules capable of completely or partially preventing, inhibiting, or neutralizing one or more biological activities of a target. For instance, an antibody as an antagonist may refer to an antibody capable of inhibiting or lowering biological activities of an antigen to which the antibody binds. The antagonist may bind to a receptor for a ligand (target) to decrease receptor phosphorylation, or incapacitating or killing a cell that is activated by the ligand. In addition, the antagonist may substantially decrease an interaction between a receptor and its ligand, by completely blocking the receptor-ligand interaction, binding to the receptor competitively with its ligand, or modifying or down-regulating three-dimensional structure of the receptor.

Term “peptibody” (derived from a combination of the terms “peptide” and “antibody”) may refer to a fusion protein wherein a peptide is fused with the whole or a part of a constant region of an antibody, such as Fc region, and the peptide acts as an antigen-binding region (e.g., a CDR or variable region of a heavy chain and/or light chain), thereby having a structure and functions similar to an antibody.

Term “nanobody” that is also called as a single-domain antibody, may refer to an antibody fragment including a single variable domain in a monomeric form and selectively binding to a specific antigen, similarly to an antibody in a complete form. The nanobody usually has a molecular weight of about 12 kDa to about 15 kDa, which is very smaller than an general molecular weight (about 150 kDa to about 160 kDa) of an antibody in a complete form (including two heavy chains and two light chains), and in some case, smaller than a molecular weight of a Fab fragment or a scFv fragment.

In a particular embodiment, the polypeptide may act as a precursor or a component of an anti-EGFR/anti-HER3 bispecific antibody.

Another embodiment provides an anti-EGFR/anti-HER3 bispecific antibody or an antigen-binding fragment thereof including the polypeptide. The antigen-binding fragment may be selected from the group consisting of scFv, (scFv)2, scFv-Fc, Fab, Fab′ and F(ab′)2.

In particular, the anti-EGFR/anti-HER3 bispecific antibody or an antigen-binding fragment thereof may include:

at least one heavy chain complementarity determining region selected from the group consisting of a CDR-H1 including the amino acid sequence of SEQ ID NO: 109, a CDR-H2 including the amino acid sequence of SEQ ID NO: 110, and a CDR-H3 including the amino acid sequence of SEQ ID NO: 111, or a heavy chain variable region including the at least one heavy chain complementarity determining region;

at least one light chain complementarity determining region selected from the group consisting of a CDR-L1 including the amino acid sequence of SEQ ID NO: 112, a CDR-L2 including the amino acid sequence of SEQ ID NO: 113, and a CDR-L3 including the amino acid sequence of SEQ ID NO: 114 or a light chain variable region including the at least one light chain complementarity determining region;

a combination of the at least one heavy chain complementarity determining region and the at least one light chain complementarity determining region; or

a combination of a heavy chain variable region and a light chain variable region.

For example, the anti-EGFR/anti-HER3 bispecific antibody or an antigen-binding fragment thereof may include a heavy chain variable region including the amino acid sequence of SEQ ID NO: 115, a light chain variable region including the amino acid sequence of SEQ ID NO: 116, or a combination thereof, or a polypeptide of SEQ ID NO: 119.

In a particular embodiment, the anti-EGFR/anti-HER3 bispecific antibody or an antigen-binding fragment thereof may be an anti-EGFR/anti-HER3 scFv including a heavy chain variable region including the amino acid sequence of SEQ ID NO: 115, and a light chain variable region including the amino acid sequence of SEQ ID NO: 116, or a combination thereof, or a polypeptide of SEQ ID NO: 119.

In the polypeptide or an anti-EGFR/anti-HER3 scFv, the heavy chain variable region and the light chain variable region may be linked with or without a linker (e.g., a peptide linker). The peptide linker may be those including any amino acids of 1 to 100, particularly 2 to 50, and any kinds of amino acids may be included without any restrictions, provided the linker does not prevent the polypeptide from binding to its target. The peptide linker may include for example, Gly, Asn and/or Ser residues, and also include neutral amino acids such as Thr and/or Ala. Amino acid sequences suitable for the peptide linker may be those known in the relevant art. Meanwhile, a length of the peptide linker may be variously determined within such a limit that the functions of the fusion protein will not be affected. For instance, the peptide linker may be formed by including a total of 1 to 100, 2 to 50, or 5 to 25 of one or more selected from the group consisting of Gly, Asn, Ser, Thr, and Ala. In one embodiment, the peptide linker may be represented as SEQ ID NO: 124 (GGGGS)n (n is a repeat number of (GGGGS, SEQ ID NO: 123), which is an integer of about 1 to about 10, particularly an integer of about 2 to about 5).

The term “antibody” may refer to a substance that specifically binds an antigen, such as an antibody. The antibody may include all of an animal antibody, a chimeric antibody, a humanized antibody, and a human antibody. In addition the antibody may include an antigen-binding fragment derived from an antibody having an antigen binding affinity. The “complementarity-determining region (CDR)” may refer to a region within a variable region, which give a binding specificity to an antigen. The antigen-binding fragment as described above may be an antibody fragment including at least one complementarity-determining region, for example, one or more selected from the group consisting of scFv, (scFv)2, scFv-Fc, Fab, Fab′, and F(ab′)2.

In the anti-EGFR/anti-HER3 bispecific antibody or an antigen-binding fragment thereof, the rest portion of the light chain and the heavy chain portion excluding the CDRs, the light chain variable region, and the heavy chain variable region as defined above, that is the light chain constant region and the heavy chain constant region, may be those from any subtype of immunoglobulin (e.g., IgA, IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), IgM, and the like).

Based on the ability of specifically binding to EGFR and HER3, the anti-EGFR/anti-HER3 bispecific antibody or an antigen-binding fragment thereof may be used in detecting EGFR and/or HER3 or confirming activation and/or overproduction (overexpression) of EGFR and/or HER3.

One embodiment provides a composition for detecting the presence of EGFR and/or HER3 including the anti-EGFR/anti-HER3 bispecific antibody or an antigen-binding fragment thereof. Another embodiment provides a method of detecting EGFR and/or HER3 including treating a biological sample with the anti-EGFR/anti-HER3 bispecific antibody or an antigen-binding fragment thereof; and detecting an antigen-antibody reaction (binding). In the method of detecting, when an antigen-antibody reaction is detected, it can be determined that EGFR and/or HER3 is present in the biological sample. Another embodiment provides a use of the anti-EGFR/anti-HER3 bispecific antibody or an antigen-binding fragment thereof for detecting EGFR and/or HER3. The biological sample may be selected from the group consisting of a cell, a tissue, a body fluid (e.g., blood, serum, etc.), and the like derived from a mammal including primates such as a human, a monkey, and the like, or a rodent such as a mouse, a rat, and the like. The biological sample may be separated from a living body. The detection of EGFR and/or HER3 may refer to detection of presence EGFR and/or HER3, expression of EGFR and/or HER3, or the level of EGFR and/or HER3.

Another embodiment provides a pharmaceutical composition for diagnosing activation and/or overproduction of EGFR and/or HER3 or a disease associated with activation and/or overproduction of EGFR and/or HER3 including the anti-EGFR/anti-HER3 bispecific antibody or an antigen-binding fragment thereof. Another embodiment provides a method of diagnosing (or determining) activation and/or overproduction of EGFR and/or HER3 or a disease associated with activation and/or overproduction of EGFR and/or HER3, including treating a biological sample derived from a patient with the anti-EGFR/anti-HER3 bispecific antibody or an antigen-binding fragment thereof, and measuring a level of an antigen-antibody reaction. In this method, when the level of the antigen-antibody reaction in the biological sample is higher than that of a normal sample, the patient from which the biological sample is derived may be determined as having activation and/or overproduction of EGFR and/or HER3 or a disease associated with activation and/or overproduction of EGFR and/or HER3. Therefore, the method may further include treating a normal sample with the anti-EGFR/anti-HER3 bispecific antibody or an antigen-binding fragment thereof, and measuring a level of an antigen-antibody reaction. Another embodiment provides a use of the anti-EGFR/anti-HER3 bispecific antibody or an antigen-binding fragment thereof for diagnosing activation and/or overproduction of EGFR and/or HER3 or a disease associated with activation and/or overproduction of EGFR and/or HER3.

The biological sample may be at least one selected from the group consisting of a cell, a tissue, fluid (e.g., blood, serum, and the like) and the like, derived from a patient to be diagnosed. The biological sample may be separated from a living body. The normal sample may be at least one selected from the group consisting of a cell, a tissue, fluid (e.g., blood, serum, and the like) and the like, derived from a patient having no condition of activation and/or overproduction of EGFR and/or HER3 or a disease associated with activation and/or overproduction of EGFR and/or HER3. The normal sample may be separated from a living body. The patient may be selected from mammal including primates such as a human, a monkey, and the like, and rodents such as a mouse, a rat, and the like.

Another embodiment provides an anti-c-Met/anti-EGFR/anti-HER3 multispecific antibody including an anti-c-Met antibody or an antigen-binding fragment thereof and an anti-EGFR/anti-HER3 bispecific antibody or an antigen-binding fragment thereof. The antigen-binding fragment thereof may be selected from the group consisting of scFv, (scFv)2, scFvFc, Fab, Fab′, and F(ab′)2.

The “c-Met protein” refers to a receptor tyrosine kinase binding to hepatocyte growth factor. The c-Met proteins may be derived from any species, for example, those derived from primates such as human c-Met (e.g., NP_000236) and monkey c-Met (e.g., Macaca mulatta, NP_001162100), or those derived from rodents such as mouse c-Met (e.g., NP_032617.2) and rat c-Met (e.g., NP_113705.1). The proteins include, for example, a polypeptide encoded by the nucleotide sequence deposited under GenBank Accession Number NM_000245, or a protein encoded by the polypeptide sequence deposited under GenBank Accession Number NM_000236, or extracellular domains thereof. The receptor tyrosine kinase c-Met is involved in several mechanisms including cancer incidence, cancer metastasis, cancer cell migration, cancer cell penetration, angiogenesis, etc.

The “EGFR (epidermal growth factor receptor)” is a member of the receptor tyrosine kinases (RTKs) of HER family. The binding of a ligand to the extracellular domain of EGFR induces receptor homo- or hetero dimerization with other ErbB receptors, which in turn results in intracellular self-phosphorylation of specific tyrosine residues. EGFR self-phosphorylation leads to downstream signal transduction networks including MAPK and PI3K/Akt activation which affects cell proliferation, angiogenesis and metastasis. Over-expression, gene amplification, mutation, or rearrangement of EGFR are frequently observed in several human malignant tumors and are related to poor prognosis of cancer treatment and bad clinical outcomes. For such reasons, the EGFR becomes an important target in anticancer therapy. The EGFR or HER2 may be derived from mammals, for example, primates such as humans and monkeys, or rodents such as rats and mice. For instance, the EGFR may be polypeptides encoded by the nucleotide sequences (mRNA) deposited under GenBank Accession Nos. JQ739160, JQ739161, JQ739162, JQ739163, JQ739164, JQ739165, JQ739166, JQ739167, NM_005228.3, NM_201284.1, NM_201282.1, or NM_201283.1.

The “HER3” is a member of the receptor tyrosine kinases (RTKs) of HER(EGFR) family. The member of the receptor tyrosine kinases of HER(EGFR) family comprises HER1 (also known as EGFR or erbB), HER2 (also known as erbB2), HER3 (also known as erbB3) and HER4 (also known as erbB4), and among them, HER3, as a transmembrane receptor, is composed of an extracellular ligand-binding domain (ECD), a dimerization domain in the ECD, an transmembrane domain (TMD), an extracellular protein Tyrosine-kinase domain (TKD), and a C-terminal phosphorylation domain, in common with original epidermal growth factor receptor. HER3, as well as EGFR and HER2, is related to tumorigenesis. HER3 is occasionally overexpressed in breast cancer, colorectal cancer, ovarian cancer, bladder cancer, prostate cancer, non-small-cell lung cancer, melanoma, pharynx cancer, pancreatic cancer, esophagus cancer, glioma, cholangiocarcinoma, biliary tract cancer, gastric cancer, endometrial cancer, gallbladder cancer, squamous cell carcinoma, or basal cell carcinoma, and the like. For instance, the HER3 may be polypeptides encoded by the nucleotide sequences (mRNA) deposited under GenBank Accession Nos. NM_001982.2, NM_001982.3, NM_001005915.1, NM_010153.1, NM_017218.2 or NM_001103105.1.

In one embodiment, the anti-c-Met/anti-EGFR/anti-HER3 multispecific antibody may include an anti-c-Met antibody or an antigen binding fragment thereof, and an anti-EGFR/anti-HER3 bispecific antibody or an antigen binding fragment thereof which is linked to the C terminus or N terminus, for example, C terminal, of the anti-c-Met antibody or the antigen binding fragment thereof.

In the anti-c-Met/anti-EGFR/anti-HER3 multispecific antibody, in order to fully perform the anti-c-Met antibody's activity to mediate intracellular migration and degradation of c-Met proteins, it may be advantageous that the anti-c-Met antibody has its own intact antibody structure. In addition, in case of the anti-EGFR/anti-HER3 bispecific antibody, its specific recognition and binding to EGFR and/or HER3 is important, and thus it will be fine that just an antigen-binding fragment recognizing EGFR and/or HER3 is included in the bispecific antibody. Therefore, the anti-c-Met/anti-EGFR/anti-HER3 multispecific antibody may be those including a complete form of an anti-c-Met antibody (e.g., IgG type antibody) and an antigen binding fragment of the anti-EGFR/anti-HER3 bispecific antibody linked to the C terminus of the anti-c-Met antibody.

In the anti-c-Met/anti-EGFR/anti-HER3 multispecific antibody, the anti-c-Met antibody or the antigen binding fragment thereof, and the anti-EGFR/anti-HER3 bispecific antibody or the antigen binding fragment thereof, may be linked via a peptide linker or without it. Furthermore, a heavy chain portion and a light chain portion within the antigen binding fragment, for example, a heavy chain variable region and a light chain variable region within the scFv fragment, may be linked via a peptide linker or without it. The peptide linker which links the anti-c-Met antibody or the antigen binding fragment thereof and the anti-EGFR/anti-HER3 bispecific antibody or the antigen binding fragment thereof, and the peptide linker which links the heavy chain portion and the light chain portion within the antigen binding fragment, may be identical or different. The peptide linker may be those including any amino acids of about 1 to about 100, particularly about 2 to about 50, and any kinds of amino acids may be included without any restrictions. The peptide linker may include for example, Gly, Asn and/or Ser residues, and also include neutral amino acids such as Thr and/or Ala. Amino acid sequences suitable for the peptide linker may be those known in the pertinent art. Meanwhile, a length of the peptide linker may be variously determined within such a limit that the functions of the fusion protein will not be affected. For instance, the peptide linker may be formed by including a total of about 1 to about 100, about 2 to about 50, or about 5 to about 25 of one or more selected from the group consisting of Gly, Asn, Ser, Thr, and Ala. In one embodiment, the peptide linker may be represented as SEQ ID NO: 124 (GGGGS)n (n is a repeat number of (GGGGS, SEQ ID NO: 123), which is an integer of about 1 to about 10, particularly an integer of about 2 to about 5).

In a particular embodiment, the anti-EGFR/anti-HER3 bispecific antibody or an antigen-biding fragment may include:

at least one heavy chain complementarity determining region selected from the group consisting of a CDR-H1 including the amino acid sequence of SEQ ID NO: 109, a CDR-H2 including the amino acid sequence of SEQ ID NO: 110, and a CDR-H3 including the amino acid sequence of SEQ ID NO: 111 or a heavy chain variable region including the at least one heavy chain complementarity determining region;

at least one light chain complementarity determining region selected from the group consisting of a CDR-L1 including the amino acid sequence of SEQ ID NO: 112, a CDR-L2 including the amino acid sequence of SEQ ID NO: 113, and a CDR-L3 including the amino acid sequence of SEQ ID NO: 114 or a light chain variable region including the at least one light chain complementarity determining region;

a combination of the at least one heavy chain complementarity determining region and the at least one light chain complementarity determining region; or

a combination of the heavy chain variable region and the light chain variable region.

For example, the anti-EGFR/anti-HER3 bispecific antibody or an antigen-binding fragment thereof may include a heavy chain variable region including the amino acid sequence of SEQ ID NO: 115, a light chain variable region including the amino acid sequence of SEQ ID NO: 116, or a combination thereof.

In a particular embodiment, the anti-EGFR/anti-HER3 bispecific antibody or an antigen-binding fragment thereof may be an anti-EGFR/anti-HER3 scFv including a heavy chain variable region including the amino acid sequence of SEQ ID NO: 115, and a light chain variable region including the amino acid sequence of SEQ ID NO: 116.

The “antigen binding fragment” refers to a fragment of a full immunoglobulin structure including parts of the polypeptide including a portion antigen-binding regions capable of binding to an antigen. For example, it may be scFv, (scFv)2, Fab, Fab′, or F(ab′)2, but not be limited thereto. In the present invention, the antigen binding fragment may be an antibody fragment including at least one complementarity determining region, for example, selected from the group consisting of scFv, (scFv)2, scFv-Fc, Fab, Fab′ and F(ab′)2.

Of the antigen binding fragments, Fab is a structure having variable regions of a light chain and a heavy chain, a constant region of the light chain, and the first constant region (CH1) of the heavy chain, and it has one antigen binding site.

Fab′ is different from Fab in that it has a hinge region including one or more cysteine residues at the C-terminal of heavy chain CH1 domain. An F(ab′)2 antibody is formed through disulfide bond of the cysteine residues at the hinge region of Fab′.

Fv is a minimal antibody piece having only a heavy chain variable region and light chain variable region, and a recombinant technique for producing the Fv fragment is well known in the pertinent art. Two-chain Fv may have a structure in which the heavy chain variable region is linked to the light chain variable region by a non-covalent bond, and single-chain Fv (scFv) may generally have a dimer structure as in the two-chain Fv in which the variable region of a heavy chain and the variable region of a light chain are covalently linked via a peptide linker or they are directly linked to each other at the C-terminal thereof. The peptide linker may be the same as described above.

The antigen binding fragments may be obtained using proteases (for example, a whole antibody is digested with papain to obtain Fab fragments, and is digested with pepsin to obtain F(ab′)2 fragments), and may be prepared by a genetic recombinant technique.

In a particular embodiment, the anti-c-Met/anti-EGFR/anti-HER3 multispecific bispecific antibody may be those including an anti-c-Met antibody, and scFv, (scFv)2, Fab, Fab′ or F(ab′)2, for example, scFv, of the anti-EGFR/anti-HER3 bispecific antibody linked to the C terminus of the anti-c-Met antibody. Hence, in a particular embodiment, the anti-c-Met/anti-EGFR/anti-HER3 multispecific bispecific antibody may be those including the anti-c-Met antibody, and scFv, (scFv)2, Fab, Fab′ or F(ab′)2 of the anti-EGFR/anti-HER3 bispecific antibody including a heavy chain variable region including the amino acid sequence of SEQ ID NO: 115 and a light chain variable region including the amino acid sequence of SEQ ID NO: 116, linked to the C terminus of the anti-c-Met antibody.

The anti-c-Met antibody may be any one recognizing a specific region of c-Met, e.g., a specific region in the SEMA domain, as an epitope. It may be any antibody or antigen-binding fragment that acts on c-Met to induce intracellular internalization and degradation of c-Met.

c-Met, a receptor for hepatocyte growth factor (HGF), may be divided into three portions: extracellular, transmembrane, and intracellular. The extracellular portion is composed of an α-subunit and a β-subunit which are linked to each other through a disulfide bond, and contains a SEMA domain responsible for binding HGF, a PSI domain (plexin-semaphorins-integrin homology domain) and an IPT domain (immunoglobulin-like fold shared by plexins and transcriptional factors domain). The SEMA domain of c-Met protein may have the amino acid sequence of SEQ ID NO: 79, and is an extracellular domain that functions to bind HGF. A specific region of the SEMA domain, that is, a region including the amino acid sequence of SEQ ID NO: 71, which corresponds to a range from amino acid residues 106 to 124 of the amino acid sequence of the SEMA domain (SEQ ID NO: 79) of c-Met protein, is a loop region between the second and the third propellers within the epitopes of the SEMA domain. The region acts as an epitope for the specific anti-c-Met antibody of the present invention.

The term “epitope” as used herein, refers to an antigenic determinant, a part of an antigen recognized by an antibody. In one embodiment, the epitope may be a region including 5 or more contiguous (consecutive or non-consecutive) amino acid residues within the SEMA domain (SEQ ID NO: 79) of c-Met protein, for instance, 5 to 19 contiguous amino acid residues within the amino acid sequence of SEQ ID NO: 71. For example, the epitope may be a polypeptide having 5 to 19 contiguous amino acids selected from among partial combinations of the amino acid sequence of SEQ ID NO: 71, wherein the polypeptide essentially includes the amino sequence of SEQ ID NO: 73 (EEPSQ) serving as an essential element for the epitope. For example, the epitope may be a polypeptide including, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 71, SEQ ID NO: 72, or SEQ ID NO: 73.

The epitope including the amino acid sequence of SEQ ID NO: 72 corresponds to the outermost part of the loop between the second and third propellers within the SEMA domain of a c-Met protein. The epitope including the amino acid sequence of SEQ ID NO: 73 is a site to which the antibody or antigen-binding fragment according to one embodiment most specifically binds.

Thus, the anti-c-Met antibody may specifically bind to an epitope which has 5 to 19 contiguous amino acids selected from among partial combinations of the amino acid sequence of SEQ ID NO: 71, including SEQ ID NO: 73 as an essential element. For example, the anti-c-Met antibody may specifically bind to an epitope including the amino acid sequence of SEQ ID NO: 71, SEQ ID NO: 72, or SEQ ID NO: 73.

In one embodiment, the anti-c-Met antibody or an antigen-binding fragment thereof may include:

at least one heavy chain complementarity determining region (CDR) selected from the group consisting of (a) a CDR-H1 including the amino acid sequence of SEQ ID NO: 4; (b) a CDR-H2 including the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 2, or an amino acid sequence having 8-19 consecutive amino acids within SEQ ID NO: 2 including amino acid residues from the 3rd to 10th positions of SEQ ID NO: 2; and (c) a CDR-H3 including the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 85, or an amino acid sequence having 6-13 consecutive amino acids within SEQ ID NO: 85 including amino acid residues from the 1st to 6th positions of SEQ ID NO: 85, or a heavy chain variable region including the at least one heavy chain complementarity determining region;

at least one light chain complementarity determining region (CDR) selected from the group consisting of (a) a CDR-L1 including the amino acid sequence of SEQ ID NO: 7, (b) a CDR-L2 including the amino acid sequence of SEQ ID NO: 8, and (c) a CDR-L3 including the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 86, or an amino acid sequence having 9-17 consecutive amino acids within SEQ ID NO: 89 including amino acid residues from the 1st to 9th positions of SEQ ID NO: 89, or a light chain variable region including the at least one light chain complementarity determining region;

a combination of the at least one heavy chain complementarity determining region and at least one light chain complementarity determining region; or

a combination of the heavy chain variable region and the light chain variable region.

Herein, the amino acid sequences of SEQ ID NOS: 4 to 9 are respectively represented by following Formulas I to VI, below:

Formula I

Xaa1-Xaa2-Tyr-Tyr-Met-Ser  (SEQ ID NO: 4),

wherein Xaa1 is absent or Pro or Ser, and Xaa2 is Glu or Asp,

Formula II

Arg-Asn-Xaa3-Xaa4-Asn-Gly-Xaa5-Thr  (SEQ ID NO: 5),

wherein Xaa3 is Asn or Lys, Xaa4 is Ala or Val, and Xaa5 is Asn or Thr,

Formula III

Asp-Asn-Trp-Leu-Xaa6-Tyr  (SEQ ID NO: 6),

wherein Xaa6 is Ser or Thr,

Formula IV

Lys-Ser-Ser-Xaa7-Ser-Leu-Leu-Ala-Xaa8-Gly-Asn-Xaa9-Xaa10-Asn-Tyr-Leu-Ala  (SEQ ID NO: 7)

wherein Xaa7 is His, Arg, Gln, or Lys, Xaa8 is Ser or Trp, Xaa9 is His or Gln, and Xaa10 is Lys or Asn,

Formula V

Trp-Xaa11-Ser-Xaa12-Arg-Val-Xaa13  (SEQ ID NO: 8)

wherein Xaa11 is Ala or Gly, Xaa12 is Thr or Lys, and Xaa13 is Ser or Pro, and

Formula VI

Xaa14-Gln-Ser-Tyr-Ser-Xaa15-Pro-Xaa16-Thr  (SEQ ID NO: 9)

wherein Xaa14 is Gly, Ala, or Gln, Xaa15 is Arg, His, Ser, Ala, Gly, or Lys, and Xaa16 is Leu, Tyr, Phe, or Met.

In one embodiment, the CDR-H1 may include an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 23, and 24. The CDR-H2 may include an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 25, and 26. The CDR-H3 may include an amino acid sequence selected from the group consisting of SEQ ID NOS: 3, 27, 28, and 85.

The CDR-L1 may include an amino acid sequence selected from the group consisting of SEQ ID NOS: 10, 29, 30, 31, 32, 33, and 106. The CDR-L2 may include an amino acid sequence selected from the group consisting of SEQ ID NOS: 11, 34, 35, and 36. The CDR-L3 may include an amino acid sequence selected from the group consisting of SEQ ID NOS: 12, 13, 14, 15, 16, 37, 86, and 89.

In another embodiment, the antibody or antigen-binding fragment may include

a heavy chain variable region comprising a polypeptide (CDR-H1) including an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 22, 23, and 24, a polypeptide (CDR-H2) including an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 25, and 26, and a polypeptide (CDR-H3) including an amino acid sequence selected from the group consisting of SEQ ID NOS: 3, 27, 28, and 85; and

a light chain variable region comprising a polypeptide (CDR-L1) including an amino acid sequence selected from the group consisting of SEQ ID NOS: 10, 29, 30, 31, 32, 33 and 106, a polypeptide (CDR-L2) including an amino acid sequence selected from the group consisting of SEQ ID NOS: 11, 34, 35, and 36, and a polypeptide (CDR-L3) including an amino acid sequence selected from the group consisting of SEQ ID NOS 12, 13, 14, 15, 16, 37, 86, and 89.

Animal-derived antibodies produced by immunizing non-immune animals with a desired antigen generally invoke immunogenicity when injected to humans for the purpose of medical treatment, and thus chimeric antibodies have been developed to inhibit such immunogenicity. Chimeric antibodies are prepared by replacing constant regions of animal-derived antibodies that cause an anti-isotype response with constant regions of human antibodies by genetic engineering. Chimeric antibodies are considerably improved in an anti-isotype response compared to animal-derived antibodies, but animal-derived amino acids still have variable regions, so that chimeric antibodies have side effects with respect to a potential anti-idiotype response. Humanized antibodies have been developed to reduce such side effects. Humanized antibodies are produced by grafting complementarity determining regions (CDR) which serve an important role in antigen binding in variable regions of chimeric antibodies into a human antibody framework.

The most important thing in CDR grafting to produce humanized antibodies is choosing the optimized human antibodies for accepting CDRs of animal-derived antibodies. Antibody databases, analysis of a crystal structure, and technology for molecule modeling are used. However, even when the CDRs of animal-derived antibodies are grafted to the most optimized human antibody framework, amino acids positioned in a framework of the animal-derived CDRs affecting antigen binding are present. Therefore, in many cases, antigen binding affinity is not maintained, and thus application of additional antibody engineering technology for recovering the antigen binding affinity is necessary.

The anti c-Met antibodies may be mouse-derived antibodies, mouse-human chimeric antibodies, humanized antibodies, or human antibodies.

An intact antibody includes two full-length light chains and two full-length heavy chains, in which each light chain is linked to a heavy chain by disulfide bonds. The antibody has a heavy chain constant region and a light chain constant region. The heavy chain constant region is of a gamma (γ), mu (μ), alpha (α), delta (δ), or epsilon (ε) type, which may be further categorized as gamma 1 (γ1), gamma 2(γ2), gamma 3(γ3), gamma 4(γ4), alpha 1(α1), or alpha 2(α2). The light chain constant region is of either a kappa (κ) or lambda (λ) type.

As used herein, the term “heavy chain” refers to full-length heavy chain, and fragments thereof, including a variable region VH that includes amino acid sequences sufficient to provide specificity to antigens, and three constant regions, CH1, CH2, and CH3, and a hinge. The term “light chain” refers to a full-length light chain and fragments thereof, including a variable region VL that includes amino acid sequences sufficient to provide specificity to antigens, and a constant region CL.

The term “complementarity determining region (CDR)” refers to an amino acid sequence found in a hyper variable region of a heavy chain or a light chain of immunoglobulin. The heavy and light chains may respectively include three CDRs (CDRH1, CDRH2, and CDRH3; and CDRL1, CDRL2, and CDRL3). The CDR may provide contact residues that play an important role in the binding of antibodies to antigens or epitopes. The terms “specifically binding” and “specifically recognized” are well known to one of ordinary skill in the art, and indicate that an antibody and an antigen specifically interact with each other to lead to an immunological activity.

The term “hinge region,” as used herein, refers to a region between CH1 and CH2 domains within the heavy chain of an antibody which functions to provide flexibility for the antigen-binding site.

When an animal antibody undergoes a chimerization process, the IgG1 hinge of animal origin is replaced with a human IgG1 hinge or IgG2 hinge while the disulfide bridges between two heavy chains are reduced from three to two in number. In addition, an animal-derived IgG1 hinge is shorter than a human IgG1 hinge. Accordingly, the rigidity of the hinge is changed. Thus, a modification of the hinge region may bring about an improvement in the antigen binding efficiency of the humanized antibody. The modification of the hinge region through amino acid deletion, addition, or substitution is well-known to those skilled in the art.

In one embodiment, the anti-c-Met antibody or an antigen-binding fragment thereof may be modified by the deletion, insertion, addition, or substitution of at least one amino acid residue on the amino acid sequence of the hinge region so that it exhibit enhanced antigen-binding efficiency. For example, the antibody may include a hinge region including the amino acid sequence of SEQ ID NO: 100 (U7-HC6), 101 (U6-HC7), 102 (U3-HC9), 103 (U6-HC8), or 104 (U8-HC5), or a hinge region including the amino acid sequence of SEQ ID NO: 105 (non-modified human hinge). In particular, the hinge region has the amino acid sequence of SEQ ID NO: 100 or 101.

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

In one embodiment, the anti-c-Met antibody may be a monoclonal antibody. The monoclonal antibody may be produced by the hybridoma cell line deposited with Accession No. KCLRF-BP-00220, which binds specifically to the extracellular region of c-Met protein (see Korean Patent Publication No. 2011-0047698, the disclosure of which is incorporated in its entirety herein by reference).

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

In the anti-c-Met antibody, the rest portion of the light chain and the heavy chain portion excluding the CDRs, the light chain variable region, and the heavy chain variable region as defined above, that is the light chain constant region and the heavy chain constant region, may be those from any subtype of immunoglobulin (e.g., IgA, IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), IgM, and the like).

By way of further example, the anti-c-Met antibody or the antibody fragment may include:

a heavy chain including the amino acid sequence selected from the group consisting of the amino acid sequence of SEQ ID NO: 62 (wherein the amino acid sequence from amino acid residues from the 1st to 17th positions is a signal peptide), or the amino acid sequence from the 18th to 462nd positions of SEQ ID NO: 62, the amino acid sequence of SEQ ID NO: 64 (wherein the amino acid sequence from the 1st to 17th positions is a signal peptide), the amino acid sequence from the 18th to 461st positions of SEQ ID NO: 64, the amino acid sequence of SEQ ID NO: 66 (wherein the amino acid sequence from the 1st to 17th positions is a signal peptide), and the amino acid sequence from the 18th to 460th positions of SEQ ID NO: 66; and

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

For example, the anti-c-Met antibody may be selected from the group consisting of:

an antibody including a heavy chain including the amino acid sequence of SEQ ID NO: 62 or the amino acid sequence from the 18th to 462nd positions of SEQ ID NO: 62 and a light chain including the amino acid sequence of SEQ ID NO: 68 or the amino acid sequence from the 21st to 240th positions of SEQ ID NO: 68;

an antibody including a heavy chain including the amino acid sequence of SEQ ID NO: 64 or the amino acid sequence from the 18th to 461st positions of SEQ ID NO: 64 and a light chain including the amino acid sequence of SEQ ID NO: 68 or the amino acid sequence from the 21st to 240th positions of SEQ ID NO: 68;

an antibody including a heavy chain including the amino acid sequence of SEQ ID NO: 66 or the amino acid sequence from the 18th to 460th positions of SEQ ID NO: 66 and a light chain including the amino acid sequence of SEQ ID NO: 68 or the amino acid sequence from the 21st to 240th positions of SEQ ID NO: 68;

an antibody including a heavy chain including the amino acid sequence of SEQ ID NO: 62 or the amino acid sequence from the 18th to 462nd positions of SEQ ID NO: 62 and a light chain including the amino acid sequence of SEQ ID NO: 70 or the amino acid sequence from the 21st to 240th positions of SEQ ID NO: 70;

an antibody including a heavy chain including the amino acid sequence of SEQ ID NO: 64 or the amino acid sequence from the 18th to 461st positions of SEQ ID NO: 64 and a light chain including the amino acid sequence of SEQ ID NO: 70 or the amino acid sequence from the 21st to 240th positions of SEQ ID NO: 70;

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

an antibody including a heavy chain including the amino acid sequence of SEQ ID NO: 62 or the amino acid sequence from the 18th to 462nd positions of SEQ ID NO: 62 and a light chain including the amino acid sequence of SEQ ID NO: 108;

an antibody including a heavy chain including the amino acid sequence of SEQ ID NO: 64 or the amino acid sequence from the 18th to 461st positions of SEQ ID NO: 64 and a light chain including the amino acid sequence of SEQ ID NO: 108; and

an antibody including a heavy chain including the amino acid sequence of SEQ ID NO: 66 or the amino acid sequence from the 18th to 460th positions of SEQ ID NO: 66 and a light chain including the amino acid sequence of SEQ ID NO: 108.

According to an embodiment, the anti-c-Met antibody may include a heavy chain including the amino acid sequence from the 18th to 460th positions of SEQ ID NO: 66 and a light chain including the sequence from the 21st to 240th positions of SEQ ID NO: 68, or a heavy chain including the amino acid sequence from the 18th to 460th positions of SEQ ID NO: 66 and a light chain including the sequence of SEQ ID NO: 108.

The polypeptide of SEQ ID NO: 70 is a light chain including human kappa (K) constant region, and the polypeptide with the amino acid sequence of SEQ ID NO: 68 is a polypeptide obtained by replacing histidine at position 62 (corresponding to position 36 of SEQ ID NO: 68 according to kabat numbering) of the polypeptide with the amino acid sequence of SEQ ID NO: 70 with tyrosine. The production yield of the antibodies may be increased by the replacement. The polypeptide with the amino acid sequence of SEQ ID NO: 108 is a polypeptide obtained by replacing serine at position 32 (position 27e according to kabat numbering in the amino acid sequence from amino acid residues 21 to 240 of SEQ ID NO: 68; positioned within CDR-L1) with tryptophan. By such replacement, antibodies and antibody fragments including such sequences exhibits increased activities, such as c-Met biding affinity, c-Met degradation activity, Akt phosphorylation inhibition, and the like.

In another embodiment, the anti-c-Met antibody may include a light chain complementarity determining region including the amino acid sequence of SEQ ID NO: 106, a light chain variable region including the amino acid sequence of SEQ ID NO: 107, or a light chain including the amino acid sequence of SEQ ID NO: 108.

The anti-c-Met/anti-EGFR/anti-HER3 multispecific antibody can not only inhibit the activity of c-Met, EGFR and HER3 by the degradation activity of anti-c-Met antibody, but also fundamentally block them by reducing the total amounts of c-Met, EGFR and HER3. Accordingly, the anti-c-Met/anti-EGFR/anti-HER3 multispecific antibody can remain effective even when applied to patients who have developed resistance against pre-existing anti-HER3 or anti-EGFR antibodies.

Another embodiment provides a pharmaceutical composition including the anti-c-Met/anti-EGFR/anti-HER3 multispecific antibody or an antigen-binding fragment thereof as an active ingredient. Another embodiment provides a pharmaceutical composition including the anti-c-Met/anti-EGFR/anti-HER3 multispecific antibody as an active ingredient.

Another embodiment provides a method of prevention and/or treatment a cancer, including administering a pharmaceutical effective amount of the anti-c-Met/anti-EGFR/anti-HER3 multispecific antibody or an antigen-binding fragment thereof to a patient in need of the prevention and/or treatment of the cancer. Another embodiment provides a method of prevention and/or treatment a cancer, including administering a pharmaceutical effective amount of the anti-c-Met/anti-HER3 bispecific antibody to a patient in need of the prevention and/or treatment of the cancer. The method of prevention and/or treatment a cancer may further comprise a step of identifying the patient in need of the prevention and/or treatment of the cancer, prior to the step of administering. For instance, a patient in need of the prevention and/or treatment of the cancer may be a subject diagnosed with cancer by a healthcare professional or determined as being at risk of cancer by a healthcare professional.

Another embodiment provides a use of the anti-c-Met/anti-EGFR/anti-HER3 multispecific antibody for preventing and/or treating a cancer.

The cancer may be a solid cancer or hematological cancer and for instance, may be, but not limited to, one or more selected from the group consisting of squamous cell carcinoma, small-cell lung cancer, non-small-cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, peritoneal carcinoma, skin cancer, melanoma in the skin or eyeball, rectal cancer, cancer near the anus, esophagus cancer, small intestinal tumor, endocrine gland cancer, parathyroid cancer, adrenal cancer, soft-tissue sarcoma, urethral cancer, chronic or acute leukemia, lymphocytic lymphoma, hepatoma, gastric cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular adenoma, breast cancer, colon cancer, large intestine cancer, endometrial carcinoma or uterine carcinoma, salivary gland tumor, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, head or neck cancer, brain cancer, and the like. In particular, the cancer may be cancer having resistance against pre-existing anticancer drugs, for example, antagonists against HER3.

In the pharmaceutical composition or method, the pharmaceutically effective amount of the anti-EGFR/anti-HER3 bispecific antibody or an antigen-binding fragment thereof or the anti-c-Met/anti-EGFR/anti-HER3 multispecific antibody may be administered along with at least one additive selected from the group consisting of a pharmaceutically acceptable carriers, diluents, and excipients.

The pharmaceutically acceptable carrier to be included in the composition may be those commonly used for the formulation of antibodies, which may be one or more selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginates, gelatin, calcium silicate, micro-crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, and mineral oil, but are not limited thereto. The pharmaceutical composition may further include one or more selected from the group consisting of a lubricant, a wetting agent, a sweetener, a flavor enhancer, an emulsifying agent, a suspension agent, and preservative.

The pharmaceutical composition or the anti-EGFR/anti-HER3 bispecific antibody or an antigen-binding fragment thereof or the anti-c-Met/anti-EGFR/anti-HER3 multispecific bispecific antibody may be administered orally or parenterally. The parenteral administration may include intravenous injection, subcutaneous injection, muscular injection, intraperitoneal injection, endothelial administration, local administration, intranasal administration, intrapulmonary administration, and rectal administration. Since oral administration leads to digestion of proteins or peptides, an active ingredient in the compositions for oral administration must be coated or formulated to prevent digestion in stomach. In addition, the compositions may be administered using an optional device that enables an active substance to be delivered to target cells.

A suitable dosage of the pharmaceutical composition, the anti-EGFR/anti-HER3 bispecific antibody or an antigen-binding fragment thereof, or the anti-c-Met/anti-EGFR/anti-HER3 multispecific antibody may be prescribed in a variety of ways, depending on factors such as formulation methods, administration methods, age of patients, body weight, gender, pathologic conditions, diets, administration time, administration route, excretion speed, and reaction sensitivity. A desirable dosage of the pharmaceutical composition or the anti-c-Met/anti-EGFR/anti-HER3 multispecific antibody may be in the range of about 0.001 to about 100 mg/kg for an adult. For example, the suitable dosage of the pharmaceutical composition, the anti-HER3 antibody or an antigen-binding fragment thereof, or the anti-c-Met/anti-EGFR/anti-HER3 multispecific antibody may be about 0.001 to about 1000 mg/kg, about 0.01 to about 100 mg/kg, or about 0.1 to about 50 mg/kg, per a day, but not be limited thereto. The term “pharmaceutically effective amount” used herein refers to an amount of the active ingredient (i.e., the anti-EGFR/anti-HER3 bispecific antibody or an antigen-binding fragment thereof, or the anti-c-Met/anti-EGFR/anti-HER3 multispecific antibody) exhibiting effects in preventing or treating cancer, and may be properly determined in a variety of ways, depending on factors such as formulation methods, administration methods, age of patients, body weight, gender, pathologic conditions, diets, administration time, administration route, excretion speed, and reaction sensitivity.

The pharmaceutical composition or the anti-c-Met/anti-HER3 bispecific antibody may be formulated with a pharmaceutically acceptable carrier and/or excipient into a unit or a multiple dosage form by a method easily carried out by a skilled person in the pertinent art. The dosage form may be a solution in oil or an aqueous medium, a suspension, syrup, an emulsifying solution, an extract, powder, granules, a tablet, or a capsule, and may further include a dispersing or a stabilizing agent.

In addition, the pharmaceutical composition or the anti-c-Met/anti-HER3 bispecific antibody may be administered as an individual drug, or together with other drugs, and may be administered sequentially or simultaneously with pre-existing drugs.

Since the pharmaceutical composition includes an antibody or an antigen binding fragment thereof, it may be formulated as an immunoliposome. The liposome containing an antibody may be prepared using a well-known method in the pertinent art. The immunoliposome is a lipid composition including phosphatidylcholine, cholesterol, and polyethyleneglycol-derivatized phosphatidylethanolamine, and may be prepared by a reverse phase evaporation method. For example, Fab′ fragments of an antibody may be conjugated to the liposome through a disulfide exchange reaction. A chemical drug such as doxorubicin may be additionally included in the liposome.

The subject to which the pharmaceutical composition is administered or the patient to which the prevention and/treatment method is applied may be mammals, for example, primates such as humans and monkeys, or rodents such as rats and mice, but are not be limited thereto. The subject or the patient may be a cancer patient having resistance against pre-existing anticancer drugs, for example, antagonists against the target cell membrane proteins.

Another embodiment provides a polynucleotide encoding a polypeptide including one amino acid sequence or a combination of two or more amino acid sequences selected from the group consisting of SEQ ID NO: 109 to SEQ ID NO: 114. In a particular embodiment, the polynucleotide may encode a polypeptide including the amino acid sequence of SEQ ID NO: 115, a polypeptide including the amino acid sequence of SEQ ID NO: 116, or a combination thereof. Another embodiment provides a recombinant vector including the polynucleotide. Another embodiment provides a recombinant cell transfected with the recombinant vector.

The term “vector” used herein refers to a means for expressing a target gene in a host cell. For example, it includes a plasmid vector, a cosmid vector, and a virus vector such as a bacteriophage vector, an adenovirus vector, a retrovirus vector and an adeno-associated virus vector. Suitable recombinant vectors may be constructed by manipulating plasmids often used in the art (for example, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, pUC19, and the like), a phage (for example, λgt4λB, λ-Charon, 1λΔz1, M13, and the like), or a virus (for example, SV40, and the like), but not be limited thereto.

In the recombinant vector, the polynucleotides may be operatively linked to a promoter. The term “operatively linked” used herein refers to a functional linkage between a nucleotide expression regulating sequence (for example, a promoter sequence) and other nucleotide sequences. Thus, the regulating sequence may regulate the transcription and/or translation of the other nucleotide sequences by being operatively linked.

The recombinant vector may be constructed typically for either cloning or expression. The expression vector may be any ordinary vectors known in the pertinent art for expressing an exogenous protein in plants, animals, or microorganisms. The recombinant vector may be constructed using various methods known in the art.

The recombinant vector may be constructed using a prokaryotic cell or a eukaryotic cell as a host. For example, when a prokaryotic cell is used as a host cell, the expression vector used generally includes a strong promoter capable of initiating transcription (for example, pLλ promoter, CMV promoter, trp promoter, lac promoter, tac promoter, T7 promoter, and the like), a ribosome binding site for initiating translation, and a transcription/translation termination sequence. When a eukaryotic cell is used as a host cell, the vector used generally includes the origin of replication acting in the eukaryotic cell, for example, a f1 replication origin, a SV40 replication origin, a pMB1 replication origin, an adeno replication origin, an AAV replication origin, or a BBV replication origin, but is not limited thereto. A promoter in an expression vector for a eukaryotic host cell may be a promoter derived from the genomes of mammalian cells (for example, a metallothionein promoter, and the like) or a promoter derived from mammalian viruses (for example, an adenovirus late promoter, a vaccinia virus 7.5K promoter, a SV40 promoter, a cytomegalovirus promoter, a tk promoter of HSV, and the like). A transcription termination sequence in an expression vector for a eukaryotic host cell may be, in general, a polyadenylation sequence.

The recombinant cell may be those obtained by transfecting the recombinant vector into a suitable host cell. Any host cells known in the pertinent art to enable stable and continuous cloning or expression of the recombinant vector may be used as the hose cell. Suitable prokaryotic host cells may be one or more selected from E. coli JM109, E. coli BL21, E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus species strains such as Bacillus subtillis, or Bacillus thuringiensis, intestinal bacteria and strains such as Salmonella typhymurum, Serratia marcescens, and various Pseudomonas species. Suitable eukaryotic host cells to be transformed may be one or more selected from yeasts, such as Saccharomyces cerevisiae, insect cells, plant cells, and animal cells, for example, Sp2/0, Chinese hamster ovary (CHO) K1, CHO DG44, PER.C6, W138, BHK, COS-7, 293, HepG2, Huh7, 3T3, RIN, and MDCK cell lines, but not be limited thereto.

The polynucleotide or the recombinant vector including the same may be transferred (transfected) into a host cell by using known transfer methods. Suitable transfer methods for prokaryotic host cells may include a method using CaCl2 and electroporation. Suitable transfer methods for eukaryotic host cells may include microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, and gene bombardment, but are not limited thereto.

A transformed host cell may be selected using a phenotype expressed by a selected marker by any methods known in the art. For example, if the selected marker is a gene that is resistant to a specific antibiotic, a transformant may be easily selected by being cultured in a medium including the antibiotic.

One or more embodiments of the present invention will now be described in further detail with reference to the following Examples. However, these examples are for the illustrative purposes only and are not intended to limit the scope of the invention.

EXAMPLES

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

1.1. Production of “AbF46”, a Mouse Antibody to c-Met

1.1.1. Immunization of Mouse

To obtain immunized mice necessary for the development of a hybridoma cell line, each of five BALB/c mice (Japan SLC, Inc.), 4 to 6 weeks old, was intraperitoneally injected with a mixture of 100 μg of human c-Met/Fc fusion protein (R&D Systems) and one volume of complete Freund's adjuvant. Two weeks after the injection, a second intraperitoneal injection was conducted on the same mice with a mixture of 50 μg of human c-Met/Fc protein and one volume of incomplete Freund's adjuvant. One week after the second immunization, the immune response was finally boosted. Three days later, blood was taken from the tails of the mice and the sera were 1/1000 diluted in PBS and used to examine a titer of antibody to c-Met by ELISA. Mice found to have a sufficient antibody titer were selected for use in the cell fusion process.

1.1.2. Cell Fusion and Production of Hybridoma

Three days before cell fusion, BALB/c mice (Japan SLC, Inc.) were immunized with an intraperitoneal injection of a mixture of 50 μg of human c-Met/Fc fusion protein and one volume of PBS. The immunized mice were anesthetized before excising the spleen from the left half of the body. The spleen was meshed to separate splenocytes which were then suspended in a culture medium (DMEM, GIBCO, Invitrogen). The cell suspension was centrifuged to recover the cell layer. The splenocytes thus obtained (1×108 cells) were mixed with myeloma cells (Sp2/0) (1×108 cells), followed by spinning to give a cell pellet. The cell pellet was slowly suspended, treated with 45% polyethylene glycol (PEG) (1 mL) in DMEM for 1 min at 37° C., and supplemented with 1 mL of DMEM. To the cells was added 10 mL of DMEM over 10 min, after which incubation was conducted in a water bath at 37° C. for 5 min. Then the cell volume was adjusted to 50 mL before centrifugation. The cell pellet thus formed was resuspended at a density of 1˜2×105 cells/mL in a selection medium (HAT medium) and 0.1 mL of the cell suspension was allocated to each well of 96-well plates which were then incubated at 37° C. in a CO2 incubator to establish a hybridoma cell population.

1.1.3. Selection of Hybridoma Cells Producing Monoclonal Antibodies to c-Met Protein

From the hybridoma cell population established in Reference Example 1.1.2, hybridoma cells which showed a specific response to c-Met protein were screened by ELISA using human c-Met/Fc fusion protein and human Fc protein as antigens.

Human c-Met/Fc fusion protein was seeded in an amount of 50 μL (2 μg/mL)/well to microtiter plates and allowed to adhere to the surface of each well. The antibody that remained unbound was removed by washing. For use in selecting the antibodies that do not bind c-Met but recognize Fc, human Fc protein was attached to the plate surface in the same manner.

The hybridoma cell culture obtained in Reference Example 1.1.2 was added in an amount of 50 μL to each well of the plates and incubated for 1 hour. The cells remaining unreacted were washed out with a sufficient amount of Tris-buffered saline and Tween 20 (TBST). Goat anti-mouse IgG-horseradish peroxidase (HRP) was added to the plates and incubated for 1 hour at room temperature. The plates were washed with a sufficient amount of TBST, followed by reacting the peroxidase with a substrate (OPD). Absorbance at 450 nm was measured on an ELISA reader.

Hybridoma cell lines which secrete antibodies that specifically and strongly bind to human c-Met but not human Fc were selected repeatedly. From the hybridoma cell lines obtained by repeated selection, a single clone producing a monoclonal antibody was finally separated by limiting dilution. The single clone of the hybridoma cell line producing the monoclonal antibody was deposited with the Korean Cell Line Research Foundation, an international depository authority located at Yungun-Dong, Jongno-Gu, Seoul, Korea, on Oct. 9, 2009, with Accession No. KCLRF-BP-00220 according to the Budapest Treaty (refer to Korean Patent Laid-Open Publication No. 2011-0047698).

1.1.4. Production and Purification of Monoclonal Antibody

The hybridoma cell line obtained in Reference Example 1.1.3 was cultured in a serum-free medium, and the monoclonal antibody (AbF46) was produced and purified from the cell culture.

First, the hybridoma cells cultured in 50 mL of a medium (DMEM) supplemented with 10% (v/v) FBS were centrifuged and the cell pellet was washed twice or more with 20 mL of PBS to remove the FBS therefrom. Then, the cells were resuspended in 50 mL of DMEM and incubated for 3 days at 37° C. in a CO2 incubator.

After the cells were removed by centrifugation, the supernatant was stored at 4° C. before use or immediately used for the separation and purification of the antibody. An AKTA system (GE Healthcare) equipped with an affinity column (Protein G agarose column; Pharmacia, USA) was used to purify the antibody from 50 to 300 mL of the supernatant, followed by concentration with an filter (Amicon). The antibody in PBS was stored before use in the following examples.

1.2. Construction of chAbF46, a Chimeric Antibody to c-Met

A mouse antibody is apt to elicit immunogenicity in humans. To solve this problem, chAbF46, a chimeric antibody, was constructed from the mouse antibody AbF46 produced in Experimental Example 1.1.4 by replacing the constant region, but not the variable region responsible for antibody specificity, with an amino sequence of the human IgG1 antibody.

In this regard, a gene was designed to include the nucleotide sequence of “EcoRI-signal sequence-VH-NheI-CH-TGA-XhoI” (SEQ ID NO: 38) for a heavy chain and the nucleotide sequence of “EcoRI-signal sequence-VL-BsiWI-CL-TGA-XhoI” (SEQ ID NO: 39) for a light chain and synthesized. Then, a DNA fragment having the heavy chain nucleotide sequence (SEQ ID NO: 38) and a DNA fragment having the light chain nucleotide sequence (SEQ ID NO: 39) were digested with EcoRI (NEB, R0101S) and XhoI (NEB, R0146S) before cloning into a pOptiVEC™-TOPO TA Cloning Kit enclosed in an OptiCHO™ Antibody Express Kit (Cat no. 12762-019, Invitrogen), and a pcDNA™3.3-TOPO TA Cloning Kit (Cat no. 8300-01), respectively.

Each of the constructed vectors was amplified using Qiagen Maxiprep kit (Cat no. 12662), and a transient expression was performed using Freestyle™ MAX 293 Expression System (invitrogen). 293 F cells were used for the expression and cultured in FreeStyle™ 293 Expression Medium in a suspension culture manner. At one day before the transient expression, the cells were provided in the concentration of 5×105 cells/ml, and after 24 hours, when the cell number reached to 1×106 cells/ml, the transient expression was performed. A transfection was performed by a liposomal reagent method using Freestyle™ MAX reagent (invitrogen), wherein in a 15 ml tube, the DNA was provided in the mixture ratio of 1:1 (heavy chain DNA:light chain DNA) and mixed with 2 ml of OptiPro™ SFM (Invitrogen) (A), and in another 15 ml tube, 100 μl (microliter) of Freestyle™ MAX reagent and 2 ml of OptiPro™ SFM were mixed (B), followed by mixing (A) and (B) and incubating for 15 minutes. The obtained mixture was slowly mixed with the cells provided one day before the transient expression. After completing the transfection, the cells were incubated in 130 rpm incubator for 5 days under the conditions of 37° C., 80% humidity, and 8% CO2.

Afterwards, the cells were incubated in DMEM supplemented with 10% (v/v) FBS for 5 hours at 37° C. under a 5% CO2 condition and then in FBS-free DMEM for 48 hours at 37° C. under a 5% CO2 condition.

After centrifugation, the supernatant was applied to AKTA prime (GE Healthcare) to purify the antibody. In this regard, 100 mL of the supernatant was loaded at a flow rate of 5 mL/min to AKTA Prime equipped with a Protein A column (GE healthcare, 17-0405-03), followed by elution with an IgG elution buffer (Thermo Scientific, 21004). The buffer was exchanged with PBS to purify a chimeric antibody AbF46 (hereinafter referred to as “chAbF46”).

1.3. Construction of Humanized Antibody huAbF46 from Chimeric Antibody chAbF46

1.3.1. Heavy Chain Humanization

To design two domains H1-heavy and H3-heavy, human germline genes which share the highest identity/homology with the VH gene of the mouse antibody AbF46 purified in Reference Example 1.2 were analyzed. An Ig BLAST (NCBI online IGBLAST tool) result revealed that VH3-71 has an identity/identity/homology of 83% at the amino acid level. CDR-H1, CDR-H2, and CDR-H3 of the mouse antibody AbF46 were defined according to Kabat numbering. A design was made to introduce the CDR of the mouse antibody AbF46 into the framework of VH3-71. Hereupon, back mutations to the amino acid sequence of the mouse AbF46 were conducted at positions 30 (S→T), 48 (V→L), 73 (D→N), and 78 (T→L). Then, H1 was further mutated at positions 83 (R→K) and 84 (A→T) to finally establish H1-heavy (SEQ ID NO: 40) and H3-heavy (SEQ ID NO: 41).

For use in designing H4-heavy, human antibody frameworks were analyzed by a BLAST search. The result revealed that the VH3 subtype, known to be most stable, is very similar in framework and sequence to the mouse antibody AbF46. CDR-H1, CDR-H2, and CDR-H3 of the mouse antibody AbF46 were defined according to Kabat numbering and introduced into the VH3 subtype to construct H4-heavy (SEQ ID NO: 42).

1.3.2. Light Chain Humanization

To design two domains H1-light (SEQ ID NO: 43) and H2-light (SEQ ID NO: 44), human germline genes which share the highest identity/homology with the VH gene of the mouse antibody AbF46 were analyzed. An Ig BLAST search result revealed that VK4-1 has a identity/homology of 75% at the amino acid level. CDR-L1, CDR-L2, and CDR-L3 of the mouse antibody AbF46 were defined according to Kabat numbering. A design was made to introduce the CDR of the mouse antibody AbF46 into the framework of VK4-1. Hereupon, back mutations to the amino acid sequence of the mouse AbF46 were conducted at positions 36 (Y→H), 46 (L→M), and 49 (Y→I). Only one back mutation was conducted at position 49 (Y→I) on H2-light.

To design H3-light (SEQ ID NO: 45), human germline genes which share the highest identity/homology with the VL gene of the mouse antibody AbF46 were analyzed by a search for BLAST. As a result, VK2-40 was selected. VL and VK2-40 of the mouse antibody AbF46 were found to have a identity/homology of 61% at an amino acid level. CDR-L1, CDR-L2, and CDR-L3 of the mouse antibody were defined according to Kabat numbering and introduced into the framework of VK4-1. Back mutations were conducted at positions 36 (Y→H), 46 (L→M), and 49 (Y→I) on H3-light.

For use in designing H4-light (SEQ ID NO: 46), human antibody frameworks were analyzed. A Blast search revealed that the Vk1 subtype, known to be the most stable, is very similar in framework and sequence to the mouse antibody AbF46. CDR-L1, CDR-L2, and CDR-L3 of the mouse antibody AbF46 were defined according to Kabat numbering and introduced into the Vk1 subtype. Hereupon, back mutations were conducted at positions 36 (Y→H), 46 (L→M), and 49 (Y→I) on H4-light.

Thereafter, DNA fragments having the heavy chain nucleotide sequences (H1-heavy: SEQ ID NO: 47, H3-heavy: SEQ ID NO: 48, H4-heavy: SEQ ID NO: 49) and DNA fragments having the light chain nucleotide sequences (H1-light: SEQ ID NO: 50, H2-light: SEQ ID NO: 51, H3-light: SEQ ID NO: 52, H4-light: SEQ ID NO: 53) were digested with EcoRI (NEB, R0101S) and XhoI (NEB, R0146S) before cloning into a pOptiVEC™-TOPO TA Cloning Kit enclosed in an OptiCHO™ Antibody Express Kit (Cat no. 12762-019, Invitrogen) and a pcDNA™3.3-TOPO TA Cloning Kit (Cat no. 8300-01), respectively, so as to construct recombinant vectors for expressing a humanized antibody.

Each of the constructed vectors was amplified using Qiagen Maxiprep kit (Cat no. 12662), and a transient expression was performed using Freestyle™ MAX 293 Expression System (invitrogen). 293 F cells were used for the expression and cultured in FreeStyle™ 293 Expression Medium in a suspension culture manner. At one day before the transient expression, the cells were provided in the concentration of 5×105 cells/ml, and after 24 hours, when the cell number reached to 1×106 cells/ml, the transient expression was performed. A transfection was performed by a liposomal reagent method using Freestyle™ MAX reagent (invitrogen), wherein in a 15 ml tube, the DNA was provided in the mixture ratio of 1:1 (heavy chain DNA:light chain DNA) and mixed with 2 ml of OptiPro™ SFM (invitrogen) (A), and in another 15 ml tube, 100 ul (microliter) of Freestyle™ MAX reagent and 2 ml of OptiPro™ SFM were mixed (B), followed by mixing (A) and (B) and incubating for 15 minutes. The obtained mixture was slowly mixed with the cells provided one day before the transient expression. After completing the transfection, the cells were incubated in 130 rpm incubator for 5 days under the conditions of 37° C., 80% humidity, and 8% CO2.

After centrifugation, the supernatant was applied to AKTA prime (GE Healthcare) to purify the antibody. In this regard, 100 mL of the supernatant was loaded at a flow rate of 5 mL/min to AKTA Prime equipped with a Protein A column (GE healthcare, 17-0405-03), followed by elution with an IgG elution buffer (Thermo Scientific, 21004). The buffer was exchanged with PBS to purify a humanized antibody AbF46 (hereinafter referred to as “huAbF46”). The humanized antibody huAbF46 used in the following examples included a combination of H4-heavy (SEQ ID NO: 42) and H4-light (SEQ ID NO: 46).

1.4. Construction of scFV Library of huAbF46 Antibody

For use in constructing an scFv of the huAbF46 antibody from the heavy and light chain variable regions of the huAbF46 antibody, a gene was designed to have the structure of “VH-linker-VL” for each of the heavy and the light chain variable region, with the linker including the amino acid sequence “GLGGLGGGGSGGGGSGGSSGVGS” (SEQ ID NO: 54). A polynucleotide sequence (SEQ ID NO: 55) encoding the designed scFv of huAbF46 was synthesized in Bioneer and an expression vector for the polynucleotide had the nucleotide sequence of SEQ ID NO: 56.

After expression, the product was found to exhibit specificity to c-Met.

1.5. Construction of Library Genes for Affinity Maturation

1.5.1. Selection of Target CDRs and Synthesis of Primers

The affinity maturation of huAbF46 was achieved. First, six complementary determining regions (CDRs) were defined according to Kabat numbering. The CDRs are given in Table 2, below.


TABLE 2
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)

For use in the introduction of random sequences into the CDRs of the antibody, primers were designed as follows. Conventionally, N codons were utilized to introduce bases at the same ratio (25% A, 25% G, 25% C, 25% T) into desired sites of mutation. In this experiment, the introduction of random bases into the CDRs of huAbF46 was conducted in such a manner that, of the three nucleotides per codon in the wild-type polynucleotide encoding each CDR, the first and second nucleotides conserved over 85% of the entire sequence while the other three nucleotides were introduced at the same percentage (each 5%) and that the same possibility was imparted to the third nucleotide (33% G, 33% C, 33% T).

1.5.2. Construction of a Library of huAbF46 Antibodies and Affinity for c-Met

The construction of antibody gene libraries through the introduction of random sequences was carried out using the primers synthesized in the same manner as in Reference Example 1.5.1. Two PCR products were obtained using a polynucleotide covering the scFV of huAbF46 as a template, and were subjected to overlap extension PCR to give scFv library genes for huAbF46 antibodies in which only desired CDRs were mutated. Libraries targeting each of the six CDRs prepared from the scFV library genes were constructed.

The affinity for c-Met of each library was compared to that of the wildtype. Most libraries were lower in affinity for c-Met, compared to the wild-type. The affinity for c-Met was retained in some mutants.

1.6. Selection of Antibody with Improved Affinity from Libraries

After maturation of the affinity of the constructed libraries for c-Met, the nucleotide sequence of scFv from each clone was analyzed. The nucleotide sequences thus obtained are summarized in Table 3 and were converted into IgG forms. Four antibodies which were respectively produced from clones L3-1, L3-2, L3-3, and L3-5 were used in the subsequent experiments.


TABLE 3
Library
con-
Clone
structed
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. Conversion of Selected Antibodies into IgG

Respective polynucleotides encoding heavy chains of the four selected antibodies were designed to have the structure of “EcoRI-signal sequence-VH-NheI-CH-XhoI” (SEQ ID NO: 38). The heavy chains of huAbF46 antibodies were used as they were because their amino acids were not changed during affinity maturation. In the case of the hinge region, however, the U6-HC7 hinge (SEQ ID NO: 57) was employed instead of the hinge of human IgG1. Genes were also designed to have the structure of “EcoRI-signal sequence-VL-BsiWI-CL-XhoI” for the light chain. Polypeptides encoding light chain variable regions of the four antibodies which were selected after the affinity maturation were synthesized in Bioneer. Then, a DNA fragment having the heavy chain nucleotide sequence (SEQ ID NO: 38) and DNA fragments having the light chain nucleotide sequences (DNA fragment including L3-1-derived CDR-L3: SEQ ID NO: 58, DNA fragment including L3-2-derived CDR-L3: SEQ ID NO: 59, DNA fragment including L3-3-derived CDR-L3: SEQ ID NO: 60, and DNA fragment including L3-5-derived CDR-L3: SEQ ID NO: 61) were digested with EcoRI (NEB, R0101S) and XhoI (NEB, R0146S) before cloning into a pOptiVEC™-TOPO TA Cloning Kit enclosed in an OptiCHO™ Antibody Express Kit (Cat no. 12762-019, Invitrogen) and a pcDNA™3.3-TOPO TA Cloning Kit (Cat no. 8300-01), respectively, so as to construct recombinant vectors for expressing affinity-matured antibodies.

Each of the constructed vectors was amplified using Qiagen Maxiprep kit (Cat no. 12662), and a transient expression was performed using Freestyle™ MAX 293 Expression System (invitrogen). 293 F cells were used for the expression and cultured in FreeStyle™ 293 Expression Medium in a suspension culture manner. At one day before the transient expression, the cells were provided in the concentration of 5×105 cells/ml, and after 24 hours, when the cell number reached to 1×106 cells/ml, the transient expression was performed. A transfection was performed by a liposomal reagent method using Freestyle™ MAX reagent (invitrogen), wherein in a 15 ml tube, the DNA was provided in the mixture ratio of 1:1 (heavy chain DNA:light chain DNA) and mixed with 2 ml of OptiPro™ SFM (invtrogen) (A), and in another 15 ml tube, 100 μl (microliter) of Freestyle™ MAX reagent and 2 ml of OptiPro™ SFM were mixed (B), followed by mixing (A) and (B) and incubating for 15 minutes. The obtained mixture was slowly mixed with the cells provided one day before the transient expression. After completing the transfection, the cells were incubated in 130 rpm incubator for 5 days under the conditions of 37° C., 80% humidity, and 8% CO2.

After centrifugation, the supernatant was applied to AKTA prime (GE Healthcare) to purify the antibody. In this regard, 100 mL of the supernatant was loaded at a flow rate of 5 mL/min to AKTA Prime equipped with a Protein A column (GE healthcare, 17-0405-03), followed by elution with an IgG elution buffer (Thermo Scientific, 21004). The buffer was exchanged with PBS to purify four affinity-matured antibodies (hereinafter referred to as “huAbF46-H4-A1 (L3-1 origin), huAbF46-H4-A2 (L3-2 origin), huAbF46-H4-A3 (L3-3 origin), and huAbF46-H4-A5 (L3-5 origin),” respectively).

1.8. Construction of Constant Region- and/or Hinge Region-Substituted huAbF46-H4-A1

Among the four antibodies selected in Reference Example 1.7, huAbF46-H4-A1 was found to be the highest in affinity for c-Met and the lowest in Akt phosphorylation and c-Met degradation degree. In the antibody, the hinge region, or the constant region and the hinge region, were substituted.

The antibody huAbF46-H4-A1 (U6-HC7) was composed of a heavy chain including the heavy chain variable region of huAbF46-H4-A1, U6-HC7 hinge, and the constant region of human IgG1 constant region, and a light chain including the light chain variable region of huAbF46-H4-A1 and human kappa constant region. The antibody huAbF46-H4-A1 (IgG2 hinge) was composed of a heavy chain including a heavy chain variable region, a human IgG2 hinge region, and a human IgG1 constant region, and a light chain including the light chain variable region of huAbF46-H4-A1 and a human kappa constant region. The antibody huAbF46-H4-A1 (IgG2 Fc) was composed of the heavy chain variable region of huAbF46-H4-A1, a human IgG2 hinge region, and a human IgG2 constant region, and a light chain including the light variable region of huAbF46-H4-A1 and a human kappa constant region. Hereupon, the histidine residue at position 36 on the human kappa constant region of the light chain was changed to tyrosine in all of the three antibodies to increase antibody production.

For use in constructing the three antibodies, a polynucleotide (SEQ ID NO: 63) encoding a polypeptide (SEQ ID NO: 62) composed of the heavy chain variable region of huAbF46-H4-A1, a U6-HC7 hinge region, and a human IgG1 constant region, a polynucleotide (SEQ ID NO: 65) encoding a polypeptide (SEQ ID NO: 64) composed of the heavy chain variable region of huAbF46-H4-A1, a human IgG2 hinge region, and a human IgG1 region, a polynucleotide (SEQ ID NO: 67) encoding a polypeptide (SEQ ID NO: 66) composed of the heavy chain variable region of huAbF46-H4-A1, a human IgG2 region, and a human IgG2 constant region, and a polynucleotide (SEQ ID NO: 69) encoding a polypeptide (SEQ ID NO: 68) composed of the light chain variable region of huAbF46-H4-A1, with a tyrosine residue instead of histidine at position 36, and a human kappa constant region were synthesized in Bioneer. Then, the DNA fragments having heavy chain nucleotide sequences were inserted into a pOptiVEC™-TOPO TA Cloning Kit enclosed in an OptiCHO™ Antibody Express Kit (Cat no. 12762-019, Invitrogen) while DNA fragments having light chain nucleotide sequences were inserted into a pcDNA™3.3-TOPO TA Cloning Kit (Cat no. 8300-01) so as to construct vectors for expressing the antibodies.

Each of the constructed vectors was amplified using Qiagen Maxiprep kit (Cat no. 12662), and a transient expression was performed using Freestyle™ MAX 293 Expression System (invitrogen). 293 F cells were used for the expression and cultured in FreeStyle™ 293 Expression Medium in a suspension culture manner. At one day before the transient expression, the cells were provided in the concentration of 5×105 cells/ml, and after 24 hours, when the cell number reached to 1×106 cells/ml, the transient expression was performed. A transfection was performed by a liposomal reagent method using Freestyle™ MAX reagent (invitrogen), wherein in a 15 ml tube, the DNA was provided in the mixture ratio of 1:1 (heavy chain DNA:light chain DNA) and mixed with 2 ml of OptiPro™ SFM (invtrogen) (A), and in another 15 ml tube, 100 μl (microliter) of Freestyle™ MAX reagent and 2 ml of OptiPro™ SFM were mixed (B), followed by mixing (A) and (B) and incubating for 15 minutes. The obtained mixture was slowly mixed with the cells provided one day before the transient expression. After completing the transfection, the cells were incubated in 130 rpm incubator for 5 days under the conditions of 37° C., 80% humidity, and 8% CO2.

After centrifugation, the supernatant was applied to AKTA prime (GE Healthcare) to purify the antibody. In this regard, 100 mL of the supernatant was loaded at a flow rate of 5 mL/min to AKTA Prime equipped with a Protein A column (GE healthcare, 17-0405-03), followed by elution with IgG elution buffer (Thermo Scientific, 21004). The buffer was exchanged with PBS to finally purify three antibodies (huAbF46-H4-A1 (U6-HC7), huAbF46-H4-A1 (IgG2 hinge), and huAbF46-H4-A1 (IgG2 Fc)). Among the three antibodies, huAbF46-H4-A1 (IgG2 Fc) was selected for the following examples, and name as L3-1Y-IgG2 (or SAIT301).

Example 1: Preparation of Anti-EGFR/Anti-HER3 scFv

An anti-EGFR/anti-HER3 bispecific antibody was designed by inserting a linker (GGGGSGGGGSGGGGS, SEQ ID NO: 121) between a heavy chain variable region of SEQ ID NO: 115 and a light chain variable region of SEQ ID NO: 116. 44th position of the heavy chain variable region of SEQ ID NO: 115 and 100th position of the light chain variable region of SEQ ID NO: 116 were substituted with cysteine. The polynucleotide encoding above designed anti-EGFR/anti-HER3 bispecific antibody designed to be conjugated to C-terminal of heavy chain of the antibody SAIT301 via a linker (GGGGSGGGGSGGGGS, SEQ ID NO: 121). The polynucleotide sequence encoding the designed anti-EGFR/anti-HER3 scFv was synthesized in Bioneer.

The obtained anti-EGFR/anti-HER3 scFv was named DL11f and used for preparing a multispecific antibody.

Example 2: Preparation of Anti-c-Met/Anti-EGFR/Anti-HER3 Multispecific Antibody

The modified anti-EGFR/anti-HER3 scFv, that is DL11f, prepared in the above Example 1 was fused at the c-terminal of Fc of the anti-c-Met antibody L3-1Y-IgG2 (or SAIT301) prepared in the above reference example. The fusion procedures are as follows:

The polynucleotide encoding anti-EGFR/anti-HER3 scFv synthesized in Example 1 was conjugated to C-terminal of heavy chain of the antibody SAIT301 using BamHI/XhoI restriction enzyme. The vector containing the polynucleotide encoding SAIT301 and the vector containing the polynucleotide encoding anti-EGFR/HER3 scFv were treated with BamHI and XhoI under 37° C. for 3 hours, and then ligated each other using T4 ligase. The vector construct was confirmed by DNA sequencing to ensure the correct ligation.

The prepared anti-c-Met/anti-EGFR/anti-HER3 multispecific antibody in which the modified anti-EGFR/anti-HER3 scFv is fused at the C-terminal of anti cMet antibody SAIT301 was named MEH3-01S.

To examine the property of the multispecific antibody MEH3-01S prepared in Example 2, 20 ug of MEH3-01 S was injected into HPLC system (WATERS 2695) equipped with TSKG3000SWXL column (Tosho) at the velocity of 0.5 ml/min, to conduct Size Exclusion Chromatography by HPLC. The obtained results are shown in FIG. 2. In FIG. 2, “1” indicates peak values of soluble dimer and “2” indicates peak values of monomer. As shown in FIG. 2, the multispecific antibody MEH3-01S forms a slight amount of soluble dimer (<1), indicating that it is very stable molecule.

Example 3: Affinity of Anti-c-Met/Anti-EGFR/Anti-HER3 Multispecific Antibody

The affinity of the anti-c-Met/anti-EGFR/anti-HER3 multispecific antibody prepared in Example 2 to EGFR and HER3 was measured. A human Fab binder (GE Healthcare) was immobilized at the surface of a CM5 chip (#BR-1005-30, GE) according to the manufacturer's specifications. About 90 to 120 RU of the MEH3-01S was captured, and the EGFR-Fc(#344-ER, R&D Systems) or HER3-Fc(#344-ER, R&D Systems) was injected at various concentrations into the captured antibody. 10 mM Glycine-HCl (pH 1.5) solution was injected thereto to regenerate the surface.

To examine affinity of the antibody, the data obtained from this experiment was fitted using BIAevaluation software (GE Healthcare, Biacore T100 evaluation software). The obtained results are shown in FIG. 3, wherein it is confirmed that the multispecific antibody binds to both EGFR and HER3 with high affinity, 0.18 mM and <0.1 mM of KD respectively.


TABLE 4
Flow
Rmax
KD
Ka
Kd
U-
T
T
Antigen
Cell
(RU)
(mM)
(1/Ms)
(1/s)
Chi2
Value
(Ka)
(Kd)
EGFR-Fc
#3-#1
97.93
0.18
1.0 × 106
 1.1 × 10−4
4.72
33
5.7 × 102
14
HER3-Fc
#2-#1
101.0
<0.01
3.4 × 106
<1.6 × 10−5
5.16
95
1.1 × 102
5.9

Example 4: Target Degradation Activity of Anti-c-Met/Anti-EGFR/Anti-HER3 Multispecific Antibody

To examine the mechanism of the prepared anti-c-Met/anti-EGFR/anti-HER3 multispecific antibody, human gastric cancer cell line SNU-638 (Korean Cell Line Bank) was incubated in RPMI1640 medium (Gibco) supplemented with 10% (v/v) FBS (Gibco) and 1% (v/v) Penicilin-Streptomycin (Gibco) under the conditions of 5% CO2 and 37° C.

After 1×105 cells were incubated in dish for 24 hours, the cells were treated with the multispecific antibody prepared in Example 2 and cultured for 24 hours. 100 μl lysis buffer (Invitrogen) was added to obtain lysate, then target expression level was determined by western blot assay using anti phospho c-Met (Cell Signaling, #3121), total c-Met (Abcam, ab14571), phospho EGFR (Cell Signaling, #2237), total EGFR (Cell Signaling, #2237), phospho HER3 (Cell Signaling, #4791), and total HER3 (Cell Signaling, #4754) with a ratio of 1:1000. GAPDH (Cell Signaling, #2118) was used as a loading control.

The obtained results are shown in FIG. 3. When HGF, EGF and NGF, which are the ligand of c-Met, EGFR and HER3, respectively, were treated, the addition of anti-cMet antibody SAIT301 remarkably reduced the expression and activation of cMet. In contrast, the addition of commercially available anti-EGFR antibody Erbitux (Merck South Korea) or DL11f did not affect the expression and activation of cMet, and reduced activation of EGFR. The addition of MEH3-01S reduced activation of HER2, as well as both expression and activation of cMet and EGFR.

Example 5: Examination of Cancer Cell Proliferation Inhibition by Anti-c-Met/Anti-EGFR/Anti-HER3 Multispecific Antibody

A human epidermoid carcinoma cell line A431 (ATCC), gastric cancer cell line SNU5 (Korean Cell Line Bank), pancreas cancer cell line BxPC3 (ATCC), lung cancer cell line NCI-H1993 (ATCC), breast cancer cell line HCC1954 (ATCC), liver cancer cell line Huh7 (ATCC), and colon cancer cell line HT29 were incubated in DMEM medium supplemented with 10% (v/v) FBS (Gibco) and 1% (v/v) Penicilin-Streptomycin (Gibco) under the conditions of 5% CO2 and 37° C. For cell proliferation assay, the cell line was sub-cultured at a concentration of 5×103 cell/well in a 96-well plate, which was treated with the multispecific antibody prepared in Example 2 and cultured for 72 hours.

After incubation, cell proliferation degrees were analyzed using Cell Counting Kit-8 assay (Dojindo Molecular Technologies, Gaithersburg, Md.) according to the manufacturer's instructions. In brief, after the incubation for 72 hours, 10 μl (microliter) of CCK8 solution was added to each well and after the additional incubation for 2.5 hours, absorption degrees were read at 450 nm using a microplate reader.

A medium with no antibody was used as a negative control (control), and anti-EGFR/anti-HER3 bispecific antibody (DL11f) treated group, anti-cMet antibody (SAIT301) treated group, co-treated group of anti-cMet antibody and anti-EGFR/anti-HER3 bispecific antibody (S+D), commercially available anti-EGFR antibody (Erbitux) treated group, co-treated group of anti-cMet antibody and anti-EGFR antibody (S+E), commercially available anti-HER2 antibody (Herceptin, Roche South Korea) treated group, and co-treated group of anti-cMet antibody, anti EGFR antibody and anti-HER2 antibody (S+E+H) were each used as positive controls.

The obtained results are shown in FIGS. 4 to 10.

As seen in FIGS. 4-7, the multispecific antibody MEH3-01S showed remarkable increases in cell proliferation inhibitory effects on human epidermoid carcinoma cell line A431 (FIG. 4), pancreas cancer cell line BxPC3 (FIG. 6) and lung cancer cell line NCI-H1993 (FIG. 7), compared to the cases treated individually with DL11f or SAIT301, and co-treatment case S+D. And as seen in FIG. 5, MEH3-01S showed remarkable increases in cell proliferation inhibitory effects on human gastric cancer cell line SNU5, compared to the cases treated individually with Erbitux or SAIT301, and co-treatment case S+E.

As seen in FIG. 8, MEH3-01S showed remarkable increases in cell proliferation inhibitory effects on human breast cancer cell line HCC1954, compared to the cases treated individually with DL11f or SAIT301.

As seen in FIGS. 9 and 10, MEH3-01S showed remarkable increases in cell proliferation inhibitory effects on human liver cancer cell line Huh7 (FIG. 9) and colon cancer cell line HT29 (FIG. 10), compared to the cases treated individually with SAIT301, Erbitux or Herceptin, and co-treatment case of S+E+H.

All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

The use of the terms “a” and “an” and “the” and “at least one” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,”“having,”“including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

<160> NUMBER OF SEQ ID NOS: 124

<210> SEQ ID NO: 1

<211> LENGTH: 5

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic heavy chain CDR1 of AbF46

<400> SEQENCE: 1

Asp Tyr Tyr Met Ser

1 5

<210> SEQ ID NO: 2

<211> LENGTH: 19

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic heavy chain CDR2 of AbF46

<400> SEQENCE: 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> SEQ ID NO: 3

<211> LENGTH: 6

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic heavy chain CDR3 of AbF46

<400> SEQENCE: 3

Asp Asn Trp Phe Ala Tyr

1 5

<210> SEQ ID NO: 4

<211> LENGTH: 6

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic heavy chain CDR1 of c-Met antibody

<220> FEATURE:

<221> NAME/KEY: misc_feature

<222> LOCATION: (1)

<223> OTHER INFORMATION: Xaa is Pro or Ser or absent

<220> FEATURE:

<221> NAME/KEY: misc_feature

<222> LOCATION: (2)

<223> OTHER INFORMATION: Xaa is Glu or Asp

<400> SEQENCE: 4

Xaa Xaa Tyr Tyr Met Ser

1 5

<210> SEQ ID NO: 5

<211> LENGTH: 8

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic heavy chain CDR2 of c-Met antibody

<220> FEATURE:

<221> NAME/KEY: misc_feature

<222> LOCATION: (3)

<223> OTHER INFORMATION: Xaa is Asn or Lys

<220> FEATURE:

<221> NAME/KEY: misc_feature

<222> LOCATION: (4)

<223> OTHER INFORMATION: Xaa is Ala or Val

<220> FEATURE:

<221> NAME/KEY: misc_feature

<222> LOCATION: (7)

<223> OTHER INFORMATION: Xaa is Asn or Thr

<400> SEQENCE: 5

Arg Asn Xaa Xaa Asn Gly Xaa Thr

1 5

<210> SEQ ID NO: 6

<211> LENGTH: 6

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic heavy chain CDR3 of c-Met antibody

<220> FEATURE:

<221> NAME/KEY: misc_feature

<222> LOCATION: (5)

<223> OTHER INFORMATION: Xaa is Ser or Thr

<400> SEQENCE: 6

Asp Asn Trp Leu Xaa Tyr

1 5

<210> SEQ ID NO: 7

<211> LENGTH: 17

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic light chain CDR1 of c-Met antibody

<220> FEATURE:

<221> NAME/KEY: misc_feature

<222> LOCATION: (4)

<223> OTHER INFORMATION: Xaa is His, Arg, Gln or Lys

<220> FEATURE:

<221> NAME/KEY: misc_feature

<222> LOCATION: (9)

<223> OTHER INFORMATION: Xaa is Ser or Trp

<220> FEATURE:

<221> NAME/KEY: misc_feature

<222> LOCATION: (12)

<223> OTHER INFORMATION: Xaa is His or Gln

<220> FEATURE:

<221> NAME/KEY: misc_feature

<222> LOCATION: (13)

<223> OTHER INFORMATION: Xaa is Lys or Asn

<400> SEQENCE: 7

Lys Ser Ser Xaa Ser Leu Leu Ala Xaa Gly Asn Xaa Xaa Asn Tyr Leu

1 5 10 15

Ala

<210> SEQ ID NO: 8

<211> LENGTH: 7

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic light chain CDR2 of c-Met antibody

<220> FEATURE:

<221> NAME/KEY: misc_feature

<222> LOCATION: (2)

<223> OTHER INFORMATION: Xaa is Ala or Gly

<220> FEATURE:

<221> NAME/KEY: misc_feature

<222> LOCATION: (4)

<223> OTHER INFORMATION: Xaa is Thr or Lys

<220> FEATURE:

<221> NAME/KEY: misc_feature

<222> LOCATION: (7)

<223> OTHER INFORMATION: Xaa is Ser or Pro

<400> SEQENCE: 8

Trp Xaa Ser Xaa Arg Val Xaa

1 5

<210> SEQ ID NO: 9

<211> LENGTH: 9

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic light chain CDR3 of c-Met antibody

<220> FEATURE:

<221> NAME/KEY: misc_feature

<222> LOCATION: (1)

<223> OTHER INFORMATION: Xaa is Gly, Ala or Gln

<220> FEATURE:

<221> NAME/KEY: misc_feature

<222> LOCATION: (6)

<223> OTHER INFORMATION: Xaa is Arg, His, Ser, Ala, Gly or Lys

<220> FEATURE:

<221> NAME/KEY: misc_feature

<222> LOCATION: (8)

<223> OTHER INFORMATION: Xaa is Leu, Tyr, Phe or Met

<400> SEQENCE: 9

Xaa Gln Ser Tyr Ser Xaa Pro Xaa Thr

1 5

<210> SEQ ID NO: 10

<211> LENGTH: 17

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic light chain CDR1 of AbF46

<400> SEQENCE: 10

Lys Ser Ser Gln Ser Leu Leu Ala Ser Gly Asn Gln Asn Asn Tyr Leu

1 5 10 15

Ala

<210> SEQ ID NO: 11

<211> LENGTH: 7

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic light chain CDR2 of AbF46

<400> SEQENCE: 11

Trp Ala Ser Thr Arg Val Ser

1 5

<210> SEQ ID NO: 12

<211> LENGTH: 9

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic light chain CDR3 of AbF46

<400> SEQENCE: 12

Gln Gln Ser Tyr Ser Ala Pro Leu Thr

1 5

<210> SEQ ID NO: 13

<211> LENGTH: 9

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic CDR-L3 derived from L3-1 clone

<400> SEQENCE: 13

Gln Gln Ser Tyr Ser Arg Pro Tyr Thr

1 5

<210> SEQ ID NO: 14

<211> LENGTH: 9

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic CDR-L3 derived from L3-2 clone

<400> SEQENCE: 14

Gly Gln Ser Tyr Ser Arg Pro Leu Thr

1 5

<210> SEQ ID NO: 15

<211> LENGTH: 9

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic CDR-L3 derived from L3-3 clone

<400> SEQENCE: 15

Ala Gln Ser Tyr Ser His Pro Phe Ser

1 5

<210> SEQ ID NO: 16

<211> LENGTH: 9

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic CDR-L3 derived from L3-5 clone

<400> SEQENCE: 16

Gln Gln Ser Tyr Ser Arg Pro Phe Thr

1 5

<210> SEQ ID NO: 17

<211> LENGTH: 117

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic heavy chain variable region of anti

c-Met humanized antibody(huAbF46-H4)

<400> SEQENCE: 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> SEQ ID NO: 18

<211> LENGTH: 114

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic light chain variable region of anti

c-Met humanized antibody(huAbF46-H4)

<400> SEQENCE: 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> SEQ ID NO: 19

<211> LENGTH: 114

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic light chain variable region of anti

c-Met humanized antibody(huAbF46-H4)

<400> SEQENCE: 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> SEQ ID NO: 20

<211> LENGTH: 114

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic light chain variable region of anti

c-Met humanized antibody(huAbF46-H4)

<400> SEQENCE: 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> SEQ ID NO: 21

<211> LENGTH: 114

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic light chain variable region of anti

c-Met humanized antibody(huAbF46-H4)

<400> SEQENCE: 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> SEQ ID NO: 22

<211> LENGTH: 6

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic CDR-H1 derived from H11-4 clone

<400> SEQENCE: 22

Pro Glu Tyr Tyr Met Ser

1 5

<210> SEQ ID NO: 23

<211> LENGTH: 6

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic CDR-H1 derived from YC151 clone

<400> SEQENCE: 23

Pro Asp Tyr Tyr Met Ser

1 5

<210> SEQ ID NO: 24

<211> LENGTH: 6

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic CDR-H1 derived from YC193 clone

<400> SEQENCE: 24

Ser Asp Tyr Tyr Met Ser

1 5

<210> SEQ ID NO: 25

<211> LENGTH: 8

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic CDR-H2 derived from YC244 clone

<400> SEQENCE: 25

Arg Asn Asn Ala Asn Gly Asn Thr

1 5

<210> SEQ ID NO: 26

<211> LENGTH: 8

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic CDR-H2 derived from YC321 clone

<400> SEQENCE: 26

Arg Asn Lys Val Asn Gly Tyr Thr

1 5

<210> SEQ ID NO: 27

<211> LENGTH: 6

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic CDR-H3 derived from YC354 clone

<400> SEQENCE: 27

Asp Asn Trp Leu Ser Tyr

1 5

<210> SEQ ID NO: 28

<211> LENGTH: 6

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic CDR-H3 derived from YC374 clone

<400> SEQENCE: 28

Asp Asn Trp Leu Thr Tyr

1 5

<210> SEQ ID NO: 29

<211> LENGTH: 17

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic CDR-L1 derived from L1-1 clone

<400> SEQENCE: 29

Lys Ser Ser His Ser Leu Leu Ala Ser Gly Asn Gln Asn Asn Tyr Leu

1 5 10 15

Ala

<210> SEQ ID NO: 30

<211> LENGTH: 17

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic CDR-L1 derived from L1-3 clone

<400> SEQENCE: 30

Lys Ser Ser Arg Ser Leu Leu Ser Ser Gly Asn His Lys Asn Tyr Leu

1 5 10 15

Ala

<210> SEQ ID NO: 31

<211> LENGTH: 17

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic CDR-L1 derived from L1-4 clone

<400> SEQENCE: 31

Lys Ser Ser Lys Ser Leu Leu Ala Ser Gly Asn Gln Asn Asn Tyr Leu

1 5 10 15

Ala

<210> SEQ ID NO: 32

<211> LENGTH: 17

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic CDR-L1 derived from L1-12 clone

<400> SEQENCE: 32

Lys Ser Ser Arg Ser Leu Leu Ala Ser Gly Asn Gln Asn Asn Tyr Leu

1 5 10 15

Ala

<210> SEQ ID NO: 33

<211> LENGTH: 17

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic CDR-L1 derived from L1-22 clone

<400> SEQENCE: 33

Lys Ser Ser His Ser Leu Leu Ala Ser Gly Asn Gln Asn Asn Tyr Leu

1 5 10 15

Ala

<210> SEQ ID NO: 34

<211> LENGTH: 7

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic CDR-L2 derived from L2-9 clone

<400> SEQENCE: 34

Trp Ala Ser Lys Arg Val Ser

1 5

<210> SEQ ID NO: 35

<211> LENGTH: 7

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic CDR-L2 derived from L2-12 clone

<400> SEQENCE: 35

Trp Gly Ser Thr Arg Val Ser

1 5

<210> SEQ ID NO: 36

<211> LENGTH: 7

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic CDR-L2 derived from L2-16 clone

<400> SEQENCE: 36

Trp Gly Ser Thr Arg Val Pro

1 5

<210> SEQ ID NO: 37

<211> LENGTH: 9

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic CDR-L3 derived from L3-32 clone

<400> SEQENCE: 37

Gln Gln Ser Tyr Ser Lys Pro Phe Thr

1 5

<210> SEQ ID NO: 38

<211> LENGTH: 1416

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic nucleotide sequence of heavy chain of

chAbF46

<220> FEATURE:

<221> NAME/KEY: misc_feature

<222> LOCATION: (1)..(6)

<223> OTHER INFORMATION: EcoRI restriction site

<220> FEATURE:

<221> NAME/KEY: misc_feature

<222> LOCATION: (7)..(66)

<223> OTHER INFORMATION: signal sequence

<220> FEATURE:

<221> NAME/KEY: misc_feature

<222> LOCATION: (67)..(417)

<223> OTHER INFORMATION: VH - heavy chain variable region

<220> FEATURE:

<221> NAME/KEY: misc_feature

<222> LOCATION: (418)..(423)

<223> OTHER INFORMATION: NdeI restriction site

<220> FEATURE:

<221> NAME/KEY: misc_feature

<222> LOCATION: (418)..(1407)

<223> OTHER INFORMATION: CH - heavy chain constant region

<220> FEATURE:

<221> NAME/KEY: misc_feature

<222> LOCATION: (1408)..(1410)

<223> OTHER INFORMATION: TGA - stop codon

<220> FEATURE:

<221> NAME/KEY: misc_feature

<222> LOCATION: (1411)..(1416)

<223> OTHER INFORMATION: XhoI restriction site

<400> SEQENCE: 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> SEQ ID NO: 39

<211> LENGTH: 759

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic nucleotide sequence of light chain of

chAbF46

<220> FEATURE:

<221> NAME/KEY: misc_difference

<222> LOCATION: (1)..(6)

<223> OTHER INFORMATION: EcoRI restriction site

<220> FEATURE:

<221> NAME/KEY: misc_difference

<222> LOCATION: (7)..(90)

<223> OTHER INFORMATION: signal sequence

<220> FEATURE:

<221> NAME/KEY: misc_difference

<222> LOCATION: (91)..(432)

<223> OTHER INFORMATION: VL - light chain variable region

<220> FEATURE:

<221> NAME/KEY: misc_difference

<222> LOCATION: (430)..(435)

<223> OTHER INFORMATION: BsiWI restriction site

<220> FEATURE:

<221> NAME/KEY: misc_difference

<222> LOCATION: (433)..(750)

<223> OTHER INFORMATION: CL - light chain constant region

<220> FEATURE:

<221> NAME/KEY: misc_difference

<222> LOCATION: (751)..(753)

<223> OTHER INFORMATION: stop codon

<220> FEATURE:

<221> NAME/KEY: misc_difference

<222> LOCATION: (754)..(759)

<223> OTHER INFORMATION: XhoI restriction site

<400> SEQENCE: 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> SEQ ID NO: 40

<211> LENGTH: 447

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic amino acid sequence of H1-heavy

<400> SEQENCE: 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> SEQ ID NO: 41

<211> LENGTH: 447

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic amino acid sequence of H3-heavy

<400> SEQENCE: 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> SEQ ID NO: 42

<211> LENGTH: 447

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic amino acid sequence of H4-heavy

<400> SEQENCE: 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> SEQ ID NO: 43

<211> LENGTH: 220

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic amino acid sequence of H1-light

<400> SEQENCE: 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> SEQ ID NO: 44

<211> LENGTH: 220

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic amino acid sequence of H2-light

<400> SEQENCE: 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> SEQ ID NO: 45

<211> LENGTH: 220

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic amino acid sequence of H3-light

<400> SEQENCE: 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> SEQ ID NO: 46

<211> LENGTH: 219

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic amino acid sequence of H4-light

<400> SEQENCE: 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> SEQ ID NO: 47

<211> LENGTH: 1350

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic nucleotide sequence of H1-heavy

<400> SEQENCE: 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> SEQ ID NO: 48

<211> LENGTH: 1350

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic nucleotide sequence of H3-heavy

<400> SEQENCE: 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> SEQ ID NO: 49

<211> LENGTH: 1350

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic nucleotide sequence of H4-heavy

<400> SEQENCE: 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> SEQ ID NO: 50

<211> LENGTH: 669

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic nucleotide sequence of H1-light

<400> SEQENCE: 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> SEQ ID NO: 51

<211> LENGTH: 669

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic nucleotide sequence of H2-light

<400> SEQENCE: 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> SEQ ID NO: 52

<211> LENGTH: 669

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic nucleotide sequence of H3-light

<400> SEQENCE: 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> SEQ ID NO: 53

<211> LENGTH: 669

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic nucleotide sequence of H4-light

<400> SEQENCE: 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> SEQ ID NO: 54

<211> LENGTH: 23

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic linker between VH and VL

<400> SEQENCE: 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> SEQ ID NO: 55

<211> LENGTH: 1088

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic polynucleotide encoding scFv of

huAbF46 antibody

<400> SEQENCE: 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> SEQ ID NO: 56

<211> LENGTH: 5597

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic expression vector including

polynucleotide encoding scFv of huAbF46 antibody

<220> FEATURE:

<221> NAME/KEY: misc_difference

<222> LOCATION: (573)..(578)

<223> OTHER INFORMATION: NheI restriction site

<220> FEATURE:

<221> NAME/KEY: misc_difference

<222> LOCATION: (588)..(938)

<223> OTHER INFORMATION: huAbF46 VH

<220> FEATURE:

<221> NAME/KEY: misc_difference

<222> LOCATION: (939)..(1007)

<223> OTHER INFORMATION: linker

<220> FEATURE:

<221> NAME/KEY: misc_difference

<222> LOCATION: (1008)..(1349)

<223> OTHER INFORMATION: huAbF46 VL

<220> FEATURE:

<221> NAME/KEY: misc_difference

<222> LOCATION: (1350)..(1355)

<223> OTHER INFORMATION: EcoRI restriction site

<220> FEATURE:

<221> NAME/KEY: misc_difference

<222> LOCATION: (1356)..(1397)

<223> OTHER INFORMATION: V5 epitope

<220> FEATURE:

<221> NAME/KEY: misc_difference

<222> LOCATION: (1398)..(1442)

<223> OTHER INFORMATION: (G4S)3 linker

<220> FEATURE:

<221> NAME/KEY: misc_difference

<222> LOCATION: (1443)..(1649)

<223> OTHER INFORMATION: Aga2

<220> FEATURE:

<221> NAME/KEY: misc_difference

<222> LOCATION: (1650)..(1652)

<223> OTHER INFORMATION: TGA(stop codon)

<220> FEATURE:

<221> NAME/KEY: misc_difference

<222> LOCATION: (1653)..(1660)

<223> OTHER INFORMATION: PmeI restriction site

<400> SEQENCE: 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> SEQ ID NO: 57

<211> LENGTH: 13

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic U6-HC7 hinge

<400> SEQENCE: 57

Glu Pro Lys Ser Cys Asp Cys His Cys Pro Pro Cys Pro

1 5 10

<210> SEQ ID NO: 58

<211> LENGTH: 435

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic polynucleotide encoding CDR-L3

derived from L3-1 clone

<400> SEQENCE: 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> SEQ ID NO: 59

<211> LENGTH: 435

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic polynucleotide encoding CDR-L3

derived from L3-2 clone

<400> SEQENCE: 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> SEQ ID NO: 60

<211> LENGTH: 435

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic polynucleotide encoding CDR-L3

derived from L3-3 clone

<400> SEQENCE: 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> SEQ ID NO: 61

<211> LENGTH: 435

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic polynucleotide encoding CDR-L3

derived from L3-5 clone

<400> SEQENCE: 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> SEQ ID NO: 62

<211> LENGTH: 462

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic polypeptide consisting of heavy chain

of huAbF46-H4-A1, U6-HC7 hinge and constant region of human IgG1

<400> SEQENCE: 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> SEQ ID NO: 63

<211> LENGTH: 1410

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic polynucleotide encoding polypeptide

consisting of heavy chain of huAbF46-H4-A1, U6-HC7 hinge and

constant region of human IgG1

<400> SEQENCE: 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> SEQ ID NO: 64

<211> LENGTH: 461

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic polypeptide consisting of heavy chain

of huAbF46-H4-A1, human IgG2 hinge and constant region of human

IgG1

<400> SEQENCE: 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> SEQ ID NO: 65

<211> LENGTH: 1407

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic polynucleotide encoding polypeptide

consisting of heavy chain of huAbF46-H4-A1, human IgG2 hinge and

constant region of human IgG1

<400> SEQENCE: 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> SEQ ID NO: 66

<211> LENGTH: 460

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic polypeptide consisting of heavy chain

of huAbF46-H4-A1, human IgG2 hinge and constant region of human

IgG2

<400> SEQENCE: 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> SEQ ID NO: 67

<211> LENGTH: 1404

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic polynucleotide encoding polypeptide

consisting of heavy chain of huAbF46-H4-A1, human IgG2 hinge and

constant region of human IgG2

<400> SEQENCE: 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> SEQ ID NO: 68

<211> LENGTH: 240

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic polypeptide consisting of light chain

of huAbF46-H4-A1(H36Y) and human kappa constant region

<400> SEQENCE: 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> SEQ ID NO: 69

<211> LENGTH: 758

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic polynucleotide encoding polypeptide

consisting of light chain of huAbF46-H4-A1(H36Y) and

human kappa constant region

<400> SEQENCE: 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> SEQ ID NO: 70

<211> LENGTH: 240

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic polypeptide consisting of light chain

of huAbF46-H4-A1 and human kappa constant region

<400> SEQENCE: 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> SEQ ID NO: 71

<211> LENGTH: 19

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic epitope in SEMA domain of c-Met

<400> SEQENCE: 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> SEQ ID NO: 72

<211> LENGTH: 10

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic epitope in SEMA domain of c-Met

<400> SEQENCE: 72

Pro Gln Ile Glu Glu Pro Ser Gln Cys Pro

1 5 10

<210> SEQ ID NO: 73

<211> LENGTH: 5

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic epitope in SEMA domain of c-Met

<400> SEQENCE: 73

Glu Glu Pro Ser Gln

1 5

<210> SEQ ID NO: 74

<211> LENGTH: 117

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic heavy chain variable region of

anti-c-Met antibody (AbF46 or huAbF46-H1)

<400> SEQENCE: 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> SEQ ID NO: 75

<211> LENGTH: 114

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic light chain variable region of

anti-c-Met antibody (AbF46 or huAbF46-H1)

<400> SEQENCE: 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> SEQ ID NO: 76

<211> LENGTH: 1416

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic nucleotide sequence of heavy chain of

anti-c-Met antibody (AbF46 or huAbF46-H1)

<220> FEATURE:

<221> NAME/KEY: misc_feature

<222> LOCATION: (1)..(6)

<223> OTHER INFORMATION: EcoRI restriction site

<220> FEATURE:

<221> NAME/KEY: misc_feature

<222> LOCATION: (7)..(66)

<223> OTHER INFORMATION: signal sequence

<220> FEATURE:

<221> NAME/KEY: misc_feature

<222> LOCATION: (67)..(417)

<223> OTHER INFORMATION: VH - heavy chain variable region

<220> FEATURE:

<221> NAME/KEY: misc_feature

<222> LOCATION: (418)..(423)

<223> OTHER INFORMATION: NdeI restriction site

<220> FEATURE:

<221> NAME/KEY: misc_feature

<222> LOCATION: (418)..(1407)

<223> OTHER INFORMATION: CH - heavy chain constant region

<220> FEATURE:

<221> NAME/KEY: misc_feature

<222> LOCATION: (1408)..(1410)

<223> OTHER INFORMATION: TGA - stop codon

<220> FEATURE:

<221> NAME/KEY: misc_feature

<222> LOCATION: (1411)..(1416)

<223> OTHER INFORMATION: XhoI restriction site

<400> SEQENCE: 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> SEQ ID NO: 77

<211> LENGTH: 759

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic nucleotide sequence of light chain of

anti-c-Met antibody (AbF46 or huAbF46-H1)

<220> FEATURE:

<221> NAME/KEY: misc_difference

<222> LOCATION: (1)..(6)

<223> OTHER INFORMATION: EcoRI restriction site

<220> FEATURE:

<221> NAME/KEY: misc_difference

<222> LOCATION: (7)..(90)

<223> OTHER INFORMATION: signal sequence

<220> FEATURE:

<221> NAME/KEY: misc_difference

<222> LOCATION: (91)..(432)

<223> OTHER INFORMATION: VL - light chain variable region

<220> FEATURE:

<221> NAME/KEY: misc_difference

<222> LOCATION: (430)..(435)

<223> OTHER INFORMATION: BsiWI restriction site

<220> FEATURE:

<221> NAME/KEY: misc_difference

<222> LOCATION: (433)..(750)

<223> OTHER INFORMATION: CL - light chain constant region

<220> FEATURE:

<221> NAME/KEY: misc_difference

<222> LOCATION: (751)..(753)

<223> OTHER INFORMATION: stop codon

<220> FEATURE:

<221> NAME/KEY: misc_difference

<222> LOCATION: (754)..(759)

<223> OTHER INFORMATION: XhoI restriction site

<400> SEQENCE: 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> SEQ ID NO: 78

<211> LENGTH: 4170

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic polynucleotide encoding c-Met protein

<400> SEQENCE: 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> SEQ ID NO: 79

<211> LENGTH: 444

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic SEMA domain of c-Met

<400> SEQENCE: 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> SEQ ID NO: 80

<211> LENGTH: 451

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic PSI-IPT domain of c-Met

<400> SEQENCE: 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> SEQ ID NO: 81

<211> LENGTH: 313

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic TyrKc domain of c-Met

<400> SEQENCE: 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> SEQ ID NO: 82

<211> LENGTH: 1332

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic polynucleotide encoding SEMA domain

of c-Met

<400> SEQENCE: 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> SEQ ID NO: 83

<211> LENGTH: 1299

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic polynucleotide encoding PSI-IPT

domain of c-Met

<400> SEQENCE: 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> SEQ ID NO: 84

<211> LENGTH: 939

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic polynucleotide encoding TyrKc domain

of c-Met

<400> SEQENCE: 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> SEQ ID NO: 85

<211> LENGTH: 13

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic heavy chain CDR3 of anti-c-Met

antibody

<400> SEQENCE: 85

Asp Asn Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val

1 5 10

<210> SEQ ID NO: 86

<211> LENGTH: 10

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic light chain CDR3 of anti-c-Met

antibody

<400> SEQENCE: 86

Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu

1 5 10

<210> SEQ ID NO: 87

<211> LENGTH: 117

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic heavy chain variable region of

monoclonal antibody AbF46

<400> SEQENCE: 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> SEQ ID NO: 88

<211> LENGTH: 114

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic light chain variable region of

anti-c-Met antibody

<400> SEQENCE: 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> SEQ ID NO: 89

<211> LENGTH: 17

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic light chain CDR3 of anti-c-Met

antibody

<400> SEQENCE: 89

Gln Gln Ser Tyr Ser Ala Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu

1 5 10 15

Glu

<210> SEQ ID NO: 90

<211> LENGTH: 117

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic heavy chain variable region of AT-VH1

<400> SEQENCE: 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> SEQ ID NO: 91

<211> LENGTH: 117

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic heavy chain variable region of AT-VH2

<400> SEQENCE: 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> SEQ ID NO: 92

<211> LENGTH: 117

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic heavy chain variable region of AT-VH3

<400> SEQENCE: 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> SEQ ID NO: 93

<211> LENGTH: 117

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic heavy chain variable region of AT-VH4

<400> SEQENCE: 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> SEQ ID NO: 94

<211> LENGTH: 117

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic heavy chain variable region of AT-VH5

<400> SEQENCE: 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> SEQ ID NO: 95

<211> LENGTH: 114

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic light chain variable region of anti

c-Met humanized antibody(huAbF46-H4)

<400> SEQENCE: 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> SEQ ID NO: 96

<211> LENGTH: 113

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic light chain variable region of AT-Vk1

<400> SEQENCE: 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> SEQ ID NO: 97

<211> LENGTH: 113

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic light chain variable region of AT-Vk2

<400> SEQENCE: 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> SEQ ID NO: 98

<211> LENGTH: 113

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic light chain variable region of AT-Vk3

<400> SEQENCE: 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> SEQ ID NO: 99

<211> LENGTH: 113

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic light chain variable region of AT-Vk4

<400> SEQENCE: 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> SEQ ID NO: 100

<211> LENGTH: 13

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic modified hinge region(U7-HC6)

<400> SEQENCE: 100

Glu Pro Ser Cys Asp Lys His Cys Cys Pro Pro Cys Pro

1 5 10

<210> SEQ ID NO: 101

<211> LENGTH: 13

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic modified hinge region(U6-HC7)

<400> SEQENCE: 101

Glu Pro Lys Ser Cys Asp Cys His Cys Pro Pro Cys Pro

1 5 10

<210> SEQ ID NO: 102

<211> LENGTH: 12

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic modified hinge region(U3-HC9)

<400> SEQENCE: 102

Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro

1 5 10

<210> SEQ ID NO: 103

<211> LENGTH: 14

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic modified hinge region(U6-HC8)

<400> SEQENCE: 103

Glu Pro Arg Asp Cys Gly Cys Lys Pro Cys Pro Pro Cys Pro

1 5 10

<210> SEQ ID NO: 104

<211> LENGTH: 13

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic modified hinge region(U8-HC5)

<400> SEQENCE: 104

Glu Lys Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

1 5 10

<210> SEQ ID NO: 105

<211> LENGTH: 15

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic human hinge region

<400> SEQENCE: 105

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

1 5 10 15

<210> SEQ ID NO: 106

<211> LENGTH: 17

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic CDR-L1 of antibody L3-11Y

<400> SEQENCE: 106

Lys Ser Ser Gln Ser Leu Leu Ala Trp Gly Asn Gln Asn Asn Tyr Leu

1 5 10 15

Ala

<210> SEQ ID NO: 107

<211> LENGTH: 114

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic amino acid sequence of light chain

variable region of antibody L3-11Y

<400> SEQENCE: 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> SEQ ID NO: 108

<211> LENGTH: 220

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic amino acid sequence of light chain of

antibody L3-11Y

<400> SEQENCE: 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> SEQ ID NO: 109

<211> LENGTH: 5

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic heavy chain CDR1 of anti-EGFR/anti-

Her3 antibody

<400> SEQENCE: 109

Gly Asp Trp Ile His

1 5

<210> SEQ ID NO: 110

<211> LENGTH: 20

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic heavy chain CDR2 of anti-EGFR/anti-

Her3 antibody

<400> SEQENCE: 110

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

1 5 10 15

Ser Val Lys Gly

20

<210> SEQ ID NO: 111

<211> LENGTH: 12

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic heavy chain CDR3 of anti-EGFR/anti-

Her3 antibody

<400> SEQENCE: 111

Glu Ser Arg Val Ser Phe Glu Ala Ala Met Asp Tyr

1 5 10

<210> SEQ ID NO: 112

<211> LENGTH: 11

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic light chain CDR1 of anti-EGFR/anti-

Her3 antibody

<400> SEQENCE: 112

Arg Ala Ser Gln Asn Ile Ala Thr Asp Val Ala

1 5 10

<210> SEQ ID NO: 113

<211> LENGTH: 7

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic light chain CDR2 of anti-EGFR/anti-

Her3 antibody

<400> SEQENCE: 113

Ser Ala Ser Phe Leu Tyr Ser

1 5

<210> SEQ ID NO: 114

<211> LENGTH: 8

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic light chain CDR3 of anti-EGFR/anti-

Her3 antibody

<400> SEQENCE: 114

Gln Gln Ser Glu Pro Glu Pro Tyr

1 5

<210> SEQ ID NO: 115

<211> LENGTH: 121

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic heavy chain variable region of

anti-EGFR/anti-Her3 antibody

<400> SEQENCE: 115

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 Leu Ser Gly Asp

20 25 30

Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Val

35 40 45

Gly Glu Ile Ser Ala Ala Gly Gly Tyr Thr Asp Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala 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 Glu Ser Arg Val Ser Phe Glu Ala Ala Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> SEQ ID NO: 116

<211> LENGTH: 107

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic light chain variable region of

anti-EGFR/anti-Her3 antibody

<400> SEQENCE: 116

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asn Ile Ala Thr Asp

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Phe Gly Cys Gly Thr Lys Val Glu Ile Lys

100 105

<210> SEQ ID NO: 117

<211> LENGTH: 363

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic nucleotide sequence encoding heavy

chain variable region of anti-EGFR/anti-Her3 antibody

<400> SEQENCE: 117

gaggttcagc tggtggagtc tggcggtggc ctggtgcagc cagggggctc actccgtttg 60

tcctgtgcag cttctggctt caccctttct ggcgactgga tacactgggt gcgtcaggcc 120

ccgggtaagt gcctggaatg ggttggagag atttctgctg cgggtggtta tactgactat 180

gccgatagcg tcaagggccg tttcactata agcgcagaca catccaaaaa cacagcctac 240

ctgcagatga acagcctgcg tgctgaggac actgccgtct attattgtgc tagagagagt 300

agggtcagct tcgaggctgc gatggactac tggggtcaag gaaccctggt caccgtctcc 360

tcg 363

<210> SEQ ID NO: 118

<211> LENGTH: 321

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic nucleotide sequence encoding light

chain variable region of anti-EGFR/anti-Her3 antibody

<400> SEQENCE: 118

gatatccaga tgacccagtc cccgagctcc ctgtccgcct ctgtgggcga tagggtcacc 60

atcacctgcc gtgccagtca gaatatcgct actgatgtag cctggtatca acagaaacca 120

ggaaaagctc cgaaactact gatttactcg gcatccttcc tctactctgg agtcccttct 180

cgcttctctg gttccggatc tgggacggat ttcactctga ccatcagcag tctgcagccg 240

gaagacttcg caacttatta ctgtcagcaa tctgagcctg aaccttatac gttcggatgc 300

ggtaccaagg tggagatcaa a 321

<210> SEQ ID NO: 119

<211> LENGTH: 243

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic amino acid sequence of

anti-EGFR/anti-Her3 scFv

<400> SEQENCE: 119

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 Leu Ser Gly Asp

20 25 30

Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Val

35 40 45

Gly Glu Ile Ser Ala Ala Gly Gly Tyr Thr Asp Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala 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 Glu Ser Arg Val Ser Phe Glu Ala Ala Met Asp Tyr Trp Gly

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

Ala Ser Gln Asn Ile Ala Thr Asp Val Ala Trp Tyr Gln Gln Lys Pro

165 170 175

Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser

180 185 190

Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

195 200 205

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

210 215 220

Gln Gln Ser Glu Pro Glu Pro Tyr Thr Phe Gly Cys Gly Thr Lys Val

225 230 235 240

Glu Ile Lys

<210> SEQ ID NO: 120

<211> LENGTH: 729

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic nucleotide acid sequence of

anti-EGFR/anti-Her3 scFv

<400> SEQENCE: 120

gaggttcagc tggtggagtc tggcggtggc ctggtgcagc cagggggctc actccgtttg 60

tcctgtgcag cttctggctt caccctttct ggcgactgga tacactgggt gcgtcaggcc 120

ccgggtaagt gcctggaatg ggttggagag atttctgctg cgggtggtta tactgactat 180

gccgatagcg tcaagggccg tttcactata agcgcagaca catccaaaaa cacagcctac 240

ctgcagatga acagcctgcg tgctgaggac actgccgtct attattgtgc tagagagagt 300

agggtcagct tcgaggctgc gatggactac tggggtcaag gaaccctggt caccgtctcc 360

tcgggtggtg gcggttcagg cggaggtggc tctggcggtg gcggatcgga tatccagatg 420

acccagtccc cgagctccct gtccgcctct gtgggcgata gggtcaccat cacctgccgt 480

gccagtcaga atatcgctac tgatgtagcc tggtatcaac agaaaccagg aaaagctccg 540

aaactactga tttactcggc atccttcctc tactctggag tcccttctcg cttctctggt 600

tccggatctg ggacggattt cactctgacc atcagcagtc tgcagccgga agacttcgca 660

acttattact gtcagcaatc tgagcctgaa ccttatacgt tcggatgcgg taccaaggtg 720

gagatcaaa 729

<210> SEQ ID NO: 121

<211> LENGTH: 15

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic peptide linker

<400> SEQENCE: 121

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> SEQ ID NO: 122

<211> LENGTH: 114

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic light chain variable region of anti

c-Met antibody

<400> SEQENCE: 122

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> SEQ ID NO: 123

<211> LENGTH: 5

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic

<400> SEQENCE: 123

Gly Gly Gly Gly Ser

1 5

<210> SEQ ID NO: 124

<211> LENGTH: 55

<212> TYPE: PRT

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Synthetic

<220> FEATURE:

<221> NAME/KEY: MISC_FEATURE

<222> LOCATION: (6)..(55)

<223> OTHER INFORMATION: Xaa can be any naturally occurring amino acid

<220> FEATURE:

<221> NAME/KEY: MISC_FEATURE

<222> LOCATION: (6)..(55)

<223> OTHER INFORMATION: Sequence can be repeated up to 10 times

<400> SEQENCE: 124

Gly Gly Gly Gly Ser Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

1 5 10 15

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

20 25 30

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

35 40 45

Xaa Xaa Xaa Xaa Xaa Xaa Xaa

50 55

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Patent Valuation

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26.0/100 Score

Market Attractiveness

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32.0/100 Score

Market Coverage

It shows the sizes of the market that is covered with the IP and in how many countries the IP guarantees protection. It reflects a market size that is potentially addressable with the invented technology/formulation with a legal protection which also includes a freedom to operate. Here we look into the size of the impacted market.

72.56/100 Score

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53.0/100 Score

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It takes the R&D behavior of the company itself into account that results in IP. During the invention phase, larger companies are considered to assign a higher R&D budget on a certain technology field, these companies have a better influence on their market, on what is marketable and what might lead to a standard.

21.0/100 Score

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Citation

Patents Cited in This Cited by
Title Current Assignee Application Date Publication Date
이중특이적 항-HER 항체 제넨테크, 인크. 19 March 2010 26 October 2011
Combination therapy with c-met and EGFR antagonists GENENTECH, INC. 06 March 2009 10 September 2009
Bispecific antibodies for inducing apoptosis of tumor and diseased cells IMMUNOMEDICS, INC. 09 August 2004 14 April 2005
二重特異的単鎖Fv抗体分子およびその使用方法 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア,フォックス チェース キャンサー センター 14 June 2006 08 January 2009
Dual Variable Domain Immumoglobulins and Uses Thereof ABBVIE INC. 28 April 2009 25 March 2010
See full citation <>

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US10000569 Anti-cMet/anti-EGFR/anti-HER3 multispecific antibodies 1 US10000569 Anti-cMet/anti-EGFR/anti-HER3 multispecific antibodies 2 US10000569 Anti-cMet/anti-EGFR/anti-HER3 multispecific antibodies 3