# Patent

## Title
C-Met targeting compound-bioactive material conjugate and use thereof

## Abstract

Disclosed is a conjugate in which a c-Met targeting compound and a bioactive material are chemically conjugated with each other, and methods of use thereof.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of Korean Patent Application No. 10-2013-0109889 filed on Sep. 12, 2013, the entire disclosure of which is hereby incorporated by reference.
INCORPORATION BY REFERENCE OF ELECTRONICALLY SUBMITTED MATERIALS
Incorporated by reference in its entirety herein is a computer-readable nucleotide/amino acid sequence listing submitted herewith and identified as follows: 135,528 ASCII (Text) file named “718402_ST25.TXT” created Sep. 12, 2014.
BACKGROUND
1. Field
Provided is a conjugate including a c-Met targeting compound and a bioactive material and methods of use of the conjugate.
2. Description of the Related Art
c-Met is a hepatocyte growth factor (HGF) receptor, encoded by the oncogene c-met, functioning to mediate a wide spectrum of signals driven by its ligand HGF which binds to an extracellular domain of the c-Met receptor tyrosine kinase to induce the promotion of division, motility, morphogenesis and angiogenesis in various normal and cancer cells. In some cases, c-Met is involved, irrespective of its ligand HGF, in a variety of tumorigenesis including oncogenesis, metastasis, cancer cell migration, cancer cell invasion, and angiogenesis, therefore attracting a keen interest as a target protein for cancer therapy.
Particularly, c-Met is recognized as being of significance for personalized therapy as it is known to play a role in drug resistance with regard to the activity of conventional anticancer agents. Erbitux and Tarceva, both representative anticancer agents which target EGFR (ERBB1), perform their functions by blocking oncogenic mechanism-related signal transduction. Herceptin, a typical therapeutic for breast cancer, functions to block a signal pathway necessary for cell growth, targeting ERBB2 (HER2). However, recent studies on patients with resistance to the drugs reported that other signaling pathways responsible for cell growth are activated by detouring the action of the anticancer agents through the overexpression of c-Met. For this reason, c-Met is a target molecule to which many pharmaceutical companies pay keen attention, attempting to develop various drugs, for example, antibodies, which are designed to bind to c-Met to inhibit HGF signaling.
These c-Met targeting drugs are known to exhibit anticancer efficiency in various c-Met positive cancers. On the contrary, some c-Met targeting drugs (for example, antibodies) are observed to act as an agonist causative of the side effect (agonism) that they combine by themselves to induce the dimerization of c-Met, irrespective of the ligand, thereby initiating oncogenic signaling. In addition, another problem is the impotence of c-Met targeting drugs (for example, antibodies) in cancer cells which express the antigen c-Met at a low level. An antibody-drug conjugate (ADC) was also developed in which an antibody is conjugated with a drug was developed to synergize the targeting of the drug, but is disadvantageously ineffective in internalization.
SUMMARY
Provided is a conjugate including a c-Met targeting compound and a bioactive material, in which the c-Met targeting compound and the bioactive material are conjugated with each other.
Another embodiment provides a conjugate including an anti-c-Met antibody and a cytotoxic agent, in which the anti-c-Met antibody and the cytotoxic agent are conjugated with each other.
Another embodiment provides a pharmaceutical composition including the conjugate.
Another embodiment provides a composition useful for the prevention and/or treatment of a cancer, including the conjugate as an active ingredient.
Another embodiment provides a method for preventing and/or treating a cancer including administering the conjugate to a subject in need thereof.
Another embodiment provides a method for preparing an anti-c-Met antibody derivative having improved efficacy, including conjugating an anti-c-Met antibody with a cytotoxic agent.
Another embodiment provides a method for improving the efficacy of an anti-c-Met antibody, including conjugating the anti-c-Met antibody with a cytotoxic agent.
Another embodiment provides a method for preparing a cytotoxic agent derivative having improved internalization efficiency, including conjugating a cytotoxic agent with an anti-c-Met antibody.
Another embodiment provides a method for improving the internalization efficiency of a cytotoxic agent, including conjugating the cytotoxic agent with an anti-c-Met antibody.
Another embodiment provides a composition for the intracellular delivery of a bioactive material, including an anti-c-Met antibody.
Still another embodiment provides a method for intracellular delivery of a bioactive material, using an anti-c-Met antibody.



BRIEF DESCRIPTION OF THE DRAWINGS
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
FIG. 1 is a series of fluorescent images showing internalization behaviors of an anti-c-Met antibody and Herceptin.
FIG. 2 is a schematic diagram showing reaction schemes designed to prepare a docetaxel (DTX) derivative for use in the preparation of an antibody-drug conjugate (ADC)
FIGS. 3A and 3B are HPLC chromatograms of docetaxel derivatives.
FIG. 4 is a set of images depicting stereostructures of antibody-drug conjugates, indicating an SH binding site and an NH2 binding site within the respective conjugates.
FIG. 5 is a schematic diagram depicting a reaction design for preparing an antibody-drug conjugate (ADC1) through an NH2 coupling linkage.
FIG. 6 is a series of HPLC chromatograms of the antibody-drug conjugate (ADC1) prepared through NH2 coupling.
FIG. 7 is a schematic diagram depicting a reaction design for preparing an antibody-drug conjugate (ADC2) through an SH coupling linkage.
FIG. 8 is a series of HPLC chromatograms of the antibody-drug conjugate (ADC2) prepared through SH coupling.
FIG. 9 is a set of graphs depicting relative cell growth rate of a c-Met-positive cancer cell (MKN45) treated with ADC1 or ADC2 indicating the cytotoxicity of ADC1 and ADC2 against the c-Met-positive cancer cell.
FIG. 10 is a set of graphs depicting relative cell growth rate of LoVo cells to which the anti-c-Met antibody exhibits agonism, when treated with ADC1 or ADC2, indicating the cytotoxicity of ADC1 and ADC2 against LoVo cells.
FIG. 11 is a graph showing relative cell viability of cells (HCT116) on which the anti-c-Met antibody alone does not inhibitory effects, but ADC1 does have an inhibitory effect, indicating the cytotoxicity of ADC1 against HCT116 cell.
FIG. 12 is a graph showing relative cell viability of human hepatocytes treated with a c-Met antibody, docetaxel, and ADC-1 and ADC-2, indicating the cytotoxicity of the c-Met antibody, docetaxel, and ADC-1 and ADC-2 against human hepatocytes.
FIG. 13 is a series of SEC-HPLC chromatograms of ADC1 and ADC2.



DETAILED DESCRIPTION
One embodiment provides a conjugate including a c-Met targeting compound and a bioactive material, wherein the c-Met targeting compound and the bioactive material are conjugated with each other.
In an embodiment, the c-Met targeting compound may be an anti-c-Met antibody. The bioactive material may be selected from the group consisting of a cytotoxic agent, a radio-isotope, a contrast agent, and a combination thereof.
Another embodiment provides an antibody-drug conjugate (ADC) in which an anti-c-Met antibody and a cytotoxic agent are conjugated with each other.
In the antibody-drug conjugate, the anti-c-Met antibody and the cytotoxic agent may be conjugated by a chemical bond, e.g., a covalent bond. For example, the anti-c-Met antibody may be linked to the cytotoxic agent by thiol coupling (SH coupling) or amine coupling (NH2 coupling). To this end, the drug may be derivatized with (further comprise) a functional group which allows for SH coupling or NH2 coupling.
The functional group may be introduced into the drug directly or via a linker. The linker is sufficiently blood stream stable to prevent the drug from segregating from the antibody during the blood circulation of the conjugate in the body so that the drug can remain in a prodrug form until encountering the target, thereby not only producing minimal damage on normal tissues, but also allowing for the cytotoxic agent to be specifically dissociated from the prodrug in cancer cells or tissues thereby exhibiting cytotoxic activity. So long as it exhibits these functions, any linker can be used.
In an embodiment, the cytotoxic agent may be a derivative into which a functional group capable of thiol (SH) coupling or amine (NH2) coupling is introduced directly or via a linker.
The anti-c-Met antibody specifically recognizes and binds to c-Met, followed by internalization into cells (see FIG. 1). Due to these characteristics, the anti-c-Met antibody can help a bioactive material conjugated thereto, such as a cytotoxic agent, a radio-isotope, and/or a contrast agent, enter target cells, improving the intracellular performance of action of the bioactive material, e.g., the cytotoxicity of the cytotoxic agent, the intended intracellular efficacy of the radio-isotope, or the contrasting efficiency of the contrast agent.
In addition, the antibody-drug conjugate produces at least additive and possibly synergistic effects of the antibody and the drug. When the bioactive material is a cytotoxic agent, the conjugate not only synergistically inhibits cancer cells against which the anti-c-Met antibody is active, but can also exert an anticancer effect on even cancer cells either for which the anti-c-Met antibody may cause a side effect (agonism) or on cancer cells resistant to the anti-c-Met antibody. Given the bioactive material, the anti-c-Met antibody can expand the spectrum of its applicability. On the other hand, when a cytotoxic agent (e.g., an anticancer agent) with the problem of, for example, hepatotoxicity, is applied as a conjugate with the anti-c-Met antibody, a significant reduction in hepatotoxicity is observed (see Example 6 and FIG. 12).
Another embodiment provides a pharmaceutical composition including the conjugate as an active ingredient.
A particular embodiment provides a pharmaceutical composition for the prevention and/or treatment of a cancer, including as an active ingredient an antibody-drug conjugate (ADC) in which an anti-c-Met antibody and a cytotoxic agent are conjugated with each other.
Still another embodiment provides a method for preventing and/or treating a cancer, including administering an antibody-drug conjugate in which an anti-c-Met antibody and a cytotoxic agent are conjugated with each other, to a subject in need of preventing and/or treating a cancer. The antibody-drug conjugate may be administered in amounts that are pharmaceutically effective, which amount may be determined by the skilled medical practitioner or medical researcher. The method may further include, prior to the administration step, a step of identifying a subject in need of preventing and/or treating a cancer. The step of identifying may be conducted by any manner and/or methods known to the relevant field for identifying whether or not a subject needs the prevention and/or treatment of cancer. For example, the step of identifying may include diagnosing a subject to have a cancer, or identifying a subject who is diagnosed as a cancer subject.
In this context, the subject is intended to encompass all animals that need the prophylaxis and/or therapy of cancer, whether urgently or potentially, and cells derived therefrom. For example, all mammals including primates such as humans and monkeys, and rodents such as mice and rats, cells or tissues derived (isolated) therefrom, and cultures of the cells or tissues may fall into the scope of the subject. For example, a patient with cancer, or cancer cells or tissues derived (isolated) from such patients, or a culture thereof may be a subject.
Taking advantage of the efficient internalization of an anti-c-Met antibody, another embodiment provides a pharmaceutical composition for the intracellular delivery of a cytotoxic agent, including an antibody-drug conjugate in which the anti-c-Met antibody and the cytotoxic agent are conjugated with each other. An additional embodiment provides a method for delivering a cytotoxic agent into a cell, including administering an antibody-drug conjugate in which an anti-c-Met antibody and the cytotoxic agent are conjugated with each other, to a subject in need of intracellular delivery of the cytotoxic agent. The delivery method may further include determining whether the subject is in need of the intracellular delivery of the cytotoxic agent before the administration.
In this context, the subject is intended to encompass all animals in need of the intracellular delivery of the cytotoxic agent, and cells or tissues derived (separated) therefrom. For example, all mammals including primates such as humans and monkeys, and rodents such as mice and rats, cells or tissues derived (isolated) therefrom, and cultures of the cells or tissues may fall into the scope of the subject. For example, a patient with cancer, or cancer cells or tissues derived (isolated) from the patients, or a culture thereof may be a subject.
As well as being effective in intracellular delivery, as elucidated herein, the antibody-drug conjugate is designed to detour problems attributed to the agonism of the anti-c-Met antibody and resistance to the drug, and to produce a significant reduction in hepatotoxicity, thereby exhibiting excellent anticancer activity against a broad spectrum of cancer cells.
Another embodiment provides a method for preparing an anti-c-Met antibody derivative (antibody-drug conjugate) by conjugating an anti-c-Met antibody with a cytotoxic agent. The conjugating step may comprise chemically (e.g., covalently) bonding an anti-c-Met antibody and a cytotoxic agent. The anti-c-Met antibody derivative may lead to not only a synergistic improvement in anticancer efficacy, but also a reduction in the side effect (agonism) of the anti-c-Met antibody and/or in resistance to the drug.
Another embodiment provides a method for potentiating (or improving) an anti-c-Met antibody, including conjugating the anti-c-Met antibody with a cytotoxic agent. The term “potentiating (or improving)” used in the context of the anti-c-Met antibody herein, is intended to pertain to increasing the anticancer activity of the anti-c-Met antibody in combination with the cytotoxic agent, and to reducing the side effect (agonism) of the anti-c-Met antibody and/or overcoming resistance to the drug.
Another embodiment provides a method for enhancing the intracellular delivery of a cytotoxic agent, including conjugating the cytotoxic agent to an anti-c-Met antibody, or a method for preparing a cytotoxic agent derivative (antibody-drug conjugate) with a high intracellular delivery potential.
The method for preparing an anti-c-Met antibody derivative, the method for potentiating an anti-c-Met antibody, and the method for enhancing the intracellular delivery of a cytotoxic agent may further include introducing a functional group capable of thiol coupling or amine coupling into an esterificable group, e.g., an OH group, of the cytotoxic agent, directly or via an intracellularly cleavable linker, prior to the conjugation of the cytotoxic agent to the anti-c-Met.
Any biocompatible substance that could perform a function in vivo may be used as the bioactive material. For example, it may be selected from the group consisting of various chemical drugs, peptides, proteins, and nucleic acids (DNA, RNA), and a combination thereof.
So long as it is cytotoxic, particularly, to cancer cells, any cytotoxic agent may be used. Various chemical drugs (e.g., anticancer agents), peptide drugs, protein drugs, and nucleic acids (e.g., antisense oligonucleotides, siRNA, shRNA, microRNA, aptamers, etc.) fall within the scope of the cytotoxic agent. Examples of the cytotoxic agent may be at least one selected from the group consisting of maytansine, auristatin based drugs, calicheamicin based drugs, pyrrolobenzodiazepine based drugs, duocarmycin, docetaxel, doxorubicin, carboplatin (paraplatin), cisplatin, cyclophosphamide, ifosfamide, nidran, nitrogen mustard, mechlorethamine HCl, bleomycin, mitomycin C, cytarabine, fluorouracil, gemcitabine, trimetrexate, methotrexate, etoposide, vinblastine, vinorelbine, alimta, altretamine, procarbazine, paclitaxel (Taxol), taxotere, topotecan, irinotecan, radio-isotopes, and the like, but are not limited thereto.
The cytotoxic agent may be derivatized by introducing a functional group capable of forming a chemical bond (e.g., an ester linkage) with the antibody, for example, a functional group capable of thiol coupling, amine coupling or reductive amination (linking between amine and aldehyde) thereinto, either directly or via a linker which is cleavable within cells.
The functional group may be selected from the group consisting of, but not limited to, maleimide compounds, pyridyldithio compounds, N-hydroxysuccinimide compounds, derivatives thereof, aldehydes, and a combination thereof. So long as it can form a chemical bond with the anti-c-Met antibody (or an amino acid residue of the anti-c-Met antibody), any compound may be used as the functional group.
By way of example, the functional group capable of thiol coupling may be a compound which can react or form a bond with the thiol group of a cysteine residue of the anti-c-Met antibody. It may be selected from the group consisting of, but not limited to, maleimide compounds, pyridyldithio compounds, and a combination thereof. The functional group capable of amine coupling may be a compound that can form an amine bond with an amino acid residue, such as lysine, of the anti-c-Met antibody. It may be at least one selected from the group consisting of, but not limited to, N-hydroxysuccinimide compounds and derivatives thereof; and aldehydes. So long as it can form an amine bond with an amino acid residue such as a primary amine, lysine, and/or N-terminal amine from the anti-c-Met antibody, any compound may be used. For example, it may be an aldehyde, but is not limited thereto.
The functional group may be introduced (grafted) to the drug either via a suitable linker or directly. As well as being sufficiently stable to blood stream to prevent the segregation of the drug from the antibody during the blood circulation of the conjugate in the body so as for the drug to remain in remain in a prodrug form until encounter with the target, thereby producing possibly minimal damage on normal tissues, the linker should be cleaved in an acidic condition such as in cancer cells or cancer tissues or by a peptidase or protease in cancer cells or cancer tissues to release the cytotoxic agent to exhibit cytotoxicity after internalization into the cancer cells or cancer tissues. For example, the linker may be selected from the group consisting of; but not limited to, an amino acid, an amino acid derivative, a peptide (for example, available as a substrate of protease or peptidase) containing 1 to about 10 amino acids (for example, 2 to about 10 amino acids), an alkyl of C1 to C12, a hydrophilic spacer containing 1 to about 12 ethylene glycol units (—CH2CH2—O—), and a combination thereof with a linkage therebetween. For example, when the linkage between the antibody and the drug is a thiol coupling, the linker may be selected from the group consisting of, but not limited to, an amino acid, a peptide (for example, available as a substrate of protease or peptidase) containing 1 to about 10 amino acids (for example, 2 to about 10 amino acids), an alkyl of C1 to C12, and a combination thereof with a linkage therebetween. For amine coupling, the linker may be, but not limited to, a hydrophilic spacer containing 1 to about 12 ethylene glycol units (—CH2CH2—O—). A reductive amination-based linker between the antibody and the drug may be selected from the group consisting of, but not limited to, an amino acid, an amino acid derivative, a peptide (for example, available as a substrate of protease or peptidase) containing 1 to about 10 amino acids (for example, 2 to about 10 amino acids), a hydrophilic spacer containing 1 to about 12 ethylene glycol units (—CH2CH2—O—), and a combination thereof with a linkage therebetween.
As for the amino acid derivative, it may be in various forms such as neurotransmitters, hormones, metabolic intermediates, etc. Examples of the amino acid derivatives include GABA (Gamma-Amino Butyric Acid), serotonin, melatonin, thyroxine, indole acetate, citrulline, ornitine, a carboxy-substituted amino acid (e.g., carboxylglutamate, etc.), a hydroxyl-substituted amino acid (e.g., hydroxyproline, hydroxylysine, allo-hydroxylysine, etc.), a phospho-substituted amino acid (e.g., phosphoserine, etc.), a C1 ̃C4 alkyl-substituted amino acid (e.g., ethylglycine, ethylasparagine, methylglycine, methylisoleucine, methyllysine, methylvaline, etc.), aminoadipic acid, aminopropionic acid, aminobutyric acid, aminocapronic acid, aminoheptanic acid, aminoisobutyric acid, aminopimelic acid, diaminobutyric acid, desmosine, isodesmosine, diaminopimelic acid, diaminopropionic acid, norvaline, norleucine, alloisoleucine, and a combination thereof, but are not limited thereto.
In one embodiment, a docetaxel derivative (III) capable of amine coupling with an antibody may be prepared through derivatization as illustrated in the following Reaction Scheme 1 (see upper panel of FIG. 2):




In another embodiment, the derivatization of docetaxel for thio coupling is carried out as illustrated in the following Reaction Scheme 2 to give a docetaxel derivative (VI) capable of thiol coupling with an antibody (see lower panel of FIG. 2):







In a particular embodiment, the anti-c-Met antibody or an antigen binding fragment thereof may be any type of antibody capable of specifically recognizing and/or binding to c-Met, or an antigen-binding fragment thereof. The antigen-binding fragment of the anti-c-Met antibody may be selected from the group consisting of a complementarity determining region (CDR), fragment including CDR and Fc region, scFv, (scFv)2, Fab, Fab′, and F(ab′)2 of the anti-c-Met antibody. The anti-c-Met antibody may also include a variant of the antibody. The variant of the antibody may be any isotype of antibodies derived from human and other animals and/or one including any Fc region of antibodies derived from human and other animals, having a mutated hinge wherein at least one amino acid is changed, deleted or added. Unless stated otherwise, the anti-c-Met antibody may include the variants of the antibody as well as the antibody with no variation. In a particular embodiment, the anti-c-Met antibody may recognize 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 c-Met intracellular internalization and degradation.
The “c-Met” or “c-Met proteins” refer to receptor tyrosine kinases that bind to hepatocyte growth factors (HGF). The c-Met proteins may be those derived from all kinds of species, particularly a mammal, for example, those derived from a primate such as human c-Met (e.g. NP_000236), monkey c-Met (e.g., Macaca mulatta, NP_001162100), and the like, or those derived from a rodent such as mouse c-Met (e.g., NP_032617.2), rat c-Met (e.g., NP_113705.1), and the like. These proteins may include, for example, polypeptides encoded by the nucleotide sequence identified as GenBank Accession Number NM_000245, or proteins encoded by the polypeptide sequence identified as 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.
c-Met, a receptor for hepatocyte growth factor, 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 amino acids 106 to 124 of the SEMA domain (SEQ ID NO: 79), is a loop region between the second and the third propellers within the epitopes of the SEMA domain. It may act as an epitope for the anti-c-Met antibody.
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 consecutive or non-consecutive amino acid residues within the SEMA domain (SEQ ID NO: 79) of c-Met protein, for instance, 5 to 19 consecutive or non-consecutive amino acid residues within the amino acid sequence of SEQ ID NO: 71 which corresponds to a range from amino acids. 106 to 124 within the SEMA domain (SEQ ID NO: 79) of a c-Met protein. For example, the epitope may be a polypeptide having 5 to 19 consecutive amino acids of the amino acid sequence of SEQ ID NO: 71, which sequence includes the amino acid sub-sequence EEPSQ (SEQ ID NO: 73) that serves as an essential element for the epitope. For example, the epitope may be a polypeptide comprising, 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, and the epitope including the amino acid sequence of SEQ ID NO: 73 is a site to which the antibody or an antigen-binding fragment most specifically binds.
Thus, the anti-c-Met antibody may specifically bind to an epitope which has 5 to 19 consecutive amino acids of the amino acid sequence of SEQ ID NO: 71, which consecutive amino acids include 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 CDR-H1 including the amino acid sequence of SEQ ID NO: 4; CDR-H2 including the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 2, or including an amino acid sequence of 8 to 19 consecutive amino acids within SEQ ID NO: 2 including amino acid residues from 3rd to 10th positions of SEQ ID NO: 2; and CDR-H3 including the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 85, or including an amino acid sequence of 6 to 13 consecutive amino acids within SEQ ID NO: 85 including amino acid residues from 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 CDR-L1 including the amino acid sequence of SEQ ID NO: 7, CDR-L2 including the amino acid sequence of SEQ ID NO: 8, and CDR-L3 including the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 15 or SEQ ID NO: 86 or SEQ ID NO: 89, or including an amino acid sequence of 9 to 17 consecutive amino acids within SEQ ID NO: 89 including amino acid residues from 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 the 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 the following Formulas I to VI, below:

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

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

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

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

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

wherein Xaa6 is Ser or Thr,

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

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

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

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

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

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 anti-c-Met antibody or an antigen-binding fragment thereof may include:
a heavy variable region including 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;
a light variable region including 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; or
a combination of the heavy variable region and the light variable region.
In one embodiment, the anti-c-Met antibody or antigen-binding fragment may include a heavy chain variable region including an amino acid sequence of SEQ ID NO: 17, 74, 87, 90, 91, 92, 93, or 94 and a light chain variable region including an amino acid sequence of SEQ ID NO: 18, 19, 20, 21, 75, 88, 95, 96, 97, 98, 99 or 107.
In one embodiment, the anti-c-Met antibody may be a monoclonal antibody. The monoclonal antibody may be produced from a hybridoma cell line deposited with Accession No. KCLRF-BP-00220, which binds specifically to the extracellular region of c-Met protein (refer to 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.
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) or the amino acid sequence from the 18th to 461st positions of SEQ ID NO: 64; and the amino acid sequence of SEQ ID NO: 66 (wherein the amino acid sequence from the 1st to 17th positions is a signal peptide), 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 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) or 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) or 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.
In particular 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 amino acid sequence from the 21st to 240th positions of SEQ ID NO: 68.
The polypeptide of SEQ ID NO: 70 is a light chain including the human kappa (κ) 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 of SEQ ID NO: 108 (corresponding to position 52 of SEQ ID NO: 68, which corresponds to 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 an 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 alight chain including the amino acid sequence of SEQ ID NO: 108.
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 are 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.
An important consideration in CDR grafting to produce humanized antibodies is choosing the optimized human antibodies for accepting CDR of animal-derived antibodies. Antibody database, 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. The antibodies or antigen-binding fragments thereof may be isolated from a living body or non-naturally occurring. The antibodies or antigen-binding fragments thereof may be recombinant or synthetic.
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” or “specifically recognized” is well known to one of ordinary skill in the art, and indicates 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 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, addition, or substitution of at least one amino acid residue on the amino acid sequence of the hinge region so that it exhibits enhanced antigen-binding efficiency. For example, the antibody may include a hinge region including the amino acid sequence of SEQ ID NO: 100, 101, 102, 103, 104, or 105. Preferably, the hinge region has the amino acid sequence of SEQ ID NO: 100 or 101.
The rest region other than the CDR region, the heavy chain variable region, and/or the light chain variable region, for example, a heavy chain constant region and a light chain constant region, may be from any subtype of immunoglobulin (e.g., IgA, IgD, IgE, IgG (IgG1, IgG2, IgG3, IgG4), IgM, etc.).
In one embodiment, the antibody may be an antigen-binding fragment selected from the group consisting of scFv, (scFv)2, Fab, Fab′, and F(ab′)2.
The term “antigen-binding fragment” used herein refers to fragments of an intact immunoglobulin including portions of a polypeptide including antigen-binding regions having the ability to specifically bind to the antigen. For example, the antigen-binding fragment may be scFv, (scFv)2, Fab, Fab′, or F(ab′)2, but is not limited thereto. Among the antigen-binding fragments, Fab that includes light chain and heavy chain variable regions, a light chain constant region, and a first heavy chain constant region CH1, has one antigen-binding site.
The Fab′ fragment is different from the Fab fragment, in that Fab′ includes a hinge region with at least one cysteine residue at the C-terminal of CH1.
The F(ab′)2 antibody is formed through disulfide bridging of the cysteine residues in the hinge region of the Fab′ fragment.
Fv is the smallest antibody fragment with only a heavy chain variable region and a light chain variable region. Recombination techniques of generating the Fv fragment are widely known in the art.
Two-chain Fv includes a heavy chain variable region and a light chain region which are linked by a non-covalent bond. Single-chain Fv generally includes a heavy chain variable region and a light chain variable region which are linked by a covalent bond via a peptide linker or linked at the C-terminals to have a dimer structure like the two-chain Fv. The peptide linker may be the same as described in the above, for example, those having the amino acid length of 1 to 100, 2 to 50, particularly 5 to 25, and any kinds of amino acids may be included without any restrictions.
The antigen-binding fragments may be attainable using protease (for example, the Fab fragment may be obtained by restricted cleavage of a whole antibody with papain, and the F(ab′)2 fragment may be obtained by cleavage with pepsin), or may be prepared by using a genetic recombination technique.
FIG. 4 shows stereostructures of antibody-drug conjugates, indicating a site at which the cytotoxic agent is conjugated to the anti-c-Met antibody while FIGS. 5 and 7 are schematic diagrams depicting processes of conjugating a drug to an antibody.
In the antibody-drug conjugate, one to ten cytotoxic agents (drug derivatives into which a functional group capable of linkage to the antibody is introduced either via a linker or directly) may be conjugated to one antibody (complete antibody or antigen-binding fragment) (see FIGS. 5 and 7). When two or more cytotoxic agents are conjugated to one antibody, they may be the same or different.
In order to increase the production yield of the conjugate with the minimalization of coagulation, oligomerization or dimerization therein, the molar ratio of antibody to drug (DAR; drug-to-antibody ratio; moles of drug/moles of antibody) in the antibody-drug conjugate may range from 1 to 10, from 1 to 8, from 1 to 5, from 3 to 8, from 3 to 5, or from 3.5 to 4.
The conjugate or the pharmaceutical composition may be provided, together with a pharmaceutical additive, such as a carrier, a diluent and/or an excipient.
A pharmaceutically acceptable carrier which is typically used for drug formulations may be available for the conjugate or the pharmaceutical composition. Examples of the carrier include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil, but are not limited thereto. In addition, the conjugate or the pharmaceutical composition may further include at least one selected from the group consisting of a diluent, an excipient, a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, and a preservative.
The conjugate or the pharmaceutical composition may be administered orally or parenterally. For parenteral administration, the administration may be carried out via intravenous, subcutaneous, intramuscular, intraperitoneal, intradermal, local, intranasal, intrapulmonary, and intrarectal routes, but is not limited thereto. For oral administration, however, the pharmaceutical composition is preferably coated or formulated to protect the active ingredient from being degraded in the stomach because proteins or peptides are digested by pepsin. In addition, the administration may be performed with the aid of an instrument adapted for delivering the pharmaceutical composition to target cells.
The term “pharmaceutically effective amount,” as used herein, refers to an amount at which the active ingredient can exert a desired effect. A dose of the conjugate or the pharmaceutical composition may vary depending on various factors including the type of formulation, the patient's age, weight, and sex, the severity of the disorder being treated, diet, the time of administration, the route of administration, the rate of excretion, and sensitivity. For example, the pharmaceutically effective amount of the active ingredient in the conjugate or the pharmaceutical composition may range in single dose from 0.001 to 100 mg/kg, or from 0.02 to 50 mg/kg, but is not limited thereto.
The single dose may be formulated into a unit dose form or distributed into separate dose forms, or may be included within a multiple dose package.
The conjugate or the pharmaceutical composition may be formulated into solutions in oil or aqueous media, suspensions, syrup, emulsions, elixirs, powders, granules, tablets, or capsules, and in this context, a dispersant or a stabilizer may be further employed.
The cancer to the prevention and/treatment of which the composition can be applied may be solid cancer or blood cancer. The cancer may be related to overexpression and/or abnormal activation of c-Met. Examples of the cancer may include squamous cell carcinoma, small-cell lung cancer, non-small-cell lung cancer, adrenocarcinoma of lung, squamous cell carcinoma of lung, peritoneal cancer, skin cancer, skin or intraocular melanoma, rectal cancer, perianal cancer, esophagus cancer, small intestine cancer, endocrine gland cancer, parathyroid cancer, adrenal cancer, soft-tissue sarcoma, urethral cancer, chronic or acute leukemia, lymphocytic lymphoma, hepatoma, gastric cancer, gastrointestinal 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, osteosarcoma, or a combination thereof. In addition, the scope of the cancer treatable with the conjugate or the composition may extend to a cancer where an anti-c-Met antibody exhibits agonism, and/or a cancer resistant to an anti-c-Met antibody, as well as c-Met expressing cancer (c-Met-positive cancer). The cancer may be a primary cancer or a metastatic cancer.
With regard to the prophylactic and/or therapeutic effect on cancer, the conjugate or the composition may have effects inhibiting migration, invasion, and/or metastasis of cancer, as well as inhibiting the growth of primary cancer cells.
Another embodiment provides an antibody-radioisotope conjugate including an anti-c-Met antibody and a radioisotope. Any radioisotope that is radioactive may be used. For example, it may be at least one selected from among the radioisotopes listed in Table 1, below. Functionally, it may be active as a cancer therapeutic or a tracer in cancer cells or tissues. The radioisotope is within the scope of a cytotoxic agent when used as a cancer therapeutic, and within the scope of a contrast agent when used as a tracer for imaging. Kinds and properties of radioisotopes available for therapy or imaging are well known in relevant arts. On the basis of the knowledge of the art, selection may be made of appropriate radioisotopes according to purposes. Representative radioisotopes are summarized, together with their properties, in Table 1, below:









TABLE 1





Radioisotope
Representative
Description







Gallium
Gallium-67
Produced in an accelerator. Used for medicinal diagnosis such as to image




tumors and inflammation



Gallium-68
Produced in a generator (Ge-68). Positron emitting isotope for use in PET,




and PET/CT


Copper
Copper-64
Produced in an accelerator. Used in the imaging analysis of the effect of




copper metabolism on genetic diseases, and the imaging analysis and




treatment of Wilson and Menke diseases, and tumor.



Copper-67
Produced in an accelerator. Used in tumor treatment, injected together 




with monoclonal antibody into tumors so as to kill tumors and help the 




antibody act in tumors


Dysprosium
Dysprosium-165
Produced in a nuclear reactor. Used to aggregate hydroxides for




radiosynovectomy


Rhenium
Rhenium-186
Produced in a nuclear reactor. Used to perform treatment and diagnosis




simultaneously because of simultaneous emitting of beta and gamma




radiation, relieving the pain of bone cancer.



Rhenium-188
Produced in a nuclear reactor. Used to irradiate beta radiation on the




coronary artery upon vascular surgery


Rubidium
Rubidium-82
Produced in a generator (Sr-82). Myocardial perfusion imaging, PET




mechanism


Lutetium
Lutetium-177
Produced in a nuclear reactor. Half life of 6.7. Emitting of beta/gamma




radiation, prepared from Lu-176. Simultaneous diagnosis/treatment,




intracranial treatment, relieving arthritis pain on synovial membrane




extension


Fluorine
Florine-18
Produced in an accelerator. Used as a tracer, a positron-emitting isotope 




for FLT, F-miso, and PET in the study of cerebral physiology and




pathogenesis, such as in epilepsy, dementia, psychosis, etc.


Bismuth
Bismuth-213
Produced in a nuclear reactor. Half life of 46  . High energy (8.4 MeV)




used to treat cancer by an alpha targeting method


Samarium
Samarium-153
Produced in a nuclear reactor. Relieving pain of secondary cancer within




bone, effective for treatment of prostate cancer and breast cancer


Oxygen
Oxygen-15
Produced in an accelerator. a positron emitting isotope for PET, used in the




study of cerebral physiology and pathology, such as in epilepsy, dementia,




psychosis, etc.


Cesium
Cesium -137
Produced in a nuclear reactor. Tumor treatment, measurement of accurate




radiation doses to patients, intracranial treatment, relieving arthritis pain




upon synovial membrane extension


Strontium
Strontium-85
Used in the study of bone structure and metabolism



Strontium-89
Produced in a nuclear reactor. Beta radiation emitting radionuclide,




effective for pain relief of prostate cancer and bone cancer.


Iodine
Erbium-169
Produced in a nuclear reactor. Relieving arthritis pain at synovitis arthritis


Iodine
Iodine-123
Produced in an accelerator. Used in the treatment of thyroid grand disease,




brain disease, and other metabolic diseases



Iodine-125
Produced in a nuclear reactor. Used in the treatment of prostate cancer,




intracranial treatment, the estimation and diagnosis of prostate cancer




clearance, the diagnosis of leg thrombosis, and as radiation diagnosis




reagent for clinical trial, and thyroid disease. Applied to biomedical study



Iodine-131
Produced in a nuclear reactor. Diagnosis and treatment of thyroid cancer,




diagnosis of abnormal liver function, impaired bladder functions, and renal




blood flow


Ytterbium
Ytterbium-169
Produced in a nuclear reactor. Used in the study of cerebrospinal fluid, and




to obtain gamma images in NDT


Yttrium
Yttrium-90
Produced in a nuclear reactor. Intracranial treatment. Relieving pain of




arthritis upon synovial membrane extension, Ce, Au, Ru also used


Gold
Au-198
Applied to vessels or tissues to obtain images. Intracranial treatment,




Relieving pain of arthritis upon synovial membrane extension


Phosphorus
Phosphorus-32
Produced in a nuclear reactor. Used in the treatment of polycythemia, and




the molecular biology and genetics study


Indium
Indium-111
Produced in an accelerator. Used in the study of brain diseases, rectal




diseases, infections, special diagnosis, etc.


Germanium
Germanium-68
Produced in an accelerator. PET, Ga-68 generator


Nitrogen
Nitrogen-13
Produced in an accelerator. Positron emitting isotope for PET. Used in the




study of cerebral physiology and pathogenesis, such as in epilepsy,




dementia, psychosis, etc.


Cobalt
Cobalt-57
Produced in an accelerator. Used as a maker for inferring organ sizes, an




intrapulmonary diagnostic reagent, and a tracer for diagnosis of pernicious




anemia



Cobalt-60
Produced in a nuclear reactor. External radiation source, used to sterilize




surgical instruments, improve the reliability and safety of industrial




petroleum burners, and investigate foods, and in radiographic examination


Krypton
Krypton-81:
Produced in a generator (Rh-81). Images of the lung of asthma patients,




diagnosis of lung function and diseases


Carbon
Carbon-11
Produced in an accelerator. Positron emitting isotope for PET, used in the




study of cerebral physiology and pathogenesis, such as in epilepsy,




dementia, psychosis, etc.


Thallium
Thallium-201
Produced in an accelerator. Used in nuclear medicine for heart diseases 




and tumors


Technetium
Technetium-99m
Produced in a generator (inclusive of Mo-99). Nuclear medicine diagnosis,




radiopharmaceuticals. Used as different forms in the study of brain, bone,




liver, kidney, and blood flow


Palladium
Palladium-103
Produced in a nuclear reactor. Treatment of early prostate cancer. 




Radiation source for permanent skin graft


Potassium
Potassium-42
Produced in a nuclear reactor. Used to determine potassium change in




coronary flow


Holmium
Holmium-166
Produced in a nuclear reactor. Diagnosis and treatment of liver cancer









Another embodiment provides an antibody-contrast agent conjugate, including an anti-c-Met antibody, and a contrast agent. Another embodiment provides a composition for intracellular imaging, including the antibody-contrast agent. Another embodiment provides a method for intracellular imaging (visualizing), including administering the anti-c-Met antibody-contrast agent conjugate to a subject and visualizing the anti-c-Met antibody-contrast agent conjugate. In this regard, the subject may be selected from the group consisting of those needed to image the inside of their cells or themselves, e.g., cells expressing c-Met (c-Met positive cells). For example, all mammals including primates such as humans and monkeys, and rodents such as mice and rats, cells or tissues derived (isolated) therefrom, and cultures of the cells or tissues may fall into the scope of the subject. For example, a patient with cancer, or cancer cells or tissues derived (isolated) from the patients, or a culture thereof may be a subject.
The anti-c-Met antibody used in the anti-c-Met antibody-contrast agent is as described above. As long as it can be used to image the inside of cells, e.g., cells expressing c-Met (c-Met positive cells), any contrast agent is available. It may be selected from among those used for MRI (Magnetic Resonance Imaging) and PET (Positron Emission Tomography). For example, iron oxide, gadolinium, or a radioisotope may be used. The radioisotope is as described above.
Based on its internalization performance, the anti-c-Met antibody can be applied to the intracellular delivery of various bioactive materials.
Another embodiment provides a pharmaceutical composition for the intracellular delivery of a bioactive material, including an anti-c-Met antibody. Another embodiment provides a method for delivering a bioactive material into cells, using an anti-c-Met antibody. Another embodiment provides an anti-c-Met antibody for delivering a bioactive material into cells. Another embodiment provides a use of an anti-c-Met antibody in delivering a bioactive material into cells or in preparing a pharmaceutical composition for the intracellular delivery of a bioactive material.
Exhibiting not only a pharmaceutical effect of the anti-c-Met antibody in synergy with the bioactive material, but also taking advantage of the internalization performance of the anti-c-Met antibody in the intracellular delivery of the bioactive material, with the concomitant subjugation of problems with individual components, e.g., agonism of the anti-c-Met antibody and resistance to the bioactive material, the anti-c-Met antibody-bioactive material conjugate is effectively applicable to the treatment of various diseases or diseased cells and to the visualization of various cells.
Hereafter, the present disclosure will be described in detail by examples.
The following examples are intended merely to illustrate the invention and are not construed to restrict the invention.
EXAMPLES
Reference Example
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, 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 tail 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 water 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.
Like this, 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. 6, 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 were 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 mice antibody AbF46 produced in Example 1 by replacing the constant region, but not the variable region responsible for antibody specificity, with an amino sequence of 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 pOptiVECTM-TOPO TA Cloning Kit enclosed in an OptiCHOTM Antibody Express Kit (Cat no. 12762-019, Invitrogen), and a pcDNATM3.3-TOPO TA Cloning Kit (Cat no. 8300-01), respectively.
Each of the vectors thus constructed was amplified with the aid of a Qiagen Maxiprep kit (Cat no. 12662). The vectors which respectively carried the heavy chain and the light chain were co-transfected at a ratio of 4:1 (80 μg:20 μg) into 293T cells (2.5×107). The transfection into 293T cells (2.5×107) was performed in the presence of 360 μL, of 2M CaCl2.
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 homology with the VH gene of the mouse antibody AbF46 purified in Reference Example 1.2 were analyzed. An Ig BLAST (http://www.ncbi.nlm.nih.gov/igblast/) result revealed that VH3-71 has a 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 search for BLAST. 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 homology with the VH gene of the mouse antibody AbF46 were analyzed. An Ig BLAST (see, e.g., NCBI's IgBlast website) search result revealed that VK4-1 has a 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 homology with the VL gene of the mouse antibody AbF46 were analyzed by a search for BLAST (see, e.g., NCBI's IgBlast website). As a result, VK2-40 was selected as well. VL and VK2-40 of the mouse antibody AbF46 were found to have a 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 pOptiVECTM-TOPO TA Cloning Kit enclosed in an OptiCHOTM Antibody Express Kit (Cat no. 12762-019, Invitrogen) and a pcDNATM3.3-TOPO TA Cloning Kit (Cat no. 8300-01), respectively, so as to construct recombinant vectors for expressing a humanized antibody.
Each of the recombinant vectors was amplified using Qiagen Maxiprep kit (Cat no. 12662). The vectors which respectively carried the heavy chain and the light chain were co-transfected at a ratio of 4:1 (80 μg:20 μg) into 293T cells (2.5×107). The transfection into 293T cells (2.5×107) was performed in the presence of 360 μL of 2M CaCl2. 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 were 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 comprised 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) coding for 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 CDR and Synthesis of Primer
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 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, as shown in FIG. 2, 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



Clone
constructed
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 coding for 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 pOptiVECTM-TOPO TA Cloning Kit enclosed in an OptiCHOTM Antibody Express Kit (Cat no. 12762-019, Invitrogen) and a pcDNATM3.3-TOPO TA Cloning Kit (Cat no. 8300-01), respectively, so as to construct recombinant vectors for expressing affinity-matured antibodies.
Each of the recombinant vectors was amplified using a Qiagen Maxiprep kit (Cat no. 12662). The vectors which respectively carried the heavy chain and the light chain were co-transfected at a ratio of 4:1 (80 μg:20 μg) into 293T cells (2.5×107). The transfection into 293T cells (2.5×107) was performed in the presence of 360 μL of 2M CaCl2. 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 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 into tyrosine in all of the three antibodies to increase antibody production.
For use in constructing the three antibodies, a polynucleotide (SEQ ID NO: 63) coding for 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) coding for 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) coding for 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) coding for 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 pOptiVECTM-TOPO TA Cloning Kit enclosed in an OptiCHOTM Antibody Express Kit (Cat no. 12762-019, Invitrogen) while DNA fragments having light chain nucleotide sequences were inserted into a pcDNATM3.3-TOPO TA Cloning Kit (Cat no. 8300-01) so as to construct vectors for expressing the antibodies.
Each of the vectors thus constructed was amplified with the aid of a Qiagen Maxiprep kit (Cat no. 12662). The vectors which respectively carried the heavy chain and the light chain were co-transfected at a ratio of 4:1 (80 μg:20 μg) into 293T cells (2.5×107). The transfection into 293T cells (2.5×107) was performed in the presence of 360 μL of 2M CaCl2. 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 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 3 antibodies, huAbF46-H4-A1(IgG2 Fc)) was exemplarily selected, named as L3-1Y/IgG2 antibody, and used in the following examples.
Example 1
Internalization of Anti-c-Met Antibody
The cell line OE33 (esophageal cancer cell line, ECACC (European Collection of Cell Cultures), #96070808), which overexpresses both c-Met and HER2, was incubated for 0, 10, 30 or 60 min with 1 ug/ml of the anti-c-Met antibody L3-1Y/IgG2, or Herceptin (Roche) to which an equal amount of the fluorescent FNR675 (BioActs, #PWS 1515) was previously labeled, and then observed under a confocal microscope (FIG. 1A). Separately, OE33 cells were incubated in the presence of both FITC-labeled anti-c-Met antibody L3-1Y/IgG2 and FNR675-labeled Herceptin, each 1 ug/ml, followed by confocal microscopy to compare internalization into the same cells therebetween (FIG. 1B).
Results are shown in FIG. 1. As can be seen in the photographs of FIG. 1, the anti-HER2 antibody Herceptin was observed not to enter the cells, but to remain on cell surfaces whereas a significant number of the anti-c-Met antibody L3-1Y/IgG2 was detected inside the cells. These results indicate the excellent internalization performance of the anti-c-Met antibody L3-1Y/IgG2.
Example 2
Derivatization of Docetaxel
Docetaxel (Sigma-Aldrich) was derivatized with a functional group capable of linking to an antibody, as illustrated in the reaction schemes of FIG. 2.
2-1. Derivatization of Docetaxel for Amine Coupling (FIG. 2, Upper)
A docetaxel derivative capable of conjugating to the primary amine of a lysine residue from an antibody was prepared according to the following two-step reaction (FIG. 2).
First, docetaxel (Sigma, 200 mg) and succinic anhydride (Sigma, excess by 2 mole times) were dissolved in dichloromethane (Sigma, 10 mg DTX/mL), and added with pyridine (Sigma, excess by 2.5 mole times of docetaxcel) before reaction at room temperature for 24 hrs.
Thereafter, the solvent was removed using a rotary evaporator, and the residue was redissolved in DMSO (Sigma), followed by semi-prep HPLC to separate docetaxel-succinate (Su-DTX). A linear gradient of a dual solvent composed of water (+0.05% (v/v) trifluoroacetic acid (TFA, Sigma)) as solvent and 0.05% TFA in acetonitrile (JT Baker) as solvent B (4 mL/min, 30 ̃90% B, 20 minutes) was loaded into a Capcell Pak column (C18, 120A, 5 um, 10*250, Shiseido). From the HPLC fraction thus obtained, Su-DTX was recovered by lyophilization (yield>70%). In the next step for the activation of Su-DTX, Su-DTX was dissolved, together with EDC (Sigma) and NHS (Sigma), each 2 times mole of Su-DTX, in DMSO (20 mg Su-DTX/mL), and allowed to react with each other at room temperature for 2 hrs. HPLC was carried out in the same manner to separate NHS-DTX, and the final product was recovered through lyophilization (yield>70%).
HPLC data of the docetaxel derivative for amine coupling is given in FIG. 3A. Both the intermediate and the product were found to have a purity of 90% or higher, as analyzed by analytical HPLC (Capcell Pak 4.6*250 column, the same eluent set, 10 ̃90% B, 15 min gradient).
2-2. Derivatization of Docetaxel for Thiol Coupling (FIG. 2, Lower)
A docetaxel derivative for thiol coupling was prepared, in the following 3-step reaction process.
In a first step, docetaxel, Fmoc-Glycine (Sigma), DCC (Sigma) and DMAP (Sigma) were dissolved in equimolar amounts in dichloromethane, and reacted at 4° C. for 2 hrs while stirring, and then at room temperature for an additional 20 hrs. After completion of the reaction, the solvent was removed by vacuum evaporation, and the residue was redissolved in DMSO. By-products were removed from the solution by filtration, and Fmoc-G-DTX was separated in a manner similar to that of Example 2-1.
A second step was of Fmoc deprotection. Fmoc-G-DTX was dissolved to a concentration of 20 mg/mL in DMSO, followed by reaction with 1,8-diazabi-cyclo[5.4.0]undec-7-ene (DBU, Sigma, 1.5 times mole) at room temperature for 5 min. The reaction mixture was isolated and purified using the same HPLC as described above. Finally, NH2-G-DTX and N-(e-maleimidocaproyloxy)succinimide ester (ME-NHS, Thermo), both obtained in the second step, were dissolved in DMSO (20 mg/mL for DTX) at room temperature for 2 hrs, and HPLC was performed in a manner similar to that of Example 2-1. Each reaction step was carried out at a production yield of approximately 70%.
HPLC data of the docetaxel derivative for thiol coupling is given in FIG. 3B. Both the intermediates and the product were found to have a purity of 90% or higher, as analyzed by analytical HPLC (Capcell Pak 4.6*250 column, the same eluent set, 10 ̃90% B, 15 min gradient).
Example 3
Preparation of Antibody-Docetaxel Conjugate
3-1. Preparation of Antibody-Drug Conjugate (ADC1) Through Amine Coupling
Referring to the reaction scheme illustrated in FIG. 5, the anti-c-Met antibody L3-1Y/IgG2 prepared in Reference Example 1 was conjugated with a docetaxel derivative for amine coupling, prepared in Example 2-1, to give an antibody-drug conjugate. In this regard, the antibody was used in a fixed amount of 2 mg while the drug was fed at various ratios (feed ratio) of 5 mole times, 10 mole times, 15 mole times, or 20 mole times.
Briefly, NHS-Docetaxel, a docetaxel derivative prepared in Example 2-1, was dissolved at a concentration of 5 mg/ml in DMSO (sigma). The antibody L3-1Y/IgG2 was dissolved to a final concentration of 2 mg/ml in a solution containing 20% DMSO (Sigma), 10 mM CHAPS (Sigma), and 80% PBS (pH 7.4) (Gibco), and fed at a molar ratio of 5, 10, 15 or 20 to the mole of NHS-Docetaxel, followed by reaction at room temperature for 1 hr. Then, only an antibody-docetaxel conjugate was isolated using the Desalting column (GE healthcare) of the AKTA Prime (GE healthcare). To this end, PBS (pH 7.4) was flowed at a rate of 5 ml/min through a desalting column installed in AKTA prime, followed by loading the reaction mixture to the column. The antibody-docetaxel conjugate was separated from docetaxel by size difference.
HPLC data of the antibody-drug conjugates prepared with various feed ratios are given in FIG. 6. Recovery rates of the antibody-drug conjugates are listed, together with their DAR (drug-to-antibody ratio), in Table 4, below.











TABLE 4





Feed Ratio
Ab feed (mg)
Recovery (mg)
DAR
Note



















5
2
1.63
4.4



10
2
1.58
7.6



15
2
1.18
—
loss during concentration


20
2
0.66
—
loss during concentration





(DAR: drug-to-antibody ratio)






At a DAR of about 8 or less (corresponding to feed ratio of 10 or less), as can be seen in Table 3, the recovery rates were relatively high, without a loss during concentration.
3-2. Preparation of Antibody-Drug Conjugate (ADC2) Through Thiol Coupling
As illustrated in the reaction scheme of FIG. 7, the anti-c-Met antibody L3-1Y/IgG2 prepared in Reference Example 1 was conjugated with a docetaxel derivative for thiol coupling, prepared in Example 2-1, to give an antibody-drug conjugate. In this regard, the antibody was used in a fixed amount of 2 mg while the drug was fed at various ratios (feed ratio) 2.5, 5, 7.5 or 10 mole times of the antibody.
Briefly, ME-G-Docetaxel, a docetaxel derivative prepared in Example 2-2, was dissolved at a concentration of 5 mg/ml in DMSO (sigma). The antibody L3-1Y/IgG2 was dissolved in excess dithiothreitol (DTT, sigma, 20 times mole), and reacted at 37° C. for 1 hr to reduce the disulfide bond of the antibody. Then, the buffer was changed with PBS (pH 7.4, Gibco) using a desalting column, followed by quantifying the number of reduced thiol using the Ellman reagent 5,5′-dithiobis(2-nitrobenzoic acid)(DTNB, sigma). The reduction was controlled so as to finally obtain 8 thiols/mAb. The antibody was dissolved at a concentration of 2 mg/ml in a solvent mixture containing 20% DMSO (Sigma), 10 mM CHAPS (Sigma), and 80% PBS (pH 7.4) (Gibco) to which the ME-G-Docetaxel solution was then fed at a mole ratio of 2.5, 5, 7.5, 10 to the mole of the antibody, and reacted at 4° C. for 2 hrs. Only the antibody-docetaxel conjugate was purified through a desalting column installed in AKTA Prime.
HPLC data of the antibody-drug conjugates prepared with various feed ratios are given in FIG. 8. Recovery rates of the antibody-drug conjugates are listed, together with their DAR (drug-to-antibody ratio), in Table 5, below.











TABLE 5





Feed ratio
Ab feed (mg)
Recovery (mg)
DAR
Oligomer (%)



















2.5
2
1.71
2.6
2.16


5
2
1.71
5.0
10.10


7.5
2
1.65
7.4
21.36


10
2
1.75
8.4
23.37





DAR: drug-to-Ab ratio






At a DAR of about 7 or less, e.g., 5, as can be seen in Table 4, purification was achieved at high efficiency because the recovery rates were relatively high with a low level of oligomer production.
3-3. Purification of Antibody-Drug Conjugate
Using the Desalting column (GE healthcare) of AKTA Prime (GE healthcare), only the antibody-docetaxel conjugates were purified. Two desalting columns were tandemly installed in AKTA prime, and then equilibrated with PBS (pH 7.4) which flowed at a rate of 5 ml/min. The reaction mixture was loaded to the columns and separated into antibody-docetaxel conjugates and docetaxel by size difference. Eluates were evaluated for purity HPLC (Waters) using a Sephdex 200 column (Tosoh) and a solvent (20 mM PB (pH 6), 500 mM NaCl, 20% isopropyl alcohol).
Results are shown in FIG. 13. As can be seen in FIG. 13, ADC1 (DAR 7.0) and ADC2 (DAR 6.5), prepared in Examples 3-1 and 3-2, respectively, were obtained with a purity of 95% or higher.
Example 4
Affinity of Antibody-Drug Conjugate for Antigen
An examination was made to see whether the antibody-drug conjugates maintain affinity for the antigen. The antibody-drug conjugate (ADC1; amine coupling at a feed molar ratio of 5) was assayed for affinity for the antibody (c-Met) using Biacore T100 (GE), and compared to the antibody alone.
Briefly, a human Fab capture (GE Healthcare) was immobilized on CM5 chip (#BR-1005-30, GE) according to the manufacturer's instruction. Approximately 90 ̃120 RU of ADC was captured, and treated with various concentrations of c-Met-Fc (#358-MT/CF, R&D Systems). The chip was regenerated with 10 mM Glycine-HCl (pH 1.5). To quantify the affinity, the data obtained above was fitted using BIAevaluation software (GE Healthcare, Biacore T100 evaluation software).
The results are shown in Table 6.













TABLE 6









Kd * 10−5
KD




Sample
Ka * 105 (1/MS)
(1/s)
(pM)
Chi2






















L3-1Y/IgG2
5.890
3.418
58.03
3.4



ADC
3.636
3.629
99.78
0.728



(DAR 3.7)














As can be seen in Table 6, the affinity of ADC for c-Met was approximately 0.1 nM (i.e. 100 pM) (Kd), which is similar to that of the antibody alone. Like this, the antibody even in the form of ADC did not undergo a significant reduction in affinity for antigen, indicating that the conjugate maintains the antibody specificity.
Example 5
Cytotoxicity of Antibody-Drug Conjugate Against c-Met-Positive Cancer Cell
ADC1 (DAR 7.0; large batch, feed ratio 10) and ADC2 (DAR 6.5; large batch, feed ratio 7.5) were assayed for cytotoxicity against MKN45 (JCRB0254, JCRB Cell Bank, Japan), a cancer cell line positive to c-Met.
Briefly, the stomach cancer cell line MKN45 was seeded at a density of 10×103 cells/well into 96-well plates (BD) and maintained for 24 hrs. Each ADC was mixed to a desired final antibody concentration (FIG. 9) in a medium, and added to each well. After 72 hrs of incubation, 10 uL of CCK-8 reagent (CK04, Dojindo) was added to each well, and placed at 37° C. for 2.5 hrs in an incubator. Thereafter, absorbance at dual wavelengths (450, 650 nm) was read on a micro-plate reader (Molecular Device).
Results are shown in FIG. 9. As is apparent from the data of FIG. 9, the anti-c-Met antibody L3-1Y/IgG2 was remarkably increased in cytotoxicity by ADC against c-Met-positive cancer cells, compared to the antibody or the drug alone.
Example 6
Cytotoxicity of Antibody-Drug Conjugate Against Cancer Cell with Anti-c-Met Antibody Serving as Agonist
ADC1 (DAR=7.0) and ADC2 (DAR=6.5) were assayed for cytotoxicity against LoVo (CCL-229, ATCC) and HCT116 (CCL-247, ATCC), both cancer cell lines for which the anti-c-Met antibody acts as an agonist.
Briefly, each of the large intestine cancer cell lines LoVo and HCT116 was seeded at a density of 5×103 cells/well into 96-well plates (#353072, BD), and maintained for 24 hrs. Each ADC was mixed to a desired final antibody concentration (FIGS. 11 and 12) in a medium, and added to each well. After 72 hrs of incubation, 10 uL of CCK-8 reagent (CK04, Dojindo) was added to each well, and placed at 37° C. for 2.5 hrs in an incubator. Thereafter, absorbance at dual wavelengths (450, 650 nm) was read on a micro-plate reader (Spectramax 340PC384, Molecular devices).
Results are shown in FIG. 10 (LoVo) and FIG. 11 (HCT116). As is apparent from the data of FIGS. 10 and 11, the anti-c-Met antibody L3-1Y/IgG2 was remarkably increased in cytotoxicity against LoVo, which is resistant to the antibody alone. The data suggest that when conjugated with a drug, the anti-c-Met antibody can expand its inhibition range to the cells on which the anti-c-Met antibody alone has no inhibitory effects.
Example 7
Assay for Hepatotoxicity of Antibody-Drug Conjugate
ADC1 (DAR=7.0) and ADC2 (DAR=6.5) were assayed for hepatotoxicity in human primary hepatocytes (Celsis, F00995, Lot# YEM). For comparison, docetaxel alone or in combination with anti-c-Met antibody was used.
Briefly, human primary hepatocytes were seeded at a density of 30,000 cells/well into collagen I-coated 96-well plates (BD), and maintained for 24 hrs. Then, the cells were treated with L3-1Y/IgG2, docetaxel, ADC1 or ADC2 at a concentration given in FIG. 13 (antibody: 10 ug/ml; docetaxel: 377 ng/ml). For Combi, the cells were incubated in the presence of both 10 ug/ml L3-1Y/1 gG2 and 377 ng/ml docetaxel. After 72 hrs of incubation, the cells were applied to Cell Titer Glo (Promega, G7573), and the human primary hepatocytes were counted in each well using the luminescent intensity, and compared to calculate cell viability.
The results are given in FIG. 12. As can be seen in FIG. 12, both ADC1 and ADC2 were remarkably low in hepatotoxicity, compared to docetaxel alone or in combination with L3-1Y/IgG2. Considering the fact that the induction of hepatotoxicity is one of the greatest barriers to the use of chemical anticancer agents, such as docetaxel, the antibody-drug conjugate can be more effectively applied to the treatment of cancer not only because of its low hepatotoxicity, compared to a drug alone or in combination with an antibody, but also because of its excellent anticancer activity.
All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.
The use of the terms “a” and “an” and “the” and “at least one” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

## Claims

1. A method of treating a cancer, comprising administering an antibody-drug conjugate comprising an anti-c-Met antibody and a cytotoxic agent, in which the anti-c-Met antibody and the cytotoxic agent are conjugated with each other, to a subject in need of cancer treatment,
wherein the anti-c-Met antibody binds to an epitope comprising a sequence of 5 to 19 consecutive amino acids of SEQ ID NO: 71 including the amino acid sequence EEPSQ (SEQ ID NO: 73) and comprises:
a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, 22, 23, or 24;
a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, 25, or 26;
a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, 27, 28, or 85;
a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 10, 29, 30, 31, 32, 33, or 106;
a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 11, 34, 35, or 36; and
a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 13, 14, 15, 16, or 37.

2. The method of claim 1, wherein the anti-c-Met antibody binds to an epitope of SEQ ID NO: 71, SEQ ID NO: 72, or SEQ ID NO: 73.

3. The method of claim 1, wherein the anti-c-Met antibody comprises:
a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3;
a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 10, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 11; and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 13, 14, 15, or 16.

4. The method of claim 1, wherein the cytotoxic agent is at least one selected from the group consisting of maytansine, auristatin based drug, calicheamycin based drug, pyrrolobenzodiazepine based drug, duocarmycin, docetaxel, doxorubicin, carboplatin, cisplatin, cyclophosphamide, ifosfamide, nidran, nitrogen mustard, mechlorethamine HCl, bleomycin, mitomycin C, cytarabine, fluorouracil, gemcitabine, trimetrexate, methotrexate, etoposide, vinblastine, vinorelbine, alimta, altretamine, procarbazine, paclitaxel, taxotere, topotecan, irinotecan, and a radio-isotope.

5. A method for delivering a cytotoxic agent into a cell, comprising administering an antibody-drug conjugate comprising an anti-c-Met antibody and a cytotoxic agent, in which the anti-c-Met antibody and the cytotoxic agent are conjugated with each other, to a subject in need of intracellular delivery of the cytotoxic agent,
wherein the anti-c-Met antibody binds to an epitope comprising a sequence of 5 to 19 consecutive amino acids of SEQ ID NO: 71 including the amino acid sequence EEPSQ (SEQ ID NO: 73) and comprises:
a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, 22, 23, or 24;
a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, 25, or 26;
a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3, 27, 28, or 85;
a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 10, 29, 30, 31, 32, 33, or 106;
a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 11, 34, 35, or 36; and
a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 13, 14, 15, 16, or 37.

6. The method of claim 5, wherein the anti-c-Met antibody binds to an epitope of SEQ ID NO: 71, SEQ ID NO: 72, or SEQ ID NO: 73.

7. The method of claim 5, wherein the anti-c-Met antibody comprises:
a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1; a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2; a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3;
a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 10, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 11; and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 13, 14, 15, or 16.

8. The method of claim 5, wherein the cytotoxic agent is at least one selected from the group consisting of maytansine, auristatin based drug, calicheamycin based drug, pyrrolobenzodiazepine based drug, duocarmycin, docetaxel, doxorubicin, carboplatin, cisplatin, cyclophosphamide, ifosfamide, nidran, nitrogen mustard, mechlorethamine HCl, bleomycin, mitomycin C, cytarabine, fluorouracil, gemcitabine, trimetrexate, methotrexate, etoposide, vinblastine, vinorelbine, alimta, altretamine, procarbazine, paclitaxel, taxotere, topotecan, irinotecan, and a radio-isotope.