# Patent

## Title
Antibodies cross-reactive to human and mouse c-Met and uses thereof

## Abstract

The antibody of the invention has high specificity to human c-Met and is cross-reactive to mouse c-Met. The antibody or its antigen-binding fragment of the invention is capable of specifically binding to human c-Met as well as mouse c-Met, more accurate preclinical results can be confirmed in the efficacy evaluation using mouse tumor models. The antibody of the invention inhibits the growth of cancer cells derived from various cancers by a considerable binding affinity to c-Met and the suppression of c-Met function therefrom, inhibits the phosphorylation of c-Met and downstream signaling molecules to suppress c-Met signaling, and inhibits neovascularization, thereby being very efficient in the prevention and treatment of cancers.

## Background
CROSS-REFERENCE TO RELATED APPLICATION AND CLAIM OF PRIORITY
This application claims the benefit of Korean Patent Application No. 10-2013-0069666, filed on Jun. 18, 2013, at the Korean Intellectual Property Office, the disclosures of which are incorporated herein in their entirety by reference.
BACKGROUND
1. Technical Field
The present invention relates to antibodies cross-reactive to human and mouse c-Met and uses thereof.
2. Description of the Related Art
Various grow factors, such as a hepatocyte growth factor (HGF), an epidermal growth factor (EGF), a vascular endothelial growth factor (VEGF), and a fibroblast growth factor (FGF), cross react with receptor tyrosine kinases (RTKs) on cellular surfaces to induce critical cell physiological regulation, such as, cell growth, differentiation, neovascularization, and tissue repair, as well as development. These growth factors and receptors, when deregulated in a physiological aspect, such as mutation, overexpression, and promotion of self activation, cause abnormal cell growth or differentiation, thereby initiating and promoting the development of cancers Lemmon M A & Schlessinger J, Cell. 141:1117-1134, 2010).
The met proto-oncogene (MET; c-Met) has been known as a proto-oncogene expressing hepatocyte growth factor (HGF)/scatter factor (SF) receptors (Dean M et al., Nature. 318:385-388, 1985, Gherardi et al., Nat. Rev. Cancer. 12:89-103, 2012), and cross-reacts with HGF, which is the only known ligand thereof, to induce mesenchymal-epithelial transition (MET) and promote cancer cell growth, invasion, and metastasis. Since c-Met is involved in mechanisms of development, metastasis, invasion, neovascularization, and the like, regardless of the ligand HGF, in the development procedure of several tumors, c-Met has been considered as an effective anti-cancer target. Based on this background, research on c-Met inhibitors, such as chemical drugs and monoclonal antibodies are being actively conducted (Comoglio P M et al., Nat. Rev. Drug. Discov. 7:504-516, 2008).
The development of antagonistic antibodies against the anticancer target c-Met is a representative strategy for anti-cancer therapy by c-Met inhibition. Anti-c-Met antibodies have been reported to inhibit the interaction between the ligand HGF and c-Met or decompose and inactivate c-Met. For example, the one-armed antagonistic antibody ‘OA-5D5’ developed as an anti-c-Met antibody is an agonist, and was developed as an antibody that is modified not to have an adverse effect, such as inducing c-Met dimerization (Martens T et al., Clin. Cancer Res. 15:6144-6152, 2006), and ‘DN30’ was developed to induce the inhibition of tumor formation by inducing the inactivation of c-Met itself to lose the function thereof (Petrelli A et al., PNAS. 103:5090-5095, 2006). However, the one-armed antagonistic antibody showed a slightly tumor suppressing effect when used alone, but a significant therapy effect when used together with chemotherapy, and the c-Met inactivating antibody was verified to be low competitive with ligand and show partial effects as an agonist. Therefore, the development of therapeutic antibodies that suppress functions of human c-Met has been continuously required.
In the development of antibodies against the anticancer target, in vitro efficacy evaluation as well as in vivo preclinical efficacy evaluation using mouse tumor models is needed. In particular, at the time of the evaluation of efficacy using mouse tumor models, therapeutic efficacies of corresponding antibodies are primarily determined through preclinical experiment results, such as the ability to reduce the confirmable tumor size and an increase in the survival period. Here, the used mouse tumor model is prepared by the injection of human-derived cancer cells overexpressing the anticancer target. In fact, it is highly possible that the correlation between preclinical and clinical results is low due to the interference of human tumor cells as well as mouse-derived cells mixed with the human tumor cells in the tumor microenvironment in the mouse at the time of the verification of antibody therapeutic effects (Talmadge J E et al., Am. J. Pathol. 170:793-804, 2007). Therefore, the combinatorial treatment of not only antibodies inhibiting only the human-derived anticancer target but also antibodies inhibiting the mouse-derived anticancer target or its ligand, or antibodies specific to human/mouse heterogeneous anticancer target can show more accurate preclinical therapy results. For example, it has been reported that, as for anti-Dll4 (delta like ligand 4) antibodies inhibiting intra-tumoral angiogenesis, when the mouse tumor model was treated with the combinatorial treatment of not only antibody against human Dll4 but also antibody against mouse Dll4, the tumor sizes were significantly reduced (Hoey T et al., Cell Stem Cell. 5:168-177, 2009). Also, as for antibodies targeting vascular endothelial growth factor receptor 2 (VEGFR-2) or vascular endothelial growth factor (VEGF), antibodies cross-reactive to human/mouse heterogeneous anticancer target exhibited high tumor inhibitory effect in the mouse tumor model, which demonstrated the necessity of the development of cross-reactive antibodies (Huang J et al., Cytotechnology. 62:61-71, 2010; Liang W-C et al., J. Biol. Chem. 281:951-961, 2006).
As described above, anti-c-Met antibodies suppressing only the function of c-Met do not have mouse c-Met receptor inhibitory action with regard to autocrine/paracrine action of human- or mouse-derived hepatocyte growth factor, and thus effects thereof are difficult to evaluate at the time of preclinical efficacy evaluation in the mouse tumor model. Since human c-Met (P08581, UniProtKB/Swiss-Prot) consists of 1,390 amino acids and mouse c-Met (P16056, UniProtKB/Swiss-Prot) consists of 1,379 amino acids, they have high amino acid sequence similarity of at least 89% therebetween (Chan A M L et al., Oncogene. 2:593-599, 1988). Also, as for the ligand hepatocyte growth factor (HGF), human HGF and mouse HGF have very high sequence similarity of at least 90% (Tashiro K et al., PNAS. 87:3200-3204, 1990). In addition, the representative sites of action of the ligand and the receptor are also the sema domain. Therefore, the possibility of development and applicability of cross-reactive antibodies is high. Therefore, it is necessary to develop antibodies cross-reactive to human/mouse c-Met, which suppress the cancer-specific ligand-receptor action in the tumoral microenvironment with regard to human/mouse c-Met, thereby confirming effective preclinical research results in the mouse tumor model.
Throughout the entire specification, many papers and patent documents are referenced and their citations are represented. The disclosures of cited papers and patent documents are entirely incorporated by reference into the present specification, and the level of the technical field within which the present invention falls and details of the present invention are explained more clearly.
SUMMARY
The present inventors have endeavored to develop antibodies that can bind to human c-Met and prevent and treat cancers. As a result, the present inventors have developed novel antibodies that are cross-reactive to human c-Met and mouse c-Met and exhibit ability to inhibit cancer cell growth and neovascularization, thereby having excellent effects in preventing and treating cancers.
Accordingly, it is an object of this invention to provide an antibody or its binding fragment against human c-Met.
It is another object of this invention to provide a nucleic acid molecule encoding a heavy chain variable region of an antibody against the human c-Met.
It is another object of this invention to provide a nucleic acid molecule encoding a light chain variable region of an antibody against the human c-Met.
It is still another object of this invention to provide a recombinant vector, comprising the above-mentioned nucleic acid molecule.
It is further object of this invention to provide a host cell transformed with the recombinant vector.
It is still further object of this invention to provide a pharmaceutical composition for preventing or treating cancer.
Other objects and advantages of the present invention will become apparent from the following detailed description together with the appended claims and drawings.
In accordance with an aspect of the present invention, there is provided an antibody to human c-Met or its antigen-binding fragment, including: (a) a heavy chain variable region having the following heavy chain complementarity determining region (CDR) amino acid sequences: CDRH1 including the amino acid sequence of SEQ ID NO: 1, CDRH2 including the amino acid sequence of SEQ ID NO: 2, and CDRH3 including the amino acid sequence of SEQ ID NO: 3; and (b) a light chain variable region having the following light chain CDR amino acid sequences: CDRL1 including the amino acid sequence of SEQ ID NO: 4, CDRL2 including the amino acid sequence of SEQ ID NO:5, and CDRL3 including the amino acid sequence of SEQ ID NO: 6.
In accordance with another aspect of the present invention, there is provided an antibody to human c-Met or its antigen-binding fragment, including: (a) a heavy chain variable region having the following heavy chain complementarity determining region (CDR) amino acid sequences: CDRH1 including the amino acid sequence of SEQ ID NO: 7, CDRH2 including the amino acid sequence of SEQ ID NO: 8, and CDRH3 including the amino acid sequence of SEQ ID NO: 9; and (b) a light chain variable region having the following light chain CDR amino acid sequences: CDRL1 including the amino acid sequence of SEQ ID NO: 10, CDRL2 including the amino acid sequence of SEQ ID NO:11, and CDRL3 including the amino acid sequence of SEQ ID NO: 12.
In accordance with still another aspect of the present invention, there is provided an antibody to human c-Met or its antigen-binding fragment, including: (a) a heavy chain variable region having the following heavy chain complementarity determining region (CDR) amino acid sequences: CDRH1 including the amino acid sequence of SEQ ID NO: 13, CDRH2 including the amino acid sequence of SEQ ID NO: 14, and CDRH3 including the amino acid sequence of SEQ ID NO: 15; and (b) a light chain variable region having the following light chain CDR amino acid sequences: CDRL1 including the amino acid sequence of SEQ ID NO: 16, CDRL2 including the amino acid sequence of SEQ ID NO:17, and CDRL3 including the amino acid sequence of SEQ ID NO: 18.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows a schematic scheme for selecting scFv antibody fragments cross-reactive to human/mouse c-Met through phage display.
FIG. 2 shows binding affinities of 25 scFv antibody fragments cross-reactive to human/mouse c-Met.
FIGS. 3A and 3B show phage-ELISA results using respective phage particles labeled by three anti-c-Met scFv antibody fragments.
FIGS. 4A and 4B show a map of phagemid vector for production of scFv antibody fragment having the linker (SEQ ID NO: 25) and Coomassie staining results of respective scFv antibody fragments expressed/purified actually.
FIGS. 5A and 5B show cross-reactive affinities to human/mouse c-Met and binding affinities to c-Met extracellular domain for scFv antibody fragment proteins.
FIGS. 6A through 6C show ELISA results confirming competitive reactions of anti-c-Met scFv antibody fragments with the ligand hepatocyte growth factor (HGF).
FIGS. 7A through 7J show FACS analysis results on binding affinities between c-Met overexpressing cell lines and scFv antibody fragments.
FIG. 8A through 8I show results confirming reductions in binding affinity of scFv antibody fragments due to c-Met expression inhibition in two c-Met overexpressing cell lines.
FIGS. 9A through 9G show results confirming growth inhibition aspects of respective cell lines due to the treatment with scFv antibody fragments in three c-Met expressing cell lines.
FIG. 10 shows immunofluorescence staining results confirming localization information of scFv antibody fragments treating the U87MG cancer line.
FIGS. 11A and 11B show western blotting results confirming the phosphorylation of downstream signaling molecules of a MET signaling pathway after U87MG and MKN54 cancer lines were treated with scFv antibody fragments.
FIGS. 12A through 12D show results confirming the neovascularization inhibition observed when human umbilical vein endothelial cells (HUVECs) in conditions with hepatocyte growth factor (9HGF) were treated with respective scFv antibody fragments. Graphs below show measurement results of blood vessel length and number of branches stretching from the blood vessel.
FIGS. 13A through 13C show sequences of 15-mer peptides (SEQ ID NOS: 33 to 256) derived from c-Met, used in epitope mapping.
FIGS. 14A through 14C show results confirming binding sites of respective scFv antibody fragments (SEQ ID NOS: 257 to 267) on c-Met through epitope mapping.
FIGS. 15A through 15C show shows sensorgram results of respective scFv clones according to concentrations with regard to c-Met through surface plasmon resonance.



DETAILED DESCRIPTION
The present inventors have endeavored to develop antibodies that can bind to human c-Met and prevent and treat cancers. As a result, the present inventors have developed novel antibodies that are cross-reactive to human c-Met and mouse c-Met and exhibit ability to inhibit cancer cell growth and neovascularization, thereby having excellent effects in preventing and treating cancers.
The antibodies of the present invention have a specific binding affinity to human c-Met. Particularly, the antibodies of the present invention are cross-reactive to human c-Met and mouse c-Met.
By “antibody” referred in this specification is meant an antibody which is capable of specifically binding human c-Met. Antibody is meant to include the entire antibody as well as any antibody fragments.
The entire antibody includes two full-length light chains and two full-length heavy chains, and each light chain is linked to the heavy chain by disulfide bond. The heavy chain constant region includes five different isotypes (γ, μ, α, δ and ε) of which the subclass is classified into γ1, γ2, γ3, γ4, α1 and α2. The light chain constant region includes two different isotypes (κ and λ) (Cellular and Molecular Immunology, Wonsiewicz, M. J., Ed., Chapter 45, pp. 41-50, W. B. Saunders Co. Philadelphia, Pa. (1991); Nisonoff, A., Introduction to Molecular Immunology, 2nd Ed., Chapter 4, pp. 45-65, sinauer Associates, Inc., Sunderland, Mass. (1984)).
Antigen binding fragment refers to any antibody fragment capable of binding antigen including Fab, F(ab′), F(ab′)2, Fv and so on. Fab has one antigen binding site which is composed of one variable domain from each heavy and light chain of the antibody, one constant region of light chain and the first constant region (CH1) of heavy chain. Fab′ is different to Fab in the senses that there is a hinge region containing one or more cysteine residues at C-terminal of CH1 domain of heavy chain. F(ab′)2 antibody is produced by forming a disulfide bond between cysteine residues of hinge region of Fab′. Fv is a minimal antibody fragment including one variable region from each heavy and light chain and recombinant technique to prepare a Fv fragment is disclosed in PCT WO 88/10649, PCT WO 88/106630, PCT WO 88/07085, PCT WO 88/07086 and PCT WO 88/09344.
Two-chain Fv is linked by non-covalent bond between one variable region of each heavy and light chain, and single-chain Fv is generally linked by covalent bond via a peptide linker between one variable region of each heavy and light chain or is directly linked to each other at C-terminal, forming a dimer such as two-chain Fv. Such antibody fragments may be obtained using a proteolytic enzymes (e.g., a whole antibody is digested with papain to produce Fab fragments, and pepsin treatment results in the production of F(ab′)2 fragments), and may be preferably prepared by genetic recombination techniques.
Preferably, the antibody in this invention is a form of scFv or entire antibody. In addition, the heavy chain constant region is selected from the isotypes consisting of γ, μ, α, δ or ε.
The term “heavy chain” refers to both a full-length heavy chain and its part, which includes variable domain (VH) containing the amino acid sequence with a variable region sequence for specifically binding to antigen and three constant domains (CH1, CH2 and CH3). The term “light chain” refers to both a full-length light chain and its part, which includes variable domain (VL) containing the amino acid sequence with a variable region sequence for specifically binding to antigen and constant domain (CL).
The “CDR (complementarity determining region)” means an amino acid sequence of hypervariable region of immunoglobulin heavy and light chain (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Ed., U.S. Department of Health and Human Services, National Institutes of Health (1987)). Three CDRs are involved in heavy chain (CH1, CH2 and CH3) and light chain (CDRL1, CDRL2 and CDRL3), respectively. CDR provides a main contacting residue to combine antibody with antigen or epitope.
Human c-Met antibody or its antigen-binding fragment may include analogs of amino acid sequences set forth in the appended Sequence Listing, which are capable of specifically recognizing human c-Met. For example, amino acid sequence of antibody may be altered to improve binding affinity and/or other biological characteristics of antibody, for example including the alterations prepared by deletion, insertion and/or substitution of amino acid residues of antibody.
Such amino acid variations may be provided on the basis of a relative similarity of amino acid side chains, e.g., hydrophobicity, hydrophilicity, charge and size. By the analysis for size, shape and type of the amino acid side chains, it could be clear that all of arginine, lysine and histidine residues are those having positive charge; alanine, glysine and serine have a similar size; phenylalanine, tryptophan and tylosin have a similar shape. Accordingly, based on these considerable factors, arginine, lysine and histidine; alanine, glysine and serine; and phenylalanine, tryptophane and tylosin may be considered to be biologically functional equivalents.
For introducing mutation, a hydropathic index of amino acids may be considered. Based on the hydrophobicity and the charge, the hydropathic index is given to each amino acid: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine (+2.5); methionine (+1.9); alanine (+1.8); glysine (−0.4); threonine (−0.7); serine (−0.8); tryptophane (−0.9); tylosin (−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagin (−3.5); lysine (−3.9); and arginine (−4.5).
For providing an interactive biological function of proteins, the hydropathic index of the amino acid is very important. It is well known to one of skill in the art that variations can possess a similar biological activity only where proteins are replaced with amino acids having similar hydropathic index. Where variations are intended to introduce based on the hydropathic index, the substitution is preferably performed between amino acid residues having no more than ±2 difference in hydropathic index values more preferably within ±1, much more preferably within ±0.5.
It would be also obvious to those of skill in the art that substitutions of amino acids with other amino acids having similar hydrophilicity values may result in the generation of variants having biologically equivalent activities. As disclosed in U.S. Pat. No. 4,554,101, each amino acid residue is assigned the following hydrophilicity values: arginine (+3.0); lysine (+3.0); aspartate (+3.0); glutamate (+3.0); serine (+0.3); asparagin (+0.2); glutamine (+0.2); glysine (0); threonine (−0.4); proline (−0.5); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tylosin (−2.3); phenylalanine (−2.5); tryptophane (−3.4).
Where variations are intended to introduce based on the hydrophilicity values, the substitution is preferably performed between amino acid residues having no more than ±2 difference in hydropathic index values more preferably within ±1, much more preferably within ±0.5.
The alteration of amino acid residues not to substantially impair protein activity is well known to one skilled in the art (H. Neurath, R. L. Hill, The Proteins, Academic Press, New York, 1979). Such amino acid alteration includes Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu and Asp/Gly, but not limited to.
Considering the afore-mentioned variations having biologically equivalent activities, it could be understood that either antibody of this invention or the nucleic acid encoding the same includes substantially identical sequences to the sequences set forth in the appended Sequence Listing. The substantially identical sequences refers to those showing preferably at least 61%, more preferably at least 70%, still more preferably at least 80%, most preferably at least 90% nucleotide similarity to the sequences of the appended Sequence Listing, as measured using one of the sequence comparison algorithms. Methods of alignment of sequences for comparison are well-known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math. 2:482 (1981); Needleman and Wunsch, J. Mol. Bio. 48:443 (1970); Pearson and Lipman, Methods in Mol. Biol. 24: 307-31 (1988); Higgins and Sharp, Gene 73:237-44 (1988); Higgins and Sharp, CABIOS 5:151-3 (1989); Corpet et al., Nuc. Acids Res. 16:10881-90 (1988); Huang et al., Comp. Appl. BioSci. 8:155-65 (1992); and Pearson et al., Meth. Mol. Biol. 24:307-31 (1994). The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215: 403-10 (1990)) is available from several sources, including the National Center for Biological Information (NBCI, Bethesda, Md.) and on the Internet, for use in connection with the sequence analysis programs blastp, blasm, blastx, tblastn and tblastx.
In addition, sequencing of framework region (FR) and CDRs in antibody variable regions may be indicated based on the sequences of IMGT generally accessible in the art.
According to an embodiment, the heavy chain variable region of 1F12 antibody includes the amino acid sequence of SEQ ID NO:19.
According to an embodiment, the light chain variable region of 1F12 antibody includes the amino acid sequence of SEQ ID NO:20.
According to an embodiment, the heavy chain variable region of 2A01 antibody includes the amino acid sequence of SEQ ID NO:21.
According to an embodiment, the light chain variable region of 2A01 antibody includes the amino acid sequence of SEQ ID NO:22.
According to an embodiment, the heavy chain variable region of 2C03 antibody includes the amino acid sequence of SEQ ID NO:23.
According to an embodiment, the light chain variable region of 2C03 antibody includes the amino acid sequence of SEQ ID NO:24.
The antibody of this invention includes, but not limited to, monoclonal antibody, polyclonal antibody, human antibody, humanized antibody, chimeric antibody, single-chain Fvs (scFV), single-chain antibody, Fab fragment, F(ab′) fragment, disulfide-linked Fvs (sdFV) and anti-idiotype (anti-Id) antibody, and epitope-binding fragment thereof.
The antibody of the present invention is basically composed of “heavy chain variable region (VH)-linker—light chain variable region (VL)”. In the scFv antibody of the present invention, the linker refers to an amino acid sequence having a predetermined length which artificially links the heavy chain and light chain variable regions.
The scFv antibody of the present invention may be expressed by VH (SEQ ID NO: 19)—linker (SEQ ID NO: 25)—VL (SEQ ID NO: 20); VH (SEQ ID NO: 21)—linker (SEQ ID NO: 25)—VL (SEQ ID NO: 22); and VH (SEQ ID NO: 23)—linker (SEQ ID NO: 25)—VL (SEQ ID NO: 24).
The antibody or its antigen-binding fragment of the present invention is specifically cross-reactive to human c-Met and mouse c-Met. Since the antibody or its antigen-binding fragment of the present invention is capable of specifically binding to human c-Met as well as mouse c-Met, more accurate preclinical results can be confirmed in the efficacy evaluation using mouse tumor models.
In another aspect of this invention, there is provided a nucleic acid molecule encoding a heavy chain variable region of an antibody against the human and mouse c-Met comprising the amino acid sequence of SEQ ID NO:19, SEQ ID NO:21 or SEQ ID NO:23.
In still another aspect of this invention, there is provided a nucleic acid molecule encoding a light chain variable region of an antibody against the human and mouse c-Met comprising the amino acid sequence of SEQ ID NO:20, SEQ ID NO:22 or SEQ ID NO:24.
The term “nucleic acid molecule” comprehensively refers to a deoxyribonucleotide (gDNA and cDNA) or ribonucleotide polymer, and the basic nucleotides of nucleic acid molecule also include analogues with modified sugar or base as well as natural nucleotides (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90:543-584 (1990)). The sequence of the present nucleic acid molecule encoding the variable region of heavy and light chain could be modified. Such modification includes addition, deletion or non-conservative or conservative substitution of nucleotide.
According to an embodiment, the nucleic acid molecule encoding the variable region of heavy chain includes the nucleotide sequence of SEQ ID NO:26, SEQ ID NO:28 or SEQ ID NO:30.
According to an embodiment, the nucleic acid molecule encoding the variable region of light chain includes the nucleotide sequence of SEQ ID NO:27, SEQ ID NO:29 or SEQ ID NO:31.
The nucleic acid molecule of this invention encoding an human c-Met antibody also includes a nucleotide sequence sharing substantial homology with the above nucleotide sequence. The substantial homology means the nucleotide sequence sharing homology of at least 80%, more preferably 90% and most preferable 95% by sequence alignment analysis using maximal alignment between the nucleotide sequence of this invention and other random sequences and algorithm ordinarily known to those skilled in the art.
In still further aspect of this invention, there is provided a recombinant vector comprising the above-described nucleic acid molecules.
The term “vector” is a tool for expressing a target gene in a host cell, including a plasmid vector; a cosmid vector; and a virus vector such as a bacteriophage vector, an adenovirus vector, a retrovirus vector and an adeno-associated virus vector, and preferably a plasmid vector.
According to a preferable embodiment, the nucleic acid molecules encoding the variable region of light and heavy chain are operatively linked to a promoter.
The term “operatively linked” refers to functional linkage between a nucleic acid expression control sequence (such as a promoter, signal sequence, or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence affects transcription and/or translation of the nucleic acid corresponding to the second sequence.
The vector system of this invention may be performed by various methods known to those skilled in the art and its practical method is described in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001), which is herein incorporated by reference.
Typically, the vector of this invention may be constructed as cloning or expression vector. In addition, the vector of this invention may be constructed using a prokaryotic or eukaryotic cell as a host cell.
For instance, in each a vector of this invention and an eukaryotic cell used as an expression vector and the host cell, the promoter derived from genome of animal cell (example: methallothionein promoter, β-actin promoter, human hemoglobin promoter and human muscle creatine promoter) or mammalian virus (example: adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, tk promoter of HSV, mouse mammary tumor virus (MMTV) promoter, LTR promoter of HIV, promoter of moloney virus, Epstein barr virus (EBV) and Rous sarcoma virus (RSV)) might be used, and polyadenylated sequence might be commonly used as the transcription termination sequence.
The vector of this invention could be fused with other sequences to purify an antibody expressed from it. For example, a fused sequence includes glutathione-S-transferase (Pharmacia, USA), maltose-binding protein (NEB, USA), FLAG (IBI, USA) and 6×His (hexahistidine; Quiagen, USA) and so on. Since the protein expressed in the vector of the present invention is antibody, expressed antibody could be also purified throughout protein A column in an easy manner without additive sequences for purification.
On the other hand, the expression vector of this invention includes an antibiotics-resistance gene known to those ordinarily skilled in the art as a selection marker, for example resistant genes against ampicillin, gentamycin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and tetracycline.
In still another aspect of this invention, there is provided a host cell transformed with the above-described recombinant vector.
The host cells in which the present vector is stably and successively cloned and expressed, also utilize any one known to those skilled in the art, for example the suitable eukaryotic host cell of the above vector includes COS7 cell (monkey kidney cell), NSO cell, SP2/0, CHO (Chinese hamster ovary) cell, W138, BHK (baby hamster kidney) cell, MDCK, myeloma cell line, HuT 78 cell and 293 cell, but not limited to.
In another aspect of this invention, there is provided a pharmaceutical composition for preventing or treating cancer, comprising: (a) a therapeutically effective amount of an antibody or its binding fragment against a human c-Met; and (b) a pharmaceutically acceptable carrier.
A pharmaceutical composition of the present invention uses, as an active ingredient, the antibody to human c-Met or its antigen-binding fragment of the present invention. Therefore, the overlapping descriptions therebetween are omitted to avoid excessive complication of the specification due to repetitive descriptions thereof.
As can be verified by the following examples, the antibody to human c-Met of the present invention inhibits the growth of cancer cells derived from various cancers by a considerable binding affinity to c-Met and the suppression of c-Met function therefrom, inhibits the phosphorylation of c-Met and downstream signaling molecules to suppress c-Met signaling, and inhibits neovascularization. Therefore, the antibody of the present invention is very useful in the prevention and treatment of cancers.
The cancers that can be prevented or treated by the composition of the invention may include various cancers known in the art, and examples thereof may include breast cancer, colon cancer, lung cancer, stomach cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, brain cancer, cervical cancer, nasopharyngeal cancer, laryngeal cancer, colon cancer, ovarian cancer, rectal cancer, colorectal cancer, vaginal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, ureter cancer, urinary tract cancer, prostate cancer, bronchial cancer, bladder cancer, kidney cancer, and marrow cancer.
Specifically, the cancers that can be prevented or treated by the composition of the invention are c-Met expressing cancers.
In the pharmaceutical compositions of this invention, the pharmaceutically acceptable carrier may be conventional one for formulation, including lactose, dextrose, sucrose, sorbitol, mannitol, starch, rubber arable, potassium phosphate, arginate, gelatin, potassium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methyl cellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, and mineral oils, but not limited to. The pharmaceutical composition according to the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, and a preservative. Details of suitable pharmaceutically acceptable carriers and formulations can be found in Remington's Pharmaceutical Sciences (19th ed., 1995), which is incorporated herein by reference.
The pharmaceutical composition according to the present invention may be administered via the parenteral. When the pharmaceutical composition of the present invention is administered parenterally, it can be done by intravenous, subcutaneous, intramuscular or intraperitoneal.
A suitable dose of the pharmaceutical composition of the present invention may vary depending on pharmaceutical formulation methods, administration methods, the patient's age, body weight, sex, severity of diseases, diet, administration time, administration route, an excretion rate and sensitivity for a used pharmaceutical composition. Preferably, the pharmaceutical composition of the present invention is administered with a daily dose of 0.001-100 mg/kg (body weight). The term “pharmaceutically effective amount” refers to an amount suitable to prevent or treat cancer
According to the conventional techniques known to those skilled in the art, the pharmaceutical composition may be formulated with pharmaceutically acceptable carrier and/or vehicle as described above, finally providing several forms including a unit dose form and a multi-dose form. Formulation may be oil or aqueous media, resuspension or emulsion, extract, powder, granule, tablet and capsule and further comprise dispersant or stabilizer.
Features and advantages of one or more embodiments of the present invention are summarized as follows:
(a) The antibody of the present invention has high specificity to human c-Met and is cross-reactive also to mouse c-Met.
(b) Since the antibody or its antigen-binding fragment of the present invention is capable of specifically binding to human c-Met as well as mouse c-Met, more accurate preclinical results can be confirmed in the efficacy evaluation using mouse tumor models.
(c) The antibody of the present invention inhibits the growth of cancer cells derived from various cancers by a considerable binding affinity to c-Met and the suppression of c-Met function therefrom, inhibits the phosphorylation of c-Met and downstream signaling molecules to suppress c-Met signaling, and inhibits neovascularization, thereby being very efficient in the prevention and treatment of cancers.
Hereinafter, the present invention will be described in detail with reference to examples. These examples are only for illustrating the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.
EXAMPLES
Example 1
Phage Display Screening for Identification of Anti-c-Met scFv Antibody Fragment
The scFv antibody fragments cross-reactive to human/mouse c-Met were identified through phage display screening by using the existing constructed synthetic scFv phage library (Yang et al., Mol. Cells. 27:225-235, 2009). The phage display screening procedure was as shown in FIG. 1.
Specifically, for the collection of phagemid vectors in a phage type, which were introduced into E. coli host ER2537, four lower sub-library samples were respectively added to 400 ml of media (SB/ampicillin/2% glucose), and then cultured for about 2 hours. The host cells cultured until OD600=0.5 were centrifuged at 5,000 g for 20 minutes to remove the supernatant, and then suspended in 400 ml of secondary media (SB/ampicillin). Then, 1012 pfu (plaque forming unit) of helper phage (VCSM13) was added, again followed by culturing for 1 hour. After that, the antibiotic Kanamycin (antibiotic gene introduced into helper phage) was added at a concentration of 70 μg/ml, followed by culturing overnight at 30° C., so that the phage library was extracellulary produced. Then, the centrifuged culture was treated with the polyethylene glycol (PEG) solution to precipitate only phage particles, thereby collecting the phage library. For the count of the phages collected from each sub-library, each sample was diluted and used for infection of host cells (ER2537). The phages were counted in the LB/ampicillin media.
Phage display screening was performed through repeated rounds of panning. The counted sub-libraries were collected to 2.0×1012 pfu, and then treated with the immunotube coated with c-Met-Fc protein diluted to 10 μg/ml in TBS. The immunotube and the phage particles before the treatment were treated with a blocking solution containing 3% skim milk for 1 hour, thereby preventing non-specific binding thereof except for c-Met. The phage libraries were treated with c-Met for 1 hour, and then the immunotube was washed with 0.1% Tween 20 in PBS (TBST). Then, 1 ml of 100 mM triethyl amine was added thereto and kept to stand for 10 minutes, so that phage particles bound to c-Met were removed and collected. For the confirmation of the number of collected phages (output), the collected solution was diluted and used for infection of the host cells, and then phage counting was performed in the medium. The remaining collected solution was plated on the 15 cm-media and then cultured, and then 5 ml of SB medium (50% glycerol) was added, followed by collection and storage (−80° C.) of colonies.
For the continuous rounds of panning, 50 μl of aliquot was taken from the stored phage solution from the previous round of panning, and subjected to phage particle amplification. The phage particles which were cultured in the host cells, added with helper phages, and collected, were prepared by PEG precipitation, and the next round of panning progressed using the phage particles by the same method as the previous round of panning. A total of four rounds of panning with mouse c-Met were progressed. After the phages collected in the fourth round of panning were again amplified, further two rounds of panning with human c-Met were performed. Phage display screening results were shown in FIG. 1.







TABLE 1







Phage display screening for mouse


(M) and human (H) c-Met (cfu/ml)











Round
1st
2nd
3rd
4th





M-Input
2.1 × 1012
2.3 × 1012
2.6 × 1012
2.8 × 1012


M-Output
7.2 × 106 
4.7 × 107 
3.8 × 107 
1.1 × 108 


H-Input
1.2 × 1011
6.2 × 1011
—
—


H-Output
1.7 × 107 
4.9 × 107 
—
—









Example 2
Sequencing and ELISA Analysis for Anti-c-Met scFv Candidate Selection and ELISA Selection
After a total of six rounds of panning with mouse/human c-Met were finished, the phage particles collected from the final round of panning were confirmed as colonies in the medium through infection of host cells. These colonies were taken and inoculated in a 96 well plate containing 200 μl of SB/ampicillin media and then cultured (37° C., within 3 hours). After that, for the induction of scFv-pIII protein expression, 1 mM final concentration IPTG was added to each well, followed by culturing overnight at 30° C. After that, the cultured plate was centrifuged to discard the supernatant. Then, for the collection of periplasmic fractions from the cultured cells in each well, the culture plate was treated with 40 μl of TES solution (20% w/v sucrose, 50 mM Tris, 1 mM EDTA, pH 8.0) maintained at 4° C., and then kept to stand at 4° C. for 30 minutes, so that the cells were lysed. After that, the cells were treated with 60 μl of 0.2×TES solution, and then kept to stand for 30 minutes. Finally, the plate was centrifuged and the supernatant was collected, thereby producing scFv-pIII protein on a small scale.
At the same time, a 96 well plate coated with human or mouse c-Met-Fc protein was prepared, and then 25 μl of aliquot was taken from the collected periplasmic fractions and added to each well, followed by binding for 1 hour. Then, after three or four times of washing procedures were performed using TBST, anti-HA-HRP antibody was added, followed by binding for 1 hour. The plate was again washed, followed by induction of a color development reaction (TMB substrate), and then the values of color development were measured at O.D. 450 nm. A total of 282 colons were analyzed, and 25 colons (binding affinity >2-fold) out of them showed a higher binding affinity to human/mouse c-Met (FIG. 2). Since antibody in which c-Met extracellular domain binds to Fc region of IgG was used at the time of the panning, commercialized antibody (Erbitux) or BSA protein was used as a control group in order to exclude the phage particles that bind to Fc region. Resultantly, it was verified that the cross-reactive affinities of respective clones to human/mouse c-Met were on average about 10-fold as compared with that of the control group.
For the scFv sequencing, phagemid DNA was collected from each clone, and DNA sequence analysis thereof was requested. As a result, it was verified that scFv sequences of clones 1D03/1D08/1E06/1F12/2B04/2D02/2DC09/3E07/3H08 were identical, and, also, scFv sequences of clones 2A01/2B10 and 2C03/3B05 were identical to each other. Considering that nine, two, and two clones out of a total of 25 colons were verified to have identical scFv sequences, respectively, the scFv antibody fragments having these sequences have higher binding affinities to specific epitope in the c-Met extracellular domain, and thus largely selected in the screening procedure. Through this, three scFv antibody fragments cross-reactive to human/mouse c-Met were identified. 2D05 clones that had no specific biding affinity to human/mouse c-Met was used as a control for further evaluation of functions of the three scFv antibody fragments.







TABLE 2







Heavy chain FR/CDR sequences of scFv antibody fragments


cross-reactive to human/mouse c-Met










Clone (V-





gene family)
1F12 (IGHV3)
2A01 (IGHV3)
2CO3 (IGHV3)





FR1
EVQLLESGG
EVQLLESGGGLVQTG
EVQLLESGGGLVQPG



GLVQPGGSLR
GSLRLSCAAS
GSLRLSCAAS



LSCAAS(SEQ ID
(SEQ ID NO: 275)
(SEQ ID NO: 282)



NO: 268)




 


CDR1
GFTFSNYA
GFTFSSYD
GFTFSNYA



(SEQ ID NO:
(SEQ ID NO:
(SEQ ID NO: 283)



269)
276)



 


FR2
MSWVRQAPG
MSWVRRAPGKGLEW
MSWVRQAPGKGLEW



KGLEWVSG
VSW (SEQ ID NO:
VSA (SEQ ID NO:



(SEQ ID NO:
277)
284)



270)




 


CDR2
ISYSGGST
ISHGGSSI
ISYDSGSI



(SEQ ID NO:
(SEQ ID NO: 278)
(SEQ ID NO: 285)



271)




 


FR3
YYADSVKGR
SYADSVKGRFTISRDN
YYADSVKGRFTISRDN



FTISRDNSKN
SKNTLYLQMNSLRAE
SKNTLYLQMNSLRAE



TLYLQMNSL
DTAVYYC
DTAVYYC



RAEDTAVYYC
(SEQ ID NO: 279)
(SEQ ID NO: 286)



(SEQ ID NO:





272)




 


CDR3
AKASRSCQRP
AKDAYPIRQETFDY
AKAARSCRNWSCSYA



ACSYANGMDV
(SEQ ID NO: 280)
NGMDV (SEQ ID 



(SEQ ID NO:

NO: 287)



273)




 


FR4
WGQGTLVTVSS
WGQGTLVTVSS
WGQGTLVTVSS



(SEQ ID NO:
(SEQ ID NO: 281)
(SEQ ID NO: 288)



274)
















TABLE 3







Light chain FR/CDR sequences of scFv antibody fragments cross-


reactive to human/mouse c-Met










Clone (V-





gene family)
1F12 (IGLV1)
2A01 (IGLV1)
2CO3 (IGLV1)





FR1
QSVLTQPPS
QSVLTQPPSASGTPGQ
QSVLTQPPSASGTPGQ



ASGTPGQRV
RVTISCSGS
RVTISCTGS



TISCTGS
(SEQ ID NO: 295)
(SEQ ID NO: 301)



(SEQ ID NO: 





289)




 


CDR1
SSNIGNNY
SSNIGNND
SSNIGSNY



(SEQ ID NO:
(SEQ ID NO: 296)
(SEQ ID NO: 302)



290)




 


FR2
VTWYQQLP
VSWYQQLPGTAPKLLIY
VSWYRQLPGTAPKLLIY



GTAPKLLIY
(SEQ ID NO: 297)
(SEQ ID NO: 303)



(SEQ ID NO:





291)




 


CDR2
YNN
PDS
SDS


 


FR3
HRPSGVPDR
QRPSGVPDRFSGSKSG
NRPSGVPDRFSGSKSG



FSGSKSGTS
TSASLAISGLRSEDEA
TSASLAISGLRSEDEA



ASLAISGLQ
DYYC
DYYC



SEDEADYYC
(SEQ ID NO: 298)
(SEQ ID NO: 304)



(SEQ ID NO:





292)




 


CDR3 
GSWDYSLSAYV
ASWDSSLSGYV
GSWDDSLSGYV



(SEQ ID NO:
(SEQ ID NO: 299)
(SEQ ID NO: 305)



293)




 


FR4
FGGGTKLTVL
FGGGTKLTVL
FGGGTKLTVL



(SEQ ID NO:
(SEQ ID NO: 300)
(SEQ ID NO: 306)



294)









Example 3
Verification on Binding Affinity to c-Met Using Anti-c-Met scFv Labeled Phages
Since the three scFV antibody fragments were selected by phage display screening, their binding affinities to c-Met were first confirmed while they are expressed in the phage structure. Respective phage particles labeling three scFV antibody fragments were individually collected, and counted according to clones (1F12: 2.58×1012 pfu, 2A01: 8.1×1011 pfu, 2C03: 8.5×1011 pfu, 2D05: 1.49×1012 pfu). After that, a 96-well plate coated with human c-Met was treated with dilutions of phage particles according to concentrations, to verify their binding affinities through ELISA analysis using anti-phage antibody. As a result, it was verified that as the number of phage particles decreases, the binding affinity tends to decrease, and thus the specificity to c-Met depending on the number of phage particles can be confirmed (FIG. 3A). Also when the plate was treated with each phage having a predetermined number of phage particles (1×1010 pfu/well), the respective phage clones were verified to show statistically significant high binding affinities as compared with the control phage (2D05) (FIG. 3B). Therefore, it was verified that the respective scFV antibody fragments selected based on the binding affinity to c-Met in the screening procedure showed binding affinities to c-Met even while they are labeled in the phage structure bodies.
Example 4
Production of Anti-c-Met scFv Proteins and Verification on Binding Affinity Thereof to c-Met
For the verification of binding affinity and function of scFv alone, expression and purification were conducted by using the protein expression strain (TOP10F′). The basic structure of phagemid can be confirmed in FIG. 4A. The host cells (ER2537) containing screened phagemid suppress the transcription stop codon (amber codon, UAG) between scFv and the pIII protein of the phage. Since expression of scFv alone is not possible therein, the expression strain (TOP10F′), which is the non-suppressor strain, was used.
Specifically, phagemid coding each antibody fragment was collected from the host cells, and then introduced into the expression strain. After that, the expression strains into which respective phagemids were successfully introduced were confirmed through DNA sequencing. A single colony was taken from the expression strains to which scFv was introduced, and inoculated in 3 ml of LB/ampicillin media, followed by culturing at 37° C. overnight. After the culturing overnight, the culture solution was transferred to 400 ml of media (SB/ampicillin), and then further cultured until OD600=0.5 to 0.5. 1 mM final concentration IPTG was added, again followed by culturing overnight at 30° C. After the culture solution was centrifuged, the expression hosts were lysed in 40 ml of TES solution, and then the periplasmic fractions were collected. The collected culture solution was filtered through 0.45 μm filter. The scFv protein in the filtered lysate was allowed to bind to 1.2 ml of Ni-NTA beads (Qiagen), which were added for His-tag purification, at room temperature for 1 hour, and then packed in the gravity column (Bio-rad), followed by wash and collection using an imidazole solution. SDS-PAGE and Coomassie blue staining results after expression and purification of each clone were shown in FIG. 4B. Each scFv was verified to have a size of about 28 kDa. The concentration of each purified scFv was determined by Bradford protein assay. Then, the purified scFv were stored, and used for later experiments.
By using the produced scFv protein, the binding affinity to human/mouse c-Met extracellular domain+Fc protein was confirmed through ELISA. Human/mouse c-Met coated on a 96-well plate were treated with 5 μg/ml of scFv proteins (1F12, 2A01, 2C03, and 2D05), followed by binding at room temperature for 1 hour. After that, the plate was washed three times with 0.1% TBST solution, and then treated with anti-HA-HRP antibody for 1 hour, to detect the scFv proteins that bind to c-Met. After that, the plate was washed three times, and then treated with 10 μl of TMB substrate, followed by standing for 5 minutes. The reaction was stopped by using sulfuric acid solution, and then the absorbance was determined by ELISA reader. As a result, it was verified that each scFv protein had significant binding affinity to human and mouse c-Met proteins as compared with the control group (IgG, BSA) (FIG. 5A). In addition, after the cells were treated with 500 ng/ml of each scFv protein using human c-Met protein to which Fc region was not bound, the same ELISA assay was conducted. Similarly, the degrees of binding affinity of the respective scFv proteins were slightly different, but all the scFv proteins successfully showed specificity to c-Met extracellular domain.
Example 5
Verification on Inhibition of Antibody Fragments on Binding Between Ligand (Hepatocyte Growth Factor) and c-Met Through Competitive Binding
For the verification of whether the selected scFv proteins have competitive binding affinity with the hepatocyte growth factor, which is the ligand of c-Met, the ligand competitive ELISA experiment was conducted. As in examples 2 and 4, the coated human c-Met was prepared, and respective wells were treated with samples in which each scFv protein was diluted at a 2-fold dilution from 500 ng/ml to about 1 ng/ml. After the treatment, the experiment procedure was conducted as in Example 4. Meanwhile, for the verification of the competitive binding affinity with the ligand, an experiment with regard to excessive treatment 2.5 μg/ml of HGF was conducted in the same manner. As a result, the 1F12 scFv and 2C03 scFv treatments showed somewhat dull competition despite a large amount of ligand, and the 2A01 scFv treatment showed sensitive competition in a concentration-dependent manner (FIGS. 6A-6C). These results verified that the 2A01 clone might bind closer to the ligand binding site of c-Met than 1F12 or 2C03 clone, thereby more effectively blocking the interaction between c-Met and the ligand. However, 1F12 and 2C03 scFv antibodies were also used for a functional evaluation using cell lines since there have been reports that antibodies having an excellent binding affinity to a receptor (or ligand) can exhibit effective treatment efficacies even though they poorly compete with the ligand to bind to the receptor.
Example 6
Verification on Binding Affinities of scFv Antibody Fragments to c-Met Overexpressing Cancer Lines
It has been so far reported that there are c-Met overexpressing cell line models in various cancers. In consideration of this, the binding degrees of scFv antibodies were compared through FACS analysis using a total of five cancer cell lines. The five cancer cell lines were respectively derived from gastric cancer (MKN45), brain tumor (U87MG), kidney cancer (Caki-1), lung cancer (H441), and liver cancer (HepG2). Each of the cell lines was cultured in media (DMEM, 10% FBS), and 5×105 cells were prepared in each tube. After that, the cells were fixed by 4% Paraformaldehyde, followed by centrifugation, and the tube was washed one time with FACS analysis solution. The prepared cells were treated with 1 μg and 5 μg of each scFv protein, and then the corresponding antibody fragment was allowed to bind to the cells through culturing at 4° C. overnight. After that, non-specifically bound scFv proteins were washed two times with FACS solution, and then fluorescence (phycoerythrin, PE)-bound anti-HA antibody was allowed to bind to the cells for 1 hour. In addition, the cells were again washed with FACS solution, and 500 μl of FACS solution was added to perform a FACS analysis. Resultantly, all of three scFv antibody fragments, 1F12, 2A01, and 2C03, showed specific binding affinities to c-Met overexpressing cell lines as compared with the control group (2D05) (FIGS. 7A-7J). Further, since the binding tendency was verified to decrease with the decrease of scFv concentration, it can be seen that the corresponding scFv antibody fragments have high specificity to the target protein c-Met.
In addition, for the verification of specificities of scFv antibody fragments to c-Met using two representative cell lines (U87MG and HepG2) of the corresponding cell lines, c-Met knockdown was induced using c-Met-siRNA. The c-Met knockdown was conducted by mixing two variants SIHK1284 and SIHK1285 Met siRNA (sigma-aldrich) at the same concentration. As for cell introduction conditions, the cells were treated with 50 pmol/ml of siRNA together with Lipofectamine (Invitrogen) of 5 μl or 2 μl per reaction, and the proteins were collected from the cells. The optimum expression reduction conditions were confirmed through western blotting (FIG. 8A). After the introduction of siRNA, FACS analysis was conducted using c-Met monoclonal antibody. As shown in FIGS. 8B and 8C, the c-Met expression was verified to be reduced in both of U87MG and HepG2 cells. In addition, as expected, it was verified that the binding affinities of the respective scFv antibody fragments of 5 μg/ml to c-Met expression-reduced cells significantly decreased (FIGS. 8C-8I). This verified that the identified scFv antibody fragments had actually specificity to the c-Met extracellular domain present on cell membranes.
Example 7
Verification on Ability of scFv Antibody Fragments to Inhibit Cell Growth in c-Met Overexpressing Cancer Line
For the verification of anti-cancer ability of the identified 1F12, 2A01, and 2C03 scFv antibody fragments through inhibition of cancer cell proliferation, cell proliferation assay was conducted. The experiment was conducted by using three cell lines, that is, UG87MG and KP-4 cell lines which act on c-Met overexpressed on cell surfaces by autocrine HGF, and MKN45 cells of which the autocrine function is not clear but in which the c-Met overexpressed on cell surfaces was hyper-activated to continuously induce c-Met signaling into the cells. Three cell lines were prepared such that 1×103 cells were present in 100 μl of media in conditions with (10 ng/ml) or without HGF. 50 μg/ml of individual scFv antibody fragment clones were prepared to treat wells containing three species of cells cultured for 24 hours. On day 0, 1, 2, 3, and 4, the degree of cell growth was measured by the EZ-Cytox cell viability assay kit (Daeil Lab. Service).
Resultantly, in the experiment without HGF, the growth inhibitory effects by all the clones were shown from day 2 in all of three cell lines (FIGS. 9A-9G). Meanwhile, in conditions with HGF, the growth inhibitory effect by the 1F12 scFv antibody fragment was the highest, and the 2A01 and 2C03 scFv antibody fragments also showed growth inhibitory effects, which were low but above the significant level. Based on these results, it was verified that the 1F12 scFv antibody fragment inhibited cell growth due to its distinctive high c-Met binding and the suppression of c-Met function therefrom.
Example 8
Verification on Permeability of Antibody Fragments into c-Met Overexpressing Cancer Cells
For the intercellular localization of anti-c-Met antibody fragments, cell immunofluorescence staining assay was conducted. A 6-well plate was covered with sterilized slide coverslips. The experimental cell line MKN45 (105 cells/well) were seeded therein, and then the cells were cultured until the cells proliferated in 60-70% of the area. After that, the culture media were treated with 5 μg/ml of respective antibody fragments, and then allowed to stand 37° C. and 4° C. for 1 hour to induce interactions between the respective antibody fragments and c-Met. After that, the cells were fixed by Paraformaldehyde and treated with 0.1% Triton X-100 to improve cell permeability. After that, the cells were treated with 1% BSA blocking solution at room temperature for 1 hour, and then treated with a red fluorescent material (Alexa-Fluor 647)-labeled mouse anti-HA antibody (Cell Signaling Technology) for intercellular localization of respective candidate antibody fragments, followed by reaction at room temperature for 1 hour. Last, DAPI staining was conducted to stain the nuclei blue, followed by final washing. Then, the coverslips were taken out and fixed on the glass slide, which was then observed by confocal microscopy.
As the result of experiment, when the cells were treated with antibody fragments and allowed to stand at 4° C., the red fluorescence was not detected in the negative control group 2D05 antibody fragment. In the other three candidate antibody fragments, the red fluorescence was detected on only cell membranes. Antibody fragments were not detected in the cytoplasm (FIG. 10). Similarly, when the cells were treated with antibody fragments and then kept to stand at 37° C., red fluorescence was not detected in the negative control group 2D05 antibody fragment. However, in 37° C. conditions, unlike 4° C. conditions, the three candidate antibody fragments were verified to be located on cell membranes as well as in the cytoplasm. Considering the degree of red fluorescence, the 1F12 antibody fragment was showed to be more permeable into the cells than the other antibody fragments (FIG. 10).
Example 9
Verification on Inhibition of Antibody Fragments on Met Signaling Pathway
For the evaluation whether each scFv clone can inhibit the phosphorylation in downstream signaling molecules of the c-Met signaling pathways, western blotting was conducted. The experimental cell lines U87MG and MKN45 were cultured in a 6-well plate and treated with 50 ng/ml of HGF together with the respective antibody fragments. After that, the respective proteins were prepared using the RIPA buffer, and then SDS-PAGE electrophoresis was conducted on 10% gel. The proteins on the electrophoresis gel were transferred to the nitrocellulose membrane, and then proteins such as AKT and ERK associated with c-MET signaling pathway were detected by respective antibodies. As a result, it was verified that all of the three antibody fragments inhibited the phosphorylation of c-Met downstream signaling molecules, such as p-AKT and p-ERT, as compared with the negative control group 2D05 antibody fragment, and it was verified that 1F12 antibody fragment among the candidate antibodies can more effectively inhibit the phosphorylation of c-Met downstream signaling molecules.
Example 10
Verification on Neovascularization Inhibition
HGF/c-Met signaling pathway has been largely linked to VEGF, and induces proliferation and migration in endothelial cells to accelerate tumor angiogenesis (Rosen E M et al. Ciba Found Symp 212:215-226, 1997). Therefore, the human umbilical vein endothelial cell (HUVEC) model cell line was treated with respective clones with or without HGF, and then the tendency of neovascularization was observed. Matrigel (BD science) was slowly melted at a low temperature. Then, 50 μl of Matrigel was used to treat a 96-well plate, and then solidified at 37° C. The EBM-2 (basal medium) was treated with 20 ng/ml of HGF. Cells were dispensed into wells at 4,000/well, and then treated with 50 μg/ml of each antibody fragment. Neovascularization was induced in a CO2 incubator at 37° C. for 6 hours, and then the formed blood vessels were photographed by optical microscopy.
As the result of experiment, the neovascularization of HUVEC cell line was verified to be accelerated by HGF treatment in the negative control group (PBS, 2D05), and it was visually verified that this acceleration of neovascularization was effectively inhibited by treatment with the candidate antibodies having a binding affinity to c-Met (FIGS. 12A and 12B). In addition, this inhibitory effect was verified to be significant as compared with the negative control group by expressing the length or number of the formed blood vessels as numerical values (FIGS. 12C and 12D).
Example 11
Verification on Identification of Binding Sites of Respective Clones Through Epitope Mapping
The epitope mapping of respective anti-c-Met antibody fragments was performed using peptide array technology. Based on the amino acid sequence expressing the extracellular domain of c-Met, a cellulose membrane on which 224 different 15-mer peptides were synthesized was prepared (JPT Peptide Technologies, FIGS. 13A to 13C). First, the prepared membrane was immersed in methanol for 1 minute to avoid the precipitation of hydrophobic peptides during the TBS washing procedure. The membrane was then washed three times with TBS for 10 minutes, and blocked at room temperature for 3 hours (5% skim milk). After that, the membrane was treated with respective antibody fragments diluted to a concentration of 1 μg/ml in the blocking buffer, and then allowed to stand at 4° C. overnight. The next day, the membrane was washed with TBST three times for 1 minute for each time, and an electric field of 1.0 mA/cm2 was applied to the membrane for 1 hour to transfer the antibody fragments, which were non-covalently bound to the peptide membrane, and to the PVDF membrane. This procedure was repeated three times. The PVDF membrane from the third time was used since the PVDF membrane from the first time and second time emits non-specific spots. The PVDF membrane from the third time was washed with TBST three times for 10 minutes for each time. The PVDF membrane to which the bound antibody fragment was transferred was again blocked (5% skim milk) for 3 hours, and then treated with anti-HA-HRP antibody at room temperature for 2 hours to label the strongly bound antibody fragments. The membrane was washed three times with TBST for 5 minutes for each time. After that, the membrane was treated with a substrate (Amersham ECL Prime Detection Reagent, GE healthcare), and then subjected to film exposure and sensitization to identify epitope.
As the result of experiment, the 1F12 antibody fragment was verified to bind to the peptide fragment coding the PSI domain of c-Met, and the 2A01 and 2C03 antibody fragments were verified to bind to the peptide fragment coding the Sema domain of c-Met (FIGS. 14A to 14C). Through these results, it can be inferred that 2A01 and 2C03 would bind to the epitope (Sema domain) of antibodies, which was previously reported, thereby inducing the inhibition of cell growth through competition with HGF. Further, it was interestingly concluded that the 1F12 antibody fragment would bind to new epitope (PSI domain), which was not previously reported, thereby exhibiting higher effects in binding affinity, cell growth inhibition, intracellular signaling inhibition, and the like.
Example 12
Determination of Coupling Constants of Respective Clones Using Surface Plasmon Resonance
For the confirmation of more accurate binding affinity (coupling constant), the binding affinity of respective clones to c-Met was verified through surface plasmon resonance analysis. Respective antibody fragments were expressed in the host E. coli, followed by purification, and then dialyzed with PBS solution. Biacore T100 (GE healthcare) was used for the analysis. The c-Met protein was fixed to a dextran medium through an amine coupling reaction in the CM5 sensor chip. The respective antibody fragments were diluted with the HBS-EP solution, and the binding affinity values (resonance unit, RU) thereof were analyzed according to concentrations (FIGS. 15A to 15C). Based on these values, kinetic analysis was conducted through the BIA-evaluation program. As a result, the KD values of the 1F12 and 2A01 clones were measured to be 6−9×10−8 M and the Kd value of 2C03 was verified to be 10−7 M (Table 4). Considering that the antibody type is a single chain antibody fragment, it can be seen that the corresponding binding affinities of the 1F12 and 2A01 clones have significantly high levels, and it was ascertained that these binding affinities of the respective clones also played critical roles in inhibiting functions of c-Met, such as inhibiting cell growth and signal transduction.







TABLE 4







Coupling constant (Kd) based on Ka and Kb


measured by surface plasmon resonance












—
KD(M)
Ka(M−1S−1)
Kd(S−1)







1F12
9.33 × 10−8
5.12 × 104
4.78 × 10−4



2A01
6.60 × 10−8
2.32 × 106
1.53 × 10−1



2C03
2.39 × 10−7
7.76 × 105
1.86 × 10−1










Although the present invention has been described in detail with reference to the specific features, it will be apparent to those skilled in the art that this description is only for a preferred embodiment and does not limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

## Claims

1. An antibody to human c-Met or its antigen-binding fragment, comprising:
(a) a heavy chain variable region having the following heavy chain complementarity determining region (CDR) amino acid sequences: CDRH1 including the amino acid sequence of SEQ ID NO: 1, CDRH2 including the amino acid sequence of SEQ ID NO: 2, and CDRH3 including the amino acid sequence of SEQ ID NO: 3; and
(b) a light chain variable region having the following light chain CDR amino acid sequences: CDRL1 including the amino acid sequence of SEQ ID NO: 4, CDRL2 including the amino acid sequence of SEQ ID NO: 5, and CDRL3 including the amino acid sequence of SEQ ID NO: 6.

2. The antibody to human c-Met or its antigen-binding fragment of claim 1, wherein the heavy chain variable region includes the amino acid sequence of SEQ ID NO: 19.

3. The antibody to human c-Met or its antigen-binding fragment of claim 1, wherein the light chain variable region includes the amino acid sequence of SEQ ID NO: 20.

4. A composition comprising: (a) the antibody to human c-Met or its antigen-binding fragment of claim 1; and (b) a pharmaceutically acceptable carrier.