KR20170012754A - Antibodies Capable of Binding Specifically to EGFR - Google Patents

Abstract

The present invention relates to a novel EGFR antibody for use in combination therapy for cancer prevention or treatment. The antibody of the present invention is an antibody that specifically binds to EGFR that is overexpressed in cancer cells (particularly, colorectal cancer), and binds to epitopes different from cetuximab. The antibodies of the present invention have a unique homology with the CDR sequences of conventional EGFR target antibodies, with very low homology. The antibody of the present invention is highly effective for the prevention or treatment of cancer (particularly, colon cancer) by killing cancer cells by a greatly improved ability to kill cancer cells when they are administered in combination with cetuximab. The superior efficacy of the conjugate administration by the antibody of the present invention can be achieved without wishing to be bound by theory that the antibody of the present invention binds to an epitope on the EGFR phase different from cetuximab, And co-operatively inhibited EGFR.

Description

≪ RTI ID = 0.0 > Antibodies < / RTI > Capable of Binding Specifically to EGFR &

The present invention relates to an EGFR antibody used for the prevention or treatment of diseases related to human EGFR (Epidermal Growth Factor Receptor), particularly cancer.

The Epidermal Growth Factor Receptor (EGFR) is a 170 kDa trans-membrane type 1 molecule and contains four structurally similar receptor tyrosine kinases, ErbB-1 (EGFR), ErbB-2 (HER- 2), ErbB-3 (HER-3), and ErbB-4 (HER-4). EGFR is activated when its ligand (EGF, TGF-α, etc.) binds to the extracellular domain, forming a dimerization with the adjacent receptor. As a result, the autophosphorylation of the tyrosine group in the intracellular domain occurs and affects cell growth and proliferation, metastasis, angiogenesis, and cell death through various signal transduction processes (Hynes NE et Nat Rev Cancer 5 (7): 341-354 (2005)).

Various EGFR inhibitors have been developed by modulating the function of EGFR and by continuing research to inhibit cancer. The EGFR inhibitor is divided into a low-molecular compound that inhibits phosphorylation of the tyrosine group present in the intracellular domain and a monoclonal antibody that inhibits the binding of the ligand to the receptor and thereby inhibits the function of EGFR. Monoclonal antibodies targeting EGFR specifically act on cancer cells overexpressing EGFR. Thus, antibodies have been developed in a variety of ways, and methods are described in the literature: Waterfield MD et al. J Cell Biochem. 20: 149-161 (1982); Gill GN et al. J Biol Chem. 259: 7755-7760 (1984); Define LH et al. J Cell Biol. 109: 2495-2507 (1989); Murthyu et al. Arch Biochem Biophys. 252: 549-560 (1987); Bellot F et al. J Cell Biol. 110: 491-502 (1990); Yang XD et al. Crit Rev Oncol Hematol. 38: 17-23 (2001); Bleeker WK et al. J Immunol. 173: 4699-4707 (2004); Schaefer G et al. Cancer cell. 20 (4): 472-486 (2011).

Among the antibodies targeting EGFR, the antibodies used are cetuximab, panitumumab, and the most successful antibodies are cetuximab (commercially available as Erbitux, US Pat. No. 6,217,866) There are many studies on this: Huang SM et al. Cancer Res. 59 (8): 1935-1940 (1999); Saleh MN et al. Cancer Biother Radiopharm. 14 (6): 451-463 (1999); Bruns CJ et al. Clin Cancer Res. 6 (5): 1936-1948 (2000); Baselga J et al. J Clin Oncol. 18 (4): 904-914 (2000); Overholser JP et al. Cancer. 89 (1): 74-82 (2000); Liang YH et al. Anticancer Res. 35 (7): 4207-4214 (2015).

Although cetuximab and panituimuthat antibodies have been successfully used commercially, only 36% of patients with colorectal cancer tend to have that effect. In addition, the effect of K-ras mutant colon cancer is 28%, and that of K-ras mutant colon cancer is 6% (Di Nicolantonio F et al. J Clin Oncol 26 (35): 5705-5712 (2008)). Thus, attempts have been made to overcome the limitations of cetuximab and improve the prognosis of cancer patients, with concomitant administration of cetuximab efficacy or spectrum of efficacy (for drug reactive cancer cell lines).

For example, WO 2009-114703 discloses an attempt to increase the anti-cancer effect by the combined administration of cetuximab and an aurora kinase inhibitor. United States Patent Application Publication No. 2010-0255108 discloses a method of coadministering cetuximab with tetraiodothyroacetic acid (tetrac), a thyroid hormone analogue having anti-angiogenic activity. United States Patent Application Publication No. 2009-0258364 discloses a method for determining cancer cell lines exhibiting anticancer efficacy by using cetuximab, pantimutham and allotinib in combination.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have sought to develop antibodies capable of preventing or treating EGFR-related diseases, particularly cancer (particularly, colon cancer). In particular, the present inventors have sought to develop an antibody that can overcome the limitation of anticancer activity of cetuximab alone when the cetuximab antibody is coadministered. As a result, the inventors of the present invention have found that, while exhibiting excellent binding ability to EGFR as a whole, the present invention can prevent cancer (particularly, colorectal cancer, more specifically EGFR-expressing colorectal cancer) Or a novel antibody capable of treating the same, thereby completing the present invention.

It is an object of the present invention to provide an antibody against EGFR (Epidermal Growth Factor Receptor) or an antigen-binding fragment thereof.

It is another object of the present invention to provide a nucleic acid molecule encoding an antibody to EGFR or an antigen-binding fragment thereof.

It is another object of the present invention to provide a recombinant vector comprising the nucleic acid molecule.

It is another object of the present invention to provide a host cell transformed with said recombinant vector.

It is another object of the present invention to provide a pharmaceutical composition for preventing or treating cancer.

It is still another object of the present invention to provide a pharmaceutical composition for inhibiting cell growth.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, there is provided an antibody against human epidermal growth factor receptor (EGFR) or antigen-binding fragment thereof, comprising:

(a) a heavy chain variable region comprising the following heavy chain CDR (complementarity determining region) amino acid sequence; CDRH1 of SEQ ID NO: 1, CDRH2 of SEQ ID NO: 2 and CDRH3 of SEQ ID NO: 3, and

(b) a light chain variable region comprising the following light chain CDR amino acid sequence; CDRL1 of Sequence Listing 4, CDRL2 of Sequence Listing 5, and CDRL3 of Sequence Listing 6.

The present inventors have found that, when cancer (particularly, colorectal cancer) can be prevented or treated and cetuximab antibody is coadministered, an antibody capable of overcoming the limit of anticancer activity of cetuximab alone treatment Efforts have been made to develop novel antibodies that can prevent or treat cancer (particularly, colorectal cancer, more specifically EGFR-expressing colorectal cancer) with greatly improved efficacy by co-administration with cetuximab.

The antibody of the present invention has a specific binding ability to EGFR. In particular, the antibody of the invention binds to an epitope that differs from the epitope bound to cetuximab among the epitopes in EGFR.

As used herein, the term " cetuximab " refers to the antibody disclosed in U.S. Patent No. 6,217,866.

The antibody of the present invention has excellent killing ability or proliferation inhibiting ability against various EGFR expressing cancer cells. As used herein, the term " kill " or " proliferation inhibition " used in reference to a cancer cell is used interchangeably.

As used herein, the term " antibody " is a specific antibody to EGFR, including the complete antibody form as well as the antigen binding fragment of the antibody molecule.

A complete antibody is a structure having two full-length light chains and two full-length heavy chains, each light chain linked by a disulfide bond with a heavy chain. The heavy chain constant region has gamma (gamma), mu (mu), alpha (alpha), delta (delta) and epsilon (epsilon) types and subclasses gamma 1 (gamma 1), gamma 2 ), Gamma 4 (gamma 4), alpha 1 (alpha 1) and alpha 2 (alpha 2). The constant region of the light chain has the kappa and lambda types (Cellular and Molecular Immunology, Wonsiewicz, MJ, Ed., Chapter 45, pp. 41-50, WB Saunders Co. Philadelphia, PA (1991); Nisonoff, A., Introduction to Molecular Immunology, 2nd Ed., Chapter 4, pp. 45-65, Sinauer Associates, Inc., Sunderland, MA (1984)).

As used herein, the term " antigen binding fragment " refers to a fragment having an antigen binding function and includes Fab, F (ab ') 2, F (ab') ₂ and Fv. Fabs in the antibody fragment have one antigen-binding site in a structure having a variable region of a light chain and a heavy chain, a constant region of a light chain, and a first constant region (C _H1 ) of a heavy chain. Fab 'differs from Fab in that it has a hinge region that contains at least one cysteine residue at the C-terminus of the heavy chain C _H1 domain. The F (ab ') ₂ antibody is produced in which the cysteine residue of the hinge region of the Fab' forms a disulfide bond. Recombinant techniques for producing Fv fragments with minimal antibody fragments having only heavy chain variable regions and light chain variable regions are described in PCT International Publication Nos. WO 88/10649, WO 88/106630, WO 88/07085, WO 88/07086, WO 88/09344. The double-chain Fv is a non-covalent bond, and the variable region of the heavy chain and the light chain variable region are connected to each other. The single-chain Fv generally shares the variable region of the heavy chain and the variable region of the short chain through the peptide linker Or directly connected at the C-terminus to form a dimer-like structure like the double-stranded Fv. Such an antibody fragment can be obtained using a protein hydrolyzing enzyme (for example, a Fab can be obtained by restriction of the whole antibody to papain, and F (ab ') ₂ fragment can be obtained by cleavage with pepsin), or Can be produced through recombinant DNA technology.

In the present invention, the antibody is preferably in the form of a Fab or a complete antibody form. In addition, the heavy chain constant region may be selected from any one of gamma (gamma), mu (mu), alpha (alpha), delta (delta) or epsilon (epsilon). Preferably, the constant region is gamma 1 (IgGl), gamma 3 (IgG3) and gamma 4 (IgG4), most preferably gamma 1 (IgGl) isotype. The light chain constant region may be a kappa or lambda form, preferably a kappa form. Thus, preferred antibodies of the invention are Fab or IgGl forms with a kappa light chain and a gamma 1 (gamma 1) heavy chain.

As used herein, the term " heavy chain " refers to a variable region domain V _H comprising an amino acid sequence having a sufficient variable region sequence to confer specificity to an antigen, and a variable region domain V _H comprising three constant region domains C _H1 , C _H2 and C _H3 Quot; means both the heavy chain and the fragment thereof. As used herein, the term " light chain " also refers to both the full length light chain and its fragments, including the variable region domain V _L and the constant region domain C _L comprising the amino acid sequence with sufficient variable region sequence to confer specificity to the antigen do.

As used herein, the term "complementarity determining region" (CDR) refers to the amino acid sequence of the hypervariable region of the immunoglobulin heavy chain and light chain (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Ed. US Department of Health and Human Services, National Institutes of Health (1987)). The heavy and light chains each contain three CDRs (heavy chain (CDRH1, CDRH2 and CDRH3) and light chains (CDRL1, CDRL2 and CDRL3)). CDRs provide the major contact residues for antibody binding to an antigen or epitope.

The EGFR antibody or antigen-binding fragment thereof of the present invention may include variants of the amino acid sequence described in the appended sequence listing within the scope of specifically recognizing EGFR. For example, the amino acid sequence of an antibody may be altered to improve the binding affinity and / or other biological properties of the antibody. Such modifications include, for example, deletion, insertion and / or substitution of the amino acid sequence residues of the antibody.

Such amino acid variations are made based on the relative similarity of the amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, and the like. By analysis of the size, shape and type of amino acid side chain substituents, arginine, lysine and histidine are both positively charged residues; Alanine, glycine and serine have similar sizes; Phenylalanine, tryptophan and tyrosine have similar shapes. Thus, based on these considerations, arginine, lysine and histidine; Alanine, glycine and serine; And phenylalanine, tryptophan and tyrosine are biologically functional equivalents.

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

The hydrophobic amino acid index is very important in imparting the interactive biological function of proteins. It is known that substitution with an amino acid having a similar hydrophobicity index can retain similar biological activities. When the mutation is introduced with reference to the hydrophobic index, substitution is made between amino acids showing preferably a hydrophobic index difference of within ± 2, more preferably within ± 1, even more preferably within ± 0.5.

On the other hand, it is also well known that the substitution between amino acids having similar hydrophilicity values leads to proteins with homogeneous biological activity. As disclosed in U.S. Patent No. 4,554,101, the following hydrophilicity values are assigned to each amino acid residue: arginine (+3.0); Lysine (+3.0); Aspartate (+ 3.0 ± 1); Glutamate (+ 3.0 ± 1); Serine (+0.3); Asparagine (+0.2); Glutamine (+0.2); Glycine (0); Threonine (-0.4); Proline (-0.5 ± 1); Alanine (-0.5); Histidine (-0.5); Cysteine (-1.0); Methionine (-1.3); Valine (-1.5); Leucine (-1.8); Isoru Isin (-1.8); Tyrosine (-2.3); Phenylalanine (-2.5); Tryptophan (-3.4).

When a mutation is introduced with reference to the hydrophilicity value, the amino acid is substituted preferably within ± 2, more preferably within ± 1, even more preferably within ± 0.5.

Amino acid exchange in proteins that do not globally alter the activity of the molecule is known in the art (H. Neurath, R. L. Hill, The Proteins, Academic Press, New York, 1979). The most commonly occurring exchanges involve amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu and Asp / Gly.

Considering the mutation having the above-mentioned biological equivalent activity, the antibody of the present invention or the nucleic acid molecule encoding the same is interpreted as including a sequence showing substantial identity with the sequence described in the sequence listing. The above-mentioned substantial identity is determined by aligning the above-described sequence of the present invention with any other sequence as much as possible and analyzing the aligned sequence using an algorithm commonly used in the art. Homology, more preferably 70% homology, even more preferably 80% homology, and most preferably 90% homology. Alignment methods for sequence comparison are well known in the art. Various methods and algorithms for alignment are described by 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 accessible from National Center for Biological Information (NBCI) It can be used in conjunction with sequence analysis programs such as blastx, tblastn and tblastx. BLSAT is available at http://www.ncbi.nlm.nih.gov/BLAST/. A method for comparing sequence homology using this program can be found at http://www.ncbi.nlm.nih.gov/BLAST/blast_help.html.

According to one embodiment of the invention, the heavy chain variable region of the invention comprises the amino acid sequence of SEQ ID NO: 12.

According to one embodiment of the invention, the light chain variable region of the invention comprises the amino acid sequence of SEQ ID NO: 14.

The antibodies of the present invention can be used in combination with monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, short chain Fvs (scFV), single chain antibodies, Fab fragments, F (ab ') fragments, disulfide- And anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of such antibodies, and the like.

On the other hand, the antibody CDR sequences of the present invention are very similar to the CDR sequences of conventional antibodies and have a unique sequence. For example, antibodies disclosed in U.S. Patent No. 6365154, which have been found to have the highest homology in the BLSAT search (http://www.ncbi.nlm.nih.gov/BLAST/), include antibodies of the invention and CDRH3 sequence homology The CDRL3 sequence homology is 78%, and the total CDR sequence homology is less than 50%. Furthermore, the antibody described in U.S. Patent No. 6365154 is an antibody that binds to Tie2, and the antibody of the present invention is different from the target thereof.

According to one embodiment of the present invention, the EGFR of the present invention is human EGFR. As demonstrated in the following example, the antibody of the present invention is an antibody specific for human EGFR, as there is no cross-species cross-reaction with mouse EGFR (Fig. 5B).

According to another aspect of the present invention, there is provided a nucleic acid molecule encoding a heavy chain variable region of an antibody against cancer cells comprising the amino acid sequence of SEQ ID NO: 12.

According to another aspect of the present invention, there is provided a nucleic acid molecule encoding a light chain variable region of an antibody against cancer cells comprising the amino acid sequence of SEQ ID NO: 14.

As used herein, the term " nucleic acid molecule " has the meaning inclusive of DNA (gDNA and cDNA) and RNA molecules. In the nucleic acid molecule, the nucleotide which is a basic constituent unit is not only a natural nucleotide, (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews , 90: 543-584 (1990)). The nucleic acid molecule sequences encoding the heavy chain variable region and the light chain variable region of the present invention may be modified. Such modifications include addition, deletion or non-conservative substitution or conservative substitution of nucleotides.

According to one embodiment of the present invention, the nucleic acid molecule encoding the heavy chain variable region comprises the nucleotide sequence of SEQ ID NO: 11.

According to one embodiment of the present invention, the nucleic acid molecule encoding the light chain variable region comprises the nucleotide sequence of SEQ ID NO: 13.

Nucleic acid molecules of the invention that encode EGFR antibodies are also interpreted to include nucleotide sequences that exhibit substantial identity to the nucleotide sequences described above. The above substantial identity is determined by aligning the nucleotide sequence of the present invention with any other sequence as much as possible and analyzing the aligned sequence using algorithms commonly used in the art. Homology, more preferably at least 90% homology, most preferably at least 95% homology.

According to another aspect of the present invention, the present invention provides a recombinant vector comprising the aforementioned nucleic acid molecule.

As used herein, the term " vector " refers to a plasmid vector as a means for expressing a gene of interest in a host cell; Cosmeptide vector; And viral vectors such as bacteriophage vectors, adenovirus vectors, retroviral vectors, and adeno-associated viral vectors.

According to one embodiment of the invention, the nucleic acid molecule encoding the light chain variable region and the nucleic acid molecule encoding the heavy chain variable region in the vector of the present invention are operatively linked to the promoter.

As used herein, the term " operably linked " means a functional linkage between a nucleic acid expression control sequence (e.g., an array of promoter or transcription factor binding sites) and another nucleic acid sequence, Regulate transcription and / or translation of other nucleic acid sequences.

The recombinant vector system of the present invention can be constructed through various methods known in the art, and specific methods for this are disclosed in Sambrook et al., Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory Press (2001) , Which is incorporated herein by reference.

The vector of the present invention can typically be constructed as a vector for cloning or as a vector for expression. In addition, the vector of the present invention can be constructed by using prokaryotic cells or eukaryotic cells as hosts.

For example, when the vector of the present invention is an expression vector and a eukaryotic cell is used as a host, a promoter derived from a genome of a mammalian cell (e.g., a metallothionine promoter, a beta -actin promoter, a human heterologous promoter, Human muscle creatine promoter) or a mammalian virus (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, HSV tk promoter, mouse breast tumor virus (MMTV) promoter, The LTR promoter of HIV, the promoter of Moloney virus, the promoter of Epstein Barr virus (EBV) and the promoter of Roussacomavirus (RSV)) can be used, and generally has a polyadenylation sequence as a transcription termination sequence.

The vector of the present invention may be fused with other sequences to facilitate purification of the antibody expressed therefrom. Fusion sequences include, for example, glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA) and 6x His (hexahistidine; Quiagen, USA).

Further, since the protein expressed by the vector of the present invention is an antibody, the expressed antibody can be easily purified through a protein A column or the like, without any additional sequence for purification.

On the other hand, the expression vector of the present invention includes, as a selection marker, an antibiotic resistance gene commonly used in the art and includes, for example, ampicillin, gentamycin, carbenicillin, chloramphenicol, streptomycin, kanamycin, There is a resistance gene for mitine and tetracycline.

According to another aspect of the present invention, the present invention provides a host cell transformed with said recombinant vector.

A host cell capable of continuously cloning and expressing the vector of the present invention in a stable manner can be any host cell known in the art, for example, a suitable eukaryotic host cell of the vector is a monkey kidney cell 7 (COS7: monkey kidney cells, NSO cells, SP2 / 0, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, MDCK, myeloma cell lines, HuT 78 cells and HEK-293 cells But is not limited thereto.

According to another aspect of the present invention, the present invention provides a pharmaceutical composition comprising (a) a pharmaceutically effective amount of the EGFR antibody of the invention or an antigen-binding fragment thereof; And (b) a pharmaceutically acceptable carrier. The present invention also provides a pharmaceutical composition for preventing or treating cancer.

Since the pharmaceutical composition of the present invention uses the above-described EGFR antibody of the present invention or an antigen-binding fragment thereof as an active ingredient, the common content between the two is that, in order to avoid the excessive complexity of the present specification by repeating substrate, It is omitted.

As demonstrated in the following examples, the EGFR antibodies of the present invention are useful for the treatment of cancer cells (particularly, colorectal cancer cells, more specifically EGFR-expressing colorectal cancer cells), in combination with cetuximab, And is very effective for the treatment of cancer (particularly, colorectal cancer, more specifically EGFR-expressing colorectal cancer). According to one embodiment of the invention, the pharmaceutical composition additionally comprises a cetuximab antibody.

Cancers which can be prevented or treated by the composition of the present invention include various cancers known in the art and include, for example, colon cancer, breast cancer, ovarian cancer, stomach cancer, lung cancer, liver cancer, bronchial cancer, Pancreatic cancer, bladder cancer, colon cancer, cervical cancer, brain cancer, prostate cancer, bone cancer, head and neck cancer, skin cancer, thyroid cancer, pituitary cancer or ureter cancer.

Specifically, the cancer that can be prevented or treated by the composition of the present invention is EGFR-expressing cancer, more specifically, EGFR-expressing colon cancer.

According to another aspect of the present invention, the present invention provides a pharmaceutical composition comprising (a) a pharmaceutically effective amount of an EGFR antibody or antigen-binding fragment thereof of the invention; And (b) a pharmaceutically acceptable carrier.

According to an embodiment of the present invention, the pharmaceutical composition of the present invention is used for preventing or treating hyperproliferative diseases by inhibiting cell growth, and the hyperproliferative diseases are cancer, hyperplasia, (Cushing's syndrome), primary aldosteronism, erythroplakia, polycythemia vera, leukoplakia, hyperplastic scar, lichen planus, Lentiginosis, atherosclerosis, atherosclerosis, restenosis or stenosis.

The pharmaceutically acceptable carriers to be contained in the pharmaceutical composition of the present invention are those conventionally used in the present invention and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, But are not limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. It is not. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention can be administered parenterally and can be administered, for example, by intravenous infusion, subcutaneous injection, muscle injection, intraperitoneal injection, topical administration, intranasal administration, intrapulmonary administration and rectal administration.

The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate and responsiveness of the patient, Usually, a skilled physician can readily determine and prescribe dosages effective for the desired treatment or prophylaxis. According to one embodiment of the present invention, the daily dosage of the pharmaceutical composition of the present invention is 0.0001-100 mg / kg. As used herein, the term "pharmaceutically effective amount" means an amount sufficient to prevent or treat cancer.

The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, suppositories, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

The antibodies of the present invention can be used to diagnose diagnoses, such as disorders, diseases or conditions related to EGFR-expression.

According to another aspect of the present invention, there is provided a diagnostic kit for an EGFR-expression-related disease, disease or condition comprising the antibody of the present invention described above.

The EGFR-expression-related disease, disease or condition is specifically a cancer, such as, for example, colon cancer, breast cancer, ovarian cancer, stomach cancer, lung cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, , Prostate cancer, bone cancer, head and neck cancer, skin cancer, thyroid cancer, pituitary cancer, or ureter cancer. Specifically, the diagnostic kit of the present invention is used for the diagnosis of EGFR-expressing cancer, more specifically, EGFR-expressing colon cancer.

In addition, the antibodies of the present invention can be used to analyze whether the antibody therapeutic of the present invention has drug responsiveness to the patient.

According to another aspect of the present invention, the present invention provides a drug responsiveness assay kit comprising the antibody of the present invention described above.

The assay kits of the invention can be used to analyze whether a particular patient is medically responsive to an antibody of the invention. For example, when an antibody of the present invention is treated in a cancer cell of a patient, when the antibody of the present invention is found to bind, the patient can be judged to have drug responsiveness to the antibody of the present invention.

Since the kit described above contains an antibody, it can be basically prepared for various immunoassays or immunostaining. The immunoassay or immunostaining can be carried out by radioimmunoassay, radioimmunoprecipitation, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), capture-ELISA, inhibition or competition assay, sandwich assay, flow cytometry, But are not limited to, immunoaffinity purification. Methods of immunoassay or immunostaining are described in Enzyme Immunoassay , ET Maggio, ed., CRC Press, Boca Raton, Florida, 1980; Gaastra, W., Enzyme-linked immunosorbent assay (ELISA), in Methods in Molecular Biology , Vol. 1, Walker, JM ed., Humana Press, NJ, 1984; And Ed Harlow and David Lane, Using Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory Press, 1999, which is incorporated herein by reference.

For example, if the method of the present invention is carried out according to the method radioactive immunoassay, radioactive isotopes (e.g., ¹⁴ C, ¹²⁵ I, ³² P And S ³⁵ ) can be used to detect EGFR receptors on the cancer cell surface. When the present invention is practiced in an ELISA fashion, a specific embodiment of the present invention comprises (i) coating a sample to be analyzed on a surface of a solid substrate; (Ii) reacting said cell lysate with an EGFR antibody as a primary antibody; (Iii) reacting the result of step (ii) with an enzyme-conjugated secondary antibody; And (iv) measuring the activity of the enzyme.

Suitable as said solid substrate are hydrocarbon polymers (e.g., polystyrene and polypropylene), glass, metal or gel, and most particularly microtiter plates.

The enzyme bound to the secondary antibody may include an enzyme catalyzing a chromogenic reaction, a fluorescence reaction, a luminescent reaction, or an infrared reaction, but is not limited thereto. For example, an alkaline phosphatase,? -Galactosidase, Radish peroxidase, luciferase, and cytochrome P ₄₅₀ . In the case where alkaline phosphatase is used as an enzyme binding to the secondary antibody, it is preferable to use, as a substrate, bromochloroindole phosphate (BCIP), nitroblue tetrazolium (NBT), naphthol-AS -Bl-phosphate and ECF (enhanced chemifluorescence) are used. When horseradish peroxidase is used, chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (10-acetyl-3,7-dihydroxyphenoxazine), HYR (p-phenylenediamine-HCl and pyrocatechol), TMB (tetramethylbenzidine), ABTS (2,2'- Azine-di [3-ethylbenzthiazoline sulfonate]), o - phenylenediamine (OPD) and naphthol / pie Ronin, glucose oxidase and t-NBT (nitroblue tetrazolium) and m-PMS a substrate such as phenzaine methosulfate may be used All.

When the present invention is practiced in a Capture-ELISA fashion, certain embodiments of the present invention comprise (i) coating the surface of a solid substrate with an EGFR antibody as a capturing antibody; (Ii) reacting the capture antibody with the sample; (Iii) reacting the result of step (ii) with an EGFR detecting antibody to which a label generating signal is attached; And (iv) measuring a signal originating from said label.

The detection antibody has a label that generates a detectable signal. Wherein the label is a chemical (e.g., biotin), an enzyme (alkaline phosphatase, β- galactosidase, horseradish peroxidase, and cytochrome P _450), the radioactive material (for example, ¹⁴ C, ¹²⁵ I, P ³² And S ³⁵ ), fluorescent materials (e.g., fluorescein), luminescent materials, chemiluminescent materials, and fluorescence resonance energy transfer (FRET). Various labels and labeling methods are described in Ed Harlow and David Lane, Using Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory Press,

In the ELISA method and the capture-ELISA method, measurement of the activity of the final enzyme or measurement of the signal can be performed according to various methods known in the art. If biotin is used as a label, it can be easily detected by streptavidin. When luciferase is used, luciferin can easily detect a signal.

Samples which may be applied to the kits of the invention include, but are not limited to, cell, tissue or tissue-derived extract, lysate or purified water, blood, plasma, serum, lymph or ascites.

The antibodies of the present invention may be used for in vivo or in vitro imaging. According to another aspect of the present invention, the present invention provides a composition for imaging comprising an antibody of the present invention described above and a label-bound conjugate that generates a detectable signal bound to the antibody.

A label that generates a detectable signal, T1 contrast materials (e. G., Gd chelate compounds), T2 contrast materials (e.g., a superparamagnetic material (e.g. _{magnetite, Fe 3 O 4, γ-} Fe 2 O 3, manganese ferrite, cobalt ferrite and nickel ferrite)), radioactive isotopes ^{(e.g., 11 C, 15 O, 13} N, P 32, S 35, 44 Sc, 45 Ti, 118 I, 136 La, 198 Tl, 200 Tl, 205 Bi and ²⁰⁶ Bi), fluorescent materials (fluorescein, phycoerythrin, rhodamine, lissamine, and Cy3 and Cy5), chemiluminescent moieties, magnetic particles, mass markers or electron dense particles But is not limited thereto.

Although the antibody of the present invention can be used alone to treat cancer, it can be provided in the form of an antibody drug conjugate (ADC) by binding to other drugs because it can target EGFR-expressing cells.

According to another aspect of the present invention, the present invention provides an antibody drug conjugate (ADC) comprising an antibody of the present invention and a drug bound to the antibody.

The drug to be bound to the antibody of the present invention is not particularly limited and includes a chemical substance, a radionuclide, an immunotherapeutic agent, a cytokine, a chemokine, a toxin, a biological agent and an enzyme inhibitor, and more specifically, But are not limited to, acyclovir, acivarin, acivaricin, acivaricin, acarbazic, acordazole, acronisin, adozelesin, alanosin, aldosterucine, allopurinol sodium, altretamine, aminoglutethimide, , Androgenase, asparaginase, 5-azacytidine, azathioprine, bacillus calmette-guerine (BCG), baker's antipol, beta- BWA773U82, BW50U83 / HCl, BW7U85 mesylate, cerasemide, carbethimer, carboplatin, carboxymethylcellulose, carboxymethylcellulose, Mustin, Chloramphenicol , Cloromycin A3, cisplatin, cladribine, corticosteroids, Corynebacterium parvum, CPT-11, quinolol, cyclocytidine, cyclophosphamide, But are not limited to, cytarabine, saimervena, davis malate, decarbazine, dactinomycin, daunorubicin HCl, diarylidines, dexarazoic acid, dianhydrogalactitol, diaziquone, dibromoducitol, B, diethyldithiocarbamate, diclercoaldehyde, dihydro-5-azacytin, doxorubicin, echinomycin, dodatrexate, edelosine, eplonin, Rubicin, esorubicin, estrameristin phosphate, estrogen, ethanidazole, ethiofos, etoposide, fadrazol, fazarabine, fenretinide, filgrastim, finasteride, flavonacet , Phloroglucine, fluodarabine phosphate, 5'-fluorouracil, Fluosol ™, flutamide, gallium nitrate, gemcitabine, gosserelin acetate, hepesulfam, hexamethylene bisacetamide, homoharringtonine, Hydrazine sulfate, hydrazine sulfate, 4-hydroxyandrostenedione, hydrosurea, dirubicin HCl, ifosfamide, 4-iipomenol, iuproplatin, isotretinoin, leucovorin calcium, luproide acetate, levamisole, liposome But are not limited to, methanol, ethanol, fructose, lecithin, daunorubicin, liposome-capturing doxorubicin, romerstine, ronidamine, mytansine, mechlorethamine hydrochloride, melphalan, menogaril, merbarone, 6- mercaptopurine, Extract, methotrexate, N-methylformamide, mifepristone, mitoguazone, mitomycin-C, mitotan, mitoxanthrone hydrochloride, monosite / But are not limited to, laparge colony-stimulating factors, napalon, napoxidine, neocarzinostatin, octreotide acetate, ormaflatatin, oxaliplatin, paclitaxel, palla, pentostatin, piperazin dione, , Pyrimethamine, pyriproxyfen, pyrroxanthrone hydrochloride, PIXY-321, plicamycin, porphimersodium, prednimustine, proccarbazine, progestins, pyrazofurin, But are not limited to, germanium, spiromustine, streptococcal, streptococcal, streptococcal, germanium, spiromustine, streptonigreen, streptozocin, sulophenur, suramin sodium, tamoxifen, taxolere, tegafur, tenifoside, terephthalamidine, Thiazepurin, topotecan, toremifene, tretinoin, tripleoperazine hydrochloride, tripleuridine, trimetrexate, tumor necrosis factor (TNF), uracil mustard, Vinblastine sulfate, vincristine sulfate, vinethine, vinorelbine, binzolidine, Yoshi 864, zorubicin, cytosine arabinoside, etoposide, melphalan, taxol and taxol.

The features and advantages of the present invention are summarized as follows:

(a) The antibody of the present invention is an antibody that specifically binds to EGFR overexpressed in cancer cells (particularly, colon cancer cells), and binds to an epitope different from cetuximab.

(b) The antibody of the present invention is very homologous to the CDR sequences of conventional EGFR target antibodies and has a unique sequence.

(c) The antibody of the present invention is highly effective for the prevention or treatment of cancer (particularly, colon cancer) by killing cancer cells with greatly improved cancer cell killing ability when the combination therapy with cetuximab is administered.

(d) The superior efficacy of the combination administration of the antibody of the present invention is not bound to theory, and the antibody of the present invention binds to an epitope on the EGFR phase different from cetuximab , Which is thought to inhibit EGFR cooperatively with cetuximab.

(e) The cell growth inhibitory activity of the antibody of the present invention can be used for prevention or treatment of hyperproliferative diseases.

(f) The antibody of the present invention can be used not only as a cancer treatment agent, but also as a cancer diagnosis, a medicament reactivity analysis, an imaging and an antibody drug conjugate (ADC).

FIG. 1 is a graph showing the proliferation inhibitory effect when the 15E3 antibody and cetuximab are treated alone or in combination with SW403 cells. CET and hIgG represent cetuximab and human IgG (negative control), respectively.
Figure 2 shows that the 15E3 antibody binds to the binding site of cetuximab and other epitopes. SPR analysis showed that the 15E3 antibody binds to one site of cetuximab and other EGFR sub-domain 3.
3 is a gene map of pcDNA3.3-IgG heavy vector and pOptiVEC-IgG Kappa vector.
FIGS. 4A and 4B are graphs showing the proliferation inhibitory effect of the hz15E3 antibody alone against the SW403 cancer cells and the LS174T cancer cell line, respectively, and the anti-proliferative effect of the combination of hz15E3 antibody and cetuximab. CET and hIgG represent cetuximab and human IgG (negative control), respectively.
FIG. 5A is an ELISA result of analyzing whether hz15E3 antibody specifically binds to EGFR among ErbB family proteins to which EGFR belongs.
FIG. 5B is an ELISA result of analyzing whether hz15E3 antibody binds to EGFR of a non-human species.
Figure 6 is a Western blotting result showing the reduction of EGFR downstream signaling by either alone or in combination with hz15E3 antibody and cetuximab.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Example 1: Antibody development against EGFR

For antibody development, extracellular domain (ECD) of EGFR protein was produced using animal cells and used as an antigen. DNA having a form in which the hinge and Fc region (CH ₂ -CH ₃ ) of human IgG1 were bound to the C-terminal of ECD was cloned into pCEP4 vector (Invitrogen, Cat. No. V044-50) using HindIII and BamHI restriction enzymes Respectively. Then, the cloned vector was transiently transfected with polyethyleneimine (Polyscience Inc., Cat. No. 23966) in FreeStyle ^TM 293F (Invitrogen, Cat. No. R790-07) EGFR-ECD Fc fusion protein was purified using Protein-A Ceramic HyperD F resin (PALL, Cat No. 20078-028). The purified protein was quantified using a protein assay die (Bio-Rad, Cat. No. 500-0006) and SDS-PAGE followed by Coomassie blue staining to confirm the concentration and purity. The purified 100 μg protein antigen was mixed with Freund's adjuvant (Sigma, Cat. No. F5506) and injected into the abdominal cavity of BALB / c mice (Korea Bio Co.). Two weeks later, 100 쨉 g of antigen was diluted with PBS and injected. Three days later, the spleen of the mouse was excised to separate the lymphocytes. The isolated lymphocytes were mixed with the myeloma cell line SP2 / 0-Ag14 (ATCC, Cat. No. CRL-1581) at a ratio of 5: 1 and analyzed using PEG-1500 (Roche, Cat. No. 783641) (fusion). The fused cells were cultured in a medium containing HAT supplement (Sigma, Cat. No. H0262) to selectively hybridize with the hybridoma cells.

The obtained hybridoma cells were confirmed to be cells producing an antibody that binds to an antigen using an ELISA method. EGFR-ECD-Fc or ChromPure human IgG (hIgG, Jackson Immunoresearch Lab, Inc., Cat No. 009-000-003) was diluted in a Costar 96-well plate (Corning, Cat. ) For 1 hour at room temperature. After three washes with TBS-T (0.05% Triton X-100), the cells were blocked with 300 μl of TBS-T / SM (2% skim milk) for 30 minutes at room temperature. After the blocked plate was washed three times, the hybridoma culture was added and the antibody was bound for 1 hour at 37 ° C. After washing three times, anti-mouse IgG-HRP (Pierce, Cat. No. 31439) was diluted 1: 5,000 in TBS-T / SM as a secondary antibody and bound for 1 hour at 37 ° C. After washing three times, TMB (SurModics, Cat. No. TMBC-1000-01) was added, color was developed at room temperature for 5 minutes, and 1 N sulfuric acid (DukSan, Cat. No. 254) was added to stop color development. Absorbance at 450 nm was measured using Victor X3 (PerkinElmer, Cat. No. 2030-0030) and antibodies that specifically bind EGFR-ECD-Fc were screened.

Since EGFR is a protein expressed on the cell surface, it was confirmed through cell-based ELISA whether the developed antibodies bind to EGFR overexpressing cells. (ATCC, Cat. No. CRL-1555), a skin cancer cell line overexpressing EGFR, was divided into 10,000 cells / well in a Costar 96-well cell culture plate (Corning, Cat. No. 3595) Respectively. The next day, the culture medium was removed, and the cells were washed three times with PBS, followed by addition of hybridoma cell culture medium and further incubation at 37 ° C for 2 hours. After washing three times with TBS-T, goat anti-mouse IgG-HRP was diluted 1: 5,000 with PBS / FBS (3% FBS) as a secondary antibody and treated at room temperature for 1 hour. After washing three times with TBS-T, the cells were developed with TMB. 43 clones with higher absorbance than SP2 / 0 cell culture were selected.

Example 2: Comparison of the inhibitory effect of the developed antibodies on the growth of colon cancer cells

Antibodies were purified from the hybridoma culture medium to perform cell viability assay to confirm the inhibitory effect on colon cancer cell proliferation. The hybridoma was cultured in a culture medium containing 3% FBS, and the IgG-type antibody was purified using Protein-A resin. Purified antibodies were quantitated by BCA analysis (Pierce, Cat. No. 23227) and SDS-PAGE followed by Coomassie blue staining to determine concentration and purity.

Cell viability analysis was performed by using SW403, a typical colon cancer cell line overexpressing EGFR, alone or in combination with cetuximab. For the combined treatment, the developed antibody and cetuximab were mixed at a 1: 1 ratio (weight ratio). SW403 (Korean Cell Line Bank, Cat No. 10230, 5,000 cells / well) cells were added to a 96-well plate and cultured for 24 hours. The purified antibodies were treated to 5 ㎍ / mL each, and the SW403 cell line was further cultured for 4 days. To measure cell viability, CCK-8 (Dojindo, Cat. No. CK-04-13) was added to a final concentration of 10%, and after 3 hours of treatment at 37 ° C, the absorbance was measured. The relative survival rate was calculated assuming that the absorbance of the well not treated with antibody was 100%. Antibodies showing good activity were sequentially diluted to a maximum of 20 ㎍ / mL to 1: 4, and cell viability analysis was performed at 5 concentrations. As a result, 15E3 showing the best activity was selected (Fig. 1).

Example 3: Comparison of the developed antibody with cetuximab

Antibody to EGFR Cetuximab is known to bind to domain-3 among the four domains of EGFR ECD. Epitope binning was carried out by surface plasmon resonance (SPR) method using a Biacore 3000 (GE Healthcare) device to check whether the developed antibodies overlap with the epitope for EGFR of cetuximab. Cetuximab was immobilized on the CM5 sensor chip (GE Healthcare, Cat. No. BR-1000-12) by amine coupling method using ECD / NHS at about 1,000 RU (response unit). The EGFR-ECD-His protein was incubated with 50 nM concentration of 4-cis ischemimab in HBS-P buffer (10 mM HEPES, 150 mM NaCl, 1 mM EDTA, 0.005% Tween-20, pH 7.4) Min and then buffered for 8 minutes to stabilize the binding between cetuximab and EGFR-ECD. Subsequently, the secondary antibody was bound at a concentration of 50 nM for 4 minutes and then the buffer was flowed. Flow rates were used at 50 μl / min for all experiments. If the secondarily bound antibody binds to the EGFR-ECD protein bound to cetuximab, it is an antibody that does not share an epitope with cetuximab.

As can be seen in FIG. 2, hIgG used as a secondary antibody does not bind to EGFR, so there is no additional binding, and cetuximab has no additional binding because it has the same epitope. On the other hand, the 15E3 antibody was found to have an epitope different from that of cetuximab because it was further bound to EGFR-ECD bound to cetuximab.

In order to identify the domain region to which the 15E3 clone binds, four sub-domains (Sub-domain 1-4) constituting the extracellular domain of the EGFR protein are separately introduced into the animal cell And purified using Protein-A. The recombinant protein produced was confirmed by ELISA for the binding of 15E3 clone, cetuximab, and the 15E3 clone bound to sub-domain 3 as cetuximab.

The above results indicate that the 15E3 clone binds to the sub-domain 3 of the ECD of the EGFR protein but binds to an epitope other than cetuximab.

Example  4: Antibody Sequence Analysis

To analyze the antibody sequences, RNA was isolated from each hybridoma using the SV Total RNA Isolation System (Promega, Cat. No. Z3100) and cDNA was synthesized. The variable region of the antibody was amplified using a known primer set (see Phage display: a laboratory manual, Carlos Barbas III, et al., Cold Spring Harbor Laboratory Press) and ligated with human Ck and CH1 to construct pComb3X vector (Barbas laboratory, The Scripps Research Institute) was cloned using SfiI restriction enzyme and transformed into ER2537 bacteria (New England Biolabs, Cat. No. 801-N). Antibodies that bind to EGFR-ECD-Fc in the colonies of each transformed antibody were identified by ELISA. The transformed bacterial colonies were cultured at 37 ° C. at an absorbance of 0.5 at 600 nm. IPTG was treated to a final concentration of 1 mM and incubated overnight at 30 ° C. to express Fab-type antibodies. Cells were harvested by centrifugation and then suspended in 0.4 ml of 1 × TES (50 mM Tris, 1 mM EDTA, 20% (v / v) sucrose, pH 8.0) and incubated at 4 ° C for 10 minutes Respectively. To this was added 0.6 mL of 0.2X TES, further treated at 4 DEG C for 30 minutes, and centrifuged to take the supernatant. TBS-T / SM (3% non) was prepared by washing the Costar 96-well half area plate (Corning Inc., Cat. No. 3690) coated with EGFR-ECD-Fc at a concentration of 1 μg / -fat skim milk, 0.05% Triton X-100) at room temperature for 1 hour. Each colony broth or periplasmic extract (Periplasm) was diluted 1: 3 with TBS-T / SM, and then treated at room temperature for 1 hour. After washing three times, anti-HA-HRP (Roche, Cat. No. 120-138-190-01) was diluted 1: 5000 as a secondary antibody, bound for 1 hour at room temperature, washed three times and developed with TMB .

Most of the colonies showed an absorbance of 0.2 or higher in cell culture medium or periplasmic extract, and the nucleotide sequences of the antibodies were analyzed for these clones. Sequence analysis revealed that the colonies derived from a single hybridoma had the same sequence. The amino acid sequence of the CDR (complementarity determining region) of the 15E3 antibody is summarized in Table 1.

The amino acid sequence of the complementarity determining region (CDR) of the 15E3 antibody Light chain Heavy chain CDR1 RASENIYSYLA TRYWLH CDR2 NARTLVE EINPSSGRTNYNEKFKS CDR3 QHHYGPPYT WDYDEGTWFAY

Example  5: Production and production of humanized antibodies

In order to make the antibody of the present invention more druggable, humanized antibodies were prepared.

The humanized antibody (hz15E3: humanize 15E3) of the mouse 15E3 antibody developed in Example 4 was developed using the CDR grafting method. The human antibodies to which the CDRs of the developed antibodies were transplanted were selected by using IMGT / V-QUEST to select V and J genes of the human germline antibody gene having high similarity to the base sequence reference (Brochet, X. et al., Nucl Acids Res. 36: 503-508 (2008)). The IGHV1-2 * 02 and IGHJ4 * 03 genes were selected as the V and J genes of the heavy chain, and their similarities were 76.04% and 78.72%, respectively. The IGKV1-9 * 01 and IGKJ2 * 01 genes were selected as the V and J genes of the light chain, and their similarities were 76.70% and 81.08%, respectively. The ClaI restriction enzyme site was inserted in the forward direction and the NheI restriction site and the BsiWI restriction enzyme site were inserted in the forward direction and the reverse direction, respectively. In the forward direction of the variable region, a signal sequence is added so that the humanized antibody can be secreted into the cell culture medium (Brochet, X. et al., Nucl Acids Res. 36: 503-508 (2008)). Each of the thus synthesized variable regions was ligated to the human constant regions Cκ and C _H , and the heavy chain portion was TA cloned into pcDNA3.3-TOPO (Invitrogen, Cat. No. K8300-01) vector. The light chain portion was pOptiVEC-TOPO (Invitrogen, Cat. No., 12744-017) vector. The nucleotide sequence and amino acid sequence of Cκ, and C _H used in the present invention are described in the Sequence Listing SEQ ID No. 15 to SEQ ID NO: 18.

A map of the finally prepared pcDNA3.3-IgG Heavy vector and pOptiVEC-IgG Kappa vector is shown in FIG.

Then, the cloned vector is transiently transfected with polyethyleneimine (Polyscience Inc., Cat. No. 23966) in an animal cell of FreeStyle ^TM 293F (Invitrogen, Cat. No. R790-07) The humanized antibody was purified from the cell culture using Protein-A Ceramic HyperD F resin (PALL, Cat No. 20078-028). Purified humanized antibodies were quantitated using BCA assay (Pierce, Cat. No. 23227) and SDS-PAGE followed by coomassie blue staining to determine concentration and purity.

Example  6: Comparison of the inhibitory effect of the developed antibodies against colon cancer growth

Cell survival analysis of EGFR overexpressing colorectal cancer cell lines SW403 and LS174T was performed to confirm the anticancer effect according to the concentration of the developed antibodies. SW403 (5,000 cells / well) and LS174T (10,000 cells / well) were dispensed into 96-well plates in an 80 [mu] l volume, incubated for 24 hours and fixed. The next day, 40 항 of antibody was added to the cells in culture. The final concentration of the treated antibody was 20 μg / mL per each antibody, and was serially diluted 1: 4 to proceed at 9 concentrations. In the case of treatment with cetuximab, the developed antibody and cetuximab were used in a 1: 1 ratio (for example, CET 1 / / mL and hz15E3 1 in the case of a dose of 1 / / mL in Figures 3a and 3b ≪ / RTI > pg / mL). SW403 and LS174T cells were further cultured for 4 days after antibody treatment, CCK-8 was added to a final concentration of 10%, and treated at 37 ° C for 3 hours. Absorbance was then measured at 450 nm using Victor X-3. Absorbance of cells not treated with antibody was set at 100% and the relative survival rate was calculated (FIGS. 4A and 4B).

The developed hz15E3 antibody showed antiproliferative activity against cetuximab-responsive SW403 (Fig. 4A) and LS174T (Fig. 4B) cell lines. Furthermore, the hz15E3 antibody was superior to cetuximab alone in inhibiting cancer cell proliferation against SW403 and LS174T cell lines in combination with cetuximab.

Example  7: Development of antibody EGFR Specificity for

The ELISA method was used to confirm whether the developed hz15E3 antibody binds specifically to EGFR or to EGFR of a non - human species among ErbB family proteins to which EGFR belongs. The EGFR, HER2, HER3 and HER4 extracellular domains belonging to the ErbB family were identified by ELISA in order to confirm whether the antibody specifically binds to EGFR among the ErbB family proteins. HER3 (R & D Systems, # 348-RB-050) and HER4 (R & D Systems, # 1131-ER-050) were produced in the same manner as EGFR-ECD- Proteins were purchased and used. In order to confirm whether the developed antibodies show interspecific cross - reactivity to EGFR proteins of other species, we confirmed by ELISA method using human and mouse EGFR extracellular domain. Human EGFR-ECD-Fc was produced by the above method, and mouse EGFR-ECD-Fc was also produced in the same manner as EGFR-ECD-Fc.

As a result, the developed hz15E3 antibody specifically binds to EGFR among human ErbB family proteins (Fig. 5A), and it can be confirmed that there is no interspecific cross-reaction with mouse EGFR (Fig. 5B).

Example  8: Development of antibody EGFR  Cell signaling inhibition efficacy

The effect of hz15E3 clone on EGFR - overexpressing colorectal cancer was investigated in order to examine the mechanism of action of anticancer efficacy in combination with cetuximab. Antibodies were treated with LS174T cells at a concentration of 10 [mu] g / mL for 48 hours. The cell extract solution (50 mM Tris, pH 7.4, 150 mM NaCl, 1% NP-40, 0.1% sodium dodecyl sulfate, 1 mM NaF, 1 mM Na ₃ VO _{4 ,} 1 mM PMSF and protease Inhibitor cocktail (Sigma)) to obtain a cell extract. Cell extracts were analyzed by Western blotting. For analysis, EGFR (# 4267), pEGFR (# 3777), HER2 (# 4290), pHER2 (# 6942), pHER3 (# 4791), AKT (# 4691), pAKT (# 4695) and pERK (# 4370) antibodies and Santa Cruz Biotechnology's HER3 (sc-285) antibodies were used. Actin (AbClon, # Abc-2002) was used as the loading control. Each protein was visualized in linear form using HRP-conjugated anti-rabbit antibody (Pierce, # 31463) and AbSignal (AbClon, # AbC-3001) reagent.

As can be seen from FIG. 6, the combination of hz15E3 and cetuximab showed a decrease in EGFR protein. As the EGFR protein decreased, the phosphorylated EGFR protein also decreased. In the case of HER2, which is known to be heterodimerized with EGFR, only the amount of phosphorylated form of HER2 protein is reduced.

This means that the activity of EGFR and HER2 can be regulated in combination with hz15E3 and cetuximab. These results indicate that AKT and ERK, which are known as EGFR subcellular signaling pathways, are regulated in a non - quantitative regulated manner.

These results suggest that the combination of hz15E3 and cetuximab inhibits cell signal transduction through HER2 and heterodimerization of EGFR.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

<110> AbClon, Inc <120> Antibodies Capable of Binding Specifically to EGFR <130> PN150325 <160> 18 <170> Kopatentin 2.0 <210> 1 <211> 6 <212> PRT <213> CDRH1 of 15E3 antibody <400> 1 Thr Arg Tyr Trp Leu His   1 5 <210> 2 <211> 17 <212> PRT <213> CDRH2 of 15E3 antibody <400> 2 Glu Ile Asn Pro Ser Ser Gly Arg Thr Asn Tyr Asn Glu Lys Phe Lys   1 5 10 15 Ser     <210> 3 <211> 11 <212> PRT <213> CDRH3 of 15E3 antibody <400> 3 Trp Asp Tyr Asp Glu Gly Thr Trp Phe Ala Tyr   1 5 10 <210> 4 <211> 11 <212> PRT <213> CDRL1 of 15E3 antibody <400> 4 Arg Ala Ser Glu Asn Ile Tyr Ser Tyr Leu Ala   1 5 10 <210> 5 <211> 7 <212> PRT <213> CDRL2 of 15E3 antibody <400> 5 Asn Ala Arg Thr Leu Val Glu   1 5 <210> 6 <211> 9 <212> PRT <213> CDRL3 of 15E3 antibody <400> 6 Gln His His Tyr Gly Pro Pro Tyr Thr   1 5 <210> 7 <211> 360 <212> DNA <213> Nucleotide sequence of mouse 15E3 heavy chain variable region <400> 7 gaggtccagc tgcaacagtc cggggctgaa ctggtgaagc ctggggcttc agtgaaactg 60 tcctgcaagg cttctggcta caccttcacc agatactggc tgcactgggt gaagcagagg 120 cctggacaag gccttgagtg gattggagag attaatccta gcagcggtcg tactaactac 180 aatgagaagt tcaagagcaa ggccacactg actgtagaca aatcctccaa cacagcctac 240 atacaactca gcagcctgac atctgaggac tctgcggtct attactgtgc aaggtgggat 300 tacgacgagg ggacctggtt tgcttactgg ggcccaggga ctctggtcac tgtctctgca 360                                                                          360 <210> 8 <211> 120 <212> PRT <213> Amino acid sequence of mouse 15E3 heavy chain variable region <400> 8 Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala   1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr              20 25 30 Trp Leu His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile          35 40 45 Gly Ile Asn Pro Ser Ser Gly Arg Thr Asn Tyr Asn Glu Lys Phe      50 55 60 Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Asn Thr Ala Tyr  65 70 75 80 Ile Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Trp Asp Tyr Asp Glu Gly Thr Trp Phe Ala Tyr Trp Gly Pro             100 105 110 Gly Thr Leu Val Thr Val Ser Ala         115 120 <210> 9 <211> 321 <212> DNA <213> Nucleotide sequence of mouse 15E3 light chain variable region <400> 9 gatattgtga tgacccaatc tccagcctcc ctctctgcat ctgtgggaga aactgtcacc 60 atcacatgtc gagcaagtga gaatatttac agttatttag catggtatca gcagaaacag 120 ggaaaatctc ctcagctcct ggtctataat gcaagaacct tagtagaggg tgtgccatca 180 aggttcagtg gcagtggatc aggcacacag ttttctctga agatcaacag cctgcagcct 240 gaagattttg ggagttattg ctgtcaacat cattatggtc ctccgtacac gttcggaggg 300 gggaccaaac tggaaatcaa a 321 <210> 10 <211> 107 <212> PRT <213> Amino acid sequence of mouse 15E3 light chain variable region <400> 10 Asp Ile Val Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly   1 5 10 15 Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Tyr              20 25 30 Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val          35 40 45 Tyr Asn Ala Arg Thr Leu Val Glu Gly Val Ser Ser Arg Phe Ser Gly      50 55 60 Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu Gln Pro  65 70 75 80 Glu Asp Phe Gly Ser Tyr Cys Cys Gln His His Tyr Gly Pro Pro Tyr                  85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys             100 105 <210> 11 <211> 360 <212> DNA <213> Nucleotide sequence of hz15E3 heavy chain variable region <400> 11 caggttcagc tcgtgcaatc aggcgctgag gttaaaaagc caggagccag tgtcaaagtg 60 tcttgtaagg cctccgggta taccttcaca aggtactggc tgcactgggt gagacaagca 120 cccggtcagg gccttgagtg gatcggggaa attaatccta gcagtggcag aaccaattat 180 aacgagaagt tcaagtctcg cgccactctg actgtagata aaagcatcag cacggcctac 240 atggagctgt ctagactgag atccgacgat accgctgtct actactgcgc gagatgggac 300 tatgacgaag gtacatggtt tgcatattgg ggccagggaa ccttggtgac agtgtcatcc 360                                                                          360 <210> 12 <211> 120 <212> PRT <213> Amino acid sequence of hz15E3 heavy chain variable region <400> 12 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala   1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr              20 25 30 Trp Leu His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile          35 40 45 Gly Ile Asn Pro Ser Ser Gly Arg Thr Asn Tyr Asn Glu Lys Phe      50 55 60 Lys Ser Arg Ala Thr Leu Thr Val Asp Lys Ser Ile Ser Thr Ala Tyr  65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Trp Asp Tyr Asp Glu Gly Thr Trp Phe Ala Tyr Trp Gly Gln             100 105 110 Gly Thr Leu Val Thr Val Ser Ser         115 120 <210> 13 <211> 321 <212> DNA <213> Nucleotide sequence of hz15E3 light chain variable region <400> 13 gacatccaga tgacccagtc acctagcttc ctgtctgctt ctgtcgggga cagagtgact 60 atcacttgtc gcgcctccga gaacatatac tcctatctcg cttggtacca acagaagccc 120 gggaaggccc caaaacttct ggtatacaat gcccggaccc tggttgaggg cgtgccttca 180 aggttcagtg ggagcggctc cggaaccgaa ttcacgttga caattagtag cctgcaaccg 240 gaggattttg caacatacta ttgccagcac cattacggcc ccccatatac ctttggacag 300 ggtacaaagg tggaaattaa a 321 <210> 14 <211> 107 <212> PRT <213> Amino acid sequence of hz15E3 light chain variable region <400> 14 Asp Ile Gln Met Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Tyr              20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Val          35 40 45 Tyr Asn Ala Arg Thr Leu Val Glu Gly Val Ser Ser Arg Phe Ser Gly      50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro  65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His His Tyr Gly Pro Pro Tyr                  85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys             100 105 <210> 15 <211> 324 <212> DNA <213> Constant region of human IgG kappa chain <400> 15 cgtacggtgg ctgcaccatc tgtcttcatc ttcccgccat ctgatgagca gttgaaatct 60 ggaactgcct ctgttgtgtg cctgctgaat aacttctatc ccagagaggc caaagtacag 120 tggaaggtgg ataacgccct ccaatcgggt aactcccagg agagtgtcac agagcaggac 180 agcaaggaca gcacctacag cctcagcagc accctgacgc tgagcaaagc agactacgag 240 aaacacaaag tctacgcctg cgaagtcacc catcagggcc tgagttcgcc cgtcacaaag 300 agcttcaaca ggggagagtg ttaa 324 <210> 16 <211> 107 <212> PRT <213> Constant region of human IgG kappa chain <400> 16 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu   1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe              20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln          35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser      50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu  65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser                  85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys             100 105 <210> 17 <211> 993 <212> DNA <213> Constant region of human IgG heavy chain <400> 17 gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60 ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120 tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180 ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240 tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300 aaatcttgcg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360 ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420 gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480 tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 540 agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600 gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660 aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 720 ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780 gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840 ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 900 cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960 cgaagagcc tctccctgtc cccgggtaaa tga 993 <210> 18 <211> 330 <212> PRT <213> Constant region of human IgG heavy chain <400> 18 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys   1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr              20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser          35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser      50 55 60 Leu Ser Ser Val Val Thr Val Ser Ser Ser Leu Gly Thr Gln Thr  65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys                  85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys             100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro         115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys     130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu                 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu             180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn         195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly     210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr                 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn             260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe         275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn     290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys                 325 330

Claims (17)

An antibody against an Epidermal Growth Factor Receptor (EGFR) or antigen-binding fragment thereof:
(a) a heavy chain variable region comprising the following heavy chain CDR (complementarity determining region) amino acid sequence; CDRH1 of SEQ ID NO: 1, CDRH2 of SEQ ID NO: 2 and CDRH3 of SEQ ID NO: 3, and
(b) a light chain variable region comprising the following light chain CDR amino acid sequence; CDRL1 of Sequence Listing 4, CDRL2 of Sequence Listing 5, and CDRL3 of Sequence Listing 6.
2. The antibody or antigen-binding fragment thereof of claim 1, wherein said heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 12.
The antibody or antigen-binding fragment thereof of claim 1, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 14.
The antibody or antigen-binding fragment thereof according to claim 1, wherein said EGFR is human EGFR.
A nucleic acid molecule encoding a heavy chain variable region of an antibody against EGFR comprising the amino acid sequence of SEQ ID NO: 12.
6. The nucleic acid molecule of claim 5, wherein the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 11.
A nucleic acid molecule encoding a light chain variable region of an antibody against EGFR comprising the amino acid sequence of SEQ ID NO: 14.
8. The nucleic acid molecule of claim 7, wherein the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 13.
9. A recombinant vector comprising the nucleic acid molecule of any one of claims 5 to 8.
A host cell transformed with the recombinant vector of claim 9.
(a) a pharmaceutically effective amount of an antibody or an antigen-binding fragment thereof for EGFR of any one of claims 1 to 4; And (b) a pharmaceutically acceptable carrier.
12. The composition of claim 11, wherein the pharmaceutical composition further comprises a cetuximab antibody.
12. The method of claim 11, wherein the cancer is selected from the group consisting of colon cancer, breast cancer, ovarian cancer, stomach cancer, lung cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, colon cancer, cervical cancer, brain cancer, prostate cancer, Skin cancer, thyroid cancer, parathyroid cancer or urethral cancer.
14. The composition of claim 13, wherein the cancer is a colon cancer.
(a) a pharmaceutically effective amount of an antibody or an antigen-binding fragment thereof for EGFR of any one of claims 1 to 4; And (b) a pharmaceutically acceptable carrier.
The pharmaceutical composition according to claim 15, wherein the pharmaceutical composition is used for preventing or treating hyperproliferative disease by inducing cell growth inhibition, wherein the hyperpigmented disease is cancer, hyperplasia, keloid, Cushing syndrome, primary aldosteronism, erythroplakia, polycythemia vera, leukoplakia, hyperplastic scar, lichen planus, lentiginosis, ), Arteriosclerosis, atherosclerosis, restenosis or stenosis.
A cancer diagnostic kit comprising an antibody against EGFR of any one of claims 1 to 4 or an antigen-binding fragment thereof.

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