KR20180039919A - Fab fragment and uses thereof - Google Patents

Abstract

The present invention relates to a Fab fragment specifically binding to an epidermal growth factor receptor (EGFR). Specifically, the Fab fragment comprises: (a) a heavy chain variable region (V_H) having an amino acid sequence represented by SEQ ID NO: 31; (b) a heavy chain constant region (C_(H1)) having an amino acid sequence represented by SEQ ID NO: 5; (c) a light chain variable region having an amino acid sequence represented by SEQ ID NO: 32 (V_L); and (d) a light chain constant region (C_L) having an amino acid sequence represented by SEQ ID NO: 8. The Fab fragment is useful for diagnosing, preventing, or treating cancer by reducing potential immunogenicity by adding a partial humanized sequence and improving production yield in E. coli.

Description

Fab fragments and uses {Fab fragment and uses thereof}

FIELD OF THE INVENTION The present invention relates to a Fab fragment and its use, and more particularly to a Fab (fragment antigen-binding) fragment specifically binding to Epidermal Growth Factor Receptor (EGFR) and a composition for diagnosis, prevention or treatment of cancer including the fragment .

EGFR (Epidermal Growth Factor Receptor; HER1) is one of the receptor HER family members on the cell surface. It binds to ligands such as EGF, TGF-alpha and Epiregulin and plays an important role in cell growth and death. . Of particular interest in cancer is the increase in EGFR expression in many types of cancer cells as a result of immunohistochemical staining, and there is a report that this increase in EGFR is closely related to prognosis (Nicholson, RI et al. Eur. J. Cancer. , 37: S9-15 (2001); Yewale, C. et al., Biomaterials , 34: 8690-707 (2013)). Thus, attempts have been made to treat cancer by inhibiting EGFR signaling, including cetuximab, an EGFR blocking antibody, and a low molecular weight EGFR tyrosine kinase inhibitor (EGFR-TK1), gefitinib ^ⓡ), ereul Loti nip (erlotinib; appearance like tarceva ^ⓡ) was to be used in clinical.

IgG, IgA, IgM, IgD and IgE are classified into five types according to the difference in the constant region (Fc portion) of the heavy chain. IgG1 and IgG3 among the isotypes of antibody are classified into ADCC, CDC IgG2 has no ADCC function, weak CDC function, IgG4 has weak ADCC function, but CDC function is not known. IgG1 isotype, which is known to have high ADCC and CDC function, has been developed the most in the case of anti-cancer antibodies. However, unlike other anticancer targets, EGFR has been developed and marketed as an IgG2-type antibody therapeutic agent as well as an IgG1-type antibody, and it can be said that the neutralizing activity due to the antibody is the main action in the case of targeting EGFR .

EGFR is present in the form of a tethered monomer or an open-ended untethered monomer. When EGF binds, the dimer forms a kinase, which activates the kinase. Therapeutic antibody Cetuximab inhibits kinase activity and downstream signals by binding to EGFR instead of the ligand. This inhibits cell growth and induces apoptosis. This binding inhibits receptor activation and signal transduction pathways, resulting in decreased penetration of tumor cells into normal tissue and tumor spread to new sites. It is also believed that tumor cells generally inhibit tumor growth by inhibiting the ability of tumor cells to repair damage due to chemotherapy and radiation therapy and inhibiting new blood vessel formation in the tumor.

However, the biggest disadvantage of antibody therapeutics is that they are expensive drugs with high production costs. If the purpose of the antibody input is to block the action of an antigen by binding to an antigen or to inhibit its function by binding to a receptor, the effector, that is, the Fc region is not necessary, and antibody fragments such as Fab are used instead of IgG. Since there is no need for carbohydrate moiety, it is essential to conduct mass production at low cost using E. coli, and to increase the production of antibody fragments required for expression of Fab in E. coli.

Chimeric antibodies that have both mouse and human-derived sequences can induce immunogenicity due to mouse-derived sequences. In this case, serious side effects such as anaphylaxis may occur, or secondary administration From then on, the efficacy may become ineffective. In the case of cetuximab, one of the chimeric antibodies, clinical hypersensitivity reactions have been clinically observed in some regions, and glycosylation of the Fab region has been attributed to it (Chung, CH et al., N. Engl. J. Med 358: 1109-17 (2008)). Therefore, the ceuximab derivatives that can be produced in Escherichia coli are not glycosylated, so that the immune response can be avoided. In addition, if the sequence is changed, the potential immunogenicity should be avoided.

1. Korean Patent Publication No. 2015-0112385 2. U.S. Published Patent Application No. 2016/0068609 3. U.S. Patent No. 7,847,432

It is an object of the present invention to provide a Fab fragment that specifically binds to EGFR (Epidermal Growth Factor Receptor).

Yet another object of the present invention is to provide a nucleic acid molecule encoding the Fab fragment.

Still another object of the present invention is a pharmaceutical composition comprising (a) a pharmaceutically effective amount of the Fab fragment; And (b) a pharmaceutically acceptable carrier. The present invention also provides a pharmaceutical composition for preventing or treating cancer.

It is still another object of the present invention to provide a diagnostic composition for cancer comprising the Fab fragment.

In order to achieve the above object, the present invention provides a Fab fragment that specifically binds to EGFR (Epidermal Growth Factor Receptor).

In one embodiment of the present invention, the Fab fragment comprises (a) a heavy chain variable region (V _H ) having the amino acid sequence of SEQ ID NO: 31; (b) a heavy chain constant region (C _H1 ) having an amino acid sequence represented by SEQ ID NO: 5; (c) a light chain variable region (V _L ) having an amino acid sequence represented by SEQ ID NO: 32; And (d) a light chain constant region (C _L ) having an amino acid sequence represented by SEQ ID NO: 8.

In one embodiment of the present invention, the Fab fragment may be a chimeric antibody or a humanized antibody.

The present invention also provides a nucleic acid molecule encoding the Fab fragment.

In one embodiment of the present invention, the nucleic acid molecule comprises: (a) a heavy chain variable region (V _H ) having the nucleotide sequence shown in SEQ ID NO: 33; (b) a heavy chain constant region (C _H1 ) having the nucleotide sequence shown in SEQ ID NO: 11; (c) a light chain variable region (V _L ) having the nucleotide sequence shown in SEQ ID NO: 34; And (d) a light chain constant region (C _L ) having the nucleotide sequence shown in SEQ ID NO: 14.

The present invention also provides a recombinant vector comprising the nucleic acid molecule.

In addition, the present invention provides a transformant transformed with said recombinant vector.

In one embodiment of the present invention, the transformant may be E. coli.

The present invention also provides a pharmaceutical composition comprising (a) a pharmaceutically effective amount of the Fab fragment; And (b) a pharmaceutically acceptable carrier. The present invention also provides a pharmaceutical composition for preventing or treating cancer.

In one embodiment of the present invention, the cancer is selected from the group consisting of breast cancer, colon cancer, lung cancer, stomach cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, brain cancer, uterine cancer, nasopharyngeal cancer, head cancer, head and neck cancer, , Colon cancer, vaginal cancer, small intestine cancer, endocrine cancer, thyroid cancer, pituitary cancer, ureter cancer, urethral cancer, prostate cancer, bronchial cancer, bladder cancer, kidney cancer and bone cancer.

The present invention also provides a diagnostic composition for cancer comprising the Fab fragment.

In one embodiment of the present invention, the cancer is selected from the group consisting of breast cancer, colon cancer, lung cancer, stomach cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, brain cancer, uterine cancer, nasopharyngeal cancer, head cancer, head and neck cancer, , Colon cancer, vaginal cancer, small intestine cancer, endocrine cancer, thyroid cancer, pituitary cancer, ureter cancer, urethral cancer, prostate cancer, bronchial cancer, bladder cancer, kidney cancer and bone cancer.

The Fab fragment according to the present invention specifically binds to Epidermal Growth Factor Receptor (EGFR), which reduces the possibility of inducing potential immunogenicity by adding a partial humanized sequence to the existing cetuximab, and further enhances production yield in Escherichia coli Thereby being useful for diagnosing, preventing or treating cancer.

Figure 1 is a schematic diagram for Fm318 and mutant production.
Figure 2 shows the PCR results for cloning.
FIG. 3 shows the results of SDS-PAGE analysis of expression of the prepared Fm318 and modified humanized derivatives in E. coli.
Figure 4 shows the PCR results for the expression of Fab antibodies.
Fig. 5 shows the results obtained by comparing production yields of Fm318 and 13 other humanized antibody fragments.
Figure 6 shows the results of ELISA for antigen binding activity against sEGFR.
Figure 7 shows the results confirmed by FACS for antigen binding activity to sEGFR.

The term, "Fab fragment" in the present invention means a fragment which has an antigen-binding function, and the heavy chain variable region (V _H), a heavy chain constant region 1 (C _H1), the light chain variable region (V _L) and a light chain constant Region (C _L ) and has one antigen-binding site. Fab 'fragments differ from Fab in that they have 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 when the cysteine residue of the hinge region of the Fab' forms a disulfide bond. Such a Fab fragment can be obtained using a protein hydrolyzing enzyme (for example, when the whole antibody is cleaved with papain, a Fab can be obtained and when it is cleaved with pepsin, an F (ab ') 2 fragment can be obtained) Can be produced through recombinant DNA technology. The present invention relates to a method of producing a Fab fragment by expressing it in Escherichia coli in order to improve the production cost which is considered to be a disadvantage in applying the antibody to the prevention or treatment of diseases.

The term "heavy chain" in the present invention includes a variable region domain V _H comprising an amino acid sequence having a sufficient variable region sequence to confer specificity to an antigen and three constant region domains C _H1 , C _H2 and C _H3 Quot; means both the full-length heavy chain and fragments thereof. The Fab fragment in the present invention is a Fab fragment comprising the heavy chain composed of the above V _H and C _H1 .

The term "light chain" in the present invention encompasses both the full-length light chain comprising the variable region domain V _L comprising the amino acid sequence having a sufficient variable region sequence for imparting specificity to the antigen and the constant region domain C _L , it means. The Fab fragment in the present invention is a Fab fragment comprising a light chain composed of the above V _L and C _L.

The Fab fragments of the present invention may contain variants of the amino acid sequences set forth in the appended sequence listing to the extent that they can specifically bind to EGFR. For example, the amino acid sequence of a Fab fragment can be altered to improve the binding affinity and / or other biological properties of the Fab fragment. Such modifications include, for example, deletion, insertion and / or substitution of the amino acid sequence residues of the Fab fragment. 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 hydropathy index of the amino acid 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 a mutation is introduced with reference to a hydrophobic index, substitution is made between amino acids showing a hydrophobic index difference preferably 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.

Fab fragments specifically binding to EGFR of the present invention additionally include amino acid sequences for the formation of disulfide bonds in C _H1 and C _L to form heterodimers of heavy and light chains.

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

The nucleic acid molecule encoding the antibody of the present invention is also interpreted to include a nucleotide sequence that exhibits substantial identity to the nucleotide sequence 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.

The expression construct produced in the present invention is constructed so as to express a desired gene in a host cell. Generally, promoters and terminators are operatively associated upstream and downstream of the expression construct, respectively.

As used herein, the term "promoter" means a DNA sequence that regulates the expression of a coding sequence or functional RNA. The target nucleotide sequence in the recombinant vector of the present invention is operatively linked to the promoter.

As used herein, the term "operatively linked" refers to a functional linkage between a nucleic acid expression control sequence (e.g., an array of promoter sequences, signal sequences, or transcription factor binding sites) Whereby the regulatory sequence regulates transcription and / or translation of the other nucleic acid sequence.

The 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). 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 using prokaryotic cells as hosts. The vector of the present invention can typically be constructed as a vector for cloning or as a vector for expression.

For example, when the vector of the present invention is an expression vector and a prokaryotic cell is used as a host, a strong promoter capable of promoting transcription (for example, T7 promoter, tac promoter, lac promoter, lacUV5 promoter, lpp promoter, A terminator such as a T7 terminator, an ADH1 terminator, a T3 terminator, a promoter, a pR? Promoter, a rac5 promoter, an amp promoter, a recA promoter, an SP6 promoter and a trp promoter) Terminators, and TonB terminators, etc.). When E. coli is used as a host cell, promoters and operator sites of the E. coli tryptophan biosynthetic pathway (Yanofsky, C., J. Bacteriol., 158: 1018-1024 (1984)) and phage λ left promoter (pL λ promoter, Herskowitz , I. and Hagen, D., Ann. Rev. Genet., 14: 399-445 (1980)).

The vectors that can be used in the present invention include plasmids such as pACYCDuet-1, pSC101, ColE1, pBR322, pUC8 / 9, pHC79, pUC19, pET etc., phage such as λgt4λB, λ-Charon ,?? z1, and M13) or a virus (e.g., SV40 or the like).

Host cells 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 and include, for example, E. coli C43 (DE3), E. coli JM109, E. coli BL21 (DE3), E. coli RR1, E. coli LE392, E. coli B, E. coli X1776, E. coli W3110, Bacillus subtilis, Bacillus subtilis, and Salmonella typhimurium , Serratia marcesensus and various Pseudomonas spp., And the like.

Methods of delivering the vector of the present invention into a host cell include the heat shock method, the CaCl ₂ method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA, 9: 2110-2114 And Hanahan, D., J. MoI. Biol., 166: 557-580 (1983)), And electroporation methods (Dower, WJ et al., Nucleic Acids Res., 16: 6127-6145 (1988)).

As used herein, the term "pharmaceutically effective amount" means an amount sufficient to achieve efficacy or activity of a Fab fragment for the EGFR described above.

When the Fab fragment of the present invention is prepared from a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. 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 orally or parenterally, and is preferably parenterally administered.

The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, . Typical dosages of the pharmaceutical compositions of this invention are in the range of 0.001 [mu] g / kg to 1000 mg / kg on an adult basis.

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, syrups or emulsions in oils or aqueous media, or in the form of excipients, powders, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

Hereinafter, the present invention will be described in detail with reference to embodiments and drawings. However, these examples are for further illustrating the present invention, and the scope of the present invention is not limited to these examples.

Example  One. Fab Construct construct

Antibody fragments platform (platform) is called Fm by making the unit Fab named and by a an amino acid sequence of setuk when Thank (Table 1) in order to clone the V _H + C _H1 and V _L + C _L domain of Fab basis of a signal peptide It requests the DNA sequence for (OmpA) + V _H + C _H1 (+ CDK) and signal peptide (OmpA) + amino acid sequence of a V _L + C _L (+ EC) (Table 2 and Table 3) course to the triangular Tech (Table 4 and Table 5). The underlined portion of the sequence of Table 1 represents the variable region. Them with V _H + C _H1 and V _L + C in the region corresponding to the _L domain PCR primers (Table 6), the production and the signal peptide (ompA) + V _H + C _H1 and the signal peptide (OmpA) + V _L + C _L were respectively cloned and cloned into a pACYCDuet-1 vector (Novagen) which is a co-expression vector of Escherichia coli through restriction enzyme treatment (Fig. 1).

- order Sequence List The anti-EGFR heavy chain QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 1 Anti-EGFR light chain DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK RT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGA SEQ ID NO: 2

- order Sequence List OmpA signal peptide KKTAIAIAVALAGFATVAQA SEQ ID NO: 3 V _H QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA SEQ ID NO: 4 C _H1 + CDK ≪ SEQ ID NO: 5

- order Sequence List OmpA signal peptide KKTAIAIAVALAGFATVAQA SEQ ID NO: 6 V _L DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK SEQ ID NO: 7 C _L + EC RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGAEC SEQ ID NO: 8

- order Sequence List OmpA signal peptide AAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTTCGCTACCGTAGCGCAGGCC SEQ ID NO: 9 V _H CAAGTCCAACTGAAACAATCGGGTCCGGGTCTGGTCCAACCGTCCCAATCACTGAGCATCACCTGTACCGTGTCGGGCTTCTCGCTGACCAATTATGGTGTGCATTGGGTTCGTCAGAGTCCGGGCAAAGGTCTGGAATGGCTGGGCGTTATTTGGTCCGGCGGTAATACCGATTACAACACCCCGTTTACGAGTCGCCTGTCCATCAATAAAGACAACTCGAAAAGCCAGGTGTTTTTCAAAATGAATTCACTGCAATCGAACGATACCGCGATTTATTACTGCGCACGTGCTCTGACGTATTACGACTATGAATTTGCCTACTGGGGCCAGGGTACCCTGGTGACGGTTAGCGCG SEQ ID NO: 10 C _H1 + CDK GCCTCTACCAAAGGTCCGAGCGTTTTCCCGCTGGCACCGAGCTCTAAATCTACCAGTGGCGGTACGGCAGCTCTGGGCTGTCTGGTGAAAGATTATTTTCCGGAACCGGTCACCGTGAGTTGGAATTCCGGTGCACTGACCAGTGGCGTCCACACGTTCCCGGCTGTGCTGCAGAGTTCCGGTCTGTATAGCCTGTCATCGGTGGTTACCGTTCCGAGCTCTAGTCTGGGCACCCAAACGTACATTTGCAATGTCAACCATAAACCGAGCAACACGAAAGTTGATAAACGTGTCGAACCGAAATCATGCGATAAA SEQ ID NO: 11

- order Sequence List OmpA signal peptide AAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTTCGCTACCGTAGCGCAGGCC SEQ ID NO: 12 V _L GATATTCTGCTGACCCAGAGCCCGGTGATCCTGAGTGTTTCCCCGGGCGAACGTGTGTCATTTTCGTGTCGCGCGAGCCAGTCTATTGGTACCAATATCCACTGGTATCAGCAACGTACGAACGGCTCTCCGCGCCTGCTGATTAAATACGCCAGTGAATCCATTTCAGGCATCCCGAGCCGCTTTTCGGGCAGCGGTTCTGGCACCGATTTCACGCTGAGTATTAACTCCGTGGAATCAGAAGATATCGCAGACTATTACTGCCAGCAAAACAATAACTGGCCGACCACGTTTGGTGCTGGCACCAAACTGGAACTGAAA SEQ ID NO: 13 C _L + EC CGTACGGTGGCGGCCCCGAGTGTTTTTATCTTCCCGCCGTCCGATGAACAGCTGAAATCGGGTACCGCCAGCGTTGTCTGTCTGCTGAATAACTTCTATCCGCGCGAAGCAAAAGTCCAGTGGAAAGTGGACAATGCTCTGCAGTCGGGCAACAGCCAAGAAAGCGTGACCGAACAAGATAGTAAAGACTCCACGTACTCACTGTCCTCAACCCTGACGCTGAGCAAAGCGGATTATGAAAAACACAAAGTGTACGCCTGCGAAGTTACCCATCAAGGTCTGAGTAGCCCGGTTACGAAATCATTCAATCGTGGTGCCGAATGC SEQ ID NO: 14

primer The sequence (5 '- > 3') Sequence List FM318 NcoI Forward GGAATTC CCATGGGC AAAAAGACAGCTATCGCGATTGCAG SEQ ID NO: 15 FM318 XhoI Reverse CCG CTCGAG TTA GCATTCGGCACCACGATTGAAT SEQ ID NO: 16

Example  2. Increase yield  for Fab  Mutant production of fragments

In order to substitute the base sequence with the Fab fragment prepared in Example 1 using a mutagenesis method, the base sequence was requested by Bioneer and gene synthesis was carried out (Table 7). Using the synthesized gene, pACYCDuet And cloned into an expression vector.

The amino acid sequences of the human-derived and mouse-derived antibodies were compared and analyzed, and as shown in Table 7 (13 kinds of partial humanized antibody fragments designed by modification from Fm318) with various amino acid mutations based on Fm318, 13 kinds of partial humanization cones And the prepared Fm318 and its modified humanized derivatives could be expressed in Escherichia coli (FIG. 3).

Construct name Mutations Light chain Heavy chain Fd FM318 none none FM307 L3Q / L4M N-term. His-tag FM308 V9S / I10S N-term. His-tag FM309 V13A / P15V N-term. His-tag FM311 none N-term. His-tag + S15G / Q16G FM323 none S15G / Q16G FM324 L3Q / L4M none FM325 V13A / P15V none FM326 L3Q / L4M / V13A / P15V none FM327 V13A / P15V S15G / Q16G FM328 L3Q / L4M / V13A / P15V S15G / Q16G FM329 L3Q / L4M S15G / Q16G

Primer Sequence (5 '- > 3') Mutagenesis 1 F (SQ- > GG) GGTGATGCTCAGTGAGCCGCCCGGTTGGACCAGACC (SEQ ID NO: 17) Mutagenesis 1 R (SQ- > GG) GGTCTGGTCCAACCGGGCGGCTCACTGAGCATCACC (SEQ ID NO: 18) Mutagenesis 2 F (TV-> AA) CTGAGCATCACCTGTGCGGCGTCGGGCTTCTCGCT (SEQ ID NO: 19) Mutagenesis 2 R (TV- > AA) AGCGAGAAGCCCGACGCCGCACAGGTGATGCTCAG (SEQ ID NO: 20) Mutagenesis 6 F (LL- > QM) CGCAGGCCGATATTCAGATGACCCAGAGCCCGG (SEQ ID NO: 21) Mutagenesis 6 R (LL- > QM) CCGGGCTCTGGGTCATCTGAATATCGGCCTGCG (SEQ ID NO: 22) Mutagenesis 7 F (VI- > SS) CTGACCCAGAGCCCCGAGCAGCCTGAGTGTTTCCCC (SEQ ID NO: 23) Mutagenesis 7 R (VI- > SS) GGGGAAACACTCAGGCTGCTCGGGCTCTGGGTCAG (SEQ ID NO: 24) Mutagenesis 8 F (VSP- > ASV) CGGTGATCCTGAGTGCGAGCGTGGGCGAACGTGTGTC (SEQ ID NO: 25) Mutagenesis 8 R (VSP- > ASV) GACACACGTTCGCCCACGCTCGCACTCAGGATCACCG (SEQ ID NO: 26) Mutagenesis 11 F (Q- > K) CCGGGTCTGGTCAAACCGTCCCAATCA (SEQ ID NO: 27) Mutagenesis 11 R (Q- > K) ACTAACCCTGCCAAACTGGTCTGGGCC (SEQ ID NO: 28) Mutagenesis 12 F (SQ- > GG) CTGGTCCAACCGGGCGGTTCACTGAGCATC (SEQ ID NO: 29) Mutagenesis 12 R (SQ- > GG) CTACGAGTCACTTGGCGGGCCAACCTGGTC (SEQ ID NO: 30)

Example  3. Fm329's  order

The Fm329 sequence according to Example 2 is shown below. Fm329 is a sequence in which the 15th and 16th serine (Ser; S) and glutamine (Gln; Q) in the amino acid sequence of V _H of Fm318 are mutated into glycine (Gly; G) and glycine (Gly; The third and fourth leucine (Leu; L) and leucine (Leu) in the amino acid sequence of V _L are mutated to glutamine (Gln; Q) and methionine (Met; M), respectively.

order Sequence List OmpA KKTAIAIAVALAGFATVAQA SEQ ID NO: 3 V _H
(SQ? GG ) QVQLKQSGPGLVQP GG SLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA SEQ ID NO: 31 C _H1 + CDK ≪ SEQ ID NO: 5

order Sequence List OmpA KKTAIAIAVALAGFATVAQA SEQ ID NO: 6 V _L
(LL? QM ) DI QM TQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK SEQ ID NO: 32 C _L + EC RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGAEC SEQ ID NO: 8

order Sequence List OmpA AAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTTCGCTACCGTAGCGCAGGCC SEQ ID NO: 9 V _H
(SQ? GG ) CAAGTCCAACTGAAACAATCGGGTCCGGGTCTGGTCCAACCG GGCGGC TCACTGAGCATCACCTGTACCGTGTCGGGCTTCTCGCTGACCAATTATGGTGTGCATTGGGTTCGTCAGAGTCCGGGCAAAGGTCTGGAATGGCTGGGCGTTATTTGGTCCGGCGGTAATACCGATTACAACACCCCGTTTACGAGTCGCCTGTCCATCAATAAAGACAACTCGAAAAGCCAGGTGTTTTTCAAAATGAATTCACTGCAATCGAACGATACCGCGATTTATTACTGCGCACGTGCTCTGACGTATTACGACTATGAATTTGCCTACTGGGGCCAGGGTACCCTGGTGACGGTTAGCGCG SEQ ID NO: 33 C _H1 + CDK GCCTCTACCAAAGGTCCGAGCGTTTTCCCGCTGGCACCGAGCTCTAAATCTACCAGTGGCGGTACGGCAGCTCTGGGCTGTCTGGTGAAAGATTATTTTCCGGAACCGGTCACCGTGAGTTGGAATTCCGGTGCACTGACCAGTGGCGTCCACACGTTCCCGGCTGTGCTGCAGAGTTCCGGTCTGTATAGCCTGTCATCGGTGGTTACCGTTCCGAGCTCTAGTCTGGGCACCCAAACGTACATTTGCAATGTCAACCATAAACCGAGCAACACGAAAGTTGATAAACGTGTCGAACCGAAATCATGCGATAAA SEQ ID NO: 11

order Sequence List OmpA AAAAAGACAGCTATCGCGATTGCAGTGGCACTGGCTGGTTTCGCTACCGTAGCGCAGGCC SEQ ID NO: 12 V _L
(LL? QM ) GATATT CAGATG ACCCAGAGCCCGGTGATCCTGAGTGTTTCCCCGGGCGAACGTGTGTCATTTTCGTGTCGCGCGAGCCAGTCTATTGGTACCAATATCCACTGGTATCAGCAACGTACGAACGGCTCTCCGCGCCTGCTGATTAAATACGCCAGTGAATCCATTTCAGGCATCCCGAGCCGCTTTTCGGGCAGCGGTTCTGGCACCGATTTCACGCTGAGTATTAACTCCGTGGAATCAGAAGATATCGCAGACTATTACTGCCAGCAAAACAATAACTGGCCGACCACGTTTGGTGCTGGCACCAAACTGGAACTGAAA SEQ ID NO: 34 C _L + EC CGTACGGTGGCGGCCCCGAGTGTTTTTATCTTCCCGCCGTCCGATGAACAGCTGAAATCGGGTACCGCCAGCGTTGTCTGTCTGCTGAATAACTTCTATCCGCGCGAAGCAAAAGTCCAGTGGAAAGTGGACAATGCTCTGCAGTCGGGCAACAGCCAAGAAAGCGTGACCGAACAAGATAGTAAAGACTCCACGTACTCACTGTCCTCAACCCTGACGCTGAGCAAAGCGGATTATGAAAAACACAAAGTGTACGCCTGCGAAGTTACCCATCAAGGTCTGAGTAGCCCGGTTACGAAATCATTCAATCGTGGTGCCGAATGC SEQ ID NO: 14

Example  4. Fab  Expression of fragment

The constructs prepared in Example 2 were transformed into E. coli expressing cell line C43 (DE3) cells (Lucigen, USA) by heat shock at 42 ° C. and induced by IPTG to approximately 44 kDa Fab 'fragments (Fig. 4). C43 (DE3) cells were shake cultured at 37 DEG C and 150 rpm.

In the lab-scale analysis using LB medium, Fm 318 and 13 mutagenized antibody fragments were produced and purified by established methods, and the yields were analyzed. As a result, it was confirmed that the production yield was the highest in Fm329 5).

In addition, production yields of Fm318 were compared with those of Fm318 produced from large-scale batch and fed-batch using the best production yield (Table 13). As a result, Fm329 was mass-produced at a high yield of 31.8 mg per 1 L of the culture solution through the pH-stat fed-batch fermentation method, which resulted in 3.5 times higher yield than the conventional Fm318.

Media composition (g / L) Seed culture Batch culture Feeding solution Tryptone
Yeast Extract
Sodium Chloride
Soy Peptone
Casein Protein
Ammonium sulfate
Potassium Phosphate monobasic
Potassium Phosphate dibasic
Glucose
Magnesium Sulfate heptahydrate
Antifoam 204
Chloramphenicol 16
10
5
-
-
-
-
-
-
-
-
0.05 -
20
-
5
5
-
5
3
20
1.2
0.2 (mL)
0.05 -
211
-
5
5
1.5
-
-
274
One
1 (mL)
0.05 Product yields Batch culture Fed-batch
(0.2 mM IPTG) Fed-batch
(1 mM IPTG)
Cultured E. coli Yield (g / L)
Protein recovery per culture (mg / L)
E. coli protein recovery per mg of protein (mg / g) FM318 / FM329
40 / 57.4
2.3 / 4.5
0.06 / 0.08 FM329
119.3
14.6
0.12 FM318 / FM329
330.6 / 143.6
9.0 / 31.8
0.03 / 0.22

Example  5. Fab  About the fragment Antigen-binding ability ELISA analysis of antigen binding activity

Antigen binding ability to the purified Fab fragments was performed using enzyme-linked immunosorbent assay (ELISA) and fluorescence-activated cell sorting (FACS).

ELISA was performed by adding 100 ng / well of sEGFR to a 96-well ELISA plate and reacting overnight at 4 ° C. The supernatant was removed, and 200 μl of blocking solution (Sigma, USA) was added to each well and then over-kneaded at 4 ° C. Cetusimab, 100 μl each of Fm318 and Fm329 were dispensed, reacted at room temperature for 1 hour, and washed three times with PBST. Anti-human IgG Fab specific antibody (goat, I5260-1ML, Sigma, USA) was diluted 1: 1000 and dispensed into each well at 100 μl. After incubation, the cells were washed 3 times with PBST, diluted 1: 3000 in the secondary antibody (anti-goat IgG whole molecule-peroxidase antibody produced in rabbit, A5420-1ML, Sigma, USA) And reacted for 1 hour. The supernatant was removed, washed three times with PBST, and 100 μl of TMB color development reagent was dispensed. The reaction was stopped by adding 100 μl of 1 M H ₂ SO ₄ to the developed wells, and the plate was measured for absorbance at 450 nm using a microplate reader.

As a result, it was confirmed that Fm329 had an efficacy without any change in the activity due to the introduced mutation as compared with the existing substances, cetuximab and Fm318 (FIG. 6 and Table 14). In particular, cetuximab is a divalent whole antibody with two antigen binding sites and Fm329 is a monovalent Fab fragment. Thus, having greater than 60% activity versus cetuximab at the same molar concentration may mean that the activity is similar or better than that based on the number of antigen binding sites. Considering that the molecular weight of cetuximab is about 153 kDa and the molecular weight of Fm329 is less than 48 kDa, it can be said that the effect is more than twice as much as considering the dosage of g / ml, not the molar concentration.

EGFR Binding affinity (%) AVER (%) STDEV (%) CTX 100 2.6 Fm318 64 2.6 Fm329 65 7.8

Example  6. Fab  About the fragment Antigen-binding ability FACS analysis results on antigen binding activity

In the FACS experiment, the cultured A431 cells were treated with 0.25% trypsin-EDTA and separated into one cell, 10 ml of PBS was added, centrifuged at 1500 rpm for 3 minutes, and the supernatant was removed. The pellet was resuspended in 10 ml of PBS and counted. Cells were diluted to a concentration of 5 x 10 ⁶ cells / ml, and then 100 μl each was added to a 5 ml tube. The purified protein was continuously diluted in a FACS solution at a concentration of 1.953125-500 nM and then 100 μl of the purified protein was added at 4 ° C for 15 minutes. After the reaction, 3 ml of PBS was added and the mixture was centrifuged at 1500 rpm for 3 minutes Separation and removal of the supernatant was repeated 3 times and washed. Then, 100 μl of anti-human IgG Fab specific-FITC antibody (goat, F5512, Sigma, USA) was diluted 1:16 in FACS solution and reacted at 4 ° C. for 15 minutes in darkness. 3 ml of PBS was added and the supernatant was discarded by centrifugation at 1500 rpm for 3 minutes. The pellet was suspended in 300 μl of PBS and measured by FACS.

As a result, the antigen binding ability was measured in the A431 cell line similar to that of cetuximab in both Fm318 and Fm329 antibody fragments (FIG. 7 and Table 15).

nM CTX Fm318 Fm329 0 0% 0% 0% 1.95313 9% 5% 7% 3.90625 16% 10% 14% 7.8125 43% 23% 36% 15.625 82% 54% 64% 31.25 95% 86% 89% 62.5 94% 97% 93% 125 95% 98% 97% 250 97% 98% 98% 500 100% 97% 99%

<110> Industry-Academic Cooperation Foundation of Konkuk University <120> Fab fragment specific binding to epidermal growth factor          receptor <130> PN1608-311 <160> 34 <170> KoPatentin 3.0 <210> 1 <211> 452 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 1 Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln   1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr              20 25 30 Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu          35 40 45 Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr      50 55 60 Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Ser Ser Ser Gln Val Phe Phe  65 70 75 80 Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala                  85 90 95 Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly             100 105 110 Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe         115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu     130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu                 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Ser Ser             180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro         195 200 205 Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Pro Lys Ser     210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu                 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser             260 265 270 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu         275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr     290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro                 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln             340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val         355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val     370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr                 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val             420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu         435 440 445 Ser Pro Gly Lys     450 <210> 2 <211> 213 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 2 Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly   1 5 10 15 Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn              20 25 30 Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile          35 40 45 Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly      50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser  65 70 75 80 Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr                  85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala             100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly         115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala     130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser                 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr             180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser         195 200 205 Phe Asn Arg Gly Ala     210 <210> 3 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 3 Lys Lys Thr Ala Ile Ala Ile Ala Val Ala Leu Ala Gly Phe Ala Thr   1 5 10 15 Val Ala Gln Ala              20 <210> 4 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 4 Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln   1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr              20 25 30 Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu          35 40 45 Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr      50 55 60 Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Ser Ser Ser Gln Val Phe Phe  65 70 75 80 Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala                  85 90 95 Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly             100 105 110 Thr Leu Val Thr Val Ser Ala         115 <210> 5 <211> 105 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 5 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 Arg Val Glu Pro Lys Ser Cys Asp Lys             100 105 <210> 6 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 6 Lys Lys Thr Ala Ile Ala Ile Ala Val Ala Leu Ala Gly Phe Ala Thr   1 5 10 15 Val Ala Gln Ala              20 <210> 7 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 7 Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly   1 5 10 15 Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn              20 25 30 Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile          35 40 45 Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly      50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser  65 70 75 80 Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr                  85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys             100 105 <210> 8 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 8 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 Ala Glu Cys             100 105 <210> 9 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 9 aaaaagacag ctatcgcgat tgcagtggca ctggctggtt tcgctaccgt agcgcaggcc 60                                                                           60 <210> 10 <211> 357 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 10 caagtccaac tgaaacaatc gggtccgggt ctggtccaac cgtcccaatc actgagcatc 60 acctgtaccg tgtcgggctt ctcgctgacc aattatggtg tgcattgggt tcgtcagagt 120 ccgggcaaag gtctggaatg gctgggcgtt atttggtccg gcggtaatac cgattacaac 180 accccgttta cgagtcgcct gtccatcaat aaagacaact cgaaaagcca ggtgtttttc 240 aaaatgaatt cactgcaatc gaacgatacc gcgatttatt actgcgcacg tgctctgacg 300 tattacgact atgaatttgc ctactggggc cagggtaccc tggtgacggt tagcgcg 357 <210> 11 <211> 315 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 11 gcctctacca aaggtccgag cgttttcccg ctggcaccga gctctaaatc taccagtggc 60 ggtacggcag ctctgggctg tctggtgaaa gattattttc cggaaccggt caccgtgagt 120 tggaattccg gtgcactgac cagtggcgtc cacacgttcc cggctgtgct gcagagttcc 180 ggtctgtata gcctgtcatc ggtggttacc gttccgagct ctagtctggg cacccaaacg 240 tacatttgca atgtcaacca taaaccgagc aacacgaaag ttgataaacg tgtcgaaccg 300 aaatcatgcg ataaa 315 <210> 12 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 12 aaaaagacag ctatcgcgat tgcagtggca ctggctggtt tcgctaccgt agcgcaggcc 60                                                                           60 <210> 13 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 13 gatattctgc tgacccagag cccggtgatc ctgagtgttt ccccgggcga acgtgtgtca 60 ttttcgtgtc gcgcgagcca gtctattggt accaatatcc actggtatca gcaacgtacg 120 aacggctctc cgcgcctgct gattaaatac gccagtgaat ccatttcagg catcccgagc 180 cgcttttcgg gcagcggttc tggcaccgat ttcacgctga gtattaactc cgtggaatca 240 gaagatatcg cagactatta ctgccagcaa aacaataact ggccgaccac gtttggtgct 300 ggcaccaaac tggaactgaa a 321 <210> 14 <211> 324 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 14 cgtacggtgg cggccccgag tgtttttatc ttcccgccgt ccgatgaaca gctgaaatcg 60 ggtaccgcca gcgttgtctg tctgctgaat aacttctatc cgcgcgaagc aaaagtccag 120 tggaaagtgg acaatgctct gcagtcgggc aacagccaag aaagcgtgac cgaacaagat 180 agtaaagact ccacgtactc actgtcctca accctgacgc tgagcaaagc ggattatgaa 240 aaacacaaag tgtacgcctg cgaagttacc catcaaggtc tgagtagccc ggttacgaaa 300 tcattcaatc gtggtgccga atgc 324 <210> 15 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 15 ggaattccca tgggcaaaaa gacagctatc gcgattgcag 40 <210> 16 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 16 ccgctcgagt tagcattcgg caccacgatt gaat 34 <210> 17 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 17 ggtgatgctc agtgagccgc ccggttggac cagacc 36 <210> 18 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 18 ggtctggtcc aaccgggcgg ctcactgagc atcacc 36 <210> 19 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 19 ctgagcatca cctgtgcggc gtcgggcttc tcgct 35 <210> 20 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 20 agcgagaagc ccgacgccgc acaggtgatg ctcag 35 <210> 21 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 21 cgcaggccga tattcagatg acccagagcc cgg 33 <210> 22 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 22 ccgggctctg ggtcatctga atatcggcct gcg 33 <210> 23 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 23 ctgacccaga gcccgagcag cctgagtgtt tcccc 35 <210> 24 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 24 ggggaaacac tcaggctgct cgggctctgg gtcag 35 <210> 25 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 25 cggtgatcct gagtgcgagc gtgggcgaac gtgtgtc 37 <210> 26 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 26 gacacacgtt cgcccacgct cgcactcagg atcaccg 37 <210> 27 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 27 ccgggtctgg tcaaaccgtc ccaatca 27 <210> 28 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 28 actaaccctg ccaaactggt ctgggcc 27 <210> 29 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 29 ctggtccaac cgggcggttc actgagcatc 30 <210> 30 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 30 ctacgagtca cttggcgggc caacctggtc 30 <210> 31 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 31 Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr              20 25 30 Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu          35 40 45 Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr      50 55 60 Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Ser Ser Ser Gln Val Phe Phe  65 70 75 80 Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala                  85 90 95 Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly             100 105 110 Thr Leu Val Thr Val Ser Ala         115 <210> 32 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 32 Asp Ile Gln Met Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly   1 5 10 15 Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn              20 25 30 Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile          35 40 45 Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly      50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser  65 70 75 80 Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr                  85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys             100 105 <210> 33 <211> 357 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 33 caagtccaac tgaaacaatc gggtccgggt ctggtccaac cgggcggctc actgagcatc 60 acctgtaccg tgtcgggctt ctcgctgacc aattatggtg tgcattgggt tcgtcagagt 120 ccgggcaaag gtctggaatg gctgggcgtt atttggtccg gcggtaatac cgattacaac 180 accccgttta cgagtcgcct gtccatcaat aaagacaact cgaaaagcca ggtgtttttc 240 aaaatgaatt cactgcaatc gaacgatacc gcgatttatt actgcgcacg tgctctgacg 300 tattacgact atgaatttgc ctactggggc cagggtaccc tggtgacggt tagcgcg 357 <210> 34 <211> 320 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 34 gatattcaga tgacccagag cccggtgatc ctgagtgttt ccccgggcga acgtgtgtca 60 ttttcgtgtc gcgcgagcca gtctattggt accaatatcc actggtatca gcaacgtacg 120 aacggctctc cgcgcctgct gattaaatac gccagtgaat ccatttcagg catcccgagc 180 cgcttttcgg gcagcggttc tggcaccgat ttcacgctga gtattaactc cgtggaatca 240 gaagatatcg cagactatta ctgccagcaa aacaataact ggccgaccac gtttggtgct 300 ggcaccaaac tggaactgaa 320

Claims (12)

A Fab fragment that specifically binds to EGFR (Epidermal Growth Factor Receptor). The method according to claim 1,
Wherein the Fab fragment comprises the following regions: &lt; RTI ID = 0.0 &gt;
(a) a heavy chain variable region (V _H ) having an amino acid sequence represented by SEQ ID NO: 31;
(b) a heavy chain constant region (C _H1 ) having an amino acid sequence represented by SEQ ID NO: 5;
(c) a light chain variable region (V _L ) having an amino acid sequence represented by SEQ ID NO: 32; And
(d) a light chain constant region (C _L ) having an amino acid sequence represented by SEQ ID NO: 8. The method according to claim 1,
Wherein the Fab fragment is a chimeric antibody or a humanized antibody. A nucleic acid molecule encoding the Fab fragment of claim 1. 5. The method of claim 4,
Wherein the nucleic acid molecule comprises the following regions:
(a) a heavy chain variable region (V _H ) having a nucleotide sequence represented by SEQ ID NO: 33;
(b) a heavy chain constant region (C _H1 ) having the nucleotide sequence shown in SEQ ID NO: 11;
(c) a light chain variable region (V _L ) having the nucleotide sequence shown in SEQ ID NO: 34; And
(d) a light chain constant region (C _L ) having the nucleotide sequence shown in SEQ ID NO: 14. A recombinant vector comprising the nucleic acid molecule of claim 4. A transformant transformed with the recombinant vector of claim 6. 8. The method of claim 7,
Wherein the transformant is Escherichia coli. (a) a pharmaceutically effective amount of the Fab fragment of claim 1; And (b) a pharmaceutically acceptable carrier. 10. The method of claim 9,
The cancer may be selected from the group consisting of breast cancer, colon cancer, lung cancer, stomach cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, brain cancer, uterine cancer, nasopharyngeal cancer, laryngeal cancer, head and neck cancer, colon cancer, ovarian cancer, Wherein the composition is any one selected from the group consisting of endometrial cancer, endometrial cancer, thyroid cancer, pituitary cancer, ureteral cancer, urethral cancer, prostate cancer, bronchial cancer, bladder cancer, kidney cancer and bone cancer. A diagnostic composition for cancer comprising the Fab fragment of claim 1. 12. The method of claim 11,
The cancer may be selected from the group consisting of breast cancer, colon cancer, lung cancer, stomach cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, brain cancer, uterine cancer, nasopharyngeal cancer, laryngeal cancer, head and neck cancer, colon cancer, ovarian cancer, Wherein the composition is any one selected from the group consisting of endometrial cancer, endometrial cancer, thyroid cancer, pituitary cancer, ureteral cancer, urethral cancer, prostate cancer, bronchial cancer, bladder cancer, kidney cancer and bone cancer.

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