WO2016171365A1 - Fab fragment specifically binding to egfr - Google Patents

Fab fragment specifically binding to egfr Download PDF

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Publication number
WO2016171365A1
WO2016171365A1 PCT/KR2015/013322 KR2015013322W WO2016171365A1 WO 2016171365 A1 WO2016171365 A1 WO 2016171365A1 KR 2015013322 W KR2015013322 W KR 2015013322W WO 2016171365 A1 WO2016171365 A1 WO 2016171365A1
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Prior art keywords
cancer
fab fragment
egfr
fab
fragment
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PCT/KR2015/013322
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French (fr)
Korean (ko)
Inventor
김영필
김혜림
유희정
이성환
정태근
하종렬
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신일제약주식회사
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Priority claimed from KR1020150126894A external-priority patent/KR101770559B1/en
Application filed by 신일제약주식회사 filed Critical 신일제약주식회사
Priority to US14/914,608 priority Critical patent/US20170145101A1/en
Publication of WO2016171365A1 publication Critical patent/WO2016171365A1/en

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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/395Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression

Definitions

  • the present invention was made by the task number A004600265 under the support of the Ministry of Trade, Industry and Energy of Korea, the research management professional agency of the project is (Foundation)
  • the present invention relates to a fragment (Fragment Antigen Binding) fragment that specifically binds to Epi dermal Growth Factor Receptor (EGFR).
  • EGFR Epi dermal Growth Factor Receptor
  • EGFRCEpidermal Growth Factor Receptor is one of the receptor HER families on the surface of the cell and plays an important role in cell growth and death by binding to ligands such as EGF, TGF-alpha, and Epiregulin.
  • ligands such as EGF, TGF-alpha, and Epiregulin.
  • immunostaining has shown increased expression of EGFR in many types of cancer cells, and reports that this increase in EGFR is closely associated with prognosis (Nicholson, RI et al. Eur. J. Cancer-, 37: S9-15 (2001); Yewale, C. et al., Biomaterials, 34: 8690—707 (2013)).
  • ISA / KR EGFR-TK1 targets the same EGFR but unlike cetuximab, lung cancer is an indication.
  • EGFR-TK1 is less effective and less relevant in cancers where EGFR expression is known to be closely associated with prognosis. This suggests that there is a mechanism different from the signal difference according to the amount of EGFR expression, and several mechanisms of action are used in order for the therapeutic antibody to show cytotoxic effects on cancer cells, most of which are ADCCCAnt i body-dependent. It is effective through the immune system through Cel lular Cytotoxic I ty or CDC (Complement Dependent Cytotox ic I ty).
  • Antibodies are structurally classified into five types, IgG, IgA, IgM, IgD, and IgE, according to differences in the constant region (Fc portion) of the heavy chain.
  • Flag of the CDC. ⁇ Strong, IgG2 has no function of ADCC, weak CDC function, IgG4 has weak ADCC function but no CDC function.
  • Antibody developed in anticancer field is known to have high ADCC and CDC function. IgGl isotypes have been the most developed.
  • EGFR has been developed and marketed as an antibody therapeutic agent in IgG2 type as well as IgGl type. This indicates that the neutralizing activity caused by antibody is the main action when targeting EGFR. It can be said.
  • EGFR exists in the form of a tethered monomer or an open structure of an untethered monomer, and when EGF binds, it forms a dimer to activate a kinase and transmit a signal.
  • Therapeutic antibody Sepusimab inhibits kinase activity and downstream signaling by binding to EGFR instead of ligand. This inhibits cell growth and induces cell death. This binding inhibits the activation of receptors and their signaling pathways, resulting in a reduction of tumor invasion into normal tissues and tumor diffusion into new sites. .
  • tumor cells are thought to inhibit tumor growth as a whole by inhibiting the ability to repair damage caused by chemotherapy and radiation therapy, and by inhibiting the formation of new blood vessels in tumors.
  • Erbitux® is used as a chemotherapeutic agent and is an EGFR chimeric antibody for the treatment of head and neck cancer and colorectal cancer .
  • the patent for cetuximab expired in 2015, and the development of biosimilars is in full swing in Korea, and various therapeutic antibodies against EGFR have been developed and approved overseas.
  • the development of anti-cancer therapies is being actively made.
  • many papers and patent documents are referenced and their citations are indicated. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.
  • the present inventors have tried to prepare a Fab fragment that can replace an anti-cancer antibody that inhibits Epidermal Growth Factor Receptor (EGFR) signal of cancer cells.
  • EGFR Epidermal Growth Factor Receptor
  • the present invention was completed by developing a Fab fragment that can be expressed in E. coli and specifically binds to EGFR, and affirming its excellent binding affinity and anticancer effect. Accordingly, it is an object of the present invention to provide Fab fragments that specifically bind to EGFR.
  • Another object of the present invention is to provide an expression construct for preparing the Fab fragment.
  • Still another object of the present invention is to provide a recombinant vector comprising the expression construct.
  • Another object of the present invention is to provide a host cell transformed with the recombinant vector.
  • Another object of the present invention to provide a pharmaceutical composition for preventing or treating cancer.
  • the invention provides a fragment ant igen-binding (fab) fragment that specifically binds to an Epidermal Growth Factor Receptor (EGFR) and comprises the following regions:
  • V H heavy chain variable region
  • Light chain constant region (C L ) comprising the amino acid sequence of SEQ ID NO: 7.
  • the present inventors have tried to prepare a Fab fragment that can replace the anti-cancer antibody that inhibits the EGFR signal of cancer cells. As a result, Fab fragments that can be expressed in E. coli and specifically bind to EGFR are developed. Binding affinity and anticancer effect were confirmed.
  • the Fab fragment of the present invention specifically binds to EGFR.
  • Fab fragment refers to a fragment having an antigen binding function, heavy chain variable region (V H ), heavy chain constant region 1 (C H1 ), light chain variable region (V L ) and light chain variable region ( C L ) with one antigen binding site.
  • V H heavy chain variable region
  • C H1 heavy chain constant region 1
  • V L light chain variable region
  • C L light chain variable region
  • F (ab ') 2 antibodies are produced by forming a bond with cysteine residues in the hinge region of Fab'.
  • Such Fab fragments can be obtained using proteolytic elements (e.g., restriction digestion of the entire antibody with papain yields Fab and cleavage with pepsin yields F (ab ') 2 fragments). Preferably it can be produced through genetic recombination technology.
  • proteolytic elements e.g., restriction digestion of the entire antibody with papain yields Fab and cleavage with pepsin yields F (ab ') 2 fragments.
  • it can be produced through genetic recombination technology.
  • the present invention is produced by expressing a fragment in E. coli, not Fab whole ant ibody.
  • variable region domain 3 ⁇ 4 VH encompasses the variable region domain 3 ⁇ 4 VH and three constant region domains C H1 , C H2 and C H3 comprising an amino acid sequence having a stratified variable region sequence for conferring specificity to the antigen. It means both full length heavy chain and fragments thereof.
  • Fab fragment of the present invention is a Fab fragment comprising a heavy chain consisting of the above V H and C H1 .
  • light chain herein also refers to the full-length light chain and fragments thereof comprising the variable region domain V L and the constant region domain C L comprising an amino acid sequence having a segmented variable region sequence for imparting specificity to the antigen. All means.
  • Fab fragments of the invention are Fab fragments comprising a light chain consisting of the above V L and CL.
  • Fab fragments of the present invention may include variants of amino acid sequences set forth in the appended sequence listing within the scope of specific binding to EGFR.
  • the binding affinity and / or other biological properties of the Fab fragment Changes can be made to the amino acid sequence of the Fab fragments for improvement.
  • modifications include, for example, deletions, insertions and / or substitutions of amino acid sequence residues in the Fab fragments.
  • amino acid variations are made based on the relative similarity of amino acid chain substituents, such as hydrophobicity, hydrophilicity, charge, size, etc.
  • arginine, lysine and histidine are all A positively charged residue; Alanine, glycine and serine have similar sizes; It can be seen that phenylalanine, tryptophan and tyrosine have a similar shape.
  • arginine, lysine and histidine; Alanine, glycine and serine; Phenylalanine, tryptophan and tyrosine are biologically the /
  • hydrophobicity index can be considered. Each amino acid is assigned a hydrophobicity index depending on its hydrophobicity and charge: isoleucine (+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); Plin (-1.6); Hintidine (-3,2); Glutamate (-3,5); Glutamine (-3.5); Aspartate (-3.5); Asparagine (-3.5); Lysine (-3.9); And arginine (-4.5).
  • hydrophobic amino acid index is of great importance in conferring the interactive biological function of proteins. It is well known that substitution with amino acids having similar hydrophobic indexes can retain similar biological activity. When introducing a mutation with reference to a hydrophobic index, substitutions are made between amino acids that exhibit a hydrophobic index difference of preferably within ⁇ 2, more preferably within 1, and even more preferably within ⁇ 0.5.
  • substitutions are made between amino acids which exhibit a hydrophilicity value difference of preferably within ⁇ 2, more preferably within 1 and even more preferably within ⁇ 0.5.
  • Amino acid exchange in proteins that do not alter the activity of the molecule as a whole is known in the art (H. Neurath, RLHi 11, The Proteins, Academic Press, New York, 1979).
  • the most commonly occurring exchanges are amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Exchange between Pro, Lys / Arg, Asp / Asn, Leu / I le, Leu / Val, Ala / Glu, Asp / Gly.
  • the antibody or nucleotide molecule encoding the same of the present invention is interpreted to include a sequence that exhibits a substantial identity with the sequence described in the sequence listing.
  • This substantial identity is at least 61% when the sequence of the present invention is aligned with any other sequence as best as possible and the aligned sequence is analyzed using algorithms commonly used in the industry. Homologous, more preferably 7% homology, even more preferably 80% homology, most preferably 90% homology. Alignment methods for sequence comparison are known in the art. Various methods and algorithms for alignment are found in Smith and Waterman, Adv. Ap l. Math. 2: 482 (1981); Needleman and Wunsch, J. Mol. Bio. 48: 443 (1970); Pearson and Lipman, Methods in Mol. Biol.
  • Fab fragments that specifically bind to EGFR of the present invention further comprise amino acid sequences for formation of disulfide bonds at C H1 and C L to form heterodimers of heavy and light chains.
  • the C L further comprises Glu-Cys at its C-terminus (SEQ ID NO: 18).
  • PEGylation refers to the conjugation of Polyethylenelylene glycol (PEG) to the protein of interest, ie the Fab fragment for the EGFR.
  • polyethylene glycol which is a highly biocompatible polymer that does not cause an immune response in vivo, is conjugated. This minimizes the degradation of Fab fragments by conjugation to sites that can minimize the effect on drug activity and maximize the PEGylation effect.
  • Pegylated Fab fragments have increased molecular weights, which can inhibit protein permeation to the manure effect in the kidneys due to renal glomerular filtration, resulting in reduced losses and in vivo protein enzymes through the stealth effect of polyethylene glycol. It shows the result of inhibiting the degradation of these compounds, which increases the half-life in vivo and prevents the access of proteolytic enzymes to the body due to the steric hindrance of polyethylene glycol. Has the effect of increasing solubility.
  • the Fab fragment further binds Thr-His-Thr—Cys-Ala-Ala to Cys-Asp—Lys at the C-terminus of its C H1 (SEQ ID NO: 23).
  • the Fab fragment is PEGylated Cys residue at Thr-His-Thr-Cys-Ala-Ala at the C-terminus of its C H1 .
  • polyethylene glycol PEG
  • PEGs suitable for the present invention are represented by the following structural formula: (0C3 ⁇ 4C) n (wherein n is an integer from 2 to 4000)
  • suitable PEG molecules for the present invention are CH 2 CH 2 0 (CH 2 CH 2 0) nCH 2 C3 ⁇ 4 ”and“ (0CH 2 CH 2 ) n0 ”.
  • PEG herein includes structures having various terminal groups and“ terminal capping ”groups.
  • the terminal group includes Maleimide *
  • PEG for? 1 in the PEGylation has a molecular weight of 5-50 kDa.
  • the PEG has a lower molecular weight of 18-38 kDa.
  • Fab fragments of the invention bind 1: 1 with one molecule of PEG.
  • Fab fragments of the invention have good half-life through PEGylation.
  • the Fab fragment has a half-life of 20-35 hours in mice (Mus musculus).
  • the present invention provides an expression construct for preparing a Fab fragment that specifically binds to EGFR:
  • a heavy chain-expressing construct comprising: (a-1) a heavy chain variable region (V H ) -encoding nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 9; And -2) a heavy chain variant region 1 (C H1 ) -encoding nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 10 sequence; And,
  • (b) a light chain-expressing construct comprising: (b-1) a light chain variable region (V L ) -encoding nucleic acid comprising the nucleotide sequence of SEQ ID NO: 11 molecule; And (b-2) a light chain constant region (C L ) -encoding nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 12.
  • Expression constructs for preparing Fab fragments that specifically bind to the EGFR of the present invention are expression constructs for preparing the Fab fragments, the contents of which are common between the two, in order to avoid excessive complexity herein. Omit.
  • the heavy chain constant region i (c H1 ) -coding nucleic acid molecules and light chain constant region ( Cl ) -coding nucleic acid molecules constituting the expression construct of the present invention are nucleotide sequences for the formation of disulfide bonds to c H1 and C L and And / or the nucleotide sequence for PEGylation of the above may be further included at the C-terminus of the nucleic acid molecule.
  • the heavy chain constant region i (c H1 ) -coding nucleic acid molecule is a nucleotide sequence of SEQ ID NO: 10, 19 or 24,
  • the light chain constant region (QJ-encoding nucleic acid molecule is a nucleotide sequence of SEQ ID NO: 12 or 20.
  • nucleic acid molecule herein refers to DNA (gDNA and cDNA) and Nucleotide, which has the meaning of encompassing RNA molecules in its entirety and which is a basic structural unit in the nucleic acid molecule, includes not only natural nucleotides: but also analogues in which sugar or base sites are modified (Schei t, Nucleotide).
  • Nucleic acid molecular sequences encoding the heavy and light chain variable regions of the Fab fragments of the invention are modified. Such modifications include additions, deletions or non-conservative substitutions or conservative substitutions of nucleotides.
  • a nucleic acid molecule encoding an antibody of the present invention is to be construed to include a nucleotide sequence which shows substantial identity to the nucleotide sequence described above.
  • the substantial identity is at least 80% when the nucleotide sequence of the present invention and any other sequence are aligned to the maximum, and the aligned sequence is analyzed using algorithms commonly used in the art.
  • One of the main features of the present invention is that the Fab fragment for EGFR. It can be produced through E. coli.
  • the nucleic acid molecule is expressed in order to express Fab fragments in E. coli,
  • the nucleotide sequence encoding the Fab fragment for EGFR was converted to the host in favor of the codon expression frequency of E. coli.
  • the expression construct produced in the present invention is constructed to express a desired gene in a host cell.
  • a desired gene in a host cell.
  • promoters and terminators operatively coupled upstream and downstream of the expression construct, respectively.
  • promoter refers to a DNA sequence that modulates the expression of a coding sequence or functional RNA. 4.
  • the target nucleotide sequence is operably linked to the promoter,
  • the term "operatively l inked” refers to the functional binding between a nucleic acid expression control sequence (e.g., a promoter sequence, a signal sequence, or an array of transcriptional regulator binding sites) and another nucleic acid sequence.
  • a nucleic acid expression control sequence e.g., a promoter sequence, a signal sequence, or an array of transcriptional regulator binding sites
  • said regulatory sequence modulates the transcription and / or translation of said other nucleic acid sequence.
  • the present invention provides a recombinant vector comprising the expression construct.
  • the vector system of the present invention can be constructed through various methods known in the art, and specific methods thereof are described in Sambrook et al. , Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001), which is incorporated herein by reference.
  • Vectors of the invention can typically be constructed as vectors for cloning or vectors for expression.
  • the vector of the present invention can be constructed using prokaryotic cells as a host.
  • Vectors of the invention can typically be constructed as vectors for cloning or vectors for expression.
  • the vector of the present invention is an expression vector and the prokaryotic cell is the host.
  • kinetic promoters e.g., T7 promoter, tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pL lambda promoter, pR lambda promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, which can promote transcription
  • Merter and trp promoters ribosome binding sites for initiation of translation and transcription / detox termination sequences (terminators such as T7 terminator, ADH1 terminator, T3 terminator and TonB terminator).
  • coli tryptophan biosynthesis pathway (Yanofsky, C, J. Bacteriol., 158: 1018-1024 (1984)) and the leftward promoter of phage ⁇ (pL ⁇ promoter, Herskowitz , I. and Hagen, D., Ann. Rev. Genet., 14: 399-445 (1980)) can be used as regulatory sites.
  • vectors that can be used in the present invention are often used in the art, such as polamide (e.g. pACYCDuet-l, pSClOl, ColEl, pBR322, pUC8 / 9, pHC79, pUC19, pET, etc.), phage (e.g., Xgt4 B , ⁇ -Charon, ⁇ ⁇ , and M13) or viruses (eg SV40). !
  • polamide e.g. pACYCDuet-l, pSClOl, ColEl, pBR322, pUC8 / 9, pHC79, pUC19, pET, etc.
  • phage e.g., Xgt4 B , ⁇ -Charon, ⁇ ⁇ , and M13
  • viruses eg SV40
  • the nucleotide sequence encoding the Fab fragment for EGFR is cloned into pACYCDuet-1.
  • the pACYCDuet-1 related information is https: // www. snapgene. com / r esour ces / p 1 asm i d_f i 1 es / pet— and— duet—vec tors— (novagen) / pACYCDuet-l /
  • the signal peptide is OmpA signal peptide, LamB signal peptide, Stll signal peptide, MalE signal peptide, Lpp signal peptide and PelB signal peptide.
  • the signal peptide is an OmpA signal peptide.
  • the OmpA signal peptide is located upstream of the nucleotide sequence encoding the heavy chain variable region.
  • the amino acid sequence encoding the OmpA signal peptide is sequence listing
  • the nucleotide sequence encoding the ⁇ signal peptide is the third sequence and is SEQ ID NO: 8. According to another aspect of the present invention, there is provided a host cell transformed with the recombination vector.
  • Host cells capable of stably and continuously cloning and expressing the vector of the present invention are known in the art and can be used with any host cell, for example, E. coli C43CDE3), E. coli JM109, E awkwardcoli BL2KDE3), Bacillus sp. Strains such as E. coli RRl, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus thuringiensis, and Salmonella typhimurium, Seri "thia Enterobacteria and strains such as Marsesons and various Pseudomonas species.
  • the method of transporting the vector of the present invention into a host cell includes a heat shock method
  • CaC12 method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA, 9: 2110-2114 (1973)), one method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA) , 9: 2110-2114 (1973); and Hanahan, D., J. Mol. Biol., 166: 557-580 (1983)) and electroporation methods (Dower, WJ et al., Nucleic. Acids Res., 16: 6127-6145 (1988)).
  • the host cell of the invention is E. coli. According to another embodiment of the invention, the host cell of the invention is E. coli
  • the method of the present invention is a method for preparing a Fab fragment for the EGFR, the content common between the two is omitted in order to avoid excessive complexity of the present specification.
  • the host cell of step (a) of the present invention can be cultured according to various culture methods known in the art.
  • the host cell is SB (Super Broth), FBC Fast idious Broth (LB), LB (Lysogeny Broth), TB (Terr if ic Broth), S0C (Super Opt imal Broth wi th Catabol ic repressor) and S0B.
  • SB Super Broth
  • LB Lysogeny Broth
  • TB Terr if ic Broth
  • S0C Super Opt imal Broth wi th Catabol ic repressor
  • S0B Super Broth Opt imal
  • the host cell is cultured in a culture medium at least one selected from the group S configuration by SB (Super Broth), FB ( Fast idious Broth) and LB Broth Lysogeny).
  • the host cell is cultured in SB (Super Broth).
  • SB Super Broth
  • the cancer is breast cancer, colon cancer, lung cancer, gastric cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, brain cancer, uterine cancer, nasopharyngeal cancer, laryngeal cancer, colon cancer, ovarian cancer, rectal cancer, colon cancer, Vaginal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, ureter cancer, urethral cancer, prostate cancer, bronchial cancer, bladder cancer, kidney cancer or bone marrow cancer.
  • the cancer is head and neck cancer.
  • pharmaceutically effective amount means an amount sufficient to achieve the efficacy or activity of the Fab fragments described above for EGFR.
  • the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are those commonly used in the preparation of lactose, textose, sucrose, sorbitol, Mannitol, starch acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyridone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxy Benzoate, talc, magnesium stearate, mineral oil, and the like.
  • the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like, in addition to the above components.
  • a lubricant e.g., talc, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, sorbibuprofate, a sorbi cal Sciences (19th ed., 1995).
  • the pharmaceutical composition of the present invention may be oral or parenteral, and is preferably applied by parenteral administration.
  • Suitable dosages of the pharmaceutical compositions of the present invention vary depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion and reaction in response to the patient. It may be prescribed. Typical dosages of the pharmaceutical compositions of the invention are in the range of 0.001 g / kg-1000 uig / kg for adults.
  • compositions of the present invention may be prepared in unit dose form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporation into a multi-dose container.
  • the formulation may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media or in the form of extracts, powders, powders, granules, tablets or capsules, and may further comprise dispersants or stabilizers.
  • the present invention is a fragment ant igen-binding (fab) fragment that specifically binds to EGFR (Epi dermal Growth Factor Receptor), an expression construct for producing the Fab fragment, a method for producing the Fab fragment and the
  • a pharmaceutical composition comprising a Fab fragment is provided.
  • Fab fragments for EGFR of the present invention are smaller in size than antibodies, and thus have good penetration into tissues or tumors and can be produced in bacteria. Less production costs. Holding
  • FIG. 1 shows the Fm301 construct and the pACYCDuet-1 vector map.
  • FIG. 2 shows the cloning of Fm301 through PCR (Polymerase Cloning React ion).
  • FIG. 3 shows the Fm302 construct and the pACYCDuet-1 vector map.
  • FIG. 5 shows the ⁇ 1 ⁇ 2306 construct and the pACYCDuet-1 vector map.
  • FIG. 6 shows the results of PCR confirming the cloning of 13 ⁇ 4306.
  • Figures 7a to 7c shows the results confirmed by SDS-PAGE and 3 ⁇ 4 Fm301 and Fm302 protein when expressed in E. coli and the result of binding to the anti-Fab antibody.
  • Figure 8 shows the results of the homogeneity fraction after purification of Fm301 and Fm302.
  • Figure 9 shows the results confirmed by SDS-PAGE Fm306 protein expressed in E. coli.
  • 10A and 10B show the yield of antibody purification by medium (Lysogeny broth; LB, Fant idious broth; FB and Super broth; SB).
  • 11 shows the results of electrophoresis of 11 ⁇ 2302 expressed in E. coli using a non-reducing die.
  • 12A to 12B show ion exchange chromatography results of Fm302.
  • FIG. 13 shows the size exclusion chromatography results of Fm302.
  • FIG. 14 shows Cys- ⁇ p-Lys and Glu- at the C-terminus of ⁇ and C L of Fm302, respectively.
  • Figure 16 shows the results of confirming the production and yield of the Fm306-PEG (20K) and Fm306-PEG (30K) conjugate.
  • Figure 17 shows the structure of the Fab 'construct.
  • Fm301, Fra302 and Fm306 All have the same four intra-chain disulfide bonds, and for 11 ⁇ 2302 and 11 ⁇ 2306 there are intrachain disulfide bonds at the C-terminus.
  • Fm306 there is an amino acid added for PEGylation at the heavy chain portion C-terminus.
  • FIG. 18A-18D show the results of confirming PEGylation of Fm306-PEG via SDS-PAGE, PEG staining and Western blot.
  • FIG. 18A shows the Coomassie blue staining results of SDS-PAGE of 11 ⁇ 2301, Fm302, and Fm306 samples and samples of construct-specific PEG.
  • FIG. 18B shows the result of PEG staining for the same sample as in FIG. 18A.
  • FIG. Figure 18c shows the results of Western blot using the anti-Fab antibody of the Fm301, Fm302 and f3 ⁇ 4306 sample and the sample reacted with PEG for each construct.
  • FIG. 18D shows Western blot results using anti-PEG antibodies of i301, Fm302, and 13 ⁇ 4) 6 samples and samples of construct-specific PEG.
  • 19A-19D show the PEGylation of Fm306-PEG confirmed via SDS-PAGE, PEG staining and Western blot after residual PEG removal.
  • 19a shows the results of coomassie blue staining of SDS-PAGE on samples from which residual PEG by Fm301, Fm302 and Fm306 constructs were removed.
  • 19B shows the result of PEG staining for the same sample as in FIG. 19A.
  • 19C shows Western blot results using anti-Fab antibodies for samples from which residual PEG was removed for each Fm301, Fm302, and Fm306 sample constructs.
  • FIG. 19D shows Western blot results using anti-PEG antibodies for samples with residual PEG removed by Fm301, Fm302 and Fm306 constructs.
  • 20A-20D show the kinetic values of Cetuximab, Sepusimab-Fab, Fm302 and Fm306FEG, respectively.
  • 21 shows sEGFR-binding affinity of 13 ⁇ 4302 and F 306PEG.
  • FIG. 22 shows the degree of EGFR phosphorylation of EGF, Sesucimab, Celuximab-Fab, Fm302, and Fm306PEG.
  • FIG. 23 shows tumor tissue growth in cecum carcinoma disease model animals of Sepusimab, Sepusimab-Fab, Fm302 and Fm306PEG.
  • FIG. 24 shows the tumor tissue size at necropsy in head and neck cancer disease model animals of cetuximab, cetuximab-Fab, Fm302 and f3 ⁇ 4306PEG.
  • intra-molecular disulfide bonds present in the V L (light chain variable region) and V H (heavy chain variable region) domains are each domain. Stabilize the structure.
  • Such intramolecular disulfide f is known to play an important role in the interaction between antibody (Ant i-EGFR) and antigen [EGFR (HERl)] [(Yang, et al., PNAS. 104 (26). ): 10813-10 ⁇ 17 (2007); Liu, H. and May, k., MAbs, 4: 17-23 (2012)], and also two cysteines present in the hinge domain.
  • Fab ' which is a monovalent Fab, is an antibody fragment platform Signal peptide (OmpA) + V H + C H1 and signal peptide (OmpA) based on the amino acid sequence of cetuximab (Table 1) for naming and cloning the V H + C H1 and V L + C L domains of Fab DNA sequences for the amino acid sequences (Tables 2 and 3) of + V L + C L were submitted to CosmoGentech for synthesis (Tables 4 and 5). Indicates.
  • PCR primers were prepared at the sites corresponding to the V H + C H1 and V L + C L domains (Table 6) and signal peptide ( om pA) + V H + C H1 and signal peptide (OmpA) + V L +
  • the genes were cloned to express C L , respectively, and cloned into the pACYCDuet-1 vector (ovagen), which is a co-expression vector of E. coli through restriction enzyme treatment (FIG. 1).
  • the amino acid sequences 'CDK' and 'EC' are inserted at the C-terminus of the C H1 and C L of the Fm301 construct (Table 7 and converted to codons that can be expressed in E. coli to clone ⁇ constructs.
  • DNA sequences (Tables 9 and 10) were commissioned by Cosmojintech to synthesize the 'CDK' and 'EC' in order to induce disulfide bonds for the light and heavy chains to form a heterodimer.
  • the PCR primers were prepared at the sites corresponding to the V H + C H1 (+ CDK) and V L + C L (+ EC) domains (Table 11), and the signal peptide ( om pA) + V H + C was used.
  • the genes were cloned to express the H1 (+ CDK) and signal peptide (0mpA) + V L + C L (+ EC) domains, respectively, and were cloned into the pACYCDuet-1 vector, which is a co-expression vector of E. coli through restriction enzyme treatment. (FIG. 3),
  • the amino acid THTCM which is a hinge region at the C-terminus of the C H1 domain, was inserted into the Fm302 gene (Table 12) and converted into codons that can be expressed in E. coli.
  • the base sequences (Tables 14 and 15) were commissioned by Cosmojintech for synthesis.
  • fCR primers were prepared at the sites corresponding to V H + C H1 + THTCAA and V L + C L + EC (Table 16), and E. coli expression PCR was performed for co-expression of E. coli through the enzyme treatment.
  • the vector was cloned into the pACYCDuet-1 vector (FIG. 5).
  • E. coli-expressing cell lines C43 (DE3) cells (Luc igen) were transformed by 42 ° C heat shock and then Fm301 and Fm302 were obtained through IPTG induction. Expression was confirmed (FIGS. 7A-7B). C4303E3) was shaken at 37 ° C and 150 rpm. To compare the expression levels of Fm301 and 302 cultured under the same culture conditions, the expression level of Fm302 was higher and to determine whether the purified Fm301 and Fm302 bind to anti-fab antibodies using Fab specific antibodies.
  • Example 4 Culture and Purification of Fra302 and Fm306 Antibody Fragments
  • the host cell for the purification of antibody fragments was E. coli C43 (DE3), which can reduce cell death due to toxicity of overexpressed recombinant protein by lowering the level of T7 RNA polymerase.
  • E. coli C43 DE3
  • Each of the two domains was expressed and an OmpA signal peptide was introduced to generate antibody fragments in the periplasm.
  • Fig. 10a Lysogeny broth (LB), Fast idious broth (FB), and Super broth (SB) were used. Cell mass and antibody fragment production were compared under the same culture conditions (37 ° C and 150 rpm shaking culture). The final OD600 values were LB 3.69, FB 6.58 and SB 11.48 with the highest SB, which means that the most cells can be obtained in SB medium.
  • SB medium was 5 times higher than LB in SB medium. The amount of antibody fragment was determined to be increased five times than the amount of antibody fragment in LB medium (Fig. 10b).
  • the antibiotic chloramphenicol was added to LB medium and incubated overnight at 37 ° C. and 150 rpm using a shaking incubator. Thereafter, the cell culture medium grown on the new SBplus medium (Gel l ix) was inoculated to about 1-2%, and the antibiotic chloramphenicol was added thereto and incubated at 37 ° C. and 150 rpm using a shaking incubator. After inoculation, when 0D 600 value was 3.0, it was induced using 1 mM IPTG and incubated for 9 hours under conditions of 25 ° C ⁇ 150 rpm. Subsequently, the culture solution was centrifuged at 4 ° C. and 8000 rpm to obtain a pel let.
  • the cells obtained in this manner were approximately 30-40 ⁇ lysis cell lysate per liter of culture medium [lxPBS (pho, sphate buf fered) sal ine), 5 mM EDTACethylenediaminetetraacetic acid) and 10% glycerol, pH 7.4] were added to the cells.
  • the cells were then lysed for 5 minutes using an ultrasonic crusher (pulse on 3 seconds, pulse off 3 seconds).
  • an ultrasonic crusher pulse on 3 seconds, pulse off 3 seconds.
  • the cell was centrifuged for 40 minutes at a speed of 20,000 rpm using a centrifuge.
  • Affinity chromatography was performed to purify only antibody fragments from the antibody fragments and protein aqueous solution separated from the cells. Fill an open column with kappa select resin (GE Heal thcare) that binds to the C L domain of the antibody fragment and equilibrate buffer (equi 1 ibrium buf fer; 50 mM Tr is-HCl, 100 mM NaCl, 5 mM EDTA, pH 7.4) was removed to equilibrate.
  • GE Heal thcare kappa select resin that binds to the C L domain of the antibody fragment
  • equilibrate buffer equi 1 ibrium buf fer; 50 mM Tr is-HCl, 100 mM NaCl, 5 mM EDTA, pH 7.4
  • Ion exchange chromatography was performed to further increase the purity of the firstly purified antibody fragments. Connect a HiTrapSP HP column (GE Heal thcare) to an A TA prime FPLC system and equilibrate buffer (100 mM glycine, 1 raM EDTA, pH 2.5) was removed to equilibrate. Resin and antibody fragments were bound to each other by pouring an aqueous solution of antibody fragment (pH 2.5) eluted during the first purification step, followed by washing with 5 CV of the complete aqueous solution 1 (50 mM MES, 1 mM EDTA, 2 mM DTT, pH).
  • the host cell and the fulllasmid were used for E-col i C43 (DE3) and pACYCDuet-1 vectors such as 11 ⁇ 2302, and antibody fragments were generated in the periplasm by using OmpA coral peptides.
  • Seed put chloramphenicol as antibiotics in LB medium and incubate overnight at 37 ° C and 200 rpm in shake incubator.
  • the culture was then centrifuged at 4 ° C and 8000 rpm to obtain a precipitate.
  • the Fm306 antibody fragments expressed in the secured 1 ⁇ 2 E-coli C43 (DE3) were suspended in about 30-40 of: cell lysis supernatant per liter of culture medium to purify with high purity.
  • the cells were suspended in a cell lysis buffer of pH 7.4 containing sodium phosphate buffer (PBS, phosphate buffered saline), 5 mM EDTA (ethylenediaminetetraacetic acid), and 10% glycerol.
  • PBS sodium phosphate buffer
  • 5 mM EDTA ethylenediaminetetraacetic acid
  • 10% glycerol 10% glycerol.
  • the cells were then lysed for 5 minutes using an ultrasonic crusher (pulse on 3 seconds, pulse off 3 seconds).
  • ion band chromatography was performed by secondary purification in order to increase purity by removing four bands (FIG. 15 # 1 lane) impurities around 17 kD of the first purified antibody fragment.
  • the open cylinder was filled with SP resin (GE Healthcare) of antibody fragments, and equilibrium complete layer solution (100 mM glycine, 1 mM EDTA, pH 2.5) was transferred to equilibrium.
  • Antibody Fragments and Proteins The resin and antibody fragments were bound to each other by pouring the aqueous solution onto the column, and then 5 CV (column volume) washing buffer (50 mM MES ⁇ 20 mM NaCl, 1 mM EDTA, pH 6 ⁇ 0) was added to non-specifically bound soloon.
  • Tr is-Cl buffer 1.5 M Tr is-Cl buffer was added to the reaction solution to adjust the pH to about 7, 5, and then fresh PEG-maleimide immediately before reaction for PEGylation.
  • NANOCS N-maleimide immediately before reaction for PEGylation.
  • Fm306-PEG liquid mixture was mixed on the reaction, the reaction time was 2 hours.
  • SP resin was used to remove free-PEG that did not bind to the antibody fragment.
  • Fm306-PEG mixed solution was lowered to about 2.5 ph using HC1 complete solution, bound to SP resin, and then stratified over 20 CV with complete solution (100 mM Glycine ph2.5, 1 mM EDTA) with the same composition. Rinse the remaining free-PEG without binding to the antibody fragment. Removed and an Fm306-PEG conjugate was obtained. The conjugate was loaded onto a Superdex 200 column (Superdex 200, GE Heal thcare) equilibrated with 10 mM sodium phosphate buffer (PBS, ph7.3), and the flow rate was 1 mi (l) per minute using the same buffer. / min) eluted from the column. Fm306-PEG conjugate (3 in FIG.
  • Fm306-PEG (30K) conjugates were also prepared using the same method as described above for Fm30 ⁇ -PEG (20K).
  • Fm306-PEG conjugate ( ⁇ of FIG. 16) was separated using the eluting property because molecular weight is relatively larger than Fm306 (8 of FIG. 16).
  • Fm306-PEG conjugate ( ⁇ of FIG. 16) was separated using the eluting property because molecular weight is relatively larger than Fm306 (8 of FIG. 16).
  • two bands were identified at about 70 kDa and 28 kDa sizes, which are thought to be the light chain and light chain portions of the PEGylated antibody fragment (about 75 kDa) and the Fm306 antibody fragment.
  • the light chain portion remaining unpegylated due to site-specific PEGylation of the repetition portion was identified.
  • one PEG is considered to be a homogeneous PEGylated antibody fragment.
  • the yield of the Fm306-PEG (30K) conjugate thus obtained was confirmed to be about 80% and the next experiment was carried out with PEGylat ionized antibody fragments (FIG. 16).
  • Fm306 which added 6 amino acid sequences (THTCAA) to the C-terminus of the C H1 domain of Fm302 was selected as a PEGylation construct.
  • PEGylation occurs through the reaction of a sul fhydryl group of cysteine residues with a maleimide group located at the end of PEG in the added amino acid sequence.
  • Cysteine residues present in different positions of Fm306 has pegil upset occur without C H1 are only upset pegil cysteine of the amino acid sequence added to the C- terminus of the pegylated domain: Fm301 to demonstrate that And a comparative experiment with the Fm302 construct.
  • 11 ⁇ 23 is a C-terminal cysteine-free construct, used as a control construct to demonstrate no pegylation of cysteine residues that make up an intra-chain disul fide bond, and Fm302 is used for each chain.
  • a construct with cysteine at the C-terminus of C was selected to demonstrate no PEGylation at that cysteine residue (FIG. 17). Production of PEGylated Fab's
  • the antibody fragment samples (eg, columns 1, 3, and 5 of FIG. 18A) that do not contain PEG for each construct and the sample that attempted PEGylation by adding PEG (FIG. 18A, 4, 6 and 6) )
  • PEG PEG
  • Samples of antibody fragments such as Fm301, Fm302 and Fm306 (columns 1 and 3 and 5 in FIG. 2A) and samples with PEG in Fm301 and Fm302 (columns 2 and 4 in FIG. 18A) are light chains of the antibody fragment samples at about 25 kDa positions.
  • the sample reacted with PEG at 13 ⁇ 4306 (column 6 in FIG. 18A) was able to identify the reprinted part and the light chain part of 25 kDa, which were PEGylated at about 65 kDa. This proves that PEGylation is specific for the cysteine of the amino acid added at the end of the C H1 domain.
  • PEG staining of the Fm301 and Fm302 sample (columns 2 and 4 of FIG. 18B) also confirmed the presence of PEG molecules by PEG staining, but did not appear in the same position as the antibody fragment and the PEG sample (column 7 of FIG. 18B).
  • the Fm306-PEG mixture was loaded on a KappaSelect resin layered column to remove residual PEG that did not bind to the antibody fragment. Subsequently, the complete layer solution (50 mM Tr is-HCl, pH 8.0, 100 mM NaCl and 5 mM EDTA) was poured in 20 CV or more to sufficiently wash the remaining PEG to remove residual PEG, and then the buffer solution (100 mM Glycine, pH 2.5, 1 mM EDTA). ) was eluted.
  • the antibody fragment and PEG were bound only to the Fm306 construct, and the antibody fragment and PEG were not bound to the Fm301 and Fm302. Therefore, it was confirmed that site-specific pegylation occurred only in the cysteine of the amino acid sequence (THTCM) introduced at 11 ⁇ 2306, and the intrachain disulfide bonds of ⁇ 1 ⁇ 2301 and the intrachain disulfide bonds and interchain disulfide bonds of Fm302, 11 ⁇ 2306 were identified. It can be seen that PEGylation did not occur in the cysteine formed. Also
  • EDMAN sequencing was requested from eMass analys is Lab to confirm purified antibody fragments and to remove N-terminal signal peptides.
  • the N answer sequence of the antibody fragment was identified by the light chain, DILLT and heavy chain QVQLK.
  • KD values were obtained by setting the standard in PBST and then making 5 series of concentrations diluted by 1/2 from EGFR-Fc 5-50 nM and sending it to the coated. After confirming the zero base using a regeneration buffer (Regeneration buffer; 50 mM NaOH), the repeated results were obtained under the optimal conditions.
  • Regeneration buffer 50 mM NaOH
  • the kD value of 11 ⁇ 2302 was determined to be somewhat high.
  • the binding affinity for EGFR seems to be considerably higher, as measured by having a lower kD value than cetuximab-Fab, and in the case of Fm306PEG, it has a lower value than the kD value of sefeximab-Fab. It means that the decrease in binding affinity by PEGylation is not large.
  • Absorbance of Fm302 was determined to be about 47.2%, indicating that the activity of antibody fragments to EGFR was well maintained, and that the binding affinity of Fm306PEG was compared to Fm302. It was reduced by about 32.2%, which means that the decrease in binding affinity due to PEGylation was not significant, so that the activity of about 703 ⁇ 4> could be maintained (FIG. 21).
  • A431 cells in culture were treated with 0.25% trypsin / EDTA, separated into single cells, inoculated with 1 x 10 6 cells in a culture plate, stabilized for 24 hours, and cultured for 8 hours in a serum-free medium. Antibodies and antibody fragments were treated. Replace the cells with culture medium and replace the cells with fresh medium. Then, the cells are recovered from the culture dish and Pathscan total EGF from cel l signaling company. Receptor sandwich EL ISA ki t based on the method proposed.
  • Head and neck cancer diseases to produce an animal model wihi thymus is the administration of 1 X 10 7 of tumor cells by T cells has not been generated cultured A431 cells in nude mice with immune deficiency associated with immune function in lacking a subcutaneous human tumor two kinds of Transplant Disease Animals were produced.
  • Antibody fragments and drug administration were started when tumor tissues formed and the size was about 50-100 rarf, and the drug was administered twice a week by intravenous injection in the tail of 0.25 mg per mouse. Total administration was administered 6 times for 3 weeks. Tumor size change was performed twice a week, prior to drug administration.
  • Tumor tissue of the disease animal model was found to be less tumor growth inhibitory than Sefeximab at 3 weeks when 0.25 treated twice a week, but 11 ⁇ 2302 showed higher inhibition of tumor growth than Sefeximab Fab.
  • I1 ⁇ 2306- PEG also showed a higher inhibitory effect on tumor growth with increased half-life than cetuximab Fab (FIG. 23).
  • Purified antibody fragments were injected intravenously into the tail by 0.25 mg per mouse in experimental animals, and blood was collected by orbital blood collection over time. The collected blood was centrifuged at 3000 rpm for 10 minutes to separate plasma and the amount of antibody fragments present in the separated plasma was measured.

Abstract

The present invention relates to: a fragment antigen-binding (Fab) fragment specifically binding to an epidermal growth factor receptor (EGFR); an expression construct for preparing the Fab fragment; a method for preparing the Fab fragment; and a pharmaceutical composition containing the Fab fragment. Since the Fab fragment specifically binding to an EGFR is small compared to an antibody, the Fab fragment has a good infiltration rate for tissues or tumors and can be prepared in bacteria, thereby lowering production costs. In addition, the Fab fragment specifically binding to an EGFR provides an increased in vivo half-life through PEGylation.

Description

【명세서】  【Specification】
【발명의 명칭】  [Name of invention]
EGFR에 특이적으로 결합하는 Fab 단편 【기술 분야】  Fab fragments that specifically bind to EGFR [Technical Field]
본 발명은 대한민국 산업통상자원부의 지원 하에서 과제번호 A004600265에 의해 이루어진 것으로서, 상기 과제의 연구관리전문기관은 (재)층청지역사업평가원 , 연구사업명은 The present invention was made by the task number A004600265 under the support of the Ministry of Trade, Industry and Energy of Korea, the research management professional agency of the project is (Foundation)
"층청광역경제권선도산업육성사업" , 연구과제명은 "표적항암치료제 Anti-EGFR 바이오베터 개발" , 주관기관은 신일제약^, 연구기간은 2012. 08. 01 ~ 2015. 04. 30 이다. "Layered Blue-Earth Economic Leading Industry Promotion Project", and the research project titled "Development of Anti-EGFR Biobetter for Target Anticancer Therapeutics", Shinil Pharm ^, and the period of research are 2012. 08. 01 ~ 2015. 04. 30.
본 특허출원은 2015년 04월 23일에 대한민국 특허청에 제출된 대한민국 특허출원 제 10-2015-0057262 호 및 2015년 09월 08일에 대한민국 특허청에 제출된 특허출원 제 10-2015-0126894호에 대하여 우선권을 주장하며, 상기 특허출원의 개시 사항은 본 명세서에 참조로서 삽입된다. 본 발명은 EGFR(Epi dermal Growth Factor Receptor)에 특이적으로 결합하는 Fab(Fragment Antigen Binding) 단편에 관한 것이다.  This patent application is filed with the Korean Patent Application No. 10-2015-0057262 filed with the Korean Patent Office on April 23, 2015 and the Korean Patent Application No. 10-2015-0126894 filed with the Korean Patent Office on September 08, 2015. Claiming priority, the disclosure of this patent application is incorporated herein by reference. The present invention relates to a fragment (Fragment Antigen Binding) fragment that specifically binds to Epi dermal Growth Factor Receptor (EGFR).
【배경 기술】 [Background technology]
EGFRCEpidermal Growth Factor Receptor; HERl)은 세포표면에 존재하는 수용체 HER 패밀리의 하나로 EGF, TGF-alpha, 에피레귤린 (Epiregulin) 등의 리간드 들과 결합하여 세포의 성장과 사멸에 있어 중요한 역할을 담당하고 있다. 암과 관련해서 특히 주목을 받게 된 것은 면역염색을 통해 조사한 결과 많은 종류의 암세포에서 EGFR 발현이 증가되어 있으며 이런 EGFR의 증가가 예후와도 밀접한 관련성이 있다는 보고들이 있다 (Nicholson, R. I. et al ., Eur. J. Cancer - , 37:S9- 15(2001); Yewale, C. et al., Biomaterials, 34:8690—707(2013)). 이에 따라 EGFR 신호를 억제하여 암을 치료하려는 시도가 있어 왔는데 EGFR 차단 항체인 세특시맙 (cetuximab) 및 저분자의 EGFR 티로신 키나제 억제제 (EGFR tyrosine kinase inhibitor; EGFR-TKl)인 제피티닙 (gef i t inib; iressa®), 에를로티닙 (erlotinib; tarceva®) 등이 등장하여 임상에 사용되게 되었다. EGFRCEpidermal Growth Factor Receptor; HERl) is one of the receptor HER families on the surface of the cell and plays an important role in cell growth and death by binding to ligands such as EGF, TGF-alpha, and Epiregulin. Of particular interest in cancer, immunostaining has shown increased expression of EGFR in many types of cancer cells, and reports that this increase in EGFR is closely associated with prognosis (Nicholson, RI et al. Eur. J. Cancer-, 37: S9-15 (2001); Yewale, C. et al., Biomaterials, 34: 8690—707 (2013)). Accordingly, attempts have been made to treat cancer by inhibiting EGFR signaling, including cetuximab, an EGFR blocking antibody, and gefitinib, an EGFR tyrosine kinase inhibitor (EGFR-TKl) iressa ® ), erlotinib (erlotinib; tarceva ® ), etc., have emerged for clinical use.
정정용지 (규칙 제 91조) ISA/KR EGFR-TK1은 같은 EGFR을 표적 하지만 세툭시맙과 달리 폐암을 그 적응증으로 하고 있는데 EGFR-TK1는 EGFR 발현이 예후와 렇접하게 관련이 있는 것으로 알려진 암들에서는 효과가 별로 없고 관련성이 아주 미약한 폐암에서 효과를 나타내어 EGFR 발현량에 따른 신호 차이와는 다른 기전이 존재함을 시사하고 있으며 치료용 항체가 암세포에 세포독성 작용을 나타내기 위해서는 몇 가지 작용기전이 복합적으로 이용되는데 대부분은 ADCCCAnt i body-Dependent Cel lular Cytotoxic i ty) 또는 CDC (Complementᅳ Dependent Cytotox ic i ty)를 통한면역 체계를 통해 효과를 나타낸다. Correction Sheet (Rule 91) ISA / KR EGFR-TK1 targets the same EGFR but unlike cetuximab, lung cancer is an indication. EGFR-TK1 is less effective and less relevant in cancers where EGFR expression is known to be closely associated with prognosis. This suggests that there is a mechanism different from the signal difference according to the amount of EGFR expression, and several mechanisms of action are used in order for the therapeutic antibody to show cytotoxic effects on cancer cells, most of which are ADCCCAnt i body-dependent. It is effective through the immune system through Cel lular Cytotoxic I ty or CDC (Complement Dependent Cytotox ic I ty).
항체는 구조적으로 중쇄 (heavy chain)의 불변 영역 (Fc 부분)의 차이에 따라 IgG, IgA, IgM, IgD 및 IgE 5가지로 분류되며, 항체의 아이소타입 ( i sotype) 증 IgGl과 IgG3는 ADCC , CDC의 기. ^이 강하고, IgG2는 ADCC의 기능이 없고, 약한 CDC의 기능이 있으며, IgG4는 약한 ADCC의 기능이 있으나 CDC의 기능은 없다고 알려져 있다ᄋ 항암분야에서 개발되어진 항체의 경우 ADCC 및 CDC 기능이 높다고 알려진 IgGl 아이소타입이 가장 많이 개발되어져 왔다.  Antibodies are structurally classified into five types, IgG, IgA, IgM, IgD, and IgE, according to differences in the constant region (Fc portion) of the heavy chain. Flag of the CDC. ^ Strong, IgG2 has no function of ADCC, weak CDC function, IgG4 has weak ADCC function but no CDC function. Antibody developed in anticancer field is known to have high ADCC and CDC function. IgGl isotypes have been the most developed.
그러나 다른 항암 표적과 달리 EGFR의 경우 IgGl 타입 뿐 아니라 IgG2 타입으로 항체 치료제가 개발되어 시판되고 있으며 이는 EGFR을 표적하는 하는 경우 항체로 인한 중화 활성 (neutral i z ing act ivi ty)이 주된 작용이라는 것을 반증한다고 블 수 있다.  However, unlike other anti-cancer targets, EGFR has been developed and marketed as an antibody therapeutic agent in IgG2 type as well as IgGl type. This indicates that the neutralizing activity caused by antibody is the main action when targeting EGFR. It can be said.
EGFR은 테더 단량체 ( tethered monomer ) 형태나 개방형 구조인 언테더 단량체 (untethered monomer ) 형태로 존재하다가 EGF가 결합하게 되면 이합체 (dimer) 형태를 이뤄 키나제 (kinase)가 활성화되어 신호를 전달하게 된다. 치료용 항체 세특시맙의 경우 리간드 대신에 EGFR에 결합함으로써 키나제 활성과 하위 신호를 억제하게 된다. 이로 인해 세포성장을 억제하고, 세포 사멸을 유도하게 되는데 이러한 결합으로 수용체의 활성화 및 그로 인한 신호전달 통로가 억제되며, 그 결과 종양세포의 정상조직 침투 및 새로운 부위로의 종양확산이 감소하게.된다. 또한 종양세포가 화학요법 및 방사선 요법으로 인한 손상을 복구하는 능력을 억제하고, 종양 내 새로운 혈관 형성을 억제함으로써 종양증식을 전반적으로 억제하는 것으로 판단된다,  EGFR exists in the form of a tethered monomer or an open structure of an untethered monomer, and when EGF binds, it forms a dimer to activate a kinase and transmit a signal. Therapeutic antibody Sepusimab inhibits kinase activity and downstream signaling by binding to EGFR instead of ligand. This inhibits cell growth and induces cell death. This binding inhibits the activation of receptors and their signaling pathways, resulting in a reduction of tumor invasion into normal tissues and tumor diffusion into new sites. . In addition, tumor cells are thought to inhibit tumor growth as a whole by inhibiting the ability to repair damage caused by chemotherapy and radiation therapy, and by inhibiting the formation of new blood vessels in tumors.
또한 EGFR을 타겟으로 하는 항체의 치료 기작이 EGFR의 신호전달을 억제함으로써 나타난다는 것의 증거로 세툭시맙 및 파니투무맙 (panitumumab)의 경우 EGFR의 하위 신호 전달 물질인 KKAS의 유전자 돌연변이에 의해 영향을 받는다는 보고들이 있다 (Li6vre, A. et al.( Cancer Res. , 66:3992-5(2006); Karapetis, C. S. et al. , N. Engl . J. Med., 359:1757-65(2008)). 이러한 KRAS 돌연변이는 대장암의 40-45%에서 발견되는데 암 환자의 생체지표 (바이오마커) 검사를 통해 'KRAS'라는 유전자돌연변이 여부를 확인해서 돌연변이가 없는 경우에 사용하고 있다. In addition, the mechanism of treatment of antibodies targeting EGFR is responsible for signaling of EGFR. Evidence that it appears by inhibition has been reported that cetuximab and panitumumab are affected by genetic mutations in KKAS, a subsignal of EGFR (Li6vre, A. et al. ( Cancer Res. , 66: 3992-5 (2006); Karapetis, CS et al., N. Engl. J. Med., 359: 1757-65 (2008)) These KRAS mutations are found in 40-45% of colorectal cancers. Biomarkers of cancer patients are checked for genetic mutations called 'KRAS' and used in the absence of mutations.
Erbitux®는 화학요법제와 같이 사용되어 두경부암 (Head and Neck cancer), 직장결장암 (colorectal cancer) 치료용 EGFR 키메라 항체 (chimeric antibody)로서, ImClQne systems사에 ; 의해 개발되었고, ImClone systems사 및 Bristol-Myers Squibb사가 북미권에서, 그리고 Merck KGaA (독일)사가 북미 외의 지역에서 Erbitux® 대한 권리를 가지고 있으며, 2004년 2월 미국 FDAChe U.S. Food, and Drug Adm.inistrat ion)에 의해 승인되었다. 이에 세툭시맙에 대한 특허는 2015년 만료되었으며, 국내에서는 이에 대한 바이오시밀러 개발이 한창 진행 중에 있으며, 해외에서는 EGFR에 대한 다양한 치료용 항체가 개발되어 승인되었거나, 현재 임상 진행 증으로 EGFR을 표적으로 하는 항암치료제 개발이 활발하게 이뤄지고 있다. 본 명세서 전체에 걸쳐 다수의 논문 및 특허문헌이 참조되고 그 인용이 표시되어 있다. 인용된 논문 및 특허문헌의 개시 내용은 그 전체로서 본 명세서에 참조로 삽입되어 본 발명이 속하는 기술 분야의 수준 및 본 발명의 내용이 보다 명확하게 설명된다. 【발명의 내용】 Erbitux® is used as a chemotherapeutic agent and is an EGFR chimeric antibody for the treatment of head and neck cancer and colorectal cancer . Was developed by ImClone systems and Bristol-Myers Squibb in North America, and Merck KGaA (Germany) has rights to Erbitux® outside of North America , and in February 2004 the US FDAChe US Food , and Drug Adm.inistrat ion Has been approved. The patent for cetuximab expired in 2015, and the development of biosimilars is in full swing in Korea, and various therapeutic antibodies against EGFR have been developed and approved overseas. The development of anti-cancer therapies is being actively made. Throughout this specification, many papers and patent documents are referenced and their citations are indicated. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained. [Content of invention]
【해결하려는 과제】  [Problem to solve]
본 발명자들은 암세포의 EGFR(Epi dermal Growth Factor Receptor) 신호를 억제하는 항암용 항체를 대체할 수 있는 Fab 단편을 제조하고자 노력하였다. 그 결과, 대장균에서 발현할 수 있고 EGFR에 특이적으로 결합하는 Fab 단편을 개발하고 이의 우수한 결합 친화도 및 항암 효과를 확민함으로써 본 발명을 완성하였다. 따라서 , 본 발명의 목적은 EGFR에 특이적으로 결합하는 Fab 단편을 제공하는 데 있다. The present inventors have tried to prepare a Fab fragment that can replace an anti-cancer antibody that inhibits Epidermal Growth Factor Receptor (EGFR) signal of cancer cells. As a result, the present invention was completed by developing a Fab fragment that can be expressed in E. coli and specifically binds to EGFR, and affirming its excellent binding affinity and anticancer effect. Accordingly, it is an object of the present invention to provide Fab fragments that specifically bind to EGFR.
본 발명의 다른 목적은 상기 Fab 단편을 제조하기 위한 발현 컨스트럭트를 제공하는 데 있다.  Another object of the present invention is to provide an expression construct for preparing the Fab fragment.
본 발명의 또다른 목적은 상기 발현 컨스트릭트를 포함하는 재조합 백터를 제공하는 데 있다.  Still another object of the present invention is to provide a recombinant vector comprising the expression construct.
본 발명의 다른 목적은 상기 재조합 백터로 형질전환된 숙주 세포를 제공하는 데 있다.  Another object of the present invention is to provide a host cell transformed with the recombinant vector.
본 발명의 또다른 목적은 EGFR에 대한 Fab 단편의 제조방법을 제공하는 데 있다.  It is another object of the present invention to provide a method for preparing Fab fragments for EGFR.
본 발명의 다른 목적은 암에 대한 예방 또는 치료용 약제학적 조성물을 제공하는 데 있다. 본 발명의 다른 목적 및 이점은 하기의 발명의 상세한 설명, 청구범위 및 도면에 의해 보다 명확하게 된다.  Another object of the present invention to provide a pharmaceutical composition for preventing or treating cancer. Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.
【과제의 해결 수단】 [Measures of problem]
본 발명의 일 양태에 따르면, 본 발명은 EGFR(Epidermal Growth Factor Receptor )에 특이적으로 결합하며 다음의 영역을 포함하는 Fab (Fragment ant igen-binding) 단편을 제공한다:  According to one aspect of the invention, the invention provides a fragment ant igen-binding (fab) fragment that specifically binds to an Epidermal Growth Factor Receptor (EGFR) and comprises the following regions:
(a) 서열목록 제 4서열의 아미노산 서열을 포함하는 중쇄 가변 영역 (VH) ; (a) a heavy chain variable region (V H ) comprising the amino acid sequence of SEQ ID NO: 4;
(b) 서열목록 제 5열의 아미노산 서열을 포함하는 중쇄 불변 영역 (c) 사열목록 제 6서열의 아미노산 서열을 포함하는 경쇄 가변 영역 (VL) ; 및 (b) a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 5 (c) a light chain variable region (V L ) comprising the amino acid sequence of SEQ ID NO: 6; And
(d) 서열목록 제 7열의 아미노산 서열을 포함하는 경쇄 불변 영역 (CL) . 본 발명자들은 암세포의 EGFR신호를 억제하는 항암용 항체를 대체할 수 있는 Fab 단편을 제조하고자 노력하였다. 그 결과, 대장균에서 발현할 수 있고 EGFR에 특이적으로 결합하는 Fab 단편을 개발하고 이의 우수한 결합 친화도 및 항암 효과를 확인하였다. 본 발명의 상기 Fab 단편은 EGFR에 특이적으로 결합한다. (d) Light chain constant region (C L ) comprising the amino acid sequence of SEQ ID NO: 7. The present inventors have tried to prepare a Fab fragment that can replace the anti-cancer antibody that inhibits the EGFR signal of cancer cells. As a result, Fab fragments that can be expressed in E. coli and specifically bind to EGFR are developed. Binding affinity and anticancer effect were confirmed. The Fab fragment of the present invention specifically binds to EGFR.
본 명세서에서 용어 "Fab 단편" 은 항원 결합 기능을 보유하고 있는 단편을 의미하며, 중쇄 가변 영역 (VH) , 중쇄 불변 영역 1(CH1) , 경쇄 가변 영역 (VL) 및 경쇄 블변 영역 (CL)을 가지는 구조로 1개의 항원 결합 부위를 가진다. Fab '는 중쇄 CH1 도메인의 C-말단에 하나 이살의 시스테인 잔기를 포함하는 힌지 영역 (hinge region)을 가진다는 점에서 Fab와 차이가 있다. F(ab' )2 항체는 Fab'의 힌지 영역의 시스테인 잔기가 디 파이드 결합을 이루면서 생성된다. 이러한 Fab 단편은 단백질 가수 분해 i소를 이용해서 얻을 수 있고 (예를 들어, 전체 항체를 파파인으로 제한 절단하면 Fab를 얻을 수 있고 펩신으로 절단하면 F(ab ' )2 단편을 얻을 수 있다) , 바람직하게는 유전자 재조합 기술을 통하여 제작할수 있다. As used herein, the term "Fab fragment" refers to a fragment having an antigen binding function, heavy chain variable region (V H ), heavy chain constant region 1 (C H1 ), light chain variable region (V L ) and light chain variable region ( C L ) with one antigen binding site. Fab 'differs from Fab in that it has a hinge region at the C-terminus of the heavy chain C H1 domain that contains one or two year-old cysteine residues. F (ab ') 2 antibodies are produced by forming a bond with cysteine residues in the hinge region of Fab'. Such Fab fragments can be obtained using proteolytic elements (e.g., restriction digestion of the entire antibody with papain yields Fab and cleavage with pepsin yields F (ab ') 2 fragments). Preferably it can be produced through genetic recombination technology.
본 발명은 항체를 질환의 예 또는 치료에 적용하는데 단점으로 여겨지는 생산 비용을 개선하고자, 홀항체 (whole ant ibody)가 아닌 Fab 형태의 단편을 대장균에서 발현하여 제조한다.  In order to improve the production cost, which is considered to be a disadvantage in applying the antibody to the example or treatment of a disease, the present invention is produced by expressing a fragment in E. coli, not Fab whole ant ibody.
본 명세서에서, 용어 "증쇄"는 항원에 특이성을 부여하기 위한 층분한 가변 영역 서열을 갖는 아미노산 서열을 포함하는 가변 영역 도메 ¾ VH 및 3개의 불변 영역 도메인 CH1 , CH2 및 CH3를 포함하는 전체길이 중쇄 및 이의 단편을 모두 의미한다. As used herein, the term “remplification” encompasses the variable region domain ¾ VH and three constant region domains C H1 , C H2 and C H3 comprising an amino acid sequence having a stratified variable region sequence for conferring specificity to the antigen. It means both full length heavy chain and fragments thereof.
본 발명의 Fab 단편은 상기 VH 및 CH1으로 구성된 중쇄를 포함하는 Fab단편이다. Fab fragment of the present invention is a Fab fragment comprising a heavy chain consisting of the above V H and C H1 .
또한 본 명세서에서 용어 "경쇄''는 항원에 특이성을 부여하기 위한 층분한 가변영역 서열을 갖는 아미노산 서열을 포함하는 가변 영역 도메인 VL 및 불변 영역 도메인 CL을 포함하는 전체길이 경쇄 및 이의 단편을 모두 의미한다. The term “light chain” herein also refers to the full-length light chain and fragments thereof comprising the variable region domain V L and the constant region domain C L comprising an amino acid sequence having a segmented variable region sequence for imparting specificity to the antigen. All means.
본 발명의 Fab 단편은 상기 VL 및 CL으로 구성된 경쇄를 포함하는 Fab 단편이다. Fab fragments of the invention are Fab fragments comprising a light chain consisting of the above V L and CL.
본 발명의 Fab 단편은 EGFR에 특이적으로 결합할 수 있는 범위 내에서 첨부한 서열목록에 기재된 아미노산 서열의 변이체를 포함할 수 있다. 예를 들면, Fab 단편의 결합 친화도 및 /또는 기타 생물학적 특성을 개선시키기 위하여 Fab 단편의 아미노산 서열에 변화를 줄수 있다ᅳ 이러한 변형은, 예를 들어 Fab 단편의 아미노산 서열 잔기의 결실, 삽입 및 /또는 치환을 포함한다. 이러한 아미노산 변이는 아미노산 결사슬 치환체의 상대적 유사성, 예컨대, 소수성, 친수성, 전하, 크기 등에 기초하여 이루어진다, 아미노산 결사슬 치환체의, 크기, 모양 및 종류에 대한 분석에 의하여, 아르기닌, 라이신과 히스티딘은 모두 양전하를 띤 잔기이고; 알라닌, 글라이신과 세린은 유사한 크기를 갖으며; 페닐알라닌, 트립토판과 타이로신은 유사한 모양을 갖는다는 것을 알 수 있다. 따라서, 이러한 고려 사항에 기초하여, 아르기닌, 라이신과 히스티딘; 알라닌, 글라이신과 세린; 그리고 페닐알라닌, 트립토판과 타이로신은 생물학적으로 /|능 균등물이라 할 수 있다. 변이를 도입하는 데 있어서, 아미노산의 소수성 인덱스Fab fragments of the present invention may include variants of amino acid sequences set forth in the appended sequence listing within the scope of specific binding to EGFR. For example, the binding affinity and / or other biological properties of the Fab fragment Changes can be made to the amino acid sequence of the Fab fragments for improvement. Such modifications include, for example, deletions, insertions and / or substitutions of amino acid sequence residues in the Fab fragments. Such amino acid variations are made based on the relative similarity of amino acid chain substituents, such as hydrophobicity, hydrophilicity, charge, size, etc. By analyzing the size, shape and type of amino acid chain substituents, arginine, lysine and histidine are all A positively charged residue; Alanine, glycine and serine have similar sizes; It can be seen that phenylalanine, tryptophan and tyrosine have a similar shape. Thus, based on these considerations, arginine, lysine and histidine; Alanine, glycine and serine; Phenylalanine, tryptophan and tyrosine are biologically the / | functional equivalents. Hydrophobicity index of amino acids in introducing mutations
(hydropathy index)가 고려될 수 있다. 각각의 아미노산은 소수성과 전하에 따라 소수성 인덱스가 부여되어 있다: 아이소루이신 (+4.5); 발린 (+4.2); 루이신 (+3.8); 페닐알라닌 (+2.8); 시스테인 /시스타인 (+2.5); 메티오닌 (+1.9); 알라닌 (+1.8); 글라이신 (-0.4); 쓰레오닌 (-0.7); 세린 (-0.8); 트립토판 (-0.9); 타이로신 (-1.3); 프를린 (-1.6); 히^티딘 (-3,2); 글루타메이트 (-3,5); 글루타민 (-3.5); 아스파르테이트 (-3.5); 아스파라긴 (-3.5); 라이신 (-3.9); 및 아르기닌 (-4.5)。 단백질의 상호적인 생물학적 기능 (interactive biological function)을 부여하 데 있어서 소수성 아미노산 인덱스는 매우 중요하다. 유사한 소수성 인텍스를 가지는 아미노산으로 치환하여야 유사한 생물학적 활성을 보유할 수 있다는 것은 공지된 사실이다. 소수성 인텍스를 참조하여 변이를 도입시키는 경우, 바람직하게는 ±2 이내, 보다 바람직하게는 士1 이내, 보다 더 바람직하게는 ±0.5 이내의 소수성 인텍스 차이를 나타내는 아미노산 사이에 치환을 한다. (hydropathy index) can be considered. Each amino acid is assigned a hydrophobicity index depending on its hydrophobicity and charge: isoleucine (+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); Plin (-1.6); Hintidine (-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 of great importance in conferring the interactive biological function of proteins. It is well known that substitution with amino acids having similar hydrophobic indexes can retain similar biological activity. When introducing a mutation with reference to a hydrophobic index, substitutions are made between amino acids that exhibit a hydrophobic index difference of preferably within ± 2, more preferably within 1, and even more preferably within ± 0.5.
한편, 유사한 친수성 값 (hydrophilicity value)을 가지는 아미노산 사이의 치환이 균등한 생물학적 활성을 갖는 단백질을 초래한다는 것도 잘 알려져 있다. 미국 특허 제 4,554,1이호에 개시된 바와 같이, 다음의 친수성 값이 각각의 아미노산 잔기에 부여되어 있다: 아르기닌 (+3.0); 라이신 (+3.0); 아스팔테이트 (+3.0土 1); 글루타메이트 (+3.0 1); 세린 (+0.3); 아스파라긴 (+0.2); 글루타민 (+0.2); 글라이신 (0); 쓰레오닌 (-0.4); 프를린 (-0.5 士 1); 알라닌 (-0.5); 히스티딘 (-0.5); 시스테인 (-1.0); 메티오닌 (-1.3); 발린 (-1.5); 루이신 (-1.8); 아이소루이신 (-1.8); 타이로신 (-2.3); 페닐알라닌 (-2.5); 트립토판 (-3.4). 친수성 값을 참조하여 변이를 도입시키는 경우, 바람직하게는 ±2 이내, 보다 바람직하게는 土 1 이내, 보다 더 바람직하게는 ±0.5 이내의 친수성 값 차이를 나타내는 아미노산 사이에 치환을 한다. 분자의 활성을 전체적으로 변경시키지 않는 단백질에서의 아미노산 교환은 당해 분야에 공지되어 있다 (H. Neurath, R.L.Hi 11 , The Proteins, Academic Press, New York, 1979). 가장 통상적으로 일어나는 교환은 아미노산 잔기 Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/I le, Leu/Val, Ala/Glu, Asp/Gly 간의 교환이다. 상술한 생물학적 균등 활성을 갖는 변이를 ^려한다면, 본 발명의 항체 또는 이를 코딩하는 핵합 분자는 서열목록에 기재된 서열과 실질적인 동일성 (substantial identity)을 나타내는 서열도 포함하는 것으로 해석된다. 상기의 실질적인 동일성은, 상기한 본 발명의 서열과 임의의 다른 서열을 최대한 대웅되도록 얼라인하고, 업계에서 통상적으로 이용되는 알고리즘을 이용하여 얼라인된 서열을 분석한 경우에ᅳ 최소 61%의 상동성, 보다 바람직하게는 7(ᅵ%의 상동성, 보다 더 바람직하게는 80%의 상동성, 가장 바람직하게는 90%의 상동성을 나타내는 서열을 의미한다. 서열비교를 위한 얼라인먼트 방법은 당업계에 공지되어 있다. 얼라인먼트에 대한 다양한 방법 및 알고리즘은 Smith and Waterman, Adv. Ap l. 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, CABI0S 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)에 개시되어 았다. NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al . , J. Mol . Biol . 215:403-10(1990))은 NBCI (National Center for Biological Information) 등에서 접근 가능하며, 인터넷 상에서 blastp, blasm, blastx, tblastn and tblastx와 같은 서열 분석 프로그램과 연동되어 이용할 수 있다. BLSAT는 http://www.ncbi.nlm. nih.gov/BLAST/에서 접속 가능하다. 이 프로그램을 이용한 서열 상동성 비교 방법은 http://丽. ncbi.nlm.nih.gov/BLAST/blast_help. html에서 확인할수 있다. 본 발명의 EGFR에 특이적으로 결합하는 Fab 단편은 중쇄 및 경쇄의 헤테로다이머 (heterodimer)를 형성하기 위해 CH1 및 CL에 이황화 결합의 형성을 위한 아미노산 서열을추가적으로 포함한다. On the other hand, it is also well known that substitutions between amino acids having similar hydrophilicity values result in proteins with equivalent biological activity. As disclosed in US Pat. No. 4,554,1, the following hydrophilicity values are assigned to each amino acid residue: arginine (+3.0); Lysine (+3.0); asphaltate (+ 3.0 × 1); Glutamate (+3.0 1); serine (+0.3); Asparagine (+0.2); Glutamine (+0.2); Glycine (0); Threonine (-0.4); Plin (-0.5 士 1); Alanine (-0.5); Histidine (-0.5); Cysteine (-1.0); Methionine (-1.3); Valine (-1.5); leucine (-1.8); Isoleucine (-1.8); Tyrosine (-2.3); Phenylalanine (-2.5); Tryptophan (-3.4). When introducing a mutation with reference to a hydrophilicity value, substitutions are made between amino acids which exhibit a hydrophilicity value difference of preferably within ± 2, more preferably within 1 and even more preferably within ± 0.5. Amino acid exchange in proteins that do not alter the activity of the molecule as a whole is known in the art (H. Neurath, RLHi 11, The Proteins, Academic Press, New York, 1979). The most commonly occurring exchanges are amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Exchange between Pro, Lys / Arg, Asp / Asn, Leu / I le, Leu / Val, Ala / Glu, Asp / Gly. In order to receive the above-described biologically equivalent variation, the antibody or nucleotide molecule encoding the same of the present invention is interpreted to include a sequence that exhibits a substantial identity with the sequence described in the sequence listing. This substantial identity is at least 61% when the sequence of the present invention is aligned with any other sequence as best as possible and the aligned sequence is analyzed using algorithms commonly used in the industry. Homologous, more preferably 7% homology, even more preferably 80% homology, most preferably 90% homology. Alignment methods for sequence comparison are known in the art. Various methods and algorithms for alignment are found in Smith and Waterman, Adv. Ap l. 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, CABI0S 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)). Biological Information) and can be used in conjunction with sequencing programs such as blastp, blasm, blastx, tblastn and tblastx on the Internet. http: //www.ncbi.nlm. Accessible at nih.gov/BLAST/. Sequence homology comparison method using this program is http: // 丽. ncbi.nlm.nih.gov/BLAST/blast_help. You can check it in html. Fab fragments that specifically bind to EGFR of the present invention further comprise amino acid sequences for formation of disulfide bonds at C H1 and C L to form heterodimers of heavy and light chains.
본 발명의 일 구현예에 따르면, 상기 ( 은 그의 C-말단에 Cys-Asp- Lys을추가적으로 포함한다 (서열목록 제 17서열).  According to an embodiment of the present invention, ((further includes Cys-Asp- Lys at its C-terminus (SEQ ID NO: 17)).
본 발명의 다른 구현예에 따르면, 상기 CL은 그의 C-말단에 Glu- Cys을추가적으로 포함한다 (서열목록 제 18서열). According to another embodiment of the invention, the C L further comprises Glu-Cys at its C-terminus (SEQ ID NO: 18).
본 발명의 특정 구현예에 따르면, 상기 ( 은 그의 C-말단에 Glu- Cys을 추가적으로 포함하며, 상기 CH1의 C-말단에 있는 Cys-Asp-Lys의 Cys 잔기와 상기 VL의 C-말단에 있는 Glu-Cys의 Cys 잔기가 이황화 결합된다. 본 발명의 주요한 특징 중 하나는 EGFR에 대한 Fab 단편의 페길화이다. According to a specific embodiment of the present invention, wherein (further includes Glu- Cys at its C-terminus, Cys residue of Cys-Asp-Lys at the C-terminus of C H1 and the C-terminus of V L Cys residues of Glu-Cys at are disulfide linked One of the main features of the present invention is the PEGylation of Fab fragments to EGFR.
본 명세서에서 용어 "페길화 (PEGylation)" 는 목적의 단백질, 즉 상기 EGFR에 대한 Fab 단편에 폴리에틸렌글리콜 (Polyethlylene glycol; PEG)을 컨쥬게이션시키는 것을 의미한다.  As used herein, the term "PEGylation" refers to the conjugation of Polyethylenelylene glycol (PEG) to the protein of interest, ie the Fab fragment for the EGFR.
본 발명의 Fab 단편의 생체 내 약효 지속성과 안 성이 홀항체에 비하여 떨어지는 문제를 개선하기 위해 생체 내에서 면역반응을 일으키지 않는 생체적합성이 뛰어난 고분자인 폴리에틸렌글리콜을 접합시킨다. 이는 약물의 활성에 대한 영향을 최소화하고 페길화 효과를 극대화시킬 수 있는 부위에 접합시켜 Fab 단편의 효능 저하를 최소화한다. 페길화 된 Fab 단편은 분자량이 증가되었으므로 신사구체 여과로 인한 신장에서의 거름효과에 대한 단백질의 투과를 억제할 수 있어 손실이 감소하고 폴리에틸렌글리콜의 스틸스 작용 (stealth effect)을 통해 생체 내 단백질 효소들의 분해 작용을 억제하는 결과를 보여 생체 내에서의 반감기를 증가시키고 폴리에틸렌글리콜의 입체장애로 체내 단백질 분해효소의 접근을 막아줌으로써 약물에 대한 안정성 증가효과 및 폴리에틸렌글리콜의 친소성 성질에 의해 수용액 증에서 용해도를 증가시키는 효과를 갖는다.  In order to improve the problem that the pharmacological efficacy and stability of Fab fragments of the present invention are inferior to those of single antibodies, polyethylene glycol, which is a highly biocompatible polymer that does not cause an immune response in vivo, is conjugated. This minimizes the degradation of Fab fragments by conjugation to sites that can minimize the effect on drug activity and maximize the PEGylation effect. Pegylated Fab fragments have increased molecular weights, which can inhibit protein permeation to the manure effect in the kidneys due to renal glomerular filtration, resulting in reduced losses and in vivo protein enzymes through the stealth effect of polyethylene glycol. It shows the result of inhibiting the degradation of these compounds, which increases the half-life in vivo and prevents the access of proteolytic enzymes to the body due to the steric hindrance of polyethylene glycol. Has the effect of increasing solubility.
본 발명의 EGFR에 대한 Fab 단편의 이 페길화 된다. 상기 Fab 단편의 페길화를 위해 아미노산 서열이 추가적으로 결합될 수 있다. This PEGylation of Fab fragments for EGFR of the invention. Fab Amino acid sequences can additionally be combined for PEGylation of the fragments.
본 발명의 일 구현예에 따르면, 상기 Fab 단편은 그의 CH1의 C- 말단에 있는 Cys-Asp— Lys에 Thr-His-Thr— Cys-Ala-Ala이 추가적으로 결합된다 (서열목록 제 23서열) . According to an embodiment of the present invention, the Fab fragment further binds Thr-His-Thr—Cys-Ala-Ala to Cys-Asp—Lys at the C-terminus of its C H1 (SEQ ID NO: 23). .
본 발명의 다른 구현예에 따르면, 상기 Fab 단편은 그의 CH1의 C- 말단부위에 있는 Thr-His-Thr-Cys-Ala-Ala에서 Cys 잔기가 페길화 된다. 본 명세서에서 용어 "폴리에틸렌글리콜 (PEG) "는 수용성 폴리 (에틸렌옥사이드)를 의미한다. 전형적으로, 본 발명에 적합한 PEG는 다음의 구조식으로 표현된다: (0C¾C )n (상기 화학식에서 n은 2 내지 4000의 정수이다) 또한, 본 발명에 적합한 PEG 분자는 CH2CH20(CH2CH20)nCH2C¾" 및 "(0CH2CH2)n0" 를 포함한하. 또한, 본 명세서에서 PEG는 다양한 말단기 및 "말단 캡핑" 그룹母 갖는 구조를 포함한다. 예를 들어, 상기 말단기는 말레이미드 (Maleimide)* 포함한다. 본 발명의 일 구현예에 따르면, 상기 페길화에 ?1용되는 PEG는 분자량이 5-50 kDa이다. According to another embodiment of the present invention, the Fab fragment is PEGylated Cys residue at Thr-His-Thr-Cys-Ala-Ala at the C-terminus of its C H1 . As used herein, the term "polyethylene glycol (PEG)" refers to water soluble poly (ethylene oxide). Typically, PEGs suitable for the present invention are represented by the following structural formula: (0C¾C) n (wherein n is an integer from 2 to 4000) Also suitable PEG molecules for the present invention are CH 2 CH 2 0 (CH 2 CH 2 0) nCH 2 C¾ ”and“ (0CH 2 CH 2 ) n0 ”. In addition, PEG herein includes structures having various terminal groups and“ terminal capping ”groups. The terminal group includes Maleimide * According to one embodiment of the present invention, PEG for? 1 in the PEGylation has a molecular weight of 5-50 kDa.
본 발명의 다른 구현예에 따르면, 상기 PEG는 붑자량이 18-38 kDa이다.  According to another embodiment of the invention, the PEG has a lower molecular weight of 18-38 kDa.
본 발명의 Fab 단편은 PEG 한 분자와 1 : 1 결합한다.  Fab fragments of the invention bind 1: 1 with one molecule of PEG.
본 발명의 Fab 단편은 페길화를 통해 우수한 반감기를 갖는다.  Fab fragments of the invention have good half-life through PEGylation.
본 발명의 일 구현예에 따르면, 상기 Fab 단편은 마우스 (Mus musculus)에서의 반감기가 20-35시간이다. 본 발명의 다른 양태에 따르면, 본 발명은 EGFR에 특이적으로 결합하는 Fab 단편을 제조하기 위한 다음을 포함하는 발현 컨스트럭트를 제공한다:  According to one embodiment of the invention, the Fab fragment has a half-life of 20-35 hours in mice (Mus musculus). According to another aspect of the present invention, the present invention provides an expression construct for preparing a Fab fragment that specifically binds to EGFR:
(a) 다음을 포함하는 중쇄 -발현 컨스트럭트: (a-1) 서열목록 제 9서열의 뉴클레오타이드 서열을 포함하는 중쇄 가변 영역 (VH)-코딩 핵산 분자; 및 -2) 서열목록 제 10서열의 뉴클레오타이드 서열을 포함하는 중쇄 블변 영역 1(CH1) -코딩 핵산분자; 그리고, (a) a heavy chain-expressing construct comprising: (a-1) a heavy chain variable region (V H ) -encoding nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 9; And -2) a heavy chain variant region 1 (C H1 ) -encoding nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 10 sequence; And,
(b) 다음을 포함하는 경쇄 -발현 컨스트럭트: (b-1) 서열목록 제 11서열의 뉴클레오타이드 서열을 포함하는 경쇄 가변 영역 (VL)-코딩 핵산 분자; 및 (b-2) 서열목록 제 12서열의 뉴클레오타이드 서열을 포함하는 경쇄 불변 영역 (CL)-코딩 핵산분자. (b) a light chain-expressing construct comprising: (b-1) a light chain variable region (V L ) -encoding nucleic acid comprising the nucleotide sequence of SEQ ID NO: 11 molecule; And (b-2) a light chain constant region (C L ) -encoding nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 12.
본 발명의 EGFR에 특이적으로 결합하는 Fab 단편을 제조하기 위한 발현 컨스트럭트는 상기 Fab 단편를 제조하기 위한 발현 컨스트력트로, 이 둘 사이에 공통된 내용은 본 명세서의 과도한 복잡성을 피하기 위하여, 그 기재를 생략한다.  Expression constructs for preparing Fab fragments that specifically bind to the EGFR of the present invention are expression constructs for preparing the Fab fragments, the contents of which are common between the two, in order to avoid excessive complexity herein. Omit.
본 발명의 발현 컨스트럭트를 구성하는 증쇄 불변 역 i(cH1)-코딩 핵산분자 및 경쇄 불변 영역 (Cl)-코딩 핵산분자는 상기 cH1 및 CL에 이황화 결합의 형성을 위한 뉴클레오타이드 서열 및 /또는 상기 ( 의 페길화를 위한 뉴클레오타이드 서열을 상기 핵산분자의 C-말단에 추가적으로 포함할 수 있다. The heavy chain constant region i (c H1 ) -coding nucleic acid molecules and light chain constant region ( Cl ) -coding nucleic acid molecules constituting the expression construct of the present invention are nucleotide sequences for the formation of disulfide bonds to c H1 and C L and And / or the nucleotide sequence for PEGylation of the above may be further included at the C-terminus of the nucleic acid molecule.
본 발명의 일 구현예에 따르면, 상기 중쇄 불변 영역 i(cH1) -코딩 핵산 분자는 서열목록 제 10서열, 제 19서열 또는 제 24서열의 뉴클레오타이드 서열이다, According to one embodiment of the invention, the heavy chain constant region i (c H1 ) -coding nucleic acid molecule is a nucleotide sequence of SEQ ID NO: 10, 19 or 24,
본 발명의 다른 구현예에 따르면 , 상기 경쇄 불변 영역 (QJ-코딩 핵산분자는 서열목록 제 12서열 또는 제 20서열의 뉴클레오타이드 서열이다. 본 명세서에서 용어 "핵산 분자" 는 DNA(gDNA 및 cDNA) 그리고 RNA 분자를 포괄적으로 포함하는 의미를 갖으며, 핵산 분자어 j서 기본 구성 단위인 뉴클레오타이드는 자연의 뉴클레오타이드뿐만 아니라:, 당 또는 염기 부위가 변형된 유사체 (analogue)도 포함한다 (Schei t , Nucleot ide Analogs , John Wi ley, New York(1980); Uhlman 및 Peyman , Chemical Reviews , 90:543-584(1990) ) . 본 발명의 Fab 단편의 중쇄 가변 영역 및 경쇄 가변 영역을 코딩하는 핵산 분자 서열은 변형될 수 있다. 상기 변형은 뉴클레오타이드의 추가, 결실 또는 비보존적 치환 또는 보존적 치환을 포함한다.  According to another embodiment of the present invention, the light chain constant region (QJ-encoding nucleic acid molecule is a nucleotide sequence of SEQ ID NO: 12 or 20. The term "nucleic acid molecule" herein refers to DNA (gDNA and cDNA) and Nucleotide, which has the meaning of encompassing RNA molecules in its entirety and which is a basic structural unit in the nucleic acid molecule, includes not only natural nucleotides: but also analogues in which sugar or base sites are modified (Schei t, Nucleotide). Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90: 543-584 (1990)) Nucleic acid molecular sequences encoding the heavy and light chain variable regions of the Fab fragments of the invention are modified. Such modifications include additions, deletions or non-conservative substitutions or conservative substitutions of nucleotides.
본 발명의 항체를 코딩하는 핵산 분자는 상기한 뉴클레오타이드 서열에 하여 실질적인 동일성을 나타내는 뉴클레오타이드 서열도 포함하는 것으로 해석된다. 상기의 실질적인 동일성은, 상기한 본 발명의 뉴클레오타이드 서열과 임의의 다른 서열을 최대한 대웅되도록 얼라인하고, 당업계에서 통상적으로 이용되는 알고리즘을 이용하여 얼라인된 서열을 분석한 경우에, 최소 80%의 상동성, 보다 바람직하게는 최소 90%의 상동성, 가장 바람직하게는 최소 95%의 상동성을 나타내는 뉴클레오타이드 서열을 의미한다。 A nucleic acid molecule encoding an antibody of the present invention is to be construed to include a nucleotide sequence which shows substantial identity to the nucleotide sequence described above. The substantial identity is at least 80% when the nucleotide sequence of the present invention and any other sequence are aligned to the maximum, and the aligned sequence is analyzed using algorithms commonly used in the art. Homology of, more preferably at least 90% Homologous, most preferably nucleotide sequence exhibiting at least 95% homology.
본 발명의 주요한 특징 중 하나는 상기 EGFR에 대한 Fab 단편은 . 대장균을 통해 제조될 수 있는 것이다.  One of the main features of the present invention is that the Fab fragment for EGFR. It can be produced through E. coli.
상기 핵산 분자는 대장균에서 Fab 단편을 발현시키기 위해, 상기 The nucleic acid molecule is expressed in order to express Fab fragments in E. coli,
EGFR에 대한 Fab 단편을 코딩하는 뉴클레오타이드 서열을 대장균의 코돈 발현 빈도를 반영하여 숙주에 유리하게 변환하였다. The nucleotide sequence encoding the Fab fragment for EGFR was converted to the host in favor of the codon expression frequency of E. coli.
본 발명에서 제작되는 발현 컨스트릭트는 숙주 세포에서 원하는 유전자를 발현할 수 있도록 구축된다. 일반적으로, 상기 발현 컨스트럭트의 업스트림 (upstream) 및 다운스트림 (downstream)에 각각 작동적으로 결합된 프로모터 및 터미네이터가위치한다.  The expression construct produced in the present invention is constructed to express a desired gene in a host cell. Generally, there are promoters and terminators operatively coupled upstream and downstream of the expression construct, respectively.
본 명세서에서 용어 "프로모터"는 코딩 서열 또는 기능적 RNA의 발현을 조절하는 DNA 서열을 의미한 4. 본 발명의 재조합 백터에서 목적 뉴클레오타이드서열은 상기 프로모터에 작동적으로 연결된다,  As used herein, the term “promoter” refers to a DNA sequence that modulates the expression of a coding sequence or functional RNA. 4. In the recombinant vector of the present invention, the target nucleotide sequence is operably linked to the promoter,
본 명세서에서 용어 "작동적으로 결합된 (operat ively l inked) "은 핵산 발현 조절 서열 (예: 프로모터 서열, 시그널 서열, 또는 전사조절인자 결합 위치의 어레이)과 다른 핵산 서 사이의 기능적인 결합을 의미하며, 이에 의해 상기 조절 서열은 상기 다른 핵산 서열의 전사 및 /또는 번역을 조절하게 된다. 본 발명의 또다른 양태에 따르면, 본 발명은 상기 발현 컨스트럭트를 포함하는 재조합 백터를 제공한다.  As used herein, the term "operatively l inked" refers to the functional binding between a nucleic acid expression control sequence (e.g., a promoter sequence, a signal sequence, or an array of transcriptional regulator binding sites) and another nucleic acid sequence. By means of which said regulatory sequence modulates the transcription and / or translation of said other nucleic acid sequence. According to another aspect of the present invention, the present invention provides a recombinant vector comprising the expression construct.
본 발명의 백터 시스템은 당업계에 공지된 다양한 방법을 통해 구축될 수 있으며, 이에 대한 구체적인 방법은 Sambrook et al . , Molecular Cloning, A Laboratory Manual , Cold Spr ing Harbor Laboratory Press(2001)에 개시되어 있으며, 이 문헌은 본 명세서에 참조로서 삽입된다. 본 발명의 백터는 전형적으로 클로닝을 위한 백터 또는 발현을 위한 백터로서 구축될 수 있다. 또한, 본 발명의 백터는 원핵 세포를 숙주로 하여 구축될 수 있다. 본 발명의 백터는 전형적으로 클로닝을 위한 백터 또는 발현을 위한 백터로서 구축될 수 있다.  The vector system of the present invention can be constructed through various methods known in the art, and specific methods thereof are described in Sambrook et al. , Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001), which is incorporated herein by reference. Vectors of the invention can typically be constructed as vectors for cloning or vectors for expression. In addition, the vector of the present invention can be constructed using prokaryotic cells as a host. Vectors of the invention can typically be constructed as vectors for cloning or vectors for expression.
예를 들어, 본 발명의 백터가 발현 백터이고, 원핵 세포를 숙주로 하는 경우에는, 전사를 진행시킬 수 있는 강력학 프로모터 (예컨대, T7 프로모터, tac 프로모터, lac 프로모터, lacUV5 프로모터, lpp 프로모터, pL λ 프로모터, pR λ 프로모터, rac5 프로모터, amp 프로모터, recA 프로모터, SP6 프로머터 및 trp 프로모터 둥), 해독의 개시를 위한 라이보좀 결합 자리 및 전사 /해독 종결 서열 (터미네이터, 예컨대, T7 터미네이터, ADH1 터미네이터, T3 터미네이터 및 TonB 터미네이터 둥)을 포함하는 것이 일반적이다. 숙주 세포로서 대장균이 이용되는 경우, 대장균 트립토판 생합성 경로의 프로모터 및 오퍼레이터 '부위 (Yanofsky, C, J. Bacteriol. , 158 :1018- 1024 (1984)) 그리고 파아지 λ의 좌향 프로모터 (pL λ 프로모터, Herskowitz, I. and Hagen, D. , Ann. Rev. Genet., 14:399- 445(1980))가조절 부위로서 이용될 수 있다. For example, the vector of the present invention is an expression vector and the prokaryotic cell is the host. In this case, kinetic promoters (e.g., T7 promoter, tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pL lambda promoter, pR lambda promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, which can promote transcription) Merter and trp promoters), ribosome binding sites for initiation of translation and transcription / detox termination sequences (terminators such as T7 terminator, ADH1 terminator, T3 terminator and TonB terminator). When E. coli is used as a host cell, the promoter and operator 'site of the E. coli tryptophan biosynthesis pathway (Yanofsky, C, J. Bacteriol., 158: 1018-1024 (1984)) and the leftward promoter of phage λ (pL λ promoter, Herskowitz , I. and Hagen, D., Ann. Rev. Genet., 14: 399-445 (1980)) can be used as regulatory sites.
한편, 본 발명에 이용될 수 있는 백터는 당업계에서 종종 사용되는 폴라스미드 (예: pACYCDuet-l, pSClOl, ColEl, pBR322, pUC8/9, pHC79, pUC19, pET 등), 파지 (예: Xgt4 B, λ -Charon, λ Δζΐ 및 M13 등) 또는 바이러스 (예: SV40등)를 조작하여 제작될 수 있다. ! On the other hand, vectors that can be used in the present invention are often used in the art, such as polamide (e.g. pACYCDuet-l, pSClOl, ColEl, pBR322, pUC8 / 9, pHC79, pUC19, pET, etc.), phage (e.g., Xgt4 B , λ-Charon, λ ζ, and M13) or viruses (eg SV40). !
본 발명의 일 구현예에 따르면, 상기 EGFR에 대한 Fab 단편을 코딩하는 뉴클레오타이드 서열은 pACYCDuet-1에 클로닝된다. 상기 pACYCDuet-1 관련 정보는 https: // www . snapgene . com/ r esour ces/p 1 asm i d_f i 1 es/pet— and— duet— vec tors— (novagen)/pACYCDuet-l/를 참조한다,  According to one embodiment of the invention, the nucleotide sequence encoding the Fab fragment for EGFR is cloned into pACYCDuet-1. The pACYCDuet-1 related information is https: // www. snapgene. com / r esour ces / p 1 asm i d_f i 1 es / pet— and— duet—vec tors— (novagen) / pACYCDuet-l /
본 발명의 Fab 단편의 대장균 내, 상세하게는 대장균 내 주변세포질 (periplasm)에서 생성되도록 재조합 백터에 신호 펩타이드 (signal peptide)를 코딩하는 뉴클레오타이드 서열을 포함한다. 본 발명의 일 구현예에 따르면, 상기 신호 펩타이드는 OmpA 신호 펩타이드, LamB 신호 펩타이드, Stll 신호 펩타이드, MalE 신호 펩타이드, Lpp신호 펩타이드 및 PelB신호펩타이드이다.  A nucleotide sequence encoding a signal peptide in a recombinant vector to be produced in the periplasm of Escherichia coli, specifically in Escherichia coli, of the Fab fragment of the present invention. According to one embodiment of the invention, the signal peptide is OmpA signal peptide, LamB signal peptide, Stll signal peptide, MalE signal peptide, Lpp signal peptide and PelB signal peptide.
본 발명의 다른 구현예에 따르면, 상기 신호 펩타이드는 OmpA 신호 펩타이드이다.  According to another embodiment of the invention, the signal peptide is an OmpA signal peptide.
상기 OmpA 신호 펩타이드는 중쇄 가변 영역을 코딩하는 뉴클레오타이드 서열의 업스트림 (upstream)에 위치한다.  The OmpA signal peptide is located upstream of the nucleotide sequence encoding the heavy chain variable region.
상기 OmpA 신호 펩타이드를 코딩하는 아미노산 서열은 서열목록 제 3서열이고, 상기 ΟπιρΑ 신호 펩타이드를 코딩하는 뉴클레호타이드 서열은 서열목록 제 8서열이다. 본 발명의 다른 양태에 따르면, 상기 재 ^합 백터로 형질전환된 숙주세포를 제공한다. The amino acid sequence encoding the OmpA signal peptide is sequence listing The nucleotide sequence encoding the ΟπιρΑ signal peptide is the third sequence and is SEQ ID NO: 8. According to another aspect of the present invention, there is provided a host cell transformed with the recombination vector.
본 발명의 백터를 안정되면서 연속적으로 클로닝 및 발현시킬 수 있는 숙주 세포는 당업계에 공지되어 어떠한 숙주 세포도 이용할 수 있으며, 예컨대, E. coli C43CDE3), E. coli JM109, E„ coli BL2KDE3), E. coli RRl, E. coli LE392 , E. coli B, E. coli X 1776, E. coli W3110, 바실러스 서브틸리스, 바실러스 츄린겐시스와 같은 바실러스 속 균주, 그리고 살모넬라 티피무리움, 세리 "티아 마르세슨스 및 다양한 슈도모나스 종과 같은 장내균과 균주 등이 있다.  Host cells capable of stably and continuously cloning and expressing the vector of the present invention are known in the art and can be used with any host cell, for example, E. coli C43CDE3), E. coli JM109, E „coli BL2KDE3), Bacillus sp. Strains such as E. coli RRl, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus thuringiensis, and Salmonella typhimurium, Seri "thia Enterobacteria and strains such as Marsesons and various Pseudomonas species.
본 발명의 백터를 숙주 세포 내로 운반하는 방법은, 열쇼크 방법, The method of transporting the vector of the present invention into a host cell includes a heat shock method,
CaC12 방법 (Cohen, S.N. et al., Proc. Natl. Acac. Sci. USA, 9:2110- 2114(1973)), 하나한 방법 (Cohen, S.N. et al., Proc. Natl. Acac. Sci. USA, 9:2110-2114(1973); 및 Hanahan, D. , J. Mol. Biol. , 166: 557- 580(1983)) 및 전기 천공 방법 (Dower, W.J. et al., Nucleic. Acids Res., 16:6127-6145(1988)) 등에 의해 실시될 수 있다ᅳ CaC12 method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA, 9: 2110-2114 (1973)), one method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA) , 9: 2110-2114 (1973); and Hanahan, D., J. Mol. Biol., 166: 557-580 (1983)) and electroporation methods (Dower, WJ et al., Nucleic. Acids Res., 16: 6127-6145 (1988)).
본 발명의 일 구현예에 따르면, 본 발명의 숙주세포는 대장균이다。 본 발명의 다른 구현예에 따르면, 본 발명의 숙주세포는 E. coli According to one embodiment of the invention, the host cell of the invention is E. coli. According to another embodiment of the invention, the host cell of the invention is E. coli
C43(DE3)이다. 상기 E. coli C43(DE3) 관련 정보는 Laurence Dumon- Seignovert et al.이 Protein Expression and Purification 37(2004) 203- 206에 게재한 The toxicity of recombinant proteins in Escherichia coli: . a comparison of ocerexpression in BL2KDE3), C41(DE3), and C43(DE3)을 참조한다. 본 발명의 또다른 양태에 따르면, 다음의 단계를 포함하는 EGFR에 특이적으로 결합하는 Fab단편의 제조방법을 제공한다: C43 (DE3). The E. coli C43 (DE3) related information, Laurence et al Dumon- Seignovert the Protein Expression and Purification 37 (2004) placed in a 203- 206 The toxicity of recombinant proteins in Escherichia coli:. See a comparison of ocerexpression in BL2KDE3), C41 (DE3), and C43 (DE3). According to another aspect of the present invention, there is provided a method for preparing a Fab fragment that specifically binds to EGFR, comprising the following steps:
(a) 상기 EGFR에 대한 Fab 단편을 코딩하는 아미노산 서열을 코딩하는 뉴클레오타이드 서열을 포함하는 발현 컨스트릭트를 포함하는 재조합 백터에 형질전환된 숙주세포를 배양하는 단계; 및 (b) 상기 숙주세포에서 EGFR에 대한 Fab 단편를 발현시키는 단계 . 본 발명의 방법은 상기 EGFR에 대한 Fab 단편의 제조방법으로, 이 둘 사이에 공통된 내용은 본 명세서의 과도한 복잡성을 피하기 위하여, 그 기재를 생략한다. (a) culturing the transformed host cell in a recombinant vector comprising an expression construct comprising a nucleotide sequence encoding an amino acid sequence encoding a Fab fragment for the EGFR; And (b) expressing a Fab fragment for EGFR in said host cell. The method of the present invention is a method for preparing a Fab fragment for the EGFR, the content common between the two is omitted in order to avoid excessive complexity of the present specification.
본발명의 단계 (a)의 상기 숙주세포는 당업계에 공지된 다양한 배양방법에 따라 배양할 수 있다.  The host cell of step (a) of the present invention can be cultured according to various culture methods known in the art.
본 발명의 일 구현예에 따르면, 상기 숙주세포는 SB(Super Broth) , FBCFast idious Broth) , LB(Lysogeny Broth) , TB(Terr i f ic Broth) , S0C( Super Opt imal Broth wi th Catabol ic repressor) 및 S0B.( Super Opt imal Broth)로 구성된 군으로부터, 선택되는 하나 이상의 배양 배지에서 배양된다. 본 발명의 다른 구현예에 따르13, 상기 숙주세포는 SB(Super Broth) , FB(Fast idious Broth) 및 LB Lysogeny Broth)로 구성 S 군으로부터 선택되는 하나 이상의 배양 배지에서 배양된다. According to one embodiment of the invention, the host cell is SB (Super Broth), FBC Fast idious Broth (LB), LB (Lysogeny Broth), TB (Terr if ic Broth), S0C (Super Opt imal Broth wi th Catabol ic repressor) and S0B. (Super Broth Opt imal), is cultured in a culture medium at least one selected from the group consisting of. Follow with another embodiment of the present invention 13, the host cell is cultured in a culture medium at least one selected from the group S configuration by SB (Super Broth), FB ( Fast idious Broth) and LB Broth Lysogeny).
본 발명의 특정 구현예에 따르면, 상기 숙주세포는 SB(Super Broth)에서 배양된다. 본 발명의 다른 양태에 따르면, (a) 상기 EGFR에 특이적으로 결합하는 Fab 단편의 약제학적 유효량; 및 (b) 약제학적으로 허용되는 담체를 포함하는 암에 대한 예방 및 치료용 약제학적 조성물을 제공한다. 본 발명의 일 구현예에 따르면, 상기 암은 유방암, 대장암, 폐암, 위암 간암, 혈액암, 골암, 췌장암, 피부암, 뇌암, 자궁암, 비인두암, 후두암, 결장암, 난소암ᅳ 직장암, 대장암, 질암, 소장암, 내분비암, 갑상선암, 부갑상선암, 요관암, 요도암, 전립선암, 기관지암, 방광암, 신장암또는 골수암이다.  According to a particular embodiment of the invention, the host cell is cultured in SB (Super Broth). According to another aspect of the invention, (a) a pharmaceutically effective amount of a Fab fragment that specifically binds to the EGFR; And (b) provides a pharmaceutical composition for the prevention and treatment of cancer comprising a pharmaceutically acceptable carrier. According to one embodiment of the present invention, the cancer is breast cancer, colon cancer, lung cancer, gastric cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, brain cancer, uterine cancer, nasopharyngeal cancer, laryngeal cancer, colon cancer, ovarian cancer, rectal cancer, colon cancer, Vaginal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, ureter cancer, urethral cancer, prostate cancer, bronchial cancer, bladder cancer, kidney cancer or bone marrow cancer.
본 발명의 다른 구현예에 따르면, 상기 암은 두경부암이다.  According to another embodiment of the invention, the cancer is head and neck cancer.
본 명세서에서 용어 "약제학적 유효량" 은 상술한 상기 EGFR에 대한 Fab 단편의 효능 또는 활성을 달성하는 데 층분한 양을 의미한다.  The term "pharmaceutically effective amount" as used herein means an amount sufficient to achieve the efficacy or activity of the Fab fragments described above for EGFR.
본 발명의 Fab 단편이 약제학적 조성물로 제조되는 경우, 본 발명의 약제학적 조성물은 약제학적으로 허용되는 담체를 포함한다. 본 발명의 약제학적 조성물에 포함되는 약제학적으로 허용되는 담체는 제제시에 통상적으로 이용되는 것으로서, 락토스, 텍스트로스, 수크로스, 솔비톨, 만니틀, 전분 아카시아 고무, 인산 칼슘, 알기네이트, 젤라틴, 규산 칼슘, 미세결정성 샐를로스, 폴리비닐피를리돈, 샐를로스, 물, 시럽, 메틸 셀를로스, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 활석, 스테아르산 마그네슘 및 미네랄 오일 등을 포함하나, 이에 한정되는 것은 아니다. 본 발명의 약제학적 조성물은 상기 성분들 이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제, 보존제 등을 추가로 포함할 수 있다. 적합한 약제학적으로 허용되는 담체 및 제제는 Remington' s Pharmaceut i cal Sciences ( 19th ed. , 1995)에 상세히 기재되어 있다. When the Fab fragment of the present invention is made into a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are those commonly used in the preparation of lactose, textose, sucrose, sorbitol, Mannitol, starch acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyridone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxy Benzoate, talc, magnesium stearate, mineral oil, and the like. The pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like, in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceut i cal Sciences (19th ed., 1995).
본 발명의 약제학적 조성물은 경구 또는 비경구 투 할 수 있으며, 바람직하게는 비경구 투여 방식으로 적용된다.  The pharmaceutical composition of the present invention may be oral or parenteral, and is preferably applied by parenteral administration.
본 발명의 약제학적 조성물의 적합한 투여량은 제제화 방법, 투여 방식, 환자의 연령, 체증, 성, 병적 상태, 음식, 투여 시간, 투여 경로, 배설 속도 및 반웅 감웅성과 같은 요인들에 의해 다양하게 처방될 수 있다. 본 발명의 약제학적 조성물의 일반적인 투여량은 성인 ᅵ준으로 0.001 g/kg - 1000 uig/kg 범위 내이다.  Suitable dosages of the pharmaceutical compositions of the present invention vary depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion and reaction in response to the patient. It may be prescribed. Typical dosages of the pharmaceutical compositions of the invention are in the range of 0.001 g / kg-1000 uig / kg for adults.
본 발명의 약제학적 조성물은 당해 발명이 속하는 기술분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있는 방법에 따라, 약제학적으로 허용되는 담체 및 /또는 부형제를 이용하여 제제화함으로써 단위 용량 형태로 제조되거나 또는 다용량 용기 내에 내입시켜 제조될 수 있다. 이때 제형은 오일 또는 수성 매질중의 용액, 현탁액, 시럽제 또는 유화액 형태이거나 엑스제, 산제, 분말제, 과립제, 정제 또는 캅셀제 형태일 수도 있으며, 분산제 또는 안정화제를추가적으로 포함할수 있다.  The pharmaceutical compositions of the present invention may be prepared in unit dose form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporation into a multi-dose container. The formulation may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media or in the form of extracts, powders, powders, granules, tablets or capsules, and may further comprise dispersants or stabilizers.
[발명의 효과】 [Effects of the Invention】
본 발명의 특징 및 이점을 요약하면 다음과 같다:  The features and advantages of the present invention are summarized as follows:
(a) 본 발명은 EGFR(Epi dermal Growth Factor Receptor)에 특이적으로 결합하는 Fab(Fragment ant igen-binding) 단편, 상기 Fab 단편을 제조하기 위한 발현 컨스트럭트, 상기 Fab 단편의 제조방법 및 상기 (A) The present invention is a fragment ant igen-binding (fab) fragment that specifically binds to EGFR (Epi dermal Growth Factor Receptor), an expression construct for producing the Fab fragment, a method for producing the Fab fragment and the
Fab 단편을 포함하는 약제학적 조성물을 제공한다. A pharmaceutical composition comprising a Fab fragment is provided.
(b) 본 발명의 EGFR에 대한 Fab 단편은 항체에 비하여 크기가 작기 때문에 조직이나 종양으로와 침투율이 좋고 박테리아에서 제조할 수 있어 생산 비용이 적게. 든다. (b) Fab fragments for EGFR of the present invention are smaller in size than antibodies, and thus have good penetration into tissues or tumors and can be produced in bacteria. Less production costs. Holding
(c) 본 발명의 EGFR에 대한 Fab 단편은 페길화 (PEGylat ion)을 통해 증가된 체내 반감기를 제공한다. 【도면의 간단한 설명】  (c) Fab fragments for EGFR of the present invention provide increased body half-life through PEGylation. [Brief Description of Drawings]
도 1은 Fm301 컨스트럭트 및 pACYCDuet-1 백터 맵을 나타낸다ᅳ 도 2는 Fm301의 클로닝을 PCR(Polymerase Cloning React ion)을 통해 확인한 결과이다.  FIG. 1 shows the Fm301 construct and the pACYCDuet-1 vector map. FIG. 2 shows the cloning of Fm301 through PCR (Polymerase Cloning React ion).
도 3은 Fm302 컨스트럭트 및 pACYCDuet-1 백터 맵을 나타낸다.  3 shows the Fm302 construct and the pACYCDuet-1 vector map.
도 4는 Fm302의 클로닝을 PCR을 통해 확인한 결과이다.  4 shows the results of cloning of Fm302 by PCR.
도 5는 ί½306 컨스트럭트 및 pACYCDuet-1 백터 맵을 낭타낸다, 도 6은 1¾306의 클로닝을 PCR을 통해 확인한 결과이다,  5 shows the ί½306 construct and the pACYCDuet-1 vector map. FIG. 6 shows the results of PCR confirming the cloning of 1¾306.
도 7a내지 7c는 대장균에서 발현시 ¾ Fm301 및 Fm302 단백질을 SDS- PAGE를 통해 확인한 결과 및 항 -Fab항체와 결합시킨 결과를 나타낸다. 도 8은 Fm301 및 Fm302의 정제 후 균질도를 분삭한 결과를 나타낸다. 도 9는 대장균에서 발현시킨 Fm306 단백질을 SDS-PAGE를 통해 확인한 결과를 나타낸다.  Figures 7a to 7c shows the results confirmed by SDS-PAGE and ¾ Fm301 and Fm302 protein when expressed in E. coli and the result of binding to the anti-Fab antibody. Figure 8 shows the results of the homogeneity fraction after purification of Fm301 and Fm302. Figure 9 shows the results confirmed by SDS-PAGE Fm306 protein expressed in E. coli.
도 10a 및 10b는 배지별 (Lysogeny broth; LB, Fant idious broth ; FB 및 Super broth; SB) 항체 정제 수율을 나타낸다.  10A and 10B show the yield of antibody purification by medium (Lysogeny broth; LB, Fant idious broth; FB and Super broth; SB).
도 11은 대장균에서 발현시킨 1½302를 비 -환원 다이를 이용해 전기영동한 결과를 나타낸다.  11 shows the results of electrophoresis of 1½302 expressed in E. coli using a non-reducing die.
도 12a 내지 12b는 Fm302의 이온 교환 크로마토그래피 결과를 나타낸다。  12A to 12B show ion exchange chromatography results of Fm302.
도 13은 Fm302의 크기 배제 크로마토그래피 결과를 나:타낸다.  FIG. 13 shows the size exclusion chromatography results of Fm302.
도 14는 Fm302의 ι 및 CL의 C-말단에 각각 Cys- ^p-Lys 및 Glu-FIG. 14 shows Cys- ^ p-Lys and Glu- at the C-terminus of ι and C L of Fm302, respectively.
Cys를추가하여 형성되는 이황화 결합을 확인한 결과를 나타낸다. The result of confirming the disulfide bond formed by adding Cys is shown.
도 15는 Fm306의 정제 결과를 SDS-PAGE를 통해 확인한 결과를 나타낸다.  15 shows the result of confirming the purification result of Fm306 through SDS-PAGE.
도 16은 Fm306-PEG(20K) 및 Fm306-PEG(30K) 결합체의 제조 및 수율을 확인한 결과를 나타낸다.  Figure 16 shows the results of confirming the production and yield of the Fm306-PEG (20K) and Fm306-PEG (30K) conjugate.
도 17은 Fab' 컨스트럭트의 구조를 나타낸다. Fm301 , Fra302 및 Fm306 모두 동일하게 4개의 쇄내 이황화 결합 (intra-chain disulfide bond)를 갖고 있고, 1½302 및 1½306의 경우 C-말단에 쇄내 이황화결합이 존재한다. Fm306의 경우 중쇄 부분 C-말단에 페길화를 위해 추가된 아미노산이 존재한다. Figure 17 shows the structure of the Fab 'construct. Fm301, Fra302 and Fm306 All have the same four intra-chain disulfide bonds, and for 1½302 and 1½306 there are intrachain disulfide bonds at the C-terminus. For Fm306 there is an amino acid added for PEGylation at the heavy chain portion C-terminus.
도 18a 내지 18d은 Fm306-PEG의 페길화를 SDS-PAGE, PEG 염색 및 웨스턴 블럿을 통해 확인한 결과를 보여준다. 도 18a는 1½301, Fm302 및 Fm306 시료와 각 컨스트럭트별 PEG를 반웅한 시료의 SDS-PAGE의 쿠마시 블루 (coomassie blue) 염색 결과를 나타낸다. 도 18b는 도 18a와 동일한 시료에 대한 PEG 염색 결과를 나타낸다. 도 18c는 Fm301, Fm302 및 f¾306 시료와 각 컨스트럭트별 PEG를 반응한 시료의 항 -Fab 항체를 이용한 웨스턴 블랏 결과를 나타낸다. 도 18d는 i301, Fm302 및 1¾ )6 시료와 각 컨스트럭트별 PEG를 반웅한 시료의 항 -PEG 항체를 이용 : 웨스턴 블랏 결과를 나타낸다.  18A-18D show the results of confirming PEGylation of Fm306-PEG via SDS-PAGE, PEG staining and Western blot. FIG. 18A shows the Coomassie blue staining results of SDS-PAGE of 1½301, Fm302, and Fm306 samples and samples of construct-specific PEG. FIG. 18B shows the result of PEG staining for the same sample as in FIG. 18A. FIG. Figure 18c shows the results of Western blot using the anti-Fab antibody of the Fm301, Fm302 and f¾306 sample and the sample reacted with PEG for each construct. FIG. 18D shows Western blot results using anti-PEG antibodies of i301, Fm302, and 1¾) 6 samples and samples of construct-specific PEG.
도 19a 내지 19d는 Fm306-PEG의 페길화를 잔여 PEG제거 후 SDS-PAGE, PEG 염색 및 웨스턴 블럿을 통해 확인함 결과를 보여준다. 19a는 Fm301, Fm302 및 Fm306 컨스트릭트별 잔여 PEG가 제거된 시료에 대한 SDS-PAGE의 쿠마시 블루 (coomassie blue) 염색 결과를 나타낸다. 도 19b는 도 19a와 동일한 시료에 대한 PEG 염색 결과를 나타낸다. 도 19c는 Fm301, Fm302 및 Fm306 시료 컨스트럭트별 잔여 PEG가 제거된 시료에 대한 항 -Fab 항체를 이용한 웨스턴 블랏 결과를 나타낸다. 도 19d는 Fm301, Fm302 및 Fm306 컨스트럭트별 잔여 PEG가 제거된 시료에 대한 항 -PEG 항체를 이용한 웨스턴 블랏 결과를 나타낸다.  19A-19D show the PEGylation of Fm306-PEG confirmed via SDS-PAGE, PEG staining and Western blot after residual PEG removal. 19a shows the results of coomassie blue staining of SDS-PAGE on samples from which residual PEG by Fm301, Fm302 and Fm306 constructs were removed. 19B shows the result of PEG staining for the same sample as in FIG. 19A. 19C shows Western blot results using anti-Fab antibodies for samples from which residual PEG was removed for each Fm301, Fm302, and Fm306 sample constructs. FIG. 19D shows Western blot results using anti-PEG antibodies for samples with residual PEG removed by Fm301, Fm302 and Fm306 constructs.
도 20a 내지 20d는 각각 세툭시맙 (Cetuximab), 세특시맙 -Fab, Fm302 및 Fm306FEG의 키네틱 (kinetic) 값을 나타낸다.  20A-20D show the kinetic values of Cetuximab, Sepusimab-Fab, Fm302 and Fm306FEG, respectively.
도 21은 1¾302 및 F 306PEG의 sEGFR-결합 친화도를 나타낸다.  21 shows sEGFR-binding affinity of 1¾302 and F 306PEG.
도 22는 EGF, 세특시맙, 세룩시맙 -Fab, Fm302 및 Fm306PEG의 EGFR 인산화 정도를 나타낸다.  FIG. 22 shows the degree of EGFR phosphorylation of EGF, Sesucimab, Celuximab-Fab, Fm302, and Fm306PEG.
도 23은 세특시맙, 세특시맙 -Fab, Fm302 및 Fm306PEG의 두경부암 질환모델동물에서의 종양조직 성장을 나타낸다.  FIG. 23 shows tumor tissue growth in cecum carcinoma disease model animals of Sepusimab, Sepusimab-Fab, Fm302 and Fm306PEG.
도 24는 세툭시맙, 세툭시맙 -Fab, Fm302 및 f¾306PEG의 두경부암 질환모델동물의 부검시점의 종양조직의 크기를 나타낸다. 【발명을 실시하기 위한 구체적인 내용】 FIG. 24 shows the tumor tissue size at necropsy in head and neck cancer disease model animals of cetuximab, cetuximab-Fab, Fm302 and f¾306PEG. [Specific contents to carry out invention]
이하, 실시 예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시 예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시 예에 의해 제한되지 않는다는 것은 당 업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다. 실시예 1: Fab 컨스트럭트 제작  Hereinafter, the present invention will be described in more detail with reference to the following examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. . Example 1: Fab Construct Construction
항체를 구성하는 도메인 중 VL( light chain variable region; 경쇄가변영역)과 VH(heavy chain variable region; 중쇄가변영역) 도메인에 각각 존재하는 분자내 이황화결합 (iptra-chain disulfide bonds)은 각 도메인의 구조를 안정화시킨다. 이와 같은 분자내 이황화결 f은 항체 (Ant i- EGFR) 및 항원 [EGFR(HERl)] 사이의 상호작용에 중요한 역 :을 하고 있는 것으로 알려져 있다 [(Yang, et al., PNAS. 104(26): 10813-10^17(2007); Liu, H. and May, k., MAbs, 4:17-23(2012)]. 또한, 힌지 도메인 (hinge domain)에 존재하는 2개의 시스테인 (cysteine)은 각 도메인 사이의 분자 내 이황화결합을 형성하는데, 이는 항체가 이형체 (dimer)를 이루는데 구조적으로 중요한 역할을 수행한다고 알려져 있다 [K. Zangger. et al., Biochem. 359:353-360(2001) , Levy, R. et al. , J. Immunol. Methods. , 394:10-21(2013)]. 하지만 본 발명에서 개발한 항 -EGFR 항체절편은 힌지 도메인을 갖고 있지 않으며, 이로 인해 발생하는 힌지 두메인의 결실로 인해 Fab'를 형성하게 되고 일가 (monovalent) 항체절편을 생산할 수 있게 된다. 한편, VH+CHi 및 VL+CL 도메인을 각각 발현시켜 대장균의 주변세포질 (periplasm)에서 폴딩 (folding)을 유도할 경우 안정적인 구조로 폴딩될 수 있는 것으로 알려져 있다 (S. Ewert. et al ., J. Mol. Biol. 325:531-553(2003). 항체절편의 각 도메인을 발현시켜 대장균의 주변세포질로 보내기 위하여 각 도메인 앞에 OmpA 신호 펩타이드 (ΟπιρΑ signal peptide)를 도입하였다. Fab' 컨스트력트 [V^H 'CDK' deletion), VL-CL : Fm301] Among the domains constituting the antibody, intra-molecular disulfide bonds present in the V L (light chain variable region) and V H (heavy chain variable region) domains are each domain. Stabilize the structure. Such intramolecular disulfide f is known to play an important role in the interaction between antibody (Ant i-EGFR) and antigen [EGFR (HERl)] [(Yang, et al., PNAS. 104 (26). ): 10813-10 ^ 17 (2007); Liu, H. and May, k., MAbs, 4: 17-23 (2012)], and also two cysteines present in the hinge domain. Forms intramolecular disulfide bonds between domains, which are known to play a structurally important role in the formation of dimers by antibodies [K. Zangger. Et al., Biochem. 359: 353-360 ( 2001, Levy, R. et al., J. Immunol.Methods., 394: 10-21 (2013)] However, the anti-EGFR antibody fragments developed in the present invention do not have a hinge domain and are caused by this. due to the two hinged to the main deletion is to form a Fab 'it is possible to produce a monovalent (monovalent) antibody fragments. on the other hand, to express the V H + C H i and V L + C L domains, respectively Induction of folding in the periplasm of E. coli is known to be able to fold into a stable structure (S. Ewert. Et al., J. Mol. Biol. 325: 531-553 (2003). In order to express each domain of the antibody fragment and send it to the periplasm of Escherichia coli, an OmpA signal peptide (ΟπιρΑ signal peptide) was introduced in front of each domain Fab 'constructor [V ^ H ' CDK 'deletion), V L -C L : Fm301]
항체절편 플랫폼 (platform)인 단가 Fab인 Fab'을 제작하여 Fm이라 명명하고 Fab의 VH+CH1 및 VL+CL 도메인을 클로닝하기 위해 세툭시맙의 아미노산 서열 (표 1)을 근거로 하여 신호 펩타이드 (OmpA)+VH+CH1 및 신호 펩타이드 (OmpA)+VL+CL의 아미노산 서열 (표 2 및 3)에 대한 DNA 염기서열을 코스모진텍에 의뢰하여 합성 (표 4 및 5)을 진행하였다, 표 1의 서열 증 밑줄 부분은 가변 영역을 나타낸다. 이를 이용해 VH+CH1 및 VL+CL 도메인에 해당하는 부위에 PCR 프라이머를 제작 (표 6)하고 신호 펩타이드 (ompA)+VH+CH1 및 신호 펩타이드 (OmpA)+VL+CL을 각각 발현되도록 유전자를 클로닝 하였고, 이를 제한효소 처리를 통해 대장균 공동 -발현 (co- express ion) 백터인 pACYCDuet-1 백터 ( ovagen)에 클로닝 하 다 (도 1) . Fab ', which is a monovalent Fab, is an antibody fragment platform Signal peptide (OmpA) + V H + C H1 and signal peptide (OmpA) based on the amino acid sequence of cetuximab (Table 1) for naming and cloning the V H + C H1 and V L + C L domains of Fab DNA sequences for the amino acid sequences (Tables 2 and 3) of + V L + C L were submitted to CosmoGentech for synthesis (Tables 4 and 5). Indicates. PCR primers were prepared at the sites corresponding to the V H + C H1 and V L + C L domains (Table 6) and signal peptide ( om pA) + V H + C H1 and signal peptide (OmpA) + V L + The genes were cloned to express C L , respectively, and cloned into the pACYCDuet-1 vector (ovagen), which is a co-expression vector of E. coli through restriction enzyme treatment (FIG. 1).
【표 1]  [Table 1]
Figure imgf000021_0001
Figure imgf000021_0001
【표 2】  Table 2
Figure imgf000021_0002
02
Figure imgf000021_0002
02
Figure imgf000022_0001
Figure imgf000022_0001
ZZCCT0/Sl0ZaM/X3d S9Cl.l/9T0Z OAV GCCTCTACCA GGTCCGAGCGTmCC GCTGGCACCGAGCT^ ZZCCT0 / Sl0ZaM / X3d S9Cl.l / 9T0Z OAV GCCTCTACCA GGTCCGAGCGTmCC GCTGGCACCGAGCT ^
CCAGTaXGGTAC(^AGCTCTOXXTGTCT(¾TGAMGAmTm  CCAGTaXGGTAC (^ AGCTCTOXXTGTCT (¾TGAMGAmTm
ACCGGTCACCGTCAG^GG TOCOTGCACTGACCAGTGGCGTCCACACG  ACCGGTCACCGTCAG ^ GG TOCOTGCACTGACCAGTGGCGTCCACACG
CHI 제 10서열 CHI sequence 10
TTCCCGaTGTGCTGCAGAGTOCOiTCTGTATAGCCTGTCATCGGTGG™ TTCCCGaTGTGCTGCAGAGTOCOiTCTGTATAGCCTGTCATCGGTGG ™
CCGmCGAGCTCTAGTCTa^ACCCAAACGTACATTTGCAATGTCAACCA rAAACCGAGCAACACGAAAGTTGATAAACGTGTCGAACCGAAATCA  CCGmCGAGCTCTAGTCTa ^ ACCCAAACGTACATTTGCAATGTCAACCA rAAACCGAGCAACACGAAAGTTGATAAACGTGTCGAACCGAAATCA
[표 5】  TABLE 5
Figure imgf000023_0001
Figure imgf000023_0001
【표 6】  Table 6
Figure imgf000023_0002
프라이머
Figure imgf000023_0002
primer
CHI도메인 CHI Domain
역방향 ATGCO CGCAAGCTTCTATGATTTCGGTTCGACACG 제 14서열 프라이머  Reverse ATGCO CGCAAGCTTCTATGATTTCGGTTCGACACG Sequence 14 Primer
VL도메인 V L domain
AAGGAGATATACATATGAAAAAGACAGCTATCGCGATTGCAGTGGCACTG  AAGGAGATATACATATGAAAAAGACAGCTATCGCGATTGCAGTGGCACTG
정방향 제 15서열  Forward 15th sequence
GCTGGTT  GCTGGTT
프라이머  primer
CL도메인 C L domain
역방향 CmACCAGACTCGAGCTAGGCACGACGAT GAATGA 제 16서열 프라이머  Reverse CmACCAGACTCGAGCTAGGCACGACGAT GAATGA Sequence 16 Primer
Fm-301 컨스트럭트를 PCR하여 로닝 결과를,확인하였다 (도 2) .  PCR of the Fm-301 construct confirmed the rowing result (FIG. 2).
Fab ' 컨스트릭트 (Vr iu, VrCL(+EC) : Fm302) Fab 'construct (Vr iu, VrC L (+ EC) : Fm302)
Fm301 컨스트력트의 CH1 및 CL의 C-말단에 각각 아미노산 염기서열 ' CDK' 및 'EC' 를 삽입 (표 7 및 φ된 컨스트럭트를 클로닝하기 위해 대장균에서 발현할 수 있는 코돈으로 변환하여 DNA 염기서열 (표 9 및 10)을 코스모진텍에 의뢰하여 합성을 진행하였다. 상기 ' CDK' 및 ' EC '는 경쇄 및 중쇄가 헤테로다이머 (heterodimer)를 형성하기 위한 이황화결합을 유도하기 위하여 추가하였다, 이를 이용해 VH+CH1(+CDK) 및 VL+CL(+EC) 도메인에 해당하는 부위에 PCR 프라이머를 제작 (표 11)하고 신호 펩타이드 (ompA)+VH+CH1(+CDK) 및 신호 펩타이드 (0mpA)+VL+CL(+EC)도메인을 각각 발현되도록 유전자를 클로닝 하였고, 이를 제한효소 처리를 통해 대장균 공동 -발현 백터인 pACYCDuet-1 백터에 클로닝 하였다 (도 3) , The amino acid sequences 'CDK' and 'EC' are inserted at the C-terminus of the C H1 and C L of the Fm301 construct (Table 7 and converted to codons that can be expressed in E. coli to clone φ constructs. DNA sequences (Tables 9 and 10) were commissioned by Cosmojintech to synthesize the 'CDK' and 'EC' in order to induce disulfide bonds for the light and heavy chains to form a heterodimer. The PCR primers were prepared at the sites corresponding to the V H + C H1 (+ CDK) and V L + C L (+ EC) domains (Table 11), and the signal peptide ( om pA) + V H + C was used. The genes were cloned to express the H1 (+ CDK) and signal peptide (0mpA) + V L + C L (+ EC) domains, respectively, and were cloned into the pACYCDuet-1 vector, which is a co-expression vector of E. coli through restriction enzyme treatment. (FIG. 3),
【표 7]  [Table 7]
Figure imgf000024_0001
ASTKGPSVFPLAPSSKSTSOTM^GaVTOYFPEPVTVS丽 SGALTSG
Figure imgf000024_0001
ASTKGPSVFPLAPSSKSTSOTM ^ GaVTOYFPEPVTVS 丽 SGALTSG
CHI+CD VHTFPAVLQSSGLYSLSSWTVPSSSLGTQTY I CNVNHKPSNTKVDKRV 제 17서열  CHI + CD VHTFPAVLQSSGLYSLSSWTVPSSSLGTQTY I CNVNHKPSNTKVDKRV Sequence 17
EPKSCD  EPKSCD
【표 8】  Table 8
- 서열 서열목록 -Sequence list
OrapA 신호 OrapA signal
MKKTAIAIAVALAGFATVAQA 제 3서열 펩타이드  MKKTAIAIAVALAGFATVAQA Sequence 3 Peptides
DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIK DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIK
VL YASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTF 제 6서열 GAGTKLELKV L YASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTF Sequence 6 GAGTKLELK
RTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQS
CL+EC GNSQESVTEQDSKDSTYSLSSTLTLSKADYE H VYACEVTHQGLSSPV 제 18서열 18 SEQ ID NO: C + L EC GNSQESVTEQDSKDSTYSLSSTLTLSKADYE H VYACEVTHQGLSSPV
TKSFNRGAEC  TKSFNRGAEC
Figure imgf000025_0001
CATCGGTGGTTACCGTTCCGAGCTCTAGTCTGGGCACCCAAACGTACAT
Figure imgf000025_0001
CATCGGTGGTTACCGTTCCGAGCTCTAGTCTGGGCACCCAAACGTACAT
ITGCAATGTCMCCAT CCGAGCMCACG GTTGATA CGTGTCITGCAATGTCMCCAT CCGAGCMCACG GTTGATA CGTGTC
GAACCGAAATCATGCGATAAA GAACCGAAATCATGCGATAAA
【표 103 Table 103
Figure imgf000026_0001
Figure imgf000027_0001
Figure imgf000026_0001
Figure imgf000027_0001
Fab' 컨스트력트 ( m+ CAA, VL+CL+EC: Fm306) Fab 'constraint (m + CAA, V L + C L + EC: Fm306)
앞서 제작한 Fm302 유전자에 부위 특이적 페길화 (site specific pegylation) 반응을 위하여 CH1 도메인 C-말단에 힌지 부위의 아미노산인 THTCM을 삽입 (표 12)하여 대장균에서 발현할 수 있는 코돈으로 변환하여 DNA 염기서열 (표 14 및 15)을 코스모진텍에 의뢰하여 합성을 진행하였다. 이를 이용하여 VH+CH1+THTCAA 및 VL+CL+EC에 해당하는 부위에 fCR프라이머를 제작 (표 16)하고 위해 대장균 발현 PCR : 수행하였고, 이를 젱한효소 처리를 통해 대장균 공동 -발현 백터인 pACYCDuet-1 백터에 클로닝 하였다 (도 5). For site specific pegylation reaction, the amino acid THTCM, which is a hinge region at the C-terminus of the C H1 domain, was inserted into the Fm302 gene (Table 12) and converted into codons that can be expressed in E. coli. The base sequences (Tables 14 and 15) were commissioned by Cosmojintech for synthesis. Using this, fCR primers were prepared at the sites corresponding to V H + C H1 + THTCAA and V L + C L + EC (Table 16), and E. coli expression PCR was performed for co-expression of E. coli through the enzyme treatment. The vector was cloned into the pACYCDuet-1 vector (FIG. 5).
ί표 12】  ί Table 12】
Figure imgf000027_0002
펩타이드
Figure imgf000027_0002
Peptide
D I LLTQSPV I LSVSPGERVSFSCRASQS I GTN I HWYQQRTNGSPRLL I Y D I LLTQSPV I LSVSPGERVSFSCRASQS I GTN I HWYQQRTNGSPRLL I Y
VL ASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGA 제 6서열 GTKLELKV L ASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGA Sequence 6 GTKLELK
RTVAAPSWIFPPSDEQLKSGTASWaL顺 FYPREAKVQWKVDNALQSG RTVAAPSWIFPPSDEQLKSGTASWaL 顺 FYPREAKVQWKVDNALQSG
CL+EC kSQESVTEQDSKDSTYSLSSTLTLSKADYEKH VYACEVTHQGLSSPVTK제 18서열  CL + EC kSQESVTEQDSKDSTYSLSSTLTLSKADYEKH VYACEVTHQGLSSPVTK
SFNRGAEC  SFNRGAEC
【표 14]  Table 14
Figure imgf000028_0001
Figure imgf000028_0001
【표 15]  Table 15
Figure imgf000028_0002
GATAmT( TGACCCAGAGCCCGGTCATCCTGAGTGTTTCCCCGGGCGA
Figure imgf000028_0002
GATAmT (TGACCCAGAGCCCGGTCATCCTGAGTGTTTCCCCGGGCGA
AO}TGTGTCATmCGTGTCGC(X:GA(XCAGTCTATTGGTACCAATATCC  AO} TGTGTCATmCGTGTCGC (X: GA (XCAGTCTATTGGTACCAATATCC
ACT(¾ TATCA(X: CGTACGMCGGCTCTCCGCGCCTGCTGATTAAATAC  ACT (¾ TATCA (X: CGTACGMCGGCTCTCCGCGCCTGCTGATTAAATAC
VL CAGTG TCCAmCAGi ATCCCGAGCCGCTmCGGGCAGCGGnC제 11서열 VL CAGTG TCCAmCAGi ATCCCGAGCCGCTmCGGGCAGCGGnC
RGGCACCGATTTCACGCTGAGTA AACTCCGTGGMTCAGAAGATATCG CAGACTA™CT(X:CA(X:AAMCMTMCTGGCCGACCACGTTTGGTGCT RGGCACCGATTTCACGCTGAGTA AACTCCGTGGMTCAGAAGATATCG CAGACTA ™ CT (X: CA (X: AAMCMTMCTGGCCGACCACGTTTGGTGCT
GGCACCAAACTGGAACTGAAA GGCACCAAACTGGAACTGAAA
CGTACOiTG(X:(^CCCGAGTGTTTrTATCTTCCCGCCGTCCGATGAACA CGTACOiTG (X: (^ CCCGAGTGTTTrTATCTTCCCGCCGTCCGATGAACA
C^TGAMTCOiGTACCGCCAGCG GTCTGTCTGCTGAATAACTTCTATC  C ^ TGAMTCOiGTACCGCCAGCG GTCTGTCTGCTGAATAACTTCTATC
CTOGMGCAA GTCCAGT∞AMGT(XiACMTGCTCT(^AGTCGGGC  CTOGMGCAA GTCCAGT∞AMGT (XiACMTGCTCT (^ AGTCGGGC
CL+EC AACAGCCAAGAAAGCGTGACCGAACAAGATAGTAAAGACTCCACGTACTC제 20서열 C L + EC AACAGCCAAGAAAGCGTGACCGAACAAGATAGTAAAGACTCCACGTACTC
ACTGTCCTCAACCCTGACGCTGAGCAAAGCGGATTATGAAAMCACAAAG  ACTGTCCTCAACCCTGACGCTGAGCAAAGCGGATTATGAAAMCACAAAG
rGTACGCCTGCGAAGmCCCATCAAGGTCTGAGTAGCCCGGTTACGAM  rGTACGCCTGCGAAGmCCCATCAAGGTCTGAGTAGCCCGGTTACGAM
TCATTCAATCGTGGTGCCGAATGC  TCATTCAATCGTGGTGCCGAATGC
[표 16]  TABLE 16
Figure imgf000029_0001
Figure imgf000029_0001
Fm-306 컨스트럭트를 PCR하여 클로닝 결과를 확인하였다 (도 6) . 실시예 2 : Fra301 및 Fra302 컨스트럭트의 대장균 발현 확인  PCR of the Fm-306 construct confirmed the cloning result (FIG. 6). Example 2 Confirmation of E. Coli Expression of Fra301 and Fra302 Constructs
Fm301 및 Fm302 컨스트릭트의 유전자의 발현을 확인하기 위해 대장균 발현세포주인 C43(DE3) 세포 (Luc igen)에 42 °C 열 충격 (heat shock)을 통해 형질전환 시킨 후 IPTG 유도를 통해 Fm301 및 Fm302 발현을 확인하였다 (도 7a내지 7b) . C4303E3)는 37 °C 및 150 rpm의 조건에서 진탕배양하였다. 동일한 배양 조건 하에서 배양한 Fm301 및 302의 발현 정도를 비교한 결과 Fm302의 발현양이 더 많았으며, 정제된 Fm301 및 Fm302가 Fab 특이적 항체를 이용하여 항 -fab 항체와 결합하는지 여부를 확인하기 위하여 정제된 단백질을 12% SDS-PAGE 겔로 전기영동한 후 NCM(ni trocel lulose membrane)에 단백질을 트랜스퍼 ( transfer) 한 후 항 -fab 항체와 반응시켜 확인한 결과, Fm3()l은 확인이 되지 않았고, Fm302만 확인되었다 (도 7c) . 두 항체절편을 균질성 (homogenei ty)을 확인하기 위하여 HPLC(SEC2000 , Shimadzu, L06AD) 분석 (이동상: PBS; 컬럼: Bio-sec 2000; 유속: 1 m«/min; 주입 용량: 25 )한 결과, Fm302가 균질성이 높음을 확인하였다 (도 8) . 실시예 3 : Fra306 컨스트릭트의 대장균 발현 확인 To confirm the expression of the genes of the Fm301 and Fm302 constructs, E. coli-expressing cell lines C43 (DE3) cells (Luc igen) were transformed by 42 ° C heat shock and then Fm301 and Fm302 were obtained through IPTG induction. Expression was confirmed (FIGS. 7A-7B). C4303E3) was shaken at 37 ° C and 150 rpm. To compare the expression levels of Fm301 and 302 cultured under the same culture conditions, the expression level of Fm302 was higher and to determine whether the purified Fm301 and Fm302 bind to anti-fab antibodies using Fab specific antibodies. Refined After electrophoresis of the protein with a 12% SDS-PAGE gel, the protein was transferred to a nitrocel lulose membrane (NCM) and then reacted with an anti-fab antibody. As a result, Fm3 () l was not confirmed. It was confirmed (FIG. 7C). HPLC (SEC2000, Shimadzu, L06AD) analysis (mobile phase: PBS; column: Bio-sec 2000; flow rate: 1 m «/ min; injection volume: 25) to confirm homogeneity of both antibody fragments It was confirmed that Fm302 had high homogeneity (FIG. 8). Example 3 Confirmation of E. Coli Expression in the Fra306 Construct
Fm306의 대장균 발현과 정제를 위하여 Fm302와 같은 대장균 Escherichia coli like Fm302 for the expression and purification of E. coli Fm306
C43(DE3)를 이용 하였으며 (실시예 2의 단백질 발현과 동일한 조건), 플라스미드는 pACYCDuet-1 백터를 사용하여 2개의 도메인을 각각 발현 하도록 하였고, 주변세포질 (per iplasm)에서 항체절편이 발현되도록 하였다 (도 9) . 실시예 4: Fra302 및 Fm306항체절편의 배양 및 정제 C43 (DE3) was used (same conditions as the protein expression of Example 2), the plasmid was used to express each of the two domains using a pACYCDuet-1 vector, and the antibody fragments were expressed in the periplasm. (FIG. 9). Example 4: Culture and Purification of Fra302 and Fm306 Antibody Fragments
Fm302 항체절편 배양및 정제  Fm302 antibody fragment culture and purification
항체절편의 정제를 위한 숙주세포는 T7 RNA 폴리머라제의 레벨을 낮춰 과다발현된 재조합 단백질의 독성으로 인한 세포 사멸을 감소시킬 수 있는 대장균 C43(DE3)를 이용하였으며, 풀라스미드는 pACYCDuet-1 백터를 사용하여 2개의 도메인을 각각 발현하도록 하였고, OmpA 신호 펩타이드를 도입하여 주변세포질에서 항체절편이 생성되도록 하였다,  The host cell for the purification of antibody fragments was E. coli C43 (DE3), which can reduce cell death due to toxicity of overexpressed recombinant protein by lowering the level of T7 RNA polymerase. Each of the two domains was expressed and an OmpA signal peptide was introduced to generate antibody fragments in the periplasm.
최적의 배양조건을 확립하기 위하여 배양액에 따른 항체절편 생산량을 비교하였다 (도 10a) · 비교시험에 사용한 배지는 Lysogeny broth(LB) , Fast idious broth(FB) 및 Super broth(SB)를 사용하였고, 동일한 배양조건 (37 °C 및 150 rpm 진탕 배양) 하에서의 세포의 질량과 항체절편 생산량을 비교하였다. 최종 OD600 값은 LB 3.69, FB 6.58 및 SB 11.48로 SB가 가장 높게 측정되었는데 이는 SB 배지에서 가장 많은 세포를 얻을 수 있음을 의미한다. 카파셀렉트 레진 (KappaSelect resin)을 이용한 친화성 크로마토그래피의 4번 레인 ( lane)들의 Fm302 샘플로 상대적 활성을 비교하였을 때 SB 배지가 LB 대비 5배의 높은 것은 SB 배지에서 존재하는 항체절편의 양이 LB배지에서 항체절편의 양 보다 5배 증가될 것으로 판단되었다 (도 10b) . In order to establish the optimal culture conditions, the antibody fragment production according to the culture was compared (Fig. 10a). As a medium used in the comparative test, Lysogeny broth (LB), Fast idious broth (FB), and Super broth (SB) were used. Cell mass and antibody fragment production were compared under the same culture conditions (37 ° C and 150 rpm shaking culture). The final OD600 values were LB 3.69, FB 6.58 and SB 11.48 with the highest SB, which means that the most cells can be obtained in SB medium. When comparing the relative activity with Fm302 samples of lane 4 of affinity chromatography using KappaSelect resin, SB medium was 5 times higher than LB in SB medium. The amount of antibody fragment was determined to be increased five times than the amount of antibody fragment in LB medium (Fig. 10b).
LB 배지에 항생제 클로람페니콜을 넣고 진탕 배양기 (shaking incubator)를 이용하여 37 °C 및 150 rpm의 조건으로 밤새 배양 하였다. 이후 새로운 SBplus 배지 (Gel l ix)에 전날 키운 세포배양배지를 약 1-2 %가 되도록 접종하고 항생제인 클로람페니콜을 넣고 진탕 배양기를 이용하여 37 °C 및 150 rpm의 조건으로 배양하였다. 접종 후 0D600 값이 3.0 일 때 1 mM IPTG을 이용하여 유도하였고, 25 °C 맟 150 rpm의 조건으로 9시간 동안 배양하였다. 이후 배양액은 4 °C , 8000 rpm으로 원심 분리하여 침전물 (pel let )을 얻었고, 이렇게 확보된 세포에 배양배지 1 L당 약 30-40 ιη£의 세포 용해 완층액 [lxPBS(pho,sphate buf fered sal ine) , 5 mM EDTACethylenediaminetetraacet ic acid) , 10% 글리세를, pH 7.4]을 첨가하여 세포와 흔합하였다. 이후 초음파 파쇄기를 이용하여 5분 동안 (pulse on 3초, pulse off 3초) 세포를 용해하였다. 수용액 상와 항체절편 및 단백질들과 세포를 분리하기 위하여 원심분리기를 이용하여 20,000 rpm의 속도로 40분 동안 원심분리를 실시하였다. The antibiotic chloramphenicol was added to LB medium and incubated overnight at 37 ° C. and 150 rpm using a shaking incubator. Thereafter, the cell culture medium grown on the new SBplus medium (Gel l ix) was inoculated to about 1-2%, and the antibiotic chloramphenicol was added thereto and incubated at 37 ° C. and 150 rpm using a shaking incubator. After inoculation, when 0D 600 value was 3.0, it was induced using 1 mM IPTG and incubated for 9 hours under conditions of 25 ° C 맟 150 rpm. Subsequently, the culture solution was centrifuged at 4 ° C. and 8000 rpm to obtain a pel let. The cells obtained in this manner were approximately 30-40 ηη lysis cell lysate per liter of culture medium [lxPBS (pho, sphate buf fered) sal ine), 5 mM EDTACethylenediaminetetraacetic acid) and 10% glycerol, pH 7.4] were added to the cells. The cells were then lysed for 5 minutes using an ultrasonic crusher (pulse on 3 seconds, pulse off 3 seconds). In order to separate the aqueous phase and the antibody fragments and the protein and the cell was centrifuged for 40 minutes at a speed of 20,000 rpm using a centrifuge.
이렇게 세포로부터 분리된 항체절편 및 단백질 수용액에서 항체절편만을 정제하기 위하여 친화성 크로마토그래피를 수행하였다. 개방 원통 (open column)에 항체절편의 CL 도메인과 결합하는 카파샐렉트 레진 (GE Heal thcare)을 충진하고, 평형 완충액 (equi 1 ibr ium buf fer ; 50 mM Tr is-HCl , 100 mM NaCl , 5 mM EDTA, pH 7.4)을 홀려주어 평형상태가 되도록 하였다. 항체절편 및 단백질 수용액을 컬럼에 홀려줌으로써 레진과 항체절편이 결합하도록 한 후, 5 CV( column volume)의 세척 완층액 (50 mM Tr i s-HCl , 100 mM NaCl , 5 mM EDTA, pH 7.4)를 홀려주어 비특이적으로 결합한 불순물들을 제거하였다. 카파셀렉트 레진으로부터 항체절편을 분리하기 위해 용출 완층액 (ehit ion buf fer ; 100 mM 글라이신, 1 mM EDTA, pH 2.5)을 홀려주었다. 실험결과에서 보듯이 Fm302 컨스트럭트를 pACYCDuetᅳ 1 백터에서 발현한 결과 항체절편은 가용성 형태로는 발현이 되었으며, 봉입체 ( inclusion body) 형태로는 발현되지 않음을 확인하였다. 1차 정제 결과 정제된 항체절편은 약 80%의 순도를 보였으며, 비환원 염색을 이용해 전기영동한 결과 경쇄 및 중쇄의 이질이량체를 형성하고 있음을 확인하였다 (도 11) . Affinity chromatography was performed to purify only antibody fragments from the antibody fragments and protein aqueous solution separated from the cells. Fill an open column with kappa select resin (GE Heal thcare) that binds to the C L domain of the antibody fragment and equilibrate buffer (equi 1 ibrium buf fer; 50 mM Tr is-HCl, 100 mM NaCl, 5 mM EDTA, pH 7.4) was removed to equilibrate. Resin and antibody fragments were bound to each other by pouring the antibody fragments and protein aqueous solution onto the column, and then washed with 5 CV (column volume) of the complete solution (50 mM Tr i s-HCl, 100 mM NaCl, 5 mM EDTA, pH 7.4). Was removed to remove nonspecifically bound impurities. Elution complete layer (ehit ion buf fer; 100 mM glycine, 1 mM EDTA, pH 2.5) was removed to separate antibody fragments from kappa select resin. As shown in the experimental results, the expression of the Fm302 construct in the pACYCDuet ᅳ 1 vector showed that the antibody fragment was expressed in soluble form and not in inclusion body form. As a result of the first purification, the purified antibody fragment showed about 80% purity, and electrophoresis using non-reduction staining resulted in the formation of heterodimers of light and heavy chains. It was confirmed (FIG. 11).
1차로 정제된 항체절편의 순도를 더욱 높이기 위하여 이온 교환 크로마토그래피를 수행하몄다, A TA 프라임 FPLC 사스템에 HiTrapSP HP 컬럼 (GE Heal thcare)을 연결하고 평형 완충액 (100 mM 글라이신, 1 raM EDTA, pH 2.5)을 홀려주어 평형상태가 되도록 하였다. 1차 정제 과정에서 용출된 항체절편 수용액 (pH 2.5)을 컬럼에 홀려줌으로써 레진과 항체절편이 결합하도록 한 후, 5 CV의 세척 완층액 1(50 mM MES, 1 mM EDTA, 2 mM DTT, pH 6.0) 및 세척완충액 2(50 mM MES, 20 mM NaCl , 1 mM EDTA, pH 6.0)를 홀려주어 비특이적으로 결합한 불순물들을 제거하:였다. 컬럼으로부터 항체절편을 분리하기 위하여 용출 완층액 (50 mM MES, 300 mM NaCl , 1 mM EDTA, pH 6.0)를 흘려주었다. 정제된 항체절편에 대하여 SDS-PAGE를 통해 확인한 결과, 세척 과정올 통해 작은 크기의 분순물들이 모두 제거되었으며, 용출 과정에서 항체절편이 모두 용출된 것을 확인하였다 (도 12a) . 이때의 순도는 90% 이상인 것으로 확인되었다 (도 12b) .  Ion exchange chromatography was performed to further increase the purity of the firstly purified antibody fragments. Connect a HiTrapSP HP column (GE Heal thcare) to an A TA prime FPLC system and equilibrate buffer (100 mM glycine, 1 raM EDTA, pH 2.5) was removed to equilibrate. Resin and antibody fragments were bound to each other by pouring an aqueous solution of antibody fragment (pH 2.5) eluted during the first purification step, followed by washing with 5 CV of the complete aqueous solution 1 (50 mM MES, 1 mM EDTA, 2 mM DTT, pH). 6.0) and Wash Buffer 2 (50 mM MES, 20 mM NaCl, 1 mM EDTA, pH 6.0) were removed to remove nonspecifically bound impurities. To separate antibody fragments from the column, eluted complete solution (50 mM MES, 300 mM NaCl, 1 mM EDTA, pH 6.0) was flowed. As a result of confirming the purified antibody fragments through SDS-PAGE, all the small-sized impurities were removed through the washing process, and it was confirmed that all the antibody fragments were eluted during the elution process (FIG. 12A). Purity at this time was confirmed to be 90% or more (FIG. 12B).
항체절편의 균질성을 확인하기 위해 크기 배제 크로마토그래피를 수행하였다. AKTA 프라임 FPLC 시스템에 HiLoad 16/600 Siiperdex 75 pg ¾럼½£ Heal thcare)을 연결하고 평형 완층액 (PBS , 10% 글리세를, 1 mM EDTA, 0.02% NaN3 , PH 7.2) )를 흘려주어 평형상태가 되도록 하였다. 2차 정제 과정에서 용출된 항체절편 수용액을 컬럼에 홀려주었고, 보유 용량 (retent ion volume)은 55-65 ^로 측정되었다. 크로마토그램에서 항체절편을 나타내는 280 nm 흡광도 및 SDS-PAGE 실험 결과로 미루어 보아, 항체절편의 균질성은 매우높은 것으로 생각된다 (도 13) . Size exclusion chromatography was performed to confirm homogeneity of the antibody fragments. AKTA prime equilibrium given a connection HiLoad 16/600 Siiperdex 75 pg column ½ ¾ £ Heal thcare) a FPLC system, and flowing a balanced complete cheungaek (PBS, 10% glycero, 1 mM EDTA, 0.02% NaN3 , P H 7.2)) It was made to be in a state. The antibody fragment solution eluted during the second purification was poured into the column, and the retention ion volume was measured at 55-65 ^. From the results of 280 nm absorbance and SDS-PAGE experiments showing the antibody fragments in the chromatogram, the homogeneity of the antibody fragments is considered to be very high (FIG. 13).
C-말단에 Cys가 없어 이황화 결합이 불가능한 Fm301과 이황화 결합이 가능한 Fm302에 대한 비환원젤 전기영동 분석을 실시한 결과 Fm302의 경우 이황화 결합으로 인해 하나의 밴드가 나타나는 것을 확인할 수 있었다 (도 14) .  As a result of non-reduction gel electrophoresis analysis of Fm301, which cannot disulfide bond and Fm302 capable of disulfide bond because there is no Cys at the C-terminus, it was confirmed that one band appeared due to disulfide bond (FIG. 14).
Fw306 항체절편의 배양및 정제 Culture and Purification of Fw306 Antibody Fragments
Fm306의 정제를 위하여 숙주세포 및 풀라스미드는 1½302와 같은 E- col i C43(DE3) , pACYCDuet-1 백터를 이용하였으며 OmpA 산호 펩타이드를 이용하여 주변세포질에서 항체절편이 생성되도록 하였다. 씨드 (seed) 배양을 위하여 LB 배지에 항생제로 클로람페니콜을 넣고 진탕 배양기에서 37 °C 및 200 rpm으로 밤새 배양한다. 본배양을 위하여 SB+ 배지에 전날 키운 세포배양배지를 약 1-2 %가 되도룸 접종하고 항생제인 클로람페니콜을 넣고 진탕 배양기에서 37 'C 및 200 rpm으로 배양한다. 접종 후 약 3- 4시간이 경과하면 흡광도로 배양정도를 확인한다. 흡광도가 약 OD600=1.5 정도 되면 배양 배지에 1 mM IPTG을 넣어 유도하고 25 V 및 200 rpm으로 설정한 진탕 배양기에서 밤새 배양하였다. For the purification of Fm306, the host cell and the fulllasmid were used for E-col i C43 (DE3) and pACYCDuet-1 vectors such as 1½302, and antibody fragments were generated in the periplasm by using OmpA coral peptides. Seed For incubation, put chloramphenicol as antibiotics in LB medium and incubate overnight at 37 ° C and 200 rpm in shake incubator. For the main culture, the cell culture medium grown on SB + medium was inoculated at about 1-2%, and the antibiotic chloramphenicol was added and incubated at 37 'C and 200 rpm in a shaker incubator. About 3 to 4 hours after inoculation, check the degree of culture by absorbance. When the absorbance was about OD600 = 1.5, 1 mM IPTG was added to the culture medium and incubated overnight in a shake incubator set at 25 V and 200 rpm.
이후 배양액은 4 °C 및 8000 rpm으로 원심 분리하여 침전물을 수득하였다. 이렇게 확보 ½ E-coli C43(DE3)에서 발현된 Fm306 항체절편을 고순도로 정제하기 위해 배양배지 1 L당 약 30-40 의: 세포 용해 완층액에 현탁시켰다. 구체적으로 인산나트륨완충액 (PBS, phosphate buffered saline), 5 mM EDTA (ethylenediaminetetraacet ic acid), 10 % 글리세롤을 포함하는 pH 7.4의 세포 용해 완충액에 세포를 현탁하였다. 이후 초음파 파쇄기를 이용하여 5분 동안 (pulse on 3초, pulse off 3초) 세포를 용해하였다. 수용액 상의 항체절편 및 단백질들과 세포를 분리하기 위하여 원심분리기를 이용하여 20,000 rpm의 속도로 40분 동안 원심붑리를 하였다. 이렇게 세포로부터 분리된 항체절편 및 단백질 수용액에서 항체절편만을 정제하기 위하여 친화성 크로마토그래피를 수행하였다. 개방 원통 (open column)에 항체절편의 CL 도메인과 결합하는 카파샐렉트 레진 (GE Healthcare)을 층진하고, 평형 완충액 (50 mM Tris-HCl, 100 mM NaCl, 5 mM EDTA, pH 7.3)을 홀려주어 평형상태가 되도록 하였다. 항체절편 및 단백질 수용액을 컬럼에 흘려줌으로써 레진과 항체절편이 결합하도록 한 후, 5 CV( column volume)의 세척 완층액 (50 mM Tris-HCl, 100 mM NaCl, 5 mM EDTA, pH 7.4)을 홀려주어 비특이적으로 결합한 블순물들을 제거하였다. 카파셀렉트 레진으로부터 항체절편을 분리하기 위해 용출 완층액 (100 mM 글라이신, 1 mM EDTA, pH 2.5)로 용출하였다 (도 15, 1 레인). The culture was then centrifuged at 4 ° C and 8000 rpm to obtain a precipitate. The Fm306 antibody fragments expressed in the secured ½ E-coli C43 (DE3) were suspended in about 30-40 of: cell lysis supernatant per liter of culture medium to purify with high purity. Specifically, the cells were suspended in a cell lysis buffer of pH 7.4 containing sodium phosphate buffer (PBS, phosphate buffered saline), 5 mM EDTA (ethylenediaminetetraacetic acid), and 10% glycerol. The cells were then lysed for 5 minutes using an ultrasonic crusher (pulse on 3 seconds, pulse off 3 seconds). In order to separate the antibody fragments and the proteins and the cells in the aqueous solution was centrifuged for 40 minutes at a speed of 20,000 rpm using a centrifuge. Affinity chromatography was performed to purify only antibody fragments from the antibody fragments and protein aqueous solution separated from the cells. Layered kappaselect resin (GE Healthcare), which binds to the C L domain of the antibody fragment, in an open column, and is poured into equilibration buffer (50 mM Tris-HCl, 100 mM NaCl, 5 mM EDTA, pH 7.3). To equilibrium. After allowing the antibody fragment and the antibody fragment to bind by flowing the antibody fragment and the protein aqueous solution to the column, 5 CV (column volume) of the washed complete solution (50 mM Tris-HCl, 100 mM NaCl, 5 mM EDTA, pH 7.4) Subjects removed nonspecifically bound impurities. To separate antibody fragments from kappaselect resin, they were eluted with an eluted supernatant (100 mM glycine, 1 mM EDTA, pH 2.5) (FIG. 15, lane 1).
이후 1차로 정제된 항체절편의 17 kD 주변의 4개 밴드 (도 15ᅳ 1 레인) 불순물을 제거하여 순도를 높이기 위하여 2차 정제로 이온 교환 크로마토그래피를 수행하였다. 개방 원통 (open cokmm)에 항체절편의 SP 레진 (GE Healthcare)을 충진하고, 평형 완층액 (100 mM glycine, 1 mM EDTA, pH 2.5)을 홀려주어 평형상태가 되도록 하였다. 항체절편 및 단백질 수용액을 컬럼에 홀려줌으로써 레진과 항체절편이 결합하도록 한 후, 5 CV( column volume)의 세척 완충액 (50 mM MESᅳ 20 mM NaCl , 1 mM EDTA, pH 6ᅳ 0)을 홀려주어 비특이적으로 결합한 블순물들을 제거하였다. 컬럼으로부터 항체절편을 분리하기 위하여 용출 완층액 (50 mM MES, 300 mM NaCl , 1 mM EDTA, pH 6.0)을 홀려주었다. 정제된 항체절편은 SDS-PAGE를 통해 확인하였다 (도 15, 2 레인) . Thereafter, ion band chromatography was performed by secondary purification in order to increase purity by removing four bands (FIG. 15 # 1 lane) impurities around 17 kD of the first purified antibody fragment. The open cylinder was filled with SP resin (GE Healthcare) of antibody fragments, and equilibrium complete layer solution (100 mM glycine, 1 mM EDTA, pH 2.5) was transferred to equilibrium. Antibody Fragments and Proteins The resin and antibody fragments were bound to each other by pouring the aqueous solution onto the column, and then 5 CV (column volume) washing buffer (50 mM MES ᅳ 20 mM NaCl, 1 mM EDTA, pH 6 ᅳ 0) was added to non-specifically bound soloon. The waters were removed. Elution buffer (50 mM MES, 300 mM NaCl, 1 mM EDTA, pH 6.0) was passed to separate antibody fragments from the column. Purified antibody fragments were confirmed by SDS-PAGE (FIG. 15, lane 2).
SDS-PAGE 결과에서 카파샐렉트 레진을 통한 1차 정제 이후, SP 레진을 이용한 이은교환 결과 타겟 단백질 이외의 17 kDa 주변의 불순물이 사라져 항체절편의 순도가 90 %이상 높아졌음을 확인할수 있다 (도 15) ,  In the SDS-PAGE results, after the first purification through kappaselect resin, it was confirmed that the impurities exchanged around 17 kDa other than the target protein disappeared as a result of the transfer by using the SP resin, thereby increasing the purity of the antibody fragment by 90% or more (Fig. 15),
Fm306-PEG결합체의 제조 Preparation of Fm306-PEG Binder
상기에서 기술한 방법으로 제조한 Fm306 항체절편의 페길화을 위하여 Fm306 정제 후 반웅 액에 1.5 M Tr is-Cl 완충액을 첨가하여 ph는 약 7, 5로 맞춘 후 페길화를 위하여 반웅 직전 신선한 PEG-maleimide(NANOCS)를 1: 10의 몰 비로 흔합하였다. 이후 실온의 교반기: 위에서 Fm306-PEG 혼합액을 흔합시키고, 반웅시간은 2시간으로 하였다. 반웅이 끝난 후 항체절편에 결합하지 않은 free-PEG를 제거하기 위하여 SP 레진을 이용하였다. Fm306-PEG 혼합액은 HC1 완층액을 이용하여 ph를 약 2.5로 낮춘 후 SP 레진에 결합시킨 후 동일한 ph의 조성올 갖는 완층용액 (100 mM Glycine ph2.5 , 1 mM EDTA)으로 20 CV 이상 층분히 세척해 주어 항체절편에 결합하지 않고 남아있는 free-PEG을. 제거하였고, Fm306-PEG 결합체를 획득하였다. 이 결합체는 반응액을 10 mM 인산나트륨 완충액 (PBS, ph7.3)으로 평형화시킨 슈퍼덱스 200 컬럼 (Superdex 200, GE Heal thcare)에 로딩 하였으며, 동일한 완충액을 이용하여 유속은 분 당 1 mi(l /min)로 상기 컬럼에서 용출 시켰다. Fm306-PEG 결합체 (도 16의 ③)는 Fm306(도 16의 ®)보다 분자량이 상대적으로 크므로 먼저 용출되는 특성을 이용하여 분리하였다. 각 분획을 SDS-PAGE를 이용한 비환원젤에서 Fni306-PEG 결합체는 약 100 kDa 이상의 사이즈로 나타났고 1½306은 45 kDa으로 확인되었다. 통상적으로 페길화 된 단백질은 PAGE 상에서 천천히 이동하여 본연의 사이즈를 나타내기 어렵다고 알려져 있다 (Anal Biochem. 1992 Feb 1; 200(2) :244-8) . 이를 환원젤로 확인한 결과 약 65 kDa , 28 kDa 사이즈에서 단백질이 확인되었는데, 이는 페길화 된 항체절편 (65 kDa)과 Fm306 항체절편의 경쇄부분으로 생각된다. 쇄부분의 부위-특이적 페길화로 인해 페길화 되지 않고 남은 경쇄부분이 확인되었으며, Fm306- PEG(30K)의 SDS-PAGE에서 중쇄부분이 70 kD에서 나타나고 이는 2개의 30 kD의 PEG이 반웅한 크기인 대략 85 kD크기로 계산되며 이보다 작은 크기로 나타나는 경향을 비교한 하였을 때 부위-특이적 페길화로 하나의 PEG 가 균질성이 있게 페길화 된 (PEGylated) 항체절편으로 생각된다. 이렇게 수득한 Fm306-PEG(20K) 결합체의 수율은 약 80% 로 확인되었으며 다음 실험은 PEGylat ion된 항체절편으로 진행하였다 (도 16) . For PEGylation of the Fm306 antibody fragment prepared by the above-described method, after the Fm306 purification, 1.5 M Tr is-Cl buffer was added to the reaction solution to adjust the pH to about 7, 5, and then fresh PEG-maleimide immediately before reaction for PEGylation. (NANOCS) were mixed in a molar ratio of 1:10. After stirring at room temperature: Fm306-PEG liquid mixture was mixed on the reaction, the reaction time was 2 hours. After the reaction, SP resin was used to remove free-PEG that did not bind to the antibody fragment. Fm306-PEG mixed solution was lowered to about 2.5 ph using HC1 complete solution, bound to SP resin, and then stratified over 20 CV with complete solution (100 mM Glycine ph2.5, 1 mM EDTA) with the same composition. Rinse the remaining free-PEG without binding to the antibody fragment. Removed and an Fm306-PEG conjugate was obtained. The conjugate was loaded onto a Superdex 200 column (Superdex 200, GE Heal thcare) equilibrated with 10 mM sodium phosphate buffer (PBS, ph7.3), and the flow rate was 1 mi (l) per minute using the same buffer. / min) eluted from the column. Fm306-PEG conjugate (3 in FIG. 16) was separated using the characteristics eluted first because the molecular weight is relatively larger than Fm306 (® in FIG. 16). In each fraction of non-reducing gel using SDS-PAGE, the Fni306-PEG conjugate was found to be about 100 kDa or more in size and 1½306 was 45 kDa. Normally, PEGylated proteins are known to migrate slowly on the PAGE, making it difficult to reveal their natural size (Anal Biochem. 1992 Feb 1; 200 (2): 244-8). As a result of confirming this as a reducing gel, about 65 kDa, 28 kDa Proteins were identified in size, which are thought to be the light chain portions of pegylated antibody fragments (65 kDa) and Fm306 antibody fragments. The site-specific PEGylation of the chain moiety resulted in the light chain moiety that remained unpegylated, and the heavy chain moiety appeared at 70 kD in the SDS-PAGE of Fm306-PEG (30K), indicating that the two 30 kD PEGs were responsive. Comparing the trends that appear to be about 85 kD in size and appearing in smaller sizes, site-specific PEGylation suggests that one PEG is homogeneously PEGylated antibody fragment. The yield of the thus obtained Fm306-PEG (20K) conjugate was confirmed to be about 80% and the next experiment proceeded to PEGylat ionized antibody fragment (Fig. 16).
Fm306-PEG(30K) 결합체도 상기 기술한 Fm30§-PEG(20K)와 동일한 방법을 이용하여 제작하였다.  Fm306-PEG (30K) conjugates were also prepared using the same method as described above for Fm30§-PEG (20K).
Fm306-PEG 결합체 (도 16의 ©)는 Fm306(도 16의 ⑧)보다 분자량이 상대적으로 크므로 먼저 용출되는 특성을 이용하여 분리하였다. 이를 환원젤로 확인한 결과 약 70 kDa 및 28 kDa 사이즈에서 두 개의 밴드가 확인되었는데, 이는 페길화 된 항체절편 (약 75 kDa) )과 Fm306 항체절편의 증쇄와 경쇄부분으로 생각된다. 상기의 Fm306-PEG(20K)와 동일하게 증쇄부분의 부위-특이적 페길화로 인해 페길화 되지 않고 남은 경쇄부분이 확인되었으며, 같은 이유로 하나의 PEG가 균질성이 있게 PEGylated 항체절편으로 생각된다. 이렇게 수득한 Fm306-PEG(30K) 결합체의 수율은 약 80%로 확인되었으며 다음 실험은 PEGylat ion 된 항체절편으로 진행하였다 (도 16) .  Fm306-PEG conjugate (© of FIG. 16) was separated using the eluting property because molecular weight is relatively larger than Fm306 (8 of FIG. 16). As a result of the reduction gel, two bands were identified at about 70 kDa and 28 kDa sizes, which are thought to be the light chain and light chain portions of the PEGylated antibody fragment (about 75 kDa) and the Fm306 antibody fragment. In the same manner as the Fm306-PEG (20K), the light chain portion remaining unpegylated due to site-specific PEGylation of the repetition portion was identified. For the same reason, one PEG is considered to be a homogeneous PEGylated antibody fragment. The yield of the Fm306-PEG (30K) conjugate thus obtained was confirmed to be about 80% and the next experiment was carried out with PEGylat ionized antibody fragments (FIG. 16).
Fab ' construct 선별 Fab 'construct screening
항체 절편 Fab' 의 CH1 도메인의 C-말단에 특이적으로 PEG를 도입하기 위하여 Fm302의 CH1 도메인 C-말단에 6개의 아미노산 서열 (THTCAA)을 추가한 Fm306을 페길화 컨스트럭트로 선정하였다. 페길화는 추가된 아미노산 서열 중 시스테인 잔기의 메르갑토기 (sul fhydryl ) 작용기와 PEG의 말단에 위치한 말레이미드 (maleimide) 작용기와의 반응을 통해 이루어진다. Fm306의 다른 위치에 존재하는 시스테인 잔기에는 페길화가 일어나지 않고 CH1 도메인의 C-말단에 페길화를 위해 추가된 아미노산 서열의 시스테인에만 페길화가 되는 : 것을 증명하기 위하여 Fm301 및 Fm302 컨스트럭트와의 비교 실험을 진행하였다. 1½3이은 C-말단에 시스테인이 없는 컨스트력트로서, 쇄내 이황화 결합 ( intra-chain disul f ide bond)을 이루는 시스테인 잔기에 페길화가 되지 않음을 증명하기 위한 대조군 컨스트럭트로 사용하였고, Fm302는 각 사슬의 C-말단에 시스테인이 있는 컨스트럭트로서 해당 시스테인 잔기에 페길화가 되지 않음을 증명하기 위해 선별하였다 (도 17) . 페길화된 Fab '의제조 In order to specifically introduce PEG into the C-terminus of the C H1 domain of the antibody fragment Fab ', Fm306 which added 6 amino acid sequences (THTCAA) to the C-terminus of the C H1 domain of Fm302 was selected as a PEGylation construct. PEGylation occurs through the reaction of a sul fhydryl group of cysteine residues with a maleimide group located at the end of PEG in the added amino acid sequence. Cysteine residues present in different positions of Fm306 has pegil upset occur without C H1 are only upset pegil cysteine of the amino acid sequence added to the C- terminus of the pegylated domain: Fm301 to demonstrate that And a comparative experiment with the Fm302 construct. 1½3 is a C-terminal cysteine-free construct, used as a control construct to demonstrate no pegylation of cysteine residues that make up an intra-chain disul fide bond, and Fm302 is used for each chain. A construct with cysteine at the C-terminus of C was selected to demonstrate no PEGylation at that cysteine residue (FIG. 17). Production of PEGylated Fab's
페길화된 Fm306(I¾306-PEG)의 제조를 위한여、 Fm306 정제 후, 반웅액에 1.5 M Tr is-HCl 완층액을 첨가하여 pH를 약 7.5로 맞추었고, Fm306와 PEG-maleimide의 비율이 1 대 10이 되도록 PEG-maleimide를 흔합하였다. 이후 실온에서 교반기를 이용하여 2시간 동안 반웅시켰다. Fm301 및 Fm302도 위와 같은 방법을 이용하여 정제 후, PEG를 첨가하여 페길화 반웅을 수행하였다. 페길화 컨스트럭트인 Fm306만 특이적으로 페길화 반웅이 이루어졌는지 확인하기 위하여 SDS^PAGE를 수행하였다 (도 18a) . 페길화 여부를 알아보기 위하여 각 컨스트럭트 별로 PEG을 반웅하지 않은 항체 절편 시료 (도 18a의 1, 3 및 5열)와 PEG을 첨강하여 페길화를 시도한 시료 (도 18a의 , 4 및 6열)를 각각 준비하였고 이를 환원젤에 로딩하였다. Fm301 , Fm302 및 Fm306 등 항체 절편 시료 (도 2a의 1ᅳ 3 및 5열) 및 Fm301과 Fm302에 PEG을 반웅한 시료 (도 18a의 2 및 4열)는 약 25 kDa 위치에서 항체 절편 시료의 경쇄부분과 증쇄부분을 확인할 수 있었으나, 1¾306에 PEG를 반웅한 시료 (도 18a의 6열)는 약 65 kDa의 페길화가 이루어진 증쇄부분과 25 kDa의 경쇄부분을 확인할 수 있었다. 이는 CH1 도메인의 말단에 추가된 아미노산의 시스테인에 특이적으로 페길화가 이루어졌음을 증명하는 결과이다. Fm301 및 Fm302에 PEG를 반웅한 시료 (도 18b의 2 및 4열)의 경우에도 PEG 분자가 존재하는 것을 PEG 염색을 통해 확인하였으나, 항체 절편과 같은 위치에서 나타나지 않았고 PEG 시료 (도 18b의 7열)와 비교하였을 때 같은 위치에서 관찰되는 것을 확인하였다 (도 18b) . 이는 PEG 분자가 항체 절편과 결합하지 않았음을 뜻하고, 따라서 항체 절편에 존재하는 쇄내 이황화 결합 및 사슬간 이황화 결합 ( inter¬ chain disul f ide bond)을 형성하는 시스테인 잔기에는 페길화가 이루어지지 않았음을 증명하는 결과이다。 동일한 시료에 대하여 항체 절편에 특이적으로 결합하는 항 -Fab 항체 및 PEG에 특이적으로 결합하는 항 -PEG 항체를 이용하여 웨스턴 블랏 (western blot )을 진행하였다. Fab 특이 항체 및 PEG 특이 항체를 사용하였을 때 모두 SDS-PAGE로 확인한 결과와 같은 위치에서 항체 절편 (도 18c의 6열) 및 PEG (도 18d의 6열)를 확인할 수 있었다。 For the preparation of PEGylated Fm 3 06 (I¾306-PEG), after Fm306 purification, the pH was adjusted to 7.5 by adding 1.5 M Tr is-HCl complete solution to the reaction solution, and the ratio of Fm306 to PEG-maleimide PEG-maleimide was mixed to make this one to ten. It was then reacted for 2 hours using a stirrer at room temperature. Fm301 and Fm302 were also purified using the same method, followed by PEGylation reaction by adding PEG. SDS ^ PAGE was performed to confirm whether the PEGylation construct Fm306 was specifically peggylated reaction (Fig. 18a). In order to determine whether pegylation is performed, the antibody fragment samples (eg, columns 1, 3, and 5 of FIG. 18A) that do not contain PEG for each construct and the sample that attempted PEGylation by adding PEG (FIG. 18A, 4, 6 and 6) ) Were prepared and loaded on a reducing gel. Samples of antibody fragments such as Fm301, Fm302 and Fm306 (columns 1 and 3 and 5 in FIG. 2A) and samples with PEG in Fm301 and Fm302 (columns 2 and 4 in FIG. 18A) are light chains of the antibody fragment samples at about 25 kDa positions. Although the part and the reprinted part could be confirmed, the sample reacted with PEG at 1¾306 (column 6 in FIG. 18A) was able to identify the reprinted part and the light chain part of 25 kDa, which were PEGylated at about 65 kDa. This proves that PEGylation is specific for the cysteine of the amino acid added at the end of the C H1 domain. PEG staining of the Fm301 and Fm302 sample (columns 2 and 4 of FIG. 18B) also confirmed the presence of PEG molecules by PEG staining, but did not appear in the same position as the antibody fragment and the PEG sample (column 7 of FIG. 18B). It was confirmed that it is observed in the same position when compared to the () (Fig. 18b). This PEG molecules, and means that you have not combined with the antibody fragment, according swaenae disulfide bond present in the chain between the antibody fragment and has a cysteine residue that forms a disulfide bond (inter ¬ chain disul f ide bond ) pegil upset been made The result was a western blot using the anti-Fab antibody that specifically binds to the antibody fragment and the anti-PEG antibody that specifically binds to PEG. When the Fab specific antibody and the PEG specific antibody were used, antibody fragments (column 6 in FIG. 18C) and PEG (column 6 in FIG. 18D) were confirmed at the same positions as confirmed by SDS-PAGE.
반웅이 끝난 후 항체 절편에 결합하지 않은 잔여 PEG를 제거하기 위하여 KappaSelect 레진이 층진된 컬럼에 Fm306-PEG 흔합액을 로딩하였다. 이후 완층용액 (50 mM Tr is-HCl , pH 8.0, 100 mM NaCl 및 5 mM EDTA)을 20 CV 이상 홀려주어 충분히 세척하여 잔여 PEG를 제전한 후 완충용액 ( 100 mM Glycine , pH 2.5, 1 mM EDTA)을 이용하여 용출하였다. 용출된 Fm306-PEG는 10 mM 인산나트륨 완충용액 (PBS, pH 7.4)으로 평형화시킨 슈퍼덱스 200 ¾럼(3叩6 6 200, GE Heal thcare)에 로딩하였으며,. 동일한 완층용액을 이용하여 유속은 분 당 1 m (l m«/min)로 상기 컬럼에서 용출시켰다. Fm301 및 Fm302에 대해서도 동일한 방법으로 잔여 PEG를 제거하였다. 용출한 시료를 SDS-PAGE를 이용하여 환원젤에서 관찰한 결과 Fn)306의 경우 (도 19a의 3열) 약 65 kDa 위치에서 PEG과 결합된 형태로 존재학는 중쇄부분이 존재함을 확인할 수 있었고, Fra301 및 Fm302의 경우 ( 19a의 1 및 2열) PEG과 결합된 항체 절편은 전혀 존재하지 않았다 (도 19a) . 위와 동일하게 PEG 염색을 통해서 Fm306 시료 (도 19b의 3열)에서만 같은 위치에 PEG가 존재함을 확인하였고, Fm301 및 Fm302 시료 (도 19b의 1 , 2열)에서는 PEG이 존재하지 않음을 확인하였다 (도 19b) . 이는 Fm301 및 1¾302의 경우 모든 PEG가 항체 절편과 결합되지 않고 정제 과정에서 제거되었음을 뜻한다, 상기 기술한 것과 동일한 방법을 이용하여 웨스턴 블랏을 진행하였고, 동일한 위치에서 항체 절편 (도 19c의 3열)과 PEG (도 19d의 3열)를 확인할 수 있었다.  After the reaction, the Fm306-PEG mixture was loaded on a KappaSelect resin layered column to remove residual PEG that did not bind to the antibody fragment. Subsequently, the complete layer solution (50 mM Tr is-HCl, pH 8.0, 100 mM NaCl and 5 mM EDTA) was poured in 20 CV or more to sufficiently wash the remaining PEG to remove residual PEG, and then the buffer solution (100 mM Glycine, pH 2.5, 1 mM EDTA). ) Was eluted. Eluted Fm306-PEG was loaded into Superdex 200 ¾ (3x6 6 200, GE Heal thcare) equilibrated with 10 mM sodium phosphate buffer (PBS, pH 7.4). Using the same complete solution, the flow rate was eluted from the column at 1 m (l m «/ min) per minute. Residual PEG was removed in the same manner for Fm301 and Fm302. As a result of observing the eluted sample on the reducing gel using SDS-PAGE, in the case of Fn) 306 (column 3 in FIG. 19A), the heavy chain portion was present in the form of binding to PEG at about 65 kDa. In the case of Fra301 and Fm302 (columns 1 and 2 of 19a), there were no antibody fragments bound to PEG (FIG. 19A). As above, PEG staining confirmed that PEG was present at the same position only in the Fm306 sample (column 3 of FIG. 19B), and PEG was not present in the Fm301 and Fm302 samples (columns 1 and 2 of FIG. 19B). (FIG. 19B). This means that in the case of Fm301 and 1¾302, all PEGs were not bound to the antibody fragments and were removed in the purification process. Western blot was performed using the same method as described above, and antibody fragments at the same position (column 3 in FIG. 19C) And PEG (column 3 of FIG. 19D) could be confirmed.
이러한 결과에 따르면 Fm306 컨스트럭트의 경우에만 항체 절편과 PEG가 결합되었고, Fm301 및 Fm302의 경우에는 항체 절편과 PEG가 결합하지 않았다. 따라서 1½306에 도입된 아미노산 서열 (THTCM)의 시스테인에만 부위-특이적 페길레이션이 일어났음을 확인하였고, ί½301의 쇄내 이황화 결합 및 Fm302 , 1½306의 쇄내 이황화 결합 및 사슬간 이황화 결합을 형성하고 있는 시스테인에는 페길화가 일어나지 않았음을 알 수 있다. 또한According to these results, the antibody fragment and PEG were bound only to the Fm306 construct, and the antibody fragment and PEG were not bound to the Fm301 and Fm302. Therefore, it was confirmed that site-specific pegylation occurred only in the cysteine of the amino acid sequence (THTCM) introduced at 1½306, and the intrachain disulfide bonds of ί½301 and the intrachain disulfide bonds and interchain disulfide bonds of Fm302, 1½306 were identified. It can be seen that PEGylation did not occur in the cysteine formed. Also
Fm306-PEG (도 18a의 6열 및 도 19a의 3열)의 경우 중쇄부분에 해당하는 밴드는 단 한 개만 존재함을 확인하였는데, 이는 단량체 (monomer i c) 페길화를 증명하는 결과이다. 따라서 상기 기술된 방법을 통해 부위-특이적 페길화 및 단량체 페길화를 확인하였다. In the case of Fm306-PEG (column 6 of FIG. 18a and 3column of FIG. 19a), only one band corresponding to the heavy chain portion was present, which is a result of promonizing monomer (monomer i c) PEGylation. Thus site-specific PEGylation and monomer PEGylation were identified via the methods described above.
EDMAN시뭔싱을 통한정제된 항체절편의 확인 Identification of Purified Antibody Fragments by EDMAN Sequencing
정제된 항체절편의 확인 및 항체절편 앞의 신호 펩타이드의 삭제유무를 확인하기 위하여 eMass analys i s Lab 사에 EDMAN 시퀀싱을 의뢰하여 정제된 항제절편의 확인과 N-말단의 신호 펩타이드의 제거를 확인하였다ᅳ 표 17에서 확인할 수 있듯이, 항체절편의 N 말답 서열은 경쇄, D-I-L-L-T및 중쇄 Q-V-Q-L-K로 확인되었다.  For confirmation of purified antibody fragments and deletion of signal peptides in front of antibody fragments, EDMAN sequencing was requested from eMass analys is Lab to confirm purified antibody fragments and to remove N-terminal signal peptides. As can be seen in Table 17, the N answer sequence of the antibody fragment was identified by the light chain, DILLT and heavy chain QVQLK.
【표 17]
Figure imgf000038_0001
실시예 5: 정제된 항체절편의 항원 결합 활성 측정
Table 17
Figure imgf000038_0001
Example 5: Determination of antigen binding activity of purified antibody fragments
정제된 단백질이 항체절편인지 확인하기 위해 CH3 항체를 이용한 웨스턴 블랏으로 확인하였고, 항원인 EGFR과의 결합능을 갖고 있는지 확인하기 위해 SPR 및 ELISA를 이용하여 결합 친화도를 측정하였다. SPR (surface plasmon resonance)^- 이용한 sEGFR-결합친화도 측정 In order to confirm whether the purified protein was an antibody fragment, it was confirmed by Western blot using a CH3 antibody, and binding affinity was measured by SPR and ELISA to confirm whether the antibody has a binding ability with EGFR. SEGFR-binding affinity measurement using surface plasmon resonance (SPR) ^-
XPR GLM 칩에 양성대조군 (세특시맙; Cetux imab)을 포함하는 항체절편 3종을 GLM 칩을 이용하여 코팅, EGFR-Fc를 홀려주어 비특이적 결합 여부를 확인 후 최적의 결합 조건을 확인하였다. 이를 바탕으로 다시 하나의 칩 위에 세특시맙 및 항체절편을 동일하게 코팅한 뒤 EGFR-Fc를 홀려주는 방식으로 결합력 평가를 수행하였다. 측정을 위하여 ProteOn XPR 36에 GLM" 칩을 50% 글리세를을 사용하여 초기화 시킨 다음 25°C 조건에서 러닝 완충액 (running buf fer ; 10 mM Na-phosphate , 150 mM NaCl 및 0.005% Tween20을 포함하는 PBS, PBST, pH 7.4)를 흘려주어 칩 안정화를 시켜주었다. GLM 칩 4개 채널에 1:1 비율의 0.04 M N-ethyl-N' -(3- dimethylaminopropyl) carbodi imide (EDC)와 0.001 M sulfo- Nhydroxysuccinimide(sulf으 NHS)을 30 / /min의 유속으로 220 μί 흘려주어 채널을 활성화 시켰다. 세특시맙 및 항체절편, 각 100 nM을 pH 5.5 아세테이트 완층액를 사용하여 30 βί/min 속도로 코팅하였다. 그 다음 1M Ethanolamine-HCl (pH 8.5)로 활성화된 칩을 불활성화 시키고 고정화 정도 (immobilization level )7> 각각 605-4340 RUC.resonance units)가 되었음을 확인하였다. PBST로 기준을 정한 다음 EGFR-Fc 5 - 50 nM로부터 1/2 씩 희석한 5가지 일련의 농도를 만들어 코팅.된 곳에 홀려보냄으로써 KD 값을 구하였다. 재생 완층액 (Regeneration buffer; 50 mM NaOH)를 이용하여 제로베이스 (zero base)를 확인 한 후 반복실험을 하여 최적 조건하에 최종 결과 값을 도출하였다. Three antibody fragments containing the positive control group (Sespecialsimab; Cetux imab) on the XPR GLM chip were coated with a GLM chip, and EGFR-Fc was cast to confirm non-specific binding and then confirmed optimal binding conditions. Based on this, the adhesion test was performed in such a way that three specific simab and antibody fragments were coated on the same chip again and EGFR-Fc was cast. For measurement, the ProteOn XPR 36 was initialized with 50% glycerol and then PBS containing running buf fer (10 mM Na-phosphate, 150 mM NaCl and 0.005% Tween20) at 25 ° C. , PBST, pH 7.4) to stabilize chips I let you. One-to-one ratio of 0.04 M N-ethyl-N '-(3-dimethylaminopropyl) carbodi imide (EDC) and 0.001 M sulfo- Nhydroxysuccinimide (NHS) in four channels of GLM chip at a flow rate of 220/30 min. μί was activated to activate the channel. Sefeximab and antibody fragments, each 100 nM, were coated at a rate of 30 βί / min using a pH 5.5 acetate complete solution. Then, the chip activated with 1M Ethanolamine-HCl (pH 8.5) was inactivated and the immobilization level7> 605-4340 RUC.resonance units, respectively. KD values were obtained by setting the standard in PBST and then making 5 series of concentrations diluted by 1/2 from EGFR-Fc 5-50 nM and sending it to the coated. After confirming the zero base using a regeneration buffer (Regeneration buffer; 50 mM NaOH), the repeated results were obtained under the optimal conditions.
세특시맙 및 항체절편을 하나의 칩 위에 동¾하게 S팅하여 EGFR을 동일한 농도로 홀려주었을 때 세툭시맙의 EGFR에 대한 결합 정도가 항체절편과는 민감도 부분에서 차이를 보여 세특시맙의 경우는 EGFR-Fc의 농도를 5 nM부터 시작하여 키네틱 (kinetic) 값,을 구하였고 다른 항체절편들은 50 nM을 시작으로 하는 EGFR의 농도에서 최적조건을 찾아 5회 이상 반복하여 평균값을 구하였다. 그 결과 세특시맙 17.5 pM, 세특시맙- Fab 3.45 nM, FM302 720 pM, FM302-GPC 1.84 nM로 확인되었다 (표 18 및 도 20a 내지 20b). 세툭시맙과 비교하였을 때, 1½302의 kD 값은 다소 높은 것으로 측정되었다. 하지만 세툭시맙 -Fab보다 낮은 kD 값을 갖는 것으로 측정된 것으로 미루어 보아 EGFR에 대한 결합 친화도는 층분히 높은 것으로 판단된다, 또한 Fm306PEG의 경우에도 세특시맙 -Fab의 kD 값보다 낮은 값을 갖는 것으로 측정되었는데, 이는 페길화에 의한 결합 친화도의 감소가 크지 않음을 의미한다 .  When three special sib and antibody fragments were simultaneously put on one chip and EGFR was injected at the same concentration, the degree of binding of cetuximab to EGFR was different in sensitivity compared to the antibody fragment. The EGFR-Fc concentration was determined from 5 nM, and the kinetic value was obtained. Other antibody fragments were determined by optimizing the EGFR concentration starting at 50 nM and repeated five times or more. As a result, it was confirmed that Sefeximab 17.5 pM, Sefeximab- Fab 3.45 nM, FM302 720 pM, FM302-GPC 1.84 nM (Table 18 and FIGS. 20A to 20B). Compared to cetuximab, the kD value of 1½302 was determined to be somewhat high. However, the binding affinity for EGFR seems to be considerably higher, as measured by having a lower kD value than cetuximab-Fab, and in the case of Fm306PEG, it has a lower value than the kD value of sefeximab-Fab. It means that the decrease in binding affinity by PEGylation is not large.
【표 18]  Table 18
Figure imgf000039_0001
Fm302 2.60 X 105 1.87 X 10"4 7.20 10"10 . 96.42 3.79
Figure imgf000039_0001
Fm302 2.60 X 10 5 1.87 X 10 "4 7.20 10 " 10 . 96.42 3.79
Fm306PEG 2.41 X 105 4.42 X 10 1.84 X 10"9 43.91 2.02 Fm306PEG 2.41 X 10 5 4.42 X 10 1.84 X 10 "9 43.91 2.02
ELISA를 이용한 sEGFR-결합친화도 측정 SEGFR-binding affinity measurement using ELISA
96 웰 ELISA 플레이트에 항원 sEGFR (100 ng/웰)을 넣고 4°C 에서 밤새 반웅하여 플레이트 표면에 항원을 고정화한 :뒤, 상층액을 제거하고 블로킹 용액 (Sigma , B6429-500ML) 200 ^를 각 웰에 분주하여 4°C 에서 밤새 블로킹 하였다. 검정곡선을 얻기 위한 표준물질인 세툼시맙과 정제된 항체절편은 PBS를 이용하여 0-125 ng/ ^의 농도를 도록 회석하였다. 이를 각각 100 ^씩 분주한 뒤 실온에서 1시간 반웅하여 항원과 결합하도록 하고, 반웅이 끝난 후 PBST(PBS, 0.05% tween 20, pH 7,4)로 3회간 세척하였다. 항 -인간 IgG(Sigma, 15260)를 1/1 , 000 희석하여 100 씩 각 웰에 분주하고 실온에서 1시간 반응한 후, 상층액을 제거하고 PBST로 3회 세척하였다. 2차 항체 항 -고트 IgG-Per oxidase (Sigma, A5420)를 l/ OQ0 회섞하여 100 ^씩 각 웰에 분주하고 실온에서 1시간 반웅하였다. 상층액을 거하고 PBST로 3회 세척한 후, TMB (발색시약을 100 ^씩 분주하였다. 발색된 웰에 1 M의 H2S04를 100 ^씩 넣어 반웅을 중지시키고, 마이크로플레이트 리더기를 이용해 450 ran 파장의 흡광도를 측정하였다. 세특시 : 대비 Fm302의 결합 친화도는 약 47.2%인 것으로 측정되었으며 이는 EGFR에 대한 항체절편의 활성이 층분히 유지되는 것을 뜻한다. 또한, Fm306PEG의 결합 친화도는 Fm302 대비 약 32.2% 감소하였는데, 이것으로 미루어보아 페길화에 의한 결합 친화도 감소가 크지 않아 약 70¾>의 활성을 유지할 수 있음을 뜻한다 (도 21) . Add antigen sEGFR (100 ng / well) to a 96 well ELISA plate and react at night at 4 ° C to immobilize the antigen on the plate surface: Remove supernatant and remove blocking solution (Sigma, B6429-500ML) 200 ^ each The wells were aliquoted and blocked overnight at 4 ° C. Septumsimab and purified antibody fragments, which were used as standard to obtain the calibration curve, were lysed to a concentration of 0-125 ng / ^ using PBS. Each 100 ^ was dispensed and reacted for 1 hour at room temperature to bind to the antigen, and after the reaction was finished, washed three times with PBST (PBS, 0.05% tween 20, pH 7,4). After diluting anti-human IgG (Sigma, 15260) 1/1, 000 in each well and reacting at room temperature for 1 hour, the supernatant was removed and washed three times with PBST. Secondary antibody anti-goat IgG-Per oxidase (Sigma, A5420) was mixed with l / OQ0 in 100 ^ aliquots into each well and reacted at room temperature for 1 hour. The supernatant was removed and washed three times with PBST , and then TMB (coloring reagent was dispensed 100 ^ each). The reaction was stopped by adding 1 M of H2S04 100 ^ to the colored wells, and the 450 ran wavelength using a microplate reader. Absorbance of Fm302 was determined to be about 47.2%, indicating that the activity of antibody fragments to EGFR was well maintained, and that the binding affinity of Fm306PEG was compared to Fm302. It was reduced by about 32.2%, which means that the decrease in binding affinity due to PEGylation was not significant, so that the activity of about 70¾> could be maintained (FIG. 21).
A431 cell line에서 정제된 Fab의 EGFR의 인산화 확인 Phosphorylation of EGFR from Fabs Purified in A431 Cell Line
배양 중인 A431 세포를 0.25% 트립신 /EDTA 처리하여 단일 세포로 떼어낸 후 배양접시에 1 X 106 개의 세포를 접종하여 24시간 동안 안정화 시킨 후 혈청이 포함되지 않은 배지로 8시간 배양 후 30 /m«의 항체 및 항체절편을 처리하였다. 배양 증인 세포에 새로운 배지로 교체해 준 다음 배양접시로부터 세포를 회수하여 cel l signal ing사의 Pathscan total EGF receptor sandwich EL ISA ki t에서 제시하는 방법에 만학다. A431 cells in culture were treated with 0.25% trypsin / EDTA, separated into single cells, inoculated with 1 x 10 6 cells in a culture plate, stabilized for 24 hours, and cultured for 8 hours in a serum-free medium. Antibodies and antibody fragments were treated. Replace the cells with culture medium and replace the cells with fresh medium. Then, the cells are recovered from the culture dish and Pathscan total EGF from cel l signaling company. Receptor sandwich EL ISA ki t based on the method proposed.
EGFR이 과발현되는 A431 세포에 항체절편을 처리하였을 때 항체에 의해 EGFR 인산화가 블로킹되어 phospho-EGF 수용체 (Try845)의 결합이 억제됨을 확인하였으며, 세툭시맙 유래 세툭시맙 Fab와 Fm302는 30 에서 35¾-40%정도의 인산화의 억제능을 확인하였다 (도 22) . 실시예 6: Fm302와 Fra306-PEG의 질환동물모델에서의 효능 확인 정제된 항체절편 Fab가 두경부암 질환동물모델 °f서의 항암효능효과를 확인하였다. 두경부암 질환동물모델을 제작하기 위히 흉선이 결핍되어 있어서 면역기능과 관련된 T 세포가 생성되지 않아 면역성이 결핍된 누드마우스에 A431 세포를 배양하여 1 X 107 개의 암세포를 피하에 투여하여 인간 종양 이종 이식 질환동물을 제작하였다. 종양 조직이 샛성되어 크기가 약 50-100 rarf일 때 항체절편 및 약물 투여를 시작하였고, 약물은 마우스 당 0.25 mg씩 꼬리에 정맥주사로 주 2회 투여하였다. 총 투여는 3주 동안 6회 투여하였다。 종양조직의 크기 변화는주 2회, 약물 투역 전에 수행하였다. 질환동물모델의 종양조직은 마우스당 주 2회 0.25 를 처리하였을 경우 3주차에서 세특시맙보다는 종양 성장억제능이 낮지만 세특시맙 Fab에 비해 1½302가 종양의 성장이 억제능 높음을 확인하였고, I½306-PEG의 경우도 세툭시맙 Fab보다 반감기의 증가로 종양 성장의 억제 효과가 높음을 확인하였다 (도 23) . When antibody fragments were treated to A431 cells overexpressing EGFR, EGFR phosphorylation was blocked by the antibody to inhibit the binding of the phospho-EGF receptor (Try845), and cetuximab-derived cetuximab Fab and Fm302 were 30 to 35¾ The inhibitory effect of phosphorylation of about -40% was confirmed (FIG. 22). Example 6 Confirmation of Efficacy of Fm302 and Fra306-PEG in Disease Animal Models The purified antibody fragment Fab confirmed the anticancer effect of the head and neck cancer disease animal model ° f. Head and neck cancer diseases to produce an animal model wihi thymus is the administration of 1 X 10 7 of tumor cells by T cells has not been generated cultured A431 cells in nude mice with immune deficiency associated with immune function in lacking a subcutaneous human tumor two kinds of Transplant Disease Animals were produced. Antibody fragments and drug administration were started when tumor tissues formed and the size was about 50-100 rarf, and the drug was administered twice a week by intravenous injection in the tail of 0.25 mg per mouse. Total administration was administered 6 times for 3 weeks. Tumor size change was performed twice a week, prior to drug administration. Tumor tissue of the disease animal model was found to be less tumor growth inhibitory than Sefeximab at 3 weeks when 0.25 treated twice a week, but 1½302 showed higher inhibition of tumor growth than Sefeximab Fab. I½306- PEG also showed a higher inhibitory effect on tumor growth with increased half-life than cetuximab Fab (FIG. 23).
두경부암질환모델의 실험 종료시점에 부검을 수행하여 종양조직의 무게를측정한 결과 종양의 성장 곡선과 비슷한 경향을 확인하였다 (도 24) . 정제된 항체절편의 약동학적 (PK; Pharmacokinetics) 분석  At the end of the experiment of the head and neck cancer disease model, an autopsy was performed and the weight of tumor tissue was measured, confirming a trend similar to that of the tumor growth curve (FIG. 24). Pharmacokinetics (PK) analysis of purified antibody fragments
정제된 항체절편을 실험동물에 마우스 당 0.25 mg씩 꼬리에 정맥주사한 후 시간별로 안와채혈을 하여 혈액을 채취하였다. 채취한 혈액을 3000 rpm에서 10분 동안 원심분리하여 혈장을 분리하였고 분리한 혈장내에 존재하는 투여된 항체절편의 양을 측정하였다.  Purified antibody fragments were injected intravenously into the tail by 0.25 mg per mouse in experimental animals, and blood was collected by orbital blood collection over time. The collected blood was centrifuged at 3000 rpm for 10 minutes to separate plasma and the amount of antibody fragments present in the separated plasma was measured.
혈액내의 항체절편의 농도측정 결과 Fab, Fm302의 경우 혈중 농도의 반감기는 투여 후 4시간 이내로 확인되었고, 페길화 된 ί¾306의 경우 혈중 농도의 반감기가 페길화 전보다 5배 이상 증가하는 것을 확인하였다. 그리고 Fm306-PEG(20 kD)는 Fm306-PEG(30 kD)의 혈중 농도의 반감기가 18.6 hr에서 28 ,31 hr로 증가함을 확인하였다 (표 19) , As a result of the measurement of antibody fragments in the blood, the half-life of blood concentration was confirmed in Fab, Fm302 within 4 hours after administration, and in the case of pegylated ί¾306, the half-life of blood concentration was increased by more than 5 times than before PEGylation. And Fm306-PEG (20 kD) was confirmed that the half-life of the blood concentration of Fm306-PEG (30 kD) increased from 18.6 hr to 28,31 hr (Table 19),
【표 19]  Table 19
Figure imgf000042_0001
이상으로 본 발명의 특정한 부분을 상세히 기专하였:는바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적인 기술은 단지 바람직한 구현예일 뿐이며, 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항과 그의 등가물에 의하여 정의된다고 할 것이다.
Figure imgf000042_0001
The specific parts of the present invention have been described in detail above, and it should be apparent to those skilled in the art that such specific techniques are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. . Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims

【특허청구범위】 [Patent Claims]
ί청구항 1】  ί claim 1
EGFR(Epi dermal Growth Factor Receptor)에 특이적으로 결합하며 다음의 영역을포함하는 Fab(Fragment ant igen-binding) 단편:  Fragment ant igen-binding (fab) fragments that specifically bind to Epi dermal Growth Factor Receptor (EGFR) and include the following regions:
(a) 서열목록 제 4 서열의 아미노산 서열을 포함하는 중쇄 가변 영역 (VH) ; (a) a heavy chain variable region (V H ) comprising the amino acid sequence of SEQ ID NO: 4;
(b) 서열목록 제 5 열의 아미노산 서열을 포함하는 증쇄 불변 영역  (b) a constant chain constant region comprising the amino acid sequence of SEQ ID NO: 5
(c) 서열목록 제 6 서열의 아미노산 서열을 포함하는 경쇄 가변 영역 (VL) ; 및 (c) a light chain variable region (V L ) comprising the amino acid sequence of SEQ ID NO: 6; And
(d) 서열목록 제 7 열의 아미노산 서열을 포함하는 경쇄 불변 영역 (CL) . (d) a light chain constant region (C L ) comprising the amino acid sequence of SEQ ID NO: 7.
【청구항 2] [Claim 2]
제 1 항에 있어서, 상기 Cm 은 그의 C-말단에 ys-Asp-Lys 올 추가적으로 포함하는 것을 특징으로 하는 Fab단편.  The Fab fragment of claim 1, wherein the Cm further comprises ys-Asp-Lys all at its C-terminus.
【청구항 3】 [Claim 3]
제 1 항에 있어서, 상기 CL은 그의 C-말단에 GkrCys 을 추가적으로 포함하는 것을 특징으로 하는 Fab 단편 . The Fab fragment of claim 1, wherein the C L further comprises GkrCys at its C-terminus.
【청구항 4】 [Claim 4]
제 2 항에 있어서, 상기 CH1은 그의 C-말단에 있는 Cys-Asp-Lys 에 Thr-His-Thr-Cys-Ala-Ala 이 추가적으로 결합된 것을 특징으로 하는 Fab 단편. The Fab fragment of claim 2, wherein the C H1 further binds to Cys-Asp-Lys at the C-terminus of Thr-His-Thr-Cys-Ala-Ala.
【청구항 5】 [Claim 5]
제 1 항 내지 제 4 항 중 어느 한 항에 있어서ᅳ 상기 Fab 단편은 페길화 (PEGylat ion) 된 것을 특징으로 하는 Fab 단편.  The Fab fragment according to any one of claims 1 to 4, wherein the Fab fragment is PEGylated (PEGylat ion).
【청구항 6] 제 5 항에 있어서, 상기 Fab 단편은 그회 (: 이 길화 된 것을 특징으로 하는 Fab단편 . [Claim 6] 6. The Fab fragment according to claim 5, wherein said Fab fragment is lengthened.
【청구항 7] [Claim 7]
제 6 항에 있어서, 상기 Fab 단편은 그의 CH1의 C-말단 부위에 있는7. The Fab fragment of claim 6 wherein the Fab fragment is at the C-terminal portion of its C H1
Thr-H i s-Thr-Cys-A la-Al a 에서 Cys 잔기가 페길화 된 것을 특징으로 하는 Fab단편 . Fab fragment characterized by pegylation of Cys residue in Thr-H i s-Thr-Cys-A la-Al a.
【청구항 8】 [Claim 8]
제 5 항에 있어서, 상기 페길화에 이용되는 PEG(Polyethyl ene glycol )는 분자량이 5-50 kDa인 것을 특징으로 하는 Fa 단 L  The method of claim 5 wherein the PEG (Polyethyl ene glycol) used for PEGylation Fa stage L, characterized in that the molecular weight of 5-50 kDa
【청구항 9] [Claim 9]
제 8항에 있어서, 상기 PEG는 분자량이 18-25 kDa인 것을 특징으로 하는 Fab단편 .  9. The Fab fragment of claim 8 wherein the PEG has a molecular weight of 18-25 kDa.
【청구항 10】 [Claim 10]
제 2 항에 있어서, 상기 VL은 그의 C-말단에 Gl -Cys 을 추가적으로 포함하며, 상기 (½의 C-말단에 있는 Cys-Asp-Lys 의 Cys 잔기와상기 VL의 C-말단에 있는 Glu-Cys 의 Cys 잔기가 이황화 결합된 것을 특징으로 하는 Fab단편 . The method of claim 2 wherein the V L is in its C- terminal and further includes a Gl -Cys, wherein (in the C- terminus of the Cys moiety of Cys-Asp-Lys at the C- terminus of the V L and ½ Fab fragment, characterized in that the Cys residues of Glu-Cys disulfide bond.
[청구항 113 [Claim 113]
제 1 항에 있어서, 상기 Fab 단편은 마우스 (Mus musculus )에서의 반감기가 20-35 시간인 것을 특징으로 하는 Fab 단편.  The Fab fragment of claim 1, wherein the Fab fragment has a half-life in mouse (Mus musculus) of 20-35 hours.
【청구항 12] [Claim 12]
EGFRCEpi dermal Growth Factor Receptor )에 특이적으로 결합하는 FabCFragment ant igen-binding) 단편을 제조하기 위한 다음을 포함하는 발현 컨스트럭트: (a) 다음을 포함하는 중쇄 -발현 컨스트럭스트: (a-1) 서열목록 제 9 서열의 뉴클레오타이드 서열을 포함하는 중쇄 가변 영역 (VH)-코딩 핵산분자; 및 (a-2) 서열목록 제 10서열의 뉴클레오타이드 선열을 포함하는 증쇄 불변 영역 1(CH1)-코딩 핵산분자; 그리고, Expression constructs for preparing FabCFragmentant igen-binding) fragments that specifically bind to EGFRCEpi dermal Growth Factor Receptor): (a) a heavy chain-expressing construct comprising: (a-1) a heavy chain variable region (V H ) -coding nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 9 sequence; And (a-2) a constant constant region 1 (C H1 ) -encoding nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO: 10 sequence; And,
(b) 다음을 포함하는 경쇄 -발현 컨스트릭스트 : (b-1) 서열목록 제 11 서열의 뉴클레오타이드 서열을 포함하는 경쇄 가변 영역 (VL)—코딩 핵산분자; 및 (b-2) 서열목록 제 12서열의 뉴클레오타이드 서열을 포함하는 경쇄 불변 영역 (CL)-코딩 핵산분자. 【청구항 13】 (b) a light chain-expressing construct comprising: (b-1) a light chain variable region (V L ) —coding nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 11 sequence; And (b-2) a light chain constant region (C L ) -encoding nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 12. [Claim 13]
상기 제 12 항의 발현 컨스트릭트를 포함하는 재:조합 백터 . ί청구항 14】  Recombinant vector comprising the expression construct of claim 12. ί claim 14】
상기 제 13 항의 재조합 백터로 형질전환된 숙주세포.  A host cell transformed with the recombinant vector of claim 13.
【청구항 15】 [Claim 15]
제 14 항에 있어서, 상기 숙주세포는 대장균인 것을 특징으로 하는 숙주세포. 【청구항 16】  15. The host cell according to claim 14, wherein the host cell is Escherichia coli. [Claim 16]
다음의 단계를 포함하는 EGFR(Epi dermal Growth Factor Receptor )에 특이적으로 결합하는 FaMFragment ant igen-binding) 단편의 제조방법 :  Method for preparing a FaMFragment ant igen-binding (EGF) fragment that specifically binds to EGFR (Epi dermal Growth Factor Receptor) comprising the following steps:
(a) 상기 제 14항 또는 제 15 항의 숙주세포를 배양하는 단계 ; 및 (a) culturing the host cell of claim 14 or 15; And
(b) 상기 숙주세포에서 EGFR에 대한 Fab단편을 발현시키는 단계 . (b) expressing a Fab fragment for EGFR in said host cell.
【청구항 17】 [Claim 17]
(a) 상기 제 1 항의 EGFR(Epidermal Growth Factor Receptor)에 특이적으로 결합하는 FaMFragment ant igen-binding) 단편의 약제학적 유효량; 및 (b) 약제학적으로 허용되는 담체를 포함하는 암에 대한 예방 또는 치료용 약제학적 조성물. 【청구항 18】 (a) a pharmaceutically effective amount of a FaMFragment ant igen-binding (EGFR) fragment that specifically binds to the Epidermal Growth Factor Receptor (EGFR) of claim 1; And (b) a pharmaceutical composition for preventing or treating cancer comprising a pharmaceutically acceptable carrier. [Claim 18]
제 17 항에 있어서, 상기 암은 유방암, 대장암, 폐암, 위암, 간암, 혈액암, 골암, 췌장암, 피부암, 뇌암, 자궁암, 비인두암, 후두암, 결장암, 난소암, 직장암, 대장암, 질암, 소장암, 내분비암, 갑상선암, 부갑상선암, 요관암, 요도암, 전립선암, 기관지암, 방광암, 신장암 또는 골수암인 것을 특징으로 하는 약제학적 조성물.  18. The method of claim 17, wherein the cancer is 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, colon cancer, ovarian cancer, rectal cancer, colon cancer, vaginal cancer, A pharmaceutical composition comprising small bowel cancer, endocrine cancer, thyroid cancer, parathyroid cancer, ureter cancer, urethral cancer, prostate cancer, bronchial cancer, bladder cancer, kidney cancer or bone marrow cancer.
PCT/KR2015/013322 2015-04-23 2015-12-07 Fab fragment specifically binding to egfr WO2016171365A1 (en)

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WO2002100348A2 (en) * 2001-06-13 2002-12-19 Genmab A/S Human monoclonal antibodies to epidermal growth factor receptor (egfr)
US20080085277A1 (en) * 2004-06-18 2008-04-10 Ambrx, Inc. Novel antigen-binding polypeptides and their uses
US20100008929A1 (en) * 2001-06-13 2010-01-14 Genmab A/S Human monoclonal antibodies to epidermal growth factor receptor (egfr)
KR20120118516A (en) * 2010-09-17 2012-10-29 주식회사 아이지세라피 A human anti-epidermal growth factor receptor fab antibody and a pharmaceutical composition for treating tumor comprising the same
KR20140126638A (en) * 2013-04-23 2014-10-31 신일제약주식회사 A New Antibody Fragment for EGFR

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* Cited by examiner, † Cited by third party
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WO2002100348A2 (en) * 2001-06-13 2002-12-19 Genmab A/S Human monoclonal antibodies to epidermal growth factor receptor (egfr)
US20100008929A1 (en) * 2001-06-13 2010-01-14 Genmab A/S Human monoclonal antibodies to epidermal growth factor receptor (egfr)
US20080085277A1 (en) * 2004-06-18 2008-04-10 Ambrx, Inc. Novel antigen-binding polypeptides and their uses
KR20120118516A (en) * 2010-09-17 2012-10-29 주식회사 아이지세라피 A human anti-epidermal growth factor receptor fab antibody and a pharmaceutical composition for treating tumor comprising the same
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