WO2016171365A1 - Fragment fab se liant spécifiquement à l'egfr - Google Patents

Fragment fab se liant spécifiquement à l'egfr Download PDF

Info

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
Authority
WO
WIPO (PCT)
Prior art keywords
cancer
fab fragment
egfr
fab
fragment
Prior art date
Application number
PCT/KR2015/013322
Other languages
English (en)
Korean (ko)
Inventor
김영필
김혜림
유희정
이성환
정태근
하종렬
Original Assignee
신일제약주식회사
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from KR1020150126894A external-priority patent/KR101770559B1/ko
Application filed by 신일제약주식회사 filed Critical 신일제약주식회사
Priority to US14/914,608 priority Critical patent/US20170145101A1/en
Publication of WO2016171365A1 publication Critical patent/WO2016171365A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction 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

La présente invention concerne : un fragment de liaison à l'antigène fragment (Fab) se liant spécifiquement à un récepteur du facteur de croissance épidermique (EGFR) ; une construction d'expression pour préparer le fragment Fab ; un procédé de préparation du fragment Fab ; et une composition pharmaceutique contenant le fragment Fab. Dans la mesure où le fragment Fab se liant spécifiquement à un EGFR est petit comparé à un anticorps, le fragment Fab présente un bon taux d'infiltration pour les tissus ou les tumeurs et peut être préparé dans des bactéries, réduisant de là les coûts de production. De plus, le fragment Fab se liant spécifiquement à un EGFR fournit une demi-vie in vivo accrue à travers la PEGylation.
PCT/KR2015/013322 2015-04-23 2015-12-07 Fragment fab se liant spécifiquement à l'egfr WO2016171365A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US14/914,608 US20170145101A1 (en) 2015-04-23 2015-12-07 Fab FRAGMENT SPECIFICALLY BINDING TO EGFR

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
KR10-2015-0057262 2015-04-23
KR20150057262 2015-04-23
KR10-2015-0126894 2015-09-08
KR1020150126894A KR101770559B1 (ko) 2015-04-23 2015-09-08 EGFR에 특이적으로 결합하는 Fab 단편

Publications (1)

Publication Number Publication Date
WO2016171365A1 true WO2016171365A1 (fr) 2016-10-27

Family

ID=57144075

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2015/013322 WO2016171365A1 (fr) 2015-04-23 2015-12-07 Fragment fab se liant spécifiquement à l'egfr

Country Status (1)

Country Link
WO (1) WO2016171365A1 (fr)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002100348A2 (fr) * 2001-06-13 2002-12-19 Genmab A/S Anticorps monoclonaux humains diriges contre le recepteur de facteur de croissance epidermique (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 (ko) * 2010-09-17 2012-10-29 주식회사 아이지세라피 인간 항-상피세포성 성장인자수용체 Ｆａｂ 항체 및 이를 포함하는 종양 치료용 약학 조성물
KR20140126638A (ko) * 2013-04-23 2014-10-31 신일제약주식회사 신규한 ｅｇｆｒ 항체 단편

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002100348A2 (fr) * 2001-06-13 2002-12-19 Genmab A/S Anticorps monoclonaux humains diriges contre le recepteur de facteur de croissance epidermique (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 (ko) * 2010-09-17 2012-10-29 주식회사 아이지세라피 인간 항-상피세포성 성장인자수용체 Ｆａｂ 항체 및 이를 포함하는 종양 치료용 약학 조성물
KR20140126638A (ko) * 2013-04-23 2014-10-31 신일제약주식회사 신규한 ｅｇｆｒ 항체 단편

Similar Documents

Publication Publication Date Title
JP6339995B2 (ja) 多機能性抗体複合体
JP6359372B2 (ja) ＤＡＲＰｉｎを含む二重特異キメラ蛋白質
CN107922477B (zh) 用于选择性制造抗体-药物缀合物的方法
CN109963876B (zh) 抗pd-1/抗her2天然抗体结构样异源二聚体形式双特异抗体及其制备
WO2020196475A1 (fr) Conjugué anticorps anti-her2/dérivé de pyrrolobenzodiazépine
US8591893B2 (en) Paratope and epitope of anti-mortalin antibody
WO2011035465A1 (fr) Protéines de liaison spécifiques et leurs utilisations
JP2020532282A (ja) Ｖｅｇｆ−ｇｒａｂタンパク質と薬物の結合体及びその用途
WO2020032165A1 (fr) Conjugué d'un dimère variant de biotine avec un pigment de phtalocyanine
WO2021160138A1 (fr) Protéine de liaison à l'antigène de l'anti-récepteur du facteur de croissance épidermique et son application
CN113754775A (zh) 一种抗pd-l1和her2的双特异性抗体
JP2013023501A (ja) がんの予防または治療用医薬組成物
KR102353086B1 (ko) 신규 면역독소 제조방법
JP2020506675A (ja) Ｖｅｇｆｒ−２抗体
WO2020108636A1 (fr) Anticorps anti-gitr complètement humanisé et son procédé de préparation
KR101770559B1 (ko) EGFR에 특이적으로 결합하는 Fab 단편
WO2016171365A1 (fr) Fragment fab se liant spécifiquement à l'egfr
KR20150097304A (ko) 항 EGFR DARPin을 포함하는 EGFR/HER2 이중 특이 항체
CN115304680A (zh) 基于Pep42构建的双特异性细胞接合器分子的制备及其应用
CN116761824A (zh) 工程化抗-trop2抗体及其抗体-药物偶联物
KR101555548B1 (ko) 신규한 ｅｇｆｒ 항체 단편
KR101884614B1 (ko) Fab 단편 및 이의 용도
US11952402B2 (en) Fusion protein containing trail and IgG binding domain and the uses thereof
KR20130057959A (ko) 항 ｈｅｒ2 단일클론항체와 ｉｌ-2를 포함하는 융합 단일클론 항체 및 이를 포함하는 암치료용 조성물
KR20190143249A (ko) 신규한 항체절편 플랫폼

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 14914608

Country of ref document: US

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15890031

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 15890031

Country of ref document: EP

Kind code of ref document: A1