WO2019107962A2 - Procédé de conjugaison d'un anticorps et d'une substance physiologiquement active - Google Patents

Procédé de conjugaison d'un anticorps et d'une substance physiologiquement active Download PDF

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WO2019107962A2
WO2019107962A2 PCT/KR2018/014939 KR2018014939W WO2019107962A2 WO 2019107962 A2 WO2019107962 A2 WO 2019107962A2 KR 2018014939 W KR2018014939 W KR 2018014939W WO 2019107962 A2 WO2019107962 A2 WO 2019107962A2
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active substance
antibody
physiologically active
peptide
conjugate
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PCT/KR2018/014939
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English (en)
Korean (ko)
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WO2019107962A3 (fr
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유태현
박지수
이유미
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아주대학교 산학협력단
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Priority claimed from KR1020180146289A external-priority patent/KR102037336B1/ko
Application filed by 아주대학교 산학협력단 filed Critical 아주대학교 산학협력단
Priority to EP18882347.0A priority Critical patent/EP3719031A4/fr
Priority to CN201880084592.6A priority patent/CN111788215B/zh
Priority to JP2020549533A priority patent/JP6969014B2/ja
Priority to US16/768,331 priority patent/US11723984B2/en
Publication of WO2019107962A2 publication Critical patent/WO2019107962A2/fr
Publication of WO2019107962A3 publication Critical patent/WO2019107962A3/fr
Priority to US18/338,832 priority patent/US11998612B2/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids

Definitions

  • the present invention relates to an Fc position-selective fused peptide in which the 5th, 10th or 11th position of the Fc position-selective binding peptide represented by the amino acid sequence of SEQ ID NO: 1 is substituted with an amino acid having a photoreactive functional group,
  • An active substance an antibody-physiologically active substance conjugate in which an antibody is bound to the physiologically active substance, and a method for producing the antibody-physiologically active substance conjugate.
  • ADC antibody-drug conjugate
  • antibody-biologically active substance conjugates should retain the properties of unbound antibodies and physiologically active substances.
  • the antibody-biologically active substance conjugate should be able to maintain affinity with the antibody prior to binding to the physiologically active substance. That is, the binding of the antibody and the bioactive substance should not inhibit the inherent antigen-antibody binding.
  • the physiologically active substance of the antibody-physiologically active substance conjugate should be able to exhibit activity after reaching the target. That is, in the antibody-physiologically active substance conjugate, the intrinsic properties of each of the monoclonal antibody and the physiologically active substance should be maintained, which is determined by the method of conjugating the two substances.
  • linker used for conjugation should be stable in the blood to prevent the biologically active substance from separating from the antibody and to reach the target tissue / cell.
  • linkers used in antibody-biologically active substance conjugates include acid-labile hydrazone, protease-labile peptide, and disulfide (reducing agent) sensitive to a reducing agent. ).
  • Non-cleavable thioether linkers are also used.
  • a method of introducing a cysteine residue into an antibody by using a functional group (lysine epsilon-amino group or cysteine thiol group) or mutation and using the reactivity of the thiol group is commonly used for conjugation of an antibody and a physiologically active substance .
  • the position where the physiologically active substance is connected can not be adjusted, and the water ratio of the antibody to the physiologically active substance is not constant, so that a heterogeneous antibody-physiologically active substance conjugate is produced.
  • the thiol group of the cysteine introduced into the antibody it is difficult to predict the effect on the structure and activity of the antibody due to the mutation in the antibody.
  • a low-molecular peptide mutant was prepared by substituting a photoreactive artificial amino acid covalently bound to an Fc domain binding peptide (US Patent Publication No. 20040253247 (2004.12.16)) that specifically binds to the Fc domain of an antibody by light irradiation .
  • a method of producing an antibody-drug conjugate in which a drug is bound to the above-prepared low molecular weight peptide mutant has been reported (Korean Patent Publication No. 10-2014-0004530 (Jan.
  • the peptide material used in the present invention must be prepared through chemical synthesis.
  • the conjugation method is limited to the case where a drug is conjugated to an antibody, and it is difficult to use it for conjugation of an antibody and a physiologically active substance such as a protein.
  • a physiologically active substance such as a protein.
  • the non-specific reactivity of the photoreactive amino acids used in the present invention has a problem in developing a conjugate of an antibody-biologically active substance into a drug (Yoshihito Tanaka et al., Molecular BioSystems, 6): 473-480, 2008).
  • an Fc position-selective peptide in which a specific position of an Fc position-selective binding peptide capable of specifically binding to an antibody Fc domain is replaced with an amino acid having a photoreactive functional group (para-benzophenylalanine (pBpa)
  • pBpa para-benzophenylalanine
  • the present invention provides an Fc position-selective fused peptide wherein the 5th, 10th or 11th position of the Fc position-selective binding peptide represented by the amino acid sequence of SEQ ID NO: 1 is substituted with an amino acid having a photoreactive functional group to provide.
  • the present invention also provides a physiologically active substance modified with a conjugate peptide in which a physiologically active substance is directly or linked to the peptide through a linker.
  • the present invention also provides an antibody-physiologically active substance conjugate wherein an antibody is bound to a physiologically active substance formulated with the conjugated peptide.
  • FIG. 1 is a schematic view showing a technique of bonding an antibody of the present invention to a physiologically active substance.
  • FIG. 2 is a result of electrophoresis after photo-reacting an antibody and a physiologically active substance formulated with the peptide to determine the substitution position of p-benzoylphenylalanine of the FcIII peptide of the present invention.
  • FIG. 3 is a schematic representation of ⁇ -lactamase (FcIII- ⁇ -lactamase) -expressing plasmid modified with the FcIII peptide of the present invention and FcIII- ⁇ -lactamase expressed in the plasmid.
  • FIG. 4 schematically shows a plasmid expressing ⁇ -lactamase zymogen (FcIII- ⁇ -lactamase zymogen) modified with the FcIII peptide of the present invention and FcIII- ⁇ -lactamase zymogen expressed in the plasmid.
  • Figure 5 schematically shows the expression plasmid PE24 (FcIII-ß-PE24) expressed in the FcIII peptide of the present invention and FcIII- ⁇ -PE24 expressed in the plasmid.
  • FIG. 6A shows a recombinant tRNA synthetase expression plasmid in the present invention.
  • 6B shows a plasmid expressing proline tRNA synthetase.
  • FIG. 7 shows the binding of the fusion protein of the present invention to the antibody (Cetuximab) by electrophoresis.
  • FIG. 8 shows the position-specific binding of the fusion protein of the present invention and the antibody (Cetuximab) by electrophoresis using a Z-domain.
  • FIG. 9 shows the location of the junction between FcIII and the antibody (Cetuximab) of the present invention by LC-MS / MS.
  • FIG. 10 shows electrophoretic results of Cetuximab-FcIII-PE24 conjugates obtained by binding the fusion protein of the present invention and an antibody (Cetuximab) at a ratio of 1: 1.
  • Figure 11 shows the results of confirming the EF2 ribosylation activity of the Cetuximab-FcIII-PE24 conjugate of the present invention.
  • FIG. 13 shows electrophoretic results of separation of Trastuzumab-FcIII-PE24 conjugate in which a fusion protein of the present invention and an antibody (Trastuzumab) are bound to each other and a fusion protein and an antibody are bound at a ratio of 1: 1.
  • a physiologically active substance which can be specifically bound to an antibody Fc domain and which is modified with an Fc position-selective peptide in which the 5th, 10th or 11th position of the Fc position-selective binding peptide is substituted with an amino acid having a photoreactive functional group (FIG. 2).
  • Fc position-selective peptide in which the 5th, 10th or 11th position of the Fc position-selective binding peptide is substituted with an amino acid having a photoreactive functional group
  • the present invention relates to an Fc position-selective fused peptide wherein the 5th, 10th, or 11th position of the Fc position-selective binding peptide represented by the amino acid sequence of SEQ ID NO: 1 is substituted with an amino acid having a photoreactive functional group will be.
  • the Fc position-selective binding peptide (FcIII) (amino acid sequence SEQ ID NO: 1, gene SEQ ID NO: 2) locally specifically binds to the CH3-CH2 interface region of the Fc domain of the human- (WL DeLano et al, Science , 2000).
  • the peptide shows a U-structure because two cysteines form a disulfide bond with each other.
  • the position substituted with the amino acid having the photoreactive functional group may be 10th.
  • a method for producing an Fc positionally-selected fused peptide substituted with an amino acid having a photoreactive functional group is characterized in that the amino acid sequence of the Fc position-selective binding peptide is identified and expressed in an expression system capable of expressing the peptide
  • a vector may be prepared and transformed into a host cell and expressed by a recombinant technique, or artificially synthesized, followed by substitution or introduction of at least one amino acid residue with an amino acid having a photoreactive functional group by a known method, or Can be synthesized by incorporating an amino acid having at least one photoreactive functional group in the artificial synthesis.
  • the introduction of the gene can be carried out by a commonly known gene manipulation method.
  • a physical method such as a method using a vector such as a virus, a non-viral method using a synthetic phospholipid or a synthetic cationic polymer, or an electrotransfer method in which a gene is introduced by applying temporary electrical stimulation to the cell membrane can be used , but is not limited thereto.
  • " amplification " in the present invention refers to amplification, substitution, or deletion of a part of the gene, introduction of a certain base, or introduction of a gene derived from another microorganism encoding the same enzyme to increase the activity of the corresponding enzyme .
  • the term " vector " means a DNA construct containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing the DNA in an appropriate host.
  • the vector may be a plasmid, phage particle, or simply a potential genome insert. Once transformed into the appropriate host, the vector may replicate and function independently of the host genome, or, in some cases, integrate into the genome itself. Because the plasmid is the most commonly used form of the current vector, the terms " plasmid " and " vector " are sometimes used interchangeably in the context of the present invention. However, the present invention includes other forms of vectors having functions equivalent to those known or known in the art.
  • &quot expression vector " is usually a recombinant carrier into which a fragment of different DNA is inserted, and generally means a fragment of double stranded DNA.
  • the heterologous DNA means a heterologous DNA that is not naturally found in the host cell.
  • the expression control sequence and the gene are contained within an expression vector containing a bacterial selection marker and a replication origin. If the expression host is a eukaryotic cell, the expression vector should further comprise a useful expression marker in the eukaryotic expression host.
  • &quot integrative vector &quot
  • integrative vector &quot means a vector in which integration or insertion into a nucleic acid is performed through integrase.
  • integrated vectors include retrovirus vector, transposon and adeno-associated viral vector. But is not limited to.
  • a host cell transformed or transfected with the vector constitutes another aspect of the present invention.
  • the term " transformation " means introducing DNA into a host and allowing the DNA to replicate as an extrachromosomal factor or by chromosomal integration. This includes any method of introducing the nucleic acid into an organism, cell, tissue or organ, and can be carried out by selecting a suitable standard technique depending on the host cell as is known in the art.
  • Such methods include electroporation, protoplast fusion, calcium phosphate (CaPO 4 ) precipitation, calcium chloride (CaCl 2 ) precipitation, agitation with silicon carbide fibers, Agrobacterium mediated transformation, PEG, dextran sulfate , Lipofectamine, and dry / inhibition-mediated transformation methods, and the like.
  • the host cell of the invention may be a prokaryotic or eukaryotic cell.
  • a host having high efficiency of introduction of DNA and high efficiency of expression of the introduced DNA is usually used.
  • Known eukaryotic and prokaryotic hosts such as Escherichia coli, Pseudomonas, Bacillus, Streptomyces, fungi and yeast, insect cells such as Spodoptera prolipida (SF9), animal cells such as CHO and mouse cells, COS 1, COS 7, BSC 1, BSC 40 and BMT 10, and tissue cultured human cells are examples of host cells that can be used.
  • the single cell host may be selected from a selected vector, the toxicity of the product encoded by the DNA sequence of the present invention, the secretion characteristics, the ability to fold the protein correctly, the culture and fermentation requirements, the product encoded by the DNA sequence of the invention And ease of purification.
  • one skilled in the art can select various vector / expression control sequences / host combinations that can express the DNA sequences of the invention in fermentation or in large animal cultures.
  • a binding method, a panning method, a film emulsion method, or the like can be applied as a screening method for cloning cDNA of NSP protein by expression cloning.
  • &quot means a technique of fusing two cells having different traits using a protoplast from which cell walls of plant cells or fungi have been removed.
  • Protoplast fusion involves the use of chemical methods such as the addition of metal ions such as calcium and magnesium to high osmotic solutions or physical exposure such as by increasing the DNA absorption of the protoplasts by exposing the protoplasts to temporary pores in the cell membrane, There is a way.
  • the "photoreactive functional group” may be a functional group capable of absorbing light of a specific wavelength upon irradiation with light and forming a covalent bond with an adjacent reactive functional group.
  • the Fc positionally-selective peptide with the photoreactive functional group of the present invention binds to or binds to the Fc domain of the antibody by its specificity as an inherent property when mixed with the antibody. Then, when the mixture is irradiated with light, it can absorb light of a specific wavelength and form a covalent bond with a reactive functional group on the adjacent antibody Fc domain through a photoreactive functional group. That is, the Fc position-selective binding peptide of the present invention can covalently bind to the specific functional group of the Fc domain through the photoreactive functional group upon light irradiation.
  • the amino acid having the photoreactive functional group may be p-benzoyl phenylalanine.
  • the artificial amino acid having a photoreactive functional group may be composed of a photoreceptor, a photomethionine or an azidophenylalanine.
  • the photoreactive functional group used in the present invention has a high wavelength band, that is, a low energy
  • the branch has specificity that the covalent bond is activated through light energy.
  • the p-benzoylphenylalanine of the present invention is represented by the following formula (1).
  • the present invention relates to a physiologically active substance which is formulated with a conjugate peptide in which a physiologically active substance is directly or linked to the peptide through a linker.
  • the physiologically active substance may be a therapeutic agent or a diagnostic agent.
  • the therapeutic agent or diagnostic agent may be an enzyme, hormone, cytokine, antibody, antibody fragment, analgesic agent, antipyretic agent, anti- A substance, an antiviral drug, an antifungal drug, a cardiovascular drug, a central nervous system drug, a kidney function and an electrolytic metabolism drug, and a chemotherapeutic agent.
  • the physiologically active substance of the present invention may be a therapeutic agent or a diagnostic / detection agent.
  • the antibody used as the physiologically active substance may be a therapeutic antibody.
  • Approximately 30 therapeutic antibodies have been approved by the FDA and their safety is very high because they closely resemble those of in vivo IgG.
  • Therapeutic antibodies have been used for a wide range of disease treatments (such as transplant rejection, cancer, autoimmune diseases and inflammation, heart disease and infectious infections, etc.).
  • the Fc domain-containing molecule is a therapeutic antibody
  • the therapeutic antibody recognizes and binds to a receptor protein or an antigenic protein specifically present in a disease-causing tissue, and therefore its specificity is very high.
  • a molecular imaging probe or a drug delivery vehicle when a molecular imaging probe or a drug delivery vehicle is combined with a therapeutic antibody as a physiologically active substance, it can be converted into a theragnosis agent capable of monitoring the therapeutic process together with a drug combination effect.
  • a molecular imaging probe or a drug delivery system by combining a molecular imaging probe or a drug delivery system with a simple targeting antibody, it is possible to develop a teraginosis preparation for diagnosis, treatment, or simultaneous diagnosis and treatment.
  • Non-limiting examples of therapeutic agents include antibodies, antibody fragments, drugs, toxins, nucleic acid hydrolases, hormones, immunomodulators, chelators, boron compounds, photoactive agents or dyes, and radioactive isotopes .
  • diagnostic / detection agents include, but are not limited to, an increase in radioisotopes, dyes (e.g., biotin-streptavidin complexes), contrast agents, fluorescent compounds or molecules and magnetic resonance imaging (MRI) (Paramagnetic ion).
  • the diagnostic agent comprises a radioactive isotope, an enhancer for use in magnetic resonance imaging, and a fluorescent compound.
  • a reactant having a long tail attached to many of the chelating groups to couple the ions.
  • the tail may be a polymer such as polylysine or polacacaride or a polymer such as ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), porphyrin, polyamine, crown ether, bis-thiosemic A chain having a pendant group capable of bonding with a chelating group such as a thiosemicarbazone, polyoximes, and a group known to be useful for the above purpose.
  • the chelate can normally be coupled to the Fc position-selective peptidic peptide by a minimal loss of immunoreactivity and a functional group capable of forming a bond to the molecule with minimal assembly and / or internal cross-linking.
  • Particularly useful metal-chelate combinations include diagnostic isotopes and 2-benzyl-DTPA and monomethyl and cyclohexyl analogues thereof used in the general energy range of 60 to 4,000 keV, for example, radioisotopes include 125 I , 131 I, 123 I, 124 I, 62 Cu, 64 Cu, 18 F, 111 In, 67 Ga, 99 mTc, 94 mTc, 11 C, 13 N, 15 O, 76 Br.
  • non-radioactive metals such as manganese, iron and gadolinium
  • the same chelate is useful for MRI when used with nanoparticles or antibodies of the present invention.
  • Macrocyclic chelates such as NOTA, DOTA, and TETA are used with a variety of metals and radioactive metals, preferably with radionuclides of gallium, yttrium and copper, respectively.
  • the metal-chelate complex can be made very stable by aligning the ring size with the metal of the object.
  • the present invention makes it possible to produce cyclic chelates such as macrocyclic polyethers useful for stably binding RAIT with nuclides such as 223Ra.
  • Immunoconjugates are conjugates of therapeutic or diagnostic agents and antibody components.
  • the diagnostic agent comprises a radioactive or non-radioactive label, a contrast agent (magnetic resonance imaging, computed tomography, or a contrast agent suitable for ultrasound), and the radioactive label includes gamma-, beta-, alpha-, It may be a positron emission isotope.
  • &quot typically stimulate immune cells that are proliferating or activating in an immune response cascade such as macrophages, B-cells, and / or T-cells.
  • An example of such an immunomodulator is a cytokine.
  • cytokine Those skilled in the art are a type of cytokine in which interleukins and interferons stimulate T-cell or other immune cell activity.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • PNA peptide nucleic acid
  • LNA locked nucleic acid
  • An Fc positionally-selected fused peptide in which the DNA, RNA, PNA or LNA is linked can be immobilized on a solid support for use as a biochip, a biosensor, an immune detection kit or a complementary self-addressable chip have.
  • the linker may include a reactive functional group, an amino acid, and a self-cleaving spacer.
  • the linker of the present invention may be in a form of linking a physiologically active substance with a specific residue in an Fc positionally selected binding peptide substituted with a photoreactive functional group and may include a nucleophilic residue For example, cysteine). ≪ / RTI >
  • the linker may comprise, for example, a reactive functional group, an amino acid, and a self-cleaving spacer that binds to an Fc positionally-selective fused peptide substituted with a photoreactive functional group.
  • the functional group may be selected from the group consisting of i) a maleimide group, an acetamide group or a derivative thereof, ii) an aziridine group, an aryl halide, an acryloyl group or a derivative thereof, iii) an alkylation reactor, an arylation reactor, pyridyldisulfide, Lysine, or a derivative thereof.
  • the linker includes, for example, i) a maleimide group or a derivative thereof-valine-citurline-para-aniline benzoic acid (PABA); Or ii) in the form of an acetamide group or a derivative thereof-valine-citurulline-para-aniline benzoic acid (PABA).
  • the binding of the Fc positionally-selective peptide and the physiologically active substance substituted with the photoreactive functional group through the linker can be carried out by a known method, for example, an alkylation, a disulfide interchange method, and a trans-esterification reaction method have.
  • a known method for example, an alkylation, a disulfide interchange method, and a trans-esterification reaction method have.
  • the conjugated peptide and the physiologically active substance can be conjugated to each other through the thiol group of the cysteine residue in the Fc positionally-selective peptide substituted with the photoreactive functional group.
  • the nucleophilic reactivity of the thiol of the cysteine residue to the maleimide group is dependent on the other amino acid functional groups present in the protein, such as the amino group of the lysine residue Terminal amino group, it can be utilized for specific binding to cysteine.
  • a physiologically active substance modulated with a maleimide group, a derivative thereof, or an acetamide group or a derivative thereof, for example, a bromoacetamide group or an iodoacetamide group is a cysteine-thioether bond and a photoreactive functional group It can be seen that the substituted Fc position selective binding peptide and the physiologically active substance are bound.
  • a method for producing a fusion protein comprising the steps of: (a) mixing a physiologically active substance formulated with a conjugated peptide with an Fc domain-containing molecule; (b) irradiating the mixture with light to generate a conjugate of an antibody-physiologically active substance to which a photoreactive functional group of the physiologically active substance modified with the conjugated peptide is bound to an Fc domain-containing molecule; And (c) a step of obtaining a conjugate of the antibody-biologically active substance.
  • the light may be 320 to 380 nm, and preferably 350 to 365 nm.
  • the present invention is not limited thereto.
  • the " Fc domain-containing molecule &quot includes, without limitation, a molecule including an Fc domain and capable of being adjacent or binding specifically recognized by an Fc position-selective junction peptide.
  • the Fc domain containing molecule includes a protein comprising an Fc domain, a peptide, a glycoprotein, a glycopeptide, an antibody, a fragment of an antibody, an immunoglobulin or an immunoglobulin fragment, and the like.
  • the antibody and immunoglobulin are heterotetrameric glycoproteins of about 150 kDa and comprise the same two light chains and the same two heavy chains.
  • Fc domain thereof may be an antibody or immunoglobulin light chain obtained by treating papain and a fragment from which the heavy chain has been removed.
  • the Fc domain-containing molecule may be a target-oriented natural or non-natural antibody capable of specifically binding to a target molecule.
  • the antibody includes both polyclonal and monoclonal antibodies as well as specific recombinant antibodies such as chimeric, humanized or human antibodies.
  • the antibody may be a receptor-specific antibody or a ligand-specific antibody.
  • the antibody may also be a receptor-specific antibody that does not prevent ligand binding but prevents receptor activation.
  • the antibody may be a therapeutic antibody, an antibody capable of binding with a separate therapeutic agent or diagnostic agent, an antibody for targeting without therapeutic effect, or an antibody capable of simply reacting with an antigen- Lt; / RTI >
  • the antibody-biomolecule conjugate can be used to maximize the advantages of antibodies such as specificity, non-toxicity in circulation and pharmacokinetics in the blood circulation, Specifically, it is a technology focused on targeting only specific tissues (for example, cancer cells).
  • an immunoconjugate includes anti-cancer drugs for " targeted chemotherapeutics ".
  • the immunoconjugate is composed of three components including a drug, a monoclonal antibody, and a linker linking the antibody and the drug.
  • the immunoconjugate technique is a technique in which a specific antigen A method for delivering a substance having physiological activity to tumor cells using an antibody that specifically binds to tumor cells.
  • the physiologically active compound is selected from the group consisting of beta-lactamase (TEM-1), beta-lactamase zymogen (Korean Patent No. 1016956840000 (2017.01. 06) and Pseudomonas exotoxin A (PE24) were used.
  • TEM-1 beta-lactamase
  • beta-lactamase zymogen Korean Patent No. 1016956840000 (2017.01. 06)
  • PE24 Pseudomonas exotoxin A
  • " ß-lactamase " (amino acid sequence number 3) is not cytotoxic in itself, but a prodrug having a beta-lactam ring is cleaved to activate the drug As a mechanism, it can be used as an effective tumor treatment drug when treated with an appropriate prodrug.
  • an inactive prodrug such as GC-mel can be used.
  • ⁇ -lactamase zymogen amino acid SEQ ID NO: 5
  • BLIP ⁇ -lactamase inhibitor protein
  • MMP-2 matrix metalloproteinase-2
  • " PE24 " (amino acid sequence number 7) binds EF-2 by ADP-ribosylation of elongation factor-2 (EF-2) And activates to induce apoptosis of cells (U.S. Patent No. 09388222 (Jul. 12, 2016)).
  • the Fc domain-containing molecule may be characterized in that it is selected from the group consisting of immunoglobulin-derived domains, combinations thereof, and Fc regions thereof.
  • IgG is selected from the group consisting of IgG, IgA, IgD, IgE, IgM, combinations thereof, and the Fc region thereof, and more preferably IgG1 or an Fc region thereof But is not limited thereto.
  • step (c) since FcIII binds to the Fc domain of the heavy chain of the antibody, when the fusion protein and the antibody are photoreactive, A form in which a physiologically active substance immobilized on one antibody, a form in which a physiologically active substance immobilized on one antibody, a form in which a physiologically active substance immobilized on one antibody, a form in which a physiologically active substance immobilized on one antibody and a physiologically active substance modulated on two antigenic peptides are bound to an antibody).
  • An antibody-biologically active substance conjugate in which a physiologically active substance formulated with one conjugated peptide is conjugated has a structure in which one neonatal Fc receptor (FcRn) is free, , It can not be decomposed and a high half-life of the drug can be expected.
  • FcRn neonatal Fc receptor
  • Antibodies that do not bind a physiologically active substance formulated with a conjugated peptide can be removed by optimizing the reaction conditions by controlling the reaction ratio of the antibody and the fusion protein and the ultraviolet irradiation time.
  • a conjugate of an antibody-physiologically active substance conjugated with a physiologically active substance conjugated with one conjugated peptide and a conjugate of an antibody-physiologically active substance conjugated with a biologically active substance conjugated with two conjugated peptides is a conjugate between the protein A and the Fc domain of the antibody Can be separated using binding affinity. Since Protein A has specific binding affinity to the CH 2 -CH 3 domain interface of the Fc domain of the antibody, affinity chromatography resins used for purification after antibody expression, .
  • antibody-biologically active substance conjugates in which the biologically active substance formulated with two conjugated peptides are bound to the antibody do not bind to the protein A resin (WL DeLano et al, Science , 287 (5456): 1279-83, 2000) .
  • the form in which the two physiologically active substances are bound to the antibody can be removed by protein A affinity chromatography and then subjected to anion chromatography successively on the resulting product to cause binding due to the difference in isoelectric point
  • a form in which a physiologically active substance formulated with one conjugated peptide is bound to the antibody can be isolated.
  • the present invention relates to a conjugate of an antibody-biologically active substance in which an antibody is bound to a physiologically active substance formulated with the conjugated peptide.
  • WST-8 and MTS essays were performed to confirm the activity of the IgG1-FcIII-PE24 conjugate.
  • the WST-8 and MTS essays are transformed into mitochondrial succinate dehydrogenase, which is produced by bacteria in the medium, into tetrazolium salt (Formazan), which can be measured at a specific absorbance , And the survival of the cells can be confirmed by measuring the absorbance.
  • the absorbance changes were measured while increasing the concentrations of the three antibody-physiologically active substance conjugates within a specific range. As a result, it was observed that cell proliferation and viability decreased as the conjugate of the antibody-biologically active substance conjugated with the physiologically active substance modified with the antibody and the conjugated peptide was treated at a high concentration. Thus, it was confirmed that the antibody-biologically active substance conjugate of the present invention can be used as a therapeutic drug.
  • trastuzumab acts specifically on HER2 that forms a heterodimer with EGFR or HER4 to inhibit the activation of ligands EGFR (EGF, TGFa) or HER4 (NRG1) Lt; / RTI > Cetuximab also inhibits the activation of EGFR, which is dependent on ligands (EGF, TGFa), and prevents its downregulation.
  • human IgG1 and FcIII peptide (amino acid sequence number 11, gene sequence number 12) in which the DNA sequence at the 5th position in the amino acid sequence was substituted with an amber codon
  • FcIII peptide (amino acid sequence SEQ. ID. NO. 13, gene SEQ. ID NO. 14) in which the DNA sequence of the position was replaced with an amber codon
  • FcIII peptide in which the DNA sequence at the 11th position was substituted with an amber codon No. 16
  • the sample was then subjected to a UV hand ramp (Lklab, U01-133-194) on 1xPBS buffer (pH 7.4) And irradiated with ultraviolet light of 365 nm for 2 hours.
  • the FcIII peptide gene in which the gene coding for the 10th amino acid was substituted with an amber codon was digested with NheI in 2.1 NEB buffer, and the gene was cut using a spin column And then cleaved by treatment with BamHI in 3.1 NEB buffer.
  • the composition of the buffer was DDW, 10x NEB buffer 3.1, DNA, restriction enzyme, and the total volume was 50 ⁇ l, and the treatment conditions were 37 ° C for 4 hours, respectively.
  • a pET21-a vector digested with sticky ends at both ends was treated with the same restriction enzymes and the cleaved FcIII gene was mixed at a molar ratio of 1: 3 to make a total volume of 10 mu l. Then, T4 DNA ligase ( NEB, England) for 2 hours at room temperature.
  • the ligation mixture was mixed with 50 ⁇ l of competent cell, E. coli DH10B (Thermosensific, C640003), and electroporation (Bio-Rad, USA) was performed. Then, the transformed strain was cultured at 37 ° C. for 12 to 14 hours on an LB agar medium containing ampicillin to obtain a transformed strain. FcIII expression plasmid-1 was obtained by DNA prep (GeneAll, mini prep kit) Respectively.
  • a plasmid (pSPEL104) containing the entire sequence of ⁇ -lactamase represented by SEQ ID NO: 4 was amplified by polymerase chain reaction (PCR) using the primer shown in Table 1 as a template, and BamHI and NotI Treated with restriction enzymes, and then ligated to the FcIII expression plasmid-1 of Example 1.
  • PCR is performed in three steps of denaturation, annealing, and amplification, and the method is as follows.
  • the reaction composition of the PCR is DDW, 10x pfu buffer, 0.2mM dNTP, 20pmol primer F / R, template, 5units Pfu polymerase, and the final reaction volume is 50 ⁇ l.
  • the reaction was carried out at 95 ° C for 2 minutes using BamhI -f and TEM1-NotI-r in Table 1, followed by 25 cycles of 95 ° C for 30 seconds, 55 ° C for 30 seconds, and 72 ° C for 1 minute, And then treated at 72 ⁇ for 10 minutes.
  • Table 1 shows PCR primers used in the present invention.
  • the two restriction enzymes were cleaved by treatment with 3.1 NEB buffer.
  • the composition of the buffer was DDW, 10x NEB buffer 3.1, DNA, restriction enzyme, total volume 50 ⁇ l, and treated at 37 ° C for 4 hours.
  • the FcIII-expressing plasmid-1 digested with sticky ends at both ends was mixed with ⁇ -lactamase at a molar ratio of 1: 3, and the total volume was adjusted to 10 ⁇ l.
  • T4 DNA ligase (NEB) Lt; RTI ID 0.0 > 25 C < / RTI > for 2 hours.
  • the ligated DNA mixture solution was added to 50 ⁇ l of competent cell E. coli DH10B (Thermosensific, C640003), followed by mixing and electroporation (Bio-Rad, USA). Then, the transformed strain was cultured at 37 ° C. for 12 to 14 hours on an LB agar medium containing ampicillin to obtain a transformed strain. FcIII- ⁇ -lactamase (amino acid sequence number 17 , Gene SEQ ID NO: 18) expression plasmids were obtained (Fig. 3).
  • a plasmid (pSPEL166) containing the ß-lactamase zymogen sequence (1353 bp) represented by SEQ ID NO: 6 was amplified by PCR using the primers shown in Table 1 under the same conditions as the above conditions and then PCR was performed using NcoI and NotI restriction After digestion with the enzyme, the FcIII expression plasmid-2 of Example 1 was ligated in the same manner as described above.
  • the ligation mixture was added to 50 ⁇ l of E. coli DH10B (Thermosensific, C640003), followed by electroporation (Bio-Rad, USA). Then, the transformant was cultured at 37 ° C. for 12 to 14 hours on an LB agar medium containing ampicillin, and the transformed strain was obtained.
  • FcIII- ⁇ -lactamase zymogen amino acid sequence No. 19, gene SEQ. ID No. 20 expression plasmids (FIG. 4).
  • PE24 expressed by de-immunized SEQ ID NO: 8 was obtained by gene synthesis (Bioneer) and then digested with BamHI and NotI restriction enzymes in the same manner as described above, and then ligated to FcIII expression plasmid-1.
  • the ligation mixture was added to 50 ⁇ l of E. coli DH10B (Thermosensific, C640003), followed by electroporation (Bio-Rad, USA). Then, the transformed strain was cultured at 37 ° C. for 12 to 14 hours on an LB agar medium containing ampicillin to obtain a transformed strain.
  • FcIII-PE24 fusion protein amino acid sequence number 21 , Gene SEQ ID NO: 22 expression plasmid was obtained (Fig. 5).
  • a tRNA sequence recognizing the TAG codon (Jason W. Chin et al., PNAS vol. 99 ) was obtained by digesting SalI and BglII with the p-benzoylphenylalanine tRNA synthetase (amino acid SEQ . , 11020-11024, 2002) was used as a vector (Fig. 6A).
  • the ligation mixture was added to 50 ⁇ l of E. coli DH10B (Thermosensific, C640003), followed by electroporation (Bio-Rad, USA). Then, the transformant was cultured at 37 ° C. for 12 to 14 hours on an LB agar medium containing chloramphenicol to obtain a transformed strain.
  • the TAG codon was recognized by DNA prep (GeneAll, mini prep kit) And a tRNA synthetase (gene SEQ. ID NO: 23) expression plasmid for inserting benzoyl phenylalanine was obtained.
  • a pBbS2K plasmid containing a proline tRNA synthetase sequence was used (Byeong Sung Lee et al., Biochimica Biophysica Acta , S0304-4165, 2017) (FIG.
  • Example 3 Expression and purification of physiologically active substance formulated with conjugated peptides
  • plasmids containing the FcIII-ß-lactamase expression plasmid, TAG codon cognitive tRNA and p-benzoylphenylalanine tRNA synthetase pair and plasmid containing proline tRNA synthetase were transformed into E. coli BL21 (DE3) (SIGMA aldrich, CMC0016), and then plated on an LB plate containing ampicillin, chloramphenicol, and kanamycin to obtain a transformed strain.
  • the obtained single colonies were seeded at 37 ° C and 180 rpm for 12 hours, seeded at a ratio of 10: 1, and incubated at 37 ° C and 180 rpm for 6 hours.
  • the culture was inoculated at a ratio of 100: 1 to 200 ml of 2xYT (including ampicillin, chloramphenicol, and kanamycin) medium and cultured at 37 ° C and 180 rpm.
  • 2xYT including ampicillin, chloramphenicol, and kanamycin
  • FcIII- ⁇ -lactamase In order to purify the expressed FcIII- ⁇ -lactamase, it was centrifuged at 4 ° C. and 9300 g for 15 minutes. The supernatant was then removed and resuspended in 5 ml of lysis buffer (0.75 M sucrose, 0.1 Tris, pH 8.0), added with 0.05 ml / l of lysozyme and 1 ml of 1 mM EDTA, And rotated for a minute. Then, 1 ml of 0.5 M MgCl 2 was added and the mixture was rotated at 4 ° C for 10 minutes. The mixture was centrifuged at 4 ° C and 9300g for 15 minutes, and the supernatant was separated.
  • lysis buffer (0.75 M sucrose, 0.1 Tris, pH 8.0
  • Example 3-2 Expression and purification of FcIII-ß-lactamase zymogen
  • FcIII-ß-lactamase zymogen For the expression of FcIII-ß-lactamase zymogen, plasmids containing the FcIII-ß-lactamase zymogen expression plasmid, TAG codon cognate tRNA and p-benzoylphenylalanine tRNA synthetase pair and plasmid containing proline tRNA synthetase were transformed into E. coli BL21 DE3) (SIGMA aldrich, CMC0016) and then expressing FcIII- ⁇ -lactamase zymogen in the same manner as the expression of FcIII- ⁇ -lactamase.
  • the purification reaction was performed under the same conditions as those for the purification of FcIII- ⁇ -lactamase to elute the 6 ⁇ His-tagged FcIII- ⁇ -lactamase zymogen.
  • FcIII-PE24 For expression of FcIII-PE24, a plasmid containing the FcIII-PE24 expression plasmid, a pair of TAG codon cognition tRNA and p-benzoylphenylalanine tRNA synthetase, and a proline tRNA synthetase was transformed into E. coli BL21 (DE3) (SIGMA aldrich, CMC0016) and then expressing FcIII-PE24 in the same manner as that of FcIII- ⁇ -lactamase.
  • E. coli BL21 DE3 (SIGMA aldrich, CMC0016)
  • Example 4 Binding of physiologically active substances formulated with cetuximab and conjugated peptides and isolation and activity of conjugates
  • Example 4-1 Confirmation of binding of physiologically active substance formulated with cetuximab and conjugated peptide
  • Example 4-2 Separation of conjugate of antibody-biologically active substance conjugated with physiologically active substance modified with cetuximab and conjugated peptide
  • an antibody-physiologically active substance conjugate conjugated with an antibody to the physiologically active substance formulated with the conjugate peptide of Example 4 After mixing with 5 ml 1 ⁇ PBS (pH 7.4), 1 ml of Protein A 50% resin slurry (CaptivA Protein A resin, Repligen) was added and the mixture was rotated at 4 ° C for 1 hour and 30 minutes. After loading the reaction solution into an empty column, the resin was completely submerged and washed by loading 30 ml of 1 ⁇ PBS (pH 7.4). Then, 5 ml of elution buffer (pH 3.0 0.1 M Glycine) was loaded to obtain a product, and 125 ⁇ l of neutralization buffer (pH 9.0 Tris) was added to titrate the pH.
  • elution buffer pH 3.0 0.1 M Glycine
  • the obtained product was a mixture of an antibody-physiologically active substance conjugate conjugated with a physiologically active substance modulated with one conjugated peptide and an antibody not conjugated (FIG. 10).
  • Example 4-3 Confirmation of EF2 ribosylation activity of Cetuximab-FcIII-PE24 conjugate
  • ADP-ribosylation activity of the Cetuximab-FcIII-PE24 conjugate was measured by the method of Zhang and Snyder.
  • the PE24 and Cetuximab-FcIII-PE24 conjugates were diluted to 1 nM in 20 mM Tris-HCl (pH 7.4), 1 mM EDTA, 1 mM DTT and incubated with wheat germ extract in the presence of 50 nM biotinylated NAD + Lt; / RTI > The reaction was then terminated with 5x sodium dodecyl sulfate (SDS) gel loading buffer. Proteins were separated on SDS-12% (w / v) polyacrylamide gel. Biotinylated EF-2 was detected by Western blot using a streptavidin-horseradish peroxidase (HRP) conjugate. Western blot images were analyzed using a ChemiDoc XRS system.
  • cell viability assay was performed using a cell line overexpressing EGFR, a specific antigen to which Cetuximab binds, on the cell surface.
  • EGFR cell line A431 (SIGMA aldrich, 85090402) was cultured in DMEM medium (10% FBS, streptomycin) and cultured in 96-well plate at 2 ⁇ 10 3 cells / well. After 24 hours, Cetuximab-FcIII-PE24 was treated at a concentration of 0 nM, 0.016 nM, 0.16 nM, 1.6 nM and 16 nM and cultured at 37 ° C, 5% CO 2 for 72 hours. Then, the MTS solution (Promega, G3580) was treated with 20 ⁇ l / well and the absorbance at 490 nm was measured after 2 hours.
  • MTS solution Promega, G3580
  • Example 5 Binding of physiologically active substances formulated with Trastuzumab and conjugated peptides and isolation and activity of conjugates
  • a conjugate of an antibody-biologically active substance conjugated with an antibody to a physiologically active substance formulated with the conjugated peptide of Example 4 (CaptivA Protein A resin, Repligen) was added to 1 ml of a Protein A 50% resin slurry, which was then rotated for 1 hour and 30 minutes at 4 ° C. The reaction solution was transferred to an empty column After loading, the resin was completely submerged and washed by loading 30 ml of 1xPBS (pH 7.4). The product was then loaded by loading 5 ml of elution buffer (pH 3.0 0.1M Glycine), and 125 ⁇ l of The pH was titrated by adding neutralizing buffer (pH 9.0 Tris).
  • the obtained product was a mixture of antibody-biomolecule conjugate conjugated with physiologically active substance modified with one conjugated peptide and antibody not conjugated (FIG. 13).
  • the resultant product was continuously mixed with 20 mM phosphate buffer (pH 7.9) and subjected to anion chromatography (mono-Q column, GE Healthcare Life Science, USA) The form in which the active substance was bound to the antibody was isolated and confirmed (Fig. 13).
  • the cell viability assay (cell viability assay (cell viability assay) was performed using cell lines overexpressing HER2, a specific antigen to which Trastuzumab binds, ) Were performed.
  • HER2 overexpressing cell line MDA-MB-231 (Korean Cell Line Bank, 60062), HCC-1954 (Korean Cell Line Bank, 9S1954), MDA-MB- Korean Cell Line Bank, 30026) were cultured in RPMI medium (10% FBS, streptomycin) and cultured in 3-5 ⁇ 10 3 cells / well on 96-well plates.
  • trastuzumab-FcIII-PE24 was treated at concentrations of 0 nM, 0.0064 nM, 0.032 nM, 0.16 nM, 0.8 nM, 4 nM and 20 nM and cultured at 37 ° C and 5% CO 2 for 72 hours.
  • the WST-8 solution (Dojindo, CK04-11) was treated with 10 ⁇ l / well, and the absorbance at 450 nm was measured after 2 hours.
  • trastuzumab-FcIII-PE24 conjugate As a result, the higher the concentration of Trastuzumab-FcIII-PE24 conjugate was, the lower the absorbance of HER2-overexpressing cells was, and the cell viability was markedly decreased as compared with the wild type trastuzumab and PE24 treated wells. In contrast, it was confirmed that the cytotoxicity of trastuzumab-FcIII-PE24 conjugate did not act in the corresponding concentration range for HER2 non-expressing cells (FIG. 14).
  • a physiologically active substance modified with a conjugated peptide is prepared by using an Fc positionally selected binding peptide in which a specific position according to the present invention is substituted with a photoreactive functional group and then the antibody and the physiologically active substance are bound through a photoreaction
  • the material can be linked to the antibody in a selective and highly efficient manner through a simple photoreaction. Therefore, the present invention can be used for the production of antibody-physiologically active substance conjugates in which various kinds of physiologically active substances and antibodies are linked, and the commercialization thereof can be accelerated.

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Abstract

La présente invention concerne un peptide conjugué à un anticorps substitué par un acide aminé ayant un groupe fonctionnel photoréactif, une substance physiologiquement active formulée avec le peptide conjugué, et un conjugué anticorps-substance physiologiquement active constitué d'un anticorps lié à la substance physiologiquement active. Quand la substance physiologiquement active formulée avec le peptide conjugué selon la présente invention est liée à l'anticorps, l'efficacité de liaison entre l'anticorps et la substance physiologiquement active est considérablement améliorée comparativement à celle de l'état de la technique classique, et par conséquent, le médicament peut être fermement lié sans affecter la spécificité de l'anticorps, permettant ainsi d'accélérer la commercialisation du conjugué anticorps-substance physiologiquement active.
PCT/KR2018/014939 2017-11-29 2018-11-29 Procédé de conjugaison d'un anticorps et d'une substance physiologiquement active WO2019107962A2 (fr)

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CN201880084592.6A CN111788215B (zh) 2017-11-29 2018-11-29 抗体和生理活性物质的缀合方法
JP2020549533A JP6969014B2 (ja) 2017-11-29 2018-11-29 抗体と生理活性物質の接合方法
US16/768,331 US11723984B2 (en) 2017-11-29 2018-11-29 Method for conjugating antibody and physiologically active substance
US18/338,832 US11998612B2 (en) 2017-11-29 2023-06-21 Method for conjugating antibody and physiologically active substance

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KR20140004530A (ko) 2012-07-03 2014-01-13 한국생명공학연구원 ＩｇＧ Ｆｃ 위치선택적 결합 펩티드 및 이를 포함하는 하이브리드 분자
US9388222B2 (en) 2013-10-06 2016-07-12 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Modified Pseudomonas exotoxin A
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