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WO2013176516A1 - Conjugué anticorps-médicament préparé au moyen de transglutaminase et son utilisation - Google Patents

Conjugué anticorps-médicament préparé au moyen de transglutaminase et son utilisation Download PDF

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WO2013176516A1
WO2013176516A1 PCT/KR2013/004571 KR2013004571W WO2013176516A1 WO 2013176516 A1 WO2013176516 A1 WO 2013176516A1 KR 2013004571 W KR2013004571 W KR 2013004571W WO 2013176516 A1 WO2013176516 A1 WO 2013176516A1
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Prior art keywords
antibody
glutamine
drug
seq
mutated
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Korean (ko)
Inventor
고민지
송대해
김영민
문경덕
박상경
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Hanwha Chemical Corp
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Hanwha Chemical Corp
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Priority claimed from KR1020120154955A external-priority patent/KR20130132236A/ko
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    • 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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • 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
    • 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
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner

Definitions

  • the present invention comprises reacting a mutated antibody comprising glutamine and a drug comprising a free amine group in the presence of transglutaminase, Method for producing, the antibody-drug conjugate prepared by the method, the antibody-drug conjugate, wherein the mutated antibody comprising glutamine and the drug comprising a free amine group are connected by an isopeptide bond, the antibody-drug conjugate
  • the present invention relates to a mutated antibody in which a peptide including glutamine and an antibody are linked.
  • Antibody-drug conjugates combine cytotoxic drugs with antibodies, and use them to enhance existing anticancer effects and apply them to cancer cells resistant to existing antibodies. Research is active. Antibody-drug conjugates currently entering the market or in clinical or research phases use chemical binding to multiple sites of lysine or cysteine in a native antibody using chemical binding to the antibody. have. Antibody-drug conjugates through these classical chemical methods are difficult to regulate in their formation and have the problem of producing heterogeneous immunomixes with different properties (KJ Hamblett et al. Clin. Cancer Res. 2004, 10). , 7063-7070).
  • cysteine-inserted antibodies are used to bind two cytotoxic substances to a desired position, but the thiol residues of artificially inserted cysteines are reactive. Since it has to be activated to have, there is a problem to follow a multi-step conjugation process that must go through the reduction and oxidation of the antibody.
  • the method is used to modify the antibody by undergoing an oxidation-reduction reaction, and thus has a weak point in the tertiary structure or function of the antibody.
  • Such weakness often results from non-specification of the antibody and incomplete reaction between accessible lysine / cysteine residues or amine groups of the antibody, often resulting in incomplete or undefined products. do.
  • Transglutaminase is an enzyme that catalyzes the acyl transfer reaction between the gamma-carboxamide group of glutamine (Q) residues and the primary epsilon amino group of lysine.
  • the bond formed through the above reaction is called an isopeptide bond, and this bond is known as a fairly stable bond such as being resistant to protease. Because of this stability, transglutaminase is generally used to link structural components of cells.
  • the recognition of glutamine residues by transglutaminase is quite stringent, and is known to recognize glutamine groups, mainly in the fluid part of proteins (Angelo Fontana et al, Advanced Drug Delivery Reviews 2008. 60. 13-28).
  • PEGylation polyethylene glycolation
  • hGH human growth hormone
  • hC-GSF human granulocyte colony stimulating factor
  • EPO erythropoietin
  • a site-specific or selective PEGylation is performed using a transglutaminase reaction instead of the problem of the chemical method by redox.
  • the pegylation reaction through transglutaminase is one in which a transglutaminase recognizes one or more glutamine groups inside a protein and connects an amino group to PEG as an amine donor substrate (Anna Mero). et al. Journal of Controlled Release . 2011. 154 27-34, Carlo Maullu et al. FEBS Journal . 2009. 276. 6741-6750, US Registered Publication No. 6995245).
  • transglutaminase is also used to bind lipids to proteins.
  • Conventional chemical reactions or EPL-mediated or saltase-mediated enzymatic reactions have been used to bind lipids to proteins, but the above methods have a long reaction time and low yields.
  • the formation of protein-lipid conjugates using transglutaminase showed more than 95% binding yield and reduced time compared to the conventional method (Hiroki Abe et al. Chem. Eur. J. 2011, 17, 14004- 14008).
  • glycosylation such as glycosylation of antibodies
  • glycosylation of antibodies is known to affect the function and pharmacokinetics of the effector of the antibody, and thus altering the sugar chain structure inside the antibody has a problem affecting the function of the antibody. Therefore, there is still an unknown field of constructing the linkage between the antibody and the drug using transglutaminase without impairing the sugar chain structure required for the in vivo function of the antibody.
  • an antibody-drug is prepared by artificially inserting a glutamine residue at a site to produce a mutated antibody, and reacting a drug having a free amine group in the presence of a transglutaminase to specifically bind the drug to a specific glutamine residue.
  • the present invention has been completed by preparing the binder.
  • One object of the present invention comprises the step of reacting a mutated antibody comprising glutamine and a drug comprising a free amine group in the presence of transglutaminase It provides a method for producing a drug conjugate.
  • Another object of the present invention to provide a pharmaceutical composition for treating cancer comprising the antibody-drug conjugate.
  • Another object of the present invention is to provide a polynucleotide encoding the mutated antibody, an expression vector comprising the polynucleotide, and a transformant into which the expression vector is introduced.
  • the antibody-drug conjugate according to the present invention has a high homogeneity as a conjugate in which a drug containing a free amine group is specifically linked to a mutated antibody including glutamine, and has a chemical reaction compared to a form in which a drug and an antibody are chemically bound. Less pass has the advantage that less deformed form is produced.
  • FIG. 1 is a representative schematic diagram showing an antibody-drug conjugate in which a modified monoclonal antibody is combined with a drug.
  • Q represents glutamine.
  • FIG. 2 shows an SDS-PAGE result of reacting mPEGamine (1KDa) with glutamine-containing IgG in the presence of transglutaminase (TGase), followed by electrophoresis and coomassie blue staining. to be.
  • (A) shows the result of the reaction with the native IgG antibody (anti-Her2 antibody) without introducing glutamine (lane 1), and the result of the reaction between the native IgG antibody (anti-Her2 antibody) without introducing glutamine and mPEGamine.
  • (Lane 2) the result of having reacted the natural IgG antibody (anti-Her2 antibody) and mPEG amine which did not introduce glutamine in the presence of TGase (lane 3).
  • Figure 2 (B) is the result of the reaction between T-KM1 and mPEGamine modified to include glutamine (lane 4), the result of the reaction with T-KM1 and TGase (lane 5), the reaction with T-KM1, mPEGamine and TGase The result (lane 6) is shown.
  • PEG is attached to the heavy chain (HC, Heavy chain) of the antibody in lane 6, it can be seen that the band is present in a higher position than the lanes 4 and 5.
  • Figure 2 (C) shows the result of the reaction between T-KM2 and mPEGamine modified to include glutamine (lane 7), the result of the reaction of T-KM2 and TGase (lane 8), the reaction of T-KM2, mPEGamine and TGase SDS- showing results (lane 9), the reaction between T-KM3 and mPEGamine (lane 10), the reaction between T-KM3 and TGase (lane 11), and the reaction with T-KM3, mPEGamine and TGase (lane 12). PAGE results.
  • Figure 2 (D) is the result of the reaction of T-KM4 and TGase modified antibody containing glutamine (lane 13), the result of the reaction between T-KM4 and mPEGamine (lane 14), the reaction of T-KM4, mPEGamine and TGase SDS- showing results (lane 15), T-KM10 and TGase (lane 16), T-KM10 and mPEGamine (lane 17), and T-KM10, mPEGamine and TGase (lane 18). PAGE results.
  • HC means heavy chain of antibody
  • TGase means transglutaminase
  • HC-PEG (1K) shows PEG bound to heavy chain of antibody.
  • Figure 3 is a graph of the antibody-drug conjugates analyzed by high performance liquid chromatography (HPLC). This was analyzed using the HIC (hydrophobicity interaction column). As shown in (B), the drug-binding form (Antibody), the drug-binding form (Ab-MMAF (1)), and the drug-binding form The morphology (Ab-MMAF (2)) is shown separated by different peaks.
  • (A) is a HPLC analysis of the antibody alone, and was used as a control to distinguish the form in which the drug is not bound when analyzing the prepared antibody-drug conjugate.
  • T-KM1-MMAF, T-KM2-MMAF, and T-KM3-MMAF are antibody-drug conjugates in which MMAF, a cytotoxic drug, is bound to T-KM1, T-KM2, and T-KM3 using glutaminase, respectively. to be.
  • Figure 5 shows the results of the efficacy test of the antibody-drug conjugate of the present invention in xenograft mice using JIMT-1 cell line.
  • the invention comprises the step of reacting a mutated antibody comprising glutamine and a drug comprising a free amine group in the presence of transglutaminase, Provided are methods for preparing antibody-drug conjugates.
  • a method for preparing an antibody-drug conjugate of the present invention comprises reacting a mutated antibody comprising glutamine and a drug comprising a free amine group in the presence of transglutaminase, for example (a) comprising glutamine Introducing the expression vector of the mutated antibody into a host cell to obtain a mutated antibody; And (b) reacting the mutated antibody obtained in step (a) in the presence of a drug containing a free amine group with a transglutaminase.
  • transglutaminase refers to an enzyme that forms a covalent bond between a free amine group and a carboxamide group of glutamine.
  • the transglutaminase may refer to an enzyme that forms a covalent bond between introduced glutamine of a mutated antibody and an amine group of a drug including a free amine group, using the transglutaminase. To form a bond between the mutated antibody comprising glutamine and the drug comprising a free amine group, thereby forming an antibody-drug conjugate.
  • transglutaminase when the transglutaminase forms a covalent bond between the carboxamide of glutamine and the epsilon amine group of lysine, an isopeptide bond may be formed through an acyl transfer reaction.
  • the transglutaminase is not limited to any protein that can catalyze the covalent bond between the gamma-carboxamide of glutamine and the free amine group present in the mutated antibody of the present invention. It may be derived from a living organism. In one embodiment of the present invention, the transglutaminase derived from bacteria was used.
  • the term “antibody-drug conjugate (ADC)” refers to a conjugate to which a mutated antibody is linked to a drug, and may also be referred to as an immunoconjugate.
  • the antibody-drug conjugate may be a conjugate obtained by reacting a mutated antibody comprising glutamine and a drug comprising a free amine group in the presence of transglutaminase.
  • the antibody-drug conjugate includes a conjugated form in which a mutated antibody including glutamine and a drug including a free amine group are linked through isopeptide bonds.
  • the conjugate of the present invention including the isopeptide bond is stable in the bloodstream and prevents the drug from being separated from the antibody, thereby prodruging the prodrug until reaching the target. To minimize the impact on normal tissues.
  • the antibody-drug conjugate binds the drug to an antibody capable of recognizing and binding an antigen present in a specific cell such as cancer cell
  • the drug can be delivered to a specific cell to which the antibody binds.
  • the antibody-drug conjugate can be used to treat a disease such as cancer.
  • the antibody of the antibody-drug conjugate is a therapeutic antibody, and may exhibit a synergistic effect compared to the case of using a therapeutic antibody and a drug in terms of therapeutic effect.
  • the antibody-drug conjugate may be a conjugate obtained by reacting a mutant antibody in which glutamine is introduced into the antibody and a drug including a free amine group in the presence of a transglutaminase. Reacting the introduced mutated antibody and mPEGamine in the presence of transglutaminase to mcMMAF containing a mutant antibody and lysine residues in which glutamine is introduced into an antibody of the IgG form or a glucosine in the presence of transglutaminase And the like obtained by the binder, but is not limited thereto.
  • the antibody-drug conjugate may refer to a form in which the drug is site-specifically bound to a specific site of the mutated antibody.
  • the antibody-drug conjugate by introducing a glutamine that specifically reacts with the transglutaminase to the antibody, it is possible to specifically bind the drug.
  • the antibody-drug conjugate of the present invention can be prepared in a form in which the drug is bound in a form that does not affect the antigen-binding ability of the antibody, a disease using the antibody-drug conjugate in which the binding ability of the antibody to the antigen is important It can be useful for the treatment of
  • An example of the form of the conjugate obtained by reacting a mutant antibody in which glutamine is introduced into the antibody and a drug containing a free amine group in the presence of transglutaminase is shown in the schematic diagram of FIG. 1.
  • antibody refers to a protein molecule that acts as a receptor for an antigen that specifically recognizes an antigen, including an immunoglobulin molecule that is immunologically reactive with a specific antigen.
  • Polyclonal antibodies monoclonal It includes all antibodies, whole antibodies and antibody fragments.
  • the total antibody is a structure having two full length light chains and two full length heavy chains, each of which is linked by a heavy chain and a disulfide bond.
  • the total antibody includes IgA, IgD, IgE, IgM and IgG, and IgG is a subtype, including IgG1, IgG2, IgG3 and IgG4.
  • the antibody fragment refers to a fragment having an antigen binding function, and includes Fab, Fab 'F (ab') 2 and Fv.
  • the Fab has one antigen binding site in a structure having a variable region of the light and heavy chains, a constant region of the light chain and a first constant region of the heavy chain (CH 1 ).
  • F (ab ') 2 antibodies are produced by disulfide bonds of cysteine residues in the hinge region of Fab'.
  • Fv (variable fragment) means a minimum antibody fragment having only the heavy chain variable region and light chain variable region.
  • Double-chain Fv is a disulfide bond, the heavy chain variable region and the light chain variable region is linked, and short-chain Fv (scFv) is generally covalently linked to the variable region of the heavy chain and the light chain through a peptide linker.
  • Such antibody fragments can be obtained using proteolytic enzymes (e.g., the entire antibody can be restricted to papain and Fab is obtained, and pepsin can yield F (ab ') 2 fragment). Can be produced through genetic recombination techniques.
  • the antibody also includes wild type antibodies, antibody fragments and genetically modified forms in which specific amino acids such as lysine (K) of the antibody or antibody fragment are removed, in particular, Lysine removed at the C or N terminus of the heavy or light chain of an IgG antibody. If the last lysine present at the C terminus of the heavy chain of the full-length antibody is not removed, the binding between the antibody heavy chains may appear preferentially rather than the binding with the drug. Preparation of the antibody; It is preferred for the preparation of antibody-drug conjugates using the prepared mutated antibodies. Such removal of lysine can be removed using conventional techniques in the art in view of the type and sequence of the antibody or antibody fragment to be used and considering the site to which the antibody and the drug are bound.
  • K lysine
  • the term “mutated antibody comprising glutamine” refers to an antibody that has been mutated to include glutamine, for example, the substitution or addition of glutamine (Q) to the antibody or glutamine. It includes a form attached to the peptide containing.
  • the mutated antibody may be a form in which a peptide including glutamine is attached to the full length antibody or antibody fragment, but is not limited thereto.
  • the mutated antibody in the form of the substitution of glutamine may be prepared by substituting residues not involved in glycosylation, such as glycosylation, and the mutated antibody binds the drug without removing the sugar chain structure of the antibody. There is an advantage to this.
  • the form in which the glutamine-containing peptide is linked to the antibody may be genetically engineered, and may be preferably in the form linked to the C or N terminal of the heavy or light chain of the antibody, more preferably the heavy chain of the antibody. Or a form linked to the C terminus of the light chain, and more preferably a form linked to the C terminus of the heavy chain of the antibody.
  • the mutated antibody comprising glutamine may be used as a substrate of transglutaminase.
  • the mutated antibody comprising glutamine of the present invention is not a form in which the amino acid involved in glycosylation is substituted with glutamine, but additionally inserts or substitutes glutamine without significantly affecting glycosylation. Or a form prepared by attaching a peptide containing glutamine, but is not limited thereto.
  • mutated antibody that introduces a specific glutamine that can react with transglutaminase at the terminal of the antibody, rather than a mutated antibody substituted with glutamine amino acids involved in glycosylation, Binding with lysine containing drug in the presence of transglutaminase.
  • Such mutated antibodies have the advantage of being able to specifically bind to the drug with little effect on glycosylation, such as glycosylation of the antibody.
  • a vector comprising a polynucleotide encoding a light chain by preparing an expression vector comprising a polynucleotide encoding a polypeptide comprising a polypeptide comprising a glutamine linked to the C terminal of the heavy chain of the IgG antibody
  • an expression vector comprising a polynucleotide encoding a polypeptide comprising a polypeptide comprising a glutamine linked to the C terminal of the heavy chain of the IgG antibody
  • a mutated antibody in which a peptide including glutamine was linked to the C terminus of the IgG antibody was prepared (Examples 1 and 2).
  • peptide including glutamine refers to a peptide containing one or more glutamine, and for the purposes of the present invention, a peptide comprising glutamine which can be linked to lysine by transglutaminase. Ramen noodles are included without limitation.
  • the peptide containing glutamine may be, for example, a peptide having an amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, but is not limited thereto.
  • Glutamine in the peptide may be present at various positions, and the peptide may comprise one or more glutamine.
  • Transglutaminase is known to recognize glutamine located in a fluid moiety in a protein (Angelo Fontana et al. Advanced Drug Delivery Reviews 2008. 60. 13-28), where the fluid moiety in the protein is chain fluid. Flexibility is part of a large protein that does not have a fixed tertiary structure or includes partially unfolded parts. Therefore, the peptide containing glutamine may be included in the peptide containing glutamine, regardless of the number of amino acids, provided that the glutamine is located in a fluid part rather than a fixed tertiary structure.
  • a representative glutamine-containing peptide consisting of eight amino acids, glutamine is located at the terminal GGGSLLQG (SEQ ID NO: 1), consisting of four amino acids LLQG (SEQ ID NO: 2), consisting of 11 amino acids and glutamine GGGGSLAQSHA (SEQ ID NO: 3) located in the middle, GQGGGSLASHA (SEQ ID NO: 4) located in front of glutamine, and NNDSTEYGLFQINNGI (SEQ ID NO: 5) consisting of 16 amino acids, have glutamine in various lengths and in various positions. Since it was confirmed that the peptide may be included in the peptide containing glutamine of the present invention, it is obvious that the peptide may be included in the peptide of the present invention regardless of the position of the glutamine or the length of the peptide.
  • the peptide containing glutamine means a peptide containing glutamine that can react with transglutaminase.
  • Carboxamide of glutamine present in the peptide may be linked to an amine group by transglutaminase.
  • the glutamine may be linked to a molecule having a free amine group by a transglutaminase at any position of the peptide such as the terminal or the middle of the peptide.
  • glutamine located in the middle or the end of the peptide is linked to mPEGamine by transglutaminase, it was confirmed that the efficacy as an antibody-drug conjugate (Fig. 2 to 5).
  • the term "drug containing a free amine group” refers to a drug having an amine group capable of reacting with a transglutaminase, and more preferably a mutated antibody comprising glutamine. It means a drug comprising an amine group that can be linked in the presence of a transglutaminase, for example mPEGamine, wherein the peptide containing glutamine can be linked in the presence of a transglutaminase and a mutated antibody in the form linked to the antibody Or a drug such as mcMMAF (maleimidocaproyl-monomethylauristatin F) prepared to include lysine, but is not limited thereto.
  • mcMMAF maleimidocaproyl-monomethylauristatin F
  • the drug containing the free amine group may be originally having an amine group, but may be synthetically or genetically engineered in a form having a free amine group capable of reacting with a transglutaminase. It can be produced by a method of synthesizing a drug to include an epsilon amine group or lysine.
  • a method for synthesizing a drug to include lysine various methods used in the art may be used, for example, using a bond between a maleimide group and a thiol group of cysteine, that is, a thioether bond, Drugs containing maleimide groups and peptides containing cysteine and lysine residues can be linked to each other to synthesize drugs containing lysine.
  • a peptide having a sequence of KGEGRGSGC SEQ ID NO: 6
  • free amine group refers to a functional group that can be linked to a carboxamide of glutamine and an acyl transfer reaction by a transglutaminase, for example, lysine (K).
  • Epsilon amine ( ⁇ -amine) present in, but is not limited thereto.
  • the term "drug” can bind to an antibody of the invention to increase the therapeutic efficiency of the therapeutic antibody itself, increase the half-life of the antibody in the blood, or reach a location targeted by the antibody, Means a substance that can be used for the treatment of diseases by killing the cancer in the target, and the like is not particularly limited, but may preferably be a cytotoxic drug, a toxin or a stabilizer.
  • the term "cytotoxic drug” may mean a drug that can be used for the treatment of a disease.
  • the cytotoxic drug may be a drug that can be combined with a mutated antibody in the presence of a transglutaminase, and may mean a drug that can be used for the treatment of a disease of an individual.
  • the cytotoxic drug is not limited thereto, but may preferably be a microtubulin structure forming inhibitor, a meiosis inhibitor, a topoisomerase inhibitor, or a DNA intercalator.
  • maytansinoid mayurisin, auristatin, dolastatin, calicheamicin, pyrrolobenzodiazepines, doxorubicin, duocamycin ), Carboplatin (paraplatin), cisplatin, cisplatin, cyclophosphamide, ifosfamide, nidran, nitrogen mustard (mecloethamine hydrochloride) nitrogen mustar (mechlorethamine HCL), bleomycin, mitomycin C, cytarabine, flurouracil, gemcitabine, trimetrexate metrexate, methotrexate, etoposide, vinpoline, vinblastine, vinorelbine, alimta, altretamine, procarbazine, It may be taxol, taxotere, topotecan or irinotecan.
  • the term "toxin” refers to a drug having a toxicity produced by an organism, and for the purpose of the present invention, the toxin binds to a mutated antibody and may be used to treat an individual's disease. Can mean.
  • the toxin may be, but is not limited to, extracellular or phytotoxic.
  • the term "stabilizer” refers to a drug capable of binding to a protein to increase the half-life of the protein in vivo, and preferably a half-life of the antibody or mutated antibody to bind to the antibody or the mutated antibody. It means a drug that can be increased, and more preferably may mean polyethylene glycol (PEG, Polyethylene glycol) or hyaluronic acid.
  • the stabilizer may be in the form of an amine (amine) to react with the transglutaminase, for example mPEGamine.
  • a mutated antibody connecting a peptide consisting of 4 to 16 amino acids including glutamine at the C-terminus of the heavy chain of the IgG antibody which has a free amine group in the presence of transglutaminase Reaction with mPEGamine or mcMMAF produced antibody-drug conjugates in which mPEGamine or mcMMAF was linked to IgG antibodies (Examples 1-5).
  • the present invention provides an antibody-drug conjugate prepared by the above method.
  • the present invention provides antibody-drug conjugates wherein the mutated antibody comprising glutamine and the drug comprising free amine groups are linked by isopeptide bonds.
  • the drug containing the mutated antibody and the free amine group are as described above.
  • isopeptide bond refers to a peptide formed between an amine group ( ⁇ -amine group) of a lysine side chain and a carboxyl group ( ⁇ or ⁇ -carboxyl group or carboxamide) of a glutamine or asparagine side chain. It means a bond, preferably a peptide bond formed between the amine group of the lysine side chain and the carboxyl group of glutamine.
  • the isopeptide bond may refer to a bond formed by transglutaminase.
  • the isopeptide bond is a stable bond having resistance to protease, which is stable to blood flow even in the blood of an individual, thereby maintaining a stable form of antibody-drug connection.
  • the antibody-drug conjugate may be a conjugate in a form in which a mutated antibody and a drug including a free amine group are linked through isopeptide bonds, and preferably a free amine that can be used for preventing or treating a disease.
  • the drug including the group may be a conjugate of a mutated antibody and a form linked through isopeptide, but is not limited thereto.
  • the present invention provides a pharmaceutical composition for preventing or treating cancer comprising the antibody-drug conjugate.
  • the antibodies, drugs and conjugates are as described above.
  • prevention means any action that inhibits or delays the onset of cancer by administration of the composition.
  • treatment means any action that improves or advantageously changes the symptoms of the cancer disease by administration of the composition.
  • cancer may refer to a cancer which can be selectively killed using the antibody-drug conjugate of the present invention, and the cancer treatable using the antibody-drug conjugate may be included without limitation.
  • skin, digestive, urinary, genital, respiratory, circulatory, brain or nervous system cancers specifically lung cancer, non-small cell lung cancer, colon cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, uterine cancer, ovarian cancer, Rectal cancer, stomach cancer, anal muscle cancer, colon cancer, breast cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal cancer , Soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocyte lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal
  • the antibody-drug conjugates of the present invention may be in the form of a site-specific binding of a cytotoxic agent, toxin or stabilizer used for the treatment of cancer to a mutated antibody for use in the treatment of cancer, which is the antigen of the original antibody. Drugs may be added to increase the efficacy of the drug more effectively without affecting cognition.
  • the mutated antibody of the present invention includes a form that has little effect on glycosylation, such as glycosylation, which plays an important role in the effector function and pharmacokinetics of the antibody, thereby binding the drug to the mutated antibody. In the case of using the antibody-drug conjugates for the treatment of cancer, there is an advantage that the effect can be further increased compared to the use of existing drugs or antibodies alone.
  • the pharmaceutical composition for treating cancer of the present invention may further include a pharmaceutically acceptable carrier, and may be formulated with the carrier.
  • a carrier or diluent that does not inhibit the biological activity and properties of the administered compound is sterile and biocompatible, which include saline, sterile water, Ringer's solution, buffered saline, albumin injectable solutions, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers and bacteriostatic agents may be added as necessary.
  • Diluents, dispersants, surfactants, binders and lubricants may also be added in addition to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.
  • composition of the present invention can be applied in any dosage form comprising it as an active ingredient, and can be prepared in an oral or parenteral dosage form.
  • Formulations for oral administration comprising the composition of the present invention as an active ingredient include, for example, tablets, troches, lozenges, water-soluble or oily suspensions, prepared powders or granules, emulsions, hard or soft capsules, syrups or elixirs can do.
  • lactose For formulation into tablets and capsules, lactose, saccharose, sorbitol, mannitol, starch, amylopectin, binders such as cellulose or gelatin, excipients such as dicalcium phosphate, disintegrating agents such as corn starch or sweet potato starch, stearic acid masne It may include a lubricating oil such as calcium, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax, and in the case of a capsule, it may further contain a liquid carrier such as fatty oil in addition to the above-mentioned materials.
  • a lubricating oil such as calcium, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax
  • a liquid carrier such as fatty oil in addition to the above-mentioned materials.
  • compositions of this invention As a dosage form for parenteral administration containing the composition of this invention as an active ingredient, it can be formulated in the form of injection, such as subcutaneous injection, intravenous injection, or intramuscular injection.
  • injection such as subcutaneous injection, intravenous injection, or intramuscular injection.
  • the compositions of the present invention may be mixed in water with stabilizers or buffers to prepare solutions or suspensions, which may be formulated for unit administration of ampoules or vials.
  • the pharmaceutical composition is administered in a pharmaceutically effective amount.
  • the term "administration” means introducing a pharmaceutical composition of the present invention to an individual in any suitable manner, and the route of administration of the composition is administered via various routes, oral or parenteral, as long as the target tissue can be reached. Specifically, it may be administered in a conventional manner via the oral, rectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, transdermal, nasal, inhalation, intraocular or intradermal routes.
  • the term "pharmaceutically effective amount” means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level is determined by the type and severity, age, sex, and cancer of the individual. It may be determined according to the type, activity of the drug, sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, factors including concurrently used drugs, and other factors well known in the medical field.
  • the compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents and may be administered sequentially or simultaneously with conventional therapeutic agents. And single or multiple administrations. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, and can be easily determined by those skilled in the art.
  • the antibody-drug conjugate is prepared by binding a mcMMAF drug containing lysine to a variant of the anti-HER2 antibody in the presence of a transglutaminase.
  • the antibody-drug conjugates showed an excellent anti-proliferative effect by specifically binding to the HER2 antigen in breast cancer cell lines expressing HER2 (Example 6).
  • the tumor suppression effect was significantly superior to that of the anti-HER2 antibody trastuzumab. (Example 7).
  • the present invention provides a mutated antibody to which a peptide comprising glutamine and an antibody are linked.
  • Peptides and antibodies comprising the glutamine are as described above.
  • the mutated antibody may refer to a peptide containing glutamine and a protein in a form in which the antibody is linked.
  • the mutated antibody may refer to a protein in which the peptide including glutamine is fused to the C or N terminus of the heavy or light chain of the antibody.
  • the peptide containing glutamine may mean a protein fused to the C terminal of the heavy chain of the antibody, but is not limited thereto.
  • the present invention provides a polynucleotide encoding the mutated antibody, an expression vector comprising the polynucleotide, and a transformant into which the expression vector is introduced.
  • Expression vectors comprising polynucleotides encoding the mutated antibodies provided herein are not particularly limited thereto, but may include mammalian cells (eg, human, monkey, rabbit, rat, hamster, mouse cells, etc.), plant cells, and the like. Can be a vector capable of replicating and / or expressing the polynucleotide in eukaryotic or prokaryotic cells, including yeast cells, insect cells or bacterial cells (e.g., E.
  • coli preferably in the host cell It may be a vector operably linked to an appropriate promoter for expression of the polynucleotide, and may comprise a vector comprising at least one selection marker, more preferably phage, plasmid, cosmid, mini-chromosome, virus, retroviral vector It may be a form in which the polynucleotide is introduced.
  • the expression vector including the polynucleotide encoding the mutated antibody includes both an expression vector comprising a polynucleotide encoding the heavy or light chain of the mutated antibody or a polynucleotide encoding the heavy or light chain of the mutated antibody, respectively. It may be an expression vector.
  • the transformant introduced with the expression vector provided by the present invention is not particularly limited thereto, but the bacterial cells such as E. coli, Streptomyces, Salmonella typhimurium transformed by introducing the expression vector; Yeast cells; Fungal cells such as Pchia pastoris; Insect cells such as Drozophila and Spodoptera Sf9 cells; Animal cells such as CHO, COS, NSO, 293T, bow melanoma cells; Or plant cells. According to one embodiment of the present invention, CHO-S cells were used as host cells.
  • introduction refers to a method of delivering a vector comprising a polynucleotide encoding the mutated antibody to a host cell.
  • introductions include calcium phosphate-DNA coprecipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroshock, microinjection, liposome fusion, lipofectamine and protoplast fusion. It can be carried out by various methods known in the art.
  • transduction refers to the delivery of a target product into cells using viral particles by means of infection.
  • the vector can be introduced into the host cell by gene bombardment or the like. Introduction in the present invention can be used interchangeably with transformation.
  • the present invention provides a method of treating cancer using the antibody-drug conjugate.
  • the method may be a method of treating cancer comprising administering a pharmaceutical composition comprising an antibody-drug conjugate, or a pharmaceutically acceptable carrier, to a subject having or suspected of having the cancer.
  • Carriers, cancers and administrations that can be used are the same as described above.
  • Such individuals include mammals, birds, and the like, including cattle, pigs, sheep, chickens, dogs, humans, and the like, and include, without limitation, individuals whose cancer is treated by administration of the composition of the present invention.
  • the composition may be administered in a single or multiple doses in a pharmaceutically effective amount.
  • the composition may be administered in the form of a liquid, powder, aerosol, capsule, enteric skin tablets or capsules or suppositories.
  • Routes of administration include, but are not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, endothelial, oral, topical, nasal, pulmonary, rectal, and the like.
  • the oral composition upon oral administration, since the peptide is digested, the oral composition must be formulated to coat the active agent or to protect it from degradation in the stomach.
  • the pharmaceutical composition may be administered by any device in which the active agent may migrate to the target cell.
  • a pharmaceutical composition comprising the antibody-drug conjugate of the present invention is administered in a pharmaceutically effective amount.
  • the pharmaceutically effective amount is as described above.
  • an anti-Her2 antibody was used as a representative antibody.
  • GGGSLLQG (SEQ ID NO: 1), LLQG (SEQ ID NO: 2), GGGGSLAQSHA (SEQ ID NO: 3), GQGGGSLASHA (SEQ ID NO: 1), a peptide containing glutamine, subtracted from the end of the C terminus of the heavy chain amino acid sequence of the anti-Her2 antibody. 4), or the amino acid sequence of the NNDSTEYGLFQINNGI (SEQ ID NO: 5) was linked to modify the amino acid sequence.
  • the antibody having a heavy chain containing GGGSLLQG (SEQ ID NO: 1) is T-KM1
  • the antibody having a heavy chain containing LLQG (SEQ ID NO: 2) is T-KM2
  • has a heavy chain comprising GGGGSLAQSHA (SEQ ID NO: 3)
  • the antibody was named T-KM3
  • the antibody having a heavy chain containing GQGGGSLASHA (SEQ ID NO: 4)
  • the antibody having a heavy chain containing NNDSTEYGLFQINNGI (SEQ ID NO: 5) was named T-KM4.
  • an expression vector comprising a polynucleotide encoding the modified heavy chain, to which the peptide containing glutamine was linked, was prepared.
  • the template DNA was used as a vector expressing the heavy chain portion of the anti-Her2 antibody.
  • the forward primer used to prepare the vector expressing the heavy chain of T-KM1 is represented by SEQ ID NO: 7 and the reverse primer is represented by SEQ ID NO: 8 (Table 1), and used to prepare the vector expressing the heavy chain of T-KM2.
  • the forward primer is represented by SEQ ID NO: 7 and the reverse primer is represented by SEQ ID NO: 9 (Table 2).
  • PCR was performed using the primers shown in Table 3 below.
  • a vector expressing the heavy chain portion of the anti-Her2 antibody was used as the template DNA.
  • the forward primer is represented by SEQ ID NO: 7
  • the reverse primer is represented by SEQ ID NO: 10.
  • the fragments amplified by PCR were cut into BsrG I, Not I and then inserted into a vector expressing an anti-Her2 heavy chain portion cut into BsrG I and Not I.
  • the vector prepared by the above method was named TG3.
  • PCR was performed again using the TG3 vector as template DNA.
  • the forward primer used is shown in SEQ ID NO: 7, and the reverse primer is shown in SEQ ID NO: 11.
  • the TG3 vector was cut back into BsrG I and BamH I, and then the PCR product obtained from the TG3 vector as a template was cut into BsrG I and BamH I and inserted.
  • a vector capable of expressing the heavy chain of T-KM3 was prepared.
  • PCR was performed using a vector expressing the heavy chain portion of the anti-Her2 antibody as template DNA.
  • the forward primer used to prepare the vector expressing the heavy chain of T-KM4 is represented by SEQ ID NO: 7 and the reverse primer is represented by SEQ ID NO: 12 (Table 4), and used to prepare the vector expressing the heavy chain of T-KM10.
  • the forward primer is represented by SEQ ID NO: 7 and the reverse primer is represented by SEQ ID NO: 13 (Table 5).
  • T-KM4 Heavy Chain Expression Vectors name order SEQ ID NO: BsrGI-201A for CCC AGG TGT ACA CCC TGC CC SEQ ID NO: 7 T-KM4 rev TCG AGC GGC CGC TCA GAT GCC GTT GTT GAT CTG GAA CAG GCC GTA CTC GGT GGA GTC GTT GTT GCC GGG GGA CAG GGA CAG SEQ ID NO: 12
  • Vectors expressing the light chain of the anti-Her2 antibody were used as is, without modification, in common for all three antibody productions.
  • Vectors expressing the heavy chain and the vector expressing the light chain of each of the T-KM1, T-KM2, T-KM3, T-KM4 and T-KM10 antibodies were transduced into CHO-S cells using PEI (Polyethylenimine) )
  • PEI Polyethylenimine
  • anti-Her2 native antibody containing no glutamine was used, and the vector expressing the heavy chain of the native antibody and the vector expressing the light chain were transduced into CHO-S cells using PEI.
  • the cells were cultured for 4 days, and then expressed recombinant T-KM1, T-KM2, T-KM3, T-KM4, T-KM10, and anti-Her2 native antibodies were recombinant protein-A Sepharose column (Hitrap MabSelect Sure). , 5 mL, GE healthcare).
  • a free amine group as the substrate.
  • Transglutaminase reaction was performed using mPEGamine (mexoxy PEG-NH 2 ) having a group). It is known that the amine groups of mPEGamine can bind to glutamine residues through the transglutaminase reaction mechanism (Anna Mero et al. Journal of Controlled Release . 2011. 154 27-34, Carlo Maullu et al. FEBS Journal .2009). 276. 6741-6750), mPEGamine was used as the substrate.
  • the light and heavy chains of the antibody appear separated around 50KDa and 25KDa, respectively.
  • mPEGamine (1K) binds to the heavy or light chain of the antibody, Since the band is raised in the band position, by checking the above phenomenon it can be confirmed the reactivity of the mutated antibody of the present invention with transglutaminase.
  • the light chain did not show a change in the band position, indicating that only the heavy chain artificially introduced with glutamine specifically forms a bond with mPEGamine (FIGS. 2 (B), (C) and (D)). .
  • cytotoxic drug conjugates using the transglutaminase reaction first a cytotoxic drug comprising lysine or a lysine derivative was synthesized.
  • mcMMAF maleimidocaproyl-monomethyl auristatin F
  • KGEGRGSGC SEQ ID NO: 6
  • cysteine binding the peptides of mcMMAF and SEQ ID NO: 6 were linked to each other to synthesize a drug including a free amine group.
  • T-KM1, 2 or 3 which is a mutated antibody comprising glutamine; Microbial transglutaminase (zedira, Germany); And the cytotoxic drugs prepared in Example 4.
  • the mixture was incubated at 37 ° C. for 6 hours, and the mixture was analyzed by HPLC (high performance liquid chromatography) to confirm the degree of drug binding to the antibody, and specifically analyzed using a Butyl NRP (4.6 * 35, TSKgel) column. Is shown in FIG. 3.
  • Figure 3A is an analysis of the antibody alone by HPLC, shows the experimental results of the control group to distinguish the form that the drug is not bound in the antibody-drug conjugate analysis prepared in the present invention
  • Figure 3B is the mutated antibody The results of analyzing the antibody mixture in which the drug was reacted in the presence of transglutaminase are shown.
  • the mutated antibody and drug of the present invention showed the result of forming an antibody-drug conjugate in the presence of transglutaminase.
  • it shows a form in which a drug is bound to an antibody (denoted as Ab-MMAF (1)) and a form in which two drugs are bound (denoted as Ab-MMAF (2)). It was suggested that can be measured.
  • an anti-proliferation assay was performed using BT474, MCF7 and JIMT-1 cell lines as follows.
  • each cell was cultured to suspend BT474 cells at 1 ⁇ 10 5 cells / ml, MCF7 and JIMT-1 cells at 2 ⁇ 10 4 cells / ml, and 100 ⁇ l were loaded into each well of a 96 well plate. Then, after 3 hours of incubation in a cell incubator, 100 ⁇ l of antibodies or antibody-drug conjugates of various concentration sections were added per well and incubated for 5 days in the cell incubator.
  • the antibody used at this time was an anti-Her2 antibody, and the antibody-drug conjugate was drug (MMAF) to T-KM1, T-KM2 or T-KM3, which was bound using transglutaminase by the method described in the above example. It was a combined form.
  • MMAF drug
  • Alamar Blue (Invitrogen, USA) was treated with 25 ⁇ l in each well, and then wrapped in foil and treated for 6 hours in a cell incubator, and fluorescence intensity was measured at 530 nm using Spectramax Geminix. The fluorescence value thus measured indicates the extent of cell growth. The percentage of living cells based on the fluorescence value is shown in FIG. 4.
  • T-KM1-MMAF, T-KM2-MMAF, and T-KM3-MMAF which are representative antibody-drug conjugates of the present invention. All three showed cell growth rates of 65-70% (FIG. 4B).
  • MCF-7 cell line with low Her2 expression used as a negative control was inhibited in cell growth when treated with anti-Her2 antibody as well as T-KM1-MMAF, T-KM2-MMAF, and T-KM3-MMAF, which are representative conjugates of the present invention. Ineffective results were shown (FIG. 4C).
  • the results indicate that the representative conjugates of the present invention T-KM1-MMAF, T-KM2-MMAF, T-KM3-MMAF specifically binds to the antigen and causes cytotoxicity, which is the transglue of the present invention. It is suggested that the antibody-drug conjugates (T-KM1-MMAF, T-KM2-MMAF, T-KM3-MMAF) prepared by using minaminase exhibit their efficacy by specifically binding to the antigen.
  • an antibody-drug conjugate was prepared by the method described in Example 5. Specifically, mutated antibodies comprising glutamine, microbial transglutaminase (zedira, Germany), and cytotoxic drugs were mixed, and these were recombinant recombinant protein-A Sepharose columns (Hitrap MabSelect Sure, 5 mL, GE healthcare). Purification yielded antibody-drug conjugates and small amounts of antibody.
  • Balb / C nu / nu mice were intraperitoneally administered 7 cells of JIMT-1 cell line 1 ⁇ 10 per mouse, and vehicle, anti-Her2 antibody (Trastuzumab, 5mg / kg) or the present invention at the time of tumor size 200mm 2 or more T-KM1-MMAF (5 mg / kg) and T-KM3-MMAF (5 mg / kg), which are antibody-drug conjugates, were administered intravenously twice a week. Then, tumor size was measured twice a week, the results are shown in FIG.

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Cited By (3)

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Publication number Priority date Publication date Assignee Title
US10941431B2 (en) 2016-06-10 2021-03-09 Eisai R&D Management Co., Ltd. Lysine conjugated immunoglobulins
US11135304B2 (en) 2015-12-18 2021-10-05 Eisai R&D Management Co., Ltd. C-terminal lysine conjugated immunoglobulins
US11786603B2 (en) 2016-02-26 2023-10-17 Regeneron Pharmaceuticals, Inc. Optimized transglutaminase site-specific antibody conjugation

Non-Patent Citations (1)

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Title
SCHNEIDER ET AL.: "Monoclonal antibody production in semi-continuous serum- and protein-free culture: Effect of glutamine concentration and culture conditions on cell growth and antibody secretion", JOURNAL OF IMMUNOLOGICAL METH SDS, vol. 129, no. ISSUE, 25 May 1990 (1990-05-25), pages 251 - 268 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11135304B2 (en) 2015-12-18 2021-10-05 Eisai R&D Management Co., Ltd. C-terminal lysine conjugated immunoglobulins
US12214050B2 (en) 2015-12-18 2025-02-04 Eisai R&D Management Co., Ltd. C-terminal lysine conjugated immunoglobulins
US11786603B2 (en) 2016-02-26 2023-10-17 Regeneron Pharmaceuticals, Inc. Optimized transglutaminase site-specific antibody conjugation
US10941431B2 (en) 2016-06-10 2021-03-09 Eisai R&D Management Co., Ltd. Lysine conjugated immunoglobulins
US11753669B2 (en) 2016-06-10 2023-09-12 Eisai R&D Management Co., Ltd. Lysine conjugated immunoglobulins

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