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WO2017073981A1 - Conjugué anticorps-médicament et son procédé de préparation - Google Patents

Conjugué anticorps-médicament et son procédé de préparation Download PDF

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Publication number
WO2017073981A1
WO2017073981A1 PCT/KR2016/012009 KR2016012009W WO2017073981A1 WO 2017073981 A1 WO2017073981 A1 WO 2017073981A1 KR 2016012009 W KR2016012009 W KR 2016012009W WO 2017073981 A1 WO2017073981 A1 WO 2017073981A1
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antibody
linker
genbank accession
drug conjugate
formula
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Korean (ko)
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박순재
정혜신
이선배
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Alteogen Inc
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Alteogen Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/407—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with other heterocyclic ring systems, e.g. ketorolac, physostigmine
    • 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

Definitions

  • the present invention relates to an antibody-drug conjugate, a method for preparing the same, and a composition for treating cancer comprising the antibody-drug conjugate, and specifically, an antibody-drug conjugate wherein a DNA alkylating agent is bound to an antibody through a linker comprising polyethylene glycol. It relates to a method for producing the same and a composition for treating cancer comprising the same.
  • Drugs used for chemotherapy are often toxic, especially bone marrow, mucosal and neurotoxic. Therefore, there is a need for the development of an anticancer agent that is safer while showing strong anticancer activity and specificity to cancer cells. Anti-cancer drugs that act only on cancer cells and reduce side effects are being developed in various ways.
  • a therapeutic agent using an antibody that specifically expresses a target i.e., an antigen, specifically binding to a specific disease
  • a target i.e., an antigen
  • methods for diagnosing and treating tumors using antibodies, such as anti-cancer antibodies, which identify tumor-associated antigens specifically expressed on the surface of cancer cells, bind to them, and inhibit cell growth or induce death are now widely used. Is also a very bright technology field.
  • anticancer antibodies have very high target specificity, but the killing effect of cancer cells is often lower than that of conventional cytotoxic drugs (anticancer drugs), that is, in combination with cytotoxic drugs and other cytostatic drugs. It is often used as a combination therapy.
  • Anticancer drugs show significantly higher cytotoxicity than anticancer antibodies, but have very high side effects compared to antibody therapeutics because of their low target specificity to cancer cells. Therefore, the combination therapy of anticancer antibody and anticancer drug shows a higher therapeutic effect than when each drug is administered separately, but has a fundamental limitation that side effects of anticancer drug are always accompanied.
  • drugs that can be used alone as anticancer drugs are limited to relatively low toxicity taxol or cisplatin-based drugs.
  • Most anticancer drugs with high cytotoxicity are virtually impossible to prescribe as single drugs because of their very high cytotoxicity.
  • the anticancer drug can be delivered only to the target cancer cells without the side effects of normal cells. Therefore, antibody-drug conjugates have been in the spotlight as a method for improving the therapeutic efficacy of anti-cancer drugs that cannot be used conventionally.
  • Antibody-drug conjugates that combine antibodies such as ZEVALIN TM and MYLOTARG TM with cytotoxic drugs or radioisotopes have been successfully developed for the treatment of non-Hodgkin's lymphoma and acute myeloid leukemia.
  • Binding toxins such as tancin (Immunogen, Inc.) and trastuzumab mertansine (Roche) to antibodies, or auristatin peptides that are dolastatin derivatives, auristatin E (AE), monomethylauristatin (MMAE), or a cytotoxic drug such as MMAF, may be used for the treatment of cBR96 (target specific to Lewis Y on carcinoma), cAC1O specific for CD30 on hematologic malignancies, CD20-expressing cancer and immunotherapy for the treatment of immune disorders. Many attempts have been actively made, such as binding to CD20 antibodies such as Rituxan, anti-EphB2R antibodies for the treatment of colorectal cancer, 2H9 and anti-IL-8, E-selectin antibodies and the like.
  • CD20 antibodies such as Rituxan, anti-EphB2R antibodies for the treatment of colorectal cancer, 2H9 and anti-IL-8, E-selectin antibodies and the like.
  • duocarmycin A, B1, B2, C1, C2, D, SA, CC-1065, etc. have been found and most show IC 50 values of several picomoles (pM).
  • Many duocarmycin derivatives based on duocarmycin have been developed, and synthetic derivatives have been reported for Adozelesin, bizelesin and carzelesin (Pavlidis et al., Clinical Trial Report, 2000, 46: 167-71).
  • Duocarmycin is conjugated to the minor groove of DNA to alkylate the N3 position of adenine (Boger, DL, Pure and Applied Chemistry, 1993, 65: 1123-32). This irreversible alkylation reaction modifies the structure of DNA, leading to the death of cancer cells.
  • the most widely used anticancer drugs used in antibody-drug conjugates, auristatin or maytansinoid family of anticancer drugs, are tubulin-binding drugs that target tubulin, which occurs during cell division. Thus, these tubulin binding drugs act only on cancer cells in the cell divider, whereas duocarmycins that bind to DNA function with or without cell dividers.
  • the very high cytotoxicity of duocarmycins is very advantageous in that low concentrations of antibody-drug conjugates can exhibit sufficient anticancer effects.
  • An effective antibody-drug conjugate maintains specificity for the cancer cell target antigen of the antibody itself, while the anti-cancer drug is stably bound to the antibody until the anti-cancer drug is delivered to the cancer cell, and is released by the released drug after it is delivered to the cancer cell. Death of cancer cells should be induced.
  • the inventors of the present application have confirmed that the antibody-drug conjugate, in which the duocarmycin, the DNA alkylating agent according to the present invention, is conjugated to the antibody through a stable linker, exhibits the desired pharmacological effect, and the antibody-drug conjugate and its The present invention has been completed for the preparation method.
  • the present invention provides an antibody-drug conjugate in which the DNA alkylating agent represented by the following structural formula (1) is bound to the antibody through a linker.
  • Ab is an antibody
  • linker is a linker comprising polyethylene glycol
  • D is a DNA alkylating agent
  • n means an integer of 1 to 20.
  • the present invention also provides a method for producing an antibody-drug conjugate, wherein the DNA alkylating agent represented by the following structural formula (1) comprising the following step is bound to the antibody through a linker.
  • Ab is an antibody
  • linker is a linker comprising polyethylene glycol
  • D is a DNA alkylating agent
  • n means an integer of 1 to 20.
  • the present invention further relates to a composition for treating cancer comprising the antibody-drug conjugate.
  • Herceptin-Duocarmycin SA conjugate M2-M-HEG-Duocarmycin compared with Herceptin through anti-proliferation assay in HER2 expressing SK-BR3 cells.
  • Herceptin biosimilar HR4-RC02-P1 Herceptin variant M2 with a linker-duocarmycin conjugate conjugated to the antibody-drug conjugate M2-M-HEG-Duoca, Duocarmycin SA itself, linker-duocarmycin conjugate M
  • the cell growth inhibitory effect of the antibody-drug conjugate M2-M-HEG-Duoca is superior to Duocarmycin SA itself or the linker-duocarmycin conjugate.
  • Figure 2 shows the caspase activation by treatment concentrations to confirm that apoptosis in HER2 expressing SK-BR3 cells is an apoptosis effect by M2 conjugated duocarmycin SA, a Herceptin cysteine variant. The results are compared with Herceptin.
  • Figure 3 shows the effect of cell growth inhibition of the paletuzumab-Duocarmycin SA conjugate, FM2-M-HEG-Duocarmycin, through parent anti-proliferation assay in folate-expressing KB cells. It shows the result confirmed compared with Jumap.
  • FM2b-M-HEG-Duoca and Duocarmycin SA which are antibody-drug conjugates in which the linker-duocarmycin conjugate is conjugated to the parent antibodies, paletuzumab and paletuzumab variants, FM2, Duocarmycin SA
  • the cell growth inhibitory effect of the antibody-drug conjugate FM2b-M-HEG-Duoca is superior to SA itself or the parent antibody.
  • Figure 4 shows the cell growth inhibition effect of FM2b-MC-vc-PAB-EDA-Duocarmycin, a paletuzumab-Duocarmycin SA conjugate, through anti-proliferation assay in folate-expressing KB cells. It shows the result confirmed by comparison with the parent antibody, paletuzumab.
  • FM2b-vc-PAB- an antibody-drug conjugate conjugated to a linker-duocarmycin conjugate made through the cleavable linker MC-vc-PAB-EDA to a parent antibody, paletuzumab, and a paletuzumab variant
  • the cell growth inhibitory effect of FM2b-vc-PAB-EDA-Duocamycin, the antibody-drug conjugate was superior to that of Duocarmycin SA itself and the parent antibody.
  • FIG. 3 show that the drug conjugate of Duocarmycin SA shows high cytotoxicity in both the non-cleavable linker and the cleavable linker.
  • the antibody-drug conjugate must be stably bound to the antibody until the anti-cancer drug is delivered to the target cancer cell.
  • Anticancer drugs delivered to target cancer cells must be released from the antibody to induce the death of cancer cells.
  • the anti-cancer drug must be stably deficient in the antibody by using a linker and have a structure of a drug-linker having sufficient cytotoxicity to induce cancer cell death when released from cancer cells.
  • the inventors of the present application use a DNA alkylating agent that binds to DNA to induce apoptosis, such as duocarmycin SA to release the cancer cell death by anti-cancer cytotoxicity of the DNA alkylating agent when released in cancer cells.
  • a DNA alkylating agent that binds to DNA to induce apoptosis, such as duocarmycin SA to release the cancer cell death by anti-cancer cytotoxicity of the DNA alkylating agent when released in cancer cells.
  • An antibody-drug conjugate based was attempted to be prepared.
  • the present invention relates to an antibody-drug conjugate in which a DNA alkylating agent represented by the following structural formula (1) is bound to an antibody through a linker.
  • Ab is an antibody
  • linker is a linker comprising polyethylene glycol
  • D is a DNA alkylating agent
  • n means an integer of 1 to 20.
  • the DNA alkylating agent may be Duocarmycin SA.
  • Duocarmycin SA has a structure represented by the following Chemical Formula 1, and consists of an alkylation part that alkylates the DNA binding part (DNA binding part) and adenine (N3) position of the DNA to the minor groove of the DNA.
  • a side chain capable of conjugating a linker to prepare an antibody-drug conjugate may be, for example, a hydroxyl group (OH) of an alkylation site.
  • the linker can be conjugated with the halide group because it is an indispensable functional group for forming a cyclopropyl group, which is an essential structure for the alkylation of DNA by Winstein cyclization of the molecular itself.
  • the linker is a site linking the antibody to the drug Duocarmycin SA, for example the linker is in a form that is cleavable under intracellular conditions, ie, in the intracellular environment, the drug can be released through cleavage of the linker.
  • the linker may be cleaved by a cleavage agent present in an intracellular environment such as a lysosomal or endosomal, for example a peptide linker that may be cleaved by an intracellular peptidase or protease enzyme such as a lysosomal or endosomal protease Can be.
  • Peptide linkers generally have at least two amino acids in length.
  • the cleavable linker is pH sensitive, and may be sensitive to hydrolysis at certain pH values.
  • pH sensitive linkers can be hydrolyzed under acidic conditions.
  • acid labile linkers that can be hydrolyzed in lysosomes such as hydrazones, semicarbazones, thiosemicarbazones, cis-aconitic amides, orthoesters, acetals, Ketal and the like.
  • the linker may be cleaved under reducing conditions, for example disulfide linkers.
  • SATA N-succinimidyl-S-acetylthioacetate
  • SPDP N-succinimidyl-3- (2-pyridyldithio) propionate
  • SPDB N-succinimidyl-3- (2-pyridyldithio) butyrate
  • SMPT N-succinimidyl-oxycarbonyl
  • alpha disulfide bonds can be formed using -alpha-methyl-alpha- (2-pyridyl-dithio) toluene).
  • the drug and / or drug-linker may be conjugated randomly via lysine of the antibody or via cysteine that is exposed when the disulfide bond chain is reduced.
  • the linker-drug may be bound via a genetically engineered tag, such as cysteine present in a peptide or protein.
  • the peptide or protein has a deletion at the carboxy terminus of the peptide or protein, or has an addition via covalent attachment of a spacer unit to the carboxy (C) terminus of the peptide or protein.
  • the peptide or protein may be directly covalently linked to the amino acid motif or covalently linked to the spacer unit to be linked to the amino acid motif.
  • linker may be, for example, a non-cleavable linker, and the drug is released through only one step of antibody hydrolysis to produce, for example, an amino acid-linker-drug complex.
  • This type of linker may be a thioether group or maleimidocaproyl, and may maintain stability in blood.
  • linker according to the present invention may be represented by the following formula (2).
  • the linker in the formula (1) may have a structure represented by the following formula (2).
  • A is a hydrocarbon or a derivative thereof, a is an integer from 1 to 20,
  • E is ethylene glycol, e is an integer from 1 to 20,
  • W is an amino acid, w is an integer of 0-20,
  • S is at least one selected from the group consisting of ethers, carbamates, carbonates and esters.
  • the linker linking the antibody and the DNA alkylating agent functions to stably bind the anti-cancer drug to the antibody until it carries the DNA alkylating agent, for example, duocarmycin SA, to the target cancer cells.
  • the hydrocarbon of A or a derivative of hydrocarbon is, for example, covalently bonded to the cysteine residue of the antibody to stably link the linker-drug to the antibody and at the same time stable between the hydrophobic drug and the antibody.
  • the hydrocarbon of A or a derivative of hydrocarbon is, for example, covalently bonded to the cysteine residue of the antibody to stably link the linker-drug to the antibody and at the same time stable between the hydrophobic drug and the antibody.
  • the substituted or unsubstituted group can be, for example, C1-C8 alkyl, for example ethyl, propyl, butyl, pentyl or octyl, aminoalkyl, aminocarbonylalkyl, carboxyalkyl, hydroxyalkyl, hydroxy or polyethylene It may be glycol.
  • aminoalkyl, aminocarbonylalkyl, carboxyalkyl, hydroxyalkyl the alkyl may be C1-C8 alkyl.
  • A means the number of repetitions of A in the formula (2), and an integer of 1-20, for example, an integer of 2-18, an integer of 3-15, an integer of 4-14, an integer of 5-10, 6 It may be an integer of -8.
  • E in the formula (2) is polyethylene glycol based on ethylene glycol monomers.
  • the linker enhances the conjugation reactivity by increasing the solubility of the DNA alkylating agent such as duocarmycin SA in the conjugation reaction in an aqueous solution, in addition to the role of stably linking the DNA alkylating agent such as duocarmycin SA to the antibody.
  • the hydrophobic (hydrophobicity) of the anticancer drug may play a role in enhancing the stability in the aqueous solution of the antibody-drug conjugate.
  • E may be polyethylene glycol in which e comprises 1 to 15, preferably 2 to 13, 2 to 12, 2 to 11, or 2 to 10 ethylene glycol.
  • the amino acid of W in the formula (2) is, for example, having a structure of a ligand of the protease overexpressed in cancer cells, specifically val-sitrulline (cit) or val-alanine (ala) yl Can be.
  • the amino acid may include a naturally occurring L ⁇ -amino acid or a residue thereof, as well as a D-amino acid and a chemically modified amino acid.
  • the peptide or analogous amino acid may comprise a natural amino acid or non-amino acid compound construct.
  • the analogous amino acid is defined as a substance exhibiting similar physical properties such as size, charge or hydrophobicity that the corresponding amino acid or peptide exhibits in spatial orientation.
  • the peptide mimetic compound may be a compound in which an amide bond present between one or more amino acids is substituted with a carbon-carbon bond or other bond known in the art.
  • W means the number of repetitions of W in the above formula (2), and if 0, W may not be included.
  • W may be an integer of 1-20, for example, an integer of 2-18, an integer of 3-15, an integer of 4-14, an integer of 5-10, or an integer of 6-8.
  • S is a functional group connecting Aa-Ee-Ww to a DNA alkylating agent such as Duocarmycin SA, and is one or more selected from the group consisting of substituted or unsubstituted ether, carbamate, carbonate, ester, Carbamate.
  • the S may allow the drug to be released from the antibody under enzyme, hydrolysis or other metabolic conditions.
  • the ether can be, for example, substituted or unsubstituted ether, specifically methyl ether or ethyl ether
  • the carbamate is, for example, substituted or unsubstituted carbamate, specifically PAB-carbamate or diamine carbame
  • the carbonate is a substituted or unsubstituted carbonate, specifically methyl-carbonate or ethyl carbonate
  • the ester is, for example, a substituted or unsubstituted ester, specifically methyl ester or ethyl Esters.
  • Ether can stably maintain duocarmycin in a conjugated state so as not to be easily separated by pH or protease, thereby improving blood stability of antibody-drug conjugates, and in the case of carbonates or esters in cancer cells through pH-sensitive degradation reactions.
  • the drug may be degraded.
  • Carbamate For example, PAB-carbamate or diamine carbamate is a structural structure in which the amino substrate of W is degraded by a protease and then separated from cancer cells with high cytotoxicity of duocarmycin through autolysis. Has characteristics.
  • linker according to the present invention may be represented by the following formula (3).
  • an antibody-drug conjugate was prepared by conjugating a drug-linker conjugate made by linking a linker to a hydroxyl group of duocarmycin SA, to a cysteine group of the antibody.
  • an 'antibody' may be used without limitation as long as it has binding ability and specificity to a specific antigen.
  • an animal-derived antibody such as a mouse antibody, a chimeric antibody, or a humanization may be used.
  • Antibodies, transgenic mice, human antibodies developed using display technology, and the like can all be used.
  • both modified antibodies such as a bispecific antibody, a fragment of an antibody, etc. can be used.
  • the linker-D in the antibody-drug conjugate represented by Structural Formula (1) may be linked to the heavy or light chain terminal of the antibody Ab, for example, the C-terminus.
  • the duocarmycin SA-linker conjugate In order to conjugate DNA alkylating agents such as duocarmycin SA to an antibody, the duocarmycin SA-linker conjugate must have the ability to bind specific amino acid residues present in the antibody.
  • the binding of the antibody and the drug in the antibody-drug conjugate may be performed by using an amine group or cysteine present in the antibody.
  • antibody-drug conjugates were prepared through conjugation to cysteines already present in the antibody or to cysteines artificially introduced into the antibody through gene mutagenesis.
  • the antibody into which the cysteine is introduced may be, for example, a modified antibody to which a motif comprising a cysteine residue represented by the following structural formula (3) is bound:
  • (M Cys ) n means a metal ion binding motif comprising a cysteine residue
  • Xa means a peptide consisting of 0 to 20 amino acid residues excluding cysteine
  • Xb n in the group consisting of amino acids A, G and S
  • n means an integer from 1 to 20,
  • the metal ion binding motif comprising the cysteine residue includes a C 2 H 2 group (Cys 2 His 2 class: Cys-X 2-4 -Cys-X 12 -His-X 3-5 -His) of a zinc finger protein.
  • Cys 2 His 2 class: Cys-X 2-4 -Cys-X 12 -His-X 3-5 -His of a zinc finger protein.
  • functional groups capable of linking the cysteine and the drug of the antibody include maleimide groups and derivatives thereof, aziridine and derivatives thereof, acryloyl and derivatives thereof, or aryl halides and derivatives thereof such as fluoro
  • maleimide groups and derivatives thereof aziridine and derivatives thereof, acryloyl and derivatives thereof, or aryl halides and derivatives thereof such as fluoro
  • aryl halides and derivatives thereof such as fluoro
  • the nucleophilic reactivity of the thiol of the cysteine residue with respect to the maleimide group is present in the protein. Because it is about 1,000 times higher than the N-terminal amino group, it is used to specifically bind to cysteine. Therefore, antibody-drug conjugates prepared using maleimide or iodine acetamide can be seen that the cysteine of the antibody binds to the drug via thioether linkage.
  • Antibodies conjugated by DNA alkylating agents e.g., duocarmycin SA, to prepare antibody-drug conjugates include all types of immunoglobulin molecules (e.g., IgG, IgE, IgM, IgD, and IgA), classes (e.g., IgG1, IgG2, IgG3, IgG4, IgAl and IgA2) or subclasses, and may be used from any species.
  • immunoglobulin molecules e.g., IgG, IgE, IgM, IgD, and IgA
  • classes e.g., IgG1, IgG2, IgG3, IgG4, IgAl and IgA2 or subclasses, and may be used from any species.
  • the antibody may be, for example, a tumor-associated antigen (TAA), cell surface receptor proteins and other cell surface molecules, transmembrane proteins, signaling proteins, cell survival regulators, cell proliferation regulators, molecules associated with tissue development or differentiation.
  • TAA tumor-associated antigen
  • cell surface receptor proteins and other cell surface molecules transmembrane proteins, signaling proteins, cell survival regulators, cell proliferation regulators, molecules associated with tissue development or differentiation.
  • TAA tumor-associated antigen
  • cell surface receptor proteins and other cell surface molecules transmembrane proteins, signaling proteins, cell survival regulators, cell proliferation regulators, molecules associated with tissue development or differentiation.
  • TAA tumor-associated antigen
  • the antibody may comprise (1) BMPR1B (bone morphogenic protein receptor-IB type, Genbank Accession No. NM_001203);
  • MPF MPF, MSLN, SMR, megakaryocyte enhancing factor, mesothelin, Genbank Accession No. NM — 005823
  • Napi3b (NAPI-3B, NPTIIb, SLC34A2, Solute Carrier Family 34 (Sodium Phosphate), Member 2, Type II Sodium-Dependent Phosphate Transporter 3b, GenBank Accession No. NM_006424);
  • Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, Semaphorin 5b Hlog, Sema Domain, 7 Thrombospondin Repeats (Type 1 and Similar Type 1), Transmembrane Domain (TM) And short cytoplasmic domain, (semaphorin) 5B, Genbank Accession No. AB040878);
  • PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12, RIKEN cDNA 2700050C12 gene, Genebank Accession No. AY358628);
  • ETBR endothelin type B receptor, Genbank Accession No. AY275463
  • MSG783 (10) MSG783 (RNF124, hypothetical protein FLJ20315, Genebank Accession No. NM_017763);
  • STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, Prostate Cancer Related Gene 1, Prostate Cancer Related Protein 1, Prostate 6 Transmembrane Epithelial Antigen 2, 6 Transmembrane Prostate Protein, Genebank Authorization number AF455138);
  • TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cation channel, subgroup M, member 4, Genbank Accession No. NM_017636);
  • CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derived growth factor, Genbank accession no. NP — 003203 or NM — 003212);
  • CD21 CR2 (complementary receptor 2) or C3DR (C3d / Epstein Barr virus receptor) or Hs.73792 Genbank Accession No. M26004);
  • CD79b (CD79B, CD79 ⁇ , IGb (immunoglobulin-associated beta), B29, Genbank Accession No. NM — 000626);
  • FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing phosphatase anchoring protein 1a), SPAP1B, SPAP1C, GenBank Accession No. NM_030764);
  • ErbB receptors selected from EGFR, HER3 and HER4
  • NCA Gene Accession No. M18728
  • PSCA Genbank Accession No. AJ297436
  • BAFF-R B cell activating factor receptor, BLyS receptor 3, BR3, NP_443177.1
  • CD22 B-cell receptor CD22-B isotype, NP-001762.1
  • CD79a, CD79A, CD79 ⁇ , and immunoglobulin-associated alpha which are covalently interacting with CD79a (Ig beta (CD79B) and forming complexes on the surface with IgM molecules, are signals involved in B cell differentiation Forwarded, Genbank approval number NP_001774.1);
  • CXCR5 (Bucket Lymphoma Receptor 1, a G protein coupled receptor activated by CXCL13 chemokines, acts on lymphocyte migration and humoral defense, participates in HIV-2 infection, and develops AIDS, lymphoma, myeloma and leukemia Considered to be related to, Genbank approval number NP_001707.1);
  • HLA-DOB beta subunit of MHC class II molecules (Ia antigen), binding to peptides and presenting in CD4 + T lymphocytes, Genbank Accession No. NP — 002111.1);
  • P2X5 purine receptor P2X ligand-gate ion channel 5, an ion channel gated by extracellular ATP, may be involved in synaptic transmission and neurogenesis, the lack of which may contribute to the pathophysiology of idiopathic detrusor instability Yes, Genbank approval number NP_002552.2);
  • CD72 B-cell differentiation antigen CD72, Lyb-2, Genbank Accession No. NP — 001773.1
  • Lymphocyte antigen 64 (RP105), a type I membrane protein of the LY64 (leucine rich repeat (LRR) family), modulates B cell activation and apoptosis, and its loss of function is attributed to systemic lupus erythematosus patients. Associated with increased disease activity, GenBank Accession No. NP_005573.1);
  • FcRH1 Fc receptor-like protein 1, a putative receptor for immunoglobulin Fc domains containing C2 Ig-like and ITAM domains, may be involved in B lymphocyte differentiation, Genbank accession number NP_443170.1)
  • IRTA2 associated gene deregulation by immunoglobulin macrophage receptor translocation, a putative immunoreceptor capable of acting on B cell development and lymphoma development, occurs in some B cell malignancies, Genbank approval number NP_112571.1); And
  • TENB2 estimated transmembrane proteoglycans associated with EGF / heregulin family of growth factors and follistatin, Genbank approval number AF179274)
  • the antibody may have binding capacity to one or more targets selected from Tenascin.
  • the antibody is, for example, trastuzumab, rituximab, bevacuzmab, cituximab, panitumumab, ipurinumap, alemtuzumab, opatumumab, gemtuzumab, brentuximab, 90Y -Ibritumobib, 131I-tocitumobib, cBR96, cAClO, anti-CD20 antibody, anti-EphB2 antibody, anti-IL-8, E-selectin antibody, anti-MUC16 antibody and anti-CD30 antibody, It may be one or more selected from the group consisting of anti-CD33 antibody, anti-CD52 antibody.
  • duocarmycin SA is bonded to a maleimide group through a linker to which a complex of ethylene glycol is connected, as shown in Formula 4.
  • the connection between duocarmycin SA and polyethylene glycol was connected to the polyethylene glycol by using a carbamate coupler with a hydroxyl group (-OH) of duocarmycin SA.
  • the polyethylene glycol connected with the duocarmycin SA connects the maleimide group with the duocarmycin SA while increasing the solubility of the duocarmycin SA.
  • the maleimide groups form strong thioether bonds with thiol groups of the antibody's cysteine residues to form antibody-drug conjugates that stably bind duocarmycin SA to the antibody.
  • HER2 is a duocarmycin SA to paletuzumab, an antibody that selectively conjugates to M2, a cysteine variant of herceptin (trastuzumab), a folate receptor overexpressed in ovarian cancer.
  • a linker-duocarmycin conjugate as in (2) was prepared and conjugated.
  • Herceptin cysteine variant M2 or paletuzumab cysteine variant FM2b is an antibody variant incorporating a cysteine-containing peptide motif at the heavy chain C-terminus of the antibody, thereby preparing antibody-drug conjugates by site-specific drug conjugation. .
  • the antibody-drug conjugate was prepared by conjugating duocarmycin SA to the heavy chain C-terminus of Herceptin or paletuzumab using a maleimide group to prepare an antibody-drug conjugate, and then using the cancer cell of the antibody-drug conjugate using duocarmycin SA. Growth inhibition and cell death effects were observed.
  • a linker-drug conjugate of formula (5) was synthesized in which the duocarmycin SA was linked to maleimide using a cleavable linker that is degraded by a protease.
  • duocarmycin SA is conjugated with cysteine after being connected with ethylene diamine group, which is a spacer which is decomposed, para-aminobenzoate (PAB), valine-citrulline, which is a ligand of protease, and ethylene glycol. Is connected to a maleimide group.
  • PAB para-aminobenzoate
  • valine-citrulline which is a ligand of protease
  • ethylene glycol ethylene glycol
  • a variant of paletuzumab an antibody that selectively binds to a folate receptor that is overexpressed in ovarian cancer cells
  • Antibody-drug conjugates were prepared.
  • the cysteine variant of paletuzumab, FM2b is a paletuzumab variant that incorporates a cysteine-containing peptide motif at the heavy chain C-terminus of paletuzumab to prepare antibody-drug conjugates by site-specific drug conjugation.
  • duocarmycin SA Conjugation of duocarmycin SA to the heavy chain C-terminus of the cysteine variant of paletuzumab using a maleimide group to prepare an antibody-drug conjugate consisting of a cleavable linker that can be cleaved to the protease, followed by duocarmycin
  • an antibody-drug conjugate consisting of a cleavable linker that can be cleaved to the protease, followed by duocarmycin
  • the present invention relates to a method for preparing an antibody-drug conjugate, wherein the DNA alkylating agent represented by the following structural formula (1) comprising the following steps is bound to the antibody via a linker comprising polyethylene glycol:
  • Ab is an antibody
  • linker is a linker comprising polyethylene glycol
  • D is a DNA alkylating agent
  • n means an integer of 1 to 20.
  • the present invention relates to a composition for treating cancer comprising the antibody-drug conjugate.
  • the present invention also provides a method of treating cancer comprising administering a therapeutically effective amount of the antibody-drug conjugate.
  • Treatable cancers in the present invention include liver cancer, stomach cancer, breast cancer, colon cancer, bone cancer, pancreatic cancer, head or neck cancer, uterine cancer, ovarian cancer, rectal cancer, esophageal cancer, small intestine cancer, anal muscle cancer, colon cancer, fallopian tube carcinoma, endometrial carcinoma,
  • the antibody-diocarmycin SA conjugate may be contacted in SK-BR-3, HER2-amplified breast cancer cells in vitro, to induce cell proliferation inhibition.
  • Cytotoxic or cell proliferative activity of the antibody-drug conjugate exposes mammalian cells with receptor proteins, such as SK-BR-3 cells or KB cells, to the antibody-drug conjugate (ADC) in cell culture medium, Cells were identified by incubating for about 6 hours to about 5 days and measuring cell viability.
  • ADC antibody-drug conjugate
  • Herceptin does not directly cause cancer cell death but instead causes her2 positive cell death through antibody dependent cellular cytotoxicity (ADCC).
  • ADCC antibody dependent cellular cytotoxicity
  • measuring caspase activity can confirm that the cytotoxicity of the antibody-drug conjugate is caspase mediated apoptosis (Bayascas, et. (2002), Cell Death and Differentiation. 9: 1078-1089; Preaudat, et al (2002), Journal of Biomolecular Screening. 7: 267-274; Phillips, et al. (2008), Cancer Research 68 (22) ): 9280-9290).
  • caspase 3 and 7 were measured to confirm the mechanism of apoptosis by the antibody-drug conjugate.
  • apoptosis by antibody-drug conjugates results in exposure of mammalian cells with receptor proteins, such as SK-BR-3 cells, to the antibody-drug conjugates in media, incubating the cells for about 2 days, and cascade It was confirmed by measuring activity.
  • M2 (refer to Korea Patent No. 1541764) purified Trastuzumab variant was added with 2-10 equivalents of TCEP, a reducing agent per 1 equivalent of antibody variant, and reacted at 4 ° C. for 30 minutes to reduce thiol groups, followed by linker-drug of Formula 2 2-10 equivalents of M-HEG-Duocarmycin, a duocarmycin SA conjugate, were added and reacted at room temperature for 2-4 hours. The reaction was terminated by adding excess cysteine, and excess M-HEG-Duocarmycin and TCEP were removed by dialysis in a centrifugal filtration filter and phosphate buffer to prepare the final purified M2-M-HEG-Duocarmycin.
  • Example 2 In vitro cell proliferation inhibitory test of SK-BR-3 cells.
  • SK-BR3 cells ⁇ Kardial, HTB-30 ⁇ were diluted in DMEM / F12 medium with 10% FBS, adjusted to 1 ⁇ 10 4 / well, and then 100 ⁇ l cell culture was 96-well Each well of the plate was added. The well plates were then incubated for 24 hours in an incubator set at 5% carbon dioxide and 37 ° C. to attach the cells to the plates.
  • M2-M-HEG-Duocarmycin conjugate a conjugate of Herceptin cysteine variant (M2) prepared in Example 1 and duocarmycin SA, Duocarmycin SA, an anticancer drug, and M-HEG-Duocarmycin with a linker linked to Duocarmycin SA
  • final concentrations 66.7 nM, 33.3 nM, 6.7 nM, 3.3 nM, 0.67 nM, 0.33 nM, 0.067 nM, and 0.0067 nM were added, together with only medium (no drug) to control wells.
  • M2-M-HEG-Duocarmycin shows a significantly higher cytotoxicity than the parent antibody Herceptin and shows that the cell activity is reduced by about 85% at high concentrations. From this, it can be seen that the antibody-Duocarmycin SA conjugate shows very good cytotoxicity compared to the parent antibody.
  • SK-BR-3 cells were diluted in RPMI 1640 medium with 10% FBS adjusted to 1 ⁇ 10 4 / well before 100 ⁇ l cell culture was added to each well of a 96-well plate. . The well plates were then incubated for 24 hours in an incubator set at 5% carbon dioxide and 37 ° C. to attach the cells to the plates.
  • Caspase-Glo 3/7 reagent [Caspase-Glo 3/7 assay at 100 ⁇ l / well; Determination of the degree of degradation of the kerose phase substrate by the activity of kease phases 3 and 7 formed in cells undergoing cell death through the caspase pathway (Caspase pathway)] was added thereto and then incubated at room temperature for 30 minutes. Luminescence was measured with a luminometer.
  • FIG. 2 shows that the cells treated with Herceptin, the parent antibody, showed almost no caspase activity, but M2-M-HEG-Duocarmycin-treated cells showed high activities of caspase 3 and 7 with increasing concentration. From the results, M2-M-HEG-Duocarmycin, unlike Herceptin, it was confirmed that the Duocarmycin SA drug released from M2-M-HEG-Duocarmycin transported intracellularly caused apoptosis through the case phase.
  • KB cells overexpressing folate, a target antigen of paletuzumab were diluted in DMEM / F12 medium with 10% FBS, adjusted to 1 ⁇ 10 4 cells / well, and then 100 ⁇ l of cell culture was prepared. Each well of the well plate was added. The well plates were then incubated for 24 hours in an incubator set at 5% carbon dioxide and 37 ° C. to attach the cells to the plates. After diluting the paletuzumab cysteine variant (FM2) prepared in Example xx with duocarmycin SA, FM2b-M-HEG-Duocarmycin conjugate, and the anticancer drug Duocarmycin SA prepared in Example xx, the final concentration was 66.7 nM.
  • FM2b-M-HEG-Duocarmycin conjugate the anticancer drug
  • Duocarmycin SA prepared in Example xx
  • FM2b-M-HEG-Duocarmycin shows a significantly higher cytotoxicity compared to herceptin, the parent antibody. From this, it can be seen that the antibody-Duocarmycin SA conjugate shows very good cytotoxicity compared to the parent antibody.
  • a conjugated antibody of Duocarmycin SA and paletuzumab was prepared to prepare FM2b (metal ion-binding motif variant of paletuzumab) -Duocarmycin SA conjugate.
  • FM2b metal ion-binding motif variant of paletuzumab
  • MC-vc-PAB-EDA-Duocarmycin is added to react at room temperature for 2 hours. Let's do it.
  • reaction was terminated by the addition of excess cysteine, and excess MC-vc-PAB-EDA-Duocarmycin and TCEP were removed via centrifugal filtration and dialysis in phosphate buffer to finally purify FM2b-MC-vc-PAB- EDA-Duocarmycin was prepared.
  • cell growth inhibitory activity was performed using KB-cells overexpressed with the folate receptor.
  • KB-cells were diluted in DMEM / F12 medium with 10% FBS adjusted to 1 ⁇ 10 4 / well and then 100 ⁇ l cell culture was added to each well of a 96-well plate. It was. The well plates were then incubated for 24 hours in an incubator set at 5% carbon dioxide and 37 ° C. to attach the cells to the plates.
  • the modified antibody-drug conjugate purified to have DAR 2 is FM2b-MC-vc-PAB-EDA-Duocarmycin SA (Diocarmycin SA drug conjugate of the modified antibody FM2b To the final concentrations of 6.45 nM, 3.23 nM, 1.61 nM, 0.806 nM, 0.403 nM, 0.202 nM, 0.101 nM, 0.0504 nM, 0.0252 nM and 0.0126 nM. Together, only the medium (no drug) was added to the control wells.
  • FM2b-MC-vc-PAB-EDA-Duocarmycin SA an antibody-drug conjugate conjugated to an antibody using Duocarmycin SA, a cleavable linker, MC-vc-PAB-EDA, is a parent antibody. It shows much higher cytotoxicity than paletuzumab. In addition, antibody-drug conjugates show significantly higher anticancer cytotoxicity than the drug Duocarmycin SA itself.
  • the antibody-drug conjugate according to the present invention can accurately deliver a DNA alkylating agent having high cytotoxicity to a target cell due to the high antigen specificity of the parent antibody, thereby enhancing the therapeutic effect, and also having a high cytotoxic DNA alkylating agent. Because it remains stable to the antibody until it is delivered into the cell, it can increase the possibility of using it for various diseases, especially as an anticancer agent, without the side effects accompanying the synthetic drug.
  • the antibody-drug conjugate according to the present invention makes it possible to use DNA alkylating agents, for example, duocarmycin SA, which cannot be used as a drug due to high cytotoxicity, such as anticancer agents.
  • DNA alkylating agents, such as duocarmycin SA carried by antibodies that are highly specific for cancer cells, can specifically induce apoptosis only in cancer cells.

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Abstract

L'invention concerne un conjugué anticorps-médicament, un procédé de préparation, et une composition de traitement du cancer contenant le conjugué anticorps-médicament. Elle concerne en particulier un conjugué anticorps-médicament dans lequel un agent d'alkylation d'ADN est lié à un anticorps par un lieur comprenant du polyéthylène-glycol; un procédé de préparation; et une composition de traitement du cancer contenant celui-ci.
PCT/KR2016/012009 2015-10-27 2016-10-25 Conjugué anticorps-médicament et son procédé de préparation Ceased WO2017073981A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003064606A2 (fr) * 2002-01-28 2003-08-07 Medarex, Inc. Anticorps monoclonaux humains de l'antigene d'enveloppe prostatique specifique (psma)
EP2354163A2 (fr) * 2005-09-26 2011-08-10 Medarex, Inc. Conjugué d'un anticorps anti-cd4 ou anti-psma et duocarmycine
US20120082616A1 (en) * 2010-09-24 2012-04-05 Mallinckrodt Llc Aptamer Conjugates for Targeting of Therapeutic and/or Diagnostic Nanocarriers
KR20130097669A (ko) * 2012-02-24 2013-09-03 (주)알테오젠 시스테인 잔기를 포함하는 모티프가 결합된 변형항체, 상기 변형항체를 포함하는 변형항체-약물 접합체 및 그 제조방법
WO2015104373A2 (fr) * 2014-01-10 2015-07-16 Synthon Biopharmaceuticals B.V. Conjugués anticorps-médicaments à base de duocarmycine utilisables dans le cadre du traitement du cancer de l'endomètre

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10550190B2 (en) * 2014-04-04 2020-02-04 Merck Sharp & Dohme Corp. Phosphate based linkers for intracellular delivery of drug conjugates

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003064606A2 (fr) * 2002-01-28 2003-08-07 Medarex, Inc. Anticorps monoclonaux humains de l'antigene d'enveloppe prostatique specifique (psma)
EP2354163A2 (fr) * 2005-09-26 2011-08-10 Medarex, Inc. Conjugué d'un anticorps anti-cd4 ou anti-psma et duocarmycine
US20120082616A1 (en) * 2010-09-24 2012-04-05 Mallinckrodt Llc Aptamer Conjugates for Targeting of Therapeutic and/or Diagnostic Nanocarriers
KR20130097669A (ko) * 2012-02-24 2013-09-03 (주)알테오젠 시스테인 잔기를 포함하는 모티프가 결합된 변형항체, 상기 변형항체를 포함하는 변형항체-약물 접합체 및 그 제조방법
WO2015104373A2 (fr) * 2014-01-10 2015-07-16 Synthon Biopharmaceuticals B.V. Conjugués anticorps-médicaments à base de duocarmycine utilisables dans le cadre du traitement du cancer de l'endomètre

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