WO2023174401A1 - Gpc3抗体药物偶联物及其用途 - Google Patents

Gpc3抗体药物偶联物及其用途 Download PDF

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WO2023174401A1
WO2023174401A1 PCT/CN2023/082141 CN2023082141W WO2023174401A1 WO 2023174401 A1 WO2023174401 A1 WO 2023174401A1 CN 2023082141 W CN2023082141 W CN 2023082141W WO 2023174401 A1 WO2023174401 A1 WO 2023174401A1
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Prior art keywords
drug conjugate
pharmaceutically acceptable
antibody drug
gpc3 antibody
isomer
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PCT/CN2023/082141
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English (en)
French (fr)
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林圣超
张禹
花海清
朱忠远
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映恩生物制药（苏州）有限公司
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Publication of WO2023174401A1 publication Critical patent/WO2023174401A1/zh

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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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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

Definitions

  • the present invention provides antibody drug conjugates that specifically bind to GPC3 and compositions containing the same. Methods and uses of the antibody drug conjugates of the invention are also provided.
  • Glypican-3 (Glypican-3, GPC3) is a 70KD membrane protein composed of 580 amino acids, anchored on the cell surface through glycosylphosphatidylinositol (GPI); GPC3 plays an important role in cell growth and Play an important role in cell differentiation and cell migration.
  • GPC3 protein is expressed in embryonic tissues (liver, kidney, placenta, etc.), but due to epigenetic silencing caused by DNA methylation, there is almost no GPC3 expression in adult tissues.
  • immunohistochemical studies have found that GPC3 is specifically highly expressed in >70% of liver cancer tissues, and clinical studies have also shown that GPC3 expression is associated with poor clinical prognosis of HCC. Therefore, GPC3 has the potential to be used as a target molecule for liver cancer treatment.
  • the anti-GPC3 monoclonal antibody Codrituzumab (development code name GC33) developed by Roche is currently the fastest-growing GPC3-targeting molecule in clinical development.
  • GC33 development code name
  • NCT01507168 phase Ib clinical study
  • NCT01507168 phase II study
  • Keji Bio reported that its GPC3-CART treated 6 patients with advanced liver cancer. One of them achieved partial response, 3 had stable disease, the disease control rate reached 50%, and the median progression-free survival was 4.2 months.
  • ADC drugs targeting GPC3 have their unique development value and clinical needs.
  • the technical problem to be solved by the present invention is to overcome the shortcoming of few anti-GPC3 antibody drug conjugates in the prior art, and provide an anti-GPC3 antibody drug conjugate and its preparation method and application.
  • the anti-GPC3 antibody-drug conjugate of the present invention has one or more advantageous effects selected from the following group: (1) better inhibitory activity on tumor cell proliferation in vitro; (2) better Targeted inhibition; (3) Better plasma stability; (4) Better tumor inhibition effect in vivo; (5) Better bystander killing effect (Bystander Effect); (6) better anti-transporter transport ability; (7) better in vivo tumor targeting ability; and (8) better and good in vivo safety.
  • the present invention mainly solves the above technical problems through the following technical means.
  • the present application provides an anti-GPC3 antibody drug conjugate, an isomer thereof, a pharmaceutically acceptable salt thereof or a mixture thereof.
  • the structure of the anti-GPC3 antibody drug conjugate is as shown in formula (I):
  • D is a cytotoxic drug
  • p represents the average number of connections, and p is an integer or decimal from 1 to 10, preferably an integer or decimal from 3 to 8;
  • Ab is an anti-GPC3 antibody or an antigen-binding fragment thereof, which includes a heavy chain variable region and a light chain variable region.
  • the heavy chain variable region includes amino acid sequences such as SEQ ID NO: 1, SEQ ID NO: 2 and SEQ respectively.
  • HCDR1, HCDR2 and HCDR3 shown in ID NO:3 and the light chain variable region includes the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 respectively.
  • the present application provides an anti-GPC3 antibody drug conjugate, an isomer thereof, a pharmaceutically acceptable salt thereof or a mixture thereof.
  • the structure of the anti-GPC3 antibody drug conjugate is as shown in formula (I):
  • -M-D is a cytotoxic drug
  • p represents the average number of connections, and p is an integer or decimal from 1 to 10, preferably an integer or decimal from 3 to 8;
  • Ab is an anti-GPC3 antibody or an antigen-binding fragment thereof, which includes a heavy chain variable region and a light chain variable region.
  • the heavy chain variable region includes amino acid sequences such as SEQ ID NO: 1, SEQ ID NO: 2 and SEQ respectively.
  • HCDR1, HCDR2 and HCDR3 shown in ID NO:3 and the light chain variable region includes the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 respectively.
  • the present application provides an anti-GPC3 antibody drug conjugate, an isomer thereof, a pharmaceutically acceptable salt thereof or a mixture thereof, wherein the structure of the anti-GPC3 antibody drug conjugate is as follows: formula (I-1) Shown:
  • M is -L 2 -L 3 -XL 1 -;
  • L 2 is -O- or -S-;
  • L 3 is -(C(R 1a )(R 1b )) m -, m is 0, 1, 2 or 3, wherein when L 3 contains a methylene unit, 0 or 1 subunits of L 3
  • the methyl unit can be replaced by -C(O)-, or -C(S)-;
  • L 1 is -(C(R 2a )(R 2b )) n -, n is selected from 1, 2 or 3, wherein when L 1 may contain a methylene unit, 0 or 1 subunits of L 1 The methyl unit can be replaced by -C(O)-, or -C(S)-;
  • X is a 3 to 6-membered saturated carbocyclyl group, a 3 to 6-membered saturated heterocyclyl group or a single bond, and the 3 to 6-membered saturated carbocyclyl group and 3 to 6-membered saturated heterocyclyl group are optionally replaced by 1 One or more R 3a substitutions;
  • each R 1a , R 1b , R 2a , R 2b , R 3a may independently be hydrogen, halogen or a C 1-6 aliphatic group optionally substituted by one or more R ;
  • each R can independently be hydrogen or halogen
  • p represents the average number of connections, and p is an integer or decimal from 1 to 10;
  • Ab is an anti-GPC3 antibody or an antigen-binding fragment thereof.
  • the heteroatoms in the 3-6-membered saturated heterocyclyl group of the present invention are selected from N, O and S, and the number of heteroatoms is 1-3.
  • the anti-GPC3 antibody or antigen-binding fragment thereof of the present invention includes a heavy chain variable region and a light chain variable region
  • the heavy chain variable region includes an amino acid sequence such as SEQ ID NO: 1, SEQ HCDR1, HCDR2 and HCDR3 shown in ID NO:2 and SEQ ID NO:3
  • the light chain variable region includes amino acid sequences as shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 respectively.
  • the anti-GPC3 antibody or antigen-binding fragment thereof of the invention comprises: the amino acid sequence is as shown in SEQ ID NO: 7 or has at least 95%, 96%, 97%, 98% or 99% identity thereto.
  • the anti-GPC3 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 7, and a light chain with an amino acid sequence as shown in SEQ ID NO: 8 Variable area.
  • the anti-GPC3 antibody or antigen-binding fragment of the invention is a murine antibody or fragment thereof, a chimeric antibody or antigen-binding fragment, a humanized antibody or antigen-binding fragment, or a fully human antibody or antigen-binding fragment. .
  • the anti-GPC3 antibody or antigen-binding fragment thereof of the invention is a humanized antibody or fragment thereof.
  • the anti-GPC3 antibody or antigen-binding fragment thereof of the invention is Fab, Fab′, Fab′-SH, Fv, scFv, F(ab′) 2 , sdAb, diabody or linear antibody.
  • the anti-GPC3 antibodies of the invention are monoclonal antibodies.
  • the antibody of the invention is an antibody in the form of IgGl, IgG2, IgG3, or IgG4.
  • the antibodies of the invention are in the form of IgG1.
  • the anti-GPC3 antibody or antigen-binding fragment thereof of the invention comprises: an amino acid sequence as shown in SEQ ID NO: 9 or at least 95%, 96%, 97%, 98% or 99% identity thereto.
  • the anti-GPC3 antibody or antigen-binding fragment thereof of the present invention includes a heavy chain with an amino acid sequence as shown in SEQ ID NO: 9, and a light chain with an amino acid sequence as shown in SEQ ID NO: 10.
  • the anti-GPC3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein said M, its L 2 end is connected to the linker unit L, L1 terminal is connected to D.
  • L 2 of the present invention is -O-.
  • L 2 of the present invention is -S-.
  • the anti-GPC3 antibody drug conjugate of the present invention isomers thereof, pharmaceutically acceptable salts thereof or mixtures thereof, wherein L 3 is -(C(R 1a )(R 1b )) m -, wherein m is 0, 1, 2 or 3; wherein each R 1a and R 1b can independently be hydrogen, halogen or C 1-6 aliphatic optionally substituted by 1, 2 or 3 R group; where each R independently can be hydrogen or halogen.
  • the anti-GPC3 antibody drug conjugate of the present invention isomers thereof, pharmaceutically acceptable salts thereof or mixtures thereof, wherein L 3 is -(C(R 1a )(R 1b )) m -, wherein m is 0, 1, 2 or 3; wherein each R 1a and R 1b is independently hydrogen, methyl, ethyl or propyl.
  • the anti-GPC3 antibody drug conjugate of the present invention isomers thereof, pharmaceutically acceptable salts thereof or mixtures thereof, wherein L 3 is -(C(R 1a )(R 1b )) m -, wherein m is 0, 1, 2 or 3; wherein each R 1a and R 1b can independently be hydrogen, halogen, CH 3 or CH 2 CH 3 .
  • the anti-GPC3 antibody drug conjugate of the present invention isomers thereof, pharmaceutically acceptable salts thereof or mixtures thereof, wherein L 3 is a single bond, -CH 2 -, -CH(CH 3 )-, -C(CH 3 ) 2 -, -CH 2 CH 2 -, -CH(CH 3 )CH 2 -, or -C(CH 3 ) 2 CH 2 -.
  • L 3 is a single bond, -CH 2 -, -CH(CH 3 )-, -C(CH 3 ) 2 -, -CH 2 CH 2 -, -CH(CH 3 )CH 2 -, or -C(CH 3 ) 2 CH 2 -.
  • the left side of the above group is preferably connected to L2 .
  • the anti-GPC3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein L 3 is The left side of the structural fragment is preferably connected to L2 .
  • the anti-GPC3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein L 3 is a single bond.
  • the anti-GPC3 antibody drug conjugate of the invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein X is optionally substituted by 1, 2 or 3 R 3a 3 to 6-membered saturated carbocyclyl or single bond; wherein each R 3a can independently be hydrogen, halogen or a C 1-6 aliphatic group optionally substituted by 1, 2 or 3 R; wherein each Each R can independently be hydrogen or halogen.
  • the anti-GPC3 antibody drug conjugate of the invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein X is optionally substituted by 1, 2 or 3 R 3a 3 to 6-membered saturated carbocyclic group or single bond; wherein each R 3a can independently be hydrogen, halogen, CH 3 or CH 2 CH 3 .
  • the anti-GPC3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein X is cyclopropyl, cyclobutyl, cyclopentyl, Cyclohexyl or single bond.
  • the anti-GPC3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein X is or single key.
  • the right side of the above group is preferably connected to L 1 .
  • the anti-GPC3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein X is a single bond.
  • the anti-GPC3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein X is Preferably, X is The right side of the above group is preferably connected to L 1 .
  • the anti-GPC3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein said -L 3 -X- is: -(C( R 1a ) (R 1b )) m -, 3 to 6-membered saturated carbocyclyl or 3 to 6-membered saturated heterocyclyl; wherein, m is 1, 2 or 3, and the 3 to 6-membered saturated carbon Ring groups and 3 to 6-membered saturated heterocyclyl groups are optionally substituted by 1, 2 or 3 R 3a ; wherein, each R 1a , R 1b , R 3a can independently be hydrogen, halogen or can be substituted by R. Selected substituted C 1-6 aliphatic groups; wherein each R can independently be hydrogen or halogen.
  • the anti-GPC3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein,
  • the -L 3 -X- is: -(C(R 1a )(R 1b )) m - or a 3- to 6-membered saturated carbocyclic group optionally substituted by 1, 2 or 3 R 3a ; for example, The -L 3 -X- is -(C(R 1a )(R 1b )) m - or cyclopropyl, cyclobutyl, cyclopentyl or cyclopropyl group optionally substituted by 1, 2 or 3 R 3a Hexyl; preferably, the -L 3 -X- is: -(C(R 1a )(R 1b )) m - or optionally substituted by 1, 2 or 3 R 3a :
  • the m is 1, 2 or 3;
  • R 1a is each independently halogen or a C 1-6 aliphatic group optionally substituted by 1, 2 or 3 R, such as methyl, ethyl or propyl;
  • R 1b and R 3a are each independently hydrogen, Halogen or a C 1-6 aliphatic group optionally substituted by 1, 2 or 3 R, such as hydrogen, methyl, ethyl or propyl; each R is independently hydrogen or halogen.
  • the anti-GPC3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein,
  • the -L 3 -X- is selected from: -(C(R 1a )(R 1b )) m - or a 3- to 6-membered saturated carbocyclic group optionally substituted by 1, 2 or 3 R 3a ; preferably Ground, said -L 3 -X- is: -(C(R 1a )(R 1b )) m -, or optionally substituted by 1, 2 or 3 R 3a
  • m is 1, 2 or 3
  • R 1a is each independently halogen or a C 1-6 aliphatic group optionally substituted by 1, 2 or 3 R
  • R 1b and R 3a are each independently hydrogen or halogen or a C 1-6 aliphatic group optionally substituted by 1, 2 or 3 R; wherein each R independently can be hydrogen or halogen.
  • the anti-GPC3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein said -L 3 -X- is:
  • the anti-GPC3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein said -L 3 -X- is:
  • the anti-GPC3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein L 1 is -C(R 2a )(R 2b )-, -C(R 2a )(R 2b )C(O)- or -C(O)-; wherein each R 2a , R 2b can independently be hydrogen, halogen or optionally selected from 1, 2 or 3 R Substituted C 1-6 aliphatic group; wherein each R can independently be hydrogen or halogen.
  • the anti-GPC3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein L 1 is -C(R 2a )(R 2b )-, -C(R 2a )(R 2b )C(O)- or -C(O)-; wherein each R 2a and R 2b can independently be hydrogen, methyl, ethyl or propyl.
  • the anti-GPC3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein L 1 is -C(R 2a )(R 2b )-, -C(R 2a )(R 2b )C(O)- or -C(O)-; wherein each R 2a and R 2b can independently be hydrogen, halogen, CH 3 or CH 2 CH 3 .
  • the anti-GPC3 antibody drug conjugate of the present invention isomers thereof, pharmaceutically acceptable salts thereof or mixtures thereof, wherein L 1 is -CH 2 C(O)-, -CH( CH 3 )C(O)- or -C(O)-.
  • the anti-GPC3 antibody drug conjugate of the present invention isomers thereof, pharmaceutically acceptable salts thereof or mixtures thereof, wherein L 1 is -C(O)-.
  • the anti-GPC3 antibody drug conjugate of the present invention isomers thereof, pharmaceutically acceptable salts thereof or mixtures thereof, wherein L 1 is -CH 2 C(O)-.
  • the anti-GPC3 antibody drug conjugates of the present invention isomers thereof, pharmaceutically acceptable salts thereof or mixtures thereof, wherein
  • the M is -L 2 -L 3 -XL 1 -;
  • L 2 is -O-
  • -L 3 -X- is a 3- to 6-membered saturated carbocyclic group optionally substituted by 1, 2 or 3 R 3a ;
  • L 1 is -C(O)-
  • each R 3a can independently be hydrogen, halogen or a C 1-6 aliphatic group optionally substituted by 1, 2 or 3 R;
  • each R independently can be hydrogen or halogen.
  • the anti-GPC3 antibody drug conjugates of the present invention isomers thereof, pharmaceutically acceptable salts thereof or mixtures thereof, wherein
  • the M is -L 2 -L 3 -XL 1 -;
  • L 2 is -O-
  • -L 3 -X- is cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl optionally substituted by 1, 2 or 3 R 3a ; preferably, the -L 3 -X- is: optional Replaced by 1, 2 or 3 R 3a :
  • L 1 is -C(O)-
  • each R 3a is independently hydrogen, halogen or a C 1-6 aliphatic group optionally substituted by 1, 2 or 3 R;
  • each R is independently hydrogen or halogen.
  • the anti-GPC3 antibody drug conjugates of the present invention isomers thereof, pharmaceutically acceptable salts thereof or mixtures thereof, wherein
  • the M is -L 2 -L 3 -XL 1 -;
  • L 2 is -O-
  • R 3a is each independently hydrogen, halogen or C 1-6 aliphatic group
  • L 1 is -C(O)-.
  • the anti-GPC3 antibody drug conjugates of the present invention isomers thereof, pharmaceutically acceptable salts thereof or mixtures thereof, wherein
  • the M is -L 2 -L 3 -XL 1 -;
  • L 2 is -O- or -S-;
  • -L 3 -X- is -(C(R 1a )(R 1b )) m -, where m is 1, 2 or 3;
  • L 1 is -C(O)-
  • R 1a , R 1b , and R 3a are each independently hydrogen, halogen or a C 1-6 aliphatic group optionally substituted by 1, 2 or 3 R;
  • Each R independently can be hydrogen or halogen.
  • the anti-GPC3 antibody drug conjugates of the present invention isomers thereof, pharmaceutically acceptable salts thereof or mixtures thereof, wherein
  • the M is -L 2 -L 3 -XL 1 -;
  • L 2 is -O- or -S-;
  • -L 3 -X- is -(C(R 1a )(R 1b )) m -, where m is 1, 2 or 3;
  • L 1 is -CH 2 C(O)-
  • R 1a is each independently halogen or a C 1-6 aliphatic group optionally substituted by 1, 2 or 3 R;
  • R 1b and R 3a are each independently hydrogen, halogen or a C 1-6 aliphatic group optionally substituted by 1, 2 or 3 R.
  • Each R independently can be hydrogen or halogen.
  • the anti-GPC3 antibody drug conjugates of the present invention isomers thereof, pharmaceutically acceptable salts thereof or mixtures thereof, wherein
  • the M is -L 2 -L 3 -XL 1 -;
  • L 2 is -O- or -S-;
  • -L 3 -X- is -CH(CH 3 )-, -C(CH 3 ) 2 - or -C(CH 3 )CH(CH 3 )-;
  • L 1 is -CH 2 C(O)-.
  • the anti-GPC3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein said M, its L 2 end is connected to the linker unit L.
  • the anti-GPC3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein said -M- is:
  • the anti-GPC3 antibody drug conjugates of the present invention isomers thereof, pharmaceutically acceptable salts thereof, or mixtures thereof, wherein the cytotoxic drug-MD choose from any of the following structures:
  • the anti-GPC3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein said L is -L a -L b -L c -,
  • the -L a - is
  • W is -(C(R wa )(R wb )) wn -
  • Y is -(OCH 2 CH 2 ) yn -O yp -
  • Z is -(C(R za )(R zb )) zn ;
  • wn 1, 2, 3 or 6
  • 0 or 1 methylene unit of W is each independently -Cyr-, -N(R wx )C(O)-, -C(O)N(R wx )-, or -C(O)- substitute;
  • yn 0, 4 or 8
  • yp is 0 or 1
  • Each methylene unit of Z is independently replaced by -Cyr-, -N(R zx )C(O)-, -C(O)N(R zx )-, or -C(O)-;
  • -Cyr- is a 3 to 10-membered saturated carbocyclylene group, wherein -Cyr- is unsubstituted or independently substituted by 1 to 3 substituents R cx ;
  • each R wa , R wb , R za , R zb , R wx , R zx , R cx is independently hydrogen, halogen, -OR r or a C 1-6 aliphatic group optionally substituted by R r ;
  • each R r is independently hydrogen, halogen or C 1-6 aliphatic group
  • the -L b - is a peptide residue composed of 2 to 7 amino acids
  • the peptide residue of -L b - is a peptide residue composed of amino acids selected from the following group: phenylalanine, glycine , alanine, valine, citrulline, lysine, serine, glutamic acid and aspartic acid; preferably, -L b - represents a peptide residue consisting of 2 to 4 amino acids
  • the peptide residue of -L b - is a peptide residue formed from amino acids selected from the group consisting of phenylalanine, glycine, alanine, valine, citrulline and lysine;
  • R L1 and R L2 are each independently selected from the following group: hydrogen, halogen, -OH and C 1-6 aliphatic group.
  • the anti-GPC3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein said L is -L a -L b -L c -,
  • the -L a - is
  • W is -(C(R wa )(R wb )) wn -
  • Y is -(OCH 2 CH 2 ) yn -O yp -
  • Z is -(C(R za )(R zb )) zn ;
  • wn 1, 2, 3 or 6
  • 0 or 1 methylene unit of W is each independently -Cyr-, -N(R wx )C(O)-, -C(O)N(R wx )-, or -C(O)- substitute;
  • yn 0, 4 or 8
  • yp is 0 or 1
  • Each methylene unit of Z is independently replaced by -Cyr-, -N(R zx )C(O)-, -C(O)N(R zx )-, or -C(O)-;
  • -Cyr- is a 3 to 10-membered saturated carbocyclylene group, wherein -Cyr- is unsubstituted or independently substituted by 1 to 3 substituents R cx ;
  • each R wa , R wb , R za , R zb , R wx , R zx , R cx is independently hydrogen, halogen, -OR r or a C 1-6 aliphatic group optionally substituted by R r ;
  • each R r is independently hydrogen, halogen or C 1-6 aliphatic group
  • Said -L b - is selected from the following group:
  • R L1 and R L2 are each independently selected from the following group: hydrogen, halogen, -OH and C 1-6 aliphatic group.
  • the anti-GPC3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein said -L a - is Preferably
  • the anti-GPC3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein -L b - is Preferably
  • the anti-GPC3 antibody drug conjugate of the present invention isomers thereof, pharmaceutically acceptable salts thereof or mixtures thereof, wherein -L c - is
  • the anti-GPC3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein the linker unit L has its L a end connected to Ab, The L c end is connected to the joint unit M.
  • the anti-GPC3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein the linker unit L has its L a end connected to Ab, The L c end is connected to M.
  • the anti-GPC3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein said L is
  • the anti-GPC3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein said L is
  • the anti-GPC3 antibody drug conjugates of the present invention isomers thereof, pharmaceutically acceptable salts thereof or mixtures thereof, wherein -LMD choose from any of the following structures:
  • the anti-GPC3 antibody drug conjugate of the present invention its isomer, a pharmaceutically acceptable salt thereof or a mixture thereof, wherein the anti-GPC3 antibody drug conjugate has a structure such as formula (II- 1) or (II-2):
  • the anti-GPC3 antibody drug conjugate of the present invention its isomer, a pharmaceutically acceptable salt thereof or a mixture thereof, wherein the anti-GPC3 antibody drug conjugate has a structure such as formula (II- 1) or (II-2):
  • p represents the average number of connections, and p is an integer or decimal from 1 to 10, preferably an integer or decimal from 3 to 8;
  • Ab is an anti-GPC3 antibody or an antigen-binding fragment thereof according to any embodiment of the invention.
  • L 2 is -O- or -S-;
  • X is a 3- to 6-membered saturated carbocyclic group optionally substituted by 1, 2 or 3 R 3a ; preferably, X is optionally substituted by 1, 2 or 3 R 3a :
  • L 3 is -C(R 1a )(R 1b )-CH 2 - or -C(R 1a )(R 1b )C(R 1a )(R 1b )-CH 2 -;
  • R 1a is each independently halogen or a C 1-6 aliphatic group optionally substituted by 1, 2 or 3 R;
  • R 1b and R 3a are each independently hydrogen, halogen or a C 1-6 aliphatic group optionally substituted by 1, 2 or 3 R;
  • each R independently can be hydrogen or halogen.
  • the anti-GPC3 antibody drug conjugate of the present invention its isomer, a pharmaceutically acceptable salt thereof or a mixture thereof, wherein the anti-GPC3 antibody drug conjugate has a structure such as formula (II- 1) or (II-2):
  • p represents the average number of connections, and p is an integer or decimal from 1 to 10, preferably an integer or decimal from 3 to 8;
  • Ab is an anti-GPC3 antibody or an antigen-binding fragment thereof according to any embodiment of the invention.
  • L 2 is -O- or -S-;
  • L 3 is -CH(CH 3 )CH 2 - or -C(CH 3 ) 2 CH 2 -.
  • the anti-GPC3 antibody drug conjugate of the present invention its isomer, a pharmaceutically acceptable salt thereof or a mixture thereof, wherein the anti-GPC3 antibody drug conjugate is selected from the following structural formula:
  • p represents the average number of connections, and p is an integer or decimal from 1 to 10, preferably an integer or decimal from 3 to 8;
  • Ab is an anti-GPC3 antibody or antigen-binding fragment thereof according to any embodiment of the invention.
  • the invention provides an anti-GPC3 antibody drug conjugate, an isomer thereof, a pharmaceutically acceptable salt thereof or a mixture thereof, wherein the anti-GPC3 antibody drug conjugate is selected from:
  • p represents the average number of connections, and p is an integer or decimal from 1 to 10, preferably an integer or decimal from 3 to 8.
  • the DB1001 is an anti-GPC3 antibody
  • the heavy chain amino acid sequence of the anti-GPC3 is shown in SEQ ID NO.: 9
  • the light chain amino acid sequence is shown in SEQ ID NO.: 10.
  • the average number of connections p described in the present invention may be an integer or a decimal number from 2 to 8.
  • the average number of connections p may be an integer or a decimal number from 3 to 8.
  • the average number of connections p may be an integer or a decimal number ranging from 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, or 9 to 10.
  • the average number of connections p is 7.51 or 7.72.
  • the invention provides an anti-GPC3 antibody drug conjugate, an isomer thereof, a pharmaceutically acceptable salt thereof or a mixture thereof, wherein the anti-GPC3 antibody drug conjugate is selected from any of the following Structure (p represents the average number of connections):
  • the DB1001 is an anti-GPC3 antibody
  • the heavy chain amino acid sequence of the anti-GPC3 is shown in SEQ ID NO.: 9
  • the light chain amino acid sequence is shown in SEQ ID NO.: 10.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the anti-GPC3 antibody drug conjugate according to any one of the present invention, its isomer, its pharmaceutically acceptable salt or its mixture, and a pharmaceutically acceptable carrier or excipient.
  • the present invention provides an anti-GPC3 antibody drug conjugate, an isomer thereof, a pharmaceutically acceptable salt thereof or a mixture or pharmaceutical composition thereof according to any one of the present invention for use in the treatment of and/or use in medicine to prevent GPC3-mediated diseases or disorders.
  • the disease or disorder is cancer, for example, GPC3-positive cancer.
  • the invention provides a method of treating and/or preventing a GPC3-mediated disease or disorder, comprising administering to a subject in need thereof an anti-GPC3 antibody pharmaceutical conjugate as described in any one of the invention.
  • the present invention provides an anti-GPC3 antibody drug conjugate as described in any one of the present invention, an isomer thereof, a pharmaceutically acceptable salt thereof or a mixture thereof or as described in any one of the present invention.
  • the disease or disorder is cancer, for example, a GPC3-positive cancer.
  • the cancer of the present invention is selected from the group consisting of liver cancer, breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, lung cancer, gastric cancer, colon cancer, bladder cancer, esophageal cancer, cervical cancer, gallbladder cancer, and gum cancer. Blastoma and melanoma.
  • the present invention provides a pharmaceutical combination comprising an antibody drug conjugate as described in any one of the present invention or a pharmaceutically acceptable salt thereof or a pharmaceutical combination as described in any one of the present invention. substance, and one or more additional therapeutic agents.
  • the present invention provides a kit, which includes the antibody-drug conjugate according to any one of the present invention, or the pharmaceutical composition according to any one of the present invention.
  • the term "about” generally refers to a variation within the range of 0.5%-10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.
  • antibody refers to any form of antibody having the desired biological activity. Therefore, it is used in the broadest sense, specifically including but not limited to monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (such as bispecific antibodies), humanized antibodies, fully human antibodies, Chimeric antibodies and camelized single domain antibodies.
  • the term "monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies, ie, the individual antibodies composing the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific and target a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically include a large number of antibodies directed against (or specific for) different epitopes.
  • the modifier "monoclonal” indicates the characteristics of an antibody obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method.
  • full-length antibody refers to an immunoglobulin molecule that contains four peptide chains when naturally occurring: two heavy (H) chains (approximately 50-70 kDa in full length) and two light (L) chains. ) chains (approximately 25 kDa in full length) are connected to each other by disulfide bonds.
  • Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH).
  • the heavy chain constant region consists of three domains, CH1, CH2 and CH3.
  • Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region consists of one domain, CL.
  • the VH and VL regions can be further subdivided into highly variable complementarity determining regions (CDRs) separated by more conservative regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH or VL region consists of 3 CDRs and 4 FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain binding domains that interact with the antigen.
  • the constant region of an antibody may mediate binding of immunoglobulins to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.
  • CDR refers to the complementarity determining regions within the variable sequences of an antibody.
  • the precise amino acid sequence boundaries of the variable region CDRs of the antibodies of the invention can be determined using any of a number of well-known protocols, including Chothia (Chothia et al. (1989)) based on the three-dimensional structure of the antibody and the topology of the CDR loops.
  • the boundaries of the CDRs of the variable regions of the same antibody obtained based on different assignment systems may be different. That is, the CDR sequences of the same antibody variable region defined under different assignment systems are different. Therefore, when referring to an antibody defined by a specific CDR sequence as defined in the invention, the scope of said antibody also encompasses antibodies whose variable region sequences comprise said specific CDR sequence but which differ due to the application of different protocols (e.g. Different assignment systems or combinations) cause the claimed CDR boundaries to be different from the specific CDR boundaries defined in the present invention.
  • the term "antigen-binding fragment" of an antibody (“parent antibody”) includes fragments or derivatives of the antibody, typically including at least one of the antigen-binding region or variable region (eg, one or more CDRs) of the parent antibody Fragments that retain at least some of the binding specificity of the parent antibody.
  • antibody-binding fragments include, but are not limited to, Fab, Fab′, F(ab′)2 and Fv fragments; diabodies; linear antibodies; single chain antibody molecules, such as scFv; nanobodies and polypeptides formed from antibody fragments specific antibodies.
  • the binding fragment or derivative When the binding activity of an antigen is expressed on a molar concentration basis, the binding fragment or derivative generally retains at least 10% of its antigen-binding activity.
  • the binding fragment or derivative retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or greater of the antigen binding affinity of the parent antibody. It is also contemplated that antigen-binding fragments of an antibody may include conservative or non-conservative amino acid substitutions that do not significantly alter its biological activity (referred to as “conservative variants” or “functionally conserved variants” of the antibody).
  • the term "chimeric antibody” is an antibody having a variable domain of a first antibody and a constant domain of a second antibody, wherein the first antibody and the second antibody are from different species.
  • the variable domains are obtained from an antibody such as a rodent (a "parent antibody")
  • the constant domain sequences are obtained from a human antibody such that the resulting chimeric antibody induces in human subjects compared to the parent rodent antibody. There is a lower likelihood of adverse immune responses.
  • humanized antibody refers to an antibody form containing sequences derived from human and non-human (eg, mouse, rat) antibodies.
  • a humanized antibody contains substantially all of at least one, and usually two, variable domains, wherein all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the
  • the framework (FR) region is the framework region of human immunoglobulin sequences.
  • the humanized antibody optionally can comprise at least a portion of a human immunoglobulin constant region (Fc).
  • the term "fully human antibody” refers to an antibody that contains only human immunoglobulin protein sequences.
  • Fully human antibodies may contain murine glycans if produced in mice, in mouse cells, or in hybridomas derived from mouse cells.
  • mouse antibody refers to an antibody that contains only mouse immunoglobulin sequences.
  • the fully human antibody may contain rat glycans if produced in rat, in rat cells, or in hybridomas derived from rat cells.
  • rat antibody refers to an antibody that contains only rat immunoglobulin sequences.
  • isotype antibody refers to the class of antibody provided by the heavy chain constant region gene (eg, IgM, IgE, IgG such as IgGl, IgG2 or IgG4). Isotypes also include modified forms of one of these classes, where modifications have been made to alter Fc function, for example to enhance or weaken effector function or binding to Fc receptors.
  • the heavy chain constant region gene eg, IgM, IgE, IgG such as IgGl, IgG2 or IgG4
  • Isotypes also include modified forms of one of these classes, where modifications have been made to alter Fc function, for example to enhance or weaken effector function or binding to Fc receptors.
  • the term "Fc region” is used to define the C-terminal region of an immunoglobulin heavy chain comprising at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxy terminus of the heavy chain.
  • the C-terminal lysine (Lys447) of the Fc region may or may not be present (numbering in this paragraph is according to the EU numbering system, also known as the EU index, as in Rabat et al., Sequences of Proteins of Immunological Interest, 5th Ed .Public Health Service, National Institutes of Health, Bethesda, MD, 1991).
  • cross-reactivity refers to the binding of antigenic fragments of the same target molecule of human, monkey, and/or murine origin (mouse or rat). Therefore, “cross-reactivity” should be understood as an inter-species reaction with the same molecule X expressed in a different species.
  • the cross-reactive specificity of monoclonal antibodies recognizing human GPC3R, monkey, and/or murine GPC3R (mouse or rat) can be determined by FACS analysis.
  • affinity refers to the inherent binding affinity that reflects the interaction between members of a binding pair.
  • the affinity of a molecule X for its partner Y can generally be represented by the equilibrium dissociation constant (KD), which is the ratio of the dissociation rate constant and the association rate constant (kdis and kon respectively).
  • KD equilibrium dissociation constant
  • kdis and kon association rate constant
  • Affinity can be measured by common methods known in the art.
  • One specific method used to measure affinity is the ForteBio kinetic binding assay herein.
  • the term "does not bind" to proteins or cells means that it does not bind to proteins or cells, or does not bind to them with high affinity, that is, the K D of binding proteins or cells is 1.0 ⁇ 10 -6 M or higher, More preferably, it is 1.0 ⁇ 10 -5 M or higher, more preferably 1.0 ⁇ 10 -4 M or higher, 1.0 ⁇ 10 -3 M or higher, and more preferably 1.0 ⁇ 10 -2 M or higher.
  • the term "high affinity" for an IgG antibody means that the K D for the antigen is 1.0 ⁇ 10 -6 M or lower, preferably 5.0 ⁇ 10 -8 M or lower, and more preferably 1.0 ⁇ 10 -8 M or less, 5.0 ⁇ 10 -9 M or less, more preferably 1.0 ⁇ 10 -9 M or less.
  • "high affinity” binding may vary.
  • "high affinity” binding of the IgM subtype means a K D of 10 -6 M or less, preferably 10 -7 M or less, more preferably 10 -8 M or less.
  • cytotoxic drug generally refers to toxic drugs that have chemical molecules in tumor cells that are powerful enough to disrupt their normal growth. Cytotoxic drugs can kill tumor cells at high enough concentrations.
  • the "cytotoxic drugs” may include toxins, such as small molecule toxins or enzymatically active toxins derived from bacteria, fungi, plants or animals, radioactive isotopes (such as At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , p 32 or Lu radioactive isotopes), toxic drugs, chemotherapeutic drugs, antibiotics and ribolytic enzymes, for example, can be toxic drugs, including but not limited to camptothecin derivatives, for example, can be camptothecin derivatives
  • Dendrine derivative isatecan chemical name: (1S, 9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H
  • linker unit or "linker structure” usually refers to a chemical structural fragment or bond that is connected to a ligand at one end and connected to a cytotoxic drug at the other end. It may also be connected to other linkers and then connected to the cytotoxic drug. .
  • the direct or indirect connection to the ligand may mean that the group is directly connected to the ligand through a covalent bond, or the ligand may be connected through a linker structure.
  • the linker structure may be the structure represented by -Lax-Lb-Lc- and or -La-Lb-Lc- described in the present invention.
  • linkers containing acid-labile linkers e.g., hydrazones
  • protease-sensitive linkers e.g., peptidase-sensitive linkers
  • photolabile linkers e.g., dimethyl linkers
  • disulfide-containing linkers e.g., linkers containing acid-labile linkers (e.g., hydrazones)
  • protease-sensitive linkers e.g., peptidase-sensitive linkers
  • photolabile linkers e.g., dimethyl linkers, or disulfide-containing linkers
  • a structure is "optionally connected to other molecular moieties” generally means that the structure is not connected to any other chemical structure, or that the structure is connected to one or more other chemical structures that are different from the structure. (for example, the ligands of the present invention) are connected (for example, connected through chemical bonds or connected through a linker structure).
  • ligand-drug conjugate generally refers to a ligand connected to a biologically active cytotoxic drug through a stable linking unit.
  • ligand-drug conjugate can be an antibody-drug conjugate (ADC).
  • ADC can refer to a monoclonal antibody or antibody fragment with a biological substance through a stable linking unit. active cytotoxic drugs.
  • methylene generally refers to a residue derived from a group of one carbon atom by removing two hydrogen atoms. Methylene groups may be substituted or unsubstituted, substituted or unsubstituted.
  • alkylene generally refers to a saturated linear or branched aliphatic hydrocarbon radical having 2 residues derived from the removal of two hydrogen atoms from the same carbon atom or two different carbon atoms of the parent alkane, which It may be a straight or branched chain group containing 1 to 20 carbon atoms, such as an alkylene group containing 1 to 12 carbon atoms, such as an alkylene group containing 1 to 6 carbon atoms.
  • Non-limiting examples of alkylene include, but are not limited to, methylene (-CH 2 -), 1,1-ethylene (-CH(CH 3 )-), 1,2-ethylene (-CH 2 CH 2 )-, 1,1-propylene (-CH(CH 2 CH 3 )-), 1,2-propylene (-CH 2 CH(CH 3 )-), 1,3-propylene (-CH 2 CH 2 CH 2 -), 1,4-butylene (-CH 2 CH 2 CH 2 CH 2 -) and 1,5-butylene (-CH 2 CH 2 CH 2 CH 2 CH 2 -) wait.
  • Alkylene groups may be substituted or unsubstituted, substituted or unsubstituted, for example when substituted the substituents may be substituted at any available point of attachment, said substituents being preferably independently optionally selected from alkanes Base, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy Substituted with one or more substituents among radicals, heterocycloalkoxy groups, cycloalkylthio groups, heterocycloalkylthio groups and oxo groups, for example, they can be hydrogen, protium, deuterium, tritium, halogen, -NO 2 , -CN, -OH, -SH, -NH 2 , -C(O)H, -CO 2 H, -C(O
  • heterocyclylene or “heterocyclyl” generally refers to a stable non-aromatic 3- to 7-membered monocyclic ring structure, a fused 7- to 10-membered bicyclic heterocyclic structure or bridge. These cyclic structures can be saturated or partially saturated. In addition to carbon atoms, these cyclic structures also contain one or more heteroatoms, among which heteroatoms The atoms can be selected from the following group: oxygen, sulfur and nitrogen. For example, it contains 1-4 heteroatoms as defined above. When used to refer to atoms of a heterocyclic ring structure, the term “nitrogen” may include substituted nitrogen. Heterocyclylene groups may be substituted or unsubstituted.
  • the term “carbocyclylene” generally refers to a residue having two residues derived from the removal of two hydrogen atoms from the same carbon atom or two different carbon atoms of the carbocyclic ring.
  • the term “carbocyclic ring” generally refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon.
  • the carbocyclic ring contains 3 to 20 carbon atoms, may contain 3 to 12 carbon atoms, may contain 3 to 10 carbon atoms, and may Contains 3 to 8 carbon atoms, for example 3 to 6 carbon atoms.
  • Non-limiting examples of monocyclic carbocycles include cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane, cyclohexene, cyclohexadiene, cycloheptane, cycloheptatriene, cyclooctane etc.; polycyclic carbocyclic rings may include spiro rings, fused rings and bridged carbocyclic rings.
  • 3- to 6-membered saturated carbocyclyl includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclopropylene, cyclobutylene, cyclopentylene or cyclohexylene.
  • Carbocyclyl and carbocyclylene groups may be substituted or unsubstituted, for example, may be substituted by one or more halogens, C 1-6 aliphatic groups, or C 1-6 aliphatic groups substituted by halogens.
  • partially unsaturated generally refers to a cyclic structure containing at least one double or triple bond between ring molecules.
  • partially unsaturated encompasses cyclic structures with multiple unsaturations, but is not intended to include aromatic or heteroaromatic rings as defined in the present invention.
  • unsaturated means that a moiety has one or more degrees of unsaturation.
  • halogen generally refers to fluorine, chlorine, bromine, iodine, for example, it can be fluorine or chlorine.
  • aliphatic group generally refers to straight-chain hydrocarbons, branched-chain hydrocarbons or hydrocarbons with a cyclic structure having 1 to 12 carbon atoms. These hydrocarbons are either fully saturated hydrocarbons; or have one or Multiple unsaturated units, but the unsaturated units are not aromatic groups.
  • suitable aliphatic groups may include substituted or unsubstituted linear, branched or cyclic alkyl groups, alkenyl groups, alkynyl groups, and mixtures of these groups; for example, (cycloalkyl)alkyl groups , (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.
  • aliphatic groups have 1-12, 1-8, 1-6, 1-4 or 1-3 carbon atoms.
  • C 1-6 aliphatic group refers to an aliphatic group as described above having 1 to 6 carbon atoms, including but not limited to straight chain, branched or Alkyl, alkenyl or alkynyl groups with cyclic structure, such as methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, hexyl, etc.
  • a heterocyclic group optionally substituted by an alkyl group means that an alkyl group may but does not have to be present. This description may include the case where the heterocyclic group is substituted by an alkyl group and the case where the heterocyclic group is not substituted by an alkyl group. situation.
  • substituted generally means that one or more hydrogen atoms in a group, for example up to 5, for example 1 to 3 hydrogen atoms, are independently substituted with a corresponding number of substituents.
  • the substituents are only in their possible chemical positions, and a person skilled in the art is able to determine (by experiment or theory) possible or impossible substitutions without undue effort.
  • an amino or hydroxyl group with a free hydrogen may be unstable when combined with a carbon atom with an unsaturated (eg, olefinic) bond.
  • one or more hydrogen atoms in the group are independently substituted with a corresponding number of substituents.
  • the substituents are only in their possible chemical positions, and a person skilled in the art is able to determine (by experiment or theory) possible or impossible substitutions without undue effort.
  • an amino or hydroxyl group with a free hydrogen may be unstable when combined with a carbon atom with an unsaturated (eg, olefinic) bond.
  • the term "pharmaceutically acceptable salt” or “pharmaceutically acceptable salt” generally refers to the salt of the drug conjugate of the present invention, which can be safe and/or effective when used in mammals. property, and may have due biological activity.
  • the antibody-drug conjugate of the present invention can form a salt with an acid.
  • pharmaceutically acceptable salts include: hydrochloride, hydrobromide, and hydroiodide.
  • sulfate hydrogen sulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, pearate, hydrogen phosphate, dihydrogen phosphate, salicylate, citric acid Hydrogen salt, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate.
  • solvate or “solvent compound” generally refers to the drug conjugate of the invention and one or more solvents.
  • agent molecules form pharmaceutically acceptable solvates.
  • solvent molecules include water, ethanol, acetonitrile, isopropyl alcohol, DMSO, and ethyl acetate.
  • the term "drug loading” usually refers to the average amount of cytotoxic drugs loaded on each ligand, and can also be expressed as the ratio of the amount of cytotoxic drugs and antibodies.
  • the range of cytotoxic drug loading can be Each ligand (Ab) connects 0-12, for example, 1-10 cytotoxic drugs.
  • the drug loading is expressed as Na, which can be an example of the average value of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.
  • the drug loading capacity of each ADC molecule after the coupling reaction can be determined by conventional methods such as UV/visible light spectroscopy, mass spectrometry, ELISA tests and HPLC characterization.
  • the term "pharmaceutically acceptable carrier” refers to ingredients in a pharmaceutical preparation or composition other than the active ingredient that are non-toxic to the subject.
  • Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.
  • the term “comprising” generally means the inclusion of explicitly specified features, but not the exclusion of other elements.
  • the terms “above” and “below” usually refer to the case where the number is included.
  • the term "subject” includes any human or non-human animal.
  • non-human animals includes all vertebrate animals, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cattle, chickens, amphibians, reptiles, and the like.
  • cyno or “cyno” refers to the cynomolgus monkey.
  • administration in combination with includes simultaneous (co) administration and sequential administration in any order.
  • the terms "therapeutically effective amount”, “therapeutically effective dose” and “effective amount” refer to the GPC3 antibody or antigen-binding fragment thereof of the present invention when administered to cells, tissues or subjects alone or in combination with other therapeutic drugs.
  • a therapeutically effective dose also refers to an amount of an antibody or antigen-binding fragment thereof sufficient to result in an improvement in symptoms, such as an amount that treats, cures, prevents, or ameliorates a related medical condition or increases the rate of treatment, cure, prevention, or amelioration of such a condition.
  • the therapeutically effective dose refers only to that ingredient.
  • a therapeutically effective dose refers to the combined amounts of the active ingredients that produce a therapeutic effect, whether combined, administered sequentially, or administered simultaneously.
  • An effective amount of the therapeutic agent will result in an improvement in diagnostic criteria or parameters of at least 10%, usually at least 20%, preferably at least about 30%, more preferably at least 40%, and most preferably at least 50%.
  • cancer is used herein to refer to a group of cells that exhibit abnormally high levels of proliferation and growth. Cancers may be benign (also called benign tumors), premalignant, or malignant. The cancer cells may be solid cancer cells or leukemia cancer cells. As used herein, the term “tumor” refers to one or more cells that contain cancer.
  • tumor growth is used herein to refer to the proliferation or growth of one or more cells comprising a cancer, which results in a corresponding increase in the size or extent of the cancer.
  • GPC3 includes any variant or isoform of GPC3 naturally expressed by cells.
  • GPC3, or any variant or isoform thereof can be isolated from the cells or tissues in which they are naturally expressed, or produced by recombinant techniques using techniques common in the art and described herein.
  • the anti-GPC3 antibody targets human GPC3 with a normal glycosylation pattern.
  • anti-GPC3 antibody refers to Sufficient to bind the GPC3 protein or fragment thereof with sufficient affinity such that the antibody can be used as a diagnostic and/or therapeutic agent in targeting GPC3.
  • the anti-GPC3 antibody or antigen-binding fragment thereof described in the present invention refers to the anti-GPC3 antibody or antigen-binding fragment thereof described in WO2006006693.
  • the CDR sequences of the antibodies used in the drug conjugates, compositions, uses or methods of the invention comprise amino acid sequences such as SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively.
  • HCDR1, HCDR2 and HCDR3 are shown, and LCDR1, LCDR2 and LCDR3 containing amino acid sequences as shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 respectively.
  • variable region sequence of the antibody used in the drug conjugates, compositions or uses of the invention includes a heavy chain variable region with an amino acid sequence such as SEQ ID NO: 7, and an amino acid sequence such as The light chain variable region shown in SEQ ID NO: 8.
  • the antibody used in the drug conjugates, compositions, uses or methods of the invention includes a heavy chain with an amino acid sequence as set forth in SEQ ID NO: 9, and an amino acid sequence as set forth in SEQ ID NO: 10 light chain shown.
  • the antibody used in the drug conjugates, compositions, uses or methods of the invention is anti-GPC3 antibody DB1001.
  • variable region of anti-GPC3 antibody DB1001 is as follows, and the CDR region is determined according to the IMGT numbering rules.
  • Antibodies of the invention may be produced using any suitable method for producing antibodies. Any suitable form of GPC3 can be used as an immunogen (antigen) to generate antibodies. By way of example and not limitation, any GPC3 variant or fragment thereof may be used as an immunogen. In some embodiments, hybridoma cells producing murine monoclonal anti-human GPC3 antibodies can be produced by methods well known in the art. Antibodies derived from rodents (e.g., mice) may cause unwanted antibody immunogenicity when used as therapeutics in vivo, and repeated use can lead to an immune response in the body against the therapeutic antibodies, which may at least result in the loss of therapeutic efficacy. , and severe cases can lead to potentially fatal allergic reactions.
  • rodents e.g., mice
  • CDR complementarity-determining region
  • chimeric or humanized antibodies of the invention can be prepared based on the sequence of a mouse monoclonal hybridoma antibody prepared.
  • DNA encoding heavy and light chain immunoglobulins can be obtained from the mouse hybridoma of interest and engineered to contain non-murine (eg, human) immunoglobulin sequences using standard molecular biology techniques.
  • the chimeric GPC3 antibodies of the invention can be operably linked to hybridoma-derived immunoglobulin heavy and light chain variable regions and human IgG constant regions using methods known in the art (see, e.g., No. 4,816,567 belonging to Cabilly et al.), a chimeric heavy chain and a chimeric light chain were obtained.
  • the constant region comprised by the chimeric antibody of the invention can be selected from any human IgG subtype, such as IgG1, IgG2, IgG3, IgG4, preferably IgG4.
  • the chimeric GPC3 antibodies of the present invention can be obtained by "mixing and matching" transfection of expression cells with chimeric light chain and chimeric heavy chain expression plasmids, and the GPC3 binding of such "mixed and matched" antibodies Testing can be performed using the binding assays described above and other conventional binding assays (eg, ELISA).
  • the mouse CDR region can be inserted into the human germline framework region using methods known in the art. See Winter et al., U.S. Patent Nos. 5,225,539 and Queen et al, U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370.
  • amino acid changes include amino acid deletions, insertions, or substitutions.
  • the anti-GPC3 antibodies or antigen-binding fragments thereof of the invention include those that have been mutated by amino acid deletions, insertions or substitutions, but are still at least identical to the above-mentioned antibodies (especially in the CDR regions depicted in the above-mentioned sequences). Those antibodies have an amino acid sequence that is about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical.
  • an antibody of the invention has no more than 1, 2, 3, 4, or 5 amino acid mutations in the CDR region by amino acid deletion, insertion, or substitution when compared to the CDR region depicted in a specific sequence.
  • Fc region variants can be generated by introducing one or more amino acid modifications into the Fc region of the antibodies provided herein.
  • Fc region variants may comprise human Fc region sequences (e.g., human IgGl, IgG2, IgG3, or IgG4 Fc regions) that comprise amino acid modifications (e.g., substitutions) at one or more amino acid positions.
  • cysteine-engineered antibodies such as "thioMAbs,” in which one or more residues of the antibody are replaced with cysteine residues.
  • the antibodies provided herein can be further modified to contain other non-proteinaceous moieties known and readily available in the art.
  • Suitable moieties for antibody derivatization include, but are not limited to, water-soluble polymers.
  • water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymer, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, polyvinylpyrrolidone, -1,3-dioxane, poly-1,3,6-triane, ethylene/maleic anhydride copolymer, polyamino acid (homopolymer or random copolymer), and dextran or poly(n-ethylene pyrrolidone) polyethylene glycol, propylene glycol homopolymer, polypropylene oxide/ethylene oxide copolymer, polyoxyethylated polyol (such as glycerol), polyvinyl
  • PEG poly
  • the present invention provides an anti-GPC3 antibody drug conjugate, an isomer thereof, a pharmaceutically acceptable salt thereof or a mixture thereof, wherein the structure of the anti-GPC3 antibody drug conjugate is as shown in formula (I-1):
  • M is -L 2 -L 3 -XL 1 -;
  • L 2 is -O- or -S-;
  • L 3 is -(C(R 1a )(R 1b )) m -, m is 0, 1, 2 or 3, wherein when L 3 contains a methylene unit, 0 or 1 subunits of L 3
  • the methyl unit can be replaced by -C(O)-, or -C(S)-;
  • L 1 is -(C(R 2a )(R 2b )) n -, n is 1, 2 or 3, wherein when L 1 may contain methylene units, 0 or 1 methylene units of L 1
  • the base unit can be replaced by -C(O)-, or -C(S)-;
  • X is a 3 to 6-membered saturated carbocyclyl group, a 3 to 6-membered saturated heterocyclyl group or a single bond, and the 3 to 6-membered saturated carbocyclyl group and 3 to 6-membered saturated heterocyclyl group are optionally replaced by 1 One or more R 3a substitutions;
  • each R 1a , R 1b , R 2a , R 2b , R 3a may independently be hydrogen, halogen or a C 1-6 aliphatic group optionally substituted by one or more R ;
  • each R can independently be hydrogen or halogen
  • p represents the average number of connections, and p is an integer or decimal from 1 to 10, preferably an integer or decimal from 3 to 8;
  • Ab is an anti-GPC3 antibody or an antigen-binding fragment thereof.
  • the anti-GPC3 antibody or antigen-binding fragment thereof of the present invention includes a heavy chain variable region and a light chain variable region
  • the heavy chain variable region includes an amino acid sequence such as SEQ ID NO: 1, SEQ HCDR1, HCDR2 and HCDR3 shown in ID NO:2 and SEQ ID NO:3
  • the light chain variable region includes amino acid sequences as shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 respectively.
  • the anti-GPC3 antibody or antigen-binding fragment thereof of the present invention includes: a heavy chain variable region with an amino acid sequence such as SEQ ID NO: 7, and an amino acid sequence such as SEQ ID NO: The light chain variable region shown in 8; more preferably, the anti-GPC3 antibody or antigen-binding fragment thereof of the present invention includes: a heavy chain with an amino acid sequence such as SEQ ID NO: 9, and an amino acid sequence such as SEQ ID NO: The light chain shown in 10.
  • the anti-GPC3 antibody drug conjugate of the present invention isomers thereof, pharmaceutically acceptable salts thereof or mixtures thereof, wherein L 3 is -(C(R 1a )(R 1b ) ) m -, wherein m is 0, 1, 2 or 3; wherein each R 1a and R 1b can independently be hydrogen, halogen or a C 1-6 aliphatic group optionally substituted by R, wherein each Each R may independently be hydrogen or halogen; preferably, L 3 is -(C(R 1a )(R 1b )) m -, where m is 0, 1, 2 or 3; wherein each R 1a and R 1b can each independently be hydrogen, halogen, CH 3 or CH 2 CH 3 ; preferably, L 3 is a single bond, -CH 2 -, -CH(CH 3 ), -C(CH 3 ) 2 , -CH 2 CH 2 -, -CH(CH 3 )CH 2 - or -C(
  • L 1 is -C(R 2a )(R 2b )-, -C(R 2a )(R 2b )C(O)- or -C(O)-; wherein each of R 2a and R 2b Each independently can be hydrogen, halogen, CH 3 or CH 2 CH 3 ; more preferably,
  • the anti-GPC3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein 6-membered saturated heterocyclyl or single bond, the 3 to 6-membered saturated carbocyclyl and 3 to 6-membered saturated heterocyclyl are optionally substituted by 1, 2 or 3 R 3a ; wherein each R 3a Each independently can be hydrogen, halogen or a C 1-6 aliphatic group optionally substituted by 1, 2 or 3 R, wherein each R independently can be hydrogen or halogen; preferably, X is optional A 3- to 6-membered saturated carbocyclic group or single bond substituted by 1, 2 or 3 R 3a ; wherein each R 3a can independently be hydrogen, halogen, CH 3 or CH 2 CH 3 ; more preferably, X is or single key.
  • the anti-GPC3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein said -L 3 -X- is: -(C( R 1a ) (R 1b )) m -, 3 to 6-membered saturated carbocyclyl or 3 to 6-membered saturated heterocyclyl; wherein, m is 1, 2 or 3, and the 3 to 6-membered saturated carbon Ring groups and 3 to 6-membered saturated heterocyclyl groups are optionally substituted by 1, 2 or 3 R 3a ; wherein, each R 1a , R 1b , R 3a can independently be hydrogen, halogen or can be substituted by R. Select a substituted C 1-6 aliphatic group, wherein each R can independently be hydrogen or halogen;
  • -L 3 -X- is: -(C(R 1a )(R 1b )) m - and a 3- to 6-membered saturated carbocyclic group optionally substituted by 1, 2 or 3 R 3a ; wherein , m is 1, 2 or 3; wherein, R 1a is each independently halogen or a C 1-6 aliphatic group optionally substituted by 1, 2 or 3 R; R 1b , R 3a is each independently hydrogen , halogen or C 1-6 aliphatic group optionally substituted by 1, 2 or 3 R; each R independently Can be hydrogen or halogen;
  • -L 3 -X- is:
  • the anti-GPC3 antibody drug conjugates of the present invention isomers thereof, pharmaceutically acceptable salts thereof or mixtures thereof, wherein
  • the M is -L 2 -L 3 -XL 1 -;
  • L 2 is -O-
  • -L 3 -X- is a 3- to 6-membered saturated carbocyclic group optionally substituted by 1, 2 or 3 R 3a ; preferably, -L 3 -X- is optionally substituted by 1, 2 or 3 R 3a replaces: Preferably, -L 3 -X- is
  • L 1 is -C(R 2a )(R 2b )-, -C(R 2a )(R 2b )C(O)- or -C(O)-; preferably, L 1 is -C(O)- ;
  • each R 2a , R 2b , R 3a is independently hydrogen, halogen or a C 1-6 aliphatic group that may be optionally substituted by R;
  • each R independently can be hydrogen or halogen.
  • the anti-GPC3 antibody drug conjugates of the present invention isomers thereof, pharmaceutically acceptable salts thereof or mixtures thereof, wherein
  • the M is -L 2 -L 3 -XL 1 -;
  • L 2 is -O- or -S-; preferably, L 2 is -O-; preferably, L 2 is -S-;
  • -L 3 -X- is -(C(R 1a )(R 1b )) m -, where m is 1, 2 or 3; preferably, -L 3 -X- is Preferably, -L 3 -X- is
  • L 1 is -C(R 2a )(R 2b )-, -C(R 2a )(R 2b )C(O)- or -C(O)-; preferably, L 1 is -CH 2 C(O )-;
  • R 1a is each independently a halogen or a C 1-6 aliphatic group optionally substituted by 1, 2 or 3 R
  • R 1b and R 3a are each independently a hydrogen, a halogen or a C 1-6 aliphatic group optionally substituted by 1, 2 or 3 R optionally substituted C 1-6 aliphatic group;
  • each R independently can be hydrogen or halogen.
  • the anti-GPC3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein -M- is:
  • the anti-GPC3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein said L is -L a -L b -L c -,
  • the -L a - is
  • W is -(C(R wa )(R wb )) wn -, where wn is 1, 2, 3 or 6, and 0 or 1 methylene units of W are each independently replaced by -Cyr-, - N(R wx )C(O)-, -C(O)N(R wx )-, or -C(O)- instead; preferably, W is -(C(R wa )(R wb )) 2 - or -(C(R wa )(R wb )) 3 -; Preferably, W is -CH 2 CH 2 CH 2 - or -CH 2 CH 2 -;
  • Y is -(OCH 2 CH 2 ) yn -O yp -, where yn is 0, 4 or 8, yp is 0 or 1; preferably, Y is a single bond;
  • Z is -(C(R za )(R zb )) zn , where zn is 1, 2 or 3, and each methylene unit of Z is independently replaced by -Cyr-, -N(R zx )C(O )-, -C(O)N(R zx )-, or -C(O)- instead, -Cyr- is a 3 to 10-membered saturated carbocyclylene group, wherein -Cyr- is unsubstituted or Independently substituted by 1 to 3 substituents R cx ; preferably, Z is -(C(R wa )(R wb )) 2 C(O)- or -(C(R wa )(R wb )) 3 C(O)-; Preferably, Z is -CH 2 CH 2 CH 2 C(O)- or CH 2 CH 2 C(O)-;
  • each R wa , R wb , R za , R zb , R wx , R zx and R cx are each independently hydrogen, halogen, -OR r or a C 1-6 aliphatic group optionally substituted by R r ;
  • each R r is independently hydrogen, halogen or C 1-6 aliphatic group
  • the -L a - is Preferably
  • the -L b - represents a peptide residue composed of 2 to 4 amino acids
  • the peptide residue of -L b - is a peptide residue composed of amino acids selected from the following group: phenylalanine, glycine , alanine, valine, citrulline and lysine;
  • -L b - is selected from the following group:
  • the -L b - is Preferably -L b - is
  • R L1 and R L2 are each independently selected from the following group: hydrogen, halogen, -OH and C 1-6 aliphatic group; preferably, the -L c - is
  • the anti-GPC3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein said L is
  • the anti-GPC3 antibody drug conjugate of the present invention its isomer, a pharmaceutically acceptable salt thereof or a mixture thereof, wherein the anti-GPC3 antibody drug conjugate has a structure such as formula (II- 1) or (II-2):
  • L 2 is -O- or -S-; preferably, L 2 is -O-, preferably, L 2 is -S-,
  • X is a 3- to 6 -membered saturated carbocyclic group optionally substituted by 1, 2 or 3 R 3a ; preferably, replaced Preferably, X is
  • L 3 is -C(R 1a )(R 1b )- or -C(R 1a )(R 1b )C(R 1a )(R 1b )-,
  • R 1a is each independently a halogen or a C 1-6 aliphatic group optionally substituted by 1, 2 or 3 R
  • R 1b and R 3a are each independently a hydrogen, a halogen or a C 1-6 aliphatic group optionally substituted by 1, 2 or 3 R
  • Optionally substituted C 1-6 aliphatic group; each R can be independently hydrogen or halogen; preferably, L 3 is -CH(CH 3 )-, -C(CH 3 ) 2 -, -CH(CH 3 )CH 2 -or-C(CH 3 ) 2 CH 2 -;
  • p represents the average number of connections, and p is an integer or decimal from 1 to 10, preferably an integer or decimal from 3 to 8;
  • Ab is an anti-GPC3 antibody or an antigen-binding fragment thereof, which includes a heavy chain variable region and a light chain variable region.
  • the heavy chain variable region includes amino acid sequences such as SEQ ID NO: 1, SEQ ID NO: 2 and SEQ respectively.
  • HCDR1, HCDR2 and HCDR3 shown in ID NO:3 and the light chain variable region includes the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 respectively.
  • the anti-GPC3 antibody or antigen-binding fragment thereof of the present invention includes: a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 7, and a light chain with an amino acid sequence as shown in SEQ ID NO: 8 Variable region; more preferably, the anti-GPC3 antibody or antigen-binding fragment thereof of the present invention includes: a heavy chain with an amino acid sequence as shown in SEQ ID NO: 9, and a light chain with an amino acid sequence as shown in SEQ ID NO: 10 .
  • the average number of connections p described in the present invention may be an integer or a decimal number from 2 to 8.
  • the average number of connections n may be an integer or a decimal number from 3 to 8.
  • the average number of connections n may be an integer or a decimal number ranging from 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, or 9 to 10.
  • compositions and pharmaceutical preparations are provided.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the anti-GPC3 antibody drug conjugate according to any one of the present invention, its isomer, its pharmaceutically acceptable salt or its mixture, and a pharmaceutically acceptable carrier or excipient.
  • anti-GPC3 antibody-drug conjugates or isomers thereof, pharmaceutically acceptable salts or mixtures thereof, or pharmaceutical compositions thereof provided by the present invention can be integrated with suitable carriers, excipients and Other agents are administered in combination to provide improved transfer, delivery, tolerance, etc.
  • the term "pharmaceutical composition” refers to a preparation that allows the active ingredients contained therein to exist in a biologically active and effective form and does not contain unacceptable toxicity to the subject to whom the preparation is administered. additional ingredients.
  • It can be prepared by combining the anti-GPC3 antibody drug conjugate of the present invention or a pharmaceutically acceptable salt thereof with the desired purity with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th Edition, Osol , A. Editor (1980)) are mixed to prepare a pharmaceutical formulation comprising an anti-GPC3 antibody as described herein, preferably in the form of an aqueous solution or a lyophilized formulation.
  • compositions or preparations of the present invention may also contain one or more other active ingredients required for the particular indication being treated, preferably those having complementary activities that do not adversely affect each other.
  • the other active ingredients are chemotherapeutic agents, immune checkpoint inhibitors, growth inhibitors, antibiotics, or various known anti-tumor or anti-cancer agents, in an appropriate amount effective for the intended use. Earth combination exists.
  • pharmaceutical compositions of the invention further comprise a composition encoding a polynucleotide encoding an anti-GPC3 antibody.
  • the present invention provides a pharmaceutical combination comprising an antibody drug conjugate according to any one of the present invention or its A pharmaceutically acceptable salt or pharmaceutical composition according to any one of the invention, and one or more additional therapeutic agents.
  • the present invention provides a kit comprising the antibody drug conjugate as described in any one of the present invention, its isomer, its pharmaceutically acceptable salt or its mixture, or the antibody drug conjugate as described herein.
  • the pharmaceutical composition described above preferably further includes a drug delivery device.
  • the present invention provides an antibody-drug conjugate according to any one of the present invention, an isomer thereof, a pharmaceutically acceptable salt thereof or a mixture thereof, or a drug according to any one of the present invention.
  • the present invention provides an antibody-drug conjugate according to any one of the present invention, an isomer thereof, a pharmaceutically acceptable salt thereof or a mixture thereof, or a drug according to any one of the present invention.
  • the invention provides a method of treating and/or preventing a GPC3-mediated disease or disorder, comprising administering to a subject in need thereof an antibody drug conjugate according to any one of the invention , its isomer, its pharmaceutically acceptable salt or its mixture, or the pharmaceutical composition according to any one of the present invention.
  • the disease or disorder is cancer.
  • the cancer is selected from the group consisting of breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, lung cancer, liver cancer, gastric cancer, colon cancer, bladder cancer, esophageal cancer, cervical cancer, gallbladder cancer, glioblastoma Cytoma and melanoma.
  • modes of administration of the present invention include, but are not limited to, oral, intravenous, subcutaneous, intramuscular, intraarterial, intraarticular (e.g., in arthritic joints), by inhalation, aerosol delivery, or intratumoral administration. wait.
  • the invention provides for co-administration to a subject of a therapeutically effective amount of one or more therapies (eg, therapeutic modalities and/or other therapeutic agents).
  • the therapy includes surgical treatment and/or radiation therapy.
  • the methods or uses provided herein also include administering to the individual one or more therapies (eg, treatment modalities and/or other therapeutic agents).
  • therapies eg, treatment modalities and/or other therapeutic agents.
  • the antibody drug conjugates of the present invention, or pharmaceutically acceptable salts thereof, may be used alone or in combination with other therapeutic agents in therapy. For example, it can be co-administered with at least one additional therapeutic agent.
  • the drug conjugate of the present invention may have inhibitory activity on the proliferation of tumor cells in vitro.
  • the inhibitory activity can be when the drug conjugate of the present invention is added to the culture medium of tumor cells.
  • the cell proliferation ability of the tumor cells is reduced by more than 1%, more than 2%, or more than 4%. , more than 5%, more than 8%, more than 10%, more than 15%, more than 18%, more than 20%, more than 25%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90 % or more, or 95% or more.
  • the inhibitory activity may be that the IC50 value (nM) for tumor cells may be below 10,000, below 5,000, below 4,000, below 3,000, below 2,000, below 1,000, below 500, below 400, below 300, below 200, below 150 Below, below 120, below 110, below 100, below 99, below 98, below 97, below 95, below 90, below 80, below 75, below 70, below 65, below 62, below 60, below 50, below 40, Below 30, below 25, below 23, below 22, below 20, below 19, below 18, below 18.5, below 17, below 15, below 12, below 10, below 9, below 8.5, below 7, below 6.7, below 6 , below 5.9, below 5.5, below 5.0, below 4.8, below 4.5, below 4.4, below 4, below 3.5, below 3, below 2.5, below 2, below 1.5, below 1.0, below 0.5, below 0.3, below 0.29, below 0.25 Below, below 0.21, below 0.20, below 0.18, below 0.17, below 0.15, below 0.12, below 0.10, below 0.09, below 0.08, below 0.07,
  • the drug conjugates of the present invention may have target inhibitory properties.
  • the target inhibitory property may be that the drug conjugate of the present invention is added to the culture medium of tumor cells with high expression of a specific target. Compared with adding a negative control or a control drug, the proliferation ability of tumor cells with high expression of the specific target is improved. Decline of more than 1%, more than 2%, more than 4%, more than 5%, more than 8%, more than 10%, more than 15%, more than 18%, more than 20%, more than 25%, more than 40%, more than 50%, more than 60 % or more, 70% or more, 80% or more, 90% or more, or 95% or more.
  • the target inhibitory property may be that the IC50 value (nM) of tumor cells with high expression of a specific target may be below 10,000, below 5,000, below 4,000, below 3,000, below 2,000, below 1,000, below 500, below 400, Below 300, below 200, below 185, below 150, below 120, below 110, below 100, below 99, below 98, below 97, below 95, below 91, below 80, below 74, below 70, below 65, below 62 , below 60, below 50, below 40, below 30, below 25, below 23, below 22, below 20, below 19, below 18, below 18.5, below 17, below 15, below 12, below 10, below 9, below 8.5 Below, below 7, below 6.7, below 6, below 5.9, below 5.5, below 5.0, below 4.8, below 4.5, below 4.4, below 4, below 3.5, below 3, below 2.5, below 2, below 1.5, below 1.0, 0.5 or less, 0.3 or less, 0.29 or less, 0.25 or less, 0.21 or less, 0.20 or less, 0.18 or less, 0.17 or less, 0.15 or
  • the drug conjugates of the present invention may have plasma stability.
  • the plasma stability can be that the drug conjugate of the present invention is added to plasma, and the cytotoxic drug released by the drug conjugate is within 1 day, 3 days, 5 days, 7 days, 14 days, 20 days or 30 days.
  • the release rate does not exceed 50%, does not exceed 40%, does not exceed 30%, does not exceed 20%, does not exceed 10%, does not exceed 7%, does not exceed 5%, does not exceed 4%, does not exceed 3%, does not exceed 2%, not more than 1.9%, not more than 1.8%, not more than 1.7%, not more than 1.6%, not more than 1.5%, not more than 1.4%, not more than 1.3%, not more than 1.2%, not more than 1.1%, not more than 1.0%, not more than 0.9%, not more than 0.8%, not more than 0.7%, not more than 0.6%, not more than 0.5%, not more than 0.4%, not more than 0.3%, not more than 0.2%, or not more than 0.1%.
  • the drug conjugates of the present invention can have anti-tumor effects in vivo.
  • the anti-tumor effect can be that when the drug conjugate of the present invention is applied to animals, compared with adding a negative control or a control drug, the tumors of the animal are better at 1 day, 3 days, 5 days, 7 days, 14 days, and 20 days.
  • the volume decreases by more than 1%, more than 2%, more than 4%, more than 5%, more than 8%, more than 10%, more than 15%, more than 18%, more than 20%, more than 25%, 40% or more, 50% or more, 55% or more, 60% or more, 70% or more, 73% or more, 75% or more, 80% or more, 90% or more, or 95% or more, or compared to administration of a negative control or control Drug, the volume of the tumor of the animal is reduced by more than 1.1 times, more than 1.3 times, more than 1.5 times, more than twice on 1 day, 3 days, 5 days, 7 days, 14 days, 20 days, 21 days or 30 days, More than three times, more than five times, more than ten times, more than twenty times, more than twenty-two times, more than thirty times, more than fifty times, more than one hundred times, more than five hundred times, more than one thousand times, or one More than 1,500 times.
  • the animal may include, but is not limited to, a mammal, for example, the animal may include, but is not limited to, a cat, a dog, a horse, a pig, a cow, a sheep, a rabbit, a mouse, a rat, a monkey, or a human.
  • the administration may include, but is not limited to, oral administration, intravenous injection, intravenous drip, intraperitoneal injection, or topical administration.
  • the drug conjugate of the present invention may have a bystander effect.
  • the bystander killing effect may be that the drug conjugate of the present invention has no obvious inhibitory effect on the cell proliferation of tumor cells with low expression of a specific target, but in co-culture of tumor cells with low expression of a specific target and tumor cells with high expression of a specific target , the drug conjugate of the present invention can simultaneously inhibit the cell proliferation of tumor cells with low expression of a specific target and tumor cells with high expression of a specific target.
  • the inhibitory activity may be that the IC50 value (nM) for the tumor cells with low expression of the specific target may be 10,000 or less, 5,000 or less, Below 4000, below 3000, below 2000, below 1000, below 500, below 400, below 300, below 200, below 185, below 150, below 120, below 110, below 100, below 99, below 98, below 97, below 95 , below 91, below 80, below 74, below 70, below 65, below 62, below 60, below 50, below 40, below 30, below 25, below 23, below 22, below 20, below 19, below 18, below 18.5 Below, below 17, below 15, below 12, below 10, below 9, below 8.5, below 7, below 6.7, below 6, below 5.9, below 5.5, below 5.0, below 4.8, below 4.5, below 4.4, below 4, 3.5 or less, 3 or less, 2.5 or less, 2 or less, 1.5 or less, 1.0 or less, 0.5 or less, 0.3
  • the expression of the specific target may be reduced by more than 1%, more than 2%, more than 4%, more than 5%, more than 8%, or more than 10%. % or more, 15% or more, 18% or more, 20% or more, 25% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more.
  • the tumor cells with high expression of the specific target may include, but are not limited to, solid tumor cells.
  • the tumor cells with high expression of the specific target may include, but are not limited to, gastric cancer cells or breast cancer cells.
  • the tumor cells with high expression of the specific target may include, but are not limited to, solid tumor cells.
  • Tumor cells may include, but are not limited to, HCC1569 cells or MDA-MB-453 cells.
  • tumor cells with low expression of the specific target may include, but are not limited to, solid tumor cells.
  • the drug conjugates of the present invention may have anti-transporter transport capabilities.
  • the anti-transport ability may be that the efflux ratio of the drug conjugate of the present invention is reduced by more than 1%, more than 2%, more than 4%, more than 5%, more than 8%, or more than 10% compared to the standard substance of the transport substrate. % or more, 15% or more, 18% or more, 20% or more, 25% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more.
  • the test of the external displacement ratio may be a commonly used method by those in the art, or may be recorded in the embodiments of the present invention.
  • the drug conjugates of the present invention can have tumor targeting ability in vivo.
  • the in vivo targeting ability may refer to applying the drug conjugate labeled with a signaling substance to an animal.
  • the distribution of the labeled drug conjugate in the tumor tissue of the animal can be better than that of other tissues and organs.
  • the signal material may be a radioactive material.
  • the signal material includes but is not limited to 125I.
  • the animal may include, but is not limited to, a mammal, for example, the animal may include, but is not limited to, a cat, a dog, a horse, a pig, a cow, a sheep, a rabbit, a mouse, a rat, a monkey, or a human.
  • the administration may include, but is not limited to, oral administration, intravenous injection, intravenous drip, intraperitoneal injection, or topical administration.
  • the tissue or organ may include, but is not limited to, heart, liver, spleen, lung, kidney, brain, or bone marrow.
  • the drug conjugates of the present invention can have good in vivo safety.
  • the in vivo safety may be that after the drug conjugate of the present invention is administered to an animal, the free toxin release rate in the animal's body does not exceed 50%, does not exceed 40%, does not exceed 30%, does not exceed 20%, or More than 10%, not more than 7%, not more than 5%, not more than 4%, not more than 3%, not more than 2%, not more than 1.9%, not more than 1.8%, not more than 1.7%, not more than 1.6%, no More than 1.5%, not more than 1.4%, not more than 1.3%, not more than 1.2%, not more than 1.1%, not more than 1.0%, not more than 0.9%, not more than 0.8%, not more than 0.7%, not more than 0.6%, no More than 0.5%, no more than 0.4%, no more than 0.3%, no more than 0.2% or no more than 0.1%.
  • the in vivo safety can be that the administration concentration of the drug conjugate of the present invention can be above 0.5 mg/kg, above 1 mg/kg, above 2 mg/kg, or above 3 mg/kg without causing toxicity in animals.
  • the animal may include, but is not limited to, cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats, monkeys, or humans.
  • the administration may include, but is not limited to, oral administration, intravenous injection, intravenous drip, intraperitoneal injection, or topical administration.
  • the anti-GPC3 antibody or its antigen-binding fragment is connected to a biologically active cytotoxic drug through a linker unit. After the antibody-drug conjugate is transferred to the tumor cell, the linker is cut off and freed out of the cell.
  • Toxic drugs H-M-D for example, small molecule compounds Preparation Example 1 to Preparation Example 9.
  • the cytotoxic drug of the present invention has significantly enhanced proliferation inhibitory activity on NCI-N87 cells, JIMT-1 cells, Colo205 cells and MDA-MB-231 cells, and can exert excellent anti-tumor effects.
  • the anti-GPC3 antibody-drug conjugate of the present invention has significantly enhanced proliferation inhibitory activity against GPC3-positive expression cells Huh7 and HepG2; the anti-GPC3 antibody-drug conjugate of the present invention has significantly enhanced inhibitory activity against HepG2 tumor-bearing mice and Huh7 tumor-bearing mice. Antitumor activity.
  • the present invention has good application prospects in GPC3-positive expression diseases (such as cancer).
  • Figure 1 Anti-tumor efficacy of antibody-drug conjugates on human hepatoma HepG2 tumor-bearing mice in vivo.
  • Figure 2 In vivo anti-tumor efficacy of antibody drug conjugates on human hepatoma Huh7 tumor-bearing mice.
  • High performance liquid chromatography Waters e2965 high performance liquid chromatography system.
  • Mobile phase B 50mM K 2 HPO 4 (pH 7.0)/isopropanol (75:25V/V);
  • Detection conditions Set the mobile phase flow rate to 1ml/min, the detection wavelength to 280nm, and the column temperature to 30°C.
  • Detection conditions Set the mobile phase flow rate to 0.5ml/min, the detection wavelength to 280nm, and the column temperature to 30°C.
  • the antibody of the present invention was prepared with reference to WO2006006693.
  • the amino acid sequence of the variable region of the anti-GPC3 antibody DB1001 is as follows, and the CDR region is determined according to the IMGT numbering rules.
  • MS-ESI m/z 522.5[M+H]+.
  • DXd was synthesized by referring to the method provided in Example 75 on page 183 of the patent "CN104755494A".
  • Linker-payload X1 Dissolve Linker-payload X1 in dry DMA (N, N-Dimethylacetamide, dimethylacetamide, manufacturer: Sinopharm Group) to prepare a 10 mg/mL linker-payload DMA solution.
  • the drug loading capacity (DAR) of the antibody drug conjugate DB1001-X1 was 7.51, and the SEC purity was 100%.
  • Linker-payload X2 Dissolve Linker-payload X2 in dry DMA (N, N-Dimethylacetamide, dimethylacetamide, manufacturer: Sinopharm) to prepare a 10 mg/mL linker-payload DMA solution.
  • the drug loading capacity (DAR) of the antibody drug conjugate DB1001-X1 was 7.72, and the SEC purity was 99.3%.
  • the linker-cytotoxin is Deruxtecan (CAS 1599440-13-7, purchased from Shanghai Haoyuan Chemical Technology Co., Ltd.), and the antibody is the antibody DB1001 of the present invention.
  • DB1001-Deruxtecan was obtained with reference to the method in Example 2.3 of the present invention.
  • CTG Luminescent Cell Viability Assay, Promega, Cat. No.: G7558
  • NCI-N87/JIMT-1/MBA-MB-231 is cultured in 10% FBS RPMI-1640 medium.
  • Cell preparation Take NCI-N87/JIMT-1/MBA-MB-231 cells in the logarithmic growth phase, wash them once with PBS, add 2-3mL trypsin for digestion for 2-3 minutes, and wait until the cells are completely digested. Add 10-15mL of cell culture medium and mix the digested After the cells are eluted, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and then add 10-20 mL of cell culture medium to resuspend the cells to make a single cell suspension.
  • Cell plating Mix the NCI-N87/JIMT-1/MBA-MB-231 single cell suspension, use cell culture medium to adjust the viable cell density to 6x10 4 cells/ml, and mix the density-adjusted cell suspension Mix well and add 50ul/well to a 96-well cell culture plate. The culture plates were incubated in an incubator for 18 hours (37°C, 5% CO2 ).
  • Compound preparation Dissolve the compound in DMSO and prepare a storage solution with an initial concentration of 10mM.
  • Sample addition operation Add the configured samples to be tested at different concentrations into the culture plate, with two duplicate wells for each sample.
  • the culture plates were incubated for 6 days in an incubator (37°C, 5% CO2 ).
  • Color development operation Take out the 96-well cell culture plate, add 50ul CTG reagent to each well, and incubate at room temperature for 10 minutes.
  • Plate reading operation Take out the 96-well cell culture plate, place it in a microplate reader, and use the microplate reader to measure chemiluminescence.
  • Data analysis Use Microsoft Excel and Graphpad Prism 5 to process and analyze the data.
  • Table 1 IC 50 values of the small molecule compounds in this application for inhibiting the proliferation of tumor cells in vitro.
  • the toxin of the drug conjugate of the present application has significantly enhanced proliferation inhibitory activity on NCI-N87 cells, JIMT-1 and MDA-MB-231 cells.
  • NCI-N87/JIMT-1/MBA-MB-231 is cultured in 10% FBSRPMI-1640 medium.
  • Cell preparation Take NCI-N87/JIMT-1/MBA-MB-231 cells in the logarithmic growth phase, wash them once with PBS, add 2-3mL trypsin for digestion for 2-3 minutes, and wait until the cells are completely digested. Add 10-15 mL of cell culture medium to elute the digested cells, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, then add 10-20 mL of cell culture medium to resuspend the cells to make a single cell suspension.
  • Cell plating Mix the NCI-N87/JIMT-1/MBA-MB-231 single cell suspension and adjust the viable cells separately with cell culture medium. Adjust the density to 6x10 4 cells/mL, mix the density-adjusted cell suspension, and add 50uL/well to a 96-well cell culture plate. The culture plates were incubated in an incubator for 18 hours (37°C, 5% CO2 ).
  • Compound preparation Dissolve the compound in DMSO and prepare a storage solution with an initial concentration of 10mM.
  • Sample addition operation Add the configured samples to be tested at different concentrations into the culture plate, with two duplicate wells for each sample.
  • the culture plates were incubated for 6 days in an incubator (37°C, 5% CO2 ).
  • Color development operation Take out the 96-well cell culture plate, add 50uL CTG reagent to each well, and incubate at room temperature for 10 minutes.
  • Plate reading operation Take out the 96-well cell culture plate, place it in a microplate reader, and use the microplate reader to measure chemiluminescence.
  • Data analysis Use Microsoft Excel and Graphpad Prism5 to process and analyze the data.
  • Cell survival rate is calculated using the formula: V sample /V vehiclecontrol x 100%. Where V sample is the reading of the drug treatment group, and V vehicle control is the average value of the solvent control group. Using GraphPad Prism software, a nonlinear regression model was used to draw a S-shaped dose-survival curve and calculate the IC 50 value.
  • the toxin of the drug conjugate in this application has significantly enhanced proliferation inhibitory activity on NCI-N87, JIMT-1 and MDA-MB-231 cells.
  • NCI-N87/Colo205 is cultured in 10% FBSRPMI-1640 medium.
  • Cell preparation Take NCI-N87/Colo205 cells in the logarithmic growth phase, wash them once with PBS, add 2-3mL trypsin for digestion for 2-3 minutes, and after the cells are completely digested, add 10-15mL cell culture medium. Elute the digested cells, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and then add 10-20 mL of cell culture medium to resuspend the cells to make a single cell suspension.
  • Cell plating Mix the NCI-N87/Colo205 single cell suspension, use cell culture medium to adjust the viable cell density to 6x10 4 cells/mL, mix the density-adjusted cell suspension, and add 50ul/well 96-well cell culture plate. The culture plates were incubated in an incubator for 18 hours (37°C, 5% CO2 ).
  • Compound preparation Dissolve the compound in DMSO and prepare a storage solution with an initial concentration of 10mM.
  • Sample addition operation Add the configured samples to be tested at different concentrations into the culture plate, with two duplicate wells for each sample.
  • the culture plates were incubated for 6 days in an incubator (37°C, 5% CO2 ).
  • Color development operation Take out the 96-well cell culture plate, add 50Ul CTG reagent to each well, and incubate at room temperature for 10 minutes.
  • Plate reading operation Take out the 96-well cell culture plate, place it in a microplate reader, and use the microplate reader to measure chemiluminescence.
  • Data analysis Use Microsoft Excel and Graphpad Prism5 to process and analyze the data.
  • Table 3 IC 50 values of small molecule compounds in this application inhibiting the proliferation of NCI-N87 and Colo205 cells in vitro.
  • the toxin of the drug conjugate of the present application has significantly enhanced proliferation inhibitory activity on NCI-N87 and Colo205 cells.
  • Example 4 In vitro anti-proliferation test of tumor cells by antibody-drug conjugates
  • Chemiluminescent cell viability assay was used to evaluate the inhibitory effect of anti-GPC3 naked antibody DB1001 and anti-GPC3 ADC DB1001-X1 on cell proliferation in GPC3-positive human hepatoma cells HepG2 and Huh7 after 7 days of treatment.
  • Cells in the logarithmic growth phase were collected and plated at a density of 4000 cells/well (HepG2 cells) and 6000 cells/well (HuH7).
  • the cell plates were cultured overnight in a 37°C, 5% CO2 incubator.
  • DB1001 and DB1001-X1 were diluted 3 times with complete culture medium to obtain 9 concentration gradients (starting with the highest concentration of 300nM).
  • 50 ⁇ L/well was added to the cell culture plate, and the complete culture medium was used as a blank.
  • For control set up 3 duplicate wells; continue to incubate in a 37°C, 5% CO2 incubator for 7 days.
  • the antibody drug conjugate DB1001-X1 of the present application has obvious proliferation inhibitory activity on GPC3-positive expression cells Huh7 and HepG2; and is significantly better than the antibody DB1001.
  • the CTG method was used to evaluate the inhibitory effect of anti-GPC3 ADC DB1001-X1 and DB1001-X2 on cell proliferation in GPC3-positive human hepatoma cells HepG2 and Huh7 after 6 days of treatment.
  • Cells in the logarithmic growth phase were collected and plated at a density of 5000 cells/well (HepG2 cells) and 5000 cells/well (HuH7).
  • the cell plates were cultured overnight in a 37°C, 5% CO2 incubator.
  • DB1001-X1 and DB1001-X2 were diluted 3 times with complete culture medium to obtain 9 concentration gradients (starting with the highest concentration of 1000nM).
  • 50 ⁇ L/well was added to the cell culture plate and the complete culture medium was obtained.
  • As a blank control set up 3 duplicate wells; continue to incubate in a 37°C, 5% CO2 incubator for 7 days.
  • Example 5 In vivo tumor inhibition test of antibody drug conjugates
  • DB1001-X1 In order to study the inhibitory effect of DB1001-X1 on tumor formation in vivo, the anti-tumor effect of DB1001-X1 was evaluated after xenograft tumors were formed in mice with GPC3-positive human hepatoma cells HepG2 or Huh7.
  • Blank control group (control group): saline
  • mice 8-week-old female BALB/c mice purchased from Jicui Yaokang Biotechnology Co., Ltd.
  • HepG2 cells were inoculated subcutaneously into the right front scapula of 8-week-old female BALB/c nude mice.
  • the tumor-bearing mice were randomly divided into StudyDirectorTM groups, and on the same day (0th day) day), administration of DB1001 and DB1001-X1 was started via intravenous (iv) injection, with a total of 1 injection, and the experiment was ended after 21 days of administration. Tumor volume and body weight were measured twice a week and the data were recorded.
  • Tumor inhibition rate 100% - (Tumor volume of the treatment group on the day of measurement - Tumor volume of the treatment group on Day 0)/(Tumor volume of the control group on the day of measurement - Tumor volume of the control group on Day 0).
  • Blank control group (control group): saline
  • mice 8-week-old female BALB/c mice purchased from Jicui Yaokang Biotechnology Co., Ltd.
  • Huh7 cells were inoculated subcutaneously into the right front scapula of 8-week-old female BALB/c nude mice.
  • the tumor-bearing mice were randomly divided into StudyDirectorTM groups and were collected on the same day (0th day). day), DB1001-X1 (3 mg/kg) and DB1001-X1 (10 mg/kg) were administered via intravenous injection (iv) for a total of once, and the experiment was ended after 21 days of administration. Tumor volume and body weight were measured twice a week and the data were recorded.
  • Tumor inhibition rate 100% - (Tumor volume of the treatment group on the day of measurement - Tumor volume of the treatment group on Day 0)/(Tumor volume of the control group on the day of measurement - Tumor volume of the control group on Day 0).
  • the experimental results are shown in Figure 2 and Table 7.
  • the antibody-drug conjugate DB1001-X1 showed significant dose-dependent anti-tumor activity after a single administration.
  • Blank control group (control group): saline
  • HepG2 cells were inoculated subcutaneously into the right front scapula of 8-week-old female BALB/c nude mice.
  • the tumor-bearing mice were randomly divided into StudyDirectorTM groups, and on the same day (0th day) day), administration of DB1001-X1 and DB1001-X2 was started via intravenous (iv.) injection, with a total of 1 injection, and the experiment was ended after 21 days of administration. Tumor volume and body weight were measured twice a week and the data were recorded.
  • Tumor inhibition rate 100% - (Tumor volume of the treatment group on the day of measurement - Tumor volume of the treatment group on Day 0)/(Tumor volume of the control group on the day of measurement - Tumor volume of the control group on Day 0).
  • Blank control group (control group): saline
  • Huh7 cells were inoculated subcutaneously into the right front scapula of 8-week-old female BALB/c nude mice.
  • the tumor-bearing mice were randomly divided into StudyDirectorTM groups and were collected on the same day (0th day).
  • Tumor inhibition rate 100% - (Tumor volume of the treatment group on the day of measurement - Tumor volume of the treatment group on Day 0)/(Tumor volume of the control group on the day of measurement - Tumor volume of the control group on Day 0).

Abstract

一种与GPC3特异性结合的抗GPC3抗体药物偶联物及其用途，其中所述抗GPC3抗体药物偶联物结构如式(I-1)所示。所述抗GPC3抗体药物偶联物具有更好的对肿瘤细胞的体外增殖的抑制活性以及更好的体内抑瘤效果。

Description

GPC3抗体药物偶联物及其用途
本申请要求申请日为2022/3/18的中国专利申请2022102734945的优先权以及申请日为2023/3/13的中国专利申请2023102386369的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明提供了与GPC3特异性结合的抗体药物偶联物和包含其的组合物。还提供了使用本发明的抗体药物偶联物的方法和用途。
背景技术
Glypican-3(磷脂酰肌醇聚糖-3，GPC3)是一种由580个氨基酸组成的70KD的膜蛋白，通过糖基磷脂酰肌醇(GPI)锚定在细胞表面；GPC3在细胞生长、细胞分化和细胞迁移中发挥重要作用。GPC3蛋白在胚胎组织(肝、肾和胎盘等)中表达，但由于DNA甲基化导致的表观遗传沉默，成人组织中几乎没有GPC3的表达。然而免疫组化研究发现GPC3在＞70％的肝癌组织中特异性高表达，临床研究也显示GPC3的表达与HCC的临床预后不良有关，因此GPC3有潜力作为肝癌治疗的靶分子。
国家癌症中心最新统计数据显示，在所有恶性肿瘤中，我国肝癌发病率位居第五位，死亡率高居第二位。我国每年新发肝癌的病例数约占全球的一半，每年约有46万的肝癌新发病例，但5年生存率仅10％，是恶性肿瘤中预后最差之一。早前对晚期肝癌患者的一线治疗药物/方案，主要为索拉非尼和系统化疗，客观缓解率较低、无进展生存期(PFS)短、不良反应较大；近年来仑伐替尼、阿替珠单抗联合贝伐单抗的治疗方案将PFS延长近3个月，但复发或进展后，肝癌的治疗手段仍有限。靶向GPC3的临床在研药物种类多样，包括单抗、双抗、ADC、CAR-T细胞疗法等。
Roche开发的anti-GPC3单抗Codrituzumab(研发代号GC33)是目前临床进展最快的GPC3靶向分子，在治疗晚期肝癌的Ib期临床研究(NCT01507168)中，对GPC3高表达的患者群体显示出疾病进展延缓的趋势，但在II期研究(NCT01507168)中没有得到有统计意义的生存期改善疗效，Roche因此暂停了对Codrituzumab的开发。科济生物在2021年的ASCO会议上，报道了其GPC3-CART治疗6名晚期肝癌患者，其中1人得到部分缓解，3人病情稳定，疾病控制率达到50％，中位无进展生存期为4.2个月。鉴于单抗药物抗肿瘤疗效较弱、CAR-T细胞治疗便利性较差，靶向GPC3的ADC药物有其独特的开发价值和临床需求。
发明内容
本发明所要解决的技术问题是为了克服现有技术中抗GPC3抗体药物偶联物少的缺陷，而提供了一种抗GPC3抗体药物偶联物及其制备方法和应用。本发明的抗GPC3抗体药物偶联物相较现有技术具有选自以下组的一种或多种优势效果：(1)更好的对肿瘤细胞的体外增殖的抑制活性；(2)更好的靶向抑制性；(3)更好的血浆稳定性；(4)更好的体内抑瘤效果；(5)更好的旁观杀伤效应(Bystander  Effect)；(6)更好的抗转运体转运能力；(7)更好的体内肿瘤靶向能力；和(8)更好的良好的体内安全性。
本发明主要是通过以下技术手段解决上述技术问题的。
一方面，本申请提供一种抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，所述抗GPC3抗体药物偶联物结构如式(I)所示：
Ab-(L-M-D)p   (I)
其中，
L和M是接头单元；
D是细胞毒性药物；
p表示平均连接数，且p为1到10的整数或小数，优选为3-8的整数或小数；
Ab为抗GPC3抗体或其抗原结合片段，其包含重链可变区和轻链可变区，所述重链可变区包含氨基酸序列分别如SEQ ID NO：1、SEQ ID NO：2和SEQ ID NO：3所示的HCDR1、HCDR2和HCDR3，和所述轻链可变区包含氨基酸序列分别如SEQ ID NO：4、SEQ ID NO：5和SEQ ID NO：6所示的LCDR1、LCDR2和LCDR3。
一方面，本申请提供一种抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，所述抗GPC3抗体药物偶联物结构如式(I)所示：
Ab-(L-M-D)p   (I)
其中，
L是接头单元；
-M-D是细胞毒性药物；
p表示平均连接数，且p为1到10的整数或小数，优选为3-8的整数或小数；
Ab为抗GPC3抗体或其抗原结合片段，其包含重链可变区和轻链可变区，所述重链可变区包含氨基酸序列分别如SEQ ID NO：1、SEQ ID NO：2和SEQ ID NO：3所示的HCDR1、HCDR2和HCDR3，和所述轻链可变区包含氨基酸序列分别如SEQ ID NO：4、SEQ ID NO：5和SEQ ID NO：6所示的LCDR1、LCDR2和LCDR3。
一方面，本申请提供一种抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述抗GPC3抗体药物偶联物结构如式(I-1)所示：
其中，
M为-L2-L3-X-L1-；
L2为-O-或-S-；
L3为-(C(R1a)(R1b))m-，m为0、1、2或3，其中当L3包含亚甲基单元时，所述L3的0个或1个亚甲基单元可以被-C(O)-、或-C(S)-替代；
L1为-(C(R2a)(R2b))n-，n选自1、2或3，其中当L1可以包含亚甲基单元时，所述L1的0个或1个亚甲基单元可以被-C(O)-、或-C(S)-替代；
X为3到6元饱和的碳环基、3到6元饱和的杂环基或单键，所述3到6元饱和的碳环基和3到6元饱和的杂环基任选被1个或多个R3a取代；
其中每个R1a，R1b，R2a，R2b，R3a各自独立地可以为氢、卤素或被1个或多个R任选取代的C1- 6脂肪族基团；
其中每个R各自独立地可以为氢或卤素；
L是接头单元；
p表示平均连接数，且p为1到10的整数或小数；
Ab为抗GPC3抗体或其抗原结合片段。
在一些实施方式中，本发明所述3-6元饱和的杂环基中的杂原子选自N、O和S，杂原子数为1-3个。
在一些实施方式中，本发明所述抗GPC3抗体或其抗原结合片段包含重链可变区和轻链可变区，所述重链可变区包含氨基酸序列分别如SEQ ID NO：1、SEQ ID NO：2和SEQ ID NO：3所示的HCDR1、HCDR2和HCDR3，和所述轻链可变区包含氨基酸序列分别如SEQ ID NO：4、SEQ ID NO：5和SEQ ID NO：6所示的LCDR1、LCDR2和LCDR3。
在一些实施方式中，本发明所述抗GPC3抗体或其抗原结合片段包含：氨基酸序列如SEQ ID NO：7所示或与其具有至少95％、96％、97％、98％或99％同一性的重链可变区，和氨基酸序列如SEQ ID NO：8所示或与其具有至少95％、96％、97％、98％或99％同一性的轻链可变区。
在一些实施方式中，本发明所述抗GPC3抗体或其抗原结合片段包含氨基酸序列如SEQ ID NO：7所示的重链可变区，和氨基酸序列如SEQ ID NO：8所示的轻链可变区。
在一些实施方式中，本发明所述抗GPC3抗体或抗原结合片段为鼠源抗体或其片段、嵌合抗体或抗原结合片段、人源化抗体或抗原结合片段、或全人抗体或抗原结合片段。
在一些实施方式中，本发明所述抗GPC3抗体或其抗原结合片段为人源化抗体或其片段。
在一些实施方式中，本发明所述抗GPC3抗体或其抗原结合片段为Fab、Fab′、Fab′-SH、Fv、scFv、F(ab′)2、sdAb、双抗体或线性抗体。
在一些实施方式中，本发明所述抗GPC3抗体为单克隆抗体。
在一些实施方式中，本发明所述抗体为IgG1形式的抗体、IgG2形式的抗体、IgG3形式的抗体或IgG4形式的抗体。
在一些实施方式中，本发明所述抗体为IgG1形式的抗体。
在一些实施方式中，本发明所述抗GPC3抗体或其抗原结合片段包含：氨基酸序列如SEQ ID NO：9所示或与其具有至少95％、96％、97％、98％或99％同一性的重链，和氨基酸序列如SEQ ID NO：10所示或与其具有至少95％、96％、97％、98％或99％同一性的轻链。
在一些实施方式中，本发明所述抗GPC3抗体或其抗原结合片段包含氨基酸序列如SEQ ID NO：9所示的重链，和氨基酸序列如SEQ ID NO：10所示的轻链。
在一些实施方式中，本发明所述抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述的M，其L2端与接头单元L相连，L1端与D相连。
在一些实施方式中，本发明L2为-O-。
在一些实施方式中，本发明L2为-S-。
在一些实施方式中，本发明所述抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中L3为-(C(R1a)(R1b))m-，其中，m为0、1、2或3；其中每个R1a和R1b各自独立地可以为氢、卤素或被1、2或3个R任选取代的C1-6脂肪族基团；其中每个R各自独立地可以为氢或卤素。
在一些实施方式中，本发明所述抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中L3为-(C(R1a)(R1b))m-，其中，m为0、1、2或3；其中每个R1a和R1b各自独立地为氢、甲基、乙基或丙基。
在一些实施方式中，本发明所述抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中L3为-(C(R1a)(R1b))m-，其中，m为0、1、2或3；其中每个R1a和R1b各自独立地可以为氢、卤素、CH3或CH2CH3。
在一些实施方式中，本发明所述抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中L3为单键、-CH2-、-CH(CH3)-、-C(CH3)2-、-CH2CH2-、-CH(CH3)CH2-或-C(CH3)2CH2-。其中上述基团的左侧优选与L2相连。
在一些实施方式中，本发明所述抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中L3为其中结构片段的左侧优选与L2相连。
在一些实施方式中，本发明所述抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中L3为单键。
在一些实施方式中，本发明所述抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中X为任选被1、2或3个R3a取代的3到6元饱和的碳环基或单键；其中每个R3a各自独立地可以为氢、卤素或被1、2或3个R任选取代的C1-6脂肪族基团；其中每个R各自独立地可以为氢或卤素。
在一些实施方式中，本发明所述抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中X为任选被1、2或3个R3a取代的3到6元饱和的碳环基或单键；其中每个R3a各自独立地可以为氢、卤素、CH3或CH2CH3。
在一些实施方式中，本发明所述的抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中X为环丙基、环丁基、环戊基、环己基或单键。
在一些实施方式中，本发明所述的抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中X为或单键。其中上述基团的右侧优选与L1相连。
在一些实施方式中，本发明所述的抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中X为单键。
在一些实施方式中，本发明所述的抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中X为优选地，X为其中上述基团的右侧优选与L1相连。
在一些实施方式中，本发明所述的抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述-L3-X-为：-(C(R1a)(R1b))m-、3到6元饱和的碳环基或3到6元饱和的杂环基；其中，m为1、2或3，所述3到6元饱和的碳环基和3到6元饱和的杂环基任选被1、2或3个R3a取代；其中，每个R1a，R1b，R3a各自独立地可以为氢、卤素或可以被R任选取代的C1-6脂肪族基团；其中每个R各自独立地可以为氢或卤素。
在一些实施方式中，本发明所述的抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中，
所述-L3-X-为：-(C(R1a)(R1b))m-或任选被1、2或3个R3a取代的3到6元饱和的碳环基；例如，所述-L3-X-为-(C(R1a)(R1b))m-或任选被1、2或3个R3a取代的环丙基、环丁基、环戊基或环己基；优选地，所述-L3-X-为：-(C(R1a)(R1b))m-或任选被1、2或3个R3a取代的：
所述m为1、2或3；
R1a各自独立为卤素或被1、2或3个R任选取代的C1-6脂肪族基团，例如为甲基、乙基或丙基；R1b，R3a各自独立地为氢、卤素或被1、2或3个R任选取代的C1-6脂肪族基团，例如为氢、甲基、乙基或丙基；R各自独立地为氢或卤素。
在一些实施方式中，本发明所述的抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中，
所述-L3-X-选自：-(C(R1a)(R1b))m-或任选被1、2或3个R3a取代的3到6元饱和的碳环基；优选地，所述-L3-X-为：-(C(R1a)(R1b))m-、或任选被1、2或3个R3a取代的 其中，m为1、2或3；R1a各自独立地为卤素或被1、2或3个R任选取代的C1-6脂肪族基团；R1b，R3a各自独立为氢、卤素或被1、2或3个R任选取代的C1-6脂肪族基团；其中每个R各自独立地可以为氢或卤素。
在一些实施方式中，本发明所述的抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述-L3-X-为：
在一些实施方式中，本发明所述的抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述-L3-X-为：
在一些实施方式中，本发明所述抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中L1为-C(R2a)(R2b)-、-C(R2a)(R2b)C(O)-或-C(O)-；其中每个R2a，R2b各自独立地可以为氢、卤素或被1、2或3个R任选取代的C1-6脂肪族基团；其中每个R各自独立地可以为氢或卤素。
在一些实施方式中，本发明所述抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中L1为-C(R2a)(R2b)-、-C(R2a)(R2b)C(O)-或-C(O)-；其中每个R2a，R2b各自独立地可以为氢、甲基、乙基或丙基。
在一些实施方式中，本发明所述抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中L1为-C(R2a)(R2b)-、-C(R2a)(R2b)C(O)-或-C(O)-；其中每个R2a，R2b各自独立地可以为氢、卤素、CH3或CH2CH3。
在一些实施方式中，本发明所述抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中L1为-CH2C(O)-、-CH(CH3)C(O)-或-C(O)-。
在一些实施方式中，本发明所述抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中L1为-C(O)-。
在一些实施方式中，本发明所述抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中L1为-CH2C(O)-。
在一些实施方式中，本发明所述抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中
所述M为-L2-L3-X-L1-；
L2为-O-；
-L3-X-为任选被1、2或3个R3a取代的3到6元饱和的碳环基；
L1为-C(O)-；
其中每个R3a各自独立地可以为氢、卤素或被1、2或3个R任选取代的C1-6脂肪族基团；
其中每个R各自独立地可以为氢或卤素。
在一些实施方式中，本发明所述抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中
所述M为-L2-L3-X-L1-；
L2为-O-；
-L3-X-为任选被1、2或3个R3a取代的环丙基、环丁基、环戊基或环己基；优选地，所述-L3-X-为：任选被1、2或3个R3a取代的：
L1为-C(O)-；
其中每个R3a各自独立地为氢、卤素或被1、2或3个R任选取代的C1-6脂肪族基团；
其中每个R各自独立地为氢或卤素。
在一些实施方式中，本发明所述抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中
所述M为-L2-L3-X-L1-；
L2为-O-；
-L3-X-为任选被1、2或3个R3a取代的R3a各自独立为氢、卤素或C1-6脂肪族基团；
L1为-C(O)-。
在一些实施方式中，本发明所述抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中
所述M为-L2-L3-X-L1-；
L2为-O-或-S-；
-L3-X-为-(C(R1a)(R1b))m-，其中，m为1、2或3；
L1为-C(O)-；
R1a，R1b，R3a各自独立地为氢、卤素或被1、2或3个R任选取代的C1-6脂肪族基团；
R各自独立地可以为氢或卤素。
在一些实施方式中，本发明所述抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中
所述M为-L2-L3-X-L1-；
L2为-O-或-S-；
-L3-X-为-(C(R1a)(R1b))m-，其中，m为1、2或3；
L1为-CH2C(O)-；
R1a各自独立地为卤素或被1、2或3个R任选取代的C1-6脂肪族基团；
R1b，R3a各自独立地为氢、卤素或被1、2或3个R任选取代的C1-6脂肪族基团。
R各自独立地可以为氢或卤素。
在一些实施方式中，本发明所述抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中
所述M为-L2-L3-X-L1-；
L2为-O-或-S-；
-L3-X-为-CH(CH3)-、-C(CH3)2-或-C(CH3)CH(CH3)-；
L1为-CH2C(O)-。
在一些实施方式中，本发明所述抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述的M，其L2端与接头单元L相连。
在一些实施方式中，本发明所述抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述-M-为：
在一些实施方案中，本发明所述抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中细胞毒性药物-M-D选自以下任一结构：

在一些实施方式中，本发明所述抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述L为-La-Lb-Lc-，
所述-La-为
其中，W为-(C(Rwa)(Rwb))wn-，Y为-(OCH2CH2)yn-Oyp-，Z为-(C(Rza)(Rzb))zn；
其中wn为1、2、3或6，
W的0个或1个亚甲基单元各自独立地被-Cyr-、-N(Rwx)C(O)-、-C(O)N(Rwx)-、或-C(O)-替代；
其中yn为0、4或8，yp为0或1；
其中zn为1、2或3，
Z的1个亚甲基单元各自独立地被-Cyr-、-N(Rzx)C(O)-、-C(O)N(Rzx)-、或-C(O)-替代；
-Cyr-为3到10元饱和的亚碳环基，其中所述-Cyr-是未取代的或独立地被1到3个取代基Rcx取代；
其中每个Rwa，Rwb，Rza，Rzb，Rwx，Rzx，Rcx各自独立地为氢、卤素、-ORr或被Rr任选取代的C1-6脂肪族基团；
其中每个Rr各自独立地为氢、卤素或C1-6脂肪族基团；
所述-Lb-为由2到7个氨基酸构成的肽残基，所述-Lb-的肽残基为由选自以下组中的氨基酸形成的肽残基：苯丙氨酸、甘氨酸、丙氨酸、缬氨酸、瓜氨酸、赖氨酸、丝氨酸、谷氨酸和天冬氨酸；优选地，-Lb-表示由2到4个氨基酸构成的肽残基，所述-Lb-的肽残基为由选自以下组中的氨基酸形成的肽残基：苯丙氨酸、甘氨酸、丙氨酸、缬氨酸、瓜氨酸和赖氨酸；
所述-Lc-为
其中RL1、RL2各自独立地选自以下组：氢、卤素、-OH和C1-6脂肪族基团。
在一些实施方式中，本发明所述抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述L为-La-Lb-Lc-，
所述-La-为
其中，W为-(C(Rwa)(Rwb))wn-，Y为-(OCH2CH2)yn-Oyp-，Z为-(C(Rza)(Rzb))zn；
其中wn为1、2、3或6，
W的0个或1个亚甲基单元各自独立地被-Cyr-、-N(Rwx)C(O)-、-C(O)N(Rwx)-、或-C(O)-替代；
其中yn为0、4或8，yp为0或1；
其中zn为1、2或3，
Z的1个亚甲基单元各自独立地被-Cyr-、-N(Rzx)C(O)-、-C(O)N(Rzx)-、或-C(O)-替代；
-Cyr-为3到10元饱和的亚碳环基，其中所述-Cyr-是未取代的或独立地被1到3个取代基Rcx取代；
其中每个Rwa，Rwb，Rza，Rzb，Rwx，Rzx，Rcx各自独立地为氢、卤素、-ORr或被Rr任选取代的C1-6脂肪族基团；
其中每个Rr各自独立地为氢、卤素或C1-6脂肪族基团；
所述-Lb-选自以下组：
所述-Lc-为
其中RL1、RL2各自独立地选自以下组：氢、卤素、-OH和C1-6脂肪族基团。
在一些实施方式中，本发明所述抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述-La-为优选为
在一些实施方式中，本发明所述抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其 混合物，其中所述-Lb-为优选为
在一些实施方式中，本发明所述抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述-Lc-为
在一些实施方式中，本发明所述抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述的接头单元L，其La端与Ab相连，Lc端与接头单元M相连。
在一些实施方式中，本发明所述抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述的接头单元L，其La端与Ab相连，Lc端与M相连。
在一些实施方式中，本发明所述抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述L为
在一些实施方式中，本发明所述的抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述L为
在一些实施方式中，本发明所述抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中-L-M-D选自以下任一结构：
在一些实施方式中，本发明所述抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述抗GPC3抗体药物偶联物结构如式(II-1)或(II-2)所示：
其中，
Ab、L2、X、p和L3分别如本发明任一项所定义。
在一些实施方式中，本发明所述抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述抗GPC3抗体药物偶联物结构如式(II-1)或(II-2)所示：
其中，
p表示平均连接数，且p为1到10的整数或小数，优选为3-8的整数或小数；
Ab为如本发明任一实施方案所述的抗GPC3抗体或其抗原结合片段；
L2为-O-或-S-；
X为任选被1、2或3个R3a取代的3到6元饱和的碳环基；优选地，X为任选被1、2或3个R3a取代的：
L3为-C(R1a)(R1b)-CH2-或-C(R1a)(R1b)C(R1a)(R1b)-CH2-；
R1a各自独立为卤素或被1、2或3个R任选取代的C1-6脂肪族基团；
R1b，R3a各自独立为氢、卤素或被1、2或3个R任选取代的C1-6脂肪族基团；
其中每个R各自独立地可以为氢或卤素。
在一些实施方式中，本发明所述抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述抗GPC3抗体药物偶联物结构如式(II-1)或(II-2)所示：
其中，
p表示平均连接数，且p为1到10的整数或小数，优选为3-8的整数或小数；
Ab为如本发明任一实施方案所述的抗GPC3抗体或其抗原结合片段；
L2为-O-或-S-；
X为
L3为-CH(CH3)CH2-或-C(CH3)2CH2-。
在一些实施方式中，本发明所述抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述抗GPC3抗体药物偶联物选自以下结构式：



其中，
p表示平均连接数，且p为1到10的整数或小数，优选为3-8的整数或小数；
Ab为如本发明任一实施方案所述的抗GPC3抗体或其抗原结合片段。
在又一个方面，本发明提供了一种抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述抗GPC3抗体药物偶联物选自：


其中，
p表示平均连接数，且p为1到10的整数或小数，优选为3-8的整数或小数。
本发明中，所述DB1001为抗GPC3抗体，所述抗GPC3的重链氨基酸序列如SEQ ID NO.：9所示，轻链氨基酸序列如SEQ ID NO.：10所示。
在一些实施方式中，本发明所述平均连接数p可以为2到8的整数或小数。例如，所述平均连接数p可以为3到8的整数或小数。例如，所述平均连接数p可以为1到2、2到3、3到4、4到5、5到6、6到7、7到8、8到9、9到10的整数或小数。例如，平均连接数p为7.51或7.72。
在又一个方面，本发明提供了一种抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述抗GPC3抗体药物偶联物选自以下任一结构(p表示平均连接数)：
其中，所述DB1001为抗GPC3抗体，所述抗GPC3的重链氨基酸序列如SEQ ID NO.：9所示，轻链氨基酸序列如SEQ ID NO.：10所示。
在又一个方面，本发明提供了一种药物组合物，其包含如本发明任一项所述的抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，和药学上可接受的载体或赋形剂。
在又一个方面，本发明提供了如本发明任一项所述的抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物或药物组合物在制备用于治疗和/或预防GPC3介导的疾病或病症的药物中的用途，优选地，所述疾病或病症为癌症，例如，GPC3阳性表达的癌症。
在又一个方面，本发明提供了一种治疗和/或预防GPC3介导的疾病或病症的方法，其包括向有需要的受试者施用如本发明任一项所述的抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物或如本发明任一项所述的药物组合物，优选地，所述疾病或病症为癌症，例如，GPC3阳性表达的癌症。
在又一个方面，本发明提供了如本发明任一项所述的抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物或如本发明任一项所述的药物组合物，其用于治疗和/或预防GPC3介导的疾病或病症，优选地，所述疾病或病症为癌症，例如，GPC3阳性表达的癌症。
在一些实施方式中，本发明所述癌症选自肝癌、乳腺癌、卵巢癌、前列腺癌、胰腺癌、肾癌、肺癌、胃癌、结肠癌、膀胱癌、食管癌、宫颈癌、胆囊癌、胶质母细胞瘤和黑色素瘤。
在又一个方面，本发明提供了一种药物组合，其包含如本发明任一项所述的抗体药物偶联物或其药学上可接受的盐或如本发明任一项所述的药物组合物，以及一种或多种另外的治疗剂。
在又一个方面，本发明提供了一种试剂盒，其包括如本发明任一项所述的抗体药物偶联物、或本如发明任一项所述的药物组合物。
术语定义
除非另有说明，本发明的实施将采用分子生物学(包括重组技术)、微生物学、细胞生物学、生物化学和免疫学的常规技术，这些都在本领域的技术范围内。
在本发明中，为了可以更容易地理解本发明，某些科技术语具体定义如下。除非本文其它部分另有明确定义，否则本文所用的科技术语都具有本发明所属领域普通技术人员通常理解的含义。关于本领域的定义及术语，专业人员具体可参考Current Protocolsin Molecular Biology(Ausubel)。氨基酸残基的缩写是本领域中所用的指代20个常用L-氨基酸之一的标准3字母和/或1字母代码。本文(包括权利要求书)所用单数形式包括其相应的复数形式，除非文中另有明确规定。
在本发明中，术语“约”通常是指在指定数值以上或以下0.5％-10％的范围内变动，例如在指定数值以上或以下0.5％、1％、1.5％、2％、2.5％、3％、3.5％、4％、4.5％、5％、5.5％、6％、6.5％、7％、7.5％、8％、8.5％、9％、9.5％、或10％的范围内变动。
在本发明中，术语“抗体”是指具有所需生物活性的任何形式的抗体。因此，其以最广义使用，具体包括但不限于单克隆抗体(包括全长单克隆抗体)、多克隆抗体、多特异性抗体(例如双特异性抗体)、人源化抗体、全人抗体、嵌合抗体和骆驼源化单结构域抗体。
在本发明中，术语“单克隆抗体”是指获自基本均质抗体群的抗体，即组成该群的各个抗体除可少量存在的可能天然存在的突变之外是相同的。单克隆抗体是高度特异性的，针对单一抗原表位。相比之下，常规(多克隆)抗体制备物通常包括大量针对不同表位(或对不同表位有特异性)的抗体。修饰语“单克隆”表明获自基本均质抗体群的抗体的特征，且不得解释为需要通过任何特定方法产生抗体。
在本发明中，术语“全长抗体”，是指在天然存在时包含四条肽链的免疫球蛋白分子：两条重(H)链(全长时约50-70kDa)和两条轻(L)链(全长时约25kDa)通过二硫键互相连接。每一条重链由重链可变区(在本文中缩写为VH)和重链恒定区(在本文中缩写为CH)组成。重链恒定区由3个结构域CH1、CH2和CH3组成。每一条轻链由轻链可变区(在本文中缩写为VL)和轻链恒定区组成。轻链恒定区由一个结构域CL组成。VH和VL区可被进一步细分为具有高可变性的互补决定区(CDR)和其间隔以更保守的称为框架区(FR)的区域。每一个VH或VL区由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。重链和轻链的可变区含有与抗原相互作用的结合结构域。抗体的恒定区可介导免疫球蛋白对宿主组织或因子(包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(Clq))的结合。
在本发明中，术语“CDR”是指抗体可变序列内的互补决定区。在重链和轻链的各个可变区中存在3个CDR，其对于各个重链和轻链可变区被命名为HCDR1、HCDR2和HCDR3或LCDR1、LCDR2 和LCDR3。本发明的所述抗体的可变区CDR的精确氨基酸序列边界可使用许多公知的方案的任何方案来确定，包括基于抗体的三维结构和CDR环的拓扑学的Chothia(Chothia等人.(1989)Nature 342：877-883；Al-Lazikani等人，“Standard conformations for the canonical structures of immunoglobulins”，Journal of Molecular Biology，273，927-948(1997))基于抗体序列可变性的Kabat(Kabat等人，Sequences of Proteins of Immunological Interest，第4版，U.S.Department of Health and Human Services，National Institutes of Health(1987))，AbM(University of Bath)，Contact(University College London)，国际ImMunoGeneTics database(IMGT)(1999 Nucleic Acids Research，27，209-212)，以及基于利用大量晶体结构的近邻传播聚类(affinity propagation clustering)的North CDR定义。本发明抗体的CDR可以由本领域的技术人员根据本领域的任何方案(例如不同的指派系统或组合)确定边界。
应该注意，基于不同的指派系统获得的同一抗体的可变区的CDR的边界可能有所差异。即不同指派系统下定义的同一抗体可变区的CDR序列有所不同。因此，在涉及用本发明定义的具体CDR序列限定抗体时，所述抗体的范围还涵盖了这样的抗体，其可变区序列包含所述的具体CDR序列，但是由于应用了不同的方案(例如不同的指派系统或组合)而导致其所声称的CDR边界与本发明所定义的具体CDR边界不同。
在本发明中，术语抗体(“亲代抗体”)的“抗原结合片段”包括抗体的片段或衍生物，通常包括亲代抗体的抗原结合区或可变区(例如一个或多个CDR)的至少一个片段，其保持亲代抗体的至少一些结合特异性。抗体结合片段的实例包括但不限于Fab，Fab′，F(ab′)2和Fv片段；双抗体；线性抗体；单链抗体分子，例如scFv；由抗体片段形成的纳米抗体(nanobody)和多特异性抗体。当抗原的结合活性在摩尔浓度基础上表示时，结合片段或衍生物通常保持其抗原结合活性的至少10％。优选结合片段或衍生物保持亲代抗体的抗原结合亲和力的至少20％、50％、70％、80％、90％、95％或100％或更高。还预期抗体的抗原结合片段可包括不明显改变其生物活性的保守或非保守氨基酸取代(称为抗体的“保守变体”或“功能保守变体”)。
在本发明中，术语“嵌合抗体”是具有第一抗体的可变结构域和第二抗体的恒定结构域的抗体，其中第一抗体和第二抗体来自不同物种。通常，可变结构域获自啮齿动物等的抗体(“亲代抗体”)，而恒定结构域序列获自人抗体，使得与亲代啮齿动物抗体相比，所得嵌合抗体在人受试者中诱导不良免疫应答的可能性较低。
在本发明中，术语“人源化抗体”是指含有来自人和非人(例如小鼠、大鼠)抗体的序列的抗体形式。一般而言，人源化抗体包含基本所有的至少一个、通常两个可变结构域，其中所有或基本所有的超变环相当于非人免疫球蛋白的超变环，而所有或基本所有的构架(FR)区是人免疫球蛋白序列的构架区。人源化抗体任选可包含至少一部分的人免疫球蛋白恒定区(Fc)。
在本发明中，术语“全人抗体”是指只包含人免疫球蛋白蛋白质序列的抗体。如在小鼠中、在小鼠细胞中或在来源于小鼠细胞的杂交瘤中产生，则全人抗体可含有鼠糖链。同样，“小鼠抗体”是指仅包含小鼠免疫球蛋白序列的抗体。或者，如果在大鼠中、在大鼠细胞中或在来源于大鼠细胞的杂交瘤中产生，则全人抗体可含有大鼠糖链。同样，“大鼠抗体”是指仅包含大鼠免疫球蛋白序列的抗体。
在本发明中，术语“同种型”抗体是指由重链恒定区基因提供的抗体种类(例如，IgM、IgE、IgG诸如IgGl、IgG2或IgG4)。同种型还包括这些种类之一的修饰形式，其中修饰已被产生来改变Fc功能，例如以增强或减弱效应子功能或对Fc受体的结合。
在本发明中，术语“Fc区”用于定义包含至少一部分恒定区的免疫球蛋白重链的C端区域。该术语包括天然序列Fc区和变异Fc区。在一些实施方案中，人IgG重链Fc区从Cys226或Pro230延伸至重链的羧基末端。但是，Fc区的C端赖氨酸(Lys447)可能存在或不存在(此段中的编号是根据EU编号系统，也称为EU索引，如Rabat等人，Sequences of Proteins of Immunological Interest，5th Ed.Public Health Service，National Institutes of Health，Bethesda，MD，1991)。
在本发明中，术语“交叉反应”指的是对人类、猴、和/或鼠源(小鼠或大鼠)相同靶分子的抗原片段的结合。因此，“交叉反应”应被理解为与在不同物种中表达的相同分子X的种属间反应。识别人GPC3R、猴、和/或鼠GPC3R(小鼠或大鼠)的单克隆抗体的交叉反应特异性可通过FACS分析确定。
在本发明中，术语“亲和力”或“结合亲和力”指反映结合对子的成员之间相互作用的固有结合亲和力。分子X对其配偶物Y的亲和力可以通常由平衡解离常数(KD)代表，平衡解离常数是解离速率常数和结合速率常数(分别是kdis和kon)的比值。亲和力可以由本领域已知的常见方法测量。用于测量亲和力的一个具体方法是本文中的ForteBio动力学结合测定法。
在本发明中，术语“不结合”蛋白或细胞是指，不与蛋白或细胞结合，或者不以高亲和力与其结合，即结合蛋白或细胞的KD为1.0×10-6M或更高，更优选1.0×10-5M或更高，更优选1.0×10-4M或更高、1.0×10-3M或更高，更优选1.0×10-2M或更高。
在本发明中，术语“高亲和性”对于IgG抗体而言，是指对于抗原的KD为1.0×10-6M或更低，优选5.0×10-8M或更低，更优选1.0×10-8M或更低、5.0×10-9M或更低，更优选1.0×10-9M或更低。对于其他抗体亚型，“高亲和性”结合可能会变化。例如，IgM亚型的“高亲和性”结合是指KD为10-6M或更低，优选10-7M或更低，更优选10-8M或更低。
在本发明中，术语“细胞毒性药物”通常指毒性药物，所述细胞毒性药物可以在肿瘤细胞内具有较强破坏其正常生长的化学分子。细胞毒性药物可以在足够高的浓度下杀死肿瘤细胞。所述“细胞毒性药物”可以包括毒素，如细菌、真菌、植物或动物来源的小分子毒素或酶活性毒素，放射性同位素(例如At211、I131、I125、Y90、Re186、Re188、Sm153、Bi212、p32或Lu的放射性同位素)，毒性药物，化疗药物，抗生素和核溶酶，例如，可以是毒性药物，包括但不限于喜树碱衍生物，例如，可以是喜树碱衍生物依沙替康(化学名：(1S，9S)-1-氨基-9-乙基-5-氟-2，3-二氢-9-羟基-4-甲基-1H，12H-苯并[de]吡喃并[3’，4’：6，7]咪唑并[1，2-b]喹啉-10，13(9H，15H)-二酮)。
在本发明中，术语“接头单元”或“接头结构”通常指指一端与配体连接而另一端与细胞毒性药物相连的化学结构片段或键，也可以连接其他接头后再与细胞毒性药物相连。所述直接或间接连接配体可以是指所述基团通过共价键直接连接配体，也可以是通过接头结构连接配体。例如，接头结构可以是本发明所述的-Lax-Lb-Lc-和或-La-Lb-Lc-所示的结构。例如，可以使用包含酸不稳定接头结构(例如腙)、蛋白酶敏感(例如肽酶敏感)接头结构、光不稳定接头结构、二甲基接头结构、或含二硫化物 接头结构的化学结构片段或键作为接头结构。
在本发明中，术语某个结构“任选地与其它分子部分相连接”通常是指该结构不与任何其它化学结构相连接，或者该结构与一个或多个不同于该结构的其它化学结构(例如本发明所述的配体)相连接(例如，通过化学键连接、或通过接头结构连接)。
在本发明中，术语“配体-药物偶联物”通常是指配体通过稳定的连接单元与具有生物活性的细胞毒性药物相连。在本发明中“配体-药物偶联物”可以为抗体-药物偶联物(antibody drug conjugate，ADC)，所述ADC可以是指把单克隆抗体或者抗体片段通过稳定的连接单元与具有生物活性的细胞毒性药物相连。
在本发明中，术语“亚甲基”通常是指1个碳原子的基团除去两个氢原子所衍生的残基。亚甲基可以是取代的或非取代的，替代或者非替代的。术语“亚烷基”通常指饱和的直链或支链脂肪族烃基，其具有2个从母体烷的相同碳原子或两个不同的碳原子上除去两个氢原子所衍生的残基，其可以为包含1至20个碳原子的直链或支链基团，例如含有1至12个碳原子，例如含有1至6个碳原子的亚烷基。亚烷基的非限制性实例包括但不限于亚甲基(-CH2-)、1，1-亚乙基(-CH(CH3)-)、1，2-亚乙基(-CH2CH2)-、1，1-亚丙基(-CH(CH2CH3)-)、1，2-亚丙基(-CH2CH(CH3)-)、1，3-亚丙基(-CH2CH2CH2-)、1，4-亚丁基(-CH2CH2CH2CH2-)和1，5-亚丁基(-CH2CH2CH2CH2CH2-)等。亚烷基可以是取代的或非取代的，替代或者非替代的，例如当被取代时，取代基可以在任何可使用的连接点上被取代，所述取代基优选独立地任选选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基和氧代基中的一个或多个取代基所取代，例如可以是氢、氕、氘、氚、卤素、-NO2、-CN、-OH、-SH、-NH2、-C(O)H、-CO2H、-C(O)C(O)H、-C(O)CH2C(O)H、-S(O)H、-S(O)2H、-C(O)NH2、-SO2NH2、-OC(O)H、-N(H)SO2H、或C1-6脂肪族基团。
在本发明中，术语“亚杂环基”或“杂环基”通常是指稳定的不具有芳香性的3元-7元单环结构，融合的7元-10元双环杂环结构或桥联的6元-10元双环杂环结构，这些环状结构即可以是饱和的，也可以是部分饱和的，除碳原子外，这些环状结构中还含有一个或多个杂原子，其中杂原子可以选自以下组：氧、硫和氮。例如是含有1-4个上述定义的杂原子。当用来表示杂环环状结构上的原子时，术语“氮”可以包括发生过取代反应的氮。亚杂环基可以是取代的或非取代的。
在本发明中，术语“亚碳环基”通常是指具有两个从碳环的相同碳原子或两个不同的碳原子上除去两个氢原子所衍生的残基。术语“碳环”通常指饱和或部分不饱和单环或多环环状烃，碳环包含3至20个碳原子，可以包含3至12个碳原子，可以包含3至10个碳原子，可以包含3至8个碳原子，例如包含3至6个碳原子。单环碳环的非限制性实例包括环丙烷、环丁烷、环戊烷、环戊烯、环己烷、环己烯、环己二烯、环庚烷、环庚三烯、环辛烷等；多环碳环可以包括螺环、稠环和桥环的碳环。例如，“3到6元饱和的碳环基”包括环丙基、环丁基、环戊基、环己基、亚环丙基、亚环丁基、亚环戊基或亚环己基。碳环基和亚碳环基可以是取代的或非取代的，例如可以被一个或多个卤素、C1-6脂肪族基团或被卤素取代的C1-6脂肪族基团所取代。
在本发明中，术语“部分不饱和的”通常是指环状结构中环分子间至少含一个双键或三键。术语“部分不饱和”涵盖带有多处不饱和的环状结构，但并非意在包括本发明所定义的芳环或杂芳环。术语″不饱和的″表示部分具有一个或多个不饱和度。
在本发明中，术语“卤素”通常是指氟、氯、溴、碘，例如可以是氟、氯。
在本发明中，术语“脂肪族基团”通常是指具有1-12个碳原子的直链烃、支链烃或环状结构的烃，这些烃或者是完全饱和烃；或者带有一个或多个不饱和单元，但不饱和单元不是芳香类基团。例如，适用的脂肪族基团可以包括取代的或未取代的直链、支链或环状结构的烷基、烯基、炔基以及这些基团的混合物；比如是(环烷基)烷基、(环烯基)烷基或(环烷基)烯基。例如，脂肪族基团具有1-12、1-8、1-6、1-4或1-3个碳原子。例如，“C1-6脂肪族基团”指的是具有1-6个碳原子的如上所述的脂肪族基团，包括但不限于具有1-6个碳原子的直链、支链或环状结构的烷基、烯基或炔基，例如甲基、乙基、丙基、异丙基、正丁基、异丁基、叔丁基、戊基、己基等。
在本发明中，术语“任选”或“任选地”通常意味着随后所描述的事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生地场合。例如，“任选被烷基取代的杂环基团”意味着烷基可以但不必须存在，该说明可以包括杂环基团被烷基取代的情形和杂环基团不被烷基取代的情形。
在本发明中，术语“取代的”通常指基团中的一个或多个氢原子，例如为最多5个，例如为1～3个氢原子彼此独立地被相应数目的取代基取代。取代基仅处在它们的可能的化学位置，本领域技术人员能够在不付出过多努力的情况下确定(通过实验或理论)可能或不可能的取代。例如，具有游离氢的氨基或羟基与具有不饱和(如烯属)键的碳原子结合时可能是不稳定的。
在本发明中，术语0个或多个(例如，0个或至少1个、0个或1个、0个)亚甲基单元被“替代”通常指当所述结构包含1个或多个亚甲基单元时，所述一个或多个亚甲基单元可以不被替代，或被一个或多个不是亚甲基的基团(例如-NHC(O)-、-C(O)NH-、-C(O)-、-OC(O)-、-C(O)O-、-NH-、-O-、-S-、-SO-、-SO2-、-PH-、-P(＝O)H-、-NHSO2-、-SO2NH-、-C(＝S)-、-C(＝NH)-、-N＝N-、-C＝N-、-N＝C-或-C(＝N2)-)所替代。
在本发明中，基团中的一个或多个氢原子，例如为最多5个，例如为1～3个氢原子彼此独立地被相应数目的取代基取代。取代基仅处在它们的可能的化学位置，本领域技术人员能够在不付出过多努力的情况下确定(通过实验或理论)可能或不可能的取代。例如，具有游离氢的氨基或羟基与具有不饱和(如烯属)键的碳原子结合时可能是不稳定的。
在本发明中，术语“药学上可接受的盐”或“可药用盐”通常是指本发明药物偶联物的盐，这类盐用于哺乳动物体内时可以具有安全性和/或有效性，且可以具有应有的生物活性，本发明抗体药物偶联物可以与酸形成盐，药学上可接受的盐的非限制性实例包括：盐酸盐、氢溴酸盐、氢碘酸盐、硫酸盐、硫酸氢盐、柠檬酸盐、乙酸盐、琥珀酸盐、抗坏血酸盐、草酸盐、硝酸盐、梨酸盐、磷酸氢盐、磷酸二氢盐、水杨酸盐、柠檬酸氢盐、酒石酸盐、马来酸盐、富马酸盐、甲酸盐、苯甲酸盐、甲磺酸盐、乙磺酸盐、苯磺酸盐、对甲苯磺酸盐。
在本发明中，术语“溶剂化物”或“溶剂化合物”通常是指本发明的药物偶联物与一种或多种溶 剂分子形成可药用的溶剂化物，溶剂分子的非限制性实例包括水、乙醇、乙腈、异丙醇、DMSO、乙酸乙酯。
在本发明中，术语“载药量”通常是指每个配体上加载的细胞毒性药物平均数量，也可以表示为细胞毒性药物和抗体量的比值，细胞毒性药物载量的范围可以是每个配体(Ab)连接0-12个，例如1-10个细胞毒性药物。在本发明的实施方式中，载药量表示为Na，示例性的可以为1，2，3，4，5，6，7，8，9，10的均值。可用常规方法如UV/可见光光谱法，质谱，ELISA试验和HPLC特征鉴定偶联反应后每个ADC分子的载药量。
在本发明中，术语“药学上可接受的载体”是指药物制剂或组合物中除活性成分以外的对受试者无毒的成分。药学上可接受的载体包括但不限于缓冲剂，赋形剂，稳定剂或防腐剂。
在本发明中，术语“包含”通常是指包括明确指定的特征，但不排除其他要素。术语“以上”、“以下”通常是指包含本数的情况。
在本发明中，术语“受试者”包括任何人或非人动物。术语“非人动物”包括所有脊椎动物，例如哺乳动物和非哺乳动物，诸如非人灵长类动物、绵羊、狗、猫、马、牛、鸡、两栖动物、爬行动物等。如本文中所用，术语“cyno”或“食蟹猴”是指食蟹猴。
在本发明中，术语“联合”一种或多种其它治疗剂的施用包括同时(共同)施用和任意次序的连续施用。
在本发明中，术语“治疗有效量”、“治疗有效剂量”和“有效量”是指本发明的GPC3抗体或其抗原结合片段当单独或与其它治疗药物组合给予细胞、组织或受试者时，有效预防或改善一种或多种疾病或病况的症状或该疾病或病况的发展的量。治疗有效剂量还指足以导致症状改善的抗体或其抗原结合片段的量，例如治疗、治愈、预防或改善相关医学病况或者提高这类病况的治疗、治愈、预防或改善的速度的量。当对个体施用单独给予的活性成分时，治疗有效剂量仅是指该成分。当组合施用时，治疗有效剂量是指引起治疗效果的活性成分的综合量，不论是组合、依次给予还是同时给予。治疗剂的有效量将导致诊断标准或参数提高至少10％，通常至少20％，优选至少约30％，更优选至少40％，最优选至少50％。
在本发明中，术语“癌症”在本文中用于指表现出异常高水平的增殖和生长的一组细胞。癌症可能是良性的(也称为良性肿瘤)，恶性前或恶性。癌细胞可以是实体癌细胞或白血病癌细胞。本文使用的术语“肿瘤”是指包含癌症的一个或多个细胞。
在本发明中，术语“肿瘤生长”在本文中用于指代包含癌症的一种或多种细胞的增殖或生长，其导致癌症的大小或程度的相应增加。
抗GPC3抗体
在本发明中，术语“GPC3”包括由细胞天然表达的GPC3的任何变体或同种型。GPC3或其任何变体或同种型可从天然表达它们的细胞或组织中分离而得，或使用本领域通用以及本文所述的那些技术通过重组技术产生。优选地，抗GPC3抗体靶向具有正常糖基化模式的人源GPC3。
在本发明中，术语“抗GPC3抗体”、“抗GPC3”、“GPC3抗体”或“结合GPC3的抗体”是指能 够以足够的亲合力结合GPC3蛋白或其片段以致所述抗体可以用作靶向GPC3中的诊断剂和/或治疗剂。
本发明所述的抗GPC3抗体或其抗原结合片段参考WO2006006693中描述的抗GPC3抗体或其抗原结合片段。
在一些实施方案中，在本发明的药物偶联物、组合物、用途或方法中使用的抗体的CDR序列包含氨基酸序列分别如SEQ ID NO：1、SEQ ID NO：2和SEQ ID NO：3所示的HCDR1、HCDR2和HCDR3，和包含氨基酸序列分别如SEQ ID NO：4、SEQ ID NO：5和SEQ ID NO：6所示的LCDR1、LCDR2和LCDR3。
在一些实施方案中，在本发明的药物偶联物、组合物或用途中使用的抗体的可变区序列包括氨基酸序列如SEQ ID NO：7所示的重链可变区，和氨基酸序列如SEQ ID NO：8所示的轻链可变区。
在一些实施方案中，在本发明的药物偶联物、组合物、用途或方法中使用的抗体包括氨基酸序列如SEQ ID NO：9所示的重链，和氨基酸序列如SEQ ID NO：10所示的轻链。在一些实施方案中，在本发明的药物偶联物、组合物、用途或方法中使用的抗体为抗GPC3抗体DB1001。
抗GPC3抗体DB1001的可变区氨基酸序列如下，CDR区依照IMGT编号规则确定。
DB1001重链可变区：
DB1001轻链可变区：
DB1001重链(IgG1)氨基酸序列
DB1001轻链(Kappa)氨基酸序列
可采用用于产生抗体的任何合适方法来产生本发明的抗体。任何合适形式的GPC3都可用作产生抗体的免疫原(抗原)。通过举例而非限制，任何GPC3变体或其片段都可用作免疫原。在一些实施方式中，产生鼠源的单克隆抗人GPC3抗体的杂交瘤细胞可通过本领域公知的方法产生。来源于啮齿动物(如小鼠)的抗体在体内用作治疗药物时可能引起不需要的抗体免疫原性，重复使用导致人体产生针对治疗性抗体的免疫应答，这类免疫应答至少导致丧失治疗功效，而严重的则导致潜在致死过敏反应。降低啮齿动物抗体的免疫原性的一种方法包括嵌合抗体的产生，其中将小鼠可变区与人恒定区融合(Liu等(1987)Proc.Natl.Acad.Sci.USA 84：3439-43)。然而，嵌合抗体中的完整啮齿动物可变区的保留仍可能在患者中引起有害的免疫原性。将啮齿动物可变结构域的互补决定区(CDR)环移植到人构架上(即人源化)已被用于进一步将啮齿动物序列减至最低(Jones等(1986)Nature 321：522；Verhoeyen等(1988)Science 239：1534)。
在一些实施方式中，本发明所述的嵌合或人源化抗体可基于所制备的鼠单克隆杂交瘤抗体的序列来制备。编码重链和轻链免疫球蛋白的DNA可以从目标鼠杂交瘤中获得，并且使用标准分子生物学技术进行工程改造以包含非鼠(例如人)免疫球蛋白序列。
在一些实施方式中，本发明所述的嵌合GPC3抗体，可使用本领域已知的方法将杂交瘤来源的免疫球蛋白重链和轻链可变区与人IgG恒定区有效连接(参见例如属于Cabilly等人的美国专利No.4,816,567)，获得嵌合型重链和嵌合型轻链来制备。在一些实施方式中，本发明的嵌合抗体包含的恒定区可选自任何人IgG亚型，如IgG1、IgG2、IgG3、IgG4，优选IgG4。
在一些实施方式中，本发明的嵌合GPC3抗体可由嵌合型轻链与嵌合型重链表达质粒“混合和匹配”转染表达细胞获得，此类“混合和匹配”的抗体的GPC3结合可使用上述结合测定和其它常规结合测定(例如，ELISA)来进行测试。
本发明所述的人源化抗体，可以使用本领域已知的方法将鼠源CDR区插入人种系框架区。参见Winter等人的美国专利No.5,225,539及Queen等人的美国专利No.5,530,101；5,585,089；5,693,762和6,180,370。
在一些实施方式中，氨基酸变化包括氨基酸缺失、插入或置换。在一些实施方式中，本发明的抗GPC3抗体或其抗原结合片段包括具有已通过氨基酸缺失、插入或置换突变的，但仍与上述抗体(特别地在上述序列中描绘的CDR区中)有至少约90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列的那些抗体。在一些实施方式中，本发明的抗体与具体序列中描绘的CDR区相比较时，在CDR区中已通过氨基酸缺失、插入或置换的氨基酸突变不超过1、2、3、4或5个。
在一些实施方式中，可在本文中所提供抗体的Fc区中引入一个或多个氨基酸修饰，以此产生Fc区变体。Fc区变体可包含在一或多个氨基酸位置处包含氨基酸修饰(例如置换)的人Fc区序列(例如人IgG1、IgG2、IgG3或IgG4 Fc区)。
在一些实施方式中，可能需要产生经半胱氨酸工程改造的抗体，例如“硫代MAb”，其中抗体的一或多个残基经半胱氨酸残基置换。
在一些实施方式中，本文中所提供的抗体可进一步经修饰为含有本领域中已知且轻易获得的其他非蛋白质部分。适合抗体衍生作用的部分包括，但不限于，水溶性聚合物。水溶性聚合物的非限制性实例包括，但不限于，聚乙二醇(PEG)、乙二醇/丙二醇共聚物、羧甲基纤维素、葡聚糖、聚乙烯醇、聚乙烯吡咯烷酮、聚-1，3-二烷、聚-1，3，6-三烷、乙烯/马来酸酐共聚物、聚氨基酸(均聚物或无规共聚物)、及葡聚糖或聚(n-乙烯基吡咯烷酮)聚乙二醇、丙二醇均聚物、聚环氧丙烷/氧化乙烯共聚物、聚氧乙基化多元醇(例如甘油)、聚乙烯醇、及其混合物。
药物偶联物
本发明提供一种抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述抗GPC3抗体药物偶联物结构如式(I-1)所示：
M为-L2-L3-X-L1-；
L2为-O-或-S-；
L3为-(C(R1a)(R1b))m-，m为0、1、2或3，其中当L3包含亚甲基单元时，所述L3的0个或1个亚甲基单元可以被-C(O)-、或-C(S)-替代；
L1为-(C(R2a)(R2b))n-，n为1、2或3，其中当L1可以包含亚甲基单元时，所述L1的0个或1个亚甲基单元可以被-C(O)-、或-C(S)-替代；
X为3到6元饱和的碳环基、3到6元饱和的杂环基或单键，所述3到6元饱和的碳环基和3到6元饱和的杂环基任选被1个或多个R3a取代；
其中每个R1a，R1b，R2a，R2b，R3a各自独立地可以为氢、卤素或被1个或多个R任选取代的C1- 6脂肪族基团；
其中每个R各自独立地可以为氢或卤素；
L是接头单元；
p表示平均连接数，且p为1到10的整数或小数，优选为3-8的整数或小数；
Ab为抗GPC3抗体或其抗原结合片段。
在一些实施方式中，本发明所述抗GPC3抗体或其抗原结合片段包含重链可变区和轻链可变区，所述重链可变区包含氨基酸序列分别如SEQ ID NO：1、SEQ ID NO：2和SEQ ID NO：3所示的HCDR1、HCDR2和HCDR3，和所述轻链可变区包含氨基酸序列分别如SEQ ID NO：4、SEQ ID NO：5和SEQ ID NO：6所示的LCDR1、LCDR2和LCDR3；优选地，本发明所述抗GPC3抗体或其抗原结合片段包含：氨基酸序列如SEQ ID NO：7所示的重链可变区，和氨基酸序列如SEQ ID NO：8所示的轻链可变区；更优选地，本发明所述抗GPC3抗体或其抗原结合片段包含：氨基酸序列如SEQ ID NO：9所示的重链，和氨基酸序列如SEQ ID NO：10所示的轻链。
在一些实施方式中，本发明所述抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中，L3为-(C(R1a)(R1b))m-，其中，m为0、1、2或3；其中每个R1a，R1b各自独立地可以为氢、卤素或被R任选取代的C1-6脂肪族基团，其中每个R各自独立地可以为氢或卤素；优选地，L3为-(C(R1a)(R1b))m-，其中，m为0、1、2或3；其中每个R1a和R1b各自独立地可以为氢、卤素、CH3或CH2CH3；优选地，L3为单键、-CH2-、-CH(CH3)、-C(CH3)2、-CH2CH2-、-CH(CH3)CH2-或-C(CH3)2CH2-。
在一些实施方式中，本发明所述抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中，L1为-(C(R2a)(R2b))n-，n为1、2或3，其中当L1可以包含亚甲基单元时，所述L1的0个或1个亚甲基单元可以被-C(O)-、或-C(＝S)-替代；其中每个R2a，R2b各自独立可以为氢、卤素或被R任选取代的C1-6脂肪族基团；其中每个R各自独立地可以为氢或卤素；优选地，L1为-C(R2a)(R2b)-、-C(R2a)(R2b)C(O)-或-C(O)-；其中每个R2a和R2b各自独立可以为氢、卤素、CH3或CH2CH3；更优选地，L1为-CH2-、-CH2C(O)-、-CH(CH3)C(O)-或-C(O)-；更优选地，L1为-CH2C(O)-；更优选地，L1为-C(O)-。
在一些实施方式中，本发明所述抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中，X为3到6元饱和的碳环基、3到6元饱和的杂环基或单键，所述3到6元饱和的碳环基和3到6元饱和的杂环基任选被1、2或3个R3a取代；其中每个R3a各自独立地可以为氢、卤素或被1、2或3个R任选取代的C1-6脂肪族基团，其中每个R各自独立地可以为氢或卤素；优选地，X为任选被1、2或3个R3a取代的3到6元饱和的碳环基或单键；其中每个R3a各自独立地可以为氢、卤素、CH3或CH2CH3；更优选地，X为或单键。
在一些实施方式中，本发明所述的抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述-L3-X-为：-(C(R1a)(R1b))m-、3到6元饱和的碳环基或3到6元饱和的杂环基；其中，m为1、2或3，所述3到6元饱和的碳环基和3到6元饱和的杂环基任选被1、2或3个R3a取代；其中，每个R1a，R1b，R3a各自独立地可以为氢、卤素或可以被R任选取代的C1-6脂肪族基团，其中每个R各自独立地可以为氢或卤素；
优选地，-L3-X-为：-(C(R1a)(R1b))m-和任选被1、2或3个R3a取代的3到6元饱和的碳环基；其中，m为1、2或3；其中，R1a各自独立地为卤素或被1、2或3个R任选取代的C1-6脂肪族基团；R1b，R3a各自独立地为氢、卤素或被1、2或3个R任选取代的C1-6脂肪族基团；每个R各自独立地 可以为氢或卤素；
更优选地，-L3-X-为：
在一些实施方式中，本发明所述抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中
所述M为-L2-L3-X-L1-；
L2为-O-；
-L3-X-为任选被1、2或3个R3a取代的3到6元饱和的碳环基；优选地，-L3-X-为任选被1、2或3个R3a取代的：优选地，-L3-X-为
L1为-C(R2a)(R2b)-、-C(R2a)(R2b)C(O)-或-C(O)-；优选地，L1为-C(O)-；
其中每个R2a，R2b，R3a各自独立地为氢、卤素或可以被R任选取代的C1-6脂肪族基团；
其中每个R各自独立地可以为氢或卤素。
在一些实施方式中，本发明所述抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中
所述M为-L2-L3-X-L1-；
L2为-O-或-S-；优选地，L2为-O-；优选地，L2为-S-；
-L3-X-为-(C(R1a)(R1b))m-，其中，m为1、2或3；优选地，-L3-X-为 优选地，-L3-X-为
L1为-C(R2a)(R2b)-、-C(R2a)(R2b)C(O)-或-C(O)-；优选地，L1为-CH2C(O)-；
其中R1a各自独立地为卤素或被1、2或3个R任选取代的C1-6脂肪族基团；R1b、R3a各自独立为氢、卤素或被1、2或3个R任选取代的C1-6脂肪族基团；
其中每个R各自独立地可以为氢或卤素。
在一些优选地实施例中，本发明所述抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述-M-为：

在一些实施方式中，本发明所述抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述L为-La-Lb-Lc-，
所述-La-为
其中，W为-(C(Rwa)(Rwb))wn-，其中wn为1、2、3或6，W的0个或1个亚甲基单元各自独立地被-Cyr-、-N(Rwx)C(O)-、-C(O)N(Rwx)-、或-C(O)-替代；优选地，W为-(C(Rwa)(Rwb))2-或-(C(Rwa)(Rwb))3-；优选地，W为-CH2CH2CH2-或-CH2CH2-；
Y为-(OCH2CH2)yn-Oyp-，其中yn为0、4或8，yp为0或1；优选地，Y为单键；
Z为-(C(Rza)(Rzb))zn，其中zn为1、2或3，Z的1个亚甲基单元各自独立地被-Cyr-、-N(Rzx)C(O)-、-C(O)N(Rzx)-、或-C(O)-替代，-Cyr-为3到10元饱和的亚碳环基，其中所述-Cyr-是未取代的或独立地被1到3个取代基Rcx取代；优选地，Z为-(C(Rwa)(Rwb))2C(O)-或-(C(Rwa)(Rwb))3C(O)-；优选地，Z为-CH2CH2CH2C(O)-或CH2CH2C(O)-；
其中每个Rwa、Rwb、Rza、Rzb、Rwx、Rzx和Rcx各自独立地为氢、卤素、-ORr或被Rr任选取代的C1-6脂肪族基团；
其中每个Rr各自独立地为氢、卤素或C1-6脂肪族基团；
优选地，所述-La-为优选为
所述-Lb-表示由2到4个氨基酸构成的肽残基，所述-Lb-的肽残基为由选自以下组中的氨基酸形成的肽残基：苯丙氨酸、甘氨酸、丙氨酸、缬氨酸、瓜氨酸和赖氨酸；
优选地，-Lb-选自以下组：
优选地；所述-Lb-为优选-Lb-为
所述-Lc-为其中RL1、RL2各自独立地选自以下组：氢、卤素、-OH和C1-6脂肪族基团；优选地，所述-Lc-为
在一些特别优选地实施例中，本发明所述抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述L为
在一些实施方式中，本发明所述抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述抗GPC3抗体药物偶联物结构如式(II-1)或(II-2)所示：
其中，
L2为-O-或-S-；优选地，L2为-O-，优选地，L2为-S-，
X为任选被1、2或3个R3a取代的3到6元饱和的碳环基；优选地，X为任选被1、2或3个R3a 取代的优选地，X为
L3为-C(R1a)(R1b)-或-C(R1a)(R1b)C(R1a)(R1b)-，
其中R1a各自独立为卤素或被1、2或3个R任选取代的C1-6脂肪族基团；R1b、R3a各自独立地为氢、卤素或被1、2或3个R任选取代的C1-6脂肪族基团；R各自独立地可以为氢或卤素；优选地，L3为-CH(CH3)-、-C(CH3)2-、-CH(CH3)CH2-或-C(CH3)2CH2-；
p表示平均连接数，且p为1到10的整数或小数，优选为3-8的整数或小数；
Ab为抗GPC3抗体或其抗原结合片段，其包含重链可变区和轻链可变区，所述重链可变区包含氨基酸序列分别如SEQ ID NO：1、SEQ ID NO：2和SEQ ID NO：3所示的HCDR1、HCDR2和HCDR3，和所述轻链可变区包含氨基酸序列分别如SEQ ID NO：4、SEQ ID NO：5和SEQ ID NO：6所示的LCDR1、LCDR2和LCDR3；优选地，本发明所述抗GPC3抗体或其抗原结合片段包含：氨基酸序列如SEQ ID NO：7所示的重链可变区，和氨基酸序列如SEQ ID NO：8所示的轻链可变区；更优选地，本发明所述抗GPC3抗体或其抗原结合片段包含：氨基酸序列如SEQ ID NO：9所示的重链，和氨基酸序列如SEQ ID NO：10所示的轻链。
在一些实施方式中，本发明所述平均连接数p可以为2到8的整数或小数。例如，所述平均连接数n可以为3到8的整数或小数。例如，所述平均连接数n可以为1到2、2到3、3到4、4到5、5到6、6到7、7到8、8到9、9到10的整数或小数。
药物组合物和药物制剂
在又一个方面，本发明提供了一种药物组合物，其包含如本发明任一项所述的抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，和药学上可接受的载体或赋形剂。
应理解，本发明提供的抗GPC3抗体药物偶联物或、其异构体、其药学上可接受的盐或其混合物，或其药物组合物可以整合制剂中合适的运载体、赋形剂和其他试剂以联合给药，从而提供改善的转移、递送、耐受等。
本发明中，术语“药物组合物”指这样的制剂，其允许包含在其中的活性成分的生物学活性有效的形式存在，并且不包含对施用所述制剂的受试者具有不可接受的毒性的另外的成分。
可以通过将具有所需纯度的本发明的抗GPC3抗体药物偶联物或其药学上可接受的盐与一种或多种任选的药用辅料(Remington′s Pharmaceutical Sciences，第16版，Osol，A.编辑(1980))混合来制备包含本文所述的抗GPC3抗体的药物制剂，优选地以水溶液或冻干制剂的形式。
本发明的药物组合物或制剂还可以包含一种或多种其它活性成分，所述活性成分是被治疗的特定适应证所需的，优选具有不会不利地影响彼此的互补活性的那些活性成分。在一些实施方式中，其它的活性成分为化疗剂、免疫检查点抑制剂、生长抑制剂、抗生素或已知的各种抗肿瘤或抗癌剂，所述活性成分以对于目的用途有效的量合适地组合存在。在一些实施方式中，本发明的药物组合物还包含编码抗GPC3抗体的多核苷酸的组合物。
在又一个方面，本发明提供了一种药物组合，其包含如本发明任一项所述的抗体药物偶联物或其 药学上可接受的盐或本发明任一项所述的药物组合物，以及一种或多种另外的治疗剂。
在又一个方面，本发明提供了一种试剂盒，其包括如本发明任一项所述的抗体药物偶联物或、其异构体、其药学上可接受的盐或其混合物或本文所述的药物组合物，优选其进一步包括给药装置。
医药用途
在又一个方面，本发明提供了如本发明任一项所述的抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，或本发明任一项所述的药物组合物在制备用于治疗和/或预防GPC3介导的疾病或病症的药物中的用途，优选地，所述疾病或病症为癌症。
在又一个方面，本发明提供了如本发明任一项所述的抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，或本发明任一项所述的药物组合物，其用于治疗和/或预防GPC3介导的疾病或病症，优选地，所述疾病或病症为癌症。
在又一个方面，本发明提供了一种治疗和/或预防GPC3介导的疾病或病症的方法，其包括向有需要的受试者施用如本发明任一项所述的抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，或本发明任一项所述的药物组合物，优选地，所述疾病或病症为癌症。
在一些实施方式中，所述癌症选自乳腺癌、卵巢癌、前列腺癌、胰腺癌、肾癌、肺癌、肝癌、胃癌、结肠癌、膀胱癌、食管癌、宫颈癌、胆囊癌、胶质母细胞瘤和黑色素瘤。
在一些实施方式中，本发明给药方式包括但不限于口服、静脉内、皮下、肌内、动脉内、关节内(例如在关节炎关节中)、通过吸入、气雾剂递送或肿瘤内给予等。
在一些实施方式中，本发明提供了向受试者联合施用治疗有效量的一种或多种疗法(例如治疗方式和/或其它治疗剂)。在一些实施方式中，所述疗法包括手术治疗和/或放射疗法。
在一些实施方式中，本发明提供的方法或用途还包括向个体施用一种或多种疗法(例如治疗方式和/或其它治疗剂)。可以单独或与疗法中的其它治疗剂组合使用本发明的抗体药物偶联物或其药学上可接受的盐。例如，可以与至少一种另外的治疗剂共施用。
本发明所述的药物偶联物可以具有对肿瘤细胞的体外增殖的抑制活性。所述抑制活性可以是本发明的药物偶联物加入肿瘤细胞的培养基中，相比于加入阴性对照或者对照药物，所述肿瘤细胞细胞增殖能力下降1％以上、2％以上、4％以上、5％以上、8％以上、10％以上、15％以上、18％以上、20％以上、25％以上、40％以上、50％以上、60％以上、70％以上、80％以上、90％以上、或95％以上。例如，所述抑制活性可以是对于肿瘤细胞的IC50值(nM)可以为10000以下、5000以下、4000以下、3000以下、2000以下、1000以下、500以下、400以下、300以下、200以下、150以下、120以下、110以下、100以下、99以下、98以下、97以下、95以下、90以下、80以下、75以下、70以下、65以下、62以下、60以下、50以下、40以下、30以下、25以下、23以下、22以下、20以下、19以下、18以下、18.5以下、17以下、15以下、12以下、10以下、9以下、8.5以下、7以下、6.7以下、6以下、5.9以下、5.5以下、5.0以下、4.8以下、4.5以下、4.4以下、4以下、3.5以下、3以下、2.5以下、2以下、1.5以下、1.0以下、0.5以下、0.3以下、0.29以下、0.25以下、0.21以下、0.20以下、0.18以下、0.17以下、0.15以下、0.12以下、0.10以下、0.09以下、0.08以下、0.07以下、0.06以下、 0.05以下、0.04以下、0.03以下、0.02以下或0.01以下。例如，所述肿瘤细胞可以包括但不限于实体瘤细胞，例如所述肿瘤细胞包括但不限于胃癌细胞、或乳腺癌细胞、肝癌细胞。
本发明所述的药物偶联物可以具有靶向抑制性。所述靶向抑制性可以是本发明的药物偶联物加入特定靶标高表达的肿瘤细胞的培养基中，相比于加入阴性对照或者对照药物，所述特定靶标高表达的肿瘤细胞细胞增殖能力下降1％以上、2％以上、4％以上、5％以上、8％以上、10％以上、15％以上、18％以上、20％以上、25％以上、40％以上、50％以上、60％以上、70％以上、80％以上、90％以上、或95％以上。例如，所述靶向抑制性可以是对于特定靶标高表达的肿瘤细胞的IC50值(nM)可以为10000以下、5000以下、4000以下、3000以下、2000以下、1000以下、500以下、400以下、300以下、200以下、185以下、150以下、120以下、110以下、100以下、99以下、98以下、97以下、95以下、91以下、80以下、74以下、70以下、65以下、62以下、60以下、50以下、40以下、30以下、25以下、23以下、22以下、20以下、19以下、18以下、18.5以下、17以下、15以下、12以下、10以下、9以下、8.5以下、7以下、6.7以下、6以下、5.9以下、5.5以下、5.0以下、4.8以下、4.5以下、4.4以下、4以下、3.5以下、3以下、2.5以下、2以下、1.5以下、1.0以下、0.5以下、0.3以下、0.29以下、0.25以下、0.21以下、0.20以下、0.18以下、0.17以下、0.15以下、0.12以下、0.10以下、0.09以下、0.08以下、0.07以下、0.06以下、0.05以下、0.04以下、0.03以下、0.02以下或0.01以下。例如，所述特定靶标高表达的肿瘤细胞可以包括但不限于实体瘤细胞，例如所述特定靶标高表达的肿瘤细胞包括但不限于胃癌细胞、或乳腺癌细胞、肝癌细胞。
本发明所述的药物偶联物可以具有血浆稳定性。所述血浆稳定性可以是本发明的药物偶联物加入血浆中，所述药物偶联物释放的细胞毒性药物在1天、3天、5天、7天、14天、20天或30天的释放率不超过50％、不超过40％、不超过30％、不超过20％、不超过10％、不超过7％、不超过5％、不超过4％、不超过3％、不超过2％、不超过1.9％、不超过1.8％、不超过1.7％、不超过1.6％、不超过1.5％、不超过1.4％、不超过1.3％、不超过1.2％、不超过1.1％、不超过1.0％、不超过0.9％、不超过0.8％、不超过0.7％、不超过0.6％、不超过0.5％、不超过0.4％、不超过0.3％、不超过0.2％或不超过0.1％。
本发明所述的药物偶联物可以具有体内抑瘤效果。所述抑瘤效果可以是本发明的药物偶联物施用于动物，相比于加入阴性对照或者对照药物，所述动物的肿瘤在1天、3天、5天、7天、14天、20天、21天或30天的体积减少1％以上、2％以上、4％以上、5％以上、8％以上、10％以上、15％以上、18％以上、20％以上、25％以上、40％以上、50％以上、55％以上、60％以上、70％以上、73％以上、75％以上、80％以上、90％以上、或95％以上，或者相比于施用阴性对照或者对照药物，所述动物的肿瘤在1天、3天、5天、7天、14天、20天、21天或30天的体积减少1.1倍以上，1.3倍以上，1.5倍以上，两倍以上，三倍以上，五倍以上，十倍以上，二十倍以上，二十二倍以上，三十倍以上，五十倍以上，一百倍以上，五百倍以上，一千倍以上，或一千五百倍以上。所述动物可以包括但不限于哺乳动物，例如所述动物可以包括但不限于猫、狗、马、猪、奶牛、羊、兔、小鼠、大鼠、猴或人。所述施用可以包括但不限于口服、静脉注射、静脉滴注、腹腔注射、或局部给药。
本发明所述的药物偶联物可以具有旁观杀伤效应(BystanderEffect)。所述旁观杀伤效应可以是本发明的药物偶联物对特定靶标低表达的肿瘤细胞的细胞增殖无明显抑制作用，而在特定靶标低表达的肿瘤细胞与特定靶标高表达的肿瘤细胞共培养中，本发明的药物偶联物可以同时抑制特定靶标低表达的肿瘤细胞与特定靶标高表达的肿瘤细胞的细胞增殖。例如，在特定靶标低表达的肿瘤细胞与特定靶标高表达的肿瘤细胞共培养中，所述抑制活性可以是对于特定靶标低表达的肿瘤细胞的IC50值(nM)可以为10000以下、5000以下、4000以下、3000以下、2000以下、1000以下、500以下、400以下、300以下、200以下、185以下、150以下、120以下、110以下、100以下、99以下、98以下、97以下、95以下、91以下、80以下、74以下、70以下、65以下、62以下、60以下、50以下、40以下、30以下、25以下、23以下、22以下、20以下、19以下、18以下、18.5以下、17以下、15以下、12以下、10以下、9以下、8.5以下、7以下、6.7以下、6以下、5.9以下、5.5以下、5.0以下、4.8以下、4.5以下、4.4以下、4以下、3.5以下、3以下、2.5以下、2以下、1.5以下、1.0以下、0.5以下、0.3以下、0.29以下、0.25以下、0.21以下、0.20以下、0.18以下、0.17以下、0.15以下、0.12以下、0.10以下、0.09以下、0.08以下、0.07以下、0.06以下、0.05以下、0.04以下、0.03以下、0.02以下或0.01以下。所述特定靶标低表达的肿瘤细胞与特定靶标高表达的肿瘤细胞相比，所述特定靶标的表达可以是降低1％以上、2％以上、4％以上、5％以上、8％以上、10％以上、15％以上、18％以上、20％以上、25％以上、40％以上、50％以上、60％以上、70％以上、80％以上、90％以上、或95％以上。例如，所述特定靶标高表达的肿瘤细胞可以包括但不限于实体瘤细胞，例如所述特定靶标高表达的肿瘤细胞包括但不限于胃癌细胞、或乳腺癌细胞，例如所述特定靶标高表达的肿瘤细胞可以包括但不限于HCC1569细胞或MDA-MB-453细胞。例如，所述特定靶标低表达的肿瘤细胞可以包括但不限于实体瘤细胞。
本发明所述的药物偶联物可以具有抗转运体转运能力。所述抗转运能力可以是相比于转运底物的标准品，本发明所述药物偶联物的外排比降低1％以上、2％以上、4％以上、5％以上、8％以上、10％以上、15％以上、18％以上、20％以上、25％以上、40％以上、50％以上、60％以上、70％以上、80％以上、90％以上、或95％以上。例如，所述外排比的测试可以为本领域人员常用方法，或记载在本发明的实施例之中。
本发明所述的药物偶联物可以具有体内肿瘤靶向能力。所述体内靶向能力可以是指将用信号物质标记的所述药物偶联物施用于动物，所述标记的药物偶联物在动物的肿瘤组织的分布，相较于其他组织和器官，可以是分布增加1％以上、2％以上、4％以上、5％以上、8％以上、10％以上、15％以上、18％以上、20％以上、25％以上、40％以上、50％以上、60％以上、70％以上、80％以上、90％以上、或95％以上，或者可以是分布增加1.1倍以上，1.3倍以上，1.5倍以上，两倍以上，三倍以上，五倍以上，十倍以上，二十倍以上，二十二倍以上，三十倍以上，五十倍以上，一百倍以上，五百倍以上，一千倍以上，或一千五百倍以上。所述信号物质可以是放射性物质，例如所述信号物质包括但不限于125I。所述动物可以包括但不限于哺乳动物，例如所述动物可以包括但不限于猫、狗、马、猪、奶牛、羊、兔、小鼠、大鼠、猴或人。所述施用可以包括但不限于口服、静脉注射、静脉滴注、腹腔注射、 或局部给药。所述组织或器官可以包含但不限于心、肝、脾、肺、肾、脑、或骨髓。
本发明所述的药物偶联物可以具有良好的体内安全性。所述体内安全性可以是本发明的药物偶联物在施用于动物后，所述动物的体内游离毒素释放率不超过50％、不超过40％、不超过30％、不超过20％、不超过10％、不超过7％、不超过5％、不超过4％、不超过3％、不超过2％、不超过1.9％、不超过1.8％、不超过1.7％、不超过1.6％、不超过1.5％、不超过1.4％、不超过1.3％、不超过1.2％、不超过1.1％、不超过1.0％、不超过0.9％、不超过0.8％、不超过0.7％、不超过0.6％、不超过0.5％、不超过0.4％、不超过0.3％、不超过0.2％或不超过0.1％。例如，所述体内安全性可以是在动物不产生毒性表现的情况下本发明所述药物偶联物的施用浓度可以是0.5mg/kg以上、1mg/kg以上、2mg/kg以上、3mg/kg以上、4mg/kg以上、5mg/kg以上、10mg/kg以上、20mg/kg以上、30mg/kg以上、50mg/kg以上、70mg/kg以上、100mg/kg以上、200mg/kg以上、500mg/kg以上、或1000mg/kg以上。例如所述动物可以包括但不限于猫、狗、马、猪、奶牛、羊、兔、小鼠、大鼠、猴或人。所述施用可以包括但不限于口服、静脉注射、静脉滴注、腹腔注射、或局部给药。
本发明的积极进步效果在于：
本发明抗GPC3抗体药物偶联物中，抗GPC3抗体或其抗原结合片段通过接头单元与具有生物活性的细胞毒性药物相连，抗体药物偶联物转移至肿瘤细胞后，接头被切断，游离出细胞毒性药物H-M-D(例如小分子化合物制备例1-制备例9)。本发明细胞毒性药物对NCI-N87细胞、JIMT-1细胞、Colo205细胞和MDA-MB-231细胞具有明显增强的增殖抑制活性，能发挥优异的抗肿瘤作用。
本发明抗GPC3抗体药物偶联物对GPC3阳性表达细胞Huh7和HepG2具有明显增强的增殖抑制活性；本发明抗GPC3抗体药物偶联物对HepG2荷瘤小鼠和Huh7荷瘤小鼠具有显著增强的抗肿瘤活性。
因此，本发明在GPC3的阳性表达疾病(例如癌症)方面有着良好的应用前景。
附图说明
图1：抗体药物偶联物对人肝癌细胞HepG2荷瘤小鼠的抗肿瘤体内药效。
图2：抗体药物偶联物对人肝癌细胞Huh7荷瘤小鼠的抗肿瘤体内药效。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
样品检测
1.ADC DAR值分析方法-HIC-HPLC(疏水色谱法)
高效液相色谱仪：沃特世e2965高效液相色谱系统。
色谱柱：Thermo MAbPacTM HIC-Butyl(4.6×100mm，5μm)；
流动相A：1.5M(NH4)2SO4+50mM K2HPO4(pH 7.0)；
流动相B：50mM K2HPO4(pH 7.0)/异丙醇(75∶25V/V)；
按照以下洗脱程序进行洗脱，
检测条件：设置流动相流速为1ml/min，检测波长为280nm，柱温30℃。
2.SEC纯度分析-SEC-HPLC(分子排阻色谱法)
高效液相色谱仪：1260安捷伦液相色谱仪。
色谱柱：Waters Xbridge BEH200 SEC(7.8×300mm，3.5μm)
流动相：50mM NaH2PO4+200mM精氨酸(pH 6.80)+10％异丙醇
检测条件：设置流动相流速为0.5ml/min，检测波长为280nm，柱温30℃。
本发明包括所叙述特定实施方式的所有组合。本发明的进一步实施方式及可应用性的完整范畴将自下文所提供的详细描述变得显而易见。然而，应理解，尽管详细描述及特定实施例指示本发明的优选实施方式，但仅以说明的方式提供这些描述及实施例，因为本发明的精神及范畴内的各种改变及修改将自此详细描述对熟悉此项技术者变得显而易见。出于所有目的，包括引文在内的本文所引用的所有公开物、专利及专利申请将以引用的方式全部并入本文。
实施例
提供以下实施例以证明并进一步解释本发明的一些优选的实施方式和方面，不应被解释为限制其范围。
实施例1：抗GPC3抗体
本发明的抗体参照WO2006006693制备，其中，抗GPC3抗体DB1001的可变区氨基酸序列如下，CDR区依照IMGT编号规则确定。
DB1001重链可变区：
DB1001轻链可变区：
根据以上序列，设计引物PCR搭建得到VH/VK基因片段，获得可变区。抗体可变区再与恒定区基因片段进行同源重组，构建DB1001完整抗体序列。经过转染CHO细胞后，按照常规表达纯化方法得到抗体DB1001。
DB1001重链(IgG1)氨基酸序列
DB1001轻链(Kappa)氨基酸序列
实施例2：抗GPC3抗体药物偶联物(ADC)的制备
2.1、毒素(payload)的制备
制备例1
第一步：
在氮气保护下，在0℃下向KI4(900mg，1.69mmol)，HATU(691mg，1.88mmol)，3a(320mg，2.00mmol)的DMF(18mL)溶液中加入DIEA(500mg，3.87mmol)。并在25℃搅拌3小时。TLC(EA)显示原料反应完全。将反应液滴加到320mL去离子水中，过滤，得灰色固体850mg，收率：87％。
第二步：
向3b(100mg，0.174mmol)的MeOH/DCM(1/1，3mL)中加入NaHCO3(42mg，0.50mmol)固体并在25℃搅拌3小时，TLC(EA)显示反应完全，将反应液过滤，低温旋干后，用aq.HCl(0.5M，10mL)打浆后过滤，经prep-HPLC(0.1％TFA)制备后冻干得到15毫克灰色固体。收率：16％。
MS m/z(ESI)：534[M+1]。
H-NMR(400MHz，DMSO-D)：8.45(d，1H)，7.81(d，1H)，7.32(s，1H)，6.52(m，1H)，5.58-5.56(m，1H)，5.44(s，2H)，5.14(dd，2H)，3.96(m，1H)，3.48(m，1H)，3.19(m，2H)，2.53-2.28(m，3H)，2.48(s，3H)，2.20-2.00(m，4H)，1.95-1.80(m，2H)，0.89(t，3H)。
制备例2
第一步：
在氮气保护下，在0℃下向KI 4(1.00g，1.88mmol)，HATU(691mg，1.88mmol)，4a(310mg，1.88mmol)的DMF(18mL)溶液中加入DIEA(486mg，3.76mmol)。并在25℃搅拌3小时。TLC(EA)显示原料反应完全。将反应液滴加到320mL去离子水中，过滤，得灰色固体910mg，收率：84％。
第二步：
向4b(100mg，1.58mmol)的MeOH/DCM(1/1，3mL)中加入NaHCO3(42mg，0.50mmol)固体并在25℃搅拌3小时，TLC(EA)显示反应完全，将反应液过滤，低温旋干后，用aq.HCl(0.5M，10mL)打浆后过滤，经prep-HPLC(0.1％TFA)制备冻干得13毫克黄色固体，收率：14％。
MS m/z(ESI)：534[M+1]。
H-NMR(400MHz，DMSO-D)：8.39(d，1H)，7.77(d，1H)，7.29(s，1H)，6.52(m，1H)，5.58-5.54(m，1H)，5.41(s，2H)，5.18-5.06(m，3H)，4.39-4.33(m，1H)，3.19-3.07(m，2H)，2.97-2.82(m，1H)，2.49-2.36(m， 2H)，2.38(s，3H)，2.20-1.96(m，4H)，1.93-1.79(m，2H)，0.87(t，3H)。
制备例3
在氮气保护下，向KI4(100mg，0.188mmol)，HATU(86mg，0.23mmol)，7a(22mg，0.21mmol)的DMF(2mL)溶液中滴加DIEA(61mg，0.47mmol)，加完后在25℃反应2.5小时。LCMS显示原料反应完全。将反应液加入到20mL水中，有固体析出，过滤得到13mg，收率：11％。
MS m/z(ESI)：522[M+1]。
H-NMR(400MHz，DMSO-D)：8.44(d，1H)，7.80(d，1H)，7.32(s，1H)，6.54(m，1H)，5.60-5.50(m，1H)，5.44(s，2H)，5.22(dd，2H)，4.10-4.00(m，1H)，3.30-3.17(m，2H)，2.41(s，3H)，2.38-2.10(m，4H)，1.96-1.80(m，2H)，1.11(d，3H)，0.89(t，3H)。
制备例4
在氮气保护下，向KI4(100mg，0.188mmol)，HATU(86mg，0.23mmol)，8a(24mg，0.21mmol) 的DMF(1mL)溶液中滴加DIEA(61mg，0.47mmol)的DMF(1mL)溶液，加完后在25℃反应3小时。TLC(EA)显示原料反应完全。将反应液制备得黄色固体17mg，收率：17％。
MS m/z(ESI)：536[M+1]。
H-NMR(400MHz，DMSO-D)：8.42(d，1H)，7.79(d，1H)，7.30(s，1H)，6.53(m，1H)，5.59-5.55(m，1H)，5.42(s，2H)，5.22(dd，2H)，4.69(s，1H)，3.20-3.11(m，2H)，2.39(sc，3H)，2.29(s，2H)，2.20-2.08(m，2H)，1.19(d，6H)，0.87(t，3H)。
制备例5
第一步
氮气保护下，向KI4(100mg，0.19mmol)，HATU(85.7mg，0.23mmol)，23a(21.5mg，0.21mmol)的DMF(2mL)溶液中滴加DIEA(60.6mg，0.47mmol)，加完后在0℃反应2小时。LCMS显示原料反应完全。将反应液滴加到20mL水中搅拌，析出固体后过滤，得60.2mg灰色固体P-III-30，收率：61％。MS-ESI：m/z 522.2[M+H]+。
1H NMR(400MHz，DMSO-d6)δ8.42(d，J＝8.7Hz，1H)，7.79(d，J＝11.0Hz，1H)，7.30(s，1H)，6.53(s，1H)，5.62-5.53(m，1H)，5.42(s，2H)，5.30-5.16(m，2H)，4.63(d，J＝4.6Hz，1H)，4.09-3.99(m，1H)，3.22-3.11(m，2H)，2.40(s，3H)，2.28(dd，J＝13.7，7.2Hz，1H)，2.22-2.08(m，3H)，1.94-1.78(m，2H)，1.08(d，J＝6.1Hz，3H)，0.87(t，J＝7.3Hz，3H)。
制备例6
第一步
将24a(19.6mg，0.188mmol)，KI4(100mg，0.188mmol)，DIEA(60.7mg，0.47mmol)加入三口瓶(100mL)中，加入DMF(2mL)溶解，N2保护置换三次并在0℃搅拌加入HATU(85.9mg，0.226mmol)，0℃反应2小时。TLC(DCM∶MeOH＝10∶1)显示反应结束。将反应液加入水(20mL)析出灰色固体，冻干再进行制备板纯化(EA∶MeOH＝10∶1)得54.5mg淡黄色色固体P-III-31，收率：56％。MS-ESI：m/z 522.5[M+H]+。
1H NMR(400MHz，DMSO-d6)δ8.44(dd，J＝8.7，2.9Hz，1H)，7.78(dd，J＝10.9，2.6Hz，1H)，7.30(s，1H)，6.53(s，1H)，5.60-5.51(m，1H)，5.42(s，2H)，5.21(dd，J＝10.9，3.4Hz，2H)，4.66(dd，J＝13.1，4.7Hz，1H)，4.10-3.98(m，1H)，3.21-3.10(m，2H)，2.39(s，3H)，2.34-2.05(m，4H)，1.93-1.77(m，2H)，1.08(dd，J＝6.2，1.6Hz，3H)，0.87(t，J＝7.3Hz，3H)。
制备例7
第一步
取KI4(50mg，0.094mmol)，25a(11mg，0.094mmol)，HATU(39mg，0.103mmol)加入DMF(3mL)中，氮气置换后加入DIEA(30mg，0.235mmol)，25℃下反应1.5小时后LCMS显示反应结束。将反应液滴入处于搅拌状态下的水(50mL)中，加完后静置5分钟，过滤，滤饼冻干后得20mg灰色固体25，收率：40％。
MS-ESI：m/z 534.3[M+H]+。
1H NMR(400MHz，DMSO-d6)δ8.26(d，J＝9.1Hz，1H)，7.72(d，J＝10.9Hz，1H)，7.28(s，1H)，6.51(s，1H)，6.12(s，1H)，5.59-5.50(m，1H)，5.40(s，2H)，5.15(d，J＝18.8Hz，1H)，4.98(d，J＝19.0Hz，1H)，3.28-3.16(m，1H)，3.15-3.02(m，1H)，2.74-2.52(m，2H)，2.36(s，3H)，2.24-2.04(m，4H)，1.92-1.79(m，4H)，0.86(t，J＝7.3Hz，3H)。
制备例8
第一步
取KI4(100mg，0.188mmol)，11a(33mg，0.188mmol)，HATU(77mg，0.203mmol)加入DMF(3mL)中，氮气置换后加入DIEA(73mg，0.564mmol)，25℃下反应1.5小时后LCMS显示反应结束。将反应液滴入处于搅拌状态下的水(50mL)中，加完后静置5分钟，过滤，滤饼冻干后得80mg灰色固体11b，收率：72％。MS-ESI：m/z 592.4[M+H]+。
第二步
取27b(80mg，0.135mmol)溶于DCM(2mL)中，N2置换后降温至0℃，加入TFA(0.5mL)，0℃下反应1.5小时后LCMS显示反应结束。将反应液低温旋干，经PTLC纯化后加乙腈和水冻干得25mg白色固体，收率：36％。
MS-ESI：m/z 508.3[M+H]+。
1H NMR(400MHz，DMSO-d6)δ8.44(d，J＝8.6Hz，1H)，7.79(d，J＝11.0Hz，1H)，7.30(s，1H)，6.53(s，1H)，5.60-5.53(m，1H)，5.42(s，2H)，5.28-5.15(m，2H)，4.59(t，J＝5.1Hz，1H)，3.72-3.63(m，2H)，3.21-3.12(m，2H)，2.40(s，3H)，2.32(t，J＝6.4Hz，2H)，2.21-2.06(m，2H)，1.92-1.79(m，2H)，0.87(t，J ＝7.3Hz，3H)。
制备例9：DXd
参照专利“CN104755494A”中说明书第183页的实施例75提供的方法合成DXd。
参照例1
第一步
取KI4(50mg，0.094mmol)，26a(18mg，0.094mmol)，HATU(39mg，0.103mmol)加入DMF(3mL)中，氮气置换后加入DIEA(48mg，0.376mmol)，25℃下反应1.5小时后LCMS显示反应结束。将反应液滴入处于搅拌状态下的水(50mL)中，加完后静置5分钟，过滤，滤饼冻干后得30mg灰色固体26b，收率：52％。MS-ESI：m/z 607.4[M+H]+。
第二步
取26b(30mg，0.049mmol)溶于DCM(2mL)中，N2置换后降温至0℃，加入TFA(0.5mL)，0℃下反应1.5小时后LCMS显示反应结束。将反应液低温旋干，用DCM带一次后加乙腈和水冻干得20mg黄色固体，收率：80％。
MS-ESI：m/z 507.1[M+H]+。
1H NMR(400MHz，DMSO-d6)δ8.67(d，J＝8.6Hz，1H)，7.86-7.66(m，4H)，7.32(s，1H)，6.56(brs，1H)，5.63-5.54(m，1H)，5.43(s，2H)，5.29(d，J＝18.9Hz，1H)，5.22(d，J＝18.9Hz，1H)，3.23-3.15(m， 2H)，3.13-3.03(m，2H)，2.57-2.51(m，2H)，2.43-2.38(m，3H)，2.27-2.08(m，2H)，1.94-1.78(m，2H)，0.87(t，J＝7.3Hz，3H)。
2.2、接头-毒素(linker-payload)的制备
接头-毒素X1
第一步
氮气保护下，向27a(5.00g，43.0mmol)，NaHCO3(10.9g，129mmol)的DMF(50mL)溶液中滴加苄溴(11.0g，64.6mmol)，并在25℃反应17小时。TLC(PE/EA＝2/1)显示反应完全，将反应液加入到500mL水中，用EA(250mL)萃取两遍，分液后经饱和氯化钠水溶液(500mL)洗涤，无水Na2SO4干燥，浓缩过柱(PE∶EA＝3∶2)得5.1g无色液体，收率：57.1％。
第二步
氮气保护下，向KI2(4.00g，10.9mmol)，TsOH(800mg，4.65mmol)的THF(30mL)溶液中，在0℃下，滴加27b(4.50g，21.8mmol)的THF(10mL)溶液，并在25℃反应2小时。TLC(PE/EA＝1/2)显示反应完全，将反应液加入到200mL水中，用EA(200mL)萃取两次分液，无水Na2SO4干燥，浓缩过柱(PE/EA＝3/2)得白色固体1.56g，收率：26％。
第三步
氢气环境下，在0℃下，向27c(800mg，1.55mmol)的EtOH(8mL)和EA(8mL)混合溶液中加入Pd/C(80mg)，在0℃搅拌2.5小时。LCMS显示反应完全。反应液经硅藻土过滤，用EA(200mL)洗涤滤饼浓缩后用THF(20mL)溶解旋干得白色固体600mg，收率：91％。
第四步
氮气保护下，在0℃下，向27d(220mg，0.515mmol)，HY-13631A(250mg，0.47mmol)和HATU(214mg，0.56mmol)的DMF(6mL)溶液中加入DIEA(152mg，1.18mmol)，并在0℃反应2小时。LCMS显示反应完全。将反应液加入柠檬酸水溶液(pH＝4)(150mL)中，过滤，并用175mL水洗涤滤饼，滤干，用油泵拉干得棕色固体260mg，收率：66％。
第五步
氮气保护下，在0℃下，向27e(260mg，0.309mmol)的DCM(30mL)溶液中滴加二乙胺(8mL)，并在0℃反应3小时。LCMS显示反应完全。将反应液加入0℃的石油醚溶液(600mL)中，有固体析出，静置待固体吸附于瓶底后，倒出溶液，用油泵拉干，得棕色固体90mg，收率：47.1％。
第六步
氮气保护下，在0℃下，向27f(90mg，0.13mmol)，KI-1(92mg，0.19mmol)和DIEA(50mg，0.39mmol)的DMF(2.5mL)溶液中加入HATU(74mg，0.19mmol)并在0℃反应2小时。LCMS显示基本反应结束。在0℃下，将反应液加入PH＝4的柠檬酸水溶液(30mL)中，有絮状固体析出，过滤，经制备板(DCM/MecOH＝10/1)得9.2mg淡黄色固体X1，收率：6％。
MS m/z(ESI)：1074[M+1]。
H-NMR(400MHz，MeOD)：7.65(d，1H)，7.62(s，1H)，7.30-7.21(m，5H)，6.79(s，2H)，5.69-5.65(m，1H)，5.57(d，1H)，5.43-5.10(m，3H)，4.70(d，2H)，4.48-4.39(m，2H)，4.10-4.05(m，1H)，4.01-3.75(m，5H)，3.46(t，2H)，3.22-3.15(m，2H)，3.07-3.00(m，1H)，2.75(m，1H)，2.62(m，1H)，2.45(s，3H)，2.37-2.20(m，6H)，2.10-2.02(m，2H)，2.00-1.92(m，2H)1.68-1.57(m，6H)，1.01(t，3H)。
接头-毒素X2
第一步
将34a(5g，48.0mmol)、K2CO3(19.9g，144.0mmol)溶于DMF(20mL)中，滴加苄溴(12.3g，72.0mmol)，25℃下反应17小时。TLC(PE/EA＝3/1)检测原料反应完全。将反应液加入水(200mL)中、用EA(250mL)萃取分液，用饱和NaCl洗涤，无水Na2SO4干燥后浓缩过柱(PE∶EA＝2∶1)得8.7g无色液体34b，收率93％。MS-ESI：m/z 195.1[M+H]+。
第二步
取34c(7.3g，19.8mmol)、TsOH(1.46g，8.5mmol)溶于THF(20mL)中，氮气保护并降温至0℃，滴加43b(7.7g，39.6mmol)的THF(10mL)溶液，加完后0℃反应2小时。TLC(PE/EA＝2/1)显示原料大部分反应。将反应液倒入100mL水中，DCM(100mL)萃取，分液并用饱和NaCl洗涤，无水Na2SO4干燥后过柱(PE/EA＝1/1)，得3.9g无色稠状物34d，收率：39％。MS-ESI：m/z 503.3[M+H]+。
第三步
氢气环境下，在0℃下，向34d(1.9g，3.78mmol)的EtOH(100mL)和EA(100mL)混合溶液中加入Pd/C(1g，10wt.％)，在0℃反应3小时。TLC(PE/EA＝2/1)显示反应完全。反应液经 硅藻土过滤，用EA/EtOH(1∶1，100mL×3)洗涤滤饼，滤液浓缩，并用THF(50mL×3)溶解旋干并重复三次得1g灰色固体34e，收率：64％。MS-ESI：m/z 435.2[M+Na]+。
第四步
在氮气保护下，0℃下向34e(426mg，1.03mmol)，KI4(500mg，0.94mmol)和HATU(429mg，1.13mmol)的DMF(20mL)溶液中滴加DIEA(303mg，2.35mmol)，加完后在0℃反应2小时。LCMS显示反应结束。将反应液滴到300mL水中，搅拌后静置5分钟，过滤，滤饼用DCM/MeOH(10∶1，100mL)的溶液溶解后，干燥旋干拌样，柱层析(EA∶MeOH＝30∶1)得600mg黄色固体34f，收率：77％。MS-ESI：m/z 830.3[M+H]+。
第五步
氮气保护下，在0℃下，向34f(150mg，0.18mmol)的DCM(5mL)溶液中滴加二乙胺(5mL)，并在0℃反应2小时。LCMS显示反应完全。将石油醚溶液(100mL×6)加入反应液中，有固体析出，静置待固体沉淀后，倒出溶液，再用油泵拉干，得120mg白色粉末34g，LCMS显示产物含量为70％，收率：76％。MS-ESI：m/z 608.3[M+H]+。
第六步
在氮气保护下，向34g(60mg，0.099mmol)、43h(51mg，0.108mmol)、和DIEA(32mg，0.25mmol)的DMF(1mL)溶液中在0℃下加入HATU(45mg，0.118mmol)的DMF(1mL)溶液，并在0℃下反应2小时。LCMS显示原料反应完全。将反应液直接过反相柱，洗脱剂((MeCN/MeOH＝1/1)∶H2O＝60％∶40％)，纯化得14.8mg黄色固体X2，收率14％。
MS-ESI：m/z 1062.4[M+H]+。
1H NMR(400MHz，Methanol-d4)δ7.69-7.61(m，2H)，7.22-7.16(m，2H)，7.16-7.09(m，3H)，6.76(s，2H)，5.70-5.64(m，1H)，5.60(d，J＝16.4Hz，1H)，5.40-5.31(m，2H)，5.26(d，J＝19.0Hz，1H)，4.65-4.50(m，7H)，4.25-4.16(m，1H)，3.87(d，J＝16.7Hz，1H)，3.83-3.76(m，3H)，3.72(d，J＝17.0Hz，2H)，3.44(t，J＝7.1Hz，2H)，3.25-3.17(m，2H)，3.10-3.02(m，1H)，2.92-2.83(m，1H)，2.45-2.39(m，5H)，2.32-2.20(m，5H)，1.97-1.89(m，2H)，1.63-1.50(m，4H)，1.34-1.20(m，6H)，0.99(t，J＝7.3Hz，3H)。
接头-毒素X3

第一步
向32a(2.00g，6.6mmol)，K2CO3(1.82g，13.2mmol)的MeCN(20mL)中加入溴丙烯(960mg，7.92mmol)，在20℃下搅拌5小时。TLC(PE/EA＝1/2)显示反应结束。将反应液倒入水100mL中，将pH值调至5，用EA(100mL)萃取三次，无水硫酸钠干燥，旋干过柱纯化(PE/EA＝2/1)得1.83g白色固体32b，收率：81％。
第二步
向32b(1.38g，4.02mmol)的DCM(10mL)中加入TFA(10mL)，在25℃下搅拌17小时。TLC(PE/EA＝1/3)显示反应结束。将反应液旋干得0.91g黄色粘状物32c，收率不计。
第三步
向32c(910mg，4.87mmol)，NaHCO3(613mg，7.3mmol)的DME/H2O(20mL/10mL)中加入41d(1.92g，4.87mmol)，在25℃下搅拌3小时。TLC(DCM/MeOH＝1/1)显示反应结束。将反应液倒入水100mL中，用aq.HCl(1N)将pH值调至5，用EA(150mL)萃取两次，无水硫酸钠干燥，旋干过柱纯化(DCM/MeOH＝20/1)得1.53g白色固体32e，收率：67％。MS-ESI：m/z 467.4[M+H]+。
第四步
向32f(3g，5.83mmol)的MeOH(50mL)中加入Pd/C(600mg)，在25℃在氢气球下搅拌5小时。TLC(EA)显示反应结束。将反应液过滤旋干得1.9g白色固体32g，收率：77％。
第五步
向32g(789mg，1.86mmol)，KI4(900mg，1.69mmol)，三乙胺(342mg，3.38mmol)的DMF(10mL)中加入HATU(707mg，1.86mmol)，在0℃下搅拌3.5小时。TLC(EA)显示反应结束。将反应液倒入H2O(80mL)，用EA(100mL)萃取两次，无水硫酸钠干燥，旋干过柱纯化(EA)得1.186g白色固体32h，收率：83％。MS-ESI：m/z 842.3[M+H]+。
第六步
将32h(1.186g，1.41mmol)的DCM/二乙胺(20mL，20/1)在25℃下搅拌17小时。TLC(DCM/MeOH＝10/1)显示反应结束。将反应液倒入石油醚(200mL)中过滤得768mg白色固体32i，收率：88％。MS-ESI：m/z 620.3[M+H]+。
第七步
向32i(676mg，1.09mmol)，32e(508mg，1.09mmol)，DIEA(423mg，3.27mmol)的DMF(10mL)中加入HATU(414mg，1.09mmol)，在20℃下搅拌17小时。TLC(PE/EA＝1/5)显示反应结束。将反应液倒入水中(30mL)过滤，滤饼过柱纯化(DCM/MeOH＝50/1)511mg白色固体32j，收率：44％。MS-ESI：m/z 1068.3[M+H]+。
第八步
将32j(482mg，0.451mmol)的二乙胺/DCM(10mL，1/5)的溶液在10℃下搅拌17小时。TLC(EA)显示反应结束。将反应液倒入PE(300mL)中过滤得301mg白色固体32k，收率不计。
第九步
向32k(301mg，0.356mmol)，Pd(PPh3)4(82mg，0.071mmol)的THF(5mL)中加入吗啡啉(93mg，1.07mmol)，在25℃下搅拌5小时。LCMS显示反应结束。将反应液制备得108mg白色固体32l，收率：38％。MS-ESI：m/z 806.3[M+H]+。
第十步
向32l(108mg，0.134mmol)，三乙胺(41mg，0.402mmol)的THF(2mL)和DMF(2mL)中加入溴乙酰溴(27mg，0.134mmol)，在0℃下搅拌1小时。TLC(DCM/MeOH＝10/1)显示反应结束。将反应液直接制备得15mg白色固体X3，收率：12％。
MS-ESI：m/z 926.3[M+H]+。
1H NMR(400MHz，DMSO-d6)δ12.11(s，1H)，8.54-8.42(m，3H)，8.27-8.16(m，2H)，7.78(d，J＝11.0Hz，1H)，7.30(s，1H)，6.53(s，1H)，5.61-5.51(m，1H)，5.42(s，2H)，5.20-5.05(m，2H)，4.56-4.42(m，2H)，4.32-4.22(m，1H)，3.96-3.87(m，3H)，3.79(d，J＝5.6Hz，2H)，3.70(d，J＝5.9Hz，2H)，3.25-3.08(m，2H)，2.61-2.53(m，2H)，2.45-2.36(m，4H)，2.36-2.22(m，3H)，2.20-2.03(m，4H)，1.99-1.68(m，4H)，0.87(t，J＝7.3Hz，3H)。
接头-毒素X4
第一步
向33a(2.00g，2.58mmol)的MeOH(20mL)中加入Pd/C(400mg，10wt.％)，在20℃下搅拌5小时。TLC(EA)显示反应结束。将反应液过滤旋干得1.3g白色固体33b，收率：74％。
第二步
向33b(0.55g，0.802mmol)，KI4(427mg，0.802mmol)和DIPEA(310mg，2.40mmol)的DMF(5mL)中加入HATU(305mg，0.802mmol)，在0℃下搅拌2小时。TLC(DCM/MeOH＝1/10)显示反应结束。将反应液倒入水(40mL)中，过滤得粗品，经柱纯化(DCM/MeOH＝20/1)得360mg黄色固体33c，收率41％。
第三步
向33c(360mg，0.326mmol)的DCM(10mL)中加入二乙胺(2mL)。在25℃下搅拌17小时。TLC(DCM/MeOH＝5/1)显示反应结束。将反应液倒入PE(100Ml)中，过滤得205mg白色固体33d，收率：71％。MS-ESI：m/z 881.3[M+H]+。
第四步
向33d(205mg，0.233mmol)和三乙胺(118mg，1.17mmol)的DMF(1mL)和水(1mL)中加入溴乙酰溴(94mg，0.446mmol)的THF(2mL)溶液，并在0度搅拌1小时，反应液直接制备得15mg白色固体X4，收率：6％。
MS-ESI：m/z 1001.2[M+H]+。
1H NMR(400MHz，DMSO-d6)δ8.57-8.50(m，1H)，8.50-8.43(m，2H)，8.35-8.29(m，1H)，8.19-8.12(m，2H)，7.80(d，J＝10.8Hz，1H)，7.27-7.14(m，7H)，6.53(s，1H)，5.59-5.51(m，1H)，5.44-5.39(m，2H)，5.20-5.07(m，2H)，4.56-4.44(m，3H)，3.92(s，3H)，3.80-3.68(m，5H)，3.41(s，1H)，3.21-3.12(m，2H)，2.83-2.74(m，1H)，2.58-2.55(m，3H)，2.39(s，4H)，2.18-2.03(m，4H)，1.93-1.78(m，2H)，0.87(t，J＝7.3Hz，3H)。
2.3、抗GPC3抗体药物偶联物的制备
抗体药物偶联物ADC DB1001-X1的制备
用超纯水分别配制还原剂和保护剂如下：2mg/ml TCEP(Tris-2-carboxyethyl-phosphine，厂家：Thermo)水溶液和100mmol/L EDTA(乙二胺四乙酸二钠厂家：Sigma)水溶液。
将Linker-payload X1溶于干燥的DMA(N，N-Dimethylacetamide，二甲基乙酰胺，厂家：国药集团)，配制成10mg/mL的linker-payload DMA溶液。
取20mg 6.28mg/ml的DB1001单抗置于50ml离心管，加入30mM His-HAc，pH5.5缓冲液稀释抗体浓度至3mg/ml，按照反应液总体积5％加入100mM EDTA水溶液，震荡混匀后加入2mg/ml TCEP水溶液进行抗体还原，TCEP与抗体的摩尔比18∶1，震荡混匀后置于制冷型恒温混匀仪上反应，37℃，2h。按照药物与抗体终浓度摩尔比12∶1加入前述Linker-payload的DMA溶液，按照反应液总体积10％补充DMA，震荡混匀后置于制冷型恒温混匀仪上反应，4℃，1h。用超滤管(MWCO30KD，厂家：密理博)置换样品保存buffer，先用含有10％DMSO的30mM His-HAc，pH5.5缓冲液超滤3次，再用无DMSO的30mM His-HAc，pH5.5超滤6次，最终用0.2μm PES膜过滤除菌，得到抗体药物偶联物DB1001-X1 12.7mg，浓度5.59mg/mL，收率63.5％。
经HIC检测，抗体药物偶联物DB1001-X1的载药量(DAR)为7.51，SEC纯度为100％。
抗体药物偶联物ADC DB1001-X2的制备
用超纯水分别配制还原剂和保护剂如下：2mg/ml TCEP(Tris-2-carboxyethyl-phosphine，厂家：Thermo)水溶液和100mmol/L EDTA(乙二胺四乙酸二钠厂家：Sigma)水溶液。
将Linker-payload X2溶于干燥的DMA(N，N-Dimethylacetamide，二甲基乙酰胺，厂家：国药集团)，配制成10mg/mL的linker-payload DMA溶液。
取20mg 6.28mg/ml的DB1001单抗置于50ml离心管，加入30mM His-HAc，pH5.5缓冲液稀释抗体浓度至3mg/ml，按照反应液总体积5％加入100mM EDTA水溶液，震荡混匀后加入2mg/ml TCEP水溶液进行抗体还原，TCEP与抗体的摩尔比18∶1，震荡混匀后置于制冷型恒温混匀仪上反应，37℃，2h。按照药物与抗体终浓度摩尔比12∶1加入前述Linker-payload的DMA溶液，按照反应液总体积10％补充DMA，震荡混匀后置于制冷型恒温混匀仪上反应，4℃，1h。用超滤管(MWCO30KD，厂家：密理博)置换样品保存buffer，先用含有10％DMSO的30mM His-HAc，pH5.5缓冲液超滤3次，再用无DMSO的30mM His-HAc，pH5.5超滤6次，最终用0.2μm PES膜过滤除菌，得到抗体药物偶联物DB1001-X2 14.3mg，浓度6.71mg/mL，收率71.5％。
经HIC检测，抗体药物偶联物DB1001-X1的载药量(DAR)为7.72，SEC纯度为99.3％。
抗体药物偶联物ADC DB1001-Deruxtecan的制备
接头-细胞毒素为Deruxtecan(CAS 1599440-13-7，购自上海皓元化学科技有限公司)，抗体为本发明抗体DB1001。参照本发明实施例2.3的方法得到DB1001-Deruxtecan。
实施例3：生物活性测试
3.1、小分子化合物(毒素)对肿瘤细胞体外增殖抑制测试
测试目的
为了检测药物化合物，对NCI-N87，JIMT-1和MBA-MB-231肿瘤细胞体外增殖的抑制活性。以不同浓度的化合物体外处理细胞，经6天培养后，采用CTG(Luminescent Cell Viability Assay，Promega，货号：G7558)试剂对细胞的增值进行检测，根据IC50值评价该化合物的体外活性。
1、细胞培养：NCI-N87/JIMT-1/MBA-MB-231用10％FBS RPMI-1640培养基培养。
2、细胞准备：取对数生长期的NCI-N87/JIMT-1/MBA-MB-231细胞，用PBS洗涤1次之后，加入2-3mL胰蛋白酶消化2-3min，待细胞消化完全后，加入10-15mL细胞培养液，将经过消化的 细胞洗脱下来，1000rpm离心5min，弃上清，接着加入10-20mL细胞培养液将细胞重悬，制成单细胞悬液。
3、细胞铺板：将NCI-N87/JIMT-1/MBA-MB-231单细胞悬液混匀，用细胞培养液分别调整活细胞密度至6x104cells/ml，将密度调整过后的细胞悬液混匀，以50ul/孔加入96孔细胞培养板。将培养板在培养箱培养18小时(37℃，5％CO2)。
4、化合物准备：用DMSO溶解化合物，配制成初始浓度为10mM的存储液。
小分子化合物共8个浓度，分别为：300，100，30，10，3，1，0.3，0.1nM。
5、加样操作：向培养板中加入配置的不同浓度的待测样品，每个样品两复孔。将培养板在培养箱孵育6天(37℃，5％CO2)。
6、显色操作：取出96孔细胞培养板，向每孔加入50ul CTG试剂，室温孵育10分钟。
7、读板操作：取出96孔细胞培养板，置于酶标仪中，用酶标仪测定化学发光。
数据分析：用Microsoft Excel，Graphpad Prism 5对数据进行处理分析。
表1本申请中的小分子化合物对肿瘤细胞体外增殖抑制的IC50值。
“-”：未检测。
结论：根据表1的结果，本申请药物偶联物的毒素对NCI-N87细胞、JIMT-1和MDA-MB-231细胞具有明显增强的增殖抑制活性。
3.2、小分子化合物(毒素)对肿瘤细胞体外增殖抑制测试
测试目的
为了检测药物化合物，对NCI-N87细胞，JIMT-1和MBA-MB-231肿瘤细胞体外增殖的抑制活性。以不同浓度的化合物体外处理细胞，经6天培养后，采用CTG( Promega，货号：G7558)试剂对细胞的增值进行检测，根据IC50值评价该化合物的体外活性。
1、细胞培养：NCI-N87/JIMT-1/MBA-MB-231用10％FBSRPMI-1640培养基培养。
2、细胞准备：取对数生长期的NCI-N87/JIMT-1/MBA-MB-231细胞，用PBS洗涤1次之后，加入2-3mL胰蛋白酶消化2-3min，待细胞消化完全后，加入10-15mL细胞培养液，将经过消化的细胞洗脱下来，1000rpm离心5min，弃上清，接着加入10-20mL细胞培养液将细胞重悬，制成单细胞悬液。
3、细胞铺板：将NCI-N87/JIMT-1/MBA-MB-231单细胞悬液混匀，用细胞培养液分别调整活细胞 密度至6x104cells/mL，将密度调整过后的细胞悬液混匀，以50uL/孔加入96孔细胞培养板。将培养板在培养箱培养18小时(37℃，5％CO2)。
4、化合物准备：用DMSO溶解化合物，配制成初始浓度为10mM的存储液。
小分子化合物共8个浓度，分别为：300，100，30，10，3，1，0.3，0.1nM。
5、加样操作：向培养板中加入配置的不同浓度的待测样品，每个样品两复孔。将培养板在培养箱孵育6天(37℃，5％CO2)。
6、显色操作：取出96孔细胞培养板，向每孔加入50uL CTG试剂，室温孵育10分钟。
7、读板操作：取出96孔细胞培养板，置于酶标仪中，用酶标仪测定化学发光。
数据分析：用MicrosoftExcel，GraphpadPrism5对数据进行处理分析。
8、数据分析：细胞存活率用公式：Vsample/Vvehiclecontrolx 100％计算。其中Vsample为药物处理组的读数，Vvehiclecontrol为溶剂对照组的平均值。应用GraphPad Prism软件，使用非线性回归模型绘制S型剂量-存活率曲线并计算IC50值。
表2本申请中的小分子化合物对NCI-N87、JIMT-1和MDA-MB-231细胞体外增殖抑制的IC50值
“-”：未检测
结论：根据表2的结果，本申请中药物偶联物的毒素对NCI-N87、JIMT-1和MDA-MB-231细胞具有明显增强的增殖抑制活性。
3.3、小分子化合物(毒素)对肿瘤细胞体外增殖抑制测试
测试目的
为了检测药物化合物，对NCI-N87细胞和Colo205肿瘤细胞体外增殖的抑制活性。以不同浓度的化合物体外处理细胞，经6天培养后，采用CTG(Promega，货号：G7558)试剂对细胞的增值进行检测，根据IC50值评价该化合物的体外活性。
1、细胞培养：NCI-N87/Colo205用10％FBSRPMI-1640培养基培养。
2、细胞准备：取对数生长期的NCI-N87/Colo205细胞，用PBS洗涤1次之后，加入2-3mL胰蛋白酶消化2-3min，待细胞消化完全后，加入10-15mL细胞培养液，将经过消化的细胞洗脱下来，1000rpm离心5min，弃上清，接着加入10-20mL细胞培养液将细胞重悬，制成单细胞悬液。
3、细胞铺板：将NCI-N87/Colo205单细胞悬液混匀，用细胞培养液分别调整活细胞密度至6x104cells/mL，将密度调整过后的细胞悬液混匀，以50ul/孔加入96孔细胞培养板。将培养板在培养箱培养18小时(37℃，5％CO2)。
4、化合物准备：用DMSO溶解化合物，配制成初始浓度为10mM的存储液。
小分子化合物共8个浓度，分别为：300，100，30，10，3，1，0.3，0.1nM。
5、加样操作：向培养板中加入配置的不同浓度的待测样品，每个样品两复孔。将培养板在培养箱孵育6天(37℃，5％CO2)。
6、显色操作：取出96孔细胞培养板，向每孔加入50Ul CTG试剂，室温孵育10分钟。
7、读板操作：取出96孔细胞培养板，置于酶标仪中，用酶标仪测定化学发光。
数据分析：用MicrosoftExcel，GraphpadPrism5对数据进行处理分析。
表3本申请中的小分子化合物对NCI-N87和Colo205细胞体外增殖抑制的IC50值。
结论：根据表3的结果，本申请药物偶联物的毒素对NCI-N87和Colo205细胞具有明显增强的增殖抑制活性。
实施例4：抗体药物偶联物对肿瘤细胞体外增殖抑制测试
4.1、DB1001和DB1001-X1的体外增殖抑制测试
采用化学发光细胞活率检测法(即CTG方法)评估anti-GPC3裸抗DB1001、anti-GPC3 ADC DB1001-X1在GPC3阳性表达的人肝癌细胞HepG2及Huh7中处理7天，对细胞增殖的抑制作用。
收集对数生长期细胞，以4000个/孔(HepG2细胞)、6000个/孔(HuH7)的密度铺板，细胞板放入37℃、5％CO2培养箱培养过夜。实验第二天，将DB1001、DB1001-X1用完全培养基按3倍稀释，获得9个浓度梯度(以300nM的最高浓度开始)药物后，50μL/孔加入细胞培养板中，完全培养基作为空白对照，设置3个复孔；继续于37℃、5％CO2培养箱内孵育7天。孵育结束，取出细胞培养板，平衡至室温后，每孔加入50μL CTG检测试剂(Promega，Cat#：G7573)，震荡混匀后放置于暗处静置10分钟后，利用酶标仪检测读取信号值。应用GraphPad Prism软件，使用非线性回归模型绘制S型剂量-反应曲线并计算IC50值。细胞存活率计算公式＝(Lum待测药-Lum空白对照)/(Lum溶剂空白对照-Lum空白对照)×100％。实验结果如下表所示：
表4：抗体、抗体偶联药物对人肝癌细胞的增殖抑制活性
结论：根据表4的结果，本申请抗体药物偶联物DB1001-X1对GPC3阳性表达细胞Huh7和HepG2具有明显的增殖抑制活性；并且显著优于抗体DB1001。
4.2、DB1001-X1和DB1001-X2的体外增殖抑制测试
采用CTG法评估anti-GPC3 ADC DB1001-X1和DB1001-X2在GPC3阳性表达的人肝癌细胞HepG2、Huh7中处理6天，对细胞增殖的抑制作用。
收集对数生长期细胞，以5000个/孔(HepG2细胞)、5000个/孔(HuH7)的密度铺板，细胞板放入37℃、5％CO2培养箱培养过夜。实验第二天，将DB1001-X1、DB1001-X2用完全培养基按3倍稀释，获得9个浓度梯度(以1000nM的最高浓度开始)药物后，50μL/孔加入细胞培养板中，完全培养基作为空白对照，设置3个复孔；继续于37℃、5％CO2培养箱内孵育7天。孵育结束，取出细胞培养板，平衡至室温后，每孔加入50μL CTG检测试剂(Promega，Cat#：G7573)，震荡混匀后放置于暗处静置10分钟后，利用酶标仪检测读取信号值。应用GraphPad Prism软件，使用非线性回归模型绘制S型剂量-反应曲线并计算IC50值。细胞存活率计算公式＝(Lum待测药-Lum空白对照)/(Lum溶剂空白对照-Lum空白对 照)×100％。实验结果如下表所示：
表5抗体、抗体偶联药物对人肝癌细胞的增殖抑制活性
结论：根据表5的结果，本申请抗体药物偶联物DB1001-X1和DB1001-X2对GPC3阳性表达细胞Huh7和HepG2具有明显增强的增殖抑制活性。
实施例5：抗体药物偶联物体内肿瘤抑制测试
为研究DB1001-X1对体内形成肿瘤的抑制作用，在小鼠体内用GPC3阳性表达的人肝癌细胞HepG2或Huh7形成移植瘤后，评估DB1001-X1的抗肿瘤效果。
5.1、抗体药物偶联物对HepG2荷瘤小鼠药效评价
1.受试药物及材料
空白对照组(对照组)：生理盐水
DB1001-X1(治疗组)：10mg/kg
DB1001(治疗组)：10mg/kg
2.配制方法：所有样品均用生理盐水稀释配制。
3.试验动物：8周龄的雌性BALB/c小鼠，购自集萃药康生物技术有限公司。
4.试验方法：
将1×107个HepG2细胞接种于8周龄的雌性BALB/c裸小鼠右前肩胛处皮下，当肿瘤长至约110mm3，对荷瘤小鼠进行StudyDirectorTM随机分组，并于当天(第0天)开始通过静脉(i.v.)注射DB1001及DB1001-X1给药，共注射1次，给药21天后结束实验。每周测量2次瘤体积和体重，记录数据。
溶媒对照组或治疗组每组6只小鼠。通过测量肿瘤体积计算抑瘤率。抑瘤率(TGI％)＝100％-(测量当天治疗组肿瘤体积-第0天治疗组肿瘤体积)/(测量当天对照组肿瘤体积-第0天对照组肿瘤体积)。
表6抗体药物偶联物对HepG2荷瘤小鼠的体内肿瘤抑制效果
实验结果如图1及表6所示，本申请抗GPC3裸抗DB1001单次给药没有显著的抑瘤作用。抗体药物偶联物DB1001-X1单次给药后表现出显著增强的抗肿瘤活性。
5.2、抗体药物偶联物对Huh7荷瘤小鼠药效评价
1.受试药物及材料
空白对照组(对照组)：生理盐水
DB1001-X1(治疗组)：3mg/kg
DB1001-X1(治疗组)：10mg/kg
2.配制方法：所有样品均用生理盐水稀释配制。
3.试验动物：8周龄的雌性BALB/c小鼠，购自集萃药康生物技术有限公司。
4.试验方法：
将5×106个Huh7细胞接种于8周龄的雌性BALB/c裸小鼠右前肩胛处皮下，当肿瘤长至约130mm3，对荷瘤小鼠进行StudyDirectorTM随机分组，并于当天(第0天)开始通过静脉注射(i.v.)给药DB1001-X1(3mg/kg)及DB1001-X1(10mg/kg)，共给药1次，给药21天后结束实验。每周测量2次瘤体积和体重，记录数据。
溶媒对照组或给药组每组6只小鼠。通过测量肿瘤体积计算抑瘤率。抑瘤率(TGI％)＝100％-(测量当天治疗组肿瘤体积-第0天治疗组肿瘤体积)/(测量当天对照组肿瘤体积-第0天对照组肿瘤体积)。
表7抗体药物偶联物对Huh7荷瘤小鼠的体内肿瘤抑制效果
实验结果如图2及表7所示。抗体药物偶联物DB1001-X1单次给药后表现出显著的剂量依赖性抑瘤活性。
5.3、抗体药物偶联物对HepG2荷瘤小鼠药效评价
(1)受试药物及材料
空白对照组(对照组)：生理盐水
DB1001-Deruxtecan(治疗组)：1mg/kg
DB1001-X2(治疗组)：1mg/kg
DB1001-Deruxtecan(治疗组)：3mg/kg
DB1001-X2(治疗组)：3mg/kg
(2)配制方法：所有样品均用生理盐水稀释配制。
(3)试验动物：8周龄的雌性BALB/c小鼠，购自集萃药康生物技术有限公司。
(4)试验方法：
将1×107个HepG2细胞接种于8周龄的雌性BALB/c裸小鼠右前肩胛处皮下，当肿瘤长至约110mm3，对荷瘤小鼠进行StudyDirectorTM随机分组，并于当天(第0天)开始通过静脉(iv.)注射DB1001-X1及DB1001-X2给药，共注射1次，给药21天后结束实验。每周测量2次瘤体积和体重，记录数据。
通过测量肿瘤体积计算抑瘤率。抑瘤率(TGI％)＝100％-(测量当天治疗组肿瘤体积-第0天治疗组肿瘤体积)/(测量当天对照组肿瘤体积-第0天对照组肿瘤体积)。
表8 DB1001-Deruxtecan和DB1001-X2对HepG2荷瘤小鼠的体内肿瘤抑制效果
+：TGI＜50％
++：60≤TGI％＜75％
+++：TGI％≥80％
实验结果表明，本申请抗体药物偶联物单次给药后表现出显著增强的抗肿瘤活性。
5.4、抗体药物偶联物对Huh7荷瘤小鼠药效评价
(1)受试药物及材料
空白对照组(对照组)：生理盐水
DB1001-Deruxtecan(治疗组)：1mg/kg
DB1001-X2(治疗组)：1mg/kg
DB1001-Deruxtecan(治疗组)：3mg/kg
DB1001-X2(治疗组)：3mg/kg
(2)配制方法：所有样品均用生理盐水稀释配制。
(3)试验动物：8周龄的雌性雌性BALB/c裸小鼠，购自集萃药康生物技术有限公司。
(4)试验方法：
将5×106个Huh7细胞接种于8周龄的雌性BALB/c裸小鼠右前肩胛处皮下，当肿瘤长至约110mm3，对荷瘤小鼠进行StudyDirectorTM随机分组，并于当天(第0天)开始通过静脉(i.v.)注射DB1001-X1 及DB1001-X2给药，共注射1次，给药21天后结束实验。每周测量2次瘤体积和体重，记录数据。
通过测量肿瘤体积计算抑瘤率。抑瘤率(TGI％)＝100％-(测量当天治疗组肿瘤体积-第0天治疗组肿瘤体积)/(测量当天对照组肿瘤体积-第0天对照组肿瘤体积)。
表9 DB1001-Deruxtecan和DB1001-X2对Huh7荷瘤小鼠的体内肿瘤抑制效果
+：TGI＜50％
++：60≤TGI％＜75％
+++：TGI％≥80％
实验结果表明，本申请抗体药物偶联物单次给药后表现出显著增强的抗肿瘤活性。
虽然以上描述了本发明的具体实施方式，但是本领域的技术人员应当理解，这些仅是举例说明，在不背离本发明的原理和实质的前提下，可以对这些实施方式做出多种变更或修改。因此，本发明的保护范围由所附权利要求书限定。

Claims (35)

  1. 一种抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，所述抗GPC抗体药物偶联物结构如式(I-1)所示：
    其中，
    M为-L2-L3-X-L1-；
    L2为-O-或-S-；
    L3为-(C(R1a)(R1b))m-，m为0、1、2或3，其中当L3包含亚甲基单元时，所述L3的0个或1个亚甲基单元可以被-C(O)-、或-C(S)-替代；
    L1为-(C(R2a)(R2b))n-，n为1、2或3，其中当L1包含亚甲基单元时，所述L1的0个或1个亚甲基单元被-C(O)-、或-C(S)-替代；
    X为3到6元饱和的碳环基、3到6元饱和的杂环基或单键，所述3到6元饱和的碳环基和3到6元饱和的杂环基任选被1个或多个R3a取代；
    其中每个R1a，R1b，R2a，R2b，R3a各自独立地为氢、卤素或被1个或多个R任选取代的C1-6脂肪族基团；
    其中每个R各自独立地为氢或卤素；
    L是接头单元；
    p表示平均连接数，且p为1到10的整数或小数；
    Ab为抗GPC3抗体或其抗原结合片段。
  2. 如权利要求1所述的抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述抗GPC3抗体或其抗原结合片段包含重链可变区和轻链可变区，所述重链可变区包含氨基酸序列分别如SEQ ID NO：1、SEQ ID NO：2和SEQ ID NO：3所示的HCDR1、HCDR2和HCDR3，和所述轻链可变区包含氨基酸序列分别如SEQ ID NO：4、SEQ ID NO：5和SEQ ID NO：6所示的LCDR1、LCDR2和LCDR3。
  3. 如权利要求1或2所述的抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述抗GPC3抗体或其抗原结合片段包含：氨基酸序列如SEQ ID NO：7所示或与其具有至少95％、96％、97％、98％或99％同一性的重链可变区，和氨基酸序列如SEQ ID NO：8所示或与其具有至少95％、96％、97％、98％或99％同一性的轻链可变区。
  4. 如权利要求1或2所述的抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述抗GPC3抗体或其抗原结合片段为鼠源抗体或其片段、嵌合抗体或其片段、人源化抗体或其片段。
  5. 如权利要求1或2所述的抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述抗GPC3抗体或其抗原结合片段为Fab、Fab′、Fab′-SH、Fv、scFv、F(ab′)2、sdAb、双抗体或线性抗体。
  6. 如权利要求1或2所述的抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述抗体为IgG1形式的抗体、IgG2形式的抗体、IgG3形式的抗体或IgG4形式的抗体。
  7. 如权利要求1或2所述的抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述抗GPC3抗体或其抗原结合片段包含：氨基酸序列如SEQ ID NO：9所示或与其具有至少95％、96％、97％、98％或99％同一性的重链，和氨基酸序列如SEQ ID NO：10所示或与其具有至少95％、96％、97％、98％或99％同一性的轻链。
  8. 如权利要求1-7中任一项所述的抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中p为3-8的整数或小数。
  9. 如权利要求1-7中任一项所述的抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中L3为-(C(R1a)(R1b))m-，其中，m为0、1、2或3；
    其中每个R1a和R1b各自独立地为氢、卤素或被1、2或3个R任选取代的C1-6脂肪族基团，优选为氢、甲基、乙基或丙基；
    其中每个R各自独立地为氢或卤素。
  10. 如权利要求9所述的抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中L3为单键、-CH2-、-CH(CH3)-、-C(CH3)2-、-CH2CH2-、-CH(CH3)CH2-或-C(CH3)2CH2-。
  11. 如权利要求1-7中任一项所述的抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中X为任选被1、2或3个R3a取代的3到6元饱和的碳环基或单键；
    其中每个R3a各自独立地为氢、卤素或被1、2或3个R任选取代的C1-6脂肪族基团；
    其中每个R各自独立地为氢或卤素。
  12. 如权利要求11所述的抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中X为3到6元饱和的碳环基或单键，优选为环丙基、环丁基、环戊基、环己基或单键，更优选为或单键。
  13. 如权利要求1-7中任一项所述的抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中-L3-X-为：-(C(R1a)(R1b))m-、3到6元饱和的碳环基或3到6元饱和的杂环基；
    其中，m为1、2或3，所述3到6元饱和的碳环基和3到6元饱和的杂环基任选被1、2或3个R3a取代；
    其中，每个R1a，R1b，R3a各自独立地为氢、卤素或可以被1、2或3个R任选取代的C1-6脂肪族 基团；
    其中每个R各自独立地为氢或卤素。
  14. 如权利要求13所述的抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中，所述-L3-X-为：-(C(R1a)(R1b))m-或任选被1、2或3个R3a取代的3到6元饱和的碳环基；例如，所述-L3-X-为-(C(R1a)(R1b))m-或任选被1、2或3个R3a取代的环丙基、环丁基、环戊基或环己基；优选地，所述-L3-X-为：-(C(R1a)(R1b))m-或任选被1、2或3个R3a取代的：
    所述m为1、2或3；
    R1a各自独立地为卤素或被1、2或3个R任选取代的C1-6脂肪族基团，例如为甲基、乙基或丙基；R1b，R3a各自独立地为氢、卤素或被1、2或3个R任选取代的C1-6脂肪族基团，例如为氢、甲基、乙基或丙基；R各自独立地为氢或卤素。
  15. 如权利要求14所述的抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述-L3-X-为：
  16. 如权利要求1-7中任一项所述的抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中L1为-C(R2a)(R2b)-、-C(R2a)(R2b)C(O)-或-C(O)-；
    其中每个R2a，R2b各自独立地为氢、卤素或被R任选取代的C1-6脂肪族基团，优选为氢、甲基、乙基或丙基；
    其中每个R各自独立地为氢或卤素。
  17. 如权利要求16所述的抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中L1为-CH2C(O)-、-CH(CH3)C(O)-或-C(O)-。
  18. 如权利要求1-17中任一项所述的抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中，
    所述M为-L2-L3-X-L1-；
    L2为-O-；
    -L3-X-为任选被1、2或3个R3a取代的3到6元饱和的碳环基；或者，-L3-X-为任选被1、2或3个R3a取代的环丙基、环丁基、环戊基或环己基；优选地，所述-L3-X-为：任选被1、2或3个R3a取代的：
    L1为-C(O)-；
    其中每个R3a各自独立地为氢、卤素或可以被R任选取代的C1-6脂肪族基团；
    其中每个R各自独立地为氢或卤素。
  19. 如权利要求18所述的抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中，
    所述M为-L2-L3-X-L1-；
    L2为-O-；
    -L3-X为：
    L1为-C(O)-。
  20. 如权利要求1-17中任一项所述的抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中，
    M为-L2-L3-X-L1-；
    L2为-O-；
    -L3-X-为-(C(R1a)(R1b))m-，其中，m为1、2或3；
    L1为-CH2C(O)-；
    其中R1a各自独立地为卤素或被1、2或3个R任选取代的C1-6脂肪族基团；R1b，R3a各自独立地为氢、卤素或被1、2或3个R任选取代的C1-6脂肪族基团；
    其中每个R各自独立地为氢或卤素。
  21. 如权利要求20所述的抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中，
    M为-L2-L3-X-L1-；
    L2为-O-；
    -L3-X-为
    L1为-CH2C(O)-。
  22. 如权利要求21所述的抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中，
    M为-L2-L3-X-L1-；
    L2为-O-；
    -L3-X-为
    L1为-CH2C(O)-。
  23. 如权利要求1-17中任一项所述的抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中-M-为：
  24. 如权利要求1-23中任一项所述的抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述L为-La-Lb-Lc-，
    所述-La-为
    其中，W为-(C(Rwa)(Rwb))wn-，Y为-(OCH2CH2)yn-Oyp-，Z为-(C(Rza)(Rzb))zn；
    其中wn为1、2、3或6，
    W的0个或1个亚甲基单元各自独立地被-Cyr-、-N(Rwx)C(O)-、-C(O)N(Rwx)-、或-C(O)-替代；
    其中yn为0、4或8，yp为0或1；
    其中zn为1、2或3，
    Z的1个亚甲基单元各自独立地被-Cyr-、-N(Rzx)C(O)-、-C(O)N(Rzx)-、或-C(O)-替代；
    -Cyr-为3到10元饱和的亚碳环基，其中所述-Cyr-是未取代的或独立地被1到3个取代基Rcx取代；
    其中每个Rwa，Rwb，Rza，Rzb，Rwx，Rzx，Rcx各自独立地为氢、卤素、-ORr或被Rr任选取代的C1-6脂肪族基团；
    其中每个Rr各自独立地为氢、卤素或C1-6脂肪族基团；
    所述-Lb-表示由2到4个氨基酸构成的肽残基，所述-Lb-的肽残基为由选自以下组中的氨基酸形成的肽残基：苯丙氨酸、甘氨酸、丙氨酸、缬氨酸、瓜氨酸和赖氨酸；
    所述-Lc-为
    其中RL1、RL2各自独立地选自以下组：氢、卤素、-OH和C1-6脂肪族基团。
  25. 如权利要求24所述的抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述-La-为
  26. 如权利要求24所述的抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合 物，其中所述-Lb-选自以下组：
    优选地，所述-Lb-为
  27. 如权利要求24所述的抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述-Lc-为
  28. 如权利要求24-27中任一项所述的抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述L为
  29. 如权利要求24-27中任一项所述的抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述L为
  30. 如权利要求1-29中任一项所述的抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述抗GPC3抗体药物偶联物结构如式(II-1)或(II-2)所示：
    其中，
    p如权利要求1或8所述；
    Ab如权利要求1-7中任一项所述；
    L2为-O-或-S-；优选为-O-；
    X为任选被1、2或3个R3a取代的3到6元饱和的碳环基；优选地，所述X为任选被1、2或3个R3a取代的：
    L3为-C(R1a)(R1b)-CH2-或-C(R1a)(R1b)C(R1a)(R1b)-CH2-；
    R1a各自独立地为卤素或被1、2或3个R任选取代的C1-6脂肪族基团；R1b，R3a各自独立地为氢、卤素或被1、2或3个R任选取代的C1-6脂肪族基团；R各自独立地为氢或卤素。
  31. 如权利要求1-30中任一项所述的抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述抗GPC3抗体药物偶联物选自以下结构式：



    其中，
    p如权利要求1或8所述；
    Ab如权利要求1-7中任一项所述。
  32. 一种抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述抗GPC3抗体药物偶联物选自：


    其中，
    p表示平均连接数，且p为1到10的整数或小数，优选为3-8的整数或小数。
  33. 一种药物组合物，其包含如权利要求1-32中任一项所述的抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，和药学上可接受的载体或赋形剂。
  34. 如权利要求1-32中任一项所述的抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物或权利要求33所述的药物组合物在制备用于治疗和/或预防GPC3介导的疾病或病症的药 物中的用途，优选地，所述疾病或病症为癌症；更优选地，所述癌症选自乳腺癌、卵巢癌、前列腺癌、胰腺癌、肾癌、肺癌、肝癌、胃癌、结肠癌、膀胱癌、食管癌、宫颈癌、胆囊癌、胶质母细胞瘤和黑色素瘤。
  35. 一种治疗和/或预防GPC3介导的疾病或病症的方法，其包括向有需要的受试者施用如权利要求1-32中任一项所述的抗GPC3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物或权利要求33所述的药物组合物，优选地，所述疾病或病症为癌症；更优选地，所述癌症选自乳腺癌、卵巢癌、前列腺癌、胰腺癌、肾癌、肺癌、肝癌、胃癌、结肠癌、膀胱癌、食管癌、宫颈癌、胆囊癌、胶质母细胞瘤和黑色素瘤。
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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014097648A1 (ja) * 2012-12-21 2014-06-26 中外製薬株式会社 Gpc3標的治療剤療法が有効である患者に投与されるgpc3標的治療剤
CN110577600A (zh) * 2018-06-07 2019-12-17 中国科学院上海药物研究所 靶向gpc3的抗体-药物偶联物及其制备方法和用途
WO2021007435A1 (en) * 2019-07-10 2021-01-14 Cybrexa 2, Inc. Peptide conjugates of cytotoxins as therapeutics
CN113444179A (zh) * 2020-03-26 2021-09-28 苏州普乐康医药科技有限公司 一种抗gpc3抗体和包含该抗体的药物组合物
WO2022068878A1 (zh) * 2020-09-30 2022-04-07 映恩生物制药（苏州）有限公司 一种抗肿瘤化合物及其制备方法和应用
WO2022109334A1 (en) * 2020-11-19 2022-05-27 Ardeagen Corporation Gpc3 binding agents, conjugates thereof and methods of using the same
CN115429894A (zh) * 2021-06-02 2022-12-06 上海翰森生物医药科技有限公司 配体-药物偶联物、其制备方法及其医药用途

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014097648A1 (ja) * 2012-12-21 2014-06-26 中外製薬株式会社 Gpc3標的治療剤療法が有効である患者に投与されるgpc3標的治療剤
CN110577600A (zh) * 2018-06-07 2019-12-17 中国科学院上海药物研究所 靶向gpc3的抗体-药物偶联物及其制备方法和用途
WO2021007435A1 (en) * 2019-07-10 2021-01-14 Cybrexa 2, Inc. Peptide conjugates of cytotoxins as therapeutics
CN113444179A (zh) * 2020-03-26 2021-09-28 苏州普乐康医药科技有限公司 一种抗gpc3抗体和包含该抗体的药物组合物
WO2022068878A1 (zh) * 2020-09-30 2022-04-07 映恩生物制药（苏州）有限公司 一种抗肿瘤化合物及其制备方法和应用
WO2022109334A1 (en) * 2020-11-19 2022-05-27 Ardeagen Corporation Gpc3 binding agents, conjugates thereof and methods of using the same
CN115429894A (zh) * 2021-06-02 2022-12-06 上海翰森生物医药科技有限公司 配体-药物偶联物、其制备方法及其医药用途

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