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WO2020063673A1 - 抗b7h3抗体-依喜替康类似物偶联物及其医药用途 - Google Patents

抗b7h3抗体-依喜替康类似物偶联物及其医药用途 Download PDF

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WO2020063673A1
WO2020063673A1 PCT/CN2019/107852 CN2019107852W WO2020063673A1 WO 2020063673 A1 WO2020063673 A1 WO 2020063673A1 CN 2019107852 W CN2019107852 W CN 2019107852W WO 2020063673 A1 WO2020063673 A1 WO 2020063673A1
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Prior art keywords
cancer
antibody
pharmaceutically acceptable
ligand
solvate
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PCT/CN2019/107852
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English (en)
French (fr)
Inventor
应华
张玲
张婷
张蕾
许建烟
陶维康
Original Assignee
江苏恒瑞医药股份有限公司
上海恒瑞医药有限公司
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Application filed by 江苏恒瑞医药股份有限公司, 上海恒瑞医药有限公司 filed Critical 江苏恒瑞医药股份有限公司
Priority to CA3114474A priority Critical patent/CA3114474A1/en
Priority to JP2021517606A priority patent/JP7408646B2/ja
Priority to CN201980049765.5A priority patent/CN112543771B/zh
Priority to BR112021004829-4A priority patent/BR112021004829A2/pt
Priority to EP19865297.6A priority patent/EP3854816A4/en
Priority to US17/281,062 priority patent/US20210347894A1/en
Priority to KR1020217010853A priority patent/KR20210068457A/ko
Priority to MX2021003446A priority patent/MX2021003446A/es
Priority to AU2019351427A priority patent/AU2019351427A1/en
Publication of WO2020063673A1 publication Critical patent/WO2020063673A1/zh
Priority to ZA2021/02696A priority patent/ZA202102696B/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—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
    • A61K47/6835—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 the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—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 the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • 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
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68037—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a camptothecin [CPT] or derivatives
    • 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
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • 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
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell

Definitions

  • the present disclosure relates to an anti-B7H3 antibody-esteticon analogue conjugate, a method for preparing the same, a pharmaceutical composition comprising the same, and its use in the manufacture of a medicament for the treatment of a B7H3-mediated disease or disorder; particularly in use Use in the preparation of anticancer drugs.
  • T cell-mediated immune response plays an extremely important role in the body's anti-tumor process.
  • T cell activation and proliferation require not only TCR recognition of the antigen signal, but also a second signal provided by a co-stimulatory molecule.
  • B7 family molecules belong to the co-stimulatory molecule immunoglobulin superfamily. More and more studies show that this family of molecules play an important regulatory role in the body's normal immune function and pathological state.
  • B7H3 is a member of the B7 family and belongs to a type I transmembrane protein. It contains a signal peptide at the amino terminus, an extracellular immunoglobulin-like variable region (IgV) and constant region (IgC), a transmembrane region, and a Contains a 45 amino acid cytoplasmic tail region (Tissue Antigens. 2007 Aug; 70 (2): 96-104). At present, there are two main types of spliceosome in B7H3, B7H3a and B7H3b.
  • the extracellular segment of B7H3a is composed of two IgV-IgC immunoglobulin domains, also known as 2IgB7H3, while the extracellular segment of B7H3b is composed of IgV-IgC-IgV-IgC, four immunoglobulin domains, also called 4IgB7H3.
  • B7H3 protein is not expressed in normal tissues or cells or is extremely low expressed, but is highly expressed in a variety of tumor tissues, and is closely related to tumor progression, patient survival and prognosis. Clinically, B7H3 has been reported in many cancers, especially in non-small cell lung cancer, kidney cancer, urinary tract epithelial cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer and pancreatic cancer. Expression (Lung Cancer. 2009 Nov; 66 (2): 245-249; Clin Cancer Res. 2008 Aug 15; 14 (16): 5150-5157).
  • B7H3 is considered as a new tumor marker and potential therapeutic target.
  • Phage display technology is the fusion expression of exogenous protein or polypeptide and phage shell protein, so that the exogenous protein is expressed on the surface of the phage.
  • the phage antibody library is an antibody library created by combining phage display technology, PCR amplification technology, and protein expression technology with comprehensive technical means.
  • the biggest advantage of the phage antibody library is to prepare fully human antibodies without in vivo immunization and simulating the three processes of antibody production in vivo.
  • the phage antibody library also has the following advantages: 1 The genotype and phenotype are unified; in addition, the experimental method is simple and fast. The traditional method of generating antibodies by hybridoma technology takes several months, and the antibody library technology It only takes a few weeks; 2 It expresses fully human antibodies and has a small molecular weight, mainly expressed in the form of active fragments Fab and scFv. Compared with intact antibodies, it has obvious advantages in tissue penetration; 3 screening Large capacity. Hybridoma technology is selected in thousands of clones.
  • Antibody library technology can select among millions or even billions of molecules. There are many types of antibodies screened. 4 It is widely used and uses prokaryotic expression system. The advantages are even more obvious in large-scale production (Curr Opin Biotechnol. 2002Dec; 13 (6): 598-602; Immunotechnology, 2013, 48 (13) 48 (13): 63-73).
  • Antibody-drug conjugate connects monoclonal antibodies or antibody fragments with biologically active cytotoxins through stable chemical linker compounds, making full use of the specificity of antibodies for binding to surface antigens of normal cells and tumor cells And the high efficiency of cytotoxic substances, while avoiding the disadvantages such as low efficacy of the former and excessive toxic and side effects of the latter. This means that, compared with traditional chemotherapy drugs, antibody-drug conjugates can bind tumor cells more accurately and reduce the impact on normal cells.
  • Kadcyla which is an ADC drug that targets Her2 to trastuzumab and DM1.
  • Kadcyla an ADC drug that targets Her2 to trastuzumab and DM1.
  • camptothecin derivative which has antitumor effects by inhibiting topoisomerase I.
  • Report of camptothecin derivative Ixatecan (chemical name: (1S, 9S) -1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H , 12H-benzo [de] pyrano [3 ', 4': 6,7] imidazo [1,2-b] quinoline-10,13 (9H, 15H) -dione) used in antibody couples
  • the literature of the combined drug (ADC) includes WO2014057687; Clinical Research (2016) 22 (20): 5097-5108; Cancer Sci (2016) 107: 1039-1046. However, further development of ADC drugs with better efficacy is still needed.
  • the present disclosure relates to an ADC of an anti-B7H3 antibody and use thereof, in which an ADC drug coupled with a monoclonal antibody or an antigen-binding fragment that binds to an amino acid sequence or a three-dimensional structure of an extracellular region of B7H3 and a cytotoxic substance ethetican analog is provided. .
  • an object of the present disclosure is to provide a ligand-drug conjugate represented by the general formula (Pc-L-Y-Dr) or a pharmaceutically acceptable salt or solvate thereof:
  • Y is selected from -O- (CR a R b ) m -CR 1 R 2 -C (O)-, -O-CR 1 R 2- (CR a R b ) m- , -O-CR 1 R 2- -NH- (CR a R b ) m -CR 1 R 2 -C (O)-or -S- (CR a R b ) m -CR 1 R 2 -C (O)-;
  • R a and R b are the same or different and are each independently selected from the group consisting of a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, an alkoxy group, a hydroxyl group, an amino group, a cyano group, a nitro group, and a hydroxyalkane Radical, cycloalkyl or heterocyclyl;
  • R a and R b together with the carbon atom to which they are attached form a cycloalkyl or heterocyclic group
  • R 1 is selected from halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl, or heteroaryl;
  • R 2 is selected from a hydrogen atom, halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl, or heteroaryl;
  • R 1 and R 2 together with the carbon atom to which they are attached form a cycloalkyl or heterocyclic group
  • R a and R 2 together with the carbon atom to which they are attached form a cycloalkyl or heterocyclic group
  • n is an integer from 0 to 4.
  • n is 1 to 10, optionally from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; can be an integer or a decimal;
  • L is a joint unit
  • Pc is an anti-B7H3 antibody or an antigen-binding fragment thereof.
  • the combined fragment contains:
  • the heavy chain HCDR1, HCDR2, HCDR3 shown in the amino acid sequences of SEQ ID NO: 8, 9, and 10, or HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 8, 9, and 10, respectively, have 3, 2 or 1 HCDR variants with amino acid differences;
  • the light chain FR region on the light chain variable region is derived from a human germline light chain sequence or a mutant sequence thereof, and / or the heavy chain FR region on the heavy chain variable region is derived from a human germline heavy chain sequence or a mutation thereof sequence.
  • amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 6 or has at least 95% sequence identity
  • amino acid sequence of the light chain variable region is shown in SEQ ID NO: 7 or has at least 95% % Sequence identity.
  • An h1702 antibody consisting of the heavy chain sequence shown in SEQ ID NO: 14 and the light chain sequence shown in SEQ ID NO: 15, and
  • the h1702DS antibody consists of the heavy chain sequence shown in SEQ ID NO: 14 and the light chain sequence shown in SEQ ID NO: 16.
  • the antigen-binding fragment is selected from Fab, Fab ', F (ab') 2, single chain antibody (scFv), dimerized V region (diabody), disulfide stabilized V region (dsFv), and antigen-binding fragment of a CDR-containing peptide.
  • the ligand-drug conjugate represented by the general formula (Pc-L-Y-Dr) or a pharmaceutically acceptable salt or solvate thereof is represented by the general formula (Pc-L-Y-Dr) or a pharmaceutically acceptable salt or solvate thereof,
  • Y is -O- (CR a R b ) m -CR 1 R 2 -C (O)-;
  • R a and R b are the same or different and are each independently selected from a hydrogen atom, a deuterium atom, a halogen or an alkyl group;
  • R 1 is haloalkyl or C 3-6 cycloalkyl
  • R 2 is selected from a hydrogen atom, a halogenated alkyl group or a C 3-6 cycloalkyl group;
  • R 1 and R 2 together with the carbon atom to which they are attached form a C 3-6 cycloalkyl group
  • m 0 or 1.
  • the ligand-drug conjugate represented by the general formula (Pc-L-Y-Dr) or a pharmaceutically acceptable salt or solvate thereof is represented by the general formula (Pc-L-Y-Dr) or a pharmaceutically acceptable salt or solvate thereof,
  • Y is -O- (CR a R b ) m -CR 1 R 2 -C (O)-;
  • R a and R b are the same or different and are each independently selected from a hydrogen atom, a deuterium atom, a halogen or an alkyl group;
  • R 1 is C 3-6 cycloalkyl
  • R 2 is selected from a hydrogen atom
  • R 1 and R 2 together with the carbon atom to which they are attached form a C 3-6 cycloalkyl group
  • L 1 is s 1 is an integer from 2 to 8;
  • L 2 is a chemical bond
  • L 3 is a tetrapeptide residue
  • L 4 is -NR 5 (CR 6 R 7 ) t-, R 5 , R 6 or R 7 is the same or different, and each is independently a hydrogen atom or an alkyl group, and t is 1 or 2.
  • L 2 is a chemical bond
  • L 3 is a tetrapeptide residue of GGFG
  • R 1 is haloalkyl or C 3-6 cycloalkyl
  • R 2 is selected from a hydrogen atom, a halogenated alkyl group or a C 3-6 cycloalkyl group;
  • R 1 and R 2 together with the carbon atom to which they are attached form a C 3-6 cycloalkyl group
  • R 5 , R 6 or R 7 are the same or different and are each independently a hydrogen atom or an alkyl group
  • s 1 is an integer from 2 to 8;
  • n is an integer from 0 to 4.
  • the ligand-drug conjugate represented by the general formula (Pc-LY-Dr) or a pharmaceutically acceptable salt or solvate thereof is the general formula (Pc-L a -Y -Dr) a ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof:
  • W is selected from C 1-8 alkyl, C 1-8 alkyl-cycloalkyl, or 1 to 8 atom straight-chain heteroalkyl, said heteroalkyl containing 1 to 3 selected from N, O, or S Hetero atom, wherein the C 1-8 alkyl, cycloalkyl, and linear heteroalkyl are each optionally further selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, Deuterated alkyl, alkoxy and cycloalkyl substituted with one or more substituents;
  • L 2 is selected from -NR 4 (CH 2 CH 2 O) p1 CH 2 CH 2 C (O)-, -NR 4 (CH 2 CH 2 O) p1 CH 2 C (O)-, -S (CH 2 ) p1 C (O) - or a bond, p 1 is an integer of from 1 to 20, preferably 1 to 6;
  • L 3 is a peptide residue composed of 2 to 7 amino acids.
  • the amino acid may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available point of attachment.
  • the substituent is one Or more are independently selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy, and cycloalkyl;
  • R 1 is selected from halogen, haloalkyl, deuterated alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl;
  • R 2 is selected from a hydrogen atom, halogen, haloalkyl, deuterated alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl;
  • R 1 and R 2 together with the carbon atom to which they are attached form a cycloalkyl or heterocyclic group
  • R 4 and R 5 are the same or different and are each independently selected from a hydrogen atom, an alkyl group, a haloalkyl group, a deuterated alkyl group, and a hydroxyalkyl group;
  • R 6 and R 7 are the same or different, and are each independently selected from a hydrogen atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, and a hydroxyalkyl group;
  • n is an integer from 0 to 4.
  • n 1 to 10, which can be an integer or a decimal
  • Pc is an anti-B7H3 antibody or an antigen-binding fragment thereof.
  • the ligand-drug conjugate represented by the general formula (Pc-LY-Dr) or a pharmaceutically acceptable salt or solvate thereof is a general formula (Pc-L b -Y -Dr) a ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof:
  • s 1 is an integer from 2 to 8; preferably 5;
  • R 1 , R 2 , R 5 to R 7 , m and n are as defined in the general formula (Pc-L a -Y-Dr).
  • the ligand-drug conjugate represented by the general formula (Pc-LY-Dr) or a pharmaceutically acceptable salt or solvate thereof is selected from the group consisting of a ligand-drug conjugate from:
  • the ligand-drug conjugate represented by the general formula (Pc-L-Y-Dr) typical in the present disclosure includes, but is not limited to:
  • n can be a non-zero integer or decimal from 0 to 10, preferably an integer or decimal from 1 to 10; more preferably 2 to 8, which can be an integer or a decimal; most preferably 3 to 8, which can Is an integer or a decimal.
  • the present disclosure further provides a method for preparing a ligand-drug conjugate represented by the general formula (Pc-L a -Y-Dr) or a pharmaceutically acceptable salt or solvate thereof, which includes the following steps:
  • Pc is an anti-B7H3 antibody or an antigen-binding fragment thereof
  • W, L 2 , L 3 , R 1 , R 2 , R 5 to R 7 , m and n are as defined in the general formula (Pc-L a -Y-Dr).
  • R 1 , R 2 , R 5 to R 7 , s 1 and m are as defined in the general formula Pc-L b -Y-Dr.
  • the ligand-drug couple represented by the general formula (Pc-L a -Y-Dr) or the general formula (Pc-L b -Y-Dr) according to the present disclosure
  • a method of a conjugate or a pharmaceutically acceptable salt or solvate thereof, wherein the compound represented by the general formula (L a -Y-Dr) or the compound represented by the general formula (L b -Y-Dr) is selected from:
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising an antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to the present disclosure, and one or more pharmaceutically acceptable excipients Formulation, diluent or carrier.
  • the present disclosure provides an antibody-drug conjugate according to the present disclosure, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition comprising the same, for use in the preparation of a B7H3-mediated disease or condition.
  • a B7H3-mediated disease or disorder is a B7H3 overexpressing cancer.
  • the present disclosure provides a ligand-drug conjugate according to the present disclosure or a pharmaceutically acceptable salt or solvate thereof or a pharmaceutical composition comprising the same in the manufacture of a medicament for treating or preventing a tumor.
  • the cancer is preferably selected from breast cancer, ovarian cancer, cervical cancer, lung cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, ovarian cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, Esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, pharyngeal cancer, lung cancer, colon cancer, rectal cancer, colorectal cancer, leukemia, bone cancer, skin cancer, thyroid cancer, pancreatic cancer or lymphoma .
  • the present disclosure further relates to a method for treating and / or preventing a tumor, the method comprising administering to a patient in need thereof a therapeutically effective dose of a ligand-drug conjugate according to the present disclosure or a pharmacy thereof
  • a therapeutically effective dose of a ligand-drug conjugate according to the present disclosure or a pharmacy thereof The above acceptable salts or solvates or pharmaceutical compositions comprising them; preferably wherein the tumor is a cancer associated with high expression of B7H3.
  • the present disclosure further relates to a method for treating or preventing cancer, the method comprising administering to a patient in need thereof a therapeutically effective dose of a ligand-drug conjugate according to the present disclosure or a pharmaceutically acceptable An accepted salt or solvate or a pharmaceutical composition comprising the same; wherein the cancer is preferably selected from breast cancer, ovarian cancer, cervical cancer, lung cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, ovarian cancer, Liver cancer, gastric cancer, endometrial cancer, salivary adenocarcinoma, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, pharyngeal cancer, lung cancer, colon cancer, rectal cancer, colorectal cancer, leukemia, bone cancer , Skin cancer, thyroid cancer, pancreatic cancer, or lymphoma.
  • the cancer is preferably selected from breast cancer, ovarian cancer, cervical cancer, lung cancer, uter
  • the active compound may be prepared in a form suitable for administration by any suitable route, and the active compound is preferably in the form of a unit dose or in such a manner that the patient can self-administer in a single dose.
  • the unit dose of a compound or composition of the present disclosure can be expressed in the form of tablets, capsules, cachets, bottled potions, powders, granules, lozenges, suppositories, reconstituted powders or liquid preparations.
  • the dosage of the compound or composition used in the methods of treatment of the present disclosure will generally vary with the severity of the disease, the weight of the patient, and the relative efficacy of the compound.
  • a suitable unit dose may be 0.1 to 1000 mg.
  • the pharmaceutical composition of the present disclosure may contain one or more excipients selected from the following ingredients: fillers (diluents), binders, wetting agents, disintegrating agents, or excipients Wait.
  • the composition may contain from 0.1 to 99% by weight of active compound.
  • FIG. 1 Endocytosis effect of B7H3 antibody on U87MG cells.
  • Figures 2A-2F Results of ADCs of the present disclosure inhibiting proliferation of different tumor cells.
  • Figure 2A is the result of the proliferation inhibition experiment of different ADCs on A498 cells;
  • Figure 2B is the result of the proliferation inhibition experiment of different ADCs on Calu-6 cells;
  • Figure 2C is the result of the proliferation inhibition experiment of different ADCs on U87 cells;
  • Figure 2D is The proliferation inhibition experiment results of different ADCs on A375 cells;
  • Figure 2E is the proliferation inhibition experiment results of different ADCs on Detroit562 cells, and
  • Figure 2F is the proliferation inhibition experiment results of different ADCs on CHOK1 cells.
  • Fig. 3 Inhibitory effect of intraperitoneal injection of ADC-8 (1mpk, 3mpk) and ADC-5 (1mpk, 3mpk) of the present disclosure on transplanted tumors of U87MG nude mice in Test Example 7.
  • Figure 4 Inhibitory effect of intraperitoneal injection of ADC-2 (1mpk, 3mpk) and ADC-1 (1mpk, 3mpk) of the present disclosure on transplanted tumors of Detroit 562 nude mice in Test Example 8.
  • FIG. 5A Proliferation inhibition rates of ADC-4, ADC-6 and ADC-7 of the present disclosure on Detroit562 cells in Test Example 9.
  • FIG. 5A Proliferation inhibition rates of ADC-4, ADC-6 and ADC-7 of the present disclosure on Detroit562 cells in Test Example 9.
  • FIG. 6 Plasma stability results of ADC-4 of the present disclosure in Test Example 10.
  • the applicant intends to include the formulation of the trade name product, the generic drug product, and the active drug portion of the trade name product.
  • drug refers to a cytotoxic drug, which is expressed as Dr. It has chemical molecules in tumor cells that strongly disrupt its normal growth. Cytotoxic drugs can, in principle, kill tumor cells at a sufficiently high concentration, but due to the lack of specificity, while killing tumor cells, it will also cause apoptosis in normal cells, leading to serious side effects.
  • the term includes toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, radioisotopes (e.g.
  • linker unit refers to a chemical structure fragment or bond connected at one end to a ligand and the other end to a drug. It may also be connected to another linker and then to the drug.
  • the preferred embodiment of the present disclosure is expressed as L and L 1 to L 4 , wherein the L 1 end is connected to a ligand, and the L 4 end is connected to a structural unit Y and then to a drug (Dr).
  • Linkers including extensions, spacers, and amino acid units, can be synthesized by methods known in the art, such as those described in US2005-0238649A1.
  • the linker may be a "cleavable linker" that facilitates the release of a drug in a cell.
  • an acid labile linker e.g., hydrazone
  • a protease sensitive linker e.g., peptidase sensitive
  • photolabile linker e.g., peptidase sensitive
  • a dimethyl linker e.g., a disulfide-containing linker
  • ligand-drug conjugate refers to a ligand attached to a biologically active drug through a stable linking unit.
  • the "ligand-drug conjugate” is preferably an antibody-drug conjugate (ADC), which means that a monoclonal antibody or an antibody fragment is connected to a biologically active toxic drug through a stable linking unit. .
  • ADC antibody-drug conjugate
  • antibody refers to an immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds.
  • the amino acid composition and arrangement order of the constant region of the immunoglobulin heavy chain are different, so their antigenicity is also different.
  • immunoglobulins can be divided into five categories, or isotypes called immunoglobulins, that is, IgM, IgD, IgG, IgA, and IgE, and the corresponding heavy chains are ⁇ , ⁇ , and ⁇ chains, respectively. , Alpha, and epsilon chains.
  • Igs of the same class can be divided into different subclasses according to the difference in the amino acid composition of the hinge region and the number and position of heavy chain disulfide bonds.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • the light chain is divided into a kappa chain or a lambda chain by different constant regions.
  • Each of the five types of Ig can have a kappa chain or a lambda chain.
  • the sequence of about 110 amino acids near the N-terminus of the heavy and light chains of the antibody varies greatly and is a variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable and are constant regions.
  • the variable region includes three hypervariable regions (HVR) and four relatively conserved backbone regions (FR). Three hypervariable regions determine the specificity of an antibody, also known as complementarity determining regions (CDRs).
  • CDRs complementarity determining regions
  • Each light chain variable region (LCVR) and heavy chain variable region (HCVR) are composed of three CDR regions and four FR regions.
  • the sequence from the amino terminal to the carboxy terminal is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the three CDR regions of the light chain are referred to as LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3.
  • the CDR amino acid residues of the LCVR region and HCVR region of the antibody or antigen-binding fragment according to the present disclosure conform to the known Kabat numbering rules (LCDR1-3, HCDR2-3) or the numbering rules of Kabat and chothia. (HCDR1).
  • Fully human antibody or “fully human antibody”, also known as “fully human monoclonal antibody”, the variable and constant regions of the antibody are of human origin, removing immunogenicity and toxic side effects.
  • the development of monoclonal antibodies has gone through four stages: murine monoclonal antibodies, chimeric monoclonal antibodies, humanized monoclonal antibodies and fully human monoclonal antibodies.
  • Relevant technologies for the production of fully human antibodies include: human hybridoma technology, EBV transformed B lymphocyte technology, phage display technology, transgenic mouse antibody production technology, and single B cell antibody production technology.
  • the "fully human antibody” in the present disclosure is obtained using phage display technology. Phage display technology can isolate B cells from human PBMC, spleen, and lymph node tissues, construct a natural single-chain phage human antibody library, or screen the obtained antibodies by immunizing transgenic mice that can express the light and heavy chains of human antibodies.
  • antigen-binding fragment refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen. It has been shown that fragments of a full-length antibody can be used to perform the antigen-binding function of the antibody.
  • binding fragments included in the "antigen-binding fragment” include (i) a Fab fragment, a monovalent fragment composed of VL, VH, CL, and CH1 domains; (ii) a F (ab ') 2 fragment, which is included on the hinge region A bivalent fragment of two Fab fragments connected by a disulfide bridge; (iii) an Fd fragment composed of VH and CH1 domains; (iv) an Fv fragment composed of one-armed VH and VL domains; (v ) A single domain or dAb fragment (Ward et al.
  • Such single chain antibodies are also intended to be included in the term "antigen-binding fragment" of an antibody.
  • antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are functionally screened in the same manner as for intact antibodies.
  • Antigen-binding moieties can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact immunoglobulins.
  • the antibodies may be antibodies of different isotypes, for example, IgG (eg, IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.
  • Fab is an antibody fragment having a molecular weight of about 50,000 and having antigen-binding activity among fragments obtained by treating an IgG antibody molecule with a protease papain (amino acid residue at position 224 of the H chain), in which the N-terminal side of the H chain About half and the entire L chain are held together by disulfide bonds.
  • protease papain amino acid residue at position 224 of the H chain
  • F (ab ') 2 is an antibody having a molecular weight of about 100,000 and having antigen-binding activity and containing two Fab regions connected at hinge positions by digesting the lower part of two disulfide bonds in the IgG hinge region with the enzyme pepsin. Fragment.
  • Fab ' is an antibody fragment having a molecular weight of about 50,000 and having an antigen-binding activity obtained by cutting the disulfide bond of the hinge region of F (ab') 2 described above.
  • the Fab ' can be produced by inserting DNA encoding a Fab' fragment of the antibody into a prokaryotic expression vector or a eukaryotic expression vector and introducing the vector into a prokaryote or eukaryote to express the Fab '.
  • single chain antibody single chain Fv or “scFv” is meant to include an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) linked by a linker Molecule.
  • Such scFv molecules may have a general structure: NH 2 -VL-linker-VH-COOH or NH 2 -VH-linker-VL-COOH.
  • Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof, for example using 1-4 repeated variants (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA90: 6444-6448) .
  • linkers useful in the present disclosure are by Alfthan et al. (1995), Protein Eng. 8: 725-731, Choi et al. (2001), Eur. J. Immuno l. 31: 94-106, Hu et al. (1996) , Cancer Res. 56: 3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293: 41-56 and Roovers et al. (2001), Cancer Immunol.
  • CDR refers to one of the six hypervariable regions within the variable domain of an antibody that primarily contributes to antigen binding.
  • 6 CDRs One of the most commonly used definitions of the 6 CDRs is provided by Kabat E.A. et al. (1991) Sequences of protein of immunological interest. NIH Publication 91-3242).
  • the Kabat definition of CDRs applies only to CDR1, CDR2, and CDR3 of the light chain variable domain (CDR1, CDR2, CDR2, CDR3, or L1, L2, L3), and heavy chain variable domain CDR2 and CDR3 (CDR H2, CDR H3 or H2, H3).
  • antibody framework refers to a portion of a variable domain VL or VH that serves as a scaffold for the antigen-binding loop (CDR) of the variable domain. In essence, it is a variable domain without a CDR.
  • epitope refers to a site on an antigen to which an immunoglobulin or antibody specifically binds.
  • An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-contiguous amino acids in a unique spatial conformation. See, for example, Epitope Mapping Protocols Methods Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996).
  • antibodies bind with an affinity (KD) of less than about 10 -7 M, such as about less than 10 -8 M, 10 -9 M, or 10 -10 M or less.
  • nucleic acid molecule refers to both DNA molecules and RNA molecules.
  • the nucleic acid molecule may be single-stranded or double-stranded, but is preferably double-stranded DNA.
  • a nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, the promoter or enhancer is operatively linked to the coding sequence.
  • expression vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • the vector is a "plasmid”, which refers to a circular double-stranded DNA loop into which an additional DNA segment can be ligated.
  • the vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome.
  • the vectors disclosed herein are capable of autonomous replication in host cells into which they have been introduced (e.g., bacterial vectors and episomal mammalian vectors with bacterial origins of replication) or can be integrated into the host cell's genome after introduction into the host cell, thereby accompanying The host genome is replicated together (eg, non-episomal mammalian vectors).
  • Antigen-binding fragments can also be prepared by conventional methods.
  • the antibody or antigen-binding fragment according to the invention is genetically engineered to add one or more human-derived FR regions to a CDR region of non-human origin.
  • the human FR germline sequence can be obtained by aligning the IMGT human antibody variable region germline gene database with MOE software from the website of ImMunoGeneTics (IMGT) http://imgt.cines.fr, or from the Journal of Immunoglobulins, 2001ISBN012441351 obtain.
  • host cell refers to a cell into which an expression vector has been introduced.
  • Host cells may include bacterial, microbial, plant or animal cells. Easily transformed bacteria include members of the enterobacteriaceae, such as strains of Escherichiacoli or Salmonella; Bacilillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae.
  • Suitable microorganisms include Saccharomycescerevisiae and Pichia pastoris.
  • Suitable animal host cell lines include CHO (Chinese Hamster Ovary Cell Line) and NSO cells.
  • the engineered antibodies or antigen-binding fragments of the present disclosure can be prepared and purified using conventional methods.
  • cDNA sequences encoding heavy and light chains can be cloned and recombined into a GS expression vector.
  • the recombinant immunoglobulin expression vector can stably transfect CHO cells.
  • mammalian expression systems cause glycosylation of antibodies, especially the highly conserved N-terminal site in the Fc region. Positive clones were expanded in serum-free medium in the bioreactor to produce antibodies.
  • the culture medium in which the antibody is secreted can be purified by conventional techniques. For example, use A or G Sepharose FF columns with adjusted buffer for purification.
  • Non-specifically bound components are washed away.
  • the bound antibody was eluted by pH gradient method, and antibody fragments were detected by SDS-PAGE and collected.
  • the antibody can be concentrated by filtration using a conventional method. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves, ion exchange.
  • the resulting product needs to be immediately frozen, such as -70 ° C, or lyophilized.
  • peptide refers to a fragment of a compound between amino acids and proteins. It is formed by the connection of two or more amino acid molecules through peptide bonds. It is a structural and functional fragment of a protein, such as hormones and enzymes. Are peptides.
  • sucrose refers to a biological macromolecule composed of three elements: C, H, and O, and can be divided into monosaccharides, disaccharides, and polysaccharides.
  • fluorescent probe refers to the characteristic fluorescence in the ultraviolet-visible-near infrared region, and its fluorescent properties (excitation and emission wavelength, intensity, lifetime, polarization, etc.) can be dependent on the nature of the environment, such as polarity, refractive index A class of fluorescent molecules that are sensitively changed by changes in viscosity, viscosity, etc., which non-covalently interact with nucleic acid (DNA or RNA), protein, or other macromolecular structures to change one or more fluorescent properties, which can be used for research The nature and behavior of macromolecular substances.
  • toxic drug refers to a substance that inhibits or prevents the function of cells and / or causes cell death or destruction. These include toxins and other compounds that can be used in the treatment of tumors.
  • alkyl refers to a saturated aliphatic hydrocarbon group, which is a straight or branched chain group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 carbon atoms, and more preferably 1 to 10 carbon atoms Atom alkyl group, most preferably an alkyl group containing 1 to 6 carbon atoms (comprising 1, 2, 3, 4, 5, or 6 carbon atoms).
  • Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1 2,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2- Methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3 -Dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2 -Methylhexyl, 3-methylhexyl, 4-methylhex
  • lower alkyl groups containing 1 to 6 carbon atoms include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl Methyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethyl Butylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl Group, 2,3-dimethylbutyl and the like.
  • the alkyl group may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available point of attachment.
  • the substituent is preferably one or more of the following groups, which are independently selected from alkane Alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkane Oxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo.
  • heteroalkyl refers to an alkyl group containing one or more heteroatoms selected from N, O or S, wherein alkyl is as defined above.
  • alkylene refers to a saturated straight or branched chain aliphatic hydrocarbon group having 2 residues derived from the same carbon atom or two different carbon atoms of the parent alkane, which is derived from A straight or branched chain group containing 1 to 20 carbon atoms, preferably containing 1 to 12 carbon atoms, more preferably containing 1 to 6 carbon atoms (including 1, 2, 3, 4, 5 Or 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.
  • the alkylene group may be substituted or unsubstituted.
  • the substituent When substituted, the substituent may be substituted at any available point of attachment, and the substituent is preferably independently optionally selected from alkyl, alkenyl, and alkynyl. , Alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy , Cycloalkylthio, heterocycloalkylthio, and oxo.
  • alkoxy refers to -O- (alkyl) and -O- (unsubstituted cycloalkyl), wherein alkyl or cycloalkyl is as defined above.
  • alkoxy include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy.
  • the alkoxy group may be optionally substituted or unsubstituted.
  • the substituent is preferably one or more of the following groups, which are independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, and alkane Thio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio , Heterocycloalkylthio.
  • cycloalkyl refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent.
  • the cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, and more preferably 3 to 10 carbon atoms. Carbon atoms, most preferably 3 to 8 carbon atoms (including 3, 4, 5, 6, 7, or 8 carbon atoms).
  • Non-limiting examples of monocyclic cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatriene Groups, cyclooctyl and the like; polycyclic cycloalkyl groups include spiro, fused and bridged cycloalkyl.
  • heterocyclyl refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent that contains 3 to 20 ring atoms, one or more of which are selected from nitrogen, oxygen, or S (O) A heteroatom of m (where m is an integer of 0, 1 or 2), excluding the ring portion of -OO-, -OS-, or -SS-, and the remaining ring atoms are carbon.
  • m is an integer of 0, 1 or 2
  • a heteroatom of m is an integer of 0, 1 or 2
  • Non-limiting examples of monocyclic heterocyclyl include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, and the like.
  • Polycyclic heterocyclic groups include spiro, fused and bridged heterocyclic groups.
  • spiroheterocyclyl refers to a 5- to 20-membered monocyclic polycyclic heterocyclic group sharing a single atom (called a spiro atom), wherein one or more ring atoms are selected from nitrogen, oxygen, or S (O ) m (where m is an integer from 0 to 2) and the remaining ring atoms are carbon. It can contain one or more double bonds, but none of the rings have a completely conjugated ⁇ -electron system. It is preferably 6 to 14 yuan, and more preferably 7 to 10 yuan.
  • Spiroheterocyclyl is divided into monospiroheterocyclyl, bisspiroheterocyclyl or polyspiroheterocyclyl according to the number of common spiro atoms between the rings, preferably monospiroheterocyclyl and bisspiroheterocyclyl. More preferred are 4-membered / 4-membered, 4-membered-5-membered, 4-membered-6-membered, 5-membered / 5-membered, or 5-membered / 6-membered monospiroheterocyclyl.
  • Non-limiting examples of spiroheterocyclyl include:
  • fused heterocyclyl refers to a 5- to 20-membered polycyclic heterocyclic group in which each ring in the system shares an adjacent pair of atoms with other rings in the system.
  • One or more rings may contain one or more Double bond, but none of the rings have a completely conjugated ⁇ electron system, where one or more ring atoms are heteroatoms selected from nitrogen, oxygen or S (O) m (where m is the integer 0, 1 or 2), The remaining ring atoms are carbon. It is preferably 6 to 14 members, and more preferably 7 to 10 members (7-, 8-, 9-, or 10-membered rings).
  • fused heterocyclyl include:
  • bridged heterocyclyl refers to a 5- to 14-membered polycyclic heterocyclic group in which any two rings share two atoms that are not directly connected, which may contain one or more double bonds, but none of the rings have a total A y-electron system of a yoke in which one or more ring atoms are heteroatoms selected from nitrogen, oxygen, or S (O) m (where m is an integer of 0, 1, or 2), and the remaining ring atoms are carbon. It is preferably 6 to 14 members, and more preferably 7 to 10 members (7-, 8-, 9-, or 10-membered rings).
  • bridged heterocyclyls include:
  • the heterocyclic ring may be fused to an aryl, heteroaryl or cycloalkyl ring, wherein the ring connected to the parent structure is a heterocyclic group, and non-limiting examples include:
  • the heterocyclic group may be optionally substituted or unsubstituted.
  • the substituent is preferably one or more of the following groups, which are independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, and alkane Thio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio , Heterocycloalkylthio, oxo.
  • aryl refers to a 6 to 14 membered, all-carbon monocyclic or fused polycyclic (ie, rings that share adjacent pairs of carbon atoms) group having a conjugated pi electron system, preferably 6 to 10 members (6 members , 7-, 8-, 9-, or 10-membered), such as phenyl and naphthyl, preferably phenyl.
  • the aryl ring may be fused to a heteroaryl, heterocyclic or cycloalkyl ring, wherein the ring connected to the parent structure is an aryl ring, and non-limiting examples thereof include:
  • an aryl group may be substituted or unsubstituted.
  • the substituent is preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, Alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycle Alkylthio.
  • heteroaryl refers to a heteroaromatic system containing 1 to 4 heteroatoms (1, 2, 3, or 4 heteroatoms), 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur And nitrogen.
  • Heteroaryl is preferably 5- to 10-membered (5-, 6-, 7-, 8-, 9-, or 10-membered heteroaryl), more preferably 5- or 6-membered, such as furyl, thienyl, pyridyl , Pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl, and the like.
  • the heteroaryl ring may be fused to an aryl, heterocyclic or cycloalkyl ring, wherein the ring connected to the parent structure is a heteroaryl ring, and non-limiting examples include:
  • Heteroaryl may be optionally substituted or unsubstituted.
  • the substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkane Thio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio , Heterocycloalkylthio.
  • amino-protecting group is to protect the amino group with an easily removable group in order to keep the amino group unchanged during the reaction of other parts of the molecule.
  • Non-limiting examples include 9-fluorenylmethoxycarbonyl, tert-butoxycarbonyl, acetyl, benzyl, allyl, p-methoxybenzyl, and the like. These groups may be optionally substituted with 1 to 3 substituents (1, 2 or 3 substituents) selected from halogen, alkoxy or nitro.
  • the amino protecting group is preferably a 9-fluorenylmethoxycarbonyl group.
  • haloalkyl refers to an alkyl group substituted with one or more halogens, where alkyl is as defined above.
  • deuterated alkyl refers to an alkyl group substituted with one or more deuterium atoms, wherein alkyl is as defined above.
  • hydroxyalkyl refers to an alkyl group substituted with one or more hydroxyl groups, wherein alkyl is as defined above.
  • hydroxy refers to the -OH group.
  • halogen refers to fluorine, chlorine, bromine or iodine.
  • amino means -NH 2.
  • nitro refers to -NO 2.
  • cyano refers to -CN.
  • amido refers to -C (O) N (alkyl) or (cycloalkyl), wherein alkyl, cycloalkyl are as defined above.
  • carboxylate refers to -C (O) O (alkyl) or (cycloalkyl), wherein alkyl and cycloalkyl are as defined above.
  • the present disclosure also includes compounds of formula (I) in various deuterated forms. Each available hydrogen atom attached to a carbon atom can be independently replaced by a deuterium atom. Those skilled in the art can refer to the related literature to synthesize compounds of formula (I) in deuterated form. Commercially available deuterated starting materials can be used in the preparation of deuterated forms of compounds of formula (I), or they can be synthesized using conventional techniques using deuterated reagents, including but not limited to deuterated borane, trideuterated Borane tetrahydrofuran solution, lithium aluminum deuteride, deuterated iodoethane, and deuterated iodomethane.
  • an heterocyclic group optionally substituted with an alkyl group means that the alkyl group may but need not exist, and this description includes a case where the heterocyclic group is substituted with an alkyl group and a case where the heterocyclic group is not substituted with an alkyl group .
  • Substituted refers to one or more hydrogen atoms in a group, preferably up to five, more preferably one, two or three hydrogen atoms independently of one another by a corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and those skilled in the art can determine (by experiment or theory) possible or impossible substitutions without undue effort. For example, an amino or hydroxyl group having free hydrogen may be unstable when combined with a carbon atom having an unsaturated (eg, olefinic) bond.
  • pharmaceutical composition means a mixture containing one or more of the compounds described herein or a physiological / pharmaceutically acceptable salt or prodrug thereof with other chemical components, as well as other components such as physiological / pharmaceutically acceptable Carriers and excipients.
  • the purpose of the pharmaceutical composition is to promote the administration to the organism, which is beneficial to the absorption of the active ingredient and then exerts the biological activity.
  • pharmaceutically acceptable salt refers to a salt of a ligand-drug conjugate of the present disclosure, or a salt of a compound described in the present disclosure, when such salts are used in mammals It is safe and effective, and has due biological activity.
  • the ligand drug conjugate of the present disclosure contains at least one amino group, and therefore can form a salt with an acid.
  • Non-limiting examples of pharmaceutically acceptable salts include: hydrochloride, Hydrobromide, hydroiodate, sulfate, bisulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, citrate, hydrogen phosphate, dihydrogen phosphate Salt, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, mesylate, ethanesulfonate, benzenesulfonate, Tosylate.
  • solvate refers to a ligand-drug conjugate of the present disclosure that forms a pharmaceutically acceptable solvate with one or more solvent molecules.
  • solvent molecules include water, ethanol, acetonitrile, isopropanol , DMSO, ethyl acetate.
  • drug loading refers to the average number of cytotoxic drugs loaded on each ligand in a molecule of formula (I), and can also be expressed as the ratio of the amount of drug to the amount of antibody.
  • the range of drug loading can be per ligand (Pc) Connect 0-12, preferably 1-10 cytotoxic drugs (D).
  • the drug loading amount is expressed as n, which may also be referred to as a DAR value, and is exemplified as an average value of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.
  • Conventional methods such as UV / visible light spectroscopy, mass spectrometry, ELISA tests, and HPLC characteristics can be used to identify the average number of drugs per ADC molecule after the coupling reaction.
  • the cytotoxic drug is coupled to the N-terminal amino group of the ligand and / or the epsilon-amino group of the lysine residue through a linking unit.
  • the The number of drug molecules will be less than the theoretical maximum.
  • carrier refers to a system that can change the way a drug enters the body and its distribution in the body, control the rate of drug release, and deliver the drug to a targeted organ.
  • Drug carrier release and targeting systems can reduce drug degradation and loss, reduce side effects, and increase bioavailability.
  • polymer surfactants that can be used as carriers can self-assemble due to their unique amphiphilic structure to form various forms of aggregates.
  • Preferred examples are micelles, microemulsions, gels, liquid crystals, vesicles, etc. . These aggregates have the ability to encapsulate drug molecules, and at the same time have good permeability to the membrane, and can be used as excellent drug carriers.
  • excipient is an additive in a pharmaceutical formulation other than the main drug and may also be referred to as an excipient.
  • excipients such as binders, fillers, disintegrating agents, lubricants in tablets; matrix parts in semi-solid preparations ointments and creams; preservatives, antioxidants, flavoring agents, fragrances in liquid preparations, Solubilizers, emulsifiers, solubilizers, osmotic pressure regulators, colorants, etc. can all be called excipients.
  • the term "diluent” is also called a bulking agent, and its main purpose is to increase the weight and volume of a tablet.
  • the addition of the diluent not only ensures a certain volume size, but also reduces the dose deviation of the main components and improves the compression moldability of the drug.
  • an absorbent to absorb the oily substance, so as to keep the "dry” state, so as to facilitate the preparation of the tablet.
  • the pharmaceutical composition may be in the form of a sterile injectable aqueous solution.
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
  • the sterile injectable preparation may be a sterile injectable oil-in-water microemulsion in which the active ingredient is dissolved in the oil phase.
  • the active ingredient is dissolved in a mixture of soybean oil and lecithin.
  • the oil solution is then added to a mixture of water and glycerol to form a microemulsion. Injections or microemulsions can be injected into a patient's bloodstream by local, large injections.
  • solutions and microemulsions are preferably administered in a manner that maintains a constant circulating concentration of the compounds of the present disclosure.
  • continuous intravenous drug delivery devices can be used.
  • An example of such a device is the Deltec CADD-PLUS.TM. 5400 intravenous pump.
  • the pharmaceutical composition may be in the form of a sterile injectable water or oily suspension for intramuscular and subcutaneous administration.
  • This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension prepared in a non-toxic parenterally acceptable diluent or solvent, for example, a solution prepared in 1,3-butanediol.
  • a sterile fixed oil can be conveniently used as a solvent or suspension medium.
  • any blended fixing oil including synthetic mono- or diesters can be used.
  • fatty acids such as oleic acid can also be prepared for injection.
  • the present disclosure relates to a class of cleavable linking arms with specific structures and actives with specific structures, and antibody drug conjugates (ADCs) composed of linking arms, actives, and antibodies.
  • ADCs antibody drug conjugates
  • This type of ADC is a complex formed by attaching a toxic substance to an antibody via a spacer.
  • the antibody-coupled drug (ADC) is degraded in the body to release active molecules, thereby playing an anti-tumor role.
  • a method for preparing a compound represented by the general formula (Pc-L a -Y-Dr) includes the following steps:
  • the reducing agent is preferably TCEP, and particularly, it is preferred to reduce the Disulfide bond
  • Pc, W, L 2 , L 3 , R 1 , R 2 , R 5 to R 7 , m and n are as defined in the general formula (Pc-L a -Y-Dr).
  • the experimental methods without specific conditions in the examples of the present disclosure generally follow the conventional conditions or the conditions recommended by the raw material or commodity manufacturers.
  • the reagents without specific sources are conventional reagents purchased on the market.
  • the structure of the compound is determined by nuclear magnetic resonance (NMR) or mass spectrometry (MS).
  • NMR nuclear magnetic resonance
  • MS mass spectrometry
  • the NMR measurement was performed using Bruker AVANCE-400 nuclear magnetic analyzer.
  • the measurement solvents were deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl 3 ), and deuterated methanol (CD 3 OD).
  • the internal standard was tetramethyl.
  • Silyl (TMS) chemical shifts are given in units of 10 -6 (ppm).
  • MS was measured using a FINNIGAN LCQAd (ESI) mass spectrometer (manufacturer: Thermo, model: Finnigan LCQ advantage MAX).
  • UV-HPLC was measured using a Thermonano2000 UV spectrophotometer.
  • a PHERA starFS microplate reader (BMG, Germany) was used to measure the proliferation inhibition rate and IC 50 value.
  • the thin layer chromatography silica gel plate uses Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plate.
  • the silica gel plate used by thin layer chromatography (TLC) uses a size of 0.15mm to 0.2mm.
  • the thin layer chromatography purification product uses a size of 0.4mm ⁇ 0.5mm silicone board.
  • the known starting materials of the present disclosure can be synthesized by or in accordance with methods known in the art, or can be purchased from ABCR, GmbH, Co.KG, Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc., Dari Chemicals and other companies.
  • reaction is performed under an argon atmosphere or a nitrogen atmosphere.
  • An argon or nitrogen atmosphere means that the reaction flask is connected to an argon or nitrogen balloon with a volume of about 1 L.
  • the hydrogen atmosphere means that a reaction balloon is connected to a hydrogen gas balloon with a volume of about 1 L.
  • Pressurized hydrogenation reaction uses Parr 3916EKX type hydrogenation instrument and clear blue QL-500 type hydrogen generator or HC2-SS type hydrogenation instrument.
  • the hydrogenation reaction is usually evacuated and charged with hydrogen, and the operation is repeated 3 times.
  • the solution in the reaction refers to an aqueous solution.
  • reaction temperature is room temperature.
  • Room temperature is the most suitable reaction temperature, and the temperature range is from 20 ° C to 30 ° C.
  • the eluent system for column chromatography and the eluent system for thin layer chromatography used to purify compounds include: A: dichloromethane and isopropanol system, B: dichloromethane and methanol system, C: petroleum ether and
  • C petroleum ether
  • the volume ratio of the solvent in the ethyl acetate system is adjusted according to the polarity of the compound, and it can also be adjusted by adding a small amount of triethylamine and acidic or basic reagents.
  • Q-TOF LC / MS Some compounds of this disclosure are characterized by Q-TOF LC / MS.
  • the Q-TOF LC / MS uses an Agilent 6530 accurate mass quadrupole-time of flight mass spectrometer and an Agilent 1290-Infinity ultra high performance liquid chromatograph (Agilent Poroshell 300SB-C8 5 ⁇ m, 2.1 ⁇ 75mm column).
  • reaction was stirred at 0-5 ° C. for 1 hour. Remove the ice-water bath and heat to 30 ° C and stir for 2 hours.
  • the reaction solution was concentrated under reduced pressure, and the obtained crude compound 2 was purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5um19 * 250mm; mobile phase: A-water (10mmol NH 4 OAc), B-acetonitrile , Gradient elution, flow rate: 18 mL / min), the corresponding components were collected and concentrated under reduced pressure to give the title product (1.5 mg, 1.5 mg).
  • the reaction solution was concentrated under reduced pressure, and the obtained crude compound 3 was purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5um 19 * 250 mm; mobile phase: A-water (10 mmol NH 4 OAc): B- Acetonitrile, gradient elution, flow rate: 18 mL / min), the corresponding fractions were collected and concentrated under reduced pressure to give the title product (1.5 mg, 1.5 mg).
  • the 1-hydroxycyclopropane-1-carboxylic acid benzyl ester 4a (104 mg, 0.54 mmol; prepared by the method disclosed in the patent application "US2005 / 20645”) and 2-((((9H-fluoren-9-yl) Methoxy) carbonyl) amino) acetamino) methyl acetate 4b (100 mg, 0.27 mmol; prepared by the method disclosed in the patent application "CN105829346A”) was added to a reaction flask, 5 mL of tetrahydrofuran was added, and replaced with argon for three times, and ice Cool the water bath to 0-5 ° C, add potassium tert-butoxide (61mg, 0.54mmol), remove the ice bath, warm to room temperature and stir for 10 minutes, add 20mL ice water, and use ethyl acetate (5mL ⁇ 2) and chloroform (5mL ⁇ 5) Extract, combine the organic phases and concentrate.
  • reaction solution was purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5um 19 * 250mm; mobile phase: A-water (10mmol NH 4 OAc): B-acetonitrile, gradient elution, flow rate: 18mL / min), the corresponding components were collected and concentrated under reduced pressure to give the title product 4 (2 mg, yield: 39.0%).
  • the obtained residue was dissolved in 4 mL of dioxane, 2 mL of water was added, sodium bicarbonate (49.2 mg, 0.586 mmol) and 9-fluorenyl methyl chloroformate (126 mg, 0.49 mmol) were added, and the mixture was stirred at room temperature for 2 hours. 20 mL of water was added, extracted with ethyl acetate (10 mL ⁇ 3), and the organic phase was washed with a saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography as a developing system C to obtain the title product 5b (48 mg, yield: 19%).
  • reaction solution was purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5um19 * 250mm; mobile phase: A-water (10mmol NH 4 OAc): B-acetonitrile, gradient elution, flow rate: 18mL / min ), The corresponding components were collected and concentrated under reduced pressure to give the title product (2.4 mg, 1.7 mg).
  • reaction solution was purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5um19 * 250mm; mobile phase: A-water (10mmol NH 4 OAc) B-acetonitrile, gradient elution, flow rate: 18mL / min)
  • the corresponding components were collected and concentrated under reduced pressure to give the title product (2.7 mg, 2.6 mg).
  • the human B7H3 shown in SEQ ID NO: 1 is used as a template for the B7H3 of the present disclosure to design the amino acid sequences of the antigens and detection proteins involved in the present disclosure.
  • the following B7H3 antigens refer to human B7H3 unless otherwise specified.
  • B7H3 (SEQ ID NO: 1):
  • the double horizontal line is the signal peptide (Signal peptide: 1-28);
  • the cross-hatched part is the extracellular domain of B7H3 (Extra cellular domain: 29-466), where 29-139 are Ig-like V-type 1 domains, and 145-238 are Ig-like C2-type 1 domains; 243-357 Are Ig-like V-type 2 domains, and 363–456 are Ig-like C2-type 2 domains;
  • the dotted line part is the part of the transmembrane region (Transmembrane domain: 467-487);
  • Italics are intracellular domains (Cytoplasmic domain: 488-534).
  • the double horizontal line is the signal peptide (Signal peptide: 1-28);
  • the cross-hatched area is the extracellular domain of B7H3 (Extra cellular domain: 29-248), where 29–139 are Ig-like V-type domains and 145–238 are Ig-like C2-type domains;
  • the dotted line part is the part of the transmembrane region (Transmembrane domain: 249-269);
  • Italics are intracellular domains (Cytoplasmic domain: 270-316).
  • the horizontal line is the extracellular area of B7H3;
  • the italics are His-tag tags.
  • the horizontal line is the extracellular area of B7H3;
  • the italics are His-tag tags.
  • the horizontal line is the extracellular area of B7H3;
  • the italics are His-tag tags.
  • PBMC Human PBMC, spleen, and lymph node tissues were used to isolate B cells, and RNA was extracted to construct a natural single-chain phage antibody library (storage capacity 3.2 ⁇ 10 10 ).
  • the constructed single-stranded phage library was packaged to form phage particles, and then sieved by liquid phase method.
  • the phage was combined with biotinylated B7H3 in liquid phase, and then separated by streptavidin magnetic beads.
  • biotinylated human B7H3 and biological Primed rat B7H3 was screened alternately.
  • the first round was screened with 2 ⁇ g / ml biotinylated human B7H3.
  • the second round was screened with 2 ⁇ g / ml biotinylated rat B7H3.
  • the third round was screened with 0.5 ⁇ g / ml.
  • ml biotinylated human B7H3 was panned.
  • the clones with OD450 values greater than 0.5 measured by ELISA and ELISA OD450 values bound to human and murine B7H3 divided by ELISA OD450 values bound to 1% BSA both greater than 2.0 were sequenced to obtain the specific sequence 1702 (in this It is also called h1702 in the publication, and the antibodies h1702 and h1702DS referred to in the present disclosure are the same as h1702 and h1702-1 in the application PCT / CN2018 / 081249, and the entire contents of the application PCT / CN2018 / 081249 are incorporated into the present disclosure).
  • the phage library screen obtained the specific sequence 1702, and the process of constructing its complete monoclonal antibody was as follows:
  • primers were designed to construct the VH / VK / VL gene fragments of each single-chain antibody sequence.
  • a heavy and light chain variable region of 1702 was obtained.
  • the sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, FR sequences in italics, and CDR sequences underlined.
  • the antibody variable region was homologously recombined with the constant region gene (CH1-FC / CL) fragment to construct a complete antibody VH-CH1-FC / VK-CL / VL-CL.
  • the constructed full-length antibody 1702 sequence is as follows:
  • amino acid mutations were made to the light chain sequence of 1702.
  • the specific mutation was the first amino acid residue Q at the N-terminus of the light chain (SEQ ID NO: 15) was replaced by D, and the deletion mutation was the first at the C-terminus.
  • Amino acid residue S to obtain a more stable and uniform monoclonal antibody 1702DS (also referred to as h1702DS in this disclosure).
  • the mutation-modified 1702DS heavy chain sequence is SEQ ID NO: 14, and the amino acid sequence of the light chain is as follows: (SEQ ID NO: 16).
  • the plasmids expressing the light and heavy chains of the antibodies were respectively transfected into HEK293E cells at a ratio of 1.5: 1. After 6 days, the expression supernatant was collected, the impurities were removed by high-speed centrifugation, and purification was performed using a Protein A column. Flush the column with PBS until the A280 reading drops to baseline. The target protein was eluted with an acidic eluent of pH 3.0-pH 3.5, and neutralized with 1 M Tris-HCl, pH 8.0-9.0. The eluted sample was appropriately concentrated, and further purified using Superdex 200 (GE) gel chromatography equilibrated to remove the polymer, and the monomer peaks were collected and used for equipment.
  • GE Superdex 200
  • UV-Vis calculated average: n 6.87.
  • UV-Vis calculated average: n 6.11.
  • UV-Vis calculated average: n 6.33.
  • the Biacore, GE instrument was used to measure the anti-B7H3 antibody, and the reaction affinity between B7H3-ADC and human 2Ig-B7H3 antigen, human 4Ig-B7H3 antigen.
  • a biosensor chip Protein A (Cat. # 29127556, GE) was used to affinity capture a certain amount of antibody / ADC to be tested, and then the human 2Ig-B7H3 antigen (Cat. # 1949-B3-050 / CF, R & D), human 4Ig-B7H3 antigen (Cat. # 11188-H08H, Sino Biological), using Biacore instrument (Biacore T200, GE) to detect reaction signals in real time to obtain binding and dissociation curves. After the dissociation of each cycle was completed, the biochip was washed and regenerated with a glycine-hydrochloric acid regeneration solution (pH 1.5) (Cat. # BR-1003-54, GE). The buffer used in the experiment was HBS-EP buffer solution (pH 7.4) (Cat. # BR-1001-88, GE).
  • Test example 2 In vitro cell endocytosis experiment
  • the fluorescence signal of the antibody in the cell is detected, and the endocytosis effect of the antibody is evaluated according to the strength of the fluorescence signal.
  • the B7-H3 antibody and APC anti-human IgG Fc (Biolegend, 409306) were mixed at a molar ratio of 1: 2 and incubated on ice for 15 minutes.
  • the antibody mixture was incubated with 2 ⁇ 10 5 U87MG cells (human brain astrocyte tumor, Chinese Academy of Sciences Cell Bank, Catalog # TCHu138) on ice for 30 minutes, then the excess antibodies were washed off, and then the cells were transferred to pre-warming Incubate at 37 ° C for 0, 15, 30, 60, and 120 minutes at 37 ° C.
  • SD rats purchased from Jiejiejie Experimental Animal Co., Ltd.
  • male and female 12/12 hours light / dark adjustment, temperature 24 ⁇ 3 °C constant temperature, 50-60% humidity, free to eat and drink.
  • SD rats were injected with the test drug B7H3 antibody / ADC in the tail vein at a dose of 3 mg / kg and an injection volume of 5 ml / kg.
  • the blood collection time points were: 5 minutes, 8 hours, 24:00 (day 2), 3 days, 5 days, 8 days, 11 days, and 15 days after the administration on the first day.
  • Blood was taken intravenously at 200 ⁇ L each time (equivalent to 100 ⁇ L of serum); the collected blood samples were allowed to stand at room temperature for half an hour to agglutinate, and then centrifuged at 10000 ⁇ g for 10 minutes at 4 ° C. The supernatant was collected and immediately stored at -80 ° C.
  • the serum B7H3 antibody concentration was detected by ELISA, and the PK analysis was performed. The results are shown in Table 3.
  • the Agilent 6530Q-TOF was analyzed by LC-MS, and the potential modification sites were analyzed by mass spectrometry (see Table 4 for the results).
  • the results showed that none of the B7H3 antibodies h1702 involved in the present disclosure had any obvious deamidation, oxidation, or isomerization trend, suggesting good physical and chemical stability.
  • h1702 and h1702DS were tested by SEC, non-reducing CE-SDS analysis and detection method (pH 9.0) and IEX analysis and detection method.
  • Samples were processed using the Beckman SDS-MW Analysis Kit. Buffer was added to 100 ⁇ g of protein and denatured by heating. Data were collected using a PA800 capillary electrophoresis instrument.
  • Test example 6 In vitro cell proliferation experiment
  • U87MG cells human brain astrocyte tumor, Chinese Academy of Sciences cell bank, Catalog # TCHu138
  • Calu-6 cells lung cancer cells, ATCC, Catalog HTB-56 TM
  • Detroit562 cells human pharyngeal carcinoma cells ATCC, Catalog CCL-138 TM
  • A498 cells renal cancer cells, ATCC, Catalog HTB-44 TM
  • MEM medium MEM medium (GE, CAT # SH30024.01)
  • NEAA Sigma, CAT # M7145-100ML
  • sodium pyruvate solution sodium pyruvate solution
  • A-375 (melanoma cells, ATCC, Catalog CRL-1619 TM ) was cultured in DMEM (GE, SH30243.01) medium containing 10% FBS, and passaged 2-3 times a week at a passage ratio of 1: 3 or 1: 6.
  • CHO-K1 (does not express human B7H3, ATCC, Catalog CCL-61 (TM )) was cultured in F12 (Gibco, 11765-054) medium containing 10% FBS and passaged 2-3 times a week at a passage ratio of 1: 4 or 1: 6.
  • the samples to be tested were sequentially diluted to 9 concentrations with PBS or DMSO at a ratio of 3 times (the initial concentration of each ADC was 500 nM).
  • the sample is added to the culture plate, the plates were incubated for 6 days in an incubator (37 °C, 5% CO 2 ).
  • an incubator 37 °C, 5% CO 2 .
  • a 96-well cell culture plate add 90 ⁇ l of CellTiter-Glo reagent to each well, and store at room temperature for 10 minutes in the dark, read the chemiluminescence signal value in Victor3, and use GraphPad software to process the data.
  • the measured IC 50 values are shown in Table 6 and Figures 2A to 2F.
  • Test Example 7 Efficacy Evaluation of ADC of the Disclosure on Human Brain Astroblastoma U87MG Nude Mice Transplantation Tumor
  • BALB / cA-nude nude mice were used as test animals to evaluate the efficacy of the ADC compounds of the present disclosure on human brain astrocyte tumor U87MG nude mice transplanted tumors.
  • BALB / cA-nude nude mice SPF, female, purchased from Shanghai Jiesijie Experimental Animal Co., Ltd.
  • nude mice BALB / cA-nude, female, 6-7 weeks were subcutaneously inoculated with human brain astrocyte tumor U87MG cells (ibid.).
  • D0 human brain astrocyte tumor U87MG cells
  • Intraperitoneal injection was started once / week for a total of 3 times.
  • Tumor volume and weight were measured 2-3 times a week, and the data were recorded .
  • Tumor volume (V) is calculated as:
  • V 1/2 ⁇ a ⁇ b 2
  • a and b represent length and width respectively.
  • Relative volume (RTV) V T / V 0
  • Tumor inhibition rate (%) (C RTV -T RTV ) / C RTV (%)
  • V 0 and V T are the tumor volume at the beginning and end of the experiment, respectively.
  • C RTV and T RTV are the relative tumor volume of the control group (blank group) and the experimental group at the end of the experiment, respectively.
  • Intraperitoneal injection was administered once a week for a total of 3 times.
  • the tumor suppression rate of ADC-8 1mpk reached 39.22% (P ⁇ 0.01); the tumor suppression rate of ADC-8 3mpk reached 80.24% (P ⁇ 0.0001); ADC-5 tumor inhibition rate of 1mpk reached 27.53% (P ⁇ 0.05); ADC-5 tumor inhibition rate of 3mpk reached 55.88% (P ⁇ 0.0001). It was observed that at 22 days (D22), the tumor inhibition rate of each administration group was further increased.
  • the tumor inhibition rate of ADC-8 1mpk reached 47.7% (P ⁇ 0.0001); the tumor inhibition rate of ADC-8 3mpk reached 89.8% ( P ⁇ 0.0001); ADC-5 tumor inhibition rate of 1mpk reached 40.6% (P ⁇ 0.0001); ADC-5 tumor inhibition rate of 3mpk reached 63.3% (P ⁇ 0.0001).
  • Test Example 8 Efficacy Evaluation of ADC of the Disclosure on Human Pharyngeal Carcinoma Pleural Effusion Metastatic Cells Detroit 562 Nude Mice Transplantation Tumors
  • BALB / cA-nude nude mice were used as test animals to evaluate the efficacy of the disclosed ADC compounds on human pharyngeal carcinoma pleural fluid metastasis cells Detroit 562 nude mice transplanted tumors.
  • Negative control ADC a ligand toxin conjugate formed by coupling a non-B7H3 target with a reference compound (Example 58 in the patent "CN104755494A”)
  • BALB / cA-nude nude mice purchased from Changzhou Cavins Experimental Animal Co., Ltd.
  • mice Female, 6-7 weeks, were subcutaneously inoculated with human pharyngeal carcinoma pleural fluid metastasis cells Detroit 562 cells. On the tenth day after the cells were inoculated, the animals were randomly divided into groups (D0), with 8 animals in each group. Intraperitoneal injection was started once / week for a total of 3 times. The tumor volume and weight were measured 2-3 times a week, and the data were recorded. . Tumor volume (V) is calculated as:
  • V 1/2 ⁇ a ⁇ b 2
  • a and b represent length and width respectively.
  • Relative volume (RTV) V T / V 0
  • Tumor inhibition rate (%) (C RTV -T RTV ) / C RTV (%)
  • V 0 and V T are the tumor volume at the beginning and end of the experiment, respectively.
  • C RTV, T RTV respectively as the control group (negative control) at the end of the experiment and the relative tumor volume in the experimental group.
  • Intraperitoneal injection was administered once a week for a total of 3 times.
  • the tumor suppression rates of the tested ADCs were: ADC-1 tumor suppression rate of 1 mg / kg (1mpk) reached 40.85%; ADC-1 3mg / kg (3mpk) tumor inhibition rate reached 62.55% (P ⁇ 0.05); ADC-2 1mg / kg (1mpk) tumor inhibition rate reached 44.26%; ADC-23mg / kg (3mpk) tumor inhibition rate reached 72.27% (P ⁇ 0.01).
  • Test Example 9 In vitro Cell Proliferation of ADCs with Different Drug Loading
  • Test example 6 is the same.
  • the ADC-4 sample was mixed with human plasma, monkey plasma (Shanghai Medic Biomedical Co., Ltd.), and 1% BSA (Shanghai Shenggong) solution at a final concentration of 100 ⁇ g / ml. After filtering and sterilizing, incubate in a 37 ° C water bath and record the day of incubation as day 0, and then take out samples on days 7, 14, and 21, respectively, for the detection of free toxins.

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Abstract

本发明披露了抗B7H3抗体-依喜替康类似物偶联物、其制备方法，以及其抗肿瘤的医药用途。

Description

抗B7H3抗体-依喜替康类似物偶联物及其医药用途 技术领域
本公开涉及抗B7H3抗体-依喜替康类似物偶联物，其制备方法，包含其的药物组合物，以及其用于制备治疗B7H3介导的疾病或病症的药物中的用途；尤其在用于制备抗癌药物中的用途。
背景技术
T细胞介导的免疫反应在机体抗肿瘤过程中发挥着极其重要的作用，而T细胞的活化和增殖不仅需要TCR识别抗原信号，还需要共刺激分子提供的第二信号。B7家族分子属于共刺激分子免疫球蛋白超家族，越来越多的研究表明，该家族分子在机体正常免疫功能和病理状态下均发挥了重要的调节作用。
B7H3是B7家族的成员之一，属于I型跨膜蛋白，包含氨基端的一个信号肽，一个细胞外的免疫球蛋白样可变区(IgV)和恒定区(IgC)，一个跨膜区和一个含有45个氨基酸的胞质尾区(Tissue Antigens.2007Aug；70(2):96-104)。目前，B7H3主要存在2种剪切体，B7H3a和B7H3b。B7H3a胞外段由IgV-IgC 2个免疫球蛋白结构域组成，又称为2IgB7H3，而B7H3b胞外段由IgV-IgC-IgV-IgC 4个免疫球蛋白结构域组成，又称为4IgB7H3。
B7H3蛋白在正常组织、细胞中不表达或极低表达，却高表达于多种肿瘤组织，并与肿瘤的进展、患者的生存及预后密切相关。临床上已经报道，B7H3在许多癌症中，特别是在非小细胞肺癌、肾癌、泌尿道上皮癌、结直肠癌、前列腺癌、多形性胶质母细胞瘤、卵巢癌和胰腺癌中过表达(Lung Cancer.2009Nov；66(2):245-249；Clin Cancer Res.2008Aug 15；14(16):5150-5157)。此外，也有文献报道，在前列腺癌中，B7H3的表达强度与临床病理学恶性(诸如肿瘤体积、前列腺外侵袭或Gleason评分)正相关，且也与癌症进展相关(Cancer Res.2007Aug15；67(16):7893-7900)。类似地，在多形性胶质母细胞瘤中，B7H3的表达与无事件存活负相关，且在胰腺癌中，B7H3的表达与淋巴结转移和病理学进展相关。因此，B7H3被认为是一种新的肿瘤标志物和潜在的治疗靶点。
目前，已有针对B7H3靶点的治疗策略用于临床前研究，如靶向小鼠B7H3的抗体会增强瘤内的浸润性的CD8-阳性的T细胞和抑制肿瘤生长( Mod Pathol.2010Aug；23(8):1104-1112)。此外，WO 2008/066691显示，识别B7H3变体B7H3a的抗体会对腺癌表现出体内抗肿瘤作用。在临床研究中，一种鼠源的B7H3抗体与放射性I 131的偶联药物可显著抑制患者成神经母细胞瘤的生长[J Neufooocol 97(3):409-l8(2010)]。但目前在研的项目都是鼠源抗体经人源化改造的人源化抗体，而人源化抗体在免疫时存在免疫原性相对较高的问题，在人体应用时是一个不利的因素。
噬菌体展示技术(phage display technology)是将外源蛋白质或多肽与噬菌体外 壳蛋白融合表达，从而将外源蛋白表达在噬菌体的表面。噬菌体抗体库是将噬菌体展示技术、PCR扩增技术、蛋白表达技术相结合的一项运用综合技术手段所建立起来的抗体库。
噬菌体抗体库最大的优点是不经体内免疫，模拟体内抗体生成的三个过程而制备出全人源的抗体。除此之外，噬菌体抗体库还具有以下优势：①实现了基因型与表型的统一；此外，实验方法简单、快速，传统的通过杂交瘤技术抗体产生方法需历经数月，而抗体库技术只需短短几周的时间；②表达的是完全人抗体，且分子量小，主要以活性片段Fab、scFv的形式表达，与完整抗体相比在组织穿透力方面都有明显优势；③筛选容量大，杂交瘤技术是在上千个克隆内筛选，抗体库技术可以在百万甚至亿万个分子中进行选择，筛选到的抗体种类多；④用途广泛，采用了原核表达系统，当大规模生产时优势更加明显(Curr Opin Biotechnol.2002Dec；13(6):598-602；Immunotechnology,2013,48(13)48(13):63-73)。
抗体-药物偶联物(antibody drug conjugate,ADC)将单克隆抗体或者抗体片段通过稳定的化学接头化合物与具有生物活性的细胞毒素相连，充分利用了抗体对正常细胞和肿瘤细胞表面抗原结合的特异性和细胞毒性物质的高效性，同时又避免了前者疗效偏低和后者毒副作用过大等缺陷。这也就意味着，与以往传统的化疗药物相比，抗体-药物偶联物能更精准地结合肿瘤细胞并降低将对正常细胞的影响。
目前已有多种ADC药物被用于临床或临床研究，如Kadcyla，是靶向Her2的曲妥珠单抗与DM1形成的ADC药物。同时，也有靶向B7H3的抗体及ADC药物的专利报道，如WO2008100934、WO2012147713、WO2014061277、WO2015184203、WO2016044383。
用于抗体药物偶联物的具有细胞毒性的小分子有几类；其中有一类是喜树碱衍生物，它们通过抑制拓卜异构酶I而具有抗肿瘤作用的。报道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)应用于抗体偶联药物(ADC)的文献有WO2014057687；Clinical Cancer Research(2016)22(20)：5097-5108；Cancer Sci(2016)107:1039-1046。但仍需进一步开发疗效更好的ADC药物。
发明内容
本公开涉及抗B7H3抗体的ADC及其用途，其中提供与B7H3的胞外区的氨基酸序列或三维结构结合的单克隆抗体或抗原结合片段与细胞毒性物质依喜替康类似物偶联的ADC药物。
因此，本公开的目的为提供一种通式(Pc-L-Y-Dr)所示的配体-药物偶联物或 其药学上可接受的盐或溶剂化物：
Figure PCTCN2019107852-appb-000001
其中：
Y选自-O-(CR aR b) m-CR 1R 2-C(O)-、-O-CR 1R 2-(CR aR b) m-、-O-CR 1R 2-、-NH-(CR aR b) m-CR 1R 2-C(O)-或-S-(CR aR b) m-CR 1R 2-C(O)-；
R a和R b相同或不同，且各自独立地选自氢原子、氘原子、卤素、烷基、卤代烷基、氘代烷基、烷氧基、羟基、氨基、氰基、硝基、羟烷基、环烷基或杂环基；
或者，R a和R b与其相连接的碳原子一起形成环烷基或杂环基；
R 1选自卤素、卤代烷基、氘代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基或杂芳基；
R 2选自氢原子、卤素、卤代烷基、氘代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基或杂芳基；
或者，R 1和R 2与其相连接的碳原子一起形成环烷基或杂环基；
或者，R a和R 2与其相连的碳原子一起形成环烷基或杂环基；
m为0至4的整数；
n为1至10，任选自约1、2、3、4、5、6、7、8、9、10；可以为整数，也可以为小数；
L为接头单元；
Pc为抗B7H3抗体或其抗原结合片段。
本公开的一些实施方案中，提供的通式(Pc-L-Y-Dr)所示的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其中所述抗B7H3抗体或其抗原结合片段包含：
分别如SEQ ID NO：8、9和10氨基酸序列所示的重链HCDR1、HCDR2、HCDR3，或与SEQ ID NO：8、9和10所示的HCDR1、HCDR2和HCDR3分别具有3、2或1个氨基酸差异的HCDR变体；和
分别如SEQ ID NO：11、12和13氨基酸序列所示的轻链LCDR1、LCDR2和LCDR3，或与SEQ ID NO：11、12和13所示的LCDR1、LCDR2和LCDR3分别具有3、2或1个氨基酸差异的LCDR变体。
本公开的一些实施方案中，通式(Pc-L-Y-Dr)所示的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其中所述抗B7H3抗体或其抗体结合片段的轻链可 变区上的轻链FR区来源于人种系轻链序列或其突变序列，和/或重链可变区上的重链FR区来源于人种系重链序列或其突变序列。
本公开的一些实施方案中，通式(Pc-L-Y-Dr)所示的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其中所述抗B7H3抗体或其抗原结合片段包含选自如下所示的重链可变区和/或轻链可变区：
其中所述重链可变区氨基酸序列如SEQ ID NO：6所示或与其具有至少95％序列同一性，所述轻链可变区氨基酸序列如SEQ ID NO：7所示或与其具有至少95％序列同一性。
本公开的一些实施方案中，通式(Pc-L-Y-Dr)所示的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其中所述抗B7H3抗体或其抗原结合片段包含抗体恒定区；所述抗体恒定区的重链恒定区来源于人IgG1、IgG2、IgG3或IgG4或与其具有至少95％序列同一性，所述抗体恒定区的轻链恒定区来源于人抗体κ、λ链或与其具有至少95％序列同一性；优选地，所述重链恒定区的氨基酸序列源于人IgG1或与其具有至少95％序列同一性。
本公开的一些实施方案中，通式(Pc-L-Y-Dr)所示的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其中所述Pc为全长抗体；其中所述的全长抗体选自：
由SEQ ID NO:14所示的重链序列和SEQ ID NO:15所示的轻链序列组成的h1702抗体，和
由SEQ ID NO:14所示的重链序列和SEQ ID NO:16所示的轻链序列组成的h1702DS抗体。
本公开的一些实施方案中，通式(Pc-L-Y-Dr)所示的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其中所述抗原结合片段选自Fab、Fab'、F(ab')2、单链抗体(scFv)、二聚化的V区(双抗体)、二硫键稳定化的V区(dsFv)和包含CDR的肽的抗原结合片段。
本公开的一些实施方案中，通式(Pc-L-Y-Dr)所示的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其中，R 1为卤代烷基或C 3-6环烷基。
本公开的一些实施方案中，通式(Pc-L-Y-Dr)所示的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其中，R 2为氢原子。
本公开的一些实施方案中，通式(Pc-L-Y-Dr)所示的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其中，R 1为C 3-6环烷基；R 2为氢原子。
本公开的一些实施方案中，通式(Pc-L-Y-Dr)所示的配体-药物偶联物或其药学上可接受的盐或溶剂化物，
其中：
Y为-O-(CR aR b) m-CR 1R 2-C(O)-；
R a和R b相同或不同，且各自独立地选自氢原子、氘原子、卤素或烷基；
R 1为卤代烷基或C 3-6环烷基；
R 2选自氢原子、卤代烷基或C 3-6环烷基；
或者，R 1和R 2与其相连接的碳原子一起形成C 3-6环烷基；
m为0或1。
本公开的一些实施方案中，通式(Pc-L-Y-Dr)所示的配体-药物偶联物或其药学上可接受的盐或溶剂化物，
其中：
Y为-O-(CR aR b) m-CR 1R 2-C(O)-；
R a和R b相同或不同，且各自独立地选自氢原子、氘原子、卤素或烷基；
R 1为C 3-6环烷基；
R 2选自氢原子；
或者，R 1和R 2与其相连接的碳原子一起形成C 3-6环烷基；
m为0。
本公开的一些实施方案中，通式(Pc-L-Y-Dr)所示的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其中Y选自：
Figure PCTCN2019107852-appb-000002
Figure PCTCN2019107852-appb-000003
优选
Figure PCTCN2019107852-appb-000004
Figure PCTCN2019107852-appb-000005
最优选
Figure PCTCN2019107852-appb-000006
本公开的一些实施方案中，通式(Pc-L-Y-Dr)所示的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其中Y的O端与接头单元L相连。
本公开的一些实施方案中，通式(Pc-L-Y-Dr)所示的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其中n为2至8，优选为5至9，最优选为7.5；非限制性实施例包括3、4、5、6、7.2、7.5、8、8.5、9。
本公开的一些实施方案中，通式(Pc-L-Y-Dr)所示的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其中接头单元-L-为-L 1-L 2-L 3-L 4-，
L 1为
Figure PCTCN2019107852-appb-000007
s 1为2至8的整数；
L 2为化学键；
L 3为四肽残基；
L 4为-NR 5(CR 6R 7)t-，R 5、R 6或R 7相同或不同，且各自独立地为氢原子或烷基，t为1或2。
本公开的一些实施方案中，通式(Pc-L-Y-Dr)所示的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其中所述的接头单元-L-，其L 1端与配体相连，L 4端与Y相连。
本公开的一些实施方案中，通式(Pc-L-Y-Dr)所示的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其中所述的L 3的四肽残基为由两个或多个选自苯丙氨酸(E)、甘氨酸(G)、缬氨酸(V)、赖氨酸(K)、瓜氨酸、丝氨酸(S)、谷氨酸(E)、天冬氨酸(N)中的氨基酸形成的氨基酸残基；优选为GGFG(甘氨酸-甘氨酸-苯丙氨酸-甘氨酸)的四肽残基。
本公开的一些实施方案中，通式(Pc-L-Y-Dr)所示的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其中所述-L-Y-为以下结构：
Figure PCTCN2019107852-appb-000008
L 2为化学键；
L 3为GGFG的四肽残基；
R 1为卤代烷基或C 3-6环烷基；
R 2选自氢原子、卤代烷基或C 3-6环烷基；
或者，R 1和R 2与其相连接的碳原子一起形成C 3-6环烷基；
R 5、R 6或R 7相同或不同，且各自独立地为氢原子或烷基；
s 1为2至8的整数；
m为0至4的整数。
本公开的一些实施方案中，通式(Pc-L-Y-Dr)所示的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其中-L-Y-选自：
Figure PCTCN2019107852-appb-000009
Figure PCTCN2019107852-appb-000010
本公开的一些实施方案中，通式(Pc-L-Y-Dr)所示的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其为通式(Pc-L a-Y-Dr)所示的配体-药物偶联物或其药学上可接受的盐或溶剂化物：
Figure PCTCN2019107852-appb-000011
其中：
W选自C 1-8烷基、C 1-8烷基-环烷基或1至8个原子的直链杂烷基，所述杂烷基包含1至3个选自N、O或S的杂原子，其中所述的C 1-8烷基、环烷基和直链杂烷基各自独立地任选进一步被选自卤素、羟基、氰基、氨基、烷基、氯代烷基、氘代烷基、烷氧基和环烷基的一个或多个取代基所取代；
L 2选自-NR 4(CH 2CH 2O) p1CH 2CH 2C(O)-、-NR 4(CH 2CH 2O) p1CH 2C(O)-、-S(CH 2) p1C(O)-或化学键，p 1为1至20的整数，优选1-6；
L 3为由2至7个氨基酸构成的肽残基，氨基酸可以是取代的或非取代的，当被取代时，取代基可以在任何可使用的连接点上被取代，所述取代基为一个或多个独立地选自卤素、羟基、氰基、氨基、烷基、氯代烷基、氘代烷基、烷氧基和环烷基；
R 1选自卤素、卤代烷基、氘代烷基、环烷基、杂环基、芳基或杂芳基；
R 2选自氢原子、卤素、卤代烷基、氘代烷基、环烷基、杂环基、芳基或杂芳基；
或者，R 1和R 2与其相连接的碳原子一起形成环烷基或杂环基；
R 4和R 5相同或不同，且各自独立地选自氢原子、烷基、卤代烷基、氘代烷基和羟烷基；
R 6和R 7相同或不同，且各自独立地选自氢原子、卤素、烷基、卤代烷基、氘代烷基和羟烷基；
m为0至4的整数；
n为1至10，可以为整数，也可以为小数；
Pc为抗B7H3抗体或其抗原结合片段。
本公开的一些实施方案中，通式(Pc-L-Y-Dr)所示的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其为通式(Pc-L b-Y-Dr)所示的配体-药物偶联物或其药学上可接受的盐或溶剂化物：
Figure PCTCN2019107852-appb-000012
其中：
s 1为2至8的整数；优选5；
R 1、R 2、R 5～R 7、m和n如通式(Pc-L a-Y-Dr)中所定义。
本公开的一些实施方案中，通式(Pc-L-Y-Dr)所示的的配体-药物偶联物或其药学上可接受的盐或溶剂化物，所述配体-药物偶联物选自：
Figure PCTCN2019107852-appb-000013
Figure PCTCN2019107852-appb-000014
Figure PCTCN2019107852-appb-000015
其中Pc和n如通式(Pc-L-Y-Dr)中所定义。
本公开典型的通式(Pc-L-Y-Dr)所示的配体-药物偶联物包括，但不限于：
Figure PCTCN2019107852-appb-000016
Figure PCTCN2019107852-appb-000017
Figure PCTCN2019107852-appb-000018
或其药学上可接受的盐或溶剂化物；
其中n可以为0至10的非零整数或小数，优选为1-10之间的整数或小数；更优选为2至8，可以为整数，也可以为小数；最优选为3至8，可以为整数，也可以为小数。
本公开进一步提供一种制备如通式(Pc-L a-Y-Dr)所示的配体-药物偶联物或其药学上可接受的盐或溶剂化物的方法，其包括以下步骤：
Figure PCTCN2019107852-appb-000019
Pc还原后，与通式(L a-Y-Dr)偶联反应，得到通式(Pc-L a-Y-Dr)所示的化合物；所述的还原剂优选TCEP；
其中：
Pc为抗B7H3抗体或其抗原结合片段；
W、L 2、L 3、R 1、R 2、R 5～R 7、m和n如通式(Pc-L a-Y-Dr)中所定义。
在另一个实施方式中，提供另一种方法，其中所述的通式L a-Y-Dr为通式L b-Y-Dr所示的化合物：
Figure PCTCN2019107852-appb-000020
或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐，
其中R 1、R 2、R 5～R 7、s 1和m如通式Pc-L b-Y-Dr中所定义。
在本公开的一个优选的实施方案中，根据本公开所述的通式(Pc-L a-Y-Dr)或通式(Pc-L b-Y-Dr)所示的配体-药物偶联物或其药学上可接受的盐或溶剂化物的方法，其中通式(L a-Y-Dr)所示的化合物或通式(L b-Y-Dr)所示的化合物选自：
Figure PCTCN2019107852-appb-000021
Figure PCTCN2019107852-appb-000022
另一方面，本公开提供一种药物组合物，其包含根据本公开所述的抗体-药物偶联物或其药学上可接受的盐或溶剂化物，以及一种或多种可药用的赋形剂、稀释剂或载体。
另一方面，本公开提供根据本公开所述的抗体-药物偶联物或其药学上可接受的盐或溶剂化物或包含其的药物组合物在制备用于治疗B7H3介导的疾病或病症的药物中的用途。其中所述B7H3介导的疾病或病症为B7H3高表达癌症。
另一方面，本公开提供根据本公开所述的配体-药物偶联物或其药学上可接受的盐或溶剂化物或包含其的药物组合物在制备用于治疗或预防肿瘤的药物中的用途；其中所述癌症优选选自乳腺癌、卵巢癌、宫颈癌、肺癌、子宫癌、前列腺癌、肾癌、尿道癌、膀胱癌、卵巢癌、肝癌、胃癌、子宫内膜癌、唾液腺癌、食道癌、黑色素瘤、神经胶质瘤、神经母细胞瘤、肉瘤、咽头癌、肺癌、结肠癌、直肠癌、结直肠癌、白血病、骨癌、皮肤癌、甲状腺癌、胰腺癌或淋巴瘤。
另一方面，本公开进一步涉及一种用于治疗和/或预防肿瘤的方法，该方法包括向需要其的患者施用治疗有效剂量的根据本公开所述的配体-药物偶联物或其药学上可接受的盐或溶剂化物或包含其的药物组合物；优选其中所述的肿瘤为与B7H3高表达相关的癌症。
另一方面，本公开进一步涉及一种用于治疗或预防癌症的方法，该方法包括向需要其的患者施用治疗有效剂量的根据本公开所述的配体-药物偶联物或其药学上可接受的盐或溶剂化物或包含其的药物组合物；其中所述癌症优选选自乳腺癌、卵巢癌、宫颈癌、肺癌、子宫癌、前列腺癌、肾癌、尿道癌、膀胱癌、卵巢癌、 肝癌、胃癌、子宫内膜癌、唾液腺癌、食道癌、黑色素瘤、神经胶质瘤、神经母细胞瘤、肉瘤、咽头癌、肺癌、结肠癌、直肠癌、结直肠癌、白血病、骨癌、皮肤癌、甲状腺癌、胰腺癌或淋巴瘤。
可将活性化合物制成适合于通过任何适当途径给药的形式，活性化合物优选是以单位剂量的方式，或者是以患者可以以单剂自我给药的方式。本公开化合物或组合物的单位剂量的表达方式可以是片剂、胶囊、扁囊剂、瓶装药水、药粉、颗粒剂、锭剂、栓剂、再生药粉或液体制剂。
本公开治疗方法中所用化合物或组合物的剂量通常将随疾病的严重性、患者的体重和化合物的相对功效而改变。不过，作为一般性指导，合适的单位剂量可以是0.1～1000mg。
本公开的药物组合物除活性化合物外，可含有一种或多种辅料，所述辅料选自以下成分：填充剂(稀释剂)、粘合剂、润湿剂、崩解剂或赋形剂等。根据给药方法的不同，组合物可含有0.1至99重量％的活性化合物。
附图说明
图1：B7H3抗体在U87MG细胞上的内吞效果。
图2A-图2F：本公开ADC对不同肿瘤细胞的增殖抑制结果。图2A为不同ADC在A498细胞上的增殖抑制实验结果；图2B为不同ADC在Calu-6细胞上的增殖抑制实验结果；图2C为不同ADC在U87细胞上的增殖抑制实验结果；图2D为不同ADC在A375细胞上的增殖抑制实验结果；图2E为不同ADC在Detroit562细胞上的增殖抑制实验结果，图2F为不同ADC在CHOK1细胞上的增殖抑制实验结果。
图3：测试例7中腹腔注射本公开ADC-8(1mpk，3mpk)和ADC-5(1mpk，3mpk)对U87MG裸小鼠移植瘤的抑制作用。
图4：测试例8中腹腔注射本公开ADC-2(1mpk，3mpk)和ADC-1(1mpk，3mpk)对Detroit 562裸小鼠移植瘤的抑制作用。
图5A：测试例9中本公开ADC-4、ADC-6和ADC-7在Detroit562细胞上的增殖抑制率。
图5B：测试例9中本公开ADC-4、ADC-6和ADC-7在Calu-6细胞上的增殖抑制率。
图5C：测试例9中本公开ADC-4、ADC-6和ADC-7在CHOK12细胞上的增殖抑制率。
图6：测试例10中本公开ADC-4的血浆稳定性结果。
具体实施方式
发明详述
一、术语
除非另有限定，本文所用的所有技术和科学术语均与本公开所属领域普通技术人员的通常理解一致。虽然也可采用与本文所述相似或等同的任何方法和材料实施或测试本公开，但本文描述了优选的方法和材料。描述和要求保护本公开时，依据以下定义使用下列术语。
当本公开中使用商品名时，申请人旨在包括该商品名产品的制剂、该商品名产品的非专利药和活性药物部分。
除非有相反陈述，在说明书和权利要求书中使用的术语具有下述含义。
术语“药物”是指细胞毒性药物，药物表示为Dr，能在肿瘤细胞内具有较强破坏其正常生长的化学分子。细胞毒性药物原则上在足够高的浓度下都可以杀死肿瘤细胞，但是由于缺乏特异性，在杀伤肿瘤细胞的同时，也会导致正常细胞的凋亡，导致严重的副作用。该术语包括毒素，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At 211、I 131、I 125、Y 90、Re 186、Re 188、Sm 153、Bi 212、P 32和Lu的放射性同位素)，毒性药物，化疗药物，抗生素和核溶酶，优选为毒性药物。
术语“接头单元(或连接片段)”是指一端与配体连接而另一端与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药物相连。本公开的优选方案表示为L和L 1至L 4，其中L 1端与配体相连，L 4端与结构单元Y相连后与药物(Dr)相连。
接头，包括延伸物、间隔物和氨基酸单元，可以通过本领域已知方法合成，诸如US2005-0238649A1中所记载的。接头可以是便于在细胞中释放药物的“可切割接头”。例如，可使用酸不稳定接头(例如腙)、蛋白酶敏感(例如肽酶敏感)接头、光不稳定接头、二甲基接头、或含二硫化物接头(Chari等,Cancer Research 52:127-131(1992)；美国专利No.5,208,020)。
术语“配体-药物偶联物”指配体通过稳定的连接单元与具有生物活性的药物相连。在本公开中“配体-药物偶联物”优选为抗体-药物偶联物(antibody drug conjugate，ADC)，指将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毒性药物相连。
本公开所用氨基酸三字母代码和单字母代码如J.biol.chem,243,p3558(1968)中所述。
术语“抗体”指免疫球蛋白，是由两条相同的重链和两条相同的轻链通过链间二硫键连接而成的四肽链结构。免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将免疫球蛋白分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、α链、和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4。轻链 通过恒定区的不同分为κ链或λ链。五类Ig中每类Ig都可以有κ链或λ链。
抗体重链和轻链靠近N端的约110个氨基酸的序列变化很大，为可变区(Fv区)；靠近C端的其余氨基酸序列相对稳定，为恒定区。可变区包括3个高变区(HVR)和4个序列相对保守的骨架区(FR)。3个高变区决定抗体的特异性，又称为互补性决定区(CDR)。每条轻链可变区(LCVR)和重链可变区(HCVR)由3个CDR区4个FR区组成，从氨基端到羧基端依次排列的顺序为：FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。轻链的3个CDR区指LCDR1、LCDR2、和LCDR3；重链的3个CDR区指HCDR1、HCDR2和HCDR3。本公开所述的抗体或抗原结合片段的LCVR区和HCVR区的CDR氨基酸残基在数量和位置符合已知的Kabat编号规则(LCDR1-3，HCDR2-3)，或者符合kabat和chothia的编号规则(HCDR1)。
术语“完全人源抗体”或“全人抗体”，也称“全人源单克隆抗体”，其抗体的可变区和恒定区都是人源的，去除免疫原性和毒副作用。单克隆抗体的发展经历了四个阶段，分别为：鼠源性单克隆抗体、嵌合性单克隆抗体、人源化单克隆抗体和全人源单克隆抗体。全人源抗体制备的相关技术主要有：人杂交瘤技术、EBV转化B淋巴细胞技术、噬菌体显示技术(phage display)、转基因小鼠抗体制备技术(transgenic mouse)和单个B细胞抗体制备技术等。本公开中的“完全人抗体”采用噬菌体展示技术获得。噬菌体展示技术，从人PBMC、脾脏、淋巴结组织分离B细胞，构建天然单链噬菌体人抗体库，或者通过免疫可表达人抗体轻重链的转基因小鼠，筛选获得的抗体。
术语“抗原结合片段”是指抗体的保持特异性结合抗原的能力的一个或多个片段。已显示可利用全长抗体的片段来进行抗体的抗原结合功能。“抗原结合片段”中包含的结合片段的实例包括(i)Fab片段，由VL、VH、CL和CH1结构域组成的单价片段；(ii)F(ab') 2片段，包含通过铰链区上的二硫桥连接的两个Fab片段的二价片段；(iii)由VH和CH1结构域组成的Fd片段；(iv)由抗体的单臂的VH和VL结构域组成的Fv片段；(v)单结构域或dAb片段(Ward等人,(1989)Nature341：544-546)，其由VH结构域组成；和(vi)分离的互补决定区(CDR)或(vii)可任选地通过合成的接头连接的两个或更多个分离的CDR的组合。此外，虽然Fv片段的两个结构域VL和VH由分开的基因编码，但可使用重组方法，通过合成的接头连接它们，从而使得其能够产生为其中VL和VH区配对形成单价分子的单个蛋白质链(称为单链Fv(scFv)；参见，例如，Bird等人(1988)Science242:423-426；和Huston等人(1988)Proc.Natl.Acad.Sci USA85:5879-5883)。此类单链抗体也意欲包括在术语抗体的“抗原结合片段”中。使用本领域技术人员已知的常规技术获得此类抗体片段，并且以与对于完整抗体的方式相同的方式就功用性筛选片段。可通过重组DNA技术或通过酶促或化学断裂完整免疫球蛋白来产生抗原结合部分。抗体可以是不同同种型的抗体，例如，IgG(例如，IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或 IgM抗体。
Fab是通过用蛋白酶木瓜蛋白酶(切割H链的224位的氨基酸残基)处理IgG抗体分子所获得的片段中的具有约50,000的分子量并具有抗原结合活性的抗体片段，其中H链N端侧的约一半和整个L链通过二硫键结合在一起。
F(ab')2是通过用酶胃蛋白酶消化IgG铰链区中两个二硫键的下方部分而获得的分子量为约100,000并具有抗原结合活性并包含在铰链位置相连的两个Fab区的抗体片段。
Fab'是通过切割上述F(ab')2的铰链区的二硫键而获得的分子量为约50,000并具有抗原结合活性的抗体片段。
此外，可以通过将编码抗体的Fab'片段的DNA插入到原核生物表达载体或真核生物表达载体中并将载体导入到原核生物或真核生物中以表达Fab'来生产所述Fab'。
术语“单链抗体”、“单链Fv”或“scFv”意指包含通过接头连接的抗体重链可变结构域(或区域；VH)和抗体轻链可变结构域(或区域；VL)的分子。此类scFv分子可具有一般结构：NH 2-VL-接头-VH-COOH或NH 2-VH-接头-VL-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成，例如使用1-4个重复的变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA90:6444-6448)。可用于本公开的其他接头由Alfthan等人(1995)，Protein Eng.8:725-731，Choi等人(2001)，Eur.J.Immuno l.31:94-106，Hu等人(1996)，Cancer Res.56:3055-3061，Kipriyanov等人(1999)，J.Mol.Biol.293:41-56和Roovers等人(2001)，Cancer Immunol.描述。
术语“CDR”是指抗体的可变结构域内主要促成抗原结合的6个高变区之一。所述6个CDR的最常用的定义之一由Kabat E.A.等人，(1991)Sequences of proteins of immunological interest.NIH Publication91-3242)提供。如本文中使用的，CDR的Kabat定义只应用于轻链可变结构域的CDR1、CDR2和CDR3(CDR L1、CDR L2、CDR L3或L1、L2、L3)，以及重链可变结构域的CDR2和CDR3(CDR H2、CDR H3或H2、H3)。
术语“抗体框架”，是指可变结构域VL或VH的一部分，其用作该可变结构域的抗原结合环(CDR)的支架。从本质上讲，其是不具有CDR的可变结构域。
术语“表位”或“抗原决定簇”是指抗原上免疫球蛋白或抗体特异性结合的部位。表位通常以独特的空间构象包括至少3、4、5、6、7、8、9、10、11、12、13、14或15个连续或非连续的氨基酸。参见，例如，Epitope Mapping Protocols in Methods in Molecular B iology，第66卷，G.E.Morris，Ed.(1996)。
术语“特异性结合”、“选择性结合”、“选择性地结合”和“特异性地结合”是指抗体对预先确定的抗原上的表位的结合。通常，抗体以大约小于 10 -7M，例如大约小于10 -8M、10 -9M或10 -10M或更小的亲和力(KD)结合。
术语“核酸分子”是指DNA分子和RNA分子。核酸分子可以是单链或双链的，但优选是双链DNA。当将核酸与另一个核酸序列置于功能关系中时，核酸是“有效连接的”。例如，如果启动子或增强子影响编码序列的转录，那么启动子或增强子有效地连接至所述编码序列。
术语“表达载体”是指能够运输已与其连接的另一个核酸的核酸分子。在一个实施方案中，载体是“质粒”，其是指可将另外的DNA区段连接至其中的环状双链DNA环。在另一个实施方案中，载体是病毒载体，其中可将另外的DNA区段连接至病毒基因组中。本文中公开的载体能够在已引入它们的宿主细胞中自主复制(例如，具有细菌的复制起点的细菌载体和附加型哺乳动物载体)或可在引入宿主细胞后整合入宿主细胞的基因组，从而随宿主基因组一起复制(例如，非附加型哺乳动物载体)。
现有技术中熟知生产和纯化抗体和抗原结合片段的方法，如冷泉港的抗体实验技术指南，5-8章和15章。抗原结合片段同样可以用常规方法制备。发明所述的抗体或抗原结合片段用基因工程方法在非人源的CDR区加上一个或多个人源FR区。人FR种系序列可以通过比对IMGT人类抗体可变区种系基因数据库和MOE软件，从ImMunoGeneTics(IMGT)的网站http://imgt.cines.fr得到，或者从免疫球蛋白杂志，2001ISBN012441351上获得。
术语“宿主细胞”是指已向其中引入了表达载体的细胞。宿主细胞可包括细菌、微生物、植物或动物细胞。易于转化的细菌包括肠杆菌科(enterobacteriaceae)的成员，例如大肠杆菌(Escherichia coli)或沙门氏菌(Salmonella)的菌株；芽孢杆菌科(Bacillaceae)例如枯草芽孢杆菌(Bacillus subtilis)；肺炎球菌(Pneumococcus)；链球菌(Streptococcus)和流感嗜血菌(Haemophilus influenzae)。适当的微生物包括酿酒酵母(Saccharomyces cerevisiae)和毕赤酵母(Pichia pastoris)。适当的动物宿主细胞系包括CHO(中国仓鼠卵巢细胞系)和NS0细胞。
本公开工程化的抗体或抗原结合片段可用常规方法制备和纯化。比如，编码重链和轻链的cDNA序列，可以克隆并重组至GS表达载体。重组的免疫球蛋白表达载体可以稳定地转染CHO细胞。作为一种更推荐的现有技术，哺乳动物类表达系统会导致抗体的糖基化，特别是在Fc区的高度保守N端位点。阳性的克隆在生物反应器的无血清培养基中扩大培养以生产抗体。分泌了抗体的培养液可以用常规技术纯化。比如，用含调整过的缓冲液的A或G Sepharose FF柱进行纯化。洗去非特异性结合的组分。再用PH梯度法洗脱结合的抗体，用SDS-PAGE检测抗体片段，收集。抗体可用常规方法进行过滤浓缩。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛、离子交换。得到的产物需立即冷冻，如-70℃，或者冻干。
术语“肽”是指介于氨基酸和蛋白质之间的化合物片段，由2个或2个以上 氨基酸分子通过肽键相互连接而成，是蛋白质的结构与功能片段，如激素、酶类等本质上都是肽。
术语“糖”是指由C、H、O三种元素组成的生物大分子，可分为单糖、二糖和多糖等。
术语“荧光探针”是指在紫外-可见-近红外区有特征荧光，并且其荧光性质(激发和发射波长、强度、寿命和偏振等)可随所处环境的性质，如极性、折射率、粘度等改变而灵敏地改变的一类荧光性分子，其与核酸(DNA或RNA)、蛋白质或其他大分子结构非共价相互作用而使一种或几种荧光性质发生改变，可用于研究大分子物质的性质和行为。
术语“毒性药物”是指抑制或防止细胞的功能和/或引起细胞死亡或破坏的物质。包括毒素和其他能用于肿瘤治疗的化合物。
术语“烷基”指饱和脂肪族烃基团，其为包含1至20个碳原子的直链或支链基团，优选含有1至12个碳原子的烷基，更优选含有1至10个碳原子的烷基，最优选含有1至6个碳原子(包含1个、2个、3个、4个、5个或6个碳原子)的烷基。非限制性实例包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基、1,1-二甲基丙基、1,2-二甲基丙基、2,2-二甲基丙基、1-乙基丙基、2-甲基丁基、3-甲基丁基、正己基、1-乙基-2-甲基丙基、1,1,2-三甲基丙基、1,1-二甲基丁基、1,2-二甲基丁基、2,2-二甲基丁基、1,3-二甲基丁基、2-乙基丁基、2-甲基戊基、3-甲基戊基、4-甲基戊基、2,3-二甲基丁基、正庚基、2-甲基己基、3-甲基己基、4-甲基己基、5-甲基己基、2,3-二甲基戊基、2,4-二甲基戊基、2,2-二甲基戊基、3,3-二甲基戊基、2-乙基戊基、3-乙基戊基、正辛基、2,3-二甲基己基、2,4-二甲基己基、2,5-二甲基己基、2,2-二甲基己基、3,3-二甲基己基、4,4-二甲基己基、2-乙基己基、3-乙基己基、4-乙基己基、2-甲基-2-乙基戊基、2-甲基-3-乙基戊基、正壬基、2-甲基-2-乙基己基、2-甲基-3-乙基己基、2,2-二乙基戊基、正癸基、3,3-二乙基己基、2,2-二乙基己基，及其各种支链异构体等。更优选的是含有1至6个碳原子的低级烷基，非限制性实施例包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基、1,1-二甲基丙基、1,2-二甲基丙基、2,2-二甲基丙基、1-乙基丙基、2-甲基丁基、3-甲基丁基、正己基、1-乙基-2-甲基丙基、1,1,2-三甲基丙基、1,1-二甲基丁基、1,2-二甲基丁基、2,2-二甲基丁基、1,3-二甲基丁基、2-乙基丁基、2-甲基戊基、3-甲基戊基、4-甲基戊基、2,3-二甲基丁基等。烷基可以是取代的或非取代的，当被取代时，取代基可以在任何可使用的连接点上被取代，所述取代基优选为一个或多个以下基团，其独立地选自烷基、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、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基、氧代基。
术语“杂烷基”指含有一个或多个选自N、O或S的杂原子的烷基，其中烷 基如上所定义。
术语“亚烷基”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，其为包含1至20个碳原子的直链或支链基团，优选含有1至12个碳原子，更优选含有1至6个碳原子(包含1个、2个、3个、4个、5个或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(-CH 2-)、1,1-亚乙基(-CH(CH 3)-)、1,2-亚乙基(-CH 2CH 2)-、1,1-亚丙基(-CH(CH 2CH 3)-)、1,2-亚丙基(-CH 2CH(CH 3)-)、1,3-亚丙基(-CH 2CH 2CH 2-)、1,4-亚丁基(-CH 2CH 2CH 2CH 2-)和1,5-亚丁基(-CH 2CH 2CH 2CH 2CH 2-)等。亚烷基可以是取代的或非取代的，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。
术语“烷氧基”指-O-(烷基)和-O-(非取代的环烷基)，其中烷基或环烷基的定义如上所述。烷氧基的非限制性实例包括：甲氧基、乙氧基、丙氧基、丁氧基、环丙氧基、环丁氧基、环戊氧基、环己氧基。烷氧基可以是任选取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自烷基、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、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基。
术语“环烷基”指饱和或部分不饱和单环或多环环状烃取代基，环烷基环包含3至20个碳原子，优选包含3至12个碳原子，更优选包含3至10个碳原子，最优选包含3至8个碳原子(包含3个、4个、5个、6个、7个或8个碳原子)。单环环烷基的非限制性实例包括环丙基、环丁基、环戊基、环戊烯基、环己基、环己烯基、环己二烯基、环庚基、环庚三烯基、环辛基等；多环环烷基包括螺环、稠环和桥环的环烷基。
术语“杂环基”指饱和或部分不饱和单环或多环环状烃取代基，其包含3至20个环原子，其中一个或多个环原子为选自氮、氧或S(O) m(其中m是整数0、1或2)的杂原子，但不包括-O-O-、-O-S-或-S-S-的环部分，其余环原子为碳。优选包含3至12个环原子，其中1～4个是杂原子(1个、2个、3个或4个杂原子)；更优选环烷基环包含3至10个环原子(包含3个、4个、5个、6个、7个、8个、9个或10个环原子)。单环杂环基的非限制性实例包括吡咯烷基、哌啶基、哌嗪基、吗啉基、硫代吗啉基、高哌嗪基等。多环杂环基包括螺环、稠环和桥环的杂环基。
术语“螺杂环基”指5至20元的单环之间共用一个原子(称螺原子)的多环杂环基团，其中一个或多个环原子为选自氮、氧或S(O) m(其中m是整数0至2)的杂原子，其余环原子为碳。其可以含有一个或多个双键，但没有一个环具有完全共轭的π电子系统。优选为6至14元，更优选为7至10元。根据环与环之间共用 螺原子的数目将螺杂环基分为单螺杂环基、双螺杂环基或多螺杂环基，优选为单螺杂环基和双螺杂环基。更优选为4元/4元、4元/5元、4元/6元、5元/5元或5元/6元单螺杂环基。螺杂环基的非限制性实例包括：
Figure PCTCN2019107852-appb-000023
术语“稠杂环基”指5至20元，系统中的每个环与体系中的其他环共享毗邻的一对原子的多环杂环基团，一个或多个环可以含有一个或多个双键，但没有一个环具有完全共轭的π电子系统，其中一个或多个环原子为选自氮、氧或S(O) m(其中m是整数0、1或2)的杂原子，其余环原子为碳。优选为6至14元，更优选为7至10元(7元、8元、9元或10元环)。根据组成环的数目可以分为双环、三环、四环或多环稠杂环基，优选为双环或三环，更优选为5元/5元或5元/6元双环稠杂环基。稠杂环基的非限制性实例包括：
Figure PCTCN2019107852-appb-000024
术语“桥杂环基”指5至14元，任意两个环共用两个不直接连接的原子的多环杂环基团，其可以含有一个或多个双键，但没有一个环具有完全共轭的π电子系统，其中一个或多个环原子为选自氮、氧或S(O) m(其中m是整数0、1或2)的杂原子，其余环原子为碳。优选为6至14元，更优选为7至10元(7元、8元、9元或10元环)。根据组成环的数目可以分为双环、三环、四环或多环桥杂环基，优选为双环、三环或四环，更优选为双环或三环。桥杂环基的非限制性实例包括：
Figure PCTCN2019107852-appb-000025
所述杂环基环可以稠合于芳基、杂芳基或环烷基环上，其中与母体结构连接在一起的环为杂环基，其非限制性实例包括：
Figure PCTCN2019107852-appb-000026
等。
杂环基可以是任选取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自烷基、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、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基、氧代基。
术语“芳基”指具有共轭的π电子体系的6至14元全碳单环或稠合多环(也就是共享毗邻碳原子对的环)基团，优选为6至10元(6元、7元、8元、9元或10元)，例如苯基和萘基，优选苯基。所述芳基环可以稠合于杂芳基、杂环基或环烷基环上，其中与母体结构连接在一起的环为芳基环，其非限制性实例包括：
Figure PCTCN2019107852-appb-000027
芳基可以是取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自烷基、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、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基。
术语“杂芳基”指包含1至4个杂原子(1个、2个、3个或4个杂原子)、5至14个环原子的杂芳族体系，其中杂原子选自氧、硫和氮。杂芳基优选为5至10元(5元、6元、7元、8元、9元或10元杂芳基)，更优选为5元或6元，例如呋喃基、噻吩基、吡啶基、吡咯基、N-烷基吡咯基、嘧啶基、吡嗪基、咪唑基、四唑基等。所述杂芳基环可以稠合于芳基、杂环基或环烷基环上，其中与母体结构连接在一起的环为杂芳基环，其非限制性实例包括：
Figure PCTCN2019107852-appb-000028
杂芳基可以是任选取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自烷基、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、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基。
术语“氨基保护基”是为了使分子其它部位进行反应时氨基保持不变，用易于脱去的基团对氨基进行保护。非限制性实施例包含9-芴甲氧羰基、叔丁氧羰基、乙酰基、苄基、烯丙基和对甲氧苄基等。这些基团可任选地被选自卤素、烷氧基或硝基中的1-3个取代基(1个、2个或3个取代基)所取代。所述氨基保护基优选为9-芴甲氧羰基。
术语“卤代烷基”指烷基被一个或多个卤素取代，其中烷基如上所定义。
术语“氘代烷基”指烷基被一个或多个氘原子取代，其中烷基如上所定义。
术语“羟烷基”指烷基被一个或多个羟基取代，其中烷基如上所定义。
术语“羟基”指-OH基团。
术语“卤素”指氟、氯、溴或碘。
术语“氨基”指-NH 2。
术语“硝基”指-NO 2。
术语“氰基”指-CN。
术语“酰氨基”指-C(O)N(烷基)或(环烷基)，其中烷基、环烷基如上所定义。
术语“羧酸酯基”指-C(O)O(烷基)或(环烷基)，其中烷基、环烷基如上所定义。
本公开还包括各种氘化形式的式(I)化合物。与碳原子连接的各个可用的氢原子可独立地被氘原子替换。本领域技术人员能够参考相关文献合成氘化形式的式(I)化合物。在制备氘代形式的式(I)化合物时可使用市售的氘代起始物质，或它们可使用常规技术采用氘代试剂合成，氘代试剂包括但不限于氘代硼烷、三氘代硼烷四氢呋喃溶液、氘代氢化锂铝、氘代碘乙烷和氘代碘甲烷等。
“任选”或“任选地”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。
“取代的”指基团中的一个或多个氢原子，优选为最多5个，更优选为1个、2个或3个氢原子彼此独立地被相应数目的取代基取代。不言而喻，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。
术语“药物组合物”表示含有一种或多种本文所述化合物或其生理学上/可药用的盐或前体药物与其他化学组分的混合物，以及其他组分例如生理学/可药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
术语“药学上可接受的盐”或“可药用盐”是指本公开配体-药物偶联物的盐，或本公开中所述的化合物的盐，这类盐用于哺乳动物体内时具有安全性和有效性，且具有应有的生物活性，本公开配体药物偶联物至少含有一个氨基，因此可以与 酸形成盐，可药用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。
术语“溶剂合物”指本公开的配体-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。
术语“载药量”是指式(I)分子中每个配体上加载的细胞毒性药物平均数量，也可以表示为药物量和抗体量的比值，药物载量的范围可以是每个配体(Pc)连接0-12个，优选1-10个细胞毒性药物(D)。在本公开的实施方案中，载药量表示为n，也可称为DAR值，示例性的为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分子的药物品均数量。
本公开的一个实施方式中，细胞毒性药物通过连接单元偶联在配体的N端氨基和/或赖氨酸残基的ε-氨基上，一般地，偶联反应中能与抗体偶联的药物分子数将小于理论上的最大值。
可以用以下非限制性方法控制配体细胞毒性药物偶联物的载量，包括：
(1)控制连接试剂和单抗的摩尔比，
(2)控制反应时间和温度，
(3)选择不同的反应试剂。
常规的药物组合物的制备见中国药典。
术语“载体”用于本公开的药物，是指能改变药物进入人体的方式和在体内的分布、控制药物的释放速度并将药物输送到靶向器官的体系。药物载体释放和靶向系统能够减少药物降解及损失，降低副作用，提高生物利用度。如可作为载体的高分子表面活性剂由于其独特的两亲性结构，可以进行自组装，形成各种形式的聚集体，优选的实例如胶束、微乳液、凝胶、液晶、囊泡等。这些聚集体具有包载药物分子的能力，同时又对膜有良好的渗透性，可以作为优良的药物载体。
术语“赋形剂”是在药物制剂中除主药以外的附加物，也可称为辅料。如片剂中的粘合剂、填充剂、崩解剂、润滑剂；半固体制剂软膏剂、霜剂中的基质部分；液体制剂中的防腐剂、抗氧剂、矫味剂、芳香剂、助溶剂、乳化剂、增溶剂、渗透压调节剂、着色剂等均可称为赋形剂。
术语“稀释剂”又称填充剂，其主要用途是增加片剂的重量和体积。稀释剂的加入不仅保证一定的体积大小，而且减少主要成分的剂量偏差，改善药物的压缩成型性等。当片剂的药物含有油性组分时，需加入吸收剂吸收油性物，使保持“干燥”状态，以利于制成片剂。如淀粉、乳糖、钙的无机盐、微晶纤维素等。
药物组合物可以是无菌注射水溶液形式。可在使用的可接受的溶媒和溶剂中 有水、林格氏液和等渗氯化钠溶液。无菌注射制剂可以是其中活性成分溶于油相的无菌注射水包油微乳。例如将活性成分溶于大豆油和卵磷脂的混合物中。然后将油溶液加入水和甘油的混合物中处理形成微乳。可通过局部大量注射，将注射液或微乳注入患者的血流中。或者，最好按可保持本公开化合物恒定循环浓度的方式给予溶液和微乳。为保持这种恒定浓度，可使用连续静脉内递药装置。这种装置的实例是Deltec CADD-PLUS.TM.5400型静脉注射泵。
药物组合物可以是用于肌内和皮下给药的无菌注射水或油混悬液的形式。可按已知技术，用上述那些适宜的分散剂或湿润剂和悬浮剂配制该混悬液。无菌注射制剂也可以是在无毒肠胃外可接受的稀释剂或溶剂中制备的无菌注射溶液或混悬液，例如1,3-丁二醇中制备的溶液。此外，可方便地用无菌固定油作为溶剂或悬浮介质。为此目的，可使用包括合成甘油单或二酯在内的任何调和固定油。此外，脂肪酸例如油酸也可以制备注射剂。
本公开涉及一类可裂解的特定结构的连接臂和特定结构的活性物，及由连接臂、活性物与抗体组成的抗体药物偶联物(ADC)。此类ADC是经由间隔物将一种毒性物质连于抗体而形成的复合物。该抗体偶联药物(ADC)在体内经降解而释放出活性分子，从而起到抗肿瘤的作用。
二、合成方法
为了完成合成目的，采用如下的合成技术方案：
通式(Pc-L a-Y-Dr)所示的化合物的制备方法，其包括如下步骤：
Figure PCTCN2019107852-appb-000029
Pc还原后，与通式(L a-Y-Dr)偶联反应，得到通式(Pc-L a-Y-Dr)所示的化合物；还原剂优选TCEP，特别地，优选还原抗体上的二硫键；
其中：
Pc、W、L 2、L 3、R 1、R 2、R 5～R 7、m和n如通式(Pc-L a-Y-Dr)中所定义。
在以上说明书中提出了本公开一种或多种实施方案的细节。虽然可使用与本文所述类似或相同的任何方法和材料来实施或测试本公开，但是以下描述优选的 方法和材料。通过说明书和权利要求书，本公开的其他特点、目的和优点将是显而易见的。在说明书和权利要求书中，除非上下文中有清楚的另外指明，单数形式包括复数指代物的情况。除非另有定义，本文使用的所有技术和科学术语都具有本公开所属领域普通技术人员所理解的一般含义。说明书中引用的所有专利和出版物都通过引用纳入。提出以下实施例是为了更全面地说明本公开的优选实施方案。这些实施例不应以任何方式理解为限制本公开的范围，本公开的范围由权利要求书限定。
本公开实施例中未注明具体条件的实验方法，通常按照常规条件，或按照原料或商品制造厂商所建议的条件。未注明具体来源的试剂，为市场购买的常规试剂。
化合物的结构是通过核磁共振(NMR)或质谱(MS)来确定的。NMR的测定是用Bruker AVANCE-400核磁仪，测定溶剂为氘代二甲基亚砜(DMSO-d6)、氘代氯仿(CDCl 3)、氘代甲醇(CD 3OD)，内标为四甲基硅烷(TMS)，化学位移是以10 -6(ppm)作为单位给出。
MS的测定用FINNIGAN LCQAd(ESI)质谱仪(生产商：Thermo,型号：Finnigan LCQ advantage MAX)。
UPLC的测定用Waters Acquity UPLC SQD液质联用仪。
HPLC的测定使用安捷伦1200DAD高压液相色谱仪(Sunfire C18 150×4.6mm色谱柱)和Waters 2695-2996高压液相色谱仪(Gimini C18 150×4.6mm色谱柱)。
UV-HPLC的测定使用Thermo nanodrop2000紫外分光光度计。
增殖抑制率及IC 50值的测定用PHERA starFS酶标仪(德国BMG公司)。
薄层层析硅胶板使用烟台黄海HSGF254或青岛GF254硅胶板，薄层色谱法(TLC)使用的硅胶板采用的规格是0.15mm～0.2mm，薄层层析分离纯化产品采用的规格是0.4mm～0.5mm硅胶板。
柱层析一般使用烟台黄海200～300目硅胶为载体。
本公开的已知的起始原料可以采用或按照本领域已知的方法来合成，或可购买自ABCR GmbH & Co.KG，Acros Organnics，Aldrich Chemical Company，韶远化学科技(Accela ChemBio Inc)、达瑞化学品等公司。
实施例中如无特殊说明，反应均在氩气氛或氮气氛下进行。
氩气氛或氮气氛是指反应瓶连接一个约1L容积的氩气或氮气气球。
氢气氛是指反应瓶连接一个约1L容积的氢气气球。
加压氢化反应使用Parr 3916EKX型氢化仪和清蓝QL-500型氢气发生器或HC2-SS型氢化仪。
氢化反应通常抽真空，充入氢气，反复操作3次。
微波反应使用CEM Discover-S 908860型微波反应器。
实施例中如无特殊说明，反应中的溶液是指水溶液。
实施例中如无特殊说明，反应的温度为室温。
室温为最适宜的反应温度，温度范围是20℃～30℃。
实施例中pH＝6.5的PBS缓冲液的配制：取KH 2PO 4 8.5g，K 2HPO 4.3H 2O 8.56g，NaCl 5.85g，EDTA 1.5g置于瓶中，定容至2L，超声波使其全部溶解，摇匀即得。
纯化化合物采用的柱层析的洗脱剂的体系和薄层色谱法的展开剂的体系包括：A：二氯甲烷和异丙醇体系，B：二氯甲烷和甲醇体系，C：石油醚和乙酸乙酯体系，溶剂的体积比根据化合物的极性不同而进行调节，也可以加入少量的三乙胺和酸性或碱性试剂等进行调节。
本公开部分化合物是通过Q-TOF LC/MS来表征的。Q-TOF LC/MS使用安捷伦6530精确质量数四级杆-飞行时间质谱仪和安捷伦1290-Infinity超高效液相色谱仪(安捷伦Poroshell 300SB-C8 5μm,2.1×75mm色谱柱)。
实施例1
N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)-1-羟基环丙烷-1-甲酰胺1
Figure PCTCN2019107852-appb-000030
向依喜替康甲磺酸盐1b(2.0mg，3.76μmol，采用专利申请“EP0737686A1”公开的方法制备而得)中添加1mL N,N-二甲基甲酰胺，冰水浴冷却至0-5℃，滴加一滴三乙胺，搅拌至反应液变澄清。向反应液中依次加入1-羟基环丙基甲酸1a(1.4mg，3.7μmol，采用公知的方法“Tetrahedron Letters,25(12),1269-72；1984”制备而得)和4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐(3.8mg，13.7μmol)，加毕，在0-5℃搅拌反应2小时。向反应液中加入5mL水淬灭反应，用乙酸乙酯 (8mL×3)萃取反应液，合并有机相，用饱和氯化钠溶液(5mL×2)洗涤，有机相用无水硫酸钠干燥，过滤，将滤液减压浓缩，用薄层层析以展开剂体系B纯化所得残余物，得到标题产物1(1.6mg，产率：82.1％)。
MS m/z(ESI):520.2[M+1]。
1H NMR(400MHz,CDCl 3):δ7.90-7.84(m,1H),7.80-7.68(m,1H),5.80-5.70(m,1H),5.62-5.54(m,2H),5.44-5.32(m,2H),5.28-5.10(m,2H),3.40-3.15(m,3H),2.44(s,3H),2.23(t,1H),2.06-1.75(m,2H),1.68-1.56(m,1H),1.22-1.18(m,2H),1.04-0.98(m,2H),0.89(t,3H)。
实施例2
(S)-2-环丙基-N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-1,2,3,9,10,12,13,15-八氢苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)-2-羟基乙酰胺2-A
(R)-2-环丙基-N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-1,2,3,9,10,12,13,15-八氢苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)-2-羟基乙酰胺2-B
Figure PCTCN2019107852-appb-000031
向1b(4mg，7.53μmol)中加入2mL乙醇和0.4mL N,N-二甲基甲酰胺，氩气置换三次，冰水浴冷却至0-5℃，滴加0.3mL N-甲基吗啉，搅拌至反应液变澄清。向反应液中依次加入2-环丙基-2-羟基乙酸2a(2.3mg，19.8μmol，采用专利申请“WO2013106717”公开的方法制备而得)、1-羟基苯并三唑(3mg，22.4μmol)和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(4.3mg，22.4μmol)，加毕，在0-5℃搅拌反应1小时。撤去冰水浴，加热至30℃搅拌2小时。反应液减压浓缩，所得到的粗品化合物2用高效液相色谱法纯化(分离条件：色谱柱：XBridge Prep C18 OBD 5um19*250mm；流动相：A-水(10mmol NH 4OAc)，B-乙腈，梯度洗脱，流速：18mL/min)，收集其相应组分，减压浓缩，得到标题产物(1.5mg，1.5mg)。
MS m/z(ESI):534.0[M+1]。
单一构型化合物2-B(较短保留时间)
UPLC分析：保留时间1.06分钟，纯度：88％(色谱柱：ACQUITY UPLC BEHC18 1.7um 2.1*50mm，流动相：A-水(5mmol NH 4OAc)，B-乙腈)。
1H NMR(400MHz,DMSO-d 6):δ8.37(d,1H),7.76(d,1H),7.30(s,1H),6.51(s,1H),5.58-5.56(m,1H),5.48(d,1H),5.41(s,2H),5.32-5.29(m,1H),3.60(t,1H),3.19-3.13(m,2H),2.38(s,3H),2.20-2.14(m,1H),1.98(q,2H),1.87-1.83(m,1H),1.50-1.40(m,1H),1.34-1.28(m,1H),0.86(t,3H),0.50-0.39(m,4H)。
单一构型化合物2-A(较长保留时间)
UPLC分析：保留时间1.10分钟，纯度：86％(色谱柱：ACQUITY UPLC BEHC18 1.7um 2.1*50mm，流动相：A-水(5mmol NH 4OAc)，B-乙腈)。
1H NMR(400MHz,DMSO-d 6):δ8.35(d,1H),7.78(d,1H),7.31(s,1H),6.52(s,1H),5.58-5.53(m,1H),5.42(s,2H),5.37(d,1H),5.32(t,1H),3.62(t,1H),3.20-3.15(m,2H),2.40(s,3H),2.25-2.16(m,1H),1.98(q,2H),1.87-1.82(m,1H),1.50-1.40(m,1H),1.21-1.14(m,1H),0.87(t,3H),0.47-0.35(m,4H)。
实施例3
(S)-N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-1,2,3,9,10,12,13,15-八氢苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)-3,3,3-三氟-2-羟基丙酰胺3-A
(R)-N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-1,2,3,9,10,12,13,15-八氢苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)-3,3,3-三氟-2-羟基丙酰胺3-B
Figure PCTCN2019107852-appb-000032
向1b(5.0mg，9.41μmol)中添加2mL乙醇和0.4mL N,N-二甲基甲酰胺，冰水浴冷却至0-5℃，滴加0.3mL N-甲基吗啡啉，搅拌至反应液变澄清。向反应液中依次加入3,3,3-三氟-2-羟基丙酸3a(4.1mg，28.4μmol，供应商Alfa)、1-羟基苯并三唑(3.8mg，28.1μmol)和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(5.4mg，28.2μmol)，加毕，在0-5℃搅拌反应10分钟。撤去冰水浴，加热至30℃搅拌8小时。反应液减压浓缩，所得到的粗品化合物3用高效液相色谱法纯化(分离条件：色谱柱：XBridge Prep C18 OBD 5um 19*250mm；流动相：A-水(10mmol NH 4OAc)：B-乙腈，梯度洗脱，流速：18mL/min)，收集其相应组分，减压浓缩，得到标题产 物(1.5mg，1.5mg)。
MS m/z(ESI):561.9[M+1]。
单一构型化合物(较短保留时间)
UPLC分析：保留时间1.11分钟，纯度：88％(色谱柱：ACQUITY UPLC BEHC18 1.7um 2.1*50mm，流动相：A-水(5mmol NH 4OAc)，B-乙腈)。
1H NMR(400MHz,DMSO-d 6):δ8.94(d,1H),7.80(d,1H),7.32(s,1H),7.20(d,1H),6.53(s,1H),5.61-5.55(m,1H),5.45-5.23(m,3H),5.15-5.06(m,1H),4.66-4.57(m,1H),3.18-3.12(m,1H),2.40(s,3H),2.26-2.20(m,1H),2.16-2.08(m,1H),2.02-1.94(m,1H),1.89-1.82(m,1H),1.50-1.40(m,1H),0.87(t,3H)。
单一构型化合物(较长保留时间)
UPLC分析：保留时间1.19分钟，纯度：90％(色谱柱：ACQUITY UPLC BEHC18 1.7um 2.1*50mm，流动相：A-水(5mmol NH 4OAc)，B-乙腈)。
1H NMR(400MHz,DMSO-d 6):δ8.97(d,1H),7.80(d,1H),7.31(s,1H),7.16(d,1H),6.53(s,1H),5.63-5.55(m,1H),5.45-5.20(m,3H),5.16-5.07(m,1H),4.66-4.57(m,1H),3.18-3.12(m,1H),2.40(s,3H),2.22-2.14(m,1H),2.04-1.95(m,2H),1.89-1.82(m,1H),1.50-1.40(m,1H),0.87(t,3H)。
实施例4
1-(((S)-7-苄基-20-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)-3,6,9,12,15-五氧代-2,5,8,11,14-五氮杂二十烷基)氧基)-N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)环丙烷-1-甲酰胺4
Figure PCTCN2019107852-appb-000033
Figure PCTCN2019107852-appb-000034
第一步
1-((2-((((9H-芴-9-基)甲氧基)羰基)氨基)乙酰氨基)甲氧基)环丙烷-1-羧酸苄酯4c
将1-羟基环丙烷-1-羧酸苄酯4a(104mg，0.54mmol；采用专利申请“US2005/20645”公开的方法制备而得)和2-((((9H-芴-9-基)甲氧基)羰基)氨基)乙酰氨基)甲基乙酸酯4b(100mg，0.27mmol；采用专利申请“CN105829346A”公开的方法制备而得)加入反应瓶，加入5mL四氢呋喃，氩气置换三次，冰水浴降温至0-5℃，加入叔丁醇钾(61mg，0.54mmol)，撤去冰浴，升至室温搅拌10分钟，加入20mL冰水，用乙酸乙酯(5mL×2)和氯仿(5mL×5)萃取，合并有机相并浓缩。所得残余物溶于3mL 1,4-二氧六环中，加入0.6mL水，加入碳酸氢钠(27mg，0.32mmol)和氯甲酸-9-芴甲酯(70mg，0.27mmol)，室温搅拌1小时。加入20mL水，用乙酸乙酯(8mL×3)萃取，有机相用饱和氯化钠溶液(20mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，用硅胶柱色谱法以展开剂体系B纯化所得残余物，得到标题产物4c(100mg，产率：73.6％)。
MS m/z(ESI):501.0[M+1]。
第二步
1-((2-((((9H-芴-9-基)甲氧基)羰基)氨基)乙酰氨基)甲氧基)环丙烷-1-羧酸4d
将4c(50mg，0.10mmol)溶于3mL四氢呋喃和乙酸乙酯(V:V＝2:1)混合溶剂中，加入钯碳(25mg，含量10％)，氢气置换三次，室温搅拌反应1小时。反应液用硅藻土过滤，滤饼用四氢呋喃淋洗，滤液浓缩，得到标题产物4d(41mg，产率：100％)。
MS m/z(ESI):411.0[M+1]。
第三步
(9H-芴-9-基)甲基(2-(((1-(((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基羰基)环丙氧基)甲基)氨基)-2-氧代乙基)氨基甲酸酯4e
将1b(7mg，0.013mmol)加入反应瓶，加入1mL N,N-二甲基甲酰胺，氩气置换三次，冰水浴降温至0-5℃，滴加一滴三乙胺，加入4d(7mg，0.017mmol)的0.5mL N,N-二甲基甲酰胺溶液，加入4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐(7mg，0.026mmol)，冰浴搅拌反应35分钟。加入10mL水，用乙酸乙酯(5mL×3)萃取，有机相用饱和氯化钠溶液(10mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，用薄层层析以展开剂体系B纯化所得残余物，得到标题产物4e(8.5mg，产率78.0％)。
MS m/z(ESI):828.0[M+1]。
第四步
1-((2-氨基乙酰氨基)甲氧基)-N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)环丙烷-1-甲酰胺4f
将4e(4mg，4.84μmol)溶于0.2mL二氯甲烷中，加入0.1mL二乙胺，室温搅拌2小时。反应液减压浓缩，加入2mL甲苯减压浓缩，重复两次，加入3mL正己烷打浆，倾倒出上层正己烷，重复三次，减压浓缩得到粗品标题产物4f(2.9mg)，产品不经纯化直接用于下一步反应。
MS m/z(ESI):606.0[M+1]。
第五步
1-(((S)-7-苄基-20-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)-3,6,9,12,15-五氧代-2,5,8,11,14-五氮杂二十烷基)氧基)-N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)环丙烷-1-甲酰胺4
将粗品4f(2.9mg，4.84μmol)溶于0.5mL N,N-二甲基甲酰胺，氩气置换三次，冰水浴降温至0-5℃，加入(S)-2(-2-(-2-(6-(2,5-二氧代-1H-吡咯-1-基)已酰氨基)乙酰氨基)乙酰氨基)-3-苯基丙酸4g(2.7mg，5.80μmol，采用专利申请“EP2907824”公开的方法制备而得)的0.3mL N,N-二甲基甲酰胺溶液，加入4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐(2.7mg，9.67μmol)，冰浴搅拌反应30分钟，撤去冰浴，升至室温搅拌15分钟。反应液进行高效液相色谱法纯化(分离条件：色谱柱：XBridge Prep C18 OBD 5um 19*250mm；流动相：A-水(10mmol NH 4OAc)：B-乙腈，梯度洗脱，流速：18mL/min)，收集其相应组分，减压浓缩得到标题产物4(2mg，产率：39.0％)。
MS m/z(ESI):1060.0[M+1]。
1H NMR(400MHz,DMSO-d 6):δ9.01(d,1H),8.77(t,1H),8.21(t,1H),8.08-7.92(m,2H),7.73(d,1H),7.28(s,1H),7.24-7.07(m,4H),6.98(s,1H),6.50(s,1H),5.61(q,1H),5.40(s,2H),5.32(t,1H),5.12(q,2H),4.62(t,1H),4.52(t,1H),4.40-4.32(m,1H),3.73-3.47(m,8H),3.16-3.04(m,2H),2.89(dd,1H),2.69-2.55(m,2H),2.37-2.23(m,4H),2.12-1.93(m,4H),1.90-1.74(m,2H),1.52-1.38(m,4H), 1.33-1.11(m,5H),0.91-0.81(m,4H)。
实施例5
N-((2R,10S)-10-苄基-2-环丙基-1-(((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基)-1,6,9,12,15-五氧代-3-氧杂-5,8,11,14-四氮杂十六-16-基)-6-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)己酰胺5-A
N-((2S,10S)-10-苄基-2-环丙基-1-(((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基)-1,6,9,12,15-五氧代-3-氧杂-5,8,11,14-四氮杂十六-16-基)-6-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)己酰胺5-B
Figure PCTCN2019107852-appb-000035
第一步
2-环丙基-2-羟基乙酸苄酯5a
将2a(1.3g，11.2mmol；采用专利申请“WO2013/106717”公开的方法制备而得)溶于50mL乙腈中，依次加入碳酸钾(6.18g，44.8mmol)、溴化苄(1.33mL，11.2mmol)和四丁基碘化铵(413mg，1.1mmol)。将反应液室温搅拌48小时，通过硅藻土过滤，滤饼用乙酸乙酯(10ml)淋洗，合并滤液减压浓缩，用硅胶柱色谱法以 展开剂体系C纯化所得残余物，得到标题产物5a(2g，产率：86.9％)。
第二步
10-环丙基-1-(9H-芴-9-基)-3,6-二氧代-2,9-二氧杂-4,7-二氮杂十一-11-酸苄酯5b
将5a(120.9mg,0.586mmol)和4b(180mg，0.489mmol)加入反应瓶，加入4mL四氢呋喃，氩气置换三次，冰水浴降温至0-5℃，加入叔丁醇钾(109mg，0.98mmol)，撤去冰浴，升至室温搅拌40分钟，加入10mL冰水，用乙酸乙酯(20mL×2)和氯仿(10mL×5)萃取，合并有机相并浓缩。所得残余物溶于4mL二氧六环中，加入2mL水，加入碳酸氢钠(49.2mg，0.586mmol)和氯甲酸-9-芴甲酯(126mg，0.49mmol)，室温搅拌2小时。加入20mL水，用乙酸乙酯(10mL×3)萃取，有机相用饱和氯化钠溶液(20mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩。用硅胶柱色谱法以展开剂体系C纯化所得残余物，得到标题产物5b(48mg，产率：19％)。
MS m/z(ESI):515.0[M+1]。
第三步
10-环丙基-1-(9H-芴-9-基)-3,6-二氧代-2,9-二氧杂-4,7-二氮杂十一-11-酸5c
将5b(20mg，0.038mmol)溶于4.5mL四氢呋喃和乙酸乙酯(V:V＝2:1)混合溶剂中，加入钯碳(12mg，含量10％，干型)，氢气置换三次，室温搅拌反应1小时。反应液用硅藻土过滤，滤饼用乙酸乙酯淋洗，滤液浓缩，得到粗品标题产物5c(13mg)，产品不经纯化直接进行下一步反应。
MS m/z(ESI):424.9[M+1]。
第四步
(9H-芴-9-基)甲基(2-(((1-环丙基-2-(((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基)-2-氧代乙氧基)甲基)氨基)-2-氧代乙基)氨基甲酸酯5d
将1b(10mg，18.8μmol)加入反应瓶，加入1mL N,N-二甲基甲酰胺，氩气置换三次，冰水浴降温至0-5℃，滴加一滴三乙胺，加入粗品5c(13mg，30.6μmol)，加入4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐(16.9mg，61.2μmol)，冰浴搅拌反应40分钟。加入10mL水，用乙酸乙酯(10mL×3)萃取，合并有机相。有机相用饱和氯化钠溶液(10mL×2)洗涤，用无水硫酸钠干燥，过滤，滤液减压浓缩。用薄层层析以展开剂体系B纯化所得残余物，得到标题产物5d(19mg，产率：73.6％)。
MS m/z(ESI):842.1[M+1]。
第五步
2-((2-氨基乙酰氨基)甲氧基)-2-环丙基-N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)乙酰胺5e
将5d(19mg，22.6μmol)溶于2mL二氯甲烷中，加入1mL二乙胺，室温搅 拌2小时。反应液减压浓缩，加入1mL甲苯并减压浓缩，重复两次。向残余物中加入3mL正己烷打浆，静置后倾倒出上层清液，保留固体。将固体残余物减压浓缩，油泵拉干得到粗品标题产物5e(17mg)，产品不经纯化直接用于下一步反应。
MS m/z(ESI):638.0[M+18]。
第六步
N-((2R,10S)-10-苄基-2-环丙基-1-(((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基)-1,6,9,12,15-五氧代-3-氧杂-5,8,11,14-四氮杂十六-16-基)-6-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)己酰胺5-A
N-((2S,10S)-10-苄基-2-环丙基-1-(((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基)-1,6,9,12,15-五氧代-3-氧杂-5,8,11,14-四氮杂十六-16-基)-6-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)己酰胺5-B
将粗品5e(13.9mg，22.4μmol)溶于0.6mL N,N-二甲基甲酰胺，氩气置换三次，冰水浴降温至0-5℃，加入4g(21.2mg，44.8μmol)的0.3mL N,N-二甲基甲酰胺溶液，加入4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐(18.5mg，67.3μmol)，冰浴搅拌反应10分钟，撤去冰浴，升至室温搅拌1小时，反应生成化合物5。反应液进行高效液相色谱法纯化(分离条件：色谱柱：XBridge Prep C18 OBD 5um19*250mm；流动相：A-水(10mmol NH 4OAc)：B-乙腈，梯度洗脱，流速：18mL/min)，收集其相应组分，减压浓缩，得到标题产物(2.4mg，1.7mg)。
MS m/z(ESI):1074.4[M+1]。
单一构型化合物5-A(较短保留时间)：
UPLC分析:保留时间1.14分钟，纯度：85％(色谱柱：ACQUITY UPLC BEHC18 1.7um 2.1*50mm，流动相：A-水(5mmol NH 4OAc)，B-乙腈)。
1H NMR(400MHz,DMSO-d 6):δ8.60(t,1H),8.51-8.49(d,1H),8.32-8.24(m,1H),8.13-8.02(m,2H),8.02-7.96(m,1H),7.82-7.75(m,1H),7.31(s,1H),7.26-7.15(m,4H),6.99(s,1H),6.55-6.48(m,1H),5.65-5.54(m,1H),5.41(s,2H),5.35-5.15(m,3H),4.74-4.62(m,2H),4.54-4.40(m,2H),3.76-3.64(m，4H),3.62-3.48(m,2H),3.20-3.07(m,2H),3.04-2.94(m,2H),2.80-2.62(m,2H),2.45-2.30(m,3H),2.25-2.15(m,2H),2.15-2.04(m,2H),1.93-1.78(m,2H),1.52-1.39(m,3H),1.34-1.12(m,5H),0.87(t,3H),0.64-0.38(m,4H)。
单一构型化合物5-B(较长保留时间)：
UPLC分析:保留时间1.16分钟，纯度：89％(色谱柱：ACQUITY UPLC BEHC18 1.7um 2.1*50mm，流动相：A-水(5mmol NH 4OAc)，B-乙腈)。
1H NMR(400MHz,DMSO-d 6):δ8.68-8.60(m,1H),8.58-8.50(m,1H),8.32-8.24(m,1H),8.13-8.02(m,2H),8.02-7.94(m,1H),7.82-7.75(m,1H),7.31(s,1H),7.26-7.13(m,4H),6.99(s,1H),6.55-6.48(m,1H),5.60-5.50(m,1H),5.41(s,2H), 5.35-5.15(m,3H),4.78-4.68(m,1H),4.60-4.40(m,2H),3.76-3.58(m,4H),3.58-3.48(m,1H),3.20-3.10(m,2H),3.08-2.97(m,2H),2.80-2.72(m,2H),2.45-2.30(m,3H),2.25-2.13(m,2H),2.13-2.04(m,2H),2.03-1.94(m,2H),1.91-1.78(m,2H),1.52-1.39(m,3H),1.34-1.12(m,5H),0.91-0.79(m,3H),0.53-0.34(m,4H)。
实施例6
N-((2S,10S)-10-苄基-2-(((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基羰基)-1,1,1-三氟-6,9,12,15-四氧代-3-氧杂-5,8,11,14-四氮杂十六-16-基)-6-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)己酰胺6-A
N-((2R,10S)-10-苄基-2-(((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基羰基)-1,1,1-三氟-6,9,12,15-四氧代-3-氧杂-5,8,11,14-四氮杂十六-16-基)-6-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)己酰胺6-B
Figure PCTCN2019107852-appb-000036
第一步
3,3,3-三氟-2-羟基丙酸苄酯6a
将3a(1.80g，12.5mmol)溶于100mL乙腈中，依次加入碳酸钾(5.17g，37.5 mmol)、溴化苄(4.48mL，37.5mmol)和四丁基碘化铵(231mg，0.63mmol)。将反应液加热至60℃搅拌5小时。将反应液冷却至室温，过滤，滤液减压浓缩，用硅胶柱色谱法以展开剂体系C纯化所得残余物，得到标题产物6a(980mg，产率：33.5％)。
1H NMR(400MHz,CDCl 3):δ7.43-7.36(m,5H),5.34(s,2H),4.53(s,1H),3.44(s,1H)。
第二步
1-(9H-芴-9-基)-3,6-二氧代-10-(三氟甲基)-2,9-二氧杂-4,7-二氮杂十一-11-酸苄酯6b
将4b(63mg,0.17mmol)和6a(80mg，0.34mmol)加入反应瓶，加入3mL四氢呋喃，氩气置换三次，冰水浴降温至0-5℃，加入叔丁醇钾(38mg，0.34mmol)，撤去冰浴，升至室温搅拌20分钟，加入10mL冰水，用乙酸乙酯(20mL×2)和氯仿(10mL×5)萃取，合并有机相并浓缩，所得残余物溶于2mL二氧六环中，加入0.4mL水，加入碳酸氢钠(19mg，0.23mmol)和氯甲酸-9-芴甲酯(49mg，0.19mmol)，室温搅拌1小时。加入20mL水，用乙酸乙酯(10mL×3)萃取，有机相用饱和氯化钠溶液(20mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，用硅胶柱色谱法以展开剂体系C纯化所得残余物，得到标题产物6b(51mg，产率：55.3％)。
MS m/z(ESI):559.9[M+18]。
第三步
1-(9H-芴-9-基)-3,6-二氧代-10-(三氟甲基)-2,9-二氧杂-4,7-二氮杂十一-11-酸6c
将6b(15mg，0.28mmol)溶于3mL四氢呋喃和乙酸乙酯(V:V＝2:1)混合溶剂中，加入钯碳(15mg，含量10％)，氢气置换三次，室温搅拌反应1小时。反应液用硅藻土过滤，滤饼用四氢呋喃淋洗，滤液浓缩，得到粗品标题产物6c(13mg)。
MS m/z(ESI):452.9[M+1]。
第四步
(9H-芴-9-基)甲基(2-((((3-(((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基)-1,1,1-三氟-3-氧代丙-2-基)氧基)甲基)氨基)-2-氧代乙基)氨基甲酸酯6d
将1b(10mg，18.8μmol)加入反应瓶，加入1mL N,N-二甲基甲酰胺，氩气置换三次，冰水浴降温至0-5℃，滴加一滴三乙胺，加入6c(13mg，28.7μmol)的0.5mL N,N-二甲基甲酰胺溶液，加入4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐(11mg，39.7μmol)，冰浴搅拌反应30分钟。加入10mL水，用乙酸乙酯(10mL×3)萃取，合并有机相，有机相用饱和氯化钠溶液(10mL×2)洗涤，用无水硫酸钠干燥，过滤，滤液减压浓缩，用薄层层析以展开剂体系B纯化所得残余物，得到标题产物6d(16mg，产率97.8％)。
MS m/z(ESI):870.0[M+1]。
第五步
2-((2-氨基乙酰氨基)甲氧基)-N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)-3,3,3-三氟丙酰胺6e
将6d(16mg，18.4μmol)溶于0.6mL二氯甲烷中，加入0.3mL二乙胺，室温搅拌2小时。反应液减压浓缩，加入2mL甲苯并减压浓缩，重复两次。向残余物中加入3mL正己烷打浆，静置后倾倒出上层清液，保留固体；重复三次。将固体残余物减压浓缩，油泵拉干得到粗品标题产物6e(12mg)，产品不经纯化直接用于下一步反应。
MS m/z(ESI):647.9[M+1]。
第六步
N-((2S,10S)-10-苄基-2-(((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基羰基)-1,1,1-三氟-6,9,12,15-四氧代-3-氧杂-5,8,11,14-四氮杂十六-16-基)-6-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)己酰胺6-A
N-((2R,10S)-10-苄基-2-(((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基羰基)-1,1,1-三氟-6,9,12,15-四氧代-3-氧杂-5,8,11,14-四氮杂十六-16-基)-6-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)己酰胺6-B
将粗品6e(12mg，18.5μmol)溶于1.0mL N,N-二甲基甲酰胺，氩气置换三次，冰水浴降温至0-5℃，加入4g(14mg，29.6μmol)的0.3mL N,N-二甲基甲酰胺溶液，加入4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐(15mg，54.2μmol)，冰浴搅拌反应30分钟，撤去冰浴，升至室温搅拌1小时，反应生成化合物6。反应液进行高效液相色谱法纯化(分离条件：色谱柱：XBridge Prep C18 OBD 5um19*250mm；流动相：A-水(10mmol NH 4OAc)B-乙腈，梯度洗脱，流速：18mL/min)，收集其相应组分，减压浓缩，得到标题产物(2.7mg，2.6mg)。
MS m/z(ESI):1102.0[M+1]。
单一构型化合物(较短保留时间)：
UPLC分析:保留时间1.18分钟，纯度：91％(色谱柱：ACQUITY UPLC BEHC18 1.7um 2.1*50mm，流动相：A-水(5mmol NH 4OAc)，B-乙腈)。
1H NMR(400MHz,DMSO-d 6):δ8.97(d,1H),8.85-8.76(m,1H),8.37-8.27(m,1H),8.12-8.02(m,1H),8.02-7.95(m,1H),7.80(d,1H),7.31(s,1H),7.26-7.10(m,4H),6.99(s,1H),6.66(s,1H),6.52(s,1H),5.65-5.54(m,1H),5.41(s,1H),5.37-5.25(m,3H),5.23-5.13(m,1H),4.81-4.68(m,2H),4.51-4.41(m,1H),3.78-3.45(m,6H),3.21-3.13(m,1H),3.02-2.93(m,1H),2.77-2.63(m,2H),2.45-2.29(m,3H),2.24-2.05(m,3H),2.04-1.93(m,5H),1.90-1.75(m,2H),1.52-1.38(m,4H),0.90-0.78(m,5H)。
单一构型化合物(较长保留时间)：
UPLC分析:保留时间1.23分钟，纯度：90％(色谱柱：ACQUITY UPLC BEHC18 1.7um 2.1*50mm，流动相：A-水(5mmol NH 4OAc)，B-乙腈)。
1H NMR(400MHz,DMSO-d 6):δ9.05(d,1H),8.97-8.88(m,1H),8.35-8.27(m,1H),8.11-8.03(m,1H),8.02-7.95(m,1H),7.80(d,1H),7.34(s,1H),7.29-7.13(m,4H),6.99(s,1H),6.66(s,1H),6.54(s,1H),5.64-5.55(m,1H),5.43(s,1H),5.36-5.20(m,3H),4.92-4.85(m,1H),4.82-4.72(m,2H),4.52-4.42(m,1H),3.77-3.48(m,6H),3.21-3.14(m,1H),3.03-2.95(m,1H),2.79-2.65(m,2H),2.47-2.28(m,3H),2.25-2.05(m,3H),2.05-1.94(m,5H),1.91-1.76(m,2H),1.52-1.37(m,4H),0.92-0.77(m,5H)。
实施例7相关抗体及其检测蛋白的制备
实施例7-1.B7H3抗原及检测用蛋白
以SEQ ID NO:1所示人B7H3作为本公开B7H3的模板，设计本公开涉及的抗原及检测用蛋白的氨基酸序列。以下B7H3抗原未特殊说明的均指人B7H3。
1.1人B7H3全长氨基酸序列：B7H3(SEQ ID NO：1)：
Figure PCTCN2019107852-appb-000037
注释：
双横线部分为信号肽(Signal peptide：1–28)；
划横线部分为B7H3胞外区(Extracellular domain：29-466)，其中29-139为Ig-样V-型1结构域，145–238为Ig-样C2-型1结构域；243-357为Ig-样V-型2结构域，363–456为Ig-样C2-型2结构域；
点划线部分为跨膜区部分(Transmembrane domain：467-487)；
斜体部分为胞内区(Cytoplasmic domain：488-534)。
1.2鼠B7H3全长氨基酸序列(SEQ ID NO：2)
Figure PCTCN2019107852-appb-000038
注释：
双横线部分为信号肽(Signal peptide：1–28)；
划横线部分为B7H3胞外区(Extracellular domain：29-248)，其中29–139为Ig-样V-型结构域，145–238为Ig-样C2-型结构域；
点划线部分为跨膜区部分(Transmembrane domain：249-269)；
斜体部分为胞内区(Cytoplasmic domain：270-316)。
1.3筛选及检测用人B7H3抗原(SEQ ID NO：3)
为商业化产品(R&D cat#1949-B3-050/CF，简称2Ig-B7H3)，序列如下：
Figure PCTCN2019107852-appb-000039
注释：
划横线部分为B7H3胞外区；
斜体部分为His-tag标记。
1.4检测用人B7H3抗原(SEQ ID NO：4)
为商业化产品(SinoBiological cat#11188-H08H，简称4Ig-B7H3)，序列如下：
Figure PCTCN2019107852-appb-000040
注释：划横线部分为B7H3胞外区；
斜体部分为His-tag标记。
1.5筛选及检测用鼠B7H3抗原(SEQ ID NO：5)
为商业化产品(R&D cat#1397-B3-050/CF)，序列如下：
Figure PCTCN2019107852-appb-000041
注释：划横线部分为B7H3胞外区；
斜体部分为His-tag标记。
实施例7-2.完全人源抗体的制备
2.1阳性序列的筛选
利用人PBMC、脾脏、淋巴结组织分离B细胞，并提取RNA，构建天然单链噬菌体抗体库(库容3.2×10 10)。将构建的天然单链噬菌体文库经过包装形成噬菌体颗粒后，采用液相法进行淘筛，噬菌体与生物素化的B7H3液相结合，再采用链霉亲和素磁珠分离。为了获得可分别与人B7H3(R&D cat#1949-B3-050/CF)和鼠B7H3(R&D cat#1397-B3-050/CF)交叉结合的阳性序列，分别采用生物素化的人B7H3和生物素化的鼠B7H3进行交替淘筛，首轮采用2μg/ml生物素化的人B7H3进行淘筛，第二轮采用2μg/ml生物素化的鼠B7H3进行淘筛，第三轮采用0.5μg/ml生物素化的人B7H3进行淘筛，经三轮淘筛后，挑取500个单克隆包装成噬菌体，用于噬菌体ELISA测试。分别测试单克隆噬菌体与人B7H3(R&D cat#1949-B3-050/CF)和鼠B7H3(R&D cat#1397-B3-050/CF)的结合活性：ELISA板上分别包被1μg/ml人B7H3或鼠B7H3以及1％BSA，加入1:1封闭缓冲液稀释的噬菌体上清，最后用anti-M13HRP检测。将ELISA测试到的OD450值大于0.5，以及结合人和鼠B7H3的ELISA OD450值除以结合1％BSA的ELISA OD450值的两个比值均大于2.0的克隆进行测序，得到特异性序列1702(在本公开中也称h1702，本公开所称抗体h1702和h1702DS与申请PCT/CN2018/081249的h1702和h1702-1相同，将申请PCT/CN2018/081249中的所有内容引入本公开)。
2.2完整单克隆抗体的构建
噬菌体库筛选得到特异性序列1702，构建其完整单克隆抗体的过程如下：
根据测序得到的单链抗体序列，设计引物PCR搭建各单链抗体序列的VH/VK/VL基因片段。获得1702的重轻链可变区。
>1702重链可变区序列
Figure PCTCN2019107852-appb-000042
>1702轻链可变区序列
Figure PCTCN2019107852-appb-000043
注：顺序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，序列中斜体为FR序列，下划线为CDR序列。
其中各抗体轻重链中CDR序列如表1所示。
表1 各重链及轻链CDR区序列
Figure PCTCN2019107852-appb-000044
Figure PCTCN2019107852-appb-000045
抗体可变区再与恒定区基因(CH1-FC/CL)片段进行同源重组，构建完整抗体VH-CH1-FC/VK-CL/VL-CL。
构建的完整全长抗体1702序列如下：
1702重链(IgG1)氨基酸序列：(SEQ ID NO：14)
Figure PCTCN2019107852-appb-000046
1702轻链氨基酸序列：Lamada(SEQ ID NO：15)
Figure PCTCN2019107852-appb-000047
为进一步提高抗体的稳定性，对1702的轻链序列进行氨基酸突变，具体突变为轻链(SEQ ID NO：15)N端第一个氨基酸残基Q替代为D，缺失突变C端第一个氨基酸残基S，以获得更加稳定和均一的单克隆抗体1702DS(在本公开中也称h1702DS)。
突变修饰后的1702DS的重链序列为SEQ ID NO：14，轻链氨基酸序列如下：(SEQ ID NO：16)。
Figure PCTCN2019107852-appb-000048
2.3全人抗体的表达与纯化
分别表达抗体轻重链的质粒以1.5:1的比例转染HEK293E细胞，6天后收集表达上清，高速离心去除杂质，用Protein A柱进行纯化。用PBS冲洗柱子，至 A280读数降至基线。用pH3.0-pH3.5的酸性洗脱液洗脱目的蛋白，用1M Tris-HCl,pH8.0-9.0中和。洗脱样品适当浓缩后，利用PBS平衡好的凝胶层析Superdex200(GE)进一步纯化，以去除聚体，收集单体峰，分装备用。
B7H3抗体-药物偶联物制备实施例
实施例8 ADC-1
Figure PCTCN2019107852-appb-000049
在37℃条件下，向抗体1702DS的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；7.3ml，13.8mg/ml，0.681μmol)加入配置好的三(2-羧乙基)膦的水溶液(10mM，0.347mL，3.47μmol)，置于水浴振荡器，于37℃振荡反应3小时，停止反应；将反应液用水浴降温至25℃，稀释至14.0ml，并取出3.3ml溶液往下反应。
将化合物4(3.0mg，2.75μmol)溶解于0.15mL DMSO中，加入到上述3.3ml溶液中，置于水浴振荡器，于25℃下振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到FADC-1通式的示例性产物ADC-1的PBS缓冲液(1.35mg/mL，13mL)，于4℃冷冻储存。
UV-HPLC计算平均值：n＝7.50。
实施例9 ADC-2
Figure PCTCN2019107852-appb-000050
在37℃条件下，向抗体1702DS的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；1.0ml，13.8mg/ml，0.093μmol)加入配置好的三(2-羧乙基)膦的水溶液(10mM，0.050mL，0.50μmol)，置于水浴振荡器，于37℃振荡反应3小时，停止反应；将反应液用水浴降温至25℃，稀释至2.0ml，并取出1.15ml溶液往下反应。
将化合物5-较短保留时间化合物5-A(1.29mg，1.02μmol)溶解于0.10mL DMSO中，加入到上述1.15ml溶液中，置于水浴振荡器，于25℃振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到FADC-2通式的示例性产物ADC-2的PBS缓冲液(2.63mg/mL，2.4mL)，于4℃冷冻储存。
UV-HPLC计算平均值：n＝7.24。
实施例10 ADC-3
Figure PCTCN2019107852-appb-000051
在37℃条件下，向抗体1702DS的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；7.3ml，13.8mg/ml，0.681μmol)加入配置好的三(2-羧乙基)膦的水溶液(10mM，0.347mL，3.47μmol)，置于水浴振荡器，于37℃下振荡反应3小时，停止反应；将反应液用水浴降温至25℃，稀释至14.0ml，并取出3.3ml溶液往下反应。
将化合物6-较长保留时间化合物(3.0mg，2.75μmol)溶解于0.15mL DMSO中，加入到上述3.3ml溶液中，置于水浴振荡器，于25℃振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到FADC-3通式的示例性产物ADC-3的PBS缓冲液(1.28mg/mL，13mL)，于4℃冷冻储存。
UV-HPLC计算平均值：n＝7.58。
实施例11 ADC-4
Figure PCTCN2019107852-appb-000052
在37℃条件下，向抗体1702DS的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/ml，2.14mL，144.60nmol)加入配置好的的三(2-羧乙基)膦(TCEP)的水溶液(10mM，73.7uL，740nmol)，置于水浴振荡器，于37℃振荡反 应3小时，停止反应。将反应液用水浴降温至25℃。
将化合物5-较短保留时间化合物5-A(3.0mg，2793nmol)溶解于150ul DMSO中，加入到上述反应液中，置于水浴振荡器，于25℃振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到FADC-2通式的示例性产物ADC-4的PBS缓冲液(1.28mg/mL，13.0mL)，于4℃冷冻储存。
UV-Vis计算平均值：n＝6.87。
实施例12 ADC-5
Figure PCTCN2019107852-appb-000053
在37℃条件下，向抗体1702DS的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/ml，0.89mL，60.14nmol)加入配置好的三(2-羧乙基)膦(TCEP)的水溶液(10mM，30.1uL，300nmol)，置于水浴振荡器，于37℃振荡反应3小时，停止反应。将反应液用水浴降温至25℃。
将化合物5-较短保留时间化合物5-A(1.02mg，950nmol)溶解于100ul DMSO中，加入到上述反应液中，置于水浴振荡器，于25℃振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到FADC-2通式的示例性产物ADC-5的PBS缓冲液(1.94mg/mL，3.5mL)，于4℃冷冻储存。
UV-Vis计算平均值：n＝6.11。
参考以上反应步骤，按本领域的常规技术手段调整反应条件，得到n值分别为2.97、4.8的FADC-2通式的示例性产物：ADC-6(n＝2.97)；ADC-7(n＝4.8)。
实施例13 ADC-8
Figure PCTCN2019107852-appb-000054
在37℃条件下，向抗体1702DS的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/ml，0.89mL，60.14nmol)加入配置好的的三(2-羧乙基)膦(TCEP)的水溶液(10mM，30.1uL，300nmol)，置于水浴振荡器，于37℃振荡反应3小时，停止反应。将反应液用水浴降温至25℃。
将化合物4(1.0mg，943nmol)溶解于100ul DMSO中，加入到上述反应液中，置于水浴振荡器，于25℃振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到FADC-1通式的示例性产物ADC-8的PBS缓冲液(1.47mg/mL，4.5mL)，于4℃冷冻储存。
UV-Vis计算平均值：n＝6.33。
生物学评价
测试例1.Biacore抗体亲和力实验
用Biacore，GE仪器测定抗B7H3抗体，及B7H3-ADC和人2Ig-B7H3抗原，人4Ig-B7H3抗原之间的反应亲和力。
用生物传感芯片Protein A(Cat.#29127556，GE)亲和捕获一定量的待测抗体/待测ADC，然后于芯片表面流经一系列浓度梯度下的人2Ig-B7H3抗原(Cat.#1949-B3-050/CF，R&D)、人4Ig-B7H3抗原(Cat.#11188-H08H，Sino Biological)，利用Biacore仪器(Biacore T200，GE)实时检测反应信号从而获得结合和解离曲线。在每个循环解离完成后，用甘氨酸-盐酸再生溶液(pH 1.5)(Cat.#BR-1003-54，GE)将生物芯片洗净再生。实验中用到的缓冲液为HBS-EP缓冲溶液(pH 7.4)(Cat.#BR-1001-88，GE)。
实验得到的数据用BIAevaluation version 4.1，GE软件以(1:1)Langmuir模型进行拟合，从而得出亲和力数值。实验结果见表2。
表2.h1702抗体和各种抗原之间的反应亲和力(单位：M)
抗体 人2Ig-B7H3 人4Ig-B7H3
h1702 7.97E-7 8.55E-9
ADC-2   7.55E-9
结论：h1702抗体与抗原具有很强的亲和力。同时，针对与人2Ig-B7H3、人4Ig-B7H3在Biacore实验上的亲和力测试表明，ADC与裸抗的亲和力相似。
测试例2.体外细胞内吞实验
本实验通过检测细胞内抗体的荧光信号，根据荧光信号强弱来评价抗体的內吞效果。将B7-H3抗体和APC anti-human IgG Fc(Biolegend，409306)以1:2的摩尔比例混合在冰上孵育15分钟。将抗体混合物与2×10 5个U87MG细胞(人脑星形胶质母细胞瘤，中科院细胞库，Catalog#TCHu138)在冰上孵育30分钟后洗 掉多余的抗体，然后将细胞转入预温37℃的培养基中，在37℃分别孵育0、15、30、60和120分钟。离心细胞并将细胞重悬在抗体洗脱液(0.05M甘氨酸，0.1M NaCl，PH2.45)中室温孵育7分钟，洗掉抗体洗脱液，使用BD Verse读取细胞内荧光信号(结果见图1)。结果表明h1702与U87MG细胞系结合后，均能有效地被内吞入细胞。
测试例3.SD大鼠T 1/2评价
SD大鼠(购自杰思捷实验动物有限公司)4只，雌雄各半，12/12小时光/暗调节，温度24±3℃恒温，湿度50-60％，自由进食饮水。实验当天对SD大鼠分别尾静脉注射受试药物B7H3抗体/ADC，给药剂量为3mg/kg，注射体积5ml/kg。
取血时间点为：第1天给药后5分钟、8时、24时(第2天)、第3天、第5天、第8天、第11天、第15天，于大鼠眼底静脉取血，每次200μL(相当于取血清100μL)；收集的血样在室温放置半小时至凝集，然后于4℃10000×g离心10分钟。收集上清，立即放置-80℃贮存。
用ELISA检测血清中的B7H3抗体浓度，进行PK分析，结果见表3。
表3.B7H3抗体在SD大鼠中的T 1/2
受试药物 给药方式 T 1/2(平均值±SD，小时)
h1702 IV(3mg/kg) 185±17
结果表明，本公开h1702在大鼠体内的半衰期约为185h(7.7天)。
测试例4.B7H3抗体的化学稳定性
抗体制备后化学修饰是导致产品稳定性的常见问题之一，尤其是CDR区域的部分氨基酸高度脱酰胺、氧化或者异构化修饰，一般选择尽量避免或者突变降低。取500μg待测抗体溶于500μl pH 7.4的PBS中，40℃水浴；分别于0、10、20天取样，用于酶解实验。将100μg不同时间点取样的样品溶于100μl 0.2M His-HCl(组氨酸盐酸缓冲液)和8M Gua-HCl(瓜氨酸盐酸缓冲液)，pH 6.0溶液中，加3μl 0.1g/mL DTT(二硫苏糖醇)，50℃水浴1小时后用0.02M His-HCl，pH 6.0的溶液超滤两次，加入3μL 0.25mg/mL的胰蛋白酶(invitrogen，CAT#25200-072)，37℃水浴酶解过夜。Agilent 6530Q-TOF进行LC-MS检测分析，对潜在的修饰位点进行质谱分析(结果见表4)。结果显示本公开中涉及的B7H3抗体h1702均无明显的脱酰胺、氧化或者异构化加剧趋势，提示良好的理化稳定性。
表4.不同抗体的化学稳定性
Figure PCTCN2019107852-appb-000055
测试例5.h1702DS抗体的稳定性
通过SEC、非还原CE-SDS分析检测方法(pH 9.0)和IEX分析检测方法，对h1702和h1702DS进行稳定性检测。
SEC检测：使用Waters e2695色谱仪，Xbridge BEH 200A SEC色谱柱，上样50μg抗体，PBS流动相等度洗脱。
CE-SDS NR方法：
使用Beckman SDS-MW Analysis Kit试剂盒处理样品。100μg蛋白中加入缓冲液，加热变性。使用PA800毛细管电泳仪采集数据。
IEX方法：
使用Waters Acquity H-Class色谱仪，Thermo MAbPac SCX-10色谱柱，CX-1pH Gradient Buffer Kit作为流动相，上样50μg抗体，线性梯度，采集280nm波长的紫外信号。
表5 h1702和h1702DS稳定性比较
  SEC CE-SDS(pH9.0) IEX
h1702 100％ 71.21％ 40.5％
h1702DS 100％ 94.67％ 86.21％
测试例6：体外细胞增殖实验
本实验通过检测细胞内ATP含量，根据IC 50大小评价B7H3-ADC对细胞增殖的抑制效果。
U87MG细胞(人脑星形胶质母细胞瘤，中科院细胞库，Catalog#TCHu138)、Calu-6细胞(肺癌细胞，ATCC，Catalog
Figure PCTCN2019107852-appb-000056
HTB-56 TM)、Detroit562细胞(人咽头癌细胞ATCC，Catalog
Figure PCTCN2019107852-appb-000057
CCL-138 TM)、A498细胞(肾癌细胞，ATCC，Catalog
Figure PCTCN2019107852-appb-000058
HTB-44 TM)培养在含10％FBS的EMEM培养基中，一周传代2-3次，传代比例1:3或1:6。EMEM培养基配置：MEM培养基(GE，CAT#SH30024.01)，NEAA(sigma，CAT#M7145-100ML)，丙酮酸钠溶液(Sodium pyruvate solution，sigma，CAT#S8636-100ML)。
A-375(黑色素瘤细胞，ATCC，Catalog
Figure PCTCN2019107852-appb-000059
CRL-1619 TM)培养在含10％FBS的DMEM(GE，SH30243.01)培养基中，一周传代2-3次，传代比例1:3或1:6。
CHO-K1(不表达人B7H3，ATCC，Catalog
Figure PCTCN2019107852-appb-000060
CCL-61 TM)培养在含10％FBS的F12(Gibco，11765-054)培养基中，一周传代2-3次，传代比例1:4或1:6。
传代时，吸掉培养基，用5ml 0.25％的胰酶冲洗细胞层，然后吸掉胰酶，将细胞放在培养箱中消化3～5分钟，加入新鲜培养基重悬细胞，计数，将细胞悬液配制为相应密度(U87MG细胞500个细胞/孔；A-498细胞500个细胞/孔；A-375 细胞300个细胞/孔；Calu-6细胞800个细胞/孔；detroit562细胞2000个细胞/孔；CHO-K1细胞500个细胞/孔)。
在96孔细胞培养板中加入180μL的细胞悬液，96孔板外围只加入200ul培养基。将培养板在培养箱中培养24小时(37℃，5％CO 2)。
将待测样品用PBS或DMSO以3倍的倍比依次稀释成9个浓度(各ADC起始浓度500nM)。将样品加入培养板中，将培养板在培养箱孵育6天(37℃，5％CO 2)。在96孔细胞培养板中，每孔加入90μl CellTiter-Glo试剂，室温避光放置10分钟，在Victor3中读取化学发光信号值，数据使用GraphPad软件处理。测得的IC 50值见表6及图2A至图2F。
表6.本公开ADC对细胞增殖的抑制效果
  A498 Calu-6 U87 A375 Detroit562 CHOK1
ADC-2 418.9* 70.6 49.1 33.3 31.6 >500
ADC-3 13.1 1.91 3.78 1.51 1.89 36.8
ADC-1 196.6 26.54 29 23.72 26.9 >500
*数值单位为nM
测试例7：本公开ADC对人脑星形胶质母细胞瘤U87MG裸小鼠移植瘤的疗效评价
一、试验目的
本实验以BALB/cA-nude裸小鼠为受试动物，评价本公开ADC化合物对人脑星形胶质母细胞瘤U87MG裸小鼠移植瘤的的疗效。
二、受试药物及材料
1、受试药物
ADC-5(1mpk，3mpk)
ADC-8(1mpk，3mpk)
空白组(blank)：PH7.4的PBS缓冲液
2、配制方法：PH7.4的PBS缓冲液。
3、试验动物
BALB/cA-nude裸小鼠：SPF，雌性，购自上海杰思捷实验动物有限责任公司。
三、试验方法
实验用BALB/cA-nude裸小鼠，雌性，6-7周，皮下接种人脑星形胶质母细胞瘤U87MG细胞(同上)。接种细胞后第十天，将动物随机分组(D0)，每组8只，开始腹腔注射给药1次/周，共给药3次，每周测2－3次瘤体积和体重，记录数据。肿瘤体积(V)计算公式为：
V＝1/2×a×b 2
其中：
a、b分别表示长、宽。
相对体积(RTV)＝V T/V 0
抑瘤率(％)＝(C RTV-T RTV)/C RTV(％)
其中V 0、V T分别为实验开始时及实验结束时的肿瘤体积。C RTV、T RTV分别为实验结束时的对照组(空白组)及实验组的相对肿瘤体积。
四、试验结果
腹腔注射(i.p.)给药每周1次，共给药3次，观察至第18天时，ADC-8 1mpk的抑瘤率达到39.22％(P<0.01)；ADC-8 3mpk的抑瘤率达到80.24％(P<0.0001)；ADC-5 1mpk的抑瘤率达到27.53％(P<0.05)；ADC-5 3mpk的抑瘤率达到55.88％(P<0.0001)。观察至22天(D22)时，各给药组的抑瘤率均进一步提高，ADC-8 1mpk的抑瘤率达到47.7％(P<0.0001)；ADC-8 3mpk的抑瘤率达到89.8％(P<0.0001)；ADC-5 1mpk的抑瘤率达到40.6％(P<0.0001)；ADC-5 3mpk的抑瘤率达到63.3％(P<0.0001)。
给药过程中各组动物体重正常，提示ADC无明显毒副作用。检测结果如表7和图3所示。所检测抗体能够有效抑制荷瘤裸鼠中U87MG移植瘤的生长，并且呈现出剂量依赖性。
表7.本公开ADC对荷瘤裸鼠U87MG移植瘤的疗效(D22)
Figure PCTCN2019107852-appb-000061
vs空白：*p<0.05，**p<0.01，***p<0.001
测试例8：本公开ADC对人咽头癌胸水转移细胞Detroit 562裸小鼠移植瘤的疗效评价
一、试验目的
本实验以BALB/cA-nude裸小鼠为受试动物，评价本公开ADC化合物对人咽头癌胸水转移细胞Detroit 562裸小鼠移植瘤的疗效。
二、受试药物及材料
1、受试药物
ADC-1(1mpk，3mpk)
ADC-2(1mpk，3mpk)
阴性对照ADC(3mpk)：非B7H3靶点与参比化合物(专利“CN104755494A”中实施例58)偶联形成的配体毒素偶联物
2、配制方法：均用PBS稀释配制。
3、试验动物
BALB/cA-nude裸小鼠：购自常州卡文斯实验动物有限责任公司。
三、试验方法
实验用BALB/cA-nude裸小鼠，雌性，6-7周，皮下接种人咽头癌胸水转移细胞Detroit 562细胞。接种细胞后第十天，将动物随机分组(D0)，每组8只，开始腹腔注射给药1次/周，共给药3次，每周测2－3次瘤体积和体重，记录数据。肿瘤体积(V)计算公式为：
V＝1/2×a×b 2
其中：
a、b分别表示长、宽。
相对体积(RTV)＝V T/V 0
抑瘤率(％)＝(C RTV-T RTV)/C RTV(％)
其中V 0、V T分别为实验开始时及实验结束时的肿瘤体积。C RTV、T RTV分别为实验结束时的对照组(阴性对照)及实验组的相对肿瘤体积。
四、试验结果
腹腔注射给药每周1次，共给药3次，观察至D28时，受试ADC抑瘤率分别是：ADC-1 1mg/kg(1mpk)的抑瘤率达到40.85％；ADC-1 3mg/kg(3mpk)的抑瘤率达到62.55％(P<0.05)；ADC-2 1mg/kg(1mpk)的抑瘤率达到44.26％；ADC-23mg/kg(3mpk)的抑瘤率达到72.27％(P<0.01)。
给药过程中各组动物体重正常，提示ADC无明显毒副作用。检测结果如表8和图4所示。所检测抗体能够有效抑制荷瘤裸鼠中Detroit 562移植瘤的生长，并且呈现出剂量依赖性。
表8.本公开ADC对荷瘤裸鼠Detroit 562移植瘤的疗效(D28)
Figure PCTCN2019107852-appb-000062
Figure PCTCN2019107852-appb-000063
测试例9：不同载药量的ADC的体外细胞增殖
对具有FADC-2结构式的ADC化合物ADC-4(N＝6.87)、ADC-6(n＝2.97)、ADC-7(n＝4.8)，检测其在体外细胞增殖实验的疗效，实验操作流程与测试例6相同。
测得的IC 50值和最大抑制率见表9及图5A、5B和5C。结果显示，不同DAR值的FADC-2均显示出抑制细胞增殖的疗效，且抑制效果与DAR值正相关，而裸抗无细胞增殖抑制的效果。
表9
Figure PCTCN2019107852-appb-000064
测试例10：血浆稳定性
将ADC-4样品，以100μg/ml的终浓度，分别与人血浆、猴血浆(上海美迪西生物医药股份有限公司)、和1％BSA(Sigma)PBS(上海生工)溶液混合均匀后，过滤除菌后置于37℃水浴锅内孵育，将孵育当天记为第0天，随后分别在第7天、14天和21天取出样品，进行游离毒素的检测。
不同时间点的样品取出后放至室温，涡旋混匀；取25时间样品至96孔板中；加入50中；内标工作液(100ng/mL喜树碱乙腈溶液)及150μl乙腈；涡旋混合5分钟，离心10分钟(4000rpm)，取上清液5取上清进行LC/MS/MS分析(美国应用生物系统公司)。
结果如图6所示，ADC-4在人和猴血浆，以及1％BSA PBS溶液中均相当稳定，游离毒素的释放率最高不超过2％，且在第14天趋于稳定。

Claims (28)

  1. 一种通式(Pc-L-Y-Dr)所示的配体-药物偶联物或其药学上可接受的盐或溶剂化物：
    Figure PCTCN2019107852-appb-100001
    其中：
    Y选自-O-(CR aR b) m-CR 1R 2-C(O)-、-O-CR 1R 2-(CR aR b) m-、-O-CR 1R 2-、-NH-(CR aR b) m-CR 1R 2-C(O)-或-S-(CR aR b) m-CR 1R 2-C(O)-；
    R a和R b相同或不同，且各自独立地选自氢原子、氘原子、卤素、烷基、卤代烷基、氘代烷基、烷氧基、羟基、氨基、氰基、硝基、羟烷基、环烷基或杂环基；
    或者，R a和R b与其相连接的碳原子一起形成环烷基或杂环基；
    R 1选自卤素、卤代烷基、氘代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基或杂芳基；
    R 2选自氢原子、卤素、卤代烷基、氘代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基或杂芳基；
    或者，R 1和R 2与其相连的碳原子一起形成环烷基或杂环基；
    或者，R a和R 2与其相连的碳原子一起形成环烷基或杂环基；
    m为0至4的整数；
    n为1至10，n是小数或整数；
    L为接头单元；
    Pc为抗B7H3抗体或其抗原结合片段。
  2. 根据权利要求1所述的通式(Pc-L-Y-Dr)所示的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其中所述抗B7H3抗体或其抗原结合片段包含：
    分别如SEQ ID NO：8、9和10氨基酸序列所示的重链HCDR1、HCDR2、HCDR3，或与SEQ ID NO：8、9和10所示的HCDR1、HCDR2和HCDR3分别具有3、2或1个氨基酸差异的HCDR变体；和
    分别如SEQ ID NO：11、12和13氨基酸序列所示的轻链LCDR1、LCDR2和LCDR3，或与SEQ ID NO：11、12和13所示的LCDR1、LCDR2和LCDR3分别具有3、2或1个氨基酸差异的LCDR变体。
  3. 根据权利要求1或2所述的通式(Pc-L-Y-Dr)所示的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其中所述抗B7H3抗体或其抗原结合片段的轻链可变区上的轻链FR区来源于人种系轻链序列或其突变序列，和/或重链可变区上的重链FR区来源于人种系重链序列或其突变序列。
  4. 根据权利要求1至3中任一项所述的通式(Pc-L-Y-Dr)所示的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其中所述抗B7H3抗体或其抗原结合片段包含选自如下所示的重链可变区和/或轻链可变区：
    其中所述重链可变区氨基酸序列如SEQ ID NO：6所示或与其具有至少95％序列同一性，所述轻链可变区氨基酸序列如SEQ ID NO：7所示或与其具有至少95％序列同一性。
  5. 根据权利要求1至4中任一项所述的通式(Pc-L-Y-Dr)所示的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其中所述抗B7H3抗体或其抗原结合片段包含抗体恒定区；所述抗体恒定区的重链恒定区来源于人IgG1、IgG2、IgG3或IgG4或与其具有至少95％序列同一性，所述抗体恒定区的轻链恒定区来源于人抗体κ、λ链或与其具有至少95％序列同一性；优选地，所述重链恒定区的氨基酸序列源于人IgG1或与其具有至少95％序列同一性。
  6. 根据权利要求1至5中任一项所述的通式(Pc-L-Y-Dr)所示的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其中所述Pc为全长抗体，其中所述的全长抗体选自：
    由SEQ ID NO:14所示的重链序列和SEQ ID NO:15所示的轻链序列组成的h1702抗体，和
    由SEQ ID NO:14所示的重链序列和SEQ ID NO:16所示的轻链序列组成的h1702DS抗体。
  7. 根据权利要求1至5中任一项所述的通式(Pc-L-Y-Dr)所示的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其中所述抗原结合片段选自Fab、Fab'、F(ab')2、单链抗体(scFv)、二聚化的V区(双抗体)、二硫键稳定化的V区(dsFv)和包含CDR的肽的抗原结合片段。
  8. 根据权利要求1至7中任一项所述的通式(Pc-L-Y-Dr)所示的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其中n为2至8，优选为5至9，n是小数或整数。
  9. 根据权利要求1至8中任一项所述的通式(Pc-L-Y-Dr)所示的配体-药物偶 联物或其药学上可接受的盐或溶剂化物，
    其中：
    Y为-O-(CR aR b) m-CR 1R 2-C(O)-；
    R a和R b相同或不同，且各自独立地选自氢原子、氘原子、卤素或烷基；
    R 1为卤代烷基或C 3-6环烷基；
    R 2选自氢原子、卤代烷基或C 3-6环烷基；
    或者，R 1和R 2与其相连接的碳原子一起形成C 3-6环烷基；
    m为0或1。
  10. 根据权利要求1至9中任一项所述的通式(Pc-L-Y-Dr)所示的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其中Y选自：
    Figure PCTCN2019107852-appb-100002
  11. 根据权利要求1至10中任一项所述的通式(Pc-L-Y-Dr)所示的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其中Y的O端与接头单元L相连。
  12. 根据权利要求1至11中任一项所述的通式(Pc-L-Y-Dr)所示的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其中接头单元-L-为-L 1-L 2-L 3-L 4-，
    L 1为
    Figure PCTCN2019107852-appb-100003
    s 1为2至8的整数；
    L 2为化学键；
    L 3为四肽残基；
    L 4为-NR 5(CR 6R 7)t-，R 5、R 6或R 7相同或不同，且各自独立地为氢原子或烷基，t为1或2。
  13. 根据权利要求1至12中任一项所述的通式(Pc-L-Y-Dr)所示的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其中所述的接头单元-L-，其L 1端与Pc相连，L 4端与Y相连。
  14. 根据权利要求1至13中任一项所述的通式(Pc-L-Y-Dr)所示的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其中所述的L 3的四肽残基为由两个或 多个选自苯丙氨酸、甘氨酸、缬氨酸、赖氨酸、瓜氨酸、丝氨酸、谷氨酸、天冬氨酸中的氨基酸形成的氨基酸残基；优选为GGFG的四肽残基。
  15. 根据权利要求1至14中任一项所述的通式(Pc-L-Y-Dr)所示的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其中所述-L-Y-为以下结构：
    Figure PCTCN2019107852-appb-100004
    L 2为化学键；
    L 3为GGFG的四肽残基；
    R 1为卤代烷基或C 3-6环烷基；
    R 2选自氢原子、卤代烷基或C 3-6环烷基；
    或者，R 1和R 2与其相连接的碳原子一起形成C 3-6环烷基；
    R 5、R 6或R 7相同或不同，且各自独立地为氢原子或烷基；
    s 1为2至8的整数；
    m为0至4的整数。
  16. 根据权利要求1至15中任一项所述的通式(Pc-L-Y-Dr)所示的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其中-L-Y-选自：
    Figure PCTCN2019107852-appb-100005
    Figure PCTCN2019107852-appb-100006
  17. 根据权利要求1至11中任一项所述的通式(Pc-L-Y-Dr)所示的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其为通式(Pc-L a-Y-Dr)所示的配体-药物偶联物或其药学上可接受的盐或溶剂化物：
    Figure PCTCN2019107852-appb-100007
    其中：
    W选自C 1-8烷基、C 1-8烷基-环烷基或1至8个原子的直链杂烷基，所述杂烷基包含1至3个选自N、O或S的杂原子，其中所述的C 1-8烷基、环烷基和直链杂烷基各自独立地任选进一步被选自卤素、羟基、氰基、氨基、烷基、氯代烷基、氘代烷基、烷氧基和环烷基的一个或多个取代基所取代；
    L 2选自-NR 4(CH 2CH 2O) p1CH 2CH 2C(O)-、-NR 4(CH 2CH 2O) p1CH 2C(O)-、-S(CH 2) p1C(O)-或化学键，p 1为1至20的整数；
    L 3为由2至7个氨基酸构成的肽残基，氨基酸可以是取代的或非取代的，当被取代时，取代基可以在任何可使用的连接点上被取代，所述取代基为一个或多个独立地选自卤素、羟基、氰基、氨基、烷基、氯代烷基、氘代烷基、烷氧基和环烷基；
    R 1选自卤素、卤代烷基、氘代烷基、环烷基、杂环基、芳基或杂芳基；
    R 2选自氢原子、卤素、卤代烷基、氘代烷基、环烷基、杂环基、芳基或杂芳基；
    或者，R 1和R 2与其相连接的碳原子一起形成环烷基或杂环基；
    R 4和R 5相同或不同，且各自独立地选自氢原子、烷基、卤代烷基、氘代烷基和羟烷基；
    R 6和R 7相同或不同，且各自独立地选自氢原子、卤素、烷基、卤代烷基、氘代烷基和羟烷基；
    m为0至4的整数；
    n为1至10，n是小数或整数；
    Pc为抗B7H3抗体或其抗原结合片段。
  18. 根据权利要求17所述的通式(Pc-L-Y-Dr)所示的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其为通式(Pc-L b-Y-Dr)所示的配体-药物偶联物或其药学上可接受的盐或溶剂化物：
    Figure PCTCN2019107852-appb-100008
    其中：
    s 1为2至8的整数；
    R 1、R 2、R 5～R 7、m和n如权利要求17中所定义。
  19. 根据权利要求1至18中任一项所述的通式(Pc-L-Y-Dr)所示的配体-药物偶联物或其药学上可接受的盐或溶剂化物，所述配体-药物偶联物选自：
    Figure PCTCN2019107852-appb-100009
    Figure PCTCN2019107852-appb-100010
    Figure PCTCN2019107852-appb-100011
    其中Pc和n如权利要求1中所定义。
  20. 根据权利要求1至19中任一项所述的通式(Pc-L-Y-Dr)所示的配体-药物偶联物或其药学上可接受的盐或溶剂化物，所述配体-药物偶联物选自：
    Figure PCTCN2019107852-appb-100012
    Figure PCTCN2019107852-appb-100013
    其中，n如权利要求1所定义。
  21. 一种制备如通式(Pc-L a-Y-Dr)所示的配体-药物偶联物或其药学上可接受的盐或溶剂化物的方法，其包括以下步骤：
    Figure PCTCN2019107852-appb-100014
    Pc还原后，与通式(L a-Y-Dr)偶联反应，得到通式(Pc-L a-Y-Dr)所示的化合物；
    其中：
    Pc为抗B7H3抗体或其抗原结合片段；
    W、L 2、L 3、R 1、R 2、R 5～R 7、m和n如权利要求17中所定义。
  22. 根据权利要求21所述的方法，其中所述的通式L a-Y-Dr为通式L b-Y-Dr所示的化合物：
    Figure PCTCN2019107852-appb-100015
    或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐，
    其中
    R 1、R 2、R 5～R 7、s 1和m如权利要求18中所定义。
  23. 根据权利要求21或22所述的方法，其中式(L a-Y-Dr)所示的化合物或式(L b-Y-Dr)所示的化合物选自：
    Figure PCTCN2019107852-appb-100016
    Figure PCTCN2019107852-appb-100017
  24. 一种药物组合物，其包含根据权利要求1至20中任一项所述的抗体-药物偶联物或其药学上可接受的盐或溶剂化物，以及一种或多种药学上可接受的赋形剂、稀释剂或载体。
  25. 根据权利要求1至20中任一项所述的抗体-药物偶联物或其药学上可接受的盐或溶剂化物或根据权利要求24所述的药物组合物在制备用于治疗B7H3介导的疾病或病症的药物中的用途。
  26. 根据权利要求25所述的用途，其中所述B7H3介导的疾病或病症为B7H3高表达癌症。
  27. 根据权利要求1至20中任一项所述的配体-药物偶联物或其药学上可接受的盐或溶剂化物或根据权利要求24所述的药物组合物在制备用于治疗或预防肿瘤的药物中的用途。
  28. 根据权利要求1至20中任一项所述的配体-药物偶联物或其药学上可接受的盐或溶剂化物或根据权利要求24所述的药物组合物在制备治疗和/或预防癌症的药物中的用途，其中所述癌症优选选自乳腺癌、卵巢癌、宫颈癌、肺癌、子宫癌、前列腺癌、肾癌、尿道癌、膀胱癌、肝癌、胃癌、子宫内膜癌、唾液腺癌、食道癌、黑色素瘤、神经胶质瘤、神经母细胞瘤、肉瘤、咽头癌、肺癌、结肠癌、直肠癌、结直肠癌、白血病、骨癌、皮肤癌、甲状腺癌、胰腺癌和淋巴瘤。
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