WO2021190583A1 - 抗psma抗体-依喜替康类似物偶联物及其医药用途 - Google Patents

抗psma抗体-依喜替康类似物偶联物及其医药用途 Download PDF

Info

Publication number
WO2021190583A1
WO2021190583A1 PCT/CN2021/082857 CN2021082857W WO2021190583A1 WO 2021190583 A1 WO2021190583 A1 WO 2021190583A1 CN 2021082857 W CN2021082857 W CN 2021082857W WO 2021190583 A1 WO2021190583 A1 WO 2021190583A1
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
cancer
group
drug conjugate
pharmaceutically acceptable
Prior art date
Application number
PCT/CN2021/082857
Other languages
English (en)
French (fr)
Inventor
应华
张小敏
杨筱莹
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
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
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 BR112022019027A priority Critical patent/BR112022019027A2/pt
Priority to EP21775717.8A priority patent/EP4130006A4/en
Priority to JP2022557789A priority patent/JP2023519261A/ja
Priority to KR1020227035634A priority patent/KR20220160016A/ko
Priority to AU2021240756A priority patent/AU2021240756A1/en
Priority to CN202180022490.3A priority patent/CN115298186A/zh
Priority to US17/914,209 priority patent/US20230140397A1/en
Priority to MX2022011808A priority patent/MX2022011808A/es
Priority to CA3177279A priority patent/CA3177279A1/en
Publication of WO2021190583A1 publication Critical patent/WO2021190583A1/zh
Priority to ZA2022/10724A priority patent/ZA202210724B/en

Links

Images

Classifications

    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/22—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains four or more hetero rings
    • 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
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A61K47/6835—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
    • A61K47/6851—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 determinant of a tumour cell
    • A61K47/6869—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 determinant of a tumour cell the tumour determinant being from a cell of the reproductive system: ovaria, uterus, testes, prostate
    • 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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3069—Reproductive system, e.g. ovaria, uterus, testes, prostate
    • 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
    • C07K2317/50—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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the present disclosure relates to an anti-PSMA antibody, an anti-PSMA antibody-Isinotecan analog conjugate, a preparation method thereof, a pharmaceutical composition containing the same, and use thereof in the preparation of a medicine for treating PSMA-mediated diseases or disorders ; Especially in the preparation of anti-cancer drugs.
  • PSMA prostate-specific membrane antigen
  • non-prostatic tissues such as the small intestine, proximal renal tubules and salivary glands, but its level is much lower than that of prostate tissue.
  • PSMA is highly expressed in prostate cancer cells, especially in metastatic disease, hormone refractory disease and high-grade disease.
  • PSMA is also highly expressed in the neovascular endothelial cells of all solid tumors, but it is not expressed in normal blood vessels, so it is also used as a target for the treatment of solid tumors (Clinical Cancer Research, Vol. 3, 81-85).
  • PSMA prostate-specific membrane antigen
  • PSMA belongs to Glutamate carboxypeptidase II (GCPII), which acts as a NAALDase in the nervous system.
  • GCPII Glutamate carboxypeptidase II
  • FPGn folyl-poly- ⁇ -glutamate
  • folic acid which is related to folate metabolism (Curr Med Chem. 2012; 19(6): 856-870. ).
  • PSMA in the prostate it is expressed on epithelial cells and is related to the occurrence of prostate cancer.
  • PSMA consists of 750 amino acids, 19 intracellular, 24 transmembrane, and 707 extracellular.
  • the crystallization results show that the extracellular part is composed of 3 domains, namely the protease-like domain, the apical domain and the C-terminal domain. All three are involved in the binding of the substrate, the former two directly bind to the substrate, and the C-terminal domain plays a role in the formation of dimers of PSMA (The EMBO Journal (2006) 25, 1375-1384).
  • Glutamate type II carboxypeptidase (GCPII) and its splice variants, paralogs are limited in research. Most of the splice variants represented by PSM' exist in the cells of normal prostate tissue.
  • Antibody-drug conjugate connects monoclonal antibodies or antibody fragments with biologically active cytotoxins through linker compounds, making full use of the specificity and cellular binding of antibodies to normal cells and tumor cell surface antigens.
  • ADC Antibody-drug conjugate
  • ADC drugs have been used in clinical or clinical research, such as Kadcyla, which is an ADC drug formed by trastuzumab targeting Her2 and DM1.
  • Kadcyla is an ADC drug formed by trastuzumab targeting Her2 and DM1.
  • ADC drugs targeting PSMA for clinical treatment research.
  • Cytogen's PSMA-ADC is in the second clinical phase.
  • MedImmune's MEDI-3726 and BZL Biologics Inc.'s MLN-2704 stopped the study due to poor efficacy after the first phase of the clinical trial.
  • the use of different strategies to develop new ADC drugs has broad prospects.
  • the present disclosure relates to an anti-PSMA antibody-ADC and its use, and provides ADC drugs coupled with an anti-PSMA antibody or an antigen-binding fragment and a cytotoxic substance exenotecan analog.
  • the present disclosure provides an antibody-drug conjugate represented by the general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt 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)- and -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 a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a halogenated alkyl group, a deuterated alkyl group, an alkoxy group, a hydroxyl group, an amino group, a cyano group, a nitro group, a hydroxyalkyl group group, cycloalkyl group and heterocyclic group; form a cycloalkyl or heterocyclic group, or, R a and R b are carbon atoms connected thereto;
  • R 1 is selected from halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclic group, aryl and heteroaryl;
  • R 2 is selected from hydrogen atom, halogen, Haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl and heteroaryl; alternatively, R 1 and R 2 form together with the carbon atom to which they are attached Cycloalkyl or heterocyclic group;
  • R a and R 2 together with the carbon atom to which they are connected form a cycloalkyl group or a heterocyclic group;
  • n is an integer from 0 to 4; non-limiting examples such as m is selected from 0, 1, 2, 3 and 4;
  • n is 1 to 10, and n is a decimal or integer; preferably, n is 1-8, more preferably 3-8, most preferably 3-7, and more preferably 6-7.
  • Pc is an anti-PSMA antibody or an antigen-binding fragment thereof.
  • the anti-PSMA antibody or an antigen-binding fragment thereof specifically binds to the extracellular domain of PSMA.
  • the chain variable region includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively.
  • the variable region and the light chain variable region wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:1 or has at least 90%-100% sequence identity therewith, including but not limited to at least 91% , At least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity; and the light chain variable region
  • the amino acid sequence is shown in SEQ ID NO: 2 or has at least 90%-100% sequence identity with it, including but not limited to at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96 %, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity.
  • the heavy chain variable region shown in SEQ ID NO: 1 and the light chain variable region shown in SEQ ID NO: 2 are sequenced.
  • the heavy chain constant region is selected from the constant regions of human IgG1, IgG2, IgG3 and IgG4 and conventional variants thereof
  • the light chain constant region is selected from the constant regions of human antibody kappa and lambda chains and Its regular variant.
  • the antibody-drug conjugate represented by the aforementioned general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof is represented by the aforementioned general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt 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, and a C 1-6 alkyl group;
  • R 1 is haloalkyl or C 3-6 cycloalkyl
  • R 2 is selected from a hydrogen atom, a halogenated alkyl group and 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 O end of Y is connected to the joint unit L.
  • L 1 is selected from -(succinimide-3-yl-N)-WC(O)-, -CH 2 -C(O)-NR 3 -WC(O)- or -C(O)-WC( O)-, wherein W is selected from C 1-8 alkyl, C 1-8 alkyl-cycloalkyl and linear heteroalkyl of 1 to 8 atoms, and the heteroalkyl contains 1 to 3 selected from N, O and S heteroatoms, wherein the C 1-8 alkylene, C 1-8 alkyl-cycloalkyl and linear heteroalkyl are each independently optionally further selected from halogen, hydroxyl, Substituted by one or more substituents of cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;
  • L 2 is selected from -NR 4 (CH 2 CH 2 O)p 1 CH 2 CH 2 C(O)-, -NR 4 (CH 2 CH 2 O)p 1 CH 2 C(O)-, -S(CH 2 ) p 1 C(O)- and chemical bond, where p 1 is an integer from 1 to 20;
  • L 3 is a peptide residue composed of 2 to 7 amino acid residues, wherein the amino acid is selected from the group consisting of phenylalanine (F), glycine (G), valine (V), and lysine (K) , Citrulline, serine (S), glutamic acid (E) and aspartic acid (D) in amino acid residues, and optionally further selected from halogen, hydroxyl, cyano, amino, alkane Substituted by one or more substituents in the group, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;
  • L 4 is selected from -NR 5 (CR 6 R 7 ) t -, -C(O)NR 5 , -C(O)NR 5 (CH 2 ) t -and chemical bonds, wherein t is an integer from 1 to 6;
  • R 3 , R 4 and R 5 are the same or different, and are each independently selected from a hydrogen atom, an alkyl group, a halogenated alkyl 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 halogenated alkyl group, a deuterated alkyl group, and a hydroxyalkyl 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; preferably, L 3 is a tetrapeptide residue of GGFG (SEQ ID NO: 13) (glycine-glycine-phenylalanine-glycine);
  • L 4 is -NR 5 (CR 6 R 7 )t-, R 5 , R 6 or R 7 are the same or different, and each independently is a hydrogen atom or an alkyl group, and t is 1 or 2;
  • the L 1 terminal is connected to Pc, and the L 4 terminal is connected to Y.
  • the antibody-drug conjugate represented by the aforementioned general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof is represented by the general formula (Pc-L a -YD)
  • W, L 2 , L 3 , R 5 , R 6 , and R 7 are as defined in the aforementioned joint unit -L-;
  • Pc, n, R 1 , R 2 , m are as defined in the general formula (Pc-LYD);
  • Pc is an anti-PSMA antibody or an antigen-binding fragment thereof
  • n is an integer from 0 to 4; non-limiting examples such as m is selected from 0, 1, 2, 3 and 4;
  • n is 1 to 10, n is a decimal or integer; preferably, n is 1-8, more preferably 3-8, most preferably 3-7, more preferably 6-7;
  • R 1 is selected from halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclic group, aryl and heteroaryl;
  • R 2 is selected from hydrogen atom, halogen, Haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl and heteroaryl; alternatively, R 1 and R 2 form together with the carbon atom to which they are attached Cycloalkyl or heterocyclic group;
  • W is selected from a C 1-8 alkyl group, a C 1-8 alkyl-cycloalkyl group or a linear heteroalkyl group of 1 to 8 atoms, and the heteroalkyl group contains 1 to 3 selected from N, O or S
  • L 2 is selected from -NR 4 (CH 2 CH 2 O)p 1 CH 2 CH 2 C(O)-, -NR 4 (CH 2 CH 2 O)p 1 CH 2 C(O)-, -S(CH 2 ) p 1 C(O)- and chemical bond, where p 1 is an integer from 1 to 20;
  • L 3 is a peptide residue composed of 2 to 7 amino acid residues, wherein the amino acid residue is selected from phenylalanine (F), glycine (G), valine (V), lysine ( K), citrulline, serine (S), glutamic acid (E) and aspartic acid (D) formed by amino acid residues, and optionally further selected from halogen, hydroxyl, cyano, amino , Alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl substituted by one or more substituents;
  • the amino acid residue is selected from phenylalanine (F), glycine (G), valine (V), lysine ( K), citrulline, serine (S), glutamic acid (E) and aspartic acid (D) formed by amino acid residues, and optionally further selected from halogen, hydroxyl, cyano, amino , Alkyl, chloroalkyl, deuterated alkyl, alkoxy
  • R 5 is selected from hydrogen atom, alkyl group, haloalkyl group, deuterated alkyl group and 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 halogenated alkyl group, a deuterated alkyl group, and a hydroxyalkyl group.
  • the antibody-drug conjugate represented by the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof as described in any one of the preceding items is of the general formula (Pc-L b -YD)
  • s 1 is an integer from 2 to 8;
  • Pc, R 1 , R 2 , R 5 , R 6 , R 7 , m, and n are as defined in the general formula (Pc-L a -YD).
  • the antibody-drug conjugate represented by the general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof as described in any one of the preceding is selected from:
  • Pc and n are as defined in the general formula (Pc-L-Y-D), specifically, Pc is an anti-PSMA antibody or an antigen-binding fragment thereof, or an anti-PSMA antibody as described in any one of the preceding items;
  • n is 1 to 10, n is a decimal or integer; preferably, n is 1-8, more preferably 3-8, most preferably 3-7; most preferably 6-7.
  • n is 1 to 8, preferably 3-8, n is a decimal or integer;
  • PM is an anti-PSMA antibody, which includes a heavy chain as shown in SEQ ID NO: 9 and a light chain as shown in SEQ ID NO: 10.
  • the present disclosure further provides a method for preparing the antibody-drug conjugate represented by the general formula (Pc-L a -YD) or a pharmaceutically acceptable salt thereof, which comprises the following steps:
  • Pc' is reduced Pc, and undergoes a coupling reaction with the compound represented by the general formula (L a -YD) to obtain the compound represented by the general formula (Pc-L a -YD);
  • Pc is an anti-PSMA antibody or an antigen-binding fragment thereof
  • n, m, W, L 2 , L 3 , R 1 , R 2 , R 5 , R 6 and R 7 are as defined in the aforementioned general formula (Pc-L a -YD).
  • the present disclosure further provides a method for preparing the antibody drug conjugate represented by the general formula (Pc-L'-D), which includes the following steps:
  • Pc is the aforementioned anti-PSMA antibody or its antigen-binding fragment; Pc' is reduced Pc,
  • n is as defined in the general formula (Pc-L-Y-D).
  • the present disclosure further provides a method for preparing the antibody drug conjugate shown in PM-9-A, which includes the following steps:
  • n is 1 to 8, preferably 3-8, n is a decimal or integer;
  • PM is an anti-PSMA antibody, which includes a heavy chain with the sequence shown in SEQ ID NO: 9 and a light chain with the sequence shown in SEQ ID NO: 10;
  • n represents the drug loading of the antibody-drug conjugate, which can also be referred to as the DAR value, which can be determined by conventional methods such as UV/visible light spectroscopy, mass spectroscopy, ELISA test and HPLC; in some schemes , N is 0-10, preferably 1-10, more preferably 1-8, or 2-8, or 2-7, or 2-4, or 3-8, or 3-7, or 3-6, or 4 -7, or 4-6, or 4-5; in some embodiments, n is the average of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising the antibody-drug conjugate according to the present disclosure or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients Agent, diluent or carrier.
  • the unit dose of the pharmaceutical composition contains 0.1 mg-3000 mg or 1 mg-1000 mg of the aforementioned anti-PSMA antibody or the aforementioned antibody-drug conjugate.
  • the present disclosure provides the use of the antibody-drug conjugate or a pharmaceutically acceptable salt thereof or a pharmaceutical composition containing the same as a medicine according to the present disclosure.
  • the present disclosure provides that the antibody-drug conjugate or a pharmaceutically acceptable salt thereof or a pharmaceutical composition containing the same according to the present disclosure is used in the preparation of a medicament for the treatment of PSMA-mediated diseases or disorders
  • the PSMA-mediated disease or disorder is preferably a cancer with high expression of PSMA, a cancer with medium expression or a cancer with low expression.
  • the present disclosure provides the use of the antibody-drug conjugate according to the present disclosure, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing the same, in the preparation of a medicament for the treatment or prevention of cancer, wherein
  • the cancer is preferably head and neck squamous cell carcinoma, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system cancer, neuroendocrine tumor, throat cancer, Nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatocellular carcinoma, hepatocellular carcinoma, hepatobiliary cancer, pancreatic cancer, gastric cancer, gastrointestinal cancer, bowel cancer, colon cancer, Colorectal cancer, kidney cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, melanoma,
  • the present disclosure further relates to a method for treating and/or preventing tumors, the method comprising administering to a subject in need thereof a therapeutically effective dose of the antibody-drug conjugate according to the present disclosure or A pharmaceutically acceptable salt or a pharmaceutically acceptable salt thereof or a pharmaceutical composition containing the same; preferably, the tumor is a cancer related to high expression of PSMA, a medium expression cancer or a low expression cancer.
  • the present disclosure further relates to a method for treating or preventing tumor or cancer, the method comprising administering to a subject in need thereof a therapeutically effective dose of the antibody-drug conjugate according to the present disclosure or a pharmacological agent thereof.
  • the tumor and cancer are preferably head and neck squamous cell carcinoma, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, Neuroblastoma, central nervous system cancer, neuroendocrine tumors, throat cancer, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatocellular carcinoma, hepatocellular carcinoma, hepatobiliary cancer , Pancreatic cancer, stomach cancer, gastrointestinal cancer, bowel cancer, colon cancer, colorectal cancer, kidney cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer , Skin cancer, melanoma, leukemia, lymphoma, bone cancer, chondrosarcoma, myeloma, multiple myeloma, myelodys
  • the present disclosure further provides the aforementioned anti-PSMA antibody or its antibody-drug conjugate as a medicine, preferably as a medicine for treating cancer or tumor, and more preferably as a medicine for treating PSMA-mediated cancer.
  • the active compound for example, the ligand-drug conjugate according to the present disclosure, or a pharmaceutically acceptable salt thereof
  • the active compound can be prepared into a form suitable for administration by any appropriate route, and the active compound is preferably in a unit dose Or it is a way that subjects can self-administer in a single dose.
  • the unit dose of the active compound or composition described in the present disclosure can be tablet, capsule, cachet, bottled syrup, powder, granule, lozenge, suppository, rejuvenated powder or liquid preparation.
  • the dosage of the active compound or composition used in the treatment methods of the present disclosure will generally vary with the severity of the disease, the weight of the subject, and the relative efficacy of the active compound.
  • a suitable unit dose can be 0.1 mg 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, disintegrants or excipients, etc. .
  • the composition may contain 0.1 to 99% by weight of the active compound.
  • the PSMA antibodies and antibody drug conjugates provided in the present disclosure have good affinity with cell surface antigens, good endocytosis efficiency and strong tumor suppression efficiency, and have a wider window of drug application and higher tumor suppression effects And therapeutic activity, better safety, pharmacokinetic properties and druggability (such as stability), more suitable for clinical drug application.
  • Figure 1A In vitro binding ability of ADCs or antibodies of the present disclosure with cellular MDAPCa.
  • Figure 1B In vitro binding ability of ADCs or antibodies of the present disclosure to cell LNCaP.
  • Figure 1C In vitro binding ability of ADCs or antibodies of the present disclosure to cell 22Rv1.
  • Figure 1D In vitro binding ability of ADCs or antibodies of the present disclosure to cell PC-3.
  • Figure 1E In vitro binding ability of ADC or antibody of the present disclosure to cell DU 145.
  • Figure 2A In vitro endocytosis experiment of antibody PM in LNCaP cells; 2K cells, 10% U-L IgG FBS.
  • Figure 2B In vitro endocytosis experiment of antibody PM in 22Rv1 cells; 2K cells, 10% U-L IgG FBS.
  • Figure 3A The killing effect of different ADCs of the present disclosure on LNCaP cells, using 2K cells, 4.5% FBS.
  • Figure 3B The killing effect of the toxin (Compound 2-B) in the present disclosure on LNCaP cells, using 2K cells, 4.5% FBS.
  • Figure 3C The killing effect of different ADCs of the present disclosure on 22Rv1 cells, using 4K cells, 4.5% FBS.
  • Figure 3D The killing effect of the toxin (Compound 2-B) in the present disclosure on 22Rv1 cells, using 4K cells, 4.5% FBS.
  • Figure 3E The killing effect of different ADCs of the present disclosure on PC-3 cells, using 4K cells, 4.5% FBS.
  • Figure 3F The killing effect of the toxin (compound 2-B) in the present disclosure on PC-3 cells, using 2K cells, 4.5% FBS.
  • Figure 4A The inhibitory activity of ADCs of different doses of the present disclosure on human prostate cancer cell 22Rv1 transplanted tumors in nude mice.
  • Figure 4B The body weight changes of the mice in the test of the inhibitory activity of ADCs of different doses of the present disclosure on human prostate cancer cell 22Rv1 transplanted tumors in nude mice.
  • Fig. 5A The inhibitory activity of ADCs of different doses of the present disclosure on human prostate cancer cell 22Rv1 transplanted tumors in nude mice.
  • Figure 5B The body weight changes of the mice in the test of the inhibitory activity of ADCs of different doses of the present disclosure on human prostate cancer cell 22Rv1 transplanted tumors in nude mice.
  • Fig. 6A The inhibitory activity of ADCs of different doses of the present disclosure on transplanted tumors of human prostate cancer cell LNCap on SCID Beighe mice.
  • Figure 6B The body weight changes of the mice in the test of the inhibitory activity of ADCs of different doses of the present disclosure on the transplanted tumor of human prostate cancer cell LNCap on SCID Beighe mice.
  • Figure 7 The pharmacokinetic stability of ADC-2 of the present disclosure, where the concentration of ADC is 100 ⁇ g/ml.
  • Figure 8 Plasma stability of ADC-5 of the present disclosure, where the concentration of ADC is 100 ⁇ g/ml.
  • Figure 9 The therapeutic effect of ADC on 22Rv1 xenograft tumor in nude mice.
  • a trade name When a trade name is used in this disclosure, it is intended to include the formulation of the trade name product, the drug and the active drug portion of the trade name product.
  • drug refers to cytotoxic drugs, chemical molecules that can strongly disrupt the normal growth of tumor cells.
  • cytotoxic drugs can kill cells at sufficiently high concentrations, but due to lack of specificity, while killing tumor cells, they can also cause normal cell apoptosis, leading to serious side effects.
  • the term includes small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, radioisotopes (e.g. At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 and Lu radioisotopes), chemotherapeutics, antibiotics and nucleolytic enzymes.
  • linker unit refers to a chemical structural fragment or bond that is connected to a ligand (antibody in this disclosure) at one end and a drug at the other end, and can also be connected to others After the linker, it is connected to the ligand or drug.
  • the joint may contain one or more joint elements.
  • exemplary linker elements include 6-maleimidohexanoyl ("MC"), maleimidopropionyl ("MP”), valine-citrulline (“val-cit” or “vc "), alanine-phenylalanine (“ala-phe”), p-aminobenzyloxycarbonyl (“PAB”), N-succinimidyl 4-(2-pyridylthio)pentanoate ( “SPP”), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1 carboxylate (“SMCC”, also referred to herein as "MCC”), and N- Succinimidyl (4-iodo-acetyl) aminobenzoate (“SIAB”).
  • MC 6-maleimidohexanoyl
  • MP maleimidopropionyl
  • val-cit valine-citrulline
  • the linker may include one or more of the following elements, or a combination thereof: an extension, a spacer, and an amino acid unit, and may 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 the drug in the cell.
  • acid-labile linkers such as hydrazone
  • protease-sensitive such as peptidase-sensitive
  • linkers light-labile linkers, dimethyl linkers, or disulfide-containing linkers
  • Joint components include but are not limited to:
  • MC 6-maleimidohexanoyl
  • Val-Cit or "vc” valine-citrulline (an exemplary dipeptide in a protease cleavable linker);
  • Citrulline 2-amino-5-ureidovaleric acid
  • PAB p-aminobenzyloxycarbonyl (an example of a "self-sacrificing" linker element);
  • Me-Val-Cit N-methyl-valine-citrulline (wherein the linker peptide bond has been modified to prevent it from being cleaved by cathepsin B);
  • MC(PEG)6-OH maleimidohexanoyl-polyethylene glycol (can be attached to antibody cysteine);
  • SPP N-succinimidyl 4-(2-pyridylthio)pentanoate
  • SPDP N-succinimidyl 3-(2-pyridyldithio)propionate
  • SMCC succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate
  • antibody-drug conjugate refers to the connection between the antibody and the biologically active drug through a linking unit.
  • ADC antibody drug conjugate
  • n is the average number of drug modules per antibody, which can be an integer or a decimal, and its range can be: for example, about 0 to about 20 drug modules per antibody, and in some embodiments, 1 to about 10 drug modules per antibody
  • Each drug module in some embodiments, is 1 to about 8 drug modules per antibody, such as 2, 3, 4, 5, 6, 7, or 8 drug modules.
  • composition of the antibody-drug conjugate mixture of the present disclosure wherein the average drug load of each antibody is about 1 to about 10, including but not limited to about 3 to about 7, and about 3 to about 6 One, about 3 to about 5, about 1 to about 9, about 7 or about 4.
  • antibody refers to an immunoglobulin.
  • a complete antibody 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 sequence of the constant region of the immunoglobulin heavy chain are different.
  • Immunoglobulins can be divided into five categories, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA and IgE, and their corresponding The heavy chains are respectively ⁇ chain, ⁇ chain, ⁇ chain, ⁇ chain, and ⁇ chain.
  • the same type of Ig can be divided into different subclasses according to 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 the difference of the constant region.
  • 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 full-length antibody heavy and light chains varies greatly and is a variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable and are a constant region.
  • the variable region includes 3 hypervariable regions (HVR) and 4 relatively conserved framework regions (FR). Three hypervariable regions determine the specificity of the antibody, also known as complementarity determining regions (CDR).
  • Each light chain variable region (LCVR) and heavy chain variable region (HCVR) is composed of 3 CDR regions and 4 FR regions.
  • the sequence from the amino terminus to the carboxy terminus is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the 3 CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.
  • fully human antibody means “fully human antibody”, “fully human antibody”, “human antibody” or “fully human antibody”, also known as “fully human monoclonal antibody”, the variable region and constant region of the antibody are all of human origin , Remove immunogenicity and toxic side effects.
  • the related technologies of fully human antibody preparation mainly include: human hybridoma technology, EBV transformed B lymphocyte technology, phage display technology (phage display), transgenic mouse antibody preparation technology (transgenic mouse) and single B cell antibody preparation technology.
  • antigen-binding fragment refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Fragments of the full-length antibody can be used to perform the antigen-binding function of the antibody.
  • the binding fragment contained in the "antigen-binding fragment” is selected from Fab, Fab', F(ab')2, single-chain antibody (scFv), dimerized V region (diabody), disulfide bond stabilized V region (dsFv) and antigen-binding fragments of peptides containing CDRs, examples include (i) Fab fragments, monovalent fragments composed of VL, VH, CL and CH1 domains; (ii) F(ab') 2 fragments, including through hinge A bivalent fragment of two Fab fragments connected by a disulfide bridge in the region; (iii) Fd fragment composed of VH and CH1 domains; (iv) Fv fragment composed of VH and VL domains of one arm of an antibody;
  • the two domains VL and VH of the Fv fragment are encoded by separate genes, recombination methods can be used to connect them through a synthetic linker so that it can be produced as a single protein in which the VL and VH regions are paired to form a monovalent molecule.
  • Chain referred to as single chain Fv (scFv); see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci USA 85: 5879-5883).
  • single chain antibodies are also intended to be included in the term "antigen-binding fragments" of antibodies.
  • the antigen-binding portion can be produced by recombinant DNA technology or by enzymatic or chemical fragmentation of the intact immunoglobulin.
  • the antibodies may be antibodies of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtype), 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 IgG antibody molecules with the protease papain (for example, cleaving the amino acid residue at position 224 of the H chain), wherein the H chain The part on the N-terminal side and the L chain are joined together by a disulfide bond.
  • protease papain for example, cleaving the amino acid residue at position 224 of the H chain
  • F(ab')2 is obtained by digesting the lower part of the disulfide bond in the hinge region of IgG with the enzyme pepsin. It has a molecular weight of about 100,000 and has antigen-binding activity and contains two Fab regions connected at the hinge position. Antibody fragments.
  • Fab' is an antibody fragment with a molecular weight of about 50,000 and antigen-binding activity obtained by cleaving the disulfide bond in the hinge region of F(ab')2.
  • the Fab' can be produced by inserting the DNA encoding the Fab' fragment into a prokaryotic expression vector or a eukaryotic expression vector and introducing the vector into a prokaryotic organism or eukaryotic organism to express Fab'.
  • single chain antibody means a molecule comprising an antibody heavy chain variable domain (or VH) and an antibody light chain variable domain (or VL) connected by a linker.
  • Such scFv molecules may have the general structure: NH 2 -VL-linker-VH-COOH or NH 2 -VH-linker-VL-COOH.
  • Suitable prior art linkers consist of a repeated GGGGS amino acid sequence or variants thereof, for example using 1-4 repeated variants (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448) .
  • linkers that can be used in the present disclosure are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 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 in the variable domain of an antibody that mainly contribute to antigen binding.
  • CDR there are three CDRs (HCDR1, HCDR2, HCDR3) in each heavy chain variable region, and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable region.
  • Any one of various well-known schemes can be used to determine the amino acid sequence boundaries of CDRs, including the "Kabat” numbering rule (see Kabat et al.
  • the CDR in the variable domain of the heavy chain (VH) The amino acid residue numbers are 31-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3); the CDR amino acid residue numbers in the light chain variable domain (VL) are 24-34 (LCDR1), 50 -56 (LCDR2) and 89-97 (LCDR3).
  • the CDR amino acids in VH are numbered 26-32 (HCDR1), 52-56 (HCDR2) and 95-102 (HCDR3); and the amino acids in VL
  • the residue numbers are 26-32 (LCDR1), 50-52 (LCDR2) and 91-96 (LCDR3).
  • the CDR is defined by the amino acid residues 26-35 (HCDR1) in human VH. ), 50-65 (HCDR2) and 95-102 (HCDR3) and the amino acid residues 24-34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3) in human VL.
  • HCDR1 amino acid residues 26-35
  • HCDR2 amino acid residues 26-65
  • HCDR3 amino acid residues 24-34
  • LCDR2 amino acid residues 24-34
  • LCDR2 amino acid residues 24-34
  • LCDR2 amino acid residues 24-34
  • LCDR2 amino acid residues 24-34
  • LCDR2 amino acid residues 24-34
  • LCDR3 amino acid residues 24-34
  • LCDR3 amino acid residues 24-34
  • LCDR3 amino acid residues 24-34
  • LCDR3 amino acid residues 24-34
  • LCDR3 amino acid residues 24-34
  • LCDR3 amino acid residues 24-34
  • LCDR3 amino acid residues
  • the CDR regions of the antibody can be determined using the program IMGT/DomainGap Align. Unless otherwise specified, the 6 CDRs described in this disclosure are all obtained according to the Kabat numbering rules. ((Kabat EA et al. (1991) Sequences of proteins of immunological intere st. NIH Publication 91-3242)). According to the Kabat rule, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3); the light chain variable domain (VL The CDR amino acid residue numbers in) are 24-34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3). Other numbering rules in the field Chothia, IMGT, etc.
  • antibody framework region refers to a part of the variable domain VL or VH, which serves as a scaffold for the antigen binding loop (CDR) of the variable domain. Essentially, it is a variable domain without CDRs.
  • epitope or "antigenic determinant” refers to a site on an antigen that is bound by an immunoglobulin or antibody. Epitopes usually include 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 in Methods in Molecular B iology, Volume 66, G.E. Morris, Ed. (1996).
  • the terms “specifically binds”, “selectively binds”, “selectively binds” and “specifically binds” refer to the binding of an antibody or antigen-binding fragment to an epitope on a predetermined antigen.
  • the antibody or antigen-binding fragment binds with an affinity (KD) of approximately less than 10 -7 M, for example, approximately less than 10 -8 M, 10 -9 M, or 10 -10 M or less.
  • KD refers to the dissociation equilibrium constant of the antibody-antigen interaction.
  • the antibody or antigen-binding fragment of the present disclosure binds to PSMA or its epitope with a dissociation equilibrium constant (KD) of less than about 10 -7 M, for example, less than about 10 -8 M or 10 -9 M, for example, in the present disclosure
  • KD dissociation equilibrium constant
  • nucleic acid molecule refers to a DNA molecule or an RNA molecule.
  • the nucleic acid molecule may be single-stranded or double-stranded, but is preferably double-stranded DNA.
  • the nucleic acid is "operably linked.” For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is effectively linked to the coding sequence.
  • sequence identity refers to the process of aligning amino acid sequences, introducing gaps when necessary to achieve the maximum percentage of sequence identity, and not considering any conservative substitutions as part of sequence identity.
  • the alignment can be achieved in a variety of ways within the technical scope of the art, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software.
  • BLAST BLAST-2
  • ALIGN ALIGN-2
  • ALIGN-2 ALIGN-2
  • ALIGN-2 ALIGN-2
  • ALIGN-2 Megalign
  • 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 additional DNA segments can be ligated.
  • the vector is a viral vector in which additional DNA segments can be ligated into the viral genome.
  • the vectors disclosed herein can replicate autonomously in the host cell into which they have been introduced (for example, bacterial vectors with a bacterial origin of replication and episomal mammalian vectors) or can be integrated into the genome of the host cell after being introduced into the host cell, so as to follow The host genome replicates together (e.g., a non-episomal mammalian vector).
  • host cell refers to a cell into which an expression vector has been introduced.
  • Host cells may include microorganisms (such as bacteria), plant or animal cells.
  • Bacteria that are easily transformed include members of the enterobacteriaceae, such as Escherichia coli or Salmonella strains; Bacillaceae such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae.
  • Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris.
  • Suitable animal host cell lines include CHO (Chinese Hamster Ovary cell line) and NS0 cells.
  • the engineered antibodies or antigen-binding fragments of the present disclosure can be prepared and purified by conventional methods.
  • the cDNA sequences encoding the heavy and light chains can be cloned and recombined into a GS expression vector.
  • the recombinant immunoglobulin expression vector can be stably transfected into CHO cells.
  • mammalian expression systems can lead to glycosylation of antibodies, especially in the highly conserved N-terminal sites of the Fc region.
  • Positive clones are expanded in the serum-free medium of the bioreactor to produce antibodies.
  • the culture medium from which the antibody is secreted can be purified by conventional techniques. For example, use A or G Sepharose FF column with adjusted buffer for purification.
  • the bound antibody was eluted by the PH gradient method, and the antibody fragment was detected by SDS-PAGE and collected.
  • the antibody can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange. The resulting product needs to be frozen immediately, such as -70°C, or lyophilized.
  • peptide refers to a molecule composed of two or more amino acid molecules connected to each other through peptide bonds, and is a structural and functional fragment of a protein.
  • alkyl refers to a saturated aliphatic hydrocarbon group, which is a straight or branched chain group containing 1 to 20 carbon atoms, preferably containing 1 to 12 (e.g. 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10, 11 and 12) carbon atoms, more preferably an alkyl group containing 1 to 10 carbon atoms, most preferably containing 1 to 6 carbon atoms (including 1, 2, 3 1, 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-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-methylhe
  • lower alkyl groups containing 1 to 6 carbon atoms More preferred are lower alkyl groups containing 1 to 6 carbon atoms.
  • Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, and sec-butyl.
  • Alkyl groups may be substituted or unsubstituted.
  • substituents When substituted, substituents may be substituted at any available attachment point.
  • the substituents are preferably one or more of the following groups, which are independently selected from alkanes Group, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkane Oxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio and oxo.
  • heteroalkyl refers to an alkyl group containing one or more heteroatoms selected from N, O or S, wherein the alkyl group is as defined above.
  • alkylene refers to a saturated linear or branched aliphatic hydrocarbon group, which has two residues derived from the removal of two hydrogen atoms from the same carbon atom or two different carbon atoms of the parent alkane, which is A straight or branched chain group containing 1 to 20 carbon atoms, preferably containing 1 to 12 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12) carbons Atom, more preferably an alkylene group containing 1 to 6 carbon atoms (including 1, 2, 3, 4, 5, or 6 carbon atoms).
  • Non-limiting examples of alkylene groups 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. When substituted, the substituent may be substituted at any available point of attachment.
  • the substituent is preferably independently optionally selected from alkyl, alkenyl, alkynyl , Alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocyclic, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy , Cycloalkylthio, heterocycloalkylthio and oxo groups are substituted by one or more substituents.
  • alkoxy refers to -O- (alkyl) and -O- (unsubstituted cycloalkyl), where the definition of alkyl or cycloalkyl is as described above.
  • alkoxy groups include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, and 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 alkyl, alkenyl, alkynyl, alkoxy, alkane Thio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio And 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, preferably 3 to 8 The carbon atom more preferably contains 3 to 6 carbon atoms.
  • Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatriene Groups, cyclooctyl, etc.; polycyclic cycloalkyls include spiro, fused, and bridged cycloalkyls.
  • the cycloalkyl ring includes the cycloalkyl as described above (including monocyclic, spiro, fused and bridged rings) fused to an aryl, heteroaryl or heterocycloalkyl ring, wherein it is connected to the parent structure
  • the ring together is a cycloalkyl group, and non-limiting examples include indanyl, tetrahydronaphthyl, benzocycloheptyl and the like; phenylcyclopentyl and tetrahydronaphthyl are preferred.
  • Cycloalkyl groups can be substituted or unsubstituted. When substituted, the substituents can be substituted at any available point of attachment.
  • the substituents are preferably independently optionally selected from hydrogen atoms, halogens, alkyl groups, Alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl, and heteroaryl are substituted by one or more substituents.
  • heterocyclyl refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent containing 3 to 20 ring atoms, one or more of which is selected from nitrogen, oxygen or S(O) P (wherein p is an integer of 0 to 2) heteroatoms, but does not include the ring part of -OO-, -OS- or -SS-, and the remaining ring atoms are carbon.
  • Non-limiting examples of monocyclic heterocyclic groups include pyrrolidinyl, tetrahydropyranyl, 1,2.3.6-tetrahydropyridinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl and Homopiperazinyl and so on.
  • Polycyclic heterocyclic groups include spiro, fused, and bridged heterocyclic groups.
  • the heterocyclyl ring includes the heterocyclic group as described above (including monocyclic, spiro heterocyclic, fused heterocyclic and bridged heterocyclic ring) fused on an aryl, heteroaryl or cycloalkyl ring, wherein it is combined with the parent
  • the rings linked together in the structure are heterocyclic groups, non-limiting examples of which include:
  • the heterocyclic group may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available point of attachment.
  • the substituents are preferably independently optionally selected from hydrogen atoms, halogens, alkyl groups, Alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl, and heteroaryl are substituted by one or more substituents.
  • aryl refers to a 6 to 14-membered all-carbon monocyclic or fused polycyclic (that is, rings sharing adjacent pairs of carbon atoms) with a conjugated ⁇ -electron system, preferably 6 to 10 members, such as benzene Base and naphthyl.
  • the aryl ring includes the aryl ring as described above 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 :
  • Aryl groups can be substituted or unsubstituted. When substituted, the substituents can be substituted at any available attachment point.
  • the substituents are preferably independently optionally selected from hydrogen atoms, halogens, alkyl groups, and alkyl groups.
  • One or more substituents of oxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl and heteroaryl are substituted.
  • heteroaryl refers to a heteroaromatic system containing 1 to 4 heteroatoms and 5 to 14 ring atoms, where the heteroatoms are selected from oxygen, sulfur, and nitrogen.
  • Heteroaryl groups are preferably 5 to 10 members, more preferably 5 or 6 members, such as furyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, pyridazinyl, Imidazolyl, pyrazolyl, triazolyl, tetrazolyl and the like.
  • the heteroaryl ring includes the above-mentioned heteroaryl group fused to an aryl, heterocyclic or cycloalkyl ring, wherein the ring connected to the parent structure is a heteroaryl ring, and non-limiting examples thereof include :
  • Heteroaryl groups can be substituted or unsubstituted. When substituted, the substituents can be substituted at any available point of attachment.
  • the substituents are preferably independently optionally selected from hydrogen atoms, halogens, alkyl groups, Alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl, and heteroaryl are substituted by one or more substituents.
  • haloalkyl refers to the replacement of hydrogen on an alkyl group with one or more halogens, where the alkyl group is as defined above.
  • deuterated alkyl refers to the replacement of hydrogen on an alkyl group with one or more deuterium atoms, where the alkyl group is as defined above.
  • hydroxyalkyl means that the hydrogen on an alkyl group is replaced by one or more hydroxy groups, where the alkyl group is as defined above.
  • hydroxy refers to the -OH group.
  • halogen refers to fluorine, chlorine, bromine or iodine.
  • amino refers to -NH 2 .
  • nitro refers to -NO 2 .
  • cyano refers to -CN.
  • heterocyclic group optionally substituted by an alkyl group means that an alkyl group may but does not have to be present, and the description includes the case where the heterocyclic group is substituted by an alkyl group and the case where the heterocyclic group is not substituted by an alkyl group.
  • Substituted refers to one or more hydrogen atoms in the group, preferably at most 5, more preferably 1, 2, or 3 hydrogen atoms are independently substituted with substituents. 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 too much effort. For example, an amino group or a hydroxyl group having free hydrogen may be unstable when combined with a carbon atom having an unsaturated (e.g., ethylenic) bond.
  • pharmaceutical composition means a mixture containing one or more of the compounds described herein or their physiologically/pharmaceutically acceptable salts or prodrugs and other chemical components, as well as other components such as physiologically/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 thus the biological activity.
  • pharmaceutically acceptable salt refers to the salt of the antibody-drug conjugate of the present disclosure. Such salt is safe and effective when used in a subject, and has due Biologically active, the antibody-drug conjugate of the present disclosure contains at least one amino group, so it can form a salt with an acid.
  • Non-limiting examples of pharmaceutically acceptable salts include: hydrochloride, hydrobromide, hydroiodide, sulfate, Bisulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, pearate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartaric acid Salt, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate.
  • Drug loading or “average drug load” is also called drug-to-antibody ratio (DAR), that is, the average number of drugs coupled to each antibody in the ADC. It can be in the range of, for example, about 1 to about 10 drugs conjugated per antibody, and in certain embodiments, in the range of about 1 to about 8 drugs conjugated per antibody, preferably from 2-8. , 2-7, 2-6, 2-5, 2-4, 3-4, 3-5, 5-6, 5-7, 5-8 and 6-8. Exemplarily, the drug loading amount may be an average value of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.
  • the ADC general formula of the present disclosure includes a collection of antibody drug conjugates within a certain drug loading range described above.
  • the drug loading amount is expressed as n, which can also be referred to as the DAR value, and exemplarily is the mean value of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.
  • Conventional methods such as UV/visible light spectroscopy, mass spectrometry, ELISA test and HPLC can be used to determine the drug loading.
  • the following non-limiting methods can be used to control the loading of the ligand-drug conjugate, including:
  • carrier is used for the drug of the present disclosure, and refers to a system that can change the way the drug enters the human body and its distribution in the body, control the release rate of the drug, and deliver the drug to the targeted organ.
  • the drug carrier release and targeting system can reduce drug degradation and loss, reduce side effects, and improve bioavailability.
  • polymer surfactants that can be used as carriers can self-assemble to form various forms of aggregates due to their unique amphiphilic structure. Preferred examples are micelles, microemulsions, gels, liquid crystals, vesicles, etc. . These aggregates have the ability to contain drug molecules and at the same time have good permeability to the membrane, and can be used as excellent drug carriers.
  • excipient is an add-on other than the main drug in a pharmaceutical preparation, and can also be referred to as an adjuvant.
  • adjuvant such as binders, fillers, disintegrants, lubricants in tablets; base parts in semi-solid preparations ointments and creams; preservatives, antioxidants, correctives, fragrances, etc. in liquid preparations
  • Cosolvents, emulsifiers, solubilizers, osmotic pressure regulators, colorants, etc. can all be called excipients.
  • diluent is also called a filler, and its main purpose is to increase the weight and volume of the tablet.
  • the addition of diluent not only guarantees a certain volume, but also reduces the deviation of the dosage of the main components and improves the compression molding of the drug.
  • an absorbent should be added to absorb the oily substance to keep it in a "dry” state to facilitate the manufacture of tablets.
  • the pharmaceutical composition may be in the form of a sterile injectable aqueous solution.
  • a sterile injectable aqueous solution Among the acceptable solvents and solvents that can be used 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 an oil phase.
  • the active ingredient is dissolved in a mixture of soybean oil and lecithin.
  • the oil solution is added to a mixture of water and glycerin to form a microemulsion.
  • the injection or microemulsion can be injected into the bloodstream of the subject by local large-volume injection.
  • a continuous intravenous delivery device 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 injection water or oil suspension for intramuscular and subcutaneous administration.
  • the suspension can be formulated according to known techniques using those suitable dispersing or wetting agents and suspending agents mentioned above.
  • the sterile injection preparation may also be a sterile injection solution or suspension prepared in a non-toxic parenterally acceptable diluent or solvent, for example, a solution prepared in 1,3-butanediol.
  • sterile fixed oil can be conveniently used as a solvent or suspending medium. For this purpose, any blended fixed oil including synthetic mono- or diglycerides can be used.
  • fatty acids such as oleic acid can also be used to prepare injections.
  • the preparation method of the compound represented by general formula (PM-9-A) includes the following steps:
  • the reducing agent is preferably TCEP, in particular, it is preferred to reduce the two Sulfur bond
  • PM is an anti-PSMA antibody or an antigen-binding fragment thereof
  • n 1 to 10
  • n is a decimal or integer
  • the antibody of the present disclosure is prepared with reference to WO2003034903A2, wherein the amino acid sequence of the variable region of AB-PG1-XG1-006 is as follows:
  • the underlined part is the CDR region determined according to the Kabat numbering rules.
  • Antibody AB-PG1-XG1-006 Heavy chain CDR1 RYGMH(SEQ ID NO: 3) Heavy chain CDR2 VIWYDGSNKYYADSVKG (SEQ ID NO: 4) Heavy chain CDR3 GGDFLYYYYYGMDV (SEQ ID NO: 5) Light chain CDR1 RASQGISNYLA (SEQ ID NO: 6) Light chain CDR2 EASTLQS(SEQ ID NO:7) Light chain CDR3 QNYNSAPFT(SEQ ID NO: 8)
  • primers are designed to construct by PCR to obtain VH/VK gene fragments and obtain variable regions.
  • the antibody variable region is then homologously recombined with the constant region gene (CH1-Fc/CL) fragment to construct a complete antibody VH-CH1-Fc/VK-CL.
  • the constructed complete full-length antibody PM sequence is as follows:
  • control antibody labetuzumab (Lmab for short) was prepared with reference to WHO Drug Information Vol. 30, No. 1, 2016, in which the amino acid sequences of the heavy and light chains are as follows:
  • the experimental methods without specific conditions in the embodiments of the present disclosure usually follow the conventional conditions or the conditions suggested by the raw material or commodity manufacturers.
  • the reagents without specific sources are the conventional reagents purchased on the market.
  • the structure of the compound is determined by nuclear magnetic resonance (NMR) or mass spectrometry (MS). NMR is measured with Bruker AVANCE-400 nuclear magnetic instrument, the solvent is deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl 3 ), deuterated methanol (CD 3 OD), and the internal standard is tetramethyl sulfoxide (DMSO-d6) Based on silane (TMS), the chemical shift is given in units of 10 -6 (ppm).
  • NMR nuclear magnetic resonance
  • MS mass spectrometry
  • HPLC determination uses Agilent 1200DAD high pressure liquid chromatograph (Sunfire C18 150 ⁇ 4.6mm chromatographic column) and Waters 2695-2996 high pressure liquid chromatograph (Gimini C18 150 ⁇ 4.6mm chromatographic column).
  • the UV-HPLC measurement uses a Thermonanodrop2000 ultraviolet spectrophotometer.
  • the thin layer chromatography silica gel plate uses Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plate, the size of the silica gel plate used for thin layer chromatography (TLC) is 0.15mm ⁇ 0.2mm, and the size of the thin layer chromatography separation and purification product is 0.4mm. ⁇ 0.5mm silica gel plate.
  • the known starting materials of the present disclosure can be synthesized by or according to methods known in the art, or can be purchased from ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc, Darui Chemicals and other companies.
  • the reactions are all carried out under an argon atmosphere or a nitrogen atmosphere.
  • the argon atmosphere or nitrogen atmosphere means that the reaction flask is connected to an argon or nitrogen balloon with a volume of about 1L.
  • the hydrogen atmosphere means that the reaction flask is connected to a hydrogen balloon with a volume of about 1L.
  • the pressure hydrogenation reaction uses Parr 3916EKX hydrogenator and Qinglan QL-500 hydrogen generator or HC2-SS hydrogenator.
  • the hydrogenation reaction is usually evacuated and filled with hydrogen, and the operation is repeated 3 times.
  • the microwave reaction uses the CEM Discover-S 908860 microwave reactor.
  • 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 20°C to 30°C.
  • the preparation of the PBS buffer with pH 6.5 in the examples: take KH 2 PO 4 8.5g, K 2 HPO 4 .3H 2 O 8.56g, NaCl 5.85g, and EDTA 1.5g in a bottle, dilute the volume to 2L, and ultrasonic Dissolve all of it, shake well and get it.
  • the eluent system of column chromatography and the developing solvent system of thin-layer chromatography used to purify compounds include: A: dichloromethane and isopropanol system, B: dichloromethane and methanol system, C: petroleum ether and In the ethyl acetate system, the volume ratio of the solvent is adjusted according to the polarity of the compound, and a small amount of triethylamine and acidic or alkaline reagents can also be added for adjustment.
  • Q-TOF LC/MS uses Agilent 6530 accurate mass quadrupole-time-of-flight mass spectrometer and Agilent 1290-Infinity ultra-high performance liquid chromatograph (Agilent Poroshell 300SB-C8 5 ⁇ m, 2.1 ⁇ 75mm column).
  • 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 5 ⁇ m 19*250mm; mobile phase: A-water (10mmol NH 4 OAc), B- Acetonitrile, gradient elution, flow rate: 18 mL/min), collect the corresponding components, and concentrate under reduced pressure to obtain the title product (2-A: 1.5 mg, 2-B: 1.5 mg).
  • reaction was quenched by adding 5 mL of water to the reaction solution, the reaction solution was extracted with ethyl acetate (10 mL ⁇ 3), the organic phases were combined, washed with saturated sodium chloride solution (5 mL ⁇ 2), and the organic phase was dried with anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the resulting residue was purified by thin layer chromatography with the developing solvent system B to obtain the title product 4 (2.5 mg, yield: 80.9%).
  • the benzyl 1-hydroxycyclopropane-1-carboxylate 8a (104mg, 0.54mmol; prepared by the method disclosed in the patent application "US2005/20645”) and 2-((((9H-fluoren-9-yl) Methoxy) carbonyl) amino) acetamido) methyl acetate 8b (100 mg, 0.27 mmol; prepared by the method disclosed in the patent application "CN105829346A”) was added to the reaction flask, 5 mL of tetrahydrofuran was added, argon replaced three times, ice The temperature of the water bath was cooled to 0-5°C, potassium tert-butoxide (61mg, 0.54mmol) was added, the ice bath was removed, and the temperature was raised to room temperature and stirred for 10 minutes.
  • 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 obtain the title product 8 (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-fluorenylmethyl 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), the organic phase was washed with 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 with the developing solvent system C to obtain the title product 9b (48 mg, yield: 19%).
  • Dissolve 9d (19 mg, 22.6 ⁇ mol) in 2 mL of dichloromethane, add 1 mL of diethylamine, and stir at room temperature for 2 hours.
  • the reaction solution was concentrated under reduced pressure, 1 mL of toluene was added and concentrated under reduced pressure, repeated twice.
  • the solid residue was concentrated under reduced pressure and pulled dry by an oil pump to obtain the crude title product 9e (17 mg), which was directly used in the next reaction without purification.
  • reaction solution was purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5 ⁇ m 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 obtain the title product (9-A: 2.4 mg, 9-B: 1.7 mg).
  • the ADC load was determined by ultraviolet spectrophotometry (UV-Vis). Instrument: Thermonanodrop2000 ultraviolet spectrophotometer. The principle is that the total absorbance of the ADC at a certain wavelength is equal to the sum of the absorbance of the drug and the monoclonal antibody at that wavelength.
  • a 280nm ⁇ mab-280 bC mab + ⁇ Drug-280 bC Drug
  • ⁇ Drug-280 The average molar extinction coefficient of the drug at 280nm is 5100;
  • ⁇ mab-280 The average molar extinction coefficient of the monoclonal antibody at 280nm is 214600;
  • C mab concentration of monoclonal antibody
  • the optical path length is 1 cm.
  • a 370nm ⁇ mab-370 bC mab + ⁇ Drug-370 bC Drug
  • ⁇ Drug-370 The average molar extinction coefficient of the drug at 370nm is 19000;
  • ⁇ mab-370 The extinction coefficient of the monoclonal antibody at 370nm is 0;
  • C mab concentration of monoclonal antibody
  • the optical path length is 1 cm.
  • the drug loading in the ADC can be calculated.
  • Drug load C Drug /C mab .
  • the following example is the preparation process of the relevant ADC of the present disclosure.
  • the antibody PM in Example 3-1 to Example 3-3 and Example 3-6 is linked by a sulfhydryl coupling on cysteine
  • Examples 3-8 to 3-10 pass through the sulfhydryl group on the cysteine of the antibody PM, and
  • the drug VcMMAE (Hanxiang Biotechnology, CAS646502-53-6) with the connecting unit was reacted to prepare the conjugate molecule PM-VcMMAE as a control.
  • antibody-drug conjugates with different DAR values can be obtained.
  • the DAR value is preferably 1-8, more preferably 3-8, and most preferably 3. -7.
  • UV-Vis calculated average value: n 6.31.
  • UV-Vis calculated average value: n 6.63.
  • TCEP tris(2-carboxyethyl)phosphine
  • UV-Vis calculated average value: n 6.9.
  • UV-Vis calculated average value: n 3.68.
  • Embodiment 3-5 ADC-5
  • UV-Vis calculated average value: n 3.89.
  • UV-Vis calculated average value: n 6.61.
  • TCEP tris(2-carboxyethyl)phosphine
  • UV-Vis calculated average: n 3.93.
  • the compound VcMMAE (2.22 mg, 1689 nmol) was dissolved in 100 ⁇ L of dimethyl sulfoxide, added to the above reaction solution, placed in a water bath shaker, and reacted with shaking at 25° C. for 3 hours to stop the reaction.
  • the reaction solution was desalted and purified with a Sephadex G25 gel column (elution phase: pH 6.5 0.05M PBS buffer aqueous solution, containing 0.001M EDTA) to obtain the exemplary product ADC of the conjugate shown in formula PM-VcMMAE -8 PBS buffer (1.76mg/mL, 12mL), stored at 4°C.
  • UV-Vis calculated average: n 4.19.
  • UV-Vis calculated average value: n 4.16.
  • UV-Vis calculated average value: n 7.07.
  • Test Example 1 In vitro cell binding experiment
  • the fluorescence signal of the antibody on the cell surface is detected, and the binding of the antibody is evaluated according to the intensity of the fluorescence signal.
  • the prepared ADC-2, ADC-10, control ADC Lmab-9-A and antibody PM were used for in vitro binding detection.
  • the serially diluted ADC-2, ADC-10 and antibody PM were combined with 1 ⁇ 10 5 cells (ATCC, MDA PCa 2b/CRL-2422, LNCaP/CRL-1740, 22Rv1/CRL-2505, PC-3/CRL- 1435, DU 145/HTB-81) After incubating at 4°C for 60 minutes, the excess ADC or antibody is washed away. Incubate the cells with FITC-labeled goat anti-human IgG (H+L) secondary antibody (Jackson Immuno Research, 109-095-003) at 4°C for 30 minutes. After washing off the excess antibody, use BD CantoII to read the fluorescence on the cell surface Signal, (the results are shown in Table 2, Figure 1A, Figure 1B, Figure 1C, Figure 1D, and Figure 1E).
  • the expression level of the antigen PSMA of the cells used for detection MDA PCa 2b> LNCaP> 22Rv1, PC-3 and DU 145 do not express PSMA.
  • ADC-2, ADC-10, control ADCLmab-9-A and antibody PM have corresponding strong or weak binding ability to cells with different PSMA antigen expression levels.
  • DT3C, 70kd is a recombinantly expressed fusion protein. It is a fusion of Fragment A (toxin part only) of diphtheria toxin and 3C fragment (IgG binding part) of group G streptococci. This protein can have a high affinity with the IgG part of antibodies. , When the antibody enters the cell when it undergoes endocytosis, it releases toxic DT under the action of intracellular furin. DT can inhibit the activity of EF2-ADP ribosylation, block the protein translation process, and ultimately lead to cell death . The DT3C that has not entered the cell has no cell-killing activity. The endocytosis activity of the antibody is evaluated according to the cell killing situation.
  • DT3C sterile filtered DT3C
  • the antibody PM antibody the molar concentration of DT3C is 6 times the molar concentration of the antibody
  • Serum culture medium is serially diluted, and added to cells (ATCC, LNCaP/CRL-1740, 22Rv1/CRL-2505) cultured in medium containing 20% low IgG FBS prepared one day in advance (2000 cells/well), 5% Incubate in a carbon dioxide incubator at 37°C for three days.
  • CellTiter-Glo Promega, G7573 was added, incubated at room temperature for 10 minutes in the dark, and chemiluminescence was read on Victor3 (the results are shown in Table 3, Figure 2A, and Figure 2B).
  • LNCaP cells were cultured in RPMI-1640 medium containing 10% FBS and passaged 2 to 3 times a week, with a passage ratio of 1:3 or 1:6.
  • aspirate the culture medium wash the cell layer with 5 mL of 0.25% trypsin, then aspirate the trypsin, place the cells in an incubator for 3 to 5 minutes, and add fresh culture medium to resuspend the cells.
  • the culture plate was cultured in an incubator for 24 hours (37°C, 5% CO 2 ).
  • the ADCs (ADC-2, ADC-4, Lmab-9-A) to be tested are prepared to the starting concentration, and 9 points are diluted 1:6 with PBS, and 0 concentration points are added. Add 20 ⁇ L to the above cell plate and incubate in an incubator for 5 days (37°C, 5% CO 2 ). For compound 2-B (i.e.
  • 22Rv1 cells were cultured in RPMI-1640 medium containing 10% FBS and passaged twice a week, with a passage ratio of 1:3 or 1:6.
  • aspirate the culture medium wash the cell layer with 5 mL of 0.25% trypsin, then aspirate the trypsin, place the cells in an incubator for 3 to 5 minutes, and add fresh culture medium to resuspend the cells.
  • the culture plate was cultured in an incubator for 24 hours (37°C, 5% CO 2 ).
  • the preparation and experimental methods of the ADC and compound 2-B to be tested are the same as above. (The results are shown in Table 4, Figure 3C, and Figure 3D).
  • PC-3 cells were cultured in F-12K medium containing 10% FBS and passaged 2-3 times a week, with a passage ratio of 1:3 or 1:6.
  • aspirate the culture medium wash the cell layer with 5 mL of 0.25% trypsin, then aspirate the trypsin, place the cells in an incubator for 3 to 5 minutes, and add fresh culture medium to resuspend the cells.
  • the culture plate was cultured in an incubator for 24 hours (37°C, 5% CO 2 ).
  • the preparation and experimental methods of the ADC and compound 2-B to be tested are the same as above. (The results are shown in Table 4, Figure 3E, Figure 3F).
  • PSMA ADC has a killing effect on LNCaP and 22RV1 cells.
  • Compound 2-B i.e. the free toxin of 9-A
  • Test Example 4 Evaluation of the efficacy of ADC drugs on human prostate cancer cell 22Rv1 transplanted tumors in nude mice
  • mice Male, 6-8 weeks old, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. (certificate number 1908120082). Feeding environment: SPF level. Nude mice were subcutaneously inoculated with human prostate cancer cells 22Rv1 (Chinese Academy of Sciences). When the average tumor volume reached 220 mm 3 , the animals were randomly divided into groups (D0), each group of 6 animals, starting intraperitoneal injection twice a week for a total of 5 Check the tumor volume and weight twice a week, and record the data.
  • Tumor volume V 1/2 ⁇ a ⁇ b 2 , where a and b represent length and width respectively.
  • T/C(%) (T-T0)/(C-C0) ⁇ 100
  • T and C are the tumor volumes of the treatment group and the control group at the end of the experiment
  • T0 and C0 are the tumors at the beginning of the experiment volume.
  • TGI (%) 1-T/C (%).
  • ADC-10 3mpk, 10mpk;
  • ADC-2 3mpk, 10mpk;
  • Blank control group PBS buffer with pH 7.4.
  • Test Example 5 Evaluation of the efficacy of ADC drugs on human prostate cancer cell 22Rv1 transplanted tumors in nude mice
  • mice Male, 6-8 weeks old, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. (certificate number 1908120082). Feeding environment: SPF level. Nude mice were subcutaneously inoculated with human prostate cancer cells 22Rv1 (Chinese Academy of Sciences). When the average tumor volume reached 210 mm 3 , the animals were randomly divided into groups (D0), each group of 8 animals, starting intraperitoneal injection twice a week, a total of 6 Check the tumor volume and weight twice a week, and record the data.
  • Tumor volume V 1/2 ⁇ a ⁇ b 2 , where a and b represent length and width respectively.
  • T/C(%) (T-T0)/(C-C0) ⁇ 100
  • T and C are the tumor volumes of the treatment group and the control group at the end of the experiment
  • T0 and C0 are the tumors at the beginning of the experiment volume.
  • TGI (%) 1-T/C (%).
  • ADC-8 10mpk
  • ADC-6 3mpk, 6mpk;
  • ADC-7 3mpk, 6mpk, 10mpk;
  • Blank control group PBS buffer with pH 7.4.
  • the tumor inhibition rate of 10mpk of positive ADC-8 was 81.6%, and the tumor inhibition rates of 6mpk and 3mpk of the tested ADC-6 were >100% and 97.8%, respectively.
  • the tumor inhibition rates of 10mpk, 6mpk and 3mpk of a tested ADC-7 were >100%, 88.4% and 80.5%, respectively. All the administration groups were significantly better than the control group.
  • the tested ADC-6 was significantly better at the same dose. In the tested ADC-7, the two tested ADCs showed a good dose-dependent relationship (Table 6, Figure 5A).
  • Test Example 6 Evaluation of the efficacy of ADC drugs on transplanted tumors of human prostate cancer cells LNCap in SCID Beighe mice
  • mice Male, 6-8 weeks old, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. (certificate number 1908120082). Feeding environment: SPF level. Mice were subcutaneously inoculated with human prostate cancer cells LNCap (ATCC). When the average tumor volume reached 160 mm 3 , the animals were randomly divided into groups (D0), each group of 7 mice, and intraperitoneal injections on D0 and D4 twice, twice a week Detect tumor volume and weight, and record data.
  • LNCap human prostate cancer cells
  • Tumor volume V 1/2 ⁇ a ⁇ b 2 , where a and b represent length and width respectively.
  • T/C(%) (T-T0)/(C-C0) ⁇ 100
  • T and C are the tumor volumes of the treatment group and the control group at the end of the experiment
  • T0 and C0 are the tumors at the beginning of the experiment volume.
  • TGI (%) 1-T/C (%).
  • ADC-3 3mpk, 10mpk;
  • ADC-5 3mpk, 6mpk, 10mpk;
  • Blank control group PBS buffer with pH 7.4.
  • Test Example 7 PMSA ADC SD rat T1/2 evaluation
  • test drugs ADC-2, ADC-5, and ADC-10 were injected into the tail vein of SD male rats at a dose of 3 mg/kg and an injection volume of 5 mL/kg.
  • the blood collection time points are: 5 minutes, 8 hours, 24 hours (day 2) after administration on day 1, day 3, day 5, day 8, day 11, day 15, day 22 and On the 29th day, blood was taken from the fundus vein of the rat, each time 300 ⁇ L (equivalent to taking 150 ⁇ L of serum); the collected blood sample was placed at room temperature for half an hour to agglutinate, and then centrifuged at 1000 ⁇ g for 15 minutes at 4°C, and the supernatant (Serum) transferred to EP tube and stored immediately at -80°C.
  • ELISA was used to detect the concentration of PSMA antibody ADC in the serum of SD rats.
  • the concentration of the PSMA antibody ADC in the serum was detected by ELISA, and the PK analysis was performed (see Table 8 for the results).
  • the half-life is approximately 219.8 hours (9.2 days), 171.2 hours (7.1 days), 172.5 hours (7.2 days); the half-life of intact ADC in rats is approximately 76.7 hours (3.2 days), 153.9 hours (6.4 days), 137.9 hours (5.7 days).
  • the T 1/2 of ADC-2 and ADC-5 is better than ADC-10.
  • Test Example 8 PSMA ADC stability study (free toxin detection)
  • Blq means: 1ng/mL; M means that the sex of the rat is male.
  • ADC-2 showed good stability in the plasma of different species at a certain temperature and within a certain period of time.
  • test operation is carried out in a sterile laboratory, and the blank plasma is sterilized by filtration with a 0.22 ⁇ m microporous membrane.
  • the linear range of compound 2-B is 1 ⁇ 5000ng/mL; BLQ means: 1ng/mL. "-1" and “-2" indicate the first and second repeat of the experiment.
  • ADC-5 showed good stability in plasma of different species, at a certain temperature and within a certain period of time.
  • test operation is carried out in a sterile laboratory, and the blank plasma is sterilized by filtration with a 0.22 ⁇ m microporous membrane.
  • 23d is only a single hole, and the linear range of compound 2-B is 2 ⁇ 5000ng/mL; BLQ means: 1ng/mL.
  • Test Example 9 Evaluation of the efficacy of ADC drugs on human prostate cancer cell 22Rv1 transplanted tumors in nude mice
  • mice The experimental male nu/nu nude mice, 6-8 weeks old, were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. (certificate number 1908120082). Feeding environment: SPF level. Nude mice were subcutaneously inoculated with human prostate cancer cells 22Rv1 (Chinese Academy of Sciences). When the average tumor volume reached 190mm 3 , the animals were randomly divided into groups (D0), each group of 8 animals, started intraperitoneal injection, 2 times a week, total administration 4 times, 2 times a week to detect tumor volume and weight, and record the data.
  • V tumor volume
  • T/C(%) (TT 0 )/(CC 0 ) ⁇ 100, where T and C are the tumor volumes of the treatment group and the control group at the end of the experiment; T 0 , C 0 are the tumor volumes at the beginning of the experiment Tumor volume.
  • TGI (%) 1-T/C (%).
  • mice The weight of the mice was stable during the administration process, indicating that the tested antibodies had no obvious toxic side effects.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • Epidemiology (AREA)
  • Cell Biology (AREA)
  • Reproductive Health (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Pregnancy & Childbirth (AREA)
  • Gynecology & Obstetrics (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)
  • Medicinal Preparation (AREA)

Abstract

提供了抗PSMA抗体-依喜替康类似物偶联物及其医药用途。具体地，提供了如通式(Pc-L-Y-D)所示的抗PSMA抗体-药物偶联物，其中Pc为抗PSMA抗体或其抗原结合片段。

Description

抗PSMA抗体-依喜替康类似物偶联物及其医药用途
本申请要求2020年3月25日提交的中国专利申请(申请号CN 202010218297.4)的优先权。
技术领域
本公开涉及抗PSMA抗体、抗PSMA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PSMA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
这里的陈述仅提供与本公开有关的背景信息，而不必然地构成现有技术。
美国癌症协会2019年统计数据显示，美国男性新增癌症病例中前列腺癌排名第一，为174650例(CA CANCER J CLIN 2019；69:7–34)。对于临床局部疾病，通常选择手术和放射治疗。对于局部晚期或转移性疾病，通常先采用手术或化学方式剥夺雄激素(ADT)治疗。对于去势抵抗(CRPC)初期可选用Sipuleucel-T细胞免疫疗法，对于转移性去势抵抗阶段(mCRPC)病人，可根据情况选用雄激素抑制剂、雄激素受体拮抗剂、针对骨转移的放疗药物以及作用于微管的化疗药物等药物治疗。但每种治疗式都只能延长几个月的存活期，需要寻求有效的治疗方式(European Urology,66(6),1190–1193；Journal of Nuclear Medicine,59(2),177–182.)。
前列腺特异性膜抗原(PSMA)除了由前列腺上皮细胞表达以外，还可以由非前列腺组织表达，例如小肠，近端肾小管和唾液腺，但其水平远低于前列腺组织。而PSMA在前列腺癌细胞中高表达，特别是在转移性疾病、激素难治性疾病和高级别病变中的表达最高。另外，PSMA还高表达于所有实体瘤的新生血管的内皮细胞中，但其在正常血管中没有表达，所以它也被作为实体瘤治疗的靶点(Clinical Cancer Research,Vol.3,81-85,January 1997；Urologic Oncology:Seminars and Original Investigations,1(1),18–28.；Clin Cancer Res.2010 November 15；16(22):5414–5423.)。雄激素剥夺或雄激素受体拮抗剂治疗均可上调前列腺特异膜抗原(PSMA)的表达(J Nucl Med 2017；58:81–84)，这为靶向疗法与传统激素疗法联合治疗提供依据。
PSMA属于谷氨酸II型羧肽酶(Glutamate carboxypeptidase II，GCPII)，它在神经系统中作为NAALDase，酶解NAAG得到谷氨酸，与谷氨酸的神经传递相关。在小肠中作为FOLH1，分解叶酸-聚-γ-谷氨酸(folyl-poly-γ-glutamate，FPGn)得到叶酸，与叶酸代谢相关(Curr Med Chem.2012；19(6):856–870.)。而在前列腺中作为PSMA，表达在上皮细胞上，与前列腺癌的发生有关。PSMA由750个氨基酸组成， 胞内19个，跨膜24个，胞外707个。结晶结果显示胞外部分由3个结构域组成，分别为蛋白酶样结构域、顶端结构域和C末端结构域。三者都参与底物的结合，前两者直接与底物结合，C末端结构域在使PSMA形成二聚体中发挥作用(The EMBO Journal(2006)25,1375–1384)。谷氨酸II型羧肽酶(GCPII)及其剪接变体，旁系同源物的研究有限。以PSM’为代表的剪接变体大多存在于正常前列腺组织细胞的胞内。研究最深入的GCPII同源物GCPIII或NAALADase II作为自身有效的药物靶标，可以弥补正常的GCPII酶促活性的不足，其与GCPII有68％序列相似性(Current Medicinal Chemistry,2012,19,1316-1322；Frontiers in Bioscience,Landmark,24,648-687,March 1,2019)。
抗体-药物偶联物(antibody drug conjugate,ADC)把单克隆抗体或者抗体片段通过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，同时又避免了抗体的疗效偏低和毒性物质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杀伤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PSMA的ADC药物的用于临床治疗研究。Cytogen公司的PSMA-ADC，处于二期临床阶段，MedImmune公司的MEDI-3726以及BZL Biologics Inc公司的MLN-2704在临床一期结束后因为药效不良停止了研究。采用不同策略开发新的ADC药物具有广阔的前景。
发明内容
本公开涉及抗PSMA抗体-ADC以及其用途，提供与抗PSMA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。
本公开提供了一种通式(Pc-L-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐：
Figure PCTCN2021082857-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)-和-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、环烷基和杂环基；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、芳基和杂芳基；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、芳基和杂芳基；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；非限制性实例如m选自0、1、2、3和4；
n为1至10，n是小数或整数；优选地，n为1-8，更优选为3-8，最优选为3-7，更优选为6-7。
L为接头单元；
Pc为抗PSMA抗体或其抗原结合片段，优选地，所述抗PSMA抗体或其抗原结合片段特异性结合PSMA胞外结构域。
在一些实施方案中，如前所述的通式(Pc-L-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐，其中所述抗PSMA抗体或其抗原结合片段包含重链可变区和轻链可变区，其中所述重链可变区包含与如SEQ ID NO：1所示的重链可变区相同序列的HCDR1、HCDR2和HCDR3，所述轻链可变区包含与如SEQ ID NO：2所示的轻链可变区相同序列的LCDR1、LCDR2和LCDR3。
在一些实施方案中，如前所述的通式(Pc-L-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐，其中所述抗PSMA抗体或其抗原结合片段包含重链可变区和轻链可变区，其中所述重链可变区包含分别如SEQ ID NO：3、SEQ ID NO：4和SEQ ID NO：5所示的HCDR1、HCDR2和HCDR3，所述轻链可变区包含分别如SEQ ID NO：6、SEQ ID NO：7和SEQ ID NO：8所示的LCDR1、LCDR2和LCDR3。
在一些实施方案中，如前所述的通式(Pc-L-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐，其中所述抗PSMA抗体是鼠源抗体、嵌合抗体、人源化抗体或人抗体。
在一些实施方案中，如前所述的通式(Pc-L-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐，其中所述抗PSMA抗体或其抗原结合片段包含重链可变区和轻链可变区，其中所述重链可变区的氨基酸序列如SEQ ID NO:1所示或与其有至少90％-100％的序列同一性，包括但不限于至少91％、至少92％、至少93％、至少94％，至少95％、至少96％、至少97％、至少98％、至少99％或至少100％的序列同一性；和所述轻链可变区的氨基酸序列如SEQ ID NO:2所示或与其有至少90％-100％的序列同一性，包括但不限于至少91％、至少92％、至少93％、至少94％，至少95％、至少96％、至少97％、至少98％、至少99％或至少100％的 序列同一性。
在一些实施方案中，如前所述的通式(Pc-L-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐，其中所述抗PSMA抗体或其抗原结合片段，包含序列如SEQ ID NO:1所示的重链可变区和序列如SEQ ID NO：2所示的轻链可变区。
在一些实施方案中，如前所述的通式(Pc-L-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐，其中所述抗PSMA抗体包含抗体重链恒定区和轻链恒定区；优选地，所述重链恒定区选自人IgG1、IgG2、IgG3和IgG4的恒定区及其常规变体，所述轻链恒定区选自人抗体κ和λ链的恒定区及其常规变体。
在一些实施方案中，如前所述的通式(Pc-L-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐，其中所述抗PSMA抗体，包含如SEQ ID NO：9所示的重链和如SEQ ID NO：10所示的轻链。
在一些实施方案中，如前所述的通式(Pc-L-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐，其中n为1至8，优选为3-8，更优选3-7，n是小数或整数。
在一些实施方案中，如前所述的通式(Pc-L-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐，
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、卤素和C 1-6烷基；
R 1为卤代烷基或C 3-6环烷基；
R 2选自氢原子、卤代烷基和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。
在一些实施方案中，如前所述的通式(Pc-L-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐，其中Y选自：
Figure PCTCN2021082857-appb-000002
其中Y的O端与接头单元L相连。
在一些实施方案中，如前所述的通式(Pc-L-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐，其中接头单元-L-为-L 1-L 2-L 3-L 4-，
L 1选自-(琥珀酰亚胺-3-基-N)-W-C(O)-、-CH 2-C(O)-NR 3-W-C(O)-或-C(O)-W-C(O)-，其中W选自C 1-8烷基、C 1-8烷基-环烷基和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和S的杂原子，其中所述的C 1-8亚烷 基、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)p 1CH 2CH 2C(O)-、-NR 4(CH 2CH 2O)p 1CH 2C(O)-、-S(CH 2)p 1C(O)-和化学键，其中p 1为1至20的整数；
L 3为由2至7个氨基酸残基构成的肽残基，其中所述的氨基酸选自苯丙氨酸(F)、甘氨酸(G)、缬氨酸(V)、赖氨酸(K)、瓜氨酸、丝氨酸(S)、谷氨酸(E)和天冬氨酸(D)中的氨基酸形成的氨基酸残基，并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、烷氧基和环烷基中的一个或多个取代基所取代；
L 4选自-NR 5(CR 6R 7) t-、-C(O)NR 5、-C(O)NR 5(CH 2) t-和化学键，其中t为1至6的整数；
R 3、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。
在一些实施方案中，如前所述的通式(Pc-L-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐，其中接头单元-L-为-L 1-L 2-L 3-L 4-，
L 1为
Figure PCTCN2021082857-appb-000003
s 1为2至8的整数；
L 2为化学键；
L 3为四肽残基；优选地，L 3为GGFG(SEQ ID NO：13)(甘氨酸-甘氨酸-苯丙氨酸-甘氨酸)的四肽残基；
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；
其中所述的L 1端与Pc相连，L 4端与Y相连。
在一些实施方案中，如前所述的通式(Pc-L-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐，其中-L-为：
Figure PCTCN2021082857-appb-000004
在一些实施方案中，如前所述的通式(Pc-L-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐，其中-L-Y-任选自：
Figure PCTCN2021082857-appb-000005
在一些实施方案中，如前所述的通式(Pc-L-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐，其为通式(Pc-L a-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐：
Figure PCTCN2021082857-appb-000006
其中：
W、L 2、L 3、R 5、R 6、R 7如前所述接头单元-L-中所定义；
Pc、n、R 1、R 2、m如通式(Pc-L-Y-D)中所定义；
具体的，Pc为抗PSMA抗体或其抗原结合片段；
m为0至4的整数；非限制性实例如m选自0、1、2、3和4；
n为1至10，n是小数或整数；优选地，n为1-8，更优选为3-8，最优选为3-7，更优选为6-7；
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、芳基和杂芳基；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、芳基和杂芳基；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；
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)p 1CH 2CH 2C(O)-、-NR 4(CH 2CH 2O)p 1CH 2C(O)-、-S(CH 2)p 1C(O)-和化学键，其中p 1为1至20的整数；
L 3为由2至7个氨基酸残基构成的肽残基，其中所述的氨基酸残基选自苯丙氨酸(F)、甘氨酸(G)、缬氨酸(V)、赖氨酸(K)、瓜氨酸、丝氨酸(S)、谷氨酸(E)和天冬氨酸(D)中的氨基酸形成的氨基酸残基，并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、烷氧基和环烷基中的一个或多个取代基所取代；
R 5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。
在一些实施方案中，如前任一项所述的通式(Pc-L-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐，其为通式(Pc-L b-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐：
Figure PCTCN2021082857-appb-000007
其中：
s 1为2至8的整数；
Pc、R 1、R 2、R 5、R 6、R 7、m和n如通式(Pc-L a-Y-D)所中所定义。
在一些实施方案中，如前任一项所述的通式(Pc-L-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐，所述抗体-药物偶联物选自：
Figure PCTCN2021082857-appb-000008
Figure PCTCN2021082857-appb-000009
其中Pc和n如通式(Pc-L-Y-D)中所定义，具体地，Pc为抗PSMA抗体或其抗原结合片段，或如前任一项所述的抗PSMA抗体；
n为1至10，n是小数或整数；优选地，n为1-8，更优选为3-8，最优选为3-7；最优选为6-7。
在一些实施方案中，如前任一项所述的通式(Pc-L-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐，所述抗体-药物偶联物为：
Figure PCTCN2021082857-appb-000010
其中：
n为1至8，优选为3-8，n是小数或整数；
PM为抗PSMA抗体，其包含如SEQ ID NO：9所示的重链和如SEQ ID NO：10所示的轻链。
本公开进一步提供一种制备如通式(Pc-L a-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐的方法，其包括以下步骤：
Figure PCTCN2021082857-appb-000011
Pc’为还原后的Pc，与通式(L a-Y-D)所示的化合物进行偶联反应，得到通式(Pc-L a-Y-D)所示的化合物；
其中：
Pc为抗PSMA抗体或其抗原结合片段；
n、m、W、L 2、L 3、R 1、R 2、R 5、R 6和R 7如前述通式(Pc-L a-Y-D)中所定义。
本公开进一步提供一种制备如通式(Pc-L’-D)所示的抗体药物偶联物的方法，其包括以下步骤：
Figure PCTCN2021082857-appb-000012
上述反应中，Pc’与式(L’-D)所示的化合物进行偶联反应，得到通式(Pc-L’-D)所示的化合物；其中：
Pc为如前所述的抗PSMA抗体或其抗原结合片段；Pc’为经还原后的Pc，
n如通式(Pc-L-Y-D)中所定义。
本公开进一步提供一种制备如PM-9-A所示的抗体药物偶联物的方法，其包括以下步骤：
Figure PCTCN2021082857-appb-000013
PM’与式9-A所示的化合物进行偶联反应，得到通式(PM-9-A)所示的化合物；其中：
n为1至8，优选3-8，n是小数或整数；
PM为抗PSMA抗体，其包含序列如SEQ ID NO：9所示的重链和序列如SEQ  ID NO：10所示的轻链；
PM’为经PM还原后得到。
在一些实施方案中，n代表抗体-药物偶联物的载药量，也可称为DAR值，其可用常规方法如UV/可见光光谱法、质谱、ELISA试验和HPLC测定；在一些是方案中，n为0-10，优选1-10，更优选1-8，或2-8，或2-7，或2-4，或3-8，或3-7，或3-6，或4-7，或4-6，或4-5的平均值；在一些实施方案中，n为1，2，3，4，5，6，7，8，9，10的平均值。
另一方面，本公开提供了一种药物组合物，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，以及一种或多种药学上可接受的赋形剂、稀释剂或载体。在一些实施方案中，所述单位剂量的药物组合物中含有0.1mg-3000mg或1mg-1000mg如前所述的抗PSMA抗体或如前所述的抗体药物偶联物。
另一方面，本公开提供了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包含其的药物组合物作为药物的用途。
另一方面，本公开提供了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包含其的药物组合物在制备用于治疗PSMA介导的疾病或病症的药物中的用途，其中所述PSMA介导的疾病或病症优选为PSMA高表达癌症，中表达癌症或低表达癌症。
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包含其的药物组合物在制备用于治疗或预防癌症的药物中的用途，其中所述癌症优选头和颈鳞状细胞癌、头和颈癌、脑癌、神经胶质瘤、多形性成胶质细胞瘤、神经母细胞瘤、中枢神经系统癌、神经内分泌肿瘤、咽喉癌、鼻咽癌、食管癌、甲状腺癌、恶性胸膜间皮瘤、肺癌、乳腺癌、肝癌、肝细胞瘤、肝细胞癌、肝胆癌、胰腺癌、胃癌、胃肠道癌、肠癌、结肠癌、结肠直肠癌、肾癌、透明细胞肾细胞癌、卵巢癌、子宫内膜癌、子宫颈癌、膀胱癌、前列腺癌、睾丸癌、皮肤癌、黑色素瘤、白血病、淋巴瘤、骨癌、软骨肉瘤、骨髓瘤、多发性骨髓瘤、骨髓异常增生综合征、库肯勃氏瘤、骨髓增生性肿瘤、鳞状细胞癌、尤因氏肉瘤、尿路上皮癌和梅克尔细胞癌，优选前列腺癌；更优选的，所述淋巴瘤选自：何杰金淋巴瘤、非何杰金淋巴瘤、弥漫性大B-细胞淋巴瘤、滤泡性淋巴瘤、原发性纵隔大B-细胞淋巴瘤、套细胞淋巴瘤、小淋巴细胞性淋巴瘤、富含T-细胞/组织细胞的大B-细胞淋巴瘤和淋巴浆细胞性淋巴瘤，所述肺癌选自：非小细胞肺癌和小细胞肺癌，所述白血病选自：慢性髓细胞样白血病、急性髓细胞样白血病、淋巴细胞白血病、成淋巴细胞性白血病、急性成淋巴细胞性白血病、慢性淋巴细胞性白血病和髓样细胞白血病。
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受试者施用治疗有效剂量的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其药学上可接受的盐或包含其的药物组合物；优选其中所述的肿瘤为与PSMA高表达相关的癌症，中表达癌症或低表达癌症。
另一方面，本公开进一步涉及一种用于治疗或预防肿瘤或癌症的方法，该方法包括向需要其的受试者施用治疗有效剂量的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包含其的药物组合物；其中所述肿瘤和癌症优选头和颈鳞状细胞癌、头和颈癌、脑癌、神经胶质瘤、多形性成胶质细胞瘤、神经母细胞瘤、中枢神经系统癌、神经内分泌肿瘤、咽喉癌、鼻咽癌、食管癌、甲状腺癌、恶性胸膜间皮瘤、肺癌、乳腺癌、肝癌、肝细胞瘤、肝细胞癌、肝胆癌、胰腺癌、胃癌、胃肠道癌、肠癌、结肠癌、结肠直肠癌、肾癌、透明细胞肾细胞癌、卵巢癌、子宫内膜癌、子宫颈癌、膀胱癌、前列腺癌、睾丸癌、皮肤癌、黑色素瘤、白血病、淋巴瘤、骨癌、软骨肉瘤、骨髓瘤、多发性骨髓瘤、骨髓异常增生综合征、库肯勃氏瘤、骨髓增生性肿瘤、鳞状细胞癌、尤因氏肉瘤、尿路上皮癌和梅克尔细胞癌；优选前列腺癌；更优选的，所述淋巴瘤选自：何杰金淋巴瘤、非何杰金淋巴瘤、弥漫性大B-细胞淋巴瘤、滤泡性淋巴瘤、原发性纵隔大B-细胞淋巴瘤、套细胞淋巴瘤、小淋巴细胞性淋巴瘤、富含T-细胞/组织细胞的大B-细胞淋巴瘤和淋巴浆细胞性淋巴瘤，所述肺癌选自：非小细胞肺癌和小细胞肺癌，所述白血病选自：慢性髓细胞样白血病、急性髓细胞样白血病、淋巴细胞白血病、成淋巴细胞性白血病、急性成淋巴细胞性白血病、慢性淋巴细胞性白血病和髓样细胞白血病。
另一方面，本公开进一步提供前述的抗PSMA抗体或其抗体-药物偶联物作为药物，优选作为治疗癌症或肿瘤的药物，更优选作为治疗PSMA介导的癌症的药物。
可将活性化合物(例如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)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，或者是以受试者能够以单剂进行自我给药的方式。本公开所述的活性化合物或组合物的单位剂量的方式可以是片剂、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活性化合物或组合物的施用剂量通常将随疾病的严重性、受试者的体重和活性化合物的相对功效而改变。作为一般性指导，合适的单位剂量可以是0.1mg～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。
本公开提供的PSMA抗体及抗体药物偶联物具有与细胞表面抗原良好的亲和力，良好的细胞内吞效率和很强的肿瘤抑制效率，并且具有更宽的药物应用窗口，更高的抑瘤效果和治疗活性，更好的安全性、药物代谢动力学特性和成药性(如 稳定性)，更适于临床的药物应用。
附图说明
图1A：本公开的ADC或抗体与细胞MDAPCa的体外结合能力。
图1B：本公开的ADC或抗体与细胞LNCaP的体外结合能力。
图1C：本公开的ADC或抗体与细胞22Rv1的体外结合能力。
图1D：本公开的ADC或抗体与细胞PC-3的体外结合能力。
图1E：本公开的ADC或抗体与细胞DU 145的体外结合能力。
图2A：抗体PM在LNCaP细胞中的体外内吞实验；2K细胞，10％U-L IgG FBS。
图2B：抗体PM在22Rv1细胞中的体外内吞实验；2K细胞，10％U-L IgG FBS。
图3A：本公开的不同ADC对LNCaP细胞杀伤作用，用2K细胞，4.5％FBS。
图3B：本公开中毒素(化合物2-B)对LNCaP细胞杀伤作用，用2K细胞，4.5％FBS。
图3C：本公开的不同ADC对22Rv1细胞杀伤作用，用4K细胞，4.5％FBS。
图3D：本公开中毒素(化合物2-B)对22Rv1细胞杀伤作用，用4K细胞，4.5％FBS。
图3E：本公开的不同ADC对PC-3细胞杀伤作用，用4K细胞，4.5％FBS。
图3F：本公开中毒素(化合物2-B)对PC-3细胞杀伤作用，用2K细胞，4.5％FBS。
图4A：本公开不同剂量的ADC对人前列腺癌细胞22Rv1裸小鼠移植瘤的抑制活性。
图4B：本公开不同剂量的ADC对人前列腺癌细胞22Rv1裸小鼠移植瘤的抑制活性测试中小鼠的体重变化。
图5A：本公开不同剂量的ADC对人前列腺癌细胞22Rv1裸小鼠移植瘤的抑制活性。
图5B：本公开不同剂量的ADC对人前列腺癌细胞22Rv1裸小鼠移植瘤的抑制活性测试中小鼠的体重变化。
图6A：本公开不同剂量的ADC对人前列腺癌细胞LNCap在SCID Beighe小鼠上的移植瘤的抑制活性。
图6B：本公开不同剂量的ADC对人前列腺癌细胞LNCap在SCID Beighe小鼠上的移植瘤的抑制活性测试中小鼠的体重变化。
图7：本公开ADC-2的药代稳定性，其中ADC的浓度为100μg/ml。
图8：本公开ADC-5的血浆稳定性，其中ADC的浓度为100μg/ml。
图9：给药ADC对荷瘤裸鼠22Rv1移植瘤的疗效。
具体实施方式
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，旨在包括该商品名产品的制剂、该商品名产品的药物和活性药物部分。
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”或“毒素”是指细胞毒性药物，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)，化疗药物，抗生素和核溶酶。
术语“接头单元”、“接头”、“连接单元”或“连接片段”是指一端与配体(本公开中为抗体)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配体或药物相连。
接头可以包含一种或多种接头元件。示例性的接头元件包括6-马来酰亚氨基己酰基(“MC”)、马来酰亚氨基丙酰基(“MP”)、缬氨酸-瓜氨酸(“val-cit”或“vc”)、丙氨酸-苯丙氨酸(“ala-phe”)、对氨基苄氧羰基(“PAB”)、N-琥珀酰亚氨基4-(2-吡啶基硫代)戊酸酯(“SPP”)、N-琥珀酰亚氨基4-(N-马来酰亚氨基甲基)环己烷-1羧酸酯(“SMCC”，在本文中也称作“MCC”)和N-琥珀酰亚氨基(4-碘-乙酰基)氨基苯甲酸酯(“SIAB”)。接头可以包含以下的元件中的一个或多个、或其组合：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)。
接头元件包括但不限于：
MC＝6-马来酰亚氨基己酰基，结构如下：
Figure PCTCN2021082857-appb-000014
Val-Cit或“vc”＝缬氨酸-瓜氨酸(蛋白酶可切割接头中的示例二肽)；
瓜氨酸＝2-氨基-5-脲基戊酸；
PAB＝对氨基苄氧羰基(“自我牺牲”接头元件的示例)；
Me-Val-Cit＝N-甲基-缬氨酸-瓜氨酸(其中接头肽键已经修饰以防止其受到组织蛋白酶B的切割)；
MC(PEG)6-OH＝马来酰亚氨基己酰基-聚乙二醇(可附着于抗体半胱氨酸)；
SPP＝N-琥珀酰亚氨基4-(2-吡啶基硫代)戊酸酯；
SPDP＝N-琥珀酰亚氨基3-(2-吡啶基二硫代)丙酸酯；
SMCC＝琥珀酰亚氨基-4-(N-马来酰亚氨基甲基)环己烷-1-羧酸酯；
IT＝亚氨基硫烷。
IT＝亚氨基硫烷。
术语“抗体药物偶联物”指抗体通过连接单元与具有生物活性的药物相连。在本公开中“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连接单元与具有生物活性的毒性药物相连。抗体可直接地或经接头地偶联至药物。n是每个抗体的平均药物模块数，可以是整数或小数，其范围可以是：例如每个抗体约0到约20个药物模块，在某些实施方案中是每个抗体1个到约10个药物模块，在某些实施方案中是每个抗体1个到约8个药物模块，如2、3、4、5、6、7、8个药物模块。本公开的抗体-药物偶联物的混合物的组合物，其中每个抗体的平均药物载荷是约1个至约10个，包括但不限于约3个至约7个，约3个至约6个，约3个至约5个，约1个至约9个，约7个或约4个。
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，完整抗体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，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。
全长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个序列相对保守的框架区(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。
术语“完全人源抗体”、“完全人抗体”、“人抗体”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。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。
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。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，示例包括(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。
通常，Fab是通过用蛋白酶木瓜蛋白酶(例如，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端侧的部分和L链通过二硫键结合在一起。
通常，F(ab')2是通过用酶胃蛋白酶消化IgG铰链区中二硫键的下方部分而获得的，分子量约为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。
通常，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VH)和抗体轻链可变结构域(或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。通常，每个重链可变区中存在三个CDR(HCDR1、HCDR2、HCDR3)，每个轻链可变区中存在三个CDR(LCDR1、LCDR2、LCDR3)。可以使用各种公知方案中的任何一种来确定CDR的氨基酸序列边界，包括“Kabat”编号规则(参见Kabat等(1991)，“Sequences of Proteins of Immunological Interest”，第5版，Public Health Service，National Institutes of Health，Bethesda，MD)、“Chothia”编号规则(参见Al-Lazikani等人，(1997)JMB 273：927-948)和ImMunoGenTics(IMGT)编号规则(Lefranc M.P.，Immunologist，7，132-136(1999)；Lefranc，M.P.等，Dev.Comp.Immunol.，27，55-77(2003)等。例如，对于经典格式，遵循Kabat规则，所述重链可变域(VH)中的CDR氨基酸残基编号为31-35(HCDR1)、50-65(HCDR2)和95-102(HCDR3)；轻链可变域(VL)中的CDR氨基酸残基编号为24-34(LCDR1)、50-56(LCDR2)和89-97(LCDR3)。遵循Chothia规则，VH中的CDR氨基酸编号为26-32(HCDR1)、52-56(HCDR2)和95-102(HCDR3)；并且VL中的氨基酸残基编号为26-32(LCDR1)、50-52(LCDR2)和91-96(LCDR3)。通过组合Kabat和Chothia两者的CDR定义，CDR由人VH中的氨基酸残基26-35(HCDR1)、50-65(HCDR2)和95-102(HCDR3)和人VL中的氨基酸残基24-34(LCDR1)、50-56(LCDR2)和89-97(LCDR3)构成。遵循IMGT规则，VH中的CDR氨基酸残基编号大致为26-35(CDR1)、51-57(CDR2)和93-102(CDR3)，VL中的CDR氨基酸残基编号大致为27-32(CDR1)、50-52(CDR2)和89-97(CDR3)。遵循IMGT规则，抗体的CDR区可以使用程序IMGT/DomainGap Align确定。除非特别说明，本公开中所述6个CDR均按照Kabat编号规则获得。((Kabat E.A.等人，(1991)Sequences of proteins of immunological interest.NIH Publication 91-3242))。按照Kabat规则，所述重链可变域(VH)中的CDR氨基酸残基编号为31-35(HCDR1)、50-65(HCDR2)和95-102(HCDR3)；轻链可变域(VL)中的CDR氨基酸残基编号为24-34(LCDR1)、50-56(LCDR2)和89-97(LCDR3)。本领域其他的编号规则Chothia、IMGT等。
术语“抗体框架区”，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”是指抗原上被免疫球蛋白或抗体结合的部位。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”是指抗体或抗原结合片段对预先确定的抗原上的表位的结合。通常，抗体或抗原结合片段以大约小于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“KD”是指抗体-抗原相互作用的解离平衡常数。通常，本公开的抗体或抗原结合片段以小于大约10 -7M，例如小于大约10 -8M或10 -9M的解离平衡常数(KD)结合PSMA或其表位，例如，在本公开中抗体与细胞表面抗原的亲和力采用FACS法测定KD值。
术语“核酸分子”是指DNA分子或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。
氨基酸序列“序列同一性”指比对氨基酸序列过程中，在必要时引入间隙，以达成最大序列同一性百分比，且不将任何保守性取代视为序列同一性的一部分，第一序列中与第二序列中的氨基酸残基相同的氨基酸残基的百分比。为测定氨基酸序列同一性百分比的目的，比对可以通过属于本领域技术的范围内的多种方式来实现，例如使用公开可得到的计算机软件，诸如BLAST、BLAST-2、ALIGN、ALIGN-2或Megalign(DNASTAR)软件。本领域技术人员可确定适用于测量比对的参数，包括在所比较的序列全长上达成最大比对所需的任何算法。
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)。
术语“宿主细胞”是指已向其中引入了表达载体的细胞。宿主细胞可包括微生物(如细菌)、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”是指由2个或2个以上氨基酸分子通过肽键相互连接而成的分子，是蛋白质的结构与功能片段。
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(例如1、2、3、4、5、6、7、8、9、10、11和12个)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。非限制性示例包括甲基、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、杂环烷硫基和氧代基。
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(例如1、2、3、4、5、6、7、8、9、10、11和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。亚烷基的非限制性示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。烷氧基的非限制性示例包括：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、环烷硫基和杂环烷硫基。
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，优选包含3至8个碳原子，更优选包含3至6个碳原子。单环环烷基的非限制性示例包括环丙基、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。
所述环烷基环包括如上所述的环烷基(包括单环、螺环、稠环和桥环)稠合于芳基、杂芳基或杂环烷基环上，其中与母体结构连接在一起的环为环烷基，非限制性实例包括茚满基、四氢萘基和苯并环庚烷基等；优选苯基并环戊基和四氢萘基。
环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氢原子、卤素、烷基、烷氧基、卤代烷基、羟基、羟烷基、氰基、氨基、硝基、环烷基、杂环基、芳基和杂芳基中的一个或多个取代基所取代。
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) P(其中p是整数0至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；更优选包含3至8个环原子，其中1-3是杂原子；更优选包含3至6个环原子，其中1-3个是杂原子；最优选包含5或6个环原子，其中1-3个是杂原子。单环杂环基的非限制性示例包括吡咯烷基、四氢吡喃基、1,2.3.6-四氢吡啶基、哌啶基、哌嗪基、吗啉基、硫代吗啉基和高哌嗪基 等。多环杂环基包括螺环、稠环和桥环的杂环基。
所述杂环基环包括如上所述的杂环基(包括单环、螺杂环、稠杂环和桥杂环)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，其非限制性示例包括：
Figure PCTCN2021082857-appb-000015
杂环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氢原子、卤素、烷基、烷氧基、卤代烷基、羟基、羟烷基、氰基、氨基、硝基、环烷基、杂环基、芳基和杂芳基中的一个或多个取代基所取代。
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，例如苯基和萘基。所述芳基环包括如上所述的芳基环稠合于杂芳基、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，其非限制性示例包括：
Figure PCTCN2021082857-appb-000016
芳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氢原子、卤素、烷基、烷氧基、卤代烷基、羟基、羟烷基、氰基、氨基、硝基、环烷基、杂环基、芳基和杂芳基中的一个或多个取代基所取代。
术语“杂芳基”指包含1至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，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、哒嗪基、咪唑基、吡唑基、三唑基、四唑基等。所述杂芳基环包括如上述的杂芳基稠合于芳基、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，其非限制性示例包括：
Figure PCTCN2021082857-appb-000017
Figure PCTCN2021082857-appb-000018
杂芳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氢原子、卤素、烷基、烷氧基、卤代烷基、羟基、羟烷基、氰基、氨基、硝基、环烷基、杂环基、芳基和杂芳基中的一个或多个取代基所取代。
术语“卤代烷基”指烷基上的氢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”指烷基上的氢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”指烷基上的氢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。
“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取代基取代。取代基仅处在它们的可能的化学位置，本领域技术人员能够在不付出过多努力的情况下确定(通过实验或理论)可能或不可能的取代。例如，具有游离氢的氨基或羟基与具有不饱和(如烯)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”是指本公开抗体药物偶联物的盐，这类盐用于受试者体内时具有安全性和有效性，且具有应有的生物活性，本公开抗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示例包括：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。
“载药量”或“平均药物载荷”也称药物抗体比例(Drug-to-Antibody Ratio，DAR)，即ADC中每个抗体所偶联的药物的平均数量。其可在例如每个抗体偶联约1至约10个药物的范围内，并且在某些实施例中，在每个抗体偶联约1至约8个药物的范围内，优选自2-8，2-7，2-6，2-5，2-4，3-4，3-5，5-6，5-7，5-8和6-8的范围。示例性的，载药量可以为1，2，3，4，5，6，7，8，9，10的平均值。本披露的ADC通式包括与前述一定载药量范围内的抗体药物偶联物的集合。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测定载药量。
可以用以下非限制性方法控制配体药物偶联物的载量，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，优选的示例如胶束、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受试者的血流中。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。这种装置的示例是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。
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(PM-9-A)所示的化合物的制备方法，其包括如下步骤：
Figure PCTCN2021082857-appb-000019
PM还原后得到PM’，PM’与通式(9-A)偶联反应，得到通式(PM-9-A)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：
PM为抗PSMA抗体或其抗原结合片段；
n为1至10，n是小数或整数。
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。
本公开实施例或测试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。参见Sambrook等，分子克隆，实验室手册，冷泉港实验室；当代分子生物学方法，Ausubel等著，Greene出版协会，Wiley Interscience，NY。未注明具体来源的试剂，为市场购买的常规试剂。
实施例1.抗体的制备
(一)PSMA抗体制备
1.1抗体序列
本公开的抗体参照WO2003034903A2制备，其中AB-PG1-XG1-006可变区的氨基酸序列如下：
AB-PG1-XG1-006重链可变区：
Figure PCTCN2021082857-appb-000020
AB-PG1-XG1-006轻链可变区：
Figure PCTCN2021082857-appb-000021
注：下划线部分为依照Kabat编号规则确定的CDR区。
表1 AB-PG1-XG1-006抗体的CDR区
抗体 AB-PG1-XG1-006
重链CDR1 RYGMH(SEQ ID NO:3)
重链CDR2 VIWYDGSNKYYADSVKG(SEQ ID NO:4)
重链CDR3 GGDFLYYYYYGMDV(SEQ ID NO:5)
轻链CDR1 RASQGISNYLA(SEQ ID NO:6)
轻链CDR2 EASTLQS(SEQ ID NO:7)
轻链CDR3 QNYNSAPFT(SEQ ID NO:8)
1.2全长抗体的构建
根据以上序列，设计引物PCR搭建得到VH/VK基因片段，获得可变区。
抗体可变区再与恒定区基因(CH1-Fc/CL)片段进行同源重组，构建完整抗体VH-CH1-Fc/VK-CL。
构建的完整全长抗体PM序列如下：
重链(IgG1)氨基酸序列：
Figure PCTCN2021082857-appb-000022
轻链(Kappa)氨基酸序列：
Figure PCTCN2021082857-appb-000023
1.3全长抗体的表达与纯化
将分别表达抗体轻、重链的质粒例转染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。
(二)对照抗体Lmab制备
对照抗体labetuzumab(简称Lmab)参照WHO Drug Information Vol.30,No.1,2016制备，其中重链和轻链氨基酸序列如下：
>抗体重链序列Lmab-HC
Figure PCTCN2021082857-appb-000024
Figure PCTCN2021082857-appb-000025
>抗体轻链序列Lmab-LC
Figure PCTCN2021082857-appb-000026
实施例2.化合物的制备
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)。
本公开抗体药物偶联物的Y-D药物部分参见PCT/CN2019/107873，相关的化合物合成及测试引用至本专利。其中的非限制性实施例合成引用如下：
实施例2-1
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 PCTCN2021082857-appb-000027
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2-2
(S)-2-环丙基-N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-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)-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-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)-2-羟基乙酰胺2-B
Figure PCTCN2021082857-appb-000028
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 5μm 19*250mm；流动相：A-水(10mmol NH 4OAc)，B-乙腈，梯度洗脱，流速：18mL/分钟)，收集其相应组分，减压浓缩，得到标题产物(2-A：1.5mg，2-B：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,2H),3.60(t,1H),3.19-3.13(m,1H),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2-3
(S)-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-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-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-2-羟基丙酰胺3-B
Figure PCTCN2021082857-appb-000029
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/分钟)，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2-4
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-羟基环戊烷-1-甲酰胺4
Figure PCTCN2021082857-appb-000030
向1b(3.0mg，5.64μmol)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-环戊烷甲酸4a(2.2mg，16.9μmol，采用专利申请“WO2013106717”公开的方法制备而得)和4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐(4.7mg，16.9μmol)，加毕，在0-5℃搅拌反应1小时。向反应液中加入5mL水淬灭反应，用乙酸乙酯(10mL×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4(2.5mg，产率：80.9％)。
MS m/z(ESI):548.0[M+1]。
1H NMR(400MHz,CDCl 3):δ7.73-7.62(m,2H),5.75-5.62(m,1H),5.46-5.32(m,2H),5.26-5.10(m,1H),3.30-3.10(m,1H),2.43(s,3H),2.28-2.20(m,2H),2.08-1.84(m,8H),1.69-1.58(m,2H),1.04-1.00(m,2H),0.89(t,3H)。
实施例2-5
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-(羟甲基)环丙烷-1-甲酰胺5
Figure PCTCN2021082857-appb-000031
向1b(2.0mg，3.76μmol)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-(羟甲基)-环戊烷甲酸5a(0.87mg，7.5μmol，采用专利申请“WO201396771”公开的方法制备而得)和4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐(2mg，7.24μmol)，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5(1.0mg，产率：50％)。
MS m/z(ESI):533.9[M+1]。
1H NMR(400MHz,CDCl 3):δ8.07(s,1H),7.23-7.18(m,2H),6.71-6.64(m,1H),6.55-6.51(m,1H),5.36-5.27(m,2H),4.67-4.61(m,2H),3.53-3.48(m,1H),3.30-3.22(m,2H),3.18-3.13(m,1H),2.71-2.61(m,2H),2.35-2.28(m,1H),2.04-1.91(m,4H),1.53-1.40(m,3H),0.91-0.75(m,4H)。
实施例2-6
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-(羟基甲基)环丁烷-1-甲酰胺6
Figure PCTCN2021082857-appb-000032
向1b(3.0mg，5.64μmol)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-(羟基甲基)环丁烷-1-甲酸6a(2.2mg，16.9μmol；采用文献“Journal of the American Chemical Society,2014,vol.136,#22,p.8138-8142”公开的方法制备而得)和4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐(4.7mg，16.9μmol)，加毕，在0-5℃搅拌反应1小时。向反应液中加入5mL水淬灭反应，用乙酸乙酯(10mL×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6(2.1mg，产率：67.9％)。
MS m/z(ESI):548.0[M+1]。
1H NMR(400MHz,DMSO-d 6):δ7.85-7.62(m,1H),6.88(br，1H),5.87-5.48(m，2H),5.47-5.33(m，1H),5.31-5.06(m，1H),4.25-3.91(m,2H),3.25(br,1H),2.60-2.32(m,3H),2.23(t,1H),2.15-1.95(m,3H),1.70-1.56(m,2H),1.41-1.17(m,9H),1.03(s,1H),0.95-0.80(m,2H)。
实施例2-7
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-羟基环丁烷-1-甲酰胺7
Figure PCTCN2021082857-appb-000033
向1b(3.0mg，5.64μmol)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1-羟基环丁烷甲酸7a(2.0mg，17.22μmol，供应商药石)，1-羟基苯并三唑(2.3mg，17.0μmol)和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(3.2mg，16.7μmol)，加毕，在0-5℃搅拌反应10分钟。撤去冰水浴，常温搅拌2小时。反应液减压浓缩，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7(2.5mg，产率：83.1％)。
MS m/z(ESI):534.0[M+1]。
1H NMR(400MHz,DMSO-d 6):δ8.28(d,1H),7.75(d,1H),7.29(s,1H),6.51(s,1H),6.12(s,1H),5.59-5.51(m,1H),5.41(s,2H),5.20-5.01(m,2H),3.27-3.17(m,1H),3.15-3.05(m,1H),2.71-2.63(m,1H),2.37(s,3H),2.12-2.05(m,1H),2.03-1.94(m,2H),1.92-1.78(m,4H),1.50-1.42(m,1H),0.90-0.83(m,4H)。
实施例2-8
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8
Figure PCTCN2021082857-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8c
将1-羟基环丙烷-1-羧酸苄酯8a(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8b(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8c(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8d
将8c(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8d(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8e
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8d(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8e(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8f
将8e(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8f(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8
将粗品8f(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8g(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8(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2-9
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9-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9-B
Figure PCTCN2021082857-appb-000035
第一步
2-环丙基-2-羟基乙酸苄酯9a
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9a(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9b
将9a(120.9mg,0.586mmol)和8b(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9b(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9c
将9b(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9c(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9d
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9c(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，有机相用无水硫酸钠干燥，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9d(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9e
将9d(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。往残余物中加入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9e(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9-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9-B
将粗品9e(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8g(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9。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 5μm 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(9-A：2.4mg，9-B：1.7mg)。
MS m/z(ESI):1074.4[M+1]。
单一构型化合物9-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 BEHC181.7um 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,1H),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,1H),2.80-2.62(m,1H),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9-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 BEHC181.7um 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,3H),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,2H),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,4H),0.91-0.79(m,3H),0.53-0.34(m,4H)。
实施例3.ADC的制备
ADC药物载量分析
实验目的及原理
采用紫外分光光度法(UV-Vis)测定ADC载量。仪器：Thermo nanodrop2000紫外分光光度计。其原理是在某波长下ADC的总吸光值等于药物与单克隆抗体在该波长下吸光值的加和。
实验方法
将装有琥珀酸钠缓冲液的比色皿分别置于参比吸收池和样品测定吸收池中后，扣除溶剂空白后，再将装有供试品溶液的比色皿置于样品测定吸收池中，测定280nm和370nm处吸光度。
结果计算：
(1)A 280nm＝ε mab-280bC mab+ε Drug-280bC Drug
ε Drug-280：药物在280nm平均摩尔消光系数5100；
C Drug：药物的浓度；
ε mab-280：单抗在280nm平均摩尔消光系数214600；
C mab：单抗的浓度；
b：光程长度为1cm。
同理可以得到样品在370nm下的总吸光值方程：
(2)A 370nm＝ε mab-370bC mab+ε Drug-370bC Drug
ε Drug-370：药物在370nm平均摩尔消光系数19000；
C Drug：药物的浓度；
ε mab-370：单抗在370nm消光系数为0；
C mab：单抗的浓度；
b：光程长度为1cm。
由⑴和⑵两种方程结合单克隆抗体和药物在两个检测波长下的消光系数和浓度数据可以计算出ADC中药物的载量。
药物载量＝C Drug/C mab。
不同DAR值的PSMA抗体-药物偶联物PM-9-A制备实施例
以下实施例为本公开相关ADC的制备过程。其中实施例3-4、实施例3-5、实施例3-7、实施例3-11中抗体PM通过半胱氨酸上的巯基偶联带有连接单元的药物9-A反应，制备抗体药物偶联PM-9-A(DAR＝约3-4)；实施例3-1至实施例3-3和实施例3-6中抗体PM通过半胱氨酸上的巯基偶联带有连接单元的药物9-A反应，制备PSMA ADC分子PM-9-A(DAR＝约6-7)；实施例3-8至实施例3-10通过抗体PM半胱氨酸上的巯基,与带有连接单元的药物VcMMAE(瀚香生物科技，CAS646502-53-6)反应制备偶联物分子PM-VcMMAE为对照。
通过对抗体和药物比例，反应量规模，以及其它条件的调整，可以获得不同DAR值(n)的抗体药物偶联物，优选DAR值为1-8，更优先为3-8，最优选3-7。
(一)不同DAR值的抗体药物偶联PM-9-A的制备
Figure PCTCN2021082857-appb-000036
实施例3-1 ADC-1
在37℃条件下，向抗体PM的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，0.27mL，18nmol)加入配置好的三(2-羧乙基)膦(TCEP)的水溶液(10mM，9.8μL，98nmol)，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℃。
将化合物9-A(0.3mg，277nmol)溶解于20μL二甲亚砜中，加入到上述反应液中，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)，得到如式PM-9-A所示偶联物的示例性产物ADC-1的PBS缓冲液(0.18mg/mL，11mL)，于4℃储存。
UV-Vis计算平均值：n＝6.31。
实施例3-2 ADC-2
在37℃条件下，向抗体PM的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，3.6mL，243nmol)加入配置好的三(2-羧乙基)膦(TCEP)的水溶液(10mM，128.9μL，1289nmol)，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℃。
将化合物9-A(3.93mg，3649nmol)溶解于200μL二甲亚砜中，加入到上述反应液中，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)，得到如式PM-9-A所示偶联物的示例性产物ADC-2的PBS缓冲液(1.93mg/mL，15.4mL)，于4℃储存。
UV-Vis计算平均值：n＝6.63。
实施例3-3 ADC-3
在37℃条件下，向抗体PM的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，10mL，676nmol)加入配置好的三(2-羧乙基)膦(TCEP)的水溶液(10mM，358.1μL，3581nmol)，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℃。
将化合物9-A(10.91mg，10135nmol)溶解于480μL二甲亚砜中，加入到上述反应液中，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)，得到如式PM-9-A所示偶联物的示例性产物ADC-3的PBS缓冲液(3.47mg/mL，25mL)，于4℃储存。
UV-Vis计算平均值：n＝6.9。
实施例3-4 ADC-4
在37℃条件下，向抗体PM的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，0.21mL，14nmol)加入配置好的三(2-羧乙基)膦(TCEP)的水溶液(10mM，3.5μL，35nmol)，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℃。
将化合物9-A(0.15mg，139nmol)溶解于20μL二甲亚砜中，加入到上述反应液中，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)，得到如式PM-9-A所示偶联物的示例性产物ADC-4的PBS缓冲液(0.28mg/mL，4.6mL)，于4℃储存。
UV-Vis计算平均值：n＝3.68。
实施例3-5 ADC-5
在37℃条件下，向抗体PM的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，5.23mL，353nmol)加入配置好的三(2-羧乙基)膦(TCEP)的水溶液(10mM，84.8μL，848nmol)，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℃。
将化合物9-A(3.8mg，3534nmol)溶解于260μL二甲亚砜中，加入到上述反应液中，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)，得到如式PM-9-A所示偶联物的示例性产物ADC-5的PBS缓冲液(2.48mg/mL，18.2mL)，于4℃储存。
UV-Vis计算平均值：n＝3.89。
实施例3-6 ADC-6
在37℃条件下，向抗体PM的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，3.5mL，236nmol)加入配置好的三(2-羧乙基)膦(TCEP)的水溶液(10mM，125.3μL，1253nmol)，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℃。
将化合物9-A(3.82mg，3547nmol)溶解于150μL二甲亚砜中，加入到上述反应液中，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)，得到如式PM-9-A所示偶联物的示例性产物ADC-6的PBS缓冲液(1.83mg/mL，14mL)，于4℃储存。
UV-Vis计算平均值：n＝6.61。
实施例3-7 ADC-7
在37℃条件下，向抗体PM的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，14.68mL，992nmol)加入配置好的三(2-羧乙基)膦(TCEP)的水溶液(10mM，238.1μL，2381nmol)，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℃。
将化合物9-A(10.67mg，9919nmol)溶解于420μl二甲亚砜中，加入到上述反应液中，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)，得到如式PM-9-A所示偶联物的示例性产物ADC-7的PBS缓冲液(3.61mg/mL，37mL)，于4℃储存。
UV-Vis计算平均值：n＝3.93。
(二)对照抗体药物偶联PM-VcMMAE的制备(参考专利WO2007002222A2)
Figure PCTCN2021082857-appb-000037
实施例3-8 ADC-8
在37℃条件下，向抗体PM的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，2.5mL，169nmol)加入配置好的三(2-羧乙基)膦(TCEP)的水溶液(10mM，42.2μL，422nmol)，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℃。
将化合物VcMMAE(2.22mg，1689nmol)溶解于100μL二甲亚砜中，加入到上述反应液中，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)，得到如式PM-VcMMAE所示偶联物的示例性产物ADC-8的PBS缓冲液(1.76mg/mL，12mL)，于4℃储存。
CE-SDS计算平均值：n＝4.47。
实施例3-9 ADC-9
在37℃条件下，向抗体PM的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，3.3mL，223nmol)加入配置好的三(2-羧乙基)膦(TCEP)的水溶液(10mM，55.7μL，557nmol)，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℃。
将化合物VcMMAE(2.93mg，2230nmol)溶解于200μL二甲亚砜中，加入到上述反应液中，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)，得到如式PM-VcMMAE所示偶联物的示例性产物ADC-9的PBS缓冲液(2.05mg/mL，17mL)，于4℃储存。
CE-SDS计算平均值：n＝4.23。
实施例3-10 ADC-10
在37℃条件下，向抗体PM的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，2.5mL，169nmol)加入配置好的三(2-羧乙基)膦(TCEP)的水溶液(10mM，42.2μL，422nmol)，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℃。
将化合物VcMMAE(2.22mg，1689nmol)溶解于150μL二甲亚砜中，加入到上述反应液中，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)，得到如式PM-VcMMAE所示偶联物的示例性产物ADC-10的PBS缓冲液(2.2mg/mL，13mL)，于4℃储存。
CE-SDS计算平均值：n＝3.92。
实施例3-11 ADC-11
在37℃条件下，向抗体PM的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，2.4mL，160nmol)加入配置好的三(2-羧乙基)膦(TCEP)的水溶液(10mM，42.2μL，422nmol)，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℃。
将化合物9-A(1.75mg，1629nmol)溶解于100μL二甲亚砜中，加入到上述反应液中，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)，得到如式PM-9-A所示偶联物的示例性产物ADC-11的PBS缓冲液(1.34mg/mL，15.5mL)，于4℃储存。
UV-Vis计算平均值：n＝4.19。
实施例3-12 ADC-12
Figure PCTCN2021082857-appb-000038
在37℃条件下，向抗体PM的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，2.4mL，160nmol)加入配置好的三(2-羧乙基)膦(TCEP)的水溶液(10mM，42.2μL，422nmol)，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℃。
将化合物58(参照专利“CN104755494A中第163页的实施例58制备，1.68mg，1624nmol)溶解于100μL二甲亚砜中，加入到上述反应液中，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)，得到如式PM-58所示偶联物的示例性产物ADC-12的PBS缓冲液(1.45mg/mL，14.8mL)，于4℃储存。
UV-Vis计算平均值：n＝4.16。
(三)对照抗体药物偶联Lmab-9-A的制备
Figure PCTCN2021082857-appb-000039
在37℃条件下，向抗体Lmab的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，2.15mL，145nmol)加入配置好的三(2-羧乙基)膦(TCEP)的水溶液(10mM，74.1μL，741nmol)，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℃。
将化合物9-A(3.20mg，2179nmol)溶解于155μL二甲亚砜中，加入到上述反应液中，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)，得到标题产物Lmab-9-A的PBS缓冲液(0.99mg/mL，15mL)，于4℃储存。
UV-Vis计算平均值：n＝7.07。
以下用生化测试方法验证本公开的抗体的活性
测试例1：体外细胞结合实验
本实验通过检测细胞表面抗体的荧光信号，根据荧光信号强弱来评价抗体的结合。制备好的ADC-2、ADC-10、对照ADC Lmab-9-A和抗体PM一起被用于进行 体外结合检测。
将梯度稀释的ADC-2、ADC-10和抗体PM与1×10 5个细胞(ATCC，MDA PCa 2b/CRL-2422，LNCaP/CRL-1740，22Rv1/CRL-2505，PC-3/CRL-1435，DU 145/HTB-81)在4℃孵育60分钟后洗掉多余的ADC或抗体。将细胞与FITC标记的山羊抗人IgG(H+L)二抗(Jackson Immuno Research，109-095-003)在4℃孵育30分钟，洗掉多余抗体后，使用BD CantoII读取细胞表面的荧光信号，(结果如表2，图1A，图1B，图1C，图1D，图1E所示)。
表2体外细胞结合活性(EC 50，nM)
  MDAPCa 2b LNCaP 22RV1 PC-3 DU145
抗体PM 1.079 1.421 0.6761 不结合 不结合
ADC-2 0.7183 0.9381 0.5714 不结合 不结合
ADC-10 0.9537 1.581 0.6894 不结合 不结合
Lmab-9-A 不结合 不结合 不结合 不结合 不结合
检测用细胞的抗原PSMA的表达水平：MDA PCa 2b>LNCaP>22Rv1，PC-3和DU 145不表达PSMA。
结果表明：ADC-2、ADC-10、对照ADCLmab-9-A和抗体PM与不同PSMA抗原表达水平的细胞具有相应强或弱的结合能力。EC 50越小代表结合能力越强。
测试例2：体外细胞内吞实验
DT3C，70kd，是重组表达的融合蛋白，由白喉毒素的Fragment A(仅毒素部分)和G群链球菌的3C片段(IgG结合部分)融合而成，该蛋白能够与抗体的IgG部分高度亲和，在抗体发生内吞时一同进入细胞，在胞内弗林蛋白酶的作用下，释放出具有毒性的DT，DT能够抑制EF2-ADP核糖基化的活性，阻断蛋白翻译过程，最终导致细胞死亡。而未进入细胞的DT3C则不具有杀伤细胞的活性。根据细胞杀伤情况来评价抗体的内吞活性。
将无菌过滤的DT3C(70KD,SB HRS3A,MEI3AU0803)和抗体PM抗体(DT3C摩尔浓度为抗体摩尔浓度的6倍)按照1:1的体积混匀，于室温下静置孵育30分钟后用无血清培养基梯度稀释，加入提前一天准备的用含20％low IgG FBS的培养基培养的细胞(ATCC，LNCaP/CRL-1740，22Rv1/CRL-2505)中(2000个细胞/孔)，5％二氧化碳培养箱中37℃孵育三天。加入CellTiter-Glo(Promega，G7573)，室温下避光孵育10分钟，Victor3上读取化学发光(结果如表3，图2A，图2B所示)。
表3抗体在不同细胞的内吞活性(IC 50，nM)
  LNCaP 22RV1
抗体PM+DT3C ～4.641e+012 0.08563
hIgG1对照+DT3C 不内吞 不内吞
仅DT3C 不内吞 不内吞
结果表明：抗体PM在阳性表达PSMA抗原的细胞上具备内吞能力。
测试例3：细胞增殖抑制实验
本实验通过检测细胞内ATP含量，根据IC 50大小评价PSMA ADC对细胞(ATCC，LNCaP/CRL-1740，22Rv1/CRL-2505，PC-3/CRL-1435)增殖的抑制效果。
待检测样品：ADC-2，ADC-4，对照ADC Lmab-9-A
1、对LNCaP细胞增殖的抑制效果
LNCaP细胞培养在含10％FBS的RPMI-1640培养基中，一周传代2～3次，传代比列1:3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。在96孔细胞培养板中加入180μL的细胞悬液，2×10 3细胞/孔，培养基为4.5％FBS的RPMI-1640，96孔板外围只加入培养基。将培养板在培养箱培养24小时(37℃，5％CO 2)。
将待测ADC(ADC-2，ADC-4，Lmab-9-A)配成起始浓度，用PBS 1：6梯度稀释9个点，另加0浓度点。取20μL加入到上述细胞板中，在培养箱中孵育5天(37℃，5％CO 2)。对于化合物2-B(即9-A的游离毒素)，先用DSMO稀释成母液并用PBS配成200μM为起始，再用PBS 1:6梯度稀释9个点，另加0浓度点，取1μL加入20μL RPMI-1640中并转至96孔细胞培养板中，在培养箱中孵育5天(37℃，5％CO 2)。在96孔细胞培养板中，每孔加入80μL CellTiter-Glo试剂，室温避光放置10-15分钟，在Victor3中读取化学发光信号值，数据使用GraphPad软件处理。(结果如表4，图3A，图3B所示)。
2、对22Rv1细胞增殖的抑制效果
22Rv1细胞培养在含10％FBS的RPMI-1640培养基中，一周传代2次，传代比列1:3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。在96孔细胞培养板中加入180μL的细胞悬液，4×10 3细胞/孔，培养基为4.5％FBS的RPMI-1640，96孔板外围只加入培养基。将培养板在培养箱培养24小时(37℃，5％CO 2)。待测ADC及化合物2-B配制及实验方法同上。(结果如表4，图3C，图3D所示)。
3、对PC-3细胞增殖的抑制效果
PC-3细胞培养在含10％FBS的F-12K培养基中，一周传代2-3次，传代比列1:3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。在96孔细胞培养板中加入180μL的细胞悬液，4×10 3细胞/孔，培养基为4.5％FBS的F-12K，96孔板外围只加入培养基。将培养板在培养箱培养24小时(37℃，5％CO 2)。待 测ADC及化合物2-B配制及实验方法同上。(结果如表4，图3E，图3F所示)。
表4对细胞杀伤作用(IC 50，nM)
  化合物2-B ADC-2 ADC-4 Lmab-9-A
LNCaP 1.695 0.7988 4.67 ～7.751e+006
22RV1 2.843 1.238 78.34 ～3.509e+007
PC-3 8.328 ～1.256e+006 ～3.140e+006 ～529.9
结论：PSMA ADC在LNCaP和22RV1细胞上具有杀伤作用。化合物2-B(即9-A的游离毒素)具有透膜杀伤作用。
测试例4：ADC药物对人前列腺癌细胞22Rv1裸小鼠移植瘤的疗效评价
一、测试方法
实验用nu/nu裸小鼠，雄性，6-8周，购自北京维通利华实验动物技术有限公司(合格证编号1908120082)。饲养环境：SPF级。裸小鼠皮下接种人前列腺癌细胞22Rv1(中科院)，当肿瘤平均体积达到220mm 3时，将动物随机分组(D0)，每组6只，开始腹腔注射给药2次/周，共给药5次，每周2次检测瘤体积和体重，记录数据。
肿瘤体积V＝1/2×a×b 2，其中a、b分别表示长、宽。
相对肿瘤增殖率T/C(％)＝(T-T0)/(C-C0)×100其中T、C为实验结束时治疗组和对照组的肿瘤体积；T0、C0为实验开始时的肿瘤体积。
抑瘤率TGI(％)＝1-T/C(％)。
二、测试对象
ADC-10：3mpk,10mpk；
ADC-2：3mpk,10mpk；
空白对照组：pH7.4的PBS缓冲液。
三、抗体ADC的抑瘤效果
观察至给药开始后第17天(D17)时，阳性ADC-10的10mpk和3mpk的抑瘤率分别为48.9％和10.0％，ADC-2的10mpk和3mpk的抑瘤率分别为>100％和91.5％，显著优于空白对照组和阳性ADC组，且呈现良好的剂量依赖关系(表5，图4A)。
给药过程中小鼠体重平稳，提示ADC-2各给药剂量没有明显的毒副作用(图4B)。
表5给药ADC对荷瘤裸鼠22Rv1移植瘤的疗效
Figure PCTCN2021082857-appb-000040
Figure PCTCN2021082857-appb-000041
vs空白对照组：*p<0.05，***p<0.001；vs ADC-10相应剂量组：###p<0.001
测试例5：ADC药物对人前列腺癌细胞22Rv1裸小鼠移植瘤的疗效评价
一、测试方法
实验用nu/nu裸小鼠，雄性，6-8周，购自北京维通利华实验动物技术有限公司(合格证编号1908120082)。饲养环境：SPF级。裸小鼠皮下接种人前列腺癌细胞22Rv1(中科院)，当肿瘤平均体积达到210mm 3时，将动物随机分组(D0)，每组8只，开始腹腔注射给药2次/周，共给药6次，每周2次检测瘤体积和体重，记录数据。
肿瘤体积V＝1/2×a×b 2，其中a、b分别表示长、宽。
相对肿瘤增殖率T/C(％)＝(T-T0)/(C-C0)×100其中T、C为实验结束时治疗组和对照组的肿瘤体积；T0、C0为实验开始时的肿瘤体积。
抑瘤率TGI(％)＝1-T/C(％)。
二、测试对象
ADC-8：10mpk；
ADC-6：3mpk,6mpk；
ADC-7：3mpk,6mpk,10mpk；
空白对照组：pH7.4的PBS缓冲液。
三、抗体ADC的抑瘤效果
观察至给药开始后第14天(D14)时，阳性ADC-8的10mpk抑瘤率为81.6％，受试ADC-6的6mpk和3mpk的抑瘤率分别为>100％和97.8％，另一个受试ADC-7的10mpk、6mpk和3mpk的抑瘤率分别为>100％、88.4％和80.5％，所有给药组均显著优于对照组，受试ADC-6在同等剂量下显著优于受试ADC-7，2个受试ADC均呈现良好的剂量依赖关系(表6，图5A)。
给药过程中小鼠体重平稳，提示各受试抗体各给药剂量没有明显的毒副作用(图5B)。
表6给药ADC对荷瘤裸鼠22Rv1移植瘤的疗效
Figure PCTCN2021082857-appb-000042
Figure PCTCN2021082857-appb-000043
vs对照组：***p<0.001；vs ADC-6相应剂量组：##p<0.01，###p<0.001
测试例6：ADC药物对人前列腺癌细胞LNCap在SCID Beighe小鼠上的移植瘤的疗效评价
一、测试方法
实验用SCID Beige小鼠，雄性，6-8周，购自北京维通利华实验动物技术有限公司(合格证编号1908120082)。饲养环境：SPF级。小鼠皮下接种人前列腺癌细胞LNCap(ATCC)，当肿瘤平均体积达到160mm 3时，将动物随机分组(D0)，每组7只，于D0和D4腹腔注射给药2次，每周2次检测瘤体积和体重，记录数据。
肿瘤体积V＝1/2×a×b 2，其中a、b分别表示长、宽。
相对肿瘤增殖率T/C(％)＝(T-T0)/(C-C0)×100其中T、C为实验结束时治疗组和对照组的肿瘤体积；T0、C0为实验开始时的肿瘤体积。
抑瘤率TGI(％)＝1-T/C(％)。
二、测试对象
ADC-9：10mpk；
ADC-3：3mpk,10mpk；
ADC-5：3mpk,6mpk,10mpk；
空白对照组：pH7.4的PBS缓冲液。
三、抗体ADC的抑瘤效果
所有给药组肿瘤在D0和D4两次给药后即开始消退，且其药效一直维持到实验终点D18。受试ADC-3和ADC-5均呈现出一定的剂量依赖关系，但它们之间并没有显著性差异(表7，图6A)。
给药过程中小鼠体重平稳，提示各受试抗体各给药剂量没有明显的毒副作用(图6B)。
表7给药ADC对荷瘤SCID Beige鼠LNCap移植瘤的疗效
Figure PCTCN2021082857-appb-000044
vs对照组：***p<0.001
测试例7：PMSA ADC SD大鼠T1/2评价
SD雄性大鼠9只，分为三组，每组3只，12/12小时光/暗调节，温度20-26℃恒温，湿度40-70％，自由进食饮水。购自浙江维通利华实验动物有限公司。实验当天对SD雄性大鼠分别尾静脉注射受试药物ADC-2，ADC-5，ADC-10，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小时、24小时(第2天)、第3天、第5天、第8天、第11天、第15天、第22天和第29天，于大鼠眼底静脉取血，每次300μL(相当于取血清150μL)；收集的血样在室温下置放半小时至凝集，然后4℃下1000×g离心15分钟，将上清(血清)转移至EP管中，立即放置-80℃贮存。采用ELISA法检测SD大鼠血清中PSMA抗体ADC的浓度。
测试方法：
总抗体(Total antibody)的检测：
1.在ELISA板(cornning)中每孔加入100μL，1μg/mL羊抗人lgG FC(abcam)，4℃隔夜孵育。
2.用洗板液PBST冲洗三遍(1×PBS,0.5％tween-20(生工)。
3.每孔加入200μL封闭液(5％牛奶，碧云天)，37℃孵育1-3小时。
4.用洗板液PBST冲洗三遍。
5.加入100μL的标准样品，质控样品和检测样品，37℃孵育1-3小时。
6.用洗板液冲洗三遍。
7.加入100μL鼠预吸附过的羊抗人lgG轻/重链HRP(1:5000，abcam)，37℃孵育1-1.5小时。
8.用洗板液冲洗三遍。
9.加入100μL TMB(KPL)，常温避光孵育10分钟。
10.加入100μL 1M稀硫酸(国药集团)终止，在450nm波长下读板(molecular  device,flexstation 3)。
完整ADC(Intact ADC)的检测：
1.在ELISA板中每孔加入100μL，1μg/mL羊抗鼠IgG Fc，4℃隔夜孵育。
2.用洗板液PBST冲洗三遍。
3.每孔加入200μL封闭液，37℃孵育1-3小时。
4.用洗板液冲洗三遍。
5.每孔加入100μL 0.2μg/mL抗毒素抗体或anti-pab-mmae(中科英沐，s-497-8)，37℃孵育1-1.5小时。
6.用洗板液冲洗三遍。
7.加入100μL的标准样品，质控样品和检测样品，37℃孵育1-3小时。
8.用洗板液冲洗三遍。
9.加入100μL鼠预吸附过的羊抗人lgG轻/重链HRP(1:5000)，37℃孵育1-1.5小时。
10.用洗板液冲洗三遍。
11.加入100μL TMB，常温避光孵育10分钟。
12.加入100μL 1M稀硫酸终止，在450nm波长下读板。
检测及分析结果：
用ELISA检测血清中的PSMA抗体ADC的浓度，进行PK分析，(结果见表8)。
表8 PSMA抗体偶联物在SD大鼠中的T 1/2
Figure PCTCN2021082857-appb-000045
结果表明，大鼠静脉给予3mg/kg受试抗体偶联物ADC-10，ADC-2，ADC-5后，总抗体(ADC中偶联的抗体和血清中游离的抗体)在大鼠体内的半衰期约为219.8小时(9.2天)，171.2小时(7.1天)，172.5小时(7.2天)；完整ADC在大鼠体内的半衰期约为76.7小时(3.2天)，153.9小时(6.4天)，137.9小时(5.7天)。ADC-2和ADC-5的T 1/2优于ADC-10。
测试例8：PSMA ADC的稳定性研究(游离毒素检测)
1.大鼠药代样品中ADC-2稳定性
运用液质联用技术，监测样品经静脉给药，在雄性大鼠体内，28天中化合物2-B(9-A的游离毒素)随时间的释放量，用以反映样品在定浓度条件下的稳定性。通过对28天中，不同时间点(5分钟，8小时，1天，2天，4天，7天，10天，14天，21天，28天)的大鼠血浆中化合物2-B的含量测定，ADC-2表现了良好的稳定性，结果如表9所示。
结果显示，ADC-2在雄性大鼠体内28天之内均显示出良好的稳定性。
表9 PSMA ADC的稳定性研究
Figure PCTCN2021082857-appb-000046
Blq表示：1ng/mL；M表示大鼠性别为雄性。
2.PSMA ADC血浆稳定性研究
运用液质联用技术，监测样品ADC-2(100μg/mL)分别在(人、猴、狗、大鼠、小鼠五个种属的血浆(肝素抗凝)以及1％BSA-PBS作为对照)中，分别在(37℃&5％CO 2孵育箱中，放置0天，7天，14天，21天)不同时间内化合物2-B随时间的释放量，用以反映样品在特定浓度(100μg/mL)条件下的稳定性。通过对不同时间点中化合物2-B的含量测定，ADC-2表现了良好的稳定性，结果如图7和表10所示。
结果显示，ADC-2在不同种属血浆中，在一定温度、一定时间内显示出良好的稳定性。
注：试验操作是在无菌实验室中进行的，空白血浆用0.22μm的微孔滤膜过滤除菌。
表10化合物2-B的含量(ng/mL)
  BSA 大鼠 小鼠 人 狗 猴
0d-1 BLQ BLQ BLQ BLQ BLQ BLQ
0d-2 BLQ BLQ BLQ BLQ BLQ BLQ
7d-1 1.83 10.7 11.7 2.15 20.2 3.67
7d-2 BLQ 9.52 12.9 3.51 18.2 3.95
14d-1 1.46 9.60 15.1 2.19 21.7 3.12
14d-2 3.74 9.61 19.2 3.92 23.0 3.59
21d-1 3.51 19.8 18.3 5.78 26.0 6.54
21d-2 4.15 17.3 16.9 4.71 27.7 5.61
化合物2-B线性范围1～5000ng/mL；BLQ表示：1ng/mL。“-1”和“-2”表示重复实验的第一次和第二次。
3.PSMA ADC血浆稳定性研究
运用液质联用技术，监测样品ADC-5分别在(人、猴、狗、大鼠、小鼠五个种属的血浆(肝素抗凝)以及1％BSA-PBS作为对照)中，分别在(37℃&5％CO 2孵育箱中，放置0天，7天，14天，23天)不同时间内化合物2-B随时间的掉落的量，用以反映样品在特定浓度(100μg/mL)条件下的稳定性。通过对不同时间点中化合物2-B的含量测定，ADC-5表现了良好的稳定性，结果如表11、图8所示。
结果显示，ADC-5在不同种属血浆，一定温度，一定时间内显示出良好的稳定性。
注：试验操作是在无菌实验室中进行的，空白血浆用0.22μm的微孔滤膜过滤除菌。
表11化合物2-B的含量(ng/mL)–ADC-5(100μg/mL)在不同血浆中37℃孵育23天
  BSA 大鼠 小鼠 人 狗 猴
0d-1 BLQ BLQ BLQ BLQ BLQ BLQ
0d-2 BLQ BLQ BLQ BLQ BLQ BLQ
7d-1 BLQ 3.74 4.69 2.46 7.14 BLQ
7d-2 BLQ 3.82 3.88 BLQ 5.89 BLQ
14d-1 BLQ 10.1 9.32 BLQ 14.2 5.09
14d-2 BLQ 10.0 10.6 2.00 13.8 6.34
23d-1 3.32 14.9 16.5 8.57 23.1 9.11
23d仅为单孔，化合物2-B线性范围2～5000ng/mL；BLQ表示：1ng/mL。
测试例9：ADC药物对人前列腺癌细胞22Rv1裸小鼠移植瘤的疗效评价
一、测试方法
实验用雄性nu/nu裸小鼠，6-8周龄，购自北京维通利华实验动物技术有限公 司(合格证编号1908120082)。饲养环境：SPF级。裸小鼠皮下接种人前列腺癌细胞22Rv1(中科院)，当肿瘤平均体积达到190mm 3时，将动物随机分组(D0)，每组8只，开始腹腔注射给药，2次/周，共给药4次，每周2次检测瘤体积和体重，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。
相对肿瘤增殖率T/C(％)＝(T-T 0)/(C-C 0)×100，其中T、C为实验结束时治疗组和对照组的肿瘤体积；T 0、C 0为实验开始时的肿瘤体积。
抑瘤率TGI(％)＝1-T/C(％)。
二、测试对象
ADC-11：5mpk
ADC-12：5mpk
空白对照组：pH7.4的PBS缓冲液
三、抗体ADC的抑瘤效果
观察至给药开始后第13天(D13)时，ADC-11和ADC-12在5mpk剂量下的抑瘤率分别为87.63％和79.82％，均显著优于对照组。ADC-11和ADC-12之间没有显著性差异(表12，图9)。
给药过程中小鼠体重平稳，提示各受试抗体没有明显的毒副作用。
表12给药ADC对荷瘤裸鼠22Rv1移植瘤的疗效
Figure PCTCN2021082857-appb-000047
vs空白对照组：**p<0.01,***p<0.001

Claims (20)

  1. 一种通式(Pc-L-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐：
    Figure PCTCN2021082857-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)-和-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、环烷基和杂环基；
    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、芳基和杂芳基；
    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、芳基和杂芳基；
    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，n是小数或整数；
    L为接头单元；
    Pc为抗PSMA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-D)所示的抗体-药物偶联物或其药学上可接受的盐，其中所述抗PSMA抗体或其抗原结合片段包含重链可变区和轻链可变区，其中：
    所述重链可变区包含分别如SEQ ID NO：3、SEQ ID NO：4和SEQ ID NO：5所示的HCDR1、HCDR2和HCDR3，所述轻链可变区包含分别如SEQ ID NO：6、SEQ ID NO：7和SEQ ID NO：8所示的LCDR1、LCDR2和LCDR3。
  3. 根据权利要求1或2中任一项所述的通式(Pc-L-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐，其中所述抗PSMA抗体是鼠源抗体、嵌合抗体、人 源化抗体或人抗体。
  4. 根据权利要求1至3中任一项所述的通式(Pc-L-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐，其中所述抗PSMA抗体或其抗原结合片段包含如SEQ ID NO：1所示的重链可变区和如SEQ ID NO：2所示的轻链可变区。
  5. 根据权利要求1至4中任一项所述的通式(Pc-L-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐，其中所述抗PSMA抗体包含抗体重链恒定区和轻链恒定区；优选地，所述重链恒定区选自人IgG1、IgG2、IgG3和IgG4的恒定区及其常规变体，所述轻链恒定区选自人抗体κ和λ链的恒定区及其常规变体；更优选地，所述抗PSMA抗体包含如SEQ ID NO：9所示的重链和如SEQ ID NO：10所示的轻链。
  6. 根据权利要求1至5中任一项所述的通式(Pc-L-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐，其中n为1至8，优选为3至8，n是小数或整数。
  7. 根据权利要求1至6中任一项所述的通式(Pc-L-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐，
    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、卤素和C 1-6烷基；
    R 1为卤代烷基或C 3-6环烷基；
    R 2选自氢原子、卤代烷基和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。
  8. 根据权利要求1至7中任一项所述的通式(Pc-L-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐，其中Y选自：
    Figure PCTCN2021082857-appb-100002
    其中Y的O端与接头单元L相连。
  9. 根据权利要求1至8中任一项所述的通式(Pc-L-Y-D)所示的抗体-药物偶 联物或其药学上可接受的盐，其中接头单元-L-为-L 1-L 2-L 3-L 4-，
    L 1选自-(琥珀酰亚胺-3-基-N)-W-C(O)-、-CH 2-C(O)-NR 3-W-C(O)-和-C(O)-W-C(O)-，其中W选自C 1-8烷基、C 1-8烷基-环烷基和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和S的杂原子，其中所述的C 1-8烷基、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)p 1CH 2CH 2C(O)-、-NR 4(CH 2CH 2O)p 1CH 2C(O)-、-S(CH 2)p 1C(O)-和化学键，其中p 1为1至20的整数；
    L 3为由2至7个氨基酸残基构成的肽残基，其中所述的氨基酸残基选自苯丙氨酸、甘氨酸、缬氨酸、赖氨酸、瓜氨酸、丝氨酸、谷氨酸和天冬氨酸中的氨基酸形成的氨基酸残基，并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、烷氧基和环烷基中的一个或多个取代基所取代；
    L 4选自-NR 5(CR 6R 7) t-、-C(O)NR 5、-C(O)NR 5(CH 2) t-和化学键，其中t为1至6的整数；
    R 3、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。
  10. 根据权利要求1至9中任一项所述的通式(Pc-L-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐，其中接头单元-L-为-L 1-L 2-L 3-L 4-，
    L 1为
    Figure PCTCN2021082857-appb-100003
    s 1为2至8的整数；
    L 2为化学键；
    L 3为四肽残基；优选地，L 3为GGFG的四肽残基；
    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；
    其中所述的L 1端与Pc相连，L 4端与Y相连。
  11. 根据权利要求1至10中任一项所述的通式(Pc-L-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐，其中-L-为：
    Figure PCTCN2021082857-appb-100004
  12. 根据权利要求1至11中任一项所述的通式(Pc-L-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐，其中-L-Y-任选自：
    Figure PCTCN2021082857-appb-100005
  13. 根据权利要求1至10中任一项所述的通式(Pc-L-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐，其为通式(Pc-L a-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐：
    Figure PCTCN2021082857-appb-100006
    其中，
    Pc为抗PSMA抗体或其抗原结合片段；
    m为0至4的整数；
    n为1至10，n是小数或整数；
    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、芳基和杂芳基；
    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、芳基和杂芳基；
    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；
    W选自C 1-8烷基、C 1-8烷基-环烷基和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和S的杂原子，其中所述的C 1-8烷基、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)p 1CH 2CH 2C(O)-、-NR 4(CH 2CH 2O)p 1CH 2C(O)-、-S(CH 2)p 1C(O)-和化学键，其中p 1为1至20的整数；
    L 3为由2至7个氨基酸残基构成的肽残基，其中所述的氨基酸残基选自苯丙氨酸、甘氨酸、缬氨酸、赖氨酸、瓜氨酸、丝氨酸、谷氨酸和天冬氨酸中的氨基酸形成的氨基酸残基，并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、烷氧基和环烷基中的一个或多个取代基所取代；
    R 5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。
  14. 根据权利要求1至10、13中任一项所述的通式(Pc-L-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐，其为通式(Pc-L b-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐：
    Figure PCTCN2021082857-appb-100007
    其中：
    s 1为2至8的整数；
    Pc、R 1、R 2、R 5、R 6和R 7、m和n如权利要求13中所定义。
  15. 根据权利要求1至14中任一项所述的通式(Pc-L-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐，所述抗体-药物偶联物选自：
    Figure PCTCN2021082857-appb-100008
    Figure PCTCN2021082857-appb-100009
    其中
    Pc为抗PSMA抗体或其抗原结合片段；
    n为1至10，n是小数或整数。
  16. 根据权利要求1至15中任一项所述的通式(Pc-L-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐，所述抗体-药物偶联物为：
    Figure PCTCN2021082857-appb-100010
    其中：
    n为1至8，优选为3至8，n是小数或整数；
    PM为抗PSMA抗体，其包含如SEQ ID NO：9所示的重链和如SEQ ID NO：10所示的轻链。
  17. 一种制备如通式(Pc-L a-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐的方法，其包括以下步骤：
    Figure PCTCN2021082857-appb-100011
    Pc’与通式(L a-Y-D)所示的化合物进行偶联反应，得到通式(Pc-L a-Y-D)所示的化合物；
    其中：
    Pc为抗PSMA抗体或其抗原结合片段；优选地，Pc为权利要求2至5中任一项所述的抗PSMA抗体或其抗原结合片段；Pc’为Pc经还原后所得；
    n、m、W、L 2、L 3、R 1、R 2、R 5、R 6和R 7如权利要求13中所定义。
  18. 一种药物组合物，其包含根据权利要求1至16中任一项所述的抗体-药物偶联物或其药学上可接受的盐，以及一种或多种药学上可接受的赋形剂、稀释剂或载体。
  19. 根据权利要求1至16中任一项所述的抗体-药物偶联物或其药学上可接受的盐、或根据权利要求18所述的药物组合物在制备用于治疗PSMA介导的疾病或病症的药物中的用途。
  20. 根据权利要求1至16中任一项所述的抗体-药物偶联物或其药学上可接受的盐、或根据权利要求18所述的药物组合物在制备用于治疗和/或预防肿瘤和癌症的药物中的用途，其中所述肿瘤和癌症优选头和颈鳞状细胞癌、头和颈癌、脑癌、神经胶质瘤、多形性成胶质细胞瘤、神经母细胞瘤、中枢神经系统癌、神经内分 泌肿瘤、咽喉癌、鼻咽癌、食管癌、甲状腺癌、恶性胸膜间皮瘤、肺癌、乳腺癌、肝癌、肝细胞瘤、肝胆癌、胰腺癌、胃癌、胃肠道癌、肠癌、结肠癌、结肠直肠癌、肾癌、透明细胞肾细胞癌、卵巢癌、子宫内膜癌、子宫颈癌、膀胱癌、前列腺癌、睾丸癌、皮肤癌、黑色素瘤、白血病、淋巴瘤、骨癌、软骨肉瘤、骨髓瘤、多发性骨髓瘤、骨髓异常增生综合征、库肯勃氏瘤、骨髓增生性肿瘤、鳞状细胞癌、尤因氏肉瘤、尿路上皮癌和梅克尔细胞癌；优选地，所述淋巴瘤选自：何杰金淋巴瘤、非何杰金淋巴瘤、弥漫性大B-细胞淋巴瘤、滤泡性淋巴瘤、原发性纵隔大B-细胞淋巴瘤、套细胞淋巴瘤、小淋巴细胞性淋巴瘤、富含T-细胞/组织细胞的大B-细胞淋巴瘤和淋巴浆细胞性淋巴瘤；所述肺癌选自：非小细胞肺癌和小细胞肺癌；所述白血病选自：慢性髓细胞样白血病、急性髓细胞样白血病、淋巴细胞白血病、成淋巴细胞性白血病、急性成淋巴细胞性白血病、慢性淋巴细胞性白血病和髓样细胞白血病。
PCT/CN2021/082857 2020-03-25 2021-03-25 抗psma抗体-依喜替康类似物偶联物及其医药用途 WO2021190583A1 (zh)

Priority Applications (10)

Application Number Priority Date Filing Date Title
BR112022019027A BR112022019027A2 (pt) 2020-03-25 2021-03-25 Conjugado de anticorpo anti-psma-análogo de exatecano e uso médico do mesmo
EP21775717.8A EP4130006A4 (en) 2020-03-25 2021-03-25 ANTI-PSMA ANTIBODY EXATECAN ANALOG CONJUGATE AND MEDICAL USE THEREOF
JP2022557789A JP2023519261A (ja) 2020-03-25 2021-03-25 抗ｐｓｍａ抗体－エキサテカン類似体複合体及びその医薬用途
KR1020227035634A KR20220160016A (ko) 2020-03-25 2021-03-25 항-psma 항체-엑사테칸 유사체 접합체 및 그의 의학적 용도
AU2021240756A AU2021240756A1 (en) 2020-03-25 2021-03-25 Anti-PSMA antibody-exatecan analogue conjugate and medical use thereof
CN202180022490.3A CN115298186A (zh) 2020-03-25 2021-03-25 抗psma抗体-依喜替康类似物偶联物及其医药用途
US17/914,209 US20230140397A1 (en) 2020-03-25 2021-03-25 Anti-psma antibody-exatecan analogue conjugate and medical use thereof
MX2022011808A MX2022011808A (es) 2020-03-25 2021-03-25 Conjugado de anticuerpo anti-psma-analogo de exatecan y uso medico del mismo.
CA3177279A CA3177279A1 (en) 2020-03-25 2021-03-25 Anti-psma antibody-exatecan analogue conjugate and medical use thereof
ZA2022/10724A ZA202210724B (en) 2020-03-25 2022-09-28 Anti-psma antibody-exatecan analogue conjugate and medical use thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010218297 2020-03-25
CN202010218297.4 2020-03-25

Publications (1)

Publication Number Publication Date
WO2021190583A1 true WO2021190583A1 (zh) 2021-09-30

Family

ID=77890968

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/082857 WO2021190583A1 (zh) 2020-03-25 2021-03-25 抗psma抗体-依喜替康类似物偶联物及其医药用途

Country Status (12)

Country Link
US (1) US20230140397A1 (zh)
EP (1) EP4130006A4 (zh)
JP (1) JP2023519261A (zh)
KR (1) KR20220160016A (zh)
CN (1) CN115298186A (zh)
AU (1) AU2021240756A1 (zh)
BR (1) BR112022019027A2 (zh)
CA (1) CA3177279A1 (zh)
MX (1) MX2022011808A (zh)
TW (1) TW202144016A (zh)
WO (1) WO2021190583A1 (zh)
ZA (1) ZA202210724B (zh)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11814394B2 (en) 2021-11-16 2023-11-14 Genequantum Healthcare (Suzhou) Co., Ltd. Exatecan derivatives, linker-payloads, and conjugates and thereof
US11999748B2 (en) 2021-11-16 2024-06-04 Genequantum Healthcare (Suzhou) Co., Ltd. Exatecan derivatives, linker-payloads, and conjugates and thereof

Citations (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5208020A (en) 1989-10-25 1993-05-04 Immunogen Inc. Cytotoxic agents comprising maytansinoids and their therapeutic use
EP0737686A1 (en) 1995-04-10 1996-10-16 Daiichi Pharmaceutical Co., Ltd. Camthothecin derivative with antitumour activity
WO2003034903A2 (en) 2001-10-23 2003-05-01 Psma Development Company, L.L.C. Psma antibodies and protein multimers
US20050020645A1 (en) 2001-06-20 2005-01-27 Daiichi Pharmaceutical Co., Ltd. Diamine derivatives
US20050238649A1 (en) 2003-11-06 2005-10-27 Seattle Genetics, Inc. Monomethylvaline compounds capable of conjugation to ligands
WO2007002222A2 (en) 2005-06-20 2007-01-04 Psma Development Company, Llc Psma antibody-drug conjugates
WO2013096771A1 (en) 2011-12-21 2013-06-27 Ardelyx, Inc. Non-systemic tgr5 agonists
WO2013106717A1 (en) 2012-01-13 2013-07-18 The General Hospital Corporation Anesthetic compounds and related methods of use
CN104755494A (zh) * 2012-10-11 2015-07-01 第一三共株式会社 抗体-药物偶联物
CN105829346A (zh) 2014-01-31 2016-08-03 第三共株式会社 抗her2抗体－药物偶联物
CN106999517A (zh) * 2014-10-07 2017-08-01 免疫医疗公司 抗体‑药物缀合物的新辅助剂用途
CN107735090A (zh) * 2012-12-13 2018-02-23 免疫医疗公司 具有cl2a接头的抗体‑sn‑38免疫缀合物
US10195175B2 (en) * 2015-06-25 2019-02-05 Immunomedics, Inc. Synergistic effect of anti-Trop-2 antibody-drug conjugate in combination therapy for triple-negative breast cancer when used with microtubule inhibitors or PARP inhibitors
WO2020063676A1 (zh) * 2018-09-26 2020-04-02 江苏恒瑞医药股份有限公司 依喜替康类似物的配体-药物偶联物及其制备方法和应用
CN112125915A (zh) * 2019-09-18 2020-12-25 四川百利药业有限责任公司 一种喜树碱衍生物及其偶联物

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015155976A1 (ja) * 2014-04-10 2015-10-15 第一三共株式会社 抗ｈｅｒ２抗体－薬物コンジュゲート

Patent Citations (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5208020A (en) 1989-10-25 1993-05-04 Immunogen Inc. Cytotoxic agents comprising maytansinoids and their therapeutic use
EP0737686A1 (en) 1995-04-10 1996-10-16 Daiichi Pharmaceutical Co., Ltd. Camthothecin derivative with antitumour activity
US20050020645A1 (en) 2001-06-20 2005-01-27 Daiichi Pharmaceutical Co., Ltd. Diamine derivatives
WO2003034903A2 (en) 2001-10-23 2003-05-01 Psma Development Company, L.L.C. Psma antibodies and protein multimers
US20050238649A1 (en) 2003-11-06 2005-10-27 Seattle Genetics, Inc. Monomethylvaline compounds capable of conjugation to ligands
WO2007002222A2 (en) 2005-06-20 2007-01-04 Psma Development Company, Llc Psma antibody-drug conjugates
WO2013096771A1 (en) 2011-12-21 2013-06-27 Ardelyx, Inc. Non-systemic tgr5 agonists
WO2013106717A1 (en) 2012-01-13 2013-07-18 The General Hospital Corporation Anesthetic compounds and related methods of use
CN104755494A (zh) * 2012-10-11 2015-07-01 第一三共株式会社 抗体-药物偶联物
EP2907824A1 (en) 2012-10-11 2015-08-19 Daiichi Sankyo Company, Limited Antibody-drug conjugate
CN107735090A (zh) * 2012-12-13 2018-02-23 免疫医疗公司 具有cl2a接头的抗体‑sn‑38免疫缀合物
CN105829346A (zh) 2014-01-31 2016-08-03 第三共株式会社 抗her2抗体－药物偶联物
CN106999517A (zh) * 2014-10-07 2017-08-01 免疫医疗公司 抗体‑药物缀合物的新辅助剂用途
US10195175B2 (en) * 2015-06-25 2019-02-05 Immunomedics, Inc. Synergistic effect of anti-Trop-2 antibody-drug conjugate in combination therapy for triple-negative breast cancer when used with microtubule inhibitors or PARP inhibitors
WO2020063676A1 (zh) * 2018-09-26 2020-04-02 江苏恒瑞医药股份有限公司 依喜替康类似物的配体-药物偶联物及其制备方法和应用
CN112125915A (zh) * 2019-09-18 2020-12-25 四川百利药业有限责任公司 一种喜树碱衍生物及其偶联物

Non-Patent Citations (24)

* Cited by examiner, † Cited by third party
Title
ALFTHAN ET AL., PROTEIN ENG, vol. 8, 1995, pages 725 - 731
AL-LAZIKANI ET AL., JMB, vol. 273, 1997, pages 927 - 948
BIRD ET AL., SCIENCE, vol. 242, 1988, pages 423 - 426
CHARI ET AL., CANCER RESEARCH, vol. 52, 1992, pages 127 - 131
CHOI ET AL., EUR. J. IMMUNOL., vol. 31, 2001, pages 94 - 106
CLIN CANCER RES., vol. 16, no. 22, 15 November 2010 (2010-11-15), pages 5414 - 5423
CLINICAL CANCER RESEARCH, vol. 3, January 1997 (1997-01-01), pages 81 - 85
CURR MED CHEM., vol. 19, no. 6, 2012, pages 856 - 870
CURRENT MEDICINAL CHEMISTRY, vol. 19, 2012, pages 1316 - 1322
HOLLIGER ET AL., PROC. NATL. ACAD. SCI. USA, vol. 90, 1993, pages 6444 - 6448
HU ET AL., CANCER RES., vol. 56, 1996, pages 3055 - 3061
HUSTON ET AL., PROC. NATL. ACAD. SCI USA, vol. 85, 1988, pages 5879 - 5883
J NUCL MED, vol. 58, 2017, pages 81 - 84
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 136, 2014, pages 8138 - 8142
LANDMARK, FRONTIERS IN BIOSCIENCE, vol. 24, 1 March 2019 (2019-03-01), pages 648 - 687
LEFRANC M. P., IMMUNOLOGIST, vol. 7, 1999, pages 132 - 136
LEFRANC, M. P. ET AL., DEV. COMP. IMMUNOL., vol. 27, 2003, pages 55 - 77
ROOVERS ET AL., CANCER IMMUNOL., 2001
See also references of EP4130006A4
TETRAHEDRON LETTERS, vol. 25, no. 12, 1984, pages 1269 - 72
THE EMBO JOURNAL, vol. 25, 2006, pages 1375 - 1384
TOSHINORI AGATSUMA: "Development of New ADC Technology with Topoisomerase I Inhibitor", THE PHARMACEUTICAL SOCIETY OF JAPAN, vol. 137, no. 5, 1 January 2017 (2017-01-01), pages 545 - 550, XP055698408, DOI: 10.1248/yakushi.16-00255-4 *
UROLOGIC ONCOLOGY: SEMINARS AND ORIGINAL INVESTIGATIONS, vol. 1, no. 1, pages 18 - 28
Y. OGITANI, T. AIDA, K. HAGIHARA, J. YAMAGUCHI, C. ISHII, N. HARADA, M. SOMA, H. OKAMOTO, M. OITATE, S. ARAKAWA, T. HIRAI, R. ATSU: "DS-8201a, Anovel HER2-Targeting ADC with a Novel DNA Topoisomerase I Inhibitor, Demonstrates a Promising Antitumor Efficacy with Differentiation from T-DM1", CLINICAL CANCER RESEARCH, vol. 22, no. 20, 29 March 2016 (2016-03-29), pages 5097 - 5108, XP055482007, ISSN: 1078-0432, DOI: 10.1158/1078-0432.CCR-15-2822 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11814394B2 (en) 2021-11-16 2023-11-14 Genequantum Healthcare (Suzhou) Co., Ltd. Exatecan derivatives, linker-payloads, and conjugates and thereof
EP4211145A4 (en) * 2021-11-16 2023-12-13 Genequantum Healthcare (Suzhou) Co., Ltd. EXATECAND DERIVATIVES, LINKER PAYLOADS AND CONJUGATES AND THEREOF
US11999748B2 (en) 2021-11-16 2024-06-04 Genequantum Healthcare (Suzhou) Co., Ltd. Exatecan derivatives, linker-payloads, and conjugates and thereof

Also Published As

Publication number Publication date
US20230140397A1 (en) 2023-05-04
CA3177279A1 (en) 2021-09-30
EP4130006A4 (en) 2024-01-17
ZA202210724B (en) 2023-12-20
TW202144016A (zh) 2021-12-01
AU2021240756A1 (en) 2022-11-17
EP4130006A1 (en) 2023-02-08
CN115298186A (zh) 2022-11-04
BR112022019027A2 (pt) 2022-11-01
KR20220160016A (ko) 2022-12-05
JP2023519261A (ja) 2023-05-10
MX2022011808A (es) 2022-12-06

Similar Documents

Publication Publication Date Title
JP7408646B2 (ja) 抗－ｂ７ｈ３抗体－エキサテカンアナログコンジュゲート及びその医薬用途
WO2021147993A1 (zh) 抗trop-2抗体-依喜替康类似物偶联物及其医药用途
WO2021148003A1 (zh) 艾日布林衍生物的药物偶联物、其制备方法及其在医药上的应用
WO2021115426A1 (zh) 抗密蛋白抗体药物偶联物及其医药用途
WO2022022508A1 (zh) 抗cd79b抗体药物偶联物、其制备方法及其医药用途
WO2021190586A1 (zh) B7h3抗体-依喜替康类似物偶联物及其医药用途
WO2021190583A1 (zh) 抗psma抗体-依喜替康类似物偶联物及其医药用途
CN113121639A (zh) 澳瑞他汀类似物及其偶联物、其制备方法及其应用
WO2021121204A1 (zh) 抗cea抗体-依喜替康类似物偶联物及其医药用途
CN113082224A (zh) 吡啶并嘧啶类衍生物偶联物、其制备方法及其在医药上的应用
WO2023001300A1 (zh) 艾日布林衍生物的药物偶联物
RU2785664C2 (ru) Конъюгат антитело к b7h3-аналог экзатекана и его применение в медицине

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21775717

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2022557789

Country of ref document: JP

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 3177279

Country of ref document: CA

REG Reference to national code

Ref country code: BR

Ref legal event code: B01A

Ref document number: 112022019027

Country of ref document: BR

ENP Entry into the national phase

Ref document number: 20227035634

Country of ref document: KR

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 2022125170

Country of ref document: RU

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 112022019027

Country of ref document: BR

Kind code of ref document: A2

Effective date: 20220922

ENP Entry into the national phase

Ref document number: 2021775717

Country of ref document: EP

Effective date: 20221025

ENP Entry into the national phase

Ref document number: 2021240756

Country of ref document: AU

Date of ref document: 20210325

Kind code of ref document: A