WO2020156513A1 - 双配体药物偶联体及其用途 - Google Patents

双配体药物偶联体及其用途 Download PDF

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WO2020156513A1
WO2020156513A1 PCT/CN2020/074117 CN2020074117W WO2020156513A1 WO 2020156513 A1 WO2020156513 A1 WO 2020156513A1 CN 2020074117 W CN2020074117 W CN 2020074117W WO 2020156513 A1 WO2020156513 A1 WO 2020156513A1
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Prior art keywords
conjugate compound
pharmaceutically acceptable
acceptable salt
cells
molecule
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PCT/CN2020/074117
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English (en)
French (fr)
Inventor
黄保华
谭巍
戴建
王中波
刘小栋
胡新礼
谢雪原
邵军
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同宜医药（苏州）有限公司
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Priority to KR1020217024254A priority Critical patent/KR20210119413A/ko
Priority to US17/426,293 priority patent/US20220175761A1/en
Priority to MX2021009199A priority patent/MX2021009199A/es
Priority to EP20748044.3A priority patent/EP3903825A4/en
Priority to CN202080011608.8A priority patent/CN113453720A/zh
Priority to BR112021015109A priority patent/BR112021015109A2/pt
Application filed by 同宜医药（苏州）有限公司 filed Critical 同宜医药（苏州）有限公司
Priority to AU2020214507A priority patent/AU2020214507A1/en
Priority to SG11202108182RA priority patent/SG11202108182RA/en
Priority to JP2021543527A priority patent/JP2022518924A/ja
Priority to CA3127903A priority patent/CA3127903A1/en
Publication of WO2020156513A1 publication Critical patent/WO2020156513A1/zh
Priority to IL285148A priority patent/IL285148A/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—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 organic compound
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • 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
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    • 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
    • A61K47/54—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 organic compound
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    • 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
    • A61K47/54—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 organic compound
    • A61K47/55—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 organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
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    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system

Definitions

  • This application relates to the field of biomedical chemistry. More specifically, the present application relates to a dual-ligand drug conjugate, a pharmaceutical composition including the dual-ligand drug conjugate, and a method for using the dual-ligand drug conjugate to deliver payload to a subject in need And use it to treat diseases.
  • ADC antibody-drug conjugates
  • PDC polypeptide-drug conjugates
  • ADC is mainly used in the field of tumor treatment.
  • the affinity of the targeting antibody to the surface antigen of cancer cells can be as high as 10 -9 ⁇ 10 -12 (Kd, mol/L), so while having high specificity for the target cell, the ADC pair Normal cells whose target cells have the same target receptor also have higher specificity.
  • ADC has a long metabolic time in the body (1 to 3 weeks), it will continue to kill normal cells during this period, which greatly increases the toxic side effects of ADC. Therefore, a more ideal indication for ADC should be a disease characterized by a very large difference in the amount of cell surface antigens between tumors and normal cells. However, very few diseases are currently known to meet this stringent requirement.
  • PDC is used in clinical or pre-clinical research to treat a variety of diseases, but these are simply connecting chemotherapeutic drugs to peptides, or adding peptides to nanoparticles or polymer materials embedded with chemotherapeutic drugs, which will cause most Polypeptides cannot enter cells due to their large molecular weight and charge. Therefore, most of these PDCs are currently only suitable for extracellular therapy, which severely limits the application range and efficacy of PDCs.
  • the drug conjugate compound can also be a ligand-drug conjugate (LDC), where the ligand can be a peptide or a small molecule.
  • LDC ligand-drug conjugate
  • problems in the application of LDC in terms of bioavailability, stability, efficacy and toxicity.
  • many ligands cannot enter cells due to their large molecular weight, lipophilicity or other properties, which limits their therapeutic applications.
  • the ligand is conjugated with conventional chemotherapeutics (for example, doxorubicin, paclitaxel, etc.), the efficacy is usually low, while if it is conjugated with high-efficiency drug molecules (for example, MMAE, DM1), the toxicity is greater , It may even lead to animal poisoning and death before reaching the therapeutically effective dose for tumor treatment.
  • conventional chemotherapeutics for example, doxorubicin, paclitaxel, etc.
  • high-efficiency drug molecules for example, MMAE, DM1
  • conjugate compound or a pharmaceutically acceptable salt thereof comprises a payload and two targeting molecules, wherein the two The target molecules are the synergistic molecule part and the prostate-specific membrane antigen ligand part.
  • conjugate compound or a pharmaceutically acceptable salt thereof comprises a payload and two targeting molecules, wherein the The two targeting molecules are respectively a synergistic molecule part and a ligand part represented by formula (I):
  • conjugate compound or a pharmaceutically acceptable salt thereof comprises a payload and two targeting molecules, wherein the The two targeting molecules are the synergistic molecular part and P10 respectively, and the payload is camptothecin and any derivatives thereof.
  • the prostate-specific membrane antigen ligand contained in the conjugate compound or a pharmaceutically acceptable salt thereof includes the following structure:
  • the prostate-specific membrane antigen ligand contained in the conjugate compound or a pharmaceutically acceptable salt thereof includes the following structure:
  • the prostate-specific membrane antigen ligand contained in the conjugate compound or a pharmaceutically acceptable salt thereof includes the following structure:
  • the prostate-specific membrane antigen ligand contained in the conjugate compound or a pharmaceutically acceptable salt thereof includes the following structure:
  • the two targeting molecules contained in the conjugate compound or a pharmaceutically acceptable salt thereof are different.
  • the synergistic molecule contained in the conjugate compound or a pharmaceutically acceptable salt thereof is a cell interaction molecule.
  • the conjugate compound or a pharmaceutically acceptable salt thereof contains two targeting molecules that interact with different cellular molecules.
  • the synergistic molecule contained in the conjugate compound or a pharmaceutically acceptable salt thereof is an endocytosis molecule capable of mediating endocytosis.
  • the synergistic molecule contained in the conjugate compound or a pharmaceutically acceptable salt thereof is combined with a molecule selected from the group consisting of FOLR1, TRPV6, FOLH1 (PMSA), GNRHR, Her2, Trop2, Her3, NECTIN4 , LRP1, GLUT1, EGFR1, AXL, CA9, CD44, Claudin18.2, APN, DLL3, CEACAM5, FZD10, TFRC, MET, IGFR1, SSTR2, CCKBR, LFA1, ICAM, GPR87, GM-CSF, GM-CSFR, TIM3 , TLR family, CD40, CD40L, OX40, OX40L, GITRL, GITR, 4-BBL, 4-1BB, CD70, CD27, ICOSL, ICOS, HHLA2, CD28, CD86/80, CD28, MHCII antigen, TCR, CTLA-4 , CD155, CD122, CD113, IGIT, PD-L
  • the conjugate compound or a pharmaceutically acceptable salt thereof comprises a prostate-specific membrane antigen ligand portion and a synergistic molecule portion that binds to a molecule selected from the group consisting of FOLR1, TRPV6, FOLH1 (PMSA), SSTR2 and GNRHR.
  • the conjugate compound or a pharmaceutically acceptable salt thereof comprises a ligand part represented by formula (I) and a synergistic molecule part, which is combined with a molecule selected from the following group: FOLR1, TRPV6, SSTR2 and GNRHR.
  • the conjugate compound or a pharmaceutically acceptable salt thereof comprises P10 and a synergistic molecular moiety that binds to a molecule selected from the group consisting of FOLR1, TRPV6, FOLH1 (PMSA) and GNRHR.
  • the synergistic molecule contained in the conjugate compound or a pharmaceutically acceptable salt thereof is folic acid or an analog thereof.
  • the folate analog is selected from the group consisting of 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, methotrexate, and 5,10-methylenetetrahydrofolate.
  • the conjugate compound or a pharmaceutically acceptable salt thereof contains one, two, three, four or more payloads.
  • the payload is selected from the group consisting of small molecule compounds, nucleotides, peptides, and proteins.
  • the payload is a small molecule compound.
  • the small molecule compound is selected from the group consisting of camptothecin and any derivative thereof, auristatin and any derivative thereof, maytansine and any derivative thereof, radionuclide complex, epoxy Catalytic enzyme-2 inhibitors, paclitaxel and any of its derivatives, epothilone and any of its derivatives, bleomycin and any of its derivatives, dactinomycin and any of its derivatives, prucamycin and any of its derivatives Derivatives, and mitomycin C.
  • the small molecule compound is camptothecin and any derivative thereof, auristatin and any derivative thereof, maytansine and any derivative thereof, a radionuclide complex or cyclooxygenase-2 Inhibitor.
  • the payload contained in the conjugate compound of the present application or a pharmaceutically acceptable salt thereof is connected to at least one of the targeting molecules through a linker.
  • the linker contained in the conjugate compound of the present application or a pharmaceutically acceptable salt thereof is a peptide linker, a disulfide linker, a pH-dependent linker, or a combination of the foregoing linkers.
  • the peptide linker can be cleaved by protease or reductively under a specific physiological environment.
  • the peptide linker is selected from the group consisting of cysteine, lysine, lysine-lysine, valine-citrulline, phenylalanine-lysine, valine Acid-lysine, cysteine-lysine, cysteine-glutamic acid, aspartic acid-aspartic acid and aspartic acid-aspartic acid-lysine, can be Optionally, the carboxylic acid in the above amino acid is amidated.
  • the disulfide linker is selected from the group consisting of DMDS, MDS, DSDM, and NDMDS.
  • the pH-dependent linker is cis-aconitic anhydride.
  • the linker of the conjugate compound of the present application or a pharmaceutically acceptable salt thereof includes the following structure:
  • linker is a combination of the above structure and a peptide linker.
  • the two targeting molecules contained in the conjugate compound of the present application or a pharmaceutically acceptable salt thereof are connected by a spacer.
  • the spacer described in the present application includes an amino acid sequence selected from the group consisting of SEQ ID NO: 1-14, Arg-Arg, Ala-Ser-Asn, Ala-Ala-Ala, Ser-Ser- Arg, Pro-Arg and Pro-Leu-Gly.
  • the conjugate compound of the present application is CB-20B, and its structural formula is as follows:
  • the conjugate compound of the present application is CB-20BK, and its structural formula is as follows:
  • the conjugate compound of the present application is CB-60S, and its structural formula is as follows:
  • the conjugate compound of the present application is CB-60SK, and its structural formula is as follows:
  • the conjugate compound of the present application is CB-20C, and its structural formula is as follows:
  • the conjugate compound of the present application is CB-1020, and its structural formula is as follows:
  • the conjugate compound of the present application is CB-1320, and its structural formula is as follows:
  • the conjugate compound of the present application is CB-1820, and its structural formula is as follows:
  • the conjugate compound of the present application is CR19428, and its structural formula is as follows:
  • the conjugate compound of the present application is 20R-SM09, and its structural formula is as follows:
  • the conjugate compound of the present application is CB-20R, and its structural formula is as follows:
  • M is a radionuclide
  • the conjugate compound of the present application is CB-18G, and its structural formula is as follows:
  • the conjugate compound of the present application is CR19426, and its structural formula is as follows:
  • the conjugate compound of the present application is CB-10S, and its structural formula is as follows:
  • the conjugate compound of the present application is CR19425, and its structural formula is as follows:
  • the conjugate compound of the present application is CB-50S, and its structural formula is as follows:
  • compositions which comprises the conjugate compound in the application or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
  • the composition is for intravenous, subcutaneous, oral, intramuscular, or intraventricular administration.
  • Another aspect of this application discloses a method for delivering a payload to a subject in need, comprising administering to the subject a therapeutically effective amount of the conjugate compound described in this application or a pharmaceutically acceptable salt thereof, Or the pharmaceutical composition described in this application.
  • Another aspect of this application discloses a method for treating a disease in a subject, comprising administering to the subject a therapeutically effective amount of the conjugate compound described in this application or a pharmaceutically acceptable salt thereof, or The pharmaceutical composition described in this application.
  • the method for treating a disease in a subject in this application further comprises combining one or more therapeutic agents with the conjugate compound or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition Combined application.
  • Another aspect of the application discloses a conjugate compound described in the application or a pharmaceutically acceptable salt thereof, or the use of the pharmaceutical composition described in the application in the preparation of a medicament for treating diseases in a subject .
  • Another aspect of the present application discloses a conjugate compound described in the present application or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described in the present application for treating diseases in a subject.
  • the disease is selected from the group consisting of cancer, immune disease, cardiovascular disease, metabolic disease, and neurological disease.
  • the cancer is selected from the group consisting of prostate cancer, breast cancer, lung cancer, kidney cancer, leukemia, ovarian cancer, stomach cancer, uterine cancer, endometrial cancer, liver cancer, colon cancer, thyroid cancer, pancreatic cancer, nodules Rectal cancer, esophageal cancer, skin cancer, lymphoma and multiple myeloma.
  • the immune disease is an autoimmune disease.
  • the autoimmune disease is selected from the group consisting of connective tissue disease, systemic sclerosis, rheumatoid arthritis, and systemic lupus erythematosus.
  • the cardiovascular disease is selected from the group consisting of angina pectoris, myocardial infarction, stroke, heart attack, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, cardiac arrhythmia, and congenital heart disease.
  • the metabolic disease is selected from the group consisting of diabetes, gout, obesity, hypoglycemia, hyperglycemia, and dyslipidemia.
  • the neurological disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, head injury, multiple sclerosis, vertigo, coma, and epilepsy.
  • Figure 1 shows the conjugate compounds CB-20B, CB-20BK, CB-60S, CB-60SK, CB-20C, CB-1020, CB-1320, CB-1820, CR19428, 20R-SM09, CB-20R, The chemical structure of CB-18G, CR19426, CB-10S, CR19425 and CB-50S.
  • Figure 2A shows the time-dependent curve of Cy5-pep-20BK binding to different cells and endocytosis (from top to bottom: LNCaP cells, SKOV3 cells, DU145 cells and NCI-H460 cells).
  • Figure 2B shows the time-dependent curve of Cy5-pep-20AK binding to different cells and endocytosis (from top to bottom: LNCaP cells, SKOV3 cells, DU145 cells and NCI-H460 cells).
  • Figure 3 shows the fluorescence photos of Cy5-FA binding and endocytosis with different cells over time.
  • the one that shows a complete circle is the fluorescence of the cell nucleus, and the one that is dotted is the fluorescence of Cy5-FA.
  • Figure 4A shows the inhibitory activity of the conjugate compound CB-20BK on the expansion of the illustrated tumor cells.
  • Figure 4B shows the inhibitory activity of the conjugate compound CB-20B on the expansion of tumor cells as shown.
  • Figure 4C shows the inhibitory activity of the conjugate compound CB-10S on the expansion of the illustrated tumor cells.
  • Figure 4D shows the inhibitory activity of the conjugate compound CB-60S on the expansion of the illustrated tumor cells.
  • Figure 4E shows the inhibitory activity of the conjugate compound CB-60SK on the expansion of the illustrated tumor cells.
  • Figure 4F shows the inhibitory activity of the conjugate compound CB-18G on the expansion of the illustrated tumor cells.
  • Figure 4G shows the inhibitory activity of the conjugate compound CB-50S on the expansion of tumor cells as shown.
  • Figures 5A-5E show the inhibitory effect of the conjugate compound CB-20BK on tumors in mice.
  • Figures 6A-6C show the inhibitory effect of the conjugate compound CB-20B on tumors in mice.
  • Figures 7A-7E show the inhibitory effect of the conjugate compound CB-18G on tumors in mice.
  • Figures 8A-8B show the effect of CBP-1018 for injection on the tumor volume of lung cancer LU2505 model and lung cancer LU1206 model.
  • analog includes structural analogs and functional analogs.
  • Structural analogs refer to a class of compounds with similar chemical structures, and one or more different atoms or one or more different functional groups may exist between them.
  • Functional analog refers to a class of compounds with the same or similar chemical, biological or pharmacological effects.
  • folate analogs include 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, methotrexate, and 5,10-methylenetetrahydrofolate.
  • derivative refers to a relatively complex compound derived from a parent compound molecule in which one or more atoms or groups of atoms are replaced by other atoms or groups of atoms .
  • camptothecin derivatives include irinotecan, SN-38, Dxd, topotecan, GI-147211C, topotecan, 9-aminocamptothecin, 7-hydroxymethylcamptothecin, 7- Aminomethylcamptothecin, 10-hydroxycamptothecin, (20S)-camptothecin, 9-nitrocamptothecin, gematican, karenitecin, silatecan, letotecan, esatecan, two Flutecan, Belotecan, Letotecan and S39625
  • conjugate compound or a pharmaceutically acceptable salt thereof comprises a payload and two targeting molecules, wherein the two The target molecules are the synergistic molecule part and the prostate-specific membrane antigen ligand part.
  • conjugate compound or a pharmaceutically acceptable salt thereof comprises a payload and two targeting molecules, wherein the The two targeting molecules are respectively a synergistic molecule part and a ligand part represented by formula (I):
  • conjugate compound or a pharmaceutically acceptable salt thereof comprises a payload and two targeting molecules, wherein the The two targeting molecules are the synergistic molecular part and P10 respectively, and the payload is camptothecin and any derivatives thereof.
  • the term "payload" as used in this application refers to a molecule or substance intended to be delivered to a target cell or tissue.
  • the payload can be any molecule or substance intended for the diagnosis, treatment, or prevention of a disease in a subject.
  • the payload has a molecular weight less than or equal to about 5 kDa.
  • the payload has a molecular weight less than or equal to about 1.5 kDa.
  • the payload is a drug or diagnostic agent that has been deemed safe and effective for use by appropriate drug approval and registration agencies (e.g., FDA, EMEA, or NMPA).
  • the payload of the present application is a small molecule compound, a nucleotide (e.g., DNA, plasmid DNA, RNA, siRNA, antisense oligonucleotide or nucleic acid aptamer, etc.), a peptide or protein (e.g., Enzyme).
  • the payload is a small molecule compound.
  • the payload of the application includes but is not limited to: anticancer drugs, radioactive substances, vitamins, anti-AIDS drugs, antibiotics, immunosuppressants, antiviral drugs, enzyme inhibitors, neurotoxins, opioids , Cell-extracellular matrix interaction modulators, vasodilators, antihypertensives, sleeping pills, antihistamines, anticonvulsants, muscle relaxants, anti-Parkinson substances, anti-spasmodics and muscle contraction drugs , Anti-parasitic and/or anti-protozoal drugs, analgesics, antipyretics, steroidal and non-steroidal anti-inflammatory drugs, anti-angiogenic factors, antisecretory factors, anticoagulants and/or anti- Thrombosis agents, local anesthetics, prostaglandins, antidepressants, antipsychotics, antiemetics, or imaging agents.
  • the payload of the present application has a free amino or carboxyl group before being connected to the conjugate compound of the present application, and the payload passes through the aforementioned amino or carboxyl group and the corresponding part of the conjugate compound (for example, a linker).
  • the group is acylated to couple to the conjugate compound.
  • modification of the aforementioned free amino or carboxyl group (for example, by coupling to the conjugate compound of the present application) will significantly reduce the activity of the payload (for example, reduce at least 50%, 60%, 70%). , 80%, 90%, 95%, 98% or 99%).
  • the "small molecule compound” used in this application refers to a compound having a molecular weight of less than or equal to about 2 kDa. In some embodiments, the small molecule compound has a molecular weight less than or equal to about 1.5 kDa. In some preferred embodiments, the small molecule compound has a molecular weight less than or equal to about 1 kDa, 800 Da, 700 Da, 600 Da, or 500 Da.
  • the small molecule compound of the present application is selected from the group consisting of camptothecin and any derivative thereof (for example, SN38 or Dxd), auristatin and any derivative thereof (for example, MMAE and MMAF), Dengsin and any derivatives thereof, cyclooxygenase-2 inhibitors (for example, celecoxib), radionuclide complexes, paclitaxel and any derivatives thereof, epothilone and any derivatives thereof, bo Leomycin and any derivatives thereof, dactinomycin and any derivatives thereof, pracamycin and any derivatives thereof, and mitomycin C.
  • camptothecin and any derivative thereof for example, SN38 or Dxd
  • auristatin and any derivative thereof for example, MMAE and MMAF
  • Dengsin and any derivatives thereof for example, cyclooxygenase-2 inhibitors (for example, celecoxib), radionuclide complexes, paclitaxel and any derivatives thereof
  • the small molecule compound is camptothecin and any derivative thereof, auristatin and any derivative thereof, a radionuclide complex or a cyclooxygenase-2 inhibitor.
  • the small molecule compounds described in this application are drugs for alleviating or treating cancer. In some embodiments, the small molecule compounds described in this application are drugs for alleviating or treating autoimmune diseases.
  • camptothecin used in this application refers to a cytotoxic alkaloid, mainly derived from the Davidia family plant Camptotheca acuminata, which shows strong anti-tumor activity.
  • the camptothecin and its derivatives in this application include camptothecin and its derivatives that already exist or will be produced later.
  • Camptothecin and its derivatives in this application include but are not limited to: camptothecin, irinotecan, SN-38, Dxd, topotecan, GI-147211C, topotecan, 9-aminocamptothecin, 7 -Hydroxymethylcamptothecin, 7-aminomethylcamptothecin, 10-hydroxycamptothecin, (20S)-camptothecin, 9-nitrocamptothecin, gematecan, karenitecin, silatecan, Le Topotecan, Isartecan, Diflutecan, Belotecan, Letotecan and S39625.
  • the term "Auristatin and any of its derivatives” used in this application refers to the natural anti-tumor product donotoxin 10 and a series of its derivatives. This type of compound interferes with microscopic self-assembly to arrest the cell in the mitotic phase, which is harmful to the cell. Has a strong lethality.
  • the auristatin and any derivatives thereof in the present application include auristatin and any derivatives that already exist or will be produced later.
  • the auristatin and its derivatives in this application include but are not limited to auristatin, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), monomethyl auristatin D (MMAD) , AFP and AFHPA.
  • cyclooxygenase-2 inhibitor used in this application is a specific type of cyclooxygenase-2 inhibitor. Cyclooxygenase-2 participates in the development and infiltration of malignant tumors through a variety of mechanisms. Cyclooxygenase-2 inhibitors can inhibit tumor cell migration and adhesion and intravascular infiltration, thereby inhibiting the occurrence and development of malignant tumors.
  • the cyclooxygenase-2 inhibitors of the present application include cyclooxygenase-2 inhibitors that already exist or will be produced later. Cyclooxygenase-2 inhibitors include, but are not limited to, celecoxib, rofecoxib, parecoxib, valdecoxib, and etuocoxib.
  • radionuclide complex refers to a special type of complex containing a radionuclide.
  • the chelating agent in the complex can chelate with the radionuclide and provide a more stable binding target The connecting part to the substance.
  • radionuclide refers to an element that can spontaneously emit radiation (for example, ⁇ -ray, ⁇ -ray, or ⁇ -ray).
  • the radionuclides in this application include all radionuclides that currently exist or are produced later that can be used for treatment and diagnosis.
  • the radionuclides in this application include but are not limited to 67 Cu, 64 Cu, 90 Y, 109 Pd, 111 Ag, 149 Pm, 153 Sm, 165 Ho, 166 Ho, 177 Lu, 186 Re, 188 Re, 99m Tc, 67 Ga, 68 Ga, 111 In, 90 Y, 177 Lu, 186 Re, 188 Re, 197 Au, 198 Au, 199 Au, 105 Rh, 161 Tb, 149 Pm, 44 Sc, 47 Sc, 70 As, 71 As, 72 As, 73 As, 74 As, 76 As, 77 As, 212 Pb, 212 Bi, 213 Bi, 225 Ac, 117m Sn, 67 Ga, 201 Tl, 123 I, 131 I, 160 Gd, 148 Nd, 89 Sr And 211 At.
  • the chelating agent is a macrocyclic chelating agent.
  • the chelating agent of this application includes but not limited to H2dedpa, H4octapa, H2azapa, DTPA, CHX-A"-DTPA, DTPA-bis anhydride, Maleimide-DTPA, DTPA(tBu)4, DiamSar CB-TE2A, Cyclam, DO2A, DOTA, OTA-GA(tBu) 4 , Maleimide-DOTA-GA, p-NCS-Bz-DOTA-GA, NH2-DOTA-GA, DOTA-GA anhydride, DOTA-tris(tBu)ester, Propargyl-DOTA-tris(tBu )ester, DO3AM-acetic acid, DO3AM-N-(2-aminoethyl)ethanamide, DO3AtBu-N-(2-aminoethyl)ethanamide, DOTA-di(tBu)ester, D
  • the conjugate compound of the present application or a pharmaceutically acceptable salt thereof includes a payload.
  • the conjugate compound of the present application or a pharmaceutically acceptable salt thereof contains two or more payloads.
  • the conjugate compound of the present application or a pharmaceutically acceptable salt thereof includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more payloads.
  • each payload may be the same or different from each other. In some embodiments, at least two payloads are different from each other.
  • targeting molecule refers to any molecule or part capable of targeting the conjugate compound of the present application to a target site, target tissue, target organ, target cell, or target intracellular region.
  • the targeting molecule allows the conjugate compound of the present application to be at the target site compared to the non-target site, non-target tissue, non-target organ, non-target cell, or non-target intracellular region.
  • Target tissues, target organs, target cells or areas within target cells are distributed more, for example, at least 10%, 20%, 50%, 80%, 100%, 150%, 200%, 300%, 400%, 500% or higher.
  • the targeting molecule allows the conjugate compound with the targeting molecule to be more distributed in the target site, target tissue, target organ, target cell, or intracellular region than without the targeting molecule. More, for example, at least 10%, 20%, 50%, 80%, 100%, 150%, 200%, 300%, 400%, 500% or more.
  • the targeting molecule can trigger or promote the specific binding of the conjugate compound containing such targeting molecule to the target molecule, trigger or promote the endocytosis of the conjugate compound by the target cell, trigger or promote the The conjoined compound is enriched around the target cell and/or enters the target cell.
  • the conjugate compound of the present application includes at least two targeting molecules. In some embodiments, the two or more targeting molecules included in the conjugate compound of the present application are the same or different. In some embodiments, at least two of the two or more targeting molecules included in the conjugate compound of the present application are different. In some embodiments, the two or more targeting molecules included in the conjugate compound of the present application are different from each other. In some embodiments, at least two of the two or more targeting molecules included in the conjugate compound of the present application can specifically bind to different cell surface proteins or markers. In some embodiments, two or more targeting molecules included in the conjugate compound of the present application can specifically bind to different cell surface proteins or markers.
  • the conjugate compound of the present application contains at least two targeting molecules, at least one of which is a synergistic molecule.
  • the term "synergistic molecule" used in this application refers to a synergistic effect with other targeting molecules contained in the conjugate compound of the application to better trigger or promote the specific binding of the conjugate compound to the target molecule , Trigger or promote the endocytosis of the target cell to the conjugate compound, trigger or promote the enrichment of the conjugate compound around the target cell and/or enter the target cell, and/or cause the conjugate compound to be specific to the target cell in other forms Any molecule or part that is sexually bound and retained.
  • the synergistic molecule allows the conjugate compound of the present application to be on target compared to non-target sites, non-target tissues, non-target organs, non-target cells or non-target intracellular regions.
  • target tissue, target organ, target cell or area within target cell is distributed more, for example, at least 10%, 20%, 50%, 80%, 100%, 150%, 200%, 300%, 400% more , 500% or higher.
  • the synergistic molecule allows the conjugate compound with the synergistic molecule to be more distributed in the target site, target tissue, target organ, target cell, or area within the target cell than without the synergistic molecule. More, for example, at least 10%, 20%, 50%, 80%, 100%, 150%, 200%, 300%, 400%, 500% or more.
  • the synergistic molecule makes the conjugate compound with the synergistic molecule more active on the target cell than the molecule without the synergistic effect, for example, at least 10%, 20%, 50% higher. %, 80%, 100%, 150%, 200%, 300%, 400%, 500% or more.
  • the synergistic molecule of the present application is a cell interacting molecule.
  • cell-interacting molecule used in this application refers to the ability to interact with the cell surface material of the target cell to trigger or promote the specific binding of the conjugate compound containing such cell-interacting molecule to the cell, triggering Or promote the endocytosis of the conjugate compound by the target cell, and/or trigger or promote the enrichment of the conjugate compound around the target cell and/or enter the target cell.
  • Cell interaction molecules can be small chemical molecules or large biological molecules.
  • the cell-interacting molecule is a small molecule compound or a polypeptide.
  • the cell-interacting molecule is a small molecule compound or includes 2-50, 2-40, 2-30, 2-25, 2-22, 2-20, 2-18, 2-15, 2 -12, 2-10, 2-8, 4-50, 5-50, 5-40, 5-30, 5-25, 5-22, 5-20, 5-18, 5-15, 5-12 , 5-10, 6, 7, 8, 9 amino acid peptides.
  • the targeting molecule is a ligand capable of binding to cell surface receptors or other molecules. In some embodiments, at least one targeting molecule is a ligand capable of binding to cell surface receptors or other molecules.
  • the ligands of the present application can include a variety of chemical or biological molecules, which can have specific binding affinity to the selected target.
  • the selected target can be, for example, cell surface receptors, cell Surface antigens, cells, tissues, organs, etc.
  • the ligand can specifically bind to a protein or marker expressed on the surface of the target cell.
  • the ligand of the present application binds to cell surface proteins or markers with an affinity of 10 -6 to 10 -11 M (K d value).
  • the ligand of the present application binds to cell surface proteins or markers with an affinity of at least 10 -7 , at least 10 -8 , and at least 10 -9 M (K d value).
  • the ligands of the present application bind to cell surface proteins or markers with an affinity of less than 10 -6 , less than 10 -7 , and less than 10 -8 M (K d value). In some embodiments, the ligand of the present application binds to cell surface proteins or markers with a certain affinity, and the certain affinity refers to the affinity with non-target cell surface proteins or markers. Compared with the ligand, the affinity of the target cell surface protein or marker is at least two times, three times, four times, five times, six times, eight times, ten times, twenty times, fifty times, one hundred times higher Or more times.
  • the expression of the cell surface protein or marker of the present application in target cells is significantly higher than that in normal cells.
  • target cells for example, cancer cells
  • the term "significant” as used in this application refers to a statistically significant difference, or a significant difference that can be recognized by those skilled in the art.
  • the expression level of the cell surface protein or marker of the present application in target cells is 2 times to 1,000,000 times the expression level in normal cells, such as in target cells (e.g., The expression level in cancer cells) is 2 times to 10 times, 2 times to 100 times, 2 times to 1,000 times, 2 times to 10,000 times, 2 times to 100,000 times, 2 times to 1,000,000 times higher than the expression level in normal cells Times (may be equal to any value in the above numerical range, including the end points of this range).
  • the expression level of the cell surface receptor in the target cell is at least 10 times, or at least 100 times, or at least 1,000 times, or at least 10,000 times higher than the expression level in normal cells , Or at least 100,000 times.
  • the level of cell surface receptors on normal cells is reduced by at least 50%, 60%, 70%, when compared with the level of cell surface proteins or markers on target cells (e.g., cancer cells). 80%, 90%, 95% or 99%.
  • the cell surface proteins or markers described in the present application cannot be detected in normal cells.
  • the cell surface protein or marker of the present application is a cell surface receptor.
  • the cell surface receptor of the present application is selected from the group consisting of transferrin receptor (TFR), low density lipoprotein receptor (LDLR), folate receptor (FR), growth hormone receptor , Urate kinase receptor, tumor necrosis factor receptor (TNFR), integrin receptor (LFA-1), SST-14 receptor (SSTR2), GNRH receptor (GNRHR), TRPV6 and integrin alpha receptor.
  • TFR transferrin receptor
  • LDLR low density lipoprotein receptor
  • FR folate receptor
  • growth hormone receptor ase receptor
  • TNFR tumor necrosis factor receptor
  • LFA-1 integrin receptor
  • SSTR2 SST-14 receptor
  • GNRHR GNRH receptor
  • TRPV6 integrin alpha receptor
  • the cell surface protein or marker of the present application is a cell surface antigen.
  • the cell surface antigen of the present application is selected from the following group: prostate specific membrane antigen, MUC1 mucin, acute lymphoblast common antigen, Thy-1 cell surface antigen, Melan-A protein, squamous cell carcinoma Antigens, Galectin 3 and Human Leukocyte Antigens.
  • the cell-interacting molecule of the present application can bind to a molecule selected from the group consisting of FOLR1, TRPV6, FOLH1 (PMSA), GNRHR, Her2, Trop2, Her3, NECTIN4, LRP1, GLUT1, EGFR1, AXL, CA9, CD44, Claudin18.2, APN, DLL3, CEACAM5, FZD10, TFRC, MET, IGFR1, SSTR2, CCKBR, LFA1, ICAM, GPR87, GM-CSF, GM-CSFR, TIM3, TLR family, CD40, CD40L, OX40 , OX40L, GITRL, GITR, 4-BBL, 4-1BB, CD70, CD27, ICOSL, ICOS, HHLA2, CD28, CD86/80, CD28, MHCII antigen, TCR, CTLA-4, CD155, CD122, CD113, IGIT, PD-L1, PD1, Galectin-9, TIM-3, H
  • the conjugate compound of the present application or a pharmaceutically acceptable salt thereof comprises a prostate-specific membrane antigen ligand portion and a synergistic molecule portion that binds to a molecule selected from the group consisting of : FOLR1, TRPV6, FOLH1 (PMSA), SSTR2 and GNRHR.
  • the conjugate compound of the present application or a pharmaceutically acceptable salt thereof comprises a ligand portion represented by formula (I) and a synergistic molecular portion, and the synergistic molecular portion is selected from the group consisting of Molecular binding: FOLR1, TRPV6, SSTR2 and GNRHR.
  • the conjugate compound of the present application or a pharmaceutically acceptable salt thereof comprises P10 and a synergistic molecule part, and the synergistic molecule binds to a molecule selected from the group consisting of FOLR1, TRPV6, FOLH1 ( PMSA) and GNRHR.
  • a synergistic molecule in the conjugate compound of the present application or a pharmaceutically acceptable salt thereof is an endocytosis molecule part capable of mediating endocytosis.
  • endocytosis refers to the ability of the conjugate compound or its pharmaceutically acceptable salt to interact with the target cell to mediate its own endocytosis, internalization or uptake into the target cell .
  • endocytosis molecule used in this application refers to a molecule that, after interacting with target cells, can mediate the endocytosis of the conjugate compound of this application or a pharmaceutically acceptable salt thereof. , Internalization or uptake into target cells.
  • the endocytosis molecule is selected from the group consisting of folic acid and its analogs, peptides capable of mediating endocytosis, and penetrating peptides.
  • the endocytosis molecule of the present application is folic acid or an analog thereof.
  • folic acid is beneficial to form chemical bonds with other groups.
  • Folic acid can bind with the folate receptor expressed on the cell surface with high affinity to mediate the uptake of folic acid by the cell.
  • the expression level of folate receptor in most normal cells is very low, it is expressed at a high level in a large number of cancer cells to meet the higher demand for folic acid in rapidly dividing cells under low folate conditions (see Kelemen LE, Int J Cancer , 2006; 119: 243-50; Kane MA, etc., J Clin Invest. 1988; 81:1398-406; Matsue H, etc., Proc Natl Acad Sci USA.
  • Folic acid can specifically bind to folate receptors on the cell surface, and it can also mediate the endocytosis of the conjugate compound or its pharmaceutically acceptable salt into target cells.
  • the analog of folic acid is selected from the group consisting of 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, methotrexate, and 5,10-methylenetetrahydrofolate.
  • the endocytosis molecule is a peptide capable of mediating endocytosis.
  • the peptide capable of mediating endocytosis comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, Arg-Gly-Asp (referred to as RGD ), and SEQ ID NO: any one of 16-18 has at least 70%, at least 80%, at least 85%, at least 90%, 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% of amino acid sequence homology, wherein the homologous peptides are functional equivalents of the peptides shown in SEQ ID NO: 16-18 .
  • the peptide capable of mediating endocytosis as described in this application compared with the sequence of SEQ ID NO: 16-20 and RGD, only has a conservative substitution of amino acid at one amino acid position. In some embodiments, the peptide capable of mediating endocytosis as described in this application is compared with the sequence of SEQ ID NO: 16-20 at 2, 3, 4, 5, 6, 7, 8, 9 or There are conservative substitutions of amino acids at 10 amino acid positions.
  • the peptide capable of mediating endocytosis as described in this application may also contain non-naturally occurring amino acids, including, for example, ⁇ -fluoroalanine, 1-methyl-histidine , ⁇ -methylene-glutamic acid, ⁇ -methyl-leucine, 4,5-dehydro-lysine, hydroxyproline, 3-fluoro-phenylalanine, 3-amino-tyrosine Acid, 4-methyl-tryptophan, etc.
  • non-naturally occurring amino acids including, for example, ⁇ -fluoroalanine, 1-methyl-histidine , ⁇ -methylene-glutamic acid, ⁇ -methyl-leucine, 4,5-dehydro-lysine, hydroxyproline, 3-fluoro-phenylalanine, 3-amino-tyrosine Acid, 4-methyl-tryptophan, etc.
  • sequences can be compared using the following publicly available tool: BLASTp software (available from the website of the National Center for Biotechnology Information (NCBI): http://blast.ncbi.nlm.nih.gov/Blast.cgi See also Altschul SF et al., J. Mol. Biol., 215:403-410 (1990); Stephen F.
  • NCBI National Center for Biotechnology Information
  • the term "functional equivalent” as used in this application refers to a derivative peptide that retains a biological activity substantially similar to that of the original peptide sequence from which the peptide is derived.
  • Functional equivalents can be natural derivatives or synthetically prepared.
  • Exemplary functional equivalents include amino acid sequences with one or more amino acid substitutions, deletions, or additions, provided that the biological activity of the peptide is maintained.
  • the substituted amino acid ideally has chemical-physical properties similar to the substituted amino acid. Ideally similar chemical-physical properties include similarities in charge, bulkiness, hydrophobicity, and hydrophilicity.
  • functional equivalents include conservative substitutions of amino acid residues.
  • Conservative substitutions of amino acid residues refer to substitutions between amino acids with similar properties, such as between polar amino acids (for example, between glutamine and asparagine), and between hydrophobic amino acids. Substitutions (e.g., substitutions between leucine, isoleucine, methionine, and valine), and substitutions between amino acids with the same charge (e.g., arginine, lysine, and histidine) Substitution between acids, or between glutamic acid and aspartic acid) and so on.
  • the endocytosis molecule is a penetrating peptide.
  • CPP Cell-Penetrating Peptides
  • PTD protein transduction domains
  • the penetrating peptide described in the present application is selected from the group consisting of tumor homing peptide, mitochondrial penetrating peptide, activatable cell penetrating peptide, and antibacterial peptide.
  • the penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 19 (RRRRRRRRR, referred to as R9) and SEQ ID NO: 20 (GRKKRRQRRRPPQ, which is a Tat peptide, that is, the HIV transcription protein trans Activating factor penetrating peptide).
  • a targeting molecule in the conjugate compound of the present application or a pharmaceutically acceptable salt thereof is a prostate-specific membrane antigen ligand portion.
  • prostate-specific membrane antigen refers to a type II transmembrane glycoprotein that exists in the membrane of prostate epithelial cells and consists of 750 amino acids. It has 19 intracellular amino acids and 24 transmembrane glycoproteins. Membrane amino acids and 707 extracellular amino acids. Prostate-specific membrane antigen is expressed in normal prostate epithelial cells, but its expression level in prostate cancer cells is much higher. Compared with the traditional prostate-specific antigen used for clinical detection, the prostate-specific membrane antigen is a more sensitive and specific prostate cancer tumor marker, especially in hormone-refractory prostate cancer and prostate cancer metastases. , It has high sensitivity and specificity in distinguishing prostate cancer from other types of malignant tumors.
  • prostate-specific membrane antigens are also highly specifically expressed on tumor vascular endothelial cells.
  • prostate-specific membrane antigen ligand used in this application refers to antibodies, nucleic acid aptamers and small molecules that can specifically recognize and bind to prostate-specific membrane antigen.
  • the prostate-specific membrane antigen ligands of the present application include the prostate-specific membrane antigen ligands that already exist or will be produced later, as well as fragments of the aforementioned ligands, as long as these fragments retain the ability to bind to the prostate-specific membrane antigen.
  • Antibody ligands are the most common prostate-specific membrane antigen ligands, which include but are not limited to monoclonal antibodies J591, J533, J415 and E99 (for example, see Liu H, Rajasekaran AK, Moy P et al.
  • Nucleic acid aptamers are single-stranded DNA or RNA that can bind to prostate-specific membrane antigens with high affinity and specificity, which are obtained through technical screening by an exponential enrichment ligand system.
  • prostate-specific membrane antigen ligands include But not limited to xPSM-A10 aptamer and its derivatives and xPSM-A9 aptamer and its derivatives (for example, see Lupoid SE et al., Identification and Characterization of RNA molecules that bind human prostate cancer cells via the state-specific membrane antigen, Cncer Res, 2002, 62(14): 4029-4033).
  • prostate-specific membrane antigen small molecule ligands Compared with antibody and nucleic acid aptamer ligands, prostate-specific membrane antigen small molecule ligands have the advantages of small molecular weight, high permeability, low immunogenicity, and ease of synthesis, including but not limited to glutamine Small molecule ligands and phosphoramidate-like small molecule ligands.
  • the prostate-specific membrane antigen small molecule ligands of the present application can be selected from the group consisting of: 2-[[methylhydroxyphosphinyl]methyl]glutaric acid; 2-[[ethyl Hydroxyphosphinyl]methyl]glutaric acid; 2-[[propylhydroxyphosphinyl]methyl]glutaric acid; 2-[[butylhydroxyphosphinyl]methyl]glutaric acid; 2- [[Cyclohexylhydroxyphosphinyl]methyl]glutaric acid; 2-[[phenylhydroxyphosphinyl]methyl]glutaric acid; 2-[[2-(tetrahydrofuranyl)hydroxyphosphinyl]methyl Glutaric acid; 2-[[(2-tetrahydropyranyl)hydroxyphosphinyl]methyl]glutaric acid; 2-[[((4-pyridyl)methyl)hydroxyphosphinyl] Methyl]glutaric acid; 2-[[[((2-pyridyl)methyl)
  • prostate-specific membrane antigen ligands of the present application also include all prostate-specific membrane antigen small molecule ligands disclosed in PCT applications WO2010/108125 and WO2006/093991, and the above two patent applications are incorporated herein in their entirety.
  • the prostate-specific membrane antigen small molecule ligand of the present application is a glutaric acid derivative. In some embodiments, the prostate-specific membrane antigen small molecule ligand of the present application is an aminocarbonyl derivative of glutaric acid.
  • the prostate-specific membrane antigen small molecule ligand of the present application includes the following structure:
  • the prostate-specific membrane antigen ligand contained in the conjugate compound or a pharmaceutically acceptable salt thereof includes the following structure:
  • the prostate-specific membrane antigen ligand contained in the conjugate compound or a pharmaceutically acceptable salt thereof includes the following structure:
  • the prostate-specific membrane antigen ligand contained in the conjugate compound or a pharmaceutically acceptable salt thereof includes the following structure:
  • the prostate-specific membrane antigen ligand contained in the conjugate compound or a pharmaceutically acceptable salt thereof includes the following structure:
  • a targeting molecule in the conjugate compound of the present application or a pharmaceutically acceptable salt thereof has a ligand moiety represented by formula (I):
  • a ligand portion with at least 70%, at least 80%, at least 85%, or at least 90% amino acid sequence homology with it, or at most 3, 2, or 1 amino acid substitutions therewith (for example, conservative substitutions).
  • a targeting molecule in the conjugate compound of the present application or a pharmaceutically acceptable salt thereof is P10 or has at least 70%, at least 80%, at least 85%, at least 90%, or at least 91% of P10. , A ligand portion with at least 92% or at least 93% amino acid sequence homology or at most 3, 2 or 1 amino acid substitutions with it (for example, conservative substitutions).
  • P10 refers to a peptide having the amino acid sequence Cys-Lys-Glu-Phe-Leu-His-Pro-Ser-Lys-Val-Asp-Leu-Pro-Arg.
  • the conjugate compound of the present application has a targeting molecule selected from the group consisting of: (1) folate ligand and prostate specific membrane antigen ligand; (2) TRPV6 ligand and prostate specific membrane Antigen ligand; (3) GNRHR ligand and prostate specific membrane antigen ligand; (4) SSTR2 ligand and prostate specific membrane antigen ligand; (5) folate ligand and SSTR2 ligand; or (6) TRPV6 Ligand and folic acid ligand.
  • a targeting molecule selected from the group consisting of: (1) folate ligand and prostate specific membrane antigen ligand; (2) TRPV6 ligand and prostate specific membrane Antigen ligand; (3) GNRHR ligand and prostate specific membrane antigen ligand; (4) SSTR2 ligand and prostate specific membrane antigen ligand; (5) folate ligand and SSTR2 ligand; or (6) TRPV6 Ligand and folic acid ligand.
  • the two targeting molecules of the conjugate compound or a pharmaceutically acceptable salt thereof provided in the present application are respectively a synergistic molecule part and a prostate-specific membrane antigen ligand part.
  • the synergistic molecule is capable of mediating endocytosis.
  • the two targeting molecules of the conjugate compound or a pharmaceutically acceptable salt thereof provided in the present application are folic acid or an analog thereof and a prostate-specific membrane antigen ligand portion, respectively.
  • the two targeting molecules of the conjugate compound or a pharmaceutically acceptable salt thereof provided in the present application are respectively a synergistic molecule part and a ligand part represented by formula (I).
  • the synergistic molecule is capable of mediating endocytosis.
  • the two targeting molecules of the conjugate compound or a pharmaceutically acceptable salt thereof provided in the present application are folic acid or an analog thereof and a ligand portion represented by formula (I), respectively.
  • the two targeting molecules of the conjugate compound or pharmaceutically acceptable salt thereof provided in the present application are the synergistic molecular part and P10, respectively.
  • the synergistic molecule is capable of mediating endocytosis.
  • the two targeting molecules of the conjugate compound or its pharmaceutically acceptable salt provided in the present application are folic acid or its analogue and P10, respectively.
  • the conjugate compound provided in the present application only contains a single payload coupled to two targeting molecules. In some embodiments, the conjugate compound provided in the present application contains multiple payloads coupled to two targeting molecules.
  • Coupled refers to the connection of two chemical groups by covalent bonds, which can be directly formed between two chemical groups, or through a linker Connect two chemical groups indirectly.
  • the conjugate compound or a pharmaceutically acceptable salt thereof comprises a payload (eg, 1) and two targeting molecules, wherein the payload is directly covalently linked to at least one targeting molecule.
  • the payload and both targeting molecules are directly covalently linked.
  • the conjugate compound or a pharmaceutically acceptable salt thereof comprises a payload (eg, 1) and two targeting molecules, wherein the payload and at least one targeting molecule are covalently linked via a linker. In some embodiments, the payload and both targeting molecules are covalently linked via a linker.
  • linker refers to a molecule or part that covalently connects a payload to a targeting molecule.
  • the linker includes a functional group for connecting the payload with at least one targeting molecule.
  • the functional group may contain two reactive moieties, one for attaching to the payload and the other for attaching to the targeting molecule.
  • the functional groups are different from each other.
  • the functional group includes a group containing a sulfhydryl reactive portion and an amine reactive portion.
  • the functional groups are the same as each other.
  • the functional group is a maleimide group.
  • the linker contains amino acids.
  • the carboxylic acid in the amino acid contained in the linker is amidated.
  • the linker contains a short chain of polyethylene glycol (e.g., including 2-10, 2-8, 3-8, 4-8, 4-7, 4-6, or 5 repeating units).
  • the linker of the present application is capable of combining at least one (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) payload and at least one Multivalent linkers for targeting molecules.
  • the payloads bound to the multivalent linker may be the same or different, and the targeting molecules bound to the multivalent linker may be the same or different.
  • the linker should be stable enough to avoid accidental release of the payload during blood circulation to increase the effective amount of the payload to the target cell or tissue and avoid toxicity. In another aspect, the linker should be able to release a payload around or inside the target cell to effectively kill the target cell or block the function of the target cell.
  • the linker includes at least one cleavable functional group. Preferably, the cleavable functional group is sufficiently stable outside the target cell, but it is cleaved after entering the target cell to release the payload. In some embodiments, the lysis efficiency of the cleavable functional group in the target cell is at least 10, 20, 30, 50, 100, or more times higher than the lysis efficiency in blood or serum.
  • the cleavable linker can be cleaved by hydrolysis, enzymatic reaction, or reduction reaction, or by pH change.
  • the linker is cleavable under a specific physiological environment (e.g., under a suitable pH environment).
  • the linker can be cleaved in an acidic environment with a pH of about 6.5 or lower, or can be cleaved by reagents such as enzymes.
  • the linker is sensitive to cleavage agents. For example, pH, redox potential, or the presence of degrading molecules.
  • the linker is non-cleavable.
  • the non-cleavable linker used in this application refers to a linker that remains substantially intact during intracellular metabolism.
  • the linker is a peptide linker, which is composed of straight or branched chain amino acids connected by peptide bonds.
  • peptide linkers can be cleaved by proteases that are highly or specifically expressed around or in target cells, such as cathepsin B in lysosomes or endosomes.
  • the peptide linker used in this application can have various lengths. Generally, the peptide linker of the present application has a length of 1 to 50 amino acids. In some embodiments, the length of the peptide linker is 1 to 45, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1.
  • the length of the peptide linker is 2 to 45, 2 to 40, 2 to 35, 2 to 30, 2 to 25, 2 to 20, 2 to 15, 2 to 10, 2 to 9, 2 To 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3 or 2 amino acids.
  • the number of amino acids of the peptide linker described in the present application can be equal to any integer value within the above numerical range, including the end point of this range.
  • the length of the peptide linker is preferably 1, 2, 3, 4, or 5 amino acids.
  • the peptide linker is cysteine, lysine, lysine-lysine, valine-citrulline, phenylalanine-lysine, valine-lysine Amino acid, cysteine-lysine, cysteine-glutamic acid aspartic acid-aspartic acid and aspartic acid-aspartic acid-lysine, optionally, the above amino acids
  • the carboxylic acid in is amidated.
  • the linker is a disulfide linker containing a disulfide bond.
  • Disulfide bonds can be cleaved in the reducing environment in the cell and remain stable in the circulatory system.
  • the disulfide linker of the present application can be DSDM, DMDS, MDS or NDMDS. The structures of these disulfide linkers are shown in Table 1 below.
  • the linker is a pH-dependent linker.
  • the pH-dependent linker described in this application can be cleaved under a specific pH environment.
  • the pH-dependent linker can be stable under alkaline conditions, but cleaved under acidic conditions (e.g., at a pH of 6.5 or lower).
  • the pH-dependent linker is cis-aconitic anhydride.
  • the linker of the conjugate compound or a pharmaceutically acceptable salt thereof is
  • peptide linker for example, a peptide linker containing 1-3 amino acids is connected to a targeting molecule.
  • the linker of the conjugate compound or a pharmaceutically acceptable salt thereof has the following structure:
  • peptide linker for example, a peptide linker containing 1-3 amino acids is connected to a targeting molecule.
  • the linker of the conjugate compound or a pharmaceutically acceptable salt thereof has the following structure:
  • peptide linker for example, a peptide linker containing 1-3 amino acids is connected to a targeting molecule.
  • the linker of the conjugate compound or a pharmaceutically acceptable salt thereof has the following structure:
  • peptide linker for example, a peptide linker containing 1-3 amino acids is connected to a targeting molecule.
  • the linker of the conjugate compound or a pharmaceutically acceptable salt thereof has the following structure:
  • peptide linker for example, a peptide linker containing 1-3 amino acids is connected to a targeting molecule.
  • the linker of the conjugate compound or a pharmaceutically acceptable salt thereof has the following structure:
  • peptide linker for example, a peptide linker containing 1-3 amino acids is connected to a targeting molecule.
  • the linker of the conjugate compound or a pharmaceutically acceptable salt thereof has the following structure:
  • peptide linker for example, a peptide linker containing 1-3 amino acids is connected to a targeting molecule.
  • the linker of the conjugate compound or a pharmaceutically acceptable salt thereof has the following structure:
  • peptide linker for example, a peptide linker containing 1-3 amino acids is connected to a targeting molecule.
  • the linker of the conjugate compound or a pharmaceutically acceptable salt thereof has the following structure:
  • peptide linker for example, a peptide linker containing 1-3 amino acids is connected to a targeting molecule.
  • the linker of the conjugate compound or a pharmaceutically acceptable salt thereof has the following structure:
  • peptide linker for example, a peptide linker containing 1-3 amino acids is connected to a targeting molecule.
  • the linker of the conjugate compound or a pharmaceutically acceptable salt thereof has the following structure:
  • peptide linker for example, a peptide linker containing 1-3 amino acids is connected to a targeting molecule.
  • the linker of the conjugate compound or a pharmaceutically acceptable salt thereof has the following structure:
  • peptide linker for example, a peptide linker containing 1-3 amino acids is connected to a targeting molecule.
  • the linker of the present application may include any one or a combination of the linkers described above.
  • the payload is directly or indirectly coupled to the first targeting molecule, and the first targeting molecule is directly or indirectly coupled to the second targeting molecule. In some embodiments, the payload is directly coupled to both the first targeting molecule and the second targeting molecule. In some embodiments, the payload is indirectly coupled to both the first targeting molecule and the second targeting molecule. In some embodiments, the payload is coupled to the first targeting molecule indirectly (eg, via a linker), and the first targeting molecule is directly or indirectly coupled to the second targeting molecule. In some embodiments, the payload is coupled to the first targeting molecule through a first linker, and the payload is coupled to the second targeting molecule through a second linker. In some embodiments, the linker is bound to at least one (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) payload and two targeting molecules Multivalent linker.
  • two targeting molecules are connected to each other by a spacer.
  • the spacer can be cleaved by a protease that is specifically expressed by the target cell or will be expressed by the target cell.
  • proteases include, for example, the proteases listed in Table 2 below.
  • the spacer comprises an amino acid sequence selected from any one of the amino acid sequences listed in Table 2 below.
  • Protease Amino acid sequence of recognition site SEQ ID NO.
  • Prostate specific antigen SSLY SEQ ID NO: 3
  • Urokinase-type plasminogen activator SSR - Activated protein C LVKR SEQ ID NO: 5 Factor Ixa LVVR SEQ ID NO: 6 Factor Vila QLTR SEQ ID NO: 7 Factor Xa LEGR SEQ ID NO: 8 Thrombin PR - Calpain-a PLFAEP SEQ ID NO: 9 Calpain-2 GLGSEP SEQ ID NO: 10 Intestinal peptidase DDDDK SEQ ID NO: 11 MMP-8 GPSG SEQ ID NO: 12 Cathepsin L PLG
  • cleavable or “cleavable” as used in this application refers to the metabolic process or reaction process performed on the conjugate compound provided in this application, thereby linking the payload with the target molecule. , Or the spacer between targeting molecules is broken to release free payload or targeting molecules.
  • the linker or spacer is cleaved by a protease or under a specific physiological environment (for example, pH environment).
  • the conjugate compound has a structure represented by the following formula I, II, III or IV, wherein n, m, p, and q are independently 0 or 1, which represents the presence or absence of a connection independently Sub or spacer.
  • the "molecule” in the following formula is an abbreviation for "targeting molecule”.
  • the conjugate compound provided in the application or a pharmaceutically acceptable salt thereof contains at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) payload, two targeting molecules as provided in this application, and optionally linkers or spacers as provided in this application.
  • the conjugate compound provided in the application or a pharmaceutically acceptable salt thereof includes a payload as provided in the application, and a specific binding to a cell surface protein or a marker as provided in the application.
  • the conjugate compound has the structure of formula V, VI, VII or VIII as shown below, wherein n, m, p, q and s are independently 0 or 1, which independently represent the presence or absence There are linkers, multivalent linkers and spacers.
  • the conjugate compound provided in the present application or a pharmaceutically acceptable salt thereof includes a payload and two targeting molecules, wherein the two targeting molecules are a part of a synergistic molecule and a prostate-specific membrane antigen.
  • Ligand parts such as CB-20B, CB-20BK, CB-60S, CB-60SK, CB-20C, CB-1020, CB-1320, CB-1820, CR19428, 20R-SM09 and CB-20R.
  • the conjugate compound provided in the present application or a pharmaceutically acceptable salt thereof contains one or more payloads and two targeting molecules, wherein the two targeting molecules are part of a synergistic molecule and have The ligand part represented by formula (I), for example, CB-18G, CB-1820 and CR19426.
  • the conjugate compound provided in the present application or a pharmaceutically acceptable salt thereof comprises a payload and two targeting molecules, wherein the two targeting molecules are the synergistic molecular part and P10, which are effective
  • the load is camptothecin and any of its derivatives, such as CB-10S, CR19425 and CB-50S.
  • the conjugate compound of the present application is selected from the group consisting of the following compounds: CB-20B, CB-20BK, CB-60S, CB-60SK, CB-20C, CB-1020, CB-1320, CB-1820, CR19428, 20R-SM09, CB-20R, CB-18G, CR19426, CB-10S, CR19425 and CB-50S (the specific structure of each conjugate compound is shown in Figure 1).
  • the conjugate compound of the present application is formed by a linker-drug moiety and a ligand moiety connected by a covalent bond.
  • the linker-drug part of the present application includes a payload and a linker
  • the ligand part of the present application includes two targeting molecules and an optional spacer or linker.
  • the two parts react to form a covalent bond to form the present application
  • the covalent bond can be formed between the linker in the linker-drug part and the ligand molecule of the ligand part, or it can be formed between the linker and the ligand part in the linker-drug part Between the spacers or linkers.
  • the conjugate compound CB-20B of the present application is formed by covalently connecting the linker-drug part LT1002 and the ligand part 20B-SM09 through a covalent bond.
  • the conjugate compound CB-20BK of the present application is formed by covalently connecting the linker-drug part LT1002 and the ligand part 20BK-SM09 through a covalent bond.
  • the conjugate compound CB-60S of this application is formed by covalently connecting the linker-drug part LT2000C and the ligand part 60S-SM09 through a covalent bond.
  • the conjugate compound CB-60SK of the present application is formed by the linker-drug part LT2000C and the ligand part 60SK-SM09 connected by a covalent bond.
  • the conjugate compound CB-20C of the present application is formed by covalently connecting the linker-drug part LD1001 and the ligand part 20BK-SM09 through a covalent bond.
  • the conjugate compound CB-1020 of the present application is formed by covalently connecting the linker-drug part LT1002 and the ligand part 1020BK-SM09 through a covalent bond.
  • the coupling body compound CB-1320 of the present application is formed by covalently connecting the linker-drug part LT1002 and the ligand part 1320BK-SM09 through a covalent bond.
  • the conjugate compound CB-1820 of the present application is formed by covalently connecting the linker-drug part LT1002 and the ligand part 1820BK-SM09.
  • the conjugate compound CR19428 of the present application is formed by the linker-drug part CR19423 and the ligand part 20BK-SM09 connected by a covalent bond.
  • the conjugate compound CB-20R of the present application is complexed with the radionuclide ion M by 20R-SM09.
  • the conjugate compound CB-18G of the present application is formed by the linker-drug part LT1002 and the ligand part 18G-SM09 connected by covalent bonds.
  • the conjugate compound CR19426 of the present application is formed by the linker-drug part CR19423 and the ligand part 18G-SM09 connected by a covalent bond.
  • the conjugate compound CB-10S of the present application is formed by covalently connecting the linker-drug part LT1000 and the ligand part CBSM09 through a covalent bond.
  • the conjugate compound CR19425 of the present application is formed by covalently connecting the linker-drug part CR19423 and the ligand part CBSM09 through a covalent bond.
  • the conjugate compound CB-50S of the present application is formed by covalently connecting the linker-drug part LT1000N3 and the ligand part 50S-SM09 through a covalent bond. Each structure is shown in Table 3 below.
  • the conjugate compound provided in the present application or a pharmaceutically acceptable salt thereof enters the blood circulation and the outside of the cell (intercellular substance). Because the linker is very stable in the extracellular environment and cannot release the drug molecule, the drug The toxicity of the molecule is blocked.
  • the conjugate is a drug with no cytotoxicity or low toxicity and will not have a toxic effect on normal cells.
  • the conjugate compound provided in the present application or a pharmaceutically acceptable salt thereof binds to multiple receptors or antigens that are simultaneously highly expressed on diseased cells, and its synergistic effect greatly increases the coupling.
  • the affinity of the body compound to target cells reduces the possibility of binding to normal cells. It can carry high-efficiency toxin drugs such as MMAE/Dxd/SN38/radionuclide complex, enhance the efficacy and broaden the treatment window to avoid drug side effects.
  • the conjugate compound provided in the present application or a pharmaceutically acceptable salt thereof enters the interior of the targeted cell, and then changes through the internal environment of the cell (specific digestion, pH change, disulfide bond reduction, etc.)
  • the linker can be cleaved to release the drug molecule (equivalent to removing the modification group of the drug molecule), which has a therapeutic effect on tumor cells.
  • the conjugate compound of the present application or a pharmaceutically acceptable salt thereof can be used to specifically deliver a payload to target cells in a target tissue environment.
  • two targeting molecules of a conjugate compound or a pharmaceutically acceptable salt thereof have three advantages.
  • the two targeting molecules can act in a variety of ways (usually synergistically) to improve the therapeutic effect while reducing side effects.
  • the combination of two targeting molecules increases the affinity or affinity of the conjugate compound or its pharmaceutically acceptable salt to the target receptor or target cell, thereby enhancing its specificity and avoiding off-target toxicity.
  • the combination of two targeting molecules can meet the multifunctional requirements usually required by drug conjugates.
  • the conjugate compound of the present application or its pharmaceutically acceptable salt has achieved unexpected technical effects, including but not limited to: (1) ligands that can bind to cell surface receptors and those that can mediate endocytosis The combination of synergistic molecules enables the conjugate compound to specifically enter the target cell; (2) the conjugate compound or a pharmaceutically acceptable salt thereof enhances the affinity and targeting specificity of the drug compound, thereby providing a patient Delivery of highly effective chemotherapeutic agents (such as MMAE) broadens the therapeutic window of such agents and avoids side effects; (3) The linker can prevent the release of payload outside the target cell (e.g., blood circulation system, intercellular substance, etc.), It ensures the stability of the conjugate compound in the blood circulation and reduces the toxicity of the drug.
  • highly effective chemotherapeutic agents such as MMAE
  • the linker After entering the target cell, the linker is cleaved and the payload is released, thereby exerting the effect of the drug. At the same time, it can avoid multiple drug resistance (MDR ); (4) A variety of drugs can be delivered in the form of the conjugate compound of the present application, thus expanding the application scope of related drugs. Therefore, the conjugate compound of the present application or a pharmaceutically acceptable salt thereof not only broadens the target range and therapeutic window of LDC drugs, but also reduces the toxicity and side effects of some drugs.
  • polypeptide used in this application may be a single amino acid or a polymer of amino acids.
  • the polypeptides, proteins or peptides described in this application may contain naturally-occurring amino acids, as well as non-naturally-occurring amino acids, or analogs and mimetics of amino acids.
  • Polypeptides, proteins or peptides can be obtained by any method well known in the art, such as but not limited to isolation and purification from natural substances, recombinant expression, chemical synthesis and the like.
  • Another aspect of the application discloses a pharmaceutical composition, which contains the conjugate compound provided in the application or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable salt refers to the relatively non-toxic, inorganic and organic acid addition salts and base addition salts of the conjugate compound of this application.
  • Representative acid addition salts include hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, oxalate, valerate, oleate, palmitate, Stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate , Naphthalate, methanesulfonate, glucoheptonate, lactobionate, sulfamate, malonate, salicylate, propionate, methylene-bis-b-hydroxynaphthalene Acid salt, gentisate, isethionate, di-p-toluoyl tartrate, methanesulfonate, ethanesulfonate, benz
  • Base addition salts include pharmaceutically acceptable metal and amine salts.
  • Suitable metal salts include sodium, potassium, calcium, barium, zinc, magnesium and aluminum salts. In some embodiments, sodium and potassium salts are preferred.
  • Suitable inorganic base addition salts are prepared from metal bases including, for example, sodium hydride, sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminum hydroxide, lithium hydroxide, magnesium hydroxide, and zinc hydroxide.
  • Suitable amine base addition salts are prepared from amines with sufficient basicity to form stable salts, and preferably include the following amines commonly used in medicinal chemistry because of their low toxicity and acceptable medical use: ammonia, ethylene Amine, N-methylglucamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine , N-benzyl phenethylamine, diethylamine, piperazine, trimethylolaminomethane, tetramethylammonium hydroxide, triethylamine, dibenzylamine, diphenylhydroxymethylamine, dehydroabietylamine, N -Ethyl piperidine, benzylamine, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, ethylamine, basic amino acids (for example,
  • pharmaceutically acceptable carrier refers to a pharmaceutically acceptable solvent, suspension or any other pharmaceutically inert carrier used to deliver the conjugate compound provided in this application to a subject. It does not interfere with the structure and properties of the conjugate compound. Certain such carriers are capable of formulating the conjugate compound into tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions, and pastilles for oral ingestion by subjects. Some of these carriers can make the conjugate compound into a preparation for injection, infusion or topical administration.
  • the pharmaceutically acceptable carriers used in the pharmaceutical compositions provided in this application include, but are not limited to, for example, pharmaceutically acceptable liquid, gel or solid carriers, aqueous carriers (for example, sodium chloride injection, Ringer Injection, isotonic dextrose injection, sterile water injection or dextrose and lactated Ringer's injection), non-aqueous carrier (for example, fixed oil of plant origin, cottonseed oil, corn oil, sesame oil Or peanut oil), antimicrobial agents, isotonic agents (for example, sodium chloride or dextrose), buffers (for example, phosphoric acid or citric acid buffers), antioxidants (for example, sodium bisulfate), anesthetics (for example, , Procaine hydrochloride), suspension/dispersant (for example, sodium carboxymethyl cellulose, hydroxypropyl methylcellulose or polyvinylpyrrolidone), chelating agent (for example, EDTA (ethylene diamine tetraacetic acid) Or EGTA (ethylene glycol
  • the pharmaceutical composition is an injection formulation.
  • Injection preparations include sterile aqueous solutions or dispersions, suspensions or emulsions. In all cases, the injection preparation should be sterile and fluid to facilitate injection. Injectable preparations should be stable under the conditions of production and storage, and the contamination of microorganisms such as bacteria and fungi must be prevented.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and/or vegetable oils. Injection preparations should maintain proper fluidity.
  • the proper fluidity can be maintained by using a coating such as lecithin, by using a surfactant, or the like.
  • a coating such as lecithin
  • a surfactant or the like.
  • Various antibacterial and antifungal agents can be used to prevent microorganisms, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like.
  • the pharmaceutical composition is an oral formulation.
  • Oral preparations include, but are not limited to, capsules, cachets, pills, tablets, lozenges (using a flavored base, usually sucrose and gum arabic or tragacanth), powders, granules, or solutions in aqueous or non-aqueous liquids Preparation or suspension, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as a pastille (using an inert base such as gelatin and glycerin, or sucrose and acacia) and/or as Mouthwash etc.
  • the conjugate compound is mixed with one or more pharmaceutically acceptable carriers, such as lemon Sodium or dicalcium phosphate, and/or any one of the following: (1) filler or extender, for example, starch, lactose, sucrose, glucose, mannitol and/or silicic acid; (2) sticky Mixtures, for example, carboxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and/or gum arabic; (3) humectants, for example, glycerin; (4) disintegrants, for example, agar, carbonic acid Calcium, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; (5) solution blockers, such as paraffin wax; (6) absorption enhancers, such as quaternary ammonium compounds; (7) wetting agents , For example, acetyl alcohol and
  • the conjugate compound is mixed with any of the following: pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs.
  • the liquid dosage form may contain inert diluents commonly used in the art, for example, water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol , Benzyl benzoate, isopropanol, 1,3-butanediol, oil (especially, cottonseed oil, peanut oil, corn oil, olive oil, castor oil and sesame oil), glycerin, tetrahydrofurfuryl alcohol, polyethylene glycol and Sorbitan fatty acid esters and mixtures thereof.
  • the oral composition may also contain adjuvants such
  • the pharmaceutical composition is an oral spray formulation or a nasal spray formulation.
  • Spray formulations include but are not limited to aqueous aerosols, non-aqueous suspensions, liposome formulations or solid particle formulations.
  • Aqueous aerosols are prepared by mixing an aqueous solution or suspension of the medicament with conventional pharmaceutically acceptable carriers and stabilizers. Carriers and stabilizers vary according to the requirements of the specific compound, but under normal circumstances, they include nonionic surfactants (Tween or polyethylene glycol), oleic acid, lecithin, amino acids, such as glycine, buffer solutions, Salt, sugar or sugar alcohol. Aerosols are usually prepared from isotonic solutions and can be delivered by spray.
  • the pharmaceutical composition can be used by mixing with one or more other drugs.
  • the pharmaceutical composition includes at least one other drug.
  • the other drugs are antitumor drugs, cardiovascular drugs, anti-inflammatory drugs, antiviral drugs, digestive system drugs, nervous system drugs, respiratory system drugs, immune system drugs, dermatological drugs, metabolic drugs, and the like.
  • the pharmaceutical composition can be administered to a subject in need through a suitable route, including but not limited to oral, injection (for example, intravenous, intramuscular, subcutaneous, intradermal, intracardiac, intrathecal, intrapleural , Intraperitoneal injection, etc.), mucosal (for example, intranasal, intraoral administration, etc.), sublingual, rectal, transdermal, intraocular, and pulmonary administration.
  • a suitable route including but not limited to oral, injection (for example, intravenous, intramuscular, subcutaneous, intradermal, intracardiac, intrathecal, intrapleural , Intraperitoneal injection, etc.), mucosal (for example, intranasal, intraoral administration, etc.), sublingual, rectal, transdermal, intraocular, and pulmonary administration.
  • the pharmaceutical composition can be administered intravenously, subcutaneously, orally, intramuscularly, or intraventricularly.
  • Another aspect of the application discloses a method of delivering a payload to a subject in need, the method comprising administering to the subject a therapeutically effective amount of the conjugate compound provided in the application, or a pharmaceutically acceptable salt thereof, Or the pharmaceutical composition provided in this application.
  • the payload described in this application can be any agent that researchers, veterinarians, doctors or other physicians are looking for to trigger a biological or medical response in tissues, systems, animal individuals or humans to prevent, inhibit, ameliorate or treat diseases.
  • Non-human animals include all vertebrates, such as mammals and non-mammals.
  • the subject can also be domestic animals, such as cattle, pigs, sheep, poultry, and horses, or domestic animals, such as dogs and cats.
  • the subject can be male (e.g., male) or female (e.g., female), and can be elderly, adult, adolescent, child, or infant.
  • People can be Caucasian, African, Asian, Semitic or other ethnic backgrounds, or a mixture of these ethnic backgrounds.
  • terapéuticaally effective amount refers to the amount of the conjugate compound or a pharmaceutically acceptable salt or pharmaceutical composition thereof that alleviates one or more symptoms of a disease or disorder in a subject to a certain extent ; An amount that partially or completely restores one or more physiological or biochemical parameters related to a disease or condition or that causes the disease or condition to normal; and/or an amount that reduces the likelihood of the disease or condition developing.
  • This amount usually changes according to a variety of factors, and those of ordinary skill in the art can determine and explain it according to the scope of the specification provided in this application. These include, but are not limited to: the specific subject and its age, weight, height, general physical condition and medical history, the specific compound used, the carrier of its formulation and the selected route of administration; and the nature and severity of the condition being treated.
  • the amount of the conjugate compound or a pharmaceutically acceptable salt or pharmaceutical composition thereof is sufficient to inhibit the disease or disorder in the subject, or prevent or prevent the onset of the disease or disorder.
  • the therapeutically effective amount can vary in different subjects, it usually ranges from 0.01 to 100 mg/kg, such as 0.01 to 90 mg/kg, 0.01 to 80 mg/kg, 0.01 to 70 mg/kg, 0.01 to 60 mg/kg, 0.01 to 50mg/kg, 0.01 to 40mg/kg, 0.01 to 30mg/kg, 0.01 to 20mg/kg, 0.01 to 10mg/kg, 0.01 to 5mg/kg, 0.01 to 4mg/kg, 0.01 to 3mg/kg, 0.01 to 2mg/ kg, 0.01 to 1 mg/kg, 0.01 to 0.1 mg/kg.
  • the therapeutically effective amount described in this application can be equal to any value within the aforementioned numerical range, including the end point of this range.
  • Another aspect of the application discloses a method for delivering a payload to a subject in need, the method comprising administering to the subject a therapeutically effective amount of the conjugate compound provided in the application or a pharmaceutically acceptable salt thereof, or The pharmaceutical composition provided in this application.
  • Another aspect of the application discloses a method for treating a disease in a subject, the method comprising administering to the subject a therapeutically effective amount of the conjugate compound provided in the application or a pharmaceutically acceptable salt thereof, or the application Provided pharmaceutical composition.
  • the disease is cancer, including but not limited to prostate cancer, breast cancer, lung cancer, kidney cancer, leukemia, ovarian cancer, stomach cancer, uterine cancer, endometrial cancer, liver cancer, thyroid cancer, pancreatic cancer, colon cancer , Colorectal cancer, esophageal cancer, skin cancer, lymphoma and multiple myeloma.
  • the cancer cells of the cancer have the expression of cell surface receptors or antigens mentioned in this application.
  • the cancer cells of the cancer have high expression of the cell surface receptors or antigens mentioned in this application (for example, according to Depmap data (see https://depmap.org/portal/), the corresponding gene expression is at least 0, 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10).
  • the cancer cell of the cancer has high expression of FOLR1 and FOLH1, TRPV6 and FOLH1, GNRHR and FOLH1, SSTR2 and FOLH1, FOLR1 and SSTR2, or TRPV6 and FOLR1.
  • the disease is an immune disease, for example, an autoimmune disease, including but not limited to connective tissue disease, systemic sclerosis, rheumatoid arthritis, and systemic lupus erythematosus.
  • the disease is a cardiovascular disease, including but not limited to angina pectoris, myocardial infarction, stroke, heart attack, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, cardiac arrhythmia, and congenital heart disease.
  • a cardiovascular disease including but not limited to angina pectoris, myocardial infarction, stroke, heart attack, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, cardiac arrhythmia, and congenital heart disease.
  • the disease is a metabolic disease, including but not limited to diabetes, gout, obesity, hypoglycemia, hyperglycemia, and dyslipidemia.
  • the disease is a neurological disease, including but not limited to Alzheimer's disease, Parkinson's disease, Huntington's disease, head injury, multiple sclerosis, vertigo, coma, and epilepsy.
  • the method provided in this application further includes the combined administration of one or more therapeutic agents with the conjugate compound or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition.
  • the therapeutic agent targets an anti-cancer treatment target, induces or enhances an immune response against cancer, or is a chemotherapeutic agent.
  • conjugate compounds CB-18G, CB-20B, CB-10S, CB-20C, FA-MMAE (the structure is shown below), and CB-20AK (the structure is shown below) can be obtained through similar steps as the above method ), CB-1020, CB-1320 and CB-1820.
  • Wang Resin Xi'an Lanxiao Technology New Materials Co., Ltd., article number 1365700-43-1
  • HPLC purity 84.6% Preparation and separation by HPLC (preparation conditions: C18 column, mobile phase A: 0.1% trifluoroacetic acid aqueous solution, B: acetonitrile, elution gradient (20-29)% B, time 60 minutes, collect fractions), will contain qualified products The fraction was freeze-dried to obtain 2.86g of 50S-SM09 with a purity of 97.6%.
  • conjugate compounds CB-60S and CB-60SK can be obtained through steps similar to the above method.
  • reaction solution was poured into acetic acid water, the solid was separated out, filtered, the filter cake was washed with acetic acid water, washed with water, and dried under vacuum to obtain 835.7mg CR19421 (structure as shown in the above reaction step), brown powder, HPLC purity: 90.0% , Yield: 90.6%.
  • Buffer HBS-EP+buffer 10X (GE, item number: BR100669), dilute 10 times with deionized water before use.
  • Regeneration reagent 10mM Glycine 2.0 (GE, article number: BR100355)
  • N/D means that no specific binding was detected.
  • Table 4 shows that CB-20BK binds specifically to FOLR1 and shows good affinity.
  • the binding affinity of CB-20BK and FOLR1 is slightly weaker than the binding affinity of FA or FA-MMAE to FOLR1.
  • CB-20AK does not have the folic acid part of CB-20BK, and no specific binding to FOLR1 was detected in the experiment.
  • Table 5 shows that CB-20BK binds to FOLH1 and shows good affinity.
  • FOLH1 binds to CB-20BK-FOLR1 complex
  • Buffer HBS-EP+buffer10X (GE, article number: BR100669), diluted 10 times with deionized water before use.
  • Regeneration reagent 10mM Glycine2.0 (GE, article number: BR100355)
  • CM5 chip coupling ligand FOLR1 (R&D System, article number 5646-FR)
  • Table 6 shows that the amount of high-concentration FOLH1 solution bound to CB-20BK-FOLR1 complex is significantly higher than that of FOLH1 and FA-MMAE-FOLR1 complex, and it continues to rise.
  • the combination of FOLH1 and FA-MMAE-FOLR1 complex tends to saturate at high concentrations. This experiment proved that CB-20BK can bind well to both FOLR1 and FOLH1 receptors at the same time.
  • Example 3 Study on binding and endocytosis of ligand conjugate to target cells
  • HPLC purity 76.3% Preparation and separation by HPLC (preparation conditions: C18 column, mobile phase A: 0.1% trifluoroacetic acid aqueous solution, B: acetonitrile, elution gradient (20-28)% B, time 50 minutes, collect fractions), will contain qualified products The fraction was lyophilized to obtain 736 mg of Cy5-pep-20BK with a purity of 93.4%.
  • Cell lines LNCaP human prostate cancer cells, Du145 human prostate cancer cells, SKOV3 human ovarian cancer cells, NCI-H460 human lung cancer cells
  • IMDM medium fetal bovine serum
  • penicillin-streptomycin solution penicillin-streptomycin solution
  • L-glutamine PBS
  • Cell culture Prepare the cells, trypsin digestion, collect and count, add the cells to several culture flasks containing complete medium, place them in a 37°C, 5% CO 2 incubator, and finally collect about 5 ⁇ 10 6 cells into the centrifuge tube.
  • RNA-Seq LNCaP SKOV3 Du145 NCI-H460 FOLR1 +/- 6+ +/- +/- FOLH1 10+ + +/- +/-
  • the data of 0 or negative is expressed as "-"
  • the data of 0.001-0.499 is expressed as "+/-”
  • the value of 0.500-1.499 is expressed as "+”
  • the data of 1.500-2.499 is expressed as "2+”
  • the fluorescence intensity of the cells was measured at 15 minutes, 30 minutes, 60 minutes, and 90 minutes of incubation, and the results are shown in Figure 2A and 2B. It can be seen that the sample Cy5-pep-20BK cells bind and endocytosis. In the LNCaP cell line with relatively high expression of FOLR1 and the SKOV3 cell line with relatively high expression of FOLH1, the fluorescence intensity of CB-20BK that binds and endocytoses is significantly higher than that of FOLR1. Fluorescence intensity in DU145 cell line and NCI-H460 cell line with relatively low expression of FOLH1. Cy5-pep-20AK has only FOLH1 ligand and no FOLR1 ligand FA.
  • Cy5-pep-20AK shows high levels of binding and endocytosis in LNCaP cells that highly express FOLH1.
  • SKOV3 cells with moderate FOLH1 expression they showed moderate levels of binding and endocytosis; in the two cell lines, Cy5-pep-20AK binding and endocytosis levels were significantly lower than Cy5-pep-CB-20BK.
  • the degree of internalization of the ligand conjugate and cell binding is positively correlated with the expression level of cell-related receptors.
  • Cell lines Hela cervical cancer cells, SKOV3 human ovarian cancer cells and A549 human lung cancer cells
  • IMDM medium fetal bovine serum
  • penicillin-streptomycin solution penicillin-streptomycin solution
  • L-glutamine penicillin-streptomycin
  • PBS anti-fluorescence quenching mounting tablets containing DAPI
  • Cell climbing film Put the climbing film in a 24-well plate. Prepare the cells, trypsin digestion, collect and count, use complete cell culture medium to dilute the cells to about 2 ⁇ 10 5 cells/ml, add 500 ⁇ l of the diluted cell solution to each well of the 24-well plate, and place at 37°C, 5% Incubate in a CO 2 incubator for 48 hours.
  • the binding and endocytosis of the sample Cy5-FA in 15 minutes and 30 minutes, the fluorescence intensity in the cell lines Hela and SKOV-3 with high FOLR1 expression is significantly higher than that in the cell lines with relatively low FOLR1 expression Fluorescence intensity in A549.
  • the degree of internalization of the ligand conjugate and cell binding is positively correlated with the expression level of cell-related receptors.
  • Cell lines KB human oral epidermoid carcinoma cells, T-47D human breast cancer cells, NCI-H460 human lung cancer cells, CALU-3 human lung adenocarcinoma cells, HuH-7 human liver cancer cells and LNCaP human prostate cancer cells
  • IMDM medium fetal bovine serum
  • penicillin-streptomycin solution penicillin-streptomycin solution
  • L-glutamine CCK8
  • the samples were diluted with culture medium to the desired concentration (see Table 8). Add 50 ⁇ l per well to a 96-well plate after overnight culture, 3 replicate wells, and set up a negative control and a blank control, and place it in a 37°C, 5% CO 2 incubator for 72 hours.
  • CB-20BK has a positive effect on the growth and expansion of KB, T-47D, NCI-H460, CALU-3, HuH-7 and LNCaP tumor cell lines. Has an inhibitory effect. Since the expression levels of the corresponding receptors of the conjugate compounds in the cells are different, the inhibition levels are different. The IC 50 of the cell lines T-47D, KB, LNCaP, HuH-7 and CALU-3 with relatively high receptor expression was significantly lower than that of the cell line NCI-H460 with relatively low receptor expression. CB-20BK shows the correlation between the anti-tumor effect and the expression level of cell-related receptors.
  • Conjugate compound CB-20BK and related compounds inhibit the expansion of tumor cell lines LNCaP (human prostate cancer cells) and 22RV1 (human prostate cancer cells)
  • FA-MMAE also has an inhibitory effect on the expansion of LNCaP and 22RV1 tumor cell lines. Because the expression level of FOLR1 of LNCaP and 22RV1 is very low, the expression in LNCaP cells (FOLR1 gene expression level is 0.3219, refer to Depmap data) is slightly higher than that in 22RV1 (FOLR1 gene expression level is 0.0704, refer to Depmap data), the difference between the IC 50 ratio of FA-MMAE between LNCaP and 22RV1 is only 1.5 times. The above data indicate that the cell proliferation inhibitory effect is positively correlated with the expression levels of cell expression receptors FOLH1 and FOLR1.
  • Conjugate compound CB-20BK and related compounds inhibit the expansion of tumor cell lines PANC-1 (human pancreatic cancer cell) and CFPAC-1 (human pancreatic cancer cell)
  • Cell lines A549 human lung cancer cells, HuH-7 human liver cancer cells, KB human oral epidermoid cancer cells, LNCaP human prostate cancer cells, DU145 human prostate cancer cells and T-47D human breast cancer cells
  • IMDM medium fetal bovine serum
  • penicillin-streptomycin solution penicillin-streptomycin solution
  • L-glutamine CCK8
  • the samples were diluted with culture medium to the desired concentration (see Table 11). Add 50 ⁇ l per well to a 96-well plate after overnight culture, 3 replicate wells, and set up a negative control and a blank control, and place it in a 37°C, 5% CO 2 incubator for 72 hours.
  • the four-parameter fit graph ( Figure 4B, wherein the C value corresponding to IC 50), CB-20B inhibited the growth of both amplification A549, HuH-7, KB, LNcap, DU145 , and T-47D tumor cell lines. Since the expression levels of the corresponding receptors of the conjugate compounds in the cells are different, the inhibition levels are different. The IC 50 of the cell lines T-47D, LNcap, KB, HuH-7 and DU145 with relatively high receptor expression was significantly lower than that of the cell line A549 with relatively low receptor expression. CB-20B shows the correlation between the anti-tumor effect and the expression level of cell-related receptors.
  • Cell lines KB human oral epidermoid carcinoma cells, NCI-H460 human lung cancer cells, RT4 human bladder cancer cells, T-47D human breast cancer cells and LNcap human prostate cancer cells
  • IMDM medium fetal bovine serum
  • penicillin-streptomycin solution penicillin-streptomycin solution
  • L-glutamine CCK8
  • the samples were diluted with culture medium to the desired concentration (see Table 12). Add 50 ⁇ l per well to a 96-well plate after overnight culture, 3 replicate wells, and set up a negative control and a blank control, and place it in a 37°C, 5% CO 2 incubator for 72 hours.
  • CB-10S has inhibitory effects on the growth and expansion of KB, LNCaP, T-47D, RT4 and NCI-H460 tumor cell lines. Since the expression levels of the corresponding receptors of the conjugate compounds in the cells are different, the inhibition levels are different. The IC 50 of the cell lines KB, LNcap, T-47D and RT4 with relatively high receptor expression was significantly lower than that of the cell line NCI-H460 with relatively low receptor expression. CB-10S shows the correlation between the anti-tumor effect and the expression level of cell-related receptors.
  • Cell lines KB human oral epidermoid carcinoma cells, T-47D human breast cancer cells, NCI-H460 human lung cancer cells, CALU-3 human lung adenocarcinoma cells, HuH-7 human liver cancer cells and LNCaP human prostate cancer cells
  • IMDM medium fetal bovine serum
  • penicillin-streptomycin solution penicillin-streptomycin solution
  • L-glutamine CCK8
  • the samples were diluted with culture medium to the desired concentration (see Table 13). Add 50 ⁇ l per well to a 96-well plate after overnight culture, 3 replicate wells, and set up a negative control and a blank control, and place it in a 37°C, 5% CO 2 incubator for 72 hours.
  • CB-60S has effects on the growth and expansion of KB, T-47D, NCI-H460, CALU-3, HuH-7 and LNCaP tumor cell lines Inhibition. Since the expression levels of the corresponding receptors of the conjugate compounds in the cells are different, the inhibition levels are different.
  • the IC 50 values of cell lines LNCaP and HuH-7 with relatively high receptor expression were significantly lower than cell lines with relatively low receptor expression NCI-H460, KB, T-47D and CALU-3.
  • CB-60S shows the correlation between the anti-tumor effect and the expression level of cell-related receptors.
  • Cell lines KB human oral epidermoid carcinoma cells, T-47D human breast cancer cells, NCI-H460 human lung cancer cells, CALU-3 human lung adenocarcinoma cells, HuH-7 human liver cancer cells and LNCaP human prostate cancer cells
  • IMDM medium fetal bovine serum
  • penicillin-streptomycin solution penicillin-streptomycin solution
  • L-glutamine CCK8
  • the samples were diluted with culture medium to the desired concentration (see Table 14). Add 50 ⁇ l per well to a 96-well plate after overnight culture, 3 replicate wells, and set up a negative control and a blank control, and place it in a 37°C, 5% CO 2 incubator for 72 hours.
  • CB-60SK has a positive effect on the growth and expansion of KB, T-47D, NCI-H460, CALU-3, HuH-7 and LNCaP tumor cell lines. Has an inhibitory effect. Since the expression levels of the corresponding receptors of the conjugate compounds in the cells are different, the inhibition levels are different. The IC 50 values of cell lines LNCaP and HuH-7 with relatively high receptor expression are significantly lower than those of cell lines T-47D, NCI-H460, CALU-3 and KB with relatively low receptor expression. CB-60SK shows the correlation between the anti-tumor effect and the expression level of cell-related receptors.
  • Cell lines A549 human lung cancer cells, Hela human cervical cancer cells, SCLC-21H small cell lung cancer cells, U-2OS human osteosarcoma cells, T-47D human breast cancer cells and NCI-H460 human lung cancer cells
  • IMDM medium fetal bovine serum
  • P penicillin-streptomycin solution P penicillin-streptomycin solution
  • L-glutamine CCK8
  • the samples were diluted with medium to the desired concentration (see Table 15). Add 50 ⁇ l per well to a 96-well plate after overnight culture, 3 replicate wells, and set up a negative control and a blank control, and place it in a 37°C, 5% CO 2 incubator for 72 hours.
  • CB-18G has a positive effect on the growth and expansion of A549, Hela, SCLC-21H, U-2OS, T-47D and NCI-H460 tumor cell lines. Has an inhibitory effect. Since the expression levels of the corresponding receptors of the conjugate compounds in the cells are different, the inhibition levels are different. The IC 50 of the cell line Hela with relatively high receptor expression is lower than that of the cell line NCI-H460 with relatively low receptor expression. CB-18G shows the correlation between the anti-tumor effect and the expression level of cell-related receptors.
  • Cell lines KB human oral epidermoid carcinoma cells, NCI-H460 human lung cancer cells, RT4 human bladder cancer cells, T-47D human breast cancer cells and LNCaP human prostate cancer cells
  • IMDM medium fetal bovine serum
  • penicillin-streptomycin solution penicillin-streptomycin solution
  • L-glutamine CCK8
  • the samples were diluted with culture medium to the desired concentration (see Table 16). Add 50 ⁇ l per well to a 96-well plate after overnight culture, 3 replicate wells, and set up a negative control and a blank control, and place it in a 37°C, 5% CO 2 incubator for 72 hours.
  • the four-parameter curve fit ( Figure 4G, the value of C which corresponds to the IC 50), CB-50S to KB, LNCaP, T-47D, RT4 , and NCI-H460 tumor cell lines are inhibited growth of amplification. Since the expression levels of the corresponding receptors of the conjugate compounds in the cells are different, the inhibition levels are different.
  • the IC 50 of the cell lines KB, LNcap, T-47D and RT4 with relatively high receptor expression was significantly lower than that of the cell line NCI-H460 with relatively low receptor expression.
  • CB-50S shows the correlation between the anti-tumor effect and the expression level of cell-related receptors.
  • both LNCaP and SK-BR-3 express TRPV6 and FOLRH1, but the expression of the two receptors of LNCap is relatively higher.
  • NCI-H226, CFPAC-1 and PANC-1 expressed low or weak expression of two receptors.
  • CB-1020 has inhibitory effects on the growth and expansion of LNCaP, SK-BR-3, NCI-H226, CFPAC-1 and PANC-1 tumor cell lines.
  • CB-1020 bis relatively high expression of receptor in the cell lines LNCaP the IC 50 ratio is above the moderate receptor expressing cell line SK-BR-3 IC 50 ratio; SK-BR-3 IC 50 is higher than the ratio of receptor has relatively low and CFPAC-1 expression were weakly expressed by the two cell lines NCI-H226, and PANC-1 IC 50 ratio.
  • the cell proliferation inhibitory effect is positively correlated with the expression levels of the two receptors in the cell.
  • LNCaP human prostate cancer cells
  • SK-BR-3 human breast cancer cells
  • MDA-MB-468 human breast cancer cells
  • CFPAC-1 Pancreatic cancer cells
  • Test CB-1820 and related compounds on the tumor cell lines LNCaP (human prostate cancer cells), MDA-MB-468 (human breast cancer cells), CFPAC-1 (pancreatic cancer cells) and PANC-1 (pancreatic cancer cells) ) Inhibition of amplification.
  • LNCaP human prostate cancer cells
  • MDA-MB-468 human breast cancer cells
  • CFPAC-1 pancreatic cancer cells
  • PANC-1 pancreatic cancer cells
  • Main cell lines KB human oral epidermoid carcinoma cells, MIA paca-2 human pancreatic cancer cells, HCC1954 human breast cancer cells, CALU-3 human lung adenocarcinoma cells and DU145 human prostate cancer cells.
  • Model construction resuscitate, culture cells, collect, count and inject into the right limb of BALB/c-nude mice subcutaneously, and when the tumor grows to 80-160mm 3 , group administration or transfer rapid expansion tumor mass to the expansion mouse Tumor model.
  • Grouping When the tumor grows to about 80-160mm 3 on average, group them into groups, and set up a model control group and different dose groups.
  • mice After tumor inoculation, routine monitoring includes the effect of tumor growth and treatment on the normal behavior of animals.
  • the specific content includes the activity of the experimental animals, food and water consumption, weight gain (weight measurement twice a week), eyes, coat and other abnormal conditions.
  • the model control group and the administration group are set up. The administration was started on the first day, and the dose was adjusted according to the weight of the mouse last time, and the dose was injected into the tail vein with a volume of 10 ⁇ l/g.
  • the monitoring includes the effects of tumor growth and treatment on the normal behavior of the animals.
  • the specific contents include the activity of the experimental animals, food and drinking conditions, weight gain or loss, eyes, coat and other abnormal conditions.
  • the weight of the mice was measured twice a week to calculate the rate of weight change; at the same time, the long diameter and short diameter of the tumor were measured with a vernier caliper, and the tumor volume, relative tumor growth rate, tumor volume inhibition rate and other indicators were calculated.
  • the test sample CB-20BK showed different degrees of tumor growth inhibition under the tail vein administration regimen.
  • LNCaP DU145 and NCI-H460 human cell line CDX models, compared with the negative control group, they all have a certain anti-tumor growth effect.
  • FOLR1 and FOLH1 dual expression model LNCaP at a dose of 3 mg/kg, administered on the 1, 8 and 15 days (D1, D8 and D15), the TGI value is 95.68%, and it has excellent anti-tumor growth
  • the effect is significantly better than the DU145 and NCI-H460 models with relatively low expression of FOLR1 and FOLH1.
  • the anti-tumor effect is related to the expression level of cell-related receptors.
  • mice Inoculate the prepared tumor tissue block into the subcutaneous front of the right limb of BALB/c-nude mice.
  • the model control group and the drug group are set up.
  • the administration was started on the first day of grouping, and the dose was adjusted according to the weight of the mouse last time.
  • the dose was injected into the tail vein with a volume of 10 ⁇ l/g and a dose of 3 mg/kg.
  • the monitoring includes the effects of tumor growth and treatment on the normal behavior of the animals.
  • the specific contents include the activity of the experimental animals, food and drinking conditions, weight gain or loss, eyes, coat and other abnormal conditions.
  • the weight of the mice was measured twice a week to calculate the rate of weight change; at the same time, the long diameter and short diameter of the tumor were measured with a vernier caliper, and the tumor volume, relative tumor growth rate, tumor volume inhibition rate and other indicators were calculated.
  • test sample CB-20BK (3 mg/kg) has a certain anti-tumor effect on LU1206, LU1380 and LU0367 human lung cancer PDX models.
  • the TGI value of the FOLR1 and FOLH1 dual expression model LU1206 is 90.89%, and the LU1380 and LU0367 single expression models have TGI values of 30.02% and 71.34%, respectively.
  • the tumor suppressor effect is correlated with the expression level of cell-related receptors. .
  • mice Inoculate the prepared tumor tissue block into the subcutaneous front of the right limb of BALB/c-nude mice.
  • the model control group and the drug group are set up.
  • the administration was started on the first day of grouping, and the dose was adjusted according to the weight of the mouse last time.
  • the dose was injected into the tail vein with a volume of 10 ⁇ l/g and a dose of 3 mg/kg.
  • the monitoring includes the effects of tumor growth and treatment on the normal behavior of the animals.
  • the specific contents include the mobility of the experimental animals, food and water consumption, weight gain or loss, eyes, coat and other abnormal conditions.
  • the weight of the mice was measured twice a week to calculate the rate of weight change; at the same time, the long diameter and short diameter of the tumor were measured with a vernier caliper, and the tumor volume, relative tumor growth rate, tumor volume inhibition rate and other indicators were calculated.
  • test sample CB-20BK has a TGI value of 96.49% and 70.74% in the FOLR1 and FOLH1 dual expression models BR1283 and BR0438 at a dose of 3 mg/kg, showing excellent anti-tumor growth effects.
  • CB-20BK has a higher expression of FOLR1 and FOLH1 in BR1283, and TGI is better.
  • Main cell lines KB human oral epidermoid carcinoma cells, PC-9 human lung cancer cells and DU145 human prostate cancer cells.
  • Model construction resuscitate, culture cells, collect, count and inject into the right limb of BALB/c-nude mice subcutaneously, and when the tumor grows to 80-160mm 3 , group administration or transfer rapid expansion tumor mass to the expansion mouse Tumor model.
  • Grouping When the tumor grows to about 80-160mm 3 on average, group them into groups, and set up a model control group and different dose groups.
  • mice After tumor inoculation, routine monitoring includes the effect of tumor growth and treatment on the normal behavior of animals.
  • the specific content includes the activity of the experimental animals, food and water consumption, weight gain (weight measurement twice a week), eyes, coat and other abnormal conditions.
  • Main cell lines KB human oral epidermoid carcinoma cells, PC-9 human lung cancer cells, SPC-A1 human lung adenocarcinoma cells, CALU-3 human lung adenocarcinoma cells and DU145 human prostate cancer cells
  • Model construction resuscitate, culture cells, collect and count cells, inject the cell fluid into the right limb of BALB/c-nude mice subcutaneously, and wait for the tumor to grow to 80-160mm 3 when the tumor grows to 80-160 mm 3 Block transfer to expand the mouse tumor model.
  • Grouping When the tumor grows to about 80-160mm 3 on average, group them into groups, and set up a model control group and different dose groups.
  • mice After tumor inoculation, routine monitoring includes the effect of tumor growth and treatment on the normal behavior of animals.
  • the specific content includes the activity of the experimental animals, food and water consumption, weight gain (weight measurement twice a week), eyes, coat and other abnormal conditions.
  • HPAF-II human pancreatic cancer cells
  • NCI-H226 human lung cancer cells
  • SCLC-21H small cell lung cancer cells
  • the model control group and the administration group are set up. The administration was started on the first day, and the dose was adjusted according to the weight of the mouse last time, and the dose was injected into the tail vein with a volume of 10 ⁇ l/g.
  • the monitoring includes the effects of tumor growth and treatment on the normal behavior of the animals.
  • the specific contents include the activity of the experimental animals, food and drinking conditions, weight gain or loss, eyes, coat and other abnormal conditions.
  • the weight of the mice was measured twice a week to calculate the rate of weight change; at the same time, the long diameter and short diameter of the tumor were measured with a vernier caliper, and the tumor volume, relative tumor growth rate, tumor volume inhibition rate and other indicators were calculated.
  • CB-1020 has obvious anti-tumor growth effects on human HPAF-II, CB-1320 on NCI-H226 and SCLC-21H, and CB-1820 on SCLC-21H cell line subcutaneous allograft models.
  • mice Inoculate the prepared cells into the subcutaneous front of the right limb of BALB/c-nude mice.
  • the model control group and the administration group are set.
  • the administration was started on 1 day, and the dosage was adjusted according to the weight of the mouse last time.
  • the administration volume was 10 ⁇ l/g via tail vein injection, and the administration was administered twice a week for 3 weeks.
  • the monitoring includes the effects of tumor growth and treatment on the normal behavior of the animals.
  • the specific contents include the activity of the experimental animals, food and drinking conditions, weight gain or loss, eyes, coat and other abnormal conditions.
  • the weight of the mice was measured twice a week to calculate the rate of weight change; at the same time, the long diameter and short diameter of the tumor were measured with a vernier caliper, and the tumor volume, relative tumor growth rate, tumor volume inhibition rate and other indicators were calculated.
  • CR-19428 is a conjugate of a ligand of FOLR1 and FOLH1 and a drug with a payload of Dxd. It can be seen from the above table that it has a significant anti-tumor growth effect on human CALU-3, SCLC-21H and SPC-A1 cell lines subcutaneous allograft models.
  • This experiment is the third generation of lung cancer PDX model LU2505 (from Asian female patients), which is a fast-growing tumor model.
  • BALB/c nude mice are subcutaneously bearing tumors, and the tumor volume is about 150mm 3.
  • Groups are divided into groups: low, medium and high test product groups, small molecule MMAE and targeting peptide 20BK-SM09 control group, and blank preparation control group, total 6 Group, 8 animals/group; administration on 1, 8 and 15 days after grouping, and continued observation for 14 days after the last administration.
  • the active substance of the test product CBP-1018 is CB-20BK, which is obtained by mixing CB-20BK with auxiliary materials and then freeze-drying.
  • CBP-1018 for injection has obvious dose correlation: the low-dose group is ineffective, the middle and high-dose group is effective, and the tumor in the high-dose group has been completely cured on day 19 (D19), and on day 29 ( D29) No signs of growth were seen.
  • the peptide group 20BK-SM09 has no obvious anti-tumor effect; it suggests that the targeted peptide alone is not enough to produce a clear anti-tumor effect.
  • the MMAE group has a clear anti-tumor effect.
  • the dose of 0.375mg/kg is that CBP-1018 contains equimolar MMAE at 1.5mg/kg.
  • the comparison shows that CBP-1018 is significantly better than the MMAE group at 1.5mg/kg, suggesting the ligand The advantage of targeting (82.60% vs 48.97%).
  • This experiment is the fifth generation of lung cancer PDX model LU1206 (from Asian female patients), which is a fast-growing tumor model.
  • BALB/c nude mice are subcutaneously bearing tumors, and the tumor volume is about 150mm 3.
  • Groups are divided into groups: low, medium and high test product groups, small molecule MMAE and targeting peptide 20BK-SM09 control group, and blank preparation control group, total 6 Group, 8 animals/group; administration on 1, 8 and 15 days after grouping, and continued observation for 14 days after the last administration.
  • CBP-1018 for injection has obvious dose correlation: the low-dose group is ineffective (the tumor inhibition rate is 8.08%), and the middle and high-dose groups are effective, and the tumor inhibition rates are 75.21% and 97.42%, respectively.
  • the difference between low and medium doses is relatively large.
  • the polypeptide group 20BK-SM09 has no obvious anti-tumor effect, suggesting that the targeted polypeptide alone is not enough to produce a clear anti-tumor effect in this model.
  • the MMAE group has a clear anti-tumor effect.
  • the dose of 0.375mg/kg means that CBP-1018 contains equimolar MMAE at 1.5mg/kg; comparison shows that CBP-1018 is significantly better than the MMAE group at 1.5mg/kg, suggesting the advantage of ligand targeting (tumor volume suppression Rate: 75.21% vs 36.03%, tumor suppression rate: 78.38% vs 54.53%).
  • the amount of radioactivity was measured using a liquid scintillation counter.
  • the amount of radioactivity measured in samples such as bile, urine, feces, cage washing and cleaning fluid, and corpses is used to calculate the percentage of the dose.
  • the amount of radioactivity in the plasma sample is used to calculate the total radioactivity in each gram of the sample.
  • WinNonLin software version 7.0, Pharsight is used to calculate the main pharmacokinetic parameters of the total plasma radioactivity according to the non-compartmental model.
  • CBP-1018 is mainly excreted through urine, which accounts for about 57% of the total dose; less than 25% is excreted through stool.
  • the result of excretion from the urine mainly through the kidney is consistent with the large amount of tissue distribution in the kidney of nude mice.
  • CBP-1008 1 ⁇ g/mL, 10 ⁇ g/mL and 100 ⁇ g/mL CBP-1008 (the active substance is LDC10B described in WO2017025047A1, CBP-1008 is obtained by mixing LDC10B with excipients and then freeze-drying. WO2017025047A1 is incorporated by reference in its entirety) And CBP-1018, incubated with plasma of different species at 37°C for 2 hours to observe the stability of the two compounds. The results show (Table 30) that the stability of CBP-1018 in various types of plasma, relative to the 0 hour concentration and the remaining percentage after 2 hours of incubation, is above 90%. While CBP-1008 is only stable in rat plasma (87.92-91.82%), and it is unstable in plasma of other species, with only 0.751%-24.52% remaining.
  • the data in the table is the percentage of plasma compound concentration at 0 hour after plasma incubation for 2 hours;

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Abstract

一种偶联体化合物或其药学上可接受的盐，所述偶联体化合物或其药学上可接受的盐包含有效载荷和两个靶向分子。还涉及一种药物组合物，所述药物组合物包含偶联体化合物或其药学上可接受的盐。另外涉及一种用于向有需要的对象递送有效载荷的方法以及一种治疗疾病的方法，所述方法均包括向所述对象施用治疗有效量的偶联体化合物或其药学上可接受的盐，或者药物组合物。

Description

双配体药物偶联体及其用途 技术领域
本申请涉及生物医药化学领域。更具体地，本申请涉及双配体药物偶联体、包括所述双配体药物偶联体的药物组合物以及使用所述双配体药物偶联体向有需要的对象递送有效载荷的方法和使用其治疗疾病的方法。
背景技术
通常，病变细胞的病理及生理特征都与正常细胞有显著不同，其中之一表现为病变细胞表面具有特异性或过度表达的物质(例如，抗原、化学信号、受体等)，这些物质在正常细胞中无表达或低表达。基于这一原理，研发了抗体-药物偶联体(ADC)和多肽-药物偶联体(PDC)来治疗疾病。目前，虽然已有一些ADC和PDC药物上市或进入临床研究，但由于这些药物设计原理的原因，ADC和PDC在临床应用中均具有很大的局限性。
由于ADC的复杂性和具有较大分子量，使其研发面临着许多困难，包括缺乏适宜的靶点、生产困难和药物稳定性较差。目前，ADC主要用于肿瘤治疗领域。在一些情况下，靶向抗体对癌细胞表面抗原的亲和性能够高达10 -9～10 -12(Kd，摩尔/升)，因此在对靶细胞具有较高特异性的同时，ADC对与靶细胞具有相同靶向受体的正常细胞也具有较高的特异性。同时，由于ADC在体内的代谢时间较长(1至3周)，在此期间其会不断地杀伤正常细胞，因而极大地增加了ADC的毒副作用。因此，ADC更理想的适应症应当是其特征为在肿瘤和正常细胞中的细胞表面抗原量相差非常悬殊的疾病。然而，目前所知满足此严格要求的疾病非常少。
PDC在临床或临床前研究中用于治疗多种疾病，但这些只是简单的把化疗药物与多肽连接，或在包埋了化疗药物的纳米颗粒或高分子材料里添加多肽，这样会使得大部分多肽由于分子量较大以及带有电荷导致其无法进入细胞，因此目前此类PDC大多数只适用于胞外治疗，严重限制了PDC的应用范围和药效。
药物偶联体化合物还可以是配体-药物偶联体(LDC)，其中配体可以是肽或小分子。然而，从生物利用度、稳定性、疗效到毒性等方面，LDC的应用存在很多问题。例如，很多配体由于其具有较大分子量、亲脂性或其他属性使其不能进入细胞，这限制了其治疗应用。此外，如果配体与常规化疗药物(例如，多柔比星、紫杉醇等)偶联，则疗效通常较低，而如果其与高效药物分子(例如，MMAE、DM1)偶联，则毒性较大，甚至可能在达到肿瘤治疗的治疗有效量之前就导致动物中毒死亡。
因此领域内还迫切需要获得改进的LDC，使其可以作用于病变细胞表面广泛存在的较高表达受体、拓宽靶向范围和治疗窗口以及增强药效避免药物副作用。
发明内容
本申请的一个方面公开了一种偶联体化合物或其药学上可接受的盐，所述偶联体化合物或其药学上可接受的盐包含有效载荷和两个靶向分子，其中所述两个靶向分子分别为协同作用分子部分以及前列腺特异性膜抗原配体部分。
本申请的另一方面公开了一种偶联体化合物或其药学上可接受的盐，所述偶联体化合物或其药学上可接受的盐包含有效载荷和两个靶向分子，其中所述两个靶向分子分别为协同作用分子部分以及具有式(I)所示的配体部分：
Figure PCTCN2020074117-appb-000001
本申请的另一方面公开了一种偶联体化合物或其药学上可接受的盐，所述偶联体化合物或其药学上可接受的盐包含有效载荷和两个靶向分子，其中所述两个靶向分子分别为协同作用分子部分以及P10，并且所述有效载荷为喜树碱及其任何衍生物。
在一些实施方式中，偶联体化合物或其药学上可接受的盐包含的前列腺特异性膜抗原配体包括如下结构：
Figure PCTCN2020074117-appb-000002
在一些实施方式中，偶联体化合物或其药学上可接受的盐包含的前列腺特异性膜抗原配体包括如下结构：
Figure PCTCN2020074117-appb-000003
在一些实施方式中，偶联体化合物或其药学上可接受的盐包含的前列腺特异性膜抗原配体包括如下结构：
Figure PCTCN2020074117-appb-000004
在一些实施方式中，偶联体化合物或其药学上可接受的盐包含的前列腺特异性膜抗原配体包括如下结构：
Figure PCTCN2020074117-appb-000005
在一些实施方式中，偶联体化合物或其药学上可接受的盐包含的两个靶向分子是不同的。
在一些实施方式中，偶联体化合物或其药学上可接受的盐包含的协同作用分子是细胞相互作用分子。
在一些实施方式中，偶联体化合物或其药学上可接受的盐包含的两个靶向分子与不同的细胞分子相互作用的细胞相互作用分子。
在一些实施方式中，偶联体化合物或其药学上可接受的盐包含的协同作用分子是能够介导内吞作用的内吞作用分子。
在一些实施方式中，偶联体化合物或其药学上可接受的盐包含的协同作用分子与选自下组的分子结合：FOLR1、TRPV6、FOLH1(PMSA)、GNRHR、Her2、Trop2、Her3、NECTIN4、LRP1、GLUT1、EGFR1、AXL、CA9、CD44、Claudin18.2、APN、DLL3、CEACAM5、FZD10、TFRC、MET、IGFR1、SSTR2、CCKBR、LFA1、ICAM、GPR87、GM-CSF、GM-CSFR、TIM3、TLR家族、CD40、CD40L、OX40、OX40L、GITRL、GITR、4-BBL、4-1BB、CD70、CD27、ICOSL、ICOS、HHLA2、CD28、CD86/80、CD28、MHCII抗原、TCR、CTLA-4、CD155、CD122、CD113、IGIT、PD-L1、PD1、Galectin-9、TIM-3、HVEM、BTLA、CD160、VISTA、B7-H4、B7-H3、磷脂酰丝氨酸、HHLA2、LAG3、Galectin-3、LILRB4、SIGLEC15、NKG2A、NKG2D、SLAMF7、KIR2DL1、KIR2DL2、KIR2DL3、FGFR1、FGFR2、FGFR4、NeuGcGM3和CXCR4。
在一些实施方式中，偶联体化合物或其药学上可接受的盐包含前列腺特异性膜抗原配体部分以及协同作用分子部分，所述协同作用分子部分与选自下组的分子结合：FOLR1、TRPV6、FOLH1(PMSA)、SSTR2和GNRHR。
在一些实施方式中，偶联体化合物或其药学上可接受的盐包含式(I)所示的配体部分以及协同作用分子部分，所述协同作用分子部分与选自下组的分子结合：FOLR1、TRPV6、SSTR2和GNRHR。
在又一些实施方式中，偶联体化合物或其药学上可接受的盐包含P10以及协同作用分子部分，所述协同作用分子与选自下组的分子结合：FOLR1、TRPV6、FOLH1(PMSA)和GNRHR。
在一些实施方式中，偶联体化合物或其药学上可接受的盐包含的协同作用分子是叶酸或其类似物。在一些实施方式中，叶酸类似物选自下组：5-甲基四氢叶酸、5-甲酰基四氢叶酸、甲氨蝶呤和5,10-亚甲基四氢叶酸。
在一些实施方式中，偶联体化合物或其药学上可接受的盐包含一个、两个、三个、四个或更多个有效载荷。在一些实施方式中，有效载荷选自下组：小分子化合物、核苷酸、肽、和蛋白。在一些实施方式中，有效载荷是小分子化合物。在一些实施方式中，小分子化合物选自下组：喜树碱及其任何衍生物、澳瑞他汀及其任何衍生物、美登素及其任何衍生物、放射性核素络合物、环氧化酶-2抑制剂、紫杉醇及其任何衍生物、埃博霉素及其任何衍生物、博来霉素及其任何衍生物、更生霉素及其任何衍生物、普卡霉素及其任何衍生物，以及丝裂霉素C。在一些实施方式中，小分子化合物是喜树碱及其任何衍生物、澳瑞他汀及其任何衍生物、美登素及其任何衍生物、放射性核素络合物或者环氧化酶-2抑制剂。
在一些实施方式中，本申请的偶联体化合物或其药学上可接受的盐包含的有效载荷通过连接子与至少一个所述靶向分子连接。
在一些实施方式中，本申请的偶联体化合物或其药学上可接受的盐包含的连接子是肽类连接子、二硫化物连接子、pH依赖型连接子或者上述连接子的组合。
在一些实施方式中，肽类连接子可在特定的生理环境下通过蛋白酶裂解或还原裂解。在一些实施方式中，肽类连接子选自下组：半胱氨酸、赖氨酸、赖氨酸-赖氨酸、缬氨酸-瓜氨酸、苯丙氨酸-赖氨酸、缬氨酸-赖氨酸、半胱氨酸-赖氨酸、半胱氨酸-谷氨酸、天冬氨酸-天冬氨酸和天冬氨酸-天冬氨酸-赖氨酸，可选的，上述氨基酸中的羧酸被酰胺化。
在一些实施方式中，二硫化物连接子选自下组：DMDS、MDS、DSDM和NDMDS。
在一些实施方式中，pH依赖型连接子是顺乌头酸酐。
在一些实施方式中，本申请的偶联体化合物或其药学上可接受的盐的连接子包括如下结构：
Figure PCTCN2020074117-appb-000006
Figure PCTCN2020074117-appb-000007
Figure PCTCN2020074117-appb-000008
或者所述连接子是上述结构与肽类连接子的组合。
在一些实施方式中，本申请的偶联体化合物或其药学上可接受的盐包含的两个靶向分子通过间隔区连接。在一些实施方式中，本申请所述的间隔区包含选自下组的氨基酸序列：SEQ ID NO：1-14、Arg-Arg、Ala-Ser-Asn、Ala-Ala-Ala、Ser-Ser-Arg、Pro-Arg和Pro-Leu-Gly。
在一些实施方式中，本申请的偶联体化合物为CB-20B，其结构式如下：
Figure PCTCN2020074117-appb-000009
在一些实施方式中，本申请的偶联体化合物为CB-20BK，其结构式如下：
Figure PCTCN2020074117-appb-000010
在一些实施方式中，本申请的偶联体化合物为CB-60S，其结构式如下：
Figure PCTCN2020074117-appb-000011
在一些实施方式中，本申请的偶联体化合物为CB-60SK，其结构式如下：
Figure PCTCN2020074117-appb-000012
在一些实施方式中，本申请的偶联体化合物为CB-20C，其结构式如下：
Figure PCTCN2020074117-appb-000013
在一些实施方式中，本申请的偶联体化合物为CB-1020，其结构式如下：
Figure PCTCN2020074117-appb-000014
在一些实施方式中，本申请的偶联体化合物为CB-1320，其结构式如下：
Figure PCTCN2020074117-appb-000015
在一些实施方式中，本申请的偶联体化合物为CB-1820，其结构式如下：
Figure PCTCN2020074117-appb-000016
在一些实施方式中，本申请的偶联体化合物为CR19428，其结构式如下：
Figure PCTCN2020074117-appb-000017
在一些实施方式中，本申请的偶联体化合物为20R-SM09，其结构式如下：
Figure PCTCN2020074117-appb-000018
在一些实施方式中，本申请的偶联体化合物为CB-20R，其结构式如下：
Figure PCTCN2020074117-appb-000019
其中M为放射性核素。
在一些实施方式中，本申请的偶联体化合物为CB-18G，其结构式如下：
Figure PCTCN2020074117-appb-000020
在一些实施方式中，本申请的偶联体化合物为CR19426，其结构式如下：
Figure PCTCN2020074117-appb-000021
在一些实施方式中，本申请的偶联体化合物为CB-10S，其结构式如下：
Figure PCTCN2020074117-appb-000022
在一些实施方式中，本申请的偶联体化合物为CR19425，其结构式如下：
Figure PCTCN2020074117-appb-000023
在一些实施方式中，本申请的偶联体化合物为CB-50S，其结构式如下：
Figure PCTCN2020074117-appb-000024
本申请的另一方面公开了一种药物组合物，其包含本申请中的偶联体化合物或其药学上可接受的盐，以及药学上可接受的载体。
在一些实施方式中，所述组合物用于静脉内、皮下、口服、肌内或心室内施用。
本申请的另一方面公开了用于向有需要的对象递送有效载荷的方法，包括向所述对象施用治疗有效量的本申请中所述的偶联体化合物或其药学上可接受的盐、或者本申请中所述的药物组合物。
本申请的另一方面公开了一种用于治疗对象中的疾病的方法，包括向所述对象施用治疗有效量的本申请中所述的偶联体化合物或其药学上可接受的盐，或者本申请中所述的药物组合物。
在一些实施方式中，本申请中用于治疗对象中的疾病的方法还包括将一种或多种治疗剂与所述偶联体化合物或其药学上可接受的盐，或者所述药物组合物联合施用。
本申请的又一方面公开了一种本申请中所述的偶联体化合物或其药学上可接受的盐，或者本申请中所述的药物组合物在制备治疗对象中疾病的药物中的用途。
本申请的又一方面公开了一种用于治疗对象中疾病的本申请中所述的偶联体化合物或其药学上可接受的盐，或者本申请中所述的药物组合物。
在一些实施方式中，疾病选自下组：癌症、免疫性疾病、心血管疾病、代谢疾病和神经疾病。
在一些实施方式中，癌症选自下组：前列腺癌、乳腺癌、肺癌、肾癌、白血病、卵巢癌、胃癌、子宫癌、子宫内膜癌、肝癌、结肠癌、甲状腺癌、胰腺癌、结直肠癌、食道癌、皮肤癌、淋巴瘤和多发性骨髓瘤。
在一些实施方式中，免疫性疾病是自身免疫性疾病。在一些实施方式中，自身免疫性疾病选自下组：结缔组织病、系统性硬化症、类风湿性关节炎和系统性红斑狼疮。
在一些实施方式中，心血管疾病选自下组：心绞痛、心肌梗死、中风、心脏病发作、高血压性心脏病、风湿性心脏病、心肌病、心脏心律失常和先天性心脏病。
在一些实施方式中，代谢疾病选自下组：糖尿病、痛风、肥胖症、低血糖症、高血糖症和血脂异常。
在一些实施方式中，神经疾病选自下组：阿尔茨海默病、帕金森病、亨廷顿病、头部损伤、多发性硬化症、眩晕、昏迷和癫痫。
附图说明
图1显示了偶联体化合物CB-20B、CB-20BK、CB-60S、CB-60SK、CB-20C、CB-1020、CB-1320、CB-1820、CR19428、20R-SM09、CB-20R、CB-18G、CR19426、CB-10S、CR19425和CB-50S的化学结构式。
图2A显示了Cy5-pep-20BK与不同细胞结合和内吞的随时间变化的曲线(从上往下依次为LNCaP细胞、SKOV3细胞、DU145细胞和NCI-H460细胞)。图2B显示了Cy5-pep-20AK与不同细胞结合和内吞的随时间变化的曲线(从上往下依次为LNCaP细胞、SKOV3细胞、DU145细胞和NCI-H460细胞)。
图3显示了Cy5-FA与不同细胞结合和内吞的随时间变化的荧光照片。其中显示完整的圆形的为细胞核的荧光，呈点状分布的为Cy5-FA的荧光。
图4A显示了偶联体化合物CB-20BK对图示的肿瘤细胞扩增的抑制活性。图4B显示了偶联体化合物CB-20B对图示的肿瘤细胞扩增的抑制活性。图4C显示了偶联体化合物CB-10S对图示的肿瘤细胞扩增的抑制活性。图4D显示了偶联体化合物CB-60S对图示的肿瘤细胞扩增的抑制活性。图4E显示了偶联体化合物CB-60SK对图示的肿瘤细胞扩增的抑制活性。图4F显示了偶联体化合物CB-18G对图示的肿瘤细胞扩增的抑制活性。图4G显示了偶联体化合物CB-50S对图示的肿瘤细胞扩增的抑制活性。
图5A-5E显示了偶联体化合物CB-20BK在小鼠体内对肿瘤的抑制效果。
图6A-6C显示了偶联体化合物CB-20B在小鼠体内对肿瘤的抑制效果。
图7A-7E显示了偶联体化合物CB-18G在小鼠体内对肿瘤的抑制效果。
图8A-8B显示了注射用CBP-1018对肺癌LU2505模型和肺癌LU1206模型肿瘤体积的影响。
具体实施方式
尽管本申请将公开各种方面和实施方式，但是显而易见的是，在不偏离本申请的主旨和范围的前提下，本领域技术人员可以对这些方面和实施方式进行各种等同改变和修改。本申请公开的各个方面和实施方式仅用于说明目的，并非旨在限制，真正的范围由所附权利要求表示。本申请引用的所有出版物、专利或专利申请通过引用整体并入本申请。除非另外说明，否则本申请中使用的所有科技术语具有的含义与本申请所属领域技术人员通常理解的含义相同。
如本文和所附权利要求中所使用的，单数形式“一”、“一个”、“一种”和“所述”包括复数对象，除非上下文清楚地表示不是这样。术语“一”(或“一个”)、“一个或 多个”和“至少一个”在本申请中可以互换使用。还应当注意的是，术语“包含”、“包括”和“具有”可以互换使用。
如本文和所附权利要求中所使用的，术语“类似物”包括结构类似物与功能类似物。其中结构类似物是指具有相似化学结构的一类化合物，它们之间可以存在一个或多个不同的原子、或一个或多个不同的官能团。功能类似物是指具有相同或相似的化学作用、生物作用或药理学作用的一类化合物。例如，叶酸类似物包括了5-甲基四氢叶酸、5-甲酰基四氢叶酸、甲氨蝶呤和5,10-亚甲基四氢叶酸。
如本文和所附权利要求中所使用的，术语“衍生物”是指针对某一母体化合物分子，其一个或多个原子或原子团被其他原子或原子团取代而衍生出的一类较复杂的化合物。例如，喜树碱衍生物包括了伊立替康、SN-38、Dxd、拓扑替康、GI-147211C、托泊替康、9-氨基喜树碱、7-羟甲基喜树碱、7-氨基甲基喜树碱、10-羟基喜树碱、(20S)-喜树碱、9-硝基喜树碱、吉马替康、karenitecin、silatecan、勒托替康、依沙替康、二氟替康、贝洛替康、勒托替康和S39625
本申请的一个方面公开了一种偶联体化合物或其药学上可接受的盐，所述偶联体化合物或其药学上可接受的盐包含有效载荷和两个靶向分子，其中所述两个靶向分子分别为协同作用分子部分以及前列腺特异性膜抗原配体部分。
本申请的另一方面公开了一种偶联体化合物或其药学上可接受的盐，所述偶联体化合物或其药学上可接受的盐包含有效载荷和两个靶向分子，其中所述两个靶向分子分别为协同作用分子部分以及具有式(I)所示的配体部分：
Figure PCTCN2020074117-appb-000025
本申请的另一方面公开了一种偶联体化合物或其药学上可接受的盐，所述偶联体化合物或其药学上可接受的盐包含有效载荷和两个靶向分子，其中所述两个靶向分子分别为协同作用分子部分以及P10，并且所述有效载荷为喜树碱及其任何衍生物。
在本申请中使用的术语“有效载荷”指的是拟递送至靶细胞或组织的分子或物质。非限制性地，有效载荷可以是旨在用于诊断、治疗或预防对象中的疾病的任意分子或物质。在一些实施方案中，所述有效载荷具有小于或等于约5kDa的分子量。在一些实施方案中，所述有效载荷具有小于或等于约1.5kDa的分子量。在一些实施方案中，所述有效载荷是已 经被适当的药物审批和注册机构(例如，FDA，EMEA或NMPA)认为使用安全且有效的药物或诊断试剂。
在一些实施方式中，本申请的有效载荷是小分子化合物、核苷酸(例如，DNA、质粒DNA、RNA、siRNA、反义寡核苷酸或核酸适体等)、肽或蛋白(例如，酶)。在一些实施方式中，有效载荷是小分子化合物。
在一些实施方式中，本申请的有效载荷包括但不限于：抗癌药、放射性物质、维生素、抗-AIDS药物、抗生素、免疫抑制剂、抗病毒药物、酶抑制剂、神经毒素、阿片样药物、细胞-胞外基质相互作用的调节剂、血管扩张剂、抗高血压药、安眠药、抗组胺剂、抗惊厥药、肌肉松弛药、抗-帕金森物质、抗-痉挛药和肌肉收缩药、抗-寄生虫和/或抗-原生动物药、镇痛药、解热剂、甾体和非甾体抗炎药、抗-血管生成因子、抗分泌因子、抗凝血剂和/或抗血栓形成剂、局部麻醉药、前列腺素、抗抑郁剂、抗精神病药物、止吐药或成像剂。
在一些实施方式中，本申请的有效载荷在连接至本申请的偶联体化合物之前具有游离的氨基或者羧基，有效载荷通过上述氨基或者羧基与偶联体化合物的相应部分(例如，连接子)的基团发生酰化反应从而偶联至偶联体化合物。在一些实施方式中，对上述游离的氨基或者羧基的修饰(例如，通过偶联至本申请的偶联体化合物)会显著降低有效载荷的活性(例如，降低至少50％、60％、70％、80％、90％、95％、98％或99％)。
在本申请中使用的“小分子化合物”指的是具有小于或等于约2kDa分子量的化合物。在一些实施方式中，小分子化合物具有小于或等于约1.5kDa的分子量。在一些优选的实施方式中，小分子化合物具有小于或等于约1kDa、800Da、700Da、600Da或500Da的分子量。在一些实施方式中，本申请的小分子化合物选自下组：喜树碱及其任何衍生物(例如，SN38或Dxd)、澳瑞他汀及其任何衍生物(例如，MMAE和MMAF)、美登素及其任何衍生物、环氧化酶-2抑制剂(例如，塞来昔布)、放射性核素络合物、紫杉醇及其任何衍生物、埃博霉素及其任何衍生物、博来霉素及其任何衍生物、更生霉素及其任何衍生物、普卡霉素及其任何衍生物，以及丝裂霉素C。在一些实施方式中，小分子化合物是喜树碱及其任何衍生物、澳瑞他汀及其任何衍生物、放射性核素络合物或者环氧化酶-2抑制剂。在一些实施方式中，本申请所述的小分子化合物是用于缓解或治疗癌症的药物。在一些实施方式中，本申请所述的小分子化合物是用于缓解或治疗自身免疫性疾病的药物。
本申请中使用的术语“喜树碱”是指一种细胞毒性生物碱，主要来源于珙桐科植物喜树，其显示出较强的抗肿瘤活性。本申请的喜树碱及其衍生物包括目前已经存在的或者之后产生的喜树碱及其衍生物。本申请的喜树碱及其衍生物包括但不限于：喜树碱、伊立替康、SN-38、Dxd、拓扑替康、GI-147211C、托泊替康、9-氨基喜树碱、7-羟甲基喜树碱、7-氨基甲基喜树碱、10-羟基喜树碱、(20S)-喜树碱、9-硝基喜树碱、吉马替康、karenitecin、silatecan、勒托替康、依沙替康、二氟替康、贝洛替康、勒托替康和S39625。
本申请中使用的术语“澳瑞他汀及其任何衍生物”是指天然抗肿瘤产品海兔毒素10以及其一系列衍生物，该类化合物通过干扰微观自组装使细胞停滞于有丝分裂期，对细胞具有很强的杀伤力。本申请的澳瑞他汀及其任何衍生物包括目前已经存在的或者之后产生的澳瑞他汀及其任何衍生物。本申请的奥瑞他汀及其衍生物包括但不限于澳瑞他汀、单甲基澳瑞他汀E(MMAE)、单甲基澳瑞他汀F(MMAF)、一甲基澳瑞他汀D(MMAD)、AFP和AFHPA。
本申请中使用的术语“环氧化酶-2抑制剂”是一类特异性的环氧化酶-2抑制剂。环氧化酶-2通过多种机制参与恶性肿瘤的发展和浸润，环氧化酶-2抑制剂能抑制肿瘤细胞的迁移和黏附以及血管内浸润，从而抑制恶性肿瘤的发生和发展。本申请的环氧化酶-2抑制剂包括目前已经存在的或者之后产生的环氧化酶-2抑制剂。环氧化酶-2抑制剂包括但不限于塞来昔布、罗非昔布、帕瑞昔布、瓦德昔布和依妥昔布。
本申请中使用的术语“放射性核素络合物”是指含有放射性核素的一类特殊络合物，络合物中的螯合剂能够与放射性核素螯合，并提供更稳定地结合靶向物质的连接部分。本申请中使用的术语“放射性核素”是指能自发地放出射线(例如α射线、β射线或γ射线等)的元素。本申请的放射性核素包括目前已经存在的或者之后产生的所有能用于治疗和诊断的放射性核素。本申请的放射性核素包括但不限于 67Cu、 64Cu、 90Y、 109Pd、 111Ag、 149Pm、 153Sm、 165Ho、 166Ho、 177Lu、 186Re、 188Re、 99mTc、 67Ga、 68Ga、 111In、 90Y、 177Lu、 186Re、 188Re、 197Au、 198Au、 199Au、 105Rh、 161Tb、 149Pm、 44Sc、 47Sc、 70As、 71As、 72As、 73As、 74As、 76As、 77As、 212Pb、 212Bi、 213Bi、 225Ac、 117mSn、 67Ga、 201Tl、 123I、 131I、 160Gd、 148Nd、 89Sr和 211At。在一些实施方式中，螯合剂是大环螯合剂。本申请的螯合剂包括但不限于H2dedpa、H4octapa、H2azapa、DTPA、CHX-A”-DTPA、DTPA-bis anhydride、Maleimide-DTPA、DTPA(tBu)4、DiamSar CB-TE2A、Cyclam、DO2A、DOTA、OTA-GA(tBu) 4、Maleimide-DOTA-GA、p-NCS-Bz-DOTA-GA、NH2-DOTA-GA、DOTA-GA anhydride、DOTA-tris(tBu)ester、Propargyl-DOTA-tris(tBu)ester、DO3AM-acetic acid、DO3AM-N-(2-aminoethyl)ethanamide、DO3AtBu-N-(2-aminoethyl)ethanamide、DOTA-di(tBu)ester、DOTA-tris(tBu)ester NHS ester、DOTA-NHS ester、Propargyl-DOTA-tris(tBu)ester、DOTADOTA-GA anhydride、DOTA-GA(tBu) 4、p-NCS-Bz-DOTA-GA、NH2-DOTA-GA、Maleimide-DOTA-GA、AGuIX、Gado-H、CYCLEN、DO2AtBu、DO3AtBu、DO3AEt、DO3AM、DOTAEt、DOTPrEt、cis-Glyoxal-Cyclen、Mono-N-Benzyl-Cyclen、trans-N-Dibenyl-Cyclen、TriBOC-Cyclen、Mono-N-Benzyl-TACN、DiBOC-TACN、Cross-bridge-Cyclam(CB-Cyclam)、(13)aneN4、TACN、TACN·3HCl、TACD、Mono-N-benzyl-TACD、DiBOC-TACD、1,7-Dioxa-4,10-diazacyclododecane、C-Methyl-Ester-Cyclam、C-Carboxylic-Acid-Cyclam、trans-N-Dimethyl-Cyclam、TETRAM、TETAEt、TETAMEt2、TETAMMe2、TETAM、CPTA、CB-Cyclam derivatives、CB-TE2A、Methylamino-(13)aneN4、Bis-(13)aneN4、Oxo-(13)aneN4、Mono-N-Benzyl-(13)aneN4、 TriBOC-(13)aneN4、TRITRAM、TRI3AEt、TRI3AtBu、TRITAM、TRITA、Mono-N-Benzyl-Cyclam、Formaldehyde-Cyclam、cis-Glyoxal-Cyclam、Dioxocyclam、Oxocyclam、trans-N-Dibenzyl-Cyclam、TriBOC-Cyclam、DOTP、DOTMA、TETA、DOTAM、DiAmSar、CB-Cyclam、CB-TE2A、NOTA、NOTAM、NH 2-NODA-GA、Iodo-NODA-GA、NCS-MP-NODA、NH2-MPAA-NODA、NODA-GA(tBu) 3、NODA-GA-NHS ester、Maleimide-NODA-GA、NOTA-NHS ester、Maleimide-NOTA、Propargyl-NOTA(tBu) 2、p-NCS-benzyl-NODA-GA、NOTA(tBu) 2、NCS-MP-NODA、NH 2-MPAA-NODA、NH 2-NODA-GA、Iodo-NODA-GA和TACN。
在一些实施方式中，本申请的偶联体化合物或其药学上可接受的盐包含一个有效载荷。在一些实施方式中，本申请的偶联体化合物或其药学上可接受的盐包含两个或两个以上有效载荷。例如，本申请的偶联体化合物或其药学上可接受的盐包含1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20个或更多个有效载荷。在含有多个有效载荷的偶联体化合物中，每个有效载荷彼此之间可以是相同的或不同的。在一些实施方式中，至少两个有效载荷彼此之间是不同的。
在本申请中使用的术语“靶向分子”指的是能够将本申请的偶联体化合物靶向至靶部位、靶组织、靶器官、靶细胞或靶细胞内区域的任意分子或部分。在一些实施方式中，靶向分子使得，相比于非靶部位、非靶组织、非靶器官、非靶细胞或非靶细胞内区域，靶向分子使得本申请的偶联体化合物在靶部位、靶组织、靶器官、靶细胞或靶细胞内区域分配的更多，例如，多至少10％、20％、50％、80％、100％、150％、200％、300％、400％、500％或更高。在一些实施方式中，靶向分子使得，相比于不带靶向分子，带有靶向分子的偶联体化合物在靶部位、靶组织、靶器官、靶细胞或靶细胞内区域分配的更多，例如，多至少10％、20％、50％、80％、100％、150％、200％、300％、400％、500％或更高。在一些实施方式中，靶向分子能够触发或促进含有此类靶向分子的偶联体化合物与靶分子的特异性结合，触发或促进靶细胞对偶联体化合物的内吞作用，触发或促进偶联体化合物在靶细胞周围富集和/或进入靶细胞。
在一些实施方式中，本申请的偶联体化合物包括至少两个靶向分子。在一些实施方式中，本申请的偶联体化合物包括的两个或更多的靶向分子是相同的或不同的。在一些实施方式中，本申请的偶联体化合物包括的两个或更多的靶向分子中至少两个靶向分子是不同的。在一些实施方式中，本申请的偶联体化合物包括的两个或更多的靶向分子彼此之间都是不同的。在一些实施方式中，本申请的偶联体化合物包括的两个或更多的靶向分子中至少两个靶向分子是能够与不同的细胞表面蛋白或标记物特异性结合。在一些实施方式中，本申请的偶联体化合物包括的两个或更多的靶向分子能够与不同的细胞表面蛋白或标记物特异性结合。
在一些实施方式中，本申请的偶联体化合物包含至少两个靶向分子，其中至少一个为协同作用分子。
在本申请中使用的术语“协同作用分子”是指能够与本申请的偶联体化合物包含的其它靶向分子协同作用，以更好的触发或促进偶联体化合物与靶分子的特异性结合，触发或促进靶细胞对偶联体化合物的内吞作用，触发或促进偶联体化合物在靶细胞周围富集和/或进入靶细胞，和/或以其他形式引起偶联体化合物与靶细胞特异性结合和保留的任意分子或部分。在一些实施方式中，协同作用分子使得，相比于非靶部位、非靶组织、非靶器官、非靶细胞或非靶细胞内区域，协同作用分子分子使得本申请的偶联体化合物在靶部位、靶组织、靶器官、靶细胞或靶细胞内区域分配的更多，例如，多至少10％、20％、50％、80％、100％、150％、200％、300％、400％、500％或更高。在一些实施方式中，协同作用分子使得，相比于不带协同作用分子，带有协同作用分子的偶联体化合物在靶部位、靶组织、靶器官、靶细胞或靶细胞内区域分配的更多，例如，多至少10％、20％、50％、80％、100％、150％、200％、300％、400％、500％或更高。在一些实施方式中，协同作用分子使得，相比于不带协同作用分子，带有协同作用分子的偶联体化合物对靶细胞的作用活性更高，例如，高至少10％、20％、50％、80％、100％、150％、200％、300％、400％、500％或更多。
在一些实施方式中，本申请的协同作用分子是细胞相互作用分子。
在本申请中使用的术语“细胞相互作用分子”指的是能够与靶细胞的细胞表面物质相互作用以触发或促进含有此类细胞相互作用分子的偶联体化合物与细胞的特异性结合，触发或促进靶细胞对偶联体化合物的内吞作用，和/或触发或促进偶联体化合物在靶细胞周围富集和/或进入靶细胞。
细胞相互作用分子可以是小的化学分子或大的生物分子。在一些实施方式中，细胞相互作用分子是小分子化合物或者是多肽。在一些实施方式中，细胞相互作用分子是小分子化合物或者是包括2-50、2-40、2-30、2-25、2-22、2-20、2-18、2-15、2-12、2-10、2-8、4-50、5-50、5-40、5-30、5-25、5-22、5-20、5-18、5-15、5-12、5-10、6、7、8、9个氨基酸的多肽。
在一些实施方式中，靶向分子是能够与细胞表面受体或其它分子结合的配体。在一些实施方式中，至少一个靶向分子是能够与细胞表面受体或其它分子结合的配体。
本申请的配体可以包括各种各样的化学或生物分子，其可以对所选定的靶点具有特异性的结合亲和性，所选定的靶点可以是例如细胞表面受体、细胞表面抗原、细胞、组织、器官等。在一些实施方式中，配体可以与靶细胞表面上表达的蛋白或标记物特异性结合。在一些实施方式中，本申请的配体以10 -6～10 -11M(K d值)的亲和性与细胞表面蛋白或标记物结合。在一些实施方式中，本申请的配体以至少10 -7、至少10 -8、至少10 -9M(K d值)的亲和性与细胞表面蛋白或标记物结合。在一些实施方式中，本申请的配体以低于10 -6、低于10 -7、低于10 -8M(K d值)的亲和性与细胞表面蛋白或标记物结合。在一些实施方式 中，本申请的配体以一定的亲和性与细胞表面蛋白或标记物结合，所述一定的亲和性是指与同非靶细胞表面蛋白或标记物的亲和性相比，配体对靶细胞表面蛋白或标记物的亲和性高至少两倍、三倍、四倍、五倍、六倍、八倍、十倍、二十倍、五十倍、一百倍或更多倍。在一些实施方式中，本申请的细胞表面蛋白或标记物在靶细胞(例如，癌细胞)中的表达显著高于在正常细胞中的表达。在本申请中使用的术语“显著”指的是统计学上的显著差异，或者可以由本领域技术人员认识到的显著性差异。
在一些实施方式中，本申请的细胞表面蛋白或标记物在靶细胞(例如，癌细胞)中的表达水平是在正常细胞中的表达水平的2倍至1,000,000倍，例如在靶细胞(例如，癌细胞)中的表达水平比在正常细胞中的表达水平高2倍至10倍、2倍至100倍、2倍至1,000倍、2倍至10,000倍、2倍至100,000倍、2倍至1,000,000倍(可以等于上述数值范围内的任意值，包括该范围的端点)。在一些实施方式中，细胞表面受体在靶细胞(例如，癌细胞)中的表达水平比在正常细胞中的表达水平高至少10倍、或者至少100倍、或者至少1,000倍、或者至少10,000倍、或者至少100,000倍。在一些实施方式中，当与靶细胞(例如，癌细胞)上的细胞表面蛋白或标记物的水平比较时，正常细胞上的细胞表面受体的水平降低至少50％、60％、70％、80％、90％、95％或99％。在一些实施方式中，本申请所述的细胞表面蛋白或标记物在正常细胞中是不能检测到的。
在一些实施方式中，本申请的细胞表面蛋白或标记物是细胞表面受体。
在一些实施方式中，本申请的细胞表面受体选自下组：转铁蛋白受体(TFR)、低密度脂蛋白受体(LDLR)、叶酸受体(FR)、生长素抑制激素受体、尿酸激酶受体、肿瘤坏死因子受体(TNFR)、整合素受体(LFA-1)、SST-14受体(SSTR2)、GNRH受体(GNRHR)、TRPV6和整合素α受体。
在一些实施方式中，本申请的细胞表面蛋白或标记物是细胞表面抗原。
在一些实施方式中，本申请的细胞表面抗原选自下组：前列腺特异性膜抗原、MUC1粘蛋白、急性淋巴母细胞共同抗原、Thy-1细胞表面抗原、Melan-A蛋白、鳞状细胞癌抗原、半乳糖凝集素3和人类白细胞抗原。
在一些实施方式中，本申请的细胞相互作用分子能与选自下组的分子结合：FOLR1、TRPV6、FOLH1(PMSA)、GNRHR、Her2、Trop2、Her3、NECTIN4、LRP1、GLUT1、EGFR1、AXL、CA9、CD44、Claudin18.2、APN、DLL3、CEACAM5、FZD10、TFRC、MET、IGFR1、SSTR2、CCKBR、LFA1、ICAM、GPR87、GM-CSF、GM-CSFR、TIM3、TLR家族、CD40、CD40L、OX40、OX40L、GITRL、GITR、4-BBL、4-1BB、CD70、CD27、ICOSL、ICOS、HHLA2、CD28、CD86/80、CD28、MHCII抗原、TCR、CTLA-4、CD155、CD122、CD113、IGIT、PD-L1、PD1、Galectin-9、TIM-3、HVEM、BTLA、CD160、VISTA、B7-H4、B7-H3、磷脂酰丝氨酸、HHLA2、LAG3、Galectin-3、LILRB4、SIGLEC15、NKG2A、NKG2D、SLAMF7、KIR2DL1、KIR2DL2、KIR2DL3、FGFR1、FGFR2、FGFR4、NeuGcGM3和CXCR4。
在一些实施方式中，本申请的偶联体化合物或其药学上可接受的盐包含前列腺特异性膜抗原配体部分以及协同作用分子部分，所述协同作用分子部分与选自下组的分子结合：FOLR1、TRPV6、FOLH1(PMSA)、SSTR2和GNRHR。
在一些实施方式中，本申请的偶联体化合物或其药学上可接受的盐包含式(I)所示的配体部分以及协同作用分子部分，所述协同作用分子部分与选自下组的分子结合：FOLR1、TRPV6、SSTR2和GNRHR。
在又一些实施方式中，本申请的偶联体化合物或其药学上可接受的盐包含P10以及协同作用分子部分，所述协同作用分子与选自下组的分子结合：FOLR1、TRPV6、FOLH1(PMSA)和GNRHR。
在一些实施方式中，本申请的偶联体化合物或其药学上可接受的盐中的一个协同作用分子是能够介导内吞作用的内吞作用分子部分。在本申请中使用的术语“内吞作用”指的是偶联体化合物或其药学上可接受的盐与靶细胞相互作用后，能够介导其自身被内吞、内化或摄取进入靶细胞。在本申请中使用的术语“内吞作用分子”指的是这样的分子，即，其与靶细胞相互作用后，能够介导本申请的偶联体化合物或其药学上可接受的盐内吞、内化或摄取进入靶细胞。
在一些实施方式中，内吞作用分子选自下组：叶酸及其类似物、能够介导内吞作用的肽和穿膜肽。
在一些实施方式中，本申请的内吞作用分子是叶酸或其类似物。
叶酸由于其分子量小、无免疫原性和稳定性好，有利于与其他基团形成化学键。叶酸能够以高亲和性与细胞表面上表达的叶酸受体结合以介导细胞摄取叶酸。虽然叶酸受体在大部分正常细胞中的表达水平非常低，但是在大量癌细胞中以高水平表达以满足在低叶酸条件下快速分裂细胞对叶酸的较高需求(参见Kelemen LE,Int J Cancer,2006；119:243-50；Kane MA等，J Clin Invest.1988；81:1398-406；Matsue H等，Proc Natl Acad Sci USA.1992；89:6006-9；Zhao R等，Annu Rev Nutr.2011；31:177-201)。叶酸能够与细胞表面上的叶酸受体特异性结合，并且其还能介导偶联体化合物或其药学上可接受的盐进入靶细胞的内吞作用。
在一些实施方式中，叶酸的类似物选自下组：5-甲基四氢叶酸、5-甲酰基四氢叶酸、甲氨蝶呤和5,10-亚甲基四氢叶酸。
在一些实施方式中，内吞作用分子是能够介导内吞作用的肽。
在一些实施方式中，能够介导内吞作用的肽包含选自下组的氨基酸序列：SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、Arg-Gly-Asp(称为RGD)、与SEQ ID NO：16-18中的任意一个具有至少70％、至少80％、至少85％、至少90％、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％的氨基酸序列同源性的同源肽，其中所述同源肽分别为SEQ ID NO：16-18所示的肽的功能性等价物。
在一些实施方式中，如本申请所述的能够介导内吞作用的肽与SEQ ID NO:16-20、RGD的序列相比，仅在一个氨基酸位点具有氨基酸的保守性取代。在一些实施方式中，如本申请所述的能够介导内吞作用的肽与SEQ ID NO:16-20的序列相比，在2、3、4、5、6、7、8、9或10个氨基酸位点具有氨基酸的保守性取代。
在不影响其生物活性的前提下，如本申请所述的能够介导内吞作用的肽还可以含有非天然存在的氨基酸，包括例如β-氟丙氨酸、1-甲基-组氨酸、γ-亚甲基-谷氨酸、α-甲基-亮氨酸、4,5-脱氢-赖氨酸、羟脯氨酸、3-氟-苯丙氨酸、3-氨基-酪氨酸、4-甲基-色氨酸等。
可以采用本领域熟知的多种方法确定同源性百分率。例如，可以通过下述可公开获得的工具对序列进行比较：BLASTp软件(可从国家生物技术信息中心(NCBI)的网站获得： http://blast.ncbi.nlm.nih.gov/Blast.cgi，亦参见Altschul S.F.等，J.Mol.Biol.,215:403–410(1990)；Stephen F.等，Nucleic Acids Res.,25:3389–3402(1997))，ClustalW2(可从欧洲生物信息研究所的网站获得： http://www.ebi.ac.uk/Tools/msa/clustalw2/，亦参见Higgins D.G.等，Methods in Enzymology,266:383-402(1996)；Larkin M.A.等，Bioinformatics(Oxford,England),23(21):2947-8(2007))以及Tcoffee(可从瑞典生物信息研究所的网站获得，亦参见Poirot O.等，Nucleic Acids Res.,31(13):3503-6(2003)；Notredame C.等，J.Mol.Boil.,302(1):205-17(2000))。如果使用软件进行序列比对，则可以使用软件中提供的默认参数，或者可以以其他方式对参数进行定制以适应比对目的。所有的这些均在本领域技术人员的知识范围内。
在本申请中使用的术语“功能性等价物”指的是保留了与衍生肽来源的原始肽序列的生物活性基本上类似的生物活性的衍生肽。功能性等价物可以是天然衍生物或合成制备的。示例性的功能性等价物包括具有一个或多个氨基酸取代、缺失或加入的氨基酸序列，条件是肽的生物活性得以保持。取代的氨基酸理想地具有与被取代的氨基酸类似的化学-物理性质。理想的类似的化学-物理性质包括电荷、蓬松性、疏水性、亲水性等的相似性。
在一些实施方式中，功能性等价物包括氨基酸残基的保守性取代。氨基酸残基的保守性取代指的是具有相似性质的氨基酸之间的取代，例如极性氨基酸之间的取代(例如，谷氨酰胺和天冬酰胺之间的取代)、疏水性氨基酸之间的取代(例如，亮氨酸、异亮氨酸、甲硫氨酸和缬氨酸之间的取代)、以及具有相同电荷的氨基酸之间的取代(例如，精氨酸、赖氨酸和组氨酸之间的取代，或谷氨酸和天冬氨酸之间的取代)等等。
在一些实施方式中，内吞作用分子是穿膜肽。穿膜肽(Cell-Penetrating Peptides,CPP)也称为蛋白转导结构域(PTD)，是短肽(通常少于40个氨基酸)，能够以不依赖于受体的方式进入细胞内部。穿膜肽与有效载荷偶联时能够介导有效载荷的跨膜转运并且具有蛋白转导活性。在一些实施方式中，本申请所述的穿膜肽选自下组：肿瘤归巢肽、线粒体穿 透肽、可活化细胞穿膜肽和抗菌肽。在一些实施方式中，穿膜肽包含选自下组的氨基酸序列：SEQ ID NO:19(RRRRRRRRR，称为R9)和SEQ ID NO:20(GRKKRRQRRRPPQ，其是Tat肽，即HIV转录蛋白反式激活因子的穿膜肽)。
在一些实施方式中，本申请的偶联体化合物或其药学上可接受的盐中的一个靶向分子是前列腺特异性膜抗原配体部分。
在本申请中使用的术语“前列腺特异性膜抗原”，是指存在于前列腺上皮细胞膜的一种Ⅱ型跨膜糖蛋白，由750个氨基酸组成，其具有19个胞内区氨基酸、24个跨膜区氨基酸和707个胞外区氨基酸。前列腺特异性膜抗原在正常前列腺上皮细胞中表达，但其在前列腺癌细胞中的表达水平要高得多。相对于传统用于临床检测的前列腺特异抗原，前列腺特异性膜抗原是一种更加敏感和特异的前列腺癌肿瘤标记物，尤其是在激素难治性前列腺癌及前列腺癌转移灶中均为高表达，在区分前列腺癌和其他类型恶性肿瘤时具有高敏感度和特异性。同时，在多种非前列腺来源的实体瘤(如肺癌、膀胱癌、胃癌、胰腺癌、肾癌和结直肠癌等)中，前列腺特异性膜抗原也高度特异地表达于肿瘤血管内皮细胞上。
在本申请中使用的术语“前列腺特异性膜抗原配体”是指能特异性识别并结合前列腺特异性膜抗原的抗体、核酸适配体和小分子等。本申请的前列腺特异性膜抗原配体包括目前已经存在的或者之后产生的前列腺特异性膜抗原配体，还包括前述配体的片段，只要这些片段还保留与前列腺特异性膜抗原结合的能力。抗体类配体是最常见的前列腺特异性膜抗原配体，其包括但不限于单克隆抗体J591、J533、J415和E99(例如，参见Liu H,Rajasekaran AK,Moy P等人Constitutive and antibody-induced internalization of prostate-specific memberane antigen[J].Cancer Res,1998,58(18):4055-4060)。核酸适配体是经过指数富集配体系统进行技术筛选得到的能与前列腺特异性膜抗原高亲和性、高特异性结合的单链DNA或RNA，该类前列腺特异性膜抗原配体包括但不限于xPSM-A10核酸适配体及其衍生物和xPSM-A9核酸适配体及其衍生物(例如，参见Lupoid SE等人，Identification and Characterization of nuclease-stabilized RNA molecules that bind human prostate cancer cells via the prostate-specific membrane antigen,Cncer Res,2002,62(14):4029-4033)。相比于抗体类和核酸适配体类配体，前列腺特异性膜抗原小分子配体具有分子量小、高渗透性、低免疫原性、易于合成等优势，其包括但不限于谷胺酰脲类小分子配体和氨基磷酸酯类小分子配体。
在一些实施方式中，本申请的前列腺特异性膜抗原小分子配体可以选自由以下组成的组：2-[[甲基羟基氧膦基]甲基]戊二酸；2-[[乙基羟基氧膦基]甲基]戊二酸；2-[[丙基羟基氧膦基]甲基]戊二酸；2-[[丁基羟基氧膦基]甲基]戊二酸；2-[[环己基羟基氧膦基]甲基]戊二酸；2-[[苯基羟基氧膦基]甲基]戊二酸；2-[[2-(四氢呋喃基)羟基氧膦基]甲基]戊二酸；2-[[(2-四氢吡喃基)羟基氧膦基]甲基]戊二酸；2-[[((4-吡啶基)甲基)羟基氧膦基]甲基]戊二酸；2-[[((2-吡啶基)甲基)羟基氧膦基]甲基]戊二酸；2-[[(苯基甲基)羟基氧膦基]甲基]戊二酸；2-[[((2-苯基乙基)甲基)羟基氧膦基]甲基]戊二酸；2-[[((3-苯基丙基)甲基)羟基氧膦基]甲基]戊二酸； 2-[[((3-苯基丁基)甲基)羟基氧膦基]甲基]戊二酸；2-[[((2-苯基丁基)甲基)羟基氧膦基]甲基]戊二酸；2-[[(4-苯基丁基)羟基氧膦基]甲基]戊二酸；和2-[[(氨甲基)羟基氧膦基]甲基]戊二酸；2-[[甲基羟基氧膦基]氧]戊二酸；2-[[乙基羟基氧膦基]氧]戊二酸；2-[[丙基羟基氧膦基]氧]戊二酸；2-[[丁基羟基氧膦基]氧]戊二酸；2-[[苯基羟基氧膦基]氧]戊二酸；2-[[((4-吡啶基)甲基)羟基氧膦基]氧]戊二酸；2-[[((2-吡啶基)甲基)羟基氧膦基]氧]戊二酸；2-[[(苯基甲基)羟基氧膦基]氧]戊二酸；和2[[((2-苯基乙基)甲基)羟基氧膦基]氧]戊二酸；2-[[(N-羟基)氨基甲酰]甲基]戊二酸；2-[[(N-羟基-N-甲基)氨基甲酰]甲基]戊二酸；2-[[(N-丁基-N-羟基)氨基甲酰]甲基]戊二酸；2-[[(N-苄基-N-羟基)氨基甲酰]甲基]戊二酸；2-[[(N-羟基-N-苯基)氨基甲酰]甲基]戊二酸；2-[[(N-羟基-N-2-苯基乙基)氨基甲酰]甲基]戊二酸；2-[[(N-乙基-N-羟基)氨基甲酰]甲基]戊二酸；2-[[(N-羟基-N-丙基)氨基甲酰]甲基]戊二酸；2-[[(N-羟基-N-3-苯基丙基)氨基甲酰]甲基]戊二酸；2-[[(N-羟基-N-4-吡啶基)氨基甲酰]甲基]戊二酸；2-[[(N-羟基)酰胺]甲基]戊二酸；2-[[N-羟基(甲基)酰胺]甲基]戊二酸；2-[[N-羟基(苄基)酰胺]甲基]戊二酸；2-[[N-羟基(苯基)酰胺]甲基]戊二酸；2-[[N-羟基(2-苯基乙基)酰胺]甲基]戊二酸；2-[[N-羟基(乙基)酰胺]甲基]戊二酸；2-[[N-羟基(丙基)酰胺]甲基]戊二酸；2-[[N-羟基(3-苯基丙基)酰胺]甲基]戊二酸；和2-[[N-羟基(4-吡啶基)酰胺]甲基]戊二酸；2-[(亚硫酰基)甲基]戊二酸；2-[(甲基亚硫酰基)甲基]戊二酸；2-[(乙基亚硫酰基)甲基]戊二酸；2-[(丙基亚硫酰基)甲基]戊二酸；2-[(丁基亚硫酰基)甲基]戊二酸；2-[(苯基亚硫酰基]甲基]戊二酸；2-[[(2-苯基乙基)亚硫酰基]甲基]戊二酸；2-[[(3-苯基丙基)亚硫酰基]甲基]戊二酸；2-[[(4-吡啶基)亚硫酰基]甲基]戊二酸；2-[(苄基亚硫酰基)甲基]戊二酸；2-[(磺酰基)甲基]戊二酸；2-[(甲磺酰基)甲基]戊二酸；2-[(乙磺酰基)甲基]戊二酸；2-[(丙磺酰基)甲基]戊二酸；2-[(丁磺酰基)甲基]戊二酸；2-[(苯基磺酰基]甲基]戊二酸；2-[[(2-苯基乙基)磺酰基]甲基]戊二酸；2-[[(3-苯基丙基)磺酰基]甲基]戊二酸；2-[[(4-吡啶基)磺酰基]甲基]戊二酸；2-[(苄基磺酰基)甲基]戊二酸；2-[(亚砜亚胺基(sulfoximinyl))甲基]戊二酸；2-[(甲基亚砜亚胺基)甲基]戊二酸；2-[(乙基亚砜亚胺基)甲基]戊二酸；2-[(丙基亚砜亚胺基)甲基]戊二酸；2-[(丁基亚砜亚胺基)甲基]戊二酸；2-[(苯基亚砜亚胺基]甲基]戊二酸；2-[[(2-苯基乙基)亚砜亚胺基]甲基]戊二酸；2-[[(3-苯基丙基)亚砜亚胺基]甲基]戊二酸；2-[[(4-吡啶基)亚砜亚胺基]甲基]戊二酸；和2-[(苄基亚砜亚胺基)甲基]戊二酸；N-[甲基羟基氧膦基]谷氨酸；N-[乙基羟基氧膦基]谷氨酸；N-[丙基羟基氧膦基]谷氨酸；N-[丁基羟基氧膦基]谷氨酸；N-[苯基羟基氧膦基]谷氨酸；N-[(苯基甲基)羟基氧膦基]谷氨酸；N-[((2-苯基乙基)甲基)羟基氧膦基]谷氨酸；和N-甲基-N-[苯基羟基氧膦基]谷氨酸。本申请的前列腺特异性膜抗原配体还包括PCT申请WO2010/108125和WO2006/093991中公开的所有前列腺特异性膜抗原小分子配体，上述两个专利申请以其全部内容并入本文。
在一些实施方式中，本申请的前列腺特异性膜抗原小分子配体是戊二酸衍生物。在一些实施方式中，本申请的前列腺特异性膜抗原小分子配体是戊二酸的氨羰基衍生物。
在一些实施方式中，本申请的前列腺特异性膜抗原小分子配体包括如下结构：
Figure PCTCN2020074117-appb-000026
在一些实施方式中，偶联体化合物或其药学上可接受的盐包含的前列腺特异性膜抗原配体包括如下结构：
Figure PCTCN2020074117-appb-000027
在一些实施方式中，偶联体化合物或其药学上可接受的盐包含的前列腺特异性膜抗原配体包括如下结构：
Figure PCTCN2020074117-appb-000028
在一些实施方式中，偶联体化合物或其药学上可接受的盐包含的前列腺特异性膜抗原配体包括如下结构：
Figure PCTCN2020074117-appb-000029
在一些实施方式中，偶联体化合物或其药学上可接受的盐包含的前列腺特异性膜抗原配体包括如下结构：
Figure PCTCN2020074117-appb-000030
在一些实施方式中，本申请的的偶联体化合物或其药学上可接受的盐中一个靶向分子是具有式(I)所示的配体部分：
Figure PCTCN2020074117-appb-000031
或者与其具有至少70％、至少80％、至少85％或至少90％的氨基酸序列同源性的配体部分或者与其具有至多3个、2个或1个氨基酸替换(例如，保守性替换)。
在一些实施方式中，本申请的偶联体化合物或其药学上可接受的盐中一个靶向分子是P10或者与其具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％或至少93％的氨基酸序列同源性的配体部分或者与其具有至多3个、2个或1个氨基酸替换(例如，保守性替换)。
在本申请中使用的术语“P10”指的是具有氨基酸序列Cys-Lys-Glu-Phe-Leu-His-Pro-Ser-Lys-Val-Asp-Leu-Pro-Arg的肽。
在一些实施方式中，本申请的偶联体化合物具有选自下组组合的靶向分子：(1)叶酸配体和前列腺特异性膜抗原配体；(2)TRPV6配体和前列腺特异性膜抗原配体；(3)GNRHR配体和前列腺特异性膜抗原配体；(4)SSTR2配体和前列腺特异性膜抗原配体；(5)叶酸配体和SSTR2配体；或(6)TRPV6配体和叶酸配体。
在一些实施方式中，本申请提供的偶联体化合物或其药学上可接受的盐的两个靶向分子分别为协同作用分子部分以及前列腺特异性膜抗原配体部分。在一些实施方式中，所述协同作用分子能够介导内吞作用。在一些实施方式中，本申请提供的偶联体化合物或其药学上可接受的盐的两个靶向分子分别为叶酸或其类似物以及前列腺特异性膜抗原配体部分。不希望受到理论的限制，选取特定的叶酸或其类似物以及前列腺特异性膜抗原配体部分，会比现有技术中的配体组合有更好的稳定性。
在一些实施方式中，本申请提供的偶联体化合物或其药学上可接受的盐的两个靶向分子分别为协同作用分子部分以及具有式(I)所示的配体部分。在一些实施方式中，所述协同作用分子能够介导内吞作用。在一些实施方式中，本申请提供的偶联体化合物或其药学上可接受的盐的两个靶向分子分别为叶酸或其类似物以及具有式(I)所示的配体部分。
在一些实施方式中，本申请提供的偶联体化合物或其药学上可接受的盐的两靶向分子分别为协同作用分子部分以及P10。在一些实施方式中，所述协同作用分子能够介导内吞作用。在一些实施方式中，本申请提供的偶联体化合物或其药学上可接受的盐的两个靶向分子分别为叶酸或其类似物以及P10。
在一些实施方式中，本申请提供的偶联体化合物仅包含与两个靶向分子偶联的单一有效载荷。在一些实施方式中，本申请提供的偶联体化合物包含与两个靶向分子偶联的多个有效载荷。
在本申请中使用的术语“偶联的”指的是通过两个化学基团共价键的连接，其可以是在两个化学基团之间直接形成共价键，也可以是通过连接子将两个化学基团间接连接。
在一些实施方式中，偶联体化合物或其药学上可接受的盐包含有效载荷(例如，1个)和两个靶向分子，其中有效载荷与至少一个靶向分子直接共价连接。在一些实施方式中，有效载荷与两个靶向分子均直接共价连接。
在一些实施方式中，偶联体化合物或其药学上可接受的盐包含有效载荷(例如，1个)和两个靶向分子，其中有效载荷与至少一个靶向分子通过连接子共价连接。在一些实施方式中，有效载荷与两靶向分子均通过连接子共价连接。
在本申请中使用的术语“连接子”指的是将有效载荷与靶向分子共价连接的分子或部分。连接子包括用于将有效载荷与至少一个靶向分子连接的官能团。在一些实施方式中，官能团可以含有两个反应性部分，一个用于与有效载荷连接，另一个用于与靶向分子连接。在一些实施方式中，官能团彼此之间是不同的。在一些实施方式中，官能团包含含有巯基反应性部分和胺反应性部分的基团。在一些实施方式中，官能团彼此之间是相同的。在一些实施方式中，官能团是马来酰亚胺基团。在一些实施方式中，连接子含有氨基酸。在一些实施方案中，连接子含有的氨基酸中的羧酸被酰胺化。在一些实施方案中，连接子含有短链的聚乙二醇(例如，包括2-10、2-8、3-8、4-8、4-7、4-6或5个重复单元)。
在一些实施方式中，本申请的连接子是能够结合至少一个(例如，1、2、3、4、5、6、7、8、9、10个或更多个)有效载荷和至少1个靶向分子的多价连接子。与多价连接子结合的有效载荷可以是相同的或不同的，并且与多价连接子结合的靶向分子可以是相同的或不同的。
在一个方面中，连接子应足够稳定以避免在血液循环过程中意外释放有效载荷，以增加有效载荷到靶细胞或组织的有效量并避免毒性。在另一个方面，连接子应能够在靶细胞周围或内部释放有效载荷以有效杀死靶细胞或阻断靶细胞的功能。在一些实施方式中，连接子包含至少一个可裂解官能团。优选地，可裂解官能团在靶细胞外足够稳定，但是其在进入靶细胞后裂解以释放有效载荷。在一些实施方式中，可裂解官能团在靶细胞中的裂解效率比在血液或血清中的裂解效率高至少10、20、30、50、100倍或更多倍。
可裂解连接子可以被水解、酶促反应、或还原反应、或通过pH改变所裂解。在一些实施方式中，连接子在特定生理环境下(例如，在适宜pH环境下)是可裂解的。在一些实施方式中，连接子可在pH约6.5或更低的酸性环境中裂解，或通过诸如酶等试剂裂解。在一些实施方式中，连接子对裂解剂敏感。例如，pH、氧化还原电位或存在降解分子。
在一些实施方式中，连接子是不可裂解的。在本申请中使用的不可裂解连接子指的是在细胞内代谢期间基本保持完整的连接子。
在一些实施方式中，连接子是肽类连接子，其由通过肽键连接的直链或支链氨基酸组成。在一些实施方式中，肽类连接子可被在靶细胞周围或靶细胞中高度或特异性表达的蛋白酶裂解，例如在溶酶体或胞内体中的组织蛋白酶B。本申请所用的肽类连接子的长度可以有多种。通常地，本申请的肽类连接子的长度为1至50个氨基酸。在一些实施方式中，肽类连接子的长度为1至45、1至40、1至35、1至30、1至25、1至20、1至15、1至10、1至9、1至8、1至7、1至6、1至5、1至4、1至3个、1至2个或1氨基酸。在一些实施方式中，肽类连接子的长度为2至45、2至40、2至35、2至30、2至25、2至20、2至15、2至10、2至9、2至8、2至7、2至6、2至5、2至4、2至3个或2个氨基酸。本申请所述的肽类连接子的氨基酸数量可以等于上述数值范围内的任意整数值，包括该范围的端点。在一些实施方式中，肽类连接子的长度优选为1、2、3、4或5个氨基酸。在一些实施方式中，肽类连接子是半胱氨酸、赖氨酸、赖氨酸-赖氨酸、缬氨酸-瓜氨酸、苯丙氨酸-赖氨酸、缬氨酸-赖氨酸、半胱氨酸-赖氨酸、半胱氨酸-谷氨酸天冬氨酸-天冬氨酸和天冬氨酸-天冬氨酸-赖氨酸，可选的，上述氨基酸中的羧酸被酰胺化。
在一些实施方式中，连接子是含有二硫键的二硫化物连接子。二硫键可以在细胞内的还原环境下裂解，而在循环系统中保持稳定。本申请的二硫化物连接子可以是DSDM、DMDS、MDS或NDMDS。这些二硫化物连接子的结构如下表1中所示。
表1：DSDM、DMDS、MDS和NDMDS的结构
Figure PCTCN2020074117-appb-000032
Figure PCTCN2020074117-appb-000033
在一些实施方式中，连接子是pH依赖型连接子。本申请所述的pH依赖型连接子可以在特定pH环境下裂解。在一些实施方式中，pH依赖型连接子可以在碱性条件下稳定，但在酸性条件下(例如，在6.5或更低的pH值下)裂解。在一些实施方式中，pH依赖型连接子是顺乌头酸酐。
在一些实施方式中，偶联体化合物或其药学上可接受的盐的连接子是
Figure PCTCN2020074117-appb-000034
或者是上述结构与肽类连接子的组合(例如，通过含有1-3个氨基酸的肽类连接子与靶向分子连接)。
在一些实施方式中，偶联体化合物或其药学上可接受的盐的连接子具有如下结构：
Figure PCTCN2020074117-appb-000035
或者是上述结构与肽类连接子的组合(例如，通过含有1-3个氨基酸的肽类连接子与靶向分子连接)。
在一些实施方式中，偶联体化合物或其药学上可接受的盐的连接子具有如下结构：
Figure PCTCN2020074117-appb-000036
或者是上述结构与肽类连接子的组合(例如，通过含有1-3个氨基酸的肽类连接子与靶向分子连接)。
在一些实施方式中，偶联体化合物或其药学上可接受的盐的连接子具有如下结构：
Figure PCTCN2020074117-appb-000037
或者是上述结构与肽类连接子的组合(例如，通过含有1-3个氨基酸的肽类连接子与靶向分子连接)。
在一些实施方式中，偶联体化合物或其药学上可接受的盐的连接子具有如下结构：
Figure PCTCN2020074117-appb-000038
或者是上述结构与肽类连接子的组合(例如，通过含有1-3个氨基酸的肽类连接子与靶向分子连接)。
在一些实施方式中，偶联体化合物或其药学上可接受的盐的连接子具有如下结构：
Figure PCTCN2020074117-appb-000039
或者是上述结构与肽类连接子的组合(例如，通过含有1-3个氨基酸的肽类连接子与靶向分子连接)。
在一些实施方式中，偶联体化合物或其药学上可接受的盐的连接子具有如下结构：
Figure PCTCN2020074117-appb-000040
或者是上述结构与肽类连接子的组合(例如，通过含有1-3个氨基酸的肽类连接子与靶向分子连接)。
在一些实施方式中，偶联体化合物或其药学上可接受的盐的连接子具有如下结构：
Figure PCTCN2020074117-appb-000041
或者是上述结构与肽类连接子的组合(例如，通过含有1-3个氨基酸的肽类连接子与靶向分子连接)。
在一些实施方式中，偶联体化合物或其药学上可接受的盐的连接子具有如下结构：
Figure PCTCN2020074117-appb-000042
或者是上述结构与肽类连接子的组合(例如，通过含有1-3个氨基酸的肽类连接子与靶向分子连接)。
在一些实施方式中，偶联体化合物或其药学上可接受的盐的连接子具有如下结构：
Figure PCTCN2020074117-appb-000043
或者是上述结构与肽类连接子的组合(例如，通过含有1-3个氨基酸的肽类连接子与靶向分子连接)。
在一些实施方式中，偶联体化合物或其药学上可接受的盐的连接子具有如下结构：
Figure PCTCN2020074117-appb-000044
或者是上述结构与肽类连接子的组合(例如，通过含有1-3个氨基酸的肽类连接子与靶向分子连接)。
在一些实施方式中，偶联体化合物或其药学上可接受的盐的连接子具有如下结构：
Figure PCTCN2020074117-appb-000045
或者是上述结构与肽类连接子的组合(例如，通过含有1-3个氨基酸的肽类连接子与靶向分子连接)。
在一些实施方式中，本申请的连接子可以包含如上文所述的任意一个连接子或其组合。
在一些实施方式中，有效载荷与第一靶向分子直接或间接偶联，并且第一靶向分子与第二靶向分子直接或间接偶联。在一些实施方式中，有效载荷与第一靶向分子和第二靶向分子均是直接偶联。在一些实施方式中，有效载荷与第一靶向分子和第二靶向分子均是间接偶联。在一些实施方式中，有效载荷与第一靶向分子间接(例如，通过连接子)偶联，并且第一靶向分子与第二靶向分子直接或间接偶联。在一些实施方式中，有效载荷与第一靶向分子通过第一连接子偶联，并且有效载荷与第二靶向分子通过第二连接子偶联。在一些实施方式中，连接子是与至少一个(例如，1、2、3、4、5、6、7、8、9、10个或更多个)有效载荷和两个靶向分子结合的多价连接子。
在一些实施方式中，两个靶向分子彼此之间通过间隔区连接。在一些实施方式中，间隔区可以被由靶细胞特异性表达或将由靶细胞表达的蛋白酶裂解。此类蛋白酶包括例如在下表2中列出的蛋白酶。在一些实施方式中，间隔区包含选自在下表2中列出的氨基酸序列的任意一个的氨基酸序列。
表2：酶促可裂解序列列表
蛋白酶 识别位点的氨基酸序列 SEQ ID NO.
组织蛋白酶B RR -
豆荚蛋白 ASN -
Matripase KSRAEDE SEQ ID NO:1
MMP-2 PLGLAG SEQ ID NO:2
前列腺特异性抗原 SSLY SEQ ID NO:3
基质溶解素-3 AAA -
TMPRSS2 LLRSLIG SEQ ID NO:4
尿激酶型纤溶酶原激活物 SSR -
活化的蛋白C LVKR SEQ ID NO:5
因子Ixa LVVR SEQ ID NO:6
因子VIIa QLTR SEQ ID NO:7
因子Xa LEGR SEQ ID NO:8
凝血酶 PR -
钙蛋白酶-a PLFAEP SEQ ID NO:9
钙蛋白酶-2 GLGSEP SEQ ID NO:10
肠肽酶 DDDDK SEQ ID NO:11
MMP-8 GPSG SEQ ID NO:12
组织蛋白酶L PLG -
原蛋白转化酶5 RSKR SEQ ID NO:13
钙蛋白酶-3 VGVF SEQ ID NO:14
在本申请中使用的术语“可裂解”或“裂解的”指的是在本申请提供的偶联体化合物上进行的代谢过程或反应过程，从而将有效载荷与靶向分子之间的连接子，或靶向分子之间的间隔区破坏以释放游离的有效载荷或靶向分子。连接子或间隔区被蛋白酶裂解或在特定生理环境(例如，pH环境)下裂解。
在一些实施方式中，偶联体化合物具有下述式I、II、III或IV所示的结构，其中n、m、p和q独立地为0或1，其代表独立地存在或不存在连接子或间隔区。下式中的“分子”为“靶向分子”的简称。
Figure PCTCN2020074117-appb-000046
Figure PCTCN2020074117-appb-000047
在一些实施方式中，本申请提供的偶联体化合物或其药学上可接受的盐包含如本申请所提供的至少一个(例如，1、2、3、4、5、6、7、8、9、10个或更多个)有效载荷、如本申请所提供的两个靶向分子以及任选地如本申请所提供的连接子或间隔区。在一些实施方式中，本申请提供的偶联体化合物或其药学上可接受的盐包含如本申请所提供的一个有效载荷、如本申请所提供的一个与细胞表面蛋白或标记物特异性结合的配体、如本申请所提供的一个协同作用分子、以及如本申请所提供的连接子或间隔区。
在一些实施方式中，偶联体化合物具有如下所示的式V、VI、VII或VIII的结构，其中n、m、p、q和s独立地为0或1，其独立地表示存在或不存在连接子、多价连接子和间隔区。
Figure PCTCN2020074117-appb-000048
在一些实施方式中，本申请提供的偶联体化合物或其药学上可接受的盐包含有效载荷和两个靶向分子，其中两个靶向分子分别为协同作用分子部分以及前列腺特异性膜抗原配体部分，例如CB-20B、CB-20BK、CB-60S、CB-60SK、CB-20C、CB-1020、CB-1320、CB-1820、CR19428、20R-SM09和CB-20R。
在一些实施方式中，本申请提供的偶联体化合物或其药学上可接受的盐包含一个或多个有效载荷和两个靶向分子，其中两个靶向分子分别为协同作用分子部分以及具有式(I)所示的配体部分，例如CB-18G、CB-1820和CR19426。
在一些实施方式中，本申请提供的偶联体化合物或其药学上可接受的盐包含一个有效载荷和两个靶向分子，其中，两个靶向分子分别为协同作用分子部分以及P10，有效载荷为喜树碱及其任何衍生物，例如CB-10S、CR19425和CB-50S。
在一些实施方式中，本申请的偶联体化合物选自由下述化合物组成的组：CB-20B、CB-20BK、CB-60S、CB-60SK、CB-20C、CB-1020、CB-1320、CB-1820、CR19428、20R-SM09、CB-20R、CB-18G、CR19426、CB-10S、CR19425和CB-50S(各偶联体化合物的具体结构如图1所示)。在一些实施方式中，本申请的偶联体化合物由连接子-药物部分与配体部分 通过共价键连接而成。本申请的连接子-药物部分包括有效载荷和连接子，并且本申请的配体部分包括两个靶向分子以及可选的间隔区或连接子，两部分通过反应形成共价键后形成本申请的偶联体化合物，该共价键可以形成于连接子-药物部分中的连接子与配体部分的配体分子之间，也可以形成于连接子-药物部分中的连接子与配体部分的间隔区或连接子之间。
本申请的偶联体化合物CB-20B由连接子-药物部分LT1002与配体部分20B-SM09通过共价键连接而成。本申请的偶联体化合物CB-20BK由连接子-药物部分LT1002与配体部分20BK-SM09通过共价键连接而成。本申请的偶联体化合物CB-60S由连接子-药物部分LT2000C与配体部分60S-SM09通过共价键连接而成。本申请的偶联体化合物CB-60SK由连接子-药物部分LT2000C与配体部分60SK-SM09通过共价键连接而成。本申请的偶联体化合物CB-20C由连接子-药物部分LD1001与配体部分20BK-SM09通过共价键连接而成。本申请的偶联体化合物CB-1020由连接子-药物部分LT1002与配体部分1020BK-SM09通过共价键连接而成。本申请的偶联体化合物CB-1320由连接子-药物部分LT1002与配体部分1320BK-SM09通过共价键连接而成。本申请的偶联体化合物CB-1820由连接子-药物部分LT1002与配体部分1820BK-SM09通过共价键连接而成。本申请的偶联体化合物CR19428由连接子-药物部分CR19423与配体部分20BK-SM09通过共价键连接而成。本申请的偶联体化合物CB-20R由20R-SM09与放射性核素离子M络合。本申请的偶联体化合物CB-18G由连接子-药物部分LT1002与配体部分18G-SM09通过共价键连接而成。本申请的偶联体化合物CR19426由连接子-药物部分CR19423与配体部分18G-SM09通过共价键连接而成。本申请的偶联体化合物CB-10S由连接子-药物部分LT1000与配体部分CBSM09通过共价键连接而成。本申请的偶联体化合物CR19425由连接子-药物部分CR19423与配体部分CBSM09通过共价键连接而成。本申请的偶联体化合物CB-50S由连接子-药物部分LT1000N3与配体部分50S-SM09通过共价键连接而成。各结构如下表3所示。
表3：连接子-药物部分和配体部分的结构
Figure PCTCN2020074117-appb-000049
Figure PCTCN2020074117-appb-000050
Figure PCTCN2020074117-appb-000051
Figure PCTCN2020074117-appb-000052
Figure PCTCN2020074117-appb-000053
Figure PCTCN2020074117-appb-000054
Figure PCTCN2020074117-appb-000055
在一些实施方式中，本申请提供的偶联体化合物或其药学上可接受的盐进入血液循环和细胞外部(胞间质)，由于连接子在胞外环境非常稳定无法释放药物分子，则药物分子的毒性被封闭。该偶联体是一个无细胞毒性或低毒性的药物，不会对正常细胞产生毒害作用。
在一些实施方式中，本申请提供的偶联体化合物或其药学上可接受的盐与病变细胞上同时有高表达的多个受体或抗原等作用分子结合，其协同效应大大增加了偶联体化合物对靶细胞的亲和性，降低了与正常细胞结合的可能。从而可以携带高效的毒素药物如MMAE/Dxd/SN38/放射性核素络合物，增强药效并拓宽治疗窗口避免药物副作用。
在一些实施方式中，本申请提供的偶联体化合物或其药学上可接受的盐进入靶向细胞内部后，通过细胞内部环境的变化(特异性酶切、pH变化、二硫键还原等)连接子可以裂解释放出药物分子(相当于去除药物分子的修饰基团)，对肿瘤细胞产生治疗作用。
在一些实施方式中，本申请的偶联体化合物或其药学上可接受的盐可用于将有效载荷特异性地递送至在靶组织环境中的靶细胞。在通常情况下，偶联体化合物或其药学上可接 受的盐的两个靶向分子有三个优点。首先，两个靶向分子能够以多种方式起作用(通常是协同作用)，从而提高治疗效果，同时降低副作用。其次，两个靶向分子结合增加了偶联体化合物或其药学上可接受的盐对靶受体或靶细胞的亲和性或亲和力，从而增强其特异性，并且避免了脱靶毒性。最后，当设计合理时，两个靶向分子的组合可以满足药物偶联体通常所需的多功能要求。
本申请的偶联体化合物或其药学上可接受的盐取得了预料不到的技术效果，包括但不限于：(1)能够与细胞表面受体结合的配体和能介导内吞作用的协同作用分子的组合使得偶联体化合物能够特异性地进入靶细胞；(2)偶联体化合物或其药学上可接受的盐增强了药物化合物的亲和性和靶向特异性，从而向患者递送高效的化疗剂(如MMAE)，拓宽了此类药剂的治疗窗并避免了副作用；(3)连接子能够阻止有效载荷在靶细胞外(例如，血液循环系统、细胞间质等)释放，确保了偶联体化合物在血液循环中的稳定性并降低了药物的毒性，进入靶细胞后，连接子被裂解，释放有效载荷，从而发挥药物的作用，同时，可以避免多重耐药性(MDR)；(4)多种多样的药物可以以本申请的偶联体化合物的形式递送，因此扩大了相关药物的应用范围。因此，本申请的偶联体化合物或其药学上可接受的盐不仅拓宽了LDC药物的目标范围和治疗窗，而且还降低了一些药物的毒性和副作用。
在本申请中使用的术语“多肽”、“蛋白”和“肽”可以是单个氨基酸或者氨基酸的聚合物。如本申请所述的多肽、蛋白或肽可以含有天然存在的氨基酸，以及非天然存在的氨基酸，或者氨基酸的类似物和模拟物。可以通过本领域熟知的任何方法获得多肽、蛋白或肽，例如但不限于从天然物质中分离和纯化、重组表达、化学合成等。
本申请的另一个方面公开了药物组合物，所述药物组合物含有本申请提供的偶联体化合物或其药学上可接受的盐，以及药学上可接受的载体。
在本申请中使用的术语“药学上可接受的”指的是在合理的医学判断范围内，其适用于与人和其他动物的细胞接触而没有不适当的毒性、刺激性、过敏反应等，并且与合理的效益/风险比是相称的。
在本申请中使用的术语“药学上可接受的盐”指的是本申请的偶联体化合物的相对无毒性的、无机和有机酸加成盐和碱加成盐。代表性的酸加成盐包括氢溴酸盐、盐酸盐、硫酸盐、硫酸氢盐、磷酸盐、硝酸盐、乙酸盐、草酸盐、戊酸盐、油酸盐、棕榈酸盐、硬脂酸盐、月桂酸盐、硼酸盐、苯甲酸盐、乳酸盐、磷酸盐、甲苯磺酸盐、柠檬酸盐、马来酸盐、富马酸盐、琥珀酸盐、酒石酸盐、萘酸盐、甲磺酸盐、葡庚糖酸盐、乳糖酸盐、氨基磺酸盐、丙二酸盐、水杨酸盐、丙酸盐、亚甲基-双-b-羟基萘甲酸盐、龙胆酸盐、羟乙基磺酸盐、二对甲苯甲酰基酒石酸盐、甲磺酸盐、乙磺酸盐、苯磺酸盐、对甲苯磺酸盐、环己基氨基磺酸盐和奎尼酸月桂基磺酸盐等。碱加成盐包括药学上可接受的金属和胺盐。适宜的金属盐包括钠、钾、钙、钡、锌、镁和铝盐。在一些实施方式中，钠盐和钾盐是优选 的。适宜的无机碱加成盐由金属碱制备，所述金属碱包括例如氢化钠、氢氧化钠、氢氧化钾、氢氧化钙、氢氧化铝、氢氧化锂、氢氧化镁和氢氧化锌。适宜的胺碱加成盐由具有足够碱性的胺制备，以形成稳定的盐，并且优选包括以下常用于药物化学的胺，因为其毒性较低并且是医疗用途可接受的：氨、乙二胺、N-甲基葡糖胺、赖氨酸、精氨酸、鸟氨酸、胆碱、N,N'-二苄基乙二胺、氯普鲁卡因、二乙醇胺、普鲁卡因、N-苄基苯乙胺、二乙胺、哌嗪、三羟甲基氨基甲烷、四甲基氢氧化铵、三乙胺、二苄胺、二苯羟甲胺、脱氢枞胺、N-乙基哌啶、苄胺、四甲基铵、四乙基铵、甲胺、二甲胺、三甲胺、乙胺、碱性氨基酸(例如，赖氨酸和精氨酸)以及二环己胺等。
在本申请中使用的术语“药学上可接受的载体”指的是用于向对象递送本申请提供的偶联体化合物的药学上可接受的溶剂、混悬剂或任意其他药学上惰性的载剂，其不干扰偶联体化合物的结构和性质。某些这样的载体能够将偶联体化合物配制成例如片剂、丸剂、胶囊剂、液体、凝胶剂、糖浆剂、浆剂、混悬剂和软锭剂，供对象口服摄入。某些这样的载体能够将偶联体化合物制成注射、输注或局部施用的制剂。
用于本申请提供的药物组合物中的药学上可接受的载体包括但不限于，例如药学上可接受的液体、凝胶或固体载体、水性载剂(例如，氯化钠注射液、林格氏注射液、等渗右旋糖注射液、无菌水注射液或者右旋糖和乳酸林格氏注射液)、非水性载剂(例如，植物来源的固定油、棉籽油、玉米油、芝麻油或花生油)、抗微生物剂、等渗剂(例如，氯化钠或右旋糖)、缓冲剂(例如，磷酸或柠檬酸缓冲剂)、抗氧剂(例如，硫酸氢钠)、麻醉剂(例如，盐酸普鲁卡因)、混悬剂/分散剂(例如，羧甲基纤维素钠、羟丙基甲基纤维素或聚乙烯吡咯烷酮)、螯合剂(例如，EDTA(乙二胺四乙酸)或EGTA(乙二醇四乙酸))、乳化剂(例如，聚山梨醇酯80(吐温-80))、稀释剂、佐剂、赋形剂、或无毒性辅助物质、本领域公知的其他成分或其各种组合。适宜的成分可以包括例如填充剂、粘合剂、缓冲剂、防腐剂、润滑剂、调味剂、增稠剂、着色剂或乳化剂。
在一些实施方式中，药物组合物是注射制剂。注射制剂包括无菌水溶液或分散剂、混悬剂或乳剂。在所有情况下，注射制剂应该是无菌的并且应该是流体以便于注射。注射制剂应在生产和储存条件下保持稳定，并且必须防止细菌和真菌等微生物的污染。载体可以是含有例如水、乙醇、多元醇(例如，甘油、丙二醇和液体聚乙二醇等)及其适宜的混合物和/或植物油的溶剂或分散介质。注射制剂应保持适当的流动性。例如，可以通过使用诸如卵磷脂的包衣、通过使用表面活性剂等保持适当的流动性。可以通过各种抗细菌剂和抗真菌剂实现阻止微生物的作用，例如对羟基苯甲酸酯、氯丁醇、苯酚、山梨酸、硫柳汞等。
在一些实施方式中，药物组合物是口服制剂。口服制剂包括但不限于胶囊、扁胶囊、丸剂、片剂、锭剂(使用调味基质，通常为蔗糖和阿拉伯胶或黄蓍胶)、粉剂、颗粒剂、或在水性或非水性液体中的溶液剂或混悬剂、或者作为水包油或油包水液体乳剂、或者作 为酏剂或糖浆、或者作为软锭剂(使用惰性基质，如明胶和甘油，或者蔗糖和阿拉伯胶)和/或作为漱口剂等。
在用于口服施用的固体剂型(例如，胶囊、片剂、丸剂、糖衣丸、散剂、颗粒剂等)中，将偶联体化合物与一种或多种药学上可接受的载体混合，如柠檬酸钠或磷酸氢二钙，和/或下述中的任意一种：(1)填充剂或增量剂，例如，淀粉、乳糖、蔗糖、葡萄糖、甘露醇和/或硅酸；(2)粘合剂，例如，羧甲基纤维素、藻酸盐、明胶、聚乙烯吡咯烷酮、蔗糖和/或阿拉伯胶；(3)保湿剂，例如，甘油；(4)崩解剂，例如，琼脂、碳酸钙、马铃薯或木薯淀粉、海藻酸、某些硅酸盐和碳酸钠；(5)溶液阻滞剂，例如，石蜡；(6)吸收促进剂，例如，季铵化合物；(7)润湿剂，例如，乙酰基醇和单硬脂酸甘油酯；(8)吸收剂，例如，高岭土和膨润土；(9)润滑剂，例如，滑石、硬脂酸钙、硬脂酸镁、固体聚乙二醇、月桂基硫酸钠及其混合物；以及(10)着色剂。
在用于口服施用的液体剂型中，将偶联体化合物与下述中的任意一种混合：药学上可接受的乳剂、微乳、溶液、混悬剂、糖浆剂和酏剂。除了偶联体化合物以外，液体剂型可以含有本领域常用的惰性稀释剂，例如，水或其他溶剂、增溶剂和乳化剂，例如，乙醇、异丙醇、碳酸乙酯、乙酸乙酯、苯甲醇、苯甲酸苄酯、异丙醇、1,3-丁二醇、油(特别是，棉籽油、花生油、玉米油、橄榄油、蓖麻油和芝麻油)、甘油、四氢糠醇、聚乙二醇和脱水山梨醇脂肪酸酯及其混合物。除了惰性稀释剂以外，口服组合物还可以包含佐剂，例如，润湿剂、乳化剂和混悬剂、甜味剂、调味剂、着色剂、增香剂和防腐剂。
在一些实施方式中，药物组合物是口腔喷雾制剂或鼻喷雾制剂。喷雾制剂包括但不限于水性气雾剂、非水性混悬剂、脂质体制剂或固体颗粒制剂等。水性气雾剂通过将药剂的水溶液或混悬液与常规药学上可接受的载体和稳定剂混合制备。载体和稳定剂根据具体化合物的要求而改变，但是在通常情况下，其包括非离子表面活性剂(吐温或聚乙二醇)、油酸、卵磷脂、氨基酸，例如，甘氨酸、缓冲溶液、盐、糖或糖醇。气雾剂通常由等渗溶液制备，并且能够通过喷雾递送。
在一些实施方式中，药物组合物可以通过与一种或多种其他药物混合使用。在一些实施方式中，药物组合物包含至少一种其他药物。在一些实施方式中，其他药物是抗肿瘤药、心血管药、抗炎药、抗病毒药、消化系统药、神经系统药、呼吸系统药、免疫系统药、皮肤病药、代谢药等。
在一些实施方式中，可以通过适宜的途径向有需要的对象施用药物组合物，包括但不限于口服、注射(例如，静脉内、肌内、皮下、皮内、心内、鞘内、胸膜内、腹膜内注射等)、粘膜(例如，鼻内、口内施用等)、舌下、直肠、经皮、眼内和肺部施用。在一些实施方式中，药物组合物可以静脉内、皮下、口服、肌内或心室内施用。
由于一些有效载荷的性质，例如高毒性、高亲水性，因而希望将有效载荷更特异性地和更有效地向有需要的对象递送。例如，在癌症治疗中，希望将化疗剂特异性地向癌细胞 递送，而不对正常细胞产生毒性。因而，本申请的另一个方面公开了向有需要的对象递送有效载荷的方法，所述方法包括向对象施用治疗有效量的本申请提供的偶联体化合物，或其药学上可接受的盐，或者本申请提供的药物组合物。本申请所述的有效载荷可以是研究者、兽医、医生或其他医师正在寻找的在组织、系统、动物个体或人中引发生物学或医学应答以预防、抑制、改善或治疗疾病的任何药剂。
在本申请中使用的术语“对象”指的是人和非人动物。非人动物包括所有的脊椎动物，例如哺乳动物和非哺乳动物。对象也可以是家畜，例如，牛、猪、羊、家禽和马，或家养动物，例如，狗和猫。对象可以是雄性(例如，男性)或雌性(例如，女性)，可以是老年，成年、青少年、儿童或婴儿。人可以是高加索人、非洲人、亚洲人、闪族人或其他种族背景人士，或者是这些种族背景的混合。
在本申请中使用的术语“治疗有效量”指的是偶联体化合物或其药学上可接受的盐或药物组合物在一定程度上减轻对象中疾病或病症的一种或多种症状的量；使与疾病或病症相关或导致疾病或病症的一个或多个生理或生化参数部分或完全恢复正常的量；和/或降低疾病或病症发病的可能性的量。这种量通常根据多种因素而改变，本领域的普通技术人员根据本申请提供的说明书的范围能够对其进行确定和说明。这些包括但不限于：特定对象及其年龄、体重、身高、一般身体状况和病史、所使用的特定化合物，以及其制剂的载体和所选择的施用途径；以及所治疗病况的性质和严重程度。
在一些实施方式中，偶联体化合物或其药学上可接受的盐，或者药物组合物的量足以在对象中抑制疾病或病症，或者预防性地抑制或阻止疾病或病症的发病。尽管治疗有效量可以在不同对象中改变，但是其范围通常为从0.01至100mg/kg，例如0.01至90mg/kg、0.01至80mg/kg、0.01至70mg/kg、0.01至60mg/kg、0.01至50mg/kg、0.01至40mg/kg、0.01至30mg/kg、0.01至20mg/kg、0.01至10mg/kg、0.01至5mg/kg、0.01至4mg/kg、0.01至3mg/kg、0.01至2mg/kg、0.01至1mg/kg、0.01至0.1mg/kg。本申请所述的治疗有效量可以等于上述数值范围内的任意值，包括该范围的端点。
本申请的另一个方面公开了一种向有需要的对象递送有效载荷的方法，所述方法包括向对象施用治疗有效量的本申请提供的偶联体化合物或其药学上可接受的盐，或者本申请提供的药物组合物。
本申请的另一个方面公开了用于治疗对象中的疾病的方法，所述方法包括通过向对象施用治疗有效量的本申请提供的偶联体化合物或其药学上可接受的盐，或者本申请提供的药物组合物。
在一些实施方式中，疾病是癌症，包括但不限于前列腺癌、乳腺癌、肺癌、肾癌、白血病、卵巢癌、胃癌、子宫癌、子宫内膜癌、肝癌、甲状腺癌、胰腺癌、结肠癌、结直肠癌、食道癌、皮肤癌、淋巴瘤和多发性骨髓瘤。
在一些实施方式中，癌症的癌症细胞具有本申请提及的细胞表面受体或抗原的表达。在一些实施方式中，癌症的癌症细胞具有本申请提及的细胞表面受体或抗原的高表达(例如，根据Depmap数据(参见https://depmap.org/portal/)，相应的基因表达至少为0、0.01、0.05、0.1、0.5、1、2、3、4、5、6、7、8、9、10或10以上)。在一些实施方式中，癌症的癌症细胞具有FOLR1和FOLH1、TRPV6和FOLH1、GNRHR和FOLH1、SSTR2和FOLH1、FOLR1和SSTR2、或TRPV6和FOLR1的高表达。在一些实施方式中，疾病是免疫性疾病，例如，自身免疫性疾病，包括但不限于结缔组织病、系统性硬化症、类风湿性关节炎和系统性红斑狼疮。
在一些实施方式中，疾病是心血管疾病，包括但不限于心绞痛、心肌梗死、中风、心脏病发作、高血压性心脏病、风湿性心脏病、心肌病、心脏心律失常和先天性心脏病。
在一些实施方式中，疾病是代谢疾病，包括但不限于糖尿病、痛风、肥胖症、低血糖症、高血糖症和血脂异常。
在一些实施方式中，疾病是神经疾病，包括但不限于阿尔茨海默病、帕金森病、亨廷顿病、头部损伤、多发性硬化症、眩晕、昏迷和癫痫。
在一些实施方式中，本申请提供的方法还包括将一种或多种治疗剂与偶联体化合物或其药学上可接受的盐，或者药物组合物联合施用。在一些实施方式中，治疗剂靶向抗癌治疗靶点，诱导或增强针对癌症的免疫应答，或者是化疗剂。
以下将通过具体实施例更详细地描述本申请。提供以下实施例仅用于说明目的，并且不旨在以任何方式限制本发明。本领域技术人员将容易地意识到可以对多种非关键参数进行改变或修改以产生基本上相同的结果。
实施例
以下实施例旨在进一步说明本申请。通过描述，本申请的优点和特征将变得清楚。然而，这些说明仅仅是示例性的，并且不应将其理解为对本申请的范围的限制。
实施例1：偶联体化合物的制备
偶联体化合物CB-20BK、CB-18G、CB-20B、CB-10S、CB-20C、FA-MMAE、CB-20AK、 CB-1020、CB-1320和CB-1820的合成
1.称取替代度为0.45mmol/g的Rink amide-am树脂(下称“Rink Resin”，西安蓝晓科技新材料股份有限公司，货号183599-10-2)10g，将其装载于固相反应柱中，加入DCM，氮气鼓泡至溶剂中，使树脂溶胀30分钟；抽掉溶剂，用DBLK脱除树脂上的Fmoc保护基团，再用DMF洗涤5次。称取Fmoc-Lys(Dde)-OH4.79g(9mmol)，HOBt1.47g(10.8mmol)，用DMF溶解，0℃冰水浴下向上述溶液中加入DIC1.67ml(10.8mmol)，混合使其活化5 分钟，将该溶液加入上述反应柱中，反应3小时后，抽干溶剂，洗涤反应柱中的树脂3遍。然后用DBLK脱除Fmoc保护基团。
2.重复上述操作，按照结构依次偶联Fmoc-Cys(Trt)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Asp(OtBu)-OH以及中间体108。
Figure PCTCN2020074117-appb-000056
3.用2％水合肼/DMF脱除Dde保护基两遍，每次10分钟，再用DMF洗涤树脂5次。依次偶联Fmoc-Glu-OtBu以及蝶酸。最后用甲醇收缩树脂两遍，抽干溶剂，获得保护肽树脂17.4g。
4.将上一步得到的肽树脂17.4g加入到250ml单口烧瓶中，预先配置裂解液139ml，TFA:H 2O:TIS＝95:3:2(体积比)，并称量2.1g DTT加入裂解液中。将裂解液加入到上述烧瓶中，室温反应2.5小时，过滤，树脂继续用30ml TFA洗涤，合并上述滤液，并加入到834ml无水乙醚中，此时有黄色固体析出，离心得到固体，无水乙醚洗涤固体，真空干燥得到黄色固体6.4g，粗产物收率93.4％。HPLC纯度82.3％。经HPLC制备分离(制备条件：C18柱子，流动相A：0.1％三氟乙酸水溶液、B：乙腈，洗脱梯度(15-25)％B，时间60分钟，收集馏分)，将含有合格产品的馏分冻干得到20BK-SM09 4.73g，纯度为98.8％。
5.称取Mc-Val-Cit-PAB-MMAE(LT1002)4.09g(3.11mmol)加入到1000ml单口烧瓶中，并加入500ml磷酸缓冲液和100ml乙腈，搅拌，保持pH＝7.2至澄清，加入中间体4.73g(3.11mmol)20BK-SM09，室温反应2小时，期间HPLC监测反应。待反应完全后，过滤，滤液通过HPLC制备分离(制备条件：C18柱子，流动相A：碳酸氢铵溶液(pH＝7.2)、B：乙腈，洗脱梯度(25-35)％B，时间60分钟，收集馏分)，将含有合格产品的馏分冻干得到CB-20BK 6.96g产物，纯度98.8％，收率为78.8％。
同理，通过与以上方法类似的步骤可获得偶联体化合物CB-18G、CB-20B、CB-10S、CB-20C、FA-MMAE(结构如下所示)、CB-20AK(结构如下所示)、CB-1020、CB-1320和CB-1820。
Figure PCTCN2020074117-appb-000057
偶联体化合物CB-50S、CB-60S和CB-60SK的合成
1.称取替代度为1.1mmol/g的王树脂(下称“Wang Resin”，西安蓝晓科技新材料股份有限公司，货号1365700-43-1)10g，将其装载于固相反应柱中，加入DMF，氮气鼓泡至溶剂，溶胀30分钟；称取Fmoc-Arg(pbf)-OH 14.3g(22mmol)，HOBt 3.56g(26.4mmol)，DMAP 0.27g(2.2mmol)，用DMF溶解，0℃冰水浴下加入4.1ml DIC(26.4mmol)，混合使其活化5分钟，将该溶液加入反应柱，反应3小时后，抽干溶剂，洗涤3遍。
2.将10.4ml醋酸酐、8.9ml吡啶溶于50ml DMF中，混合加入以上洗涤后的树脂，室温封闭5小时，DMF洗涤三次，甲醇收缩后抽干树脂，得到Fmoc-Arg(pbf)-Wang Resin，检测替代度为0.53mmol/g。
3.称取Fmoc-Arg(pbf)-Wang Resin(Sub＝0.53mmol/g)3.8克(2mmol)于反应柱中，用DMF清洗3次，再加入DMF使树脂溶胀30分钟。然后用DBLK脱除Fmoc保护基团，再用DMF洗涤6次。称取Fmoc-Pro-OH2.0g(6mmol)，HOBt 0.97g(7.2mmol)，用DMF溶解，0℃冰水浴下加入1.1ml DIC(7.2mmol)，混合使其活化5分钟，加入反应柱，反应2小时，然后用DBLK脱除Fmoc保护基团。
4.重复上述操作，按照结构依次偶联Fmoc-Leu-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Val-OH、Fmoc-Lys(Boc)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Pro-OH、Fmoc-His(Trt)-OH、Fmoc-Leu-OH、Fmoc-Phe-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Lys(Boc)-OH、Fmoc-propargyl-Gly-OH、Fmoc-Glu-OtBu以及蝶酸。用甲醇收缩两次，抽干溶剂，获得肽树脂8.4g。
5.将上一步得到的肽树脂8.4g加入到250ml单口烧瓶中，预先配置裂解液67ml，TFA:H 2O:TIS＝95:3:2(体积比)，并称量0.92g DTT加入裂解液中。将裂解液加入到烧瓶中，室温反应2.5小时，滤掉树脂，树脂用20ml TFA洗涤，合并滤液，加入到402ml无水乙醚中析出黄色固体，离心得到固体，无水乙醚洗涤固体，真空干燥得到黄色固体4.06g，粗产物收率97.3％。HPLC纯度84.6％。经HPLC制备分离(制备条件：C18柱子，流动相A：0.1％三氟乙酸水溶液、B：乙腈，洗脱梯度(20-29)％B，时间60分钟，收集馏分)，将含有合格产品的馏分冻干得到50S-SM09 2.86g，纯度为97.6％。
6.称取LT1000N3 1.29g(1.37mmol)加入到500ml单口烧瓶中，并加入270ml混合溶剂(ACN:H 2O＝1:4)，CuBr 393mg(2.74mmol)，搅拌。加入中间体2.86g(1.37mmol)50S-SM09，室温反应2-3小时，期间HPLC监测反应。反应完全后，过滤，通过HPLC制备分离(制备条件：C18柱子，流动相A：0.1％三氟乙酸水溶液、B：乙腈，洗脱梯度(22-40)％B，时间60分钟，收集馏分)，将含有合格产品的馏分冻干得到CB-50S 3.17g，纯度98.6％，收率为76.4％。
同理，通过与以上方法类似的步骤可获得偶联体化合物CB-60S和CB-60SK。
CB-20R的合成
1.称取替代度为0.45mmol/g的Rink Resin 5g，将其装载于固相反应柱中，加入DCM，氮气鼓泡至溶剂，使树脂溶胀30分钟；抽掉溶剂，用DBLK脱除树脂上的Fmoc保护基团，再用DMF洗涤5次。称取Fmoc-Lys(Dde)-OH 2.4g(4.5mmol)，HOBt 0.74g(5.4mmol)，用DMF溶解，0℃冰水浴下加入DIC 0.84ml(5.4mmol)，混合使其活化5分钟，加入反应柱，反应3小时后，抽干，洗涤3遍。然后用DBLK脱除Fmoc保护基团。
2.重复上述操作，按照结构依次偶联Fmoc-Cys(Trt)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Asp(OtBu)-OH以及中间体108。
3.用2％水合肼/DMF脱除Dde保护基两遍，每次10分钟，再用DMF洗涤5次。偶联DOTA-tris(tBu)ester。再用2％水合肼/DMF脱除Dde保护基两遍，每次10分钟，再用DMF洗涤5次。依次偶联Fmoc-Glu-OtBu和蝶酸，最后用甲醇收缩两遍，抽干，获得保护肽树脂9.2g。
4.将上一步得到的肽树脂9.2g加入到250ml单口烧瓶中，预先配置裂解液74ml，TFA:H 2O:TIS＝95:3:2(体积比)，并称量1.05g DTT加入裂解液中。将裂解液加入到烧瓶 中，室温反应2.5小时，滤掉树脂，树脂用20ml TFA洗涤，合并滤液，加入到560ml无水乙醚中析出黄色固体，离心，无水乙醚洗涤固体，真空干燥得到黄色固体3.8g，粗产物收率87.4％。HPLC纯度81.2％。经HPLC制备分离(制备条件：C18柱子，流动相A：0.1％三氟乙酸水溶液、B：乙腈，洗脱梯度(15-25)％B，时间60分钟，收集馏分)，将含有合成产品的馏分冻干得到20R-SM09 2.9g，纯度为97.8％。
5.将20R-SM09与放射性核素离子M络合获得CB-20R。具体的，将放射性标记物 177Lu(约50MBq)与100μl 0.5M的乙酸钠缓冲液(pH＝5)混合。将40μl溶于10％DMSO水溶液的1mM CB-20R溶液、2μl饱和抗坏血酸溶液和100μl含 177Lu溶液进行混合，加热至95℃并保持10分钟。所述标记通过radio-HPLC检验(在5分钟以内，0-100％ACN水溶液，C18柱)。
化合物CR19425的合成
Figure PCTCN2020074117-appb-000058
Figure PCTCN2020074117-appb-000059
1.N 2保护下，往反应瓶中加入441.4mg CR19420(结构如上反应步骤中所示)、8.0mLDMF，搅拌溶解，冰浴降温，加入459.2mg HATU，加入380μL DIPEA，搅拌半小时；加入500.0mg CR19419(结构如上反应步骤中所示)，加入190μL DIPEA，室温反应直至完毕。反应完毕后，将反应液倒入醋酸水中，析出固体，过滤，滤饼用醋酸水洗涤，水洗，真空干燥得到835.7mg CR19421(结构如上反应步骤中所示)，棕色粉末，HPLC纯度：90.0％，收率：90.6％。
2.N 2保护下，往反应瓶中加入835.7mg CR19421、16mL10％哌啶DMF溶液，室温下反应半小时。反应完毕后，将反应液倒入TFA/MTBE中，析出固体，过滤，滤饼用MTBE洗涤，真空干燥得到599.7mg CR19422(结构如上反应步骤中所示)，土灰色粉末，HPLC纯度：84.7％，收率：83.0％。
3.N 2保护下，往反应瓶中加入599.7mg CR19422、586.7mg CR19424(结构如上反应步骤中所示)、15mL DMF，搅拌溶解，冰浴降温，加入495.7mg HATU，加入410μLDIPEA，室温反应。反应完毕后，将反应液制备纯化，制备纯品液减压除去乙腈，二氯甲烷甲醇混合溶剂萃取，浓缩干燥得674.9mg CR19423(结构如上反应步骤中所示)，黄色粉末，HPLC纯度：88.4％，收率：63.1％。
4.N 2保护下，往反应瓶中加入14.8mg CR19423，3.0mL pH＝6.6的PBS缓冲液，3.0mL乙腈，搅拌溶解，加入32.9mg CBSM09，用Na 2HPO 4调pH至6.6～6.8，反应半小时。反应完毕后，将反应液制备纯化，制备纯品冷冻干燥得21.8mg CR19425，黄色粉末，HPLC纯度：95.6％，收率：48.8％。
化合物CR19426的合成
Figure PCTCN2020074117-appb-000060
1.N 2保护下，往反应瓶中加入25.2mg CR19423，3.0mL pH＝6.6的PBS缓冲液，3.0mL乙腈，搅拌溶解，加入55.5mg18G-SM09，用Na 2HPO 4调pH至6.6～6.8，反应半小时。反应完毕后，将反应液制备纯化，制备纯品冷冻干燥得44.6mg CR19426，黄色粉末，HPLC纯度：95.4％，收率：55.2％。
化合物CR19428的合成
Figure PCTCN2020074117-appb-000061
1.N 2保护下，往反应瓶中加入486mg CR19423，3.0mL pH＝6.6的PBS缓冲液，3.0mL乙腈，搅拌溶解，加入712mg20BK-SM09，用Na 2HPO 4调pH至6.6～6.8，反应半小时。反应完毕后，将反应液制备纯化，制备纯品冷冻干燥得508mg CR19428，黄色粉末，HPLC纯度：96.7％，收率：42.3％。
实施例2：偶联体化合物与靶蛋白的亲和力测定
1.CB-20BK与蛋白FOLR1结合的亲和力测定
实验仪器、材料及试剂
BIAcore T200(GE)
CM5芯片(GE，货号：29104988)
缓冲液：HBS-EP+buffer 10X(GE，货号：BR100669)，使用前用去离子水稀释10倍。
氨基偶联试剂盒(GE，货号：BR100050)
再生试剂：10mM Glycine 2.0(GE，货号：BR100355)
10mM Glycine 3.0(GE，货号：BR100357)
实验步骤
按照BIAcore T200(GE)使用手册进行实验，测定分析物CB-20BK、CB-20AK、叶酸(FA)和FA-MMAE与FOLR1的亲和力。其中CM5芯片偶联配体FOLR1(R&D System，货号5646-FR)。实验结果如表4所示。
表4.CB-20BK和相关化合物与FOLR1结合的亲和力
  ka kd KD
FA(叶酸) 3.34×10 6 M -1s -1 2.26×10 -4 s -1 6.77×10 -11 M
FA-MMAE 1.79×10 6 M -1s -1 1.15×10 -4 s -1 6.43×10 -11 M
CB-20AK N/D N/D N/D
CB-20BK 1.03×10 6 M -1s -1 1.31×10 -4 s -1 1.27×10 -10 M
“N/D”表示未检测到特异性结合。
表4显示，CB-20BK和FOLR1特异性结合，并显示出良好的亲和力。CB-20BK和FOLR1结合的亲和力比FA或FA-MMAE与FOLR1结合的亲和力稍弱。CB-20AK没有CB-20BK中的叶酸部分，在实验中未检测出它与FOLR1特异性结合。
2.CB-20BK与蛋白FOLH1结合的亲和力测定
实验仪器、材料及试剂
Gator TM(Probe Life)
SA探针(Probe Life，货号1906018)
缓冲液：Q buffer(Probe Life)，10mM，pH＝7.4
实验步骤
(1)分析物Biotin-CB-20BK的合成步骤
1)称取替代度为0.45mmol/g的Rink Resin 5.1g，将其装载于固相反应柱中，加入DCM，氮气鼓泡溶胀30分钟；抽掉溶剂，用DBLK脱除Fmoc保护基团，再用DMF洗涤5次。称取Fmoc-Lys(Dde)-OH 2.45g，HOBt 0.75g，用DMF溶解，0℃冰水浴下加入DIC 0.83ml， 活化5分钟，加入反应柱，反应3小时后，抽干，洗涤3遍。然后用DBLK脱除Fmoc保护基团。
2)重复上述操作，按照结构依次偶联Fmoc-Cys(Trt)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Asp(OtBu)-OH以及中间体108。
3)用2％水合肼/DMF脱除Dde保护基两遍，每次10分钟，再用DMF洗涤5次。依次偶联Fmoc-Lys(Biotin)-OH、Fmoc-Glu-OtBu以及蝶酸。偶联蝶酸后，用DMF洗涤2次，最后用甲醇收缩两遍，抽干，获得保护肽树脂9.7g。
4)将上一步得到的肽树脂9.7g加入到250ml单口烧瓶中，预先配置裂解液77.6ml，TFA:H2O:TIS＝95:3:2(体积比)，并称量1.1g DTT加入裂解液中。将裂解液加入到烧瓶中，室温反应2.5小时，滤掉树脂，树脂用20ml TFA洗涤，合并滤液，加入到466ml甲叔醚中析出黄色固体，离心，甲叔醚洗涤固体，真空干燥得到黄色固体4.6g，HPLC纯度83.2％。经HPLC制备分离，冻干得到2.1g、纯度95％以上的Biotin-20BK-SM09。
5)称取Mc-Val-Cit-PAB-MMAE(LT1002)500mg加入到250ml单口烧瓶中，并加入65ml磷酸缓冲液和20ml乙腈，搅拌，保持pH＝7.2至澄清，加入中间体785mgBiotin-20BK-SM09，室温反应2小时，期间HPLC监测反应。反应完全后，过滤，通过HPLC制备分离，冻干得到Biotin-CB-20BK723mg，纯度96.8％，收率为59.4％。
(2)按照Gator TM(Probe Life)使用手册进行试验，测定Biotin-CB-20BK与FOLH1结合的亲和力。其中SA探针结合配体Biotin-CB-20BK，分析物为FOLH1(Sino Biologicals，货号，15877-H07H)。实验结果如表5所示。
表5.CB-20BK与FOLH1结合的亲和力
  ka kd KD
FOLH1 1.19×10 4M -1s -1 1.70×10 -4s -1 6.98×10 -9M
表5显示，CB-20BK和FOLH1结合，并显示出良好的亲和力。
3.FOLH1与CB-20BK-FOLR1复合物结合
实验材料及试剂
BIAcore T200(GE)
CM5芯片(GE，货号：29104988)
缓冲液：HBS-EP+buffer10X(GE，货号：BR100669)，使用前用去离子水稀释10倍。
氨基偶联试剂盒(GE，货号：BR100050)
再生试剂：10mM Glycine2.0(GE，货号：BR100355)
10mM Glycine3.0(GE，货号：BR100357)
实验步骤
CM5芯片偶联配体：FOLR1(R&D System，货号5646-FR)
分析物：CB-20BK，FA-MMAE和FOLH1(Sino Biologicals，货号：15877-H07H)
按照BIAcore T200(GE)操作手册，将FOLR1偶联到CM5芯片上，先进样CB-20BK或FA-MMAE，然后，进样FOLH1，检测FOLH1与CB-20BK-FOLR1复合物的结合。实验结果如表6所示。
表6.不同浓度的FOLH1溶液与CB-20BK-FOLR1复合物结合
浓度(μM) FA-MMAE(RU) CB-20BK(RU)
0.5 9.8 23.9
0.25 8.4 14.1
0.125 4.2 4.6
0.0625 -1 -0.9
表6显示，高浓度的FOLH1溶液与CB-20BK-FOLR1复合物结合的量显著高于FOLH1与FA-MMAE-FOLR1复合物结合的量，而且呈继续上升的趋势。FOLH1与FA-MMAE-FOLR1复合物结合在高浓度时趋于饱和。该实验证明，CB-20BK可以同时与FOLR1和FOLH1两个受体良好的结合。
实施例3：配体偶联物与靶细胞的结合和内吞研究
1.偶联体化合物CB-20BK的细胞结合和内吞实验
标记样品Cy5-pep-20BK、Cy5-FA和Cy5-pep-20AK的合成
(1)称取替代度为0.45mmol/g的Rink Resin2g，将其装载于固相反应柱中，加入DCM，氮气鼓泡至溶剂，使树脂溶胀30分钟；抽掉溶剂，用DBLK脱除Fmoc保护基团，再用DMF洗涤5次。称取Fmoc-Lys(Dde)-OH 0.96g(1.8mmol)，HOBt 0.3g(2.2mmol)，用DMF溶解，0℃冰水浴下加入DIC 0.33ml(2.2mmol)，混合使其活化5分钟，加入反应柱，反应3小时后，抽干，洗涤3遍。然后用DBLK脱除Fmoc保护基团。
(2)重复上述操作，按照结构依次偶联Fmoc-Lys(Boc)-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Asp(OtBu)-OH以及中间体108：
(3)用2％水合肼/DMF脱除Dde保护基两遍，每次10分钟，再用DMF洗涤5次。依次偶联Fmoc-Lys(Dde)-OH、Fmoc-Glu-OtBu以及蝶酸。
(4)然后用2％水合肼/DMF脱除Dde保护基两遍，每次10分钟，再用DMF洗涤5次，偶联Cy5-COOH后，用DMF洗涤2次，最后用甲醇收缩两遍，抽干，获得保护肽树脂3.6g。
(5)将上一步得到的肽树脂3.6g加入到50ml单口烧瓶中，预先配置裂解液29ml，TFA:H 2O:TIS＝95:3:2(体积比)，并称量0.4g DTT加入裂解液中。将裂解液加入到烧瓶中，室温反应2.5小时，滤掉树脂，树脂用8ml TFA洗涤，合并滤液，加入到173ml无水乙醚中析出黄色固体，离心，无水乙醚洗涤固体，真空干燥得到蓝色固体1.9g，粗产物收率89.6％。HPLC纯度76.3％。经HPLC制备分离(制备条件：C18柱子，流动相A：0.1％三氟乙酸水溶液、B：乙腈，洗脱梯度(20-28)％B，时间50分钟，收集馏分)，将含有合格产品的馏分冻干得到Cy5-pep-20BK736mg，纯度为93.4％。
同理，通过与以上方法类似的步骤可获得化合物Cy5-FA和Cy5-pep-20AK。
Figure PCTCN2020074117-appb-000062
Figure PCTCN2020074117-appb-000063
流式细胞仪法检测细胞结合和内吞Cy5-pep-20AK/Cy5-pep-20BK样品实验
样品信息：Cy5-pep-20AK、Cy5-pep-20BK
细胞系：LNCaP人前列腺癌细胞、Du145人前列腺癌细胞、SKOV3人卵巢癌细胞、NCI-H460人肺癌细胞
主要试剂：IMDM培养基、胎牛血清、青霉素-链霉素溶液、L-谷氨酰胺、PBS
实验操作：
(1)细胞培养：准备好细胞，Trypsin消化、收集计数，将细胞加入数个含完全培养基的培养瓶中，置于37℃，5％CO 2培养箱中培养，最终收集约5×10 6个细胞至离心管中。
(2)样品孵育：使用PBS将Cy5-pep-20AK/Cy5-pep-20BK样品稀释至8nmol/L。1000转离心细胞悬液5分钟，去除上清，PBS清洗一次，均匀悬浊细胞，按实验方案等量分装到不同离心管中，离心去除PBS，每管加入200μl样品工作液，37℃分别孵育15、30、60和90分钟，留取1管只加PBS，作为空白对照。全程避光操作。
(3)清洗上机：1000转离心5分钟去除工作液，用PBS清洗细胞3遍，加适量PBS悬浊细胞至1×10 6个细胞/ml。提前打开Beckman CytoFLEX流式细胞仪，完成开机清洗流程，将细胞样本依次上机，在APC通道下读取10000个活细胞的荧光量。全程避光操作。
(4)数据分析：分别获取每个细胞样本APC荧光的绝对MFI(平均荧光强度)，以及相对空白对照的相对MFI，并根据相对MFI完成数据折线图。
结果与分析
表7.FOLR1和FOLH1在不同细胞系中的表达水平
(参考Depmap的数据，来源：https://depmap.org/portal/)
RNA-Seq LNCaP SKOV3 Du145 NCI-H460
FOLR1 +/- 6+ +/- +/-
FOLH1 10+ + +/- +/-
根据Depmap的数据，其中数据为0或者负数的表示为“-”，0.001-0.499的表示为“+/-”，0.500-1.499的表示为“+”，1.500-2.499的为“2+”，以此类推。
分别在孵育15分钟、30分钟、60分钟和90分钟时检测细胞的荧光强度，将结果制图示于图2A和2B。可知样品Cy5-pep-20BK细胞的结合并内吞，在FOLR1相对高表达的LNCaP细胞系和FOLH1相对高表达SKOV3细胞系中，结合和内吞的CB-20BK的荧光强度明显高于它在FOLR1和FOLH1相对低表达的DU145细胞系和NCI-H460细胞系中的荧光强度。Cy5-pep-20AK只有FOLH1配体，没有FOLR1的配体FA，所以，Cy5-pep-20AK在高表达FOLH1的LNCaP细胞中显示高水平的结合和内吞。在中度表达FOLH1的SKOV3细胞中，则显示中度水平的结合和内吞；在两个细胞系中，Cy5-pep-20AK结合和内吞水平较Cy5-pep-CB-20BK有显著下降。配体偶联物与细胞结合内化的程度与细胞相关受体表达水平呈现正相关性。
2.单配体偶联物Cy5-FA与细胞的结合和内吞实验
样品信息：Cy5-FA
细胞系：Hela宫颈癌细胞、SKOV3人卵巢癌细胞和A549人肺癌细胞
主要试剂：IMDM培养基、胎牛血清、青霉素-链霉素溶液、L-谷氨酰胺、PBS、含DAPI的防荧光淬灭封片剂
实验操作：
(1)细胞爬片：在24孔板中放入爬片。准备好细胞，Trypsin消化、收集计数，使用完全细胞培养基将细胞稀释成约2×10 5个细胞/ml，在24孔板每个孔加入500μl稀释的细胞溶液，置于37℃，5％CO 2培养箱培养48小时。
(2)样品孵育：使用IMDM培养基将Cy5-FA样品稀释至73nmol/L。甩去24孔板中培养基，每孔细胞爬片加入样品工作液200μl，37℃分别孵育15、30、60分钟，留取1孔只加IMDM培养基，作为空白对照。全程避光操作。
(3)清洗封片：甩去24孔板中工作液，用37℃预热的PBS清洗3遍，夹出细胞爬片，在载玻片上滴加5μl含DAPI的防荧光淬灭封片剂，将细胞爬片覆盖在封片剂上，完成封片。全程避光操作。
(4)样本读片：打开Leica DM2500荧光显微镜预热荧光激发器15分钟，在相应荧光通道下分别拍摄同一位置细胞核和Cy5荧光素，并在软件中完成图片融合。
根据图3显示可知，样品Cy5-FA在15分钟、30分钟与细胞的结合及内吞，在FOLR1高表达的细胞系Hela和SKOV-3中的荧光强度明显高于FOLR1相对低表达的细胞系A549 中的荧光强度。配体偶联物与细胞结合内化的程度与细胞相关受体表达水平呈现正相关性。
实施例4：偶联体化合物的细胞扩增抑制实验
1.CB-20BK肿瘤细胞扩增抑制实验
样品信息：CB-20BK
细胞系：KB人口腔表皮样癌细胞、T-47D人乳腺癌细胞、NCI-H460人肺癌细胞、CALU-3人肺腺癌细胞、HuH-7人肝癌细胞和LNCaP人前列腺癌细胞
主要试剂：IMDM培养基、胎牛血清、青霉素-链霉素溶液、L-谷氨酰胺、CCK8
实验操作：
1)细胞铺板
提前准备好细胞，Trypsin消化、收集计数，使用完全细胞培养基将KB细胞稀释成2.5×10 4个细胞/ml，T-47D细胞稀释成1.3×10 4个细胞/ml，NCI-H460细胞稀释成3.5×10 4个细胞/ml，CALU-3细胞稀释成4.0×10 4个细胞/ml，HuH-7细胞稀释成3.0×10 4个细胞/ml，LNCaP细胞稀释成8.0×10 4个细胞/ml，铺96孔板，每个孔加入100μl稀释的细胞溶液，每块板设置阴性及空白对照孔。将加好细胞的96孔板置于37℃，5％CO 2培养箱培养过夜。
2)稀释加样
将样品用培养基稀释到所需浓度(参见表8)。按照每孔50μl加入到过夜培养后的96孔板中，3个复孔，并设立阴性对照和空白对照，置于37℃，5％CO 2培养箱培养72小时。
表8：样品(CB-20BK)稀释后浓度
管号 稀释后浓度μmol/L
1 201
2 50
3 13
4 3.1
5 0.8
6 0.2
7 0.05
8 0.01
9 0.003
10 0.0008
3)显色读板
取CCK-8显色液，每孔加入15μl(孔内液体体积的10％)，37℃孵育适宜时间(尽量使OD值在1.0-2.0范围内)，96孔板去掉培养板盖，置于酶标仪(Molecular Devices Spectra MAX Plus)，450nm读数数值。
4)数据处理
用SoftMax Pro进行数据编辑，绘四参数拟合曲线。
5)实验结果与分析
根据四参数拟合曲线图(图4A，其中C值对应于IC 50)，CB-20BK对KB、T-47D、NCI-H460、CALU-3、HuH-7以及LNCaP肿瘤细胞系生长扩增都有抑制作用。由于细胞中偶联体化合物相应的受体表达水平有不同，所以抑制水平有所差异。针对受体相对高表达的细胞株T-47D、KB、LNCaP、HuH-7和CALU-3的IC 50明显低于受体相对低表达的细胞株NCI-H460。CB-20BK显示出了抑瘤效果与细胞相关受体表达水平的相关性。
2.偶联体化合物CB-20BK及相关化合物对肿瘤细胞系LNCaP(人前列腺癌细胞)和22RV1(人前列腺癌细胞)扩增的抑制实验
由于不同细胞对配体偶联物中的有效载荷的细胞增殖抑制毒性的敏感度不同，偶尔会干扰定量分析。我们挑选了一系列不同配体的受体表达水平已知的细胞系，同时检测它们对配体偶联物和单独有效载荷细胞增殖抑制作用的反应。取单独有效载荷与配体偶联物在一个细胞系中的IC 50的比值，从而去除这种干扰。
相关样品：MMAE、CB-20BK、CB-20AK和FA-MMAE
实验操作：将提前准备好的LNCaP和22RV1计数，分别以4×10 4细胞/ml和2.0×10 4细胞/ml的细胞密度铺96孔细胞培养板，100μl/孔，每块板设置相应的阴性及空白对照孔，贴壁过夜后，按50μl/孔加入稀释好的待测样品，放置于37℃，5％CO 2培养箱中，培养68-72小时，加入CCK8显色液，每孔加入15μl(孔内液体体积的10％)，37℃孵育45-70分钟，置于酶标仪450nm读取数值。用SoftMax Pro进行数据编辑，绘4-P拟合曲线。计算出的数据如表9所示。
表9.MMAE、FA-MMAE、CB-20BK和CB-20AK对细胞系增殖的抑制作用和细胞系中受体基因的表达水平(参考Depmap数据)
Figure PCTCN2020074117-appb-000064
Figure PCTCN2020074117-appb-000065
由上表可知，LNCaP和22RV1的FOLR1表达水平差距不大，LNCaP的FOLH1表达量显著高于22RV1的FOLH1表达量。CB-20BK(有FOLH1的配体和FOLR1配体)和CB-20AK(只有FOLH1的配体)对LNCaP和22RV1肿瘤细胞系扩增均有抑制作用。CB-20BK和CB-20AK对于受体FOLH1表达水平有不同的细胞抑制程度有所差异，在受体FOLH1相对高表达细胞株LNCaP中，IC 50比值高于受体相对低表达细胞株22RV1的IC 50比值2-4倍。FA-MMAE对LNCaP和22RV1肿瘤细胞系扩也有抑制作用，由于LNCaP和22RV1的FOLR1表达水平很低，在LNCaP细胞中的表达(FOLR1基因表达水平为0.3219，参考Depmap数据)略高于在22RV1中的表达(FOLR1基因表达水平为0.0704，参考Depmap数据)，FA-MMAE在LNCaP和22RV1的IC 50比值的差距只有1.5倍。上述数据表明细胞增殖抑制效果与细胞表达受体FOLH1和FOLR1表达水平呈现正相关性。
3.偶联体化合物CB-20BK及相关化合物对肿瘤细胞系PANC-1(人胰腺癌细胞)和CFPAC-1(人胰腺癌细胞)扩增的抑制实验
相关样品：MMAE和CB-20BK
实验操作：提前准备好细胞计数，PANC-1和CFPAC-1以4×10 4细胞/ml的细胞密度铺96孔板，100μl/孔，每块培养板设立相应的阴性及空白对照孔，贴壁过夜后，按50μl/孔加入稀释好的待测样品，放置于37℃，5％CO 2培养箱中，培养68-72小时，加入CCK8显色液，每孔加入15μl(孔内液体体积的10％)，37℃孵育45-70分钟，置于酶标仪450nm读取数值。用SoftMax Pro进行数据编辑，绘4-P拟合曲线。计算出的数据如下表所示：
表10.CB-20BK对细胞系增殖的抑制作用和细胞系受体基因的表达水平
(参考Depmap数据)
Figure PCTCN2020074117-appb-000066
由表10可知，CFPAC-1的FOLH1表达水平很低，CFPAC-1的FOLR1表达量显著高于PANC1细胞系的表达量。CB-20BK对PANC-1和CFPAC-1肿瘤细胞系扩增均有抑制作用。对于受体FOLR1表达水平有不同的细胞抑制程度有所差异，在受体FOLR1相对高表达细胞株CFPAC-1中，IC 50比值显著高于受体FOLR1相对低表达PANC-1的IC 50比值，细胞增殖抑制效果与细胞相关受体FOLR1表达水平呈现正相关性。
实验2和3证明，在CB-20BK中两个配体和表达在细胞表面的它们的受体(FOLR1和FOLH1)在该化合物抑制肿瘤细胞增殖的过程中起重要作用。
4.CB-20B肿瘤细胞扩增抑制实验
样品信息：CB-20B
细胞系：A549人肺癌细胞、HuH-7人肝癌细胞、KB人口腔表皮样癌细胞、LNCaP人前列腺癌细胞、DU145人前列腺癌细胞和T-47D人乳腺癌细胞
主要试剂：IMDM培养基、胎牛血清、青霉素-链霉素溶液、L-谷氨酰胺、CCK8
实验操作：
1)细胞铺板
提前准备好细胞，Trypsin消化、收集计数，使用完全细胞培养基，按A549细胞、HuH-7细胞、KB细胞和DU145细胞以2×10 4个细胞/ml密度铺板；T-47D细胞以1×10 5个细胞/ml密度铺板；LNCaP细胞以6×10 4个细胞/ml密度铺96孔板，每个孔加入100μl稀释的细胞溶液，每块板设置阴性及空白对照孔。将加好细胞的96孔板置于37℃，5％CO 2培养箱培养过夜。
2)稀释加样
将样品用培养基稀释到所需浓度(参见表11)。按照每孔50μl加入到过夜培养后的96孔板中，3个复孔，并设立阴性对照和空白对照，置于37℃，5％CO 2培养箱培养72小时。
表11：样品(CB-20B)稀释后浓度
管号 稀释后浓度μmol/L
1 200
2 66.7
3 22.2
4 7.4
5 2.5
6 0.8
7 0.3
8 0.09
9 0.03
10 0.01
3)显色读板
取CCK-8显色液，每孔加入15μl(孔内液体体积的10％)，37℃孵育适宜时间(尽量使OD值在1.0-2.0范围内)，96孔板去掉培养板盖，置于酶标仪(Molecular Devices Spectra MAX Plus)，450nm读数数值。
4)数据处理
用SoftMax Pro进行数据编辑，绘四参数拟合曲线。
5)实验结果与分析
根据四参数拟合曲线图(图4B，其中C值对应于IC 50)，CB-20B对A549、HuH-7、KB、LNcap、DU145和T-47D肿瘤细胞系生长扩增都有抑制作用。由于细胞中偶联体化合物相应的受体表达水平有不同，所以抑制水平有所差异。针对受体相对高表达的细胞株T-47D、LNcap、KB、HuH-7和DU145的IC 50明显低于受体相对低表达的细胞株A549。CB-20B显示出了抑瘤效果与细胞相关受体表达水平的相关性。
5.CB-10S肿瘤细胞扩增抑制实验
样品信息：CB-10S
细胞系：KB人口腔表皮样癌细胞、NCI-H460人肺癌细胞、RT4人膀胱癌细胞、T-47D人乳腺癌细胞和LNcap人前列腺癌细胞
主要试剂：IMDM培养基、胎牛血清、青霉素-链霉素溶液、L-谷氨酰胺、CCK8
实验操作：
1)细胞铺板
提前准备好细胞，Trypsin消化、收集计数，使用完全细胞培养基将KB细胞稀释成3.5×10 4个细胞/ml，LNCaP细胞稀释成8.0×10 4个细胞/ml，T-47D细胞稀释成1.2×10 4个细胞/ml，NCI-H460细胞稀释成2.5×10 4个细胞/ml，RT4细胞稀释成1.2×10 4个细胞/ml，取96孔板，每个孔加入100μl稀释的细胞溶液，每块板设置阴性及空白对照孔。将加好细胞的96孔板置于37℃，5％CO 2培养箱培养过夜。
2)稀释加样
将样品用培养基稀释到所需浓度(参见表12)。按照每孔50μl加入到过夜培养后的96孔板中，3个复孔，并设立阴性对照和空白对照，置于37℃，5％CO 2培养箱培养72小时。
表12：样品(CB-10S)稀释后浓度
管号 稀释后浓度μmol/L
1 302
2 43
3 6
4 0.9
5 0.1
6 0.02
7 0.003
8 0.0004
9 0.00005
10 0.000007
3)显色读板
取CCK-8显色液，每孔加入15μl(孔内液体体积的10％)，37℃孵育适宜时间(尽量使OD值在1.0-2.0范围内)，96孔板去掉培养板盖，置于酶标仪(Molecular Devices Spectra MAX Plus)，450nm读数数值。
4)数据处理
用SoftMax Pro进行数据编辑，绘四参数拟合曲线。
5)实验结果与分析
根据四参数拟合曲线图(图4C，其中C值对应于IC 50)，CB-10S对KB、LNCaP、T-47D、RT4和NCI-H460肿瘤细胞系生长扩增都有抑制作用。由于细胞中偶联体化合物相应的受体表达水平有不同，所以抑制水平有所差异。对于受体相对高表达的细胞株KB、LNcap、T-47D和RT4的IC 50明显低于受体相对低表达细胞株NCI-H460。CB-10S显示出了抑瘤效果与细胞相关受体表达水平的相关性。
6.CB-60S肿瘤细胞扩增抑制实验
样品信息：CB-60S
细胞系：KB人口腔表皮样癌细胞、T-47D人乳腺癌细胞、NCI-H460人肺癌细胞、CALU-3人肺腺癌细胞、HuH-7人肝癌细胞和LNCaP人前列腺癌细胞
主要试剂：IMDM培养基、胎牛血清、青霉素-链霉素溶液、L-谷氨酰胺、CCK8
实验操作：
1)细胞铺板
提前准备好细胞，Trypsin消化、收集计数，使用完全细胞培养基将KB细胞稀释成2.0×10 4个细胞/ml，T-47D细胞稀释成1.5×10 4个细胞/ml，NCI-H460细胞稀释成2.0×10 4个细胞/ml，CALU-3细胞稀释成3.0×10 4个细胞/ml，HuH-7细胞稀释成4.0×10 4个细胞/ml， LNCaP细胞稀释成8.0×10 4个细胞/ml，取96孔板，每个孔加入100μl稀释的细胞溶液，每块板设置阴性及空白对照孔。将加好细胞的96孔板置于37℃，5％CO 2培养箱培养过夜。
2)稀释加样
将样品用培养基稀释到所需浓度(参见表13)。按照每孔50μl加入到过夜培养后的96孔板中，3个复孔，并设立阴性对照和空白对照，置于37℃,5％CO 2培养箱培养72小时。
表13：样品(CB-60S)稀释后浓度
管号 稀释后浓度μmol/L
1 320
2 80
3 20
4 5
5 1.3
6 0.3
7 0.08
8 0.02
9 0.005
10 0.001
3)显色读板
取CCK-8显色液，每孔加入15μl(孔内液体体积的10％)，37℃孵育适宜时间(尽量使OD值在1.0-2.0范围内)，96孔板去掉培养板盖，置于酶标仪(Molecular Devices Spectra MAX Plus)，450nm读数数值。
4)数据处理
用SoftMax Pro进行数据编辑，绘四参数拟合曲线。
5)实验结果与分析
四参数拟合曲线图(图4D，其中C值对应于IC 50)，CB-60S对KB、T-47D、NCI-H460、CALU-3、HuH-7和LNCaP肿瘤细胞系生长扩增都有抑制作用。由于细胞中偶联体化合物相应的受体表达水平有不同，所以抑制水平有所差异。对于受体相对高表达的细胞株LNCaP和HuH-7的IC 50值明显低于受体相对低表达的细胞株NCI-H460、KB、T-47D和CALU-3。CB-60S显示出了抑瘤效果与细胞相关受体表达水平的相关性。
7.CB-60SK肿瘤细胞扩增抑制实验
样品信息：CB-60SK
细胞系：KB人口腔表皮样癌细胞、T-47D人乳腺癌细胞、NCI-H460人肺癌细胞、CALU-3人肺腺癌细胞、HuH-7人肝癌细胞和LNCaP人前列腺癌细胞
主要试剂：IMDM培养基、胎牛血清、青霉素-链霉素溶液、L-谷氨酰胺、CCK8
实验操作：
1)细胞铺板
提前准备好细胞，Trypsin消化、收集计数，使用完全细胞培养基将KB细胞稀释成2.5×10 4个细胞/ml，T-47D细胞稀释成1.3×10 4个细胞/ml，NCI-H460细胞稀释成3.5×10 4个细胞/ml，CALU-3细胞稀释成4.0×10 4个细胞/ml，HuH-7细胞稀释成3.0×10 4个细胞/ml，LNCaP细胞稀释成8.0×10 4个细胞/ml，每个孔加入100μl稀释的细胞溶液，每块板设置阴性及空白对照孔。将加好细胞的96孔板置于37℃，5％CO 2培养箱培养过夜。
2)稀释加样
将样品用培养基稀释到所需浓度(参见表14)。按照每孔50μl加入到过夜培养后的96孔板中，3个复孔，并设立阴性对照和空白对照，置于37℃，5％CO 2培养箱培养72小时。
表14：样品(CB-60SK)稀释后浓度
管号 稀释后浓度μmol/L
1 289
2 72
3 18
4 4.5
5 1.1
6 0.3
7 0.07
8 0.02
9 0.004
10 0.001
3)显色读板
取CCK-8显色液，每孔加入15μl(孔内液体体积的10％)，37℃孵育适宜时间(尽量使OD值在1.0-2.0范围内)，96孔板去掉培养板盖，置于酶标仪(Molecular Devices Spectra MAX Plus)，450nm读数数值。
4)数据处理
用SoftMax Pro进行数据编辑，绘四参数拟合曲线。
5)实验结果与分析
根据四参数拟合曲线图(图4E，其中C值对应于IC 50)，CB-60SK对KB、T-47D、NCI-H460、CALU-3、HuH-7和LNCaP肿瘤细胞系生长扩增都有抑制作用。由于细胞中偶联体化合物相应的受体表达水平有不同，所以抑制水平有所差异。对于受体相对高表达的细胞株LNCaP和HuH-7的IC 50值明显低于受体相对低表达的细胞株T-47D、NCI-H460、CALU-3和KB。CB-60SK显示出了抑瘤效果与细胞相关受体表达水平的相关性。
8.CB-18G肿瘤细胞扩增抑制实验
样品信息：CB-18G
细胞系：A549人肺癌细胞、Hela人宫颈癌细胞、SCLC-21H小细胞肺癌细胞、U-2OS人骨肉瘤细胞、T-47D人乳腺癌细胞和NCI-H460人肺癌细胞
主要试剂：IMDM培养基、胎牛血清、P青霉素-链霉素溶液、L-谷氨酰胺、CCK8
实验操作：
1)细胞铺板
提前准备好细胞，Trypsin消化、收集计数，使用完全细胞培养基，按A549细胞、Hela细胞、SCLC-21H细胞和U-2OS细胞以2×10 4个细胞/ml密度铺板；T-47D细胞和NCI-H460细胞以3×10 4个细胞/ml密度铺96孔板，取96孔板，每个孔加入100μl稀释的细胞溶液，每块板设置阴性及空白对照孔。将加好细胞的96孔板置于37℃，5％CO 2培养箱培养过夜。
2)稀释加样
将样品用培养基稀释到所需浓度(参见表15)。按照每孔50μl加入到过夜培养后的96孔板中，3个复孔，并设立阴性对照和空白对照，置于37℃，5％CO 2培养箱培养72小时。
表15：样品(CB-18G)稀释后浓度
管号 稀释后浓度μmol/L
1 100
2 25
3 6.25
4 1.56
5 0.39
6 0.10
7 0.02
8 0.006
9 0.002
10 0.0004
3)显色读板
取CCK-8显色液，每孔加入15μl(孔内液体体积的10％)，37℃孵育适宜时间(尽量使OD值在1.0-2.0范围内)，96孔板去掉培养板盖，置于酶标仪(Molecular Devices Spectra MAX Plus)，450nm读数数值。
4)数据处理
用SoftMax Pro进行数据编辑，绘四参数拟合曲线。
5)实验结果与分析
根据四参数拟合曲线图(图4F，其中C值对应于IC 50)，CB-18G对A549、Hela、SCLC-21H、U-2OS、T-47D和NCI-H460肿瘤细胞系生长扩增都有抑制作用。由于细胞中偶联体化合物相应的受体表达水平有不同，所以抑制水平有所差异。对于受体相对高表达的细胞株Hela的IC 50低于受体相对低表达的细胞株NCI-H460。CB-18G显示出了抑瘤效果与细胞相关受体表达水平的相关性。
9.CB-50S肿瘤细胞扩增抑制实验
样品信息：CB-50S
细胞系：KB人口腔表皮样癌细胞、NCI-H460人肺癌细胞、RT4人膀胱癌细胞、T-47D人乳腺癌细胞和LNCaP人前列腺癌细胞
主要试剂：IMDM培养基、胎牛血清、青霉素-链霉素溶液、L-谷氨酰胺、CCK8
实验操作：
1)细胞铺板
提前准备好细胞，Trypsin消化、收集计数，使用完全细胞培养基将KB细胞稀释成3.5×10 4个细胞/ml，LNCaP细胞稀释成8.0×10 4个细胞/ml，T-47D细胞稀释成1.2×10 4个细胞/ml，NCI-H460细胞稀释成2.5×10 4个细胞/ml，RT4细胞稀释成1.2×10 5个细胞/ml，取96孔板，每个孔加入100μl稀释的细胞溶液，每块板设置阴性及空白对照孔。将加好细胞的96孔板置于37℃，5％CO 2培养箱培养过夜。
2)稀释加样
将样品用培养基稀释到所需浓度(参见表16)。按照每孔50μl加入到过夜培养后的96孔板中，3个复孔，并设立阴性对照和空白对照，置于37℃，5％CO 2培养箱培养72小时。
表16：样品(CB-50S)稀释后浓度
管号 稀释后浓度μmol/L
1 314
2 45
3 6
4 0.9
5 0.1
6 0.02
7 0.003
8 0.0004
9 0.00005
10 0.000008
3)显色读板
取CCK-8显色液，每孔加入15μl(孔内液体体积的10％)，37℃孵育适宜时间(尽量使OD值在1.0-2.0范围内)，96孔板去掉培养板盖，置于酶标仪(Molecular Devices Spectra MAX Plus)，450nm读数数值。
4)数据处理
用SoftMax Pro进行数据编辑，绘四参数拟合曲线。
5)实验结果与分析
根据四参数拟合曲线图(图4G，其中C值对应于IC 50)，CB-50S对KB、LNCaP、T-47D、RT4和NCI-H460肿瘤细胞系生长扩增都有抑制作用。由于细胞中偶联体化合物相应的受体表达水平有不同，所以抑制水平有所差异。对于受体相对高表达的细胞株KB、LNcap、T-47D和RT4的IC 50明显低于受体相对低表达的细胞株NCI-H460。CB-50S显示出了抑瘤效果与细胞相关受体表达水平的相关性。
10.偶联体化合物CB-1020肿瘤细胞扩增抑制实验
实验目的：测试CB-1020及相关化合物对肿瘤细胞系LNCaP(人前列腺癌细胞)、SK-BR-3(人乳腺癌细胞)、NCI-H226(人肺癌细胞)、CFPAC-1(胰腺癌细胞)和PANC-1(胰腺癌细胞)扩增的抑制作用。
相关样品：MMAE和CB-1020
实验操作：将提前准备好的LNCaP、SK-BR-3、NCI-H226、CFPAC-1和PANC-1计数，用完全培养基(IMDM+10％FBS+1X L-Glutamine+1X P/S)将细胞LNCaP、SK-BR-3和CFPAC-1稀释成4×10 4细胞/ml，NCI-H226稀释成2.0×10 4细胞/ml，PANC-1稀释成3.0×10 4细胞/ml，100μl/孔铺96孔板，每板设阴性及空白对照孔，贴壁过夜后，按50μl/孔 加入稀释好的待测样品，放置于37℃，5％CO 2培养箱中，培养68-72小时，加入CCK8显色液，每孔加入15μl(孔内液体体积的10％)，37℃孵育45-70分钟，置于酶标仪450nm读取数值。用SoftMax Pro进行数据编辑，绘4-P拟合曲线。数据如下表所示：
表17.CB-1020对细胞系增殖的抑制作用和细胞系受体基因的表达水平
(参考Depmap数据)
Figure PCTCN2020074117-appb-000067
由上表可知，LNCaP和SK-BR-3均表达TRPV6和FOLRH1，但LNCap两个受体的表达量相对高一些。NCI-H226、CFPAC-1和PANC-1低表达或微弱表达两个受体。CB-1020对LNCaP、SK-BR-3、NCI-H226、CFPAC-1和PANC-1肿瘤细胞系生长扩增均有抑制作用。CB-1020在受体相对双高表达细胞系LNCaP的IC 50比值高于受体中度表达细胞系SK-BR-3的IC 50比值；SK-BR-3的IC 50比值有高于受体相对低的CFPAC-1和两个受表达均为微弱表达的细胞株NCI-H226和PANC-1的IC 50比值。细胞增殖抑制效果与细胞中两个受体表达水平呈现正相关性。
11.偶联体化合物CB-1320肿瘤细胞扩增抑制实验
实验目的：测试CB-1320及相关化合物对肿瘤细胞系LNCaP(人前列腺癌细胞)、SK-BR-3(人乳腺癌细胞)、MDA-MB-468(人乳腺癌细胞)和CFPAC-1(胰腺癌细胞)扩增的抑制作用。
相关样品：MMAE和CB-1320
实验操作：将提前准备好的LNCaP、SK-BR-3、MDA-MB-468和CFPAC-1计数，用完全培养基(IMDM+10％FBS+1X L-Glutamine+1X P/S)将细胞LNCaP、SK-BR-3、MDA-MB-468和CFPAC-1稀释成4×10 4细胞/ml，100μl/孔铺96孔板，每板设立阴性及空白对照孔，贴壁过夜后，按50μl/孔加入稀释好的待测样品，放置于37℃，5％CO 2培养箱中，培养68-72小时，加入CCK8显色液，每孔加入15μl(孔内液体体积的10％)，37℃孵育45-70分钟，置于酶标仪450nm读取数值。用SoftMax Pro进行数据编辑，绘4-P拟合曲线。具体数据如下表所示：
表18.CB-1320对细胞系增殖的抑制作用和细胞系受体基因的表达水平
(参考Depmap数据)
Figure PCTCN2020074117-appb-000068
由上表可知，4细胞系的GNRHR表达水平大体相当，LNCaP的FOLH1表达量显著高于其他细胞系的表达量。CB-1320对4个肿瘤细胞系扩增均有抑制作用。CB-1320对于受体FOLH1表达水平有不同的细胞抑制程度有所差异，在受体FOLH1相对高表达细胞株LNCap中，IC 50比值显著高于受体相对低表达低的其他细胞株的IC 50比值。
12.偶联体化合物CB-1820肿瘤细胞扩增抑制实验
实验目的：测试CB-1820及相关化合物对肿瘤细胞系LNCaP(人前列腺癌细胞)、MDA-MB-468(人乳腺癌细胞)、CFPAC-1(胰腺癌细胞)和PANC-1(胰腺癌细胞)扩增的抑制作用。
相关样品：MMAE和CB-1820
实验操作：将提前准备好的LNCaP、MDA-MB-468、CFPAC-1和PANC-1计数，用完全培养基(IMDM+10％FBS+1X L-Glutamine+1X P/S)将细胞LNCaP、MDA-MB-468和CFPAC-1稀释成4×10 4细胞/ml；PANC-1稀释成3.0×10 4细胞/ml，100μl/孔铺96孔板，每板设立阴性及空白对照孔，贴壁过夜后，按50μl/孔加入稀释好的待测样品，放置于37℃，5％CO 2培养箱中，培养68-72小时，加入CCK8显色液，每孔加入15μl(孔内液体体积的10％)，37℃孵育45-70分钟，置于酶标仪450nm读取数值。用SoftMax Pro进行数据编辑，绘4-P拟合曲线。具体数值如下表所示：
表19.CB-1820对细胞系增殖的抑制作用和细胞系受体基因的表达水平
(参考Depmap数据)
Figure PCTCN2020074117-appb-000069
由上表可知，LNCap、MDA-MB-468、CFPAC-1和PANC-1的SSTR2表达水平大体相当，LNCaP的FOLH1表达量显著高于其他细胞的FOLH1表达量。CB-1820对4株肿瘤细胞系扩增均有抑制作用。CB-1820对于受体FOLH1表达水平有不同的细胞抑制程度有所差异，在受体相对高表达细胞株LNCaP中，IC 50比值显著高于受体相对低表达细胞株其他细胞系的IC 50比值，细胞增殖抑制效果与细胞相关受体 FOLH1表达水平呈现正相关性。
13.偶联体化合物CR19425、CR19426和CR19428肿瘤细胞扩增抑制实验
实验目的：测试CR19428、CR19425、CR19426及相关化合物对肿瘤细胞系NCI--H226(人肺癌细胞)、CFPAC-1(人胰腺癌细胞)和MDA-MB-468(人乳腺癌细胞)扩增的抑制作用。
相关样品：SN-38、Dxd0017、CR19428、CR19425和CR19426
实验操作：将提前准备好的NCI-H226、CFPAC-1和MDA-MB-468计数，细胞MDA-MB-468和CFPAC-1铺板密度均为2×10 4细胞/ml，NCI-H226铺板密度均为1×10 4细胞/ml，100μl/well铺96孔板，每块培养板设立阴性及空白对照孔，贴壁过夜后，按50μl/孔加入稀释好的待测样品，放置于37℃，5％CO 2培养箱中，培养68-72小时，加入CCK8显色液，每孔加入15μl(孔内液体体积的10％)，37℃孵育45-70m分钟，置于酶标仪450nm读取数值。用SoftMax Pro进行数据编辑，绘4-P拟合曲线。具体数据如下表所示：
表20.CR19428、CR19425和CR19426对细胞系增殖的抑制作用
Figure PCTCN2020074117-appb-000070
实施例5：偶联体化合物在CDX模型中的药效学研究
1.CB-20BK在CDX模型中的药效学研究1
1)样品准备：
称取CB-20BK冻干粉，用PBS溶解配成样品母液，用注射用生理盐水稀释母液到工作浓度样品溶液，备用。
2)CDX模型构建
主要细胞系：KB人口腔表皮样癌细胞、MIA paca-2人胰腺癌细胞、HCC1954人乳腺癌细胞、CALU-3人肺腺癌细胞和DU145人前列腺癌细胞。
模型构建：复苏、培养细胞，收集、计数注射于BALB/c-nude小鼠右肢皮下，等肿瘤生长扩增到80-160mm 3时分组给药或取快速扩增肿瘤块转接扩建小鼠肿瘤模型。
3)分组给药观察
分组：待肿瘤平均长至约80-160mm 3进行分组，设置模型对照组和不同剂量给药组。
给药：尾静脉注射。
实验观察与测量：肿瘤接种后，常规监测包括了肿瘤生长及治疗对动物正常行为的影响。具体内容包括实验动物的活动性，摄食和饮水情况，体重增减(体重每周测量2次)情况，眼睛、被毛及其它异常情况。
测量小鼠体重及瘤块的长径(a)和短径(b)，每周2次。肿瘤体积(tumor volume，TV)计算公式为：TV＝1/2×a×b 2。
4)实验结果与分析
根据小鼠肿瘤体积的变化(图5A-5E)，可见CB-20BK对小鼠KB、MIA aca-2、HCC1954、CALU-3和DU145细胞系CDX肿瘤模型均有较好的抑制作用。
2.CB-20BK在CDX模型中的药效学研究2
实验目的：偶联物化合物CB-20BK在人源LNCaP、DU145和NCI-H460细胞株皮下异体移植模型(CDX)中的药效研究
主要CDX模型：LNCaP、DU145和NCI-H460
实验方案：
将准备好的细胞或肿瘤组织块，接种于BALB/c-nude小鼠右肢前部皮下，当肿瘤体积扩增到80-160mm 3时随机分组，设置模型对照组和给药组，在分组第1天开始给药，根据小鼠最近一次的体重调整给药量，尾静脉注射，给药体积为10μl/g。分组给药后，监测包括了肿瘤生长及治疗对动物正常行为的影响，具体内容有实验动物的活动性，摄食和饮水情况，体重增加或降低情况，眼睛、被毛及其它异常情况。分组给药后，每周测量2次小鼠体重，计算体重变化率；同时以游标卡尺测量肿瘤长径、短径，并计算肿瘤体积、相对肿瘤增值率、肿瘤体积抑瘤率等指标。肿瘤体积公式为TV＝0.5a×b 2，其中a是肿瘤的长径，b是肿瘤的短径。
表21.CB-20BK对CDX模型的生长抑制作用和各个模型受体基因的表达水平
(参考中美冠科数据)
Figure PCTCN2020074117-appb-000071
由上表可知，测试样品CB-20BK的尾静脉给药方案下表现出不同程度的肿瘤生长抑制作用。对于LNCaP、DU145和NCI-H460人源细胞系CDX模型，与阴性对照组相比， 都有一定抗肿瘤生长的作用。FOLR1和FOLH1双表达模型LNCaP，以3mg/kg的剂量，在第1天、第8天和第15天(D1、D8和D15)给药，TGI值为95.68％，有优异的抗肿瘤生长的作用，明显优于FOLR1和FOLH1相对低表达的DU145和NCI-H460模型。抑瘤效果与细胞相关受体表达水平呈现一定相关性。
3.偶联体化合物
Figure PCTCN2020074117-appb-000072
异体移植PDX模型的抑瘤实验1
实验目的：偶联体化合物CB-20BK在PDX模型上的药效学研究
主要模型：LU1206、LU1380和LU0367
实验方案：将准备好的肿瘤组织块，接种于BALB/c-nude小鼠右肢前部皮下，当肿瘤体积扩增到80-160mm 3时随机分组，设置模型对照组和给药组，在分组第1天开始给药，根据小鼠最近一次的体重调整给药量，尾静脉注射，给药体积为10μl/g，给药剂量为3mg/kg。分组给药后，监测包括了肿瘤生长及治疗对动物正常行为的影响，具体内容有实验动物的活动性，摄食和饮水情况，体重增加或降低情况，眼睛、被毛及其它异常情况。分组给药后，每周测量2次小鼠体重，计算体重变化率；同时以游标卡尺测量肿瘤长径、短径，并计算肿瘤体积、相对肿瘤增值率、肿瘤体积抑瘤率等指标。肿瘤体积公式为TV＝0.5a×b 2，其中a是肿瘤的长径，b是肿瘤的短径。
表22.CB-20BK对肺癌PDX模型生长的抑制作用和各个模型受体基因的表达水平
(参考中美冠科数据)
Figure PCTCN2020074117-appb-000073
由表22数据可知，测试样品CB-20BK(3mg/kg)对于LU1206、LU1380和LU0367人源肺癌PDX模型，均表现出一定的抗肿瘤效果。FOLR1和FOLH1双表达模型LU1206的TGI值为90.89％，LU1380和LU0367单表达模型，TGI值分别为30.02％和71.34％，在抑瘤实验中抑瘤效果与细胞相关受体表达水平呈现一定相关性。
4.偶联体化合物
Figure PCTCN2020074117-appb-000074
异体移植PDX模型的抑瘤实验2
实验目的：偶联体化合物CB-20BK在PDX模型上的药效学研究
主要模型：BR1283和BR0438
实验方案：将准备好的肿瘤组织块，接种于BALB/c-nude小鼠右肢前部皮下，当肿瘤体积扩增到80-160mm 3时随机分组，设置模型对照组和给药组，在分组第1天开始给药，根据小鼠最近一次的体重调整给药量，尾静脉注射，给药体积为10μl/g，给药剂量为3mg/kg。分组给药后，监测包括了肿瘤生长及治疗对动物正常行为的影响， 具体内容有实验动物的活动性，摄食和饮水情况，体重增加或降低情况，眼睛、被毛及其它异常情况。分组给药后，每周测量2次小鼠体重，计算体重变化率；同时以游标卡尺测量肿瘤长径、短径，并计算肿瘤体积、相对肿瘤增值率、肿瘤体积抑瘤率等指标。肿瘤体积公式为TV＝0.5a×b 2，其中a是肿瘤的长径，b是肿瘤的短径。
表23.CB-20BK对乳腺癌PDX模型生长的抑制作用和各个模型受体基因的表达水平
(参考中美冠科数据)
Figure PCTCN2020074117-appb-000075
由上表可知，测试样品CB-20BK以3mg/kg的剂量在FOLR1和FOLH1双表达模型BR1283和BR0438中TGI值分别为96.49％、70.74％，显现出优异的抗肿瘤生长的作用。而且，CB-20BK在FOLR1和FOLH1表达较高的表达BR1283中，TGI更优。
5.CB-20B在CDX模型中的药效学研究
1)样品准备：
称取CB-20B冻干粉，用PBS溶解配成样品母液，用注射用生理盐水稀释母液到工作浓度样品溶液，备用。
2)CDX模型构建
主要细胞系：KB人口腔表皮样癌细胞、PC-9人肺癌细胞和DU145人前列腺癌细胞。
模型构建：复苏、培养细胞，收集、计数注射于BALB/c-nude小鼠右肢皮下，等肿瘤生长扩增到80-160mm 3时分组给药或取快速扩增肿瘤块转接扩建小鼠肿瘤模型。
3)分组给药观察
分组：待肿瘤平均长至约80-160mm 3进行分组，设置模型对照组和不同剂量给药组。
给药：尾静脉注射。
实验观察与测量：肿瘤接种后，常规监测包括了肿瘤生长及治疗对动物正常行为的影响。具体内容包括实验动物的活动性，摄食和饮水情况，体重增减(体重每周测量2次)情况，眼睛、被毛及其它异常情况。
测量小鼠体重及瘤块的长径(a)和短径(b)，每周2次。肿瘤体积(tumor volume，TV)计算公式为：TV＝1/2×a×b 2。
4)实验结果与分析
根据小鼠肿瘤体积的变化(图6A-6C)，可见CB-20B对小鼠KB、PC-9和DU145细胞系CDX肿瘤模型均有较好的抑制作用。
6.CB-18G在CDX模型中的药效学研究
1)样品准备：
称取CB-18G冻干粉，用PBS溶解配成样品母液，用注射用生理盐水稀释母液到工作浓度样品溶液，备用。
2)CDX模型构建
主要细胞系：KB人口腔表皮样癌细胞、PC-9人肺癌细胞、SPC-A1人肺腺癌细胞、CALU-3人肺腺癌细胞和DU145人前列腺癌细胞
模型构建：复苏、培养细胞，收集、进行细胞计数，将细胞液注射于BALB/c-nude小鼠右肢皮下，等肿瘤生长扩增到80-160mm 3时分组给药或取快速扩增肿瘤块转接扩建小鼠肿瘤模型。
3)分组给药观察
分组：待肿瘤平均长至约80-160mm 3进行分组，设置模型对照组和不同剂量给药组。
给药：尾静脉注射。
实验观察与测量：肿瘤接种后，常规监测包括了肿瘤生长及治疗对动物正常行为的影响。具体内容包括实验动物的活动性，摄食和饮水情况，体重增减(体重每周测量2次)情况，眼睛、被毛及其它异常情况。
测量小鼠体重及瘤块的长径(a)和短径(b)，每周2次。肿瘤体积(TV)计算公式为：TV＝1/2×a×b 2。
4)实验结果与分析
根据小鼠肿瘤体积的变化(图7A-7E)，可见CB-18G对小鼠KB、PC-9、SPC-A1、CALU-3和DU145细胞系CDX肿瘤模型均有较好的抑制作用。
7.偶联体化合物CB-1020、CB-1320和CB-1820对人源HPAF-II、NCI-H226和SCLC-21H细胞株皮下异体移植模型(CDX)的抑瘤实验
实验目的：配体偶联物CB-1020、CB-1320和CB-1820在CDX模型上的药效学研究
主要CDX模型：HPAF-II(人胰腺癌细胞)、NCI-H226(人肺癌细胞)和SCLC-21H(小细胞肺癌细胞)
实验方案：
将准备好的细胞或肿瘤组织块，接种于BALB/c-nude小鼠右肢前部皮下，当肿瘤体积扩增到80-160mm 3时随机分组，设置模型对照组和给药组，在分组第1天开始给药，根据小鼠最近一次的体重调整给药量，尾静脉注射，给药体积为10μl/g。分组给药后，监测包 括了肿瘤生长及治疗对动物正常行为的影响，具体内容有实验动物的活动性，摄食和饮水情况，体重增加或降低情况，眼睛、被毛及其它异常情况。分组给药后，每周测量2次小鼠体重，计算体重变化率；同时以游标卡尺测量肿瘤长径、短径，并计算肿瘤体积、相对肿瘤增值率、肿瘤体积抑瘤率等指标。肿瘤体积公式为TV＝0.5a×b 2，其中a是肿瘤的长径，b是肿瘤的短径。
表24.CB-1020、CB-1320和CB-1820对CDX模型的抑制作用和各个模型受体基因的表达
水平(参考中美冠科数据)
Figure PCTCN2020074117-appb-000076
CB-1020对人源HPAF-II，CB-1320对NCI-H226和SCLC-21H，CB-1820对SCLC-21H细胞株皮下异体移植模型，都有较明显的抗肿瘤生长作用。
8.偶联体化合物CR19428对人源CALU-3、SCLC-21H和SPC-A1细胞株皮下异体移植模型(CDX)的抑瘤实验
实验方案：将准备好的细胞，接种于BALB/c-nude小鼠右肢前部皮下，当肿瘤体积扩增到80-160mm 3时随机分组，设置模型对照组和给药组，在分组第1天开始给药，根据小鼠最近一次的体重调整给药量，尾静脉注射，给药体积为10μl/g，每周给药2次连续给药3周。分组给药后，监测包括了肿瘤生长及治疗对动物正常行为的影响，具体内容有实验动物的活动性，摄食和饮水情况，体重增加或降低情况，眼睛、被毛及其它异常情况。分组给药后，每周测量2次小鼠体重，计算体重变化率；同时以游标卡尺测量肿瘤长径、短径，并计算肿瘤体积、相对肿瘤增值率、肿瘤体积抑瘤率等指标。肿瘤体积公式为TV＝0.5a×b 2，其中a是肿瘤的长径，b是肿瘤的短径。
表25.CR19428对肺癌PDX模型的抑制作用
Figure PCTCN2020074117-appb-000077
CR-19428为FOLR1和FOLH1的配体和有效载荷Dxd的药物偶联体。由上表可知，它对人源CALU-3、SCLC-21H和SPC-A1细胞株皮下异体移植模型，都有较明显的抗肿瘤生长作用。
9.偶联体化合物CBP-1018肺癌LU2505模型有效性研究(PDX模型)
本实验为肺癌PDX模型LU2505(来自亚洲女性患者)第3代，属于快速生长的肿瘤模型。BALB/c裸小鼠皮下荷瘤，肿瘤体积约150mm 3时进行分组：供试品低、中和高组，小分子MMAE和靶向多肽20BK-SM09对照组，以及空白制剂对照组，共6组，8只/组；分组后第1、8和15天给药，末次药后继续观察14天。供试品CBP-1018的活性物质为CB-20BK，通过将CB-20BK与辅料混合后冻干获得。
表26.注射用CBP-1018在肺癌LU2505模型的药效学结果(首次试验)
Figure PCTCN2020074117-appb-000078
注：1)因动物福利要求，组内平均瘤体积达到2000mm 3，动物需要进行安乐死，因此每组的处死时间不同。2)体重、瘤体积、抑瘤率的数据是第22天(D22)的数据。
如表26和图8A所示：
所有组别动物给药后未见异常临床表现，未见死亡；各组动物体重缓慢增长。
空白制剂对照组、20BK-SM09组、CBP-1018低剂量组的动物因平均瘤体积超过2000mm 3分别在第22天、第22天和第26天(D26)进行了安乐死。因此表26中体重、瘤体积、抑瘤率数据均是D22的数据。
注射用CBP-1018的有效性具有明显的剂量相关性：低剂量组无效，中和高剂量组有效，高剂量组的肿瘤在第19天(D19)就已经被完全治愈，在第29天(D29)未见生长迹象。
多肽组20BK-SM09未见明显的抑瘤作用；提示单独的靶向性多肽不足以产生明确的抗肿瘤作用。
MMAE组具有明确的抑瘤作用，0.375mg/kg的剂量是CBP-1018在1.5mg/kg含有等摩尔的MMAE，对比发现，CBP-1018在1.5mg/kg明显优于MMAE组，提示配体靶向的优势(82.60％vs48.97％)。
10.偶联体化合物CBP-1018肺癌LU1206模型有效性研究(PDX模型)
本实验为肺癌PDX模型LU1206(来自亚洲女性患者)第5代，属于快速生长的肿瘤模型。BALB/c裸小鼠皮下荷瘤，肿瘤体积约150mm 3时进行分组：供试品低、中和高组，小分子MMAE和靶向多肽20BK-SM09对照组，以及空白制剂对照组，共6组，8只/组；分组后第1、8和15天给药，末次药后继续观察14天。
表27.注射用CBP-1018在肺癌LU1206模型的药效学结果(首次试验)
Figure PCTCN2020074117-appb-000079
如表27和图8B所示：
所有组别动物给药后未见异常临床表现，未见死亡，所有动物均在第29天进行安乐死。各组动物体重基本维持不变，均稍高于给药的第一天。
注射用CBP-1018的有效性具有明显的剂量相关性：低剂量组无效(抑瘤率8.08％)，中和高剂量组有效，抑瘤率分别在75.21％和97.42％。低和中剂量的差距比较大。
多肽组20BK-SM09未见明显的抑瘤作用，提示单独的靶向性多肽在此模型也不足以产生明确的抗肿瘤作用。
MMAE组具有明确的抑瘤作用。0.375mg/kg的剂量是CBP-1018在1.5mg/kg含有等摩尔的MMAE；对比发现，CBP-1018在1.5mg/kg明显优于MMAE组，提示配体 靶向的优势(瘤体积抑瘤率：75.21％vs36.03％，瘤重抑瘤率：78.38％vs54.53％)。
11.荷瘤小鼠组织分布(PDX模型LU2505)
12只接种
Figure PCTCN2020074117-appb-000080
肺癌LU2505模型瘤块的雌性荷瘤鼠和12只健康雄性BALB/c裸鼠，单次经尾静脉注射给予1.5mg/140μCi/kg的[ 3H]CBP-1018(同位素标记在MMAE上)。分别于0.5小时、2小时、6小时和24小时取3雄3雌，置于诱导盒内吸入适量二氧化碳麻醉后经心脏穿刺采集血液，然后立即实施安乐死取材。
表28.单次静脉给予[ 3H]CBP-1018后不同时间点各组织中的总放射性
Figure PCTCN2020074117-appb-000081
由上表可知：动物组织分布未见雌雄差异。给药后，药物主要分布于肾脏、全血(主要分布在血浆)、肝脏和肺脏。各组织0.5小时最高，随后各组织药物迅速消除，肝脏和肿瘤消除最慢。肿瘤内药物的缓慢消除可以解释CBP-1018在有效性方面的优势。
12.大鼠排泄试验
6只正常SD大鼠，均雌雄各半，单次尾静脉注射给予0.75mg/70μCi/kg的[ 3H]CBP-1018。在指定时间间隔收集了整体大鼠给药前和给药后0～168小时的尿液、粪便、笼具冲洗/清洗 液及尸体等样品，以及BDC大鼠给药前和给药后0～72小时的胆汁、尿液、粪便及笼具冲洗/清洗液等样品。上述样品均在低温冰箱(-10℃～-30℃)冷冻保存。
每份样品加入适量闪烁液混匀后，使用液体闪烁计数仪测定放射性量。胆汁、尿液、粪便、笼具冲洗及清洗液、尸体等样品中测定的放射性量用于计算占给药量的百分比。血浆样品中的放射性量用于计算每克样品中的总放射性，应用WinNonLin软件(7.0版，Pharsight)按照非房室模型计算血浆总放射性的主要药代动力学参数。
表29.整体大鼠给药后物料平衡研究结果
Figure PCTCN2020074117-appb-000082
由上表可知，CBP-1018主要经过尿液排出，约占到总给药量的约57％；经过粪便排出不足25％。主要经过肾脏从尿液排出的结果与裸鼠的组织分布中在肾脏大量分布是一致的。
13.血浆稳定性
1μg/mL、10μg/mL和100μg/mL浓度的CBP-1008(其活性物质为WO2017025047A1中记载的LDC10B，通过将LDC10B与辅料混合后冻干获得CBP-1008。WO2017025047A1通过引用的方式全文并入)和CBP-1018，与不同种属的血浆在37℃下孵育2小时，观察两个化合物的稳定性。结果显示(表30)，CBP-1018在各种属血浆稳定性，相对0小时的浓度，2小时孵育后剩余百分比均在90％以上。而CBP-1008仅在大鼠血浆稳定(87.92-91.82％)，在其他种属血浆中均不稳定，仅剩余0.751％-24.52％。
试验结果提示CBP-1018的血浆稳定性优于CBP-1008。
表30.不同浓度的CBP-1008和CBP-1018在各种属的血浆稳定性数据汇总
(相对0小时的％)
Figure PCTCN2020074117-appb-000083
注：
1)表内数据为血浆孵育2小时后血浆化合物浓度是0小时浓度的百分比；
2)由于本次数据分属于两个试验，小数点后面的有效位数不一致，为了数据的可溯源性和真实性，直接引用报告中数据，未进行统一。
14.PK中的半衰期
和血浆稳定性比较一致的是，CBP-1018在大鼠和食蟹猴的药代动力学半衰期(约1小时)明显长于CBP-1008(约20分钟)，提示CBP-1018的稳定性优于CBP-1008。
表31.在大鼠PK中CBP-1008和CBP-1018的半衰期(小时)比较
Figure PCTCN2020074117-appb-000084

Claims (46)

  1. 一种偶联体化合物或其药学上可接受的盐，所述偶联体化合物或其药学上可接受的盐包含有效载荷和两个靶向分子，其中所述两个靶向分子分别为协同作用分子部分以及前列腺特异性膜抗原配体部分。
  2. 根据权利要求1所述的偶联体化合物或其药学上可接受的盐，其中所述前列腺特异性膜抗原配体包括如下结构：
    Figure PCTCN2020074117-appb-100001
  3. 根据权利要求1所述的偶联体化合物或其药学上可接受的盐，其中所述前列腺特异性膜抗原配体包括如下结构：
    Figure PCTCN2020074117-appb-100002
  4. 根据权利要求1所述的偶联体化合物或其药学上可接受的盐，其中所述前列腺特异性膜抗原配体包括如下结构：
    Figure PCTCN2020074117-appb-100003
  5. 根据权利要求1所述的偶联体化合物或其药学上可接受的盐，其中所述前列腺特异性膜抗原配体包括如下结构：
    Figure PCTCN2020074117-appb-100004
  6. 一种偶联体化合物或其药学上可接受的盐，所述偶联体化合物或其药学上可接受的盐包含有效载荷和两个靶向分子，其中所述两个靶向分子分别为协同作用分子部分以及具有式(I)所示的配体部分：
    Figure PCTCN2020074117-appb-100005
  7. 一种偶联体化合物或其药学上可接受的盐，所述偶联体化合物或其药学上可接受的盐包含有效载荷和两个靶向分子，其中所述两个靶向分子分别为协同作用分子部分以及P10，并且所述有效载荷为喜树碱及其任何衍生物。
  8. 根据权利要求1-7中任一项所述的偶联体化合物或其药学上可接受的盐，其中所述两个靶向分子是不同的。
  9. 根据权利要求1-8中任一项所述的偶联体化合物或其药学上可接受的盐，其中所述协同作用分子是细胞相互作用分子。
  10. 根据权利要求1-9中任一项所述的偶联体化合物或其药学上可接受的盐，其中所述两个靶向分子是与不同的细胞分子相互作用的细胞相互作用分子。
  11. 根据权利要求1-10中任一项所述的偶联体化合物或其药学上可接受的盐，其中所述协同作用分子是能够介导内吞作用的内吞作用分子。
  12. 根据权利要求1-11中任一项所述的偶联体化合物或其药学上可接受的盐，其中所述协同作用分子与选自下组的分子结合：FOLR1、TRPV6、FOLH1(PMSA)、GNRHR、Her2、Trop2、Her3、NECTIN4、LRP1、GLUT1、EGFR1、AXL、CA9、CD44、Claudin18.2、APN、DLL3、CEACAM5、FZD10、TFRC、MET、IGFR1、SSTR2、CCKBR、LFA1、ICAM、GPR87、GM-CSF、GM-CSFR、TIM3、TLR家族、CD40、CD40L、OX40、OX40L、GITRL、GITR、4-BBL、4-1BB、CD70、CD27、ICOSL、ICOS、HHLA2、CD28、CD86/80、CD28、MHCII抗原、TCR、CTLA-4、CD155、CD122、CD113、IGIT、PD-L1、PD1、Galectin-9、TIM-3、HVEM、BTLA、CD160、VISTA、B7-H4、B7-H3、磷脂酰丝氨酸、HHLA2、LAG3、Galectin-3、LILRB4、SIGLEC15、NKG2A、NKG2D、SLAMF7、KIR2DL1、KIR2DL2、KIR2DL3、FGFR1、FGFR2、FGFR4、NeuGcGM3和CXCR4。
  13. 根据权利要求1-12中任一项所述的偶联体化合物或其药学上可接受的盐，其中所述协同作用分子与选自下组的分子结合：FOLR1、TRPV6、FOLH1(PMSA)、SSTR2和GNRHR。
  14. 根据权利要求1所述的偶联体化合物或其药学上可接受的盐，其中所述协同作用分子与选自下组的分子结合：FOLR1、TRPV6、SSTR2和GNRHR。
  15. 根据权利要求6所述的偶联体化合物或其药学上可接受的盐，其中所述协同作用分子与选自下组的分子结合：FOLR1、TRPV6、FOLH1(PMSA)和GNRHR。
  16. 根据权利要求1-15中任一项所述的偶联体化合物或其药学上可接受的盐，其中所述协同作用分子是叶酸或其类似物。
  17. 根据权利要求16所述的偶联体化合物或其药学上可接受的盐，其中所述叶酸类似物选自下组：5-甲基四氢叶酸、5-甲酰基四氢叶酸、甲氨蝶呤和5,10-亚甲基四氢叶酸。
  18. 根据权利要求1或6中所述的偶联体化合物或其药学上可接受的盐，所述偶联体化合物或其药学上可接受的盐包含一个、两个、三个、四个或更多个有效载荷。
  19. 根据权利要求1或6中所述的偶联体化合物或其药学上可接受的盐，其中所述有效载荷选自下组：小分子化合物、核苷酸、肽和蛋白。
  20. 根据权利要求19所述的偶联体化合物或其药学上可接受的盐，其中所述有效载荷是小分子化合物。
  21. 根据权利要求20所述的偶联体化合物或其药学上可接受的盐，其中所述小分子化合物选自下组：喜树碱及其任何衍生物、澳瑞他汀及其任何衍生物、美登素及其任何衍生物、放射性核素络合物、环氧化酶-2抑制剂、紫杉醇及其任何衍生物、埃博霉素及其任何衍生物、博来霉素及其任何衍生物、更生霉素及其任何衍生物、普卡霉素及其任何衍生物，以及丝裂霉素C。
  22. 根据权利要求21所述的偶联体化合物或其药学上可接受的盐，其中所述小分子化合物是喜树碱及其任何衍生物、澳瑞他汀及其任何衍生物、放射性核素络合物或者环氧化酶-2抑制剂。
  23. 根据权利要求1-22中任一项所述的偶联体化合物或其药学上可接受的盐，其中所述有效载荷通过连接子与至少一个所述靶向分子连接。
  24. 根据权利要求23所述的偶联体化合物或其药学上可接受的盐，其中所述连接子是肽类连接子、二硫化物连接子、pH依赖型连接子或者上述连接子的组合。
  25. 根据权利要求24所述的偶联体化合物或其药学上可接受的盐，其中所述肽类连接子可在特定的生理环境下通过蛋白酶裂解或还原裂解。
  26. 根据权利要求24或25所述的偶联体化合物或其药学上可接受的盐，其中所述肽类连接子选自下组：半胱氨酸、赖氨酸、赖氨酸-赖氨酸、缬氨酸-瓜氨酸、苯丙氨酸-赖氨酸、缬氨酸-赖氨酸、半胱氨酸-赖氨酸、半胱氨酸-谷氨酸、天冬氨酸-天冬氨酸和天冬氨酸-天冬氨酸-赖氨酸，可选的，上述氨基酸中的羧酸被酰胺化。
  27. 根据权利要求24所述的偶联体化合物或其药学上可接受的盐，其中所述二硫化物连接子选自下组：DMDS、MDS、DSDM和NDMDS。
  28. 根据权利要求24所述的偶联体化合物或其药学上可接受的盐，其中所述pH依赖型连接子是顺乌头酸酐。
  29. 根据权利要求23所述的偶联体化合物或其药学上可接受的盐，其中所述连接子包括如下结构：
    Figure PCTCN2020074117-appb-100006
    Figure PCTCN2020074117-appb-100007
    Figure PCTCN2020074117-appb-100008
    Figure PCTCN2020074117-appb-100009
    或者所述连接子是上述结构与肽类连接子的组合。
  30. 根据权利要求1-29中任一项所述的偶联体化合物或其药学上可接受的盐，其中所述两个靶向分子通过间隔区连接。
  31. 根据权利要求30所述的偶联体化合物或其药学上可接受的盐，其中所述间隔区包含选自下组的氨基酸序列：SEQ ID NO：1-14、Arg-Arg、Ala-Ser-Asn、Ala-Ala-Ala、Ser-Ser-Arg、Pro-Arg和Pro-Leu-Gly。
  32. 根据权利要求1所述的偶联体化合物或其药学上可接受的盐，其中所述偶联体化合物选自由下述化合物组成的组：
    Figure PCTCN2020074117-appb-100010
    Figure PCTCN2020074117-appb-100011
    Figure PCTCN2020074117-appb-100012
    Figure PCTCN2020074117-appb-100013
    Figure PCTCN2020074117-appb-100014
    Figure PCTCN2020074117-appb-100015
    Figure PCTCN2020074117-appb-100016
    Figure PCTCN2020074117-appb-100017
    Figure PCTCN2020074117-appb-100018
    Figure PCTCN2020074117-appb-100019
    Figure PCTCN2020074117-appb-100020
    其中M为放射性核素。
  33. 根据权利要求6所述的偶联体化合物或其药学上可接受的盐，其中所述偶联体化合物为：
    Figure PCTCN2020074117-appb-100021
    Figure PCTCN2020074117-appb-100022
    Figure PCTCN2020074117-appb-100023
  34. 根据权利要求7所述的偶联体化合物或其药学上可接受的盐，其中所述偶联体化合物为
    Figure PCTCN2020074117-appb-100024
    Figure PCTCN2020074117-appb-100025
    Figure PCTCN2020074117-appb-100026
  35. 一种药物组合物，所述药物组合物包含根据权利要求1-34中任意一项所述的偶联体化合物或其药学上可接受的盐，以及药学上可接受的载体。
  36. 根据权利要求35所述的药物组合物，其中，所述组合物用于静脉内、皮下、口服、肌内或心室内施用。
  37. 一种用于向有需要的对象递送有效载荷的方法，所述方法包括向所述对象施用治疗有效量的根据权利要求1-34中任意一项所述的偶联体化合物或其药学上可接受的盐，或者根据权利要求35或36所述的药物组合物。
  38. 一种用于治疗对象中的疾病的方法，所述方法包括向所述对象施用治疗有效量的根据权利要求1-34中任意一项所述的偶联体化合物或其药学上可接受的盐，或者根据权利要求35或36所述的药物组合物。
  39. 根据权利要求38所述的方法，其中所述疾病选自下组：癌症、免疫性疾病、心血管疾病、代谢疾病和神经疾病。
  40. 根据权利要求39所述的方法，其中所述癌症选自下组：前列腺癌、乳腺癌、肺癌、肾癌、白血病、卵巢癌、胃癌、子宫癌、子宫内膜癌、肝癌、结肠癌、甲状腺癌、胰腺癌、结直肠癌、食道癌、睾丸癌、皮肤癌、淋巴瘤和多发性骨髓瘤。
  41. 根据权利要求39所述的方法，其中所述免疫性疾病是自身免疫性疾病。
  42. 根据权利要求41所述的方法，其中所述自身免疫性疾病选自下组：结缔组织病、系统性硬化症、类风湿性关节炎和系统性红斑狼疮。
  43. 根据权利要求39所述的方法，其中所述心血管疾病选自下组：心绞痛、心肌梗死、中风、心脏病发作、高血压性心脏病、风湿性心脏病、心肌病、心脏心律失常和先天性心脏病。
  44. 根据权利要求39所述的方法，其中所述代谢疾病选自下组：糖尿病、痛风、肥胖症、低血糖症、高血糖症和血脂异常。
  45. 根据权利要求39所述的方法，其中所述神经疾病选自下组：阿尔茨海默病、帕金森病、亨廷顿病、头部损伤、多发性硬化症、眩晕、昏迷和癫痫。
  46. 根据权利要求38-45中任意一项所述的方法，其中所述方法还包括将一种或多种治疗剂与所述偶联体化合物或其药学上可接受的盐，或者所述药物组合物联合施用。
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WO2022268202A1 (zh) * 2021-06-25 2022-12-29 同宜医药（苏州）有限公司 配体药物偶联物及其应用
US11806405B1 (en) 2021-07-19 2023-11-07 Zeno Management, Inc. Immunoconjugates and methods
WO2023066251A1 (zh) * 2021-10-19 2023-04-27 同宜医药（苏州）有限公司 偶联体药物制剂及其制备方法和用途
WO2023202654A1 (zh) * 2022-04-20 2023-10-26 同宜医药（苏州）有限公司 化合物及用途
WO2023209591A1 (en) * 2022-04-27 2023-11-02 Daiichi Sankyo Company, Limited Combination of antibody-drug conjugate with ezh1 and/or ezh2 inhibitor
WO2024051794A1 (zh) * 2022-09-09 2024-03-14 同宜医药（苏州）有限公司 放射性核素偶联药物及其药物组合物和应用

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SG11202108182RA (en) 2021-08-30
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JP2022518924A (ja) 2022-03-17
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