[go: up one dir, main page]

WO2025113659A1 - Lieur trisaccharide, lieur-charge utile comprenant un lieur trisaccharide, et conjugué anticorps-médicament remodelé à chaîne glycane, leurs procédés de préparation et leurs utilisations - Google Patents

Lieur trisaccharide, lieur-charge utile comprenant un lieur trisaccharide, et conjugué anticorps-médicament remodelé à chaîne glycane, leurs procédés de préparation et leurs utilisations Download PDF

Info

Publication number
WO2025113659A1
WO2025113659A1 PCT/CN2024/135760 CN2024135760W WO2025113659A1 WO 2025113659 A1 WO2025113659 A1 WO 2025113659A1 CN 2024135760 W CN2024135760 W CN 2024135760W WO 2025113659 A1 WO2025113659 A1 WO 2025113659A1
Authority
WO
WIPO (PCT)
Prior art keywords
linker
antibody
adc
compound
gly
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/CN2024/135760
Other languages
English (en)
Chinese (zh)
Inventor
熊美军
秦刚
敖佳铭
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Genequantum Healthcare Suzhou Co Ltd
Original Assignee
Genequantum Healthcare Suzhou Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Genequantum Healthcare Suzhou Co Ltd filed Critical Genequantum Healthcare Suzhou Co Ltd
Publication of WO2025113659A1 publication Critical patent/WO2025113659A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/475—Quinolines; Isoquinolines having an indole ring, e.g. yohimbine, reserpine, strychnine, vinblastine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/537—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines spiro-condensed or forming part of bridged ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/07—Tetrapeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids

Definitions

  • the present invention relates to a trisaccharide linker. It also relates to a linker-carrier compound comprising a trisaccharide group, wherein the trisaccharide group is connected to the rest of the compound through an amide bond.
  • the present invention also relates to an antibody-drug conjugate comprising the linker-carrier compound, wherein the trisaccharide group in the linker-carrier compound is used to reshape the sugar chain in the antibody.
  • the present invention also relates to a preparation method and use of the above-mentioned substance.
  • Antibody-drug conjugates are a new type of drug that connects antibodies to physiologically active effector molecule payloads. They make full use of the targeting of antibodies and the efficient physiological activity of effector molecules to achieve targeted delivery of effector molecules, reduce off-target toxicity caused by drug distribution in non-target organs and tissues, and further improve the limited efficacy of traditional antibody drugs on solid tumors. At present, more than ten antibody-drug conjugates with small molecule toxins have been approved for marketing worldwide, bringing better survival benefits to cancer patients (Dumontet C, Reichert JM, Senter PD, et.al. Antibody–drug conjugates come of age in oncology. Nat. Rev. Drug. Discov. 2023, 22, 641.).
  • trastuzumab monoclonal antibody drug targeted therapy represented by trastuzumab has brought a revolution in breast cancer and even cancer treatment methods, which has significantly reduced the mortality rate of breast cancer patients and has benefited millions of breast cancer patients.
  • trastuzumab also has its own limitations, that is, among all breast cancer patients, it is only effective for HER2-positive patients, accounting for about 20%, while the higher proportion of HER2 low-expressing breast cancer patients is difficult to benefit from trastuzumab treatment.
  • Enhertu an antibody-drug conjugate based on trastuzumab and topoisomerase I inhibitor DXd, has brought another revolution in targeted therapy: for HER2-positive breast cancer patients who have received at least two or more HER2 targeted therapies and have developed drug resistance, Enhertu has achieved an overall remission rate of 60.3%; and for patients with metastatic breast cancer with low HER2 expression who have no targeted drugs available, Enhertu can reduce the risk of death or tumor progression by 50% compared with standard chemotherapy.
  • antibody-drug conjugates fully utilize the advantages of antibodies and effector molecules through the organic connection of both, improve the targeting, safety and efficacy of treatment, and bring about changes in cancer treatment.
  • the present invention provides a novel trisaccharide linker, and a preparation method and application thereof. Also provided is a linker-carrier compound comprising a trisaccharide group, wherein the trisaccharide group is connected to the rest of the compound via an amide bond. Specifically, the present invention provides a linker-carrier compound having formula (I): D——L——(P) t
  • L is a linker, and L is directly connected to D via the terminal -NH- therein, wherein when L is an unbranched linker, it is connected to 1 P, and t is 1, and when L is a branched linker, each branch can be connected to 1 P, and t is an integer greater than 1 (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10);
  • D is a trisaccharide unit structure, which contains a first hexose unit or a derivative thereof, a second hexose unit or a derivative thereof, and a third hexose unit or a derivative thereof;
  • the 6-OH of the first hexose unit or its derivative is oxidized to -C(O)- and connected to the terminal -NH- of L;
  • the first hexose unit or its derivative part is connected to the second hexose unit or its derivative part via an ⁇ -(1 ⁇ 6) glycosidic bond;
  • the second hexose unit or its derivative part is connected to the third hexose unit or its derivative part via a ⁇ -(1 ⁇ 4) glycosidic bond;
  • the third hexose unit or its derivative part has the following structure:
  • L can be disconnected from P to release P chemically (eg, hydrolytically) or biologically (eg, enzymatically).
  • the present invention also relates to an antibody-drug conjugate comprising the linker-load compound, wherein the sugar chain in the antibody is reshaped using the trisaccharide group in the linker-load compound.
  • the present invention provides an antibody-drug conjugate having formula (II) based on the site-specific attachment of the N-glycosylation site in the Fc region of the antibody:
  • R is hydrogen or ⁇ -L-fucosyl
  • q 1 or 2;
  • Ab is an antibody or antigen-binding fragment
  • the first hexose unit or its derivative part is connected to the second hexose unit or its derivative part via an ⁇ -(1 ⁇ 6) glycosidic bond;
  • the second hexose unit or its derivative is linked to the ⁇ -D-N-acetylglucosamine portion via a ⁇ -(1 ⁇ 4) glycosidic bond;
  • the 6-OH of the first hexose unit or its derivative is oxidized to -C(O)-;
  • L is a linker, and L is directly connected to the -C(O)- in the first hexose unit or its derivative portion via the terminal -NH- therein; wherein when L is an unbranched linker, it is connected to 1 P, and t is 1, and when L is a branched linker, each branch can be connected to 1 P, and t is an integer greater than 1 (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10).
  • the first hexose unit or its derivative moiety is selected from glucosyl, mannosyl, galactosyl, fructosyl, gulosyl, idosyl or derivatives thereof.
  • the second six-carbon sugar unit or its derivative moiety is selected from glucosyl, mannosyl, galactosyl, fructosyl or their derivatives.
  • the -NHC(O)CH 2 - in formula (II) that is linked to Ab is derived from asparagine at position 297 of the Fc region of the antibody.
  • the invention also relates to a preparation method and application of the substance.
  • FIG1 and FIG2 respectively show the results of hydrophobic interaction high performance liquid chromatography (HIC-HPLC) and size exclusion chromatography (SEC-HPLC) analysis of antibody-drug conjugate ADC-1.
  • HIC-HPLC hydrophobic interaction high performance liquid chromatography
  • SEC-HPLC size exclusion chromatography
  • FIG3 and FIG4 respectively show the results of hydrophobic interaction high performance liquid chromatography (HIC-HPLC) and size exclusion chromatography (SEC-HPLC) analysis of antibody-drug conjugate ADC-2.
  • HIC-HPLC hydrophobic interaction high performance liquid chromatography
  • SEC-HPLC size exclusion chromatography
  • FIG5 and FIG6 respectively show the results of hydrophobic interaction high performance liquid chromatography (HIC-HPLC) and size exclusion chromatography (SEC-HPLC) analysis of the antibody-drug conjugate ADC-3.
  • HIC-HPLC hydrophobic interaction high performance liquid chromatography
  • SEC-HPLC size exclusion chromatography
  • FIG7 and FIG8 respectively show the results of the hydrophobic interaction high performance liquid chromatography (HIC-HPLC) and size exclusion chromatography (SEC-HPLC) analysis of the antibody-drug conjugate ADC-4.
  • HIC-HPLC hydrophobic interaction high performance liquid chromatography
  • SEC-HPLC size exclusion chromatography
  • Figures 9-11 show the in vitro activities of antibody-drug conjugates ADC-1 to ADC-4 on SKBR-3, NCI-N87, and MDA-MB-468 cell lines, respectively.
  • Figures 12-14 show the in vitro activities of antibody-drug conjugates ADC-5 to ADC-8 on SKBR-3, NCI-N87, and MDA-MB-468 cell lines, respectively.
  • Figures 15-17 show the in vitro activities of antibody-drug conjugates ADC-9 to ADC-11 on SKBR-3, NCI-N87, and MDA-MB-468 cell lines, respectively.
  • Figures 18-20 show the in vitro activities of antibody-drug conjugates ADC-12 to ADC-14 on SKBR-3, NCI-N87, and MDA-MB-468 cell lines, respectively.
  • FIG. 21 shows the affinity detection of ADC-5 to ADC-8 to cell surface HER2.
  • FIG. 22 shows the in vivo efficacy evaluation of ADC-6 to ADC-8.
  • FIG. 23 shows the ADCC effects of ADC-5 to ADC-8.
  • targeting molecule refers to a molecule that has affinity for a specific target (e.g., a receptor, a cell surface protein, a cytokine, a tumor-specific antigen, etc.).
  • Targeting molecules can deliver cargo to a specific site in the body by targeted delivery.
  • Targeting molecules can recognize one or more targets.
  • a specific target is defined by the target it recognizes.
  • a targeting molecule that targets a receptor can deliver a cytotoxin to a site containing a large number of receptors.
  • Examples of targeting molecules include, but are not limited to, antibodies, binding proteins for a given antigen, antibody mimics, scaffold proteins with affinity for a given target, ligands, etc.
  • Targets recognized by targeted molecules include but are not limited to CD19, CD22, CD25, CD30/TNFRSF8, CD33, CD37, CD44v6, CD56, CD70, CD71, CD74, CD79b, CD117/KIT, CD123, CD138, CD142, CD174, CD227/MUC1, CD352, CLDN18.2, DLL3, ErbB2/HER2, ErbB3/HER3, CN33, GPNMB, ENPP3, Nectin-4, EGFRvIII, SLC44A4/AGS-5, CEACAM5, PSMA, TIM1, LY6E, LIV1, Nec tin4, SLITRK6, HGFR/cMet, SLAMF7/CS1, EGFR, BCMA, AXL, NaPi2B, GCC, STEAP1, MUC16, Mesothelin, ETBR, EphA2, 5T4, FOLR1, LAMP1, Cadherin 6, FGFR2, FGFR3, CA6, CanAg, Integrin ⁇ V, TD
  • the term "antibody” is used in a broad manner, and its definition covers conventional antibodies, recombinant antibodies/genetically engineered antibodies, particularly complete monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies) and antibody fragments, as long as they have the desired biological activity.
  • the antibody can be any subtype (e.g., IgG, IgE, IgM, IgD, and IgA) or subclass, and can be derived from any suitable species.
  • the antibody is human or mouse-derived.
  • the antibody can also be a fully human antibody, a humanized antibody, or a chimeric antibody prepared by a recombinant method.
  • monoclonal antibodies refer to antibodies obtained from a substantially homogeneous antibody population, i.e., the individual antibodies constituting the population are identical except for a few possible natural mutations. Monoclonal antibodies are highly specific for a single antigenic site.
  • the term "monoclonal” refers to the characteristic of an antibody being derived from a substantially homogeneous antibody population and should not be construed as requiring some specific method to produce the antibody.
  • An intact antibody or full-length antibody essentially comprises an antigen-binding variable region and a light chain constant region ( CL ) and a heavy chain constant region ( CH ), which may include CH1 , CH2 , CH3 and CH4 , depending on the subtype of the antibody.
  • An antigen-binding variable region (also referred to as a fragment variable region, Fv fragment) generally comprises a light chain variable region ( VL ) and a heavy chain variable region ( VH ).
  • the constant region may be a constant region with a native sequence (e.g., a constant region with a human native sequence) or an amino acid sequence variant thereof.
  • the variable region recognizes and interacts with a target antigen.
  • the constant region can be recognized and interacted with by the immune system.
  • Antibody fragments may comprise a portion of an intact antibody, preferably an antigen binding region or variable region thereof.
  • antibody fragments include Fab, Fab', F(ab')2, Fd fragments consisting of VH and CH1 domains, Fv fragments, single domain antibody (dAb) fragments, and isolated complementarity determining regions (CDRs).
  • Fab fragments are antibody fragments obtained by digestion of full-length immunoglobulins with papain, or fragments having the same structure produced, for example, by recombinant expression.
  • Fab fragments comprise a light chain (comprising VL and CL ) and another chain, wherein the other chain comprises the variable region ( VH ) of the heavy chain and one constant region ( CH1 ) of the heavy chain.
  • F(ab')2 fragments are antibody fragments obtained by digestion of immunoglobulins with pepsin at pH 4.0-4.5, or fragments having the same structure produced, for example, by recombinant expression.
  • F(ab')2 fragments essentially comprise two Fab fragments, wherein each heavy chain portion comprises several additional amino acids, including cysteine that forms a disulfide bond linking the two fragments.
  • a Fab' fragment is a fragment that contains half of a F(ab')2 fragment (a heavy chain and a light chain).
  • the antibody fragment may contain multiple chains linked together, for example, by disulfide bonds and/or by peptide linkers.
  • antibody fragments also include single-chain Fv (scFv), Fv, dsFv, bispecific antibodies, Fd and Fd' fragments, and other fragments, including modified fragments.
  • Antibody fragments typically contain at least or about 50 amino acids, typically at least or about 200 amino acids.
  • Antigen-binding fragments may include any antibody fragment that, when inserted into an antibody framework (e.g., by replacing the corresponding region), can obtain an antibody that immunospecifically binds to an antigen.
  • the antibody-drug conjugate of the present application is based on any site containing the natural N-glycosylation modification of the antibody Fc region for site-specific conjugation, and molecules containing sugar chains in the antibody Fc region (including but not limited to antibodies/bispecific antibodies/Fc fusion proteins/single-chain antibodies/nanoantibodies, etc.) can be prepared in a one-step method by the trisaccharide-containing linker-load of the present application. Therefore, the antibody of the present application is not particularly limited, and its Fc region can contain sugar chains, which can be natural antibodies. Drugs are not limited to small molecule therapeutic drugs, as long as they have therapeutic, diagnostic, and preventive effects. Including but not limited to oligonucleotides (such as ASO, siRNA, etc.), polypeptides, cytokines, etc.
  • the antibodies of the present invention can also be prepared using techniques well known in the art, such as the following techniques or combinations thereof: recombinant technology/genetic engineering technology, phage display technology, synthetic technology, or other techniques known in the art.
  • genetically engineered recombinant antibodies can be expressed by a suitable culture system (e.g., E. coli or mammalian cells).
  • the genetic engineering can refer to, for example, the introduction of a ligase-specific recognition sequence at its end.
  • conjugates include, but are not limited to, antibody-drug conjugates.
  • Small molecule compounds refer to molecules of a size comparable to organic molecules commonly used in drugs.
  • the term does not cover biomacromolecules (e.g., proteins, nucleic acids, etc.), but covers low molecular weight peptides or derivatives thereof, such as dipeptides, tripeptides, tetrapeptides, pentapeptides, etc.
  • the molecular weight of a typical small molecule compound can be, for example, about 100-about 2000Da, about 200-about 1000Da, about 200-about 900Da, about 200-about 800Da, about 200-about 700Da, about 200-about 600Da, about 200-about 500Da.
  • Cytotoxin refers to a substance that inhibits or prevents the expression activity of a cell, a cell function, and/or causes cell destruction.
  • the cytotoxins currently commonly used in antibody-drug conjugates are more toxic than chemotherapeutic drugs.
  • Examples of cytotoxins include, but are not limited to, drugs targeting the following targets: microtubule cytoskeleton, DNA, RNA, kinesin-mediated protein transport, and regulation of apoptosis.
  • Drugs targeting the microtubule cytoskeleton can, for example, be microtubule stabilizers or microtubule polymerization inhibitors. Examples of microtubule stabilizers include, but are not limited to, taxanes.
  • microtubule polymerization inhibitors include, but are not limited to, maytansinoids, auristatins, vinblastines, colchicines, and dolastatins.
  • Drugs targeting DNA can, for example, be drugs that directly destroy DNA structure or topoisomerase inhibitors.
  • drugs that directly destroy DNA structure include, but are not limited to, DNA double strand breakers, DNA alkylating agents, and DNA intercalators.
  • DNA double-strand disruptors can be, for example, enediyne antibiotics, including but not limited to danemycin, esperamicin, neocarcin, uncialamycin, etc.
  • DNA alkylating agents can be, for example, DNA bis-alkylators (i.e., DNA cross-linkers) or DNA mono-alkylators.
  • DNA alkylating agents include but are not limited to pyrrolo[2,1-c][1,4]benzodiazepines PBD dimer, 1-(chloromethyl)-2,3-dihydro-1H-benzo[e]indole (CBI) dimer, CBI-PBD heterodimer, dihydroindole benzodiazepine (IGN) dimers, duocarmycin-like compounds, etc.
  • topoisomerase inhibitors include, but are not limited to, exatecan and its derivatives (e.g., DX8951f, DXd-(1) and DXd-(2), the structures of which are described below), camptothecins and anthracyclines.
  • Drugs targeting RNA may, for example, be drugs that inhibit splicing, examples of which include, but are not limited to, pladienolide.
  • Drugs targeting kinesin-mediated protein transport may, for example, be mitotic kinesin inhibitors, including, but not limited to, spindle kinesin (KSP) inhibitors.
  • KSP spindle kinesin
  • Small molecule compounds may also include, for example, small molecule drugs with various mechanisms of action, including various traditional small molecule drugs, photoacoustic dynamic therapy drugs, photothermal therapy drugs, etc., such as chemotherapeutic drugs, small molecule targeted drugs, immune agonists, etc., such as traditional cytotoxic drugs, such as cisplatin, paclitaxel, 5-fluorouracil, cyclophosphamide and bendamustine, etc.; small molecule targeted drugs, such as imatinib mesylate, gefitinib and anlotinib, etc.; immune agonists, such as STING agonists, TLR agonists, etc.).
  • small molecule drugs with various mechanisms of action including various traditional small molecule drugs, photoacoustic dynamic therapy drugs, photothermal therapy drugs, etc., such as chemotherapeutic drugs, small molecule targeted drugs, immune agonists, etc., such as traditional cytotoxic drugs, such as cisplatin, paclitaxel, 5-
  • the load of the present invention may also be a nucleic acid and a nucleic acid analog, a tracer molecule, including a fluorescent molecule, biotin, a fluorophore, a chromophore, a spin resonance probe and a radioactive label, etc., or may be a short peptide, a polypeptide, a peptidomimetic and a protein.
  • a tracer molecule including a fluorescent molecule, biotin, a fluorophore, a chromophore, a spin resonance probe and a radioactive label, etc.
  • Spacer refers to a structure located between different structural modules that can separate the structural modules in space. The definition of a spacer does not limit whether it has a certain function or whether it can be cut or degraded in vivo. Examples of spacers include, but are not limited to, amino acids and non-amino acid structures, wherein the non-amino acid structure can be, but is not limited to, an amino acid derivative or analog.
  • Spacer sequence refers to an amino acid sequence that serves as a spacer, and examples thereof include, but are not limited to, a single amino acid, a sequence containing multiple amino acids, such as a sequence containing two amino acids, such as GA, or, for example, GGGGS (SEQ ID No.
  • Self-excision spacers are covalent components that cause two chemical bonds to cleave successively after activation of the protective portion in the precursor: the protective portion (such as the cleavable sequence) is removed after activation, triggering a cascade of decomposition reactions, resulting in the release of smaller molecules in a sequential order.
  • self-immolative spacers include, but are not limited to, PABC (p-aminobenzyloxycarbonyl), acetals, heteroacetals, and combinations thereof.
  • amino acid includes “natural amino acids” and “unnatural amino acids”.
  • natural amino acids refers to amino acids, which are protein-constituting amino acids, including the common twenty amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine), as well as the less common selenocysteine and pyrrolysine.
  • amino acids which are protein-constituting amino acids, including the common twenty amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine
  • unnatural amino acid refers to an amino acid that is not a protein-constituting amino acid. Specifically, the term refers to an amino acid that is not a natural amino acid as defined above.
  • alkyl refers to a straight or branched saturated aliphatic hydrocarbon group consisting of carbon atoms and hydrogen atoms, which is connected to the rest of the molecule by a single bond.
  • the alkyl group may have 1 to 20 carbon atoms, referring to "C 1 -C 20 alkyl", such as C 1 -C 4 alkyl, C 1 -C 3 alkyl, C 1 -C 2 alkyl, C 3 alkyl, C 4 alkyl, C 3 -C 6 alkyl.
  • alkyl include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl or 1,2-dimethylbutyl, or isomers thereof.
  • a divalent free radical refers to a group obtained by removing a hydrogen atom from a carbon atom having free valence electrons of a corresponding monovalent free radical.
  • a divalent free radical has two attachment sites connected to the rest of the molecule.
  • alkylene or “alkylene” refers to a saturated straight or branched divalent hydrocarbon radical.
  • alkylene examples include, but are not limited to, methylene (—CH 2 —), ethylene (—C 2 H 4 —), propylene (—C 3 H 6 —), butylene (—C 4 H 8 —), pentylene (—C 5 H 10 —), hexylene (—C 6 H 12 —), 1-methylethylene (—CH(CH 3 )CH 2 —), 2-methylethylene (—CH 2 CH(CH 3 )-), methylpropylene or ethylpropylene, and the like.
  • cycloalkyl refers to a cyclic saturated aliphatic group consisting of carbon atoms and hydrogen atoms, which is connected to the rest of the molecule by a single bond.
  • a cycloalkyl group may have 3 to 10 carbon atoms, i.e., a "C 3 -C 10 cycloalkyl group", such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, or cyclodecyl.
  • Cycloalkylene refers to a divalent cycloalkyl group.
  • heterocyclyl means that one or more carbon atoms in the above cycloalkyl are replaced by heteroatoms selected from nitrogen, oxygen and sulfur, such as azepine, oxa- or thiirane, azepine, oxa- or thiol-cycloalkyl, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, tetrahydrofuranyl, tetrahydrothienyl, piperidinyl, piperazinyl, tetrahydropyranyl or tetrahydrothiopyranyl.
  • Heterocyclylene means a divalent cycloalkyl.
  • the relevant substituent is selected from alkyl, halogen, amino, monoalkylamino, dialkylamino, nitro, cyano, formyl, alkylcarbonyl, carboxy, alkyloxycarbonyl, alkylcarbonyloxy, aminocarbonyl, monoalkylaminocarbonyl, dialkylaminocarbonyl, formylamino, alkylcarbonylamino, formyl(monoalkyl)amino or alkylcarbonyl(monoalkyl)amino.
  • connection between the groups can be linear or branched, provided that a chemically stable structure is formed.
  • the structure formed by such a combination can be connected to the rest of the molecule through any suitable atom in the structure, preferably through a specified chemical bond.
  • the two or more divalent groups can form a linear connection with each other, such as -CR1R2 -C1-10 alkylene-(CO)-, -CR1R2 -C4-10 cycloalkylene- (CO)-, -CR1R2 - C4-10 cycloalkylene- C1-10 alkylene-(CO)-, -CR1R2 - CR1R2 ' -(CO)-, -CR1R2 - CR'R2' - CR1 " R2 "-(CO)-, etc.
  • the resulting divalent structure can be further connected to other parts of the molecule.
  • multiple identical letters representing chemical groups appear in the same chemical structure, they are selected independently and are not necessarily the same.
  • multiple M in Formula I-2 are independently selected from LKa-L 2 ⁇ L 1 ⁇ B ⁇ P; and multiple L 2 are also independent of each other and are not necessarily the same.
  • two a or i appear in the same structure at the same time, they are also independently selected and are not necessarily the same.
  • the present invention provides a linker-carrier compound having formula (I): D——L——(P) t
  • L is a linker, and L is directly connected to D via the terminal -NH- therein, wherein when L is an unbranched linker, it is connected to 1 P, and t is 1, and when L is a branched linker, each branch can be connected to 1 P, and t is an integer greater than 1 (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10);
  • D is a trisaccharide unit structure, which contains a trisaccharide unit structure of a first hexose unit or a derivative thereof, a second hexose unit or a derivative thereof, and a third hexose unit or a derivative thereof;
  • the 6-OH of the first hexose unit or its derivative is oxidized to -C(O)- and connected to the terminal -NH- of L;
  • the first hexose unit or its derivative part is connected to the second hexose unit or its derivative part via an ⁇ -(1 ⁇ 6) glycosidic bond;
  • the second hexose unit or its derivative part is connected to the third hexose unit or its derivative part via a ⁇ -(1 ⁇ 4) glycosidic bond;
  • the third hexose unit or its derivative has the following structure:
  • R 1 -R 12 are selected from -OH, or H;
  • the two substituents on the same carbon atom are different;
  • the two substituents on the same carbon atom are different; and R 3 is H, and R 4 is -OH;
  • the two substituents on the same carbon atom are different; and R 3 is H, R 4 is -OH; R 9 is H, R 10 is -OH;
  • R 3 is H, R 4 is -OH;
  • R 9 is H, R 10 is -OH;
  • R 8 is H, R 7 is -OH;
  • R 12 is H, R 11 is -OH;
  • L and P refer to those of formula (I).
  • formula (III) is as shown in formula (III-1):
  • R 1 , R 2 , R 5 , and R 6 are selected from -OH, or H; and the two substituents on the same carbon atom are different.
  • the first hexose unit or its derivative portion is selected from glucosyl, mannosyl, galactosyl, fructosyl, gulosyl, idosyl or their derivatives; wherein the 6-hydroxyl group of the first hexose unit or its derivative portion is oxidized to -C(O)- form and connected to the terminal -NH- of the L.
  • the first hexose unit or its derivative moiety is selected from in, represents the site of connection to L; * represents the site of connection to the second hexose unit or its derivative.
  • the second hexose unit or its derivative part is selected from glucosyl, mannosyl, galactosyl, fructosyl or their derivatives; wherein the first hexose unit or its derivative part is connected to the second hexose unit or its derivative part through an ⁇ -(1 ⁇ 6) glycosidic bond; the second hexose unit or its derivative part is connected to the third hexose unit or its derivative part through a ⁇ -(1 ⁇ 4) glycosidic bond.
  • the second hexose unit or its derivative moiety is selected from
  • the derivatives in the hexose unit or its derivative part are independently selected from uronic acid or monosaccharide derivatives in which the hydroxyl group is replaced by an acylamino group (e.g., an alkanoylamino group, such as formylamino, acetylamino, propionylamino, etc., especially acetylamino).
  • an acylamino group e.g., an alkanoylamino group, such as formylamino, acetylamino, propionylamino, etc., especially acetylamino.
  • the trisaccharide unit structure has the following structure:
  • -L-(P) t is -L 2 -L 1 -BP, that is, formula (I) is: D———L2——L1——B——P
  • B is independently absent, or is 1) below, or is 2) below, or is a combination of 1) and 2) below: 1) a self-immolative spacer Sp1; 2) a divalent group, or a combination of two or more divalent groups, wherein the divalent group is selected from: -CR 1 R 2 -, C 1-10 alkylene, C 4-10 cycloalkylene, C 4-10 heterocyclylene, and -(CO)-;
  • L2 is independently absent; or is the following 1); or is the following 2); or is a combination of the following 1) and 2):
  • B, L 1 and L 2 do not exist at the same time;
  • R1 , R2 , R3 , R4 , R5 , R6 , R7 , R8 , R9 are each independently selected from hydrogen, halogen, substituted or unsubstituted -C1-10 alkyl, C4-10 cycloalkylene; or R1 and R2 together with the carbon atom to which they are attached form a 3-6 membered cycloalkylene, and/or R3 and R4 together with the carbon atom to which they are attached form a 3-6 membered cycloalkylene;
  • P is a load connected to part B, or part L1 , or part L2 ;
  • Ld2 and each Ld1 are independently a bond; or are selected from -NH-C 1-20 alkylene-(CO)-, -NH-(PEG) i -(CO)-; or are natural amino acids or oligomeric natural amino acids having a degree of polymerization of 2-10 (i.e., 2, 3, 4, 5, 6, 7, 8, 9 or 10) which are each independently unsubstituted or substituted with -CO-(PEG) j -R 11 on the side chain;
  • -(PEG) i - and -(PEG) j - are each a PEG fragment comprising a specified number of consecutive -(OC 2 H 4 )- structural units or consecutive -(C 2 H 4 -O)- structural units, optionally with a C 1-10 alkylene group appended at one end;
  • M is hydrogen or LKa-L 2 ⁇ L 1 ⁇ B ⁇ P;
  • Q is NH 2 or L 2 ⁇ L 1 ⁇ B ⁇ P
  • M is hydrogen and at the same time Q is NH 2 ;
  • Each LKa is independently selected from
  • Each L2 is independently absent; or is the following 1); or is the following 2); or is a combination of the following 1) and 2):
  • an amino acid residue sequence i.e., -*(AA) n **-, wherein n is an integer from 1 to 100, AA is independently an amino acid residue at each occurrence, * represents the N-terminus of the corresponding amino acid, ** represents the C-terminus of the corresponding amino acid, and -(C 2 H 4 -O) m -(CH 2 ) p - is optionally present between the amino group and the ⁇ -carbon of an amino acid, wherein m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; p is 0, 1, 2 or 3, and the * terminus forms an amide bond with the carbonyl group in the trisaccharide structure; preferably, -Gly-Gly-Gly-;
  • L1 is absent independently; or is a non-cleavable sequence, for example, coupling the load to the antibody through a thioether bond; or is a cleavable sequence comprising an amino acid sequence cleavable by an enzyme, wherein the amino acid sequence cleavable by the enzyme comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids;
  • Each B is independently absent, or is 1) below, or is 2) below, or is a combination of 1) and 2) below: 1) a self-immolative spacer Sp1; 2) a divalent group, or a combination of two or more divalent groups, wherein the divalent group is selected from: -CR 1 R 2 -, C 1-10 alkylene, C 4-10 cycloalkylene, C 4-10 heterocyclylene, and -(CO)-;
  • B, L 1 and L 2 do not exist at the same time;
  • P is a load connected to part B, or part L1 , or part L2 ;
  • R1 , R2 , R3 , R4 , R5 , R6 , R7 , R8 , R9 are each independently selected from hydrogen, halogen, substituted or unsubstituted -C1-10 alkyl, C4-10 cycloalkylene; or R1 and R2 together with the carbon atom to which they are attached form a 3-6 membered cycloalkylene, and/or R3 and R4 together with the carbon atom to which they are attached form a 3-6 membered cycloalkylene;
  • R 11 is C 1-10 alkyl
  • d 0, 1, 2, 3, 4, 5, or 6;
  • Each i is independently an integer of 1-100 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10), preferably 1-20; preferably, each i is independently an integer of 1-12; more preferably 2-8; especially 4;
  • Each j is independently an integer of 1-100 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10), preferably 1-20; preferably, each j is independently an integer of 1-12; more preferably 8-12; especially 8, 9, 12 or 13.
  • At least one of B, L1 and L2 is not absent.
  • L2 is an amino acid residue sequence, i.e. -*(AA) n **-, n is an integer from 1 to 100, AA is independently an amino acid residue at each occurrence, * represents the N-terminus of the corresponding amino acid, ** represents the C-terminus of the corresponding amino acid, and -( C2H4 - O ) m- ( CH2 ) p- , wherein m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and p is 0, 1, 2 or 3, and the *terminus forms an amide bond with the carbonyl group in the trisaccharide structure.
  • AA is independently any one of Phe, Lys, Gly, Ala, Leu, Asn, Val, Ile, Pro, Trp, Ser, Tyr, Cys, Met, Asp, Gln, Glu, Thr, Arg, His or any combination thereof at each occurrence.
  • n is an integer of 1-50, preferably an integer of 1-30, preferably an integer of 1-20, preferably an integer of 1-10, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.
  • L comprises a cleavable sequence of an amino acid sequence cleavable by an enzyme, wherein the amino acid sequence cleavable by the enzyme comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids.
  • the amino acid sequence cleavable by the enzyme is selected from -Val-Ala-, -Gly-Gly-Phe-Gly-, -Phe-Lys-, -Val-Cit-, -Val-Lys-, -Gly-Phe-Leu-Gly-, -Ala-Leu-Ala-Leu-, -Ala-Ala-Ala- and combinations thereof; the preferred amino acid sequence cleavable by the enzyme is -Gly-Gly-Phe-Gly-.
  • L 1 is any one of Val, Cit, Phe, Lys, Gly, Ala, Leu, Asn or any combination thereof, preferably, -Val-Ala-, -Gly-Gly-Phe-Gly-, -Phe-Lys-, -Val-Cit-, -Val-Lys-, -Gly-Phe-Leu-Gly-, -Ala-Leu-Ala-Leu-, -Ala-Ala-Ala- and combinations thereof.
  • L 1 represents -Val-Cit-.
  • Sp1 is selected from PABC (p-aminobenzyloxycarbonyl), acetal, heteroacetal and a combination thereof; preferably, Sp1 is acetal, heteroacetal or PABC; further preferably, the heteroacetal is selected from N,O-heteroacetal; more preferably, Sp1 is -O-CH 2 -U- or -NH-CH 2 -U-, wherein -O- or -NH- is linked to an amino acid sequence cleavable by an enzyme, U is absent, or is CH 2 , O, S or NH, preferably O or S; further preferably, Sp1 is PABC.
  • PABC p-aminobenzyloxycarbonyl
  • B is absent.
  • B is 1), 2) or a combination of 1) and 2): 1) a self-immolative spacer Sp1; 2) a divalent group, or a combination of two or more divalent groups, wherein the divalent group is selected from: -CR 1 R 2 -, C 1-10 alkylene and -(CO)-.
  • B is connected to the load via an amide bond, an ester bond or an ether bond.
  • B is selected from: (-PABC-), -NH-CH2 -U- or -NH-CH2- U- ( CH2 ) g- (CO)-; wherein g is 1, 2, 3, 4, 5 or 6; U is absent or is CH2 , O, S or NH, preferably O or S.
  • -L 1 -B- represents -Val-Cit-PABC-.
  • -L 1 -B- represents -Gly-Gly-Phe-Gly-.
  • -L2 - L1- B- represents -Gly-Gly-Gly-Val-Cit-PABC-.
  • -L 2 -L 1 -B- represents -HN-(C 2 H 4 -O) m -(CH 2 ) p -Gly-Gly-Phe-Gly-.
  • Ld2 and each Ld1 are independently selected from a bond, or
  • Each i, j and k is independently selected from an integer of 1-100.
  • each i, j and k is independently selected from an integer from 1 to 20. In one embodiment, each i, j and k is independently selected from an integer from 1 to 12.
  • each i is independently selected from an integer of 2-8; particularly 4.
  • each j is independently selected from an integer of 8-12; particularly 8 or 12.
  • each k is independently selected from an integer of 1-7; in particular 1 or 3 or 5.
  • -(PEG) i - comprises -(OC 2 H 4 ) i - or -(C 2 H 4 -O) i -, and optionally a C 1-10 alkylene group is appended at one terminal;
  • -(PEG) j - comprises -(OC 2 H 4 ) j - or -(C 2 H 4 -O) j -, and optionally a C 1-10 alkylene group is appended at one terminal.
  • -(PEG) i - comprises -C 2 H 4 -(OC 2 H 4 ) i - or -(C 2 H 4 -O) i -C 2 H 4 -.
  • the load can be selected from the group consisting of small molecule compounds (e.g., small molecule drugs of various mechanisms of action, including various traditional small molecule drugs, photoacoustic dynamic therapy drugs, photothermal therapy drugs, etc., such as chemotherapeutic drugs, small molecule targeted drugs, immune agonists, etc., such as traditional cytotoxic drugs, such as cisplatin, paclitaxel, 5-fluorouracil, cyclophosphamide and bendamustine, etc.; small molecule targeted drugs, such as imatinib mesylate, gefitinib and anlotinib, etc.; immune agonists such as STING agonists, TLR agonists, etc.), nucleic acids and nucleic acid analogs, tracer molecules (including fluorescent molecules, biotin, fluorophores, chromophores, spin resonance probes and radioactive labels, etc.), short peptides, polypeptides, peptidomimetics and proteins.
  • small molecule compounds
  • the cargo is a cytotoxin or a fragment thereof, with optional derivatization for attachment to the L moiety in formula (I), or the B moiety, L2 or L1 moiety in a compound of formula (I-1) or formula (I-2).
  • the cytotoxin is selected from the group consisting of drugs that target the microtubule cytoskeleton.
  • the cytotoxin is selected from the group consisting of taxanes, maytansinoids, auristatins, epothilones, combretastatin A-4 phosphate, combretastatin A-4 and its derivatives, indole-sulfonamides, vinca alkaloids such as vinblastine, vincristine, vindesine, vinorelbine
  • the cytotoxin is selected from the group consisting of DNA topoisomerase inhibitors such as camptothecins and their derivatives, mitoxantrone, and mitoguanidine.
  • the cytotoxin is selected from the group consisting of nitrogen mustards such as chlorambucil, naphthyl nitrogen mustard, cholophosphamide, estramustine, ifosfamide, nitrogen mustard, nitrogen oxide mustard hydrochloride, melphalan, new nitrogen mustard, methionine, phenylephrine, prednimustine, trofosfamide, uramustine.
  • the cytotoxin is selected from the group consisting of nitrosoureas such as carmustine, flubenzuron, formoterol, lomustine, nimustine, ranimustine.
  • the cytotoxin is selected from the group consisting of aziridines.
  • the cytotoxin is selected from the group consisting of benzodopa, carboquinone, resetpa and uredepa. In one embodiment, the cytotoxin is selected from the group consisting of antitumor antibiotics. In a preferred embodiment, the cytotoxin is selected from the group consisting of enediyne antibiotics. In a more preferred embodiment, the cytotoxin is selected from the group consisting of dynemicin, esperamicin, neocarcin, aclarubicin.
  • the cytotoxin is selected from the group consisting of actinomycin, anthramycin, bleomycins, actinomycin C, carabicin, carminomycin, carminomycin, actinomycin D, daunorubicin, detorubicin, doxorubicin, epirubicin, esorubicin, idarubicin, mexiromycin, mitomycins, noramycin, olivemycin, peplomycin, porfiromycin, puromycin, ferroxorubicin, rhodorubicin, streptozocin, streptozocin, netastatin, zorubicin.
  • the cytotoxin is selected from the group consisting of trichothecenes. In a more preferred embodiment, the cytotoxin is selected from the group consisting of T-2 toxin, verracurin A, baculozolin A and anguidine. In one embodiment, the cytotoxin is selected from the group consisting of anti-tumor amino acid derivatives. In a preferred embodiment, the cytotoxin is selected from the group consisting of ubenimex, azaserine, 6-diazo-5-oxo-L-norleucine. In another embodiment, the cytotoxin is selected from the group consisting of folic acid analogs.
  • the cytotoxin is selected from the group consisting of dimethylfolate, methotrexate, pteroxine, trimetrexate, edatrexate. In one embodiment, the cytotoxin is selected from the group consisting of purine analogs. In a preferred embodiment, the cytotoxin is selected from the group consisting of fludarabine, 6-mercaptopurine, thiopurine, thioguanine. In another embodiment, the cytotoxin is selected from the group consisting of pyrimidine analogs.
  • the cytotoxin is selected from the group consisting of ancitabine, gemcitabine, enocitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, floxuridine.
  • the cytotoxin is selected from the group consisting of androgens.
  • the cytotoxin is selected from the group consisting of captestosterone, drostanolone propionate, cyclothiodine, mestanolidine, testolactone.
  • the cytotoxin is selected from the group consisting of anti-adrenal.
  • the cytotoxin is selected from the group consisting of aminoglutethimide, mitotane, trilostane. In one embodiment, the cytotoxin is selected from the group consisting of anti-androgens. In a preferred embodiment, the cytotoxin is selected from the group consisting of flutamide, nilutamide, bicalutamide, leuprolide acetate and goserelin. In yet another embodiment, the cytotoxin is selected from the group consisting of protein kinase inhibitors and proteasome inhibitors.
  • the cytotoxin is selected from the group consisting of vinca alkaloids, colchicines, taxanes, auristatins, maytansinoids, calicheamicin, doxonubicin, duocarmucin, SN-38, cryptophycin analog, deruxtecan, duocarmazine, calicheamicin, centanamycin, dolastansine, pyrrolobenzodiazepine (PBD), and exatecan.
  • the cytotoxin is selected from the group consisting of vinca alkaloids, colchicines, taxanes, auristatins, and maytansinoids.
  • the cytotoxin is exatecan or a derivative thereof, such as DX8951f and the like.
  • the cytotoxin is a maytansinoid, such as DM1, etc.
  • the sulfhydryl moiety can react with a maleimide moiety to form a sulfosuccinimide, such as a maytansinoid, such as DM1, and the cytotoxin can be directly linked via the sulfosuccinimide.
  • the load and the sulfhydryl moiety together constitute the cytotoxin, so in this case, the load represents the remainder of the cytotoxin molecule except the sulfhydryl moiety.
  • the cytotoxin is an auristatin, such as MMAE (monomethyl auristatin E), MMAF (monomethyl auristatin F), MMAD (monomethyl auristatin D), etc.
  • the cytotoxin is MMAE.
  • the synthesis and structure of auristatin compounds are described in US20060229253, the entire disclosure of which is incorporated herein by reference.
  • the support contains an active group that can react with the active group in the compound of formula (I) and thus covalently conjugate the support to the compound of formula (I).
  • Compounds without active groups require appropriate derivatization to obtain the support.
  • the cytotoxin is a compound of formula (i)
  • g is any integer from 1 to 6;
  • g is any integer from 1 to 3, preferably 1.
  • the cytotoxin is selected from the following compounds 1-16; wherein the wavy bond represents the attachment site to the compound of formula (I).
  • linker-carrier compound having formula (I) is:
  • the present invention provides a linker-carrier compound obtained by a method comprising the steps of:
  • step (ii) reacting the carboxyl group in the intermediate compound (a) obtained in step (i) with the reactive group in the linker-carrier compound (b) having a reactive group at the end to obtain the linker-carrier compound.
  • the first six-carbon sugar unit is selected from glucosyl, mannosyl, galactosyl, fructosyl, gulosyl or idosyl; and/or
  • the second six-carbon sugar unit is selected from glucosyl, mannosyl, galactosyl or fructosyl; and/or
  • the first hexose unit or its derivative part in the trisaccharide structure is connected to the second hexose unit or its derivative part via an ⁇ -(1 ⁇ 6) glycosidic bond; and/or
  • the second hexose unit or its derivative in the trisaccharide is linked to the ⁇ -D-N-acetylglucosamine portion or the ⁇ -D-glucose oxazoline portion via a ⁇ -(1 ⁇ 4) glycosidic bond; and/or
  • the derivatives are independently selected from the derivatives in which the hydroxyl groups of the monosaccharide are replaced by acylamino groups (eg alkanoylamino groups, such as formylamino, acetylamino, propionylamino, etc., especially acetylamino groups).
  • acylamino groups eg alkanoylamino groups, such as formylamino, acetylamino, propionylamino, etc., especially acetylamino groups).
  • the first six-carbon sugar unit is selected from mannose, glucosyl or galactosyl; and/or
  • the second six-carbon sugar unit is selected from mannosyl, glucosyl or galactosyl.
  • the trisaccharide in step (i) has the following structure:
  • the trisaccharide in step (i) has the following structure:
  • the trisaccharide in step (i) has the following structure:
  • the trisaccharide in step (i) has the following structure:
  • the trisaccharide in step (i) has the following structure:
  • the trisaccharide in step (i) has the following structure:
  • the trisaccharide in step (i) has the following structure:
  • the trisaccharide in step (i) has the following structure:
  • the trisaccharide in step (i) has the following structure:
  • the reactive group of the linker-carrier compound (b) having a reactive group at the terminal in step (ii) is an amino group.
  • the method may also include the step of converting the intermediate compound (a) having a carboxyl group into an acyl halide, and further reacting it with a linker-carrier compound (b) having a reactive group at the end to obtain the linker-carrier compound.
  • the linker-carrier compound is a linker-carrier compound as defined in the first aspect or various embodiments thereof.
  • the invention provides a method of preparing a linker-carrier compound having formula (I), wherein unless otherwise indicated, the various variables are as defined in the first aspect or various embodiments thereof.
  • the method comprises the step of reacting D with L'-(P) t to form D-L-(P) t by amide formation, wherein L' is the same as L defined in the first aspect or its various embodiments except that the -NH- in L attached to D is H2N- in L'.
  • the method can further include Condensation
  • the condensation is carried out in the presence of water, a base and 2-chloro-1,3-dimethylimidazolinium chloride (DMC, cas: 37091-73-9).
  • the base is an inorganic base or an organic base.
  • the inorganic base is potassium carbonate, potassium phosphate, etc.
  • the organic base is an amine, such as a tertiary amine, such as triethylamine (Et 3 N).
  • the amide formation reaction is carried out in the presence of an organic solvent, an organic base and a condensation reagent.
  • the organic solvent is selected from N,N-dimethylformamide (DMF) and N,N-dimethylacetamide (DMA).
  • the organic base is selected from diisopropylethylamine (DIPEA) and N-methylmorpholine (NMM).
  • the preparation is carried out under the condition of debenzylation of the substrate.
  • the deprotection is carried out in the presence of hydrogen and a palladium catalyst.
  • the palladium catalyst is selected from palladium carbon and palladium hydroxide carbon.
  • the palladium catalyst is palladium carbon.
  • the preparation is carried out under alkaline conditions, and then the reaction system is adjusted to acidic conditions for purification.
  • the alkaline conditions are the pH of the reaction system is about 11-12, and the acidic conditions are the pH of the reaction system is about 6-7.
  • the preparation is carried out in the presence of thioacetic acid (AcSH) and an organic solvent.
  • the organic solvent is selected from chloroform and pyridine or a mixture of the two.
  • the preparation is carried out under glycosidic bond forming conditions.
  • the glycosidic bond forming conditions include using N-iodosuccinimide and silver trifluoromethanesulfonate in an anhydrous state.
  • the preparation comprises using dibutylboron trifluoromethanesulfonate and borane-tetrahydrofuran.
  • the preparation comprises the use of acetic anhydride.
  • the preparation comprises the use of methyl iodide.
  • the preparation comprises the use of 2,2,6,6-tetramethylpiperidinoxide (TEMPO) and iodophenyldiacetic acid.
  • TEMPO 2,2,6,6-tetramethylpiperidinoxide
  • iodophenyldiacetic acid 2,2,6,6-tetramethylpiperidinoxide
  • the preparation is carried out in the presence of an acid.
  • the acid is p-toluenesulfonic acid.
  • the preparation comprises using a catalyst and acetic anhydride.
  • the catalyst is 4-dimethylaminopyridine (DMAP).
  • the present invention provides an antibody-drug conjugate having formula (II) based on site-directed attachment of the N-glycosylation site in the Fc region of an antibody:
  • R is hydrogen or ⁇ -L-fucosyl
  • q 1 or 2;
  • Ab is an antibody or an antigen-binding fragment (for example, the -NHC(O)CH 2 - linked to Ab in formula (II) comes from asparagine at position 297 in the Fc region of an antibody);
  • the first hexose unit or its derivative part is connected to the second hexose unit or its derivative part via an ⁇ -(1 ⁇ 6) glycosidic bond;
  • the second hexose unit or its derivative is linked to the ⁇ -D-N-acetylglucosamine portion via a ⁇ -(1 ⁇ 4) glycosidic bond;
  • the 6-OH of the first hexose unit or its derivative is oxidized to -C(O)-;
  • L is a linker, and L is directly connected to the -C(O)- in the first hexose unit or its derivative portion via the terminal -NH- therein; wherein when L is an unbranched linker, it is connected to 1 P, and t is 1, and when L is a branched linker, each branch can be connected to 1 P, and t is an integer greater than 1 (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10).
  • the first six-carbon sugar unit or its derivative moiety is selected from glucosyl, mannosyl, galactosyl, fructosyl, gulosyl, idosyl or derivatives thereof.
  • the first hexose unit or its derivative moiety is selected from in, represents the site of connection to L; * represents the site of connection to the second hexose unit or its derivative.
  • the second six-carbon sugar unit or its derivative part is selected from glucosyl, mannosyl, galactosyl, fructosyl or their derivatives. In one embodiment, the second six-carbon sugar unit or its derivative part is selected from
  • the derivatives in the hexose unit or its derivative part are independently selected from uronic acid or monosaccharide derivatives in which the hydroxyl group is replaced by an acylamino group (e.g., an alkanoylamino group, such as formylamino, acetylamino, propionylamino, etc., especially acetylamino).
  • an acylamino group e.g., an alkanoylamino group, such as formylamino, acetylamino, propionylamino, etc., especially acetylamino.
  • the -NHC(O)CH 2 - in formula (II) that is attached to Ab is derived from asparagine at position 297 of the Fc region of the antibody.
  • Ab is a monoclonal antibody. In some embodiments, Ab is an anti-human HER2 antibody or its antigen-binding fragment. Examples of anti-human HER2 antibodies include but are not limited to Pertuzumab and Trastuzumab. In one embodiment, Ab is Trastuzumab.
  • formula (II) is formula (II-2):
  • formula (II) is formula (II-3):
  • formula (II) is formula (II-4):
  • formula (II) is formula (II-5):
  • formula (II) is formula (II-6):
  • formula (II) is formula (II-7):
  • formula (II) is formula (II-8):
  • formula (II) is formula (II-9):
  • R is hydrogen or ⁇ -L-fucosyl
  • q 1 or 2;
  • Ab is an antibody or an antigen-binding fragment (for example, the -NHC(O)CH 2 - linked to Ab in formula (II-1) is derived from asparagine at position 297 in the Fc region of an antibody);
  • Ab is an anti-ErbB2/HER2 antibody (eg, trastuzumab);
  • R in formula (II) is ⁇ -L-fucosyl
  • the -NHC(O)CH 2 - in formula (II) that is linked to Ab is derived from asparagine at position 297 in the Fc region of the antibody.
  • the present invention provides a method for preparing an antibody-drug conjugate having formula (II), comprising conjugating a linker-load compound having formula (I) to an antibody Ab, wherein unless otherwise indicated, each variable is as defined in the third aspect or its various embodiments.
  • the method comprises the steps of:
  • the antibody Ab is cleaved from its N-sugar chain to obtain an antibody whose Fc region N-glycosylation site is modified with N-acetylglucosamine or fucosyl- ⁇ -1,6-N-acetylglucosamine;
  • step b) under the catalysis of glycosidase or its mutant, the modified antibody obtained in step a) is coupled with the above-mentioned linker-load compound;
  • glycosidase or its mutant used in step a) and step b) may be the same or different.
  • the glycosidase or mutant thereof used in steps a) and b) is fucosylase, N-acetylglucosamine endohydrolase or mutants thereof.
  • the N-acetylglucosamine endohydrolase comprises at least one selected from Endo-S (Streptococcus pyogenes endoglycosidase-S), Endo-F3 (Elizabethkingia miricola endoglycosidase-F3), Endo-S2 (Endoglycosidase-S2, Streptococcus pyogenes endoglycosidase-S2), Endo-Sd (Endoglycosidase-Sd, Streptococcus pyogenes endoglycosidase-Sd) and Endo-CC (Endoglycosidase-CC, Streptococcus pyogenes endoglycosidase
  • steps a) and b) are performed by one-pot enzymatic catalysis.
  • steps a) and b) are performed by one-pot enzymatic catalysis and the enzyme is Endo S2.
  • R 1 -R 12 are selected from -OH, or H;
  • the two substituents on the same carbon atom are different;
  • the two substituents on the same carbon atom are different; and R 3 is H, and R 4 is -OH;
  • the two substituents on the same carbon atom are different; and R 3 is H, R 4 is -OH; R 9 is H, R 10 is -OH;
  • R 3 is H, R 4 is -OH;
  • R 9 is H, R 10 is -OH;
  • R 8 is H, R 7 is -OH;
  • R 12 is H, R 11 is -OH;
  • L and P refer to those of formula (I).
  • formula (IV) is as shown in formula (IV-1):
  • R 1 , R 2 , R 5 , and R 6 are selected from -OH, or H; and the two substituents on the same carbon atom are different.
  • the trisaccharide structure of the formula (IV) or (IV-1) is combined with an amino group-containing fragment to form an amide bond to link the drug fragment, or is connected to a linker with a bioorthogonal functional group in the form of an amide bond, and then reacted with a drug fragment having the aforementioned bioorthogonal functional group to form an antibody drug conjugate.
  • compositions and pharmaceutical preparations are provided.
  • Another object of the present invention is to provide a pharmaceutical composition comprising a preventive or therapeutically effective amount of the antibody-drug conjugate of the present invention and at least one pharmaceutically acceptable carrier.
  • the pharmaceutical composition of the present invention can be administered in any manner as long as it achieves the effect of preventing, alleviating, preventing or treating the symptoms of humans or animals.
  • various suitable dosage forms can be prepared according to the route of administration, particularly injections such as lyophilized powder injections, injections or sterile injection powders.
  • pharmaceutically acceptable means that it does not produce undue toxicity, irritation or allergic response when in contact with patient tissues within the scope of normal medical judgment, has a reasonable ratio of advantages to disadvantages, and is effective for the intended use.
  • pharmaceutically acceptable carrier refers to those carrier materials that are pharmaceutically acceptable and do not interfere with the biological activity and performance of the antibody-drug conjugate.
  • aqueous carriers include, but are not limited to, buffered saline, etc.
  • Pharmaceutically acceptable carriers also include carrier substances that make the composition close to physiological conditions, such as pH adjusters and buffers, toxicity regulators, etc., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc.
  • the drug/antibody ratio (DAR) of the pharmaceutical composition of the present invention is an integer or non-integer of 1-20, such as about 1-about 10, about 1-about 8, about 1-about 6, about 1-about 4, about 1-3 about, about 1-about 2.5, about 1-about 2.
  • the DAR of the antibody-drug conjugate of the present invention is about 2, about 4, about 6 or about 8.
  • the DAR of the antibody-drug conjugate of the present invention is about 2.
  • the DAR of the antibody-drug conjugate of the present invention is about 1.65.
  • the antibody-drug conjugates of the present invention can be used to treat tumors and/or autoimmune diseases.
  • Tumors that are sensitive to antibody-drug conjugates include tumors characterized by specific tumor-associated antigens or cell surface receptors, and these tumor cells can be recognized by the targeting molecules in the antibody-drug conjugates and then killed by the payload/cytotoxin in the antibody-drug conjugates.
  • the present invention also provides use of the antibody-drug conjugate of the present invention or the pharmaceutical composition of the present invention in the preparation of a medicament for treating a disease, disorder or condition selected from a tumor or an autoimmune disease.
  • the present invention provides the antibody-drug conjugate of the present invention or the pharmaceutical composition of the present invention for use in treating tumors or autoimmune diseases.
  • the present invention provides a method for treating tumors or autoimmune diseases, the method comprising administering an effective amount of the antibody-drug conjugate of the present invention or the pharmaceutical composition of the present invention to an individual in need thereof.
  • the antibody-drug conjugate formed by connecting the anti-human HER2 antibody provided by the present invention to a small molecule cytotoxin can specifically bind to HER2 on the surface of tumor cells and selectively kill tumor cells expressing HER2.
  • the present invention provides the use of the antibody-drug conjugate of the present invention or the pharmaceutical composition of the present invention in the preparation of a drug for treating a disease, disorder or condition, wherein the disease, disorder or condition is selected from a HER2-positive tumor.
  • the disease, disorder or condition is selected from the group consisting of breast cancer, gastric cancer, lung cancer, ovarian cancer and urothelial carcinoma.
  • the dosage of the antibody-drug conjugate administered to a subject can be adjusted to a considerable extent.
  • the dosage can vary depending on the specific route of administration and the needs of the subject and can be subject to the judgment of a healthcare professional.
  • the present invention expands the scope of antibody-drug conjugates obtained by sugar chain remodeling technology due to the use of a novel trisaccharide structure that forms an amide bond.
  • a unique trisaccharide carboxylic acid substrate e.g., ) can be well connected with amine-containing compounds through a mild and compatible amide bonding method to achieve one-step connection, simplifying and accelerating the synthesis of linker-load compounds, without the generation of adverse by-products, and enriching the source of substrates.
  • the present invention utilizes modular combinatorial chemistry to efficiently synthesize a series of linkers-loads based on trisaccharide structures, and verifies that it can achieve efficient coupling of different payloads. Due to the introduction of trisaccharides, its water solubility has increased to a certain extent, which has a meaningful improvement on the stability of antibody-drug conjugate products.
  • the above linker-loads based on trisaccharide structures are a further supplement and improvement to the one-step sugar chain fixed-point coupling technology based on disaccharides, especially enriching the source of substrates, improving the connection mode between disaccharides and effector molecules, enriching the sugar chain pattern of the resulting antibody-drug conjugates, and improving the water solubility of antibody-drug conjugates. It avoids the limitations of the prior art that coupling still requires a certain amount of organic solvents to promote dissolution, and further improves the coupling efficiency of some substrates.
  • the present invention further developed a series of trisaccharide molecular libraries with novel structures and differentiated characteristics, and based on the molecular library, a group of antibody-drug conjugate molecular libraries containing structurally differentiated glycoforms in the Fc region of the antibody were constructed.
  • These molecular libraries exhibit unique antibody-dependent cell-mediated cytotoxicity (ADCC) activity, which is significantly different from the existing ADC technology based on disaccharide linkers.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • the use of disaccharide linkers in traditional technologies usually causes antibody-drug conjugates such as ADCs to lose their ADCC function, and the technology we developed enables the new molecular library to meet the diverse drug molecule design needs through the introduction of trisaccharide linkers.
  • trisaccharide molecules have stronger hydrophilicity and better adaptability to hydrophobic load molecules. This feature not only improves the water solubility of ADC molecules, but also broadens the scope of application of ADC molecule design. Therefore, by adopting structurally diverse trisaccharide linkers, ADC molecules with differentiated Fc glycoforms and diversified physical and chemical properties can be easily constructed, thereby better meeting the diverse needs of ADC drug development.
  • compound 1-1d (201 mg, 0.54 mmol), DMF (2 mL), cesium carbonate, and iodomethane were added to a 25 mL single-mouth bottle in sequence and dissolved, and stirred at room temperature for about 2 hours.
  • water (15 mL) was added to quench the reaction, and the reaction system was extracted with ethyl acetate (15 mL ⁇ 3).
  • compound 1-2a (20 g, 51.2 mmol, CAS No.: 604-68-2) was synthesized through 4 steps to finally obtain compound 1-2 (7.1 g, total yield 30%, colorless oily liquid).
  • compound 1-1 (6 g, 14.1 mmol), acetone (30 mL) and N-bromosuccinimide were added to a 250 mL single-mouth bottle in sequence and stirred at room temperature for about 1 hour.
  • a saturated sodium thiosulfate solution (10 mL) was added to quench the reaction, and the excess acetone was removed by concentration under reduced pressure.
  • the reaction system was extracted with ethyl acetate (50 mL ⁇ 3).
  • compound 2-2a 5 g, 10.5 mmol, CAS number: 342640-42-0
  • compound 1-2 (equivalent to 0.1-5 equivalents of 2-2a)
  • 4A molecular sieves 4A molecular sieves
  • dichloromethane 100 mL
  • the reaction system was cooled to -60 ° C, N-iodosuccinimide and trifluoromethanesulfonic acid were added thereto, and stirred at this temperature for 2 hours.
  • compound 2-2b (6.6 g, 8.15 mmol), borane tetrahydrofuran complex and dibutylboron trifluoromethanesulfonate were added to a 250 mL Schlenk reaction bottle in sequence, and after dissolving, it was stirred at 0°C for about 40 minutes. After TLC monitoring that the reaction of the starting materials was complete, triethylamine (6 mL) and methanol (6 mL) were added to the reaction system, and the mixture and methanol were concentrated under reduced pressure three times.
  • compound 2-2a 5 g, 10.5 mmol, CAS number: 342640-42-0
  • compound 1-3 (equivalent to 0.1-5 equivalents of 2-2a)
  • 4A molecular sieves 4A molecular sieves
  • dichloromethane 100 mL
  • the reaction system was cooled to -60 ° C, N-iodosuccinimide and trifluoromethanesulfonic acid were added thereto, and stirred at this temperature for 2 hours.
  • compound 2-3a (6.6 g, 8.15 mmol), borane tetrahydrofuran complex and dibutylboron trifluoromethanesulfonate were added to a 250 mL Schlenk reaction bottle in sequence, and after dissolving, it was stirred at 0°C for about 40 minutes.
  • compound 1-4 0.1-10 equivalents relative to compound 2-2
  • compound 2-2 1.2 g, 1.48 mmol
  • toluene 100 mL
  • scandium trifluoromethanesulfonate was added to the system, and the mixture was heated to 60°C and stirred for about 2 hours.
  • water 100 mL was added to quench the reaction, and the mixture was extracted with dichloromethane (100 mL ⁇ 2).
  • compound 3-2b 550 mg, 0.481 mmol
  • methanol 10 mL
  • 1 M sodium hydroxide solution (1 mL) was slowly added (10 minutes) and stirred at room temperature for 2 hours until the reaction was detected to be complete by HPLC.
  • Acetic acid (1 mL) was added to the reaction system to quench the reaction, and the crude compound 3-2c (300 mg, yield 68%, colorless oily liquid) was obtained after concentration under reduced pressure.
  • compound 1-4 0.1-10 equivalents relative to compound 2-3
  • compound 2-3 1.2 g, 1.48 mmol
  • toluene 100 mL
  • scandium trifluoromethanesulfonate was added to the system, and the mixture was heated to 60°C and stirred for about 2 hours.
  • water 100 mL was added to quench the reaction, and the mixture was extracted with dichloromethane (100 mL ⁇ 2).
  • linker-load compound 4-1 The structure of the linker-load compound 4-1 is as follows:
  • linker-load compound 4-2 The structure of the linker-load compound 4-2 is as follows:
  • linker-load compound 4-3 The structure of the linker-load compound 4-3 is as follows:
  • linker-load compound 4-4 The structure of the linker-load compound 4-4 is as follows:
  • linker-load compound 4-5 is as follows:
  • linker-load compounds 4-6 are as follows:
  • linker-load compounds 4-7 are as follows:
  • linker-load compounds 4-8 are as follows:
  • the linker cargo 4-8 was synthesized using similar steps to the synthesis of 4-5.
  • linker-load compound 4-9 is as follows:
  • linker-load compound 4-10 is as follows:
  • linker-load compound 4-11 is as follows:
  • the linker cargo 4-11 was synthesized using similar steps to the synthesis of 4-9.
  • linker-load compound 4-12 is as follows:
  • Step 4 Coupling of Fmoc-PEG 2 -CH 2 CH 2 COOH
  • Step 5 Resin cleavage
  • linker-load compound 4-13 is as follows:
  • linker-load compound 4-14 is as follows:
  • the linker cargo 4-14 was synthesized using similar steps to the synthesis of 4-12.
  • linker-load compound 4-15 is as follows:
  • 4-15-1a (0.1-10 equivalents relative to 4-15-1b) and 4-15-1b (1 g, 4.582 mmol) were placed in a 100 mL round-bottom flask, and DMF (25 mL) was added. After ice bathing to 0°C, DIEA was added, and HATU was added after stirring for 5 minutes. The mixture was stirred at room temperature for 1 hour until the reaction was complete as detected by HPLC. Water (150 mL) was added to the reaction solution, and the mixture was washed with ethyl acetate (150 mL ⁇ 3).
  • the antibody-drug conjugate prepared by the substrate and conjugation method of the present invention can be selected as any molecule containing a sugar chain in the Fc region of an antibody, including but not limited to antibodies/bispecific antibodies/Fc fusion proteins/single-chain antibodies, etc.
  • trastuzumab can be selected, which is commercially available.
  • antibodies with engineered modifications may also be selected.
  • the production, purification and identification of anti-human ErbB2/HER2 antibody mAb-1 refer to Example 1 of patent CN 106856656 B, the entire disclosure of which is incorporated herein by reference.
  • the sugar chain at the Fc end of the antibody mAb-1 is remodeled by endoglycosidase, and the linker-load is specifically coupled to the antibody to form the corresponding ADC drug.
  • Treatment of mAb-1 Treat the antibody using ultrafiltration, dialysis or desalting column method, and replace its storage buffer with 50mM Tris-HCl (pH between 5-8) and 150mM NaCl.
  • ADC-1 was prepared by catalyzing the coupling reaction between the antibody mAb-1 and the linker-load compound 4-1 with the endoglycosidase Endo S2.
  • 1 ⁇ endoglycosidase buffer the antibody mAb-1 and the compound 4-1 were fully mixed at an appropriate molar ratio (1:1 to 1:100), and then the endoglycosidase Endo S2 was added and mixed. Under the mixing state, the coupling reaction was carried out at 4-40°C for 0.5-20 hours. After the reaction was completed, purification was performed.
  • the purified ADC-1 was stored in 1 ⁇ PBS (pH7.4) at 4°C or -80°C.
  • the test results are shown in Figure 1.
  • the unconjugated cytotoxic antibody is less than 5%; the conjugated product is mainly DAR2, and the overall DAR value of the ADC drug is about 1.88 (under better conjugation conditions).
  • a TSKgel G3000SWXL 7.8mm I.D.*30cm, 5 ⁇ m column (manufacturer: Tosoh, PN: 0008541) was used, 2*PBS: acetonitrile 9:1 (v/v) as the mobile phase, and the flow rate was 1.0mL/min at room temperature for 15min isocratic operation. 280nm was selected as the detection wavelength to analyze the high molecular weight aggregation of ADC drugs.
  • the test results are shown in FIG2 .
  • the high molecular weight polymers in the ADC drug are less than 6%, and the ADC sample (8.03 min) mainly exists in the form of monomers.
  • the damage of the coupling reaction to the antibody is almost negligible.
  • the sugar chain at the Fc end of the antibody mAb-1 is remodeled by endoglycosidase, and the linker-load is specifically coupled to the antibody to form the corresponding ADC drug.
  • Treatment of mAb-1 Treat the antibody using ultrafiltration, dialysis or desalting column method, and replace its storage buffer with 50mM Tris-HCl (pH between 5-8) and 150mM NaCl.
  • ADC-2 was prepared by catalyzing the coupling reaction between the antibody mAb-1 and the linker-load compound 4-2 using the endoglycosidase Endo S2.
  • 1 ⁇ endoglycosidase buffer the antibody mAb-1 and compound 4-2 were fully mixed at an appropriate molar ratio (1:1 to 1:100), and then the endoglycosidase Endo S2 was added and mixed. Under the mixing state, the coupling reaction was carried out at 4-40°C for 0.5-20 hours. After the reaction was completed, purification was performed. The purified ADC-2 was stored in 1 ⁇ PBS (pH7.4) at 4°C or -80°C.
  • the test results are shown in FIG3 .
  • the unconjugated cytotoxic antibody is less than 1%; the conjugated product is mainly DAR2, and the overall DAR value of the ADC drug is about 1.90.
  • the test results are shown in FIG4 .
  • the high molecular weight polymers in the ADC drug are less than 2%, and the ADC sample (8.54 min) mainly exists in the form of monomers.
  • the damage of the coupling reaction to the antibody is almost negligible.
  • the preparation and characterization were performed according to the method described in Example 27, except that the linker-payload used was 4-3.
  • the characterization data of the antibody-drug conjugate ADC-3 are described below.
  • hydrophobic interaction high performance liquid chromatography (HIC-HPLC) detection and analysis of ADC-3 the detection results are shown in Figure 5, the unconjugated cytotoxic antibody is ⁇ 1%; the conjugated product is mainly DAR2, and the overall DAR value of the ADC drug is about 1.90.
  • ADC-3 was analyzed by high performance liquid phase size exclusion chromatography (SEC-HPLC). The test results are shown in FIG6 .
  • the high molecular weight polymer in the ADC drug is less than 2%, and the ADC sample (8.53 min) mainly exists in the form of monomers. The damage of the coupling reaction to the antibody is almost negligible.
  • the preparation and characterization were performed according to the method described in Example 27, except that the linker-payload used was 4-4.
  • the characterization data of the antibody-drug conjugate ADC-4 are described below.
  • hydrophobic interaction high performance liquid chromatography (HIC-HPLC) detection and analysis of ADC-4 the detection results are shown in Figure 7, the unconjugated cytotoxic antibody is ⁇ 1%; the conjugated product is mainly DAR2, and the overall DAR value of the ADC drug is about 1.95.
  • ADC-4 was analyzed by high performance liquid phase size exclusion chromatography (SEC-HPLC). The test results are shown in FIG8 .
  • the high molecular weight polymer in the ADC drug is less than 2%, and the ADC sample (8.53 min) mainly exists in the form of monomers. The damage of the coupling reaction to the antibody is almost negligible.
  • the preparation and characterization were performed according to the method described in Example 27, except that the linker-payload used was 4-5.
  • the characterization data of the antibody-drug conjugate ADC-5 are described below.
  • hydrophobic interaction high performance liquid chromatography (HIC-HPLC) analysis of ADC-5 showed that the unconjugated cytotoxic antibody was less than 1%; the conjugated product was mainly DAR4, and the overall DAR value of the ADC drug was about 3.82.
  • the preparation and characterization were performed according to the method described in Example 27, except that the linker-payload used was 4-6.
  • the characterization data of the antibody-drug conjugate ADC-6 are described below.
  • hydrophobic interaction high performance liquid chromatography (HIC-HPLC) analysis of ADC-6 showed that the unconjugated cytotoxic antibody was less than 1%; the conjugated product was mainly DAR4, and the overall DAR value of the ADC drug was about 3.91.
  • the preparation and characterization were performed according to the method described in Example 27, except that the linker-payload used was 4-7.
  • the characterization data of the antibody-drug conjugate ADC-7 are described below.
  • hydrophobic interaction high performance liquid chromatography (HIC-HPLC) analysis of ADC-7 showed that the unconjugated cytotoxic antibody was less than 1%; the conjugated product was mainly DAR4, and the overall DAR value of the ADC drug was about 3.98.
  • the preparation and characterization were performed according to the method described in Example 27, except that the linker-payload used was 4-8.
  • the characterization data of the antibody-drug conjugate ADC-8 are described below.
  • hydrophobic interaction high performance liquid chromatography (HIC-HPLC) analysis of ADC-8 showed that the unconjugated cytotoxic antibody was less than 1%; the conjugated product was mainly DAR4, and the overall DAR value of the ADC drug was about 3.87.
  • the preparation and characterization were performed according to the method described in Example 27, except that the linker-payload used was 4-9.
  • the characterization data of the antibody-drug conjugate ADC-9 are described below.
  • hydrophobic interaction high performance liquid chromatography (HIC-HPLC) analysis of ADC-9 showed that the unconjugated cytotoxic antibody was less than 1%; the conjugated product was mainly DAR4, and the overall DAR value of the ADC drug was about 3.76.
  • the preparation and characterization were performed according to the method described in Example 27, except that the linker-payload used was 4-10.
  • the characterization data of the antibody-drug conjugate ADC-10 are described below.
  • hydrophobic interaction high performance liquid chromatography (HIC-HPLC) analysis of ADC-10 showed that the unconjugated cytotoxic antibody was less than 2%; the conjugated product was mainly DAR4, and the overall DAR value of the ADC drug was about 3.72.
  • the preparation and characterization were performed according to the method described in Example 27, except that the linker-payload used was 4-11.
  • the characterization data of the antibody-drug conjugate ADC-11 are described below.
  • hydrophobic interaction high performance liquid chromatography (HIC-HPLC) analysis of ADC-11 showed that the unconjugated cytotoxic antibody was less than 1%; the conjugated product was mainly DAR4, and the overall DAR value of the ADC drug was about 3.86.
  • the preparation and characterization were performed according to the method described in Example 27, except that the linker-payload used was 4-12.
  • the characterization data of the antibody-drug conjugate ADC-12 are described below.
  • hydrophobic interaction high performance liquid chromatography (HIC-HPLC) analysis of ADC-12 showed that the unconjugated cytotoxic antibody was less than 1%; the conjugated product was mainly DAR4, and the overall DAR value of the ADC drug was about 3.64.
  • the preparation and characterization were performed according to the method described in Example 27, except that the linker-payload used was 4-13.
  • the characterization data of the antibody-drug conjugate ADC-13 are described below.
  • hydrophobic interaction high performance liquid chromatography (HIC-HPLC) analysis of ADC-13 showed that the unconjugated cytotoxic antibody was less than 1%; the conjugated product was mainly DAR4, and the overall DAR value of the ADC drug was about 3.74.
  • the preparation and characterization were performed according to the method described in Example 27, except that the linker-payload used was 4-14.
  • the characterization data of the antibody-drug conjugate ADC-14 are described below.
  • hydrophobic interaction high performance liquid chromatography (HIC-HPLC) analysis of ADC-14 showed that the unconjugated cytotoxic antibody was less than 1%; the conjugated product was mainly DAR4, and the overall DAR value of the ADC drug was about 3.84.
  • the preparation and characterization were performed according to the method described in Example 27, except that the linker-payload used was 4-15.
  • the characterization data of the antibody-drug conjugate ADC-15 are described below.
  • hydrophobic interaction high performance liquid chromatography (HIC-HPLC) analysis of ADC-15 showed that the unconjugated cytotoxic antibody was less than 1%; the conjugated product was mainly DAR2, and the overall DAR value of the ADC drug was about 1.92.
  • HER2-positive human breast cancer cells SKBR-3, HER2-positive human gastric cancer cells NCI-N87, and HER2-negative human breast cancer cells MDA-MB-468 were inoculated into 96-well cell plates at 100 ⁇ L per well (containing 1,000-10,000 cells) and cultured overnight in a cell culture incubator at 37° C., 5% CO 2 , 95% air, and 100% humidity.
  • ADC-1, ADC-2, ADC-3 and ADC-4 were added to the HER2 positive cells and negative cells cultured overnight, and a non-administered group and a 5 ⁇ M Puromycin group were set up as 100% cell viability and 0% cell viability controls, respectively. After administration, the cells were placed in a cell culture incubator and incubated at 37°C for 72-120 hours.
  • ADC-1, ADC-2, ADC-3 and ADC-4 have significant inhibitory effects on HER2-positive cells, and there is no significant difference in efficacy between disaccharide ADC-4 and trisaccharides ADC-1, ADC-2 and ADC-3 under the same DAR.
  • ADC-1, ADC-2, ADC-3 and ADC-4 have no inhibitory effect on HER2-negative cells.
  • ADC-5, ADC-6, ADC-7 and ADC-8 were tested as follows:
  • the antibody-drug conjugates ADC-5, ADC-6, ADC-7 and ADC-8 were tested for their effects on tumor cell proliferation at different HER2 expression levels.
  • the results of the inhibitory effects of different drugs on tumor cell proliferation are shown in Table 2 and Figures 12-14, where ADC-5, ADC-6, ADC-7 and ADC-8 had significant inhibitory effects on HER2-positive cells, and there was no significant difference in efficacy between the disaccharide ADC-8 and the trisaccharide ADC-5, ADC-6 and ADC-7 under the same DAR conditions.
  • ADC-5, ADC-6, ADC-7 and ADC-8 had no inhibitory effect on HER2-negative cells.
  • the antibody-drug conjugates ADC-9, ADC-10 and ADC-11 were tested for their proliferation effects on tumor cells at different HER2 expression levels using the method described in Reference Example 41.
  • the results of the inhibitory effects of different drugs on tumor cell proliferation are shown in Table 3 and Figures 15-17, where ADC-9, ADC-10 and ADC-11 had significant inhibitory effects on HER2-positive cells, and there was no significant difference in efficacy between the disaccharide ADC-11 and the trisaccharide ADC-9 and ADC-10 under the same DAR conditions.
  • ADC-9, ADC-10 and ADC-11 had no inhibitory effect on HER2-negative cells.
  • ADC-12, ADC-13, and ADC-14 were tested as follows:
  • the antibody-drug conjugates ADC-12, ADC-13 and ADC-14 were tested for their effects on tumor cell proliferation at different HER2 expression levels.
  • the results of the inhibitory effects of different drugs on tumor cell proliferation are shown in Table 4 and Figures 18-20, where ADC-12, ADC-13 and ADC-14 had significant inhibitory effects on HER2-positive cells, and there was no significant difference in efficacy between the disaccharide ADC-14 and the trisaccharides ADC-12 and ADC-13 under the same DAR conditions.
  • ADC-12, ADC-13 and ADC-14 had no inhibitory effect on HER2-negative cells.
  • Collect cells After the adherent cells are detached from the culture flask using 0.25% trypsin in a cell digestion solution, the cell suspension is placed in a 15 mL centrifuge tube. Centrifuge the cell suspension at 1000 rpm for 5 min, remove the supernatant and resuspend the cells with an appropriate amount of FACS buffer. Use trypan blue staining to determine the cell viability.
  • Cell dilution Take an appropriate amount of cell suspension and add FACS buffer to prepare 10mL of 1-2 ⁇ 10 6 Cells/mL cell suspension.
  • V-bottom plate to plate the cells. Invert and mix the cells several times before plating. Add 100 ⁇ L of cell solution to each well. Add 100 ⁇ L of FACS buffer to the cell control group only, and 100 ⁇ L of antibody solution to other experimental groups. Immediately incubate on ice for 60 min.
  • Binding of secondary antibody Resuspend the cells with 500-fold diluted Goat anti-Human IgG (H+L), Superclonal TM Recombinant Secondary Antibody, Alexa Fluor TM Plus 647 at 100 ⁇ L/well and incubate on ice for 60 min in a dark place. Repeat step 2 to wash the unbound antibodies. Resuspend the cells with 100 ⁇ L/well buffer and store at 4°C in a dark place until detection by flow cytometry.
  • the binding results of different drugs to tumor cells are shown in Table 5 and Figure 21.
  • the binding affinity of disaccharide ADC-8 is comparable to that of trisaccharides ADC-5, ADC-6, ADC-7 and monoclonal antibody OL1302, and the binding behavior is concentration-dependent.
  • the negative control antibody Isotype has no obvious specific binding on NCI-N87 cells.
  • group dosing began.
  • the animals were randomly divided into a vehicle control group, a 5 mg/kg ADC-6 group, a 5 mg/kg ADC-7 group, and a 5 mg/kg ADC-8 group, with 5 animals in each group, and the animals were injected via the tail vein.
  • the tumor volume of each group of animals was measured twice a week after administration, and the tumor volume of animals on the 21st day was compared between groups.
  • T/C% TRTV / CRTV ⁇ 100% ( TRTV : treatment group RTV; CRTV : vehicle control group RTV).
  • Relative tumor volume (RTV) was calculated based on the results of tumor measurement.
  • TGI (%) [1-(average tumor volume at the end of drug administration in a treatment group - average tumor volume at the beginning of drug administration in the treatment group)/(average tumor volume at the end of treatment in the vehicle control group - average tumor volume at the beginning of treatment in the vehicle control group)] ⁇ 100%.
  • bT/C% TRTV / CRTV x 100% ( TRTV : RTV of treatment group; CRTV : RTV of vehicle control group).
  • TGI (%) [1-(T 21 -T 0 )/(V 21 -V 0 )] ⁇ 100) calculation;
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • HER2-positive SK-BR-3 cells were used as target cells and human primary NK cells were used as effector cells.
  • the two were combined into a cell co-culture system at a ratio of 1:5 (SK-BR-3:NK). After the effector cells and target cells were incubated with the test article for 4 hours, the ADCC effect of the test drug was reflected by detecting the release of LDH.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicinal Preparation (AREA)

Abstract

La présente invention concerne un composé lieur-charge utile comprenant un groupe trisaccharide, le groupe trisaccharide étant lié au reste du composé au moyen d'une liaison amide. La présente invention concerne en outre un conjugué anticorps-médicament comprenant le composé lieur-charge utile, une chaîne glycane dans un anticorps étant remodelée à l'aide du groupe trisaccharide dans le composé lieur-charge utile. La présente invention concerne en outre des procédés de préparation et des utilisations des substances décrites.
PCT/CN2024/135760 2023-12-01 2024-11-29 Lieur trisaccharide, lieur-charge utile comprenant un lieur trisaccharide, et conjugué anticorps-médicament remodelé à chaîne glycane, leurs procédés de préparation et leurs utilisations Pending WO2025113659A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202311634308.7 2023-12-01
CN202311634308 2023-12-01

Publications (1)

Publication Number Publication Date
WO2025113659A1 true WO2025113659A1 (fr) 2025-06-05

Family

ID=95896277

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2024/135760 Pending WO2025113659A1 (fr) 2023-12-01 2024-11-29 Lieur trisaccharide, lieur-charge utile comprenant un lieur trisaccharide, et conjugué anticorps-médicament remodelé à chaîne glycane, leurs procédés de préparation et leurs utilisations

Country Status (1)

Country Link
WO (1) WO2025113659A1 (fr)

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018124758A2 (fr) * 2016-12-28 2018-07-05 주식회사 인투셀 Composé portant un lieur auto-immolable à bêta-galactoside introduit
CN114949236A (zh) * 2021-02-22 2022-08-30 中国科学院上海药物研究所 二糖连接子、二糖-小分子药物偶联物以及糖链定点抗体-药物偶联物，其制备方法及用途
CN115197297A (zh) * 2021-04-14 2022-10-18 启德医药科技（苏州）有限公司 连接子、缀合物及其应用
CN115429889A (zh) * 2021-06-04 2022-12-06 上海大学 新型亲水性糖基化连接子及其合成和应用
CN115703845A (zh) * 2021-08-05 2023-02-17 中国科学院上海药物研究所 寡糖连接子与侧链亲水性片段组合的糖链定点抗体-药物偶联物，其制法及用途
CN115916802A (zh) * 2020-08-21 2023-04-04 岩唐生物科技(杭州)有限责任公司 位点特异性抗体偶联物及其制备方法
CN116688143A (zh) * 2023-06-05 2023-09-05 南京鼓楼医院 一种PD-1抗体和iRGD的偶联物、制备方法及其在制备免疫治疗药物中的应用

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018124758A2 (fr) * 2016-12-28 2018-07-05 주식회사 인투셀 Composé portant un lieur auto-immolable à bêta-galactoside introduit
CN115916802A (zh) * 2020-08-21 2023-04-04 岩唐生物科技(杭州)有限责任公司 位点特异性抗体偶联物及其制备方法
CN114949236A (zh) * 2021-02-22 2022-08-30 中国科学院上海药物研究所 二糖连接子、二糖-小分子药物偶联物以及糖链定点抗体-药物偶联物，其制备方法及用途
CN115197297A (zh) * 2021-04-14 2022-10-18 启德医药科技（苏州）有限公司 连接子、缀合物及其应用
CN115429889A (zh) * 2021-06-04 2022-12-06 上海大学 新型亲水性糖基化连接子及其合成和应用
CN115703845A (zh) * 2021-08-05 2023-02-17 中国科学院上海药物研究所 寡糖连接子与侧链亲水性片段组合的糖链定点抗体-药物偶联物，其制法及用途
CN116688143A (zh) * 2023-06-05 2023-09-05 南京鼓楼医院 一种PD-1抗体和iRGD的偶联物、制备方法及其在制备免疫治疗药物中的应用

Similar Documents

Publication Publication Date Title
CN115197297B (zh) 连接子、缀合物及其应用
CA3093327C (fr) Anticorps cd73 cible et conjugue anticorps-medicament, procede de preparation associe et utilisations correspondantes
WO2022188740A1 (fr) Conjugué anticorps-agoniste immun et ses applications
US12318457B2 (en) Oligosaccharide linker, linker-payload comprising the same and glycan chain-remodeled antibody-drug conjugate, preparation methods and uses thereof
CN116194149A (zh) 具有溶解性标签的分子和相关方法
WO2024012566A9 (fr) Anticorps, lieurs, charge utile, conjugués et leurs applications
JP2025523117A (ja) リンカー、コンジュゲート及びその用途
WO2024078612A1 (fr) Composé de charge utile et de lieur, conjugués et applications associées
WO2025113659A1 (fr) Lieur trisaccharide, lieur-charge utile comprenant un lieur trisaccharide, et conjugué anticorps-médicament remodelé à chaîne glycane, leurs procédés de préparation et leurs utilisations
JP2025531032A (ja) 抗体薬物複合体の医薬組成物
WO2024199432A1 (fr) Modalité de bioconjugué orthogonal spécifique à un site et son application
WO2024199442A9 (fr) Lieur disaccharide, lieur-charge utile le contenant et conjugué anticorps-médicament à chaîne glycane remodelée, procédé de préparation correspondant et utilisations associées

Legal Events

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

Ref document number: 24896739

Country of ref document: EP

Kind code of ref document: A1