WO2024255873A1 - Procédé de préparation d'un conjugué anticorps-médicament - Google Patents

Procédé de préparation d'un conjugué anticorps-médicament Download PDF

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Publication number
WO2024255873A1
WO2024255873A1 PCT/CN2024/099341 CN2024099341W WO2024255873A1 WO 2024255873 A1 WO2024255873 A1 WO 2024255873A1 CN 2024099341 W CN2024099341 W CN 2024099341W WO 2024255873 A1 WO2024255873 A1 WO 2024255873A1
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formula
alkyl
group
substituted
antibody
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Chinese (zh)
Inventor
周伟
朱会凯
陈宇航
王珍珍
谭小钉
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Jiangsu Mabwell Health Pharmaceutical R & D Co Ltd
Mabwell Shanghai Bioscience Co Ltd
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Jiangsu Mabwell Health Pharmaceutical R & D Co Ltd
Mabwell Shanghai Bioscience Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68037—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a camptothecin [CPT] or derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68031—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being an auristatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68033—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a maytansine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/20—Partition-, reverse-phase or hydrophobic interaction chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Definitions

  • the present invention belongs to the field of biomedicine, and more specifically, the present invention relates to a new method for preparing antibody-drug conjugates.
  • Antibody-drug conjugates are a new form of drug that connects small molecule drugs to monoclonal antibodies. They use the targeting function of monoclonal antibodies to transport small molecule drugs to target sites to exert their efficacy, reduce toxic side effects, and increase the therapeutic window. In recent years, antibody-drug conjugates have been one of the hot directions in precision tumor treatment, bringing hope to tumor treatment.
  • Antibody-drug conjugates are composed of antibody drugs targeting specific antigens and small molecule cytotoxic drugs coupled through linkers. They have both the powerful killing effect of traditional small molecule chemotherapy and the tumor targeting of antibody drugs. So far, more than a dozen ADC drugs have been approved for marketing worldwide.
  • Antibody-drug conjugates consist of several parts, such as antibodies, linkers, and payloads. Among them, antibodies and drug-linkers can be connected in different coupling modes.
  • the coupling methods are mainly divided into non-fixed site coupling and fixed site coupling. In the early days, non-fixed site coupling methods were used, mainly lysine coupling and cysteine coupling, which directly coupled drugs to amino acid residues on antibodies using chemical methods, without involving antibody transformation or modification. The number of toxin molecules and coupling sites coupled to them cannot be determined, and the uniformity is poor.
  • the currently commonly used fixed-site coupling method is to perform specific coupling through genetic engineering sites or special connectors to achieve more uniform coupling, and can achieve the connection of cytotoxins at specific sites.
  • Antibody-drug conjugates produced by fixed-site coupling can reduce the fluctuations in efficacy, pharmacokinetics, and quality control caused by different coupling sites and coupling numbers.
  • common fixed-site coupling methods include THIOMAB technology, non-natural amino acid coupling technology, glutamine enzymatic coupling technology, Sortase transpeptidase coupling technology, and Thiobridge technology.
  • the modification of antibodies by antibody engineering or enzymatic coupling may have a certain impact on the structural stability of the antibody, and at the same time have certain requirements for CMC.
  • the Thiobridge technology using chemical coupling also has certain defects, such as the replacement of DBM (dibromomaleimides)-type linkers and other thiol-containing biological groups, which are unstable in plasma, resulting in reduced efficacy and increased toxic side effects (Chem.-Eur.J, 2019, 25, 43-59.).
  • DBM dibromomaleimides
  • the drug-to-antibody ratio is a unique and important quality attribute of ADC drugs, which represents the average number of small molecule drugs coupled to antibodies.
  • the degree of drug coupling will affect the stability and aggregation tendency of ADC drugs. Therefore, the DAR value is a key quality attribute for ADC drug analysis and an important quality control link in the ADC drug development process.
  • Fixed-site coupling technology can achieve fixed-site and quantitative coupling of antibodies and small molecule toxins.
  • the ADC obtained by this technology has a suitable drug-to-antibody ratio (DAR), high uniformity, good stability, high batch-to-batch reproducibility, better activity and pharmacokinetic properties, and is also more suitable for large-scale production of ADC.
  • ADC drugs use site-specific coupling methods to couple highly active small molecule drugs to antibodies to form site-specific DAR2 ADCs, such as the approved targeted CD19-ADC (loncastuximab tesirine, Zynlonta) that couples PBD small molecules to antibodies through glycosylation site-specific coupling technology to achieve site-specific DAR2, as well as targeted HER2 ADC (ARX788) and targeted FR ⁇ ADC (MORAb-202). All of them use the method of non-natural amino acid mutation to achieve site-specific DAR2.
  • these methods either require the use of enzymes or the modification of antibody sequences, and the manufacturing process is complicated.
  • WO2022253033A1 discloses the following method for preparing an ADC conjugate: dilute the antibody with a solution of disodium edetate, then adjust the pH with a Na 2 HPO 4 solution, add a TCEP (tri(2-carboxyethyl)phosphine) solution and mix well, and leave at room temperature for 2 hours. Add 5-10 times the amount of the antibody to the above solution system, mix well, and let stand at room temperature for 20 hours. After completion, use a NAP-5 gel column (Cytiva) to replace the buffer with a 10mM histidine buffer solution at pH 6.0 to obtain an ADC product. According to sample measurement, the DAR of the ADC conjugate prepared by the above non-site-specific coupling method in this patent is 4, and there is no record of a DAR of 2.
  • WO2022078524A2 discloses a site-specific coupling method for antibody conjugates. After reducing and incubating the antibody with a variety of zinc complexes and TCEP, a payload/linker complex is introduced. Cysteine is then added to consume excess TCEP and excess payload. Free thiol groups in the protein are oxidized. Finally, the reaction mixture is purified using a desalting column or UF/DF or ion exchange chromatography. The results of the DAR analysis of the conjugates using HIC-HPLC showed that the proportion of conjugates with a DAR of 4 was about 60-70%, while the proportion of conjugates with a DAR of 2 was only 5-10%. It can be seen that the DAR of the antibody conjugates prepared by this patented method is mainly concentrated in 4, while the conjugate products with a DAR of 2 account for only a minority.
  • WO2020164561A1 prepares an antibody-drug conjugate (ADC) with improved homogeneity, and the coupling method used comprises the following steps:
  • a reducing agent e.g., tris(2-carboxyethyl)phosphine (TCEP)
  • TCEP tris(2-carboxyethyl)phosphine
  • a buffer system e.g., Hepes, histidine buffer, PBS, MES, etc.
  • transition metal ions e.g., Zn2+, etc.
  • a payload carrying a reactive group e.g., a maleimide-linked drug
  • an oxidant e.g., dehydroascorbic acid (DHAA)
  • the DAR value is the key quality attribute of ADC.
  • high DAR can bring more small molecule drugs to the target site and may have a better therapeutic effect
  • the design of high DAR will sacrifice the stability and uniformity of ADC drugs, which may cause the aggregation of antibodies, thereby affecting the efficacy of ADC drugs and causing greater toxic side effects.
  • the preparation of ADCs with high uniformity and/or purity of DAR2 can avoid the adverse effects of high DAR and can be beneficial to the stability and efficacy of ADC drugs.
  • the prior art can achieve fixed-point DAR2 through glycosylation fixed-point coupling technology. However, these methods either require the use of enzymes or the modification of antibody sequences, and the manufacturing process is complicated.
  • the chemical fixed-point coupling method currently reported for the preparation of ADC although the process is relatively simple, can only achieve ADC products with a main DAR of 4.
  • the present invention provides a novel method for preparing an antibody-drug conjugate, which obtains highly homogeneous antibody conjugate molecules through a specific chemical site-specific coupling method, and the DAR2 main peak accounts for more than 99%.
  • the method is simple to operate and is conducive to industrial amplification.
  • the prepared product has few impurities and high purity, which significantly improves the safety of medication and reduces production costs.
  • the anti-tumor drug prepared by the coupling method of the present invention has excellent anti-tumor effect and safety (including higher stability and less side effects).
  • the present invention provides a method for preparing an antibody-drug conjugate (ADC), the method comprising the following steps:
  • the metal in the metal salt or metal complex described in step (a) can be selected from Zn, Cd, Hg, and the like.
  • the metal salt or metal complex in step (a) is selected from one or more of the following: hydrochloride or sulfate of Zn, Cd and Hg,
  • the Zn salts or Zn complexes disclosed in WO2022078524A2 include but are not limited to
  • the metal salt or metal complex in step (a) is selected from ZnCl 2 , CdCl 2 , HgCl 2 , One or more of .
  • the reducing agent in step (a) is selected from tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT), mercaptoethylamine and mercaptoethanol, preferably TCEP.
  • TCEP tris(2-carboxyethyl)phosphine
  • DTT dithiothreitol
  • mercaptoethylamine mercaptoethanol
  • the concentration of the antibody in step (a) is 0.5-150 mg/mL, such as 1-75 mg/mL, 1-50 mg/mL, 2-40 mg/mL, 3-30 mg/mL, 4-30 mg/mL or 5-15 mg/mL, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120 or 150 mg/mL.
  • the amount of the reducing agent used in step (a) is 1-6 times the antibody molar equivalent, such as 2-6, 3-5, 3.5-5.0 or 4.0-4.5 times the antibody molar equivalent, such as 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5 times the antibody molar equivalent.
  • the amount of the metal salt or metal complex used in step (a) is 1-5 times the antibody molar equivalent, such as 1-4, 1-3 or 1.5-2.5 times the antibody molar equivalent, such as 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5 times the antibody molar equivalent.
  • the buffer solution in step (a) has a pH of 5-8, such as a pH of 5.5-7.5, 6.5-7.5 or 6.5-7.4, such as a pH of 5.0, 5.5, 6.0, 6.5, 6.9, 7.0, 7.5 or 8.0, preferably a pH of 6.9.
  • the buffer solution in step (a) is selected from phosphate buffer, acetate buffer, citrate buffer, succinate buffer, preferably disodium hydrogen phosphate-sodium dihydrogen phosphate buffer.
  • the buffer concentration in the buffer solution is 5-100mM, such as 10-90mM, 20-80mM, 30-70mM, 40-60mM, 10-30 or mM, such as 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100mM.
  • the incubation in step (a) is performed at 0-30°C, such as 0-15°C or 5-15°C.
  • the incubation in step (a) is performed for 1-48 hours, such as 2-24 hours, 2-12 hours, 3-10 hours, 4-8 hours or 5-24 hours, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or 35 hours.
  • the amount of the drug-containing linker in step (b) is 1-8 times the antibody molar equivalent, such as 2-6, 2.5-5.5, 3-5 or 3.5-4.5 times the antibody molar equivalent, such as 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7 or 7.5 times the antibody molar equivalent.
  • the coupling reaction temperature in step (b) is 0-30°C, such as 0-15°C or 3-15°C.
  • the reaction solution in step (b) has a pH of 5-8, such as a pH of 5.5-7.5, 6.5-7.5, or 6.5-7.4, such as a pH of 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, or 8.0.
  • the drug-containing linker is provided as a DMA solution.
  • the coupling reaction of step (b) is carried out for 0.2-24 hours, such as 0.5-12 hours, 1-10 hours, 1.5-5 hours, 1-3 hours or 1-4 hours, such as 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9 or 10 hours. Hour.
  • the hydrophobic gel filler in step (c) is selected from Butyl-650M, Butyl-4FF, Butyl-650S, Butyl-ImpRes and Phenyl Sepharose HP.
  • the type of salt in step (c) is selected from sulfates, chlorides, etc., such as alkali metal (such as lithium, sodium, potassium) salts of sulfuric acid or hydrochloric acid, alkaline earth metal (such as calcium, magnesium) salts, salts formed by amines or ammonia and sulfuric acid or hydrochloric acid, preferably ammonium sulfate and sodium chloride; preferably, the salt concentration is 0.1-5 mol/L, such as 0.2-4 mol/L, 0.3-3 mol/L, 0.5-2 mol/L, 0.5-1.5 mol/L, 0.6-1.2 mol/L or 0.6-1.0 mol/L, such as 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.5 or 2.0 mol/L.
  • alkali metal such as lithium, sodium, potassium
  • alkaline earth metal such as calcium, magnesium
  • salt concentration is 0.1-5 mol/L, such as 0.2-4 mol
  • the salt solution described in step (c) is used as mobile phase A for hydrophobic interaction chromatography.
  • the hydrophobic interaction chromatography in step (c) uses phosphate buffer as mobile phase B, preferably, the phosphate buffer has a phosphate concentration of 5-200 mM and a pH of 7.0-7.6, such as 7.1, 7.2, 7.3, 7.4 or 7.5.
  • mobile phase A and/or mobile phase B may further contain other solvents such as isopropanol.
  • step (b) is followed by step (b'): adding a metal ion chelator such as ethylenediaminetetraacetic acid (EDTA) to react.
  • a metal ion chelator such as ethylenediaminetetraacetic acid (EDTA)
  • step (b') is performed at room temperature, e.g., for 5 min-5 hours, 10 min-45 min, or 30 min.
  • the amount of metal ion chelator used is 1-8 times the antibody molar equivalent, such as 2-6, 2.5-5.5, 3-5 or 3.5-4.5 times the antibody molar equivalent, such as 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7 or 7.5 times the antibody molar equivalent.
  • step (b') is followed by step (b"): adding dehydroascorbic acid (DHAA) to react.
  • DHAA dehydroascorbic acid
  • step (b") is performed at 25-35°C, for example 0.2-24 hours, for example 0.5-12 hours, 1-10 hours, 1.5-5 hours, 1-3 hours or 1-4 hours, for example 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9 or 10 hours.
  • the amount of DHAA used is 1-16 times the antibody molar equivalent, such as 2-10, 2-6, 6-16 or 6-12 times the antibody molar equivalent, such as 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 14 or 16 times the antibody molar equivalent.
  • the DAR2-ADC obtained by the method of the present invention has a purity greater than 90%, such as greater than 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5%, as measured, for example, by HIC-HPLC.
  • the antibody is selected from the group consisting of a humanized antibody, a murine antibody, a human antibody, a chimeric antibody, a single chain antibody, and a bispecific antibody.
  • the antibody is a monoclonal antibody or a polyclonal antibody.
  • the antibody is an antibody that binds to the following antigens: HER2, B7H3, HER3, CD19, CD20, CD22, CD30, CD33, CD37, CD45, CD56, CD66e, CD70, CD74, CD73, CD79b, CD138, CD147, CD223, EpCAM, Mucin 1, STEAP1, GPNMB, FGF2, FOLR1, EGFR, EGFRvIII, tissue factor, c-MET, FGFR, Nectin 4, AGS-16, Guanylyl cyclase C, Mesothelin, SLC44A4, PSMA, EphA2, AGS-5, GPC-3, c-KIT, RoR1, PD-L1, CD27L, 5T4, Mucin16, NaPi2b, STEAP, SLITRK6, ETBR, BCMA, Trop-2, CEACAM5, SC-16, SLC39A6, Delta-like protein3, Claudin 18.2.
  • the antibody is an anti-HER2 antibody.
  • the antibody comprises one or more CDRs (preferably 3 CDRs, i.e., HCDR1, HCDR2H and HCDR3; or LCDR1, LCDR2 and LCDR3, more preferably 6 CDRs, i.e., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3) of adalimumab, bevacizumab, cetuximab, trastuzumab, pertuzumab, nimotuzumab, rituximab, h23-12 or hH2L1, or comprises the VH and/or VL of the antibody, or comprises the heavy chain and/or light chain of the antibody.
  • CDRs preferably 3 CDRs, i.e., HCDR1, HCDR2H and HCDR3; or LCDR1, LCDR2 and LCDR3, more preferably 6 CDRs, i.e., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and
  • the antibody is selected from adalimumab, bevacizumab, cetuximab, trastuzumab, pertuzumab, nimotuzumab, rituximab, h23-12, and hH2L1.
  • the antibody comprises one or more CDRs (preferably 3 CDRs, i.e., HCDR1, HCDR2H and HCDR3; or LCDR1, LCDR2 and LCDR3, more preferably 6 CDRs, i.e., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3) of trastuzumab, Pertuzumab, h23-12 or hH2L1, or comprises the VH and/or VL of trastuzumab, Pertuzumab, h23-12 or hH2L1, or comprises the heavy chain and/or light chain of the antibody.
  • CDRs preferably 3 CDRs, i.e., HCDR1, HCDR2H and HCDR3; or LCDR1, LCDR2 and LCDR3, more preferably 6 CDRs, i.e., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3
  • trastuzumab preferably 3 CDRs
  • the antibody is selected from trastuzumab, pertuzumab, h23-12, and hH2L1.
  • the drug-containing linker in step (b) is a compound represented by "L 1 -AL 2 -D" or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof.
  • L1 represents a linker unit for connecting to an antibody
  • A represents one or more (e.g. 1, 2, 3 or 4) amino acids
  • L2 represents a linker unit for connecting to a small molecule drug
  • D is a small molecule drug.
  • the drug-containing linker in step (b) is a compound represented by Formula AI or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof,
  • E is selected from the following groups, wherein The bond of represents the attachment site to M:
  • n is an integer selected from 1-10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • R 6 and R 7 are independently halogen or Ar'S-
  • Ar' is phenyl or phenyl substituted by one or more substituents, in the substituted phenyl, the substituents are selected from alkyl (e.g. C1-C6 alkyl, preferably C1-C4 alkyl), alkoxy (e.g. C1-C6 alkoxy, preferably C1-C4 alkoxy, preferably methoxy), 5-6 membered heterocyclic group -CO-, halogen, ester group, amide group and cyano group.
  • Ar' is phenyl, 4-methylcarbamoylphenyl or 4-formylmorpholine substituted phenyl
  • M is a phenylene group or a phenylene group substituted by one or more substituents, or a chemical bond; in the substituted phenylene group, the substituent group is selected from an alkyl group (e.g., a C1-C6 alkyl group, preferably a C1-C4 alkyl group), a haloalkyl group (e.g., a halo C1-C6 alkyl group, preferably a halo C1-C4 alkyl group, such as a trifluoromethyl group), an alkoxy group (e.g., a C1-C6 alkoxy group, preferably a C1-C4 alkoxy group, preferably a methoxy group), a halogen group, an ester group, an amide group and a cyano group; preferably, M is a halogen-substituted phenylene group.
  • an alkyl group e.g., a C1
  • SP 1 is selected from C1-8 alkylene, C1-8 alkylene phenylene-, C1-8 cycloalkylene or C1-21 (preferably C1-16, more preferably C1-11) straight chain heteroalkylene, the C1-21 straight chain heteroalkylene containing 1-11 (preferably 1-6) heteroatoms selected from N, O or S, wherein the C1-8 alkylene, C1-8 cycloalkylene and C1-21 straight chain heteroalkylene are each independently optionally substituted by one or more substituents selected from hydroxy, oxo, amino, sulfonic acid and cyano;
  • SP 2 is selected from -NH(CH2CH2O)aCH2CH2CO-, -NH(CH2CH2O)aCH2CO-, -S(CH2)aCO- or a chemical bond, wherein a is an integer of 1-20, preferably an integer of 1-10, more preferably an integer of 1-6;
  • A represents 2 to 4 amino acids, wherein when A represents 2 amino acids, it can be NH-Phe-Lys-CO, NH-Val-Ala-CO, NH-Val-Lys-CO, NH-Ala-Lys-CO, NH-Val-Cit-CO, NH-Phe-Cit-CO, NH-Leu-Cit-CO, NH-Phe-Arg-CO or NH-Gly-Val-CO, preferably NH-Phe-Lys-CO, NH-Val-Ala-CO or NH-Val-Cit-CO.
  • A when A represents 3 amino acids, it can be NH-Glu-Val-Ala-CO, NH-Glu-Val-Cit-CO or NH-Ala-Ala-Ala-CO, preferably NH-Glu-Val-Ala-CO or NH-Ala-Ala-Ala-CO; when A represents 4 amino acids, it can be NH-Gly-Gly-Phe-Gly-CO or NH-Gly-Phe-Gly-Gly-CO, preferably NH-Gly-Gly-Phe-Gly-CO.
  • A is NH-Val-Ala-CO, NH-Gly-Gly-Phe-Gly-CO or NH-Ala-Ala-Ala-CO;
  • A is selected from the group consisting of NH-Val-Cit-CO, NH-Val-Ala-CO and NH-Gly-Gly-Phe-Gly-CO;
  • NH on the left side of A is the amino group in the left amino acid in A
  • CO on the right side of A is the carbonyl group in the right amino acid in A
  • L 2 is selected from In some embodiments, M is selected from
  • q is an integer selected from 1-10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • p is an integer selected from 1-20, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20;
  • D is selected from the group consisting of camptothecins, calicheamicins, maytansinoids, dolastatins, auristatins, and trichothecenes.
  • the maytansinoid derivative is selected from DM1, DM3 and DM4; the auristatin compound is selected from MMAE and MMAF; the camptothecin compound is selected from camptothecin, 10-hydroxycamptothecin, exatecan, SN-38, topotecan and camptothecin derivatives.
  • the drug-containing linker is a compound of Formula III or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof.
  • E, M, SP 1 , SP 2 and A are as defined above, and CPT is a camptothecin compound.
  • the drug-containing linker is a compound represented by Formula III' or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof,
  • E, M, SP 1 , SP 2 and A are as defined above, and D' is an auristatin compound.
  • the compound of formula III is further a compound of formula IIIA:
  • R 6 and R 7 are independently halogen or Ar'S-
  • Ar' is phenyl or phenyl substituted by one or more substituents, in the substituted phenyl, the substituent is selected from alkyl (e.g. C1-C6 alkyl, preferably C1-C4 alkyl), alkoxy (e.g. C1-C6 alkoxy, preferably C1-C4 alkoxy, preferably methoxy), 5-6 membered heterocyclic group -CO-, halogen, ester group, amide group and cyano group.
  • Ar' is phenyl, 4-methylcarbamoylphenyl or 4-morpholinoformylphenyl wherein M, SP 1 , SP 2 , A and CPT are as defined above.
  • the compound of formula III' is further a compound of formula IIIA':
  • R 6 , R 7 , M, SP 1 , SP 2 , A and D′ are as defined above.
  • CPT is a compound as shown in Formula I or Formula IA below, or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, and the corresponding specific compound:
  • R 1 , R 2 , R 3 , R 4 are independently hydrogen, halogen, Hydroxyl, C1-6 alkoxy, amino or substituted amino, C1-7 alkyl or substituted C1-7 alkyl, or any two of R 1 , R 2 , R 3 , R 4 together with the carbon atoms to which they are connected constitute a C3-6 cyclic alkyl.
  • R 1 , R 2 , R 3 , R 4 are independently C1-6 alkoxy
  • the C1-6 alkoxy includes a straight or branched C1-6 alkoxy, preferably a straight or branched C1-3 alkoxy, more preferably a methoxy.
  • R 1 , R 2 , R 3 , R 4 are independently substituted amino
  • the substituted amino is an amino substituted by one or more substituents selected from methyl and ethyl.
  • R 1 , R 2 , R 3 , and R 4 are independently C1-7 alkyl or substituted C1-7 alkyl
  • the C1-7 alkyl or substituted C1-7 alkyl includes linear or branched C1-7 alkyl or substituted C1-7 alkyl
  • the substituted C1-7 alkyl is C1-7 alkyl substituted by one or more substituents selected from cyclopropyl and cyclobutyl; or, the linear or branched C1-7 alkyl or substituted C1-7 alkyl is preferably C1-3 alkyl or substituted C1-3 alkyl, such as methyl, halogenated methyl (preferably trifluoromethyl).
  • G is hydrogen, halogen, methyl or methoxy.
  • G is hydrogen, fluorine or chlorine.
  • Y is oxygen, sulfur, sulfone, sulfoxide, methylene or substituted methylene.
  • the substituted methylene may be a methylene in which one hydrogen is substituted or two hydrogens are substituted simultaneously, and the substituent may be a benzyl or an alkyl group; when the substituent is an alkyl group, the alkyl group and R 3 and/or R 4 and the carbon atoms to which they are attached may form a C3-6-membered cyclic or spirocyclic structure.
  • Y is a substituted methylene group
  • the substituent of the substituted methylene group is preferably an alkyl group, more preferably a linear or branched C1-4 alkyl group.
  • Y is oxygen, sulfur, sulfone or sulfoxide; or, preferably, Y is oxygen, sulfur or methylene.
  • X is oxygen or sulfur
  • n 0 or 1.
  • R 1 , R 2 , R 3 , and R 4 are independently hydrogen, halogen (e.g., fluorine), C1-7 alkyl, or substituted C1-7 alkyl, or any two of R 1 , R 2 , R 3 , and R 4 together with the carbon atoms to which they are attached constitute a C3-6 cyclic alkyl (e.g., a C3-5 cyclic alkyl).
  • R 1 and R 2 may be the same; and/or, R 3 and R 4 may be the same.
  • Y is a methylene group substituted by an alkyl group, and the alkyl group, R3 and/or R4, and the carbon atoms to which they are connected can form a C3-6-membered cyclic or spirocyclic structure.
  • X may be oxygen
  • X is oxygen
  • G is hydrogen
  • halogen eg fluorine or chlorine
  • Y and R 1 , R 2 , R 3 , R 4 are as defined above.
  • X is oxygen
  • G is fluorine
  • Y is methylene or substituted methylene, oxygen or sulfur
  • R 1 , R 2 , R 3 and R 4 are as defined above.
  • X is oxygen
  • G is chlorine
  • Y is methylene or substituted methylene, oxygen or sulfur
  • R 1 , R 2 , R 3 and R 4 are as defined above.
  • X is oxygen
  • G is methyl
  • Y is methylene or substituted methylene, oxygen or sulfur
  • R 1 , R 2 , R 3 and R 4 are as defined above.
  • X is oxygen
  • G is methoxy
  • Y is methylene or substituted methylene, oxygen or sulfur
  • R 1 , R 2 , R 3 and R 4 are as defined above.
  • X is oxygen
  • G is hydrogen
  • Y is oxygen, sulfone or sulfoxide
  • R 1 , R 2 , R 3 , R 4 are as defined above.
  • X is oxygen
  • G is hydrogen
  • Y is sulfone or sulfoxide
  • R 1 , R 2 , R 3 , R 4 are as defined above.
  • the compound represented by formula I is further represented by formula IA:
  • R 1 , R 2 , R 3 , and R 4 are the same as defined in formula I above.
  • R 1 , R 2 , R 3 , and R 4 may be hydrogen at the same time or not at the same time.
  • the camptothecin compound is a compound represented by Formula II or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof:
  • R 5 is C1-5 alkyl or C1-5 alkyl substituted by one or more substituents, C3-6 cyclic alkyl or C3-6 cyclic alkyl substituted by one or more substituents, phenyl or substituted phenyl.
  • R 5 is C1-5 alkyl or substituted C1-5 alkyl
  • the C1-5 alkyl includes straight or branched C1-5 alkyl.
  • R 5 is C1-4 straight chain alkyl.
  • R 5 When R 5 is substituted C1-5 alkyl or substituted C3-6 cyclic alkyl, the substituent is selected from halogen, hydroxyl, methoxy, trifluoromethyl, amino or substituted amino, mesyl and C3-6 cyclic alkyl; and wherein the substituted amino is an amino substituted by one or more substituents selected from methyl and ethyl.
  • R 5 When R 5 is substituted phenyl, the substituent is selected from alkyl (e.g. C1-6 alkyl, preferably C1-3) or halogen.
  • G is hydrogen, halogen (eg fluorine), methyl or methoxy.
  • G is hydrogen, fluorine or chlorine.
  • X is oxygen or sulfur.
  • n 0 or 1.
  • the compound represented by formula II is further represented by formula IIA:
  • R 5 is the same as the definition of the group R 5 in Formula II above.
  • R 5 may not be n-butyl.
  • R 5 may be n-butyl.
  • camptothecin compound (CPT) has the following structure:
  • the compound represented by Formula I or Formula IA is connected to the carboxyl group of A through an amide bond via its amino group (see Formula I or Formula IA, respectively), that is, the amino group of the compound represented by Formula I or Formula IA forms an amide bond with the carboxyl group of A in Formula III or IIIA.
  • the compound represented by formula II or formula IIA is connected to the carboxyl group of A through an amide bond via its amino group (see formula II or formula IIA, respectively), that is, the amino group of the compound represented by formula II or formula IIA forms an amide bond with the carboxyl group of A in formula III or IIIA.
  • CPT pharmaceutically acceptable salts, stereoisomers, solvates, or prodrugs thereof set forth above
  • CPT pharmaceutically acceptable salts, stereoisomers, solvates, or prodrugs thereof set forth above
  • the amino group is linked to the carboxyl group of A in formula III or IIIA via an amide bond.
  • CPT in Formula III and Formula IIIA, can be an exteacan derivative shown in Formula IV or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof:
  • R 8 is hydrogen, trifluoromethyl, C 1-5 alkyl or C 1-5 alkyl substituted by one or more substituents, C 3-6 cyclic alkyl or C 3-6 cyclic alkyl substituted by one or more substituents, or halogen.
  • R8 is substituted C1-5 alkyl or substituted C3-6 cyclic alkyl
  • the substituent is selected from halogen, hydroxy, methoxy, trifluoromethyl, amino or substituted amino, methylsulfonyl and C3-6 cyclic alkyl; and wherein the substituted amino is an amino substituted by one or more substituents selected from methyl and ethyl.
  • the compound represented by formula IV is connected to the carboxyl group of A through a self-releasing structure via its hydroxyl group connected to the same carbon as R 8 (see formula IV), and the self-releasing structure is, for example
  • the solid line indicates the site of attachment to the carboxyl group of A in Formula III or Formula IIIA, and the wavy line indicates the site of attachment to the hydroxyl group in Formula IV.
  • M is preferably a phenylene or substituted phenylene
  • SP1 is a C1-21 (preferably C1-16, more preferably C1-11) straight chain heteroalkylene, wherein the straight chain heteroalkylene contains 1-11 (preferably 1-6) heteroatoms selected from N, O or S.
  • D' is a compound as shown in the following Formula I' or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, and the corresponding specific compound:
  • Ra1 , Ra2 , Ra3 , Ra4 , Ra5 and Ra8 are each independently selected from C1-8 alkyl; preferably C1-4 alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or sec-butyl;
  • R a6 and R a7 are each independently selected from C 1-8 alkoxy, such as methoxy, ethoxy or propoxy;
  • R a9 is selected from C 1-8 alkyl and COOH; preferably C 1-4 alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, Isobutyl or sec-butyl;
  • R a10 is selected from OH and H.
  • the compound of formula I' is further a compound of formula IA':
  • R a1 -R a10 are as defined above;
  • the compound of Formula I' is selected from:
  • the compound represented by formula I’ is connected to the carboxyl group of A via an amide bond via its amino group, that is, the left secondary amino group of the compound represented by formula I’ forms an amide bond with the carboxyl group of A in formula III’ or IIIA’.
  • the compound represented by Formula III and Formula IIIA is further represented by Formula V.
  • R 6 and R 7 are independently Ar'S
  • Ar' is phenyl or phenyl substituted by one or more substituents, in the substituted phenyl, the substituent is selected from alkyl (e.g. C1-C6 alkyl, preferably C1-C4 alkyl), alkoxy (e.g. C1-C6 alkoxy, preferably C1-C4 alkoxy, preferably methoxy), 5-6 membered heterocyclic group -CO-, halogen, ester group, amide group and cyano group.
  • Ar' is phenyl, 4-methylcarbamoylphenyl or 4-morpholinoformylphenyl
  • Xh and Yh are independently hydrogen, halogen, haloalkyl (e.g. haloC1-C6 alkyl, preferably haloC1-C4 alkyl, such as trifluoromethyl) or alkoxy (e.g. C1-C6 alkoxy, preferably C1-C4 alkoxy, such as methoxy).
  • haloalkyl e.g. haloC1-C6 alkyl, preferably haloC1-C4 alkyl, such as trifluoromethyl
  • alkoxy e.g. C1-C6 alkoxy, preferably C1-C4 alkoxy, such as methoxy
  • m is any integer from 1 to 10, preferably from 1 to 5, more preferably from 3 to 5.
  • A represents 2 to 4 amino acids, as defined above.
  • the compound of formula IIIA' is further a compound of formula V',
  • R 6 , R 7 , Xh, Yh, m and A are as defined above (especially in formula V), and D′ is as defined above.
  • the compound of formula V' is further a compound of formula V-A',
  • the compound of formula V is further a compound of formula VA:
  • G is hydrogen, fluorine or chlorine.
  • Y is methylene, sulfur or oxygen.
  • the compound represented by Formula VA is further represented by Formula VA-1:
  • the compound represented by formula VB is further represented by formula VB-1:
  • A represents 2 to 4 amino acids, as defined above.
  • CPT is a camptothecin compound.
  • the definition of CPT and its connection relationship in Formula VI are the same as those of CPT in Formula III and Formula IIIA, as defined above.
  • the compound represented by Formula VI provided by the present invention is further represented by Formula VI-A:
  • G is hydrogen, fluorine or chlorine.
  • Y is methylene, sulfur or oxygen.
  • the compound represented by formula VI-A is further represented by formula VI-A-1:
  • formula VC can be further structured as shown in formula VI-C.
  • the drug-containing linker is selected from the following structures:
  • the present invention also relates to the specific methods disclosed in the embodiments of the present invention.
  • the technical solutions obtained by replacing or combining certain technical features in the specific embodiments of the present invention with the technical features in the above-mentioned embodiments are also considered in the present invention. middle.
  • Some small molecule drugs, drug-containing linkers and/or intermediates thereof, and/or antibody-drug conjugates in the present invention are recorded in Chinese patent application 202211627164.8, and the entire content of the patent application is introduced into this application as part of the content of this application.
  • the steps of the above preparation method are interrelated and interact with each other to produce a technical effect of improving the purity of the DAR2-ADC conjugate, and this technical effect is produced by the steps as a whole.
  • the present invention provides an antibody drug conjugate having the general formula The structure shown, wherein mAb represents an antibody or antibody fragment, such as the antibody defined above, and groups M, SP1 , SP2 , A, L2 and D are as defined above in the first aspect.
  • y is 1-10, preferably 1-8 (such as 1-5), more preferably 1-3, and most preferably 2.
  • EL is selected from the following groups, wherein, Indicates that it is linked to the cysteine in the mAb, and the other The key indicates connection with M:
  • n is as defined above in variable E.
  • mAb may be an IgG type antibody or a fragment thereof, preferably an IgG1 subtype antibody or a fragment thereof.
  • the antibody drug conjugate is prepared by the method of the present invention as described in the first aspect.
  • the antibody drug conjugate has an average DAR of about 2.
  • the antibody drug conjugate has a purity greater than 90%, such as greater than 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5%, such as measured by HIC-HPLC.
  • the antibody drug conjugate has the structure shown in the following general formula VII, VII', VIII, VIII':
  • y is 1-10, preferably 1-8 (eg, 1-5), and more preferably 1, 2 or 3.
  • the present invention provides the small molecule drugs, drug-containing linkers and/or intermediates thereof (including but not limited to formula A-I, I, I', I-A, IA', II, II-A, III, III', III-A, III-A', V, V-A, V-A-1, V-B, V-B-1, V-C, VI, VI-A, VI-A-1, VI-B, VI-B-1, VI-C) described in the preparation method of the first aspect of the present invention, preferably including but not limited to the compounds disclosed in the embodiments.
  • formula A-I, I, I', I-A, IA', II, II-A, III, III', III-A, III-A', V, V-A, V-A-1, V-B, V-B-1, V-C, VI, VI-A, VI-A-1, VI-B, VI-B-1, VI-C described in the preparation method of the first aspect of the present invention, preferably including but not limited to the compounds disclosed in
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the small molecule drug, drug-containing linker, intermediate and/or antibody-drug conjugate described in the first to third aspects of the present invention or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof.
  • the pharmaceutical composition is a liquid preparation or a lyophilized preparation.
  • the pharmaceutical composition comprises a buffer, a stabilizer, and a surfactant.
  • the present invention provides use of a compound according to the present invention or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, an antibody-drug conjugate or a pharmaceutical composition thereof in the preparation of a medicament for treating a tumor.
  • the tumor is cancer.
  • the tumor is associated with tumor-associated antigens such as HER2, B7H3, HER3, CD19, CD20, CD22, CD30, CD33, CD37, CD45, CD56, CD66e, CD70, CD74, CD73, CD79b, CD138, CD147, CD223, EpCAM, Mucin 1, STEAP1, GPNMB, FGF2, FOLR1, EGFR, EGFRvIII, Tissue factor, c-MET, FGFR, Nectin 4, AG It is associated with positive or high expression of S-16, guanylate cyclase C, mesothelin, SLC44A4, PSMA, EphA2, AGS-5, GPC-3, c-KIT, RoR1, PD-L1, CD27L, 5T4, Mucin16, NaPi2b, STEAP, SLITRK6, ETBR, BCMA, Trop-2, CEACAM5, SC-16, SLC39A6, Delta-like protein3, and C
  • the tumor is colorectal cancer, bladder cancer, breast cancer, pancreatic cancer, liver cancer, ovarian cancer, endometrial cancer, fallopian tube cancer, gastric cancer, prostate cancer, small cell lung cancer, non-small cell lung cancer, esophageal squamous cell carcinoma, head and neck squamous cell carcinoma, melanoma, leukemia, lymphoma, glioma, glioblastoma.
  • the present invention provides a method for treating tumors using a compound according to the present invention or a pharmaceutically acceptable salt, stereoisomer, solvate or prodrug thereof, an antibody-drug conjugate or a pharmaceutical composition thereof, the method comprising administering the compound or antibody-drug conjugate to a subject in need thereof.
  • the tumor is cancer.
  • the tumor is associated with tumor-associated antigens such as HER2, B7H3, HER3, CD19, CD20, CD22, CD30, CD33, CD37, CD45, CD56, CD66e, CD70, CD74, CD73, CD79b, CD138, CD147, CD223, EpCAM, Mucin 1, STEAP1, GPNMB, FGF2, FOLR1, EGFR, EGFRvIII, Tissue factor, c-MET, FGFR, Nectin 4, AG It is associated with positive or high expression of S-16, guanylate cyclase C, mesothelin, SLC44A4, PSMA, EphA2, AGS-5, GPC-3, c-KIT, RoR1, PD-L1, CD27L, 5T4, Mucin16, NaPi2b, STEAP, SLITRK6, ETBR, BCMA, Trop-2, CEACAM5, SC-16, SLC39A6, Delta-like protein3, and C
  • the tumor is colorectal cancer, bladder cancer, breast cancer, pancreatic cancer, liver cancer, ovarian cancer, endometrial cancer, fallopian tube cancer, gastric cancer, prostate cancer, small cell lung cancer, non-small cell lung cancer, esophageal squamous cell carcinoma, head and neck squamous cell carcinoma, melanoma, leukemia, lymphoma, glioma, glioblastoma.
  • the subject is a mammal, preferably a primate, more preferably a human.
  • the method and/or product of the present invention has the following advantages and/or benefits:
  • the method of the present invention can provide highly homogeneous antibody conjugate molecules, the main peak of which accounts for more than 99% of DAR2;
  • the preparation method of the present invention has few steps and is simple to operate, which is conducive to industrial scale-up production. At the same time, the prepared product has few impurities and high purity, which significantly improves the safety of drug use and reduces production costs; and/or
  • the anti-tumor drug prepared by the coupling method of the present invention has excellent anti-tumor effect and safety, including high stability. Qualitative and smaller side effects, expand the treatment window, improve treatment effects and reduce toxic reactions.
  • ADC antibody drug conjugate
  • the term "antibody drug conjugate” or "ADC” refers to a substance obtained by connecting a bioactive compound fragment (small molecule drug) to an antibody or its antigen-binding fragment.
  • the bioactive compound fragment is connected to the targeting portion through a linker.
  • the linker can be broken in a specific environment (e.g., a low pH environment in the cell) or under a specific action (e.g., the action of a lysosomal protease), thereby separating the bioactive compound fragment from the antibody or its antigen-binding fragment.
  • the linker comprises a cleavable or non-cleavable unit, such as a peptide or a disulfide bond.
  • the bioactive compound fragment is directly connected to the antibody or its antigen-binding fragment through a covalent bond, and the covalent bond can be broken under a specific environment or action, thereby separating the bioactive compound fragment from the antibody or its antigen-binding fragment.
  • linker refers to a fragment that connects a biologically active compound fragment (small molecule drug) to an antibody portion.
  • the linker has a functional group that can form a bond with a functional group of the antibody or its antigen-binding fragment before being attached to the antibody or its antigen-binding fragment (i.e., a linker precursor).
  • small molecule drug refers to a low molecular weight compound that can regulate biological processes.
  • Small molecule is defined as a molecule with a molecular weight less than 10kD, usually less than 2kD and preferably less than 10kD.
  • Small molecules include but are not limited to inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimics and antibody mimics. As therapeutic agents, small molecules can be more permeable to cells, less susceptible to degradation and less prone to eliciting immune responses than macromolecules.
  • Small molecule drugs include but are not limited to cytotoxic agents, such as camptothecin compounds, calicheamicin (calicheamicin), maytansinoids (maytansinoids), dolastatin (dolastatin), auristatin compounds and trichothecene (trichothecene).
  • cytotoxic agents such as camptothecin compounds, calicheamicin (calicheamicin), maytansinoids (maytansinoids), dolastatin (dolastatin), auristatin compounds and trichothecene (trichothecene).
  • the maytansinoid derivative is selected from DM1, DM3 and DM4;
  • the auristatin compound is selected from the auristatin compounds mentioned in the embodiments of the present invention, including but not limited to MMAE and MMAF;
  • the camptothecin compound is selected from camptothecin, 10-hydroxycamptothecin, exitecan, SN-38 and topotecan and the camptothecin compounds mentioned in the embodiments of the present invention.
  • a small molecule drug when a small molecule drug is a part of an ADC or a drug-containing linker, it refers to a structural fragment from a small molecule drug, such as a structural fragment formed by bonding a hydroxyl group or an amino group of a small molecule drug to a linker, which can form a part (fragment or radical) of a biologically active drug (such as a small molecule cytotoxic drug, wherein the drug includes a group after losing an atom or a group of atoms) or a derivative (such as a precursor thereof) after the linker is cleaved/degraded/enzymatically cleaved between tumor tissues or in tumor cells.
  • a biologically active drug such as a small molecule cytotoxic drug, wherein the drug includes a group after losing an atom or a group of atoms
  • a derivative such as a precursor thereof
  • drug-containing linker refers to a compound formed by linking a bioactive compound fragment (small molecule drug) to a linker, which is sometimes referred to herein as a "drug-linker conjugate”.
  • Metal ion chelators are compounds that, through their strong binding with metal ions, encapsulate metal ions into the interior of the chelator, turning them into stable compounds with larger molecular weight, thereby preventing the metal ions from taking effect.
  • Metal ion chelators include but are not limited to ethylenediaminetetraacetic acid,
  • composition refers to a composition that is in a form that permits the active ingredient contained therein to be effective for biological activity, and that contains no additional ingredients that are unacceptably toxic to a subject to which the composition would be administered.
  • prevention includes inhibition of the occurrence or development of a disease or disorder or symptoms of a particular disease or disorder.
  • subjects with a family history of cancer are candidates for preventive regimens.
  • prevention refers to the administration of a drug before the signs or symptoms of cancer occur, particularly in a subject at risk for cancer.
  • alkyl refers to a straight chain, branched, fully saturated hydrocarbon group, preferably C1 - C10 , more preferably C1 - C8 , C1 - C6 , or C1 - C4 alkyl.
  • alkyl groups are methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl or n-decyl, and the like.
  • alkylene refers to a divalent group formed by removing a hydrogen atom from an alkyl group as defined above.
  • cycloalkyl refers to a cyclic alkyl group, preferably a C3 - C15 cycloalkyl, such as a C3 - C8 cycloalkyl, a C3 - C6 cycloalkyl.
  • Examples of cycloalkyl are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexyl or cycloheptyl and the like.
  • cycloalkylene refers to a divalent group formed by removing a hydrogen atom from a cycloalkyl group as defined above.
  • heteroalkylene refers to an alkyl group as defined above, which contains one or more heteroatoms selected from N, O or S in the chain. It should be understood that heteroalkylene does not include a group formed by two or more N, O or S heteroatoms directly connected to each other.
  • heteroalkylene is a C1-21 (e.g., C1-10, C1-6) straight chain heteroalkylene, which contains 1-11 (preferably 1-6, 1-3) heteroatoms selected from N, O or S.
  • heterocyclyl refers to a partially unsaturated or fully unsaturated 3-10-membered cyclic group, preferably a 3-10-membered cyclic group, more preferably a 3-6-membered cyclic group, and more preferably a 5-6-membered cyclic group, which may be optionally substituted and contains one or more (e.g., 1, 2, 3 or 4 ) heteroatoms selected from N, O, S, SO, SO2 or P; the heterocyclyl group contains 1-9 carbon atoms, preferably 2-8 carbon atoms, and more preferably 3-5 carbon atoms.
  • heterocyclyl groups include: 1,3-dioxolanyl, 1,3-dioxanyl, 1,4-dioxanyl, imidazolinyl, indolinyl, morpholinyl, pyridone, 2-pyrrolidone, piperazinyl, homopiperazinyl, piperidinyl, pyrazinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydroquinolinyl, tetrahydrothienyl, oxanyl, oxathiolanyl, thianyl, and the like.
  • “Chemical bond” refers to a bond connecting two groups on both sides. For example, for the group -AL 2 -D, when L 2 is a chemical bond, the group can be written as -AD.
  • halogen refers to F, Cl, Br, I.
  • reducing agent refers to an agent capable of selectively reducing disulfide bonds.
  • the reducing agent is selected from but not limited to tris(2-carboxyethyl)phosphine (TCEP) (including tris(2-carboxyethyl)phosphine hydrochloride), dithiothreitol (DTT), mercaptoethylamine, mercaptoethanol, and combinations thereof.
  • TCEP tris(2-carboxyethyl)phosphine
  • DTT dithiothreitol
  • mercaptoethylamine mercaptoethanol
  • antibody molar equivalent refers to the molar equivalent relative to the antibody.
  • DAR drug to antibody ratio
  • D drug to antibody ratio
  • mAb antibody moiety
  • the DAR of an ADC can be in the range of 1 to 10, but higher loadings are possible depending on the number of attachment sites on the antibody.
  • DAR can be used when referring to the number of drugs loaded onto a single antibody.
  • the DAR can also be calculated as the average DAR of a population of molecules in a product, i.e., the overall ratio (molar ratio) of the small molecule drug moiety (D) coupled to the mAb moiety described herein to the Ab moiety in a product measured by a detection method (e.g., by conventional methods such as mass spectrometry, ELISA assay, electrophoresis and/or HPLC), which DAR is referred to herein as the average DAR.
  • a detection method e.g., by conventional methods such as mass spectrometry, ELISA assay, electrophoresis and/or HPLC
  • the average DAR value of the antibody drug conjugate of the invention is 1.0-10.0, such as 4.0-10.0, 5.0-9.0, 6.0-8.0, 1.0-8.0, 2.0-6.0, such as 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.
  • DAR2-ADC refers to an ADC with a DAR of 2, i.e., an ADC in which an antibody is linked to two small molecule drugs.
  • the term “comprising” or “including” means including the elements, integers or steps described, but does not exclude any other elements, integers or steps.
  • the term “comprising” or “including” when used, unless otherwise specified, it also covers the combination of the elements, integers or steps described.
  • an antibody variable region “comprising” a specific sequence when referring to an antibody variable region “comprising” a specific sequence, it is also intended to cover the antibody variable region consisting of the specific sequence.
  • the disclosure is further described below by describing specific embodiments, but this is not a limitation of the disclosure. Those skilled in the art can make various modifications or improvements based on the teachings of the disclosure without departing from the basic idea and scope of the disclosure.
  • the instruments or equipment used without indicating the source are all products that can be obtained commercially.
  • the reagents used without indicating the source are products that can be obtained commercially or synthesized by conventional methods.
  • MWC-1 was prepared using B-1 as an intermediate. It was a yellow solid.
  • LC-MS (ESI): [M+1]+ 404
  • the BL20E compound is prepared by the method disclosed in WO2018/095422A1 and the BL linker compound VC-PABC-MMAE is obtained by condensation, and the synthetic route is as follows:
  • the MWD-L1 compound is synthesized from the BL linker compound and GGFG-Dxd.
  • the synthesis route is as follows:
  • BL linker compound (857 mg, 1 mmol), GGFG-Dxd [synthesized according to the method described in the patent disclosure document (US20190151328A1)] (840 mg, 1 mmol, 1 eq), DIPEA (323 mg, 2.5 mmol, 2.5 eq), HATU (570 mg, 1.5 mmol, 1.5 eq), dissolved in 30 ml DCM, stirred for 2 h.
  • the reaction solution was cooled to 5-10 ° C, 1N hydrochloric acid (20 ml) was added, and stirred for 0.5 h.
  • the liquid was separated, the aqueous phase was extracted with DCM (30 ml * 2), and the organic phases were combined.
  • BL linker compound (857mg, 1mmol), HoSu (138mg, 1.2mmol, 1.2eq), DCC (310mg, 1.5mmol, 1.5eq) were dissolved in 30ml DCM, stirred at room temperature for 3h, and the reaction solution was filtered. The filtrate was the DCM solution of A-Osu. The filtrate was added to the mixed solution of the crude intermediate 2-2, DIPEA (323mg, 2.5mmol, 2.5eq) and DCM (30ml), and the reaction was stirred for 3h. The reaction solution was cooled to 10°C, 1N hydrochloric acid (20ml) was added, and stirred for 0.5h.
  • Boc-Gly-OH (175 mg, 1 mmol), A1 compound (176 mg, 1 mmol, 1 eq), DIPEA (322 mg, 2.5 mmol, 2.5 eq), HATU (456 mg, 1.2 mmol, 1.2 eq) were dissolved in 30 ml DCM and stirred for 2 h.
  • BL linker compound (318 mg, 0.37 mmol), HoSu (51 mg, 0.44 mmol, 1.2 eq), DCC (114 mg, 0.56 mmol, 1.5 eq) were dissolved in 30 ml DCM, stirred at room temperature for 3 h, and the reaction solution was filtered. The filtrate was added to a mixed solution of the crude intermediate 3-4, DIPEA (120 mg, 0.93 mmol, 2.5 eq), and DCM (30 ml), and the reaction was stirred for 3 h. The reaction solution was cooled to 10 ° C, 1N hydrochloric acid (20 ml) was added, and stirred for 0.5 h.
  • MWF-L6 was prepared by replacing compound A1 with compound A7.
  • the product was an orange-red solid.
  • LC-MS (ESI): M+1 1450.
  • compound A7-2 (2.45 g, 7.7 mmol, 1 eq) was dissolved in tetrahydrofuran (200 ml), and the reaction solution was cooled to -70 to -60 ° C with a dry ice-acetone bath, and KHMDS (1 M, 31 ml, 31.0 mmol, 4 eq) was slowly added dropwise. After the addition, the mixture was stirred at -70 to -60 ° C for 10 minutes; a tetrahydrofuran solution of NFSI (7.35 g, 23 mmol, 3 eq) was added dropwise.
  • DT-2-A compound was used to replace the BL linker compound to synthesize DT-2, which was a light yellow solid.
  • LC-MS (ESI): [M+1]+ 1339.7, wherein the DT-2-A compound was synthesized according to the literature Chem. Sci., 2022, 13, 8781–8790.
  • MC-VC-PAB-MMAE used in the examples was purchased from MCE.
  • the trastuzumab antibody solution was changed to 20mM sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer solution at pH 6.5-7.4 and diluted to 5-15mg/mL.
  • Different metal ion aqueous solutions and tris(2-carboxyethyl)phosphine hydrochloride (TCEP) aqueous solutions were added in sequence and reacted at 0-15°C overnight.
  • the reaction equivalents are shown in Table 1-1.
  • the concentration of the above reaction solution was adjusted to about 5-15 mg/mL, 8 times the antibody equivalent of dehydroascorbic acid (DHAA) was added, and the solution was changed to 50 mM disodium hydrogen phosphate-sodium dihydrogen phosphate buffer at pH 7.4 after reacting at 25-35°C for 2 hours.
  • the antibody drug conjugate product was obtained. Samples were taken for analysis, and the antibody coupling ratio (DAR) was determined. The test results are shown in Table 1-1.
  • the trastuzumab antibody was exchanged into a 20mM sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer at pH 6.5-7.4 and diluted to 5-15 mg/mL.
  • a ZnCl 2 aqueous solution of 2 times the antibody molar equivalent and a tris(2-carboxyethyl)phosphine hydrochloride (TCEP) aqueous solution of 4.0-4.5 times the antibody molar equivalent were added in sequence, and after overnight reaction at 0-15°C, the solution was exchanged into a 20mM sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer solution of different pH values.
  • TCEP tris(2-carboxyethyl)phosphine hydrochloride
  • a DMA solution of BL20E (C-3) of 3 times the antibody molar equivalent was added to the above reaction solution.
  • the coupling reaction temperature and the pH of the coupling solution are shown in Table 1-2.
  • the solution was exchanged into a 20mM sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer solution of pH 6.5-7.4.
  • the concentration of the above reaction solution was adjusted to about 5-15 mg/mL, 4 times the antibody equivalent of ethylenediaminetetraacetic acid (EDTA) was added, and the solution was changed to 20 mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution with a pH of 7.4-8.0 after reacting at room temperature for 30 minutes.
  • EDTA ethylenediaminetetraacetic acid
  • the concentration of the above reaction solution was adjusted to about 5-15 mg/mL, 8 times the antibody molar equivalent of dehydroascorbic acid (DHAA) was added, and the solution was changed to 50 mM sodium dihydrogen phosphate-disodium dihydrogen phosphate buffer with a pH of 7.4 after reacting at 25-35°C for 2 hours.
  • the antibody drug conjugate product was obtained. Samples were taken for analysis, and the antibody coupling ratio (DAR) and purity were determined. The test results are shown in Table 1-2.
  • the HIC peak value of DAR2-ADC can be achieved to be 61-78% by using the above coupling reaction temperature and coupling solution pH in the preparation method of the present application.
  • the trastuzumab antibody solution was changed to 20 mM sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer at pH 6.5-7.4 and diluted to 5-15 mg/mL.
  • a 2-fold antibody molar equivalent of ZnCl 2 aqueous solution and a 4.0-4.5-fold antibody molar equivalent of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) aqueous solution were added in sequence and reacted at 0-15°C overnight.
  • the concentration of the above reaction solution was adjusted to about 5-15 mg/mL, and 8 times the antibody equivalent of dehydroascorbic acid (DHAA) was added, and the reaction was carried out at 25-35°C for 2 hours. After the reaction was completed, the solution was changed to a 50mM sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution with a pH of 7.5, diluted to about 5-20 mg/mL, and ammonium sulfate solution was added to adjust the conductivity.
  • the antibody drug conjugate product was purified by hydrophobic interaction chromatography (HIC). The purification conditions are shown in Table 1-3. The purified product was sampled for HIC analysis. The HIC test results are shown in Table 1-3.
  • the antibody solution was changed to 20mM sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer at pH 6.5-7.4 and diluted to 5-15mg/mL.
  • a ZnCl 2 aqueous solution with a molar equivalent of 2 times the antibody and a tris(2-carboxyethyl)phosphine hydrochloride (TCEP) aqueous solution with a molar equivalent of 4.0-4.5 times the antibody were added in sequence, and reacted at 0-15°C overnight.
  • TCEP tris(2-carboxyethyl)phosphine hydrochloride
  • a DMA solution containing a drug linker with a molar equivalent of 2.5-5.5 times the antibody was added to the above reaction solution, and the solution was changed to 20mM sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer solution with a pH of 6.5-7.4 after reacting at 0-15°C for 2h.
  • the concentration of the above reaction solution was adjusted to about 5-15mg/mL, and ethylenediaminetetraacetic acid (EDTA) with a molar equivalent of 4 times the antibody was added.
  • EDTA ethylenediaminetetraacetic acid
  • antibody h23-12 was prepared according to the method described in patent WO2021068949A1
  • antibody hH2L1 was prepared according to the method described in patent CN113527486A.
  • reaction mixture was purified using a desalting column (Model: 40K, 0.5 mL, REF: 87766, Lot: SJ251704, Thermo).
  • Sample injection analysis For samples with a concentration exceeding 5 mg/mL, dilute the sample with 50% phase A solution, filter through a 0.2 ⁇ m filter, and then perform sample injection analysis; for samples with a concentration of 5 mg/mL or less, filter directly before sample injection analysis.
  • the method was determined according to the monoclonal antibody molecular size variant determination method in General Chapter 3127 of the Chinese Pharmacopoeia Volume IV.
  • This experiment used human gastric cancer cells NCI-N87 (purchased from ATCC). After adjusting the density of NCI-N87 cells to 15 ⁇ 10 4 with complete medium (RPMI 1640 medium 45ml, 5ml FBS, mixed before use), 100 ⁇ l/well was added to the cell culture plate and cultured overnight. On the second day, the ADC samples were diluted with the above complete medium to 50ug/ml, and then diluted 4 times in a gradient, with a total of 9 gradients plus zero point, and all samples were set up with 3 replicates. Set up negative control (cells + culture medium) and blank control (no cells, pure culture medium); add the above diluted ADC samples to the cell culture plate cultured overnight in sequence, 100 ⁇ l/well.
  • complete medium RPMI 1640 medium 45ml, 5ml FBS, mixed before use

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Abstract

La présente invention concerne un procédé de préparation d'un conjugué anticorps-médicament. Selon le procédé, une molécule conjuguée d'anticorps hautement homogène est obtenue au moyen d'un procédé de conjugaison dirigée par site chimique spécifique, et la proportion d'un pic principal de DAR2 de celle-ci atteint 99% ou plus. Le procédé de préparation de la présente invention présente peu d'étapes, et est facile à utiliser, et est ainsi bénéfique pour une production à l'échelle industrielle. De plus, le produit préparé présente peu d'impuretés et une pureté élevée, la sécurité du médicament est évidemment améliorée, et le coût de production est également réduit.
PCT/CN2024/099341 2023-06-14 2024-06-14 Procédé de préparation d'un conjugué anticorps-médicament Ceased WO2024255873A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105899235A (zh) * 2014-01-10 2016-08-24 斯索恩生物制药有限公司 用于纯化cys连接的抗体-药物缀合物的方法
CN106729743A (zh) * 2015-11-23 2017-05-31 四川科伦博泰生物医药股份有限公司 抗ErbB2抗体-药物偶联物及其组合物、制备方法和应用
CN109600993A (zh) * 2016-06-08 2019-04-09 艾伯维公司 抗egfr抗体药物偶联物
CN113423730A (zh) * 2019-02-15 2021-09-21 上海药明生物技术有限公司 用于制备具有改善的同质性的抗体-药物缀合物的方法
WO2022078524A2 (fr) * 2021-11-03 2022-04-21 Hangzhou Dac Biotech Co., Ltd. Conjugaison spécifique d'un anticorps

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105899235A (zh) * 2014-01-10 2016-08-24 斯索恩生物制药有限公司 用于纯化cys连接的抗体-药物缀合物的方法
CN106729743A (zh) * 2015-11-23 2017-05-31 四川科伦博泰生物医药股份有限公司 抗ErbB2抗体-药物偶联物及其组合物、制备方法和应用
CN109600993A (zh) * 2016-06-08 2019-04-09 艾伯维公司 抗egfr抗体药物偶联物
CN113423730A (zh) * 2019-02-15 2021-09-21 上海药明生物技术有限公司 用于制备具有改善的同质性的抗体-药物缀合物的方法
WO2022078524A2 (fr) * 2021-11-03 2022-04-21 Hangzhou Dac Biotech Co., Ltd. Conjugaison spécifique d'un anticorps

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