WO2021051720A1 - Classe de conjugué de médicament anticorps anti-egfr humain et son procédé de préparation et son application - Google Patents

Classe de conjugué de médicament anticorps anti-egfr humain et son procédé de préparation et son application Download PDF

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WO2021051720A1
WO2021051720A1 PCT/CN2020/000225 CN2020000225W WO2021051720A1 WO 2021051720 A1 WO2021051720 A1 WO 2021051720A1 CN 2020000225 W CN2020000225 W CN 2020000225W WO 2021051720 A1 WO2021051720 A1 WO 2021051720A1
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antibody
linker
mmae
human egfr
synthesis
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Chinese (zh)
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李卓荣
胡馨月
江海伦
刘睿
白炜琪
刘秀筠
苗庆芳
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中国医学科学院医药生物技术研究所
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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
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6817—Toxins
    • 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
    • A61K38/08—Peptides having 5 to 11 amino acids
    • 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
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6873—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting an immunoglobulin; the antibody being an anti-idiotypic antibody
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to an anti-human epidermal growth factor receptor (EGFR) antibody drug conjugate (ADC), including a substance using the ADC, a preparation method and its application in the field of solid tumor treatment.
  • ADC anti-human epidermal growth factor receptor
  • the invention belongs to the field of biotechnology medicine.
  • EGFR epidermal growth factor receptor
  • EGFR epidermal growth factor receptor
  • Anti-EGFR antibody is the main targeted therapy, and its mechanism of action is mainly to compete with endogenous ligands to bind EGFR, inhibit the activation of tyrosine kinase, and promote the internalization of EGFR to produce anti-tumor effects.
  • antibody drug research has its own unique advantages and good market prospects, the curative effects of existing EGFR drugs are limited. In most cases targeting EGFR, long-term use leads to drug resistance.
  • ADC Antibody-drug conjugate
  • EGFR-ADC also has certain challenges. Among them, the two ADCs that target EGFR, IMGN-289 and AMG-595 are also due to toxicity (such as skin toxicity, gastrointestinal toxicity, etc.) or poor efficacy. The clinical research was forced to terminate, so the research opportunities and challenges of ADC targeting EGFR coexist.
  • ADC generally consists of three parts: antibody, warhead and linker.
  • SCT-200 antibody is a new type of fully human anti-EGFR monoclonal antibody targeted for the treatment of metastatic colorectal cancer (Patent Publication Number: CN101058609A). It is a high-affinity 100% human antibody obtained through screening of human phage library , Has an original sequence targeting EGFR.
  • the current research on the treatment of metastatic colorectal cancer and head and neck squamous cell carcinoma is in clinical phase II, and its clinical application may have better therapeutic effects and safety than chimeric antibodies and humanized antibodies.
  • the cytotoxicity of warhead molecules must be extremely high, and EC 50 is generally required to be less than 1 nmol/L.
  • MMAE is based on the active ingredient dolastatins 10 (dolastatins 10) in the natural marine product dolastatins extracted from the censored sea hare. It is a type of linear pentapeptide cytotoxic molecule and is used in a variety of human cancer cell lines. , IC 50 is 10 -9 ⁇ 10 -11 mol/L, which is 200 times the activity of vinblastine.
  • MMAE as an ADC warhead molecule has three main advantages: (1) high cytotoxicity; (2) N-terminal single methyl substitution, which can be connected to a linker; (3) bystander effect: it is especially beneficial for the treatment of solid tumors.
  • the linker connected to MMAE generally uses Val-Cit dipeptide type.
  • the combination with MMAE is called MC-VC-PABC-MMAE.
  • This combination is the most widely used Linker in ADC clinical trials.
  • Drug the listed ADC Brentuximab Vedotin (SGN-35, Adcetris) chose this kind of Linker-Drug combination to cleave it in cathepsin B, thereby completely releasing MMAE.
  • SGN-35 Adcetris
  • the p-aminobenzyloxycarbonyl spacer group is similar to an ester bond, and its stability in plasma needs to be further improved.
  • the present invention makes full use of the advantages of the SCT200 antibody, designs different types of linkers to connect with MMAE, and finally prepares a novel SCT200-Linker-MMAE antibody drug conjugate.
  • the prepared ADCs are comparable to SCT200 in terms of affinity and endocytosis, and can up-regulate the expression of tumor apoptosis-related proteins.
  • the ADCs of the present invention all exhibit stronger anti-tumor activity than SCT200.
  • the purpose of the present invention is to provide a type of anti-human EGFR antibody drug conjugate and its preparation method and anti-tumor application.
  • the anti-human EGFR antibody drug conjugate of the present invention has high efficiency targeting, high endocytosis activity, high affinity activity and high anti-tumor activity to EGFR-positive tumor tissues.
  • the present invention adopts the following technical means:
  • a type of anti-human EGFR antibody drug conjugate of the present invention has a structure shown in formula I:
  • M is the part of the spacer that can react with the cysteine sulfhydryl group of the antibody, and its structure is one of the following structures:
  • C is a part that can be hydrolyzed under the action of an enzyme, and is selected from the dipeptide sequence Val-Cit, Val-Ala, Val-Lys or Val-Arg; or the tripeptide sequence Ala-Val-Ala, Gly-Phe-Gly, Gly-Phe-Lys or Ala-Phe-Lys; or one of the tetrapeptide sequence Gly-Gly-Phe-Gly or Gly-Phe-Leu-Gly;
  • R is the self-decomposing part, and its structure is one of the following structures:
  • the antibody is SCT-200 monoclonal antibody.
  • the present invention also proposes a method for preparing the anti-human EGFR antibody conjugated drug, which includes: the Linker-MMAE having the structure of formula II and the interchain disulfide bond of the anti-human EGFR antibody undergo an addition reaction Preparing the anti-human EGFR antibody drug conjugate;
  • the method includes the following steps:
  • step (2) Add anti-human EGFR antibody to the reducing agent stock solution described in step (2), and incubate for 1-2h;
  • step (1) Add the linker-cytotoxic drug stock solution of step (1) to the reaction solution obtained in step (4), and incubate for 1-2 hours to prepare an anti-human EGFR antibody drug conjugate;
  • step (3) Add the L-Cys stock solution of step (3) to the reaction solution obtained in step (5) to stop the reaction.
  • the reducing agent in the step (2) is TCEP, and the molar amount of the anti-human EGFR antibody is used as a reference when mixing, and the molar amount of the reducing agent is 2-4 times the molar amount of the antibody.
  • the molar amount of the anti-human EGFR antibody is used as a reference when mixing in the step (5), and the molar amount of Linker-MMAE added is 4-8 times the molar amount of the antibody.
  • the reactions in the steps (4) and (5) are carried out under the protection of nitrogen, and the reaction temperature in the step (4) is 35-40°C, preferably 37°C.
  • the AKTA purifier protein purification system is used to collect the peak of the required antibody-drug conjugate; after the collection is completed, a 30kDa ultrafiltration tube is used for ultrafiltration Filter, centrifuge, concentrate, filter through a sterile filter membrane, and store at low temperature.
  • the present invention also proposes the use of the antibody-drug conjugate in the preparation of drugs for tumor targeted therapy, wherein the tumor is an EGFR-positive solid tumor, including squamous cell carcinoma, Esophageal cancer, nasopharyngeal cancer, lung cancer, breast cancer, pancreatic cancer, prostate cancer, head and neck cancer, colon cancer, etc.
  • a pharmaceutical composition for targeted tumor therapy using the antibody-drug conjugate as an active ingredient is also within the protection scope of the present invention, and the pharmaceutical composition contains a pharmaceutically effective amount of the present invention.
  • Antibody drug conjugates and pharmaceutically acceptable adjuvants are also within the protection scope of the present invention, and the pharmaceutical composition contains a pharmaceutically effective amount of the present invention.
  • the present invention prepares a series of Linker, which shows better plasma stability and enzyme cleavage rate, and reacts with MMAE in one step to prepare Linker-MMAE.
  • the present invention uses the SCT-200 fully humanized monoclonal antibody to couple with Linker-MMAE through the chemical method of reducing the disulfide bond between the antibody chains to construct a type of solid tumor therapy targeting EGFR.
  • Anti-human EGFR antibody drug conjugate Preferably, TCEP is used as a reducing agent.
  • the average drug-antibody coupling ratio (DAR) is prepared within the range of 4.0 ⁇ 0.5, and the quality is stable and controllable, with good repeatability and considerable yield.
  • the novel antibody drug conjugate SCT-200-Linker-MMAE proposed by the present invention does not affect the affinity, endocytic activity and targeting of the antibody, and better retains its biology Function;
  • IC 50 is in the nM level;
  • Western Blot experiment shows that compared to SCT-200, SCT-200-Linker-MMAE ADCs can Significantly inhibit the expression of related proteins in the process of tumor apoptosis; in the evaluation of in vivo activity, compared with SCT-200, SCT-200-Linker-MMAE ADCs have greatly enhanced tumor suppressing effect.
  • Figure 1 is a synthetic route diagram of Fmoc-dipeptide sequence
  • Figure 2 is a synthetic route diagram of Fmoc-AVA
  • Figure 3 is a synthetic route diagram of Fmoc-GFG
  • Figure 4 is a synthetic route diagram of Fmoc-GGFG
  • Figure 5 is a synthetic route diagram of Fmoc-GFLG
  • Figure 6 is a linker-MMAE synthesis route diagram of spacer and hydrolysis zone types
  • Figure 7 is a linker-MMAE synthesis route diagram of the self-decomposition zone type
  • Fig. 7A is a linker synthesis route diagram of the self-decomposition zone type
  • Fig. 7B is a synthesis route diagram of the Linker and MMAE condensation reaction of the self-decomposition zone type
  • Figure 8 is the HIC-HPLC spectrum of the spacer type SCT-200-Linker-MMAE
  • Figure 9 is the HIC-HPLC spectrum of the hydrolysis zone type SCT-200-Linker-MMAE
  • Figure 10 shows the HIC-HPLC spectrum of the self-decomposition zone type SCT-200-Linker-MMAE
  • Figure 11 is the ESI-MS spectrum of SCT-200-Linker-MMAE
  • Figure 12 is a graph of the affinity of SCT-200-Linker-MMAE ADCs measured by ELISA
  • Figure 13 is a diagram showing the endocytosis rate of SCT-200-Linker-MMAE ADCs measured by flow cytometry;
  • Figure 14 is a Western Blot determination of EGFR expression levels in different cells
  • Figure 15 is a diagram showing the influence of SCT200-Linker-MMAE ADCs on the expression of apoptosis-related proteins
  • Figure 16 shows the tumor inhibition curve (A) and weight change curve (B) of SCT200-Linker-MMAE ADCs on the A431 nude mouse xenograft tumor model.
  • the Buchner funnel is suction filtered until the filtrate is clear, and then filtered The cake is drained and dried in a vacuum drying oven. After drying, the white solid was ground, washed with ether for several times, and filtered with suction to obtain 1a, 15.1 g of the white solid, with a yield of 83%.
  • the 1a reaction product (7.6g, 15.3mmol) and PABOH (3.76g, 30.6mmol) were dissolved in 140mL CH 2 Cl 2 and 70 mL of CH 3 OH mixed solvent, and EEDQ (7.5 g, 30.6 mmol).
  • the synthesis method is the same as that of 4a, to obtain a yellow solid with a yield of 62%.
  • the solubility of the product in this step is not good, and it is directly washed with a mixed solvent of diethyl ether and dichloromethane and filtered by suction, and directly proceed to the next step of the reaction.
  • the Linker-MMAE to be tested is prepared as a 10mM DMSO stock solution, ready for use. Add 4 ⁇ L of DMSO stock solution to 996 ⁇ L of blank rat plasma, mix well and incubate in a 37°C water bath. Sampling 50 ⁇ L each at 0, 30, 60, 180, and 360 minutes to terminate the reaction. Two parallel samples at each reaction time point were stored at -20°C and processed after sampling. The sample was centrifuged to settle the protein, the supernatant was taken, and the concentration of MMAE was detected by LC-MS/MS, and the concentration unit (nM) was detected. Draw a curve of the release amount of MMAE released over time, and calculate the release rate of MMAE in plasma. The release rate is shown in Table 1.
  • Linker-MMAE linker-cytotoxic drug
  • step 4 Add the SCT-200 antibody to the reducing agent stock solution described in step 2), and incubate at 37°C for 1.5 hours under nitrogen protection; the molar amount of the reducing agent is 3 times the molar amount of the antibody;
  • step 5 Add the linker-cytotoxic drug stock solution of step 1) to the reaction solution of step 4), and incubate at 37°C for 1-2.5 hours under nitrogen protection to prepare the SCT-200 antibody anti-human EGFR antibody drug conjugate;
  • the molar amount of the linker-cytotoxic drug is 4-8 times the molar amount of the antibody;
  • step 6) Add the stock solution of step 3) to the reaction solution obtained in step 5) to stop the reaction.
  • the step of further purifying the obtained antibody-conjugated drug SCT-200-Linker-MMAE, preferably the AKTA purifier protein purification system, collect the required antibody-conjugated drug component peaks; after collection, use a 30kDa ultrafiltration tube for ultrafiltration Filter, centrifuge, concentrate, filter through a sterile filter membrane, and store at low temperature.
  • Flow rate 0.5mL/min; injection volume: 8 ⁇ L; analysis time: 17min; column temperature: 25°C; detection wavelength: 280nm and 248nm;
  • the liquid system adopts ACQUITY UPLC H-Class (Waters), the chromatographic column is ACQUITY UPLC Protein, BEH SEC, 1.7 ⁇ m, 2.1mm ⁇ 100mm, column temperature 25°C, mobile phase is 100mM ammonium acetate aqueous solution, isocratic elution, flow rate is 0.1ml/min.
  • the mass detection system is: Xevo G2-XS Qtof (Waters), the detection mode is positive ion, and the full scan mode.
  • MassLynx 4.1 Waters
  • the model parameter setting resolution is 2-3.5Da.
  • the minimum intensity ratio is set to 60%, and the output resolution is set to 1Da.
  • the algorithm iteration parameter is set to 20, and the result is shown in Figure 11.
  • Coating antigen Dilute the antigen with PBS to 2 ⁇ g/mL, add 50 ⁇ L coating to each well, and wrap it with fresh-keeping film at 4°C overnight. Wash the plate: Shake the liquid in the plate, add 200 ⁇ L of PBST/well, wash 3 times, shake for 3-5 min each time. Blocking: Add 1% BSA to each well, 200 ⁇ L/well, overnight at 4°C. Wash the plate: Shake the liquid in the plate, add 200 ⁇ L of PBST/well, wash 3 times, shake for 3-5 min each time. Add the sample to be tested, PBST as a diluent, add 50 ⁇ L to each well, 3 times in parallel for each concentration, and incubate at 37°C for 1h.
  • wash the plate spin dry the liquid in the plate, add 200 ⁇ L of PBST/well, wash 3 times, shake 3-5 min each time, and pat dry the ELISA plate.
  • Incubation of secondary antibody Add 50 ⁇ L of secondary antibody (diluted at 1:5000) to each well, and incubate at 37°C for 1 hour. Wash the plate: spin dry the liquid in the plate, add 200 ⁇ L of PBST/well, wash 3 times, shake 3-5 min each time, and pat dry the ELISA plate.
  • the results show that the results are shown in Figure 12.
  • the naked antibody SCT-200 and ADCs have a concentration-dependent affinity to the antigen, indicating that the affinity of the SCT200-Linker-MMAE ADCs prepared based on the cysteine coupling method is comparable to that of the antibody SCT-200
  • the affinity is similar, and the affinity of the original antigen and antibody is basically maintained.
  • the affinity between the antigen and the antibody comes from the ADC antibody itself, and the change of the linker basically has no effect on it.
  • KYSE520 cells were trypsinized, and the cells were pelleted by centrifugation at 4°C for 5 min. The cell resuspended concentration was 1 ⁇ 10 7 /mL. The cells were divided into 50 ⁇ L/tube, and each group had 3 samples in parallel.
  • Primary antibody binding Dilute the primary antibody sample with FACS staining buffer so that the final concentration of the primary antibody is 20 ⁇ g/mL. After incubating for 1 hour in an ice bath, the cells were washed with FACS staining buffer and centrifuged at 4°C for 5 minutes to remove the supernatant.
  • Antibody internalization add 200 ⁇ L of FACS staining buffer to each centrifuge tube and incubate at 37°C for 2h.
  • Flow cytometry was used to detect the internalization rate of SCT200-Linker-MMAE ADCs, and the internalization rate of each ADCs in KYSE520 cells was determined.
  • the ADCs were incubated at 4°C for 30min and 37°C for 2h, respectively.
  • the internalization level of the antibody bound to the cell surface was calculated by calculating the average fluorescence intensity (MFI) reduction level of the 37°C incubation sample compared to the 4°C incubation control.
  • MFI average fluorescence intensity
  • %MFI at time point t MFI of the sample incubated at 37°C ⁇ MFI of the sample incubated at 100/4°C;
  • the protein sample to be tested is loaded, and the protein loading amount is 30 ⁇ g/well.
  • the cells in the logarithmic growth phase were resuspended and counted by centrifugation, and seeded in 96 wells at a rate of 1 ⁇ 10 4 to 3 ⁇ 10 4 /well, and cultured at 37°C for 2 hours. Then, different concentrations of LR004-VC-MMAE (LR004 and MMAE are used as controls) were added, and 3 parallel holes were set for each drug concentration. After 72 hours of incubation at 37°C, add 20 ⁇ L of CCK8 reagent to each well and continue to incubate for 1-2 hours. Observe the color reaction, and measure the absorbance at 450nm with a microplate reader.
  • cell survival rate (additional group A450 value-blank group A450 value)/(control group) A450 value-blank group A450 value) ⁇ 100%.
  • the IC 50 value is calculated using SPSS software.
  • Example 11 The effect of SCT-200-Linker-MMAE ADCs on the expression of related proteins in the process of tumor apoptosis
  • SCT200-M-2, SCT200-C-2 and SCT200-C-4 were selected for the correlation study of apoptosis proteins based on the above experimental results.
  • the A431 cells were treated with the above-mentioned ADCs at 0.01, 0.1 and 1 ⁇ g/mL, 1 ⁇ g/mL SCT200 and 1 nM MMAE for 24 hours, and the samples were collected and tested by Western Blot.
  • the expression level of cleaved-PARP is different, the control and SCT200 bands are not obvious, the MMAE band is shallow, and ADCs 1 ⁇ g/mL can significantly increase the expression level of cleaved-PARP, and it is concentration-dependent.
  • ADCs can induce cell apoptosis through P-P53, caspase-3 and cleaved-PARP-dependent apoptotic pathways.
  • mice were injected with 5 ⁇ 10 6 /100 ⁇ L A431 cells subcutaneously into the right armpit.
  • the tumor volume of nude mice is about 110mm 3 , and they are randomly divided into 5 groups, which are the control group, SCT-200 (12mg/kg), SCT-200-M-1 (4mg/kg) , SCT-200-M-2 (4mg/kg), SCT-200-C-2 (4mg/kg) and SCT-200-C-4 (4mg/kg) each group of 6 nude mice, every 3 days
  • the tail vein was administered once for a total of 4 times.
  • the tumor suppression curve is shown in Figure 16.

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Abstract

L'invention concerne une classe de conjugués de médicament anticorps anti-EGFR humain et son procédé de préparation et son application. Le conjugué de médicament anticorps anti-EGFR humain possède la structure représentée par la formule (I). Dans la présente invention, au moyen d'un anticorps monoclonal entièrement humanisé SCT-200, et par le couplage avec le MMAE de liaison au moyen du procédé chimique de réduction des liaisons disulfure entre les chaînes d'anticorps, une classe de conjugués de médicament anticorps anti-EGFR humain ciblant l'EGFR pour le traitement de tumeurs solides est mise au point. Le nouveau conjugué de médicament anticorps SCT-200-MMAE de liaison décrit par la présente invention, par rapport au SCT-200 proprement dit, n'affecte pas l'affinité, l'activité endocytique, et le ciblage de l'anticorps, il conserve mieux sa fonction biologique, et, en comparaison avec le SCT-200, l'activité est significativement améliorée, et peut inhiber significativement l'expression de protéines apparentées pendant le processus d'apoptose tumorale, ce qui améliore considérablement l'effet d'inhibition de la tumeur.
PCT/CN2020/000225 2019-09-19 2020-09-17 Classe de conjugué de médicament anticorps anti-egfr humain et son procédé de préparation et son application WO2021051720A1 (fr)

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CN116199740A (zh) * 2021-12-01 2023-06-02 上海生物制品研究所有限责任公司 抗体药物偶联物及其用途
WO2023222108A1 (fr) * 2022-05-20 2023-11-23 上海迈晋生物医药科技有限公司 Procédé de préparation d'un conjugué anticorps-médicament
CN117917248A (zh) * 2022-10-20 2024-04-23 英诺湖医药(杭州)有限公司 酶裂解连接子及包含其的配体-艾瑞布林偶联物

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