WO2019169953A1 - Anticorps monoclonal ciblant cd24, conjugué de sel de diéthylamine azo d'onium diol et utilisation associée - Google Patents

Anticorps monoclonal ciblant cd24, conjugué de sel de diéthylamine azo d'onium diol et utilisation associée Download PDF

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WO2019169953A1
WO2019169953A1 PCT/CN2019/071523 CN2019071523W WO2019169953A1 WO 2019169953 A1 WO2019169953 A1 WO 2019169953A1 CN 2019071523 W CN2019071523 W CN 2019071523W WO 2019169953 A1 WO2019169953 A1 WO 2019169953A1
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antibody
compound
diol
azo
diethylamine
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张娟
黄张建
张鑫荣
王旻
王阳
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中国药科大学
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/655—Azo (—N=N—), diazo (=N2), azoxy (>N—O—N< or N(=O)—N<), azido (—N3) or diazoamino (—N=N—N<) compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
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    • 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
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    • 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
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    • 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
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    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/44—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having three double bonds between ring members or between ring members and non-ring members
    • C07D207/444—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having three double bonds between ring members or between ring members and non-ring members having two doubly-bound oxygen atoms directly attached in positions 2 and 5
    • C07D207/448—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having three double bonds between ring members or between ring members and non-ring members having two doubly-bound oxygen atoms directly attached in positions 2 and 5 with only hydrogen atoms or radicals containing only hydrogen and carbon atoms directly attached to other ring carbon atoms, e.g. maleimide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/44—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having three double bonds between ring members or between ring members and non-ring members
    • C07D207/444—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having three double bonds between ring members or between ring members and non-ring members having two doubly-bound oxygen atoms directly attached in positions 2 and 5
    • C07D207/448—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having three double bonds between ring members or between ring members and non-ring members having two doubly-bound oxygen atoms directly attached in positions 2 and 5 with only hydrogen atoms or radicals containing only hydrogen and carbon atoms directly attached to other ring carbon atoms, e.g. maleimide
    • C07D207/452—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having three double bonds between ring members or between ring members and non-ring members having two doubly-bound oxygen atoms directly attached in positions 2 and 5 with only hydrogen atoms or radicals containing only hydrogen and carbon atoms directly attached to other ring carbon atoms, e.g. maleimide with hydrocarbon radicals, substituted by hetero atoms, directly attached to the ring nitrogen atom

Definitions

  • the invention belongs to the field of bioengineering, and particularly relates to a novel antibody drug conjugate HN-01 which can specifically bind to a leukocyte differentiation antigen CD24 molecule overexpressing a tumor cell surface, which is composed of a monoclonal antibody G7 mAb and a novel monooxidation.
  • the nitrogen donor diethylamine azo diol salt molecule is coupled by a maleimide-disulfide bond. It utilizes the specific targeting of G7mAb to enrich the molecules of diethylamine azo diol diol onto the surface of tumor cells, increasing the therapeutic index of the nitric oxide donor molecule and avoiding low targeting when used alone.
  • the toxic side effects caused by sex on normal tissue cells solve the problem of targeted release of nitric oxide donors, provide a new choice for the treatment of liver cancer, and can also be extended to other malignant tumors overexpressing CD24 molecules. in.
  • ADC antibody drug conjugate
  • ADC Antibody drug conjugate
  • ADC drugs enable the complementary advantages of chemical-based chemotherapy and monoclonal antibody-based immunotherapy, with a high degree of specificity and a therapeutic window for highly toxic cytotoxins.
  • the overall structure of the ADC drug is complex and consists of three main components: the "missile" antibody, the Linker Linker, and the cytotoxin.
  • ADC drugs are transported to the tumor site by blood circulation, and the antibody recognizes and binds to the target antigen on the surface of the tumor cell, and the formed antigen/ADC drug complex is internalized into the target cell, in the cell lysosomal enzyme, etc.
  • Linker breaks under action, releasing active small molecules to kill tumors.
  • the target cells that have absorbed the ADC drug are lysed, and the toxin molecules are released into the nearby tumor cells to further exert the killing effect, that is, the "bystander effect”.
  • ADC drugs have been optimized for physicochemical properties of ADC drugs and advances in technology have led to rapid advances in ADC drugs in the last decade.
  • ADC drugs still faces many challenges and challenges: only less than one percent of ADC drugs reach target cells, and only fewer cytotoxic drugs play an anti-tumor effect; Linker design is not reasonable enough to occur in vivo Early breakage or failure to break; and ADC drug batches vary widely, it is difficult to develop a evaluation criteria for drug analysis and in vitro and in vivo activity investigation.
  • small molecule drugs have strong hydrophobicity, and ADCs coupled with a large number of drugs (DAR is 4 or more) are prone to coagulation and have reduced stability.
  • the first ADC drug developed by Wyeth, Mylotarg was decomposed and decomposed in the blood circulation process because it did not reach the tumor tissue as the Linker's sputum bond.
  • the cluster of differentiation 24 is a highly glycosylated adhesion molecule with integrin properties. Studies have shown that CD24 molecule is a potential carcinogenic factor that is overexpressed in various tumor tissues such as colorectal cancer, breast cancer, liver cancer, and small cell lung cancer, but not in normal tissues. As an important marker on the surface of liver cancer cells, CD24 has an important influence on the survival, proliferation and carcinogenesis of tumors, and has the potential to be developed as a molecular target.
  • the currently used anti-CD24 antibody SWA11 was developed using hybridoma technology. Studies have shown that SWA11 has good targeting and inhibits proliferation, invasion and metastasis of tumor cells both in vivo and in vitro. The Barbara A.
  • Froesch team used the targeting of SWA11 to transport doxorubicin to human small cell lung cancer tumor lesions.
  • the Shiran Shapira team used this design for new types of tumor tissue.
  • the team's immunotoxin SWA11-ZZ-PE38 can target CD24 antigen, while the PE38 group can induce tumor cell apoptosis and effectively inhibit it. Growth of CD24-positive xenografts in a nude mouse model.
  • the ADC design of the monoclonal antibody SWA11 demonstrates that the CD24 target can be used to develop ADC drugs.
  • a NO donor drug generally refers to a prodrug formed by a NO donor and related drugs or an active compound through various linking groups.
  • NO donors such as nitrosothiols, nitrates, NO-metal complexes (nitroso salts), furoxan N-oxides, azodiolates, etc.
  • Diazenium diolates have significant advantages in the selective and targeted release of NO.
  • the azo-nonanediol salt is highly unstable under physiological conditions, and can automatically release 1 to 2 molecules of NO, and its half-life ranges from several seconds to several hours.
  • the diazonium salt diol bit O 2 (O attached to the nitrogen ion called O.
  • Nitric oxide is a water-soluble, free-radical-bearing gas in living organisms that has redox properties and plays an important role in physiology and pathology. Studies have shown that NO is a potential anti-tumor agent that regulates tumor-related processes, including angiogenesis, apoptosis, invasion and metastasis, and cell cycle. The direct mechanism of NO on tumor cells is still unclear. There are three main aspects reported at present: (1) NO reacts with intracellular superoxide anion to form peroxynitrite, which is protonated and decomposed into NO 2 and hydroxyl radicals.
  • Hydroxyl radicals can bind to various molecules of tumor cells, causing tumor cell damage, such as lipid peroxidation, protein, amino acid cross-linking reaction; (2) NO is very easy to form Fe with protein Fe containing Fe-S center NO, which causes degradation of Fe-S prosthetic group and aconitase in the mitochondrial respiratory chain, thereby preventing intracellular energy synthesis and inducing apoptosis; (3) NO can directly act on ribonucleic acid reductase, affecting tumor cells DNA replication can also cause nitrosation of DNA and inhibit tumor cell proliferation.
  • NO-mediated macrophage The killing effect on tumor cells, the specific mechanism is: T cell recognition antigen ⁇ T cell secretion cytokine ⁇ cytokine stimulation NO ⁇ NO is produced to kill tumor cells.
  • NK cytotoxicity is also NO dependent. Therefore, the design and research of NO prodrugs has become one of the important strategies for anti-tumor drug innovation.
  • NCI National Cancer Institute
  • some world-renowned pharmaceutical companies such as Merk, Pfizer, NicOx, and NitroMed have all invested in the development of NO donor drugs.
  • the development of NO donor drugs has also made great progress, and NCI has included the anti-tumor drug JS-K in the rapid development program.
  • the present invention passes the laboratory-made targeting CD24 monoclonal antibody G7 mAb and the nitric oxide donor diethylamine azo diol salt molecule (NO donor) through maleimide-double
  • a novel antibody drug conjugate HN-01 was designed by coupling sulfur bonds.
  • the design of HN-01 combines the targeting of antibodies with the anti-tumor effect of nitric oxide donor molecules, and significantly reduces the accompanying effects of nitric oxide donor molecules on tumor cell inhibition and killing. The toxic side effects of normal tissue cells. Therefore, the development of the antibody drug conjugate HN-01 will provide a new candidate ADC drug for tumor immunotherapy.
  • the present invention provides an anti-CD24 antibody drug conjugate HN-01 having an antitumor effect.
  • the antibody drug conjugate of the invention is characterized in that the monoclonal antibody G7 mAb and the nitric oxide donor diethylamine azo diol salt are coupled by a maleimide-disulfide bond, and can simultaneously play a monoclonal
  • the targeting of the antibody G7 mAb and the cytotoxicity of the nitric oxide donor diethylamine azo diol salt molecule can inhibit the proliferation of CD24-positive liver cancer cells Huh-7 and BEL-7402 in vitro.
  • a molecular conjugate of an anti-CD24 monoclonal antibody and a diethylamine azo diol diol salt characterized in that the conjugate is chemically coupled based on an anti-CD24 monoclonal antibody and a nitric oxide donor molecule.
  • the nitric oxide donor molecule is dimethylamine arsenazo Alkoxide, diethylamine azo diol hydride, N-methylethanolamine azo diol diol salt, diethanolamine azo diol diol salt, pyrrolazo arsenazodiol salt, piperidinium azo hydrazide salt
  • the light chain amino acid sequence of the anti-CD24 monoclonal antibody is SEQ NO. 1, and the heavy chain amino acid sequence thereof is represented by SEQ NO.
  • HL-2 obtained by reacting a molecule of diethylamine azo hydrazide salt with a maleimide-disulfide bond, and the structure is as follows
  • the preparation method of the compound HL-2 is characterized by the following steps:
  • Step a 435 mL of a 0.1 M aqueous sodium hydrogencarbonate solution was slowly added to 80 mL of an aqueous solution of Compound 1 having a concentration of 0.55 mol/L via a dropping funnel. Reactive reaction at room temperature for 20 min; dried by an oil pump, and then finally dried on a freeze-drying apparatus to obtain a compound 2;
  • Step b 12.4g of compound 3 was added to a 250mL reaction flask, adding 100mL of benzene, stirring, slowly adding 26.6g of NBS, reaction at room temperature for 5h, TLC detection reaction is complete, to obtain compound 4;
  • Step c 0.82 g of azobisisobutyronitrile was added to the reaction flask of the compound 4, and 26.6 g of NBS was added thereto, and the reaction was refluxed at 80 ° C for 20 hours; the solvent was removed, and then extracted with ethyl acetate, and washed with saturated brine. aqueous Na 2 SO 4 dried, concentrated and column chromatography to give compound 5 as a white solid;
  • Step d 202 mg of compound 5 was dissolved in 3.5 mL of DMF, and 69.75 mg of diethylamine arsenazodiol salt was added thereto, and the reaction was carried out under nitrogen at -5 ° C for 4 hours, and the reaction was completely confirmed by TLC. Poured into 50mL ethyl acetate extracts were washed with brine, dried over anhydrous Na 2 SO 4, and concentrated by column chromatography to give compound 6 as a colorless oil.
  • Step e 455 mg of Compound 6 was dissolved in 10 mL of a mixed solution of acetone:DMF (2:1). 205 mg of compound 5 was slowly added to the reaction flask, protected with nitrogen, and refluxed at 45 ° C for 3.5 h; The acetone was removed, and the mixture was extracted with ethyl acetate (100 mL), washed with saturated brine, dried over anhydrous Na 2 SO 4 and concentrated to give the compound 7 as a colorless oil (Z)-1-(( 4-((4-((4-((4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoyl)oxy)methyl)phenyl)disulfa nyl)benzyl)oxy)-1- (N,N-diethylamino)diazen-1-iu-m-1,2-diolate.
  • the method is characterized in that the step c, the step d and the step e are carried out by extracting with ethyl acetate, washing with saturated brine, drying with anhydrous Na 2 SO 4 and concentration column chromatography to purify the reaction product.
  • An antibody drug conjugate prepared from an anti-CD24 monoclonal antibody and an HL-2 molecule comprising the following steps:
  • Step 1 After anti-CD24 monoclonal antibody was purified by Protein A column affinity chromatography and glucose gel G25FF desalting column molecular sieve chromatography to replace the antibody solution system, the antibody concentration was determined by BCA method, and the reducing agent TCEP was compared with the antibody molar ratio 2.5. :1, mixed, slowly stirred evenly, reacted at 4 ° C for 1 h, 4000 rpm, low temperature ultrafiltration to remove residual TCEP, replaced with 1M antioxidant diethylene triamine pentaacetic acid in PBS, pH 7.0 system to prepare the coupling reaction;
  • Step 2 Coupling the reduced anti-CD24 monoclonal antibody with the compound of claim 2 by a molar ratio of 1:15, solubilizing the compound of claim 2 with DMSO, and slowly adding 1 mg/mL of anti-CD24 monoclonal In the antibody, gently stir evenly, and then reacted at 4 ° C for 1 h, ultrafiltration; that is, an anti-CD24 monoclonal antibody and a diethylamine azodiol diol salt molecular conjugate;
  • Step 3 The integrity of the conjugate structure was initially identified by 8% non-reducing SDS-PAGE gel electrophoresis. After the concentration of the conjugate was determined by BCA method, it was stored at -70 °C.
  • Anti-CD24 antibody drug conjugate HN-01 Monoclonal antibody G7mAb exerts a targeting effect Molecularly toxic thymidine azodiol salt molecules are specifically displayed on the surface of CD24-expressing tumor cells . Molecular cleavage of diethylamine azo diol salt releases NO molecules and exerts its inhibitory and toxic effects on tumor cells.
  • the present invention synthesizes a novel compound HL-2 for the first time, and develops it into a novel antibody drug conjugate by utilizing the characteristics of its coupling with an anti-CD24 antibody.
  • the present invention utilizes hybridoma technology to obtain an expressible anti-CD24 monoclonal antibody.
  • Antibody cell line G7, antibody G7 mAb was prepared by ascites inoculation method; antigen-specific targeting of G7 mAb in vitro and in vivo was demonstrated by flow cytometry and near-infrared imaging technology, so G7 mAb was selected as the antibody drug conjugate of the present invention.
  • Biological warheads Further, the present invention uses HL-2 as a small molecule cytotoxin.
  • HL-2 consists of a nitric oxide donor diethylamine azo diol salt molecule and a maleimide-disulfide bonder. It has the following characteristics: (1) 2 molecules can be released after each molecule is cleaved NO; (2) One end of HL-2 is a maleimide structure, which can react with a cysteine sulfhydryl group formed by reduction to form a thioether bond; (3) HL-2 is a disulfide bond structure in the middle. It is also the key to the release of NO molecules by this molecule. Compared to the hydrazone Linker, the disulfide bond is stable in the circulatory system and is decomposed in an intracellular reducing environment.
  • the present invention utilizes a reducing agent TCEP to reduce the interchain disulfide bond of an antibody chain to achieve coupling of a toxin with an antibody, and selectively couples two pairs of disulfide bonds in the hinge region of the antibody by controlling the amount of TCEP, and plans to couple each antibody 2 ⁇ 4 small molecules.
  • the maleimide structure of the HL-2 molecule forms a stable thioether bond with the antibody cysteine thiol group, and the binding of the small molecule to the antibody is achieved, and the antibody drug conjugate HN-01 is obtained, and the structure is as shown in FIG. HN-01 is endocytosed by tumor cells. Under the action of intracellular glutathione, the disulfide bond cleavage releases the active effector molecule NO.
  • the specific mechanism is as follows:
  • HN-01 is endocytosed by tumor cells, and the disulfide bond cleavage by intracellular GSH (glutathione) releases the active effector molecule NO.
  • Figure 1 shows a schematic representation of the structure of the antibody drug conjugate HN-01.
  • the diethylamine azo diol salt molecule is bound to the cysteine sulfhydryl group in the reduced state by the maleimide-disulfide bond in the hinge region of the G7 mAb.
  • Figure 2 is a non-reducing SDS-PAGE protein electropherogram of antibody drug conjugate HN-01.
  • HN-01 has a distinct main band, and the position of the band is flush with the parent antibody G7mAb, because the molecular weight of the coupled Linker-drug is 558Da, coupling small molecule toxin does not significantly increase the molecular weight of the antibody. (150KDa), so the position of HN-01 in the electropherogram is basically the same as that of G7mAb. And due to the glycosylation of the antibody, the position of the two in the electropherogram is higher than the position of the 150KDa marker.
  • the lane of HN-01 has two shallow bands in addition to the main band.
  • the analysis may be that TCEP reduces the disulfide bond between the heavy and light chains of the antibody, resulting in a very small part of the conjugate antibody being light. The absence of a chain or heavy chain.
  • Figure 3 shows the antibody drug conjugate HN-01 peak obtained by HIC-HPLC (hydrophobic chromatography-high performance liquid chromatography). Taking the peak time of naked anti-G7mAb under the same analysis conditions as a reference (Fig. 4A), we found that there is no corresponding peak in Fig. 4B at the bare anti-peak time of about 10 min, indicating that there is no naked anti-analysis in the analysis product, that is, all the analytical products. All of them are coupled with small molecules; since the target is to couple 2 to 4 small molecules, we can judge from left to right that the peaks from left to right are 2-drug, 4-drug, 6-drug, and 8-drug.
  • Figure 4 is a graph showing the results of MTT assay of HN-01 showing the inhibitory effect of HN-01 on the proliferation of CD24-positive tumor cells Huh-7 and BEL-7402.
  • the proliferation inhibition rate of CDn-positive cells Huh-7 and BEL-7402 by 1000nM HN-01 was 41.00% and 41.16%, respectively, which was significantly improved compared with the same concentration of parental monoclonal antibody G7mAb group (1000nM).
  • the inhibition rates of G7mAb on the two positive cells after 48h were 21.96% and 24.92%, respectively.
  • the comprehensive analysis showed that the inhibitory effect of conjugate HN-01 on CD24-positive cell line increased with the increase of dose.
  • Figure 5 is a flow scatter plot of the apoptosis assay of HN-01 showing the effect of HN-01 on apoptosis in Huh-7 and BEL-7402 cells.
  • the apoptosis rates of Huh-7 cells induced by 100NM and 500nM HN-01 were 13.79% and 21.43%, respectively; the same concentration of G7mAb did not induce cell apoptosis, corresponding apoptosis.
  • the rates were 9.78% and 11.3%, respectively; the apoptotic rate of the small molar HL-2 was 13.63% and 17.12%, respectively; the corresponding apoptotic rate of the blank control under the same conditions was 8.02%.
  • the apoptosis rate of BEL-7402 cells induced by HN-01 was 3.77% and 25.81% after treatment with 100NM and 500nM HN-01 for 48h.
  • the same concentration of G7mAb did not induce cell apoptosis, and the corresponding apoptotic rate. They were 4.58% and 5.42%, respectively; the apoptotic rates induced by the equivalent molarity of HL-2 were 5.88% and 7.55%, respectively; the corresponding apoptotic rate of the blank control group under the same conditions was 2.24%.
  • Figure 6 is a statistical analysis of the results of the apoptosis test of HN-01 (statistical analysis is shown in the mean ⁇ SD form based on three independent experimental results; ***p ⁇ 0.001: very significant).
  • the results of T-test showed that the apoptosis rate of Huh-7 and BEL-7402 cells induced by high dose of HN-01 (500nM) was significantly different from that of G7mAb and HL-2 groups.
  • Step a 435 mL of a 0.1 M aqueous sodium hydrogencarbonate solution was slowly added to 80 mL of an aqueous solution of Compound 1 having a concentration of 0.55 mol/L via a dropping funnel. Reactive reaction at room temperature for 20 min; dried by an oil pump, and then finally dried on a freeze-drying apparatus to obtain a compound 2;
  • Step b 12.4g of compound 3 was added to a 250mL reaction flask, adding 100mL of benzene, stirring, slowly adding 26.6g of NBS, reaction at room temperature for 5h, TLC detection reaction is complete, to obtain compound 4;
  • Step c 0.82 g of azobisisobutyronitrile was added to the reaction flask of the compound 4, and 26.6 g of NBS was added thereto, and the reaction was refluxed at 80 ° C for 20 hours; the solvent was removed, and then extracted with ethyl acetate, and washed with saturated brine. aqueous Na 2 SO 4 dried, concentrated and column chromatography to give compound 5 as a white solid;
  • Step d 202 mg of compound 5 was dissolved in 3.5 mL of DMF, and 69.75 mg of diethylamine arsenazodiol salt was added thereto, and the reaction was carried out under nitrogen at -5 ° C for 4 hours, and the reaction was completely confirmed by TLC. Poured into 50mL ethyl acetate extracts were washed with brine, dried over anhydrous Na 2 SO 4, and concentrated by column chromatography to give compound 6 as a colorless oil.
  • Step e 455 mg of Compound 6 was dissolved in 10 mL of a mixed solution of acetone:DMF (2:1). 205 mg of compound 5 was slowly added to the reaction flask, protected with nitrogen, and refluxed at 45 ° C for 3.5 h; The acetone was spun off, poured into 100 mL of ethyl acetate and extracted with saturated brine, dried over anhydrous Na 2 SO 4
  • a 0.1 M aqueous solution of sodium hydrogencarbonate was prepared by dissolving sodium hydrogencarbonate (8.391 g, 0.1 mmol) in 1 L of water. Among them, 435 mL of a 0.1 M aqueous sodium hydrogencarbonate solution was slowly added to a solution of 4-maleimidobutyric acid 13 (8.014 g, 43.75 mmol) in water (80 mL) via a dropping funnel. The reaction was carried out at room temperature for 20 min. It is dried by an oil pump and then subjected to final drying on a freeze-drying device. Compound 2 (9.06 g; yield 100%) was obtained as an off white solid.
  • Anti-CD24 monoclonal antibody G7mAb (prepared according to the literature) After purification by Protein A column affinity chromatography and glucose gel G25FF desalting column molecular sieve chromatography to replace the antibody solution system, the antibody concentration is determined by BCA method.
  • the reducing agent TCEP was mixed with the antibody by a molar ratio of 2.5:1. After the addition, the mixture was slowly stirred uniformly. The reaction was carried out at 4 ° C for 1 h, 4000 rpm, and the residual TCEP was removed by low-temperature ultrafiltration, and replaced with PBS containing 1 M antioxidant diethylene triamine pentaacetic acid ( The pH 7.0) system was prepared for the coupling reaction.
  • the reduced G7mAb was coupled with the Linker-NO donor molecule HL-2 by a molar ratio of 1:15, and the small molecule dissolved in DMSO was added to the 1 mg/mL antibody slowly and gently, and gently stirred evenly. The reaction was carried out at 4 ° C for 1 h, and excess HL-2 molecules were removed by ultrafiltration. The antibody drug conjugate HN-01 was obtained.
  • the integrity of the conjugate structure was initially identified by 1.3.8% non-reducing SDS-PAGE gel electrophoresis.
  • the concentration of the conjugate was determined by BCA method and stored at -70 °C.
  • Sample preparation Take several EP tubes, add 6 ⁇ L of 5 ⁇ Loading Buffer containing 250 mM Tris-HCl (pH 6.8), 10% SDS, 0.5% bromophenol blue, 50% glycerol, and add 24 ⁇ L samples in each tube. Mix the solution, boil water for 5 min, 8000 rpm, centrifuge for 3 min, and set aside.
  • Matching glue Fix the double-layer glass plate to the rubber mold, and use the single-distilled water to check the leak, and separately arrange the lower layer separation glue and the upper layer layer glue.
  • the separation gel was first added to a double-layer glass plate and immediately flattened with 1 mL of absolute ethanol.
  • the lower layer of the separation gel solidified in about 25 minutes, followed by the addition of the layered glue and the insertion of the comb teeth.
  • the plastic mold is disassembled, the double-layer glass plate is taken out, installed in the electrophoresis tank, and a certain volume of the electrophoresis buffer containing Tris, Glycine, SDS, pH 8.0 is poured into the electrophoresis tank. To the lowest level.
  • Loading Load with a micro syringe, Marker's loading is 5 ⁇ L, and the HN-01 and G7mAb samples are loaded with 25 ⁇ L. After the sample is finished, turn on the electrode and start electrophoresis.
  • Electrophoresis Constant voltage current, the initial voltage is 80V. When the blue strip front migrates to the junction of the laminated glue and the separation glue, the voltage is adjusted to 120V. When the blue strip runs out of the separation gel, the electrophoresis is stopped and the glue is peeled off.
  • Dyeing and Decolorization Peel the glue into the box, add the staining solution, boil, and place it on a bleaching shaker for 30 min. The staining solution is recovered and rinsed with tap water. Add the decolorizing solution to the box, boil it, then place it on a decolorizing shaker and shake it for 20 minutes, and decolorize it twice until the strip is clearly visible.
  • Imaging Carefully placed on a white porcelain plate with glue and placed under a gel imager to observe the bands of Marker and HN-01 and G7 mAb. The experimental results are shown in Figure 2.
  • Many of the small molecule toxins coupled to the antibody are hydrophobic, so the hydrophobicity of the conjugates with different coupling ratios is quite different, and can be analyzed by hydrophobic chromatography (HIC) tandem ultraviolet detector.
  • HIC hydrophobic chromatography
  • the distribution of peaks can determine the number of small molecule drugs coupled to the antibody.
  • the peak area can be used to determine the proportion of the total mixture of conjugates coupled with different stoichiometric small molecule drugs.
  • HIC column TSKgel Butyl-NPR (2.5 ⁇ m particle size, 4.6mm inner diameter, length 10cm)
  • Table 1 shows the weighted average DAR (drug antibody coupling ratio) obtained by HIC-HPLC analysis.
  • ADC drugs utilizes the enrichment of cytotoxins on the surface of tumor cells, improving the therapeutic effect of small molecules while reducing the toxicity of the system.
  • MTT assay was used to detect the inhibitory effect of conjugate HN-01 on the proliferation of CD24-positive cells. The specific methods are as follows:
  • Experimental group setting blank control group (without cells), negative control group (containing cells, no drug), experimental group (G7mAb, HN-01 group, HL-2 group), each group set 3 complex hole.
  • the mother liquor of the drug was prepared by using a medium containing 2% FBS, and the concentration of the drug G7mAb, HN-01 and NO donor molecule HL-2 was diluted by 6 times.
  • G7mAb and HN-01 set the concentration gradients of 0.8, 4, 20, 100, 500, 1000nM.
  • HL-2 was based on the coupling ratio of 3.0, and the corresponding molar concentration was applied: 2.4, 12, 60, 300, 1500. , 3000nM six concentrations.
  • Detection After 48 hours of dosing, the 96-well plate was taken out, and 11 ⁇ L of MTT (5 mg/mL) was added to each well in the dark, and the culture was continued at 37 ° C for 4 h. After 4 hours, the plate was centrifuged, and the plate centrifuge was centrifuged at 3000 rpm for 5 min. The supernatant in the well was aspirated with a 1 mL syringe, 150 ⁇ L of DMSO was added to each well, and the micro-oscillator was shaken for 10 min. The microplate reader detects absorbance at wavelengths of 570 nm and 630 nm.
  • Annexin V/PI double staining method was used to detect the apoptosis of tumor cells induced by HN-01.
  • the experimental procedure was based on the instruction of apoptosis detection kit (purchased from Vazyme). The specific process is as follows:
  • Huh-7 and BEL-7402 cells with good logarithmic phase were selected, digested, resuspended, counted, adjusted to a cell suspension concentration of 2x10 5 cells/mL, and 1.5 mL of the suspension was added to the six-well plate. The medium was cultured overnight at 37 °C.

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Abstract

L'invention concerne un procédé de préparation d'un conjugué anticorps anti-CD24-médicament se rapportant au domaine de la bio-ingénierie et son utilisation. La procédé fourni par la présente invention consiste à conjuguer des molécules de sel de diéthylamine azo d'onium diol, en tant que nouveaux donneurs d'oxyde nitrique, avec des régions constantes de chaîne lourde d'un anticorps monoclonal anti-CD24 G7mAb par l'intermédiaire de liaisons maléimide-disulfure au moyen d'une technologie de couplage chimique, ce qui permet de préparer un conjugué anticorps-médicament HN-01 ; ledit procédé consiste également à enrichir des molécules donneuses d'oxyde nitrique sur des surfaces de cellules tumorales au moyen d'effets de ciblage spécifiques d'anticorps et à les internaliser dans des cellules pour libérer de manière directionnelle de l'oxyde nitrique, ce qui permet d'augmenter les indices thérapeutiques intratumoraux et, en même temps, de réduire les effets toxiques et secondaires sur des tissus normaux. Des résultats d'expériences in vivo et in vitro permettent de démontrer que le conjugué anticorps-médicament HN-01 utilise suffisamment le ciblage d'anticorps et la spécificité antitumorale de l'oxyde nitrique, permet de résoudre le problème de l'administration de ciblage de tumeur des donneurs d'oxyde nitrique, et présente une bonne valeur d'utilisation clinique.
PCT/CN2019/071523 2018-03-09 2019-01-14 Anticorps monoclonal ciblant cd24, conjugué de sel de diéthylamine azo d'onium diol et utilisation associée WO2019169953A1 (fr)

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CN111378006A (zh) * 2018-12-28 2020-07-07 联宁（苏州）生物制药有限公司 一种用于抗体偶联药物的新型双臂中间体lnd1026-035及其合成方法
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CN110964118A (zh) * 2019-11-27 2020-04-07 中国药科大学 一种双特异性融合抗体及其在肿瘤免疫治疗中的应用
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