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WO2019034177A1 - 具有两种不同药物的抗体药物偶联物 - Google Patents

具有两种不同药物的抗体药物偶联物 Download PDF

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WO2019034177A1
WO2019034177A1 PCT/CN2018/101215 CN2018101215W WO2019034177A1 WO 2019034177 A1 WO2019034177 A1 WO 2019034177A1 CN 2018101215 W CN2018101215 W CN 2018101215W WO 2019034177 A1 WO2019034177 A1 WO 2019034177A1
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antibody
drug
pharmaceutically acceptable
acceptable salt
drug conjugate
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PCT/CN2018/101215
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English (en)
French (fr)
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朱义
李�杰
卓识
万维李
余永国
李刚锐
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四川百利药业有限责任公司
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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
    • A61K47/6835—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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to an antibody drug conjugate having two different drugs.
  • antibody-conjugated drugs generally consist of three parts: antibody or antibody ligands, small molecule drugs, and linkers that couple ligands to drugs.
  • the antibody-conjugated drug utilizes the specific recognition of the antigen by the antibody, transports the drug molecule to the vicinity of the target cell and effectively releases the drug molecule for therapeutic purposes.
  • FDA US Food and Drug Administration
  • Adecteis TM developed by Seattle Genes for the treatment of Hodgkin's lymphoma and relapsed large cell lymphoma (ALCL). The safety and efficacy of such drugs.
  • the ADC drug antibody mainly functions as a targeted delivery function, and finally the drug effect is the coupled drug molecule.
  • the drug molecules currently used in the field of ADC are classified according to different mechanisms of action: microtubule inhibitors, DNA damaging agents, topoisomerase inhibitors, RNA polymerase inhibitors, protein translation inhibitors, and the like.
  • ADC drugs In ADC drugs, antibodies carry drug molecules to the vicinity of tumor cells, releasing drug molecules around or within the tumor cells. At the same time, however, tumors have developed different mechanisms against drug molecules during this treatment, including pumping toxin molecules out of the cell with a PgP protein pump to escape killing.
  • ADC drug development mainly focuses on single antibody linking single-acting mechanism drugs. Modern medicine has proved that the combination of different drugs in the chemotherapy process can significantly enhance the efficacy. However, as the number and type of drugs in the ADC increase, the lipid solubility of the molecule increases, the stability in plasma is significantly reduced, and the efficacy is not improved, but also causes side effects.
  • the C-LOCK technique often results in a DAR of 2-4, and the K-LOCK technique also has obvious limitations: when the lysine-linked drug antibody coupling ratio (DAR) is >2 Since a single antibody molecule contains about 88 lysines, such a large amount of lysine results in poor amino coupling selectivity, and the number of couplings and the coupling position are difficult to determine, although a certain degree of control can be achieved by coupling conditions.
  • DAR lysine-linked drug antibody coupling ratio
  • the patent CN106132431 does not report the pharmacodynamic data and plasma stability in animals.
  • ADC drugs an innovative coupling method is needed to achieve plasma stabilization, antibody-conjugated drugs coupled to two different drugs, and a more convenient combination of drugs is achieved.
  • the present invention surprisingly meets the above needs.
  • the present invention aims to provide an antibody-drug conjugate having two different drugs. Two different drug molecules are linked to the same antibody by designing the conjugate. Due to the combined use of different mechanisms of action, the conjugate can effectively improve the efficacy.
  • the present invention provides an antibody drug conjugate comprising a different drug, or a pharmaceutically acceptable salt thereof, of Formula I
  • L1, L2 are optional linking units connectable to the drug
  • D1 and D2 are drug units
  • n and n are integers of 2-8.
  • the drugs D1 and D2 are antitumor drugs having different mechanisms of action.
  • the drugs D1, D2 are preferably selected from the group consisting of tubulin binding agents, DNA alkylating agents, DNA intercalating agents, enzyme inhibitors, immunomodulators, peptides and nucleotides, respectively.
  • the m is preferably 2, 4.
  • the drug D1 is preferably attached to the antibody in a site-directed coupling manner, and the D2 is coupled to the antibody in a non-targeted manner.
  • the preferred site-directed coupling of drug D1 is a half-pointed mutant antibody original amino acid to cysteine or by insertion of a cysteine or a cysteine-containing polypeptide, which is introduced by the above method.
  • the cystine thiol is coupled to the linker-toxin.
  • a preferred non-targeted coupling of D2 is the coupling of the original interchain disulfide bond of the antibody to the linker-toxin.
  • L1 and L2 have the following formula 2:
  • C is an optional extendable unit at the end
  • E is an optional cleavable unit
  • F is a spacer unit
  • subscript e, f is 0 or 1.
  • the wavy line indicates the attachment site to the succinimide and the drug unit.
  • the cleavable unit described by E is achieved by a tumor-associated protease or an acidic pH with a drug unit D or a spacer unit F
  • F is selected from the group consisting of p-aminobenzyl alcohol or with ethylenediamine units and derivatives thereof.
  • the drug-ligand conjugate compound of any of the present invention comprises a pharmaceutically acceptable salt thereof, and a pharmaceutical composition of a pharmaceutically acceptable diluent carrier or excipient.
  • the invention comprises administering to the patient the drug-ligand conjugate of any of the preceding claims, wherein the patient has a tumor, an autoimmune disease or an infectious disease, and
  • the antibody-ligand conjugate antibody specifically binds to the target cell of the cancer, autoimmune disease
  • Figure 1 shows the results of the Payload S MS-TOF test.
  • FIG. 2 shows the results of the Payload E MS-TOF test.
  • Figure 3 shows a graph of the results of the ADC and control ADCs with different drugs.
  • the antibody drug conjugate represented by the following formula is provided by the present invention.
  • the trade name includes the product formulation, generic drug, and active pharmaceutical ingredient of the trade name product.
  • a “derivative" of a compound as used herein refers to a chemical structure that is similar to a compound but also contains at least one chemical group that is not present in the compound and/or lacks the chemical group present in at least one of the compounds.
  • the substance of the regiment. The compounds to which the derivatives are compared are referred to as "parent” compounds.
  • a “derivative" can be produced from a parent compound in one or more chemical reaction steps.
  • antibody or “antibody unit” is within the scope of its disclosure, including any portion of the antibody structure.
  • This unit can bind, reactively associate, or complex a receptor, antigen, or other receptor unit that the cell population has.
  • the antibody can be any protein or proteinaceous molecule that can bind, complex, or react with a portion of the cell population to be treated or bioengineered.
  • the linker is covalently attached to the sulfur atom of the antibody.
  • the sulfur atom is a sulfur atom of a cysteine residue that forms an interchain disulfide bond of the antibody.
  • the sulfur atom is a sulfur atom that has been introduced into the cysteine residue of the ligand unit, which forms an interchain disulfide bond of the antibody.
  • the sulfur atom is a sulfur atom that has been introduced into the cysteine residue of the ligand unit (eg, by site-directed mutagenesis or chemical reaction).
  • the linker-bonded sulfur atom is selected from a cysteine residue that forms an interchain disulfide bond of the antibody or a cysteine residue that has been introduced into the ligand unit (eg, by site-directed mutagenesis or chemical reaction).
  • the EU index in Kabat Kabat (Kabat EA et al, (1991)) "Sequences of proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242" Numbering system.
  • the antibody constituting the antibody drug conjugate of the present invention preferably retains the antigen binding ability in its original wild state. Therefore, the antibody of the present invention can, preferably, specifically bind to an antigen.
  • Antigens involved include, for example, tumor associated antigens (TAAs), cell surface receptor proteins and other cell surface molecules, cell survival regulators, cell proliferation regulators, molecules associated with tissue growth and differentiation (as known or predicted) Functional), lymphokines, cytokines, molecules involved in cell cycle regulation, molecules involved in angiogenesis, and molecules involved in angiogenesis (as known or predicted to be functional).
  • TAAs tumor associated antigens
  • cell survival regulators e.g., cell survival regulators, cell proliferation regulators, molecules associated with tissue growth and differentiation (as known or predicted) Functional)
  • lymphokines cytokines
  • molecules involved in cell cycle regulation e.g., cytokines, molecules involved in cell cycle regulation, molecules involved in angiogenesis, and molecules involved in angiogenesis (as known or predicted to be functional).
  • Antibodies for use in antibody drug conjugates described herein include, but are not limited to, antibodies directed against cell surface receptors and tumor associated antigens. Such tumor associated antigens are well known in the art and can be prepared by antibody preparation methods and information well known in the art.
  • tumor associated antigens are well known in the art and can be prepared by antibody preparation methods and information well known in the art.
  • transmembrane or other tumor-associated polypeptides are capable of being specifically expressed on the surface of one or more cancer cells with little or no expression on the surface of one or more non-cancer cells.
  • tumor-associated polypeptides are more overexpressed on the surface of cancer cells relative to the surface of non-cancer cells. Confirmation of such tumor-associated factors can greatly enhance the specific targeting characteristics of cancer-based treatment of cancer.
  • Tumor-associated antigens include, but are not limited to, tumor-associated antigens well known in the art. Nucleic acid and protein sequences corresponding to tumor associated antigens can be found in public databases such as Genbank. Antibody-targeting corresponding tumor-associated antigens include all amino acid sequence variants and isoforms, having at least 70%, 80%, 85%, 90%, or 95% homology to the sequences identified in the references, or The tumor-associated antigen sequences cited in the literature have completely identical biological properties and characteristics.
  • inhibiting means reducing the detectable amount or completely preventing it.
  • cancer refers to a physiological condition or disease characterized by dysregulated cell growth.
  • Tumors include cancer cells.
  • autoimmune disease is a disease or disorder that results from tissue or protein directed against the individual's own body.
  • site-directed coupling is preferably a cysteine introduced by the above method, either by the original amino acid of the site-directed mutant antibody to cysteine or by the insertion of a cysteine or a cysteine-containing polypeptide into the antibody.
  • the thiol group is coupled to the succinimide in the linker.
  • the "non-site-coupled” mode is coupled to the linker-toxin by utilizing the original interchain disulfide bond of the antibody.
  • pharmaceutically acceptable salt refers to a pharmaceutically acceptable organic or inorganic salt of a compound (eg, a drug, a drug-linker or a ligand-linker-drug conjugate).
  • the compound may contain at least one amino or carboxyl group and thus may form an addition salt with the corresponding acid or base.
  • Exemplary salts include, but are not limited to, sulfates, trifluoroacetates, citrates, acetates, oxalates, chlorides, bromides, iodides, nitrates, hydrogen sulfates, phosphates, acids Phosphate, isonicotinic acid, lactate, salicylate, acidic citrate, tartrate, oleate, tannic acid, pantothenate, hydrogen tartrate, ascorbate, salicylate, Formate, orthoformate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, potassium salt, sodium salt, and the like.
  • pharmaceutically acceptable salts have more than one dotted atom in the structure.
  • An example in which a plurality of charged atoms are part of a pharmaceutically acceptable salt can have multiple counterexamples.
  • a pharmaceutically acceptable salt has one or more charged atoms and/or one or more counter atoms.
  • Drugs refer to: a cytotoxic drug used in the treatment of cancer, including but not limited to maytansine or maytansinoid, doxatin 10 (Dolastatin 10) analogs, calicheamicin drugs Pyrrolo[2,1-c][1,4]benzodi-azepines (PBDs) or PBD dimers (PBD dimmers) and derivatives amanita or derivatives thereof
  • PBDs Pyrrolo[2,1-c][1,4]benzodi-azepines
  • PBD dimers PBD dimers
  • Medium active drugs include, but are not limited to, benzopyrrolidone (duocarmycins, CC-1065, etc.) camptothecin compounds including camptothecin, hydroxycamptothecin, SN-38, ezetidine, irinotecan Wait.
  • the drug is not limited to the above-mentioned categories, but also includes all drugs that can be used for antibody drug conjugates.
  • linking unit or “linking unit of antibody drug conjugate” can be divided into two categories: non-cleavable linking unit and cleavable linking unit.
  • the drug release mechanism is: after the conjugate is bound to the antigen and endocytosed by the cell, the antibody is hydrolyzed in the lysosome, and the drug is released by the small molecule drug.
  • An active molecule composed of an amino acid residue of an antibody. The resulting change in the molecular structure of the drug does not diminish its cytotoxicity, but since the active molecule is charged (amino acid residues), it cannot penetrate into adjacent cells. Therefore, such active drugs cannot kill tumor cells (bystander effect) adjacent to the non-expressing target antigen (antigen-negative cells) (Ducry et al, 2010, Bioconjugate Chem. 21: 5-13).
  • a cleavable linker unit can cleave in the target cell and release the active drug (small molecule drug itself).
  • Breakable linkers can be divided into two main classes: chemically labile linkers and enzyme labile linkers.
  • Chemically labile linkers can be selectively cleaved due to differences in plasma and cytoplasmic properties. Such properties include pH, glutathione concentration, and the like.
  • pH sensitive linkers are often referred to as acid cleavage linkers. Such a linker is relatively stable in the neutral environment of blood (pH 7.3-7.5), but will be in the weakly acidic endosomes (pH 5.0-6.5) and lysosomes (pH 4.5-5.0). hydrolysis. Most of the first generation of antibody drug conjugates use such linkers, such as hydrazine, carbonate, acetal, ketal. Antibody drug conjugates based on such linkers typically have a shorter half-life (2-3 days) due to the limited plasma stability of the acid-cleaved linker. This shorter half-life limits the use of pH-sensitive linkers in a new generation of antibody drug conjugates to some extent.
  • disulfide bond For glutathione-sensitive linkers, it is also called disulfide bond. Drug release is based on a difference between the high concentration (in millimolar range) of intracellular glutathione and the relatively low concentration of glutathione (micromolar range) in the blood. This is especially true for tumor cells, where low oxygen levels result in enhanced reductase activity, resulting in higher glutathione concentrations. Disulfide bonds are thermodynamically stable and therefore have better stability in plasma.
  • Enzyme-labile linkers such as peptide linkers, provide better control of drug release.
  • Peptide linkers are capable of being efficiently cleaved by lysosome in vivo proteases such as cathepsin B or plasmin (an increase in the amount of such enzymes in some tumor tissues). This peptide linkage is believed to be very stable in the plasma circulation because the extracellular pH is inappropriate and the serum protease inhibitors cause the protease to be generally inactive.
  • enzyme-labile linkers are widely used as cleavable linkers for antibody drug conjugates.
  • Typical enzyme-labile linkers include Val-Cit (vc), Phe-Lys, and the like.
  • Suicide linkers are typically chimeric between the cleavable linker and the active drug, or are themselves part of a cleavable linker.
  • the mechanism of action of the suicide linker is that when the cleavable linker is broken under suitable conditions, the suicide linker can spontaneously rearrange the structure and release the active drug attached thereto.
  • Common suicide linkers include p-aminobenzyl alcohols (PAB) and beta-glucuronides.
  • This patent may use the following abbreviations and have the specified definitions: Boc, tert-butoxycarbonyl; DCC, cyclohexylcarbodiimide; DCM: dichloromethane; DIPEA: diisopropylcarbodiimide; DMF: N , N-dimethylformamide; DMAP: 4-(N,N-dimethylamino)pyridine; HATU: 2-(7-oxobenzotriazole)-N,N,N',N'- Tetramethylurea hexafluorophosphate; HPLC: high performance liquid chromatography; PEG: polyethylene glycol; TFA: trifluoroacetic acid; THF: tetrahydrofuran; PBS: phosphate buffer solution (pH 7.0-7.5).
  • the pharmaceutically acceptable excipients include any carrier, diluent, adjuvant or excipient such as preservatives and antioxidants, fillers, disintegrating agents, wetting agents, emulsifying agents, suspending agents, solvents, dispersion media. , coating agents, antibacterial and antifungal agents and absorption delaying agents, and the like.
  • the use of such media and agents for the use of pharmaceutically active substances is well known in the art.
  • any conventional media or agents that are incompatible with the active ingredients their use in therapeutic compositions is also contemplated.
  • the additional active ingredient can also be incorporated into the compositions.
  • the antibody drug conjugate provided by the invention with two different drugs can effectively enhance the drug effect and obtain better therapeutic effect because of different action mechanism of the drug.
  • the method of the present invention provides a combination of site-directed coupling and non-spotting coupling, and the antibody drug conjugate containing two different drugs can be obtained by simple coupling.
  • the PCR reaction system (50 ⁇ L) was prepared in the following proportions, mixed thoroughly and then centrifuged instantaneously.
  • the antibody molecules having a monomeric ratio of more than 95% after preliminary purification were exchanged into a phosphate buffer solution containing EDTA using an ultrafiltration centrifuge tube at a concentration of 10 mg/ml.
  • TCEP was added 10 times the number of molecules of the antibody, and reacted at room temperature for 2 h.
  • the solution was changed to a phosphate buffer of pH 6.5 using an ultrafiltration centrifuge tube, and DHAA was added 10 times the number of molecules of the antibody, and reacted at room temperature for 2 hours. Then, a payload 1 of 3 times the number of molecules of the antibody was added, and the reaction was carried out for 4 hours at room temperature.
  • the solution was exchanged into a phosphate buffer containing EDTA using an ultrafiltration centrifuge tube having a molecular weight cut off of 30 KDa, and the unconjugated payload 1 was removed.
  • TCEP was added 10 times the number of molecules of the antibody, and reacted at room temperature for 8 hours.
  • the interchain disulfide bond was opened, and the number of free thiol groups was determined by the Ellman method to determine whether the disulfide bonds were all open. Then, add 2 times the antibody molar number of payload 2, and react at room temperature for 8 hours.
  • the solution was exchanged into PBS using an ultrafiltration centrifuge tube having a molecular weight cut off of 30 KDa, and unconjugated payload 2 was removed.
  • ADC hydrophobic interaction chromatography
  • MPB mobile phase B
  • MPB consisting of 1.5 M ammonium sulfate and .025 M sodium phosphate
  • MPB consisting of 0.025 M sodium phosphate, 25% isopropanol.
  • the sample loading was approximately 20 ⁇ g and the gradient elution was completed in 15 minutes.
  • UV 280 nm was used for detection, the stronger the water-transfer sample, the later the peak.
  • ADC sample Take a certain amount of ADC sample, add it to human plasma from which human IgG has been removed, repeat three tubes of each ADC, incubate in a 37 °C water bath, incubate for 0h, 72h, and then take out the ADC sample, and add ProteinA (MabSelect SuReTM) to each tube.
  • LX Lot: #10221479GE washed with PBS 100 ul, shaking with a vertical mixer for 2 h, after washing and eluting steps to obtain the ADC after incubation.
  • RP-HPLC detection of ADC samples for specific time of incubation. Plasma stability.
  • the colloid is formed in the reaction solution and attached to the bottom of the bottle; the colloid and the reaction solution are taken, dissolved in DCM, and the plate is washed with water, and the reaction is complete after 008, and the post-treatment is carried out.
  • reaction mixture was poured into 100 ml of water, and a large amount of solid was precipitated, which was extracted with DCM (60 ml ⁇ 3), and the organic phase was washed three times with brine, dried over anhydrous sodium sulfate and evaporated.
  • the VC-PABA obtained in the above step was dissolved in dry DMF, added with McOSu, DIEA, stirred at room temperature for 2 h, and the reaction was monitored by HPLC. After adding isopropyl ether at room temperature, stirring and crystallization for 2 h, then cooling to 0 ° C, stirring for 1 h, filtering, filter cake Wash twice with isopropyl ether and dry under reduced pressure
  • Mc-VC-PABA 8 g, 14 mmol was dissolved in 120 mL dry DMF, and bis(4-nitrophenyl) carbonate (8.5 g, 28 mmol, 2 eq), DIEA (3.66 ml) , 21.0 mmol, 1.5 eq).
  • the reaction mixture was stirred at room temperature for 1 h, then the mixture was evaporated, evaporated, evaporated, evaporated, evaporated, evaporated,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,
  • a single-mutation cetux antibody was prepared by the general method A, and then the general procedure A was repeated to obtain a double-mutation cetux antibody.
  • a single-mutation cetux antibody was prepared by the general method A, and then the general procedure A was repeated to obtain a double-mutation cetux antibody.
  • the Cetuximab antibody having a monomeric ratio of more than 95% after preliminary purification was exchanged into a phosphate buffer solution containing EDTA at a concentration of 10 mg/ml using an ultrafiltration centrifuge tube.
  • TCEP was added 10 times the number of molecules of the antibody, and reacted at room temperature for 8 hours.
  • the interchain disulfide bond was opened, and the number of free thiol groups was determined by the Ellman method to determine whether the disulfide bonds were all open. Then, a payload 10 times the number of molecules of the antibody was added, and the reaction was carried out for 8 hours at room temperature.
  • the SEC assay was performed on the ADC described in the present invention according to the general method.
  • the data is summarized in the following table. From the results, it can be seen that the ADC ratio of the ADC drug with different toxins is slightly lower than that of the single toxin.
  • Human pharyngeal squamous cell carcinoma Fadu was cultured in vitro and inoculated subcutaneously in the back of nude mice according to the number of cells 5 ⁇ 10 6 . After the tumor grew to 70-90 mm 3 , the mice were given a single dose of ADC drug 5 mg/kg (tail vein injection). The vehicle control group was weighed regularly, the tumor volume was measured, and the drug efficacy against the Fadu model was evaluated by investigating the antitumor efficacy of the ADC drug. The results show that ADC drugs with different toxins have better in vivo efficacy than single toxin ADC drugs.

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Abstract

本发明公开了一种具有两种不同药物的抗体药物偶联物，本发明将一种药物以定点偶联方式连接到抗体的半胱氨酸残基上，并将第二种不同作用机制的细胞毒性药物以非定点偶联的方式连接到抗体的半胱氨酸残基上。

Description

具有两种不同药物的抗体药物偶联物 技术领域
本发明涉及一种具有两种不同药物的抗体药物偶联物.
背景技术
抗体偶联药物(ADC)作为新型的靶向药物，一般由三部分组成：抗体或抗体类配体，小分子药物以及将配体和药物偶联起来的连接子。抗体偶联药物利用抗体对抗原的特异性识别，将药物分子运输至靶细胞附近并有效释放药物分子，达到治疗目的。2011年8月，美国食品药品监督管理局(FDA)批准西雅图基因公司研制的用于治疗霍奇金淋巴瘤以及复发性变性大细胞淋巴瘤(ALCL)的ADC新药Adecteis TM上市，临床应用证明了此类药物的安全性和有效性。
ADC药物抗体主要作用为靶向传递功能，最终发挥药效为所偶联的药物分子。目前应用于ADC领域的药物分子根据作用机制不同分为：微管抑制剂、DNA损伤剂、拓扑异构酶抑制剂、RNA聚合酶抑制剂、蛋白翻译抑制剂等。
在ADC药物中，抗体将药物分子靶向携带至肿瘤细胞附近，在肿瘤细胞周围或细胞内释放药物分子。然而同时，肿瘤在此治疗过程中也发展出对抗药物分子的不同机制，包括以PgP蛋白泵将毒素分子泵出细胞，从而逃避被杀灭。当前，ADC药物开发主要集中于单个抗体连接单一作用机制药物，现代医学已经证明，化疗过程中的不同药物联合用药可明显增强药效。但是ADC药物随着所带药物数量及种类的增加，分子的脂溶性增大，在血浆中稳定性会明显降低，药效不仅没得到提高，还会带来副作用。
在当前已知文献中，美国索伦托治疗有限公司专利CN106132431(A)通过分别利用抗体分子中赖氨酸上氨基残基(又称K-LOCK技术)和半胱氨酸巯基残基(C-LOCK技术)实现将不同药物与抗体连接。由于其偶联方式的局限性，C-LOCK技术往往得到的DAR是2-4，而且K-LOCK技术也具有明显的局限性：在赖氨酸连接药物抗体偶联比(DAR)＞2时，由于单个抗体分子中含有赖氨酸约88个，如此多数量的赖氨酸导致氨基偶联选择性差，偶联数量和偶联位置难以确定，虽然可以通过偶联条件进行一定程度的控制，但是在制备带有两种不同药物的复杂的抗体偶联物时会带来极大的挑战。并且专利CN106132431并未给出动物体内药效数据及血浆稳定性的报道。
在ADC药物中，亟需一种创新的偶联方法以获得血浆稳定，偶联了两种不同药物的抗体偶联药物，并且实现药物数量更方便的组合。本发明惊人的满足了以上需求。
发明内容
本发明旨在提供一种具有两种不同药物的抗体-药物偶联物。通过设计偶联物，将两种不同药物分子连接至同一抗体。由于不同作用机制药物的联合使用，偶联物可有效提高药效。
具体的，本发明提供了一种如式I的包括不同药物的抗体药物偶联物或其药学上可接受的盐
Figure PCTCN2018101215-appb-000001
其中Ab为抗体部分；
L1、L2为任选的可连接至药物的连接单元；
D1、D2为药物单元；
m、n为2-8的整数。
优选的，药物D1、D2为作用机制不同的抗肿瘤药物。
更优选的，药物D1、D2分别优选自微管蛋白结合剂、DNA烷化剂、DNA嵌入剂、酶抑制剂、免疫调控剂、肽和核苷酸。
在一些优选例中，所述的m优选为2、4。
本发明的一个方面，药物D1优选以定点偶联方式，D2以非定点偶联方式连接至抗体
在一些实施例中，药物D1优选的定点偶联方式为定点突变抗体原有氨基酸至半胱氨酸或通过插入一个半胱氨酸或一段含半胱氨酸的多肽，通过以上方法引入的半胱氨酸巯基与linker-毒素相偶联。D2优选的非定点偶联方式为通过抗体原有的链间二硫键与linker-毒素相偶联。
优选的，本发明中L1、L2具有下式二：
Figure PCTCN2018101215-appb-000002
其中C是末端任选的可延伸单元，E为任选的可断裂单元，F为间隔单元,下标e，f为0或1。波浪线表示到丁二酰亚胺和药物单元的连接位点。
较优选的，所述的抗体药物偶联物，可断裂单元E在e＝0时不存在，在e＝1时存在。当存在时，E所述的可断裂单元通过肿瘤相关蛋白酶或酸性PH实现与药物单元D或间隔单元F
较优选的，所述的抗体药物偶联物，间隔单元F在f＝0时不存在，在f＝1时存在。当F存在时，F选自对氨基苯甲醇或与乙二胺单元及其衍生物组成的组。
较优选的，本发明任一项所述的药物-配体偶联物化合物包含其药学上可接受的盐，以及药学上可接受的稀释剂运载体或赋形剂的药物组合物。
较优选的，本发明包括向所述患者给予前述权利要求中任一项所述的药物-配体偶联物，其中所述患者患有肿瘤、自身免疫疾病或感染性疾病，并且所述的药物-配体偶联物的抗体特异性结合至所述癌症、自身免疫疾病的靶细胞
附图说明
图一为Payload S MS-TOF检测结果图。
图二为Payload E MS-TOF检测结果图。
图三显示了为带不同药物的ADC和对照ADC检测结果图。
具体实施方式
发明人经过广泛而深入的研究，惊奇的发现具有两种不同作用机制药物的抗体-药物偶联物相比于传统的单一作用机制的ADC药物具有更好的体内外药效。
具体的，本发明提供的如下式所示的抗体药物偶联物
Figure PCTCN2018101215-appb-000003
缩写和定义
除非另有说明，否则如本文所用的以下术语和短语旨在具有以下含义。当本文中使用商标名称时，除非上下文中另有指明，否则商标名称包括所述商标名称产品的产品配方、通用药物和活性药物成分。
除非本文中另有说明，文中所用的化合物的“衍生物”是指具有与化合物相似的化学结构但还含有至少一个化合物中不存在的化学基团和/或缺少至少一个化合物中存在的化学基团的物质。衍生物所比较的化合物被称为“母体”化合物。通常，“衍生物”可在一个或多个化学反应步骤中由母体化合物产生。
如本文所用，“抗体”或“抗体单元”在其所属的范围内，包括抗体结构的任何部分。这一单元可以结合，反应性关联，或者络合一个受体，抗原，或者靶向细胞群体具有的其它受 体单元。抗体可以是任何蛋白或蛋白类分子，它可以结合，络合，或者与待治疗或生物改造的细胞群体的一部分发生反应。在一些实施方式中，连接子共价连接至抗体的硫原子。在一些方面中，硫原子是半胱氨酸残基的硫原子，其形成抗体的链间二硫键。在另一方面中，硫原子是已经导入配体单元的半胱氨酸残基的硫原子，其形成抗体的链间二硫键。在另一方面中，硫原子是已经导入配体单元的半胱氨酸残基的硫原子(例如，通过定点诱变或化学反应)。在其他方面中，连接子结合的硫原子选自形成抗体的链间二硫键的半胱氨酸残基或已经引入配体单元的额半胱氨酸残基(例如，通过定点诱变或化学反应)。在一些实施方式中，按照Kabat(Kabat E.A等，(1991))《免疫学感兴趣的蛋白质序列》(Sequences of proteins of Immunological Interest),第五版，NIH出版物91-3242)中的EU索引编号系统。
本发明中组成抗体药物偶联物的抗体最好保持其原有野生状态时的抗原结合能力。因此，本发明中的抗体能够，最好专一性的与抗原结合。涉及的抗原包括，例如，肿瘤相关抗原(TAA)，细胞表面受体蛋白和其他细胞表面分子，细胞存活调节因子，细胞增殖调节因子，与组织生长与分化相关的分子(如已知或预知的具有功能性的)，淋巴因子，细胞因子，参与细胞循环调节的分子，参与血管生成的分子，以及与血管生成有关的分子(如已知或预知的具有功能性的)。肿瘤相关因子可以是簇分化因子(如CD蛋白)。
本文所述应用在抗体药物偶联物中的抗体包括，但不局限于，针对细胞表面受体和肿瘤相关抗原的抗体。这样的肿瘤相关抗原是业内所熟知的，可以通过业内熟知的抗体制备方法和信息来制备。为了开发可用于癌症诊断与治疗的有效的细胞水平目标物，研究人员力图找寻跨膜或其他肿瘤相关多肽。这些目标物能够特异性的表达在一种或多种癌细胞表面，而在一种或多种非癌细胞表面表达很少或不表达。通常，相对于非癌细胞表面而言，这样的肿瘤相关多肽在癌细胞表面更加过度表达。确认这样的肿瘤相关因子，可大大提高基于抗体治疗癌症的专一靶向特性。
肿瘤相关抗原包括但不局限于业内所熟知的肿瘤相关抗原。与肿瘤相关抗原对应的核酸和蛋白序列可参见公开数据库，例如Genbank。抗体靶向对应的肿瘤相关抗原包括所有的氨基酸序列变种和同种，与参考文献中确认的序列具有至少70％，80％，85％，90％，或者95％的同源性，或者具备与引用文献中的肿瘤相关抗原序列具有完全一致的生物性质和特征。
术语“抑制”或“的抑制”指，减少了可检测的量，或完全阻止。
术语“癌症”指的是以失调的细胞生长为特征的生理病症或疾病。“肿瘤”包括癌细胞。
术语“自身免疫疾病”是源自针对个体自身的组织或蛋白质的疾病或紊乱。
本文中所用“定点偶联”优选为定点突变抗体原有氨基酸至半胱氨酸或在抗体中插入一 个半胱氨酸或一段含半胱氨酸的多肽，通过以上方法引入的半胱氨酸巯基与连接子中的丁二酰亚胺相偶联。“非定点偶联”方式为通过利用抗体原有的链间二硫键与连接子-毒素相偶联。
本文中所用的短语“药学上可接受的盐”指的是，化合物(例如，药物，药物-接头或配体-接头-药物偶联物)的药学上可接收到有机或无机盐。该化合物可含有至少一个氨基或羧基，并且因此可与相应的酸或碱形成加成盐。示例性的盐包括但不限于：硫酸盐、三氟乙酸盐、柠檬酸盐、乙酸盐、草酸盐、氯化物、溴化物、碘化物、硝酸盐、硫酸氢盐、磷酸盐、酸性磷酸盐、异烟酸盐、乳酸盐、水杨酸盐、酸性柠檬酸盐、酒石酸盐、油酸盐、单宁酸盐、泛酸盐、酒石酸氢盐、抗坏血酸盐、水杨酸盐、甲酸盐、本甲酸盐、谷氨酸盐、甲磺酸盐、乙磺酸盐、苯磺酸盐、对甲苯磺酸盐，钾盐、钠盐等。另外，药学上可接受的盐在结构中具有超过一个的带点原子。其中多个带电原子是药学上可接受的盐的一部分的示例能有多个抗衡例子。例如，药学上可接受的盐具有一个或多个带电原子和/或一个或多个抗衡原子。
药物是指：一种用于癌症治疗的细胞毒性药物,高活性药物包括但不局限于美登素或类美登素,海兔毒素10(Dolastatin10)的类似物，卡奇霉素类药物阿霉素类吡咯并苯二氮唑类(pyrrolo[2,1-c][1,4]benzodi-azepines,PBDs)或者PBD二聚体类(PBD dimmers)以及衍生物鹅膏毒素或者其衍生物，中等活性药物包括但不局限于苯并二吡咯类抗生素(duocarmycins，CC-1065等)喜树碱类化合物包括喜树碱，羟基喜树碱，SN-38，依喜替康，伊立替康等。
另一方面，药物并不仅仅局限于上述提到的类别，还包括所有可用于抗体药物偶联物的药物。
按照在细胞内药物释放的机制，如本文所用，“连接单元”或“抗体药物偶联物的连接单元”可被分为两类：不可断裂连接单元和可断裂连接单元。
对于含有不可断裂连接单元的抗体药物偶联物，其药物释放机制为：偶联物与抗原结合并被细胞内吞后，抗体在溶酶体中被酶解，释放出由小分子药物，连接子，和抗体氨基酸残基共同组成的活性分子。由此带来的药物分子结构改变并不减弱其细胞毒性，但由于活性分子是带电荷的(氨基酸残基)，从而导致其不能渗入邻近细胞。因此，此类活性药物不能杀死邻近不表达靶向抗原(抗原阴性细胞)的肿瘤细胞(旁观者效应，bystander effect)(Ducry等，2010，Bioconjugate Chem.21:5-13)。
可断裂连接单元，顾名思义，可以在目标细胞内断裂并释放出活性药物(小分子药物本身)。可断裂连接子可分为两个主要的类别：化学不稳定连接子和酶不稳定连接子。
化学不稳定连接子可以由于血浆和细胞质性质的不同而选择性的断裂。这样的性质包括pH值，谷胱甘肽浓度等。
对pH值敏感的连接子，通常又称为酸断裂连接子。这样的连接子在血液的中性环境下相对稳定(pH7.3-7.5)，但是在弱酸性的内涵体(pH5.0-6.5)和溶酶体(pH4.5-5.0)内将会被水解。第一代的抗体药物偶联物大多应用这类连接子，例如腙，碳酸酯，缩醛，缩酮类。由于酸断裂连接子有限的血浆稳定性，基于此类连接子的抗体药物偶联物通常具有较短的半衰期(2-3天)。这种较短的半衰期在一定程度上限制了pH敏感连接子在新一代抗体药物偶联物中的应用。
对于谷胱甘肽敏感的连接子，又称二硫键连接子。药物释放是基于细胞内谷胱甘肽的高浓度(毫摩尔范围)与血液中相对较低的谷胱甘肽浓度(微摩尔范围)差异引起的。对于肿瘤细胞而言尤其如此，其低含氧量导致还原酶的活性增强，因而导致更高的谷胱甘肽浓度。二硫键具有热力学稳定性，因此在血浆中具有较好的稳定性。
酶不稳定连接子，如肽连接子，能够更好的控制药物释放。肽连接子能够被溶酶体内蛋白酶，如组织蛋白酶(Cathepsin B)或纤溶酶(在一些肿瘤组织中此类酶含量增加)，有效的切断。这种肽连接被认为在血浆循环中非常稳定，这是因为细胞外不合宜的pH值及血清蛋白酶抑制剂导致蛋白酶通常不具备活性。鉴于较高的血浆稳定性和良好的细胞内断裂选择性和有效性，酶不稳定连接子被广泛用做抗体药物偶联物的可断裂连接子。典型的酶不稳定性连接子包括Val-Cit(vc)，Phe-Lys等。
自杀式连接子一般嵌合在可断裂连接子与活性药物之间，或者本身就是可断裂连接子的一部分。自杀式连接子的作用机制是：当可断裂连接子在合宜的条件下断裂后，自杀式连接子能够自发的进行结构重排，进而释放与之连接的活性药物。常见的自杀式连接子包括对氨基苄醇类(PAB)和β-葡萄糖醛酸苷类(β-Glucuronide)等。
本专利可以使用以下缩写并具有指定的定义：Boc，叔丁氧基羰基；DCC，环二己基碳二亚胺；DCM：二氯甲烷；DIPEA：二异丙基碳二亚胺；DMF：N、N-二甲基甲酰胺；DMAP:4-(N、N-二甲基氨基)吡啶；HATU:2-(7-氧化苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯；HPLC:高效液相色谱；PEG：聚乙二醇；TFA:三氟乙酸；THF:四氢呋喃；PBS:磷酸盐缓冲溶液(PH7.0-7.5)。
药学上可接受的赋形剂包括任何载体，稀释剂，佐剂或赋形剂，如防腐剂和抗氧剂，填充剂，崩解剂，湿润剂，乳化剂，悬浮剂，溶剂，分散介质，包衣剂，抗细菌剂和抗真菌剂和吸收延迟剂等。这样的介质和药剂用于药物活性物质的使用在本领域时公知的。除了任何常规的介质或试剂与活性成分不相容以外，其在治疗组合物中的用途也被考虑到。作为合适的治疗组合，还可以将补充的活性成分掺入组合物中。
本发明的主要优点在于：
1、本发明提供的具有两种不同药物的抗体药物偶联物，因为其药物不同作用机制，可有效增强药效，获得更好的治疗效果。
2、本发明提供的采用定点偶联与非定点偶联结合的方法，可简单易得的偶联获得含两种不同药物的抗体药物偶联物。
下面结合具体实施例，进一步阐述本发明，应理解，这些实施例只用于说明本发明，而不用于限制本发明的范围。下列实施例中未注明具体条件的试验方法，通常按照常规条件或按照制造厂商所建议的条件。除非另外说明，否则所有的百分数、比例、比率、或份数按重量计。
除非另行定义，文中所使用的所有专业和科学用于与本领域熟练人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法及材料皆可应用于本发明方法中。文中所述的较佳实施方法与材料仅作示范之用。
本发明下列实施例中采用的通用步骤是：
通用步骤A 抗体定点突变
按以下比例配制PCR反应体系(50μL)，充分混匀后瞬时离心，体系包含：
Figure PCTCN2018101215-appb-000004
PCR扩增后，加入1μL DpnI(10U/μL)，37℃孵育3h；取1μL DpnI处理后的PCR产物，进行E.coli转化实验，如下：冰浴30min；热激42℃30sec；冰浴2min；加入150μL LB液体培养基，37℃150rpm培养30min；3000×g RT离心3min后，去掉上清，留下50μL培养上清悬浮菌体后，涂布于含Amp的LB固体培养基上；37℃培养16h；挑取单菌落，转接于5mL含Amp(pTT5)的LB液体培养基中，过夜培养后提取质粒；进行PCR测序鉴定。 通用步骤B偶联不同毒素制备ADC
将通过初步的纯化后单体率大于95％的抗体分子，使用超滤离心管换液至含有EDTA的磷酸盐缓冲液中，浓度10mg/ml。加入10倍于抗体摩尔分子数的TCEP，室温下反应2h。使用超滤离心管换液至pH6.5的磷酸缓冲液中，再加入10倍于抗体摩尔分子数的DHAA，室温下反应2h。然后加入3倍于抗体摩尔分子数的payload 1，室温下反应4h。反应结束后，使用截留分子量为30KDa的超滤离心管换液至含有EDTA的磷酸盐缓冲液中，并去除未偶联的payload 1。加入10倍于抗体摩尔分子数的TCEP，室温下反应8h。打开抗体链间二硫键，并用Ellman方法测定游离巯基数，判断二硫键是否全部打开。然后加入10倍于抗体摩尔分子数的payload 2，室温下反应8h。反应结束后，使用截留分子量为30KDa的超滤离心管换液至PBS中，并去除未偶联的payload 2。
通用步骤C
药代动力学研究
检测血清中抗体的ELISA方法：抗抗体2ug/ml 4℃包被过夜，PBST洗板3次，1％BSA+PBST 37℃封闭1hr；孵育血清样品，PBST洗板3次；37℃孵育检测抗体(抗Fc的单抗或多抗(HRP标记))1hr，PBST洗板3次，TMB显色，2M H2SO4终止，酶标仪读值。通用步骤D
疏水性相互作用色谱(HIC)测定
使用疏水性相互作用色谱(HIC)来进行对ADC的分析。通过0-100％流动相B(MPB)脱，其中流动相A(MPA)由1.5M硫酸铵和.025M磷酸钠组成，并且MPB由0.025M的磷酸钠、25％异丙醇组成。样品上样量约为20μg，梯度洗脱在15分钟完成。用UV280nm进行检测，输水性越强的样品越晚出峰。
通用步骤E
血浆稳定性研究
取一定量的ADC样品,加入到已去除人IgG的人血浆中,每种ADC重复三管，放置37℃水浴中孵育,分别孵育0h、72h后，取出ADC样品，每管加入ProteinA(MabSelect SuReTM LX Lot:#10221479GE，用取PBS洗涤过的)100ul，垂直混合仪晃动吸附2h,经过洗涤洗脱步骤,获得孵育后的ADC.对孵育特定时间的ADC样品进行RP-HPLC检测.判定样品的血浆稳定性。
实施例1-6
Figure PCTCN2018101215-appb-000005
实施例1 化合物1的合成
Figure PCTCN2018101215-appb-000006
单口瓶中加入Fmoc-Lys(mmt)-OH(2g，3.2mmol，1eq)，PABA(788.16mg，6.4mmol，2eq)，HATU(1.34g，3.52mmol，1.1eq)，DIEA(1.24g，9.6mmol，3eq)及20mlDMF，搅拌溶解后氮气保护，25℃反应；点板检测，赖氨酸反应完全。向反应液中加入200ml水，有大量固体析出，EA(60mlx3)萃取，有机相用盐水洗涤三次，无水硫酸钠干燥，旋干得黄色油状物 2.85g。将上步中间体用20ml二乙胺溶解，氮气保护，25℃反应；点板检测，原料反应完全。将溶剂旋干并加入100ml EA溶解，盐水洗涤三次，无水硫酸钠干燥，旋干得产品浅棕色油状物2.58g。柱层析纯化：以DCM：MeOH＝100:1至50:1为流动相剂进行梯度洗脱，得产品白色泡状固体750mg。
H 1NMR(400MHz,CDCl 3):8.01(s,1H),7.63-7.65(m,2H),7.32-7.40(m,6H),7.29-7.31(m,8H),6.87-6.89(m,2H),4.62(s,2H),3.84(s,3H),3.37(t,1H),2.55(m,2H),1.89(m,2H),1.40(m,2H),1.24(m,2H)。
实施例2 化合物2的合成
Figure PCTCN2018101215-appb-000007
单口瓶中加入化合物1(300mg，0.574mmol，1eq)N 3PEG 8COOH(350mg，0.631mmol，1.1eq)，HOBT(85mg，0.631mmol，1.1eq)DIEA(149mg，1.15mmol，2.0eq)及4ml干燥DMF，氮气保护，冰水浴降温搅拌溶清；加入HATU(240mg，0.631mmol，1.1eq)，25℃反应；点板检测，005反应完全，后处理。向反应液中加入50ml水，EA(50mlx3)萃取，有机相用盐水洗涤三次，无水硫酸钠干燥，旋干得浅黄色油状物920mg。薄层板制备纯化：用DCM：MeOH＝15:1为展开剂展开一次，纯化得产品淡黄色油状物500mg。
实施例3 化合物3的合成
Figure PCTCN2018101215-appb-000008
单口瓶中加入化合物2250mg，0.5mmol，1.5eq)、DMAP(183mg，1.5mmol，4.5eq)及重蒸的10ml DCM，溶清后氮气保护，加入三光气(52mg，0.175mmol，0.52eq)，室温反应，反应液变浑浊，搅拌30s后澄清；点板检测，002反应完全，加入EL-006(353mg，0.333mmol，1.0eq)；点板检测，EL-006反应完全，加入3ml甲醇淬灭，旋干得黄色固体900mg。薄层板制备纯化：用DCM：MeOH＝10:1为展开剂展开一次，纯化得产品微黄色固体350mg。
实施例4 化合物4的合成
Figure PCTCN2018101215-appb-000009
单口瓶中加入化合物3(350mg，0.215mmol，1.0eq)及DCM，溶解后氮气保护；加入1M TBAF溶液(0.537ml，2.5eq)，反应液变为橙黄色，加入HAc(1.07mmol)后颜色变为金黄色，25℃反应；点板检测，007反应完全，后处理。反应液加入100mlDCM稀释，盐水洗涤三次，无水硫酸钠干燥，旋干得产品410mg。
实施例5 化合物5的合成
Figure PCTCN2018101215-appb-000010
单口瓶中加入化合物4(400mg，0.273mmol，1eq)、SMCC-氨基乙炔(186mg，0.682mmol，2.5eq)及10mlDMSO，搅拌溶解后氮气保护，加入CuBr(78mg，0.546mmol，2eq)及2.5ml水，25℃搅拌反应；
反应液中有胶体生成，附于瓶底；取胶体及反应液，DCM溶解，水洗后点板，008反应完全，后处理。
反应液倒入100ml水中，有大量固体析出，DCM(60mlx3)萃取，有机相用盐水洗涤三次，无水硫酸钠干燥旋干得黄色固体550mg。
实施例6 化合物6(Payload S)的合成
Figure PCTCN2018101215-appb-000011
单口瓶中加入550mg EL-009、1ml苯甲醚及10mlDCM，氮气保护，溶清，冰水浴降温；加入2ml二氯乙酸，保持冰水浴，反应液变为黄色；升至25℃反应；点板检测，EL-009反应完全。30℃旋去大部分溶剂，加入20ml甲叔醚析晶，有大量黄色固体析出，冰水浴降温固化1h，过滤，油泵干燥，得粗品黄色粉末350mg。HPLC制备：以乙腈-水(0.9％TFA)为流动相进行梯度洗脱，冻干后共得产品黄色粉末130mg(M+H＝1480.69)。
实施例7-12
Figure PCTCN2018101215-appb-000012
实施例7化合物7的合成
Figure PCTCN2018101215-appb-000013
于500mL三口瓶中加入10g Fmoc-甘氨酰-甘氨酸(SN-100,0.028mol，1eq)、四氢呋喃300mL，甲苯100mL，搅拌溶清，再加入17.5g四乙酸铅(0.0395mol，1.4eq)，氮气保护，室温搅拌至析出固体，加入2.7mL吡啶(0.0338mol，1.2eq)，升至回流反应(外温80℃)，TLC监测反应进程，约3h反应结束。反应液浓缩，残余物加入水，用乙酸乙酯萃取，合并有机层，用饱和氯化钠洗涤，无水硫酸钠干燥，浓缩得粗品。经硅胶柱纯化(PE/EA＝5/1-1/1)得白色固体8g。
H 1NMR(400MHz,CDCl 3):8.11(s,1H),8.08(s,1H),7.87-7.89(m,2H),7.56-7.58(m,2H),7.28-7.33(m,4H),6.03(s,2H),4.70(d,2H),4.47(t,1H),3.86(s,2H),2.22(s,3H).
实施例8化合物8的合成
Figure PCTCN2018101215-appb-000014
于250mL单口瓶中加入5gSN-101(13.6mmol，1eq)，150mLTHF，搅拌溶清，再加入4.5g羟乙酸苄酯(27.2mmol，2eq)及0.52g对甲苯磺酸一水合物(2.71mmol，0.2eq)，室温反应，TLC监测。反应结束，加入饱和碳酸氢钠溶液，用乙酸乙酯萃取，合并有机层，用无水硫酸钠干燥，浓缩得粗品。经硅胶柱纯化(PE/EA＝5/1-1/1)得白色固体2.8g。LC-MS：474.1[M+H] +
H 1NMR(400MHz,CDCl 3):8.13(s,1H),8.10(s,1H),7.87-7.89(m,2H),7.56-7.58(m,4H),7.28-7.36(m,7H),5.21(s,2H),4.70(d,2H),4.47(t,1H),4.35(s,2H),3.86(s,2H),3.82(s,2H)。
实施例9：化合物9的合成
Figure PCTCN2018101215-appb-000015
于250mL单口瓶中加入5gFmoc-甘氨酰-甘氨酸(SN-100,14mmol，1eq)、5g对硝基酚(28mmol，2.5eq)、7.34gDCC(28mmol，2.5eq)及80mLTHF，室温搅拌，TLC监控。反应结束后，过滤，滤液减压浓缩得粗品，经硅胶纯化(DCM/MeOH＝500/1-100/1-50/1)得较纯品6.8g。
将上步得到的较纯品(14mmol，1eq)加入至250mL单口瓶中，再加入90mLTHF，碳酸钠水溶液(28mmol，2.4g，2eq，30mL水)，1.88gL-苯丙氨酸(11mmol，0.8eq)，氮气保护，室温反应，TLC监测。反应结束后，加入5％柠檬酸水溶液调节pH至3左右，用乙酸乙酯萃取，合并有机层，用无水硫酸钠干燥，浓缩得粗品。经硅胶柱纯化(DCM/MeOH＝200/1-100/1-50/1)得白色固体1.5g。LC-MS：502.2[M+H] +
实施例10 化合物13的合成
Figure PCTCN2018101215-appb-000016
于50mL单口瓶中加入1.5gSN-102(0.032mol，1eq)，8mLDMF及哌啶0.8mL，室温反应约1h，TLC监控。反应结束，减压浓缩除去DMF及哌啶。向粗品中加入15DMF，1.58gSN-106(0.032mol，1eq)、3.29gPyBOP(0.064mol，2eq)及1mLDIEA(0.064mol，2eq)，室温反应，TLC监测。反应结束后，制备纯化，冻干，得白色固体1g。LC-MS：758.2[M+Na] +
实施例11 化合物16的合成
Figure PCTCN2018101215-appb-000017
于25mL单口瓶中加入150mg化合物13(0.2mmol，1eq)，8mL甲醇及1mLDMF，溶清，再加入45mg 5％Pd/C，氢化反应，TLC监控，约2h完成。过滤，滤液中加入1.8mL二乙胺，室温搅拌约2h，反应结束。减压浓缩得粗品。粗品中加入4mLDMF，溶清，加入62mgMC-OSu(0.2mmol，1eq)，66uLDIEA(0.4mmol，2e)，HPLC监测反应。制备纯化，冻干，得产品30mg。LC-MS：512.2(碎片峰)
实施例12 化合物17的合成
Figure PCTCN2018101215-appb-000018
于25mL单口瓶中加入30mg化合物16(0.048mmol，1eq)、25mg依喜替康甲磺酸盐(0.048mmol，1eq)，50mgPyBOP(0.097mmol，2eq)，16uLDIEA(0.097mmol，2eq)及2mLDMF，室温反应。HPLC监测，反应结束，反应液直接半制备纯化，冻干得产品13mg。LC-MS：1035.1[M+H] +
实施例13-17
Figure PCTCN2018101215-appb-000019
实施例13化合物19(Fmoc-VC-PABA)合成：
Figure PCTCN2018101215-appb-000020
将1.5g化合物18(Fmoc-Val-Cit)溶于14ml二氯甲烷和7ml甲醇混合溶剂中，加入4-氨基苄醇(445.2mg，3.62mmol)，随后加入EEDQ(1.5g，6mmol)，将反应液室温搅拌过夜，溶剂浓缩除去后于残留物中加入异丙醚搅洗30min，将固体过滤后加入异丙醚再次搅洗30min，过滤，得Fmoc-VC-PABA 1.5g，收率82％。M(+1)＝602.6.
实施例14 化合物20合成
Figure PCTCN2018101215-appb-000021
将2g Fmoc-VC-PABA溶于10mlDMF中，加入2ml哌啶，室温搅拌30min，TLC监测反应完全后以高真空油泵减压浓缩得黄色固体，不经纯化直接用于下一步反应。
实施例15 化合物21合成
Figure PCTCN2018101215-appb-000022
上步所得VC-PABA溶于干燥DMF中，加入McOSu，DIEA，室温搅拌2h，HPLC监控反应完全后加入异丙醚室温搅拌析晶2h，后降温至0℃继续搅拌1h，过滤，滤饼以异丙醚洗涤两次，减压干燥的
实施例16：化合物22的合成
Figure PCTCN2018101215-appb-000023
将类白色固体Mc-VC-PABA(8g，14mmol)溶于120ml干燥DMF中，向所得溶液中加入双(4-硝基苯基)碳酸酯(8.5g，28mmol，2eq)，DIEA(3.66ml，21.0mmol，1.5eq)。反应液室温搅拌1h，通过HPLC监控反应完全后反应液浓缩，以石油醚/乙酸乙酯析晶，过滤，减压干燥得产物9.7g棕黄色固体(94％)，不经进一步纯化直接用于下一步反应。
实施例17 化合物23的合成
Figure PCTCN2018101215-appb-000024
将上步所得500mg MC-VC-PAB-PNP溶于10ml干燥DMF中，向所得溶液中依次加入DIEA(0.2ml)及300mg MMAE，加完继续室温搅拌，HPLC监控反应至MMAE全部反应完全，HPLC制备纯化所得产品，冷冻干燥得白色固体300mg(M+1＝1302.3)，收率62％。
实施例18 Cet-4S-2M制备
以通法A制备得到单突变西妥昔抗体，并依照通法B完成payload M(DAR＝2)及payload S(DAR＝4)不同毒素ADC的制备编号Cet-4S-2M。
Figure PCTCN2018101215-appb-000025
实施例20 Cet-8S-4M制备
以通法A制备得到单突变西妥昔抗体，后重复通法A操作，获得双突变西妥昔抗体。依照通法B完成payload M(DAR＝4)及payload S(DAR＝8)不同毒素ADC的制备编号Cet-8S-4M。
Figure PCTCN2018101215-appb-000026
实施例21 Cet-8E-2M制备
以通法A制备得到单突变西妥昔抗体后依照通法B完成payload M(DAR＝2)及payload E(DAR＝8)不同毒素ADC的制备编号Cet-8E-2M。
Figure PCTCN2018101215-appb-000027
实施例22 Cet-4M的制备
以通法A制备得到单突变西妥昔抗体，后重复通法A操作，获得双突变西妥昔抗体。依照通法B完成带payload M(DAR＝4)毒素ADC Cet-4M。
Figure PCTCN2018101215-appb-000028
实施例23 Cet-8E的制备
将通过初步的纯化后单体率大于95％的西妥昔抗体，使用超滤离心管换液至含有EDTA的磷酸盐缓冲液中，浓度10mg/ml。加入10倍于抗体摩尔分子数的TCEP，室温下反应8h。打开抗体链间二硫键，并用Ellman方法测定游离巯基数，判断二硫键是否全部打开。然后加入10倍于抗体摩尔分子数的payload，室温下反应8h。反应结束后，使用截留分子量为30KDa的超滤离心管换液至PBS中，并去除未偶联的payload。制备得到非定点偶连payload E(DAR＝8)编号Cet-8E。
Figure PCTCN2018101215-appb-000029
实施例24 Cet-8S的制备
依实施例23中方法完成制备得到非定点偶连payload E(DAR＝8)编号Cet-8E。
Figure PCTCN2018101215-appb-000030
实施例25 抗体药物偶联物DAR测定
ADC编号 非定点偶联平均DAR1 定点偶联平均DAR2
Cet-4S-2M 3.81(S) 1.78(M)
Cet-8S-4M 6.35(S) 2.97(M)
Cet-8E-2M 7.54(E) 1.78(M)
Cet-4M -- 3.67(M)
Cet-8S 7.56(S) --
Cet-8E 7.92(E) --
实施例26 抗体药物偶联物SEC测定
依照通法对本发明中所述ADC完成SEC测定，数据总结如下表所示，从结果可知，带不同毒素的ADC药物与带单一毒素比较，单体率略有降低。
目标偶联物 单体率
Cet-4S-2M 84.60％
Cet-8S-4M 83.87％
Cet-8E-2M 92.91％
Cet-4M 99.82％
实施例27 体外细胞实验
ADC编号 IC 50(Fadu)(nm) IC 50(A431)(nm)
Cet-8S-2M 1.85 4.23
Cet-8S-4M 1.18 0.15
Cet-8E-2M 200～1000 0.08
Cet-4M 200～1000 0.06
Cet-8S 1.02 8.49
Cet-8E ～200 >1000
实施例28 体内药效实验
体外培养人咽鳞癌细胞Fadu，按细胞数量5×10 6接种于裸鼠背部皮下，待肿瘤长到70～90mm 3后，分组，单次给予ADC药物5mg/kg(尾静脉注射)，同时设溶媒对照组，定期称体重、测量肿瘤体积，通过考察ADC药物的抑瘤疗效等指标，来评价药物对于Fadu模型的药效。结果表明，带不同毒素的ADC药物相比于单一毒素ADC药物具有更好的体内药效。

Claims (17)

  1. 一种如式I的包括不同药物的抗体药物偶联物或其药学上可接受的盐
    Figure PCTCN2018101215-appb-100001
    其中Ab为抗体部分；
    L1、L2为任选的可连接至药物的连接单元；
    D1、D2为药物单元；
    m、n分别选自2-8的整数。
  2. 如权利要求1所述的包括不同药物的抗体药物偶联物或其药学上可接受的盐，其特征在于，D1、D2为不同的抗肿瘤药物。
  3. 如权利要求2所述的抗体药物偶联物，包括不同药物的抗体药物偶联物或其药学上可接受的盐，其特征在于，D1选自微管蛋白结合剂、DNA烷化剂、DNA嵌入剂、酶抑制剂、免疫调控剂、肽或核苷酸。
  4. 如权利要求2所述的包括不同药物的抗体药物偶联物或其药学上可接受的盐，其特征在于，D2选自微管蛋白结合剂、DNA烷化剂、DNA嵌入剂、酶抑制剂、免疫调控剂、肽或核苷酸。
  5. 如权利要求1所述包括不同药物的抗体药物偶联物或其药学上可接受的盐，其特征在于，药物D1以定点偶联方式，D2以非定点偶联方式连接至抗体。
  6. 如权利要求5所述包括不同药物的抗体药物偶联物或其药学上可接受的盐，其特征在于，药物D1的定点偶联方式为定点突变抗体原有氨基酸成为半胱氨酸或通过插入一个半胱氨酸或一段含半胱氨酸的多肽，通过以上方法引入的半胱氨酸巯基与linker-毒素相偶联；D2的非定点偶联方式为通过抗体原有的链间二硫键与linker-毒素相偶联。
  7. 如权利要求1所述的包括不同药物的抗体药物偶联物或其药学上可接受的盐，其特征在于，m优选为2、4、6、8；n优先为4、6、8。
  8. 如权利要求1所述的包括不同药物的抗体药物偶联物或其药学上可接受的盐，其特征在于，其特征在于，L1、L2是可裂解连接子或不可裂解连接子。
  9. 如权利要求1所述的包括不同药物的抗体药物偶联物或其药学上可接受的盐，其特征在于，L1、L2具有下式二：
    Figure PCTCN2018101215-appb-100002
    其中D是丁二酰亚胺或水解开环丁二酰亚胺，C是末端任选的可延伸单元，E为任选的可断裂单元，F为间隔单元，下标e，f为0或1；左侧波浪线表示到抗体巯基的连接位点，右侧波浪线表示到药物单元的连接位点。
  10. 如权利要求9所述的包括不同药物的抗体药物偶联物或其药学上可接受的盐，其特征在于，可断裂单元E在e＝0时不存在，在e＝1时存在；当存在时，E所述的可断裂单元通过肿瘤相关蛋白酶或酸性PH实现与药物单元D 1/D 2或间隔单元F间的断裂。
  11. 如权利要求9所述的包括不同药物的抗体药物偶联物或其药学上可接受的盐，其特征在于，间隔单元F在f＝0时不存在，在f＝1时存在；当F存在时，F选自对氨基苯甲醇或与乙二胺单元及其衍生物组成的组。
  12. 如权利要求1所述的包括不同药物的抗体药物偶联物或其药学上可接受的盐，其特征在于，其中所述抗体轻链包括kappa或λ同种型。
  13. 如权利要求1所述的包括不同药物的抗体药物偶联物或其药学上可接受的盐，其特征在于，其中所述抗体重链包括IgG1、IgG2、IgG3或IgG4同种型。
  14. 如权利要求12所述的包括不同药物的抗体药物偶联物或其药学上可接受的盐，其特征在于，半胱氨酸定点引入抗体，其中巯基(-SH)能够进行化学偶联。
  15. 一种包括权利要求1-13中任一项所述的包括不同药物的抗体药物偶联物或其药学上可接受的盐，以及药学上可接受的稀释剂运载体或赋形剂的药物组合物。
  16. 一种权利要求1-13中任一项所述的包括不同药物的抗体药物偶联物或其药学上可接受的盐，治疗肿瘤、自身免疫疾病或感染性疾病的用途。
  17. 如权利要求16用途，其特征在于，所述的包括不同药物的抗体药物偶联物或其药学上可接受的盐的抗体特异性结合至所述癌症、自身免疫疾病的靶细胞。
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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113941007A (zh) * 2020-07-16 2022-01-18 成都科岭源医药技术有限公司 一种串联的双药物链接组装单元及其应用
US11446292B2 (en) 2019-03-29 2022-09-20 Medimmune Limited Compounds and conjugates thereof
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RU2787617C2 (ru) * 2019-08-07 2023-01-11 Мабплекс Интернешнал Ко., Лтд. Конъюгаты антитело-лекарственное средство и их применение
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Publication number Priority date Publication date Assignee Title
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MX2019013690A (es) 2017-05-18 2020-01-27 Regeneron Pharma Conjugados de farmaco-proteina con ciclodextrina.
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WO2019136487A2 (en) 2018-01-08 2019-07-11 Regeneron Pharmaceuticals, Inc. Steroids and antibody-conjugates thereof
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WO2021190581A1 (zh) 2020-03-25 2021-09-30 江苏恒瑞医药股份有限公司 一种含抗体药物偶联物的药物组合物及其用途
CA3175048A1 (en) 2020-03-25 2021-09-30 Jiangsu Hengrui Pharmaceuticals Co., Ltd. Preparation method for antibody medicament conjugate
CN111643676B (zh) * 2020-07-10 2023-06-30 荣昌生物制药(烟台)股份有限公司 一种双特异性二聚体、双特异性二聚体-药物偶联物及其应用
WO2022194257A1 (zh) * 2021-03-17 2022-09-22 江苏恒瑞医药股份有限公司 一种喜树碱衍生物的制备方法
JP2023552733A (ja) 2021-05-08 2023-12-19 レメゲン シーオー．，エルティーディー． 抗クローディン－１８．２抗体及びその抗体薬物複合体

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106132431A (zh) * 2013-10-15 2016-11-16 索伦托治疗有限公司 具有靶向分子和两种不同药物的药物偶联物
CN106866822A (zh) * 2016-12-25 2017-06-20 四川百利药业有限责任公司 半胱氨酸改造的抗体‑毒素偶联物

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
MX2016009862A (es) * 2014-02-17 2016-10-31 Seattle Genetics Inc Conjugados de anticuerpo-farmaco hidrofilicos.
CN106467575B (zh) * 2015-08-18 2020-07-31 四川百利药业有限责任公司 半胱氨酸改造的抗体-毒素偶联物

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106132431A (zh) * 2013-10-15 2016-11-16 索伦托治疗有限公司 具有靶向分子和两种不同药物的药物偶联物
CN106866822A (zh) * 2016-12-25 2017-06-20 四川百利药业有限责任公司 半胱氨酸改造的抗体‑毒素偶联物

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RU2787617C2 (ru) * 2019-08-07 2023-01-11 Мабплекс Интернешнал Ко., Лтд. Конъюгаты антитело-лекарственное средство и их применение
CN113941007A (zh) * 2020-07-16 2022-01-18 成都科岭源医药技术有限公司 一种串联的双药物链接组装单元及其应用
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US12186403B2 (en) 2021-07-19 2025-01-07 Immunome, Inc. Immunoconjugates and methods

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