WO2018149413A1 - 靶向cd20的抗体偶联药物制剂 - Google Patents

靶向cd20的抗体偶联药物制剂 Download PDF

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WO2018149413A1
WO2018149413A1 PCT/CN2018/076835 CN2018076835W WO2018149413A1 WO 2018149413 A1 WO2018149413 A1 WO 2018149413A1 CN 2018076835 W CN2018076835 W CN 2018076835W WO 2018149413 A1 WO2018149413 A1 WO 2018149413A1
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antibody
conjugated
pharmaceutical preparation
mab
drug
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French (fr)
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吴幼玲
张玉杰
卢佳丽
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浙江特瑞思药业股份有限公司
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Priority to US16/486,862 priority Critical patent/US20210130487A1/en
Publication of WO2018149413A1 publication Critical patent/WO2018149413A1/zh

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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
    • A61K47/6849—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 receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/07—Tetrapeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68031—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being an auristatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—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
    • A61K47/6883—Polymer-drug antibody conjugates, e.g. mitomycin-dextran-Ab; DNA-polylysine-antibody complex or conjugate used for therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • C07K16/2887—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/06034—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms
    • C07K5/06052—Val-amino acid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered

Definitions

  • the present invention is in the field of biomedicine, and in particular, the present invention relates to an antibody-conjugated pharmaceutical preparation that targets CD20.
  • Lymphoma is a group of malignant tumors originating from lymph nodes and other extranodal reticular tissues. It has a wide variety and high incidence. Every year, tens of thousands of people in China lose their lives. CD20 is a non-glycosylated quadruple transmembrane phosphoprotein specifically expressed on the surface of B lymphocytes, which plays an important regulatory role in the differentiation and proliferation of B lymphocytes. The stable and specific expression of CD20 on the surface of B cells makes it an ideal target for the treatment of B cell lymphoma.
  • anti-CD20 monoclonal antibodies marketed by the FDA for the treatment of B cell lymphoma include Rituximab, Zevalin, Bexxar, and the like.
  • ADC Monoclonal-drug conjugate
  • Another object of the present invention is to provide a preparation method and use of the above antibody-conjugated pharmaceutical preparation.
  • an antibody-conjugated pharmaceutical preparation comprising:
  • mAb represents recombinant anti-CD20 mAb, and the recombinant anti-CD20 mAb is rituximab or a biosimilar thereof;
  • D represents a small molecule toxin, and D is one or more monomethylaustatatin;
  • L is a linker that links the antibody to the small molecule toxin
  • n is the average number of couplings of the small molecule toxins coupled to the antibody, and n is an integer or a non-integer of 4.2 ⁇ 1;
  • n is an integer or a non-integer of 4.2 ⁇ 0.5.
  • n is an integer or a non-integer of 4.2 ⁇ 0.3.
  • D is monomethyl auristatin-E (MMAE), monomethyl auristatin-D (MMAD), monomethyl auristatin-F (MMAF), or a combination thereof.
  • MMAE monomethyl auristatin-E
  • MMAD monomethyl auristatin-D
  • MMAF monomethyl auristatin-F
  • L is maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl.
  • the small molecule toxin is linked by a sulfhydryl group formed by reduction of a disulfide bond between the linker and the chain of the antibody.
  • the structure of the antibody-conjugated drug is as follows:
  • the formulation is an injection.
  • the formulation is a solution or lyophilizate.
  • the carrier or excipient is selected from the group consisting of a pH buffer, an osmotic pressure regulator, a lyophilized powder excipient, a protein protectant, a solubilizer, and water for injection.
  • the pH buffer comprises histidine hydrochloride.
  • the protein protectant comprises trehalose.
  • the osmotic pressure adjusting agent comprises mannitol.
  • the solubilizing agent comprises polysorbate 80.
  • the method for preparing the antibody-conjugated drug comprises the steps of:
  • a recombinant anti-CD20 mAb and a reducing agent are subjected to a reduction reaction, thereby containing a reaction system of the reduced recombinant anti-CD20 mAb; and the molar ratio of the monoclonal antibody to the reducing agent is 1:2.9 to 1:3.1; with
  • a reduction reaction is carried out in a buffer.
  • the reduced recombinant anti-CD20 mAb is a recombinant anti-CD20 mAb which is reduced to a thiol group by an interchain disulfide bond.
  • the reducing agent is tris(2-carboxyethyl)phosphine.
  • the buffer in the step (1), is a PB reaction buffer (pH 7.6).
  • the molar ratio of the monoclonal antibody to the reducing agent is 1:2.95 to 1:3.05; preferably, 1:2.99 to 1:3.01.
  • the reduction reaction is carried out at 25 ⁇ 1 °C.
  • the reduction reaction is carried out for 90 ⁇ 10 minutes.
  • the molar ratio of the monoclonal antibody to the small molecule toxin in the step (1) is 1:7.2 to 1:7.7; preferably, 1:7.4 to 1:7.6. .
  • step (2) the volume ratio of acetonitrile to water is 1:1.
  • the coupling reaction is carried out at 4 ⁇ 0.5 °C.
  • the coupling reaction is carried out for 60 ⁇ 10 minutes.
  • the mixing is a dropwise addition of a solution of a small molecule toxin in acetonitrile and water to the reaction system of the step (1).
  • the use of the antibody-conjugated pharmaceutical preparation of the first aspect of the invention which is characterized in that it is used for the preparation of an antitumor drug.
  • the tumor is a lymphoma or leukemia.
  • the tumor is a B cell non-Hodgkin's lymphoma or a chronic lymphocytic leukemia.
  • a method for non-therapeutic inhibition of tumor cells comprising the step of: adding the antibody-conjugated pharmaceutical preparation of the first aspect of the invention to a system comprising tumor cells.
  • the tumor cells are CD20-positive tumor cells, for example, Raji, Ramos, Daudi cells, and the like.
  • the present invention also provides a method of treating or preventing a tumor, the method comprising the step of administering to a subject in need thereof the antibody-conjugated pharmaceutical preparation of the first aspect of the invention.
  • Figure 1 is a hydrophobic interaction chromatogram identifying the drug loading of the antibody-conjugated drug TRS005.
  • Figure 2 shows the possible toxin binding sites and drug loadings of the formed monoclonal antibody-conjugated drugs.
  • An antibody suitable for use in the present invention is an antibody that targets CD20 and is a monoclonal antibody against recombinant anti-CD20.
  • the recombinant anti-CD20 mAb may be rituximab or a biosimilar.
  • Small molecule toxins suitable for use in the present invention are compounds having high cytotoxicity.
  • the small molecule toxin is one or more monomethylaustatatin; more preferably, the small molecule toxin is monomethyl auristatin-E (MMAE), monomethyl au Rustatin-D (MMAD), monomethyl auristatin-F (MMAF), or a combination thereof.
  • MMAE monomethyl auristatin-E
  • MMAD monomethyl au Rustatin-D
  • MMAF monomethyl auristatin-F
  • Linkers (L) suitable for use in the present invention are used to link the antibodies and small molecule toxins of the present invention.
  • the linker is a maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl group, for example, as shown in the following formula:
  • the present invention provides an antibody-conjugated drug comprising (a) a recombinant anti-CD20 monoclonal antibody and (b) a cytotoxic small molecule toxin linked together by a linker (L).
  • antibody-conjugated drug of the invention or “ADC of the invention” are used interchangeably and refer to a conjugate of an antibody of the invention and a small molecule toxin that binds via a linker.
  • the antibody-conjugated drug structure is as shown in Formula I:
  • mAb represents an antibody of the invention
  • D represents a small molecule toxin of the present invention
  • L is a linker that links the antibody to the small molecule toxin
  • n is the average number of couplings of the small molecule toxins coupled to the antibody, and n is an integer or a non-integer of 4.2 ⁇ 1;
  • n is an integer or a non-integer of 4.2 ⁇ 0.5.
  • n is an integer or a non-integer of 4.2 ⁇ 0.3.
  • the structure of the antibody-conjugated drug is as follows:
  • the invention provides a method for coupling an antibody-conjugated drug, which couples a small molecule toxin to an antibody through a specific linker, and greatly improves the lethality of the antibody against the tumor cell without changing the affinity of the antibody.
  • small molecule toxins and linkers are linked by conventional chemical synthesis methods, and commercially available small molecule toxins linked to a linker can also be used;
  • the free thiol group formed by reduction of the recombinantly expressed monoclonal antibody molecule by four pairs of interchain disulfide bonds is linked to the maleimide group in the linker to which the small molecule toxin has been attached.
  • the formed monoclonal antibody-conjugated drug has a mixture of a plurality of drug-dosing amounts in the solution due to the heterogeneity of the reaction sites.
  • the possible toxin binding sites and drug carrying amounts of the formed monoclonal antibody-conjugated drugs are shown in Figure 2.
  • the antibody-conjugated drug of the present invention has an DAR value of an integer or a non-integer of 4.2 ⁇ 1; preferably, an integer or a non-integer of 4.2 ⁇ 0.5.
  • the invention also provides an antibody-conjugated pharmaceutical formulation comprising (a) an effective amount of an antibody-conjugated drug of the invention (as an active ingredient), and (b) at least one pharmaceutically acceptable carrier or excipient.
  • the active ingredient is usually mixed with a carrier or excipient, or diluted in a carrier or excipient or in a carrier in the form of a capsule or sachet.
  • a carrier or excipient acts as a diluent, it can be employed as a solid, semi-solid or liquid material. Therefore, the preparation may be a solution, a sterile injection solution or the like.
  • Suitable excipients include: lactose, glucose, sucrose, sorbitol, mannitol, starch, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, etc.; the formulation may also include: wetting agents, emulsifiers, preservatives (such as methyl hydroxybenzoate and propyl ester) and the like.
  • the formulations may be formulated in unitary or multi-dose dosage forms, each dosage form comprising a predetermined amount of the antibody-conjugated drug of the invention calculated to produce the desired therapeutic effect, as well as suitable pharmaceutical excipients.
  • the formulations may be administered by conventional routes including, but not limited to, intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, topical, and the like.
  • a safe and effective amount of the antibody-conjugated drug is administered to a human, wherein the safe and effective amount is preferably in the range of 0.001-3 mg/kg body weight, more preferably 0.01-2 mg/kg body weight.
  • the specific dose should also consider the route of administration, the health of the patient and other factors, which are within the skill of the skilled physician.
  • the antibody-conjugated pharmaceutical preparation of the present invention may further comprise other therapeutic agents including, but not limited to, cyclophosphamide, doxorubicin, vincristine, prednisone, PD1 antibody, CTLA4 inhibitor, or a combination thereof.
  • the invention also provides a method of treating a mammalian condition using an antibody-conjugated pharmaceutical formulation of the invention.
  • the disease is a CD20-targeted disease, such as a tumor, such as a lymphoma (eg, B-cell non-Hodgkin's lymphoma) or leukemia (chronic lymphocytic leukemia).
  • a tumor such as a lymphoma (eg, B-cell non-Hodgkin's lymphoma) or leukemia (chronic lymphocytic leukemia).
  • the present invention provides a preparation comprising an antibody-conjugated drug with optimized drug loading and a suitable carrier, diluent or excipient, which has high medicinal activity, good stability, low toxicity, and medicine Good sex.
  • the coding sequence is fully synthesized based on the amino acid sequence and nucleotide sequence of the light and heavy chains of rituximab.
  • the monoclonal antibody light chain (LC) and heavy chain (HC) double expression plasmids, pcDNA3-, were constructed using the suspension-accidentated CHO-K1 as a host cell (ATCC from the United States) using a pcDNA3.0-based expression vector. RX-Neo and pcDNA3-RX-GS. Both expression plasmids carry the light and heavy chains of the antibody (the TRS001 light and heavy chains are identical to the light and heavy chain sequences of the commercially available rituximab) and carry the corresponding screening markers.
  • the two expression plasmids were co-transfected into host cells, and the stably transfected cells were screened by double screening markers. Then, multiple candidate monoclonal cell lines were screened by semi-solid medium cloning method, and then screened by shake flask and reactor culture. Used to produce antibody (TRS001) monoclonal cell lines.
  • the monoclonal cell strain was sequenced, and the sequencing results showed that the sequence encoding the light and heavy chains of TRS001 was completely identical to the commercially available rituximab, and the rituximab biosimilar was produced.
  • the physical and chemical indicators such as molecular weight are the same as rituximab.
  • the monoclonal antibody cell line selected in the above step is used as a cell line for final production, and a recombinant anti-human CD20 monoclonal antibody TRS001 is prepared according to the production method provided by the rituximab antigen research patent (CN93121424.6), and used in the subsequent examples. .
  • Buffer A 0.05 mol/L PB Reaction Buffer (pH 7.6)
  • a solution of 0.2 mol/L NaH 2 PO 4 .H 2 O and 0.2 mol/L Na 2 HPO 4 .7H 2 O was used to prepare 0.2 mol/L PB storage buffer (pH 7.6), 0.22 ⁇ m membrane filtration (Nalgene Rapid-Flow unit) and stored at 4 ° C, RT equilibrated and diluted to 0.05 mol / L as a reduction buffer.
  • TECP tris(2-carboxyethyl)phosphine
  • Buffer B 10 mg/mL L-cysteine Stop Buffer
  • a 50% acetonitrile solution was prepared by mixing the same volume of ACN and ultrapure water, filtered through a 0.22 ⁇ m membrane and stored at 4 °C.
  • Buffer C HT Formulation Buffer
  • the 1 L HT preparation buffer contained 3.18 g of L-histidine, 70 g of trehalose dihydrate and 800.2 mL of Tween, and the pH was adjusted to 6.5 using a 0.5 mol/L hydrochloric acid, and the membrane was filtered at 0.22 ⁇ m, and stored at 4 ° C.
  • TRS001 reduction (molar ratio: mAb: TCEP ⁇ 1:3)
  • the TRS001 stock solution (55 mg/mL) prepared in Example 1 was thawed overnight (stability chamber) at 25 °C.
  • a reactor reactor prefilled with 0.1 mol/L NaOH for more than 24 hours and washed
  • 1616 mL of Buffer A was added, followed by 2 mL of 100 mmol/L TCEP solution and mixed (100 rpm, 5 minutes).
  • 182 mL of the TRS001 stock solution was added, stirred at 100 rpm, and the reduction reaction was maintained at 25 ° C for 90 min.
  • the temperature controlled reaction bath was set to 4 ° C (added with ice cooling). Maintain the stirring speed at 100-200 rpm. 13.2 mL vcMMAE (50 mg/mL) (purchased from Levena Biopharma) was added to 186.8 mL of 50% ACN, then carefully added through the sample addition tube and mixed very slowly (control sample addition time within 10 min), gently to the above steps All materials were dropped into the reaction system. The reaction time was maintained for 1 h after the vcMMAE solution was completely fed.
  • the coupling solution was transferred to a TFF system (pre-soaked with 0.1 mol/L NaOH and washed). Buffer A was changed to buffer C and then filtered through a 0.22 microliter Rapid-Flow device. The residual ratio of buffer A in the final solution should be less than 0.25%.
  • the displaced TRS005 stock solution can be stored at 2-8 °C.
  • the antibody-conjugated drug TRS005 having a DAR of about 4.2 was finally obtained, and the drug-loading amount of the antibody-conjugated drug was shown in Fig. 1.
  • Formulation formulations include:
  • the obtained preparation was tested by stability test and showed that it completely met the requirements of the pharmaceutical preparation.

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Abstract

一种靶向CD20的抗体偶联药物制剂，具体地是一种包含如通式I所示的靶向CD20的抗体偶联药物和赋形剂的制剂。所述抗体偶联药物制剂具有显著的抗肿瘤效果。

Description

靶向CD20的抗体偶联药物制剂 技术领域
本发明属于生物医药领域，具体地说，本发明涉及一种靶向CD20的抗体偶联药物制剂。
背景技术
淋巴瘤是一组起源于淋巴结和结外其他淋网状组织的恶性肿瘤，其种类繁多且发病率高，每年我国有数万人因此失去生命。CD20是一种特异性表达于B淋巴细胞表面的非糖基化四重跨膜磷蛋白，对B淋巴细胞的分化和增殖具有重要的调节作用。CD20在B细胞表面的稳定和特异性表达，使得其成为治疗B细胞淋巴瘤理想的靶点。目前，经FDA批准上市用于治疗B细胞淋巴瘤的抗CD20单抗有Rituximab、Zevalin、Bexxar等。
单抗-毒素偶联药物(antibody-drug conjugate，ADC)属于近年来发展起来的新型抗癌药物，是将抗体和细胞毒性药物通过偶联子连接起来，抗体的靶向作用将细胞毒性药物靶向到肿瘤部位，通过内吞作用，ADC药物进入肿瘤细胞后释放毒素，杀死靶细胞，从而降低化疗中常见的药物非特异性的全身毒性。
因此，本领域技术人员致力于开发新的、更有效的靶向CD20的抗体偶联药物。
发明内容
本发明的目的是提供一种靶向CD20的抗体偶联药物制剂。
本发明的另一目的是提供上述抗体偶联药物制剂的制备方法和用途。
本发明的第一方面，提供了一种抗体偶联药物制剂，所述制剂包含：
(a)结构如式I所示的抗体偶联药物：
mAb-(L-D)n           I
其中，
mAb表示重组抗CD20单抗，且所述重组抗CD20单抗为利妥昔单抗或其生物类似药；
D表示小分子毒素，且D为一种或多种单甲基澳瑞他汀(monomethylauristatin)；
L为连接抗体和小分子毒素的接头；
n为偶联于所述抗体的所述小分子毒素的平均偶联数量，且n为4.2±1的整数或非整数；和
“-”为键；和
(b)载体或赋形剂。
在另一优选例中，n为4.2±0.5的整数或非整数。
在另一优选例中，n为4.2±0.3的整数或非整数。
在另一优选例中，D为单甲基澳瑞他汀-E(MMAE)、单甲基澳瑞他汀-D(MMAD)、单甲基澳瑞他汀-F(MMAF)，或其组合。
在另一优选例中，L为马来酰亚胺己酰基-缬氨酸-瓜氨酸–对氨基苄氧羰基。
在另一优选例中，所述小分子毒素通过接头与抗体的链间的二硫键还原后形成的巯基连接。
在另一优选例中，所述抗体偶联药物的结构如下式所示：
Figure PCTCN2018076835-appb-000001
在另一优选例中，所述制剂为注射剂。
在另一优选例中，所述制剂为溶液剂或冻干剂。
在另一优选例中，所述载体或赋形剂选自下组：pH缓冲剂、渗透压调节剂、冻干粉赋形剂、蛋白质保护剂、增溶剂、注射用水。
在另一优选例中，所述pH缓冲剂包括盐酸组氨酸。
在另一优选例中，所述蛋白质保护剂包括海藻糖。
在另一优选例中，所述渗透压调节剂包括甘露醇。
在另一优选例中，所述增溶剂包括聚山梨酯80。
在另一优选例中，所述的抗体偶联药物的制备方法包括步骤：
(1)将重组抗CD20单抗和还原剂进行还原反应，从而含有经还原的重组抗CD20单抗的反应体系；且所述单抗和还原剂的摩尔比为1：2.9～1：3.1；和
(2)将步骤(1)的反应体系和小分子毒素在乙腈和水中的溶液进行偶联反应，从而形成权利要求1所述的抗体偶联药物；且步骤(1)中的单抗与小分子毒素的摩尔比为1：7.0～1：8.0。
在另一优选例中，步骤(1)中，在缓冲液中进行还原反应。
在另一优选例中，步骤(1)中，所述经还原的重组抗CD20单抗为链间二硫键被还原为巯基的重组抗CD20单抗。
在另一优选例中，步骤(1)中，所述还原剂为三(2-羧乙基)膦。
在另一优选例中，步骤(1)中，所述缓冲液为PB反应缓冲液(pH7.6)。
在另一优选例中，步骤(1)中，所述单抗和还原剂的摩尔比为1：2.95～1：3.05；较佳地，为1：2.99～1：3.01。
在另一优选例中，步骤(1)中，所述还原反应在25±1℃下进行。
在另一优选例中，步骤(1)中，所述还原反应进行90±10分钟。
在另一优选例中，步骤(2)中，所述步骤(1)中的单抗与小分子毒素的摩尔比为1：7.2～1：7.7；较佳地，1：7.4～1：7.6。
在另一优选例中，步骤(2)中，所述乙腈和水的体积比例为1：1。
在另一优选例中，步骤(2)中，所述偶联反应在4±0.5℃下进行。
在另一优选例中，步骤(2)中，所述偶联反应进行60±10分钟。
在另一优选例中，步骤(2)中，所述混合是将小分子毒素在乙腈和水中的溶液滴加至步骤(1)的反应体系中。
本发明的第二方面，提供了本发明第一方面所述的抗体偶联药物制剂的用途，其特征在于，用于制备抗肿瘤的药物。
在另一优选例中，所述肿瘤为淋巴瘤或白血病。
在另一优选例中，所述肿瘤为B细胞非霍奇金淋巴瘤或慢性淋巴细胞白血病。
本发明的第三方面，提供了一种非治疗性的抑制肿瘤细胞的方法，所述方法包括步骤：在含有肿瘤细胞的体系中加入本发明第一方面所述的抗体偶联药物制 剂。
在另一优选例中，所述肿瘤细胞为CD20阳性肿瘤细胞，例如，Raji、Ramos、Daudi细胞等。
本发明还提供了一种治疗或预防肿瘤的方法，所述方法包括步骤：给需要的对象施用本发明第一方面所述的抗体偶联药物制剂。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1为鉴定抗体偶联药物TRS005载药量的疏水作用色谱。
图2显示了形成的单抗偶联药物可能的毒素结合位点和携药数量。
具体实施方式
本发明人通过广泛而深入的研究，意外发现一种高效的且负载量经过优化的靶向CD20的抗体偶联药物的制剂。在此基础上完成了本发明。
抗体
适用于本发明的抗体为靶向CD20的抗体，为重组抗CD20的单抗。所述重组抗CD20单抗可为利妥昔单抗，也可为其生物类似药。
小分子毒素
适用于本发明的小分子毒素为具有高细胞毒性的化合物。具体地，所述小分子毒素为一种或多种单甲基澳瑞他汀(monomethylauristatin)；更佳地，所述小分子毒素为单甲基澳瑞他汀-E(MMAE)、单甲基澳瑞他汀-D(MMAD)、单甲基澳瑞他汀-F(MMAF)，或其组合。
其中，MMAE分子结构如下图所示：
Figure PCTCN2018076835-appb-000002
接头
适用于本发明的接头(L)用于连接本发明的抗体和小分子毒素。具体地，所述接头为马来酰亚胺己酰基-缬氨酸-瓜氨酸–对氨基苄氧羰基，例如如下式所示：
Figure PCTCN2018076835-appb-000003
抗体偶联药物
本发明提供了一种抗体偶联药物，它包括通过接头(L)连接在一起的(a)重组抗CD20的单抗与(b)具有细胞毒性的小分子毒素。
如本文所用，术语“本发明的抗体偶联药物”或“本发明的ADC”可互换使用，是指本发明抗体和小分子毒素的通过接头结合的偶联体。
具体地，所述抗体偶联药物结构如式I所示：
mAb-(L-D)n            I
其中，
mAb表示本发明的抗体；
D表示本发明的小分子毒素；
L为连接抗体和小分子毒素的接头；
n为偶联于所述抗体的所述小分子毒素的平均偶联数量，且n为4.2±1的整数或非整数；和
“-”为键。
在另一优选例中，n为4.2±0.5的整数或非整数。
在另一优选例中，n为4.2±0.3的整数或非整数。
在另一优选例中，所述抗体偶联药物的结构如下式所示：
Figure PCTCN2018076835-appb-000004
抗体偶联药物的制备方法
本发明提供了一种抗体偶联药物的偶联方法，其将小分子毒素通过特定接头偶联到抗体上，在不改变抗体亲和性的基础上，大幅提高抗体对肿瘤细胞的杀伤力。
首先，通过常规的化学合成方法连接小分子毒素与接头，也可采用市售可得的连接有接头的小分子毒素；
然后，将重组表达的单抗分子通过四对链间二硫键还原后形成的自由巯基与已经连接小分子毒素的接头中马来酰亚胺基团连接。
形成的单抗偶联药物因为反应位点的不均一性在溶液中存在多种带药数量的混合物。形成的单抗偶联药物可能的毒素结合位点和携药数量如图2所示。
由上可知，理论上形成的单抗偶联药物共有5种携带不同药物数量的形式，分别为带0个、2个、4个、6个、8个药物分子。为了评估其载药数量，可使用了国际通用的评估方式，即统计其平均毒素药物和抗体的摩尔比值(Drug Antibody Ratio,DAR)。
本发明的抗体偶联药物的DAR值为4.2±1的整数或非整数；较佳地，为4.2±0.5的整数或非整数。
抗体偶联药物制剂
本发明还提供了一种抗体偶联药物制剂，所述制剂包含(a)有效量的本发明的抗体偶联药物(作为活性成分)，以及(b)至少一种药学上可接受的载体或赋形剂。
制备时，通常将活性成分与载体或赋形剂混合，或用载体或赋形剂稀释，或包在可以胶囊或药囊形式存在的载体中。当赋形剂起稀释剂作用时，它可采 用固体、半固体或液体材料作为赋形剂。因此，所述制剂可以是溶液剂、灭菌注射溶液等。
合适的赋形剂包括：乳糖、葡萄糖、蔗糖、山梨醇、甘露醇、淀粉、微晶纤维素、聚乙烯吡咯烷酮、纤维素、水等；制剂还可包括：湿润剂、乳化剂、防腐剂(如羟基苯甲酸甲酯和丙酯)等。
所述制剂可制成单元或多元剂型，各剂型包含为了产生所期望的疗效而计算出预定量的本发明的抗体偶联药物，以及合适的药剂学赋形剂。
所述制剂可以通过常规途径进行给药，包括(但并不限于)：肌内、腹膜内、静脉内、皮下、皮内、局部给药等。
使用该制剂时，是将安全有效量的所述抗体偶联药物施用于人，其中该安全有效量的范围优选为0.001-3毫克/千克体重，更优选为0.01-2毫克/千克体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是在熟练医师技能范围之内的。
此外，本发明的抗体偶联药物制剂还可包含其他治疗药物，所述其它治疗药物包括(但并不限于)：环磷酰胺、多柔比星、长春新碱、强的松、PD1抗体、CTLA4抑制剂，或其组合。
用途
本发明还提供了一种使用本发明的抗体偶联药物制剂治疗哺乳动物疾病的方法。优选地，所述的疾病为靶向CD20的疾病，例如肿瘤，如淋巴瘤(例如B细胞非霍奇金淋巴瘤)或白血病(慢性淋巴细胞白血病)。
与现有技术相比，本发明的主要有益效果包括：
本发明提供了一种包含载药量经过优化的抗体偶联药物和合适的载体、稀释剂或赋形剂的制剂，该制剂既具有较高的药物活性，而且稳定性好，毒性低，成药性佳。
下面结合具体实施，进一步阐述本发明。应理解，这些实施例仅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实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York: Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。本发明实施例中所用原料、试剂、细胞株或仪器，若非特别说明，均市售可得或常规的。
实施例1抗体(TRS001)的制备
基于利妥昔单抗的轻链和重链的氨基酸序列和核苷酸序列，全合成编码序列。以经悬浮驯化的CHO-K1为宿主细胞(来自美国的ATCC)，利用以pcDNA3.0为基础的表达载体，构建单克隆抗体轻链(LC)和重链(HC)双表达质粒，pcDNA3-RX-Neo和pcDNA3-RX-GS。两个表达质粒均带有抗体的轻链、重链(所述TRS001轻、重链与市售的利妥昔单抗的轻、重链序列完全一致)基因并携带相应的筛选标记。两种表达质粒共转染宿主细胞经双筛选标记对稳转细胞进行筛选，之后利用半固体培养基克隆法逐级筛选出多个候选单克隆细胞株，再通过摇瓶和反应器培养筛选出用于生产抗体(TRS001)单克隆细胞株。
对所述单克隆细胞株进行测序，测序结果表明，编码TRS001轻链和重链的序列与市售的利妥昔单抗完全一致，且生产出的利妥昔单抗生物类似药(biosimilar)的分子量等理化指标与利妥昔单抗相同。
以上述步骤选出的单克隆细胞株作为最终生产用细胞株，参照利妥昔单抗原研药专利(CN93121424.6)提供的生产方法制备重组抗人CD20的单抗TRS001，用于后续实施例。
实施例2抗体偶联药物(TRS005：DAR约为4.2)制备
1.溶液和材料的制备
缓冲液A：0.05mol/L PB反应缓冲液(pH7.6)
使用0.2mol/L NaH 2PO 4.H 2O和0.2mol/L Na 2HPO 4.7H 2O的溶液(使用Millipore纯化水)以制备0.2mol/L PB储存缓冲液(pH 7.6)，0.22μm膜过滤(Nalgene Rapid-Flow单位)并在4℃下保存，RT平衡并稀释至0.05mol/L作为还原缓冲液。
还原剂：100mmol/L TCEP溶液
溶解1.43g TECP(三(2-羧乙基)膦)在50mL纯水中得到100mmol/L溶液，等分1ml/瓶并储存在-80℃。
缓冲液B：10mg/mL L-半胱氨酸终止缓冲液
在100ml 0.1mM DTPA(二乙烯三胺五乙酸)溶液中溶解1g L-半胱氨酸，0.22μm膜过滤并储存于-80℃。
50％ACN溶液
混合相同体积的ACN和超纯水制备50％乙腈溶液，0.22μm膜过滤并在4℃下储存。
缓冲液C：HT制剂缓冲液
1L HT制剂缓冲液含有L-组氨酸3.18g，海藻糖二水合物70g和吐温800.2mL，使用0.5mol/L盐酸调节pH至6.5，0.22μm膜过滤，4℃保存。
2.TRS001还原(摩尔比：mAb：TCEP≈1:3)
在25℃条件下将实施例1制备的TRS001原液(55mg/mL)解冻过夜(稳定室)。在反应器(反应器预填充0.1mol/L NaOH超过24小时并洗涤干净)中加入1616mL缓冲液A，然后加入2mL 100mmol/L TCEP溶液并混合(100rpm，5分钟)。添加182mL TRS001原液，以100rpm搅拌，在25℃使还原反应保持90min。
3.结合(摩尔比：mAb：vcMMAE≈1:7.5)
将温度控制反应浴设置为4℃(用冰冷却添加)。保持搅拌速度在100-200rpm。将13.2mL vcMMAE(50mg/mL)(购自Levena Biopharma公司)加入186.8mL 50％ACN中，然后通过样品添加管小心加入并非常缓慢地混合(控制样品添加时间10min内)，轻轻向上述步骤的反应体系中滴入所有物料。在vcMMAE溶液完全进料后反应时间保持1h。
4.停止反应
在10分钟内向反应器中加入10mL缓冲液B停止偶联反应，停止偶联反应后进行下一步缓冲液置换。
5.缓冲液置换
转移偶联溶液至TFF系统(用0.1mol/L NaOH预先浸泡并洗涤干净)。将缓 冲液A更改为缓冲液C和然后用0.22微升Rapid-Flow装置过滤。最终溶液中缓冲液A的残留率应小于0.25％。置换后的TRS005原液可储存在2-8℃。
最终得到DAR约为4.2的抗体偶联药物TRS005，抗体偶联药物的载药量鉴定结果如图1所示。
实施例3重组抗CD20单抗偶联药物冻干制剂
制剂配方包括：
利妥昔单抗、盐酸组氨酸、海藻酸、甘露醇、聚山梨醇80、和注射用水。
所得制剂经稳定性实验检测表明，完全符合药用制剂的要求。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种抗体偶联药物制剂，其特征在于，所述制剂包含：
    (a)结构如式I所示的抗体偶联药物：
    mAb-(L-D)n    I
    其中，
    mAb表示重组抗CD20单抗，且所述重组抗CD20单抗为利妥昔单抗或其生物类似药；
    D表示小分子毒素，且D为一种或多种单甲基澳瑞他汀(monomethyl auristatin)；
    L为连接抗体和小分子毒素的接头；
    n为偶联于所述抗体的所述小分子毒素的平均偶联数量，且n为4.2±1的整数或非整数；和
    “-”为键；和
    (b)载体或赋形剂。
  2. 如权利要求1所述的抗体偶联药物制剂，其特征在于，D为单甲基澳瑞他汀-E(MMAE)、单甲基澳瑞他汀-D(MMAD)、单甲基澳瑞他汀-F(MMAF)，或其组合。
  3. 如权利要求1所述的抗体偶联药物制剂，其特征在于，L为马来酰亚胺己酰基-缬氨酸-瓜氨酸–对氨基苄氧羰基。
  4. 如权利要求1所述的抗体偶联药物制剂，其特征在于，所述抗体偶联药物的结构如下式所示：
    Figure PCTCN2018076835-appb-100001
  5. 如权利要求1所述的抗体偶联药物制剂，其特征在于，所述制剂为注射剂。
  6. 如权利要求1所述的抗体偶联药物制剂，其特征在于，所述制剂为溶液剂或冻干剂。
  7. 如权利要求1所述的抗体偶联药物制剂，其特征在于，所述载体或赋形剂选自下组：pH缓冲剂、渗透压调节剂、冻干粉赋形剂、蛋白质保护剂、增溶剂、注射用水。
  8. 如权利要求1所述的抗体偶联药物制剂，其特征在于，所述的抗体偶联药物的制备方法包括步骤：
    (1)将重组抗CD20单抗和还原剂进行还原反应，从而含有经还原的重组抗CD20单抗的反应体系；且所述单抗和还原剂的摩尔比为1：2.9～1：3.1；和
    (2)将步骤(1)的反应体系和小分子毒素在乙腈和水中的溶液进行偶联反应，从而形成权利要求1所述的抗体偶联药物；且步骤(1)中的单抗与小分子毒素的摩尔比为1：7.0～1：8.0。
  9. 如权利要求1-8任一项所述的抗体偶联药物制剂的用途，其特征在于，用于制备抗肿瘤的药物。
  10. 一种非治疗性的抑制肿瘤细胞的方法，其特征在于，所述方法包括步骤：在含有肿瘤细胞的体系中加入权利要求1-7任一项所述的抗体偶联药物制剂。
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