WO2021223048A1 - 抗体药物偶联物及其制剂 - Google Patents

抗体药物偶联物及其制剂 Download PDF

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WO2021223048A1
WO2021223048A1 PCT/CN2020/088564 CN2020088564W WO2021223048A1 WO 2021223048 A1 WO2021223048 A1 WO 2021223048A1 CN 2020088564 W CN2020088564 W CN 2020088564W WO 2021223048 A1 WO2021223048 A1 WO 2021223048A1
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antibody
lymphoma
drug conjugate
hodgkin
cell
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PCT/CN2020/088564
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English (en)
French (fr)
Inventor
胡朝红
李虎
肖莉莉
刘文超
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Shanghai Miracogen Inc
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Shanghai Miracogen Inc
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Priority to EP20934690.7A priority Critical patent/EP4148070A4/en
Priority to CN202511665218.3A priority patent/CN121422248A/zh
Priority to PCT/CN2020/088564 priority patent/WO2021223048A1/zh
Priority to US17/997,775 priority patent/US20230293709A1/en
Priority to JP2022567138A priority patent/JP7560575B2/ja
Priority to CN202511665211.1A priority patent/CN121422247A/zh
Priority to CN202080100380.XA priority patent/CN115485304B/zh
Publication of WO2021223048A1 publication Critical patent/WO2021223048A1/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
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • 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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
    • 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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • 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
    • A61K47/6849—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
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2887—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies

Definitions

  • the present invention relates to the field of biomedicine, in particular to antibody-drug conjugates, preparations containing the antibody-drug conjugates, compositions containing the antibody-drug conjugates, and medical uses of the antibody-drug conjugates.
  • non-Hodgkin's lymphoma includes traditional surgical treatment, chemotherapy, radiotherapy, bone marrow or hematopoietic stem cell transplantation, immunity and targeted therapy.
  • Traditional chemotherapy has a certain curative effect on NHL patients, but its systemic side effects are large, the tolerance is poor, and it is prone to relapse, which limits the use of patients.
  • Targeted drugs, especially antibody drugs have attracted much attention due to their remarkable curative effect and low systemic side effects during treatment.
  • CD20-targeted monoclonal antibody drugs have been developed (such as rituximab, ), and its anti-tumor activity has been verified in clinical trials.
  • CD20-targeted monoclonal antibodies represented by rituximab are far from meeting the needs of clinical practice for the treatment of relapsed or refractory B-cell NHL (including DLBCL and FL, etc.).
  • the main purpose of the development of the CD20-targeted ADC drug ADC-1 is to provide a treatment with better curative effect, lower toxic and side effects, and less likely to develop drug resistance.
  • the inventor of the present invention has prepared an anti-CD20 antibody drug conjugate through a lot of experiments and creative work, and confirmed that it has good biological activity and formulation stability, thus completing the present invention.
  • the present invention provides antibody-drug conjugates, the antibody-drug conjugates having the structure shown in formula I,
  • Ab is an anti-CD20 monoclonal antibody
  • the anti-CD20 monoclonal antibody is any antibody that targets CD20, such as rituximab or a biological analog thereof;
  • cytotoxic agent D is a cytotoxic agent, and the cytotoxic agent is Monomethyl auristatin E (MMAE);
  • MMAE Monomethyl auristatin E
  • L is a linker for connecting the anti-CD20 monoclonal antibody and the cytotoxic agent, and the linker is 6-maleimidohexanoyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-vc -PAB);
  • p 3.6-4.0.
  • p is 3.7-3.9.
  • p is 3.8.
  • L-D described in Formula I is vc-MMAE, and its structure is shown in the following formula:
  • the structure of the antibody-drug conjugate is shown in the following formula:
  • Ab is an anti-CD20 monoclonal antibody
  • the anti-CD20 monoclonal antibody is any antibody that targets CD20, such as rituximab or its biological analogues,
  • p 3.6-4.0.
  • p is 3.7-3.9.
  • p is 3.8.
  • an antibody drug conjugate preparation which includes:
  • Antibody drug conjugate the concentration of which is 1-60mg/mL (such as 1mg/mL, 2mg/mL, 3mg/mL, 4mg/mL, 5mg/mL, 6mg/mL, 7mg/mL, 8mg/mL, 9mg/mL mL, 10mg/mL, 13mg/mL, 15mg/mL, 17mg/mL, 19mg/mL, 20mg/mL, 25mg/mL, 30mg/mL, 35mg/mL, 40mg/mL, 45mg/mL, 50mg/mL, 55mg/mL or 60mg/mL);
  • 5-35mM (such as 5mM, 8mM, 9mM, 10mM, 11mM, 12mM, 15mM, 20mM, 25mM, 30mM or 35mM) histidine buffer, pH 5.0-6.2 (such as 5.0, 5.1, 5.2, 5.3, 5.4 , 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1 or 6.2);
  • sucrose a maltitol syrup
  • sucrose a maltitol syrup
  • the antibody-drug conjugate has the structure shown in Formula II,
  • Ab is an anti-CD20 monoclonal antibody
  • the anti-CD20 monoclonal antibody is any antibody that targets CD20, such as rituximab or a biological analog thereof;
  • cytotoxic agent D is a cytotoxic agent, and the cytotoxic agent is Monomethyl auristatin E (MMAE);
  • MMAE Monomethyl auristatin E
  • L is a linker for connecting the anti-CD20 monoclonal antibody and the cytotoxic agent, and the linker is 6-maleimidohexanoyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-vc -PAB);
  • q is 3.3-4.3 (such as 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2 or 4.3).
  • q is 3.6-4.0.
  • q is 3.7-3.9.
  • q is 3.8.
  • the concentration of the antibody drug conjugate is 1-20 mg/mL.
  • the concentration of the antibody drug conjugate is 1-10 mg/mL.
  • the concentration of the antibody drug conjugate is 3-7 mg/mL.
  • the concentration of the antibody drug conjugate is 5 mg/mL.
  • the concentration of the histidine buffer is 5-15 mM.
  • the concentration of the histidine buffer is 8-12 mM.
  • the concentration of the histidine buffer is 10 mM.
  • the pH of the histidine buffer is 5.8-6.2.
  • the pH of the histidine buffer is 5.6-6.0.
  • the pH of the histidine buffer is 5.4-6.0.
  • the pH of the histidine buffer is 5.4-6.2.
  • the concentration of sucrose is 3-9%.
  • the concentration of sucrose is 4-8%.
  • the concentration of sucrose is 5-7%.
  • the concentration of sucrose is 6%.
  • the concentration of Tween 80 is 0.02-0.06%.
  • the concentration of Tween 80 is 0.02-0.05%.
  • the concentration of Tween 80 is 0.03-0.04%.
  • the concentration of Tween 80 is 0.035%.
  • the formulation includes:
  • the antibody drug conjugate has a concentration of 5 mg/mL
  • sucrose means mass volume concentration (w/v%), which means that per 1000 mL of the antibody drug conjugate preparation, the mass of sucrose is 20-100 g.
  • 0.01-0.1% Tween 80 means mass volume concentration (w/v%), and its meaning can be similarly understood with reference to the aforementioned "2-10% sucrose”.
  • the concentration of "histidine buffer” refers to the concentration of histidine and histidine hydrochloride, and its pH is obtained by adjusting the ratio of histidine and histidine hydrochloride.
  • the antibody-drug conjugate preparation when the pH is 5.0-6.2 (for example, 5.6-6.0), can be stable for 6 weeks under the conditions of 25 ⁇ 2°C/60% ⁇ 5% RH.
  • the antibody-drug conjugate preparation when the pH is 5.0-6.2 (for example, 5.6-6.0), has a colorless and clear appearance when placed at 25°C for 6 weeks.
  • the pH when the pH is 5.0-6.2 (for example, 5.6-6.0), when the antibody-drug conjugate preparation is placed at 25°C for 6 weeks, the pH does not change.
  • the concentration does not change.
  • the SEC monomer% content decreases by no more than 2% (or Not more than 1%).
  • the SEC high polymer% content does not increase by more than 2% (Or no more than 1%).
  • the DAR value when the pH is 5.0-6.2 (for example, 5.6-6.0), when the antibody-drug conjugate preparation is placed at 25°C for 6 weeks, the DAR value does not change significantly.
  • the antibody-drug conjugate preparation when the pH is 5.0-6.2 (for example, 5.6-6.0), has a colorless and clear appearance when placed at 40°C for 4 weeks.
  • the pH when the pH is 5.0-6.2 (for example, 5.6-6.0), when the antibody-drug conjugate preparation is placed at 40°C for 4 weeks, the pH does not change.
  • the concentration does not change.
  • the SEC monomer% content does not decrease by more than 5% (or Not more than 4%).
  • the SEC high polymer% content does not increase by more than 5% (Or no more than 4%).
  • the DAR value when the pH is 5.0-6.2 (for example, 5.6-6.0), the DAR value does not change significantly when the antibody-drug conjugate preparation is placed at 40°C for 4 weeks.
  • no significant change or “no change” refers to no statistically significant change.
  • antibody-drug conjugate refers to a composition containing ADC molecules with the same or different DAR values.
  • the present invention provides a composition comprising a plurality of anti-CD20 ADC molecules.
  • each ADC in the composition described herein contains the same number of one or more drug molecules.
  • each ADC in the composition described herein contains a different number of one or more drug molecules.
  • each anti-CD20 antibody can be conjugated with 1, 2, 3, 4, 5, 6, 7, 8 or more drug molecules (preferably 2, 4, 6, or 8, more preferably 2 or 4).
  • the above-mentioned drug-to-antibody ratio refers to the number of molecules of the anti-cancer drug coupled to the anti-CD20 antibody.
  • the number of molecules of the anticancer drug contained in the ADC described herein is usually an integer, when the number of molecules of the anticancer drug contained in the ADC described herein (for example, p in formula I or q in formula II) is When scoring, the score refers to the average number of anti-cancer drug molecules per anti-CD20 antibody in a composition containing multiple ADC molecules.
  • the present invention provides a preparation method of the aforementioned antibody-drug conjugate preparation, including:
  • the anti-CD20 monoclonal antibody and the reducing agent are subjected to a reduction reaction to obtain a reduced anti-CD20 monoclonal antibody
  • the anti-CD20 monoclonal antibody is any antibody that targets CD20, such as rituximab or a biological analog thereof;
  • the quenched coupling reaction product is subjected to buffer replacement to obtain the antibody-drug conjugate preparation, and the antibody-drug conjugate preparation includes:
  • the concentration of the antibody drug conjugate is 1-60mg/mL (e.g. 1mg/mL, 2mg/mL, 3mg/mL, 4mg/mL, 5mg/mL, 6mg/mL, 7mg/mL, 8mg/mL, 9mg /mL, 10mg/mL, 13mg/mL, 15mg/mL, 17mg/mL, 19mg/mL, 20mg/mL, 25mg/mL, 30mg/mL, 35mg/mL, 40mg/mL, 45mg/mL, 50mg/mL , 55mg/mL or 60mg/mL);
  • 5-35mM (such as 5mM, 8mM, 9mM, 10mM, 11mM, 12mM, 15mM, 20mM, 25mM, 30mM or 35mM) histidine buffer, pH 5.0-6.2 (such as 5.0, 5.1, 5.2, 5.3, 5.4 , 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1 or 6.2);
  • sucrose a maltitol syrup
  • sucrose a maltitol syrup
  • 0.01-0.1% (such as 0.01%, 0.015%, 0.02%, 0.025%, 0.03%, 0.031%, 0.032%, 0.033%, 0.034%, 0.035%, 0.036%, 0.037%, 0.038%, 0.039%, 0.04% , 0.045%, 0.05%, 0.055%, 0.06%, 0.065%, 0.07%, 0.08%, 0.09% or 0.1%) Tween 80.
  • the concentration of the antibody drug conjugate is 1-20 mg/mL.
  • the concentration of the antibody drug conjugate is 1-10 mg/mL.
  • the concentration of the antibody drug conjugate is 3-7 mg/mL.
  • the concentration of the antibody drug conjugate is 5 mg/mL.
  • the concentration of the histidine buffer is 5-15 mM.
  • the concentration of the histidine buffer is 8-12 mM.
  • the concentration of the histidine buffer is 10 mM.
  • the pH of the histidine buffer is 5.8-6.2.
  • the pH of the histidine buffer is 5.6-6.0.
  • the pH of the histidine buffer is 5.4-6.0.
  • the pH of the histidine buffer is 5.4-6.2.
  • the concentration of sucrose is 3-9%.
  • the concentration of sucrose is 4-8%.
  • the concentration of sucrose is 5-7%.
  • the concentration of sucrose is 6%.
  • the concentration of Tween 80 is 0.02-0.06%.
  • the concentration of Tween 80 is 0.02-0.05%.
  • the concentration of Tween 80 is 0.03-0.04%.
  • the concentration of Tween 80 is 0.035%.
  • the reducing agent is DTT.
  • the preparation method includes:
  • the present invention provides a composition containing the aforementioned antibody-drug conjugate, or the aforementioned antibody-drug conjugate preparation, or the antibody-drug conjugate preparation prepared by the aforementioned method.
  • the composition further contains at least one pharmaceutically acceptable carrier, diluent or excipient.
  • the composition also contains known chemotherapy drugs for the treatment of tumors, such as adriamycin (Adriamycin), cyclophosphamide and taxanes [paclitaxel (Taxol) And docetaxel (Taxotere), capecitabine (Xeloda), gemcitabine (Gemzar), vinorelbine (Navelbine), tamoxifen, aromatase inhibitors (Rinimide, Frond, Aroxin) , 5-FU plus folinic acid, irinotecan (camptosar), oxaliplatin, cisplatin, carboplatin, estramustine, mitoxantrone (Novantrone), prednisone, vincristine (Oncovin), Doxorubicin, prednisone, etc., or a combination of them.
  • chemotherapy drugs for the treatment of tumors such as adriamycin (Adriamycin), cyclopho
  • the composition further comprises an immunosuppressant selected from: (1) glucocorticoids, such as cortisone and prednisone; (2) microbial metabolites, such as Cyclosporine and tacrolimus, etc.; (3) Antimetabolites, such as azathioprine and 6-mercaptopurine, etc.; (4) Polyclonal and monoclonal anti-lymphocyte antibodies, such as anti-lymphocyte globulin and OKT3, etc. ; (5) Alkylating agents, such as cyclophosphamide.
  • an immunosuppressant selected from: (1) glucocorticoids, such as cortisone and prednisone; (2) microbial metabolites, such as Cyclosporine and tacrolimus, etc.; (3) Antimetabolites, such as azathioprine and 6-mercaptopurine, etc.; (4) Polyclonal and monoclonal anti-lymphocyte antibodies, such as anti-lymphocyte globulin and OKT3, etc. ;
  • the immunosuppressive agent is, for example, methylprednisolone, prednisone, azathioprine, prolactin, zenapax, sule, cyclosporine, tacrolimus, rapamycin Sulfate, mycophenolate mofetil, mizoribine, cyclophosphamide, fingolimod, etc.
  • the present invention provides the foregoing antibody drug conjugate, or the foregoing antibody drug conjugate preparation, or the antibody drug conjugate preparation prepared by the foregoing method, or the foregoing composition is used in the preparation of prevention and/or Use in medicines for treating cancers or immune diseases expressing CD20.
  • the CD20-expressing cancer is lymphoma or leukemia.
  • the lymphoma is non-Hodgkin's lymphoma, B-cell non-Hodgkin's lymphoma, follicular non-Hodgkin's lymphoma, small lymphocytic lymphoma, or diffuse large B-cell lymphoma .
  • the leukemia is chronic lymphocytic leukemia, hairy cell leukemia, B-cell young lymphocytic leukemia, or acute lymphocytic leukemia.
  • the CD20-expressing immune disease is rheumatoid arthritis, granulomatous polyangiitis, Wegener's granulomatosis, microscopic polyangiitis, or multiple sclerosis.
  • the rheumatoid arthritis is severely active rheumatoid arthritis that has failed treatment with at least one TNF antagonist.
  • the CD20-expressing cancer is CD20-positive B-cell lymphoma.
  • the CD20-positive B-cell lymphoma is an anti-CD20 monoclonal antibody (eg, rituximab) resistant CD20-positive B-cell lymphoma.
  • an anti-CD20 monoclonal antibody eg, rituximab
  • the lymphoma is non-Hodgkin's lymphoma.
  • the lymphoma is anti-CD20 monoclonal antibody (eg, rituximab) resistant non-Hodgkin's lymphoma.
  • anti-CD20 monoclonal antibody eg, rituximab
  • the non-Hodgkin's lymphoma is diffuse large B-cell lymphoma.
  • the non-Hodgkin's lymphoma is an anti-CD20 monoclonal antibody (eg, rituximab) resistant diffuse large B-cell lymphoma.
  • an anti-CD20 monoclonal antibody eg, rituximab
  • the present invention provides a method for preventing and/or treating CD20-expressing cancer or immune disease, which comprises administering to a subject in need thereof a preventive and/or therapeutically effective amount of the aforementioned antibody-drug conjugate, Or the aforementioned antibody-drug conjugate preparation, or the antibody-drug conjugate preparation prepared by the aforementioned method, or the aforementioned composition.
  • the CD20-expressing cancer is lymphoma or leukemia.
  • the lymphoma is non-Hodgkin's lymphoma, B-cell non-Hodgkin's lymphoma, follicular non-Hodgkin's lymphoma, small lymphocytic lymphoma, or diffuse large B-cell lymphoma .
  • the leukemia is chronic lymphocytic leukemia, hairy cell leukemia, B-cell young lymphocytic leukemia, or acute lymphocytic leukemia.
  • the CD20-expressing immune disease is rheumatoid arthritis, granulomatous polyangiitis, Wegener's granulomatosis, microscopic polyangiitis, or multiple sclerosis.
  • the rheumatoid arthritis is severely active rheumatoid arthritis that has failed treatment with at least one TNF antagonist.
  • the CD20-expressing cancer is CD20-positive B-cell lymphoma.
  • the CD20-positive B-cell lymphoma is an anti-CD20 monoclonal antibody (eg, rituximab) resistant CD20-positive B-cell lymphoma.
  • an anti-CD20 monoclonal antibody eg, rituximab
  • the lymphoma is non-Hodgkin's lymphoma.
  • the lymphoma is anti-CD20 monoclonal antibody (eg, rituximab) resistant non-Hodgkin's lymphoma.
  • anti-CD20 monoclonal antibody eg, rituximab
  • the non-Hodgkin's lymphoma is diffuse large B-cell lymphoma.
  • the non-Hodgkin's lymphoma is an anti-CD20 monoclonal antibody (eg, rituximab) resistant diffuse large B-cell lymphoma.
  • an anti-CD20 monoclonal antibody eg, rituximab
  • the dose administered to the subject in need is 0.3-10 mg/kg (such as 0.3 mg/kg, 1 mg/kg, 3 mg/kg, 10 mg/kg ).
  • the dose administered to a subject in need is 0.3-3 mg/kg, 1-3 mg/kg, 1-10 mg/kg, or 3-10 mg/kg .
  • the dose administered to the subject in need is 0.1-5 mg/kg (such as 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg, 3 mg/kg or 5 mg /kg).
  • a prophylactic and/or therapeutically effective amount of the aforementioned antibody-drug conjugate or the aforementioned antibody-drug conjugate is administered to a subject in need every 1, 2, 3, 4, 5 or 6 weeks A formulation, or an antibody-drug conjugate formulation prepared by the foregoing method, or the foregoing composition.
  • a prophylactic and/or therapeutically effective amount of the aforementioned antibody drug conjugate, or the aforementioned antibody drug conjugate preparation, or the antibody prepared by the aforementioned method is administered to a subject in need every 18-24 days Drug conjugate formulation, or the aforementioned composition.
  • it also includes administering additional chemotherapeutic drugs or immunosuppressive agents for the treatment of tumors to subjects in need.
  • the chemotherapeutic drugs are, for example, Adriamycin (Adriamycin), cyclophosphamide and taxanes [Taxol (Taxol) and docetaxel (Taxotere)], capecitabine (Xeloda), Gemcitabine (Gemzar), Vinorelbine (Navelbine), Tamoxifen, Aromatase Inhibitors (Raininide, Furlong, Aroxin), 5-FU plus Folinic Acid, Irinotecan (camptosar), Osha Liplatin, cisplatin, carboplatin, estramustine, mitoxantrone (Novantrone), prednisone, vincristine (Oncovin), doxorubicin, prednisone, etc., or a combination thereof.
  • Adriamycin Adriamycin
  • cyclophosphamide and taxanes [Taxol (Taxol) and docetaxel (
  • the immunosuppressive agent is selected from: (1) glucocorticoids, such as cortisone and prednisone; (2) microbial metabolites, such as cyclosporin and tacrolimus; 3) Anti-metabolites, such as azathioprine and 6-mercaptopurine; (4) Polyclonal and monoclonal anti-lymphocyte antibodies, such as anti-lymphocyte globulin and OKT3, etc.; (5) Alkylating agents, such as cyclo Phosphoramide.
  • glucocorticoids such as cortisone and prednisone
  • microbial metabolites such as cyclosporin and tacrolimus
  • Anti-metabolites such as azathioprine and 6-mercaptopurine
  • Polyclonal and monoclonal anti-lymphocyte antibodies such as anti-lymphocyte globulin and OKT3, etc.
  • Alkylating agents such as cyclo Phosphoramide.
  • the immunosuppressive agent is, for example, methylprednisolone, prednisone, azathioprine, prolactin, zenapax, sule, cyclosporine, tacrolimus, rapamycin Sulfate, mycophenolate mofetil, mizoribine, cyclophosphamide, fingolimod, etc.
  • the present invention provides the foregoing antibody drug conjugate, or the foregoing antibody drug conjugate preparation, or the antibody drug conjugate preparation prepared by the foregoing method, or the foregoing composition, which is used for prevention and /Treatment of CD20-expressing cancer or immune diseases.
  • the CD20-expressing cancer is lymphoma or leukemia.
  • the lymphoma is non-Hodgkin's lymphoma, B-cell non-Hodgkin's lymphoma, follicular non-Hodgkin's lymphoma, small lymphocytic lymphoma, or diffuse large B-cell lymphoma .
  • the leukemia is chronic lymphocytic leukemia, hairy cell leukemia, B-cell young lymphocytic leukemia, or acute lymphocytic leukemia.
  • the CD20-expressing immune disease is rheumatoid arthritis, granulomatous polyangiitis, Wegener's granulomatosis, microscopic polyangiitis, or multiple sclerosis.
  • the rheumatoid arthritis is severely active rheumatoid arthritis that has failed treatment with at least one TNF antagonist.
  • the CD20-expressing cancer is CD20-positive B-cell lymphoma.
  • the CD20-positive B-cell lymphoma is an anti-CD20 monoclonal antibody (eg, rituximab) resistant CD20-positive B-cell lymphoma.
  • an anti-CD20 monoclonal antibody eg, rituximab
  • the lymphoma is non-Hodgkin's lymphoma.
  • the lymphoma is anti-CD20 monoclonal antibody (eg, rituximab) resistant non-Hodgkin's lymphoma.
  • anti-CD20 monoclonal antibody eg, rituximab
  • the non-Hodgkin's lymphoma is diffuse large B-cell lymphoma.
  • the non-Hodgkin's lymphoma is an anti-CD20 monoclonal antibody (eg, rituximab) resistant diffuse large B-cell lymphoma.
  • an anti-CD20 monoclonal antibody eg, rituximab
  • the dose administered is 0.3-10 mg/kg (such as 0.3 mg/kg, 1 mg/kg, 3 mg/kg, 10 mg/kg).
  • the dose administered is 0.3-3 mg/kg, 1-3 mg/kg, 1-10 mg/kg, or 3-10 mg/kg.
  • the dose administered is 0.1-5 mg/kg (such as 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg, 3 mg/kg, or 5 mg/kg).
  • ADC-1 is an ADC that is coupled to the CD20 receptor on the surface of tumor cells, endocytosis, and releases MMAE to kill CD20-positive non-Hodgkin’s lymphoma ( NHL) cells to control tumor growth and cause tumor regression;
  • ADC-1 has a significant inhibitory effect on tumor cell growth in a variety of CD20-expressing NHL cell lines and a variety of CD20-expressing human NHL PDX tumor models, especially for The drug-resistant NHL PDX model also showed obvious tumor growth inhibitory effect;
  • ADC-1 has good formulation stability.
  • Fig. 1 is the HIC-HPLC chart of ADC-1 of the embodiment of the present invention, where Minutes represents minutes;
  • Figure 2 shows the ADC-1 and ADC-1 of the embodiment of the present invention A representative cell killing curve in the Daudi cell line, where Inhibition represents the inhibition rate, and Concentration represents the concentration;
  • Figure 3 shows the ADC-1 and ADC-1 of the embodiment of the present invention A representative cell killing curve in the Jeko-1 cell line, where Inhibition represents the inhibition rate, and Concentration represents the concentration;
  • Fig. 4 shows ADC-1 and ADC-1 according to an embodiment of the present invention Representative cell killing curve in Raji cell line, where Inhibition represents the inhibition rate and Concentration represents the concentration;
  • Fig. 5 shows ADC-1 and ADC-1 of an embodiment of the present invention Representative cell killing curve in Ramos cell line, where Inhibition represents the inhibition rate, and Concentration represents the concentration;
  • Fig. 6 shows ADC-1 and ADC-1 of an embodiment of the present invention Schematic diagram of the effect on CD20 positive human lymphoma (NHL) PDX model LYM#004 tumor volume;
  • FIG. 7 shows the ADC-1 and ADC-1 of an embodiment of the present invention Schematic diagram of the effect on CD20 positive human lymphoma (NHL) PDX model LYM#013 tumor volume;
  • FIG. 8 shows the ADC-1 and ADC-1 of an embodiment of the present invention Schematic diagram of the effect on CD20 positive human lymphoma (NHL) PDX model LYM#016 tumor volume;
  • Figure 9 is a schematic diagram of the effect of pH on the SEC stability of the active substance ADC-1 (10mM histidine buffer system).
  • ADC-1 is in a 10mM histidine buffer system with different pH.
  • the left picture shows that ADC-1 is at 25°C.
  • the right figure shows the change trend of SEC high polymer content of ADC-1 at 25 degrees for 6 weeks and 40 degrees for 4 weeks.
  • any concentration range, percentage range, ratio range or numerical range should be understood to include any integer value within the stated range, and include fractional values within the stated range when appropriate.
  • antibody refers to an immunoglobulin molecule usually composed of two pairs of identical polypeptide chains (each pair has a "light” (L) chain and a “heavy” (H) chain).
  • the light chains of antibodies can be divided into two types: kappa and lambda.
  • Heavy chains can be divided into five types: mu, delta, gamma, alpha or epsilon, and antibodies can be divided into five types: IgM, IgD, IgG, IgA, and IgE according to the difference of heavy chains.
  • variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 3 or more amino acids.
  • Each heavy chain is composed of a heavy chain variable region (V H ) and a heavy chain constant region (C H ).
  • the heavy chain constant region is comprised of three domains (C H 1, C H 2 and C H 3) components.
  • Each light chain is comprised of a light chain variable region (V L) and a light chain constant region (C L) components.
  • the light chain constant region is comprised of one domain, C L composition.
  • the constant region of the antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (for example, effector cells) and component C1q of the complement system.
  • V L, V H regions may be subdivided into hypervariability regions (termed complementarity determining regions (CDR)), interspersed with the more conserved regions referred to as framework regions (FR) of.
  • CDR complementarity determining regions
  • FR framework regions
  • Each V H and V L the following order: FR1, CDR1, FR2, CDR2 , FR3, CDR3, FR4 from the amino terminus to the carboxy terminus arranged three four FR and CDR components.
  • the antibody suitable for the present invention is an antibody that targets CD20
  • the anti-CD20 monoclonal antibody is any antibody that targets CD20, such as rituximab or a biological analog thereof.
  • biological analogs refer to antibody products that have the same sequence as rituximab, physicochemical properties, and biological activity, and clinical safety and effectiveness are also consistent with rituximab.
  • MMAE the structure of MMAE is:
  • the drug: antibody ratio (DAR) or drug loading (loading) is represented by p or q, that is, in the molecule of formula I: Ab-(LD) p or formula II: Ab-(LD) q
  • the average number of drug modules (ie, cytotoxic agents) per antibody can be an integer or a fraction.
  • the ADC of the general formula I includes a collection of antibodies coupled with a certain range (3.6-4.0) drug modules
  • the ADC of the general formula II includes a collection of antibodies coupled with a certain range (3.3-4.3) drug modules.
  • the average number of drug modules per antibody in the ADC preparation from the coupling reaction can be verified by conventional means, such as mass spectrometry, ELISA assay, HIC, and HPLC.
  • drug moieties that are less than the theoretical maximum in the coupling reaction are coupled to the antibody.
  • antibodies do not contain many free and reactive cysteine thiol groups, which can be connected to drug modules; in fact, most of the cysteine thiol groups in antibodies exist in the form of disulfide bridges.
  • the antibody can be reduced with a reducing agent such as dithiothreitol (DTT) or tricarbonyl ethyl phosphine (TCEP) under partial or complete reducing conditions to produce a reactive cysteine thiol group.
  • DTT dithiothreitol
  • TCEP tricarbonyl ethyl phosphine
  • the antibody drug conjugate of the present invention can be used in combination with known chemotherapeutic drugs or immunosuppressive agents for the treatment of tumors, such as adriamycin (Adriamycin), cyclophosphamide and taxanes [paclitaxel (Taxol and docetaxel (Taxotere)), capecitabine (Xeloda), gemcitabine (Gemzar), vinorelbine (Navelbine), tamoxifen, aromatase inhibitors (Raininide, Fron, Anoxin), 5-FU plus leucovorin, irinotecan (camptosar), oxaliplatin, cisplatin, carboplatin, estramustine, mitoxantrone (Novantrone), prednisone, vincristine (Oncovin), doxorubicin, prednisone, etc., or a combination thereof; the immunosuppressant is selected from: (1)
  • the immunosuppressive agent is, for example, methylprednisolone, prednisone, azathioprine, prolactin, zenapax, sule, cyclosporine, tacrolimus, rapamycin Sulfate, mycophenolate mofetil, mizoribine, cyclophosphamide, fingolimod, etc.
  • treatment refers to clinical intervention that attempts to change the natural course of the individual or cell to be treated, and may be for prevention or in the course of clinical pathology.
  • the desired effects of treatment include preventing the occurrence or recurrence of the disease, alleviating symptoms, weakening any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the disease state, and avoiding or improving the prognosis.
  • the antibody or antibody-drug conjugate of the present invention is used to delay the occurrence of a disease or disorder, or to slow the progression of a disease or disorder.
  • efficacy can be measured, for example, by assessing the time to disease progression (TTP) and/or determining the response rate (RR).
  • vertebrates refer to mammals. Mammals include, but are not limited to, livestock (such as cattle), pets (such as cats, dogs, and horses), primates, mice, and rats. In certain embodiments, the mammal refers to a human.
  • an amount refers to an amount effective to achieve the desired therapeutic or preventive effect at the necessary dose and time.
  • the "therapeutically effective amount” of the substance/molecule of the present invention may vary depending on factors such as the individual's disease state, age, sex, and weight, and the ability of the substance/molecule to elicit a desired response in the individual.
  • a therapeutically effective amount also encompasses an amount in which the therapeutically beneficial effects of the substance/molecule outweigh any toxic or harmful consequences.
  • “Prophylactically effective amount” refers to an amount effective to achieve the desired preventive effect at the necessary dose and time.
  • the preventive effective dose will be lower than the therapeutically effective dose.
  • the therapeutically effective dose of the drug can reduce the number of cancer cells; reduce the tumor volume; inhibit (ie slow down to a certain extent, preferably stop) the infiltration of cancer cells into the surrounding organs; inhibit (ie slow down to a certain extent, preferably stop) ) Tumor metastasis; inhibit tumor growth to a certain extent; and/or reduce one or more symptoms related to cancer to a certain extent.
  • the appropriate dosage of the antibody-drug conjugate of the present invention (when used alone or in combination with one or more other therapeutic agents such as chemotherapeutics) will depend on the type of disease to be treated, The type of antibody drug conjugate, the severity and progress of the disease, whether the antibody drug conjugate is administered for prevention or treatment purposes, previous therapies, the patient's clinical history and reactivity to the antibody drug conjugate, and the attending physician The judgment of the teacher.
  • the antibody drug conjugate is administered to the patient at one time or through a series of treatments.
  • An exemplary dose of the antibody drug conjugate can range from about 0.1 mg/kg to about 5 mg/kg.
  • one or more doses of the antibody drug conjugate at about 0.1 mg/kg, 0.3 mg/kg, 1.0 mg/kg, 3.0 mg/kg, or 5 mg/kg (or any combination thereof) can be administered to the patient.
  • “Pharmaceutically acceptable carrier” when used in the present invention includes pharmaceutically acceptable carriers, excipients or stabilizers, which are non-toxic to the cells or mammals exposed to them at the dose and concentration used.
  • the physiologically acceptable carrier is a pH buffered aqueous solution.
  • physiologically acceptable carriers include buffers, such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or Immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose Sugar, sucrose, trehalose or dextrin; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt-forming counterions, such as sodium; and/or non-ionic surfactants, such as TWEEN TM , polyethylene Glycol (PEG) and PLURONICS TM .
  • buffers such as phosphate, citrate, and other organic acids
  • antioxidants including ascorbic acid
  • the monoclonal cell line was subjected to genetic sequencing, and the sequencing results showed that the sequences encoding the light chain and heavy chain of MAB801 were completely consistent with the commercially available rituximab.
  • the preparation of MAB801 adopts the flow-added cell culture process widely used in the antibody field.
  • the process starts with the recovery of cell seeds in the working cell bank.
  • the main steps include inoculation in the shake flask, cell expansion in the shake flask, and stepwise cell expansion and culture in the reactor, and then inoculated into the production reactor for fed-batch culture.
  • Then through the purification process including fermentation broth clarification (deep filtration), Protein A affinity chromatography, low pH virus inactivation and deep filtration, PB incubation, anion exchange chromatography, cation exchange chromatography, virus removal filtration, and cutting
  • test results show that the physicochemical indicators such as molecular weight of the produced rituximab biosimilar drug (biosimilar) are highly similar to rituximab.
  • the prepared antibody-drug conjugate ADC-1 was analyzed by HIC-HPLC (Ouyang-2013) to determine the drug/antibody ratio (DAR), as shown in Figure 1.
  • the average drug loading is calculated based on the peak area of the map The DAR is 3.8.
  • the IC 50 value is the average value of the IC 50 calculated in two 96-well plates; ND indicates that the highest proliferation inhibition percentage (inhibition%) is less than 50%, so the IC 50 value cannot be calculated.
  • PDX model Human-derived tumor tissue xenotransplantation model
  • NHL PDX model Human-derived tumor tissue xenotransplantation model
  • Table 3 PDX model information used in in vivo pharmacodynamic experiments
  • LYM#004 is a The resistant DLBCL PDX model, the experimental results are shown in Figure 6.
  • the T/C (%) on Day 18 was 45.90% (P>0.05), 4.46% (P ⁇ 0.001) and 2.05% (P ⁇ 0.001), respectively , TGI% are 54.10%, 95.54% and 97.95%, respectively.
  • ADC-1 was administered at 3 mg/kg, all tumors partially resolved on Day 18; after ADC-1 was administered at 10 mg/kg, 5/8 tumors were partially resolved and 3/8 tumors were completely resolved on Day 18, respectively.
  • the T/C (%) of Day 18 were 50.76% (P>0.05) and 51.50% (P>0.05), and the TGI% were 49.24% and 48.50%, respectively.
  • ADC-1 (3 and 10mg/kg) has significant tumor growth inhibitory activity
  • ADC-1 (1mg/kg) and reference drug (3 and 10mg/kg) have no significant anti-tumor activity.
  • Tumor-bearing mice against ADC-1 and All are well tolerated.
  • LYM#013 is a The experimental results of the resistant DLBCL PDX model are shown in Figure 7.
  • T/C (%) on Day 28 was 31.90% (P ⁇ 0.05), 0.00% (P ⁇ 0.001) and 0.00% (P ⁇ 0.001), respectively , TGI% were 68.10%, 100.00%, and 100.00%, respectively, 2/8, 0/8, and 0/8 tumors were partially regressed, and 0/8, 8/8, and 8/8 tumors were completely regressed.
  • the T/C (%) on Day 28 was 79.86% (P>0.05), and the TGI% was 20.14%.
  • LYM#016 is a DLBCL PDX model, and the experimental results are shown in Figure 8.
  • T/C (%) on Day 14 was 37.76% (P>0.05), 4.27% (P ⁇ 0.01) and 1.91% (P ⁇ 0.01), respectively , TGI% are 62.24%, 95.73% and 98.09%, respectively.
  • ADC-1 was administered at 1 mg/kg, all tumors partially resolved on Day 14.
  • ADC-1 was administered at 3 mg/kg, 6/8 of the tumors were partially resolved and 2/8 of the tumors were completely resolved on Day 14. Subside.
  • the T/C (%) on Day 14 was 30.98% (P>0.05), and the TGI% was 69.02%.
  • Table 4 summarizes the in vivo efficacy test results of ADC-1 in the three human lymphoma (NHL) PDX models with CD20 positive.
  • Relative tumor growth rate T/C(%) (administration group RTV/Vehicle group RTV) ⁇ 100%.
  • Tumor growth inhibition rate TGI% (average tumor volume in vehicle group-average tumor volume in administration group)/average tumor volume in vehicle group ⁇ 100%.
  • the inventor of the present application based on the formulation research and past experience of ADC-1, finally determined that the formulation formulation was 5mg/mL antibody-drug conjugate (naked antibody-drug conjugate without buffer and other components), 10mM Histidine buffer (pH5.8), 6% sucrose, 0.035% Tween 80 (PS80).
  • W means week, that is, 2W means 2 weeks; ⁇ means detection.
  • the stability of the sample showed the same trend, but the amplitude of the change increased; when the pH was 6.7, 7.0, the appearance of the sample changed after one week at 40°C, and opalescence appeared.
  • the degree of opalescence increased with the increase of pH. Intensified, due to the presence of particulate matter (opalescence), the calculated protein content deviates from the actual value due to the distortion of the readings due to the presence of particles (opalescence); the purity of SEC decreases, and the content of high polymer and low molecular weight fragments (LMW) increases, and the speed of change increases with the increase of pH. ;
  • the purity of iCIEF decreases, the content of acidic peaks increases, and the content of alkaline peaks decreases, and the speed of change increases with the increase of pH.

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Abstract

提供一种抗CD20抗体药物偶联物，包含该抗体药物偶联物的制剂，包含该抗体药物偶联物的组合物，以及该抗体药物偶联物的医药用途。

Description

抗体药物偶联物及其制剂 技术领域
本发明涉及生物医药领域，具体涉及抗体药物偶联物，包含该抗体药物偶联物的制剂，包含该抗体药物偶联物的组合物，以及该抗体药物偶联物的医药用途。
背景技术
目前，非霍奇金淋巴瘤(NHL)的治疗包括传统的手术治疗、化疗、放疗、骨髓或造血干细胞移植、免疫及靶向治疗。传统化学药物治疗对NHL患者有一定的疗效，但其产生的系统性毒副作用大、耐受性差，且易复发，限制了患者的使用。靶向药物，尤其是抗体药物，因疗效显著且治疗过程中产生的系统性毒副作用小等优点备受关注。
近年来有多个CD20靶向的单抗药物被开发出来(如利妥昔单抗，
Figure PCTCN2020088564-appb-000001
)，并且其抗肿瘤活性在临床试验中得到了验证。
但是随着临床应用的深入，利妥昔单抗治疗的局限性逐渐显露出来，即在治疗过程中存在病人对药物的原发性和获得性耐药，导致利妥昔单抗治疗效果降低或无效。另外，现有CD20靶向单抗单药治疗肿瘤的有效率并不理想，需要与化疗药物联用以提高抗肿瘤效果，这无疑增大了毒性的风险。由此看来，以利妥昔单抗为代表的已上市CD20靶向单抗药物还远远不能满足临床实践中对复发或难治的B细胞NHL(包括DLBCL和FL等)治疗的需求。
鉴于上述现有CD20靶向单抗药物存在的种种问题，提供疗效更好、毒副作用更低，且不容易产生耐药的治疗手段，是开发CD20靶向ADC药物ADC-1的主要目的。
发明内容
本发明的发明人通过大量实验和创造性劳动，制备得到了抗CD20抗体药物偶联物，并证实其具有良好的生物学活性和制剂稳定性，由此完成了本发明。
为此，在本发明的第一方面，本发明提供了抗体药物偶联物，所述抗体药物偶联物具有式Ⅰ所示的结构，
Ab-(L-D) p
式Ⅰ
其中：
Ab为抗CD20单抗，所述抗CD20单抗为任何靶向CD20的抗体，例如利妥昔单抗或其生物类似物；
D为细胞毒剂，所述细胞毒剂为Monomethyl auristatin E(MMAE)；
L为接头，用于连接所述抗CD20单抗和所述细胞毒剂，所述接头为6-马来酰亚氨基己酰基-缬氨酸-瓜氨酸-对氨基苄氧羰基(MC-vc-PAB)；
p为3.6-4.0。
在一些实施方案中，p为3.7-3.9。
在一些实施方案中，p为3.8。
在一些实施方案中，式I中所述的L-D为vc-MMAE，其结构如下式所示：
Figure PCTCN2020088564-appb-000002
在一些实施方案中，所述抗体药物偶联物的结构如下式所示：
Figure PCTCN2020088564-appb-000003
其中：
Ab为抗CD20单抗，所述抗CD20单抗为任何靶向CD20的抗体，例如利妥昔单抗或其生物类似物，
p为3.6-4.0。
在一些实施方案中，p为3.7-3.9。
在一些实施方案中，p为3.8。
在本发明的第二方面，本发明提供了抗体药物偶联物制剂，其包括：
抗体药物偶联物，其浓度为1-60mg/mL(如1mg/mL、2mg/mL、3mg/mL、4mg/mL、5mg/mL、6mg/mL、7mg/mL、8mg/mL、9mg/mL、10mg/mL、13mg/mL、15mg/mL、17mg/mL、19mg/mL、20mg/mL、25mg/mL、30mg/mL、35mg/mL、40mg/mL、45mg/mL、50mg/mL、 55mg/mL或60mg/mL)；
5-35mM(如5mM、8mM、9mM、10mM、11mM、12mM、15mM、20mM、25mM、30mM或35mM)的组氨酸缓冲液，pH5.0-6.2(如5.0、5.1、5.2、5.3、5.4、5.5、5.6、5.7、5.8、5.9、6.0、6.1或6.2)；
2-10％(如2％、3％、4％、5％、6％、7％、8％、9％或10％)的蔗糖；和
0.01-0.1％(如0.01％、0.015％、0.02％、0.025％、0.03％、0.031％、0.032％、0.033％、0.034％、0.035％、0.036％、0.037％、0.038％、0.039％、0.04％、0.045％、0.05％、0.055％、0.06％、0.065％、0.07％、0.08％、0.09％或0.1％)的吐温80；
所述抗体药物偶联物具有式ⅠI所示的结构，
Ab-(L-D) q
式ⅠI
其中：
Ab为抗CD20单抗，所述抗CD20单抗为任何靶向CD20的抗体，例如利妥昔单抗或其生物类似物；
D为细胞毒剂，所述细胞毒剂为Monomethyl auristatin E(MMAE)；
L为接头，用于连接所述抗CD20单抗和所述细胞毒剂，所述接头为6-马来酰亚氨基己酰基-缬氨酸-瓜氨酸-对氨基苄氧羰基(MC-vc-PAB)；
q为3.3-4.3(如3.3、3.4、3.5、3.6、3.7、3.8、3.9、4.0、4.1、4.2或4.3)。
在一些实施方案中，q为3.6-4.0。
在一些实施方案中，q为3.7-3.9。
在一些实施方案中，q为3.8。
在一些实施方案中，所述抗体药物偶联物的浓度为1-20mg/mL。
在一些实施方案中，所述抗体药物偶联物的浓度为1-10mg/mL。
在一些实施方案中，所述抗体药物偶联物的浓度为3-7mg/mL。
在一些实施方案中，所述抗体药物偶联物的浓度为5mg/mL。
在一些实施方案中，所述组氨酸缓冲液的浓度为5-15mM。
在一些实施方案中，所述组氨酸缓冲液的浓度为8-12mM。
在一些实施方案中，所述组氨酸缓冲液的浓度为10mM。
在一些实施方案中，所述组氨酸缓冲液的pH为5.8-6.2。
在一些实施方案中，所述组氨酸缓冲液的pH为5.6-6.0。
在一些实施方案中，所述组氨酸缓冲液的pH为5.4-6.0。
在一些实施方案中，所述组氨酸缓冲液的pH为5.4-6.2。
在一些实施方案中，所述蔗糖的浓度为3-9％。
在一些实施方案中，所述蔗糖的浓度为4-8％。
在一些实施方案中，所述蔗糖的浓度为5-7％。
在一些实施方案中，所述蔗糖的浓度为6％。
在一些实施方案中，所述吐温80的浓度为0.02-0.06％。
在一些实施方案中，所述吐温80的浓度为0.02-0.05％。
在一些实施方案中，所述吐温80的浓度为0.03-0.04％。
在一些实施方案中，所述吐温80的浓度为0.035％。
在一些实施方案中，所述制剂包括：
所述抗体药物偶联物，浓度为5mg/mL；
10mM组氨酸缓冲液，pH5.8；
6％蔗糖；和
0.035％吐温80。
需要说明的是，“2-10％蔗糖”表示质量体积浓度(w/v％)，指的是每1000mL的所述抗体药物偶联物制剂中，蔗糖质量为20-100g。“0.01-0.1％吐温80”表示质量体积浓度(w/v％)，其含义可以参照前述“2-10％蔗糖”进行类似的理解。
另外，“组氨酸缓冲液”的浓度指的是组氨酸和盐酸组氨酸的浓度，其pH是通过调整组氨酸和盐酸组氨酸比例得到的。
在一些实施方案中，pH为5.0-6.2(例如5.6-6.0)时，所述抗体药物偶联物制剂在25±2℃/60％±5％RH条件下可稳定6周。
在一些实施方案中，pH为5.0-6.2(例如5.6-6.0)时，所述抗体药物偶联物制剂在25℃条件下放置6周时，外观无色澄清。
在一些实施方案中，pH为5.0-6.2(例如5.6-6.0)时，所述抗体药物偶联物制剂在25℃条件下放置6周时，pH未发生变化。
在一些实施方案中，pH为5.0-6.2(例如5.6-6.0)时，所述抗体药物偶联物制剂在25℃条件下放置6周时，浓度未发生变化。
在一些实施方案中，pH为5.0-6.2(例如5.6-6.0)时，所述抗体药物偶联物制剂在25℃条件下放置6周时，SEC单体％含量的降低不超过2％(或者不超过1％)。
在一些实施方案中，pH为5.0-6.2(例如5.6-6.0)时，所述抗体药物偶 联物制剂在25℃条件下放置6周时，SEC高聚体％含量的升高不超过2％(或者不超过1％)。
在一些实施方案中，pH为5.0-6.2(例如5.6-6.0)时，所述抗体药物偶联物制剂在25℃条件下放置6周时，DAR值未出现明显变化。
在一些实施方案中，pH为5.0-6.2(例如5.6-6.0)时，所述抗体药物偶联物制剂在40℃条件下放置4周时，外观无色澄清。
在一些实施方案中，pH为5.0-6.2(例如5.6-6.0)时，所述抗体药物偶联物制剂在40℃条件下放置4周时，pH未发生变化。
在一些实施方案中，pH为5.0-6.2(例如5.6-6.0)时，所述抗体药物偶联物制剂在40℃条件下放置4周时，浓度未发生变化。
在一些实施方案中，pH为5.0-6.2(例如5.6-6.0)时，所述抗体药物偶联物制剂在40℃条件下放置4周时，SEC单体％含量的降低不超过5％(或者不超过4％)。
在一些实施方案中，pH为5.0-6.2(例如5.6-6.0)时，所述抗体药物偶联物制剂在40℃条件下放置4周时，SEC高聚体％含量的升高不超过5％(或者不超过4％)。
在一些实施方案中，pH为5.0-6.2(例如5.6-6.0)时，所述抗体药物偶联物制剂在40℃条件下放置4周时，DAR值未出现明显变化。
需要说明的是，术语“无明显变化”或“无变化”指的是无统计学上的明显变化。
另外，需要说明的是，上述“抗体药物偶联物”指的是含有相同或不同DAR值的ADC分子的组合物。
具体地，本发明提供了包含多个抗CD20ADC分子的组合物。在某些情况下，本文所述的组合物中的每个ADC包含相同数目的一个或多个药物分子。在其他情况下，本文所述的组合物中的每个ADC包含不同数目的一个或多个药物分子。
本文所述的抗体药物偶联物中，每个抗CD20抗体可以偶联有1个、2个、3个、4个、5个、6个、7个、8个或更多药物分子(优选2个、4个、6个或8个，更优选为2个或4个)。
上述药物抗体比(DAR)是指偶联到抗CD20抗体的抗癌药物的分子数。本文所述的ADC中包含的抗癌药物的分子数通常是整数，当本文所述的ADC中包含的抗癌药物的分子数(例如，式I中的p，或者式II中的q)是分数时，该分数指的是包含多个ADC分子的组合物中，每个抗CD20抗体的平 均抗癌药物分子数量。
在本发明的第三方面，本发明提供了前述抗体药物偶联物制剂的制备方法，包括：
将抗CD20单抗和还原剂进行还原反应，获得被还原的抗CD20单抗，所述抗CD20单抗为任何靶向CD20的抗体，例如利妥昔单抗或其生物类似物；
将所述被还原的抗CD20单抗和vcMMAE进行偶联反应；
淬灭所述偶联反应；
将所述淬灭后的偶联反应产物进行缓冲液置换，获得所述抗体药物偶联物制剂，所述抗体药物偶联物制剂包括：
所述抗体药物偶联物的浓度为1-60mg/mL(如1mg/mL、2mg/mL、3mg/mL、4mg/mL、5mg/mL、6mg/mL、7mg/mL、8mg/mL、9mg/mL、10mg/mL、13mg/mL、15mg/mL、17mg/mL、19mg/mL、20mg/mL、25mg/mL、30mg/mL、35mg/mL、40mg/mL、45mg/mL、50mg/mL、55mg/mL或60mg/mL)；
5-35mM(如5mM、8mM、9mM、10mM、11mM、12mM、15mM、20mM、25mM、30mM或35mM)的组氨酸缓冲液，pH5.0-6.2(如5.0、5.1、5.2、5.3、5.4、5.5、5.6、5.7、5.8、5.9、6.0、6.1或6.2)；
2-10％(如2％、3％、4％、5％、6％、7％、8％、9％或10％)的蔗糖；和
0.01-0.1％(如0.01％、0.015％、0.02％、0.025％、0.03％、0.031％、0.032％、0.033％、0.034％、0.035％、0.036％、0.037％、0.038％、0.039％、0.04％、0.045％、0.05％、0.055％、0.06％、0.065％、0.07％、0.08％、0.09％或0.1％)吐温80。
在一些实施方案中，所述抗体药物偶联物的浓度为1-20mg/mL。
在一些实施方案中，所述抗体药物偶联物的浓度为1-10mg/mL。
在一些实施方案中，所述抗体药物偶联物的浓度为3-7mg/mL。
在一些实施方案中，所述抗体药物偶联物的浓度为5mg/mL。
在一些实施方案中，所述组氨酸缓冲液的浓度为5-15mM。
在一些实施方案中，所述组氨酸缓冲液的浓度为8-12mM。
在一些实施方案中，所述组氨酸缓冲液的浓度为10mM。
在一些实施方案中，所述组氨酸缓冲液的pH为5.8-6.2。
在一些实施方案中，所述组氨酸缓冲液的pH为5.6-6.0。
在一些实施方案中，所述组氨酸缓冲液的pH为5.4-6.0。
在一些实施方案中，所述组氨酸缓冲液的pH为5.4-6.2。
在一些实施方案中，所述蔗糖的浓度为3-9％。
在一些实施方案中，所述蔗糖的浓度为4-8％。
在一些实施方案中，所述蔗糖的浓度为5-7％。
在一些实施方案中，所述蔗糖的浓度为6％。
在一些实施方案中，所述吐温80的浓度为0.02-0.06％。
在一些实施方案中，所述吐温80的浓度为0.02-0.05％。
在一些实施方案中，所述吐温80的浓度为0.03-0.04％。
在一些实施方案中，所述吐温80的浓度为0.035％。
在一些实施方案中，所述还原剂为DTT。
在一些实施方案中，所述制备方法包括：
1)取10毫克的抗CD20单抗，使用15mL的30KD超滤装置置换到还原缓冲液(25mM硼酸钠,pH8.0,25mM NaCl,5mM EDTA)中，共三次置换；终体积约为1mL,转移至新的Eppendorf离心管(称重)中，并称重；检测蛋白浓度，计算蛋白总量；
2)向抗体中加入2.0-3.0倍摩尔数的DTT,室温保温2小时，连续混匀；使用15ml的30KD超滤装置置换到偶联缓冲液(50mM Tris,pH7.2,150mM NaCl,5mM EDTA)中，共三次置换；取浓缩液，以A 280(在280nm波长处的吸光度)测定蛋白浓度，并称重，计算蛋白总量；取10μl样品，以Ellman’s方法测定自由巯基数；
并以下列公式计算其自由巯基的摩尔浓度(其中，A 412表示在412nm波长处的吸光度)：
Figure PCTCN2020088564-appb-000004
b:比色皿光路长度(通常1cm)
根据自由巯基的摩尔浓度和总蛋白溶液体积计算自由巯基摩尔数；
3)向还原后的抗体中加入1.0-1.5倍于自由巯基摩尔数的vc-MMAE(溶于DMSO)，混匀后室温反应2小时，间断混匀；向反应体系中加入20倍于所投入之vc-MMAE摩尔数的N-乙酰半胱氨酸于反应液中，混匀，静置5分钟；
4)使用15ml的30KD超滤装置置换到偶联物储存液(10mM组氨酸(Histidine),6％蔗糖(Sucrose),0.035％PS80,pH5.8)中，共三次置换，即得到所述抗体药物偶联物制剂，于4℃保存。
在本发明的第四方面，本发明提供了组合物，其含有前述抗体药物偶联物，或者前述抗体药物偶联物制剂，或者前述方法制备的抗体药物偶联物制剂。
在一些实施方案中，所述组合物还含有至少一种药学上可接受的载体、稀释剂或赋形剂。
在一些实施方案中，所述组合物中还包含已知的用于治疗肿瘤的化疗药物，所述化疗药物例如为阿霉素(Adriamycin)、环磷酰胺和紫杉烷类[紫杉醇(Taxol)和多西他赛(Taxotere)、卡培他滨(Xeloda)、吉西他滨(Gemzar)、长春瑞滨(Navelbine)、他莫昔芬、芳香酶抑制剂(瑞宁得、弗隆、阿诺新)、5-FU加亚叶酸、伊立替康(camptosar)、奥沙利铂、顺铂、卡铂、雌莫司汀、米托蒽醌(Novantrone)、泼尼松、长春新碱(Oncovin)、多柔比星、强的松等，或它们的组合。
在一些实施方案中，所述组合物中还包含免疫抑制剂，所述免疫抑制剂选自：(1)糖皮质激素类，如可的松和强的松；(2)微生物代谢产物，如环孢菌素和藤霉素等；(3)抗代谢物，如硫唑嘌呤和6-巯基嘌呤等；(4)多克隆和单克隆抗淋巴细胞抗体，如抗淋巴细胞球蛋白和OKT3等；(5)烷化剂类，如环磷酰胺。具体地，所述免疫抑制剂例如为甲基强的松龙，强的松，硫唑嘌呤，普乐可复，赛尼哌，舒莱，环孢菌素，他克莫司，雷帕霉素，霉酚酸酯，咪唑立宾，环磷酰胺，芬戈莫德等。
在本发明的第五方面，本发明提供了前述抗体药物偶联物，或者前述抗体药物偶联物制剂，或者前述方法制备的抗体药物偶联物制剂，或者前述组合物在制备预防和/或治疗表达CD20的癌症或免疫疾病的药物中的用途。
在一些实施方案中，所述表达CD20的癌症为淋巴瘤或白血病。
在一些实施方案中，所述淋巴瘤为非霍奇金淋巴瘤、B细胞非霍奇金淋巴瘤、滤泡性非霍奇金淋巴瘤、小淋巴细胞性淋巴瘤或弥漫大B细胞淋巴瘤。
在一些实施方案中，所述白血病为慢性淋巴细胞白血病、毛细胞白血病、B细胞幼淋巴细胞性白血病或急性淋巴细胞性白血病。
在一些实施方案中，所述表达CD20的免疫疾病为类风湿性关节炎、肉芽肿性多血管炎、Wegener氏肉芽肿、显微镜多血管炎或多发性硬化。
在一些实施方案中，所述类风湿性关节炎为经至少一种TNF拮抗剂治疗失败的严重活动性类风湿性关节炎。
优选地，在一些实施方案中，所述表达CD20的癌症为CD20阳性B细胞 性淋巴瘤。
在一些实施方案中，所述CD20阳性B细胞性淋巴瘤为抗CD20单抗(例如，利妥昔单抗)耐药性CD20阳性B细胞性淋巴瘤。
在一些实施方案中，所述淋巴瘤为非霍奇金淋巴瘤。
在一些实施方案中，所述淋巴瘤为抗CD20单抗(例如，利妥昔单抗)耐药性非霍奇金淋巴瘤。
在一些实施方案中，所述非霍奇金淋巴瘤为弥漫大B细胞淋巴瘤。
在一些实施方案中，所述非霍奇金淋巴瘤为抗CD20单抗(例如，利妥昔单抗)耐药性弥漫大B细胞淋巴瘤。
在本发明的第六方面，本发明提供了预防和/治疗表达CD20的癌症或免疫疾病的方法，其包括给予有需要的受试者预防和/或治疗有效量的前述抗体药物偶联物，或者前述抗体药物偶联物制剂，或者前述方法制备的抗体药物偶联物制剂，或者前述的组合物。
在一些实施方案中，所述表达CD20的癌症为淋巴瘤或白血病。
在一些实施方案中，所述淋巴瘤为非霍奇金淋巴瘤、B细胞非霍奇金淋巴瘤、滤泡性非霍奇金淋巴瘤、小淋巴细胞性淋巴瘤或弥漫大B细胞淋巴瘤。
在一些实施方案中，所述白血病为慢性淋巴细胞白血病、毛细胞白血病、B细胞幼淋巴细胞性白血病或急性淋巴细胞性白血病。
在一些实施方案中，所述表达CD20的免疫疾病为类风湿性关节炎、肉芽肿性多血管炎、Wegener氏肉芽肿、显微镜多血管炎或多发性硬化。
在一些实施方案中，所述类风湿性关节炎为经至少一种TNF拮抗剂治疗失败的严重活动性类风湿性关节炎。
优选地，在一些实施方案中，所述表达CD20的癌症为CD20阳性B细胞性淋巴瘤。
在一些实施方案中，所述CD20阳性B细胞性淋巴瘤为抗CD20单抗(例如，利妥昔单抗)耐药性CD20阳性B细胞性淋巴瘤。
在一些实施方案中，所述淋巴瘤为非霍奇金淋巴瘤。
在一些实施方案中，所述淋巴瘤为抗CD20单抗(例如，利妥昔单抗)耐药性非霍奇金淋巴瘤。
在一些实施方案中，所述非霍奇金淋巴瘤为弥漫大B细胞淋巴瘤。
在一些实施方案中，所述非霍奇金淋巴瘤为抗CD20单抗(例如，利妥昔单抗)耐药性弥漫大B细胞淋巴瘤。
在一些实施方案中，受试者为NHL PDX模型时，给予有需要的受试者 的给药剂量为0.3-10mg/kg(如0.3mg/kg、1mg/kg、3mg/kg、10mg/kg)。
在一些实施方案中，受试者为NHL PDX模型时，给予有需要的受试者的给药剂量为0.3-3mg/kg、1-3mg/kg、1-10mg/kg或3-10mg/kg。
在一些实施方案中，受试者为人时，给予有需要的受试者的给药剂量为0.1-5mg/kg(如0.1mg/kg、0.3mg/kg、1mg/kg、3mg/kg或5mg/kg)。
在一些实施方案中，向有需要的受试者每1、2、3、4、5或6周给予一次预防和/或治疗有效量的前述抗体药物偶联物，或者前述抗体药物偶联物制剂，或者前述方法制备的抗体药物偶联物制剂，或者前述的组合物。
在一些实施方案中，向有需要的受试者每18-24天给予一次预防和/或治疗有效量的前述抗体药物偶联物，或者前述抗体药物偶联物制剂，或者前述方法制备的抗体药物偶联物制剂，或者前述的组合物。
在一些实施方案中，还包括向有需要的受试者给予另外的用于治疗肿瘤的化疗药物或免疫抑制剂。
在一些实施方案中，所述化疗药物例如为阿霉素(Adriamycin)、环磷酰胺和紫杉烷类[紫杉醇(Taxol)和多西他赛(Taxotere)]、卡培他滨(Xeloda)、吉西他滨(Gemzar)、长春瑞滨(Navelbine)、他莫昔芬、芳香酶抑制剂(瑞宁得、弗隆、阿诺新)、5-FU加亚叶酸、伊立替康(camptosar)、奥沙利铂、顺铂、卡铂、雌莫司汀、米托蒽醌(Novantrone)、泼尼松、长春新碱(Oncovin)、多柔比星、强的松等，或它们的组合。
在一些实施方案中，所述免疫抑制剂选自：(1)糖皮质激素类，如可的松和强的松；(2)微生物代谢产物，如环孢菌素和藤霉素等；(3)抗代谢物，如硫唑嘌呤和6-巯基嘌呤等；(4)多克隆和单克隆抗淋巴细胞抗体，如抗淋巴细胞球蛋白和OKT3等；(5)烷化剂类，如环磷酰胺。具体地，所述免疫抑制剂例如为甲基强的松龙，强的松，硫唑嘌呤，普乐可复，赛尼哌，舒莱，环孢菌素，他克莫司，雷帕霉素，霉酚酸酯，咪唑立宾，环磷酰胺，芬戈莫德等。
在本发明的第七方面，本发明提供了前述抗体药物偶联物，或者前述抗体药物偶联物制剂，或者前述方法制备的抗体药物偶联物制剂，或者前述组合物，其用于预防和/治疗表达CD20的癌症或免疫疾病。
在一些实施方案中，所述表达CD20的癌症为淋巴瘤或白血病。
在一些实施方案中，所述淋巴瘤为非霍奇金淋巴瘤、B细胞非霍奇金淋巴瘤、滤泡性非霍奇金淋巴瘤、小淋巴细胞性淋巴瘤或弥漫大B细胞淋巴瘤。
在一些实施方案中，所述白血病为慢性淋巴细胞白血病、毛细胞白血病、 B细胞幼淋巴细胞性白血病或急性淋巴细胞性白血病。
在一些实施方案中，所述表达CD20的免疫疾病为类风湿性关节炎、肉芽肿性多血管炎、Wegener氏肉芽肿、显微镜多血管炎或多发性硬化。
在一些实施方案中，所述类风湿性关节炎为经至少一种TNF拮抗剂治疗失败的严重活动性类风湿性关节炎。
优选地，在一些实施方案中，所述表达CD20的癌症为CD20阳性B细胞性淋巴瘤。
在一些实施方案中，所述CD20阳性B细胞性淋巴瘤为抗CD20单抗(例如，利妥昔单抗)耐药性CD20阳性B细胞性淋巴瘤。
在一些实施方案中，所述淋巴瘤为非霍奇金淋巴瘤。
在一些实施方案中，所述淋巴瘤为抗CD20单抗(例如，利妥昔单抗)耐药性非霍奇金淋巴瘤。
在一些实施方案中，所述非霍奇金淋巴瘤为弥漫大B细胞淋巴瘤。
在一些实施方案中，所述非霍奇金淋巴瘤为抗CD20单抗(例如，利妥昔单抗)耐药性弥漫大B细胞淋巴瘤。
在一些实施方案中，受试者为NHL PDX模型时，其给药剂量为0.3-10mg/kg(如0.3mg/kg、1mg/kg、3mg/kg、10mg/kg)。
在一些实施方案中，受试者为NHL PDX模型时，其给药剂量为0.3-3mg/kg、1-3mg/kg、1-10mg/kg或3-10mg/kg。
在一些实施方案中，受试者为人时，其给药剂量为0.1-5mg/kg(如0.1mg/kg、0.3mg/kg、1mg/kg、3mg/kg或5mg/kg)。
有益效果：
1、ADC-1是由Rituximab生物类似物MAB801通过vcMMAE偶联而成的ADC，通过与肿瘤细胞表面CD20受体的结合、内吞、释放MMAE，以杀伤CD20阳性的非霍奇金淋巴瘤(NHL)细胞，从而控制肿瘤的生长和引起肿瘤消退；
2、ADC-1在多种CD20表达的NHL细胞株、以及多种CD20表达的人NHL PDX肿瘤模型中均表现了显著的抑制肿瘤细胞生长的药效作用，尤其对
Figure PCTCN2020088564-appb-000005
耐药的NHL PDX模型也表现出明显的肿瘤生长抑制作用；
3、ADC-1的制备工艺中，整个反应过程只需在一个容器里顺序完成，无需中间纯化步骤，优于其他制备工艺；
4、ADC-1的制备工艺中，整个工艺过程只需一次超滤纯化步骤，无 需层析工艺，优于其他制备工艺；
5、ADC-1具有良好的制剂稳定性。
附图说明
图1为本发明实施例的ADC-1的HIC-HPLC图谱，其中，Minutes表示分钟；
图2为本发明实施例的ADC-1与
Figure PCTCN2020088564-appb-000006
在Daudi细胞株中代表性的细胞杀伤作用曲线，其中，Inhibition表示抑制率，Concentration表示浓度；
图3为本发明实施例的ADC-1与
Figure PCTCN2020088564-appb-000007
在Jeko-1细胞株中代表性的细胞杀伤作用曲线，其中，Inhibition表示抑制率，Concentration表示浓度；
图4为本发明实施例的ADC-1与
Figure PCTCN2020088564-appb-000008
在Raji细胞株中代表性的细胞杀伤作用曲线，其中，Inhibition表示抑制率，Concentration表示浓度；
图5为本发明实施例的ADC-1与
Figure PCTCN2020088564-appb-000009
在Ramos细胞株中代表性的细胞杀伤作用曲线，其中，Inhibition表示抑制率，Concentration表示浓度；
图6为本发明实施例的ADC-1和
Figure PCTCN2020088564-appb-000010
对CD20阳性人淋巴癌(NHL)PDX模型LYM#004肿瘤体积的影响示意图；
图7为本发明实施例的ADC-1和
Figure PCTCN2020088564-appb-000011
对CD20阳性人淋巴癌(NHL)PDX模型LYM#013肿瘤体积的影响示意图；
图8为本发明实施例的ADC-1和
Figure PCTCN2020088564-appb-000012
对CD20阳性人淋巴癌(NHL)PDX模型LYM#016肿瘤体积的影响示意图；
图9为pH对活性物质ADC-1(10mM组氨酸缓冲体系)的SEC稳定性的影响示意图，其中，ADC-1在10mM组氨酸不同pH体系中，左图表示ADC-1在25度6周和40度4周的SEC单体含量变化趋势，右图表示ADC-1在25度6周和40度4周的SEC高聚体含量变化趋势。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件 进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的蛋白质和核酸化学、分子生物学、细胞和组织培养、微生物学、免疫学相关术语和实验室操作步骤均为相应领域内广泛使用的术语和常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
除非另有说明，在本发明中，任何浓度范围，百分比范围，比例范围或数值范围应理解为包括所述范围内的任何整数数值，以及在适当时包括所述范围内的分数数值。
在本发明中，术语“抗体”是指通常由两对相同的多肽链(每对具有一条“轻”(L)链和一条“重”(H)链)组成的免疫球蛋白分子。抗体的轻链可分为κ和λ两类。重链可分为μ、δ、γ、α或ε五种，依据重链的不同可将抗体分为IgM、IgD、IgG、IgA和IgE五类。在轻链和重链内，可变区和恒定区通过大约12或更多个氨基酸的“J”区连接，重链还包含大约3个或更多个氨基酸的“D”区。各重链由重链可变区(V H)和重链恒定区(C H)组成。重链恒定区由3个结构域(C H1、C H2和C H3)组成。各轻链由轻链可变区(V L)和轻链恒定区(C L)组成。轻链恒定区由一个结构域C L组成。抗体的恒定区可介导免疫球蛋白与宿主组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和补体系统的组分C1q的结合。V H和V L区还可被细分为具有高变性的区域(称为互补决定区(CDR))，其间散布有较保守的称为骨架区(FR)的区域。各V H和V L由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。各重链/轻链对的可变区(V H和V L)分别形成抗体结合部位。氨基酸至各区域或结构域的分配遵循Kabat Sequences of Proteins of Immunological Interest(National Institutes of Health,Bethesda,Md.(1987and 1991))，或Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883的定义。
适用于本发明的抗体为靶向CD20的抗体，所述抗CD20单抗为任何靶向CD20的抗体，例如利妥昔单抗或其生物类似物。其中，生物类似物指的是序列与利妥昔单抗相同、理化性质以及生物活性，临床安全性有效性也与利妥昔一致的抗体产品。
在本发明中，MMAE的结构为：
Figure PCTCN2020088564-appb-000013
在本发明中，药物抗体比(drug∶antibodyratio，DAR)或药物载荷(loading)由p或q表示，即式Ⅰ：Ab-(L-D) p或式ⅠI：Ab-(L-D) q的分子中每个抗体的平均药物模块(即细胞毒剂)数，可以为整数，也可以为分数。通式I的ADC包括偶联有一定范围(3.6-4.0个)药物模块的抗体的集合，通式II的ADC包括偶联有一定范围(3.3-4.3个)药物模块的抗体的集合。来自偶联反应的ADC制备物中每个抗体的平均药物模块数可以通过常规手段来验证，诸如质谱、ELISA测定法、HIC和HPLC。还可以测定ADC在p或q方面的定量分布。在有些情况中，将p或q为某数值的同质ADC从具有其它药物载荷的ADC中分离、纯化和验证可以通过诸如反相HPLC或电泳的手段来实现。
在某些实施方案中，在偶联反应中少于理论最大值的药物模块偶联至抗体。一般而言，抗体不包含许多游离的和反应性的半胱氨酸硫醇基，其可连接药物模块；事实上，抗体中的大多数半胱氨酸硫醇基以二硫桥形式存在。在某些实施方案中，可以在部分或完全还原性条件下用还原剂诸如二硫苏糖醇(DTT)或三羰基乙基膦(TCEP)还原抗体以产生反应性半胱氨酸硫醇基。
本发明的抗体药物偶联物可以与已知的用于治疗肿瘤的化疗药物或免疫抑制剂联用，所述化疗药物例如为阿霉素(Adriamycin)、环磷酰胺和紫杉烷类[紫杉醇(Taxol)和多西他赛(Taxotere)]、卡培他滨(Xeloda)、吉西他滨(Gemzar)、长春瑞滨(Navelbine)、他莫昔芬、芳香酶抑制剂(瑞宁得、弗隆、阿诺新)、5-FU加亚叶酸、伊立替康(camptosar)、奥沙利铂、顺铂、卡铂、雌莫司汀、米托蒽醌(Novantrone)、泼尼松、长春新碱(Oncovin)、多柔比星、强的松等，或它们的组合；所述免疫抑制剂选自：(1)糖皮质激素类，如可的松和强的松；(2)微生物代谢产物，如环孢菌素和藤霉素等；(3)抗代谢物，如硫唑嘌呤和6-巯基嘌呤等；(4)多克隆和单克隆抗淋巴细胞抗体，如抗淋巴细胞球蛋白和OKT3等；(5)烷化剂类，如环磷酰胺。具体地，所述免疫抑制剂例如为甲基强的松龙，强的松，硫唑嘌呤，普乐可复，赛尼哌，舒莱，环孢菌素，他克莫司，雷帕霉素，霉酚酸酯，咪唑立宾，环磷酰胺，芬戈莫德等。
在本发明中，“治疗”指试图改变所治疗个体或细胞的自然进程的临床干预，可以是为了预防或在临床病理学的进程中进行。治疗的期望效果包括预防疾病的发生或复发、缓解症状、削弱疾病的任何直接或间接病理学后果、预防转移、减缓疾病进展的速率、改善或减轻疾病状态、及免除或改善预后。在有些实施方案中，本发明的抗体或抗体药物偶联物用于延迟疾病或病症的发生，或用于减缓疾病或病症的进展。用于评估疾病的成功治疗和改善的上述参数可以容易的通过内科医师所熟悉的常规规程来测量。对于癌症治疗，可通过例如评估疾病进展前时间(TTP)和/或测定反应率(RR)来测量功效。
在本发明中，“受试者”指脊椎动物。在某些实施方案中，脊椎动物指哺乳动物。哺乳动物包括，但不限于，牲畜(诸如牛)、宠物(诸如猫、犬、和马)、灵长类动物、小鼠和大鼠。在某些实施方案中，哺乳动物指人。
在本发明中，“有效量”指在必需的剂量和时间上有效实现期望的治疗或预防效果的量。本发明的物质/分子的“治疗有效量”可根据诸如个体的疾病状态、年龄、性别和体重及该物质/分子在个体中引发期望应答的能力等因素而变化。治疗有效量还涵盖该物质/分子的治疗有益效果胜过任何有毒或有害后果的量。“预防有效量”指在必需的剂量和时间上有效实现期望的预防效果的量。通常而非必然，由于预防剂量是在疾病发作之前或在疾病的早期用于受试者的，因此预防有效量会低于治疗有效量。在癌症的情况中，药物的治疗有效量可减少癌细胞数；缩小肿瘤体积；抑制(即一定程度的减缓，优选停止)癌细胞浸润到周围器官中；抑制(即一定程度的减缓，优选停止)肿瘤转移；一定程度的抑制肿瘤生长；和/或一定程度的减轻与癌症有关的一种或多种症状。
对于疾病的预防或治疗，本发明的抗体药物偶联物的适宜剂量(在单独地或联合一种或多种其它别的治疗剂诸如化疗剂地使用时)会取决于待治疗疾病的类型、抗体药物偶联物的类型、疾病的严重程度和进程、施用抗体药物偶联物是出于预防还是治疗目的、先前的疗法、患者的临床病史和对抗体药物偶联物的反应性、及主治医师的判断。合适的是，一次性或通过一系列治疗将抗体药物偶联物施用于患者。抗体药物偶联物的例示剂量的范围可以是约0.1mg/kg至约5mg/kg。如此，可以对患者施用一剂或多剂约0.1mg/kg、0.3mg/kg、1.0mg/kg、3.0mg/kg或5mg/kg(或其任意组合)的抗体药物偶联物。
“药学上可接受的载体”在用于本发明时包括药学可接受的载体、赋形剂或稳定剂，它们在所采用的剂量和浓度对暴露于其的细胞或哺乳动物是无毒 的。通常，生理学可接受的载体是pH缓冲水溶液。生理学可接受载体的例子包括缓冲剂，诸如磷酸盐、柠檬酸盐和其它有机酸；抗氧化剂，包括抗坏血酸；低分子量(少于约10个残基)多肽；蛋白质，诸如血清清蛋白、明胶或免疫球蛋白；亲水性聚合物，诸如聚乙烯吡咯烷酮；氨基酸，诸如甘氨酸、谷氨酰胺、天冬酰胺、精氨酸或赖氨酸；单糖、二糖和其它碳水化合物，包括葡萄糖、甘露糖，蔗糖，海藻糖或糊精；螯合剂，诸如EDTA；糖醇，诸如甘露醇或山梨醇；成盐反荷离子，诸如钠；和/或非离子表面活性剂，诸如TWEEN TM、聚乙二醇(PEG)和PLURONICS TM。
在本发明中，20种常规氨基酸和其缩写遵从常规用法。参见Immunology-A Synthesis(第2版,E.S.Golub和D.R.Gren,Eds.,Sinauer Associates,Sunderland,Mass.(1991)),其通过引用合并入本文。
下面结合具体实施例对本发明进行进一步的解释说明。
实施例1 抗体MAB801(用于抗体偶联药物ADC-1的裸抗部分)的生产细胞株构建
通过LC/MS/MS分析市售药物美罗华(
Figure PCTCN2020088564-appb-000014
利妥昔单抗)，确定了抗体轻重链的氨基酸序列，根据专利(Anderson-1998，专利号：US5736137A)序列信息，进一步确认了轻链和重链的全基因序列，通过基因合成分别获得了MAB801的轻链和重链基因DNA，再利用双质粒载体表达系统(来源于Invitrogen公司)，分别构建的轻链(Light Chain,LC)和重链(Heavy Chain,HC)基因表达质粒，两个质粒共转染哺乳动物宿主细胞CHO DG44(来源于Invitrogen公司)，经过两步加压筛选得到了同时整合有轻链和重链基因序列的细胞群，评估细胞群的抗体表达能力符合要求，利用半固体培养基进行了单细胞克隆的筛选，将获得的单细胞克隆经过批次补料实验筛选出抗体表达量最高的克隆细胞株，并以此细胞株为基础制备了MAB801的生产细胞库。
对所述单克隆细胞株进行基因测序，测序结果表明，编码MAB801轻链和重链的序列与市售的利妥昔单抗完全一致。
实施例2 抗体MAB801的制备(用于抗体偶联药物ADC-1的裸抗部分)
MAB801的制备采用目前在抗体领域中广泛使用的流加细胞培养工艺。该工艺，从工作细胞库细胞种子复苏开始，包括的主要步骤有摇瓶接 种，摇瓶细胞扩增，反应器逐级细胞扩增培养，然后接种到生产反应器中做流加培养。接着通过纯化工艺，包括发酵液澄清处理(深层过滤)、Protein A亲和层析、低pH病毒灭活及深层过滤、PB孵育、阴离子交换层析、阳离子交换层析、除病毒过滤、以及切向流超滤渗滤等步骤后，过滤分装而成。
测试结果表明，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高度相似。
实施例3 ADC-1—靶向CD20抗体偶联物的制备方法
1)取10毫克的MAB801抗体，使用15mL的30KD超滤装置置换到还原缓冲液(25mM硼酸钠,pH8.0,25mM NaCl,5mM EDTA)中，共三次置换；终体积约为1mL,转移至新的Eppendorf离心管(称重)中，并称重；检测蛋白浓度，计算蛋白总量。
2)向抗体中加入2.5倍摩尔数的DTT,室温保温2小时，连续混匀；使用15ml的30KD超滤装置置换到偶联缓冲液(50mM Tris,pH7.2,150mM NaCl,5mM EDTA)中，共三次置换。取浓缩液，以A 280测定蛋白浓度，并称重，计算蛋白总量；取10μl样品，以Ellman’s方法测定自由巯基数；
并以下列公式计算其自由巯基的摩尔浓度：
Figure PCTCN2020088564-appb-000015
b:比色皿光路长度(通常1cm)
根据自由巯基的摩尔浓度和总蛋白溶液体积计算自由巯基摩尔数。
3)向还原后的抗体中加入1.1倍于自由巯基摩尔数的vc-MMAE(溶于DMSO)，混匀后室温反应2小时，间断混匀。向反应体系中加入20倍于所投入之vc-MMAE摩尔数的N-乙酰半胱氨酸于反应液中，混匀，静置5分钟。
4)使用15ml的30KD超滤装置置换到偶联物储存液(10mM组氨酸(Histidine),6％蔗糖(Sucrose),0.035％PS80,pH5.8)中，共三次置换，即得到抗体药物偶联物ADC-1于4℃保存，其中，活性成分 -抗体药物偶联物的浓度为5mg/mL。
实施例4 ADC-1中药物/抗体比例的测定
将制备好的抗体药物偶联物ADC-1经过HIC-HPLC分析(Ouyang-2013)以测定药物/抗体比例(drug antibody ratio,DAR),见图1，根据图谱峰面积计算得到平均载药数DAR为3.8。
实施例5 ADC-1对CD20表达的NHL细胞的体外杀伤活性
为了研究ADC-1对CD20表达的NHL细胞的杀伤作用，选用了4种CD20表达的NHL细胞株Daudi，Jeko-1，Raji和Ramos，采用细胞增殖抑制法，使用
Figure PCTCN2020088564-appb-000016
检测试剂，测定了ADC-1及市售参比药物
Figure PCTCN2020088564-appb-000017
对以上4株细胞的杀伤作用，研究结果如表2和图2、图3、图4和图5所示。
表2：ADC-1与
Figure PCTCN2020088564-appb-000018
在4种CD20表达的NHL细胞株中的杀伤作用
Figure PCTCN2020088564-appb-000019
注：CD20表达水平参考文献Barth-2015，Law-2004。IC 50值为2块96孔板中计算出的IC 50的平均值；ND表示最高增殖抑制百分比(inhibition％)小于50％，因而IC 50值无法计算。
实验结果显示，ADC-1对4种CD20表达的NHL细胞株均有显著的细胞杀伤作用，且效果都明显优于市售参比药物
Figure PCTCN2020088564-appb-000020
实施例6 ADC-1在NHL PDX模型中的抑瘤效果
人源性肿瘤组织异种移植模型(PDX模型)是利用人的原代肿瘤组织在免疫缺陷小鼠中建立的肿瘤模型，最大程度上保留了原代肿瘤的异质性、分子多样性和组织学特性，对药物的临床治疗效果具有较高的预测价值，近年来越来越多地被应用于癌症研究(Hidalgo-2014)。为了有效地评估 ADC-1在未来临床适应症中的药效，分别在3个CD20表达的人淋巴癌(NHL)PDX模型中进行了实验。在所有实验中，均同时使用市售CD20靶点单抗药物
Figure PCTCN2020088564-appb-000021
作为参比药物来比较肿瘤抑制活性。在这3个NHL PDX模型中，有2个对
Figure PCTCN2020088564-appb-000022
耐药。
研究中使用的3个PDX模型信息如表3所示。
表3：体内药效学实验所用的PDX模型信息
Figure PCTCN2020088564-appb-000023
1、ADC-1在CD20阳性NHL PDX模型LYM#004中的疗效研究
LYM#004是一个
Figure PCTCN2020088564-appb-000024
耐药的DLBCL PDX模型，实验结果如图6所示。ADC-1按1、3和10mg/kg剂量给药后，Day 18时T/C(％)分别为45.90％(P>0.05)、4.46％(P<0.001)和2.05％(P<0.001)，TGI％分别为54.10％、95.54％和97.95％。ADC-1按3mg/kg给药后，Day 18时所有肿瘤部分消退；ADC-1按10mg/kg给药后，Day 18时分别有5/8的肿瘤部分消退，及3/8的肿瘤完全消退。
Figure PCTCN2020088564-appb-000025
按3和10mg/kg剂量给药后，Day 18的T/C(％)分别为50.76％(P>0.05)和51.50％(P>0.05)，TGI％分别为49.24％和48.50％。
实验结果表明，ADC-1(3和10mg/kg)具有显著抑制肿瘤生长的活性，ADC-1(1mg/kg)和参比药物
Figure PCTCN2020088564-appb-000026
(3和10mg/kg)均无显著抗肿瘤活性。荷瘤小鼠对ADC-1和
Figure PCTCN2020088564-appb-000027
均有很好的耐受性。
2、ADC-1在CD20阳性NHL PDX模型LYM#013中的疗效研究
LYM#013是一个
Figure PCTCN2020088564-appb-000028
耐药的DLBCL PDX模型，实验结果如图7所示。ADC-1按0.3、1和3mg/kg剂量给药后，Day 28时T/C(％)分别为31.90％(P<0.05)、0.00％(P<0.001)和0.00％(P<0.001)，TGI％分别为68.10％、100.00％和100.00％，分别有2/8、0/8和0/8的肿瘤部分消退，及0/8、8/8和8/8的肿瘤完全消退。
Figure PCTCN2020088564-appb-000029
按3mg/kg剂量给药后，Day 28时T/C(％)为79.86％(P>0.05)，TGI％为20.14％。
实验结果表明，ADC-1(0.3、1和3mg/kg)具有显著抑制肿瘤生长的作用，且有一定的剂量依赖性，而参比药物
Figure PCTCN2020088564-appb-000030
(3mg/kg)无显著抗肿瘤活性。荷瘤小鼠对ADC-1和
Figure PCTCN2020088564-appb-000031
均有很好的耐受性。
3、ADC-1在CD20阳性NHL PDX模型LYM#016中的疗效研究
LYM#016是一个DLBCL PDX模型，实验结果如图8所示。ADC-1按0.3、1和3mg/kg剂量给药后，Day 14时T/C(％)分别为37.76％(P>0.05)、4.27％(P<0.01)和1.91％(P<0.01)，TGI％分别为62.24％、95.73％和98.09％。ADC-1按1mg/kg给药后，Day 14时所有肿瘤部分消退；ADC-1按3mg/kg给药后，Day 14时分别有6/8的肿瘤部分消退，及2/8的肿瘤完全消退。
Figure PCTCN2020088564-appb-000032
按3mg/kg剂量给药后，Day 14时T/C(％)为30.98％(P>0.05)，TGI％为69.02％。
实验结果表明，ADC-1(1和3mg/kg)具有显著抑制肿瘤生长的作用，而ADC-1(0.3mg/kg)及参比药物
Figure PCTCN2020088564-appb-000033
(3mg/kg)无显著抗肿瘤活性。荷瘤小鼠对ADC-1和
Figure PCTCN2020088564-appb-000034
均有很好的耐受性。
ADC-1在CD20阳性的3种人源性淋巴癌(NHL)PDX模型中的体内药效试验结果汇总如表4。
表4：ADC-1(i.v.,q4d×4)在CD20阳性的人淋巴癌PDX模型中体内药效结果
Figure PCTCN2020088564-appb-000035
Figure PCTCN2020088564-appb-000036
Figure PCTCN2020088564-appb-000037
注：“/”表示不适用。肿瘤体积(Tumor Volume，TV)的计算公式为：TV＝l×w 2/2。其中l、w分别代表肿瘤测量长和宽。根据测量结果计算出相对肿瘤体积(relative tumor volume，RTV)，RTV＝V f/V0。其中V0为分组给药时(即Day 0)测量所得肿瘤体积，V f为最后一天测量的肿瘤体积。相对肿瘤增殖率T/C(％)＝(给药组RTV/Vehicle组RTV)×100％。肿瘤生长抑制率TGI％＝(Vehicle组平均肿瘤体积-给药组平均肿瘤体积)/Vehicle组平均肿瘤体积×100％。“++++”表示T/C(％)≥0且≤10％，“+++”表示T/C(％)>10％且≤20％，“++”表示T/C(％)>20％且≤40％，“+”表示T/C(％)>40％。ADC-1及
Figure PCTCN2020088564-appb-000038
抑瘤活性的强弱划分是根据在该模型中最高剂量的T/C(％)值。
综上，体内药效试验结果显示，在人淋巴癌(NHL)的3个PDX模型(所有模型均为DLBCL)中，ADC-1均表现出显著的肿瘤生长抑制效果，且荷瘤鼠对ADC-1显示出很好的耐受性。值得指出的是，这3个DLBCL PDX模型中有2个对
Figure PCTCN2020088564-appb-000039
耐药。
实施例7 pH对ADC-1制剂稳定性的影响
本申请的发明人基于对ADC-1的制剂处方研究和以往经验，最终确定制剂处方为5mg/mL抗体药物偶联物(不含缓冲液等其他成分的裸的抗体药物偶联物)，10mM组氨酸缓冲液(pH5.8)、6％蔗糖、0.035％吐温80(PS80)。
为了考察ADC-1在10mM组氨酸溶液中不同的pH对其质量的影响，将其换液至待考察处方F1～F4(pH：5.8～7.0)中，置于25±2℃/60％±5％RH和40±2℃/75％±5％RH条件下分别考察6周和4周，于设定的时间点取样分析，检测项包括外观、pH、蛋白浓度、SEC、iCIEF和HIC，如样品出现较明显乳光，将不进行后续相关分析。具体实验设计见表5。
表5：pH(5.8～7.0)对ADC-1(10mM组氨酸缓冲液)稳定性影响实验设计
Figure PCTCN2020088564-appb-000040
注：W表示周，即2W为2周；√表示检测。
实验结果表明：当ADC-1在10mM的组氨酸缓冲液中，其稳定性随着pH的升高而降低。
在25℃条件下放置6周，所有样品均为无色澄清溶液，pH、浓度未发生变化；iCIEF纯度下降，酸性峰含量增加，变化速度随pH升高而加快；SEC纯度呈现下降趋势，高聚体(HMW)含量增加，变化速度随pH升高而加快；DAR值未出现明显变化。
在40℃条件下放置4周，样品稳定性呈相同趋势，但变化幅度加剧；当pH为6.7、7.0时，40℃一周其外观就发生变化，出现乳光，乳光程度随pH升高而加剧，UV检测结果由于颗粒物的存在(乳光)读数失真导致计算所得蛋白含量偏离实际值；SEC纯度降低，高聚体、低分子量片段(LMW)含量均增加，变化速度随pH升高而加快；iCIEF纯度降低，酸性峰含量增加，碱性峰含量降低，变化速度随pH升高而加快。
样品随pH变化的SEC纯度的趋势图见图9。
尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将 会理解，根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。

Claims (13)

  1. 抗体药物偶联物，所述抗体药物偶联物具有式Ⅰ所示的结构，
    Ab-(L-D) p
    式Ⅰ
    其中：
    Ab为抗CD20单抗，所述抗CD20单抗为任何靶向CD20的抗体，例如利妥昔单抗或其生物类似物；
    D为细胞毒剂，所述细胞毒剂为Monomethyl auristatin E(MMAE)；
    L为接头，用于连接所述抗CD20单抗和所述细胞毒剂，所述接头为6-马来酰亚氨基己酰基-缬氨酸-瓜氨酸-对氨基苄氧羰基(MC-vc-PAB)；
    p为3.6-4.0，优选3.7-3.9，更优选3.8。
  2. 抗体药物偶联物制剂，其包括：
    抗体药物偶联物，其浓度为1-60mg/mL(例如浓度为1-20mg/mL，或者1-10mg/mL，或者3-7mg/mL，或者5mg/mL)；
    5-35mM组氨酸缓冲液(例如5-15mM，或者8-12mM，或者10mM组氨酸缓冲液)，pH5.0-6.2(例如pH5.8-6.2，或者pH5.4-6.0，或者pH5.4-6.2，或者pH5.6-6.0)；
    2-10％蔗糖(例如3-9％，或者4-8％，或者5-7％，或者6％蔗糖)；和
    0.01-0.1％吐温80(0.02-0.06％，0.02-0.05％，0.03-0.04％或者0.035％吐温80)；
    所述抗体药物偶联物具有式ⅠI所示的结构，
    Ab-(L-D) q
    式ⅠI
    其中：
    Ab为抗CD20单抗，所述抗CD20单抗为任何靶向CD20的抗体，例如利妥昔单抗或其生物类似物；
    D为细胞毒剂，所述细胞毒剂为Monomethyl auristatin E(MMAE)；
    L为接头，用于连接所述抗CD20单抗和所述细胞毒剂，所述接头为6-马来酰亚氨基己酰基-缬氨酸-瓜氨酸-对氨基苄氧羰基(MC-vc-PAB)；
    q为3.3-4.3(例如3.6-4.0，或者3.7-3.9，或者3.8)。
    优选地，所述制剂包括：
    所述抗体药物偶联物，其浓度为5mg/mL；
    10mM组氨酸缓冲液，pH5.8；
    6％蔗糖；和
    0.035％吐温80。
  3. 权利要求2所述的抗体药物偶联物制剂的制备方法，包括：
    步骤1：将抗CD20单抗和还原剂进行还原反应，获得被还原的抗CD20单抗，所述抗CD20单抗为任何靶向CD20的抗体，例如利妥昔单抗或其生物类似物；
    步骤2：将所述被还原的抗CD20单抗和vcMMAE进行偶联反应；
    步骤3：淬灭所述偶联反应；
    步骤4：将所述淬灭后的偶联反应产物进行缓冲液置换，获得所述抗体药物偶联物制剂，所述抗体药物偶联物制剂包括：
    1-60mg/mL所述抗体药物偶联物(例如1-20mg/mL，或者1-10mg/mL，或者3-7mg/mL，或者5mg/mL所述抗体药物偶联物)，
    5-35mM组氨酸缓冲液(例如5-15mM，或者8-12mM，或者10mM组氨酸缓冲液)，pH5.0-6.2(例如pH5.8-6.2，或者pH5.4-6.0，或者pH5.4-6.2，或者5.6-6.0)，
    2-10％蔗糖(例如3-9％，或者4-8％，或者5-7％，或者6％蔗糖)，和
    0.01-0.1％吐温80(例如0.02-0.06％，0.02-0.05％，0.03-0.04％或者0.035％吐温80)。
  4. 权利要求3的制备方法，其中，所述方法包括：
    1)取10毫克的抗CD20单抗，使用15mL的30KD超滤装置置换到还原缓冲液(25mM硼酸钠,pH8.0,25mM NaCl,5mM EDTA)中，共三次置换；终体积约为1mL,转移至新的Eppendorf离心管(称重)中，并称重；检测蛋白浓度，计算蛋白总量；
    2)向抗体中加入2.0-3.0倍摩尔数的DTT,室温保温2小时，连续混匀；使用15ml的30KD超滤装置置换到偶联缓冲液(50mM Tris,pH7.2,150mM NaCl,5mM EDTA)中，共三次置换；取浓缩液，以A 280(在280nm波长处的吸光度)测定蛋白浓度，并称重，计算蛋白总量；取10μl样品，以Ellman’s方法测定自由巯基数；
    并以下列公式计算其自由巯基的摩尔浓度：
    Figure PCTCN2020088564-appb-100001
    b:比色皿光路长度(通常1cm)
    根据自由巯基的摩尔浓度和总蛋白溶液体积计算自由巯基摩尔数；
    3)向还原后的抗体中加入1.0-1.5倍于自由巯基摩尔数的vc-MMAE(溶于DMSO)，混匀后室温反应2小时，间断混匀；向反应体系中加入20倍于所投入之vc-MMAE摩尔数的N-乙酰半胱氨酸于反应液中，混匀，静置5分钟；
    4)使用15ml的30KD超滤装置置换到偶联物储存液(10mM组氨酸(Histidine),6％蔗糖(Sucrose),0.035％PS80,pH5.8)中，共三次置换，即得到所述抗体药物偶联物制剂，于4℃保存；
    优选地，所述制备方法包括：
    1)取10毫克的抗CD20单抗，使用15mL的30KD超滤装置置换到还原缓冲液(25mM硼酸钠,pH8.0,25mM NaCl,5mM EDTA)中，共三次置换；终体积约为1mL,转移至新的Eppendorf离心管(称重)中，并称重；检测蛋白浓度，计算蛋白总量；
    2)向抗体中加入2.5倍摩尔数的DTT,室温保温2小时，连续混匀；使用15ml的30KD超滤装置置换到偶联缓冲液(50mM Tris,pH7.2,150mM NaCl,5mM EDTA)中，共三次置换；取浓缩液，以A 280测定蛋白浓度，并称重，计算蛋白总量；取10μl样品，以Ellman’s方法测定自由巯基数；
    并以下列公式计算其自由巯基的摩尔浓度：
    Figure PCTCN2020088564-appb-100002
    b:比色皿光路长度(通常1cm)
    根据自由巯基的摩尔浓度和总蛋白溶液体积计算自由巯基摩尔数；
    3)向还原后的抗体中加入1.1倍于自由巯基摩尔数的vc-MMAE(溶于DMSO)，混匀后室温反应2小时，间断混匀；向反应体系中加入20倍于所投入之vc-MMAE摩尔数的N-乙酰半胱氨酸于反应液中，混匀，静置5分钟；
    4)使用15mL的30KD超滤装置置换到偶联物储存液(10mM组氨酸(Histidine),6％蔗糖(Sucrose),0.035％PS80,pH5.8)中，共三次置换，即得到所述抗体药物偶联物制剂，于4℃保存，其中，活性成分-抗体药物偶联物的浓度为5mg/mL。
  5. 组合物，其含有权利要求1所述的抗体药物偶联物，或者权利要求2所述的抗体药物偶联物制剂，或者权利要求3-4任一项所述的方法制备的抗体药物偶联物制剂，任选地，还含有至少一种药学上可接受的载体、稀释剂或赋形剂。
  6. 权利要求1所述的抗体药物偶联物，或者权利要求2所述的抗体药物偶联物制剂，或者权利要求3-4任一项所述的方法制备的抗体药物偶联物制剂，或者权利要求5所述的组合物在制备预防和/或治疗表达CD20的癌症或免疫疾病的药物中的用途。
  7. 权利要求6所述的用途，其中，所述表达CD20的癌症或免疫疾病为淋巴瘤(例如，非霍奇金淋巴瘤，B细胞非霍奇金淋巴瘤，滤泡性非霍奇金淋巴瘤，小淋巴细胞性淋巴瘤，弥漫大B细胞淋巴瘤)、白血病(例如，慢性淋巴细胞白血病，毛细胞白血病，B细胞幼淋巴细胞性白血病，急性淋巴细胞性白血病)、类风湿性关节炎(例如，经至少一种TNF拮抗剂治疗失败的严重活动性类风湿性关节炎)、肉芽肿性多血管炎、Wegener氏肉芽肿、显微镜多血管炎或多发性硬化；
    优选地，所述表达CD20的癌症为CD20阳性B细胞性淋巴瘤，优选抗CD20单抗(例如，利妥昔单抗)耐药性CD20阳性B细胞性淋巴瘤，
    更优选地，所述淋巴瘤为非霍奇金淋巴瘤，优选抗CD20单抗(例如，利妥昔单抗)耐药性非霍奇金淋巴瘤，
    进一步优选地，所述非霍奇金淋巴瘤为弥漫大B细胞淋巴瘤，优选抗CD20单抗(例如，利妥昔单抗)耐药性弥漫大B细胞淋巴瘤。
  8. 预防和/治疗表达CD20的癌症或免疫疾病的方法，其包括给予有需要的受试者预防和/或治疗有效量的权利要求1所述的抗体药物偶联物，或者权利要求2所述的抗体药物偶联物制剂，或者权利要求3-4任一项所述的方法制备的抗体药物偶联物制剂，或者权利要求5所述的组合物。
  9. 权利要求8所述的方法，其中，所述表达CD20的癌症或免疫疾病为淋巴瘤(例如，非霍奇金淋巴瘤，B细胞非霍奇金淋巴瘤，滤泡性非霍奇金淋巴瘤，小淋巴细胞性淋巴瘤，弥漫大B细胞淋巴瘤)、白血病(例如，慢性淋巴细胞白血病，毛细胞白血病，B细胞幼淋巴细胞性白血病，急性淋巴细 胞性白血病)、类风湿性关节炎(例如，经至少一种TNF拮抗剂治疗失败的严重活动性类风湿性关节炎)、肉芽肿性多血管炎、Wegener氏肉芽肿、显微镜多血管炎或多发性硬化；
    优选地，所述表达CD20的癌症为CD20阳性B细胞性淋巴瘤，优选抗CD20单抗(例如，利妥昔单抗)耐药性CD20阳性B细胞性淋巴瘤，
    更优选地，所述淋巴瘤为非霍奇金淋巴瘤，优选抗CD20单抗(例如，利妥昔单抗)耐药性非霍奇金淋巴瘤，
    进一步优选地，所述非霍奇金淋巴瘤为弥漫大B细胞淋巴瘤，优选抗CD20单抗(例如，利妥昔单抗)耐药性弥漫大B细胞淋巴瘤。
  10. 权利要求8所述的方法，其中，受试者为人时，给予有需要的受试者的给药剂量为0.1-5mg/kg(如0.1mg/kg、0.3mg/kg、1mg/kg、3mg/kg、5mg/kg)；
    或者，受试者为NHL PDX模型时，给予有需要的受试者的给药剂量为0.3-10mg/kg(如0.3mg/kg、1mg/kg、3mg/kg、10mg/kg)，优选0.3-3mg/kg、1-3mg/kg、1-10mg/kg或3-10mg/kg。
  11. 权利要求1所述的抗体药物偶联物，或者权利要求2所述的抗体药物偶联物制剂，或者权利要求3-4任一项所述的方法制备的抗体药物偶联物制剂，或者权利要求5所述的组合物，其用于预防和/治疗表达CD20的癌症或免疫疾病。
  12. 权利要求11所述的抗体药物偶联物，或者抗体药物偶联物制剂，或者组合物，其中，所述表达CD20的癌症或免疫疾病为淋巴瘤(例如，非霍奇金淋巴瘤，B细胞非霍奇金淋巴瘤，滤泡性非霍奇金淋巴瘤，小淋巴细胞性淋巴瘤，弥漫大B细胞淋巴瘤)、白血病(例如，慢性淋巴细胞白血病，毛细胞白血病，B细胞幼淋巴细胞性白血病，急性淋巴细胞性白血病)、类风湿性关节炎(例如，经至少一种TNF拮抗剂治疗失败的严重活动性类风湿性关节炎)、肉芽肿性多血管炎、Wegener氏肉芽肿、显微镜多血管炎或多发性硬化；
    优选地，所述表达CD20的癌症为CD20阳性B细胞性淋巴瘤，优选抗CD20单抗(例如，利妥昔单抗)耐药性CD20阳性B细胞性淋巴瘤，
    更优选地，所述淋巴瘤为非霍奇金淋巴瘤，优选抗CD20单抗(例如，利 妥昔单抗)耐药性非霍奇金淋巴瘤，
    进一步优选地，所述非霍奇金淋巴瘤为弥漫大B细胞淋巴瘤，优选抗CD20单抗(例如，利妥昔单抗)耐药性弥漫大B细胞淋巴瘤。
  13. 权利要求11所述的抗体药物偶联物，或者抗体药物偶联物制剂，或者组合物，其中：
    受试者为人时，其给药剂量为0.1-5mg/kg(如0.1mg/kg、0.3mg/kg、1mg/kg、3mg/kg、5mg/kg)；
    或者，受试者为NHL PDX模型时，其给药剂量为0.3-10mg/kg(如0.3mg/kg、1mg/kg、3mg/kg、10mg/kg)，优选0.3-3mg/kg、1-3mg/kg、1-10mg/kg或3-10mg/kg。
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WO2025061123A1 (zh) * 2023-09-21 2025-03-27 浙江特瑞思药业股份有限公司 治疗非霍奇金淋巴瘤的药物组合及其用途
WO2025061140A1 (zh) 2023-09-21 2025-03-27 浙江特瑞思药业股份有限公司 治疗非霍奇金淋巴瘤的药物组合及其用途
WO2025261276A1 (zh) * 2024-06-17 2025-12-26 浙江特瑞思药业股份有限公司 含抗cd20抗体药物偶联物的药物组合及其治疗非霍奇金淋巴瘤的用途

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