WO2022068914A1 - 一种含抗体药物偶联物的药物组合物及其用途 - Google Patents

一种含抗体药物偶联物的药物组合物及其用途 Download PDF

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WO2022068914A1
WO2022068914A1 PCT/CN2021/122031 CN2021122031W WO2022068914A1 WO 2022068914 A1 WO2022068914 A1 WO 2022068914A1 CN 2021122031 W CN2021122031 W CN 2021122031W WO 2022068914 A1 WO2022068914 A1 WO 2022068914A1
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antibody
seq
pharmaceutical composition
cancer
variable region
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PCT/CN2021/122031
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English (en)
French (fr)
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王志万
吴婷婷
刘洵
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江苏恒瑞医药股份有限公司
上海恒瑞医药有限公司
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Application filed by 江苏恒瑞医药股份有限公司, 上海恒瑞医药有限公司 filed Critical 江苏恒瑞医药股份有限公司
Priority to BR112023005789A priority Critical patent/BR112023005789A2/pt
Priority to CN202180055118.2A priority patent/CN116096896A/zh
Priority to IL301658A priority patent/IL301658A/en
Priority to CA3193104A priority patent/CA3193104A1/en
Priority to MX2023003448A priority patent/MX2023003448A/es
Priority to US18/029,075 priority patent/US20240024498A1/en
Priority to EP21874568.5A priority patent/EP4223785A1/en
Priority to AU2021354827A priority patent/AU2021354827A1/en
Priority to KR1020237012680A priority patent/KR20230079096A/ko
Priority to JP2023519391A priority patent/JP2023545382A/ja
Publication of WO2022068914A1 publication Critical patent/WO2022068914A1/zh

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    • A—HUMAN NECESSITIES
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    • 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
    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4375—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having nitrogen as a ring heteroatom, e.g. quinolizines, naphthyridines, berberine, vincamine
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    • 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
    • A61K47/68037—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a camptothecin [CPT] or derivatives
    • 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
    • A61K47/68—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
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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
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6859—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from liver or pancreas cancer cell
    • 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
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • 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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present disclosure belongs to the field of pharmaceutical preparations, and particularly relates to a pharmaceutical composition comprising an antibody-drug conjugate and its use as an anticancer drug.
  • Claudin 18 (Claudin-18, CLDN18) is a protein encoded by the Claudin18 gene in humans and belongs to the cellular tight junction protein family, which can control the flow of molecules between lamina cells.
  • the Claudin protein structure includes four transmembrane regions and two extracellular loops (the N- and C-termini of which are in the cytoplasm).
  • Claudin-18 has two splice variants, Claudin 18.1 and Claudin 18.2, which differ by only eight amino acids in the first extracellular loop.
  • the expression distribution of Claudin 18.1 and Claudin 18.2 is different, Claudin 18.1 is selectively expressed in normal lung cells, Claudin 18.2 is highly restricted in normal cells, but is expressed in various tumors (gastric, lung and pancreatic cancer, etc.) Frequent ectopic activation and overexpression.
  • Claudin18.2 is considered as a potential therapeutic target for gastric cancer and other cancer types, and the discovery of this target also provides a new option for the treatment of gastric cancer.
  • Antibody drug conjugates link monoclonal antibodies or antibody fragments with biologically active cytotoxins through stable chemical linker compounds, making full use of the specificity of antibodies binding to normal and tumor cell surface antigens And the high efficiency of cytotoxic substances, while avoiding the defects of low efficacy of the former and excessive toxic and side effects of the latter. This means that, compared with traditional chemotherapy drugs, antibody-drug conjugates can bind tumor cells more precisely and reduce the impact on normal cells.
  • ADCs have a more complex heterostructure than antibodies, ADC formulations for therapeutic purposes present greater challenges.
  • the present disclosure relates to pharmaceutical formulations containing anti-Claudin18.2 antibody drug conjugates and uses thereof.
  • the preparation has the advantages of good stability and good freeze-dried form.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising an anti-Claudin18.2 antibody drug conjugate and a buffer, wherein the anti-Claudin18.2 antibody in the anti-Claudin18.2 antibody drug conjugate comprises a heavy chain variable region and A light chain variable region, wherein:
  • the heavy chain variable region has HCDR1, HCDR2 and HCDR3 having the same amino acid sequence as the heavy chain variable region shown in SEQ ID NO: 5, and the light chain variable region comprises the same amino acid sequence as the heavy chain variable region shown in SEQ ID NO: 6
  • the indicated light chain variable regions have LCDR1, LCDR2 and LCDR3 having the same amino acid sequence; or
  • variable region of the heavy chain has HCDR1, HCDR2 and HCDR3 having the same amino acid sequence as the variable region of the heavy chain as shown in SEQ ID NO: 3, and the variable region of the light chain has the same amino acid sequence as the variable region of the light chain as shown in SEQ ID NO: 4;
  • the light chain variable regions shown have LCDR1, LCDR2 and LCDR3 of the same amino acid sequence;
  • the buffer is a histidine buffer.
  • the pharmaceutical composition of any of the above, wherein the buffer is a histidine-acetate buffer.
  • the pharmaceutical composition of any of the above, wherein the anti-Claudin18.2 antibody comprises a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17, respectively, and the light chain variable region comprises SEQ ID NO: 17, respectively LCDR1, LCDR2 and LCDR3 as set forth in NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20; or
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, respectively, and the light chain variable region comprises SEQ ID NO: 11, respectively LCDR1, LCDR2 and LCDR3 shown in NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14.
  • the pharmaceutical composition of any of the above, wherein the anti-Claudin18.2 antibody is a murine, chimeric, or humanized antibody.
  • the pharmaceutical composition of any one of the above, wherein the anti-Claudin18.2 antibody comprises a heavy chain variable region and a light chain variable region, wherein: comprising a heavy chain variable region and a light chain variable region variable area, where:
  • variable region of the heavy chain is shown in SEQ ID NO: 3 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% therewith , 99% or 100% identity, and said light chain variable region amino acid sequence as shown in SEQ ID NO: 4 or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity;
  • variable region of the heavy chain is shown in SEQ ID NO: 24 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% therewith , 99% or 100% identity, and said light chain variable region amino acid sequence as shown in SEQ ID NO: 21 or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity;
  • variable region of the heavy chain is as shown in SEQ ID NO: 5 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% therewith , 99% or 100% identity, and said light chain variable region amino acid sequence as shown in SEQ ID NO: 6 or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity; or
  • variable region of the heavy chain is as shown in SEQ ID NO: 31 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% therewith , 99% or 100% identity, and said light chain variable region amino acid sequence as shown in SEQ ID NO: 28 or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.
  • the pharmaceutical composition of any of the above, wherein the anti-Claudin18.2 antibody is a humanized antibody comprising a framework region derived from a human antibody or a framework region variant thereof , the framework region variants are back mutations of up to 10 amino acids in the light chain framework region and/or heavy chain framework region of a human antibody, respectively.
  • the light chain variable region comprises one or more amino acid back-mutations optionally selected from 22S, 85I and 87H, and/or the heavy chain variable region comprises optionally 48I, 82T and One or more amino acid backmutations in 69M; or
  • variable region of the light chain comprises one or more amino acid back-mutations selected from 4L or 22S, and/or the variable region of the heavy chain comprises 38K, 40R, One or more amino acid backmutations in 48I, 66K, 67A, 69L, 71L and 73K.
  • 82 in 82T of the heavy chain variable region is the 82A position of Kabat's rule.
  • the pharmaceutical composition of any of the above, wherein the anti-Claudin18.2 antibody comprises a heavy chain variable region and a light chain variable region as shown in any of the following:
  • variable region of the heavy chain is shown in SEQ ID NO:3 and the variable region of the light chain is shown in SEQ ID NO:4;
  • variable region of the heavy chain is shown in SEQ ID NO: 31, 32, 33 or 34 and the variable region of the light chain is shown in SEQ ID NO: 28, 29 or 30;
  • the pharmaceutical composition of any of the above, wherein the anti-Claudin18.2 antibody comprises a heavy chain variable region and a light chain variable region as shown in any of the following:
  • the pharmaceutical composition of any of the above, wherein the anti-Claudin 18.2 antibody comprises an antibody heavy chain constant region and a light chain constant region.
  • the heavy chain constant regions are selected from human IgGl, IgG2, IgG3 and IgG4 constant regions and conventional variants thereof, and the light chain constant regions are selected from human antibody kappa and lambda chain constant regions and conventional variants thereof body.
  • the antibody comprises a heavy chain constant region as set forth in SEQ ID NO:7 and a light chain constant region as set forth in SEQ ID NO:8.
  • the antibody comprises: having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, A heavy chain of 97%, 98%, 99% or 100% identity, and at least 90%, 91%, 92%, 93%, 94% with a light chain having the amino acid sequence shown in SEQ ID NO: 36 or 39 , 95%, 96%, 97%, 98%, 99% or 100% identical to a light chain; or at least 90%, 91%, 92% to a heavy chain having the amino acid sequence shown in SEQ ID NO: 37 or 49 %, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical heavy chains and light chains having the amino acid sequence shown in SEQ ID NO: 38 or 46 have at least Light chains of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.
  • the pharmaceutical composition of any of the above, wherein the anti-Claudin18.2 antibody comprises a heavy chain and a light chain as shown in any of the following:
  • the pharmaceutical composition of any of the above, wherein the anti-Claudin18.2 antibody comprises a heavy chain and a light chain as shown in any of the following:
  • the pharmaceutical composition of any one of the above, wherein the anti-Claudin18.2 antibody drug conjugate has a structure as shown in the general formula (Pc-L-Y-D):
  • Y is selected from -O-(CR a R b ) m -CR 1 R 2 -C(O)-, -O-CR 1 R 2 -(CR a R b ) m -, -O-CR 1 R 2 - , -NH-(CR a R b ) m -CR 1 R 2 -C(O)- and -S-(CR a R b ) m -CR 1 R 2 -C(O)-;
  • R a and R b are the same or different, and are each independently selected from hydrogen atoms, deuterium atoms, halogen, alkyl, haloalkyl, deuterated alkyl, alkoxy, hydroxy, amino, cyano, nitro, hydroxyalkane radicals, cycloalkyls and heterocyclyls;
  • R a and R b together with the carbon atom to which they are attached form a cycloalkyl or heterocyclyl;
  • R 1 is selected from halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl and heteroaryl;
  • R is selected from hydrogen atom, halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl and heteroaryl;
  • R 1 and R 2 together with the carbon atom to which they are attached form a cycloalkyl or heterocyclyl group
  • R and R together with the carbon atom to which they are attached form a cycloalkyl or heterocyclyl ;
  • n is an integer from 0 to 4.
  • n 1 to 10, n is a decimal or an integer
  • Pc is anti-Claudin18.2 antibody
  • the pharmaceutical composition of any one of the above, wherein the anti-Claudin18.2 antibody drug conjugate has a structure as shown in the general formula (Pc-L-Y-D),
  • Y is -O-(CR a R b ) m -CR 1 R 2 -C(O)-;
  • R a and R b are the same or different, and are each independently selected from a hydrogen atom, a deuterium atom, a halogen or a C 1-6 alkyl;
  • R 1 is halogenated C 1-6 alkyl or C 3-6 cycloalkyl
  • R 2 is selected from hydrogen atom, halogenated C 1-6 alkyl or C 3-6 cycloalkyl;
  • R 1 and R 2 together with the carbon atom to which they are attached form a C 3-6 cycloalkyl
  • m 0 or 1.
  • the pharmaceutical composition of any one of the above, wherein the anti-Claudin18.2 antibody drug conjugate has a structure as shown in the general formula (Pc-L-Y-D), wherein Y is selected from:
  • L 1 is selected from -(succinimide-3-yl-N)-WC(O)-, -CH 2 -C(O)-NR 3 -WC(O)- or -C(O)-WC ( O)-, wherein W is selected from C 1-8 alkyl, C 1-8 alkyl-cycloalkyl or straight chain heteroalkyl of 1 to 8 chain atoms, said heteroalkyl containing 1 to 3 optional A heteroatom from N, O or S, wherein said C 1-8 alkyl, C 1-8 alkyl-cycloalkyl or straight chain heteroalkyl of 1 to 8 chain atoms are each independently optionally further is selected from halogen, hydroxy, cyano, amino, C1-6 alkyl, chloro C1-6 alkyl, deuterated C1-6 alkyl, C1-6 alkoxy and C3-6 ring substituted with one or more substituents of an alkyl group;
  • L 2 is selected from -NR 4 (CH 2 CH 2 O)p 1 CH 2 CH 2 C(O)-, -NR 4 (CH 2 CH 2 O)p 1 CH 2 C(O)-, -S(CH 2 ) p 1 C(O)- or a chemical bond, wherein p 1 is an integer from 1 to 20;
  • L3 is a peptide residue consisting of 2 to 7 amino acids, wherein the amino acid residue is selected from the group consisting of phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid and amino acid
  • An amino acid residue formed from an amino acid in partic acid, and optionally further selected from halogen, hydroxy, cyano, amino, C 1-6 alkyl, chloro C 1-6 alkyl, deuterated C 1-6 substituted by one or more substituents in alkyl, C 1-6 alkoxy and C 3-6 cycloalkyl;
  • L 4 is selected from -NR 5 (CR 6 R 7 ) t -, -C(O)NR 5 -, -C(O)NR 5 (CH 2 ) t - or a chemical bond, wherein t is an integer from 1 to 6;
  • R 3 , R 4 and R 5 are the same or different, and are each independently selected from a hydrogen atom, a C 1-6 alkyl group, a halogenated C 1-6 alkyl group, a deuterated C 1-6 alkyl group, and a C 1-6 alkyl group Hydroxyalkyl;
  • R 6 and R 7 are the same or different, and are each independently selected from a hydrogen atom, halogen, C 1-6 alkyl, halogenated C 1-6 alkyl, deuterated C 1-6 alkyl and C 1-6 hydroxy alkyl.
  • L1 is s 1 is an integer from 2 to 8;
  • L 2 is a chemical bond
  • L3 is a tetrapeptide residue; preferably the tetrapeptide residue of GGFG (SEQ ID No: 55);
  • L 4 is -NR 5 (CR 6 R 7 )t-, R 5 , R 6 or R 7 are the same or different, and each is independently a hydrogen atom or a C 1-6 alkyl group, and t is 1 or 2;
  • the L 1 end is connected with Pc, and the L 4 end is connected with Y.
  • composition of any of the above, wherein -L- is:
  • composition of any of the above, wherein L-Y- is optionally selected from:
  • the pharmaceutical composition of any one of the above, wherein the anti-Claudin18.2 antibody drug conjugate is selected from the structures shown in any of the following:
  • the pharmaceutical composition of any one of the above, wherein the anti-Claudin18.2 antibody drug conjugate has the structure shown in the following formula:
  • n 2 to 8, n is a decimal or an integer
  • Pc is anti-Claudin18.2 antibody.
  • the surfactant is selected from the group consisting of polysorbates (eg, polysorbate 20, polysorbate 80), polyhydroxyalkenes, Triton, sodium lauryl sulfonate, sodium lauryl sulfonate, Sodium octylglycoside, lauryl-sulfobetaine, myristyl-sulfobetaine, linoleyl-sulfobetaine, stearyl-sulfobetaine, lauryl-sarcosine, myristine yl-sarcosine, linoleyl-sarcosine, stearyl-sarcosine, linoleyl-betaine, myristyl-betaine, cetyl-betaine, lauroamidopropyl-beet Alkali, Coca-Amidopropyl
  • the surfactant is a polysorbate. In some embodiments, the surfactant is polysorbate 80 or polysorbate 20. In some embodiments, the surfactant is polysorbate 80.
  • the surfactant concentration is 0.05 mg/mL, 0.1 mg/mL, 0.15 mg/mL, 0.18 mg/mL, 0.19 mg/mL, 0.2 mg/mL, 0.21 mg/mL, 0.22 mg /mL, 0.3 mg/mL, 0.4 mg/mL, 0.5 mg/mL.
  • the surfactant concentration is 0.2 mg/mL.
  • the pharmaceutical composition of any of the above, wherein the composition further comprises a sugar further comprises a sugar.
  • the sugar is selected from the group consisting of conventional compositions (CH2O ) n and derivatives thereof, including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, and the like .
  • the sugar can be selected from glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerol, erythritol, glycerol, arabitol, sylitol, sorbitol, mannitol, mili Disaccharide, melezitose, raffinose, mannose, stachyose, maltose, lactulose, maltulose, sorbitol, maltitol, lactitol, iso-maltulose, etc.
  • the sugar is selected from the group consisting of sucrose, mannitol, and trehalose.
  • the sugar is sucrose.
  • the pharmaceutical composition of any of the above, wherein the sugar concentration is 20 mg/mL to 100 mg/mL or 40 mg/mL to 80 mg/mL. In some embodiments, the sugar concentration is 20 mg/mL, 30 mg/mL, 40 mg/mL, 50 mg/mL, 60 mg/mL, 70 mg/mL, 80 mg/mL, 90 mg/mL, or 100 mg/mL. In some embodiments, the sugar concentration is 40 mg/mL.
  • the pharmaceutical composition of any of the above, wherein the concentration of the antibody drug conjugate is 1 mg/mL to 100 mg/mL as a protein (ie antibody) concentration or 10 mg/mL as a protein concentration to 30 mg/mL.
  • the antibody drug conjugate concentration is 5 mg/mL, 10 mg/mL, 20 mg/mL, 30 mg/mL, 40 mg/mL, 50 mg/mL, 60 mg/mL, 70 mg/mL as protein concentration , 80mg/mL, 90mg/mL or 100mg/mL.
  • the in vivo drug conjugate concentration is 20 mg/mL as protein concentration.
  • the pharmaceutical composition of any of the above, wherein the concentration of the buffer is 5 mM to 50 mM or 10 mM to 30 mM. In some embodiments, the concentration of the buffer is 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, or 50 mM. In some embodiments, the concentration of the buffer is 30 mM.
  • the pharmaceutical composition of any of the above, wherein the pH of the pharmaceutical composition is 5.0-6.5 or 5.0-5.5.
  • the pH of the pharmaceutical composition is 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5.
  • the pH of the pharmaceutical composition is 5.0-5.4.
  • the pH of the pharmaceutical composition is 5.0-5.3.
  • composition of any of the above comprising the following components:
  • composition of any of the above comprising the following components:
  • composition of any of the above comprising the following components:
  • the pharmaceutical composition of any one of the above, the anti-Claudin18.2 antibody drug conjugate has the structure shown in the following formula:
  • n 2 to 8, n is a decimal or an integer
  • Pc is an anti-Claudin18.2 antibody, which comprises the heavy chain shown in SEQ ID NO: 49 and the light chain shown in SEQ ID NO: 47; the pharmaceutical composition contains 20 mg/mL of the antibody in terms of protein concentration drug conjugates;
  • the pharmaceutical composition also includes the following components:
  • the pH of the pharmaceutical composition is 5.0-5.3.
  • the pharmaceutical composition of any of the above, the pharmaceutical composition is a liquid formulation.
  • the solvent of the liquid formulation is water.
  • the present disclosure also provides a lyophilized preparation containing the antibody-drug conjugate, characterized in that the preparation can form the pharmaceutical composition described in any one of the above after reconstitution.
  • the present disclosure also provides a lyophilized formulation that is a lyophilized form of the pharmaceutical composition of any of the above.
  • the present disclosure also provides a method for preparing a lyophilized formulation containing an antibody-drug conjugate, which comprises the step of lyophilizing the pharmaceutical composition according to any one of the above.
  • the present disclosure also provides a lyophilized formulation containing the antibody drug conjugate obtained by lyophilizing the pharmaceutical composition according to any one of the above.
  • freeze-drying as described in any of the above includes the steps of pre-freezing, primary drying, and secondary drying in sequence.
  • the lyophilization procedure is as follows: pre-freezing at 5°C; pre-freezing at -45°C; primary drying at -20°C and vacuum at 20 Pa; secondary drying at temperature It is 25 °C, and the vacuum degree is 1Pa.
  • the lyophilization procedure is as follows: pre-freezing, the temperature is 5°C, and the time is 10 minutes; pre-freezing, the temperature is -45°C, and the time is 50 minutes; The time is 120min; the temperature of the secondary drying is 25°C, the vacuum degree is 1Pa, and the time is 60min.
  • the lyophilized formulation is stable at 2-8°C for at least 3 months, at least 6 months, at least 12 months, at least 18 months, or at least 24 months. In some embodiments, the lyophilized formulation is stable at 40°C for at least 7 days, at least 14 days, or at least 28 days.
  • the present disclosure also provides a lyophilized formulation, which is a reconstituted form of the lyophilized formulation as described in any of the above.
  • the present disclosure also provides a reconstituted solution containing an antibody-drug conjugate, characterized in that the reconstituted solution is prepared by reconstituting the freeze-dried preparation described in any of the above.
  • the reconstitution solution as described above comprises the following components:
  • the reconstitution solution as described above comprises the following components:
  • the reconstitution solution as described above comprises the following components:
  • the present disclosure also provides an article of manufacture comprising a container containing the pharmaceutical composition of any of the above, the lyophilized formulation of any of the above, or the reconstitution solution of any of the above.
  • the present disclosure also provides a method of treating a tumor or cancer, comprising administering to a subject an effective amount of the pharmaceutical composition of any of the above, the lyophilized formulation of any of the above, or the reconstituted solution of any of the above , or the article of any of the above.
  • the present disclosure also provides the pharmaceutical composition of any of the above, the lyophilized formulation of any of the above, the reconstituted solution of any of the above, or the preparation of any of the above in the manufacture of a treatment Use in the medicament of tumors or cancer.
  • the present disclosure also provides the pharmaceutical composition of any of the above, the lyophilized formulation of any of the above, the reconstituted solution of any of the above, or the above of any of the above for use as a medicament product.
  • the tumor or cancer is preferably head and neck squamous cell carcinoma, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system Systemic cancer, neuroendocrine tumor, throat cancer, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatocellular carcinoma, hepatocellular carcinoma, hepatobiliary cancer, pancreatic cancer, gastric cancer, stomach cancer Intestinal cancer, bowel cancer, colon cancer, colorectal cancer, kidney cancer, clear cell renal cell cancer, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, melanoma, Leukemia, Lymphoma, Bone Cancer, Chondrosarcoma, Myeloma, Multiple Myeloma, Myelodysplastic Syndrome, Keuk
  • the lymphoma is selected from the group consisting of: Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, primary mediastinal large B-cell lymphoma tumor, mantle cell lymphoma, small lymphocytic lymphoma, T-cell/histiocytic-rich large B-cell lymphoma, and lymphoplasmacytic lymphoma.
  • the lung cancer is selected from the group consisting of: non-small cell lung cancer and small cell lung cancer.
  • the leukemia is selected from the group consisting of: chronic myeloid leukemia, acute myeloid leukemia, lymphocytic leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and myeloid cells leukemia.
  • Figure 1 FACS detection results of humanized antibody binding to human Claudin18.2 at the cellular level.
  • FIG. 1 NUGC4 endocytosis experiments with humanized antibodies.
  • Figures 3A to 3C Detection of ADCC effect of antibodies in NUGC4 cells with different levels of Claudin18.2 expression.
  • Figure 3A is the detection of ADCC effect of the antibody in wild-type NUGC4 cells (low expression of Claudin18.2);
  • Figure 3B is the detection of the ADCC effect of the antibody in NUGC4 cells with moderate expression of Claudin18.2;
  • Figure 3C is the detection of the antibody in high expression of Claudin18.2 ADCC effect detection in NUGC4 cells.
  • Figure 4 Results of tumor inhibition experiments of ADC-1 of the present disclosure.
  • Figure 5 Results of tumor inhibition experiments of ADC-2 of the present disclosure.
  • Antibody drug conjugate is to link an antibody or antibody fragment with a biologically active cytotoxin or a small molecule drug with cell killing activity through a stable chemical linker compound. Cell-specific or highly expressed antigen binding specificity and high cytotoxicity, avoiding toxic side effects on normal cells. Compared with traditional chemotherapy drugs, the antibody-drug conjugates can precisely bind tumor cells and reduce the impact on normal cells.
  • Buffer refers to a buffer that resists changes in pH through the action of its acid-base conjugated component.
  • buffers to control pH in the appropriate range include acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumaric acid Salt, glycylglycine and other organic acid buffers.
  • a "histidine buffer” is a buffer containing histidine ions.
  • histidine buffers include histidine-hydrochloride, histidine-acetate, histidine-phosphate, histidine-sulfate and the like; preferably histidine-acetate buffer.
  • Histidine-acetate buffer is prepared from histidine and acetic acid
  • histidine hydrochloride buffer is prepared from histidine and hydrochloric acid.
  • citrate buffer is a buffer that includes citrate ions.
  • citrate buffers include citrate-sodium citrate, citrate-potassium citrate, citrate-calcium citrate, citrate-magnesium citrate, and the like.
  • a preferred citrate buffer is citrate-sodium citrate.
  • succinate buffer is a buffer that includes succinate ions.
  • succinate buffers include succinate-sodium succinate, succinate-potassium succinate, succinate-calcium succinate, and the like.
  • a preferred succinate buffer is succinate-sodium succinate.
  • the succinic acid-sodium succinate can be prepared from succiplatinic acid and sodium hydroxide, or can be prepared from succiplatinic acid and sodium succinate.
  • a “phosphate buffer” is a buffer that includes phosphate ions.
  • phosphate buffers include disodium hydrogen phosphate-sodium dihydrogen phosphate, disodium hydrogen phosphate-potassium dihydrogen phosphate, disodium hydrogen phosphate-citric acid, and the like.
  • the preferred phosphate buffer is disodium hydrogen phosphate-sodium hydrogen phosphate.
  • acetate buffer is a buffer that includes acetate ions.
  • acetate buffers include acetate-sodium acetate, acetate-histidine, acetate-potassium acetate, calcium acetate, acetate-magnesium acetate, and the like.
  • the preferred acetate buffer is acetic acid-sodium acetate.
  • “Pharmaceutical composition” means a mixture comprising one or more of the antibody drug conjugates described herein, or a physiological/pharmaceutically acceptable salt or prodrug thereof, and other chemical components, such as physiological/pharmaceutical Pharmaceutically acceptable carriers and excipients.
  • the purpose of the pharmaceutical composition is to maintain the stability of the active ingredient, facilitate the administration to the organism, and facilitate the absorption of the active ingredient to exert biological activity.
  • the solvent therein is all water.
  • “Lyophilized formulation” means a pharmaceutical composition in liquid or solution form or a formulation or pharmaceutical composition obtained after a liquid or solution formulation has been subjected to a vacuum freeze-drying step.
  • the pharmaceutical composition described in the present disclosure can achieve a stable effect: a pharmaceutical composition in which the antibody-drug conjugate substantially retains its physical stability and/or chemical stability and/or biological activity after storage, Preferably, the pharmaceutical composition substantially retains its physical and chemical stability and its biological activity upon storage.
  • the shelf life is generally selected based on the intended shelf life of the pharmaceutical composition. There are a variety of analytical techniques for measuring protein stability, which can measure stability after storage at a selected temperature for a selected period of time.
  • a stable formulation is one in which no significant change is observed when stored at refrigerated temperature (2-8°C) for at least 3 months, preferably 6 months, more preferably 1 year, and even more preferably up to 2 years .
  • stable liquid formulations include liquid formulations that exhibit desirable characteristics after storage at temperatures including 25°C for periods of time including 1 month, 3 months, and 6 months.
  • Typical example of stability Typically no more than about 10%, preferably no more than about 5% of the antibody monomers aggregate or degrade as measured by SEC-HPLC. By visual analysis, the formulation was a pale yellow near colorless clear liquid or colorless, or clear to slightly opalescent. The formulations had no more than ⁇ 10% variation in concentration, pH and osmolality. A reduction of no more than about 10%, preferably no more than about 5%, is generally observed. Typically no more than about 10% aggregates are formed, preferably no more than about 5% aggregates.
  • Antibody-drug conjugates do not show significant increases in aggregation, precipitation, if after visual inspection of color and/or clarity, or by UV light scattering, size exclusion chromatography (SEC) and dynamic light scattering (DLS) and/or denaturation, then the antibody drug conjugate "retains its physical stability" in the pharmaceutical formulation. Changes in protein conformation can be assessed by fluorescence spectroscopy (which determines protein tertiary structure) and by FTIR spectroscopy (which determines protein secondary structure).
  • An antibody "retains its chemical stability" in a pharmaceutical formulation if the antibody drug conjugate does not exhibit significant chemical changes.
  • Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein.
  • Degradation processes that frequently change the chemical structure of proteins include hydrolysis or truncation (as assessed by methods such as size exclusion chromatography and CE-SDS), oxidation (by peptide mapping such as combined with mass spectrometry or MALDI/TOF/MS, etc.) methods), deamidation (evaluated by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide mapping, isoaspartic acid measurement, etc.), and isomerization (by measuring isoaspartic acid content, Peptide Mapping, etc.).
  • An Antibody Drug Conjugate "retains its biological activity" in a pharmaceutical formulation if its biological activity at a given time is within a predetermined range of the biological activity exhibited at the time of preparation of the pharmaceutical formulation.
  • antibody in this disclosure is used in the broadest sense and encompasses a variety of antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), full length antibodies or antigen binding thereof Fragments (also referred to as "antigen-binding portions”) so long as they exhibit the desired antigen-binding activity.
  • Full-length antibodies are immunoglobulins (Ig) comprising at least two heavy chains and two light chains interconnected by disulfide bonds. The amino acid composition and sequence of the immunoglobulin heavy chain constant region are different, so their antigenicity is also different.
  • immunoglobulins can be divided into five classes, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, whose corresponding heavy chains are ⁇ , ⁇ , and ⁇ chains, respectively. , alpha chains, and epsilon chains.
  • the same type of Ig can be divided into different subclasses according to the difference in the amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • Light chains are classified into kappa chains or lambda chains by the difference in the constant region.
  • Each of the five classes of Ig can have a kappa chain or a lambda chain.
  • Each heavy chain consists of a heavy chain variable region (abbreviated as VH) and a heavy chain constant region (abbreviated as CH).
  • the heavy chain constant region contains three domains, CH1, CH2 and CH3.
  • Each light chain consists of a light chain variable region (abbreviated as VL) and a light chain constant region (abbreviated as CL).
  • the heavy and light chain variable regions include hypervariable regions (also called complementarity determining regions, abbreviated as CDRs or HVRs) and framework regions (also called framework regions, abbreviated as FRs) with relatively conserved sequences.
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VL and VH consists of 3 CDRs and 4 FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the three CDR regions of the light chain are referred to as LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3.
  • inventions of the human antibody heavy chain constant region and the human antibody light chain constant region mentioned in the present disclosure refer to the human-derived heavy chain constant regions disclosed in the prior art that do not alter the structure and function of the antibody variable region or variants of the light chain constant region
  • exemplary variants include IgG1, IgG2, IgG3 or IgG4 heavy chain constant region variants with site-directed reengineering and amino acid substitutions of the heavy chain constant region, specifically replacing YTE as known in the art Mutations, L234A and/or L235A mutations, S228P mutations, 265A (eg D265A) and/or 297A (eg N297A), and/or mutations to obtain a knob-into-hole structure (so that the antibody heavy chain has knob-Fc and hole- Fc combination), these mutations have been shown to confer novel properties of the antibody without altering the function of the variable region of the antibody.
  • antigen-binding fragment or “functional fragment” or “antigen-binding portion” refers to one or more fragments of an intact antibody that retain the ability to specifically bind an antigen. Fragments of full-length antibodies have been shown to perform the antigen-binding function of antibodies.
  • binding fragments encompassed by the term "antigen-binding fragment” include: (i) Fab fragments, a monovalent fragment consisting of VL, VH, CL and CH1 domains; (ii) F(ab') 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge on the hinge region, (iii) an Fd fragment consisting of VH and CH1 domains; (iv) a VH and Fv fragment composed of VL domains; (v) dsFv, a stable antigen-binding fragment formed by VH and VL via interchain disulfide bonds; (vi) diabodies and bispecific antibodies comprising scFv, dsFv, Fab and other fragments and multispecific antibodies.
  • Antigen binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.
  • amino acid difference or “amino acid mutation” refers to the presence of amino acid changes or mutations in a variant protein or polypeptide compared to the original protein or polypeptide, including 1, 2, 3, or Insertion, deletion or substitution of more amino acids.
  • antibody framework or "FR region” refers to the portion of a variable domain VL or VH that serves as a scaffold for the antigen binding loops (CDRs) of the variable domain. Essentially, it is a variable domain without CDRs.
  • CDR complementarity determining region
  • HCDR1, HCDR2, HCDR3 three CDRs in each heavy chain variable region and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable region.
  • LCDR1, LCDR2, LCDR3 three CDRs in each light chain variable region.
  • the amino acid sequence boundaries of CDRs can be determined using any of a variety of well-known schemes, including the "Kabat” numbering convention (see Kabat et al.
  • VH variable domain
  • VL variable domain
  • CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50 -56 (LCDR2) and 89-97 (LCDR3).
  • CDR amino acids in VH are numbered 26-32 (HCDR1), 52-56 (HCDR2) and 95-102 (HCDR3); and amino acids in VL Residue numbers are 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3).
  • the CDRs are defined by amino acid residues 26-35 in human VH (HCDR1 ), 50-65 (HCDR2) and 95-102 (HCDR3) and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3) in human VL.
  • VH The CDR amino acid residue numbers in VL are approximately 26-35 (CDR1), 51-57 (CDR2) and 93-102 (CDR3), and the CDR amino acid residue numbers in VL are approximately 27-32 (CDR1), 50-52 (CDR2) and 89-97 (CDR3).
  • the CDR regions of the antibody can be determined using the program IMGT/DomainGap Align.
  • the CDR amino acids in VH are numbered 26-32 (HCDR1), 50-58 ( HCDR2) and 95-102 (HCDR3); and the amino acid residues in VL are numbered 24-34 (LCDR1), 50-56 (LCDR2 ) and 89-97 (LCDR3).
  • the heavy and light chain variable regions of the antibodies of the present disclosure and their CDRs conform to the Kabat numbering convention.
  • amino acid sequence identity refers to the percentage of amino acid residues in a first sequence that are identical to those in a second sequence when the amino acid sequences are aligned (allowing gaps to be introduced if necessary to achieve maximum percent sequence identity); wherein conservation Substitutions are not considered part of sequence identity.
  • alignment can be accomplished in a variety of ways that are within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign ( DNASTAR) software.
  • One of skill in the art can determine parameters suitable for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • the engineered antibodies or antigen-binding fragments of the present disclosure can be prepared and purified using conventional methods.
  • cDNA sequences encoding heavy and light chains can be cloned and recombined into a GS expression vector.
  • the recombinant immunoglobulin expression vector can stably transfect CHO cells.
  • mammalian-like expression systems lead to glycosylation of the antibody, especially at the highly conserved N-terminal site of the Fc region.
  • Stable clones are obtained by expressing antibodies that bind to the antigen. Positive clones were expanded in serum-free medium in bioreactors for antibody production.
  • the antibody-secreted culture medium can be purified by conventional techniques.
  • a or G Sepharose FF column with adjusted buffer. Non-specifically bound components are washed away. The bound antibody was eluted by pH gradient method, and the antibody fragments were detected by SDS-PAGE and collected. Antibodies can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves, ion exchange. The obtained product should be frozen immediately, eg -70°C, or lyophilized.
  • Constant modification or “conservative substitution or substitution” refers to the replacement of amino acids in a protein by other amino acids with similar characteristics (eg, charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.) such that frequent Changes are made without altering the biological activity of the protein.
  • Those skilled in the art are aware that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., 224, (4th ed.).
  • substitution of structurally or functionally similar amino acids is unlikely to disrupt biological activity. Exemplary conservative substitutions are as follows:
  • naked antibody refers to an antibody that is not conjugated to a heterologous moiety (eg, a cytotoxic moiety) or a radiolabel.
  • the content of the antibody drug conjugate is based on the protein concentration, ie, the weight/volume of the protein (antibody moiety) in the conjugate.
  • linker unit refers to a chemical structural fragment or bond which is linked to an antibody or an antigen-binding fragment thereof at one end and a drug at the other end. Other linkers can also be linked to the drug.
  • the preferred embodiment of the present disclosure is represented by L and L 1 to L 4 , wherein the L 1 end is connected to the antibody, and the L 4 end is connected to the structural unit Y and then connected to the compound or toxin.
  • Linkers including stretchers, spacers and amino acid units, can be synthesized by methods known in the art, such as described in US2005-0238649A1.
  • the linker can be a "cleavable linker" that facilitates the release of the drug in the cell.
  • acid-labile linkers eg, hydrazones
  • protease-sensitive linkers eg, peptidase-sensitive linkers
  • photolabile linkers dimethyl linkers
  • disulfide-containing linkers can be used (Chari et al., Cancer Research 52:127-131 (1992); US Patent No. 5,208,020).
  • alkyl refers to a saturated aliphatic hydrocarbon group, which is a straight or branched chain group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 carbon atoms, more preferably 1 to 10 carbon atoms atomic alkyl groups, most preferably those containing 1 to 6 carbon atoms.
  • Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1 ,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2- Methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3 -Dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2 -Methylhexyl, 3-methylhexyl, 4-methylhe
  • lower alkyl groups containing 1 to 6 carbon atoms include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec- Butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl , 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylpropyl Methylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methyl pentyl, 2,3-dimethylbutyl, etc.
  • Alkyl groups may be substituted or unsubstituted, and when substituted, substituents may be substituted at any available point of attachment, preferably one or more of the following groups, independently selected from alkanes group, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkane Oxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo.
  • substituents may be substituted at any available point of attachment, preferably one or more of the following groups, independently selected from alkanes group, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocyclo
  • heteroalkyl refers to an alkyl group containing one or more heteroatoms selected from N, O, or S, wherein alkyl is as defined above.
  • alkylene refers to a saturated straight or branched chain aliphatic hydrocarbon group having two residues derived by removing two hydrogen atoms from the same or two different carbon atoms of the parent alkane.
  • An alkylene group is a straight or branched chain group containing 1 to 20 carbon atoms, preferably an alkylene group containing 1 to 12 carbon atoms, more preferably an alkylene group containing 1 to 6 carbon atoms.
  • Non-limiting examples of alkylene groups include, but are not limited to, methylene ( -CH2- ), 1,1-ethylene (-CH( CH3 )-), 1,2-ethylene ( -CH2) CH 2 )-, 1,1-propylene (-CH(CH 2 CH 3 )-), 1,2-propylene (-CH 2 CH(CH 3 )-), 1,3-propylene ( -CH2CH2CH2- ), 1,4 - Butylene ( -CH2CH2CH2CH2- ) and 1,5 - Butylene ( -CH2CH2CH2CH2CH2- ) Wait.
  • Alkylene groups can be substituted or unsubstituted.
  • substituents may be substituted at any available point of attachment, preferably independently optionally selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino , halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio and substituted with one or more substituents in the oxo group.
  • alkoxy refers to -O-(alkyl) and -O-(unsubstituted cycloalkyl), wherein alkyl or cycloalkyl is as defined above.
  • alkoxy groups include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy.
  • Alkoxy can be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkoxy Thio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio , Heterocycloalkylthio.
  • cycloalkyl refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent. Cycloalkyl rings contain 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 10 carbon atoms, and most preferably 3 to 7 carbon atoms.
  • Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatriene
  • Polycyclic cycloalkyl groups include spiro, fused and bridged cycloalkyl groups.
  • heterocyclyl refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent containing from 3 to 20 ring atoms, one or more of which is selected from nitrogen, oxygen or S(O) m (where m is an integer from 0 to 2) heteroatoms, excluding ring moieties of -OO-, -OS- or -SS-, the remaining ring atoms being carbon.
  • the heterocyclyl group preferably contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably the cycloalkyl ring contains 3 to 10 ring atoms.
  • Non-limiting examples of monocyclic heterocyclyl groups include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, and the like.
  • Polycyclic heterocyclyls include spiro, fused and bridged heterocyclyls.
  • spiroheterocyclyl refers to a 5- to 20-membered polycyclic heterocyclic group in which one atom (called a spiro atom) is shared between the monocyclic rings, wherein one or more ring atoms are selected from nitrogen, oxygen or S(O) m (where m is an integer from 0 to 2) heteroatoms and the remaining ring atoms are carbon. It may contain one or more double bonds, but none of the rings have a fully conjugated pi electron system. Preferably it is 6 to 14 yuan, more preferably 7 to 10 yuan.
  • spiroheterocyclyls are classified into mono-spiroheterocyclyl, bis-spiroheterocyclyl or poly-spiroheterocyclyl, preferably mono-spiroheterocyclyl and bis-spiroheterocyclyl. More preferably, it is a 4-membered/4-membered, 4-membered/5-membered, 4-membered/6-membered, 5-membered/5-membered or 5-membered/6-membered monospiroheterocyclyl group.
  • Non-limiting examples of spiroheterocyclyl include:
  • fused heterocyclyl refers to a 5- to 20-membered polycyclic heterocyclic ring system in which each ring shares a pair of adjacent atoms with the other rings in the system.
  • one or more rings may contain one or more double bonds, but none of the rings have a fully conjugated pi-electron system, wherein one or more ring atoms are selected from nitrogen, oxygen, or S ( O) heteroatoms of m (where m is an integer from 0 to 2) and the remaining ring atoms are carbon.
  • the fused heterocyclic group is preferably 6 to 14 members, more preferably 7 to 10 members.
  • the fused heterocyclic group can be divided into bicyclic, tricyclic, tetracyclic or polycyclic; preferably bicyclic or tricyclic; more preferably 5-membered/5-membered, or 5-membered/6-membered bicyclic fused heterocyclic ring base.
  • fused heterocyclyl groups include:
  • bridged heterocyclyl refers to a 5 to 14 membered polycyclic heterocyclic group in which any two rings share two atoms that are not directly connected. Bridged heterocyclyl groups may contain one or more double bonds, but none of the rings have a fully conjugated pi-electron system, wherein one or more ring atoms are selected from nitrogen, oxygen, or S(O) m (where m is an integer 0 to 2) and the remaining ring atoms are carbon.
  • the bridged heterocyclyl group is preferably 6 to 14 membered, more preferably 7 to 10 membered.
  • Bridged heterocyclyl groups can be classified as bicyclic, tricyclic, tetracyclic or polycyclic according to the number of constituent rings; preferably bicyclic, tricyclic or tetracyclic; more preferably bicyclic or tricyclic.
  • Non-limiting examples of bridged heterocyclyl groups include:
  • heterocyclyl ring can be fused to an aryl, heteroaryl or cycloalkyl ring, wherein the ring attached to the parent structure is a heterocyclyl, non-limiting examples of which include:
  • Heterocyclyl groups can be optionally substituted or unsubstituted.
  • the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro group, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo.
  • aryl refers to a 6 to 14 membered all carbon monocyclic or fused polycyclic (ie, rings that share adjacent pairs of carbon atoms) groups having a conjugated pi electron system.
  • Aryl is preferably 6 to 10 membered, such as phenyl and naphthyl, preferably phenyl.
  • the aryl ring may be fused to a heteroaryl, heterocyclyl or cycloalkyl ring, wherein the ring linked to the parent structure is an aryl ring, non-limiting examples of which include:
  • Aryl groups can be substituted or unsubstituted.
  • the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro , cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio.
  • heteroaryl refers to a heteroaromatic system comprising 1 to 4 heteroatoms, 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur and nitrogen.
  • Heteroaryl is preferably 5 to 10-membered, more preferably 5- or 6-membered, such as furanyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, tetra azolyl, etc.
  • the heteroaryl ring can be fused to an aryl, heterocyclyl or cycloalkyl ring, wherein the ring linked to the parent structure is a heteroaryl ring, non-limiting examples of which include:
  • Heteroaryl groups can be optionally substituted or unsubstituted.
  • the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro , cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio.
  • amino protecting group is a group that protects the amino group with a group that is easily removed in order to keep the amino group intact when the rest of the molecule is reacted.
  • Non-limiting examples include 9-fluorenylmethoxycarbonyl, t-butoxycarbonyl, acetyl, benzyl, allyl, p-methoxybenzyl, and the like. These groups may be optionally substituted with 1-3 substituents selected from halogen, alkoxy or nitro.
  • the amino protecting group is preferably 9-fluorenemethoxycarbonyl.
  • cycloalkylalkyl refers to the substitution of a hydrogen on an alkyl group with one or more cycloalkyl groups, preferably with one cycloalkyl group, wherein alkyl is as defined above, wherein cycloalkyl is as defined above.
  • haloalkyl refers to the replacement of a hydrogen on an alkyl group with one or more halogens, wherein alkyl is as defined above.
  • deuterated alkyl refers to the replacement of a hydrogen on an alkyl group with one or more deuterium atoms, wherein alkyl is as defined above.
  • hydroxy refers to the -OH group.
  • halogen refers to fluorine, chlorine, bromine or iodine.
  • amino refers to -NH2 .
  • nitro refers to -NO2 .
  • Substituted means that one or more hydrogen atoms in a group, preferably up to 5, more preferably 1 to 3 hydrogen atoms, independently of one another, are substituted by the corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and the person skilled in the art can determine (either experimentally or theoretically) possible or impossible substitutions without undue effort. For example, amino or hydroxyl groups with free hydrogens may be unstable when combined with carbon atoms with unsaturated (eg, olefinic) bonds.
  • drug load refers to the average amount of cytotoxic drug loaded on each antibody or antigen-binding fragment thereof in the ADC molecule, and can also be expressed as the ratio of the amount of drug to the amount of antibody, and the drug load can range from each antibody 0-12, preferably 1-10, more preferably 2-8, most preferably 3.5-4.5 cytotoxic drugs (D) are linked to the antigen-binding fragment (Pc) thereof.
  • the drug load is denoted as n, and an exemplary n may be one or more of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 calculated from mean value of .
  • the average amount of drug per ADC molecule after conjugation reaction can be characterized by conventional methods such as UV/Vis spectroscopy, mass spectrometry, ELISA assay and HPLC.
  • the cytotoxic drug is coupled to the N-terminal amino group, the ⁇ -amino group of lysine residues, and/or the sulfhydryl group of the antibody or antigen-binding fragment thereof through a linker unit.
  • the number of drug molecules that can be conjugated to the antibody in the conjugation reaction will be less than the theoretical maximum.
  • the cytotoxic drug load can be controlled by the following non-limiting methods, including:
  • carrier refers to a system capable of altering the way a drug enters the human body and its distribution in the body, controlling the rate of drug release, and delivering the drug to targeted organs.
  • Drug carrier release and targeting systems can reduce drug degradation and loss, reduce side effects, and improve bioavailability.
  • polymer surfactants that can be used as carriers can self-assemble to form aggregates in various forms due to their unique amphiphilic structure.
  • Preferred examples are micelles, microemulsions, gels, liquid crystals, vesicles, etc. . These aggregates have the ability to encapsulate drug molecules, and at the same time have good permeability to the membrane, and can be used as excellent drug carriers.
  • administering when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to exogenous drugs, therapeutic agents, diagnostic agents, or compositions that interact with the animal. , contact of humans, subjects, cells, tissues, organs or biological fluids.
  • administering can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods.
  • Treatment of cells includes contact of reagents with cells, and contact of reagents with fluids, wherein the fluids are in contact with cells.
  • administering also mean in vitro and ex vivo treatment of, eg, a cell by an agent, diagnostic, binding composition, or by another cell.
  • Treatment when applied to human, veterinary or research subjects refers to therapeutic treatment, prophylactic or preventive measures, research and diagnostic applications.
  • Treatment means administering an internal or external therapeutic agent, eg, a composition comprising any of the binding compounds of the present disclosure, to a patient having one or more disease symptoms for which the therapeutic agent is known to have Therapeutic effect.
  • the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of a disease in a patient or population to be treated, to induce regression of such symptoms or to inhibit the progression of such symptoms to any clinically measurable extent.
  • the amount of a therapeutic agent effective to relieve symptoms of any particular disease can vary depending on factors such as the patient's disease state, age and weight, and the ability of the drug to produce the desired effect in the patient.
  • Whether symptoms of a disease have been alleviated can be assessed by any clinical test commonly used by doctors or other health care professionals to assess the severity or progression of the symptoms. Although embodiments of the present disclosure (eg, methods of treatment or articles of manufacture) may be ineffective in alleviating symptoms of each target disease, the method of The U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determine that it should reduce symptoms of the target disease in a statistically significant number of patients.
  • H test Kruskal-Wallis test
  • Jonckheere-Terpstra test Jonckheere-Terpstra test
  • Wilcoxon test determine that it should reduce symptoms of the target disease in a statistically significant number of patients.
  • an "effective amount” includes an amount sufficient to ameliorate or prevent a symptom or disorder of a medical disease.
  • An effective amount also means an amount sufficient to allow or facilitate diagnosis.
  • the effective amount for a particular patient or veterinary subject may vary depending on, for example, the condition being treated, the general health of the patient, the method, route and dosage of administration, and the severity of the side effect.
  • An effective amount can be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.
  • Displacement refers to the displacement of the solvent system that dissolves the antibody or ADC, eg, the physical manipulation of a high-salt or hypertonic solvent system containing the antibody or ADC using the buffer system of the stable formulation such that the antibody protein is present in the stable formulation middle.
  • the so-called physical manipulations include, but are not limited to, ultrafiltration, dialysis or reconstitution after centrifugation.
  • Example 1-1 Construction of a cell line highly expressing Claudin18.2
  • the pCDH-hClaudin18.2 lentiviral expression vector plasmid and pVSV-G or pCMV-dR8.91 lentiviral system packaging vector were transfected into virus packaging cells 293T; the medium supernatant containing virus was collected , filter and perform ultracentrifugation; use the concentrated virus to infect human gastric signet ring cell cancer cell line NUGC4, screen for two to three weeks with puromycin, and then perform FACS single-cell sorting.
  • the degree of Claudin18.2 expression was differentiated according to tumor IHC score.
  • the cells with the same level of Claudin18.2 expression as the tumor with a tumor IHC score of 3 are high-expressing cells, and the cells with the same level of Claudin18.2 expression as a tumor with a tumor IHC score of 2 are medium-expressing cells.
  • the NUGC4/hClaudin18.2 monoclonal cell line with high Claudin18.2 expression was selected.
  • the expression of Claudin18.2 on the surface of wild-type NUGC4 cells was detected by FACS, and the NUGC4 clone cell line with moderate expression of Claudin18.2 was selected.
  • the selected monoclonal cell lines are expanded and cultured, and the library is frozen for subsequent experiments.
  • Example 1-2 Anti-human claudin18.2 monoclonal antibody production
  • Anti-human Claudin18.2 monoclonal antibody was produced by immunizing mice.
  • mice SJL white mice for experiments, female, 6-8 weeks old (Charles River Laboratory Animal Technology Co., Ltd., Beijing; animal production license number: SCXK (Beijing) 2012- 0001). Breeding environment: SPF grade. After the mice were purchased, they were reared in a laboratory environment for 1 week, regulated by a 12/12 hour light/dark cycle, with a temperature of 20-25°C and a humidity of 40-60%. The acclimated mice were immunized according to the following protocol. The immunization antigen was huClaudin18.2-HEK293 cells (HEK-293 stable cell line transfected with human Claudin18.2 plasmid).
  • IP Intraperitoneally
  • Boosters were injected intraperitoneally (IP) with 1 x 10< 7 > cells 3 days prior to splenocyte fusion.
  • a PEG-mediated fusion procedure was used to combine spleen lymphocytes with myeloma cells Sp2/0 cells ( CRL-8287 TM ) was fused to obtain hybridoma cells.
  • Hybridoma cells were resuspended in complete medium (IMDM medium containing 20% FBS, 1 ⁇ HAT, 1 ⁇ OPI) at a density of 0.5-1 ⁇ 10 6 /ml, and 100 ⁇ l/well were seeded in 96-well plates, 37 After incubating for 3-4 days at °C and 5% CO 2 , supplement 100 ⁇ l/well of HAT complete medium, and continue to culture for 3-4 days until colonies are formed. Remove the supernatant, add 200 ⁇ l/well of HT complete medium (IMDM medium containing 20% FBS, 1 ⁇ HT and 1 ⁇ OPI), culture at 37° C. and 5% CO 2 for 3 days before ELISA detection.
  • IMDM medium containing 20% FBS, 1 ⁇ HT and 1 ⁇ OPI
  • the culture supernatant was detected by binding ELISA method.
  • the hybridoma clones were obtained by the above-mentioned experimental screening, and the antibody was further prepared by serum-free cell culture method, and the antibody was purified according to the purification example for use in the detection example.
  • the monoclonal hybridoma cell lines mAb1901 and mAb1902 with high in vitro activity were selected; the monoclonal antibody sequences were cloned, and then humanization, recombinant expression and activity evaluation were carried out.
  • the procedure for cloning sequences from hybridomas is as follows. Hybridoma cells in logarithmic growth phase were collected, RNA was extracted with Trizol (Invitrogen, 15596-018) (according to the kit instructions) and reverse transcribed (PrimeScript TM Reverse Transcriptase, Takara, cat#2680A). The cDNA obtained by reverse transcription was amplified by PCR using mouse Ig-Primer Set (Novagen, TB326 Rev. B 0503), and sent to a sequencing company for sequencing. The amino acid sequences corresponding to the obtained DNA sequences are shown in SEQ ID NOs: 3-6:
  • mAb1901 murine heavy chain variable region (SEQ ID NO: 3)
  • mAb1901 murine light chain variable region (SEQ ID NO: 4)
  • mAb1902 murine heavy chain variable region (SEQ ID NO: 5)
  • mAb1902 murine light chain variable region (SEQ ID NO: 6)
  • the above-mentioned murine heavy chain variable regions and light chain variable regions are respectively linked to the heavy chain constant regions and human kappa light chain constant regions of the following human IgG1 antibodies to form chimeric antibodies ch1901 and ch1902.
  • the constant region of each antibody is selected from the following sequences:
  • Murine monoclonal antibodies were humanized as described in many literatures in the art. Briefly, CDR grafting was performed using human constant domains in place of the parental (murine antibody) constant domains, and human germline antibody sequences selected based on the homology of the murine and human antibodies. In the present invention, candidate molecules with good activity are selected for humanization, and the results are as follows.
  • amino acid residues of the VH/VL CDRs in Table 1 were identified and annotated by the Kabat numbering system.
  • the sequences of the variable regions of the heavy and light chains were compared with the antibody Germline database to obtain a human germline template with high homology.
  • the human germline light chain framework region is derived from the human kappa light chain gene.
  • the corresponding heavy chain variable region in the above table is connected with the human IgG1 heavy chain constant region shown in SEQ ID NO: 7 to form the heavy chain of the full-length antibody, and the light chain variable region is connected with the human kappa light shown in SEQ ID NO: 8.
  • the chain constant regions are linked to form the light chain of a full-length antibody.
  • the heavy and light chain variable regions can also be linked to other heavy and light chain constant regions, respectively, to form full-length antibodies.
  • the corresponding heavy chain variable region in the above table is connected with the human IgG1 heavy chain constant region shown in SEQ ID NO: 7 to form the heavy chain of the full-length antibody, and the light chain variable region is connected with the human kappa light shown in SEQ ID NO: 8.
  • the chain constant regions are linked to form the light chain of a full-length antibody.
  • ch1901 heavy chain (SEQ ID NO:35)
  • ch1902 heavy chain (SEQ ID NO:37)
  • H1 H2 H3 H4 L1 h1901-1 h1901-2 h1901-3 h1901-4 L2 h1901-5 h1901-6 h1901-7 h1901-8 L3 h1901-9 h1901-10 h1901-11 h1901-12
  • the positive control antibody of the present disclosure is IMAB-362 (from WO2016166122).
  • the above antibodies were cloned, expressed and purified by conventional gene cloning and recombinant expression methods, respectively.
  • the drug moiety of the anti-Claudin18.2 ADC conjugates in this disclosure can be any suitable drug. Particularly suitable drugs are described, for example, in PCT Publication No. WO2020063676A1 (hereby incorporated by reference in its entirety).
  • Compound 9-A of the present disclosure is N-((2R,10S)-10-benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy -4-Methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6, 7] Indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentoxo-3-oxa-5,8,11,14- Tetrazahexadec-16-yl)-6-(2,5-dioxo-2,5-dihydr
  • UV spectrophotometry using instrument: Thermo nanodrop2000 UV spectrophotometer was used to determine the ADC stock solution loading.
  • the principle is that the total absorbance of the ADC stock solution at a certain wavelength is equal to the absorbance of the drug and monoclonal antibody at the wavelength.
  • a 280nm ⁇ mab-280 bC mab + ⁇ Drug-280 bC Drug
  • ⁇ Drug-280 The average molar extinction coefficient of the drug at 280nm is 5100;
  • ⁇ mab-280 The average molar extinction coefficient of single antigen solution at 280nm is 214600;
  • C mab concentration of single antigen solution
  • the optical path length is 1 cm.
  • a 370nm ⁇ mab-370 bC mab + ⁇ Drug-370 bC Drug
  • ⁇ Drug-370 The average molar extinction coefficient of the drug at 370nm is 19000;
  • ⁇ mab-370 The extinction coefficient of single antigen solution at 370nm is 0;
  • C mab concentration of single antigen solution
  • the optical path length is 1 cm.
  • the drug loading can be calculated from the two equations (1) and (2) combined with the extinction coefficient and concentration data of the monoclonal antibody and drug at two detection wavelengths.
  • Drug load C Drug /C mab .
  • Naked antibody and ADC samples to be tested were added with 4 ⁇ l DDT (sigma) for reduction, water bath at 37°C for 1 hour, and taken out into the inner tube after the end.
  • High performance liquid chromatograph Agilent 1200 was used for detection, the chromatographic column was Agilent PLRP-S 1000A 8 ⁇ m 4.6*250mm, column temperature: 80°C; DAD detector wavelength 280nm; flow rate: 1mL/min; injection volume: 40 ⁇ L;
  • Preparation example Take 5.78mg of DTT, add 150 ⁇ l of purified water to fully dissolve it, prepare a 0.25M DTT solution, and store it at -20°C.
  • Preparation example Measure 1000ml of purified water in a graduated cylinder, add 1mL of TFA (sigma), mix thoroughly before use, and store at 2-8°C for 14 days.
  • Preparation example measure 1000ml of acetonitrile in a graduated cylinder, add 1ml of TFA, mix thoroughly before use, and store at 2-8°C for 14 days.
  • the positions of the light chain and the heavy chain are distinguished, and then the spectrum of the detection sample is integrated to calculate the DAR value.
  • LC peak area sum LC peak area + LC + 1 peak area
  • HC peak areas HC peak area + HC + 1 peak area + HC + 2 peak area + HC + 3 peak area;
  • LC DAR ⁇ (number of connected drugs*percentage of peak area)/sum of LC peak area
  • HC DAR ⁇ (number of connected drugs*peak area percentage)/sum of HC peak areas
  • TCEP tris(2-carboxyethyl)phosphine
  • reaction solution was cooled to 25°C with a water bath, and compound 9-A (350mg, 303mol) was dissolved in 13.2ml of acetonitrile and 6.6ml of DMSO and added to the reaction solution, placed on a water bath shaker, and the reaction was shaken at 25°C. After 3 hours, the reaction was stopped.
  • the prepared TCEP aqueous solution (10 mM, 10.1 ⁇ L, 101 nmol), placed in a water bath shaker, and shaken at 37° C. for 3 hours to stop the reaction.
  • the reaction solution was cooled to 25°C with a water bath.
  • the prepared TCEP aqueous solution (10 mM, 16.9 ⁇ L, 169 nmol), placed in a water bath shaker, and shaken at 37° C. for 3 hours to stop the reaction.
  • the reaction solution was cooled to 25°C with a water bath.
  • the reaction solution was cooled to 25°C with a water bath.
  • the prepared TCEP aqueous solution (10 mM, 10.9 ⁇ L, 10 mM, 10.9 ⁇ L, 109 nmol), placed in a water bath shaker, and shaken at 37° C. for 3 hours to stop the reaction.
  • the reaction solution was cooled to 25°C with a water bath.
  • the prepared TCEP aqueous solution (10 mM, 18.3 ⁇ L, 183 nmol), placed in a water bath shaker, and shaken at 37° C. for 3 hours to stop the reaction.
  • the reaction solution was cooled to 25°C with a water bath.
  • the prepared TCEP aqueous solution (10 mM, 38.7 ⁇ L, 387 nmol), placed in a water bath shaker, and shaken at 37° C. for 3 hours to stop the reaction.
  • the reaction solution was cooled to 25°C with a water bath.
  • the cation eluate was subjected to 7-fold equal volume ultrafiltration (the ultrafiltration membrane package was a 30KD polycellulose membrane package) to obtain the product ADC-9.
  • RP-HPLC calculated mean value: n 4.1.
  • a cell-based ELISA assay was used to examine the binding properties of the Claudin18.2 antibody.
  • the NUGC4 cells stably expressing Claudin18.2 were cultured in a 96-well cell plate (Corning, 3599), and when the cells were grown to 90% density, 4% paraformaldehyde was added to fix the cells for 1 hour, and PBST buffer (pH 7.4 PBS containing After washing the plate 3 times with 0.05% Tween-20), add 200 ⁇ l/well of blocking solution of 5% skim milk (bright skim milk powder) diluted with PBS, incubate at 37°C for 2.5 hours or place at 4°C overnight (16-18 hours) to be closed.
  • PBST buffer pH 7.4 PBS containing After washing the plate 3 times with 0.05% Tween-20
  • sample diluent pH 7.4 PBS containing 1% skim milk
  • sample diluent pH 7.4 PBS containing 1% skim milk
  • HRP-labeled goat anti-human secondary antibody Jackson Immuno Research, 109-035-003
  • TMB chromogenic substrate KPL, 52-00-03
  • 1M H 2 SO 4 50 ⁇ l/well of 1M H 2 SO 4
  • MD Versa Max TM microplate reader reads the absorbance at 450nm, and calculates the binding EC50 value of Claudin18.2 antibody to Claudin18.2.
  • Antibody Emax EC50(nM) IMAB362 1.115 0.086 h1901-2 1.039 0.076 h1901-3 1.1055 0.22 h1901-4 0.986 0.201 h1901-6 0.937 0.091 h1901-7 0.921 0.166 h1901-8 1.047 0.091 h1901-11 1.44 0.076 h1901-12 1.22 0.116
  • Antibody Emax EC50(nM) IMAB362 0.88 0.187 h1902-1 0.87 0.113 h1902-2 0.88 0.107 h1902-3 0.84 0.175 h1902-4 0.82 0.087 h1902-5 0.9 0.098 h1902-6 0.78 0.141 h1902-7 0.75 0.121 h1902-8 0.89 0.132 h1902-9 0.75 0.137 h1902-10 0.89 0.133
  • NUGC4 cells stably expressing Claudin18.2 were prepared into 1 ⁇ 10 6 /ml cell suspension with FACS buffer (2% fetal bovine serum (Gibco, 10099141) pH7.4PBS (Sigma, P4417-100TAB)), 100 ⁇ l /well was added to a 96-well round bottom plate (Corning, 3795). After removing the supernatant by centrifugation, 50 ⁇ l/well of Claudin18.2 antibody to be tested at different concentrations diluted with FACS buffer was added, and incubated in a refrigerator at 4°C for 1 hour in the dark.
  • FACS buffer 2% fetal bovine serum (Gibco, 10099141) pH7.4PBS (Sigma, P4417-100TAB)
  • the Claudin18.2 antibody to be tested pre-labeled with DyLight 488 NHS Ester was added to 1 ⁇ 10 6 /ml NUGC4 cells stably expressing Claudin18.2 at a final concentration of 5 ⁇ g/ml, and placed on ice to avoid Incubate in light for 1 hour, centrifuge and wash 3 times with pre-cooled FACS buffer (pH 7.4 PBS, 2% fetal bovine serum), remove the supernatant, add pre-warmed complete medium, and place in 37°C 5% CO 2 for cell culture box. Cells were removed after 0, 0.5, 1, 2, and 4 hours, respectively, and stored on ice to protect from light.
  • Test Example 4 Determination of Antibody Affinity Based on Flow Cytometry
  • HEK293/hClaudin18.2 cells were collected in a U-bottom 96-well plate, with 1 ⁇ 10 5 to 2 ⁇ 10 5 cells per well.
  • Claudin18.2 antibody with an initial concentration of 5 ⁇ g/ml, 2 ⁇ gradient dilution (12 concentration points), and incubate at 4°C for 1 hour.
  • the positive control is IMAB362, and a negative control without antibody is set at the same time.
  • the antibody was removed by centrifugation, then 100 ⁇ l/well of FITC anti-human IgG Fc antibody (200 ⁇ ) was added, incubated at 4°C for 30 minutes in the dark, washed twice with PBS+2% FBS, and ready for flow cytometry detection.
  • the antibody was diluted in the above-mentioned phenol red-free medium to prepare a 3 ⁇ antibody dilution, and 25 ⁇ l/well of the antibody was added to the cell plate. Incubate for 0.5 h in a 37 °C, 5% CO2 incubator.
  • the effector cells (FcrR3A-V158-NFAT-RE-Jurkat cells) were collected, centrifuged at 1000 rpm, resuspended and counted. Cells were resuspended in phenol red-free RPMI 1640 supplemented with 10% FBS (New Zealand ultra-low IgG fetal bovine serum) at a density of 3 x 10 6 cells/ml, and 25 ⁇ l of cells (7.5 x 10 4 ) were added to each well of the assay plate. cells/well). Incubate for 6 h in a 37 °C, 5% CO2 incubator.
  • FBS New Zealand ultra-low IgG fetal bovine serum
  • ADCC effector unit IC50 (ng/ml) of antibodies in NUGC4 cells with different expression levels of Claudin18.2
  • the CellTiter-Glo Luminescence Cell Viability Assay was used to detect the killing effect of ADC molecules on human gastric cancer cell lines in vitro.
  • collect NUGC4-claudin18.2 low-expressing, NUGC4-claudin18.2-expressing, and NUGC4-claudin18.2 high-expressing cells adjust the density to 2.5 ⁇ 10 4 /ml, and add 90 ⁇ l/ml to a 96-well white transparent bottom plate. well, approximately 2500 cells per well. Incubate overnight in a 37°C, 5% CO2 incubator.
  • the samples were diluted in a U-bottom 96-well plate, the initial concentration was 5 ⁇ M, 4 ⁇ gradient dilution, 9 concentration points, and 10 ⁇ l/well of the diluted samples were added to the cell plate. Incubate for 6 days at 37°C, 5% CO2 . On the eighth day, take out the cell culture plate, add 50 ⁇ l/well Cell Titer-Glo Reagent, leave at room temperature for 2 to 3 minutes, and read the luminescence value on a PHERAstar FS microplate reader. Data analysis was performed using GraphPad Prism software. See Table 16.
  • Balb/c was subcutaneously inoculated with human gastric cancer cells NUGC4 (Claudin18.2 medium expression) cells (5 ⁇ 10 6 containing 50% matrigel/matrigel) in the right flank, and divided into groups on the 0th day, 8 animals/group, a total of 8 groups .
  • the average tumor volume was about 84.41 mm 3 .
  • the ADC was injected intraperitoneally for 3 times, and each animal was injected with 10g/0.1ml according to body weight, and it was administered on the 0th, 4th, and 11th days respectively.
  • ADC was injected intraperitoneally on the day of grouping, and administered 4 times in total, with an interval of 5 days. Each animal was injected with 10 g/0.1 ml of body weight.
  • Tumor volume and body weight were measured twice a week and the data were recorded.
  • V 1/2 ⁇ L length ⁇ L short 2
  • Relative volume (RTV) VT/V0
  • Tumor inhibition rate (%) (CRTV-TRTV)/CRTV (%)
  • V0 and VT are the tumor volume at the beginning of the experiment (the day of the first administration is the 0th day) and at the end of the experiment, respectively.
  • CRTV and TRTV are the relative tumor volumes of the blank control group (Vehicle) and the experimental group at the end of the experiment, respectively. The results are shown in Table 17 and Figures 4 and 5.
  • SEC% SEC monomer content percentage
  • a monomer is the peak area of the main peak monomer in the sample, A total is the sum of all peak areas).
  • Purity percentage of reduced CE A main peak/A total*100% (A main peak is the peak area of light chain main peak+heavy chain main peak in the sample, A total is the sum of all peak areas.
  • the freezing point method is used to measure the osmotic pressure. Based on the proportional relationship between the freezing point drop value and the molar concentration of the solution, a high-sensitivity temperature sensing element is used to measure the freezing point of the solution and convert it into osmotic pressure through electricity. Instrument manufacturer Roser Loser, model OM815.
  • A280 Cd* ⁇ 280d+Cmab* ⁇ 280mab
  • Cd represents the concentration of the drug
  • Cmab represents the concentration of the protein
  • ⁇ 280d represents the extinction coefficient of the drug at 280 nm
  • ⁇ 280mab represents the extinction coefficient of the protein at 280 nm
  • ⁇ 370d represents the extinction coefficient of the drug at 370 nm.
  • ⁇ 280mab 1.49mg-1*cm-1*ml
  • ⁇ 280d 5000 (280nm drug molar extinction coefficient)/1074.13
  • drug molecular weight 4.65mg-1*cm-1*mL
  • ⁇ 370d 19000 (370nm drug molar extinction coefficient) )/1074.13
  • drug molecular weight 17.69mg-1*cm-1*mL
  • the above extinction coefficient is the mass extinction coefficient.
  • Protein concentration determination instrument UV-Vis spectrophotometer, model: Nano Drop oneC, optical path is 1mm.
  • Example 2-1 Screening of formulation buffer system and pH value
  • Formulations were formulated containing 20 mg/mL (protein concentration) of ADC-9 and the following different buffer systems, and 0.1 mg/mL polysorbate 80 (PS80).
  • the sample No. 7 is 10mM His-AA, and the pH 5.0 preparation is superior to the other preparations in terms of appearance and various chemical detection items, so 10mM His-AA, pH 5.0 is selected as the final buffer.
  • D in the table means days, for example, D3 means 3 days, and so on; D0 means the beginning of the experiment, the same below.
  • Example 2-2 Screening of Surfactant Types and Concentrations
  • Formulations were prepared containing different types and concentrations of polysorbate and containing 10 mM His-AA, buffer at pH 5.0, 80 mg/mL sucrose and ADC-9 at a protein concentration of 20 mg/mL. Each formulation was filtered, filled, stoppered, and capped. The samples were subjected to high temperature stability studies (40°C) and freeze-thaw studies. Wherein the freeze-thaw study was placed at room temperature for three days (25°C D3) after 5 cycles of freeze-thaw (FT5C, 35°C-2 to 8°C), and the appearance, SEC, and reduced CE were investigated. The specific formulation design is shown in Table 19.
  • the type and concentration of surfactant was determined as 0.2mg/ml PS80.
  • PS20 means polysorbate 20.
  • M in the table means month
  • M1 means 1 month
  • Formulations were prepared containing sucrose, trehalose, mannitol, respectively, which also contained 10 mM His-AA (pH 5.0) buffer, 0.2 mg/mL PS80, and 20 mg/mL (protein concentration) ADC-9. Each formulation was filtered, filled, stoppered, and capped. The samples were subjected to high temperature stability studies (40°C), freeze-thaw cycles at -35°C/4°C, and placed at room temperature for 3 days to investigate appearance, SEC, and reduced CE.
  • the results are shown in Table 21.
  • the appearance of samples with sucrose was better than that with trehalose or mannitol under freeze-thaw conditions of different sugar species preparations.
  • the SEC test results show that the samples using sucrose or trehalose are better than mannitol; after being placed at 40 °C for one month, the appearance of the samples using sucrose is better than the samples using trehalose or mannitol, and the test results of SEC and reduced CE also show that The samples with sucrose were slightly better than those with trehalose.
  • Formulations were prepared containing sucrose, trehalose, mannitol, respectively, which also contained 10 mM His-AA (pH 5.0) buffer, 0.2 mg/mL PS80, and 20 mg/mL (protein concentration) ADC-9. Each formulation was filtered, filled, semi-stoppered, lyophilized, stoppered, and capped, and placed for high temperature stability studies (40°C) to investigate appearance, SEC, and reduced CE. For the lyophilization process, see lyophilization process parameter 1 in Table 22.
  • Freeze-drying process parameters Set temperature (°C) Setting time (min) Hold time (h) Vacuum (Pa) pre-frozen 5 10 1 N/A pre-frozen -45 50 2.5 N/A
  • test results of SEC show that the sample using sucrose is slightly better than the sample using trehalose, and better than the sample using mannitol.
  • the test results of reduced CE show that , the samples using sucrose were comparable to those using trehalose, and were superior to those using mannitol.
  • the lyophilized sample was a white powder with a flat surface, but the bottom edge of the powder was slightly reduced.
  • the sugar concentration of the sample was further lowered to 60 mg/mL, and the stock solution was prepared according to 10 mM His-AA, pH 5.0, 60 mg/mL sucrose, 0.2 mg/mL PS80 and 20 mg/mL (protein concentration) ADC-9, after filtering and filling Lyophilize according to lyophilization process parameter 1 in Table 22. After freeze-drying, the surface of the powder is flat without shrinkage, and the bottom edge of the powder is slightly shrunk.
  • the sugar concentration was reduced to 40mg/ml, but the osmotic pressure of the finished product was too low at this time, and there was a risk of low osmotic pressure during clinical administration.
  • the ionic strength of the buffer was increased to 30mM.
  • Example 2-5 Sample stability after lyophilization
  • the stability results are shown in Table 25.
  • the lyophilized sample of preparation No. 3 reduces CE by about 3.7% under the condition of 40 °C M1, and the chemical detection items of the other two preparations have no significant changes, and the stability is significantly better than that of preparation No. 3.
  • the pH of the stock solution of No. 2 formulation before lyophilization was 5.04, and the pH of the reconstituted solution after lyophilization was 5.27.
  • Formulations were prepared according to the formulations in Table 26, filtered, filled, stoppered, capped, followed by a freeze-thaw cycle at -35°C/4°C and placed at room temperature for 3 days, shaking for 11 days, and high temperature stability studies (40°C). ), the appearance, SEC and reduction CE changes of the samples under the corresponding conditions were investigated.

Abstract

提供一种含抗体药物偶联物的药物组合物及其用途。具体提供一种药物组合物，其包含抗密蛋白抗体药物偶联物。

Description

一种含抗体药物偶联物的药物组合物及其用途
本申请要求2020年09月30日提交的中国专利申请(202011061863.1)和2021年09月13日提交的中国专利申请(CN202111069020.0)的优先权。
技术领域
本披露属于药物制剂领域，具体涉及一种包含抗体药物偶联物的药物组合物，以及其作为抗癌药物的用途。
背景技术
这里的陈述仅提供与本披露有关的背景信息，而不必然地构成现有技术。
密蛋白18(Claudin-18，CLDN18)是一种在人类中由Claudin18基因编码的蛋白质，属于细胞紧密连接蛋白家族，可以控制层细胞之间的分子流动。Claudin蛋白结构中包括四个跨膜区域、两个细胞外环(其N末端和C末端在胞浆内)。
Claudin-18具有两个剪接变体，分别为Claudin 18.1和Claudin 18.2，两者序列之间仅在第一个细胞外环有八个氨基酸的差异。Claudin 18.1和Claudin 18.2的表达分布有所不同，Claudin 18.1在正常肺的细胞中选择性表达，Claudin 18.2在正常细胞中表达高度受限，但在多种肿瘤(胃癌、肺癌和胰腺癌等)中频繁异位激活和过表达。Claudin18.2被认为是胃癌和其他癌症类型的潜在治疗靶点，此靶点的发现也为胃癌的治疗提供了一种新的选择。
抗体药物偶联物(antibody drug conjugate，ADC)将单克隆抗体或者抗体片段通过稳定的化学接头化合物与具有生物活性的细胞毒素相连，充分利用了抗体对正常细胞和肿瘤细胞表面抗原结合的特异性和细胞毒性物质的高效性，同时又避免了前者疗效偏低和后者毒副作用过大等缺陷。这也就意味着，与以往传统的化疗药物相比，抗体药物偶联物能更精准地结合肿瘤细胞并降低将对正常细胞的影响。
目前已有靶向Claudin18.2的抗体及ADC药物的专利报道，如WO2016166122和WO2016165762。
由于ADC具有比抗体更复杂的异质结构，因此，对用于治疗目的ADC制剂提出了更大的挑战。
发明内容
本披露涉及含抗Claudin18.2抗体药物偶联物的药物制剂及其用途。该制剂具有稳定性好，冻干形态好等优势。
本披露提供一种药物组合物，其包含抗Claudin18.2抗体药物偶联物和缓冲剂，其中所述抗Claudin18.2抗体药物偶联物中的抗Claudin18.2抗体包含重链可变区和 轻链可变区，其中：
i)所述重链可变区与如SEQ ID NO：5所示的重链可变区具有相同氨基酸序列的HCDR1、HCDR2和HCDR3，所述轻链可变区包含与如SEQ ID NO：6所示的轻链可变区具有相同氨基酸序列的LCDR1、LCDR2和LCDR3；或
ii)所述重链可变区与如SEQ ID NO：3所示的重链可变区具有相同氨基酸序列的HCDR1、HCDR2和HCDR3，所述轻链可变区与如SEQ ID NO：4所示的轻链可变区具有相同氨基酸序列的LCDR1、LCDR2和LCDR3；
所述缓冲剂是组氨酸盐缓冲剂。
在一些实施方案中，如上任一项所述的药物组合物，其中所述缓冲剂为组氨酸-醋酸盐缓冲剂。
在一些实施方案中，如上任一项所述的药物组合物，其中所述抗Claudin18.2抗体包含重链可变区和轻链可变区，其中：
iii)所述重链可变区包含分别如SEQ ID NO：15、SEQ ID NO：16和SEQ ID NO：17所示的HCDR1、HCDR2和HCDR3，所述轻链可变区包含分别如SEQ ID NO：18、SEQ ID NO：19和SEQ ID NO：20所示的LCDR1、LCDR2和LCDR3；或
iv)所述重链可变区包含分别如SEQ ID NO：9、SEQ ID NO：10和SEQ ID NO：11所示的HCDR1、HCDR2和HCDR3，所述轻链可变区包含分别如SEQ ID NO：12、SEQ ID NO：13和SEQ ID NO：14所示的LCDR1、LCDR2和LCDR3。
在一些实施方案中，如上任一项所述的药物组合物，其中所述抗Claudin18.2抗体是鼠源抗体、嵌合抗体或人源化抗体。
在一些实施方案中，如上任一项所述的药物组合物，其中所述抗Claudin18.2抗体包含重链可变区和轻链可变区，其中：包含重链可变区和轻链可变区，其中：
(1)所述重链可变区氨基酸序列如SEQ ID NO：3所示或与其有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的同一性，和所述轻链可变区氨基酸序列如SEQ ID NO：4所示或与其有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的同一性；
(2)所述重链可变区氨基酸序列如SEQ ID NO：24所示或与其有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的同一性，和所述轻链可变区氨基酸序列如SEQ ID NO：21所示或与其有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的同一性；
(3)所述重链可变区氨基酸序列如SEQ ID NO：5所示或与其有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的同一性，和所述轻链可变区氨基酸序列如SEQ ID NO：6所示或与其有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的同一性；或
(4)所述重链可变区氨基酸序列如SEQ ID NO：31所示或与其有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的同一性，和所述轻链可变区氨基酸序列如SEQ ID NO：28所示或与其有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的同一性。
在一些实施方案中，如上任一项所述的药物组合物，其中所述抗Claudin18.2抗体为人源化抗体，所述人源化抗体包含来源自人抗体的框架区或其框架区变体，所述框架区变体为在人抗体的轻链框架区和/或重链框架区上分别具有至多10个氨基酸的回复突变。
在一些实施方案中，如上任一项所述的药物组合物，其中所述框架区变体包含选自以下(a)或(b)所述的突变：
(a)所述轻链可变区中包含任选自22S、85I和87H中的一个或更多个氨基酸回复突变，和/或所述重链可变区中包含任选自48I、82T和69M中的一个或更多个氨基酸回复突变；或
(b)所述轻链可变区中包含选自任选自4L或22S中的一个或更多个氨基酸回复突变，和/或所述重链可变区中包含任选自38K、40R、48I、66K、67A、69L、71L和73K中的一个或更多个氨基酸回复突变。
在一些实施方案中，如上任一项所述的药物组合物，其中所述框架区变体包含选自以下所述的突变：
(a-1)所述轻链可变区中包含22S、85I和87H的氨基酸回复突变，和所述重链可变区中包含48I和82T的氨基酸回复突变；或
(b-1)所述轻链可变区中包含4L的氨基酸回复突变；
其中，所述重链可变区的82T中的82为Kabat规则的第82A位。
在一些实施方案中，如上任一项所述的药物组合物，其中抗Claudin18.2抗体包含如下任一所示的重链可变区和轻链可变区：
(vii)所述重链可变区如SEQ ID NO：3所示和所述轻链可变区如SEQ ID NO：4所示；
(viii)所述重链可变区如SEQ ID NO：24、25、26或27所示和所述轻链可变区如SEQ ID NO：21、22或23所示；
(ix)所述重链可变区如SEQ ID NO：5所示和所述轻链可变区如SEQ ID NO：6所示；或
(x)所述重链可变区如SEQ ID NO：31、32、33或34所示和所述轻链可变区如SEQ ID NO：28、29或30所示；
在一些实施方案中，如上任一项所述的药物组合物，其中抗Claudin18.2抗体包含如下任一所示的重链可变区和轻链可变区：
(xi)所述重链可变区如SEQ ID NO：31所示和所述轻链可变区如SEQ ID NO：29所示；或
(xii)所述重链可变区如SEQ ID NO：26所示和所述轻链可变区如SEQ ID NO：23所示。
在一些实施方案中，如上任一项所述的药物组合物，其中抗Claudin18.2抗体包含抗体重链恒定区和轻链恒定区。
在一些实施方案中，所述重链恒定区选自人IgG1、IgG2、IgG3和IgG4恒定区及其常规变体，所述轻链恒定区选自人抗体κ和λ链恒定区及其常规变体。在一些实施方案中，所述抗体包含如SEQ ID NO：7所示的重链恒定区和如SEQ ID NO：8所示的轻链恒定区。
在一些实施方案中，所述抗体包含：与具有SEQ ID NO：35或42所示氨基酸序列的重链具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的重链，和与具有SEQ ID NO：36或39所示氨基酸序列的轻链有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的轻链；或与具有SEQ ID NO：37或49所示氨基酸序列的重链具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的重链，和与具有SEQ ID NO：38或46所示氨基酸序列的轻链有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的轻链。
在一些实施方案中，如上任一项所述的药物组合物，其中抗Claudin18.2抗体包含如下任一所示的重链和轻链：
(c)序列如SEQ ID NO：35所示的重链和序列如SEQ ID NO：36所示的轻链；
(d)序列如SEQ ID NO：42、43、44或45所示的重链和序列如SEQ ID NO：39、40或41所示的轻链；
(e)序列如SEQ ID NO：37所示的重链和序列如SEQ ID NO：38所示的轻链；或
(f)序列如SEQ ID NO：49、50、51或52所示的重链和序列如SEQ ID NO：46、47或48所示的轻链。
在一些实施方案中，如上任一项所述的药物组合物，其中抗Claudin18.2抗体包含如下任一所示的重链和轻链：
SEQ ID NO：44所示的重链，和SEQ ID NO：41所示的轻链；或
SEQ ID NO：49所示的重链，和SEQ ID NO：47所示的轻链。
在一些实施方案中，如上任一项所述的药物组合物，其中所述抗Claudin18.2抗体药物偶联物具有如通式(Pc-L-Y-D)所示的结构：
Figure PCTCN2021122031-appb-000001
其中：
Y选自-O-(CR aR b) m-CR 1R 2-C(O)-、-O-CR 1R 2-(CR aR b) m-、-O-CR 1R 2-、-NH-(CR aR b) m-CR 1R 2-C(O)-和-S-(CR aR b) m-CR 1R 2-C(O)-；
R a和R b相同或不同，且各自独立地选自氢原子、氘原子、卤素、烷基、卤代烷基、氘代烷基、烷氧基、羟基、氨基、氰基、硝基、羟烷基、环烷基和杂环基；
或者，R a和R b与其相连接的碳原子一起形成环烷基或杂环基；
R 1选自卤素、卤代烷基、氘代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基和杂芳基；
R 2选自氢原子、卤素、卤代烷基、氘代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基和杂芳基；
或者，R 1和R 2与其相连接的碳原子一起形成环烷基或杂环基；
或者，R a和R 2与其相连接的碳原子一起形成环烷基或杂环基；
m为0至4的整数；
n为1至10，n是小数或整数；
L为接头单元；
Pc为抗Claudin18.2抗体；
在一些实施方案中，如上任一项所述的药物组合物，其中n是小数或整数，可以是2至8、3至7、3.5至4.5、2、3、3.5、4、4.5、5、6、7、8、9。
在一些实施方案中，如上任一项所述的药物组合物，其中所述抗Claudin18.2抗体药物偶联物具有如通式(Pc-L-Y-D)所示的结构，
其中：
Y为-O-(CR aR b) m-CR 1R 2-C(O)-；
R a和R b相同或不同，且各自独立地选自氢原子、氘原子、卤素或C 1-6烷基；
R 1为卤代C 1-6烷基或C 3-6环烷基；
R 2选自氢原子、卤代C 1-6烷基或C 3-6环烷基；
或者，R 1和R 2与其相连接的碳原子一起形成C 3-6环烷基；
m为0或1。
在一些实施方案中，如上任一项所述的药物组合物，其中所述抗Claudin18.2 抗体药物偶联物具有如通式(Pc-L-Y-D)所示的结构，其中Y选自：
Figure PCTCN2021122031-appb-000002
其中Y的O端与接头单元L相连。
在一些实施方案中，如上任一项所述的药物组合物，其中接头单元-L-为-L 1-L 2-L 3-L 4-，
L 1选自-(琥珀酰亚胺-3-基-N)-W-C(O)-、-CH 2-C(O)-NR 3-W-C(O)-或-C(O)-W-C(O)-，其中W选自C 1-8烷基、C 1-8烷基-环烷基或1至8个链原子的直链杂烷基，所述杂烷基包含1至3个选自N、O或S的杂原子，其中所述的C 1-8烷基、C 1-8烷基-环烷基或1至8个链原子的直链杂烷基各自独立地任选进一步被选自卤素、羟基、氰基、氨基、C 1-6烷基、氯代C 1-6烷基、氘代C 1-6烷基、C 1-6烷氧基和C 3-6环烷基的一个或多个取代基所取代；
L 2选自-NR 4(CH 2CH 2O)p 1CH 2CH 2C(O)-、-NR 4(CH 2CH 2O)p 1CH 2C(O)-、-S(CH 2)p 1C(O)-或化学键，其中p 1为1至20的整数；
L 3为由2至7个氨基酸构成的肽残基，其中所述的氨基酸残基选自苯丙氨酸、甘氨酸、缬氨酸、赖氨酸、瓜氨酸、丝氨酸、谷氨酸和天冬氨酸中的氨基酸形成的氨基酸残基，并任选进一步被选自卤素、羟基、氰基、氨基、C 1-6烷基、氯代C 1-6烷基、氘代C 1-6烷基、C 1-6烷氧基和C 3-6环烷基中的一个或多个取代基所取代；
L 4选自-NR 5(CR 6R 7) t-、-C(O)NR 5-、-C(O)NR 5(CH 2) t-或化学键，其中t为1至6的整数；
R 3、R 4和R 5相同或不同，且各自独立地选自氢原子、C 1-6烷基、卤代C 1-6烷基、氘代C 1-6烷基和C 1-6羟烷基；
R 6和R 7相同或不同，且各自独立地选自氢原子、卤素、C 1-6烷基、卤代C 1-6烷基、氘代C 1-6烷基和C 1-6羟烷基。
在一些实施方案中，如上任一项所述的药物组合物，其中接头单元-L-为-L 1-L 2-L 3-L 4-，
L 1为
Figure PCTCN2021122031-appb-000003
s 1为2至8的整数；
L 2为化学键；
L 3为四肽残基；优选为GGFG的四肽残基(SEQ ID No：55)；
L 4为-NR 5(CR 6R 7)t-，R 5、R 6或R 7相同或不同，且各自独立地为氢原子或C 1-6烷基，t为1或2；
其中所述的L 1端与Pc相连，L 4端与Y相连。
在一些实施方案中，如上任一项所述的药物组合物，其中-L-为：
Figure PCTCN2021122031-appb-000004
在一些实施方案中，如上任一项所述的药物组合物，其中L-Y-任选自：
Figure PCTCN2021122031-appb-000005
在一些实施方案中，如上任一项所述的药物组合物，其中所述抗Claudin18.2抗体药物偶联物选自如下任一所示的结构：
Figure PCTCN2021122031-appb-000006
Figure PCTCN2021122031-appb-000007
其中Pc和n如通式(Pc-L-Y-D)中所定义。
在一些实施方案中，如上任一项所述的药物组合物，其中所述抗Claudin18.2抗体药物偶联物具有如下式所示的结构：
Figure PCTCN2021122031-appb-000008
其中：
n为2至8，n是小数或整数；
Pc为抗Claudin18.2抗体。
在一些实施方案中，如上任一项所述的药物组合物，其中所述药物组合物还包含表面活性剂。在一些实施方案中，所述表面活性剂选自聚山梨酯(如聚山梨酯20、聚山梨酯80)、聚羟亚烃、Triton、十二烷基磺酸钠、月桂基磺酸钠、辛基糖甙钠、月桂基-磺基甜菜碱、肉豆蔻基-磺基甜菜碱、亚油基-磺基甜菜碱、硬脂基-磺基甜菜碱、月桂基-肌氨酸、肉豆蔻基-肌氨酸、亚油基-肌氨酸、硬脂基-肌氨酸、亚油基-甜菜碱、肉豆蔻基-甜菜碱、鲸蜡基-甜菜碱、月桂酰胺基丙基-甜菜碱、柯卡酰胺基丙基-甜菜碱、亚油酰胺基丙基-甜菜碱、肉豆蔻酰胺基丙基-甜菜碱、棕榈酰胺基丙基-甜菜碱、异硬脂酰胺基丙基-甜菜碱、肉豆蔻酰胺基丙基-二甲基胺、棕榈酰胺基丙基-二甲基胺、异硬脂酰胺基丙基-二甲基胺、甲基可可酰基钠、 甲基油基牛磺酸钠、聚乙二醇、聚丙二醇、乙烯与丙烯二醇的共聚物等。
在一些实施方案中，所述表面活性剂为聚山梨酯。在一些实施方案中，所述表面活性剂为聚山梨酯80或聚山梨酯20。在一些实施方案中，所述表面活性剂为聚山梨酯80。
在一些实施方案中，如上任一项所述的药物组合物，其中所述表面活性剂浓度为0.05mg/mL至0.5mg/mL或0.1mg/mL至0.2mg/mL。在一些实施方案中，所述表面活性剂浓度为0.05mg/mL、0.1mg/mL、0.15mg/mL、0.18mg/mL、0.19mg/mL、0.2mg/mL、0.21mg/mL、0.22mg/mL、0.3mg/mL、0.4mg/mL、0.5mg/mL。在一些实施方案中，所述表面活性剂浓度为0.2mg/mL。
在一些实施方案中，如上任一项所述的药物组合物，其中所述组合物还包含糖。在一些实施方案中，所述糖选自常规组合物(CH 2O) n及其衍生物，包括单糖、二糖、三糖、多糖、糖醇、还原性糖、非还原性糖等等。所述的糖可选自葡萄糖、蔗糖、海藻糖、乳糖、果糖、麦芽糖、右旋糖苷、甘油、赤藻糖醇、丙三醇、阿拉伯糖醇、sylitol、山梨糖醇、甘露醇、密里二糖、松三糖、蜜三糖、甘露三糖、水苏糖、麦芽糖、乳果糖、麦芽酮糖、山梨醇、麦芽糖醇、乳糖醇、异-麦芽酮糖等。在一些实施方案中，所述糖选自蔗糖、甘露醇和海藻糖。在一些实施方案中，所述糖为蔗糖。
在一些实施方案中，如上任一项所述的药物组合物，其中所述糖浓度为20mg/mL至100mg/mL或40mg/mL至80mg/mL。在一些实施方案中，所述糖浓度为20mg/mL、30mg/mL、40mg/mL、50mg/mL、60mg/mL、70mg/mL、80mg/mL、90mg/mL或100mg/mL。在一些实施方案中，所述糖浓度为40mg/mL。
在一些实施方案中，如上任一项所述的药物组合物，其中所述抗体药物偶联物浓度为以蛋白(即抗体)浓度计1mg/mL至100mg/mL或以蛋白浓度计10mg/mL至30mg/mL。在一些实施方案中，所述抗体药物偶联物浓度为以蛋白浓度计5mg/mL、10mg/mL、20mg/mL、30mg/mL、40mg/mL、50mg/mL、60mg/mL、70mg/mL、80mg/mL、90mg/mL或100mg/mL。在一些实施方案中，所述体药物偶联物浓度为以蛋白浓度计20mg/mL。
在一些实施方案中，如上任一项所述的药物组合物，其中所述缓冲剂的浓度为5mM至50mM或10mM至30mM。在一些实施方案中，所述缓冲剂的浓度为5mM、10mM、15mM、20mM、25mM、30mM、35mM、40mM、45mM或50mM。在一些实施方案中，所述缓冲剂的浓度为30mM。
在一些实施方案中，如上任一项所述的药物组合物，其中所述药物组合物的pH为5.0-6.5或5.0-5.5。在一些实施方案中，所述药物组合物的pH为5.0、5.1、5.2、5.3、5.4、5.5、5.6、5.7、5.8、5.9、6.0、6.1、6.2、6.3、6.4或6.5。在一些实施方案中，所述药物组合物的pH为5.0-5.4。在一些实施方案中，所述药物组合物的pH为5.0-5.3。
在一些实施方案中，如上任一项所述的药物组合物，其包含如下组分：
(a)以蛋白浓度计10mg/mL至30mg/mL的所述抗Claudin18.2抗体药物偶联物，(b)0.1mg/mL至0.2mg/mL的聚山梨酯，(c)40mg/mL至80mg/mL的糖，和(d)10mM至30mM的组氨酸盐缓冲剂；所述药物组合物的pH为约5.0-5.5。
在一些实施方案中，如上任一项所述的药物组合物，其包含如下组分：
(a)以蛋白浓度计10mg/mL至30mg/mL的所述抗Claudin18.2抗体药物偶联物，(b)0.1mg/mL至0.2mg/mL的聚山梨酯80，(c)40mg/mL至80mg/mL的蔗糖，和(d)10mM至30mM的组氨酸盐缓冲剂；所述药物组合物的pH为约5.0-5.5。
在一些实施方案中，如上任一项所述的药物组合物，其包含如下组分：
(a)以蛋白浓度计20mg/mL的所述抗Claudin18.2抗体药物偶联物，(b)0.2mg/mL的聚山梨酯80，(c)40mg/mL的蔗糖，和(d)30mM组氨酸-醋酸盐缓冲剂，所述药物组合物的pH为5.0-5.3。
在一些实施方案中，如上任一项所述的药物组合物，所述抗Claudin18.2抗体药物偶联物具有如下式所示的结构：
Figure PCTCN2021122031-appb-000009
其中：
n为2至8，n是小数或整数；
Pc为抗Claudin18.2抗体，其包含SEQ ID NO：49所示的重链，和SEQ ID NO：47所示的轻链；所述药物组合物含有以蛋白浓度计20mg/mL的所述抗体药物偶联物；
所述药物组合物还包含以下组分：
0.2mg/mL的聚山梨酯80、40mg/mL的蔗糖和30mM组氨酸-醋酸盐缓冲剂，所述药物组合物的pH为5.0-5.3。
在一些实施方案中，如上任一项所述的药物组合物，所述药物组合物是液体制剂。在一些实施方案中，所述液体制剂的溶剂是水。
本披露还提供一种含抗体药物偶联物的冻干制剂，其特征在于所述制剂复溶后可形成如上任一项所述的药物组合物。
本披露还提供一种的冻干制剂，其为如上任一项所述的药物组合物的冻干形式。
本披露还提供一种制备含抗体药物偶联物的冻干制剂的方法，其中包括将如上任一项所述的药物组合物进行冷冻干燥的步骤。
本披露还提供一种含抗体药物偶联物的冻干制剂，所述制剂通过将如上任一项所述的药物组合物经冷冻干燥获得。
在一些实施方案中，如上任一项所述冷冻干燥依次包括预冻、一次干燥和二次干燥的步骤。
在一些实施方案中，冻干程序如下所示：预冻，温度为5℃；预冻，温度为-45℃；一次干燥，温度为-20℃，真空度为20Pa；二次干燥的的温度为25℃，真空度为1Pa。在一些实施方案中，冻干程序如下所示：预冻，温度为5℃，时间10min；预冻，温度为-45℃，时间50min；一次干燥，温度为-20℃，真空度为20Pa，时间120min；二次干燥的的温度为25℃，真空度为1Pa，时间60min。
在一些实施方案中，冻干制剂于2-8℃稳定至少3个月，至少6个月，至少12个月，至少18个月或至少24个月。在一些实施方案中，该冻干制剂于40℃稳定至少7天，至少14天或至少28天。
本披露还提供一种的冻干制剂，其为如上任一项所述的冻干制剂的复溶形式。
本披露还提供一种含抗体药物偶联物的复溶溶液，其特征在于所述复溶溶液是通过将如上任一项所述的冻干制剂经复溶制备获得。
在一些实施方案中，如上所述的复溶溶液包含如下组分：
(a)以蛋白浓度计10mg/mL至30mg/mL的所述抗Claudin18.2抗体药物偶联物，(b)0.1mg/mL至0.2mg/mL的聚山梨酯，(c)40mg/mL至80mg/mL的糖，和(d)10mM至30mM的组氨酸盐缓冲剂；所述复溶溶液的pH为约5.0-5.5。
在一些实施方案中，如上所述的复溶溶液包含如下组分：
(a)以蛋白浓度计10mg/mL至30mg/mL的所述抗Claudin18.2抗体药物偶联物，(b)0.1mg/mL至0.2mg/mL的聚山梨酯80，(c)40mg/mL至80mg/mL的蔗糖，和(d)10mM至30mM的组氨酸盐缓冲剂；所述复溶溶液的pH为约5.0-5.5。
在一些实施方案中，如上所述的复溶溶液包含如下组分：
(a)以蛋白浓度计20mg/mL的所述抗Claudin18.2抗体药物偶联物，(b)0.2mg/mL的聚山梨酯80，(c)40mg/mL的蔗糖，和(d)30mM组氨酸-醋酸盐缓冲剂，所述复溶溶液的pH为5.0-5.3。
本披露还提供一种制品，其包括容器，该容器中装有如上任一项所述的药物组合物、如上任一项所述的冻干制剂或如上任一项所述的复溶溶液。
本披露还提供一种治疗肿瘤或癌症的方法，包括向受试者施用有效量的如上任一项所述的药物组合物、如上任一项的冻干制剂、如上任一项的复溶溶液、或如上任一项所述的制品。
在一些实施方案中，本披露还提供如上任一项所述的药物组合物、如上任一项的冻干制剂、如上任一项的复溶溶液或如上任一项所述的制品在制备治疗肿瘤 或癌症的药物中的用途。
在一些实施方案中，本披露还提供用作药物的如上任一项所述的药物组合物、如上任一项的冻干制剂、如上任一项的复溶溶液或如上任一项所述的制品。
在一些实施方案中，所述肿瘤或癌症优选为头和颈鳞状细胞癌、头和颈癌、脑癌、神经胶质瘤、多形性成胶质细胞瘤、神经母细胞瘤、中枢神经系统癌、神经内分泌肿瘤、咽喉癌、鼻咽癌、食管癌、甲状腺癌、恶性胸膜间皮瘤、肺癌、乳腺癌、肝癌、肝细胞瘤、肝细胞癌、肝胆癌、胰腺癌、胃癌、胃肠道癌、肠癌、结肠癌、结肠直肠癌、肾癌、透明细胞肾细胞癌、卵巢癌、子宫内膜癌、子宫颈癌、膀胱癌、前列腺癌、睾丸癌、皮肤癌、黑色素瘤、白血病、淋巴瘤、骨癌、软骨肉瘤、骨髓瘤、多发性骨髓瘤、骨髓异常增生综合征、库肯勃氏瘤、骨髓增生性肿瘤、鳞状细胞癌、尤因氏肉瘤、全身性轻链淀粉样变性和梅克尔细胞癌。
在一些实施方案中，所述淋巴瘤选自：何杰金淋巴瘤、非何杰金淋巴瘤、弥漫性大B-细胞淋巴瘤、滤泡性淋巴瘤、原发性纵隔大B-细胞淋巴瘤、套细胞淋巴瘤、小淋巴细胞性淋巴瘤、富含T-细胞/组织细胞的大B-细胞淋巴瘤和淋巴浆细胞性淋巴瘤。
在一些实施方案中，所述肺癌选自：非小细胞肺癌和小细胞肺癌。
在一些实施方案中，所述白血病选自：慢性髓细胞样白血病、急性髓细胞样白血病、淋巴细胞白血病、成淋巴细胞性白血病、急性成淋巴细胞性白血病、慢性淋巴细胞性白血病和髓样细胞白血病。
附图说明
图1：人源化抗体在细胞水平与人Claudin18.2的结合FACS检测结果。
图2：人源化抗体的NUGC4细胞内吞实验。
图3A至图3C：抗体在不同Claudin18.2表达程度的NUGC4细胞中ADCC效应检测。图3A为抗体在野生型NUGC4细胞(Claudin18.2低表达)中的ADCC效应检测；图3B为抗体在Claudin18.2中等表达NUGC4细胞中的ADCC效应检测；图3C为抗体在Claudin18.2高表达NUGC4细胞中的ADCC效应检测。
图4：本披露ADC-1的抑瘤实验结果。
图5：本披露ADC-2的抑瘤实验结果。
具体实施方式
术语
为了更容易理解本披露，以下具体定义了某些技术和科学术语。除非在本文中另有明确定义，本文使用的所有其它技术和科学术语都具有本披露所属领域的一般技术人员通常理解的含义。
“抗体药物偶联物(antibody drug conjugate，ADC)”是把抗体或者抗体片段通 过稳定的化学接头化合物与具有生物活性的细胞毒素或具有细胞杀伤活性的小分子药物相连，充分利用了抗体对肿瘤细胞特异或高表达抗原结合的特异性和细胞毒素的高效性，避免对正常细胞的毒副作用。与以往传统的化疗药物相比，抗体药物偶联物能精准地结合肿瘤细胞并降低将对正常细胞的影响。
“缓冲剂”指通过其酸-碱共轭组分的作用而耐受pH变化的缓冲剂。将pH控制在适当范围中的缓冲剂的例子包括醋酸盐、琥珀酸盐、葡萄糖酸盐、组氨酸盐、草酸盐、乳酸盐、磷酸盐、枸橼酸盐、酒石酸盐、延胡索酸盐、甘氨酰甘氨酸和其它有机酸缓冲剂。
“组氨酸盐缓冲剂”是包含组氨酸根离子的缓冲剂。组氨酸盐缓冲剂的实例包括组氨酸-盐酸盐，组氨酸-醋酸盐，组氨酸-磷酸盐，组氨酸-硫酸盐等缓冲剂；优选组氨酸-醋酸盐缓冲剂。组氨酸-醋酸盐缓冲剂是组氨酸与醋酸配制而成，组氨酸盐酸盐缓冲剂是组氨酸与盐酸配制而成。
“枸橼酸盐缓冲剂”是包括枸橼酸根离子的缓冲剂。枸橼酸盐缓冲剂的实例包括枸橼酸-枸橼酸钠、枸橼酸-枸橼酸钾、枸橼酸-枸橼酸钙、枸橼酸-枸橼酸镁等。优选的枸橼酸盐缓冲剂是枸橼酸-枸橼酸钠。
“琥珀酸盐缓冲剂”是包括琥珀酸根离子的缓冲剂。琥珀酸盐缓冲剂的实例包括琥珀酸-琥珀酸钠、琥珀酸-琥珀酸钾、琥珀酸-琥珀酸钙盐等。优选的琥珀酸盐缓冲剂是琥珀酸-琥珀酸钠。示例性的，所述的琥珀酸-琥珀酸钠可由琥铂酸与氢氧化钠配制而成，或由琥铂酸与琥珀酸钠配制而成。
“磷酸盐缓冲剂”是包括磷酸根离子的缓冲剂。磷酸盐缓冲剂的实例包括磷酸氢二钠-磷酸二氢钠、磷酸氢二钠-磷酸二氢钾、磷酸氢二钠-枸橼酸等。优选的磷酸盐缓冲剂是磷酸氢二钠-磷酸二氢钠。
“醋酸盐缓冲剂”是包括醋酸根离子的缓冲剂。醋酸盐缓冲剂的实例包括醋酸-醋酸钠、醋酸组氨酸盐、醋酸-醋酸钾、醋酸醋酸钙、醋酸-醋酸镁等。优选的醋酸盐缓冲剂是醋酸-醋酸钠。
“药物组合物”表示含有一种或多种本文所述抗体药物偶联物或其生理学上/可药用的盐或前体药物与其他化学组分的混合物，所述其他组分例如生理学/可药用的载体和赋形剂。药物组合物的目的是保持活性成分的稳定性，促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
本披露中，“药物组合物”和“制剂”并不互相排斥。
本披露中所述药物组合物的溶液形式，若无特殊说明，其中的溶剂均为水。
“冻干制剂”表示液体或溶液形式的药物组合物或液体或溶液制剂经真空冷冻干燥步骤之后获得的制剂或药物组合物。
尽管本披露提供了含量范围或含量值，但本领域一般技术人员理解，所述含量范围或含量值涵盖了所测定具体值的可接受误差范围。
本披露所述的药物组合物能够达到一种稳定的效果：其中的抗体药物偶联物 在贮藏后基本上保留其物理稳定性和/或化学稳定性和/或生物学活性的药物组合物，优选地，药物组合物在贮藏后基本上保留其物理和化学稳定性以及其生物学活性。贮藏期一般基于药物组合物的预定保存期来选择。目前有多种测量蛋白质稳定性的分析技术，可测量在选定温度贮藏选定时间段后的稳定性。
稳定的制剂是在下述情况下没有观察到显著变化的制剂：在冷藏温度(2-8℃)保存至少3个月、优选6个月、更优选1年，且甚至更优选地多达2年。另外，稳定的液体制剂包括这样的液体制剂：其在包括25℃的温度保存包括1个月、3个月、6个月在内的时段后表现出期望的特征。稳定性的典型的例子：通过SEC-HPLC测得，通常不超过约10％、优选不超过约5％的抗体单体发生聚集或降解。通过视觉分析，制剂是淡黄色近无色澄明液体或者无色，或澄清至稍微乳白色。所述制剂的浓度、pH和重量克分子渗透压浓度具有不超过±10％变化。通常观察到不超过约10％、优选不超过约5％的减少。通常形成不超过约10％、优选不超过约5％的聚集。
如果在目检颜色和/或澄清度后，或者通过UV光散射、尺寸排阻色谱法(SEC)和动态光散射(DLS)测得，抗体药物偶联物没有显示出显著的聚集增加、沉淀和/或变性，那么所述抗体药物偶联物在药物制剂中“保留它的物理稳定性”。蛋白构象的变化可以通过荧光光谱法(其确定蛋白三级结构)和通过FTIR光谱法(其确定蛋白二级结构)来评价。
如果抗体药物偶联物没有显示出显著的化学改变，那么所述抗体在药物制剂中“保留它的化学稳定性”。通过检测和定量化学上改变的形式的蛋白，可以评估化学稳定性。经常改变蛋白化学结构的降解过程包括水解或截短(通过诸如尺寸排阻色谱法和CE-SDS等方法来评价)、氧化(通过诸如与质谱法或MALDI/TOF/MS结合的肽谱法等方法来评价)、脱酰胺作用(通过诸如离子交换色谱法、毛细管等电聚焦、肽谱法、异天冬氨酸测量等方法来评价)和异构化(通过测量异天冬氨酸含量、肽谱法等来评价)。
如果抗体药物偶联物在给定时间的生物活性是在制备药物制剂时表现出的生物活性的预定范围内，那么所述抗体药物偶联物在药物制剂中“保留它的生物活性”。
本披露所用氨基酸三字母代码和单字母代码如J.biol.chem,243,p3558(1968)中所述。
本披露的术语“抗体”以最广义使用，其涵盖各种抗体结构，包括但不限于单克隆抗体，多克隆抗体，多特异性抗体(例如双特异性抗体)，全长抗体或其抗原结合片段(也称“抗原结合部分”)，只要它们展现出期望的抗原结合活性。全长抗体是包含由二硫键互相连接的至少两条重链和两条轻链的免疫球蛋白(Ig)。免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将免疫球蛋白分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和 IgE，其相应的重链分别为μ链、δ链、γ链、α链、和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4。轻链通过恒定区的不同分为κ链或λ链。五类Ig中每类Ig都可以有κ链或λ链。
全长抗体重链和轻链靠近N端的约110个氨基酸的序列变化很大，为可变区(缩写为Fv区)；靠近C端的其余氨基酸序列相对稳定，为恒定区。每条重链由重链可变区(缩写为VH)和重链恒定区(缩写为CH)组成。重链恒定区包含CH1、CH2和CH3三个结构域。每条轻链由轻链可变区(缩写为VL)和轻链恒定区(缩写为CL)组成。重链可变区和轻链可变区包括高变区(也称为互补性决定区，缩写为CDR或HVR)和序列相对保守的骨架区(也称框架区，缩写为FR)。每个VL和VH由从氨基末端排到羧基末端按以下顺序排列的3个CDR 4个FR组成：FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。轻链的3个CDR区指LCDR1、LCDR2、和LCDR3；重链的3个CDR区指HCDR1、HCDR2和HCDR3。
本披露中所述人抗体重链恒定区和人抗体轻链恒定区的“常规变体”是指现有技术已公开的来源于人的不改变抗体可变区结构和功能的重链恒定区或轻链恒定区的变体，示例性变体包括对重链恒定区进行定点改造和氨基酸替换的IgG1、IgG2、IgG3或IgG4重链恒定区变体，具体替换如现有技术已知的YTE突变，L234A和/或L235A突变，S228P突变，265A(例如D265A)和/或297A(例如N297A)，和/或获得knob-into-hole结构的突变(使得抗体重链具有knob-Fc和hole-Fc组合)，这些突变已被证实使得抗体具有新的性能，但不改变抗体可变区的功能。
术语“抗原结合片段”或“功能片段”或“抗原结合部分”是指保持特异性结合抗原的能力的完整抗体的一个或更多个片段。已显示可利用全长抗体的片段来进行抗体的抗原结合功能。示例性的，涵盖在术语“抗原结合片段”的结合片段的实例包括：(i)Fab片段，一种由VL、VH、CL和CH1结构域组成的单价片段；(ii)F(ab') 2片段，一种包含通过铰链区上的二硫桥连接的两个Fab片段的二价片段，(iii)由VH和CH1结构域组成的Fd片段；(iv)由抗体的单臂的VH和VL结构域组成的Fv片段；(v)dsFv，由VH和VL经链间二硫键形成的稳定的抗原结合片段；(vi)包含scFv、dsFv、Fab等片段的双抗体、双特异性抗体和多特异性抗体。此外，虽然Fv片段的两个结构域VL和VH由分开的基因编码，但可使用重组方法将这两个结构域通过能够使它们形成为单条蛋白质链的人工肽接头来接合，其中VL和VH配对形成单价分子，称为单链Fv(scFv)(参见，例如，Bird等人(1988)Science242:423-426；和Huston等人(1988)Proc.Natl.Acad.Sci USA85:5879-5883))。此类单链抗体也包括在术语抗体的“”抗原结合片段”中。使用本领域技术人员已知的常规技术获得此类抗体片段，并且以与对于完整抗体的方式相同的方式就功用性筛选片段。可通过重组DNA技术或通过酶促或化学断裂完整免疫球蛋白来产生抗原结合部分。
术语“氨基酸差异”或“氨基酸突变”是指相较于原蛋白质或多肽，变体蛋白质或多肽存在氨基酸的改变或突变，包括在原蛋白质或多肽的基础上发生1个、2个、3个或更多个氨基酸的插入、缺失或替换。
术语“抗体框架”或“FR区”，是指可变结构域VL或VH的一部分，其用作该可变结构域的抗原结合环(CDR)的支架。从本质上讲，其是不具有CDR的可变结构域。
术语“互补决定区”、“CDR”或“高变区”是指抗体的可变结构域内主要促成抗原结合的6个高变区之一。通常，每个重链可变区中存在三个CDR(HCDR1、HCDR2、HCDR3)，每个轻链可变区中存在三个CDR(LCDR1、LCDR2、LCDR3)。可以使用各种公知方案中的任何一种来确定CDR的氨基酸序列边界，包括“Kabat”编号规则(参见Kabat等(1991)，“Sequences of Proteins of Immunological Interest”，第5版，Public Health Service，National Institutes of Health，Bethesda，MD)、“Chothia”编号规则(参见Al-Lazikani等人，(1997)JMB 273：927-948)和ImMunoGenTics(IMGT)编号规则(Lefranc M.P.，Immunologist，7，132-136(1999)；Lefranc，M.P.等，Dev.Comp.Immunol.，27，55-77(2003)等。例如，对于经典格式，遵循Kabat规则，所述重链可变域(VH)中的CDR氨基酸残基编号为31-35(HCDR1)、50-65(HCDR2)和95-102(HCDR3)；轻链可变域(VL)中的CDR氨基酸残基编号为24-34(LCDR1)、50-56(LCDR2)和89-97(LCDR3)。遵循Chothia规则，VH中的CDR氨基酸编号为26-32(HCDR1)、52-56(HCDR2)和95-102(HCDR3)；并且VL中的氨基酸残基编号为26-32(LCDR1)、50-52(LCDR2)和91-96(LCDR3)。通过组合Kabat和Chothia两者的CDR定义，CDR由人VH中的氨基酸残基26-35(HCDR1)、50-65(HCDR2)和95-102(HCDR3)和人VL中的氨基酸残基24-34(LCDR1)、50-56(LCDR2)和89-97(LCDR3)构成。遵循IMGT规则，VH中的CDR氨基酸残基编号大致为26-35(CDR1)、51-57(CDR2)和93-102(CDR3)，VL中的CDR氨基酸残基编号大致为27-32(CDR1)、50-52(CDR2)和89-97(CDR3)。遵循IMGT规则，抗体的CDR区可以使用程序IMGT/DomainGap Align确定。遵循AbM规则，VH中的CDR氨基酸编号为26-32(HCDR1)、50-58(HCDR2)和95-102(HCDR3)；并且VL中的氨基酸残基编号为24-34(LCDR1)、50-56(LCDR2)和89-97(LCDR3)。本披露的抗体的重链可变区和轻链可变区及其CDR符合Kabat编号规则。
氨基酸序列“同一性”是指：氨基酸序列比对时(必要时允许引入间隙以达成最大的序列同一性百分比)，第一序列中与第二序列中的相同氨基酸残基的百分比；其中保守性取代不视为序列同一性的一部分。为测定氨基酸序列同一性百分比，比对可以通过属于本领域技术的范围内的多种方式来实现，例如使用公开可得到的计算机软件，诸如BLAST、BLAST-2、ALIGN、ALIGN-2或Megalign(DNASTAR)软件。本领域技术人员可确定适用于测量比对的参数，包括在所比较的序列全长 上达成最大比对所需的任何算法。
本披露工程化的抗体或抗原结合片段可用常规方法制备和纯化。比如，编码重链和轻链的cDNA序列，可以克隆并重组至GS表达载体。重组的免疫球蛋白表达载体可以稳定地转染CHO细胞。作为一种更推荐的现有技术，哺乳动物类表达系统会导致抗体的糖基化，特别是在Fc区的高度保守N端位点。通过表达与抗原结合的抗体得到稳定的克隆。阳性的克隆在生物反应器的无血清培养基中扩大培养以生产抗体。分泌了抗体的培养液可以用常规技术纯化。比如，用含调整过的缓冲液的A或G Sepharose FF柱进行纯化。洗去非特异性结合的组分。再用pH梯度法洗脱结合的抗体，用SDS-PAGE检测抗体片段，收集。抗体可用常规方法进行过滤浓缩。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛、离子交换。得到的产物需立即冷冻，如-70℃，或者冻干。
“保守修饰”或“保守置换或取代”是指具有类似特征(例如电荷、侧链大小、疏水性/亲水性、主链构象和刚性等)的其它氨基酸置换蛋白中的氨基酸，使得可频繁进行改变而不改变蛋白的生物学活性。本领域技术人员知晓，一般而言，多肽的非必需区域中的单个氨基酸置换基本上不改变生物学活性(参见例如Watson等(1987)Molecular Biology of the Gene，The Benjamin/Cummings Pub.Co.，第224页，(第4版))。另外，结构或功能类似的氨基酸的置换不大可能破环生物学活性。示例性保守取代如下：
表a.氨基酸的保守取代
原始残基 保守取代
Ala(A) Gly；Ser
Arg(R) Lys；His
Asn(N) Gln；His；Asp
Asp(D) Glu；Asn
Cys(C) Ser；Ala；Val
Gln(Q) Asn；Glu
Glu(E) Asp；Gln
Gly(G) Ala
His(H) Asn；Gln
Ile(I) Leu；Val
Leu(L) Ile；Val
Lys(K) Arg；His
Met(M) Leu；Ile；Tyr
Phe(F) Tyr；Met；Leu
Pro(P) Ala
Ser(S) Thr
Thr(T) Ser
Trp(W) Tyr；Phe
Tyr(Y) Trp；Phe
Val(V) Ile；Leu
术语“裸抗体”，是指未与异源模块(例如细胞毒性模块)或放射性标记物缀合的抗体。本披露中，抗体药物偶联物的含量以蛋白浓度计，即以偶联物中蛋白(抗体部分)的重量/体积计。
术语“接头单元”或“接头”是指一端与抗体或其抗原结合片段连接而另一端与药物相连的化学结构片段或键，也可以连接其他接头后再与药物相连。本披露的优选方案表示为L和L 1至L 4，其中L 1端与抗体相连，L 4端与结构单元Y相连后与化合物或毒素相连。接头，包括延伸物、间隔物和氨基酸单元，可以通过本领域已知方法合成，诸如US2005-0238649A1中所记载的。接头可以是便于在细胞中释放药物的“可切割接头”。例如，可使用酸不稳定接头(例如腙)、蛋白酶敏感(例如肽酶敏感)接头、光不稳定接头、二甲基接头、或含二硫化物接头(Chari等，Cancer Research 52:127-131(1992)；美国专利No.5,208,020)。
术语“烷基”指饱和脂肪族烃基团，其为包含1至20个碳原子的直链或支链基团，优选含有1至12个碳原子的烷基，更优选含有1至10个碳原子的烷基，最优选含有1至6个碳原子的烷基。非限制性实例包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基、1,1-二甲基丙基、1,2-二甲基丙基、2,2-二甲基丙基、1-乙基丙基、2-甲基丁基、3-甲基丁基、正己基、1-乙基-2-甲基丙基、1,1,2-三甲基丙基、1,1-二甲基丁基、1,2-二甲基丁基、2,2-二甲基丁基、1,3-二甲基丁基、2-乙基丁基、2-甲基戊基、3-甲基戊基、4-甲基戊基、2,3-二甲基丁基、正庚基、2-甲基己基、3-甲基己基、4-甲基己基、5-甲基己基、2,3-二甲基戊基、2,4-二甲基戊基、2,2-二甲基戊基、3,3-二甲基戊基、2-乙基戊基、3-乙基戊基、正辛基、2,3-二甲基己基、2,4-二甲基己基、2,5-二甲基己基、2,2-二甲基己基、3,3-二甲基己基、4,4-二甲基己基、2-乙基己基、3-乙基己基、4-乙基己基、2-甲基-2-乙基戊基、2-甲基-3-乙基戊基、正壬基、2-甲基-2-乙基己基、2-甲基-3-乙基己基、2,2-二乙基戊基、正癸基、3,3-二乙基己基、2,2-二乙基己基，及其各种支链异构体等。更优选的是含有1至6个碳原子的低级烷基，非限制性的实施例包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基、1,1-二甲基丙基、1,2-二甲基丙基、2,2-二甲基丙基、1-乙基丙基、2-甲基丁基、3-甲基丁基、正己基、1-乙基-2-甲基丙基、1,1,2-三甲基丙基、1,1-二甲基丁基、1,2-二甲基丁基、2,2-二甲基丁基、1,3-二甲基丁基、2-乙基丁基、2-甲基戊基、3-甲基戊基、4-甲基戊基、2,3-二甲基丁基等。烷基可以是取代的或非取代的，当被取代时，取代基可以在任何可使用的连接点上被取代，所述取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基、氧代基。
术语“杂烷基”指含有一个或多个选自N、O或S的杂原子的烷基，其中烷基如上所定义。
术语“亚烷基”指饱和的直链或支链脂肪族烃基，其具有两个从母体烷的相同碳原子或两个不同的碳原子上除去两个氢原子所衍生的残基。亚烷基为包含1至20个碳原子的直链或支链基团，优选含有1至12个碳原子，更优选含有1至6个碳原子的亚烷基。亚烷基的非限制性实例包括但不限于亚甲基(-CH 2-)、1,1-亚乙基(-CH(CH 3)-)、1,2-亚乙基(-CH 2CH 2)-、1,1-亚丙基(-CH(CH 2CH 3)-)、1,2-亚丙基(-CH 2CH(CH 3)-)、1,3-亚丙基(-CH 2CH 2CH 2-)、1,4-亚丁基(-CH 2CH 2CH 2CH 2-)和1,5-亚丁基(-CH 2CH 2CH 2CH 2CH 2-)等。亚烷基可以是取代的或非取代的。当被取代时，取代基可以在任何可使用的连接点上被取代，所述取代基优选独立地任选选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基和氧代基中的一个或多个取代基所取代。
术语“烷氧基”指-O-(烷基)和-O-(非取代的环烷基)，其中烷基或环烷基的定义如上所述。烷氧基的非限制性实例包括：甲氧基、乙氧基、丙氧基、丁氧基、环丙氧基、环丁氧基、环戊氧基、环己氧基。烷氧基可以是任选取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基。
术语“环烷基”指饱和或部分不饱和单环或多环环状烃取代基。环烷基环包含3至20个碳原子，优选包含3至12个碳原子，更优选包含3至10个碳原子，最优选包含3至7个碳原子。单环环烷基的非限制性实例包括环丙基、环丁基、环戊基、环戊烯基、环己基、环己烯基、环己二烯基、环庚基、环庚三烯基、环辛基等；多环环烷基包括螺环、稠环和桥环的环烷基。
术语“杂环基”指饱和或部分不饱和单环或多环环状烃取代基，其包含3至20个环原子，其中一个或多个环原子为选自氮、氧或S(O) m(其中m是整数0至2)的杂原子，但不包括-O-O-、-O-S-或-S-S-的环部分，其余环原子为碳。杂环基优选包含3至12个环原子，其中1至4个是杂原子；更优选环烷基环包含3至10个环原子。单环杂环基的非限制性实例包括吡咯烷基、哌啶基、哌嗪基、吗啉基、硫代吗啉基、高哌嗪基等。多环杂环基包括螺环、稠环和桥环的杂环基。
术语“螺杂环基”指单环之间共用一个原子(称螺原子)的5至20元多环杂环基团，其中一个或多个环原子为选自氮、氧或S(O) m(其中m是整数0至2)的杂原子，其余环原子为碳。其可以含有一个或多个双键，但没有一个环具有完全共轭的π电子系统。优选为6至14元，更优选为7至10元。根据环与环之间共用螺原子的数目将螺杂环基分为单螺杂环基、双螺杂环基或多螺杂环基，优选为单螺杂环基和双螺杂环基。更优选为4元/4元、4元/5元、4元/6元、5元/5元或5元/6元 单螺杂环基。螺杂环基的非限制性实例包括：
Figure PCTCN2021122031-appb-000010
术语“稠杂环基”指5至20元多环杂环系统，其中的每个环与系统中的其他环共享一对毗邻的原子。在稠杂环基中，一个或多个环可以含有一个或多个双键，但没有一个环具有完全共轭的π电子系统，其中一个或多个环原子为选自氮、氧或S(O) m(其中m是整数0至2)的杂原子，其余环原子为碳。稠杂环基优选为6至14元，更优选为7至10元。根据组成环的数目，稠杂环基可以分为双环、三环、四环或多环；优选为双环或三环；更优选为5元/5元、或5元/6元双环稠杂环基。稠杂环基的非限制性实例包括：
Figure PCTCN2021122031-appb-000011
术语“桥杂环基”指5至14元的多环杂环基团，其中任意两个环共用两个不直接连接的原子。桥杂环基可以含有一个或多个双键，但没有一个环具有完全共轭的π电子系统，其中一个或多个环原子为选自氮、氧或S(O) m(其中m是整数0至2)的杂原子，其余环原子为碳。桥杂环基优选为6至14元，更优选为7至10元。根据组成环的数目，桥杂环基可以分为双环、三环、四环或多环；优选为双环、三环或四环；更优选为双环或三环。桥杂环基的非限制性实例包括：
Figure PCTCN2021122031-appb-000012
所述杂环基环可以稠合于芳基、杂芳基或环烷基环上，其中与母体结构连接在一起的环为杂环基，其非限制性实例包括：
Figure PCTCN2021122031-appb-000013
等。
杂环基可以是任选取代的或非取代的。当被取代时，取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基、氧代基。
术语“芳基”指具有共轭的π电子体系的6至14元全碳单环或稠合多环(也就是共享毗邻碳原子对的环)基团。芳基优选为6至10元，例如苯基和萘基，优选苯基。所述芳基环可以稠合于杂芳基、杂环基或环烷基环上，其中与母体结构连接在一起的环为芳基环，其非限制性实例包括：
Figure PCTCN2021122031-appb-000014
芳基可以是取代的或非取代的。当被取代时，取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基。
术语“杂芳基”指包含1至4个杂原子、5至14个环原子的杂芳族体系，其中杂原子选自氧、硫和氮。杂芳基优选为5至10元，更优选为5元或6元，例如呋喃基、噻吩基、吡啶基、吡咯基、N-烷基吡咯基、嘧啶基、吡嗪基、咪唑基、四唑基等。所述杂芳基环可以稠合于芳基、杂环基或环烷基环上，其中与母体结构连接在一起的环为杂芳基环，其非限制性实例包括：
Figure PCTCN2021122031-appb-000015
杂芳基可以是任选取代的或非取代的。当被取代时，取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基。
术语“氨基保护基”是这样一种基团：当分子其它部位进行反应时，为了使氨基保持不变，用易于脱去的基团对氨基进行保护。非限制性的实施例包含9-芴甲氧羰基、叔丁氧羰基、乙酰基、苄基、烯丙基和对甲氧苄基等。这些基团可任选地被选自卤素、烷氧基或硝基中的1-3个取代基所取代。所述氨基保护基优选为9-芴甲氧羰基。
术语“环烷基烷基”指烷基上的氢被一个或多个环烷基取代，优选被一个环烷基取代，其中烷基如上所定义，其中环烷基如上所定义。
术语“卤代烷基”指烷基上的氢被一个或多个卤素取代，其中烷基如上所定义。
术语“氘代烷基”指烷基上的氢被一个或多个氘原子取代，其中烷基如上所定义。
术语“羟基”指-OH基团。
术语“卤素”指氟、氯、溴或碘。
术语“氨基”指-NH 2。
术语“硝基”指-NO 2。
“任选”或“任选地”意味着随后所描述地事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生的场合。例如，“任选包含1-3个抗体重链可变区”意味着特定序列的抗体重链可变区可以但不必须存在。
“取代的”指基团中的一个或多个氢原子，优选为最多5个，更优选为1至3个氢原子彼此独立地被相应数目的取代基取代。不言而喻，取代基仅处在它们的可能的化学位置，本领域技术人员能够在不付出过多努力的情况下确定(通过实验或理论)可能或不可能的取代。例如，具有游离氢的氨基或羟基与具有不饱和(如烯属)键的碳原子结合时可能是不稳定的。
术语“载药量”是指ADC分子中每个抗体或其抗原结合片段上加载的细胞毒性药物平均数量，也可以表示为药物量和抗体量的比值，药物载量的范围可以是每个抗体或其抗原结合片段(Pc)连接0-12个，优选1-10个，更优选2-8个，最优选3.5-4.5个细胞毒性药物(D)。在本披露的实施方式中，载药量表示为n，示例性的n可以为1，2，3，4，5，6，7，8，9，或10中的一个或多个经计算所得的均值。可用常规方法如UV/可见光光谱法、质谱、ELISA试验和HPLC特征鉴定偶联反应后每个ADC分子的药物平均数量。
本披露的一个实施方式中，细胞毒性药物通过接头单元偶联在抗体或其抗原结合片段的N端氨基、赖氨酸残基的ε-氨基和/或巯基上。一般地，偶联反应中能与抗体偶联的药物分子数将小于理论上的最大值。
可以用以下非限制性方法控制细胞毒性药物的载量，包括：
(1)控制连接试剂和单抗的摩尔比，
(2)控制反应时间和温度，
(3)选择不同的反应试剂。
常规的药物组合物的制备见中国药典。
术语“载体”用于本披露的药物组合物，是指能改变药物进入人体的方式和在体内的分布、控制药物的释放速度并将药物输送到靶向器官的体系。药物载体释放和靶向系统能够减少药物降解及损失，降低副作用，提高生物利用度。如可作为载体的高分子表面活性剂由于其独特的两亲性结构，可以进行自组装，形成各种形式的聚集体，优选的实例如胶束、微乳液、凝胶、液晶、囊泡等。这些聚集体具有包载药物分子的能力，同时又对膜有良好的渗透性，可以作为优良的药物载体。
“施用”、“给予”和“处理”当应用于动物、人、实验受试者、细胞、组织、器官或生物流体时，是指外源性药物、治疗剂、诊断剂或组合物与动物、人、受试者、细胞、组织、器官或生物流体的接触。“施用”、“给予”和“处理”可以指例如治疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触，以及试剂与流体的接触，其中所述流体与细胞接触。“施用”、“给予”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞体外和离体处理例如细胞。“处理”当应用于人、兽医学或研究受试者时，是指治疗处理、预防或预防性措施，研究和诊断应用。
“治疗”意指给予患者内用或外用治疗剂，例如包含本披露的任一种结合化合物的组合物，所述患者具有一种或多种疾病症状，而已知所述治疗剂对这些症状具有治疗作用。通常，在受治疗患者或群体中以有效缓解一种或多种疾病症状的量给予治疗剂，以诱导这类症状退化或抑制这类症状发展到任何临床可测量的程度。有效缓解任何具体疾病症状的治疗剂的量(也称作“治疗有效量”)可根据多种因素变化，例如患者的疾病状态、年龄和体重，以及药物在患者产生需要疗效的能力。通过医生或其它专业卫生保健人士通常用于评价该症状的严重性或进展状况的任何临床检测方法，可评价疾病症状是否已被减轻。尽管本披露的实施方案(例如治疗方法或制品)在缓解每个目标疾病症状方面可能无效，但是根据本领域已知的任何统计学检验方法如Student t检验、卡方检验、依据Mann和Whitney的U检验、Kruskal-Wallis检验(H检验)、Jonckheere-Terpstra检验和Wilcoxon检验确定，其在统计学显著数目的患者中应当减轻目标疾病症状。
“有效量”包含足以改善或预防医学疾病的症状或病症的量。有效量还意指足以允许或促进诊断的量。用于特定患者或兽医学受试者的有效量可依据以下因素而变化：例如，待治疗的病症、患者的总体健康情况、给药的方法途径和剂量以及副作用严重性。有效量可以是避免显著副作用或毒性作用的最大剂量或给药方案。
“置换”是指溶解抗体或ADC的溶剂体系的置换，例如，使用稳定制剂的缓冲体系经物理操作方式将含抗体或ADC的高盐或高渗溶剂体系置换，从而使抗体蛋白存在于稳定制剂中。所称物理操作方式包括但不限于超滤、透析或离心后复溶。
实施例
以下结合实施例进一步描述本披露，但这些实施例并非是对本披露范围的限制。本披露实施例中未注明具体条件的实验方法，通常按照常规条件，如参照冷泉港实验室出版的《抗体技术实验手册》，《分子克隆手册》；或按照原料或商品制造厂商所建议的条件。未注明具体来源的试剂，为市场购买的常规试剂。
一、抗体药物偶联物
抗Claudin18.2抗体的制备
实施例1-1：构建高表达Claudin18.2的细胞株
用Lipofectamine 3000转染试剂，将pCDH-hClaudin18.2慢病毒表达载体质粒与pVSV-G或pCMV-dR8.91慢病毒系统包装载体转染至病毒包装细胞293T中；收集含有病毒的培养基上清，过滤并进行超高速离心；使用浓缩后的病毒感染人胃印戒细胞癌细胞株NUGC4，经puromycin筛选两至三周，再进行FACS单细胞分选。
根据肿瘤IHC评分来区分Claudin18.2表达程度。与肿瘤IHC评分为3分的肿瘤Claudin18.2表达水平相当的细胞为高表达细胞，与肿瘤IHC评分为2分的肿瘤Claudin18.2表平水平相当的细胞为中等表达细胞。
根据通过FACS检测慢病毒感染的NUGC4细胞表面的Claudin18.2表达，挑选出Claudin18.2表达量高的NUGC4/hClaudin18.2单克隆细胞株。同时通过FACS检测野生型NUGC4细胞表面的Claudin18.2表达，挑选出Claudin18.2表达量中等的NUGC4克隆细胞株，野生型NUGC4为Claudin18.2低表达量细胞。
将挑选出的单克隆细胞株扩大培养，冻存备库以便后续实验。
Claudin18.2序列Genbank:NP_001002026(SEQ ID NO：1)：
Figure PCTCN2021122031-appb-000016
Claudin18.2 DNA序列(SEQ ID NO：2)：
Figure PCTCN2021122031-appb-000017
Figure PCTCN2021122031-appb-000018
Figure PCTCN2021122031-appb-000019
实施例1-2：抗人claudin18.2单克隆抗体产生
1免疫
通过免疫小鼠产生抗人Claudin18.2单克隆抗体。
实验用SJL白小鼠，雌性，6-8周龄(北京维通利华实验动物技术有限公司Charles River Laboratory Animal Technology Co.,Ltd.，Beijing；动物生产许可证号：SCXK(京)2012-0001)。饲养环境：SPF级。小鼠购进后，实验室环境饲养1周，12/12小时光/暗周期调节，温度20-25℃；湿度40-60％。将已适应环境的小鼠按以下方案免疫。免疫抗原为huClaudin18.2-HEK293细胞(转染人Claudin18.2质粒的HEK-293稳转细胞株)。
免疫方案：首次免疫细胞前，用
Figure PCTCN2021122031-appb-000020
Gold Adjuvant(Sigma Cat No.T2684)0.1ml/只注射小鼠腹膜内(IP)；半小时后每只小鼠腹膜内(IP)注射0.1ml生理盐水稀释至1×10 8/ml浓度的细胞液。细胞吹散均匀后进行接种，时间为第0、14、28、42、56天。于第21，35，49，63天取血，用ELISA方法确定小鼠血清中的抗体滴度。在第4-5次免疫以后，选择血清中抗体滴度高并且滴度趋于平台的小鼠进行脾细胞融合。在进行脾细胞融合前3天加强免疫，腹膜内(IP)注射1×10 7细胞。
2脾细胞融合
采用PEG介导的融合步骤将脾淋巴细胞与骨髓瘤细胞Sp2/0细胞(
Figure PCTCN2021122031-appb-000021
CRL-8287 TM)进行融合得到杂交瘤细胞。杂交瘤细胞以0.5-1×10 6/ml的密度用完全培养基(含20％FBS、1×HAT、1×OPI的IMDM培养基)重悬，100μl/孔种于96孔板中，37℃，5％CO 2孵育3-4天后，补充HAT完全培养基100μl/孔，继续培养3-4天至形成克隆。去除上清，加入200μl/孔的HT完全培养基(含20％FBS、1×HT和1×OPI的IMDM培养基)，37℃，5％CO 2培养3天后进行ELISA检测。
3杂交瘤细胞筛选
根据杂交瘤细胞生长密度，用结合ELISA方法检测培养上清。选择与huClaudin18.2-HEK293细胞结合能力强，同时与HEK293细胞没有结合的细胞，及时进行扩增冻存；经过二到三次亚克隆直至获得单细胞克隆。
每次亚克隆细胞均需进行细胞结合实验。通过以上实验筛选得到杂交瘤克隆，用无血清细胞培养法进一步制备抗体，按纯化实例纯化抗体，供在检测例中使用。
实施例1-3：鼠源抗体的人源化
挑选出体外活性高的单克隆杂交瘤细胞株mAb1901，mAb1902；克隆其中的单克隆抗体序列，再进行人源化、重组表达和活性评价。
从杂交瘤中克隆序列的过程如下。收集对数生长期杂交瘤细胞，用Trizol(Invitrogen，15596-018)提取RNA(按照试剂盒说明书步骤)并进行反转录(PrimeScript TM Reverse Transcriptase，Takara，cat#2680A)。将反转录得到的cDNA采用mouse Ig-Primer Set(Novagen，TB326 Rev.B 0503)进行PCR扩增，送测序公司测序。得到的DNA序列对应的氨基酸序列SEQ ID NO：3-6所示：
mAb1901鼠源重链可变区(SEQ ID NO：3)
Figure PCTCN2021122031-appb-000022
mAb1901鼠源轻链可变区(SEQ ID NO：4)
Figure PCTCN2021122031-appb-000023
mAb1902鼠源重链可变区(SEQ ID NO：5)
Figure PCTCN2021122031-appb-000024
mAb1902鼠源轻链可变区(SEQ ID NO：6)
Figure PCTCN2021122031-appb-000025
上述鼠源重链可变区和轻链可变区分别与下述人IgG1抗体的重链恒定区和人源κ轻链恒定区连接形成嵌合抗体ch1901和ch1902。
各抗体的恒定区选自以下序列：
人IgG1抗体的重链恒定区：(SEQ ID NO：7)
Figure PCTCN2021122031-appb-000026
人源κ轻链恒定区：(SEQ ID NO：8)
Figure PCTCN2021122031-appb-000027
如本领域许多文献公示的方法，对鼠源单克隆抗体进行人源化。简言之，使用人恒定结构域替代亲本(鼠源抗体)恒定结构域，根据鼠源抗体和人抗体的同源性选择人种系抗体序列，进行CDR移植。本发明选择活性好的候选分子进行人源化，结果如下。
1、鼠源抗体的CDR区
表1中VH/VL CDR的氨基酸残基由Kabat编号系统确定并注释。
鼠源抗体的CDR序列如表1所述：
表1.鼠源抗体的CDR序列
抗体 mAb1901
HCDR1 DYGIH(SEQ ID NO：9)
HCDR2 YISRGSSTIYYADTVKG(SEQ ID NO：10)
HCDR3 GGYDTRNAMDY(SEQ ID NO：11)
LCDR1 KSSQSLLNSGNQKNYLA(SEQ ID NO：12)
LCDR2 GASTRAS(SEQ ID NO：13)
LCDR3 QNDLYYPLT(SEQ ID NO：14)
抗体 mAb1902
HCDR1 SYWMH(SEQ ID NO：15)
HCDR2 MIHPNSGSTNYNEKFKGR(SEQ ID NO：16)
HCDR3 LKTGNSFDY(SEQ ID NO：17)
LCDR1 KSSQSLLNSGNQKNYLT(SEQ ID NO：18)
LCDR2 WASTRES(SEQ ID NO：19)
LCDR3 QNAYTYPFT(SEQ ID NO：20)
2、选择人种系FR区序列
在所获得的鼠源抗体VH/VLCDR典型结构的基础上，将重、轻链可变区序列与抗体Germline数据库比较，获得同源性高的人种系模板。其中人类种系轻链框架区来自人κ轻链基因。
2.1 mAb1901的人源化改造和回复突变设计
选择适当的人抗体种系，对mAb1901鼠源抗体进行人源化改造，将鼠源抗体mAb1901的CDR区移植到选择的人源化模板上，替换人源化可变区，再与IgG恒定区重组，形成完整抗体。同时，对人源化抗体的V区中FR区进行回复突变，示例性回复突变方式及组合如下：
表2.mAb1901人源化抗体及回复突变 *
Figure PCTCN2021122031-appb-000028
*表格中所有氨基酸位置编号为Kabat编号规则的编号，重链可变区的N82T中，82为Kabat规则的第82A位。
表3.mAb1901人源化抗体轻链可变区和重链可变区序列
Figure PCTCN2021122031-appb-000029
上表中对应重链可变区与SEQ ID NO：7所示的人IgG1重链恒定区连接形成全长抗体的重链，轻链可变区与SEQ ID NO：8所示的人κ轻链恒定区连接形成全长抗体的轻链。在其他实施方案中，重链可变区和轻链可变区也可与其他重链恒定区和轻链恒定区分别连接形成全长抗体。
2.2 mAb1902的人源化改造和回复突变设计
选择适当的人抗体种系，对mAb1902鼠源抗体进行人源化改造，将鼠源抗体mAb1902的CDR区移植到选择的人源化模板上，替换人源化可变区，再与IgG恒定区重组，形成完整抗体。同时，对人源化抗体的V区中FR区进行回复突变，示例性回复突变方式及组合如下：
表4.mAb1902人源化抗体及其回复突变设计 *
Figure PCTCN2021122031-appb-000030
*表格中所有氨基酸位置编号为Kabat编号规则的编号。
表5.mAb1902人源化抗体轻链可变区和重链可变区序列
Figure PCTCN2021122031-appb-000031
上表中对应重链可变区与SEQ ID NO：7所示的人IgG1重链恒定区连接形成全长抗体的重链，轻链可变区与SEQ ID NO：8所示的人κ轻链恒定区连接形成全长抗体的轻链。
嵌合抗体ch1901
ch1901重链：(SEQ ID NO：35)
Figure PCTCN2021122031-appb-000032
ch1901轻链：(SEQ ID NO：36)
Figure PCTCN2021122031-appb-000033
Figure PCTCN2021122031-appb-000034
嵌合抗体ch1902
ch1902重链：(SEQ ID NO：37)
Figure PCTCN2021122031-appb-000035
ch1902轻链：(SEQ ID NO：38)
Figure PCTCN2021122031-appb-000036
表6.mAb1901人源化抗体
轻重链 H1 H2 H3 H4
L1 h1901-1 h1901-2 h1901-3 h1901-4
L2 h1901-5 h1901-6 h1901-7 h1901-8
L3 h1901-9 h1901-10 h1901-11 h1901-12
全长抗体轻重链序列如下所示：
表7.mAb1901人源化抗体轻链和重链序列
Figure PCTCN2021122031-appb-000037
Figure PCTCN2021122031-appb-000038
Figure PCTCN2021122031-appb-000039
表8.mAb1902人源化抗体
轻重链 H11 H12 H13 H14
L11 h1902-1 h1902-2 h1902-3 h1902-4
L12 h1902-5 h1902-6 h1902-7 h1902-8
L13 h1902-9 h1902-10 h1902-11 h1902-12
全长抗体轻重链序列如下所示：
表9.mAb1901人源化抗体轻链和重链序列
Figure PCTCN2021122031-appb-000040
Figure PCTCN2021122031-appb-000041
本披露阳性对照抗体为IMAB-362(来自WO2016166122)。
重链(SEQ ID NO：53)
Figure PCTCN2021122031-appb-000042
轻链(SEQ ID NO：54)
Figure PCTCN2021122031-appb-000043
用常规基因克隆、重组表达的方法分别克隆、表达、纯化上述抗体。
抗Claudin18.2 ADC偶联物的制备
药物
本披露中抗Claudin18.2 ADC偶联物的药物部分可以是任意适宜的药物。特别适宜的药物描述于例如PCT公开号WO2020063676A1(通过援引完整收入本文)。本披露的化合物9-A是N-((2R,10S)-10-苄基-2-环丙基-1-(((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基)-1,6,9,12,15-五氧代-3-氧杂-5,8,11,14-四氮杂十六-16-基)-6-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)己酰胺，其具有如下的结构：
Figure PCTCN2021122031-appb-000044
ADC原液药物载量分析
1.UV-HPLC方法
将装有琥珀酸钠缓冲液的比色皿分别置于参比吸收池和样品测定吸收池中后，扣除溶剂空白后，再将装有待测样品溶液的比色皿置于样品测定吸收池中，测定280nm和370nm处吸光度。
结果计算：采用紫外分光光度法(使用仪器：Thermo nanodrop2000紫外分光光度计)测定ADC原液载量，其原理是在某波长下ADC原液的总吸光值等于药物与单克隆抗体在该波长下吸光值的加和，即：
(1)A 280nm＝ε mab-280bC mab+ε Drug-280bC Drug
ε Drug-280：药物在280nm平均摩尔消光系数5100；
C Drug：药物的浓度；
ε mab-280：单抗原液在280nm平均摩尔消光系数214600；
C mab：单抗原液的浓度；
b：光程长度为1cm。
同理可以得到样品在370nm下的总吸光值方程：
(2)A 370nm＝ε mab-370bC mab+ε Drug-370bC Drug
ε Drug-370：药物在370nm平均摩尔消光系数19000；
C Drug：药物的浓度；
ε mab-370：单抗原液在370nm消光系数为0；
C mab：单抗原液的浓度；
b：光程长度为1cm。
由(1)和(2)两种方程结合单克隆抗体和药物在两个检测波长下的消光系数和浓度数据可以计算出药物的载量。
药物载量＝C Drug/C mab。
2.RP-HPLC方法
裸抗体和待测ADC样品(浓度为1mg/ml)，加入4μl DDT(sigma)还原，37℃水浴1小时，结束后取出到内插管中。使用高效液相色谱仪Agilent 1200进行检测，色谱柱选用Agilent PLRP-S 1000A 8μm 4.6*250mm，柱温：80℃；DAD检测器波长280nm；流速：1mL/min；进样量为：40μL；之后通过样品与裸抗体的谱图比对，区分出轻重链的位置，然后对检测样品的谱图进行积分，计算出DAR值。
溶液配制：
1)0.25M DTT溶液：
配制示例：取DTT 5.78mg，加入150μl纯化水充分溶解后，配得0.25M DTT溶液，-20℃保存。
2)流动相A(0.1％TFA水溶液)：
配制示例：量筒量取1000ml纯化水，加入1mL TFA(sigma)，充分混匀后使用，2-8℃保存14天。
3)流动相B(0.1％TFA乙腈溶液)：
配制示例：量筒量取1000ml乙腈，加入1mL TFA，充分混匀后使用，2-8℃保存14天。
数据分析：
通过样品与裸抗体的谱图比对，区分出轻链与重链的位置，然后对检测样品的谱图进行积分，计算出DAR值。
计算公式如下：
表10.ADC轻链与重链载药标记表
名称 连接药物数
LC 0
LC+1 2
HC 0
HC+1 2
HC+2 4
HC+3 6
LC峰面积总和＝LC峰面积+LC+1峰面积；
HC峰面积总和＝HC峰面积+HC+1峰面积+HC+2峰面积+HC+3峰面积；
LC DAR＝Σ(连接药物数*峰面积百分比)/LC峰面积总和；
HC DAR＝Σ(连接药物数*峰面积百分比)/HC峰面积总和；
DAR＝LC DAR+HC DAR。
实施例1-4：ADC-1/ADC-2
在37℃条件下，向含抗体h1902-5的PBS缓冲液(pH＝6.5、0.05M的PBS缓冲液；10.0mg/mL，320.0mL，21.62μmol)加入三(2-羧乙基)膦(TCEP)的水溶液(10mM，11.03mL，110.3μmol)，置于水浴振荡器，于37℃下振荡反应3小时，停止反应。
将反应液用水浴降温至25℃，再将化合物9-A(350mg，303mol)溶解于13.2ml乙腈和6.6ml DMSO中后加入到反应液中，置于水浴振荡器，于25℃下振荡反应3小时，停止反应。
反应液通过超滤膜纯化，除去小分子。纯化先后采用5L的50mM pH＝6.5PBS缓冲液(4％乙腈2％DMSO)和5L的10mM pH＝5.3琥珀酸缓冲液。随后在经纯化的溶液中加入蔗糖至60mg/mL、吐温-20至0.2mg/mL，制备得到ADC-1(10mM、pH＝5.3的琥珀酸缓冲液；10mg/mL，2.626g)，收率：81.81％。后制成20mg/瓶的冻干粉。
UV-HPLC计算平均值：n＝6.8。
使用上述方法，可以用抗体h1901-11代替h1902-5，和化合物9-A制备得到ADC-2，n＝7.1。
实施例1-5：ADC-3
在37℃条件下，向含抗体h1901-11的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，1mL，67.5nmol)加入配置好的TCEP水溶液(10mM，10.1μL，101nmol)，置于水浴振荡器，于37℃下振荡反应3小时，停止反应。将反应液用水浴降温至25℃。
将化合物9-A(0.58mg，540nmol)溶解于34μl DMSO中，加入到上述反应液中，置于水浴振荡器，于25℃下振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到ADC-3的PBS缓冲液(0.72mg/mL，11.2mL)，于4℃储存。RP-HPLC计算平均值：n＝2.51。
实施例1-6：ADC-4
在37℃条件下，向含抗体h1901-11的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，1mL，67.5nmol)加入配置好的TCEP水溶液(10mM，16.9μL，169nmol)，置于水浴振荡器，于37℃下振荡反应3小时，停止反 应。将反应液用水浴降温至25℃。
将化合物9-A(0.73mg，680nmol)溶解于43μl DMSO中，加入到上述反应液中，置于水浴振荡器，于25℃下振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到ADC-4的PBS缓冲液(0.62mg/mL，12.5mL)，于4℃储存。RP-HPLC计算平均值：n＝4.06。
实施例1-7：ADC-5
在37℃条件下，向抗体h1901-11的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，1mL，67.5nmol)加入配置好的TCEP水溶液(10mM，35.8μL，358nmol)，置于水浴振荡器，于37℃下振荡反应3小时，停止反应。将反应液用水浴降温至25℃。
将化合物9-A(1.09mg，1015nmol)溶解于64μl DMSO中，加入到上述反应液中，置于水浴振荡器，于25℃下振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到ADC-5的PBS缓冲液(0.54mg/mL，12.5mL)，于4℃储存。RP-HPLC计算平均值：n＝6.8。
实施例1-8：ADC-6
在37℃条件下，向抗体h1902-5的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，1.08mL，72.9nmol)加入配置好的TCEP水溶液(10mM，10.9μL，109nmol)，置于水浴振荡器，于37℃下振荡反应3小时，停止反应。将反应液用水浴降温至25℃。
将化合物9-A(0.63mg，587nmol)溶解于40μl DMSO中，加入到上述反应液中，置于水浴振荡器，于25℃下振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到ADC-6的PBS缓冲液(0.7mg/mL，13.0mL)，于4℃储存。RP-HPLC计算平均值：n＝2.69。
实施例1-9：ADC-7
在37℃条件下，向抗体h1902-5的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，1.08mL，72.9nmol)加入配置好的TCEP水溶液(10mM，18.3μL，183nmol)，置于水浴振荡器，于37℃下振荡反应3小时，停止反应。将反应液用水浴降温至25℃。
将化合物9-A(0.79mg，736nmol)溶解于50μl DMSO中，加入到上述反应液中，置于水浴振荡器，于25℃下振荡反应3小时，停止反应。将反应液用Sephadex  G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到ADC-7的PBS缓冲液(0.6mg/mL，14.0mL)，于4℃储存。RP-HPLC计算平均值：n＝4.25。
实施例1-10：ADC-8
在37℃条件下，向抗体h1902-5的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，1.08mL，72.9nmol)加入配置好的TCEP水溶液(10mM，38.7μL，387nmol)，置于水浴振荡器，于37℃下振荡反应3小时，停止反应。将反应液用水浴降温至25℃。
将化合物9-A(1.18mg，1099nmol)溶解于70μl DMSO中，加入到上述反应液中，置于水浴振荡器，于25℃下振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到ADC-8的PBS缓冲液(0.56mg/mL，14.2mL)，于4℃储存。RP-HPLC计算平均值：n＝7.01。
实施例1-11：ADC-9
在12℃条件下，向含抗体h1902-5的组氨酸-醋酸-Tris/EDTA缓冲液(pH 7.2的10mM组氨酸-醋酸-Tris和2.5mM EDTA的缓冲液；20.6g/L，6.49L，0.91mmol)中加入配制好的TCEP组氨酸缓冲液(10mM组氨酸缓冲液；1.717mM，1.16L 1.99mmol)，置于恒温水浴锅中，于12℃下搅拌反应2小时，停止反应，得到中间体I溶液。
将化合物9-A(4.72g，4.39mmol)溶解于0.38L DMSO中，生成化合物9-A的DMSO溶液。向上述中间体I溶液中预加0.38L DMSO，再加入上述化合物9-A的DMSO溶液，置于恒温水浴锅中，于12℃下搅拌反应1小时，停止反应。
将上述反应液经Capto S Impact阳离子层析柱纯化，分别用9个柱体积的含有10％(v/v)DMSO的0.05M醋酸缓冲液(pH＝5.0)和6个柱体积0.05M醋酸缓冲液(pH＝5.0)洗涤，再用0.05M醋酸、0.30M氯化钠缓冲液(pH＝5.5)进行洗脱，去除反应液中游离毒素和残留溶剂。在22℃下，将阳离子洗脱液进行7倍体积等体积超滤(超滤膜包采用30KD的聚纤维素膜包)得到产物ADC-9。RP-HPLC计算平均值：n＝4.1。
生物学评价
测试例1：细胞Cell水平ELISA结合实验
基于细胞的ELISA实验被用来检测Claudin18.2抗体的结合特性。将稳转表达Claudin18.2的NUGC4细胞培养于96孔细胞板(Corning，3599)中，待生长至90％密度时加入4％多聚甲醛固定细胞1小时，用PBST缓冲液(pH 7.4PBS含0.05％ Tween-20)洗板3次后，加入用PBS稀释的5％脱脂牛奶(光明脱脂奶粉)封闭液200μl/孔，37℃孵育箱孵育2.5小时或4℃放置过夜(16-18小时)进行封闭。封闭结束后，弃去封闭液，并用PBST缓冲液洗板3次后，加入50μl/孔用样品稀释液(pH7.4PBS含1％脱脂乳)稀释的不同浓度待测抗体，放于37℃孵育箱孵育2小时。孵育结束后用PBST洗板5次，加入100μl/孔用样品稀释液稀释的HRP标记的羊抗人二抗(Jackson Immuno Research，109-035-003)，37℃孵育1小时。用PBST洗板6次后，加入50μl/孔TMB显色底物(KPL，52-00-03)，于室温孵育10-15min，加入50μl/孔1M H 2SO 4终止反应，用MD Versa Max TM酶标仪在450nm处读取吸收值，计算Claudin18.2抗体对Claudin18.2的结合EC50值。
表11.抗体的结合活性
抗体 IMAB362 ch1901 ch1902
Emax 1.175 1.399 1.272
EC50(nM) 0.108 0.098 0.074
表12.mAb1901人源化抗体结合活性
抗体 Emax EC50(nM)
IMAB362 1.115 0.086
h1901-2 1.039 0.076
h1901-3 1.1055 0.22
h1901-4 0.986 0.201
h1901-6 0.937 0.091
h1901-7 0.921 0.166
h1901-8 1.047 0.091
h1901-11 1.44 0.076
h1901-12 1.22 0.116
表13.mAb1902人源化抗体结合活性
抗体 Emax EC50(nM)
IMAB362 0.88 0.187
h1902-1 0.87 0.113
h1902-2 0.88 0.107
h1902-3 0.84 0.175
h1902-4 0.82 0.087
h1902-5 0.9 0.098
h1902-6 0.78 0.141
h1902-7 0.75 0.121
h1902-8 0.89 0.132
h1902-9 0.75 0.137
h1902-10 0.89 0.133
测试例2：抗体细胞水平结合实验
将稳转表达Claudin18.2的NUGC4细胞用FACS缓冲液(2％胎牛血清(Gibco， 10099141)pH7.4PBS(Sigma，P4417-100TAB))制备成1×10 6/ml的细胞悬液，100μl/孔加入96孔圆底板(Corning，3795)中。离心去除上清后加入50μl/孔用FACS缓冲液稀释的不同浓度待测Claudin18.2抗体，放于4℃冰箱中避光孵育1小时。以FACS缓冲液300g离心洗涤3次后，加入工作浓度的Alexa Fluor 488包被的抗人IgG(H+L)(invitrogen，A-11013)，放于4℃冰箱中避光孵育40分钟。以FACS缓冲液300g离心洗涤3次后，在BD FACS CantoII流式细胞仪上检测几何平均数荧光强度，计算Claudin18.2抗体对稳转表达Claudin18.2的NUGC4细胞的结合EC50值，结果见图1。
测试例3：抗体内吞实验
将预标记了DyLight 488 NHS Ester(thermofisher，46403)的待测Claudin18.2抗体，以5μg/ml终浓度加入1×10 6/ml稳转表达Claudin18.2的NUGC4细胞中，放于冰上避光孵育1小时，以预冷的FACS缓冲液(pH7.4PBS，2％胎牛血清)离心洗涤3次，去上清后加入预热的完全培养基，放入37℃ 5％CO 2细胞培养箱。分别在0、0.5、1、2、4小时后取出细胞，放置于冰上避光保存。待样品全部收集后，300g低温离心去除上清，加入洗脱缓冲液(pH1.7 0.05M甘氨酸，0.1M氯化钠)后，室温孵育7分钟，以FACS缓冲液300g离心洗涤1次，在BD FACS CantoII流式细胞仪上检测几何平均数荧光强度，计算Claudin18.2抗体对稳转表达Claudin18.2的NUGC4细胞的内吞效率。结果显示(见图2)，人源化抗体具有良好的细胞内吞效率。
测试例4：基于流式细胞技术测定抗体亲和力
实验当天收集HEK293/hClaudin18.2细胞于U底96孔板中，每孔1×10 5至2×10 5个细胞。加入起始浓度5μg/ml，2×梯度稀释(12个浓度点)的Claudin18.2抗体，4℃孵育1小时，阳性对照为IMAB362，同时设置不加抗体的阴性对照。离心去除抗体，再加入100μl/孔FITC抗人IgG Fc抗体(200×)，4℃避光孵育30分钟，用PBS+2％FBS清洗两遍后准备进行流式细胞检测。启动BD FACS CantoII，预热完成后打开BD FACSDiva软件，建立一个新的实验，检测HEK293/hClaudin18.2阴性对照样品，调节FSC及SSC电压至适当的数值并保存。根据Quantum TM FITC-5 MESF Kit说明书，分别检测空白样品B及标准曲线1，调节FITC电压至适当的数值并保存。在保存的电压下检测U底96孔板中的样品，记录数据。使用Flowjo软件分析实验数据得到Geo Mean数值，根据Quantum TM FITC-5MESF Kit说明书拟合MESF-Geo Mean标准曲线，根据FITC抗人IgG Fc抗体的浓度荧光值计算出与HEK293/hClaudin18.2细胞结合的Claudin18.2抗体的摩尔浓度及游离抗体浓度，利用Scatchard作图法计算抗体的Bmax和解离常数KD。结果见表14。
表14.人源化抗体细胞水平亲和力
抗体 IMAB362 h1901-11 h1902-5
KD(nM) 10.2 6.8 1.64
测试例5：抗体的ADCC效应评价
消化各种NUGC4细胞(高中低表达Claudin18.2)，1000rpm离心后，重悬计数。将细胞以3×10 5细胞/ml的密度重悬在添加10％FBS(新西兰超低IgG胎牛血清，Gibco，1921005PJ)的无酚红RPMI 1640中(Gibco，11835-030)。在96孔板(Corning，3903)中，每孔加入25μl细胞(7500个/孔)。将抗体稀释在上述无酚红培养基中，配制成3×的抗体稀释液，向细胞板中加入25μl/孔的抗体。在37℃、5％CO 2培养箱中孵育0.5小时。
收集效应细胞(FcrR3A-V158-NFAT-RE-Jurkat细胞)，1000rpm离心后，重悬计数。将细胞以3×10 6细胞/ml的密度重悬在添加10％FBS(新西兰超低IgG胎牛血清)的无酚红RPMI 1640中，在实验板中每孔加入25μl细胞(7.5×10 4个细胞/孔)。在37℃、5％CO 2培养箱中孵育6小时。
向实验板的每个孔中加入75μl/孔的Bright-Glo(Promega，E2610)，用酶标仪(PerkinElmer，VITOR3)检测化学发光(luminescence)。
结果显示(见表15和图3A-图3C)，在低(图3A)-中(图3B)-高(图3C)不同程度Claudin18.2表达的NUGC4细胞中，抗体h1901-11和h1902-5均显示出很强的ADCC活性。
表15.抗体在Claudin18.2不同表达程度的NUGC4细胞中的ADCC效应单位IC50(ng/ml)
Claudin18.2表达程度 h1901-11 h1902-5 IMAB362
低表达 22.42 35.46 183.4
中等表达 15.35 30.00 210.4
高表达 26.17 32.16 132.6
测试例6：ADC分子细胞活性实验
本实验通过利用CellTiter-Glo Luminescence Cell Viability Assay检测ADC分子在体外对人胃癌细胞株的杀伤作用。第一天，收集NUGC4-claudin18.2低表达，NUGC4-claudin18.2中表达，NUGC4-claudin18.2高表达细胞，调整密度为2.5×10 4/ml，在96孔白色透明底板中加入90μl/孔，约为2500个细胞每孔。37℃，5％CO 2培养箱过夜培养。第二天，在U底96孔板中稀释样品，起始浓度为5μM，4×梯度稀释，9个浓度点，向细胞板中加入10μl/well稀释好的样品。37℃，5％CO 2培养6天。第八天，取出细胞培养板，加入50μl/well Cell Titer-Glo Reagent，室温放置2至3分钟，在PHERAstar FS microplate reader上读取luminescence数值。利用GraphPad Prism软件进行数据分析。见表16。
表16.ADC体外细胞杀伤实验
Figure PCTCN2021122031-appb-000045
测试例7：ADC分子体内药效评价
Balb/c裸在右肋部皮下接种人胃癌细胞NUGC4(Claudin18.2中等表达)细胞(5×10 6含50％matrigel基质胶/只)，第0日分组，8只/组，共8组。平均瘤体积约84.41mm 3。
ADC腹腔注射，共给药3次，每只按体重注射10g/0.1ml，分别于第0日，4日，11日给药。
分组当天ADC腹腔注射，共给药4次，间隔5天给药，每只按体重注射10g/0.1ml。
每周测量2次瘤体积和体重，记录数据。
使用Excel 2003统计软件：平均值以avg计算；SD值以STDEV计算；SEM值以STDEV/SQRT计算；组间差异P值以TTEST计算。
肿瘤体积(V)计算公式为：V＝1/2×L 长×L 短 2
相对体积(RTV)＝VT/V0
抑瘤率(％)＝(CRTV-TRTV)/CRTV(％)
其中V0、VT分别为实验开始时(首次给药当天为第0天)及实验结束时的肿瘤体积。CRTV、TRTV分别为实验结束时的空白对照组(Vehicle)及实验组的相对肿瘤体积。结果见表17和图4、图5。
表17.ADC抑瘤实验结果
Figure PCTCN2021122031-appb-000046
vs空白对照组：**p<0.01。
二、制剂
制剂制备与检测过程中使用的设备及结果计算方法如下：
SEC分子排阻色谱法：
根据凝胶孔隙的孔径大小与高分子样品分子的线团尺寸间的相对关系而对溶质进行分离的分析的方法。
SEC％(SEC单体含量百分比)＝A单体/A总*100％(A单体为样品中主峰单体的峰面积，A总为所有峰面积之和)。
SEC测定用仪器：安捷伦1260；柱子：waters，XBrige
Figure PCTCN2021122031-appb-000047
SEC(300×7.8mm 3.5μm)。
CE毛细管凝胶电泳：
将凝胶移到毛细管中作为支持介质进行的一种电泳，并在一定的电压下根据样品分子量的大小进行分离的方法。
还原CE纯度百分比＝A主峰/A总*100％(A主峰为样品中轻链主峰+重链主峰的峰面积，A总为所有峰面积之和。
CE测定用仪器：Beckman型号plus800。
渗透压测定：
冰点法测定渗透压，以冰点下降值与溶液的摩尔浓度成正比例关系为基础，采用高灵敏度感温元件，测定溶液结冰点，通过电量转化为渗透压。仪器厂家罗泽Loser，型号OM815。
蛋白浓度测定：
因为抗体药物偶联物中的药物在280nm下有吸收，用以下公式对蛋白浓度进行校正，
A280＝Cd*ε280d+Cmab*ε280mab；
A370＝Cd*ε370d；
Cd代表药物的浓度，Cmab代表蛋白的浓度，ε280d代表药物在280nm下的消光系数，ε280mab代表蛋白在280nm下的消光系数，ε370d代表药物在370nm下的消光系数。ε280mab＝1.49mg-1*cm-1*ml，ε280d＝5000(280nm药物摩尔消光系数)/1074.13(药物分子量)＝4.65mg-1*cm-1*mL，ε370d＝19000(370nm药物摩尔消光系数)/1074.13(药物分子量)＝17.69mg-1*cm-1*mL，以上消光系数为质量消光系数。
蛋白浓度测定仪器：紫外可见分光光度计，型号：Nano Drop oneC，光程为1mm。
实施例2-1：制剂缓冲体系与pH值的筛选
配制含有20mg/mL(蛋白浓度)的ADC-9和以下不同缓冲体系，以及0.1mg/mL聚山梨酯80(PS80)的制剂。
1)10mM柠檬酸-柠檬酸钠(CA)，pH 5.5
2)10mM琥珀酸-琥珀酸钠(SA)，pH 5.0
3)10mM琥珀酸-琥珀酸钠，pH 5.5
4)10mM组氨酸盐酸盐(His-HCl)，pH 5.5
5)10mM组氨酸盐酸盐，pH 6.0
6)10mM组氨酸盐酸盐，pH 6.5
7)10mM组氨酸-醋酸盐(His-AA)，pH 5.0
8)10mM组氨酸-醋酸盐，pH 5.5
9)10mM磷酸盐(PB)，pH 6.5。
将每种制剂过滤，灌装，加塞，轧盖，取样品进行高温稳定性(40℃)、振摇(25℃，300rpm)研究，考察外观、SEC、还原CE。结果见表18。
振摇11天后，只有2)、3)、7)、9)号样品外观澄明；40℃放置15天后，1)、2)、3)、7)、8)号样品外观澄明。即从外观的角度看，2)、3)、7)号样品的制剂较优。
40℃放置15天后，采用SEC检测样品，结果显示：4)、5)、6)、7)、8)号样品的单体降低幅度在4％左右；其他制剂单体降低幅度为7％-10％。
40℃放置15天后，采用还原CE检测样品，结果显示：4)、5)、7)、8)号样品的主峰下降幅度在1％-2％左右，优于其他样品。
综合以上数据，7)号样品即10mM His-AA，pH 5.0制剂从外观及各化学检测项均优于其余制剂，因此选择10mM His-AA，pH 5.0为最终缓冲液。
表18.pH和缓冲液稳定性结果
Figure PCTCN2021122031-appb-000048
Figure PCTCN2021122031-appb-000049
注：表中“D”表示天，例如D3表示3天，以此类推；D0表示实验开始时，下同。
实施例2-2：表面活性剂种类及浓度筛选
制备含有不同型号和浓度聚山梨酯，且含有10mM His-AA，pH 5.0的缓冲液、80mg/mL蔗糖和蛋白浓度为20mg/mL的ADC-9的制剂。将每种制剂过滤，灌装，加塞，轧盖。将样品进行高温稳定性研究(40℃)和冻融研究。其中冻融研究为冻融5次循环(FT5C，35℃-2至8℃)后室温放置三天(25℃ D3)，考察外观、SEC、还原CE，具体制剂设计见表19。
结果见表20，实验结果显示采用0.2mg/ml PS80的制剂在各条件下外观和化学检测项为最优。
综合以上结果，表面活性剂种类及浓度定为0.2mg/ml PS80。
表19.聚山梨酯种类及浓度筛选制剂
Figure PCTCN2021122031-appb-000050
注：PS20表示聚山梨酯20。
表20.聚山梨酯种类筛选结果
Figure PCTCN2021122031-appb-000051
Figure PCTCN2021122031-appb-000052
备注：表中“M”表示月，M1表示1个月，以此类推，下同。
实施例2-3：糖种类筛选
制备分别含有蔗糖、海藻糖、甘露醇的制剂，其还含有10mM His-AA(pH 5.0)缓冲液、0.2mg/mL PS80和20mg/mL(蛋白浓度)的ADC-9。将每种制剂过滤，灌装，加塞，轧盖。将样品进行高温稳定性研究(40℃)、-35℃/4℃冻融循环并在室温放置3天研究，考察外观、SEC、还原CE。
结果见表21。在不同糖种类制剂冻融条件下，采用蔗糖的样品的外观优于采用海藻糖或甘露醇的样品。SEC检测结果显示，采用蔗糖或海藻糖的样品优于甘露醇；40℃放置一个月后，采用蔗糖的样品的外观优于采用海藻糖或甘露醇的样品，SEC和还原CE的检测结果也显示采用蔗糖的样品略优于采用海藻糖的样品。
表21.糖种类溶液剂筛选结果
Figure PCTCN2021122031-appb-000053
制备分别含有蔗糖、海藻糖、甘露醇的制剂，其还含有10mM His-AA(pH 5.0)缓冲液、0.2mg/mL PS80和20mg/mL(蛋白浓度)的ADC-9。将每种制剂过滤、灌装、半加塞、冻干、加塞、轧盖，放置高温稳定性研究(40℃)考察外观、SEC、还原CE。冻干工艺参见表22的冻干工艺参数1。
表22.冻干工艺参数1
冻干工艺参数 设定温度(℃) 设定时间(min) 保持时间(h) 真空度(Pa)
预冻 5 10 1 N/A
预冻 -45 50 2.5 N/A
一次干燥 -20 120 45 20
二次干燥 25 60 8 1
40℃放置一个月后，SEC的检测结果(见表23)显示采用蔗糖的样品略优于采用海藻糖的样品，更优于采用甘露醇的样品，还原CE的检测结果(见表23)显示，采用蔗糖的样品和采用海藻糖的样品相当，同时优于采用甘露醇的样品。
表23.糖种类冻干剂筛选结果
Figure PCTCN2021122031-appb-000054
实施例2-4：冻干后样品外观优化实验
按照10mM His-AA，pH 5.0、80mg/mL蔗糖、0.2mg/mL PS80和20mg/mL(蛋白浓度)ADC-9配制原液，过滤灌装后按照冻干工艺参数1冻干(见表22)，考察冻干后样品外观。冻干样品为表面平整白色粉饼，但粉饼底部边缘略有缩小。
将样品的糖浓度进一步下调至60mg/mL，按照10mM His-AA，pH5.0、60mg/mL蔗糖、0.2mg/mL PS80和20mg/mL(蛋白浓度)ADC-9配制原液，过滤灌装后按照表22的冻干工艺参数1冻干。冻干后，粉饼表面平整无皱缩，粉饼底部边缘略有缩底。
将糖浓度降至40mg/ml，但此时成品渗透压过低，在临床给药时有渗透压偏低的风险，为了保证成品渗透压，将缓冲液离子强度提高至30mM。按照30mM His-AA，pH 5.0、40mg/mL蔗糖、0.2mg/mL PS80和20mg/mL(蛋白浓度)ADC-9配制原液，过滤灌装后继续按照冻干工艺参数1冻干。冻干后样品粉饼表面平整无塌陷，粉饼底部边缘完整。
三种制剂的结果见表24。
表24.冻干后样品外观
Figure PCTCN2021122031-appb-000055
Figure PCTCN2021122031-appb-000056
实施例2-5：冻干后样品稳定性
按照表25中的制剂配制原液，过滤灌装后按照冻干工艺参数1冻干(见表22)，将冻干后样品于40℃ M1条件下放置后复溶检测其稳定性变化。
稳定性结果显示见表25，3号制剂的冻干样品在40℃ M1条件下还原CE下降约3.7％，其余两制剂各化学检测项均无明显变化，稳定性明显优于3号制剂。2号制剂冻干前原液的pH为5.04，冻干后复溶溶液的pH为5.27。
表25.制剂稳定性结果
Figure PCTCN2021122031-appb-000057
实施例2-6：冻干制剂溶液剂稳定性
按照表26中的制剂制备制剂，过滤，灌装，加塞，轧盖后进行-35℃/4℃冻融循环并在室温放置3天研究，振摇11天研究和高温稳定性研究(40℃)，考察对应条件下样品外观，SEC和还原CE变化。
稳定性结果显示见表26，在降低蔗糖浓度和提高缓冲液离子强度后，2号制剂在外观和纯度项变化上与1号制剂无明显差异，在高温条件下还原CE降幅略优于1号制剂。
表26.制剂稳定性结果
Figure PCTCN2021122031-appb-000058

Claims (18)

  1. 一种药物组合物，包含抗Claudin18.2抗体药物偶联物和缓冲剂，其中所述抗Claudin18.2抗体药物偶联物中的抗Claudin18.2抗体包含重链可变区和轻链可变区，其中：
    i)所述重链可变区与如SEQ ID NO：5所示的重链可变区具有相同氨基酸序列的HCDR1、HCDR2和HCDR3，所述轻链可变区包含与如SEQ ID NO：6所示的轻链可变区具有相同氨基酸序列的LCDR1、LCDR2和LCDR3；或
    ii)所述重链可变区与如SEQ ID NO：3所示的重链可变区具有相同氨基酸序列的HCDR1、HCDR2和HCDR3，所述轻链可变区与如SEQ ID NO：4所示的轻链可变区具有相同氨基酸序列的LCDR1、LCDR2和LCDR3；
    所述缓冲剂是组氨酸盐缓冲剂，优选为组氨酸-醋酸盐缓冲剂。
  2. 根据权利要求1所述的药物组合物，其中所述抗Claudin18.2抗体包含重链可变区和轻链可变区，其中：
    iii)所述重链可变区包含分别如SEQ ID NO：15、SEQ ID NO：16和SEQ ID NO：17所示的HCDR1、HCDR2和HCDR3，所述轻链可变区包含分别如SEQ ID NO：18、SEQ ID NO：19和SEQ ID NO：20所示的LCDR1、LCDR2和LCDR3；或
    iv)所述重链可变区包含分别如SEQ ID NO：9、SEQ ID NO：10和SEQ ID NO：11所示的HCDR1、HCDR2和HCDR3，所述轻链可变区包含分别如SEQ ID NO：12、SEQ ID NO：13和SEQ ID NO：14所示的LCDR1、LCDR2和LCDR3；
    优选地，所述抗Claudin18.2抗体包含重链可变区和轻链可变区，其中：
    v)所述重链可变区如SEQ ID NO：31所示和所述轻链可变区如SEQ ID NO：29所示；或
    vi)所述重链可变区如SEQ ID NO：26所示和所述轻链可变区如SEQ ID NO：23所示；
    更优选地，所述抗Claudin18.2抗体包含：
    vii)SEQ ID NO：49所示的重链，和SEQ ID NO：47所示的轻链；或
    viii)SEQ ID NO：44所示的重链，和SEQ ID NO：41所示的轻链。
  3. 根据权利要求1或2所述的药物组合物，其中所述抗Claudin18.2抗体药物偶联物具有如通式(Pc-L-Y-D)所示的结构：
    Figure PCTCN2021122031-appb-100001
    其中：
    Y选自-O-(CR aR b) m-CR 1R 2-C(O)-、-O-CR 1R 2-(CR aR b) m-、-O-CR 1R 2-、-NH-(CR aR b) m-CR 1R 2-C(O)-和-S-(CR aR b) m-CR 1R 2-C(O)-；
    R a和R b相同或不同，且各自独立地选自氢原子、氘原子、卤素、烷基、卤代烷基、氘代烷基、烷氧基、羟基、氨基、氰基、硝基、羟烷基、环烷基和杂环基；
    或者，R a和R b与其相连接的碳原子一起形成环烷基或杂环基；
    R 1选自卤素、卤代烷基、氘代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基和杂芳基；
    R 2选自氢原子、卤素、卤代烷基、氘代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基和杂芳基；
    或者，R 1和R 2与其相连接的碳原子一起形成环烷基或杂环基；
    或者，R a和R 2与其相连接的碳原子一起形成环烷基或杂环基；
    m为0至4的整数；
    n为1至10，n是小数或整数；
    L为接头单元；
    Pc为抗Claudin18.2抗体；
    优选地，所述抗Claudin18.2抗体药物偶联物具有如下式所示的结构：
    Figure PCTCN2021122031-appb-100002
    其中：
    n为2至8，n是小数或整数；
    Pc为抗Claudin18.2抗体。
  4. 根据权利要求1至3任一项所述的药物组合物，其中所述药物组合物还包含表面活性剂；
    优选地，所述表面活性剂为聚山梨酯；
    更优选地，所述表面活性剂为聚山梨酯80或聚山梨酯20；
    最优选地，所述表面活性剂为聚山梨酯80。
  5. 根据权利要求4所述的药物组合物，其中所述表面活性剂浓度为0.05mg/mL至0.5mg/mL，优选为0.1mg/mL至0.2mg/mL，更优选为0.2mg/mL。
  6. 根据权利要求1至5任一项所述的药物组合物，其中所述组合物还包含糖；
    优选地，所述糖选自蔗糖、甘露醇和海藻糖；
    更优选地，所述糖为蔗糖。
  7. 根据权利要求6所述的药物组合物，其中所述糖浓度为20mg/mL至100mg/mL，优选为40mg/mL至80mg/mL，更优选为40mg/mL。
  8. 根据权利要求1至7任一项所述的药物组合物，其中所述抗Claudin18.2抗体药物偶联物浓度为以蛋白浓度计1mg/mL至100mg/mL，优选为以蛋白浓度计10mg/mL至30mg/mL，更优选为以蛋白浓度计20mg/mL。
  9. 根据权利要求1至8任一项所述的药物组合物，其中所述缓冲剂的浓度为5mM至50mM，优选为10mM至30mM，更优选为30mM。
  10. 根据权利要求1至9任一项所述的药物组合物，其中所述药物组合物的pH为5.0-6.5，优选为5.0-5.5，更优选为5.0-5.3。
  11. 根据权利要求1至10任一项所述的药物组合物，其包含如下组分：
    (a)以蛋白浓度计10mg/mL至30mg/mL的所述抗Claudin18.2抗体药物偶联物，(b)0.1mg/mL至0.2mg/mL的聚山梨酯，(c)40mg/mL至80mg/mL的糖，和(d)10mM至30mM的组氨酸盐缓冲剂；所述药物组合物的pH为5.0-5.5；
    优选地，所述药物组合物包含如下组分：
    (a)以蛋白浓度计20mg/mL的所述抗Claudin18.2抗体药物偶联物，(b)0.2mg/mL的聚山梨酯80，(c)40mg/mL的蔗糖，和(d)30mM组氨酸-醋酸盐缓冲剂，所述药物组合物的pH为5.0-5.3。
  12. 一种药物组合物，其包含：
    以蛋白浓度计20mg/mL的抗Claudin18.2抗体药物偶联物、
    0.2mg/mL的聚山梨酯80、
    40mg/mL的蔗糖、和
    30mM组氨酸-醋酸盐缓冲剂；
    所述药物组合物的pH为5.0至5.3；
    所述抗Claudin18.2抗体药物偶联物具有如下式所示的结构：
    Figure PCTCN2021122031-appb-100003
    其中：
    n为2至8，n是小数或整数；
    Pc为抗Claudin18.2抗体，其包含如SEQ ID NO：49所示的重链，和如SEQ ID NO：47所示的轻链。
  13. 一种含抗体药物偶联物的冻干制剂，其特征在于所述制剂复溶后可形成权利要求1至12任一项所述的药物组合物。
  14. 一种制备含抗体药物偶联物的冻干制剂的方法，其中包括将权利要求1至12中任一项所述的药物组合物进行冷冻干燥的步骤。
  15. 一种含抗体药物偶联物的冻干制剂，所述制剂通过将权利要求1至12中任一项所述的药物组合物经冷冻干燥获得。
  16. 一种含抗体药物偶联物的复溶溶液，其特征在于所述复溶溶液是通过将权利要求13或15所述的冻干制剂经复溶制备获得；
    优选地，所述复溶溶液包含如下组分：
    (a)以蛋白浓度计10mg/mL至30mg/mL的所述抗Claudin18.2抗体药物偶联物，(b)0.1mg/mL至0.2mg/mL的聚山梨酯，(c)40mg/mL至80mg/mL的糖，和(d)10mM至30mM的组氨酸盐缓冲剂；所述复溶溶液的pH为约5.0-5.5；
    更优选地，所述药物组合物包含如下组分：
    (a)以蛋白浓度计20mg/mL的所述抗Claudin18.2抗体药物偶联物，(b)0.2mg/mL的聚山梨酯80，(c)40mg/mL的蔗糖，和(d)30mM组氨酸-醋酸盐缓冲剂， 所述复溶溶液的pH为5.0-5.3。
  17. 一种制品，其包括容器，该容器中装有如权利要求1至12任一项所述的药物组合物、权利要求13或15所述的冻干制剂或权利要求16所述的复溶溶液。
  18. 一种治疗肿瘤或癌症的方法，包括给予受试者有效量的权利要求1至12任一项所述的药物组合物、或权利要求13或15所述的冻干制剂、或权利要求16所述的复溶溶液、或权利要求17所述制品；
    其中，所述肿瘤或癌症优选选自：头和颈鳞状细胞癌、头和颈癌、脑癌、神经胶质瘤、多形性成胶质细胞瘤、神经母细胞瘤、中枢神经系统癌、神经内分泌肿瘤、咽喉癌、鼻咽癌、食管癌、甲状腺癌、恶性胸膜间皮瘤、肺癌、乳腺癌、肝癌、肝细胞瘤、肝胆癌、胰腺癌、胃癌、胃肠道癌、肠癌、结肠癌、结肠直肠癌、肾癌、透明细胞肾细胞癌、卵巢癌、子宫内膜癌、子宫颈癌、膀胱癌、前列腺癌、睾丸癌、皮肤癌、黑色素瘤、白血病、淋巴瘤、骨癌、软骨肉瘤、骨髓瘤、多发性骨髓瘤、骨髓异常增生综合征、库肯勃氏瘤、骨髓增生性肿瘤、鳞状细胞癌、尤因氏肉瘤、全身性轻链淀粉样变性和梅克尔细胞癌；
    更优选地，所述淋巴瘤选自：何杰金淋巴瘤、非何杰金淋巴瘤、弥漫性大B-细胞淋巴瘤、滤泡性淋巴瘤、原发性纵隔大B-细胞淋巴瘤、套细胞淋巴瘤、小淋巴细胞性淋巴瘤、富含T-细胞/组织细胞的大B-细胞淋巴瘤和淋巴浆细胞性淋巴瘤；所述肺癌选自：非小细胞肺癌和小细胞肺癌；所述白血病选自：慢性髓细胞样白血病、急性髓细胞样白血病、淋巴细胞白血病、成淋巴细胞性白血病、急性成淋巴细胞性白血病、慢性淋巴细胞性白血病和髓样细胞白血病。
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BR112022019042A2 (pt) * 2020-03-25 2022-11-01 Jiangsu Hengrui Pharmaceuticals Co Ltd Método de preparação para conjugado anticorpo-fármaco
JP2023518583A (ja) * 2020-03-25 2023-05-02 江蘇恒瑞医薬股▲ふん▼有限公司 抗体薬物複合体を含む医薬組成物及びその使用 本願は、２０２０年３月２５日に提出された中国特許出願（出願番号ｃｎ ２０２０１０２１９６０１．７）及び２０２１年３月１７日に提出された中国特許出願（出願番号ｃｎ ２０２１１０２８７０１２．７）の優先権を主張する。

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