WO2023046131A1 - 抗cd40抗体的应用 - Google Patents

抗cd40抗体的应用 Download PDF

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Publication number
WO2023046131A1
WO2023046131A1 PCT/CN2022/121184 CN2022121184W WO2023046131A1 WO 2023046131 A1 WO2023046131 A1 WO 2023046131A1 CN 2022121184 W CN2022121184 W CN 2022121184W WO 2023046131 A1 WO2023046131 A1 WO 2023046131A1
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Prior art keywords
antibody
amino acid
seq
antigen
cancer
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Ceased
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PCT/CN2022/121184
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English (en)
French (fr)
Inventor
沈忱
吕鹏
田心
杨玲
张喜全
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Chia Tai Tianqing Pharmaceutical Group Co Ltd
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Chia Tai Tianqing Pharmaceutical Group Co Ltd
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Application filed by Chia Tai Tianqing Pharmaceutical Group Co Ltd filed Critical Chia Tai Tianqing Pharmaceutical Group Co Ltd
Priority to EP22872175.9A priority Critical patent/EP4406552A4/en
Priority to CA3232304A priority patent/CA3232304A1/en
Priority to US18/690,886 priority patent/US20240398942A1/en
Priority to AU2022351098A priority patent/AU2022351098A1/en
Priority to CN202280060359.0A priority patent/CN118103066A/zh
Publication of WO2023046131A1 publication Critical patent/WO2023046131A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • 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
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • 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
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4709—Non-condensed quinolines and containing further heterocyclic rings
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39541—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against normal tissues, cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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 [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • 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
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • 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
    • C07K2317/75—Agonist effect on antigen
    • 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 invention relates to the field of biomedicine, in particular to a combined medicine for treating cancer.
  • CD40 is a member of the tumor necrosis factor receptor (TNFR) superfamily, present on antigen-presenting cells (APC) such as B cells, dendritic cells (DC) and macrophages, and activated on other hematopoietic cells (such as T cells) There are also expressions later.
  • APC antigen-presenting cells
  • B cells non-Hodgkin's lymphoma, acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloma, etc.
  • epithelial tumors prostate cancer, liver cancer, lung cancer, breast cancer, etc.
  • CD40 The natural ligand for CD40 (known as CD154 or CD40L) is mainly expressed on activated T lymphocytes and mast cells.
  • the CD40/CD40L signaling pathway participates in the body's humoral and cellular immune responses, and plays a key regulatory role in T cell-dependent antibody immune responses and inflammatory responses such as the activation, proliferation and differentiation of B cells, antibody production, and antibody class switching. Therefore, treatment with a CD40 agonist (such as an agonistic anti-CD40 antibody) can enhance the body's anti-tumor immune response.
  • a CD40 agonist such as an agonistic anti-CD40 antibody
  • Tyrosine kinases are a group of enzymes that catalyze the phosphorylation of protein tyrosine residues. They play an important role in intracellular signal transduction. They are involved in the regulation, signal transmission and development of normal cells, and also with the tumor cells Proliferation, differentiation, migration and apoptosis are closely related. Many receptor tyrosine kinases are associated with the formation of tumors, which can be divided into epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR), fibroblast growth factor receptor (FGFR), etc.
  • EGFR epidermal growth factor receptor
  • PDGFR platelet-derived growth factor receptor
  • VEGFR vascular endothelial growth factor receptor
  • FGFR fibroblast growth factor receptor
  • Anlotinib is a quinoline derivative tyrosine kinase inhibitor, which acts as a multi-target tyrosine kinase inhibitor (TKI) in affecting tumor angiogenesis and proliferation signal transduction.
  • Primary targets include: receptor tyrosine kinases vascular endothelial growth factor receptor (VEGFR) 1 to 3, epidermal growth factor receptor (EGFR), fibroblast growth factor receptor (FGFR) 1 to 4, platelet-derived growth Factor receptors (PDGFR) alpha and beta, and stem cell factor receptors (SCFR) 7, 8 and 9.
  • the present application provides a pharmaceutical combination comprising (a) an anti-CD40 antibody or an antigen-binding portion thereof, and (b) a second therapeutic agent.
  • the second therapeutic agent has cancer treatment efficacy.
  • the second therapeutic agent comprises a tyrosine kinase inhibitor.
  • the second therapeutic agent comprises a VEGFR tyrosine kinase inhibitor.
  • the second therapeutic agent comprises anlotinib or a pharmaceutically acceptable salt thereof.
  • a tyrosine kinase inhibitor refers to any substance or agent capable of inhibiting (eg, blocking, destroying, or inactivating) a tyrosine kinase or downstream signal transduction from a tyrosine kinase, including the small molecule tyrosine Kinase inhibitors and antibodies.
  • Tyrosine kinase inhibitors include receptor tyrosine kinase inhibitors and non-receptor tyrosine kinase inhibitors.
  • the receptor tyrosine kinase inhibitors include, but are not limited to, EGFR tyrosine kinase inhibitors, VEGFR tyrosine kinase inhibitors, FGFR tyrosine kinase inhibitors, SCFR tyrosine kinase inhibitors agents and PDGFR tyrosine kinase inhibitors, etc.
  • VEGFR tyrosine kinase inhibitor refers to any substance or agent that inhibits (eg, blocks, destroys, or inactivates) VEGF receptor tyrosine kinase or downstream signal transduction from VEGF receptor tyrosine kinase, Includes small molecule VEGFR tyrosine kinase inhibitors and antibodies.
  • VEGFR tyrosine kinase inhibitors include, but are not limited to, Anlotinib, Vandetanib, Sorafenib, Cediranib, Vataranib ( Vatalanib), Pazopanib, Motesanib, Lenvatinib, Sunitinib, Bevacizumab, Ramucirumab ( Ramucirumab), or a pharmaceutically acceptable salt thereof, or any combination of the foregoing.
  • the drug combination further includes a third therapeutic agent having cancer treatment efficacy.
  • the third therapeutic agent may be a cancer therapeutic agent known in the art, such as a chemotherapeutic drug, a radiotherapeutic drug, surgery, or a combination thereof.
  • the pharmaceutical combination includes (a) an anti-CD40 antibody, or antigen-binding portion thereof, and (b) a tyrosine kinase inhibitor; optionally, further includes (c) a chemotherapeutic agent.
  • the pharmaceutical combination includes (a) an anti-CD40 antibody, or antigen-binding portion thereof, and (b) a VEGFR tyrosine kinase inhibitor; optionally, further includes (c) a chemotherapeutic agent.
  • the pharmaceutical combination comprises (a) an anti-CD40 antibody or an antigen-binding portion thereof, and (b) anlotinib or a pharmaceutically acceptable salt thereof; optionally, further comprising (c )Chemotherapy drugs.
  • the pharmaceutical combination comprises (a) a pharmaceutical composition of an anti-CD40 antibody or antigen-binding portion thereof, and (b) a pharmaceutical composition of a tyrosine kinase inhibitor; optionally, further comprising (c )Chemotherapy drugs.
  • the pharmaceutical combination includes (a) a pharmaceutical composition of an anti-CD40 antibody or an antigen-binding portion thereof, and (b) a pharmaceutical composition of a VEGFR tyrosine kinase inhibitor; optionally, further comprising ( c) Chemotherapy drugs.
  • the pharmaceutical combination includes (a) a pharmaceutical composition of an anti-CD40 antibody or an antigen-binding portion thereof, and (b) a pharmaceutical composition of anlotinib or a pharmaceutically acceptable salt thereof;
  • (c) chemotherapy drugs are also included.
  • the pharmaceutical combination is packaged in the same kit, the kit further comprising instructions for using the anti-CD40 antibody or antigen-binding portion thereof in combination with a tyrosine kinase inhibitor to treat cancer.
  • the anti-CD40 antibody or antigen-binding portion thereof and the tyrosine kinase inhibitor in the pharmaceutical combination are packaged separately in separate kits, and the kits further include an anti-CD40 antibody or antigen-binding portion thereof Instructions for use in combination with tyrosine kinase inhibitors in the treatment of cancer.
  • the pharmaceutical combination is packaged in the same kit, the kit further comprising instructions for using the combination of an anti-CD40 antibody or antigen-binding portion thereof, a tyrosine kinase inhibitor, and a chemotherapeutic agent to treat cancer.
  • the anti-CD40 antibody or antigen-binding portion thereof, the tyrosine kinase inhibitor, and the chemotherapeutic drug in the pharmaceutical combination are packaged separately in respective kits, and the kits further include the anti-CD40 antibody or its antigen-binding portion. Illustration of combinations of antigen binding moieties, tyrosine kinase inhibitors, and chemotherapeutic agents for the treatment of cancer.
  • the pharmaceutical combination is packaged in the same kit, and the kit further includes instructions for using an anti-CD40 antibody or antigen-binding portion thereof in combination with a VEGFR tyrosine kinase inhibitor to treat cancer.
  • the anti-CD40 antibody or antigen-binding portion thereof and the VEGFR tyrosine kinase inhibitor in the pharmaceutical combination are packaged separately in separate kits, and the kits further include an anti-CD40 antibody or an antigen-binding portion thereof. Indications for use in combination with VEGFR tyrosine kinase inhibitors in the treatment of cancer.
  • the pharmaceutical combination is packaged in the same kit, and the kit further includes instructions for combined use of an anti-CD40 antibody or antigen-binding portion thereof, a VEGFR tyrosine kinase inhibitor, and a chemotherapeutic agent for the treatment of cancer.
  • the anti-CD40 antibody or antigen-binding portion thereof, the VEGFR tyrosine kinase inhibitor, and the chemotherapeutic drug in the pharmaceutical combination are packaged separately in respective kits, and the kits further include the anti-CD40 antibody or Instructions for combination use of an antigen binding portion thereof, a VEGFR tyrosine kinase inhibitor, and a chemotherapeutic agent for the treatment of cancer.
  • the pharmaceutical combination is packaged in the same kit, and the kit further includes an anti-CD40 antibody or its antigen-binding portion and anlotinib or a pharmaceutically acceptable salt thereof used in combination to treat cancer.
  • the anti-CD40 antibody or antigen-binding portion thereof and anlotinib or a pharmaceutically acceptable salt thereof in the pharmaceutical combination are packaged separately in respective kits, and the kits further include anti-CD40 Instructions for use of an antibody or antigen-binding portion thereof in combination with anlotinib or a pharmaceutically acceptable salt thereof for the treatment of cancer.
  • the drug combination is packaged in the same kit, and the kit further includes an anti-CD40 antibody or an antigen-binding portion thereof, anlotinib or a pharmaceutically acceptable salt thereof, and chemotherapy drugs for use in combination to Illustration of treatment for cancer.
  • the anti-CD40 antibody or antigen-binding portion thereof, anlotinib or a pharmaceutically acceptable salt thereof, and chemotherapy drugs in the pharmaceutical combination are packaged separately in respective kits, and the kits also include Included are instructions for the combined use of an anti-CD40 antibody or antigen-binding portion thereof, anlotinib or a pharmaceutically acceptable salt thereof, and a chemotherapeutic agent for the treatment of cancer.
  • the drug combination is a fixed combination.
  • the fixed combination is in the form of a solid pharmaceutical composition or a liquid pharmaceutical composition.
  • the drug combination is a non-fixed combination.
  • the anti-CD40 antibody or antigen-binding portion thereof and the tyrosine kinase inhibitor in the non-fixed combination are each in the form of a pharmaceutical composition.
  • the anti-CD40 antibody or antigen-binding portion thereof, tyrosine kinase inhibitor, and chemotherapeutic agent in the non-fixed combination are each in the form of a pharmaceutical composition.
  • the anti-CD40 antibody or antigen-binding portion thereof and the VEGFR tyrosine kinase inhibitor in the non-fixed combination are each in the form of a pharmaceutical composition.
  • the anti-CD40 antibody or antigen-binding portion thereof, VEGFR tyrosine kinase inhibitor, and chemotherapeutic agent in the non-fixed combination are each in the form of a pharmaceutical composition.
  • the anti-CD40 antibody or antigen-binding portion thereof and anlotinib or a pharmaceutically acceptable salt thereof in the non-fixed combination are each in the form of a pharmaceutical composition.
  • the anti-CD40 antibody or antigen-binding portion thereof in the non-fixed combination is in the form of a liquid pharmaceutical composition, and the anlotinib or a pharmaceutically acceptable salt thereof in the non-fixed combination In the form of a solid pharmaceutical composition.
  • the anti-CD40 antibody or its antigen-binding portion in the non-fixed combination is in the form of an injection, and the anlotinib or a pharmaceutically acceptable salt thereof in the non-fixed combination is in the form of a solid In the form of an oral preparation.
  • the anti-CD40 antibody or its antigen-binding portion in the non-fixed combination is in the form of an injection, and the anlotinib or a pharmaceutically acceptable salt thereof in the non-fixed combination is in the form of a capsule form.
  • the anti-CD40 antibody or antigen-binding portion thereof, anlotinib or a pharmaceutically acceptable salt thereof, and the chemotherapeutic agent in the non-fixed combination are each in the form of a pharmaceutical composition.
  • the present application also provides the use of the drug combination of the first aspect in the preparation of a drug for treating cancer.
  • the present application also provides the use of the anti-CD40 antibody or its antigen-binding part and the tyrosine kinase inhibitor in the preparation of a medicament for treating cancer.
  • the present application also provides the use of the anti-CD40 antibody or its antigen-binding part, the tyrosine kinase inhibitor and the chemotherapeutic drug in the preparation of a drug for treating cancer.
  • the present application also provides the use of the anti-CD40 antibody or its antigen-binding part and the VEGFR tyrosine kinase inhibitor in the preparation of a drug for treating cancer.
  • the present application also provides the use of the anti-CD40 antibody or its antigen-binding part, the VEGFR tyrosine kinase inhibitor and the chemotherapeutic drug in the preparation of a drug for treating cancer.
  • the present application also provides the use of an anti-CD40 antibody or its antigen-binding part and anlotinib or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating cancer.
  • the present application also provides the use of the anti-CD40 antibody or its antigen-binding part, anlotinib or its pharmaceutically acceptable salt, and chemotherapy drugs in the preparation of drugs for treating cancer.
  • the present application also provides a method for treating cancer in a subject, which includes administering an effective amount of the drug combination of the first aspect to the subject.
  • the present application provides a method of treating cancer in a subject, comprising administering to the subject an effective amount of an anti-CD40 antibody or an antigen-binding portion thereof and an effective amount of a tyrosine kinase inhibitor; optionally Optionally, an effective amount of a chemotherapeutic drug is also included.
  • the present application provides methods of treating cancer in a subject, comprising administering to the subject an effective amount of an anti-CD40 antibody, or an antigen-binding portion thereof, and an effective amount of a VEGFR tyrosine kinase inhibitor; either Optionally, an effective amount of chemotherapeutic drugs is also included.
  • the present application provides a method for treating cancer in a subject, comprising administering to the subject an effective amount of an anti-CD40 antibody or an antigen-binding portion thereof and an effective amount of anlotinib or a pharmaceutically acceptable accepted salts; optionally, an effective amount of a chemotherapeutic drug is also included.
  • the present application provides a kit, which includes the drug combination of the first aspect.
  • the composition comprises (a) a first pharmaceutical composition, wherein an anti-CD40 antibody or an antigen-binding portion thereof is used as an active ingredient; and (b) a second pharmaceutical composition, wherein a tyrosine kinase inhibitor is used as an active ingredient.
  • Components Optionally, (c) a chemotherapeutic agent is also included.
  • the present invention comprises (a) a first pharmaceutical composition, wherein an anti-CD40 antibody or an antigen-binding portion thereof is used as an active ingredient; and (b) a second pharmaceutical composition, wherein a VEGFR tyrosine kinase inhibitor As an active ingredient; optionally, (c) chemotherapy drugs are also included.
  • the composition contains (a) a first pharmaceutical composition, wherein an anti-CD40 antibody or an antigen-binding portion thereof is used as an active ingredient; and (b) a second pharmaceutical composition, wherein anlotinib or its pharmaceutically Acceptable salts are used as active ingredients; optionally, (c) chemotherapeutic drugs are also included.
  • the kit is used to treat cancer.
  • the present application also provides a method for treating cancer, which comprises administering to a subject an effective amount of the anti-CD40 antibody of the present application or an antigen-binding portion thereof.
  • the present application also provides the use of the anti-CD40 antibody or the antigen-binding portion thereof of the present application for treating cancer.
  • the present application also provides the use of the anti-CD40 antibody or the antigen-binding part thereof in the preparation of a medicament for treating cancer.
  • the anti-CD40 antibody or its antigen-binding portion and the tyrosine kinase inhibitor are each in the form of a pharmaceutical composition, and can be administered simultaneously, sequentially and/or alternately.
  • the anti-CD40 antibody or its antigen-binding portion, the tyrosine kinase inhibitor and the chemotherapeutic drug are each in the form of a pharmaceutical composition, which can be administered simultaneously, sequentially and/or alternately.
  • the anti-CD40 antibody or its antigen-binding portion and the VEGFR tyrosine kinase inhibitor are each in the form of a pharmaceutical composition, and can be administered simultaneously, sequentially and/or alternately.
  • the anti-CD40 antibody or its antigen-binding portion, the VEGFR tyrosine kinase inhibitor and the chemotherapeutic drug are each in the form of a pharmaceutical composition, which can be administered simultaneously, sequentially and/or alternately.
  • the anti-CD40 antibody or its antigen-binding portion and anlotinib or its pharmaceutically acceptable salt are each in the form of a pharmaceutical composition, which can be administered simultaneously, sequentially and/or alternately. medicine.
  • the anti-CD40 antibody or its antigen-binding portion, anlotinib or its pharmaceutically acceptable salt, and the chemotherapeutic drug are each in the form of a pharmaceutical composition, which can be simultaneously, sequentially and/or or alternatively.
  • the anti-CD40 antibody or antigen-binding portion thereof and the tyrosine kinase inhibitor are each in the form of a pharmaceutical composition, and are administered at intervals.
  • the anti-CD40 antibody or its antigen-binding portion, the tyrosine kinase inhibitor, and the chemotherapeutic drug are each in the form of a pharmaceutical composition, and are administered at intervals.
  • the anti-CD40 antibody or its antigen-binding portion and the VEGFR tyrosine kinase inhibitor are each in the form of a pharmaceutical composition, and are administered at intervals.
  • the anti-CD40 antibody or its antigen-binding portion, the VEGFR tyrosine kinase inhibitor, and the chemotherapeutic drug are each in the form of a pharmaceutical composition, and are administered at intervals.
  • the anti-CD40 antibody or its antigen-binding portion and anlotinib or a pharmaceutically acceptable salt thereof are each in the form of a pharmaceutical composition, and are administered at intervals. medicine.
  • the anti-CD40 antibody or its antigen-binding portion, anlotinib or a pharmaceutically acceptable salt thereof, and the chemotherapeutic drug are each in the form of a pharmaceutical composition, and each is administered at intervals way of administering drugs.
  • the interval dosing includes a dosing period and a dosing period, and in the dosing period, it can be given once or more than once a day.
  • the drug is administered every day during the administration period, and then the administration is stopped for a period of time during the drug withdrawal period, followed by the administration period, and then the drug withdrawal period, which can be repeated many times.
  • the anti-CD40 antibody or antigen-binding portion thereof and the tyrosine kinase inhibitor are administered in the same or different dosage regimens, respectively. In some embodiments, in the above uses or methods, the anti-CD40 antibody or antigen-binding portion thereof and the tyrosine kinase inhibitor are administered in different dosage regimens. In some embodiments, in the above-mentioned uses or methods, the anti-CD40 antibody or its antigen-binding portion, the tyrosine kinase inhibitor and the chemotherapeutic drug are administered in the same or different dosage regimens, respectively. In some embodiments, in the above uses or methods, the anti-CD40 antibody or antigen-binding portion thereof, the tyrosine kinase inhibitor and the chemotherapy drug are administered in different dosage regimens.
  • the anti-CD40 antibody or its antigen-binding portion and the VEGFR tyrosine kinase inhibitor are administered in the same or different dosage regimens, respectively. In some embodiments, in the above uses or methods, the anti-CD40 antibody or antigen-binding portion thereof and the VEGFR tyrosine kinase inhibitor are administered in different dosage regimens. In some embodiments, in the above uses or methods, the anti-CD40 antibody or its antigen-binding portion, the VEGFR tyrosine kinase inhibitor, and the chemotherapeutic drug are administered in the same or different dosage regimens, respectively. In some embodiments, in the above uses or methods, the anti-CD40 antibody or the antigen-binding portion thereof, the VEGFR tyrosine kinase inhibitor and the chemotherapeutic drug are administered in different dosage regimens.
  • the anti-CD40 antibody or antigen-binding portion thereof and anlotinib or a pharmaceutically acceptable salt thereof are administered in the same or different dosage regimens, respectively. In some embodiments, in the above uses or methods, the anti-CD40 antibody or antigen-binding portion thereof and anlotinib or a pharmaceutically acceptable salt thereof are administered in different dosage regimens, respectively. In some embodiments, in the above uses or methods, the anti-CD40 antibody or antigen-binding portion thereof, anlotinib or a pharmaceutically acceptable salt thereof, and chemotherapy drugs are administered in the same or different dosage regimens, respectively. In some embodiments, in the above uses or methods, the anti-CD40 antibody or antigen-binding portion thereof, anlotinib or a pharmaceutically acceptable salt thereof, and chemotherapy drugs are administered in different dosage regimens.
  • the anti-CD40 antibody or antigen-binding portion thereof may be administered once every week (q1w), every 2 weeks (q2w), every 3 weeks (q3w), or every 4 weeks (q4w) .
  • the anti-CD40 antibody or antigen-binding portion thereof may be administered every week ( ⁇ 3 days), every 2 weeks ( ⁇ 3 days), every 3 weeks ( ⁇ 3 days), or every 4 weeks Administer once a week ( ⁇ 3 days).
  • the anti-CD40 antibody or antigen-binding portion thereof may be administered every 3 weeks ( ⁇ 3 days).
  • the anti-CD40 antibody or antigen-binding portion thereof is administered at a dose of 0.001 to 100 mg/kg body weight.
  • the anti-CD40 antibody or antigen-binding portion thereof is administered in a uniform dose effective to treat said cancer.
  • the unitary dose is in the range of about 1 mg to about 1000 mg of an anti-CD40 antibody or antigen-binding portion thereof.
  • anlotinib or a pharmaceutically acceptable salt thereof is treated every 1 week, every 2 weeks, every 3 weeks, or every 4 weeks as a treatment cycle. In some embodiments, in the above-mentioned uses or methods, anlotinib or a pharmaceutically acceptable salt thereof is administered every 1 week ( ⁇ 3 days), every 2 weeks ( ⁇ 3 days), every 3 weeks ( ⁇ 3 days) ) or every 4 weeks ( ⁇ 3 days) as a treatment cycle. In a specific embodiment, anlotinib or a pharmaceutically acceptable salt thereof is treated every 3 weeks ( ⁇ 3 days) as a treatment cycle.
  • the anlotinib or a pharmaceutically acceptable salt thereof can be administered at intervals, for example, continuous administration for 2 weeks with rest for 2 weeks, continuous administration for 2 weeks with rest 1 week of administration, or continuous administration for 5 days and withdrawal for 2 days; the interval administration method can be repeated several times.
  • the daily dose of anlotinib or a pharmaceutically acceptable salt thereof administered is 3 mg to 30 mg. In some embodiments, anlotinib or a pharmaceutically acceptable salt thereof is administered at a daily dose of 5 mg to 20 mg. In some embodiments, anlotinib or a pharmaceutically acceptable salt thereof is administered at a daily dose of 8 mg to 16 mg. In some embodiments, the daily dose of anlotinib or a pharmaceutically acceptable salt thereof administered is 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg and 16 mg .
  • anlotinib or a pharmaceutically acceptable salt thereof is administered at a daily dose of 8 mg. In a specific embodiment, the daily dose of anlotinib or a pharmaceutically acceptable salt thereof administered is 10 mg. In a specific embodiment, the daily dose of anlotinib or a pharmaceutically acceptable salt thereof administered is 12 mg. In some embodiments, anlotinib or a pharmaceutically acceptable salt thereof may be administered one or more times daily. In some embodiments, anlotinib or a pharmaceutically acceptable salt thereof may be administered once daily. In some embodiments, anlotinib or a pharmaceutically acceptable salt thereof is administered once daily in an oral solid formulation.
  • anlotinib or a pharmaceutically acceptable salt thereof can be dosed once a day at 6 mg, 8 mg, 10 mg or 12 mg, administered continuously for 2 weeks and administered for 1 week Program dosing.
  • anlotinib or a pharmaceutically acceptable salt thereof can be administered once a day at a dose of 6 mg, 8 mg, 10 mg or 12 mg for 2 consecutive weeks with a break of 2 weeks. Program dosing.
  • the anti-CD40 antibody or antigen-binding portion thereof and the tyrosine kinase inhibitor have the same or different treatment cycles, respectively.
  • the anti-CD40 antibody or its antigen-binding portion and the tyrosine kinase inhibitor have the same treatment cycle, for example, every 1 week ( ⁇ 3 days), every 2 weeks ( ⁇ 3 days) ), every 3 weeks ( ⁇ 3 days) or every 4 weeks ( ⁇ 3 days) is a treatment cycle.
  • the anti-CD40 antibody or antigen-binding portion thereof and the tyrosine kinase inhibitor are treated every 3 weeks ( ⁇ 3 days) as a treatment cycle.
  • the anti-CD40 antibody or antigen-binding portion thereof, the tyrosine kinase inhibitor and the chemotherapy drug have the same or different treatment cycles, respectively.
  • the anti-CD40 antibody or its antigen-binding portion, the tyrosine kinase inhibitor and the chemotherapeutic drug have the same treatment cycle, for example, every 1 week ( ⁇ 3 days), every 2 weeks ( ⁇ 3 days), every 3 weeks ( ⁇ 3 days) or every 4 weeks ( ⁇ 3 days) is a treatment cycle.
  • the anti-CD40 antibody or antigen-binding portion thereof, the tyrosine kinase inhibitor and the chemotherapeutic drug are treated every 3 weeks ( ⁇ 3 days) as a treatment cycle.
  • the anti-CD40 antibody or antigen-binding portion thereof and the VEGFR tyrosine kinase inhibitor have the same or different treatment periods, respectively.
  • the anti-CD40 antibody or its antigen-binding portion and the VEGFR tyrosine kinase inhibitor have the same treatment cycle, for example, every 1 week ( ⁇ 3 days), every 2 weeks ( ⁇ 3 days), days), every 3 weeks ( ⁇ 3 days) or every 4 weeks ( ⁇ 3 days) was a treatment cycle.
  • the anti-CD40 antibody or antigen-binding portion thereof and the VEGFR tyrosine kinase inhibitor are treated every 3 weeks ( ⁇ 3 days) as a treatment cycle.
  • the anti-CD40 antibody or antigen-binding portion thereof, the VEGFR tyrosine kinase inhibitor and the chemotherapy drugs have the same or different treatment cycles, respectively.
  • the anti-CD40 antibody or its antigen-binding portion, the VEGFR tyrosine kinase inhibitor and the chemotherapy drug have the same treatment cycle, for example, every 1 week ( ⁇ 3 days), every 2 weeks ( ⁇ 3 days), every 3 weeks ( ⁇ 3 days) or every 4 weeks ( ⁇ 3 days) is a treatment cycle.
  • the anti-CD40 antibody or antigen-binding portion thereof, the VEGFR tyrosine kinase inhibitor and chemotherapy drugs are treated every 3 weeks ( ⁇ 3 days) as a treatment cycle.
  • the anti-CD40 antibody or its antigen-binding portion and anlotinib or a pharmaceutically acceptable salt thereof have the same or different treatment cycles, respectively.
  • the anti-CD40 antibody or its antigen-binding portion and anlotinib or a pharmaceutically acceptable salt thereof have the same treatment cycle, for example, every 1 week ( ⁇ 3 days), every 2 weeks ( ⁇ 3 days), every 3 weeks ( ⁇ 3 days) or every 4 weeks ( ⁇ 3 days) was a treatment cycle.
  • the anti-CD40 antibody or antigen-binding portion thereof and anlotinib or a pharmaceutically acceptable salt thereof are treated every 3 weeks ( ⁇ 3 days) as a treatment cycle.
  • the anti-CD40 antibody or antigen-binding portion thereof, anlotinib or a pharmaceutically acceptable salt thereof, and chemotherapy drugs have the same or different treatment cycles, respectively.
  • the anti-CD40 antibody or its antigen-binding portion, anlotinib or a pharmaceutically acceptable salt thereof, and the chemotherapeutic drug have the same treatment cycle, for example, every week ( ⁇ 3 days) ), every 2 weeks ( ⁇ 3 days), every 3 weeks ( ⁇ 3 days) or every 4 weeks ( ⁇ 3 days) is a treatment cycle.
  • anti-CD40 antibody or antigen-binding portion thereof, anlotinib or a pharmaceutically acceptable salt thereof, and chemotherapy drugs are treated every 3 weeks ( ⁇ 3 days) as a treatment cycle.
  • the anti-CD40 antibody or antigen-binding portion thereof comprises:
  • VH CDR1, VH CDR2 and VH CDR3 comprise and SEQ ID NOs respectively :
  • the amino acid sequences shown in 1, 4 and 7 have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% %, 98%, 99% or 100% identity amino acid sequence;
  • V H CDR1, V H CDR2 and V H CDR3 respectively comprise amino acid sequences with SEQ ID NOs: 2, 5 and 8 at least 85 %, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to amino acids or
  • VH CDR1, VH CDR2, and VH CDR3 comprise at least 85%, 86%, 87%, 88%, and 89% of the amino acids
  • V L CDR1, V L CDR2 and V L CDR3 comprise and SEQ ID NOs respectively :
  • the amino acid sequences shown in 10, 13 and 16 have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% %, 98%, 99% or 100% identity amino acid sequence;
  • V L CDR1, V L CDR2 and V L CDR3 respectively comprise the amino acid sequence shown in SEQ ID NOs: 11, 14 and 17 with at least 85 %, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to amino acids sequence;
  • V L CDR1, V L CDR2 and V L CDR3 respectively comprise at least 85%, 86%, 87%, 88%, 89%
  • the anti-CD40 antibody, or antigen-binding portion thereof comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising VH CDR1, VH CDR2, and VH CDR3, wherein The light chain variable region comprises V L CDR1, V L CDR2 and V L CDR3, wherein, (1) V H CDR1, V H CDR2, V H CDR3, V L CDR1, V L CDR2 and V L CDR3 respectively comprise and The amino acid sequences shown in SEQ ID NOs: 1, 4, 7, 10, 13 and 16 have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% %, 95%, 96%, 97%, 98%, 99% or 100% identity amino acid sequence; (2) VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL The CDR3 comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
  • the anti-CD40 antibody or antigen-binding portion thereof comprises a heavy chain variable region and a light chain variable region comprising: (1) the same as SEQ ID NO:
  • the anti-CD40 antibody, or antigen-binding portion thereof comprises a heavy chain comprising a heavy chain variable region and a heavy chain constant region, and a light chain comprising a light chain variable region and a light chain
  • the light chain constant region wherein the C-terminal of the heavy chain variable region is connected to the N-terminal of the heavy chain constant region, and the C-terminal of the light chain variable region is connected to the N-terminal of the light chain constant region, wherein the heavy chain variable region and the light chain
  • the chain variable region comprises the above-mentioned amino acid sequence
  • the heavy chain constant region can be a human IgG2 constant region having the amino acid sequence shown in SEQ ID NO:28, or a human IgG1 constant region having the amino acid sequence shown in SEQ ID NO:27
  • the light chain constant region may be a human kappa constant region having an amino acid sequence as shown in SEQ ID NO:29.
  • Heavy chain constant regions, such as the Fc fragment can be engineered to have reduced or
  • the anti-CD40 antibody comprises two heavy chains and two light chains, or consists of two heavy chains and two light chains, wherein each heavy chain comprises the above-mentioned heavy chain constant region, heavy chain variable region or CDR sequences, and each light chain comprises the light chain constant region, light chain variable region or CDR sequences described above.
  • the anti-CD40 antibody of the present application may be a full-length antibody, such as a full-length antibody of the IgG1, IgG2 or IgG4 isotype.
  • the anti-CD40 antibodies of the present application, or antigen-binding portions thereof may be single chain variable region (scFv) antibodies, or antibody fragments, such as Fab or F(ab')2 fragments.
  • an anti-CD40 antibody of the disclosure, or an antigen-binding portion thereof binds CD40 (eg, human CD40 and monkey CD40). In some embodiments, an anti-CD40 antibody of the present application, or an antigen-binding portion thereof, blocks the binding of CD40 to CD40L. In some embodiments, an anti-CD40 antibody of the present application, or an antigen-binding portion thereof, activates CD40 signaling.
  • CD40 eg, human CD40 and monkey CD40
  • an anti-CD40 antibody of the present application, or an antigen-binding portion thereof blocks the binding of CD40 to CD40L. In some embodiments, an anti-CD40 antibody of the present application, or an antigen-binding portion thereof, activates CD40 signaling.
  • the anti-CD40 antibody of the present application is a humanized monoclonal antibody.
  • the anti-CD40 antibodies of the present application may be, for example, chimeric monoclonal antibodies.
  • the amino acid sequence IDs of the heavy/light chain variable regions of exemplary anti-CD40 antibodies or antigen-binding portions thereof of the present application are summarized in Table 1 below, some antibodies having the same VH or VL .
  • the heavy chain constant region of the antibody may be a human IgG1 or IgG2 heavy chain constant region, which have the amino acid sequences shown in SEQ ID NO: 27 and 28, respectively, and the light chain constant region of the antibody may be a human kappa constant region, which has Amino acid sequence as shown in SEQ ID NO:29.
  • These antibodies may also comprise a mouse IgGl or IgG2 heavy chain constant region, and/or a mouse kappa constant region.
  • CDR regions may also be determined by other numbering systems such as Chothia, IMGT, AbM or Contact numbering systems/methods based on heavy/light chain variable region sequences, as is well known in the art.
  • VH and VL sequences (or CDR sequences) of other anti-CD40 antibodies that bind to human CD40 can be "mixed and paired" with the VH and VL sequences (or CDR sequences) of the anti-CD40 antibodies of the present application.
  • VH and VL chains (or CDRs in these chains) are mixed and paired, the VH sequence from a particular VH / VL pair is replaced by a structurally similar VH sequence.
  • an antibody of the present application or an antigen-binding portion thereof, comprises:
  • an antibody of the present application or an antigen-binding portion thereof, comprises:
  • an antibody of the present application comprises the heavy chain variable region CDR2 of an anti-CD40 antibody of the present application and the CDRs of other antibodies that bind human CD40, such as heavy chains from another anti-CD40 antibody.
  • the CDR3 domain alone can determine the binding specificity of an antibody for a cognate antigen, and based on the common CDR3 sequence can predictively generate antibodies with the same Multiple antibodies with binding specificities.
  • the antibody of the present application comprises the heavy chain variable region CDR2 of the anti-CD40 antibody of the present application and at least the heavy chain variable region and/or light chain variable region CDR3 of the anti-CD40 antibody of the present application , or the heavy chain variable region and/or the light chain variable region CDR3 of another anti-CD40 antibody, wherein the antibody specifically binds human CD40.
  • These antibodies preferably (a) compete for CD40 binding with the anti-CD40 antibodies of the application; (b) retain functional properties; (c) bind the same epitope; and/or (d) have similar binding affinities.
  • the antibody of the present application may also comprise the light chain variable region CDR2 of the anti-CD40 antibody of the present application, or the light chain variable region CDR2 of another anti-CD40 antibody, wherein the antibody specifically binds to human CD40.
  • the antibody of the present application may also comprise the heavy chain variable region and/or the light chain variable region CDR1 of the anti-CD40 antibody of the present application, or the heavy chain variable region and the light chain variable region of another anti-CD40 antibody. and/or light chain variable region CDR1, wherein the antibody specifically binds human CD40.
  • the antibody of the present application comprises a heavy chain variable region and/or a light chain variable region, and the heavy chain variable region and the light chain variable region respectively comprise CDR1, CDR2 and CDR3, wherein these The CDR sequences differ from the CDR sequences of the anti-CD40 antibodies of the present application by one or more conservative modifications. It is understood in the art that certain conservative sequence modifications do not abolish antigen binding.
  • the antibody of the present application comprises a heavy chain variable region and/or a light chain variable region, said heavy chain variable region and light chain variable region comprising CDR1, CDR2 and CDR3 sequences, respectively, wherein :
  • the heavy chain variable region CDR1 sequence comprises the sequences listed in Table 1, and/or conservative modifications thereof; and/or
  • the heavy chain variable region CDR2 sequence comprises the sequences listed in Table 1, and/or conservative modifications thereof; and/or
  • the heavy chain variable region CDR3 sequence comprises the sequences listed in Table 1, and/or conservative modifications thereof; and/or
  • light chain variable region CDR1, and/or CDR2, and/or CDR3 sequence comprises the sequence listed in Table 1, and/or its conservative modification;
  • the antibody specifically binds human CD40.
  • conservative sequence modification refers to amino acid modifications that do not significantly affect or alter the binding properties of an antibody. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the antibodies of the present application by standard techniques known in the art, such as point mutations and PCR-mediated mutations. Conservative amino acid substitutions refer to the substitution of amino acid residues for amino acid residues with similar side chains. Groups of amino acid residues having similar side chains are known in the art.
  • amino acid residues include those with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., For example, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (for example, alanine, valine, leucine acid, isoleucine, proline, phenylalanine, methionine), ⁇ -branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains ( For example, amino acids of tyrosine, phenylalanine, tryptophan, histidine). Therefore, one or more amino acid residues in the CDR regions of the antibodies of the present application can be replaced by other amino acid residues from the same side chain group, and the resulting antibodies can be tested to
  • the anti-CD40 antibody or antigen-binding portion thereof is selected from the group consisting of: Dacetuzumab, Lucatumumab, APX005M, CDX-1140 (Celldex), GEN-1042 (BioNTech; Genmab), Selicrelumab, SEA-CD40 (Seagen) , 2141-v11 (AbbVie), ChiLob7/4 (BioNTech), Mitazalimab, Vanalimab, Giloralimab, LVGN-7409 (Lijin Bio), LVGN-7408 (Lijin Bio), YH003 (Yuhe Pharmaceutical), SHR-1704 ( Hengrui Medicine), MIL97 (Tianguangshi Biology), Iscalimab, Bleselumab, Ravagalimab, or the antigen-binding portion of the above antibodies.
  • compositions of anti-CD40 antibodies or antigen-binding portions thereof are provided.
  • the single dose of the pharmaceutical composition of an anti-CD40 antibody or antigen-binding portion thereof comprises 2 mg to 20 mg, for example 2 mg, 4 mg, 6 mg, 8 mg, 10 mg, 12 mg, 14 mg, 16 mg, 18 mg or 20 mg of the anti-CD40 antibody.
  • CD40 antibodies or antigen-binding portions thereof comprises 2 mg to 20 mg, for example 2 mg, 4 mg, 6 mg, 8 mg, 10 mg, 12 mg, 14 mg, 16 mg, 18 mg or 20 mg of the anti-CD40 antibody.
  • the pharmaceutical composition of an anti-CD40 antibody or antigen-binding portion thereof comprises an anti-CD40 antibody or antigen-binding portion thereof and any number of excipients.
  • Excipients that can be used include carriers, surfactants, thickening or emulsifying agents, solid binders, dispersion or suspending agents, solubilizers, coloring agents, flavoring agents, coatings, disintegrants, lubricants, Sweeteners, preservatives, isotonic agents and combinations thereof.
  • the selection and use of suitable excipients is taught in Gennaro, ed., Remington: The Science and Practice of Pharmacy, 20th Ed. (Lippincott Williams & Wilkins 2003), the disclosure of which is incorporated herein by reference.
  • compositions of anti-CD40 antibodies or antigen-binding portions thereof are suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (eg, by injection or infusion).
  • the active ingredient can be coated in a material that protects it from acids and other natural conditions that might inactivate it.
  • parenteral administration refers to modes of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intrathecal, intraorbital, cardiac Intratracheal, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.
  • compositions of anti-CD40 antibodies or antigen-binding portions thereof may be administered parenterally, eg, topically, epidermally or mucosally, such as intranasally, orally, vaginally, rectally, sublingually or topically. Administration can also be performed, eg, once, multiple times, and/or over one or more extended periods of time.
  • the pharmaceutical composition of the anti-CD40 antibody or an antigen-binding portion thereof is a water-soluble injection
  • the water-soluble injection includes but is not limited to a non-lyophilized water-soluble preparation or a lyophilized powder. structured water-soluble preparations.
  • the pharmaceutical composition of the anti-CD40 antibody or antigen-binding portion thereof is a lyophilized formulation.
  • the lyophilized preparation refers to a preparation in which an aqueous solution undergoes a freeze-drying process, in which the substance is first frozen, then the amount of solvent is reduced by sublimation (primary drying process), and then the amount of solvent is reduced by desorption (secondary drying process) until the amount of solvent reaches a value that no longer supports biological activity or chemical reaction.
  • the lyophilized formulations of the present application can also be dried by other methods known in the art, such as spray drying and bubble drying.
  • anlotinib ie, the compound of formula (I)
  • the chemical name of the anlotinib is 1-[[[4-(4-fluoro-2-methyl-1H-indol-5-yl ) oxy-6-methoxyquinolin-7-yl] oxy] methyl] cyclopropylamine, which has the following structural formula:
  • Anlotinib can be administered in its free base form or in the form of its salts, hydrates and prodrugs which are converted into the free base form of anlotinib in vivo.
  • pharmaceutically acceptable salts of anlotinib which can be produced from various organic and inorganic acids according to methods well known in the art, are also within the scope of the present invention.
  • the pharmaceutically acceptable salt of anlotinib is a salt formed by the compound of formula (I) and any of the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, trifluoroacetic acid, propionic acid, Caproic acid, heptanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, almond acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, p-chlorobenzenesulfonic acid, p-toluenesulfonic acid, 3-phenylpropionic acid, trimethyl G
  • the administration is as anlotinib hydrochloride. In some embodiments, the administration is as anlotinib monohydrochloride. In some embodiments, the administration is as anlotinib dihydrochloride. In some embodiments, the crystalline form of anlotinib hydrochloride is administered. In a specific embodiment, the crystalline form of anlotinib dihydrochloride is administered.
  • anlotinib or its salts involved in this application are calculated based on the free base of anlotinib.
  • composition of anlotinib or pharmaceutically acceptable salt thereof is provided.
  • the single dose of the pharmaceutical composition of anlotinib or a pharmaceutically acceptable salt thereof comprises 6 mg, 8 mg, 10 mg, or 12 mg of anlotinib.
  • the pharmaceutical composition of anlotinib or a pharmaceutically acceptable salt thereof includes, but is not limited to, formulations suitable for oral, parenteral, and topical administration.
  • the pharmaceutical composition of anlotinib or a pharmaceutically acceptable salt thereof is preferably a formulation suitable for oral administration, including tablets, capsules, powders, granules, dropping pills, pastes, powders, etc. , preferably tablets and capsules.
  • the tablet can be ordinary tablet, dispersible tablet, effervescent tablet, sustained-release tablet, controlled-release tablet or enteric-coated tablet
  • the capsule can be ordinary capsule, sustained-release capsule, controlled-release capsule or enteric-coated capsule.
  • the oral preparations can be prepared by conventional methods using pharmaceutically acceptable carriers known in the art.
  • Pharmaceutically acceptable carriers include fillers, absorbents, wetting agents, binders, disintegrants, lubricants and the like.
  • Fillers include starch, lactose, mannitol, microcrystalline cellulose, etc.; absorbents include calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.; wetting agents include water, ethanol, etc.; binders include hypromellose, poly Vitone, microcrystalline cellulose, etc.; disintegrants include croscarmellose sodium, crospovidone, surfactants, low-substituted hydroxypropyl cellulose, etc.; lubricants include magnesium stearate, talc powder, polyethylene glycol, sodium lauryl sulfate, micronized silica gel, talcum powder, etc. Pharmaceutical excipients also include colorants, sweeteners, etc.
  • the pharmaceutical composition of anlotinib or a pharmaceutically acceptable salt thereof is a solid preparation suitable for oral administration.
  • the pharmaceutical composition of anlotinib or a pharmaceutically acceptable salt thereof may be in the form of tablets or capsules.
  • the pharmaceutical composition of anlotinib or a pharmaceutically acceptable salt thereof is in the form of a capsule.
  • pharmaceutically acceptable carriers for oral solid formulations include mannitol, microcrystalline cellulose, hyprolose, magnesium stearate.
  • the third therapeutic agent may be a cancer therapeutic agent known in the art, such as a chemotherapeutic drug.
  • the chemical agent is an alkylating agent, podophyllin-type drugs, platinum-type drugs, fluoropyrimidine derivatives, cytosine derivatives, taxane-type drugs, camptothecin-type drugs, anthracycline-type drugs , one or more of vinblastine drugs.
  • Chemotherapy drugs described in the application also include one or more of pemetrexed, mitomycin, methotrexate, mitoxantrone, bendamustine, temozolomide, Sapacitabine, S-1 and encequidar kind.
  • the alkylating agent is one or more of cyclophosphamide, ifosfamide, carmustine, and melphalan.
  • the podophylloid drug is one or more of etoposide, teniplatin, and epipodophyllotoxin glucopyranoside.
  • the cytosine derivative is one or more of cytarabine, gemcitabine, and azacitidine.
  • the platinum drug is one or more of oxaliplatin, cisplatin, carboplatin, nedaplatin and dicycloplatin.
  • the fluoropyrimidine derivative is one or more of gemcitabine, capecitabine, fluorouracil, bisfururacil, doxifluridine, tegafur, carmofur and trifluridine kind.
  • the taxane is one or more of paclitaxel, albumin-bound paclitaxel, and docetaxel.
  • the camptothecins are one or more of camptothecin, hydroxycamptothecin, irinotecan and topotecan.
  • the anthracycline is one or more of doxorubicin, epirubicin, daunorubicin, pirarubicin, amrubicin and aclarmycin.
  • the vinblastines are one or more of vinorelbine, vinblastine, vincristine, vinblastine and vinblastine.
  • the chemotherapeutic agent is administered according to known administration methods (including dosage, route of administration, and regimen).
  • cancer refers to any malignant and/or invasive growth or tumor arising from abnormal cell growth, including solid tumors, blood, bone marrow, or lymphatic system named after the type of cells from which they form.
  • cancer includes, but is not limited to, primary cancers that arise from a specific site in the body, metastatic cancers that have spread from their original location to other parts of the body, recurrences of the original primary cancer after remission, and second primary cancers ( A new primary cancer in a person with a previous history of a different type of cancer).
  • the cancer described herein is a solid tumor.
  • the solid tumors include, but are not limited to: skin cancers such as melanoma and sarcoma, pancreatic cancer, colorectal cancer such as colorectal adenocarcinoma, esophageal cancer, gastrointestinal cancer, prostate cancer, bladder cancer, renal cancer, ovarian cancer, uterine cancer such as cervical cancer and endometrial cancer, breast cancer such as triple-negative breast cancer, lung cancer such as small cell lung cancer and non-small cell lung cancer, thyroid cancer, nasopharyngeal cancer and liver cancer.
  • the cancer described herein is a B cell malignancy.
  • B-cell malignancy includes any malignancy derived from cells of the B-cell lineage.
  • the B-cell malignancies include leukemias and lymphomas, including but not limited to non-Hodgkin's lymphoma, chronic lymphocytic leukemia, multiple myeloma, B-cell lymphoma, high-grade B-cell lymphoma , intermediate-grade B-cell lymphoma, low-grade B-grade lymphoma, B-cell acute lymphoblastic leukemia, Hodgkin's lymphoma, plasmacytoma, follicular lymphoma, follicular small-cleaved lymphoma, follicular large Cellular lymphoma, mixed follicular lymphoma, diffuse small lymphocytic lymphoma, small lymphocytic lymphoma, prolymphocytic leukemia, lymphoplasm
  • the cancer described herein is a non-B cell hematological malignancy.
  • the non-B cell hematological malignancies include, but are not limited to, chronic myeloid leukemia and acute myeloid leukemia.
  • the components in the pharmaceutical combination of the present application can be administered independently, or some or all of them can be administered in suitable various ways, including but not limited to, oral or parenteral (by intravenous, intramuscular, topical or subcutaneous route) ) application.
  • the components of the pharmaceutical combination of the present application can be administered independently, or some or all of them can be administered orally or injected together, such as intramuscular injection, intravenous injection, subcutaneous injection, intratumoral injection or intraperitoneal injection.
  • the components in the pharmaceutical combination of the present application can be prepared independently, or part or all of them together into suitable dosage forms, including but not limited to, tablets, buccal tablets, pills, capsules (such as hard capsules, soft capsules, enteric-coated capsules, microcapsules), elixirs, granules, syrups, injections (such as intramuscular, intravenous, subcutaneous, intratumoral or intraperitoneal), granules, emulsions, suspensions, solutions, dispersions and Sustained-release preparations for oral or parenteral administration.
  • suitable dosage forms including but not limited to, tablets, buccal tablets, pills, capsules (such as hard capsules, soft capsules, enteric-coated capsules, microcapsules), elixirs, granules, syrups, injections (such as intramuscular, intravenous, subcutaneous, intratumoral or intraperitoneal), granules, emulsions, suspensions, solutions, dispersions and Sustained
  • the components in the pharmaceutical combination of the present application may each independently, or part or all of them jointly contain pharmaceutically acceptable carriers and/or excipients.
  • the treated patients have a longer survival period (such as median survival period, progression-free survival period or overall survival period);
  • CD40 refers to tumor necrosis factor receptor superfamily member 5 (TNFR5).
  • TNFR5 tumor necrosis factor receptor superfamily member 5
  • CD40 includes variants, isoforms, homologs, orthologs and paralogs.
  • an antibody specific for human CD40 protein may cross-react with CD40 protein of a species other than human (eg, monkey).
  • antibodies specific for human CD40 protein may be fully specific for human CD40 protein and not cross-reactive with proteins of other species or types, or may be specific for human CD40 protein derived from certain but not all other species. Species CD40 cross-reacts.
  • human CD40 refers to a CD40 protein having a human amino acid sequence, for example, the amino acid sequence of human CD40 with Genbank accession number NP_001241.1 (Amini M et al., (2020) Life Sci 254:117774).
  • the terms “monkey or rhesus CD40” and “mouse CD40” refer to monkey CD40 sequence and mouse CD40 sequence, respectively, eg, which have the amino acid sequences of Genbank Accession Nos. NP_001252791.1 and NP_035741.2, respectively.
  • immune response refers to the selective damage, destruction or elimination in the body of lymphocytes, antigen-presenting cells, phagocytes, granulocytes, and soluble macromolecules (including antibodies, cytokines, and complement) produced by these cells or the liver.
  • soluble macromolecules including antibodies, cytokines, and complement
  • antibody refers to an immunoglobulin molecule that recognizes and specifically binds a target (eg, CD40) through at least one antigen binding site, typically located within the variable region of the immunoglobulin molecule.
  • a target eg, CD40
  • antibody encompasses intact polyclonal antibodies, intact monoclonal antibodies, single chain Fv (scFv) antibodies, heavy chain antibodies (HCAbs), light chain antibodies (LCAbs), multispecific antibodies, bispecific antibodies , monospecific antibodies, monovalent antibodies, fusion proteins comprising the antigen binding site of an antibody, and any other modified immunoglobulin molecule comprising an antigen binding site (e.g., a dual variable domain immunoglobulin molecule), It is sufficient as long as the antibody exhibits the desired biological activity.
  • scFv single chain Fv
  • HCAbs heavy chain antibodies
  • LCAbs light chain antibodies
  • multispecific antibodies bispecific antibodies , monospecific antibodies, monovalent antibodies, fusion proteins comprising the antigen binding site of an antibody,
  • Antibodies also include, but are not limited to, mouse antibodies, chimeric antibodies, humanized antibodies, and human antibodies.
  • Antibodies can be of any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, depending on the characteristics of the constant regions of their heavy chains (termed alpha, delta, epsilon, gamma, and mu, respectively). or a subclass (isotype) thereof (eg IgGl, IgG2, IgG3, IgG4, IgAl and IgA2).
  • Different classes of immunoglobulins have different subunit structures and three-dimensional configurations that are well known in the art.
  • Antibodies can be naked antibodies or conjugated to other molecules, including but not limited to toxins and radioisotopes. Unless expressly stated otherwise, the term "antibody” as used herein includes an "antigen-binding portion" of an intact antibody.
  • IgG is a glycoprotein comprising two heavy (H) chains and two light (L) chains, wherein the heavy and light chains are linked by disulfide bonds.
  • Each heavy chain is composed of a heavy chain variable region (abbreviated as VH ) and a heavy chain constant region.
  • the heavy chain constant region consists of three domains, CH1 , CH2 and CH3 .
  • Each light chain is composed of a light chain variable region (abbreviated as V L ) and a light chain constant region.
  • the light chain constant region consists of one domain, CL .
  • the VH and VL regions can be further divided into hypervariable regions called complementarity determining regions (CDRs), which are separated by more conserved framework region (FR) regions.
  • CDRs complementarity determining regions
  • FR conserved framework region
  • Each VH and VL consists of three CDRs and four FRs, arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminal to the carboxyl terminal.
  • the variable regions of the heavy and light chains contain the binding domains that interact with the antigen.
  • the constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various immune system cells (eg, effector cells) and the first component (Clq) of the classical complement system.
  • the "antigen-binding portion" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (eg, CD40 protein). It has been demonstrated that the antigen-binding function of antibodies can be achieved by fragments of full-length antibodies.
  • binding fragments encompassed within the "antigen-binding portion" of an antibody include: (i) Fab fragments, monovalent fragments composed of VL, VH , CL and CH1 domains; ( ii) F(ab') 2 fragments, a bivalent fragment comprising two Fab fragments connected by a disulfide bond at the hinge region; (iii) an Fd fragment composed of VH and CH1 domains; (iv) an antibody single-arm VL and VH (v) dAb fragments (Ward et al., (1989) Nature 341:544-546) composed of VH domains; (vi) isolated complementarity determining regions (CDRs); and ( vii) Nanobodies, ie a heavy chain variable region comprising a single variable domain and two constant domains.
  • the two domains VL and VH of the Fv fragment are encoded by different genes, recombination can be used to link VH and VL into a single protein chain through a synthetic linker, in which the VL and VH regions are paired to form Monovalent molecules (termed single-chain Fv (scFv); see for example Bird et al., (1988) Science 242:423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879 -5883).
  • scFv single-chain Fv
  • Such single chain antibodies are also encompassed within the term "antigen-binding portion" of an antibody.
  • These antibody fragments can be obtained by common techniques known to those skilled in the art, and can be functionally screened in the same manner as whole antibodies.
  • isolated antibody refers to an antibody that is substantially free of other antibodies having different antigenic specificities (eg, an isolated antibody that specifically binds a CD40 protein is substantially free of antibodies that specifically bind an antigen other than a CD40 protein).
  • An isolated antibody that specifically binds human CD40 protein may, however, have cross-reactivity to other antigens, eg, CD40 proteins from other species.
  • an isolated antibody is substantially free of other cellular material and/or chemicals.
  • mouse antibody refers to an antibody in which both the framework and CDR regions of the variable domains are derived from mouse germline immunoglobulin sequences. Furthermore, if the antibody comprises a constant region, the constant region also is derived from mouse germline immunoglobulin sequences.
  • the mouse antibodies of the invention may comprise amino acid residues not encoded by mouse germline immunoglobulin sequences (eg, mutations introduced by random or point mutations in vitro, or by somatic mutation in vivo). However, the term “mouse antibody” as used herein does not include antibodies in which CDR sequences from other mammalian species are inserted into the sequence of the mouse framework region.
  • chimeric antibody refers to an antibody obtained by combining genetic material of non-human origin and genetic material of human origin. Or more generally, chimeric antibodies are antibodies that combine genetic material from one species with genetic material from another species.
  • humanized antibody refers to an antibody derived from a non-human species but whose protein sequence has been modified to increase its similarity to natural human antibodies.
  • Antibody that recognizes an antigen and "antibody specific/specific for an antigen” are used interchangeably herein with the term “antibody that specifically binds an antigen”.
  • an antibody or antigen-binding portion thereof that "specifically binds human CD40” refers to an antibody that binds human CD40 protein (and possibly CD40 protein of one or more non-human species) but does not substantially bind non-CD40 protein.
  • the antibody binds human CD40 protein with "high affinity", ie, binds human CD40 protein with a KD of 5.0 x 10 -8 M or less, preferably 1.0 x 10 -8 M or less.
  • substantially does not bind proteins or cells means does not bind to proteins or cells, or does not bind to them with high affinity, i.e. with 1.0 x 10 -6 M or greater, preferably 1.0 x 10 -5 M or greater, More preferably 1.0 x 10 -4 M or greater, more preferably 1.0 x 10 -3 M or greater, even more preferably 1.0 x 10 -2 M or greater KD binding protein or cell.
  • high affinity for an IgG antibody means that the antibody has 1.0 ⁇ 10 -6 M or less, preferably 9.0 ⁇ 10 -9 M or less, more preferably 5.0 ⁇ 10 -9 M for the target antigen or less, even more preferably a K D of 1.0 ⁇ 10 ⁇ 9 M or less, even more preferably 5.0 ⁇ 10 ⁇ 10 M or less.
  • high affinity binding may be different.
  • high affinity means that the antibody has a KD of 10" 6M or less, preferably 10 "7M or less, more preferably 10" 8M or less.
  • KD refers to the dissociation constant, which is the ratio of Kd to Ka (ie, Kd / Ka ) and is expressed in molarity (M).
  • KD value of an antibody can be determined using methods well known in the art. A preferred method of determining the KD value of an antibody is using surface plasmon resonance technology, preferably using a biosensor system, such as the Biacore TM system.
  • EC50 also known as the half-maximal effect concentration
  • IC50 refers to the concentration of an antibody that inhibits a specific biological or biochemical function by 50% relative to the absence of the antibody.
  • agonistic CD40 antibody or “agonistic anti-CD40 antibody” refers to an anti-CD40 antibody that binds to CD40 and activates or induces CD40 signaling (thus, for example, promoting immune cell activation and proliferation and cytokine and chemokine production) .
  • antagonist CD40 antibody/antagonist anti-CD40 antibody refers to an anti-CD40 antibody that blocks or inhibits CD40 signal transduction that may be induced by CD40L.
  • Agonistic CD40 antibodies by enhancing the antigen-presenting ability of APCs, activating tumor-specific CD4 + and CD8 + T cells, stimulating lymphocytes and monocytes to secrete cytokines and chemokines, enhancing tumorigenesis of cytotoxic lymphocytes and NK cells Cell killing effect, etc., to promote the innate and adaptive immune responses of tumor subjects to tumors.
  • Percent “identity” as used herein in the context of two or more nucleic acids or polypeptides means, when compared and aligned (with gaps introduced, if necessary) for the highest correspondence, which will or will not be conserved In the case of amino acid substitutions as part of sequence identity, two or more sequences or subsequences have the same or a specified percentage of nucleotide or amino acid residues that are identical. Percent identity can be measured using sequence alignment software or algorithms or by visual inspection. Various software and algorithms that can be used for the alignment of amino acid or nucleotide sequences are well known in the art, including but not limited to BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and variations thereof.
  • two nucleic acids or polypeptides described herein are substantially identical, meaning that when compared and aligned for highest correspondence, as measured using a sequence alignment algorithm or by visual inspection, the two nucleic acids or polypeptides are substantially identical.
  • Polypeptides have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments at least 95%, 96%, 97%, 98%, 99% nucleotide or amino acid Residue identity.
  • the term “pharmaceutical combination” refers to two or more active ingredients administered simultaneously or sequentially (administered in the form of the respective active ingredients themselves, or in the form of their respective pharmaceutically acceptable salts or esters, etc. administration in the form of derivatives, prodrugs or compositions).
  • drug combination and “drug combination” are used interchangeably herein.
  • the active ingredients may be administered to the patient each simultaneously as a single formulation, or each sequentially as a single formulation in any order.
  • treatment generally refers to manipulations to achieve a desired pharmacological and/or physiological effect.
  • the effect may be prophylactic in terms of complete or partial prevention of the disease or its symptoms; and/or therapeutic in terms of partial or complete stabilization or cure of the disease and/or side effects due to the disease.
  • treatment encompasses any treatment of a disease in a patient, including: (a) inhibiting the symptoms of the disease, ie arresting its development; or (b) relieving the symptoms of the disease, ie causing regression of the disease or symptoms.
  • the term "effective amount" means (i) treating a particular disease, condition or disorder, (ii) alleviating, ameliorating or eliminating one or more symptoms of a particular disease, condition or disorder, or (iii) preventing or delaying the The amount of a compound of the present application for the onset of one or more symptoms of a particular disease, condition or disorder as described.
  • the effective amount of the drug reduces the number of cancer cells, reduces the size of the tumor, inhibits (ie slows to some extent, preferably stops) the infiltration of cancer cells into surrounding organs, inhibits (ie slows to some extent, preferably stops) Tumor metastasis, some inhibition of tumor growth, and/or some alleviation of one or more symptoms associated with cancer.
  • a drug can inhibit the growth of cancer cells and/or kill existing cancer cells, it can be cytostatic and/or cytotoxic.
  • the amount of an active substance (such as a compound of the present application) that constitutes an "effective amount" can vary depending on factors such as the disease state, age, sex, and weight of the individual, and whether the therapeutic agent or combination of therapeutic agents elicits the desired response in the individual Ability. Effective amounts can also be routinely determined by those skilled in the art based on their own knowledge and this disclosure.
  • administering means physically introducing a composition comprising a therapeutic agent into a subject using any of a variety of methods and delivery systems known to those skilled in the art.
  • flat dose refers to a dose administered to a patient regardless of the patient's weight or body surface area (BSA).
  • Unit doses are therefore specified as absolute amounts of agent (eg, anti-CD40 antibody), rather than as mg/kg doses. For example, a 60kg person and a 100kg person will receive the same dose of antibody.
  • fixed combination means that the active ingredients (eg, anti-CD40 antibody or compound of formula (I)) are administered to a subject simultaneously in a fixed total dose or dose ratio, or in the form of a single entity, pharmaceutical composition or formulation.
  • active ingredients eg, anti-CD40 antibody or compound of formula (I)
  • non-fixed combination refers to the simultaneous, concurrent or sequential administration of two or more active components as separate entities (eg, pharmaceutical composition, formulation) to a subject without specific time limitation, wherein the administration to the subject The active ingredient reaches the therapeutically effective amount level.
  • examples of non-fixed combinations are cocktail therapy, eg administration of 3 or more active ingredients.
  • the individual active ingredients may be packaged, sold or administered as entirely separate pharmaceutical compositions.
  • the "non-fixed combination” also includes the combined use of "fixed combinations" or independent entities of any one or more active components.
  • pharmaceutically acceptable refers to those compounds, materials, compositions and/or dosage forms which, within the scope of sound medical judgment, are suitable for use in contact with human and animal tissues without excessive Toxicity, irritation, allergic reaction or other problems or complications, commensurate with a reasonable benefit/risk ratio.
  • salts of alkali ions and free acids or salts of acid ions and free bases including, for example, hydrochloride, hydrobromide, nitrate, sulfate, phosphate, ethyl salt, trifluoroacetate, fumarate, maleate, citrate, succinate, methanesulfonate, benzenesulfonate or p-toluenesulfonate, preferably hydrochloride, Hydrobromide, sulfate, acetate, trifluoroacetate, fumarate, maleate, methanesulfonate, p-toluenesulfonate, sodium salt, potassium salt, ammonium salt, Amino acid salts, etc.
  • the term “subject” or “patient” is used interchangeably herein.
  • the term “subject” or “patient” is a mammal.
  • the subject or patient is a mouse.
  • the subject or patient is a human.
  • “combination” or “combined use” means that two or more active substances may be administered to a subject simultaneously, each as a single formulation, or sequentially, each as a single formulation, in any order.
  • single dose refers to the smallest packaging unit containing a certain amount of medicine.
  • a box of medicine has seven capsules, and each capsule is a single dose; or each bottle of injection is a single dose.
  • multiple dose consists of a number of single doses.
  • pharmaceutical composition refers to a mixture of one or more active ingredients of the present application or its pharmaceutical combination and pharmaceutically acceptable excipients.
  • the purpose of the pharmaceutical composition is to facilitate administration of a compound of the present application, or a pharmaceutical combination thereof, to a subject.
  • pharmaceutical composition and “preparation” have the same meaning and are used interchangeably.
  • any concentration range, percentage range, ratio range, or integer range should be understood to include the value of any integer within the recited range and, where appropriate, fractions thereof (such as one-tenth of an integer). and percent), unless otherwise noted.
  • about or “approximately” means within an acceptable error range for a particular value as judged by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, about” Or “about” can mean within 1 or more than 1 standard deviation according to the practice in the art. Alternatively, “about” or “approximately” may mean a range of up to ⁇ 5%, such as within ⁇ 2%, within ⁇ 1%, or within ⁇ 0.5% of a given specific numerical range. When the application or When a specific value is given in the claims, unless otherwise stated, the meaning of about” or “approximately” should be considered as being within an acceptable error range for the specific value.
  • values of step parameters or conditions are modified by "about” by default.
  • Figures 1A-1C show the binding ability of chimeric antibodies 1A3 (A), 1D1 (B) and C1H1 (C) to human CD40 in a capture ELISA.
  • Figures 2A-2C show the activity of chimeric antibodies 1A3 (A), 1D1 (B) and C1H1 (C) to activate CD40 signaling in a cell-based reporter assay.
  • Figure 3 shows the binding ability of humanized antibodies huC1H1-V1 and huC1H1-V2 to human CD40 in capture ELISA.
  • Figure 4 shows the binding ability of humanized antibodies huC1H1-V1 and huC1H1-V2 to human CD40 expressing 293T cells in cell-based binding FACS.
  • Figure 5 shows the ability of humanized antibodies huC1H1-V1 and huC1H1-V2 to block the binding of human CD40 to CD40L in a competition ELISA.
  • Figure 6 shows the ability of humanized antibodies huC1H1-V1 and huC1H1-V2 to block reference binding to human CD40 in a competition ELISA.
  • Figure 7 shows the activity of humanized antibodies huC1H1-V1 and huC1H1-V2 to activate CD40 signaling in a cell-based reporter gene assay.
  • Figures 8A-B show the individual tumor volume (A) and individual tumor weight (B) of each group of mice at the end of the experiment (D14).
  • ** P ⁇ 0.01 and ** P ⁇ 0.001 are compared with IgG2 group by Mann-Whitney test; ** P ⁇ 0.01 and ** * P ⁇ 0.001 are compared with huC1H1-V2+Anluo by two-tailed Student's t test Compared with the dihydrochloride group of Tini; P ⁇ 0.05 was defined as a statistically significant difference.
  • the mouse anti-CD40 antibody or mouse anti-CD40 monoclonal antibody in the examples was produced by hybridoma technology according to the method described in the PCT/CN2021084013 patent document and obtained after in vitro functional screening.
  • the anti-CD40 antibody huC1H1-V2 in this application is the anti-CD40 antibody huC1H1-V2 in PCT/CN2021084013.
  • the affinity and binding kinetics of the anti-CD40 antibodies of the present application were characterized by Biacore T200 system (GE Healthcare, Pittsburgh, PA, USA).
  • goat anti-human IgG (GE healthcare, catalog BR100839, Human Antibody Capture Kit) was synthesized via the primary amine group using a standard amine coupling kit provided by Biacore (GE Healthcare, Pittsburgh, PA, USA). Covalently attached to a CM5 chip (carboxymethylated dextran-coated chip from GE healthcare catalog BR100530). Block the unreacted portion of the biosensor surface with ethanolamine.
  • CM5 chip carboxymethylated dextran-coated chip from GE healthcare catalog BR100530
  • the anti-CD40 antibody of the present application with a concentration of 66.67nM and two reference antibody (BM1 (Dacetuzumab, Genentech Inc., also known as CD40-BM1, internally prepared, the amino acid sequences of the heavy chain and the light chain were respectively as shown in this paper)
  • BM1 Denstuzumab, Genentech Inc., also known as CD40-BM1
  • BM2 Selicrelumab, Abgenix Inc., also known as CD40-BM2
  • internal preparation the amino acid sequences of the heavy chain and the light chain are respectively as the SEQ ID NOs of the application: 42 and 43)
  • Binding activity of anti-CD40 antibodies was determined by capture ELISA and flow cytometry (FACS).
  • affinity purified goat anti-human IgG F(ab') 2 fragment specific, Jackson Immuno Research, catalog 109-005-097
  • PBS PBS+0.05% v/v Tween-20, PBST
  • PBST blocking buffer
  • the binding of anti-CD40 antibody to 293T-CD40 cells was detected by flow cytometry (FACS), wherein 293T-CD40 cells were prepared in-house by Biosion, and their cell membranes stably express full-length human CD40 (uniprot#P25942-1, SEQ ID NO:30 ).
  • FACS flow cytometry
  • the pCMV-TP plasmid with the CD40 coding sequence inserted between the EcoRI and XbaI sites was transfected into 293T cells to prepare 293T-CD40 cells.
  • FACS buffer phosphate buffered saline (PBS) containing 2% v/v fetal bovine serum.
  • PBS phosphate buffered saline
  • Example 3 The blocking activity of anti-CD40 antibodies to CD40-CD40L binding or CD40-reference substance binding
  • the ability of anti-CD40 antibodies to block the binding of CD40 to CD40L was determined by competition ELISA.
  • the human CD40-Fc protein (the 21st-193rd amino acid residues of SEQ ID NO:30 connected at the concentration of 2 ⁇ g/mL) dissolved in the coating buffer (carbonate/bicarbonate buffer) to the N-terminus of amino acid residues 99-330 of SEQ ID NO:28) was coated in a 96-well plate at 100 ⁇ L/well, and incubated overnight at 4°C.
  • washing buffer PBS+0.05% v/v Tween-20, PBST
  • PBST 5% w/v skimmed milk
  • the ability of the anti-CD40 antibody of the present application to block the binding of the reference substance to human CD40 was determined by competition ELISA. Briefly, 100 ⁇ L/well of BM2 dissolved in PBS at a concentration of 1 ⁇ g/mL was coated in a 96-well plate and incubated overnight at 4°C. The next day, the plate was washed once with washing buffer, and blocking buffer (PBST containing 5% w/v skim milk) was added to block at 37°C for 2 hours.
  • blocking buffer PBST containing 5% w/v skim milk
  • human CD40-Fc protein labeled with biotin (the 21-193 amino acid residues of SEQ ID NO:30 are connected to the N of the 99-330 amino acid residues of SEQ ID NO:28 end, dissolved in PBST containing 2.5% w/v skimmed milk, the concentration is 0.23nM) to dilute the anti-CD40 antibody of the application or the reference substance, with 66.7nM as the initial concentration, 5-fold serial dilution, and then incubate at room temperature for 40 minute. After washing the plate 4 times, 100 ⁇ L/well of the antibody/biotin-labeled human CD40-Fc mixture was added to the BM2-coated plate.
  • the CD40 expression reporter cell line 293T-NF- ⁇ B-Luc-CD40 stably expressing full-length human CD40 was used to further measure the agonistic activity of the anti-CD40 antibody of the present application.
  • lipofectamine 3000 transfection reagent Thermo Fisher
  • the pGL4.32[luc2P NF- ⁇ B-RE Hygro] vector Promega, Accession number: EU581860
  • pCMV-TP plasmid CD40 coding sequence inserted between EcoRI and XbaI sites
  • 293T-NF- ⁇ B-Luc-CD40 cells in logarithmic phase were resuspended in DMEM medium (Gibco Inc., catalog 10566-016) containing 10% FBS (Gibco, catalog 10099-141). ), and inoculated into a 384-well cell culture plate (Corning, product catalog 3707) at 20 ⁇ L/well (containing 5 ⁇ 10 3 cells).
  • mice anti-CD40 monoclonal antibodies were sequenced and summarized in Table 1.
  • the heavy and light chain variable regions of mouse anti-CD40 monoclonal antibodies C1H1, 1D1 and 1A3 were cloned into fragments containing human IgG1 heavy chain constant region (SEQ ID NO: 27) and human ⁇ light chain fragments, respectively.
  • SEQ ID NO: 27 human IgG1 heavy chain constant region
  • SEQ ID NO: 29 fragment of the chain constant region
  • chimeric antibody C1H1 has higher binding affinity and ability to human CD40 and higher binding affinity to cynomolgus CD40 compared to BM1.
  • Mouse anti-CD40 antibody C1H1 was selected for humanization and further research. Humanization of this antibody was carried out using the well-established CDR grafting method, as described below.
  • mouse anti-CD40 antibody C1H1 In order to select the acceptor framework for the humanization of mouse anti-CD40 antibody C1H1, the sequences of the light and heavy chain variable regions of mouse anti-CD40 antibody C1H1 were BLAST compared with the human immunoglobulin gene database. The human germline antibody with the highest homology to the mouse anti-CD40 antibody C1H1 was selected as the acceptor backbone for humanization. The heavy/light chain variable region CDRs of mouse antibodies were inserted into the selected framework, and the residues in the framework were further mutated to obtain more candidate heavy/light chain variable regions. A total of 4 humanized C1H1 antibodies, namely huC1H1-V1 to huC1H1-V4, whose heavy/light chain variable region sequences are shown in Table 1, were obtained.
  • a vector comprising nucleotides encoding the heavy chain variable region linked to a human IgG2 heavy chain constant region (SEQ ID NO:28), and comprising a human nucleotide encoding nucleotide linked to a human kappa light chain constant region (SEQ ID NO:29)
  • the nucleotide-derived vector of the light chain variable region was transiently transfected into 50 mL 293F suspension cells with 1 mg/mL PEI at a ratio of 60%:40% light chain construct:heavy chain construct.
  • the cell supernatants containing humanized antibodies huC1H1-V1 to huC1H1-V4 were harvested, and using the Octet system (Fortebio, Octet RED 96), the antibody and human CD40 binding affinity.
  • Octet system Formebio, Octet RED 96
  • anti-human IgG Fc capture (AHC) biosensors from ForteBio were pre-soaked with 10 mM glycine (pH 1.5) for 3 s and then immersed in running buffer (PBST with 0.5% w/v BSA) 3 seconds, the steps of soaking and immersion are repeated 3 times.
  • the senor was immersed in cell supernatant containing humanized anti-CD40 antibody, chimeric antibody C1H1 dissolved in HBS-EP + buffer at a concentration of 5 ⁇ g/mL, or dissolved in HBS-EP + buffer at a concentration of 5 ⁇ g/mL reference for 120 seconds, followed by immersion in running buffer for 5 minutes.
  • the new baseline was run in running buffer for 180 seconds.
  • the sensor was then immersed in the human CD40-his protein (Acro biosystems, catalog CD0-H5228, starting at 40 nM, 2-fold serial dilution) diluted in running buffer for 120 seconds, and then immersed in running buffer for 10 minutes to establish a baseline.
  • the senor was pre-soaked with 10 mM glycine (pH 1.5) for 3 seconds, then immersed in running buffer for 3 seconds, and the steps of soaking and immersion were repeated 3 times. Association and dissociation curves were fitted to a 1:1 Langmuir binding model using ForteBio Data Analysis 8.1. Ka , Kd and KD values were determined and summarized in Table 3 below.
  • the humanized antibodies huC1H1-V1 and huC1H1-V2 were purified by protein A sepharose column and passed through Biacore, capture ELISA, cell-based binding FACS, competition ELISA, cell-based Reporter gene assays and protein thermal shift assays were used to characterize huC1H1-V1 and huC1H1-V2.
  • thermostability assay was also performed on the humanized antibody huC1H1-V2.
  • Tm melting temperature
  • GloMelt TM thermal shift protein stability kit Biotium, catalog 33022-T. Briefly, GloMelt TM dye was thawed to room temperature. Vortex and centrifuge the vial containing the dye. Then, prepare 10X dye by adding 5 ⁇ L of 200X dye to 95 ⁇ L of PBS. Add 2 ⁇ L of 10 ⁇ dye and 10 ⁇ g of humanized antibody to the reaction system, and add PBS to a total reaction volume of 20 ⁇ L.
  • the centrifuge tube containing the dye and the antibody was briefly centrifuged and placed in a real-time PCR thermal cycler (Roche, LightCycler 480II).
  • the parameters of the Melt Curve program in the thermal cycler are shown in Table 4.
  • humanized antibodies huC1H1-V1, huC1H1-V2 and chimeric antibody C1H1 have comparable binding affinities to human CD40 and cynomolgus monkey CD40.
  • the binding affinity of huC1H1-V1 and huC1H1-V2 to human CD40 and cynomolgus CD40 was higher than that of BM1 and BM2 to human CD40 and cynomolgus CD40.
  • Figure 5 shows that the humanized antibodies huC1H1-V1 and huC1H1-V2 of the present application can block the binding of CD40 and CD40L, and their blocking activity is equivalent to or slightly lower than that of BM1 and BM2.
  • the humanized antibodies huC1H1-V1 and huC1H1-V2 of the present application can block the binding of human CD40 to BM2, indicating that the antibodies huC1H1-V1 and huC1H1-V2 of the present application may bind similar epitopes to BM2 .
  • the agonistic activities of the humanized antibodies huC1H1-V1 and huC1H1-V2 of the present application were higher than those of BM1 and BM2.
  • Example 8 In vivo anti-tumor efficacy of huC1H1-V2 antibody in Ramos xenograft model of human B-cell lymphoma
  • the in vivo antitumor activity of the huC1H1-V2 antibody was determined using NOD-SCID mice. Briefly, 3 ⁇ 10 7 human B-cell lymphoma Ramos cells (Chinese Academy of Sciences) were injected subcutaneously in the right underarm of NOD-SCID mice. The tumor volume was measured using an electronic vernier caliper, and the calculation formula was (length ⁇ width 2 )/2. When the average tumor volume reached about 100-150 mm, 40 tumor-bearing mice were selected and randomly divided into 4 groups (10 mice in each group), and the date of animal grouping was designated as day 0.
  • mice were injected with isotype control antibody (anti-HEL-Human IgG2 Isotype-control, also known as IgG2, Biointron Inc) or huC1H1-V2 antibody into tail vein from day 0 .
  • isotype control antibody anti-HEL-Human IgG2 Isotype-control, also known as IgG2, Biointron Inc
  • huC1H1-V2 antibody into tail vein from day 0 .
  • N number of animals in each group; Q2D: once every 2 days; iv: intravenous injection.
  • mice in group 3 The results are summarized in Table 7. All treatments were well tolerated by tumor-bearing animals without significant weight loss or other symptoms.
  • huC1H1-V2 antibody (5 mg/kg) treatment showed the strongest antitumor activity, and at day 14, the mean tumor volume of this group (group 3) of mice was 230.2 mm 3 , which was statistically smaller than that of the isotype In the control group, the tumor inhibition rate (TGI) was 96%.
  • TGI tumor inhibition rate
  • huC1H1-V2 antibody (15 mg/kg) treatment did not show further enhanced efficacy, that is, the tumor volume of mice in group 4 was not significantly different from that in mice in group 3.
  • cT/C(%) (TT 0 )/(CC 0 ) ⁇ 100%, where T and C refer to the average tumor volume of the treatment group and the isotype control group on day 14, T 0 and C 0 refers to the average tumor volume of the treatment group and the isotype control group on day 0, respectively;
  • TGI (%) (1-T/C) ⁇ 100%.
  • Example 9 In vivo anti-tumor efficacy of huC1H1-V2 antibody in mouse colon cancer MC38 xenograft model
  • CD40 humanized transgenic mice also called hCD40 mice
  • hCD40 mice were used to further measure the in vivo anti-tumor activity of the huC1H1-V2 antibody.
  • 1 ⁇ 10 6 mouse colon cancer MC38 cells (Shanghai Lanli Biotechnology Co., Ltd.) were injected subcutaneously on the right back of hCD40 mice.
  • the tumor volume was measured using an electronic vernier caliper, and the calculation formula was (length ⁇ width 2 )/2.
  • 18 tumor-bearing mice were selected and randomly divided into 3 groups of 6 mice, and the day when the animals were grouped was designated as day 0.
  • mice were injected with solvent (phosphate buffered saline, also called PBS) or huC1H1-V2 antibody into tail vein.
  • solvent phosphate buffered saline
  • N number of animals in each group; Q2D: once every 2 days; iv: intravenous injection.
  • mice in this group were 1169mm 3 , which was statistically smaller than that in the vehicle group, and tumor inhibition The rate (TGI) was 64%.
  • huC1H1-V2 antibody (10mg/kg) treatment also showed significant anti-tumor activity.
  • the average tumor volume of this group (Group 3) mice was 1446mm 3 , which was statistically smaller than that of the vehicle group.
  • Tumor rate (TGI) was 56%.
  • a mean ⁇ standard error
  • b tumor volume on the 14th day, which was compared with the solvent control group by student's t test, * indicates P ⁇ 0.05, ** indicates P ⁇ 0.01
  • cT/C(%) T RTV /C RTV ⁇ 100%
  • T RTV and C RTV refer to the average RTV of the treatment group and the control group respectively
  • RTV V t /V 0
  • V t refers to the average tumor volume on the 14th day
  • V 0 refers to the average tumor volume on the 14th day Average tumor volume at day 0
  • TGI (%) (1-T/C) ⁇ 100%.
  • Human B-cell lymphoma Ramos cells purchased from the Cell Bank of the Chinese Academy of Sciences
  • RPMI1640 medium containing 10% fetal bovine serum and penicillin-streptomycin.
  • mice used in this experiment were 6-week-old NOD-Scid mice (purchased from Shanghai Lingchang Biotechnology Co., Ltd.), which were bred under specific pathogen-free conditions and adjusted for 12/12 hours of light/dark. Animal health was monitored daily.
  • Each mouse was subcutaneously inoculated with 1 ⁇ 10 7 Ramos cells, and when the tumor grew to 80-100 mm 3 , they were divided into groups according to the tumor volume, with 10 mice in each group.
  • the dosing regimen is shown in Table 10.
  • the first day of administration is D0.
  • Both huC1H1-V2 and IgG2 are prepared with normal saline to the required concentration, and the dihydrochloride of anlotinib is prepared with distilled water. Prepared to the desired concentration.
  • D14 the animals were sacrificed under CO 2 anesthesia, and then the tumors were dissected and photographed.
  • T0 or C ⁇ C0 it was defined as partial tumor regression (PR), and if the tumor disappeared completely, it was defined as complete tumor regression (CR).
  • mice in groups 4 were significantly lower than those in groups 2 and 3 (P ⁇ 0.001).
  • P value is compared with tumor volume of IgG2 group by Mann-Whitney test; *** means P ⁇ 0.001, compared with tumor volume of huC1H1-V2+anlotinib dihydrochloride group by two-tailed Student's t test; P ⁇ 0.05 was defined as a statistically significant difference.

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Abstract

提供抗CD40抗体的应用，具体而言，提供用于治疗癌症的联用药物，所述联用药物包含抗CD40抗体或其抗原结合部分和酪氨酸激酶抑制剂，具有良好的抗肿瘤活性和安全性。

Description

抗CD40抗体的应用 发明领域
本发明涉及生物医药领域，具体涉及治疗癌症的联用药物。
发明背景
CD40属于肿瘤坏死因子受体(TNFR)超家族成员，存在于B细胞、树突状细胞(DC)和巨噬细胞等抗原递呈细胞(APC)上，在其他造血细胞(如T细胞)活化后也有表达。此外，CD40在广泛的肿瘤细胞上表达，例如B细胞肿瘤(非霍奇金淋巴瘤、急性淋巴细胞白血病、慢性淋巴细胞白血病、骨髓瘤等)细胞和上皮肿瘤(前列腺癌、肝癌、肺癌、乳腺癌等)细胞等。
CD40的天然配体(称为CD154或CD40L)主要表达在活化的T淋巴细胞和肥大细胞上。CD40/CD40L信号通路参与机体的体液免疫和细胞免疫反应，在B细胞的活化、增殖与分化，抗体的产生、抗体类别转换等T细胞依赖的抗体免疫反应及炎症反应中起关键调节作用。因此，通过CD40激动剂(例如激动性抗CD40抗体)进行治疗可以增强机体的抗肿瘤免疫应答。
酪氨酸激酶是一组催化蛋白质酪氨酸残基磷酸化的酶，在细胞内的信号转导中起着重要的作用，它参与正常细胞的调节、信号传递和发育，也与肿瘤细胞的增殖、分化、迁移和凋亡密切相关。许多受体酪氨酸激酶都与肿瘤的形成相关，根据其细胞外区域结构的不同可分为表皮生长因子受体(EGFR)、血小板衍生生长因子受体(PDGFR)、血管内皮生长因子受体(VEGFR)、成纤维细胞生长因子受体(FGFR)等。
安罗替尼(Anlotinib)是一种喹啉衍生物类酪氨酸激酶抑制剂，其作为多靶点酪氨酸激酶抑制剂(TKI)在影响肿瘤血管生成和增殖信号转导方面发挥作用，主要靶点包括：受体酪氨酸激酶血管内皮生长因子受体(VEGFR)1至3，表皮生长因子受体(EGFR)，成纤维细胞生长因子受体(FGFR)1至4，血小板衍生生长因子受体(PDGFR)α和β，以及干细胞因子受体(SCFR)7、8和9。
尽管任何一种现有治疗对患者可能有益，但还需要更多降低毒性和改善治疗结果的方法供临床使用。
发明内容
第一方面，本申请提供一种药物组合，其包括(a)抗CD40抗体或其抗原结合部分，和(b)第二治疗剂。在一些实施方案中，所述第二治疗剂具有癌症治疗功效。在一些实施方案中，所述第二治疗剂包括酪氨酸激酶抑制剂。在一些实施方案中，所述第二治疗剂包括VEGFR酪氨酸激酶抑制剂。在一些具体的实施方案中，所述第二治疗剂包括安罗替尼或其药学上可接受的盐。
酪氨酸激酶抑制剂是指任何能够抑制(例如，阻断、破坏或使其失活)酪氨酸激酶或来自酪氨酸激酶的下游信号转导的物质或试剂，包括小分子酪氨酸激酶抑制剂和抗体。酪氨酸激酶抑制剂包括受体酪氨酸激酶抑制剂和非受体酪氨酸激酶抑制剂。在一些实施方案中，所述受体酪氨酸激酶抑制剂包括但不限于EGFR酪氨酸激酶抑制剂、VEGFR酪氨酸激酶抑制剂、FGFR酪氨酸激酶抑制剂、SCFR酪氨酸激酶抑制剂和PDGFR酪氨酸激酶抑制剂等。
VEGFR酪氨酸激酶抑制剂是指任何抑制(例如，阻断、破坏或使其失活)VEGF受体酪氨酸激酶或来自VEGF受体酪氨酸激酶的下游信号转导的物质或试剂，包括小分子VEGFR酪氨酸激酶抑制剂和抗体。VEGFR酪氨酸激酶抑制剂的实例包括但不限于安罗替尼(Anlotinib)、凡德他尼(Vandetanib)、索拉非尼(Sorafenib)、西地尼布(Cediranib)、伐他拉尼(Vatalanib)、帕唑帕尼(Pazopanib)、莫替沙尼(Motesanib)、仑伐替尼(Lenvatinib)、舒尼替尼(Sunitinib)、贝伐珠单抗(Bevacizumab)、雷莫芦 单抗(Ramucirumab)、或其药学上可接受的盐、或前述任意组合。
任选地，所述药物组合还包括具有癌症治疗功效的第三治疗剂。在一些实施方案中，所述第三治疗剂可以是本领域已知的癌症治疗剂，例如化疗药物、放疗药物、手术或其组合。
在一些实施方案中，所述药物组合包括(a)抗CD40抗体或其抗原结合部分，和(b)酪氨酸激酶抑制剂；任选地，还包括(c)化疗药物。在一些实施方案中，所述药物组合包括(a)抗CD40抗体或其抗原结合部分，和(b)VEGFR酪氨酸激酶抑制剂；任选地，还包括(c)化疗药物。在一些具体的实施方案中，所述药物组合包括(a)抗CD40抗体或其抗原结合部分，和(b)安罗替尼或其药学上可接受的盐；任选地，还包括(c)化疗药物。
在一些实施方案中，所述药物组合包括(a)抗CD40抗体或其抗原结合部分的药物组合物，和(b)酪氨酸激酶抑制剂的药物组合物；任选地，还包括(c)化疗药物。在一些实施方案中，所述药物组合包括(a)抗CD40抗体或其抗原结合部分的药物组合物，和(b)VEGFR酪氨酸激酶抑制剂的药物组合物；任选地，还包括(c)化疗药物。在一些具体的实施方案中，所述药物组合包括(a)抗CD40抗体或其抗原结合部分的药物组合物，和(b)安罗替尼或其药学上可接受的盐的药物组合物；任选地，还包括(c)化疗药物。
在一些实施方案中，所述药物组合包装于同一试剂盒中，所述试剂盒还包括抗CD40抗体或其抗原结合部分和酪氨酸激酶抑制剂联合使用以治疗癌症的说明。在一些实施方案中，所述药物组合中的抗CD40抗体或其抗原结合部分和酪氨酸激酶抑制剂分开包装于各自的药盒中，所述药盒还包括抗CD40抗体或其抗原结合部分和酪氨酸激酶抑制剂联合使用以治疗癌症的说明。
在一些实施方案中，所述药物组合包装于同一试剂盒中，所述试剂盒还包括抗CD40抗体或其抗原结合部分、酪氨酸激酶抑制剂和化疗药物联合使用以治疗癌症的说明。在一些实施方案中，所述药物组合中的抗CD40抗体或其抗原结合部分、酪氨酸激酶抑制剂和化疗药物分开包装于各自的药盒中，所述药盒还包括抗CD40抗体或其抗原结合部分、酪氨酸激酶抑制剂和化疗药物联合使用以治疗癌症的说明。
在一些实施方案中，所述药物组合包装于同一试剂盒中，所述试剂盒还包括抗CD40抗体或其抗原结合部分和VEGFR酪氨酸激酶抑制剂联合使用以治疗癌症的说明。在一些实施方案中，所述药物组合中的抗CD40抗体或其抗原结合部分和VEGFR酪氨酸激酶抑制剂分开包装于各自的药盒中，所述药盒还包括抗CD40抗体或其抗原结合部分和VEGFR酪氨酸激酶抑制剂联合使用以治疗癌症的说明。
在一些实施方案中，所述药物组合包装于同一试剂盒中，所述试剂盒还包括抗CD40抗体或其抗原结合部分、VEGFR酪氨酸激酶抑制剂和化疗药物联合使用以治疗癌症的说明。在一些实施方案中，所述药物组合中的抗CD40抗体或其抗原结合部分、VEGFR酪氨酸激酶抑制剂和化疗药物分开包装于各自的药盒中，所述药盒还包括抗CD40抗体或其抗原结合部分、VEGFR酪氨酸激酶抑制剂和化疗药物联合使用以治疗癌症的说明。
在一些实施方案中，所述药物组合包装于同一试剂盒中，所述试剂盒还包括抗CD40抗体或其抗原结合部分和安罗替尼或其药学上可接受的盐联合使用以治疗癌症的说明。在一些实施方案中，所述药物组合中的抗CD40抗体或其抗原结合部分和安罗替尼或其药学上可接受的盐分开包装于各自的药盒中，所述药盒还包括抗CD40抗体或其抗原结合部分和安罗替尼或其药学上可接受的盐联合使用以治疗癌症的说明。
在一些实施方案中，所述药物组合包装于同一试剂盒中，所述试剂盒还包括抗CD40抗体或其抗原结合部分、安罗替尼或其药学上可接受的盐和化疗药物联合使用以治疗癌症的说明。在一些实施方案中，所述药物组合中的抗CD40抗体或其抗原结合部分、安罗替尼或其药学上可接受的盐和化疗药物分开包装于各自的药盒中，所述药盒还包括抗CD40抗体或其抗原结合部分、安罗替尼或其药学上可接受的盐 和化疗药物联合使用以治疗癌症的说明。
在一些实施方案中，所述药物组合是固定组合。在一些实施方案中，所述固定组合呈固体药物组合物的形式或液体药物组合物的形式。
在一些实施方案中，所述药物组合是非固定组合。
在一些实施方案中，所述非固定组合中的抗CD40抗体或其抗原结合部分和酪氨酸激酶抑制剂各自呈药物组合物的形式。在一些实施方案中，所述非固定组合中的抗CD40抗体或其抗原结合部分、酪氨酸激酶抑制剂和化疗药物各自呈药物组合物的形式。
在一些实施方案中，所述非固定组合中的抗CD40抗体或其抗原结合部分和VEGFR酪氨酸激酶抑制剂各自呈药物组合物的形式。在一些实施方案中，所述非固定组合中的抗CD40抗体或其抗原结合部分、VEGFR酪氨酸激酶抑制剂和化疗药物各自呈药物组合物的形式。
在一些实施方案中，所述非固定组合中的抗CD40抗体或其抗原结合部分和安罗替尼或其药学上可接受的盐各自呈药物组合物的形式。在一些具体的实施方案中，所述非固定组合中的抗CD40抗体或其抗原结合部分呈液体药物组合物的形式，所述非固定组合中的安罗替尼或其药学上可接受的盐呈固体药物组合物的形式。在一些具体的实施方案中，所述非固定组合中的抗CD40抗体或其抗原结合部分呈注射液的形式，所述非固定组合中的安罗替尼或其药学上可接受的盐呈固体口服制剂的形式。在一些具体的实施方案中，所述非固定组合中的抗CD40抗体或其抗原结合部分呈注射液的形式，所述非固定组合中的安罗替尼或其药学上可接受的盐呈胶囊的形式。在一些实施方案中，所述非固定组合中的抗CD40抗体或其抗原结合部分、安罗替尼或其药学上可接受的盐和化疗药物各自呈药物组合物的形式。
第二方面，本申请还提供了第一方面的药物组合在制备用于治疗癌症的药物中的用途。另外，本申请还提供了抗CD40抗体或其抗原结合部分和酪氨酸激酶抑制剂在制备用于治疗癌症的药物中的用途。另外，本申请还提供了抗CD40抗体或其抗原结合部分、酪氨酸激酶抑制剂和化疗药物在制备用于治疗癌症的药物中的用途。另外，本申请还提供了抗CD40抗体或其抗原结合部分和VEGFR酪氨酸激酶抑制剂在制备用于治疗癌症的药物中的用途。另外，本申请还提供了抗CD40抗体或其抗原结合部分、VEGFR酪氨酸激酶抑制剂和化疗药物在制备用于治疗癌症的药物中的用途。另外，本申请还提供了抗CD40抗体或其抗原结合部分和安罗替尼或其药学上可接受的盐在制备用于治疗癌症的药物中的用途。另外，本申请还提供了抗CD40抗体或其抗原结合部分、安罗替尼或其药学上可接受的盐和化疗药物在制备用于治疗癌症的药物中的用途。
第三方面，本申请还提供在受试者中治疗癌症的方法，其包括向受试者给予有效量的第一方面的药物组合。在一些实施方案中，本申请提供在受试者中治疗癌症的方法，其包括向受试者给予有效量的抗CD40抗体或其抗原结合部分和有效量的酪氨酸激酶抑制剂；任选地，还包括有效量的化疗药物。在一些实施方案中，本申请提供在受试者中治疗癌症的方法，其包括向受试者给予有效量的抗CD40抗体或其抗原结合部分和有效量的VEGFR酪氨酸激酶抑制剂；任选地，还包括有效量的化疗药物。在一些实施方案中，本申请提供在受试者中治疗癌症的方法，其包括向受试者给予有效量的抗CD40抗体或其抗原结合部分和有效量的安罗替尼或其药学上可接受的盐；任选地，还包括有效量的化疗药物。
第四方面，本申请提供一种试剂盒，其包括第一方面的药物组合。在一些实施方案中，所述含有(a)第一药物组合物，其中抗CD40抗体或其抗原结合部分作为活性成份；和(b)第二药物组合物，其中酪氨酸激酶抑制剂作为活性成份；任选地，还包括(c)化疗药物。在一些实施方案中，本所述含有(a)第一药物组合物，其中抗CD40抗体或其抗原结合部分作为活性成份；和(b)第二药物组合物，其中VEGFR酪氨酸激酶抑制剂作为活性成份；任选地，还包括(c)化疗药物。在一些实施方案中，所述含有(a)第一药物组合物，其中抗CD40抗体或其抗原结合部分作为活性成份；和(b)第二药物组合物，其中安罗替尼或其药学上可接受的盐作为活性成份；任选地，还包括(c)化疗药物。在一些实施方案 中，所述试剂盒用于治疗癌症。
第五方面，本申请还提供治疗癌症的方法，其包括向受试者给予有效量的本申请的抗CD40抗体或其抗原结合部分。本申请还提供本申请的抗CD40抗体或其抗原结合部分用于治疗癌症的用途。本申请还提供本申请的抗CD40抗体或其抗原结合部分在制备用于治疗癌症的药物中的用途。
药物组合的给药/治疗方案
在一些实施方案中，上述用途或者方法中，抗CD40抗体或其抗原结合部分和酪氨酸激酶抑制剂各自呈药物组合物的形式，可同时、先后和/或交替给药。在一些实施方案中，上述用途或者方法中，抗CD40抗体或其抗原结合部分、酪氨酸激酶抑制剂和化疗药物各自呈药物组合物的形式，可同时、先后和/或交替给药。在一些实施方案中，上述用途或者方法中，抗CD40抗体或其抗原结合部分和VEGFR酪氨酸激酶抑制剂各自呈药物组合物的形式，可同时、先后和/或交替给药。在一些实施方案中，上述用途或者方法中，抗CD40抗体或其抗原结合部分、VEGFR酪氨酸激酶抑制剂和化疗药物各自呈药物组合物的形式，可同时、先后和/或交替给药。在一些实施方案中，上述用途或者方法中，抗CD40抗体或其抗原结合部分和安罗替尼或其药学上可接受的盐各自呈药物组合物的形式，可同时、先后和/或交替给药。在一些实施方案中，上述用途或者方法中，抗CD40抗体或其抗原结合部分、安罗替尼或其药学上可接受的盐和化疗药物各自呈药物组合物的形式，可同时、先后和/或交替给药。
在一些实施方案中，上述用途或者方法中，抗CD40抗体或其抗原结合部分和酪氨酸激酶抑制剂各自呈药物组合物的形式，且各自以间隔给药的方式给药。在一些实施方案中，上述用途或者方法中，抗CD40抗体或其抗原结合部分、酪氨酸激酶抑制剂和化疗药物各自呈药物组合物的形式，且各自以间隔给药的方式给药。在一些实施方案中，上述用途或者方法中，抗CD40抗体或其抗原结合部分和VEGFR酪氨酸激酶抑制剂各自呈药物组合物的形式，且各自以间隔给药的方式给药。在一些实施方案中，上述用途或者方法中，抗CD40抗体或其抗原结合部分、VEGFR酪氨酸激酶抑制剂和化疗药物各自呈药物组合物的形式，且各自以间隔给药的方式给药。在一些实施方案中，上述用途或者方法中，抗CD40抗体或其抗原结合部分和安罗替尼或其药学上可接受的盐各自呈药物组合物的形式，且各自以间隔给药的方式给药。在一些实施方案中，上述用途或者方法中，抗CD40抗体或其抗原结合部分、安罗替尼或其药学上可接受的盐和化疗药物各自呈药物组合物的形式，且各自以间隔给药的方式给药。所述的间隔给药包括给药期和停药期，在给药期内可以每天一次或多次给予。例如在给药期内每天给药，然后停药期内停止给药一段时间，接着给药期，然后停药期，如此可以反复进行多次。
在一些实施方案中，上述用途或者方法中，抗CD40抗体或其抗原结合部分和酪氨酸激酶抑制剂分别以相同或者不同的给药方案进行给药。在一些实施方案中，上述用途或者方法中，抗CD40抗体或其抗原结合部分和酪氨酸激酶抑制剂分别以不同的给药方案进行给药。在一些实施方案中，上述用途或者方法中，抗CD40抗体或其抗原结合部分、酪氨酸激酶抑制剂和化疗药物分别以相同或者不同的给药方案进行给药。在一些实施方案中，上述用途或者方法中，抗CD40抗体或其抗原结合部分、酪氨酸激酶抑制剂和化疗药物分别以不同的给药方案进行给药。
在一些实施方案中，上述用途或者方法中，抗CD40抗体或其抗原结合部分和VEGFR酪氨酸激酶抑制剂分别以相同或者不同的给药方案进行给药。在一些实施方案中，上述用途或者方法中，抗CD40抗体或其抗原结合部分和VEGFR酪氨酸激酶抑制剂分别以不同的给药方案进行给药。在一些实施方案中，上述用途或者方法中，抗CD40抗体或其抗原结合部分、VEGFR酪氨酸激酶抑制剂和化疗药物分别以相同或者不同的给药方案进行给药。在一些实施方案中，上述用途或者方法中，抗CD40抗体或其抗原结合部分、VEGFR酪氨酸激酶抑制剂和化疗药物分别以不同的给药方案进行给药。
在一些实施方案中，上述用途或者方法中，抗CD40抗体或其抗原结合部分和安罗替尼或其药学上可接受的盐分别以相同或者不同的给药方案进行给药。在一些实施方案中，上述用途或者方法中，抗 CD40抗体或其抗原结合部分和安罗替尼或其药学上可接受的盐分别以不同的给药方案进行给药。在一些实施方案中，上述用途或者方法中，抗CD40抗体或其抗原结合部分、安罗替尼或其药学上可接受的盐和化疗药物分别以相同或者不同的给药方案进行给药。在一些实施方案中，上述用途或者方法中，抗CD40抗体或其抗原结合部分、安罗替尼或其药学上可接受的盐和化疗药物分别以不同的给药方案进行给药。
在一些实施方案中，上述用途或者方法中，抗CD40抗体或其抗原结合部分可以每周(q1w)、每2周(q2w)、每3周(q3w)、或者每4周(q4w)施用一次。在一些实施方案中，上述用途或者方法中，抗CD40抗体或其抗原结合部分可以每周(±3天)、每2周(±3天)、每3周(±3天)、或者每4周(±3天)施用一次。在一个具体的实施方案中，抗CD40抗体或其抗原结合部分可以每3周(±3天)施用一次。
在一些实施方案中，上述用途或者方法中，抗CD40抗体或其抗原结合部分以0.001至100mg/kg体重的剂量施用。
在一些实施方案中，上述用途或者方法中，抗CD40抗体或其抗原结合部分以能够有效治疗所述癌症的统一剂量施用。在一些具体实施方式中，所述统一剂量在大约1mg至大约1000mg抗CD40抗体或其抗原结合部分的范围内。
在一些实施方案中，上述用途或者方法中，安罗替尼或其药学上可接受的盐以每1周、每2周、每3周、或者每4周为一个治疗周期。在一些实施方案中，上述用途或者方法中，安罗替尼或其药学上可接受的盐以每1周(±3天)、每2周(±3天)、每3周(±3天)或者每4周(±3天)为一个治疗周期。在一个具体的实施方案中，安罗替尼或其药学上可接受的盐以每3周(±3天)为一个治疗周期。
在一些实施方案中，上述用途或者方法中，所述安罗替尼或其药学上可接受的盐可以采用间隔给药方式，例如连续给药2周停药2周、连续给药2周停药1周、或连续给药5天停药2天；所述间隔给药方式可以反复进行多次。
在一些实施方案中，上述用途或者方法中，给予安罗替尼或其药学上可接受的盐的日剂量为3mg至30mg。在一些实施方案中，给予安罗替尼或其药学上可接受的盐的日剂量为5mg至20mg。在一些实施方案中，给予安罗替尼或其药学上可接受的盐的日剂量为8mg至16mg。在一些实施方案中，给予安罗替尼或其药学上可接受的盐的日剂量为3mg、4mg、5mg、6mg、7mg、8mg、9mg、10mg、11mg、12mg、13mg、14mg、15mg和16mg。在一个特定的实施方案中，给予安罗替尼或其药学上可接受的盐的日剂量为8mg。在一个特定的实施方案中，给予安罗替尼或其药学上可接受的盐的日剂量为10mg。在一个特定的实施方案中，给予安罗替尼或其药学上可接受的盐的日剂量为12mg。在一些实施方案中，安罗替尼或其药学上可接受的盐可以每日施用一次或多次。在一些实施方案中，安罗替尼或其药学上可接受的盐可以每天施用一次。在一些实施方案中，安罗替尼或其药学上可接受的盐以口服固体制剂每天施用一次。
在一些具体的实施方案中，上述用途或者方法中，安罗替尼或其药学上可接受的盐可以每日一次6mg、8mg、10mg或者12mg的剂量，连续用药2周停1周的给药方案给药。在一些具体的实施方案中，上述用途或者方法中，安罗替尼或其药学上可接受的盐可以每日一次6mg、8mg、10mg或者12mg的剂量，连续用药2周停2周的给药方案给药。
在一些实施方案中，上述用途或者方法中，抗CD40抗体或其抗原结合部分和酪氨酸激酶抑制剂分别具有相同或者不同的治疗周期。在一些实施方案中，上述用途或者方法中，抗CD40抗体或其抗原结合部分和酪氨酸激酶抑制剂具有相同的治疗周期，例如每1周(±3天)、每2周(±3天)、每3周(±3天)或者每4周(±3天)为一个治疗周期。在一个具体的实施方案中，抗CD40抗体或其抗原结合部分和酪氨酸激酶抑制剂以每3周(±3天)为一个治疗周期。在一些实施方案中，上述用途或者方法 中，抗CD40抗体或其抗原结合部分、酪氨酸激酶抑制剂和化疗药物分别具有相同或者不同的治疗周期。在一些实施方案中，上述用途或者方法中，抗CD40抗体或其抗原结合部分、酪氨酸激酶抑制剂和化疗药物具有相同的治疗周期，例如每1周(±3天)、每2周(±3天)、每3周(±3天)或者每4周(±3天)为一个治疗周期。在一个具体的实施方案中，抗CD40抗体或其抗原结合部分、酪氨酸激酶抑制剂和化疗药物以每3周(±3天)为一个治疗周期。
在一些实施方案中，上述用途或者方法中，抗CD40抗体或其抗原结合部分和VEGFR酪氨酸激酶抑制剂分别具有相同或者不同的治疗周期。在一些实施方案中，上述用途或者方法中，抗CD40抗体或其抗原结合部分和VEGFR酪氨酸激酶抑制剂具有相同的治疗周期，例如每1周(±3天)、每2周(±3天)、每3周(±3天)或者每4周(±3天)为一个治疗周期。在一个具体的实施方案中，抗CD40抗体或其抗原结合部分和VEGFR酪氨酸激酶抑制剂以每3周(±3天)为一个治疗周期。在一些实施方案中，上述用途或者方法中，抗CD40抗体或其抗原结合部分、VEGFR酪氨酸激酶抑制剂和化疗药物分别具有相同或者不同的治疗周期。在一些实施方案中，上述用途或者方法中，抗CD40抗体或其抗原结合部分、VEGFR酪氨酸激酶抑制剂和化疗药物具有相同的治疗周期，例如每1周(±3天)、每2周(±3天)、每3周(±3天)或者每4周(±3天)为一个治疗周期。在一个具体的实施方案中，抗CD40抗体或其抗原结合部分、VEGFR酪氨酸激酶抑制剂和化疗药物以每3周(±3天)为一个治疗周期。
在一些实施方案中，上述用途或者方法中，抗CD40抗体或其抗原结合部分和安罗替尼或其药学上可接受的盐分别具有相同或者不同的治疗周期。在一些实施方案中，上述用途或者方法中，抗CD40抗体或其抗原结合部分和安罗替尼或其药学上可接受的盐具有相同的治疗周期，例如每1周(±3天)、每2周(±3天)、每3周(±3天)或者每4周(±3天)为一个治疗周期。在一个具体的实施方案中，抗CD40抗体或其抗原结合部分和安罗替尼或其药学上可接受的盐以每3周(±3天)为一个治疗周期。在一些实施方案中，上述用途或者方法中，抗CD40抗体或其抗原结合部分、安罗替尼或其药学上可接受的盐和化疗药物分别具有相同或者不同的治疗周期。在一些实施方案中，上述用途或者方法中，抗CD40抗体或其抗原结合部分、安罗替尼或其药学上可接受的盐和化疗药物具有相同的治疗周期，例如每1周(±3天)、每2周(±3天)、每3周(±3天)或者每4周(±3天)为一个治疗周期。在一个具体的实施方案中，抗CD40抗体或其抗原结合部分、安罗替尼或其药学上可接受的盐和化疗药物以每3周(±3天)为一个治疗周期。
抗CD40抗体或其抗原结合部分
如本申请所用，所述抗CD40抗体或其抗原结合部分包含：
i)重链可变区，所述重链可变区包含V H CDR1、V H CDR2和V H CDR3，其中，(1)V H CDR1、V H CDR2和V H CDR3分别包含与SEQ ID NOs:1、4和7所示的氨基酸序列具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列；(2)V H CDR1、V H CDR2和V H CDR3分别包含与SEQ ID NOs:2、5和8所示的氨基酸序列具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列；或(3)V H CDR1、V H CDR2和V H CDR3分别包含与SEQ ID NOs:3、6和9所示的氨基酸序列具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列；和/或
ii)轻链可变区，所述轻链可变区包含V L CDR1、V L CDR2和V L CDR3，其中，(1)V L CDR1、V L CDR2和V L CDR3分别包含与SEQ ID NOs:10、13和16所示的氨基酸序列具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列；(2)V L CDR1、V L CDR2和V L CDR3分别包含与SEQ ID NOs:11、14和17所示的氨基酸序列具有至少85％、 86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列；或(3)V L CDR1、V L CDR2和V L CDR3分别包含与SEQ ID NOs:12、15和18所示的氨基酸序列具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列。
在一些实施方案中，所述抗CD40抗体或其抗原结合部分包含重链可变区和轻链可变区，所述重链可变区包含V H CDR1、V H CDR2和V H CDR3，所述轻链可变区包含V L CDR1、V L CDR2和V L CDR3，其中，(1)V H CDR1、V H CDR2、V H CDR3、V L CDR1、V L CDR2和V L CDR3分别包含与SEQ ID NOs:1、4、7、10、13和16所示的氨基酸序列具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列；(2)V H CDR1、V H CDR2、V H CDR3、V L CDR1、V L CDR2和V L CDR3分别包含与SEQ ID NOs:2、5、8、11、14和17所示的氨基酸序列具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列；或(3)V H CDR1、V H CDR2、V H CDR3、V L CDR1、V L CDR2和V L CDR3分别包含与SEQ ID NOs:3、6、9、12、15和18所示的氨基酸序列具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列。
在一些实施方案中，所述抗CD40抗体或其抗原结合部分包含重链可变区，所述重链可变区包含与SEQ ID NOs:19、20、21或22所示的氨基酸序列具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列；其中，SEQ ID NO:20中的X1＝A或S。
在一些实施方案中，所述抗CD40抗体或其抗原结合部分包含轻链可变区，所述轻链可变区包含与SEQ ID NOs:23、24、25或26所示的氨基酸序列具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列；其中，SEQ ID NO:24中的X1＝K和X2＝F，或X1＝Y和X2＝Y。
在一些实施方案中，所述抗CD40抗体或其抗原结合部分包含重链可变区和轻链可变区，所述重链可变区和轻链可变区包含：(1)分别与SEQ ID NOs:19和23所示的氨基酸序列具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列；(2)分别与SEQ ID NOs:20和24所示的氨基酸序列具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列，其中SEQ ID NO:20中的X1＝A，且SEQ ID NO:24中的X1＝K和X2＝F；(3)分别与SEQ ID NOs:20和24所示的氨基酸序列具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列，其中SEQ ID NO:20中的X1＝S，且SEQ ID NO:24中的X1＝K和X2＝F；(4)分别与SEQ ID NOs:20和24所示的氨基酸序列具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列，其中SEQ ID NO:20中的X1＝A，且SEQ ID NO:24中的X1＝Y和X2＝Y；(5)分别与SEQ ID NOs:20和24所示的氨基酸序列具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列，其中SEQ ID NO:20中的X1＝S，且SEQ ID NO:24中的X1＝Y和X2＝Y；(6)分别与SEQ ID NOs:21和25所示的氨基酸序列具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列；或(7)分别与SEQ ID NOs:22和26所示的氨基酸序列具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列。
在一些实施方案中，所述抗CD40抗体或其抗原结合部分包含重链和轻链，所述重链包含重链可变 区和重链恒定区，所述轻链包含轻链可变区和轻链恒定区，其中重链可变区的C末端连接至重链恒定区的N末端，轻链可变区的C末端连接至轻链恒定区的N末端，其中重链可变区和轻链可变区包含上述的氨基酸序列，重链恒定区可以是具有如SEQ ID NO:28所示氨基酸序列的人IgG2恒定区，或具有如SEQ ID NO:27所示氨基酸序列的人IgG1恒定区，轻链恒定区可以是具有如SEQ ID NO:29所示氨基酸序列的人κ恒定区。可以对重链恒定区如Fc片段进行改造，以使其具有降低的或增强的FcR结合亲和力。
在一些具体的实施方案中，所述抗CD40抗体或其抗原结合部分包含重链和轻链，其中，(1)重链包含SEQ ID NOs：19和27所示的氨基酸序列，和轻链包含SEQ ID NOs：23和29所示的氨基酸序列；(2)重链包含SEQ ID NOs：20和28所示的氨基酸序列，和轻链包含SEQ ID NOs：24和29所示的氨基酸序列，其中SEQ ID NO:20中的X1＝A，且SEQ ID NO:24中的X1＝K和X2＝F；(3)重链包含SEQ ID NOs：20和28所示的氨基酸序列，和轻链包含SEQ ID NOs：24和29所示的氨基酸序列，其中SEQ ID NO:20中的X1＝S，且SEQ ID NO:24中的X1＝K和X2＝F；(4)重链包含SEQ ID NOs：20和28所示的氨基酸序列，和轻链包含SEQ ID NOs：24和29所示的氨基酸序列，其中SEQ ID NO:20中的X1＝A，且SEQ ID NO:24中的X1＝Y和X2＝Y；(5)重链包含SEQ ID NOs：20和28所示的氨基酸序列，和轻链包含SEQ ID NOs：24和29所示的氨基酸序列，其中SEQ ID NO:20中的X1＝S，且SEQ ID NO:24中的X1＝Y和X2＝Y；(6)重链包含SEQ ID NOs：21和27所示的氨基酸序列，和轻链包含SEQ ID NOs：25和29所示的氨基酸序列；或(7)重链包含SEQ ID NOs：22和27所示的氨基酸序列，和轻链包含SEQ ID NOs：26和29所示的氨基酸序列。
在一些实施方案中，所述抗CD40抗体包含两条重链和两条轻链，或由两条重链和两条轻链组成，其中每条重链包含上述的重链恒定区、重链可变区或CDR序列，并且每条轻链包含上述的轻链恒定区、轻链可变区或CDR序列。本申请的抗CD40抗体可以是全长抗体，例如IgG1、IgG2或IgG4同种型全长抗体。在其他实施方案中，本申请的抗CD40抗体或其抗原结合部分可以是单链可变区(scFv)抗体，或抗体片段，例如Fab或F(ab')2片段。
在一些实施方案中，本申请的抗CD40抗体或其抗原结合部分结合CD40(例如，人CD40和猴CD40)。在一些实施方案中，本申请的抗CD40抗体或其抗原结合部分阻断CD40与CD40L的结合。在一些实施方式中，本申请的抗CD40抗体或其抗原结合部分激活CD40信号转导。
优选地，本申请的抗CD40抗体是人源化单克隆抗体。此外，或者，本申请的抗CD40抗体可以是例如，嵌合单克隆抗体。
本申请示例性的抗CD40抗体或其抗原结合部分的重/轻链可变区的氨基酸序列ID汇总于下表1中，一些抗体具有相同的V H或V L。抗体的重链恒定区可以是人IgG1或IgG2重链恒定区，其分别具有如SEQ ID NO:27和28所示的氨基酸序列，且抗体的轻链恒定区可以是人κ恒定区，其具有如SEQ ID NO:29所示的氨基酸序列。这些抗体也可以包含小鼠IgG1或IgG2重链恒定区，和/或小鼠κ恒定区。
表1.重/轻链可变区的氨基酸序列ID
Figure PCTCN2022121184-appb-000001
表1中的重链可变区CDR和轻链可变区CDR已由Kabat编号系统定义。然而，如本领域所熟知的，CDR区也可以基于重/轻链可变区序列由其他编号系统例如Chothia、IMGT、AbM或Contact编号系统/方法确定。本领域技术人员应当理解的是，除非另有规定，否则术语给定抗体或其区(例如可变区)的“CDR”及“互补决定区”应了解为涵盖通过任何一种已知方案界定的互补决定区。虽然本公开中请求保护的范围是基于表1示出的序列，但是根据其它CDR编号系统所定义的氨基酸序列也应当落在本公开的保护范围中。
与人CD40结合的其他抗CD40抗体的V H和V L序列(或CDR序列)可以与本申请的抗CD40抗体的V H和V L序列(或CDR序列)“混合并配对”。优选地，当V H和V L链(或这些链中的CDR)混合并配对时，来自特定V H/V L对中的V H序列被结构相似的V H序列取代。同样地，优选将来自特定V H/V L对中的V L序列替换为结构相似的V L序列。
因此，在一个实施方案中，本申请的抗体或其抗原结合部分包含：
(a)重链可变区，其包含列于表1中的氨基酸序列；和
(b)轻链可变区，其包含列于表1中的氨基酸序列，或另一种抗CD40抗体的V L，其中所述抗体特异性结合人CD40。
在另一个实施方案中，本申请的抗体或其抗原结合部分包含：
(a)列于表1中的重链可变区CDR1、CDR2和CDR3；和
(b)列于表1中的轻链可变区CDR1、CDR2和CDR3，或另一种抗CD40抗体的CDR，其中所述抗体特异性结合人CD40。
在另一个实施方案中，本申请的抗体或其抗原结合部分包括本申请的抗CD40抗体的重链可变区CDR2以及其他结合人CD40的抗体的CDR，例如来自另一种抗CD40抗体的重链可变区CDR1和/或CDR3，和/或轻链可变区CDR1、CDR2和/或CDR3。
此外，本领域所熟知的是，不依赖于CDR1和/或CDR2结构域，单独的CDR3结构域可以确定抗体对同源抗原的结合特异性，并且基于共同的CDR3序列可以预测性地产生具有相同结合特异性的多种抗体。
因此，在另一个实施方案中，本申请的抗体包含本申请的抗CD40抗体的重链可变区CDR2以及至少本申请的抗CD40抗体的重链可变区和/或轻链可变区CDR3，或另一种抗CD40抗体的重链可变区和/或轻链可变区CDR3，其中该抗体特异性结合人CD40。这些抗体优选与本申请的抗CD40抗体(a)竞争结合CD40；(b)保留功能特性；(c)结合相同表位；和/或(d)具有相似的结合亲和力。在另一个实施方案中，本申请的抗体还可以包含本申请的抗CD40抗体的轻链可变区CDR2，或者另一种抗CD40抗体的轻链可变区CDR2，其中该抗体特异性结合人CD40。在另一个实施方案中，本申请的抗体还可以包括本申请的抗CD40抗体的重链可变区和/或轻链可变区CDR1，或另一种抗CD40抗体的重链可变区和/或轻链可变区CDR1，其中该抗体特异性结合人CD40。
在另一个实施方案中，本申请的抗体包含重链可变区和/或轻链可变区，所述重链可变区和轻链可变区分别包含CDR1、CDR2和CDR3，其中，这些CDR序列不同于本申请的抗CD40抗体的CDR序列，所述不同是源于一个或多个保守修饰。本领域中应理解，某些保守的序列修饰不会使抗原结合性消失。
因此，在一个实施方案中，本申请的抗体包含重链可变区和/或轻链可变区，所述重链可变区和轻链可变区分别包含CDR1、CDR2和CDR3序列，其中：
(a)重链可变区CDR1序列包含表1中列出的序列，和/或其保守修饰；和/或
(b)重链可变区CDR2序列包含表1中列出的序列，和/或其保守修饰；和/或
(c)重链可变区CDR3序列包含表1中列出的序列，和/或其保守修饰；和/或
(d)轻链可变区CDR1、和/或CDR2、和/或CDR3序列包含表1中列出的序列，和/或其保守修饰；
和(e)该抗体特异性结合人CD40。
术语“保守序列修饰”是指不会显著影响或改变抗体结合特性的氨基酸修饰。这样的保守修饰包括氨基酸替换、添加和缺失。可以通过本领域已知的标准技术将修饰引入本申请的抗体中，例如点突变和PCR介导的突变。保守氨基酸替换是指将氨基酸残基替换为具有相似侧链的氨基酸残基。具有相似侧链的氨基酸残基组在本领域中已知。这些氨基酸残基组包括具有碱性侧链(例如，赖氨酸、精氨酸、组氨酸)、酸性侧链(例如，天冬氨酸、谷氨酸)、不带电极性侧链(例如，甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如，丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、β-支链侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳香族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。因此，本申请的抗体的CDR区中的一个或多个氨基酸残基可以被来自相同侧链组的其他氨基酸残基替换，且得到的抗体可以使用本申请所述的功能检测对其进行保留功能测试。
在另一些实施方案中，所述抗CD40抗体或其抗原结合部分选自：Dacetuzumab、Lucatumumab、APX005M、CDX-1140(Celldex)、GEN-1042(BioNTech；Genmab)、Selicrelumab、SEA-CD40(Seagen)、2141-v11(AbbVie)、ChiLob7/4(BioNTech)、Mitazalimab、Vanalimab、Giloralimab、LVGN-7409(礼进生物)、LVGN-7408(礼进生物)、YH003(祐和医药)、SHR-1704(恒瑞医药)、MIL97(天广实生物)、Iscalimab、Bleselumab、Ravagalimab，或以上抗体的抗原结合部分。
抗CD40抗体或其抗原结合部分的药物组合物
在一些实施方案中，所述抗CD40抗体或其抗原结合部分的药物组合物的单剂量包括2mg至20mg，例如2mg、4mg、6mg、8mg、10mg、12mg、14mg、16mg、18mg或20mg的抗CD40抗体或其抗原结合部分。
在一些实施方案中，所述抗CD40抗体或其抗原结合部分的药物组合物包含抗CD40抗体或其抗原结合部分和任何数量的赋形剂。可以使用的赋形剂包括载体、表面活性剂、增稠或乳化剂、固体粘合剂、分散或混悬剂、增溶剂、着色剂、矫味剂、包衣、崩解剂、润滑剂、甜味剂、防腐剂、等渗剂及其组合。合适的赋形剂的选择和使用在Gennaro,ed.,Remington:The Science and Practice of Pharmacy,20th Ed.(Lippincott Williams&Wilkins 2003)中教导，其公开的内容通过引用的方式并入本文。
抗CD40抗体或其抗原结合部分的药物组合物适合于静脉内、肌内、皮下、肠道外、脊柱或表皮施用(例如通过注射或输注)。基于给药途径，可以将活性成分包被在材料中，以保护其不受酸和可能使其失活的其他自然条件的影响。本文所用的短语“肠道外施用”是指除肠内和局部施用以外的施用方式，通常采用注射，并且包括但不限于静脉内、肌内、动脉内、鞘内、囊内、眶内、心内、皮内、腹膜内、经气管、皮下、表皮下、关节内、囊下、蛛网膜下、脊柱内、硬膜外和胸骨内注射和输注。或者，抗CD40抗体或其抗原结合部分的药物组合物可以通过非肠道外途径施用，例如局部、表皮或粘膜途径施用，如鼻内、口服、阴道、直肠、舌下或局部施用。还可以执行施用，例如，一次、多次，和/或在一个或多个延长的时间段中。
在一个具体实施方案中，所述抗CD40抗体或其抗原结合部分的药物组合物为水溶性注射液，所述水溶性注射液包括但不限于未经冻干的水溶性制剂或冻干粉重构的水溶性制剂。在另一些实施方案中，所述抗CD40抗体或其抗原结合部分的药物组合物为冻干制剂。所述冻干制剂是指水溶液经历冻干过程制备的制剂，在该过程中物质首先被冷冻，然后先通过升华降低溶剂数量(初级干燥过程)，然后通过脱附作用降低溶剂数量(二级干燥过程)，直到溶剂数量为不再支持生物学活性或化学反应的值。本申请的冻干制剂还可以通过本领域已知的其它方法干燥，如喷雾干燥和鼓泡干燥(bubble drying)。
安罗替尼或其药学上可接受的盐
如本申请所用，所述安罗替尼(即，式(I)的化合物)的化学名为1-[[[4-(4-氟-2-甲基-1H-吲哚-5-基)氧基-6-甲氧基喹啉-7-基]氧基]甲基]环丙胺，其具有如下的结构式：
Figure PCTCN2022121184-appb-000002
本申请中，凡是涉及安罗替尼，均是指式(I)化合物。
安罗替尼可以以其游离碱的形式给药，也可以以其盐、水合物和前药的形式给药，该前药在体内转换成安罗替尼的游离碱形式。例如，安罗替尼的药学上可接受的盐也在本发明的范围内，可按照本领域公知的方法由不同的有机酸和无机酸产生所述盐。
进一步的，安罗替尼的药学上可接受的盐为式(I)化合物与任意如下酸所形成的盐：盐酸、氢溴酸、硫酸、硝酸、磷酸、乙酸、三氟乙酸、丙酸、己酸、庚酸、环戊烷丙酸、乙醇酸、丙酮酸、乳酸、丙二酸、琥珀酸、苹果酸、马来酸、富马酸、酒石酸、柠檬酸、苯甲酸、肉桂酸、扁桃酸、甲磺酸、乙磺酸、1,2-乙二磺酸、2-羟基乙磺酸、苯磺酸、对氯苯磺酸、对甲苯磺酸、3-苯基丙酸、三甲基乙酸、叔丁基乙酸、十二烷基硫酸、葡糖酸、谷氨酸、羟基萘甲酸、水杨酸、硬脂酸；优选为盐酸盐或马来酸盐的形式，更优选为二盐酸盐。
在一些实施方案中，以安罗替尼盐酸盐的形式给药。在一些实施方案中，以安罗替尼一盐酸盐的形式给药。在一些实施方案中，以安罗替尼二盐酸盐的形式给药。在一些实施方案中，以安罗替尼盐酸盐的晶体形式给药。在特定的实施方案中，以安罗替尼二盐酸盐的晶体形式给药。
本申请中涉及的安罗替尼或其盐的剂量，除非另有说明，均基于安罗替尼游离碱计算。
安罗替尼或其药学上可接受的盐的药物组合物
在一些实施方案中，所述安罗替尼或其药学上可接受的盐的药物组合物的单剂量包括6mg、8mg、10mg、或12mg的安罗替尼。
在一些实施方案中，安罗替尼或其药学上可接受的盐的药物组合物包括但不限于适合口服、肠道外、局部给药的制剂。在一些实施方案中， 安罗替尼或其药学上可接受的盐的药物组合物优选为适于口服的制剂，包括片剂、胶囊剂、粉剂、颗粒剂、滴丸、糊剂、散剂等，优选片剂和胶囊剂。其中片剂可以是普通片剂、分散片、泡腾片、缓释片、控释片或肠溶片，胶囊剂可以是普通胶囊、缓释胶囊、控释胶囊或肠溶胶囊。所述的口服的制剂可使用本领域公知的药学上可接受的载体通过常规方法制得。药学上可接受的载体包括填充剂、吸收剂、润湿剂、粘合剂、崩解剂、润滑剂等。填充剂包括淀粉、乳糖、甘露醇、微晶纤维素等；吸收剂包括硫酸钙、磷酸氢钙、碳酸钙等；润湿剂包括水、乙醇等；粘合剂包括羟丙甲纤维素、聚维酮、微晶纤维素等；崩解剂包括交联羧甲基纤维素钠、交联聚维酮、表面活性剂、低取代羟丙基纤维素等；润滑剂包括硬脂酸镁、滑石粉、聚乙二醇、十二烷基硫酸钠、微粉硅胶、滑石粉等。药用辅料还包括着色剂、甜味剂等。
在一个实施方案中，安罗替尼或其药学上可接受的盐的药物组合物是适于口服的固体制剂。在一个实施方案中，安罗替尼或其药学上可接受的盐的药物组合物可以是片剂或胶囊的形式。在一个特定实施方案中，安罗替尼或其药学上可接受的盐的药物组合物是胶囊的形式。在一个特定实施方案中，口服固体制剂的药学上可接受的载体包括甘露醇、微晶纤维素、羟丙纤维素、硬脂酸镁。
化疗药物
如本申请所用，所述第三治疗剂可以是本领域已知的癌症治疗剂，例如化疗药物。
在一个实施方式中，所述化药物为烷化剂、鬼臼类药物、铂类药物、氟嘧啶衍生物、胞嘧啶衍生物、紫杉烷类药物、喜树碱类药物、蒽环类药物、长春碱类药物中的一种或多种。本申请所述的化疗药物还包括培美曲塞、丝裂霉素、甲氨蝶呤、米托蒽醌、苯达莫司汀、替莫唑胺、Sapacitabine、替吉奥和encequidar中的一种或多种。在一个实施方案中，所述烷化剂为环磷酰胺、异环磷酰胺、卡氮芥、美法仑中的一种或多种。在一个实施方案中，所述鬼臼类药物为依托泊苷、替尼铂苷、表鬼臼毒吡喃葡萄糖甙中的一种或多种。在一个实施方案中，所述胞嘧啶衍生物为阿糖胞苷、吉西他滨、阿扎胞苷中的一种或多种。在一个实施方案中，所述铂类药物为奥沙利铂、顺铂、卡铂、奈达铂和双环铂中的一种或多种。在一个实施方案中，所述氟嘧啶衍生物为吉西他滨、卡培他滨、氟尿嘧啶、双呋氟尿嘧啶、去氧氟尿苷、替加氟、卡莫氟和三氟尿苷中的一种或多种。在一个实施方案中，所述紫杉烷类为紫杉醇、白蛋白结合的紫杉醇以及多烯紫杉醇中的一种或多种。在一个实施方案中，所述喜树碱类为喜树碱、羟基喜树碱、伊立替康和拓扑替康中的一种或多种。在一个实施方案中，所述蒽环类为阿霉素、表阿霉素、柔红霉素、吡柔比星、氨柔比星和阿克拉霉素中的一种或多种。在一个实施方案中，所述的长春碱类为长春瑞滨、长春碱、长春新碱、长春地和长春富宁中的一种或多种。
在一些实施方案中，所述化疗药物按照已知的给药方式(包括给药剂量、给药途径和给药方案)给药。
癌症
本申请所述“癌症”是指由异常细胞生长引起的任何恶性和/或侵袭性生长或肿瘤，包括以形成它们的细胞类型命名的实体瘤、血癌、骨髓癌或淋巴系统癌。术语“癌症”包括但不限于源于体内特定部位的原发癌、已从其起始位置扩散到身体其它部位的转移癌、原始原发癌在缓解后的复发、和第二原发癌(之前有不同类型的癌症史的人的新发原发癌)。
在某些方面中，本申请所述癌症为实体瘤。在一些实施方案中，所述实体瘤包括但不限于：皮肤癌如黑色素瘤和肉瘤、胰腺癌、结直肠癌如结直肠腺癌、食管癌、胃肠道癌、前列腺癌、膀胱癌、肾癌、卵巢癌、子宫癌如子宫颈癌和子宫内膜癌、乳腺癌如三阴性乳腺癌、肺癌如小细胞肺癌和非小细胞肺癌、甲状腺癌、鼻咽癌和肝癌。
在某些方面中，本申请所述癌症为B细胞恶性肿瘤。术语“B细胞恶性肿瘤”包括从B细胞谱系的细胞衍生的任何恶性肿瘤。在一些实施方案中，所述B细胞恶性肿瘤包括白血病和淋巴瘤，其中包括但不限于非霍奇金淋巴瘤、慢性淋巴细胞白血病、多发性骨髓瘤、B细胞淋巴瘤、高级B细胞淋巴瘤、中级B细胞淋巴瘤、低级B级淋巴瘤、B细胞急性淋巴细胞白血病、霍奇金氏淋巴瘤、浆细胞瘤、滤泡性淋巴瘤、滤泡性小裂性淋巴瘤、滤泡性大细胞淋巴瘤、滤泡混合性小裂性淋巴瘤、弥漫性小裂性细胞淋巴瘤、小淋巴细胞性淋巴瘤、幼淋巴细胞白血病、淋巴浆细胞性淋巴瘤、边缘区淋巴瘤、黏膜相关淋巴组织淋巴瘤、单核细胞样B细胞淋巴瘤、脾脏淋巴瘤、毛细胞白血病、弥漫性大B细胞淋巴瘤、纵隔大B细胞淋巴瘤、淋巴瘤样肉芽肿病、血管内淋巴瘤、混合性淋巴瘤、免疫母细胞淋巴瘤、伯基特淋巴瘤、艾滋病相关淋巴瘤、瓦尔登斯特伦巨球蛋白血症和套细胞淋巴瘤。
在某些方面中，本申请所述癌症为非B细胞血液恶性肿瘤。在一些实施方案中，所述非B细胞血液恶性肿瘤包括但不限于慢性髓性白血病和急性髓性白血病。
施用方式
下述内容并非限制本申请药物组合的施用方式。
本申请的药物组合中的组分可以各自独立地、或者其中的部分或全部共同以适合的各种途径施用，包括但不限于，口服或肠胃外(通过静脉内、肌内、局部或皮下途径)施用。在一些实施方案中，本申请的药物组合的组分可以各自独立地、或者其中的部分或全部共同口服施用或注射施用，例如肌肉注射、 静脉注射、皮下注射、瘤内注射或腹腔注射。
本申请的药物组合中的组分可以各自独立地、或者其中的部分或全部共同制备为适合的剂型，包括但不限于，片剂、含片、丸剂、胶囊剂(例如硬胶囊、软胶囊、肠溶胶囊、微囊剂)、酏剂、颗粒剂、糖浆剂、注射剂(例如肌肉内、静脉内、皮下、瘤内或腹腔内)、颗粒剂、乳剂、悬浮液、溶液、分散剂和用于口服或非口服给药的缓释制剂的剂型。
本申请的药物组合中的组分可以各自独立地、或者其中的部分或全部共同含有药学上可接受的载体和/或赋形剂。
技术效果
通常，使用上述的本申请的药物组合将有助于：
(1)与单独给予该药物组合中的任一药物相比，在减少肿瘤的生长或甚至消除肿瘤方面产生更好的疗效；
(2)与该药物组合中的任一药物单独给药相比，提供更少量的给药；
(3)提供在患者中具有良好耐受的治疗，与单一给予该组合中的任一药物相比，其不良反应和/或并发症更少；
(4)提供在所治疗患者之中的更好的疾病控制率；
(5)提供在所治疗的患者具有更长的生存期(例如中位生存期、无进展生存期或总生存期)；
(6)相比于标准的化疗而言，所治疗患者具有更长的生存期(例如中位生存期、无进展生存期或总生存期)；
(7)提供更长时间的疾病缓解持续时间(DOR)；和/或
(8)与单独给予该药物组合中的任一药物相比，具有良好的抗癌活性，表现出更优异的抗癌协同作用。
定义和说明
为了更好地理解本发明，首先定义一些术语。其他定义则贯穿详述部分而列出。除非另有说明，本申请中所用的下列术语具有下列含义。一个特定的术语在没有特别定义的情况下不应该被认为是不确定的或不清楚的，而应该按照本领域普通的含义去理解。当本申请中出现商品名时，意在指代其对应的商品或其活性成分。
术语“CD40”是指肿瘤坏死因子受体超家族成员5(TNFR5)。术语“CD40”包括变体、亚型、同系物、直系同源物和旁系同源物。例如，在某些情况下，对人CD40蛋白具有特异性的抗体可与人以外的物种(例如猴)的CD40蛋白发生交叉反应。在其他实施方案中，对人CD40蛋白具有特异性的抗体可以对人CD40蛋白完全特异，并且不与其他物种或其他类型的蛋白发生交叉反应，或者可以与来源于某些其他物种但并非所有其他物种的CD40发生交叉反应。
术语“人CD40”是指具有人的氨基酸序列的CD40蛋白，例如Genbank登录号为NP_001241.1的人CD40的氨基酸序列(Amini M et al.,(2020)Life Sci 254:117774)。术语“猴或恒河猴CD40”和“小鼠CD40”分别是指猴CD40序列和小鼠CD40序列，例如，其分别具有Genbank登录号为Nos.NP_001252791.1和NP_035741.2的氨基酸序列。
术语“免疫应答”是指如淋巴细胞、抗原呈递细胞、吞噬细胞、粒细胞和由上述细胞或肝脏产生的可溶性大分子(包括抗体、细胞因子和补体)在人体中选择性损害、破坏或清除侵入病原体、感染了病原体的细胞或组织、癌细胞、或者自体免疫或病理性炎症的情况下的正常人细胞或组织的作用。
术语“抗体”是指通过至少一个抗原结合位点识别并特异性结合靶标(如CD40)的免疫球蛋白分子，其中所述抗原结合位点通常位于免疫球蛋白分子的可变区内。如本文所用，术语“抗体”涵盖完整多克隆抗体、完整单克隆抗体、单链Fv(scFv)抗体、重链抗体(HCAbs)、轻链抗体(LCAbs)、多特异性抗体、双特异性抗体、单特异性抗体、单价抗体、包含抗体的抗原结合位点的融合蛋白、以及任 何其他包含抗原结合位点的经修饰的免疫球蛋白分子(例如，双可变结构域免疫球蛋白分子)，只要该抗体展现出所需的生物学活性即可。抗体还包括但不限于小鼠抗体、嵌合抗体、人源化抗体、人抗体。根据抗体重链恒定区(分别被称为α、δ、ε、γ和μ)的特征，抗体可以是免疫球蛋白五种主要类别中的任何一种：IgA、IgD、IgE、IgG和IgM，或其亚类(同种型)(例如IgG1、IgG2、IgG3、IgG4、IgA1和IgA2)。不同种类的免疫球蛋白具有不同且本领域熟知的亚基结构和三维构型。抗体可以是裸抗体或与其他分子缀合的，包括但不限于毒素和放射性同位素。除非另有明确说明，否则本文所用的术语“抗体”包括完整抗体的“抗原结合部分”。IgG是包含两条重(H)链和两条轻(L)链的糖蛋白，其中重链和轻链通过二硫键连接。每条重链由重链可变区(简称V H)和重链恒定区构成。重链恒定区由三个结构域构成，即C H1、C H2和C H3。每条轻链由轻链可变区(简称V L)和轻链恒定区构成。轻链恒定区由一个结构域C L构成。V H和V L区还可以进一步划分为称作互补决定区(CDR)的高变区，其由较为保守的骨架区(FR)区分隔开。每个V H和V L由三个CDR以及四个FR构成，从氨基端到羧基端以FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4的顺序排布。重链和轻链的可变区包含与抗原相互作用的结合结构域。抗体的恒定区可以介导免疫球蛋白与宿主组织或因子的结合，所述宿主组织或因子包括多种免疫系统细胞(例如，效应细胞)和经典补体系统的第一组分(C1q)。
抗体的“抗原结合部分”(或简称“抗体部分”)，是指抗体中保留特异性结合抗原(例如，CD40蛋白)能力的一个或多个片段。已证实，抗体的抗原结合功能可以通过全长抗体的片段来实现。涵盖在抗体的“抗原结合部分”中的结合片段的例子包括：(i)Fab片段，由V L、V H、C L和C H1结构域构成的单价片段；(ii)F(ab') 2片段，包含在铰链区通过二硫键连接的两个Fab片段的二价片段；(iii)由V H和C H1结构域构成的Fd片段；(iv)由抗体单臂V L和V H结构域构成的Fv片段；(v)由V H结构域构成的dAb片段(Ward et al.,(1989)Nature 341:544-546)；(vi)分离的互补决定区(CDR)；以及(vii)纳米抗体，即一种包含单个可变结构域和两个恒定结构域的重链可变区。此外，尽管Fv片段的两个结构域V L和V H由不同的基因编码，可以利用重组的方法通过合成接头将V H和V L连接成单蛋白链，其中V L和V H区配对形成单价分子(称为单链Fv(scFv)；参见例如Bird et al.,(1988)Science 242:423-426；和Huston et al.,(1988)Proc.Natl.Acad.Sci.USA 85:5879-5883)。这样的单链抗体也涵盖在术语抗体的“抗原结合部分”中。这些抗体片段可以通过本领域技术人员已知的常用技术获得，且可以通过与完整抗体相同的方式进行功能筛选。
术语“分离的抗体”是指基本上不含具有不同抗原特异性的其他抗体的抗体(例如，特异性结合CD40蛋白的分离抗体基本上不含特异性结合CD40蛋白以外抗原的抗体)。但是，特异性结合人CD40蛋白的分离抗体可能对其他抗原例如其他物种的CD40蛋白具有交叉反应性。此外，分离的抗体基本上不含其他细胞材料和/或化学物质。
术语“小鼠抗体”是指可变区中骨架区和CDR区均来自小鼠种系免疫球蛋白序列的抗体。此外，如果抗体包含恒定区，则恒定区也来自小鼠种系免疫球蛋白序列。本发明的小鼠抗体可以包含不由小鼠种系免疫球蛋白序列编码的氨基酸残基(例如，通过体外随机突变或点突变、或通过体内体细胞突变而引入的突变)。但本文所用的术语“小鼠抗体”不包括在小鼠骨架区序列中插入来自其他哺乳动物物种的CDR序列的抗体。
术语“嵌合抗体”是指通过组合非人来源的遗传物质与人来源的遗传物质而得来的抗体。或者更笼统地说，嵌合抗体是指组合某一物种的遗传物质与另一物种遗传物质的抗体。
术语“人源化抗体”是指来源于非人物种但其蛋白序列已经被修改以增加其与人天然抗体的相似度的抗体。
“识别抗原的抗体”以及“对抗原特异的抗体/对抗原具有特异性的抗体”在本文中与术语“特异性结合抗原的抗体”可互换使用。
“特异性结合人CD40”的抗体或其抗原结合部分是指与人CD40蛋白(可能还包括一个或多个非人物种的CD40蛋白)结合但基本不与非CD40蛋白结合的抗体。优选地，抗体以“高亲和力”结合人CD40蛋白，即以5.0×10 -8M或更小，优选为1.0×10 -8M或更小的K D结合人CD40蛋白。
术语“基本不结合”蛋白或细胞是指不与蛋白或细胞结合，或者不以高亲和力与其结合，即以1.0×10 -6M或更大，优选为1.0×10 -5M或更大，更优选为1.0×10 -4M或更大，更优选为1.0×10 -3M或更大，甚至更优选为1.0×10 -2M或更大的K D结合蛋白或细胞。
术语“高亲和力”对于IgG抗体而言，是指对于目标抗原，抗体具有1.0×10 -6M或更小，优选为9.0×10 -9M或更小，更优选为5.0×10 -9M或更小，甚至更优选为1.0×10 -9M或更小，甚至更优选为5.0×10 -10M或更小的K D。但是对于其他抗体同种型，“高亲和力”结合可能有所不同。例如，对于IgM同种型，“高亲和力”是指抗体具有10 -6M或更小，优选为10 -7M或更小，更优选为10 -8M或更小的K D。
术语“K assoc”或“K a”是指特定抗体-抗原相互作用的缔合速率，而本文所用的术语“K dis”或“K d”是指特定抗体-抗原相互作用的解离速率。本文所用的术语“K D”是指解离常数，其为K d与K a之比(即K d/K a)并用摩尔浓度(M)表示。抗体的K D值可以使用本领域熟知的方法测定。测定抗体K D值的优选方法是使用表面等离子体共振技术，优选使用生物传感器系统，例如Biacore TM系统进行测定。
术语“EC50”，又叫半数最大效应浓度，是指在特定暴露时间后，诱导反应达到基线和最大值之间的一半的抗体浓度。术语“IC50”，又叫半数最大抑制浓度，是指相对于不存在抗体时，将特异性生物学或生化功能抑制50％的抗体浓度。
术语“激动性CD40抗体”或“激动性抗CD40抗体”是指与CD40结合并激活或诱导CD40信号转导(从而例如促进免疫细胞活化和增殖以及细胞因子和趋化因子产生)的抗CD40抗体。而术语“拮抗性CD40抗体/拮抗性抗CD40抗体”是指阻断或抑制可能由CD40L参与诱导的CD40信号转导的抗CD40抗体。激动性CD40抗体通过提高APC的抗原递呈能力、活化肿瘤特异性CD4 +和CD8 +T细胞、刺激淋巴细胞和单核细胞分泌细胞因子和趋化因子、增强细胞毒性淋巴细胞和NK细胞的肿瘤细胞杀伤作用等，促进肿瘤受试者对肿瘤的先天性和适应性免疫应答。
如本文在两个或多个核酸或多肽的背景中所用的百分比“同一性”是指，当以最高对应性进行比较和比对(如果需要的话，引入空位)时，在将或不将保守氨基酸替换作为序列统一性的一部分的情况下，两个或多个具有相同的或具有特定百分比相同的核苷酸或氨基酸残基的序列或子序列。百分比同一性可以使用序列比对软件或算法或通过目测来测量。可用于氨基酸或核苷酸序列比对的各种软件和算法是本领域熟知的，包括但不限于BLAST、ALIGN、Megalign、BestFit、GCG Wisconsin Package及其变换形式。在一些实施方案中，本发明所述的两种核酸或多肽基本上是相同的，是指当以最高对应性进行比较和比对时，使用序列比对算法或目测进行测量，两种核酸或多肽具有至少70％、至少75％、至少80％、至少85％、至少90％，并且在一些实施方案中具有至少95％、96％、97％、98％、99％的核苷酸或氨基酸残基同一性。
如文本所用，术语“药物组合”是指同时或先后施用的两种或两种以上的活性成分(以各自的活性成分本身的形式施用，或者以其各自的药学上可接受的盐或酯等衍生物、前药或组合物的形式施用)的组合。在本文中，术语“联用药物”和“药物组合”可互换使用。所述活性成分可以各自作为单一制剂同时地、或各自作为单一制剂以任何顺序依次地施用于患者。
术语“治疗”一般是指获得需要的药理和/或生理效应的操作。该效应根据完全或部分地预防疾病或其症状，可以是预防性的；和/或根据部分或完全稳定或治愈疾病和/或由于疾病产生的副作用，可以是治疗性的。本文使用的“治疗”涵盖了对患者疾病的任何治疗，包括：(a)抑制疾病的症状，即阻止其发展；或(b)缓解疾病的症状，即，导致疾病或症状退化。
术语“有效量”意指(i)治疗特定疾病、病况或障碍，(ii)减轻、改善或消除特定疾病、病况或障碍的一种或多种症状，或(iii)预防或延迟本文中所述的特定疾病、病况或障碍的一种或多种症状发作的本申请的化合物的用量。在癌症的情况中，药物的有效量可减少癌细胞数、缩小肿瘤体积、抑制(即一定程度的减缓，优选停止)癌细胞浸润到周围器官中、抑制(即一定程度的减缓，优选停止)肿瘤转移、一定程度的抑制肿瘤生长、和/或一定程度的减轻与癌症有关的一种或多种症状。就药物可抑制癌细胞生长和/或杀死现有癌细胞的程度而言，它可以是抑制细胞的和/或毒害细胞的。构成“有效量”的活性物质(例如本申请的化合物)的量可根据一些因素而变化，诸如个体的疾病状态、年龄、性别和重量，以及治疗剂或治疗剂组合在个体中引发所需应答的能力。有效量也可例行性地由本领域技术人员根据其自身的知识及本公开内容而确定。
术语“施用”表示使用本领域技术人员已知的多种方法和递送系统中的任一种，向主体物理引入包含治疗剂的组合物。
术语“统一剂量(flat dose)”的应用是指，不考虑患者的重量或体表面积(BSA)施用给患者的剂量。因此将统一剂量规定为药剂(例如，抗CD40抗体)的绝对量，而不是规定为mg/kg剂量。例如，60kg人和100kg人将接受相同剂量的抗体。
术语“固定组合”指活性组分(例如抗CD40抗体或式(I)化合物)以固定总剂量或剂量比例，或以单一实体、药物组合物或制剂的形式同时给予受试者。
术语“非固定组合”指两种或以上活性组分作为独立的实体(例如药物组合物、制剂)同时、并行或依序且无具体时间限制地给予受试者，其中所述给予受试者的活性成份达到治疗有效量水平。非固定组合可列举的例子是鸡尾酒疗法，例如给予3种或以上之活性组分。在非固定组合中，所述各个活性组分可以作为完全独立的药物组合物进行包装、销售或给药。所述“非固定组合”也包括“固定组合”之间、或“固定组合”与任一或多种活性组分的独立实体的联合使用。
术语“药学上可接受的”是针对那些化合物、材料、组合物和/或剂型而言，它们在可靠的医学判断的范围之内，适用于与人类和动物的组织接触使用，而没有过多的毒性、刺激性、过敏性反应或其它问题或并发症，与合理的利益/风险比相称。
术语“药学上可接受的盐”包括碱根离子与自由酸形成的盐或酸根离子与自由碱形成的盐，例如包括盐酸盐、氢溴酸盐、硝酸盐、硫酸盐、磷酸盐、乙酸盐、三氟乙酸盐、富马酸盐、马来酸盐、柠檬酸盐、琥珀酸盐、甲磺酸盐、苯磺酸盐或对甲基苯磺酸盐，优选盐酸盐、氢溴酸盐、硫酸盐、乙酸盐、三氟乙酸盐、富马酸盐、马来酸盐、甲磺酸盐、对甲基苯磺酸盐、钠盐、钾盐、铵盐、氨基酸盐等。
在本文中，术语“受试者”或“患者”可互换使用。在一些实施方案中，术语“受试者”或“患者”是哺乳动物。在部分实施方案中，所述受试者或患者是小鼠。在部分实施方案中，所述受试者或患者是人。
如本文所用，“联用”或“联合使用”意指两种或更多种活性物质可以各自作为单一制剂同时地、或各自作为单一制剂以任何顺序依次地施用于受试者。
术语“单剂量”是指含有一定量药品的最小包装单元，例如一盒药有七粒胶囊，则每个胶囊为单剂量；或者每瓶注射液为单剂量。术语“多剂量”由多个单剂量组成。
术语“药物组合物”是指一种或多种本申请的活性成分或其药物组合与药学上可接受的辅料组成的混合物。药物组合物的目的是有利于对受试者给予本申请的化合物或其药物组合。在本文中，术语“药物组合物”和“制剂”具有相同的含义，并可互换使用。
如本文中所述的，任何浓度范围、百分比范围、比率范围或整数范围应当理解为包括在列举的范围内的任意整数的值，且当适当时，包括其分数(诸如整数的十分之一和百分之一)，除非另外指出。
如本文所用，大约”或“约”表示在本领域普通技术人员判定的对特定值可以接受的误差范围内， 其部分取决于如何测量或测定该值，即测量系统的限制。例如，大约”或“约”按照本领域实践可表示1倍或超过1倍标准偏差以内。或者，大约”或“约”可以表示多至±5％的范围，例如在所给定的具体数值范围±2％范围内、±1％范围内或±0.5％范围内波动。当本申请或权利要求中给出特定值时，除非另有说明，大约”或“约”的含义应认为是在该特定值的可接受的误差范围内。在本文中，除非另有说明，步骤参数或条件的值默认均由“约”修饰。
附图说明
图1A-1C示出在捕获ELISA中，嵌合抗体1A3(A)、1D1(B)和C1H1(C)与人CD40的结合能力。
图2A-2C示出在基于细胞的报告基因检测中，嵌合抗体1A3(A)、1D1(B)和C1H1(C)激活CD40信号转导的活性。
图3示出在捕获ELISA中，人源化抗体huC1H1-V1和huC1H1-V2与人CD40的结合能力。
图4示出在基于细胞的结合FACS中，人源化抗体huC1H1-V1和huC1H1-V2与表达人CD40的293T细胞的结合能力。
图5示出在竞争ELISA中，人源化抗体huC1H1-V1和huC1H1-V2阻断人CD40与CD40L结合的能力。
图6示出在竞争ELISA中，人源化抗体huC1H1-V1和huC1H1-V2阻断参照品与人CD40结合的能力。
图7示出在基于细胞的报告基因检测中，人源化抗体huC1H1-V1和huC1H1-V2激活CD40信号转导的活性。
图8A-B示出实验结束时(D14)各组小鼠的个体肿瘤体积(A)和个体肿瘤重量(B)。其中，＊ ＊P<0.01和 ＊＊＊P<0.001是通过Mann-Whitney检验与IgG2组比较；* *P<0.01和** *P<0.001是通过双尾Student’s t检验与huC1H1-V2+安罗替尼的二盐酸盐组比较；P<0.05定义为有统计学显著性差异。
实施例
为清楚起见，进一步用实施例来阐述本申请，但是实施例并非限制本申请的范围。
本申请中抗CD40抗体或其抗原结合部分的制备、纯化方法已在公开号为PCT/CN2021084013的专利申请文件中记载，PCT/CN2021084013专利申请文件的全部内容均引入本申请。
实施例中的小鼠抗CD40抗体或小鼠抗CD40单克隆抗体是按照PCT/CN2021084013专利文件中记载方法，通过杂交瘤技术产生并经体外功能筛选后获得。本申请中的抗CD40抗体huC1H1-V2即PCT/CN2021084013中的抗CD40抗体huC1H1-V2。
实施例1使用BIACORE表面等离子共振技术对抗CD40抗体进行亲和力测定
通过Biacore T200系统(GE Healthcare，Pittsburgh，PA，USA)对本申请的抗CD40抗体的亲和力和结合动力学进行表征。
简而言之，使用Biacore(GE Healthcare，Pittsburgh，PA，USA)提供的标准胺偶联试剂盒，通过伯胺基团将山羊抗人IgG(GE healthcare，产品目录BR100839，人抗体捕获试剂盒)共价连接至CM5芯片(来自GE healthcare产品目录为BR100530的羧甲基化葡聚糖涂层芯片)上。用乙醇胺封闭生物传感器表面未反应的部分。然后，将浓度为66.67nM的本申请的抗CD40抗体和两个参照品抗体(BM1(Dacetuzumab，Genentech Inc.，也称为CD40-BM1，内部制备，重链和轻链的氨基酸序列分别如本申请的SEQ ID NOs:40和41所示)和BM2(Selicrelumab，Abgenix Inc.，也称为CD40-BM2，内部制备，重链和轻链的氨基酸序列分别如本申请的SEQ ID NOs:42和43所示))以10μL/分钟的流速流过芯片。然后，将梯度稀释的重组人CD40-his(Acro biosystems，产品目录CD0-H5228，以80nM为起始浓度，在HBS-EP +缓冲液(由Biacore提供)中2倍梯度稀释)或食蟹猴CD40-his蛋白(Acro biosystems，产品目录CD0-C52H6，以80nM为起始浓度，在HBS-EP +缓冲液(由Biacore提供)中2倍梯度稀释) 以30μL/分钟的流速流过芯片。追踪抗原-抗体结合动力学2分钟，并追踪解离动力学10分钟。使用BIAcore evaluation软件将结合和解离曲线拟合至1:1Langmuir结合模型。测定K D、K a和K d值。
实施例2抗CD40抗体的结合活性
通过捕获ELISA和流式细胞术(FACS)测定抗CD40抗体的结合活性。
对于捕获ELISA，溶于PBS的浓度为2μg/mL的亲和纯化山羊抗人IgG(F(ab') 2片段特异性，Jackson Immuno Research，产品目录109-005-097)以100μL/孔包被在96孔板中，于4℃孵育过夜。用洗涤缓冲液(PBS+0.05％v/v Tween-20，PBST)洗涤板1次，然后加入200μL/孔的封闭缓冲液(含5％w/v脱脂牛奶的PBST)，于37℃封闭2小时。洗涤板4次，每孔加入100μL梯度稀释(以66.7nM为起始浓度，在含2.5％w/v脱脂牛奶的PBST中5倍梯度稀释)的本申请的抗CD40抗体、BM1、BM2和阴性对照品hIgG(用于静脉注射的人免疫球蛋白(pH 4)，Hualan Biological Engineering Inc.)，于37℃孵育40分钟，然后再次洗涤板4次。在捕获有抗CD40抗体的96孔板中加入100μL/孔的生物素标记的人CD40-Fc蛋白(SEQ ID NO:30的第21-193位氨基酸残基连接至SEQ ID NO:28的第99-330位氨基酸残基的N末端，溶于含2.5％w/v脱脂牛奶的PBST中，浓度为1.15nM)，于37℃孵育40分钟，洗涤板4次，然后加入100μL/孔的HRP标记链霉亲和素(在PBST中以1:10000的比例稀释，Jackson Immuno Research，产品目录016-030-084)，于37℃孵育40分钟。最后一次洗涤后，加入100μL/孔的ELISA底物TMB(Innoreagents，产品目录TMB-S-002)于室温进行孵育。3-10分钟后，加入50μL/孔的1M H 2SO 4终止反应，并在酶标仪上用双波长模式(TMB检测波长450nm，参比波长630nm)读取每个孔的吸光度值，然后用OD(450-630)值与抗体浓度作图。使用Graphpad Prism软件分析数据，并得到EC50值。
通过流式细胞术(FACS)检测抗CD40抗体与293T-CD40细胞的结合，其中293T-CD40细胞由Biosion内部制备，其细胞膜稳定表达全长人CD40(uniprot#P25942-1，SEQ ID NO:30)。按照lipofectamine 3000转染试剂(Thermo Fisher)的说明，将EcoRI和XbaI位点之间插入了CD40编码序列的pCMV-T-P质粒转染至293T细胞，来制备293T-CD40细胞。具体而言，从细胞培养瓶中收获293T-CD40细胞，洗涤两次后重悬于FACS缓冲液(含有2％v/v胎牛血清的磷酸盐缓冲液(PBS))中。在含有2×10 5个细胞/孔的96孔板中加入100μL/孔FACS缓冲液梯度稀释(以80nM为起始浓度，4倍梯度稀释)的本申请的抗CD40抗体或对照品，冰浴40分钟。将细胞用FACS缓冲液洗涤两次后，每孔加入100μLR-藻红蛋白标记的亲和纯化山羊抗人IgG(Fcγ片段特异性，Jackson Immuno Research，产品目录109-115-098，在FACS缓冲液中以1:1000的比例稀释)。于4℃避光孵育40分钟后，将细胞洗涤3次后重悬于FACS缓冲液中。使用Becton Dickinson FACS Canto II-HTS测量荧光值，并用荧光值与抗体浓度作图。使用Graphpad Prism软件分析数据，并得到EC50值。
实施例3抗CD40抗体对CD40-CD40L结合或CD40-参照品结合的阻断活性
3.1配体阻断ELISA
通过竞争ELISA测定抗CD40抗体阻断CD40与CD40L结合的能力。简而言之，溶于包被缓冲液(碳酸盐/碳酸氢盐缓冲液)的浓度为2μg/mL的人CD40-Fc蛋白(SEQ ID NO:30的第21-193位氨基酸残基连接至SEQ ID NO:28的第99-330位氨基酸残基的N末端)以100μL/孔包被于96孔板中，于4℃孵育过夜。第二天，用洗涤缓冲液(PBS+0.05％v/v Tween-20，PBST)洗涤板1次，并加入含5％w/v脱脂牛奶的PBST，于37℃中封闭2小时。然后，用洗涤缓冲液洗涤板4次。
用含2.5％w/v脱脂牛奶的PBST梯度稀释(以200nM为起始浓度，5倍梯度稀释)本申请的抗CD40抗体或对照品，并将梯度稀释的抗CD40抗体或对照品以100μL/孔添加到包被有CD40-Fc蛋白的板中， 于37℃孵育40分钟。再次用洗涤缓冲液洗涤板4次，然后每孔加入100μL浓度为95ng/mL的生物素标记的人CD40L-his蛋白(Sino biological Inc.，产品目录10239-H08E)，于37℃孵育40分钟。再次用洗涤缓冲液洗涤板。接着，将100μL/孔的HRP标记链霉亲和素(Jackson Immunoresearch，产品目录016-030-084，在PBST缓冲液中以1:10000的比例稀释)加到板中，于37℃孵育40分钟。再次用洗涤缓冲液洗涤板。最后，加入TMB，并用1M H 2SO 4终止反应，并在酶标仪上用双波长模式(TMB检测波长450nm，参比波长630nm)读取每个孔的吸光度值，然后用OD(450-630)值与抗体浓度作图。使用Graphpad Prism软件分析数据，并得到IC50值。
3.2参照品阻断ELISA
通过竞争ELISA测定本申请的抗CD40抗体阻断参照品与人CD40结合的能力。简而言之，溶于PBS的浓度为1μg/mL的BM2以100μL/孔包被于96孔板中，于4℃孵育过夜。第二天，用洗涤缓冲液洗涤板1次，并加入封闭缓冲液(含5％w/v脱脂牛奶的PBST)，于37℃封闭2小时。在封闭96孔板期间，用生物素标记的人CD40-Fc蛋白(SEQ ID NO:30的第21-193位氨基酸残基连接至SEQ ID NO:28的第99-330位氨基酸残基的N末端，溶于含2.5％w/v脱脂牛奶的PBST中，浓度为0.23nM)稀释本申请的抗CD40抗体或对照品，以66.7nM为起始浓度，5倍梯度稀释，然后于室温孵育40分钟。洗涤板4次后，将100μL/孔的抗体/生物素标记的人CD40-Fc混合物加到包被有BM2的板中。于37℃孵育40分钟后，再次用洗涤缓冲液洗涤板4次。然后将100μL/孔的HRP标记链霉亲和素加到板中，于37℃孵育40分钟，以检测结合到板上的生物素标记的人CD40-Fc。再次用洗涤缓冲液洗涤板4次。最后，加入TMB，并用1M H 2SO 4终止反应，并在酶标仪上用双波长模式(TMB检测波长450nm，参比波长630nm)读取吸光度值，然后用OD(450-630)值与抗体浓度作图。使用Graphpad Prism软件分析数据，并得到IC50值。
实施例4抗CD40抗体基于细胞的报告基因检测
使用稳定表达全长人CD40(uniprot No.P25942-1，SEQ ID NO:30)的CD40表达报告基因细胞系293T-NF-κB-Luc-CD40进一步测定本申请的抗CD40抗体的激动活性。按照lipofectamine 3000转染试剂(Thermo Fisher)的说明，先后将pGL4.32[luc2P NF-κB-RE Hygro]载体(Promega，
Figure PCTCN2022121184-appb-000003
登录号：EU581860)和pCMV-T-P质粒(EcoRI和XbaI位点之间插入了CD40编码序列)转染至293T细胞，制备293T-NF-κB-Luc-CD40细胞。当CD40激动剂与细胞接触后，其中CD40信号转导被激活，荧光素酶的表达上调，并且能够用发光检测测定出来。
简而言之，将对数期的293T-NF-κB-Luc-CD40细胞重悬于含10％FBS(Gibco，产品目录10099-141)的DMEM培养基(Gibco Inc.，产品目录10566-016)中，并以20μL/孔(含5×10 3个细胞)接种至384孔细胞培养板(Corning，产品目录3707)。然后，每孔加入20μL梯度稀释(以200nM为起始浓度，在培养基中3倍梯度稀释)的本申请的抗CD40抗体或对照品(包括内部制备的阴性对照品抗CD22抗体)，于37℃孵育6小时。然后，每孔加入30μL ONE-GloTM荧光素酶检测系统(Promega，产品目录E6120)的试剂，并于室温孵育5分钟。使用Tecan
Figure PCTCN2022121184-appb-000004
200Pro测量化学发光值。使用Graphpad Prism软件分析数据，并得到EC50值。
实施例5嵌合抗体的产生和表征
将小鼠抗CD40单克隆抗体的重链可变区和轻链可变区进行了测序并汇总于表1中。
将小鼠抗CD40单克隆抗体C1H1、1D1和1A3的重链可变区和轻链可变区分别克隆至含人IgG1重链恒定区(SEQ ID NO:27)的片段中和含人κ轻链恒定区(SEQ ID NO:29)的片段中，其中可变区的C末端与相应恒定区的N末端相连。
包含编码连接至人IgG1重链恒定区的重链可变区的核苷酸的载体，和包含编码连接至人κ轻链恒定区的轻链可变区的核苷酸的载体，按1.1:1的轻链构建体：重链构建体的比例，用1mg/mL PEI瞬时转染至50mL 293F悬浮细胞中。
在摇瓶中培养六天后收获细胞上清液，通过离心沉淀上清液中的细胞，然后通过蛋白A琼脂糖柱从细胞上清液中纯化嵌合抗体。按照前述实施例中的方案，通过捕获ELISA、BIAcore亲和力测定和基于细胞的报告基因检测纯化的嵌合抗体。结果如表2、图1A-1C和图2A-2C所示。
结果显示，嵌合抗体与其亲本抗体具有相似的结合能力和激动活性。特别地，与BM1相比，嵌合抗体C1H1对人CD40具有更高的结合亲和力和能力，且对食蟹猴CD40具有更高的结合亲和力。
表2.嵌合抗体的结合亲和力
Figure PCTCN2022121184-appb-000005
实施例6小鼠抗CD40单克隆抗体C1H1的人源化
选择小鼠抗CD40抗体C1H1进行人源化和进一步的研究。该抗体的人源化是采用业已成熟的CDR移植方法进行的，如下所述。
为了选择用于小鼠抗CD40抗体C1H1人源化的接收体骨架，将小鼠抗CD40抗体C1H1轻链可变区和重链可变区的序列与人免疫球蛋白基因数据库进行BLAST比对。选择与小鼠抗CD40抗体C1H1具有最高同源性的人种系抗体作为人源化的接收体骨架。将小鼠抗体的重/轻链可变区CDR插入到选出的骨架中，并进一步突变骨架中的残基以获得更多候选的重/轻链可变区。总共获得4个人源化C1H1抗体，即huC1H1-V1至huC1H1-V4，其重/轻链可变区序列如表1中所示。
包含编码连接至人IgG2重链恒定区(SEQ ID NO:28)的重链可变区的核苷酸的载体，和包含编码连接至人κ轻链恒定区(SEQ ID NO:29)的人源化轻链可变区的核苷酸的载体，按60％:40％的轻链构建体：重链构建体的比例，用1mg/mL PEI瞬时转染至50mL 293F悬浮细胞中。
实施例7人源化抗体的表征
在摇瓶中培养六天后，收获分别含有人源化抗体huC1H1-V1至huC1H1-V4的细胞上清液，并使用Octet系统(Fortebio，Octet RED 96)，按照下述方案通过Octet测定抗体与人CD40的结合亲和力。简而言之，将抗人IgG Fc捕获(AHC)的生物传感器(来自ForteBio)用10mM甘氨酸(pH 1.5)预浸泡3秒，再浸入运行缓冲液(含0.5％w/v BSA的PBST)中3秒，浸泡和浸入的步骤重复3次。然后，将传感器浸入含有人源化抗CD40抗体的细胞上清液、溶于HBS-EP +缓冲液的浓度为5μg/mL的嵌合抗体C1H1、或溶于HBS-EP +缓冲液的浓度为5μg/mL的参照品中120秒，之后浸入运行缓冲液中5分钟。新基线在运行缓冲液中运行180秒。然后将传感器浸入以运行缓冲液梯度稀释的人CD40-his蛋白(Acro biosystems，产品目录CD0-H5228，以40nM为起始浓度，2倍梯度稀释)中120秒，然后浸入运行缓冲液中10分钟以建立基线。最后，将传感器用10mM甘氨酸(pH 1.5)预浸泡3秒，再浸入运行缓冲液中3秒，浸泡和浸入的步骤重复3次。使用ForteBio Data Analysis 8.1将结合和解离曲线拟合至1:1Langmuir结合模型。测定K a、K d和K D值，并汇总于下表3中。
结果显示，huC1H1-V1、huC1H1-V2与嵌合抗体C1H1具有相似的人CD40结合亲和力，并且其结合亲和力要优于BM2。
表3.人源化C1H1单克隆抗体的亲和力
Figure PCTCN2022121184-appb-000006
通过蛋白A琼脂糖柱纯化人源化抗体huC1H1-V1和huC1H1-V2，并按照前述实施例中的方案和下述方案，通过Biacore、捕获ELISA、基于细胞的结合FACS、竞争ELISA、基于细胞的报告基因检测和蛋白热位移测定法对huC1H1-V1和huC1H1-V2进行表征。
还对人源化抗体huC1H1-V2进行了热稳定性测定。使用GloMelt TM热位移蛋白稳定性试剂盒(Biotium，产品目录33022-T)，通过蛋白热位移分析法测定Tm(熔解温度)。简而言之，GloMelt TM染料解冻至室温。将含有染料的小瓶涡旋并离心。然后，将5μL 200×染料添加到95μL PBS中制备10×染料。反应体系中加入2μL 10×染料和10μg人源化抗体，并添加PBS至总反应体积为20μL。将含有染料和抗体的离心管短暂离心，并置于实时PCR热循环仪(Roche，LightCycler 480II)中，该热循环仪中Melt Curve程序的参数如表4所示。
表4.Melt Curve程序的参数
Profile步骤 温度 升温速率 持续时间
Initial hold 25℃ NA 30s
Melt curve 25-99℃ 0.1℃/s NA
测定结果如表5和图3-7所示。
如表5所示，人源化抗体huC1H1-V1、huC1H1-V2与嵌合抗体C1H1与人CD40和食蟹猴CD40具有相当的结合亲和力。换而言之，huC1H1-V1和huC1H1-V2与人CD40和食蟹猴CD40的结合亲和力，并且比BM1和BM2与人CD40和食蟹猴CD40的结合亲和力更高。
图5显示本申请的人源化抗体huC1H1-V1和huC1H1-V2能够阻断CD40与CD40L的结合，其阻断活性与BM1和BM2相当或略低。
根据图6所示，本申请的人源化抗体huC1H1-V1和huC1H1-V2能够阻断人CD40与BM2的结合，表明本申请的抗体huC1H1-V1和huC1H1-V2可能与BM2结合相似的表位。
如图7所示，在基于细胞的报告基因检测中，本申请的人源化抗体huC1H1-V1和huC1H1-V2的激动活性高于BM1和BM2。
表5.人源化C1H1抗体的结合和功能活性
Figure PCTCN2022121184-appb-000007
*未检测。
实施例8人B细胞淋巴瘤Ramos异种移植模型中huC1H1-V2抗体的体内抗肿瘤疗效
采用NOD-SCID小鼠测定huC1H1-V2抗体的体内抗肿瘤活性。简而言之，在NOD-SCID小鼠的右侧腋下皮下注射3×10 7个人B细胞淋巴瘤Ramos细胞(中国科学院)。使用电子游标卡尺测量肿瘤体积，计算公式为(长度×宽度 2)/2。当肿瘤平均体积达到约100-150mm 3时，选出40只荷瘤小鼠并随机分成4组(每组10只小鼠)，将动物分组的日期指定为第0天。根据表6所示的给药方案，从第0天起，对小鼠尾静脉注射同种型对照抗体(anti-HEL-Human IgG2Isotype-control，也称为IgG2，Biointron Inc)或huC1H1-V2抗体。
表6.人B细胞淋巴瘤Ramos异种移植模型的实验设计
Figure PCTCN2022121184-appb-000008
注：N：每组动物数量；Q2D：每2天一次；iv：静脉注射。
结果汇总于表7中。荷瘤动物对所有治疗均具有良好的耐受，没有出现明显的体重减轻或其他症状。huC1H1-V2抗体(5mg/kg)治疗显示出最强的抗肿瘤活性，在第14天，该组(第3组)小鼠的平均肿瘤体积为230.2mm 3，在统计学上小于同种型对照组，抑瘤率(TGI)为96％。但是，huC1H1-V2抗体(15mg/kg)治疗并未显示出进一步增强的疗效，即第4组小鼠的肿瘤体积与第3组小鼠的肿瘤体积无明显差异。
表7.huC1H1-V2抗体在人B细胞淋巴瘤Ramos异种移植模型中的抗肿瘤疗效
Figure PCTCN2022121184-appb-000009
注：a.平均值±标准误；b.第14天的肿瘤体积，其通过student’s t检验与同种型对照组进行比较，*表 示P≤0.05，**表示P<0.01，***表示P<0.001；c.T/C(％)＝(T-T 0)/(C-C 0)×100％，其中T和C分别指第14天治疗组和同种型对照组的平均肿瘤体积，T 0和C 0分别指第0天治疗组和同种型对照组的平均肿瘤体积；d.TGI(％)＝(1-T/C)×100％。
实施例9小鼠结肠癌MC38异种移植模型中huC1H1-V2抗体的体内抗肿瘤疗效
采用CD40人源化转基因小鼠(也称为hCD40小鼠)进一步测定huC1H1-V2抗体的体内抗肿瘤活性。简而言之，在hCD40小鼠右侧背部皮下注射1×10 6个小鼠结肠癌MC38细胞(上海兰立生物科技有限公司)。使用电子游标卡尺测量肿瘤体积，计算公式为(长度×宽度 2)/2。当肿瘤平均体积达到约100mm 3时，选出18只荷瘤小鼠并随机分成3组，每组6只小鼠，将动物分组的日期指定为第0天。根据表8所示的给药方案，从第1天起，对小鼠尾静脉注射溶剂(磷酸盐缓冲液，也称为PBS)或huC1H1-V2抗体。
表8.MC38异种移植模型的实验设计
Figure PCTCN2022121184-appb-000010
注：N：每组动物数量；Q2D：每2天一次；iv：静脉注射。
结果汇总于表9中。荷瘤动物对所有治疗均具有良好的耐受，没有出现明显的体重减轻或症状。huC1H1-V2抗体(3mg/kg)治疗显示出明显的抗肿瘤活性，在第14天，该组(第2组)小鼠的平均肿瘤体积为1169mm 3，在统计学上小于溶剂组，抑瘤率(TGI)为64％。huC1H1-V2抗体(10mg/kg)治疗也显示出明显的抗肿瘤活性，在第14天，该组(第3组)小鼠的平均肿瘤体积为1446mm 3，在统计学上小于溶剂组，抑瘤率(TGI)为56％。
表9.huC1H1-V2抗体在MC38异种移植模型中的抗肿瘤疗效
Figure PCTCN2022121184-appb-000011
注：a.平均值±标准误；b.第14天的肿瘤体积，其通过student’s t检验与溶剂对照组比较，*表示P≤0.05，**表示P<0.01；c.T/C(％)＝T RTV/C RTV×100％，其中T RTV和C RTV分别指治疗组和对照组的平均RTV，RTV＝V t/V 0，其中V t指第14天的平均肿瘤体积，V 0指第0天的平均肿瘤体积；d.TGI(％)＝(1-T/C)×100％。
实施10抗CD40抗体和安罗替尼或其药学上可接受的盐在人B细胞淋巴瘤Ramos小鼠皮下移植瘤模型中的疗效
人B细胞淋巴瘤Ramos细胞(购自中国科学院细胞库)用含有10％胎牛血清和青霉素-链霉素的RPMI1640培养基进行培养。于37℃、含5％CO 2的培养箱中培养至指数生长期，收集细胞进行接种。
本实验采用的小鼠为6周龄左右的NOD-Scid小鼠(购自上海灵畅生物科技有限公司)，饲养于无特定病原体的条件下，以12/12小时光/暗调节。每天监测动物的健康状况。
每只小鼠皮下接种1×10 7个Ramos细胞，待肿瘤长到80～100mm 3，根据肿瘤体积进行分组，每组 10只。给药方案见表10，给药第一天为D0，huC1H1-V2和IgG2(Biointron,Cat.No.B107803)均用生理盐水配制成所需浓度，安罗替尼的二盐酸盐用蒸馏水配制成所需浓度。实验结束时(D14)，CO 2麻醉处死动物，随后解剖取瘤并拍照。
每周用游标卡尺测量肿瘤直径2次，肿瘤体积(V)＝1/2×a×b 2其中a、b分别表示长、宽；T/C(％)＝(T-T0)/(C-C0)×100，其中T、C分别指第14天治疗组和IgG2对照组的肿瘤体积，T0、C0分别指第0天治疗组和IgG2对照组的肿瘤体积；肿瘤生长抑制率％(TGI％)＝100-T/C(％)，当肿瘤出现消退时，肿瘤生长抑制率％(TGI％)＝100-(T-T0)/T0×100，如果肿瘤比起始体积缩小，即T<T0或C<C0时，即定义为肿瘤部分消退(PR)，如果肿瘤完全消失，即定义为肿瘤完全消退(CR)。
结果如表11和图8A(肿瘤体积)和8B(肿瘤重量)所示，在第14天，抗CD40抗体huC1H1-V2(0.5mg/kg)和安罗替尼的二盐酸盐(1mg/kg)均对人B细胞淋巴瘤Ramos小鼠皮下移植瘤的生长有明显抑制作用，肿瘤生长抑制率分别为58％和56％；二者联用时，肿瘤生长抑制率提高到84％；并且第4组小鼠的肿瘤体积和肿瘤重量均显著低于第2组和第3组(P<0.001)。荷瘤小鼠对以上药物均能够较好耐受，没有明显体重下降等症状发生，说明huC1H1-V2和安罗替尼的二盐酸盐的联用具有显著的协同增效作用，且毒性没有增加。
表10.给药方案
Figure PCTCN2022121184-appb-000012
注：在第4组中，0.5mg/kg huC1H1-V2通过腹腔注射，每2天一次，共8次；1mg/kg安罗替尼的二盐酸盐通过灌胃给药，每天一次，共15次。
表11.在人B细胞淋巴瘤Ramos小鼠皮下移植瘤模型中的疗效
Figure PCTCN2022121184-appb-000013
注：P值是通过Mann-Whitney检验与IgG2组肿瘤体积比较；***表示P<0.001，通过双尾Student’s t检验与huC1H1-V2+安罗替尼的二盐酸盐组肿瘤体积比较；P<0.05定义为有统计学显著性差异。
本申请的序列信息总结于下表12中。
表12.序列信息
Figure PCTCN2022121184-appb-000014
Figure PCTCN2022121184-appb-000015
Figure PCTCN2022121184-appb-000016
Figure PCTCN2022121184-appb-000017
Figure PCTCN2022121184-appb-000018
尽管本发明已结合一个或多个实施方案进行了描述，应当理解的是，本发明不仅涵盖这些实施方案，还意在涵盖包含在所附权利要求的精神和范围内的所有可替代、修饰和等同物。本文引用的所有文献均通过引用的方式全部并入本文。

Claims (15)

  1. 一种药物组合，其包括(a)抗CD40抗体或其抗原结合部分，和(b)第二治疗剂，所述第二治疗剂为酪氨酸激酶抑制剂。
  2. 根据权利要求1所述的药物组合，其中，所述第二治疗剂为VEGFR酪氨酸激酶抑制剂，所述VEGFR酪氨酸激酶抑制剂包括安罗替尼、凡德他尼、索拉非尼、西地尼布、伐他拉尼、帕唑帕尼、莫替沙尼、仑伐替尼、舒尼替尼、贝伐珠单抗、雷莫芦单抗、或其药学上可接受的盐。
  3. 根据权利要求1或2所述的药物组合，其中，所述第二治疗剂为安罗替尼或其药学上可接受的盐；可选地，所述安罗替尼的药学上可接受的盐为盐酸盐或二盐酸盐。
  4. 根据权利要求1-3中任一项所述的药物组合，其中，所述抗CD40抗体或其抗原结合部分包含重链可变区和轻链可变区，所述重链可变区包含V H CDR1、V H CDR2和V H CDR3，所述轻链可变区包含V L CDR1、V L CDR2和V L CDR3，其中，(1)V H CDR1、V H CDR2、V H CDR3、V L CDR1、V L CDR2和V L CDR3分别包含与SEQ ID NOs:1、4、7、10、13和16所示的氨基酸序列具有至少85％同一性的氨基酸序列；(2)V H CDR1、V H CDR2、V H CDR3、V L CDR1、V L CDR2和V L CDR3分别包含与SEQ ID NOs:2、5、8、11、14和17所示的氨基酸序列具有至少85％同一性的氨基酸序列；或(3)V H CDR1、V H CDR2、V H CDR3、V L CDR1、V L CDR2和V L CDR3分别包含与SEQ ID NOs:3、6、9、12、15和18所示的氨基酸序列具有至少85％同一性的氨基酸序列。
  5. 根据权利要求1-4中任一项所述的药物组合，其中，所述抗CD40抗体或其抗原结合部分包含重链可变区，所述重链可变区包含与SEQ ID NOs:19、20、21或22所示的氨基酸序列具有至少85％同一性的氨基酸序列，其中SEQ ID NO:20中的X1＝A或S。
  6. 根据权利要求1-5中任一项所述的药物组合，其中，所述抗CD40抗体或其抗原结合部分包含轻链可变区，所述轻链可变区包含与SEQ ID NOs:23、24、25或26所示的氨基酸序列具有至少85％同一性的氨基酸序列，其中SEQ ID NO:24中的X1＝K和X2＝F，或X1＝Y和X2＝Y。
  7. 根据权利要求1-6中任一项所述的药物组合，其中，所述抗CD40抗体或其抗原结合部分包含重链可变区和轻链可变区，所述重链可变区和轻链可变区包含：(1)分别与SEQ ID NOs:19和23所示的氨基酸序列具有至少85％同一性的氨基酸序列；(2)分别与SEQ ID NOs:20和24所示的氨基酸序列具有至少85％同一性的氨基酸序列，其中SEQ ID NO:20中的X1＝A，且SEQ ID NO:24中的X1＝K和X2＝F；(3)分别与SEQ ID NOs:20和24所示的氨基酸序列具有至少85％同一性的氨基酸序列，其中SEQ ID NO:20中的X1＝S，且SEQ ID NO:24中的X1＝K和X2＝F；(4)分别与SEQ ID NOs:20和24所示的氨基酸序列具有至少85％同一性的氨基酸序列，其中SEQ ID NO:20中的X1＝A，且SEQ ID NO:24中的X1＝Y和X2＝Y；(5)分别与SEQ ID NOs:20和24所示的氨基酸序列具有至少85％同一性的氨基酸序列，其中SEQ ID NO:20中的X1＝S，且SEQ ID NO:24中的X1＝Y和X2＝Y；(6)分别与SEQ ID NOs:21和25所示的氨基酸序列具有至少85％同一性的氨基酸序列；或(7)分别与SEQ ID NOs:22和26所示的氨基酸序列具有至少85％同一性的氨基酸序列。
  8. 根据权利要求4-7中任一项所述的药物组合，其中，所述抗CD40抗体或其抗原结合部分包含连接至所述重链可变区的重链恒定区，和连接至所述轻链可变区的轻链恒定区，所述重链恒定区包含SEQ ID NOs:27或28所示的氨基酸序列，所述轻链恒定区包含SEQ ID NO:29所示的氨基酸序列。
  9. 根据权利要求1-8中任一项所述的药物组合，其中，所述药物组合还包含第三治疗剂。
  10. 根据权利要求9所述的药物组合，其中，所述第三治疗剂包含化疗药物；可选地，所述化疗药物包含烷化剂、鬼臼类药物、铂类药物、氟嘧啶衍生物、胞嘧啶衍生物、紫杉烷类药物、喜树碱类药物、蒽环类药物、长春碱类药物中的一种或多种。
  11. 一种试剂盒，其包含权利要求1-10中任一项所述的药物组合。
  12. 权利要求1-10中任一所述的药物组合或权利要求11所述的试剂盒在制备用于治疗癌症的药物中的用途。
  13. 根据权利要求12所述的用途，其中，所述抗CD40抗体或其抗原结合部分和第二治疗剂各自呈药物组合物的形式，可同时、先后和/或交替给药；可选地，所述抗CD40抗体或其抗原结合部分、第二治疗剂和第三治疗剂各自呈药物组合物的形式，可同时、先后和/或交替给药。
  14. 根据权利要求12或13所述的用途，其中，所述癌症为实体瘤；可选地，所述实体瘤包括皮肤癌、胰腺癌、结直肠癌、食管癌、胃肠道癌、前列腺癌、膀胱癌、肾癌、卵巢癌、子宫癌、乳腺癌、肺癌、甲状腺癌、鼻咽癌和肝癌。
  15. 根据权利要求12或13所述的用途，其中，所述癌症为B细胞恶性肿瘤；可选地，所述B细胞恶性肿瘤包括非霍奇金淋巴瘤、慢性淋巴细胞白血病、多发性骨髓瘤、B细胞淋巴瘤、高级B细胞淋巴瘤、中级B细胞淋巴瘤、低级B级淋巴瘤、B细胞急性淋巴细胞白血病、霍奇金氏淋巴瘤、浆细胞瘤、滤泡性淋巴瘤、滤泡性小裂性淋巴瘤、滤泡性大细胞淋巴瘤、滤泡混合性小裂性淋巴瘤、弥漫性小裂性细胞淋巴瘤、小淋巴细胞性淋巴瘤、幼淋巴细胞白血病、淋巴浆细胞性淋巴瘤、边缘区淋巴瘤、黏膜相关淋巴组织淋巴瘤、单核细胞样B细胞淋巴瘤、脾脏淋巴瘤、毛细胞白血病、弥漫性大B细胞淋巴瘤、纵隔大B细胞淋巴瘤、淋巴瘤样肉芽肿病、血管内淋巴瘤、混合性淋巴瘤、免疫母细胞淋巴瘤、伯基特淋巴瘤、艾滋病相关淋巴瘤、瓦尔登斯特伦巨球蛋白血症和套细胞淋巴瘤；可选地，所述癌症为非B细胞血液恶性肿瘤；可选地，所述非B细胞血液恶性肿瘤包括慢性髓性白血病和急性髓性白血病。
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