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WO2020063660A1 - 抗ox40抗体、其抗原结合片段及其医药用途 - Google Patents

抗ox40抗体、其抗原结合片段及其医药用途 Download PDF

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Publication number
WO2020063660A1
WO2020063660A1 PCT/CN2019/107787 CN2019107787W WO2020063660A1 WO 2020063660 A1 WO2020063660 A1 WO 2020063660A1 CN 2019107787 W CN2019107787 W CN 2019107787W WO 2020063660 A1 WO2020063660 A1 WO 2020063660A1
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seq
antibody
cancer
antigen
variable region
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PCT/CN2019/107787
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English (en)
French (fr)
Inventor
廖成
徐祖朋
蒋家骅
叶鑫
张连山
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江苏恒瑞医药股份有限公司
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Priority to CA3113541A priority Critical patent/CA3113541A1/en
Priority to JP2021517014A priority patent/JP2022502417A/ja
Priority to KR1020217011102A priority patent/KR20210069058A/ko
Priority to AU2019347934A priority patent/AU2019347934A1/en
Priority to CN201980050221.0A priority patent/CN112513088B/zh
Priority to EP19867097.8A priority patent/EP3858857A1/en
Priority to US17/278,259 priority patent/US20210355229A1/en
Priority to MX2021003168A priority patent/MX2021003168A/es
Priority to BR112021005169-4A priority patent/BR112021005169A2/pt
Publication of WO2020063660A1 publication Critical patent/WO2020063660A1/zh

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    • C07—ORGANIC CHEMISTRY
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], 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
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • 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
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    • 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
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the invention belongs to the field of biomedicine, and relates to an anti-OX40 antibody, an antigen-binding fragment thereof, a chimeric antibody containing a CDR region of the anti-OX40 antibody, a humanized antibody, and a medicine containing a human anti-OX40 antibody and an antigen-binding fragment thereof. Composition and its use as an anticancer drug.
  • Cancer is a severe health challenge that human society is facing for a long time.
  • traditional surgery, chemotherapy, and radiation therapy often have little effect.
  • Tumor immunotherapy is a continuing hot spot in the field of tumor therapy. Recent studies have demonstrated that enhanced anti-tumor T cell function can be used to fight cancer. There is a lot of evidence that tumor cells "escape" the immune system by inducing active immune tolerance mediated primarily by regulatory T lymphocytes (Treg; Quezda et al. Immunol Rev 2011; 241: 104-118). Therefore, the balance between effector T lymphocytes (Teff) and tolerogenic Tregs is essential for effective anti-tumor immunotherapy. Therefore, an effective anti-tumor immune response can be obtained by enhancing the effector function of tumor-specific Teff and / or by weakening the inhibitory function of tumor-specific Treg.
  • Treg regulatory T lymphocytes
  • the CD134 (OX40) receptor has been shown to be a key receptor that mediates these responses (Sugamura, K, Ishii, N, Weinberg, A. Therapeutic targeting of theeffector T-cell co-stimulatory molecule OX40. Nature Rev Rev Imm 2004; 4: 420-431).
  • OX40 is a member of the tumor necrosis factor receptor (TNFR) superfamily. It is a glycoprotein with a molecular weight of about 50 kDa expressed on the cell surface.
  • the extracellular ligand-binding domain of OX40 consists of four cysteine-rich domains (CRD).
  • the natural ligand of OX40 is OX40L (CD252), and the two form an OX40-OX40L complex.
  • OX40 is mainly expressed on activated T cells.
  • OX40 is a secondary costimulatory molecule that is expressed 24-72 hours after activation.
  • the ligand of OX40, OX40L is mainly expressed on activated antigen-presenting cells.
  • OX40-expressing T lymphocytes have been shown to be present in the draining lymph nodes of various human malignancies and cancer patients.
  • SCID severe combined immunodeficiency
  • OX40 antibody activates immunity through specific stimulation, improves the patient's own immune system response to tumors, and achieves the purpose of killing tumor cells.
  • Related patents such as WO2013038191, WO2015153513, WO2016179517, WO2017096182, CN110078825A, WO2016196228, etc. So far, anti-OX40 antibodies developed by AstraZeneca, BMS and other companies have been in Phase II clinical trials, and related products of Genentech, GSK and other companies have also been in clinical trials.
  • the present disclosure provides an anti-OX40 antibody or an antigen-binding fragment thereof, which specifically binds to human OX40 and includes a CDR as shown below:
  • the heavy chain HCDR1, HCDR2, HCDR3 shown in SEQ ID NO: 11, 33, 13 or HCDR1, HCDR2, HCDR3 shown in SEQ ID NO: 11, 33, 13 respectively have 3, 2 or 1 HCDR variants with amino acid differences; and / or,
  • the heavy chain HCDR1, HCDR2, HCDR3 shown in SEQ ID NO: 11, 34, 13 or HCDR1, HCDR2, HCDR3 shown in SEQ ID NO: 11, 34, 13 respectively have 3, 2 or 1 HCDR variants with amino acid differences; and / or,
  • the anti-OX40 antibody or antigen-binding fragment thereof is a murine antibody, a chimeric antibody, a humanized antibody, a human antibody, or a fragment thereof, specifically, a humanized antibody.
  • the CDRs (including 3 heavy chain CDRs and 3 light chain CDRs) of the monoclonal antibody or antigen-binding fragment are allowed to contain 3, 2 or 1 amino acid differences (ie, CDR variants),
  • the body was obtained by affinity maturation screening.
  • the monoclonal antibody or antigen-binding fragment has an affinity (KD) to OX40 of less than 10 -8 M, less than 10 -9 M, or less than 10 -10 M.
  • the murine antibody comprises a heavy chain variable region and a light chain variable region as shown below:
  • variable region of the heavy chain is as shown in SEQ ID NO: 1; or at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 91% , At least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity; and / or,
  • amino acid sequence of the light chain variable region is shown in SEQ ID NO: 2; or at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92% with SEQ ID NO: 2 %, At least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity;
  • amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO: 9; or at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 91% of SEQ ID ID: 9 , At least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity; and / or,
  • the light chain variable region amino acid sequence is shown in SEQ ID NO: 10; or at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92 %, At least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity.
  • the humanized antibody comprises a FR region derived from a human germline or a mutant sequence thereof.
  • the humanized antibody comprises a member selected from:
  • a heavy chain variable region which is shown in any one of SEQ ID NO: 17, 18, 31, 32; or has at least 70%, at least 75% of SEQ ID NO: 17, 18, 31, 32 At least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% of the sequence identity Sex; or
  • a heavy chain variable region which is shown in any one of SEQ ID NO: 26, 27, 28, 29, 30; or has at least 70% of SEQ ID NO: 26, 27, 28, 29, 30 , At least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity.
  • the anti-OX40 antibody or antigen-binding fragment thereof comprises:
  • a light chain variable region which is shown in SEQ ID NO: 19 or 20; or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90% with SEQ ID NO: 19 or 20 , At least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity; or
  • a light chain variable region which is shown in any one of SEQ ID NO: 21, 22, 23, 24, 25; or has at least 70% of SEQ ID NO: 21, 22, 23, 24, 25 , At least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity.
  • the anti-OX40 antibody or antigen-binding fragment thereof comprises:
  • the anti-OX40 antibody or antigen-binding sheet thereof comprises a variable region as shown below:
  • variable region of the heavy chain is shown in SEQ ID NO: 17, and the variable region of the light chain is shown in SEQ ID NO: 19;
  • variable region of the heavy chain is shown in SEQ ID NO: 18, and the variable region of the light chain is shown in SEQ ID NO: 19;
  • variable region of the heavy chain is shown in SEQ ID NO: 31, and the variable region of the light chain is shown in SEQ ID NO: 19;
  • variable region of the heavy chain is shown in SEQ ID NO: 32, and the variable region of the light chain is shown in SEQ ID NO: 19;
  • variable region of the heavy chain is shown in SEQ ID NO: 17, and the variable region of the light chain is shown in SEQ ID NO: 20;
  • variable region of the heavy chain is shown in SEQ ID NO: 18, and the variable region of the light chain is shown in SEQ ID NO: 20;
  • variable region of the heavy chain is shown in SEQ ID NO: 31, and the variable region of the light chain is shown in SEQ ID NO: 20;
  • variable region of the heavy chain is shown in SEQ ID NO: 32
  • variable region of the light chain is shown in SEQ ID NO: 20.
  • the anti-OX40 antibody or antigen-binding sheet thereof comprises a variable region as shown below:
  • variable region of the heavy chain is shown in SEQ ID NO: 26 and the variable region of the light chain is shown in SEQ ID NO: 21;
  • variable region of the heavy chain is shown in SEQ ID NO: 26 and the variable region of the light chain is shown in SEQ ID NO: 22;
  • variable region of the heavy chain is shown in SEQ ID NO: 26 and the variable region of the light chain is shown in SEQ ID NO: 23;
  • variable region of the heavy chain is shown in SEQ ID NO: 26, and the variable region of the light chain is shown in SEQ ID NO: 24;
  • variable region of the heavy chain is shown in SEQ ID NO: 26, and the variable region of the light chain is shown in SEQ ID NO: 25;
  • variable region of the heavy chain is shown in SEQ ID NO: 27, and the variable region of the light chain is shown in SEQ ID NO: 21;
  • variable region of the heavy chain is shown in SEQ ID NO: 27, and the variable region of the light chain is shown in SEQ ID NO: 22;
  • variable region of the heavy chain is shown in SEQ ID NO: 27, and the variable region of the light chain is shown in SEQ ID NO: 23;
  • variable region of the heavy chain is shown in SEQ ID NO: 27, and the variable region of the light chain is shown in SEQ ID NO: 25;
  • variable region of the heavy chain is shown in SEQ ID NO: 28, and the variable region of the light chain is shown in SEQ ID NO: 21;
  • the heavy chain variable region is shown in SEQ ID NO: 28, and the light chain variable region is shown in SEQ ID NO: 22;
  • variable region of the heavy chain is shown in SEQ ID NO: 28, and the variable region of the light chain is shown in SEQ ID NO: 23;
  • variable region of the heavy chain is shown in SEQ ID NO: 28, and the variable region of the light chain is shown in SEQ ID NO: 24;
  • variable region of the heavy chain is shown in SEQ ID NO: 28, and the variable region of the light chain is shown in SEQ ID NO: 25;
  • variable region of the heavy chain is shown in SEQ ID NO: 29 and the variable region of the light chain is shown in SEQ ID NO: 21;
  • variable region of the heavy chain is shown in SEQ ID NO: 29, and the variable region of the light chain is shown in SEQ ID NO: 22;
  • variable region of the heavy chain is shown in SEQ ID NO: 29
  • variable region of the light chain is shown in SEQ ID NO: 23;
  • variable region of the heavy chain is shown in SEQ ID NO: 29
  • variable region of the light chain is shown in SEQ ID NO: 24;
  • variable region of the heavy chain is shown in SEQ ID NO: 29, and the variable region of the light chain is shown in SEQ ID NO: 25;
  • variable region of the heavy chain is shown in SEQ ID NO: 30, and the variable region of the light chain is shown in SEQ ID NO: 21;
  • variable region of the heavy chain is shown in SEQ ID NO: 30, and the variable region of the light chain is shown in SEQ ID NO: 22;
  • variable region of the heavy chain is shown in SEQ ID NO: 30, and the variable region of the light chain is shown in SEQ ID NO: 23;
  • variable region of the heavy chain is shown in SEQ ID NO: 30 and the variable region of the light chain is shown in SEQ ID NO: 24;
  • variable region of the heavy chain is shown in SEQ ID NO: 30, and the variable region of the light chain is shown in SEQ ID NO: 25.
  • the anti-OX40 antibody comprises a constant region; in some specific embodiments, the antibody is a chimeric antibody or a humanized antibody, and the heavy chain constant region of the antibody is derived from human IgG1, IgG2, IgG3 or IgG4 or a mutant sequence thereof, and the light chain constant region is derived from a human kappa, lambda chain or a mutant sequence thereof; in other specific embodiments, the amino acid sequence of the heavy chain constant region is as shown in SEQ ID NO: 35 SEQ ID NO: 35 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity, the amino acid sequence of the light chain constant region is shown in SEQ ID NO: 36 or at least 70%, at least 75% with SEQ ID NO: 36 , At least 80%, at
  • the amino acid sequence of an anti-OX40 antibody heavy chain is as shown in SEQ ID NO: 39 or 37 or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity, and / or, the anti-OX40
  • the amino acid sequence of the antibody light chain is as shown in SEQ ID NO: 40 or 38 or at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 70%, or at least 70% with SEQ ID NO: 40 or 38. 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity.
  • the anti-OX40 antibody comprises:
  • a heavy chain whose amino acid sequence is shown in SEQ ID NO: 37 or has at least 85% sequence identity with SEQ ID NO: 37, and
  • the heavy chain whose amino acid sequence is shown in SEQ ID NO: 39 and has at least 85% sequence identity with SEQ ID NO: 39, and
  • the light chain has an amino acid sequence such as SEQ ID NO: 40 or has at least 85% sequence identity with SEQ ID NO: 40.
  • the present disclosure also provides an anti-OX40 antibody or an antigen-binding fragment thereof, which anti-OX40 antibody or an antigen-binding fragment thereof competitively binds human OX40 with the antibody or the antigen-binding fragment of any one of the foregoing, or with any one of the foregoing
  • the antibody or antigen-binding fragment thereof described in item 5 binds to the same OX40 epitope.
  • the present disclosure also provides a pharmaceutical composition
  • a pharmaceutical composition comprising:
  • the pharmaceutical composition may be prepared in a unit dose form, and the unit dose may contain 0.01 to 99% by weight of an anti-OX40 antibody or an antigen-binding fragment thereof; or a unit dose of the pharmaceutical composition contains a single
  • the amount of the cloned antibody or antigen-binding fragment thereof is 0.1-2000 mg, and in some embodiments, the amount of the monoclonal antibody or antigen-binding fragment thereof is 1-1000 mg.
  • the monoclonal antibody or antigen-binding fragment thereof may be the sole active ingredient in the composition; in other specific embodiments, the monoclonal antibody or antigen-binding fragment thereof is used in combination with other active ingredients.
  • the present disclosure also provides an isolated nucleic acid molecule encoding an anti-OX40 antibody or an antigen-binding fragment thereof as described above.
  • the complementary sequence of such a nucleic acid molecule is also included in the scope of the present application.
  • the present disclosure also provides a vector comprising the aforementioned nucleic acid molecule, and the vector may be a eukaryotic expression vector, a prokaryotic expression vector, or a viral vector.
  • the present disclosure also provides a host cell transformed with a vector as described above, the host cell being selected from a prokaryotic cell and a eukaryotic cell.
  • the host cell is a eukaryotic cell. In some embodiments, it is a mammalian cell, wherein the mammalian cell includes, but is not limited to, CHO, HEK293, NSO. It should be understood that the host cell of the present disclosure does not involve any cell capable of developing into a human.
  • the present disclosure also provides a method for preparing an anti-OX40 antibody or an antigen-binding fragment thereof as described above, the method comprising:
  • the present disclosure also provides a method for detecting or determining human OX40, the method comprising:
  • the present disclosure also provides a reagent for detecting or measuring human OX40, the reagent comprising the anti-OX40 antibody or an antigen-binding fragment thereof according to any one of the above.
  • the present disclosure also provides a diagnostic agent for a human OX40-related disease, the diagnostic agent comprising the anti-OX40 antibody or an antigen-binding fragment thereof according to the above.
  • the present disclosure also provides a method for diagnosing a disease related to human OX40, the method comprising detecting or measuring human OX40 or OX40 positive cells using the above-mentioned anti-OX40 antibody or an antigen-binding fragment thereof.
  • the present disclosure also provides use of the above-mentioned anti-OX40 antibody or antigen-binding fragment thereof in the preparation of a diagnostic agent for a disease related to human OX40.
  • the present disclosure also provides a method of treating a disease or disorder, comprising administering to a subject an effective amount of the above-mentioned anti-OX40 antibody or antigen-binding fragment thereof, or a pharmaceutical composition comprising the above-mentioned OX40 antibody or fragment thereof.
  • the disease or disorder may be cancer or a cell proliferative disease.
  • the cancer is lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, colon cancer, colorectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, Melanoma, kidney cancer, squamous cell carcinoma, hematological cancer, or any other disease or condition characterized by uncontrolled cell growth.
  • the hematological cancers include, but are not limited to, acute and chronic myelogenous leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, myeloproliferative diseases, multiple myeloma, He Jiejin Hodgkin's disease, non-Hodgkin's lymphoma, B-cell lymphoma, T-cell lymphoma, follicular center cell lymphoma, chronic myeloid leukemia.
  • the present disclosure also provides the use of an anti-OX40 antibody or an antigen-binding fragment thereof as described above, or a pharmaceutical composition comprising the above-mentioned OX40 antibody or a fragment thereof in the manufacture of a medicament for enhancing an immune response in a human subject.
  • the present disclosure also provides the use of an anti-OX40 antibody or an antigen-binding fragment thereof as described above, or a pharmaceutical composition comprising the above-mentioned OX40 antibody or a fragment thereof in the manufacture of a medicament for treating / preventing cancer.
  • the present disclosure also provides the use of an anti-OX40 antibody or an antigen-binding fragment thereof as described above, or a pharmaceutical composition comprising the above-mentioned OX40 antibody or a fragment thereof in a preparation kit.
  • the enhanced immune response includes an increase in immune stimulatory / effectoral functions of T effector cells, and / or a downregulation of immune suppressive functions of T regulatory cells.
  • the increase may be a result of cell proliferation
  • the down-regulation may be a result of no increase in cell number (or decrease in cell number).
  • the anti-human OX40 antibody or fragment is provided in the present disclosure for use in one or more of the following: inhibition of Treg function (for example, suppression of Treg's suppressive function), killing of cells expressing OX40 (for example, cells expressing high levels of OX40) , Improve effector T cell function and / or improve memory T cell function, reduce tumor immunity, enhance T cell function and / or reduce cells expressing OX40.
  • Treg function for example, suppression of Treg's suppressive function
  • killing of cells expressing OX40 for example, cells expressing high levels of OX40
  • Improve effector T cell function and / or improve memory T cell function reduce tumor immunity
  • enhance T cell function and / or reduce cells expressing OX40 for use in one or more of the following: inhibition of Treg function (for example, suppression of Treg's suppressive function), killing of cells expressing OX40 (for example, cells expressing high levels of OX40) , Improve effector T cell function and / or improve memory T cell function, reduce tumor immunity,
  • the present disclosure provides the use of the anti-OX40 antibody or fragment for the following purposes: treating cancer, stimulating an immune response in a subject, stimulating an antigen-specific T cell response, activating or co-stimulating T cells, increasing cytokines in T cells (Eg, IL-2 and / or IFN- ⁇ ) production, and / or T cell proliferation, reducing or depleting the number of T regulatory cells in a tumor, and / or inhibiting tumor cell growth.
  • cytokines in T cells Eg, IL-2 and / or IFN- ⁇
  • the present disclosure also provides the use of the anti-OX40 antibody to prepare a medicament for stimulating an immune response in a subject, stimulating an antigen-specific T cell response, activating or co-stimulating T cells, and increasing IL in T cells.
  • -2 and / or IFN- ⁇ production and / or T cell proliferation reducing or depleting the number of T regulatory cells in the tumor and / or inhibiting tumor cell growth.
  • Figures 1A to 1B Affinity ELISA test results of mouse-derived antibodies and chimeric antibodies with human OX40.
  • Figure 1A is the affinity results of the mouse antibody m2G3 and the chimeric antibody ch2G3, m2G3-NC and ch2G3-NC are negative controls
  • Figure 1B is the affinity results of the mouse antibody m4B5 and the chimeric antibody ch4B5, m4B5-NC and ch4B5 -NC is a negative control.
  • Figure 2 An experiment in which anti-OX40 antibodies stimulate T cells to secrete IFN- ⁇ . The results show that the test OX40 antibody can reach the maximum stimulation effect at the concentration of 10ng / mL antibody.
  • Figure 3A to Figure 3B Antitumor effect of mice with anti-OX40 antibody in vivo
  • Figure 3A shows the change of tumor volume in different mice after administration
  • Figure 3B shows the effect of different anti-OX40 antibodies on tumor quality in mice after administration .
  • the results show that on the 20th day after the administration, when the dose was 3 mg / kg, the tumor inhibition rate of the 2G3 antibody was as high as 97%.
  • enhancing T cell function means inducing, causing, or stimulating effector or memory T cells to have a newer, sustained, or amplified biological function.
  • Examples of enhanced T-cell function include increased ⁇ -interferon secretion from CD8 + effector T cells relative to pre-intervention levels, increased ⁇ -interferon secretion from CD4 + memory and / or effector T cells, and CD4 Increased + effector and / or memory T cell proliferation, increased CD8 + effector T cell proliferation, and enhanced antigen responsiveness (eg, clearance).
  • enhancing immune response refers to a response that stimulates, provokes, increases, improves or enhances the immune system of a mammal.
  • the immune response can be a cellular response (ie, cell-mediated, such as cytotoxic T lymphocytes) or a humoral response (ie, antibody-mediated response), and can be a primary or re-immune response.
  • Examples of enhanced immune responses include increased CD4 + helper T cell activity and production of cytotoxic T cells.
  • Enhancement of the immune response can be assessed using some in vitro or in vivo measurements known to those skilled in the art, including but not limited to cytotoxic T lymphocyte assays, cytokine release (e.g., IL-2 production), tumor regression, tumor-bearing animals Survival, antibody production, immune cell proliferation, cell surface marker expression and cytotoxicity.
  • the method enhances a cellular immune response, particularly a cytotoxic T cell response.
  • Tumor immunity refers to the process by which a tumor evades immune recognition and clearance. As a treatment concept, when the tumor's ability to evade immune recognition and clearance is weakened, the tumor is recognized and attacked by the immune system, and the patient is treated. Examples of tumor recognition include tumor binding, tumor shrinkage, and tumor clearance.
  • T effector cell refers to T cells (eg, CD4 + and CD8 + T cells) and T helper (Th) cells that have cytolytic activity. Teff secretes cytokines and activates and guides other immune cells. , But does not include regulatory T cells (Treg cells).
  • the anti-OX40 antibodies described in this disclosure can activate Teff cells, such as CD4 + and CD8 + Teff cells.
  • Treg cell means a specialized type of CD4 + T cell that can suppress the response of other T cells.
  • Treg cells are characterized by expressing the CD4, IL-2 receptor alpha subunit (CD25), and the transcription factor forkhead box P3 (FOXP3) (Sakaguchi, Annu Rev Immunol 22,531-62 (2004)), and induce and maintain peripheral autologous It plays a crucial role in tolerance, which is directed against tumor-expressed antigens.
  • OX40 refers to a receptor that binds to the OX40 ligand (OX40-L) and is a member of the TNF-receptor superfamily. OX40 is also known as tumor necrosis factor receptor superfamily member 4 (TNFRSF4), ACT35, IMD16, TXGP1L, and CD134.
  • TNFRSF4 tumor necrosis factor receptor superfamily member 4
  • ACT35 tumor necrosis factor receptor superfamily member 4
  • IMD16 ACT35
  • TXGP1L TXGP1L
  • CD134 tumor necrosis factor receptor superfamily member 4
  • OX40 includes any OX40 variant or isoform that is naturally expressed by a cell. Therefore, the OX40 antibodies or fragments described in the present disclosure can cross-react with OX40 (eg, cynomolgus OX40) from species other than humans. Alternatively, the OX40 antibody or fragment may be specific for human OX40 and not exhibit cross-reactivity with other species.
  • OX40 or its variants and isoforms are isolated from their naturally expressed cells or tissues, or produced recombinantly using techniques well known in the art and / or techniques described in this disclosure.
  • OX40 can be from any vertebrate source, including mammals such as primates (eg, humans) and rodents (eg, mice and rats) natural OX40.
  • the term encompasses "full length", unprocessed OX40, and any form of OX40 due to processing in the cell.
  • the term also encompasses naturally occurring variants of OX40, such as splice variants or allelic variants.
  • OX40 activation refers to activation of the OX40 receptor. Generally, OX40 activation results in signal transduction.
  • Anti-OX40 antibody or "OX40-binding antibody” refers to an antibody that is capable of binding OX40 with sufficient affinity such that the antibody can be used as a diagnostic and / or therapeutic agent for targeting OX40.
  • subtracted cells expressing OX40 refers to an anti-OX40 antibody or a fragment thereof that kills or subtracts cells expressing OX40. Subtraction of OX40-expressing cells can be achieved by a variety of mechanisms, such as antibody-dependent cells (ADCC) -mediated cytotoxicity and / or phagocytosis.
  • ADCC antibody-dependent cells
  • cytokine is a general term for a class of proteins that is released by a population of cells and acts on another cell as an intercellular medium.
  • cytokines are lymphokines, monocytes; interleukins (IL) such as IL-1, IL-1 ⁇ , IL-2, IL-3, IL-4, IL-5, IL-6, IL- 7, IL-8, IL-9, IL-11, IL-12, IL-15; tumor necrosis factor, such as TNF- ⁇ or TNF- ⁇ ; and other polypeptide factors, including LIF and kit ligand (KL) and ⁇ -interferon.
  • IL interleukins
  • IL-1 ⁇ interleukins
  • IL-6 interleukins
  • IL-8 interleukins
  • IL-9 tumor necrosis factor
  • TNF- ⁇ or TNF- ⁇ tumor necrosis factor
  • other polypeptide factors including LIF and kit ligand (KL) and ⁇ -interferon.
  • the term cytokine
  • amino acid three-letter codes and one-letter codes used in the present disclosure are as described in J. Biol. Chem, 243, p3558 (1968).
  • antibody is not limited by any particular method of producing antibodies. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies.
  • the antibodies may be antibodies of different isotypes, for example, IgG (eg, IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.
  • variable region The sequence of about 110 amino acids near the N-terminus in the heavy and light chains of the antibody varies greatly, and is the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable, and are the constant region (C region).
  • the variable region includes three hypervariable regions (HVR) and four relatively conserved backbone regions (FR). Three hypervariable regions determine the specificity of an antibody, also known as complementarity determining regions (CDRs).
  • Each light chain variable region (VL) and heavy chain variable region (VH) is composed of three CDR regions and four FR regions.
  • the sequence from the amino terminal to the carboxy terminal is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the three CDR regions of the light chain are referred to as LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3.
  • the CDR amino acid residues of the LCVR region and HCVR region of the antibody or antigen-binding fragment according to the present disclosure conform to the known Kabat numbering rules (LCDR1-3, HCDR1-3) in number and position.
  • recombinant human antibody includes human antibodies prepared, expressed, created, or isolated by recombinant methods, and the techniques and methods involved are well known in the art, such as:
  • transgenic, transchromosome animal such as a mouse
  • hybridoma prepared from the human immunoglobulin gene
  • an antibody isolated from a host cell transformed to express the antibody
  • Such recombinant human antibodies contain variable and constant regions that utilize specific human germline immunoglobulin sequences encoded by germline genes, but also include rearrangements and mutations that subsequently occur, such as during antibody maturation.
  • the antibodies of the present disclosure include murine antibodies, chimeric antibodies, humanized antibodies, and human antibodies.
  • In some embodiments are humanized antibodies.
  • murine antibody is a monoclonal antibody raised against human OX40 prepared according to the knowledge and skill in the art.
  • test subject is injected with OX40 antigen, and then spleen cells (B lymphocytes) expressing antibodies with the desired sequence or functional characteristics are isolated, and the B lymphocytes are fused with myeloma cells to obtain corresponding hybridoma cells.
  • B lymphocytes spleen cells
  • the mouse-derived OX40 antibody or antigen-binding fragment thereof may further comprise a light chain constant region of a mouse-derived ⁇ , lambda chain or a variant thereof, or further comprise a mouse-derived IgG1, IgG2, IgG3 or IgG4 Or a variant of the heavy chain constant region.
  • human antibody includes antibodies having variable and constant regions of human germline immunoglobulin sequences.
  • the human antibodies of the present disclosure may include amino acid residues that are not encoded by human germline immunoglobulin sequences (such as mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo).
  • the term “human antibody” does not include antibodies in which a CDR sequence derived from the germline of another mammalian species, such as a mouse, has been grafted onto a human backbone sequence (ie, a "humanized antibody”) .
  • humanized antibody refers to an antibody produced by transplanting non-human species CDR sequences into a human antibody variable region framework, that is, different types of human germline antibody framework sequences. It can overcome the heterogeneous response induced by the chimeric antibody because it carries a large amount of heterologous protein components.
  • framework sequences can be obtained from a public DNA database including germline antibody gene sequences or published references.
  • germline DNA sequences of human heavy and light chain variable region genes can be obtained in the "VBase" human germline sequence database (www.mrccpe.com.ac.uk/vbase), and in Kabat, EA, etc., 1991 Sequences of Proteins of Immunological Interest, 5th edition.
  • human antibody variable region framework sequence may be subjected to minimal reverse mutation or back mutation to maintain the activity.
  • the humanized antibodies of the present disclosure also include humanized antibodies further obtained by affinity maturation of CDRs from phage display or yeast display.
  • the antibody light chain of the OX40 humanized antibody further comprises a light chain constant region of a human-derived ⁇ , ⁇ chain, or a variant thereof.
  • the antibody heavy chain of the OX40 humanized antibody further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or a variant thereof, specifically, a human IgG1 heavy chain constant region.
  • back mutation refers to mutating the amino acid residues of the FR region derived from human antibodies to the amino acid residues at the corresponding positions of the original source antibodies, usually to avoid the decrease in immunogenicity caused by humanized antibodies and the activity caused Decreased, the humanized antibody variable region can be subjected to minimal back mutation to maintain the activity of the antibody.
  • chimeric antibody refers to an antibody that fuses the variable region of a non-human antibody with the constant region of a human antibody, and can reduce the immune response response induced by the non-human antibody.
  • a chimeric antibody you must choose to establish a hybridoma that secretes a mouse-specific monoclonal antibody, and then clone the variable region gene from the mouse hybridoma cell. Then, clone the constant region gene of the human antibody as needed.
  • the variable region gene is linked to the human constant region gene to form a chimeric gene and inserted into a human vector.
  • the chimeric antibody molecule is expressed in a eukaryotic industrial system or a prokaryotic industrial system.
  • the constant region of a human antibody may be selected from the heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4, or a variant thereof, and specifically, contains the human IgG1 heavy chain constant region.
  • antigen-binding fragment or “functional fragment” of an antibody refers to one or more fragments in an antibody that retain the ability to specifically bind to an antigen (eg, OX40). It has been shown that fragments of a full-length antibody can be used to achieve the antigen-binding function of the antibody. Examples of binding fragments included in the term "antigen-binding fragment" of an antibody include:
  • Fab fragment a monovalent fragment consisting of VL, VH, CL, and CH1 domains
  • CDR complementarity determining region
  • the two domains VL and VH of the Fv fragment are encoded by separate genes, recombinant methods can be used to link them through a synthetic linker so that they can produce a single protein chain (called a single-chain Fv (scFv), In which the VL and VH regions are paired to form a monovalent molecule; see, eg, 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 intended to be included in the term "antigen-binding fragment" of an antibody.
  • Antigen-binding moieties can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact immunoglobulins.
  • the antibodies may be antibodies of different isotypes, for example, IgG (eg, IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.
  • antigen-binding fragment refers to Fab, Fv, sFv, F (ab ') 2, linear antibody, single-chain antibody, scFv, sdAb, sdFv, Nanobody, peptide antibody (peptibody) having antigen-binding activity.
  • Domain antibodies and multispecific antibodies bispecific antibodies, diabody, triabody and tetrabody, tandem two-scFv, tandem tri-scFv).
  • Fab is an antibody fragment having a molecular weight of about 50,000 and having an antigen-binding activity in a fragment obtained by treating an IgG antibody molecule with a protease papain (amino acid residue at position 224 of the H chain), in which the N-terminal side of the H chain About half and the entire L chain are held together by disulfide bonds.
  • the Fab of the present disclosure can be produced by treating a monoclonal antibody of the present disclosure that specifically recognizes human OX40 and binds to the amino acid sequence of the extracellular region or its three-dimensional structure with papain.
  • the Fab can be produced by inserting DNA encoding the Fab of the antibody into a prokaryotic expression vector or a eukaryotic expression vector and introducing the vector into a prokaryote or eukaryote to express the Fab.
  • F (ab ') 2 is an antibody fragment obtained by digesting the two disulfide bonds downstream of the IgG hinge region with pepsin. It has a molecular weight of about 100,000, has antigen-binding activity, and includes hinge positions. Two Fab regions.
  • F (ab ') 2 of the present disclosure can be produced by treating a monoclonal antibody of the present disclosure with pepsin.
  • the F (ab ') 2 can be produced by connecting a Fab' described below with a thioether bond or a disulfide bond.
  • Fab ' is an antibody fragment having a molecular weight of about 50,000 and having an antigen-binding activity obtained by cutting the disulfide bond of the hinge region of F (ab') 2 described above.
  • the Fab 'of the present disclosure can be produced by treating the F (ab') 2 of the present disclosure with a reducing agent such as dithiothreitol.
  • the Fab ' can be produced by inserting DNA encoding a Fab' fragment of an antibody into a prokaryotic expression vector or a eukaryotic expression vector and introducing the vector into a prokaryote or eukaryote to express the Fab '. .
  • single chain antibody single chain Fv or “scFv” is meant to include an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) linked by a linker Molecule.
  • Such scFv molecules may have a general structure: NH 2 -VL-linker-VH-COOH or NH 2 -VH-linker-VL-COOH.
  • Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof, for example using 1-4 repeated variants (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA90: 6444-6448) .
  • linkers useful in the present disclosure are by Alfthan et al. (1995), Protein Eng. 8: 725-731, Choi et al. (2001), Eur. J. Immuno l. 31: 94-106, Hu et al. (1996) , Cancer Res. 56: 3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293: 41-56 and Roovers et al. (2001), Cancer Immunol.
  • the scFv of the present disclosure can be produced by obtaining the cDNA encoding the monoclonal antibodies VH and VL of the present disclosure, constructing a DNA encoding the scFv, inserting the DNA into a prokaryotic expression vector or a eukaryotic expression vector, and then The expression vector is introduced into a prokaryote or a eukaryote to express scFv.
  • a “diabody” is an antibody fragment in which scFv is dimerized, and is an antibody fragment having bivalent antigen-binding activity. In a bivalent antigen-binding activity, the two antigens may be the same or different.
  • the diabody of the present disclosure can be produced by obtaining the cDNA encoding VH and VL of the monoclonal antibody of the present disclosure, constructing a scFv-encoding DNA such that the amino acid sequence of the peptide linker is 8 residues or less, The DNA is inserted into a prokaryotic or eukaryotic expression vector, and the expression vector is then introduced into a prokaryotic or eukaryotic organism to express a diabody.
  • DsFv is obtained by linking a polypeptide in which one amino acid residue in each of VH and VL is replaced by a cysteine residue via a disulfide bond between the cysteine residues.
  • the amino acid residues substituted with cysteine residues can be selected according to a known method (Protein Engineering, 7,697 (1994)) based on the three-dimensional structure prediction of the antibody.
  • the dsFv of the present disclosure can be produced by obtaining the cDNA encoding VH and VL of the monoclonal antibody of the present disclosure, constructing a DNA encoding dsFv, inserting the DNA into a prokaryotic expression vector or a eukaryotic expression vector, and then The expression vector is introduced into a prokaryote or a eukaryote to express dsFv.
  • a "CDR-containing peptide” is constituted by one or more regions in a CDR comprising VH or VL. Peptides containing multiple CDRs can be linked directly or via a suitable peptide linker.
  • the CDR-containing peptide of the present disclosure can be produced by constructing a DNA encoding the CDRs of VH and VL of the monoclonal antibody of the present disclosure, inserting the DNA into a prokaryotic expression vector or a eukaryotic expression vector, and then The expression vector is introduced into a prokaryote or a eukaryote to express the peptide.
  • the CDR-containing peptide can also be produced by a chemical synthesis method such as the Fmoc method or the tBoc method.
  • antibody framework (FR) refers to a part of a variable domain VL or VH, which serves as a scaffold for the antigen-binding loop (CDR) of the variable domain. In essence, it is a variable domain without a CDR.
  • amino acid difference refers to the difference between a polypeptide and a variant thereof at a certain amino acid position or positions on a polypeptide fragment, wherein the variant can be replaced, inserted or Missing amino acids were obtained.
  • epitope refers to a site on an antigen that specifically binds to an immunoglobulin or antibody.
  • An epitope can be formed by adjacent amino acids, or non-adjacent amino acids juxtaposed by the tertiary folding of a protein. An epitope formed by adjacent amino acids is usually maintained after exposure to a denaturing solvent, while an epitope formed by tertiary folding is usually lost after treatment with a denaturing solvent.
  • An epitope usually includes at least 3-15 amino acids in a unique spatial conformation. Methods for determining epitopes are well known in the art and include immunoblotting and immunoprecipitation detection analysis. Methods for determining the spatial conformation of an epitope include techniques in the art and techniques described in this disclosure, such as X-ray crystal analysis and two-dimensional nuclear magnetic resonance.
  • the term "specific binding” refers to the binding of an antibody to an epitope on a predetermined antigen.
  • the antibody is measured by surface plasmon resonance (SPR) technology in an instrument with an equilibrium dissociation constant of approximately less than 10 -7 M or even less (K D ) binds to a predetermined antigen, and has an affinity for the predetermined antigen that is at least twice that of a non-specific antigen (such as BSA, etc.) other than the predetermined antigen (or closely related antigen).
  • SPR surface plasmon resonance
  • K D surface plasmon resonance
  • the term "antibody that recognizes an antigen” is used interchangeably with the term “antibody that specifically binds” in this disclosure.
  • KD refers to the dissociation equilibrium constant of a particular antibody-antigen interaction.
  • antibodies of the present disclosure bind to OX40 with a dissociation equilibrium constant (KD) of less than about 10 -7 M, such as less than about 10 -8 M, 10 -9 M, or 10 -10 M or less, for example, using a surface Plasmon resonance (SPR) technology was measured in a BIACORE instrument.
  • SPR surface Plasmon resonance
  • the term “competition” in the case of competing for antigen-binding proteins (such as neutralizing antigen-binding proteins or neutralizing antibodies) for the same epitope, it means competition between antigen-binding proteins, which is determined by the following assays:
  • the antigen-binding protein eg, antibody or functional fragment thereof
  • the antigen-binding protein to be detected prevents or inhibits (eg, reduces) specific binding of a reference antigen-binding protein (eg, ligand or reference antibody) to a common antigen (eg, OX40 antigen or fragment thereof).
  • RIA solid-phase direct or indirect radioimmunoassay
  • EIA solid-phase direct or indirect enzyme immunoassay
  • Sandwich competition assay see, for example, Stahli et al., 1983, Methodsin Enzymology 9: 242-253
  • solid phase direct biotin-avidin EIA see, for example, Kirkland et al., 1986, J. Immunol.
  • solid Phase direct labeling assay solid phase direct labeling sandwich assay (see, eg, Harlow and Lane, 1988, Antibodies, Laboratory Manual, Cold Spring Harbor Press); direct labeling with solid phase of I-125 label RIA (see, eg, Morel et al., 1988, Molec. Immunol. 25: 7-15); and directly labeled RIAs (Moldenhauer et al., 1990, Scand. J. Immunol. 32: 77-82).
  • the assay involves the use of purified antigens that are capable of binding to unlabeled detection antigen binding proteins and labeled reference antigen binding proteins (the antigens are on a solid surface or a cell surface).
  • Antigen-binding proteins identified by competitive assays include: antigen-binding proteins that bind to the same epitope as the reference antigen-binding protein; and tables that are sufficiently close to the epitope bound by the reference antigen-binding protein Binding antigen binding protein, the two epitopes spatially prevent each other from binding. Additional details on methods for determining competitive binding are provided in the examples of this disclosure.
  • a competitive antigen binding protein when present in excess, it will inhibit (e.g., reduce) at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70 -75% or more specific binding of a reference antigen binding protein to a common antigen. In some cases, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97% or more.
  • cross-reactivity refers to the ability of an antibody of the invention to bind to OX40 from different species.
  • an antibody of the invention that binds human OX40 can also bind OX40 of another species.
  • Cross-reactivity is measured by detecting specific reactivity with purified antigens in binding assays, such as SPR and ELISA, or binding or functional interaction with cells that physiologically express OX40.
  • binding assays such as SPR and ELISA
  • Methods to determine cross-reactivity include standard binding assays as described in this disclosure, such as surface plasmon resonance (SPR) analysis, or flow cytometry.
  • SPR surface plasmon resonance
  • inhibiting or “blocking” are used interchangeably and encompass both partial and complete inhibition / blocking.
  • inhibiting growth (for example, involving a cell) is intended to include any measurable reduction in cell growth.
  • inducing an immune response and “enhancing the immune response” are used interchangeably and refer to the stimulation (i.e., passive or adaptive) of a particular antigen by an immune response.
  • induction with respect to inducing CDC or ADCC refers to stimulating a specific direct cell killing mechanism.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • FcR Fc receptor
  • cytotoxic cells such as NK cells, neutrophils, and macrophages.
  • Secreted immunoglobulins allow these cytotoxic effector cells to specifically bind to target cells that carry the antigen, and subsequently use cytotoxins to kill the cytotoxic form of the target cells.
  • the major cells (NK cells) that mediate ADCC only express FcyRIII, while monocytes express FcyRI, FcyRII and FcyRIII. Ravetch and Kinet, Annu. Rev. Immunol.
  • the modification refers to mutation in the constant region of the heavy chain of the antibody, such as N297A, L234A, L235A selected from IgG1; IgG2 / 4 chimera, F235E, or L234A / E235A mutation of IgG4.
  • complement-dependent cytotoxicity refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the first component of the complement system (C1q) -binding antibody (of a suitable subclass) that has bound to its associated antigen.
  • C1q complement system
  • CDC assays can be performed, such as those described in Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996).
  • Polypeptide variants having altered Fc region amino acid sequences (polypeptides with mutated Fc regions) and increased or decreased C1q binding capacity are described, for example, in US Patent No. 6,194,551 B1 and WO 1999/51642. See also, for example, Idusogie et al., J. Immunol. 164: 4178-4184 (2000).
  • nucleic acid molecule refers to DNA molecules and RNA molecules.
  • the nucleic acid molecule may be single-stranded or double-stranded, but specifically, double-stranded DNA.
  • a nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, the promoter or enhancer is operatively linked to the coding sequence.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked.
  • the vector is a "plasmid", which refers to a circular double-stranded DNA loop into which an additional DNA segment can be ligated.
  • the vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome.
  • Vectors in the present disclosure are capable of autonomous replication in host cells into which they have been introduced (e.g., bacterial vectors and episomal mammalian vectors with bacterial origins of replication) or can be integrated into the host cell's genome after introduction into the host cell, thereby The host genome is replicated together (eg, non-episomal mammalian vectors).
  • a mouse can be immunized with human OX40 or a fragment thereof.
  • the resulting antibody can be renatured, purified, and amino acid sequenced by conventional methods.
  • Antigen-binding fragments can also be prepared by conventional methods.
  • the antibody or antigen-binding fragment according to the invention is genetically engineered to add one or more human-derived FR regions to a CDR region of non-human origin.
  • the human FR germline sequence can be obtained by aligning the IMGT human antibody variable region germline gene database with MOE software from the website of ImMunoGeneTics (IMGT) http://imgt.cines.fr, or from the Journal of Immunoglobulins, 2001ISBN012441351 obtain.
  • IMGT ImMunoGeneTics
  • the antibodies or antigen-binding fragments of the present disclosure can be prepared and purified by conventional methods.
  • cDNA sequences encoding heavy and light chains can be cloned and recombined into a GS expression vector.
  • the recombinant immunoglobulin expression vector can stably transfect CHO cells.
  • mammalian expression systems cause glycosylation of antibodies, especially in the highly conserved N-terminus of the FC region.
  • Stable clones are obtained by expressing antibodies that specifically bind to human-derived antigens. Positive clones were expanded in serum-free medium in the bioreactor to produce antibodies.
  • the culture medium in which the antibody is secreted can be purified and collected by conventional techniques.
  • the antibody can be concentrated by filtration using a conventional method. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves, ion exchange. The resulting product needs to be immediately frozen, such as -70 ° C, or lyophilized.
  • a “monoclonal antibody” refers to an antibody obtained from a single cloned cell line, which is not limited to eukaryotic, prokaryotic, or phage cloned cell lines.
  • Monoclonal antibodies or antigen-binding fragments can be recombined using techniques such as hybridoma technology, recombinant technology, phage display technology, synthetic technology (such as CDR-grafting), or other existing technologies.
  • host cell refers to a cell into which an expression vector has been introduced.
  • Host cells may include microorganisms (e.g., bacteria), plant or animal cells. Easily transformed bacteria include members of the enterobacteriaceae, such as strains of Escherichiacoli or Salmonella; Bacilillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae.
  • Suitable microorganisms include Saccharomycescerevisiae and Pichia pastoris.
  • Suitable animal host cell lines include CHO (Chinese Hamster Ovary Cell Line), NSO cells, 293 cells.
  • Constant modification or “conservative substitution or substitution” refers to the replacement of amino acids in proteins by other amino acids with similar characteristics (such as charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation and rigidity, etc.) Changes are made without altering the biological activity of the protein.
  • Those skilled in the art know that, in general, a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub.Co. ,, P. 224, (4th edition)).
  • structural or functionally similar amino acid substitutions are unlikely to disrupt biological activity.
  • an "effective amount” includes an amount sufficient to ameliorate or prevent the symptoms or conditions of a medical disease.
  • An effective amount also means an amount sufficient to allow or facilitate diagnosis.
  • the effective amount for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the patient's overall health, the route and dose of administration, and the severity of the side effects.
  • An effective amount can be the maximum dose or dosage regimen to avoid significant side effects or toxic effects.
  • Exogenous refers to a substance that is produced outside the organism, cell, or human as appropriate.
  • Endogenous refers to a substance that is produced in an organism, cell, or human body as appropriate.
  • “Mutated sequence” refers to the nucleotide sequence and amino acid sequence of the present disclosure, which have different percentages of sequence identity with the nucleotide sequence and amino acid sequence of the present disclosure in the case of appropriate modification such as substitution, insertion, or deletion. Nucleotide sequence and amino acid sequence. The sequence identity may be at least 85%, 90%, or 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, and 93%. , 94%, 95%, 96%, 97%, 98%, 99%, 100%. Sequence comparisons and percent identity determinations between two sequences can be performed using the default settings of the BLASTN / BLASTP algorithm available on the National Center for Biotechnology Institute website.
  • homology refers to sequence similarity between two polynucleotide sequences or between two polypeptides.
  • a position in two compared sequences is occupied by the same base or amino acid monomer subunit, for example, if each position of two DNA molecules is occupied by adenine, then the molecules are homologous at that position .
  • the percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the total number of positions compared x 100.
  • sequences are optimally aligned, if 10 positions in the two sequences have 6 matches or are homologous, then the two sequences are 60% homologous; if 100 positions in the two sequences have 95 matches Or homologous, then the two sequences are 95% homologous. In general, comparisons are made when the two sequences are aligned for the greatest percentage of homology.
  • Polymerase chain reaction or "PCR” as used in the present disclosure refers to a procedure or technique in which a specific amount of nucleic acid, RNA and / or DNA is amplified as described in, for example, US Patent No. 4,683,195. Generally speaking, it is necessary to obtain sequence information from the end of the target region or beyond so that oligonucleotide primers can be designed; these primers are identical or similar in sequence to the corresponding strands of the template to be amplified. The 5 'terminal nucleotides of the two primers may coincide with the ends of the material to be amplified.
  • PCR can be used to amplify specific RNA sequences, specific DNA sequences from the total genome, and cDNA, phage, or plasmid sequences transcribed from total cellular RNA. See generally Mullis et al. (1987) Cold Spring Harbor Symp. Ouant. Biol. 51: 263; edited by Erlich, (1989) PCR TECHNOLOGY (Stockton Press, N.Y.).
  • PCR used in the present disclosure is considered as an example, but not the only example, of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample, and the method includes the use of known nucleic acids and nucleic acid polymerases as primers to amplify Or produce a specific portion of a nucleic acid.
  • “Pharmaceutical composition” means a mixture containing one or more of the antibodies or antigen-binding fragments thereof of the present disclosure with other chemical components, such as physiological / pharmaceutically acceptable carriers or excipients.
  • the purpose of the pharmaceutical composition is to promote the administration to the organism, which is beneficial to the absorption of the active ingredient and then exerts the biological activity.
  • cancer refers to or describes a physiological condition in mammals characterized by unregulated cell growth.
  • examples of cancer include, but are not limited to, cancer, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancy.
  • cancers include, but are not limited to, squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma, and squamous cell carcinoma of the lung), peritoneal cancer, liver Cell cancer, gastric cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, liver tumor, breast cancer, colon cancer, rectal cancer , Colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, anal cancer, penile cancer, melanoma, superficial diffuse melanoma, malignant freckles Melanoma, acral melanoma, nodular melanoma, multiple myeloma and B-cell lymphoma, chronic lymphoc
  • cancers suitable for treatment by the OX40 antibodies or fragments of the present disclosure include breast cancer, colorectal cancer, rectal cancer, non-small cell lung cancer, glioblastoma, non-Hodgkin's lymphoma (NHL), renal cell carcinoma, prostate cancer, liver cancer, pancreatic cancer, soft tissue sarcoma, Kaposi's sarcoma, carcinoid carcinoma, head and neck cancer, ovarian cancer, mesothelioma, and multiple bone marrow tumor.
  • breast cancer colorectal cancer, rectal cancer, non-small cell lung cancer, glioblastoma, non-Hodgkin's lymphoma (NHL), renal cell carcinoma, prostate cancer, liver cancer, pancreatic cancer, soft tissue sarcoma, Kaposi's sarcoma, carcinoid carcinoma, head and neck cancer, ovarian cancer, mesothelioma, and multiple bone marrow tumor.
  • NDL non-Hodgkin's lympho
  • the cancer is selected from the group consisting of: non-small cell lung cancer, glioblastoma, neuroblastoma, melanoma, breast cancer (eg, triple negative breast cancer), gastric cancer, colorectal cancer (CRC), and Hepatocellular carcinoma. Also, in some embodiments, the cancer is selected from the group consisting of: non-small cell lung cancer, colorectal cancer, glioblastoma, and breast cancer (eg, triple negative breast cancer), including those metastatic forms of cancer.
  • cell proliferative disorder and “proliferative disorder” refer to disorders associated with a certain degree of abnormal cell proliferation.
  • a cell proliferative disorder refers to cancer.
  • tumor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • administering when applied to an animal, human, subject, cell, tissue, organ, or biological fluid refer to an exogenous drug, therapeutic agent, diagnostic agent, or composition and animal, Human, subject, cell, tissue, organ or biological fluid contact.
  • administering may refer to, for example, treatment, pharmacokinetics, diagnostics, research, and experimental methods.
  • Treatment of a cell includes contact of a reagent with a cell, and contact of a reagent with a fluid, wherein the fluid is in contact with the cell.
  • administering “administering,” and “treating” also mean treating cells in vitro and ex vivo by an agent, diagnostic, binding composition, or by another cell.
  • Treatment when applied to a human, veterinary or research subject refers to therapeutic treatment, preventive or prophylactic measures, research and diagnostic applications.
  • Treatment means the administration to a patient of an internal or external therapeutic agent, such as a composition comprising any one of the antibodies or antigen-binding fragments thereof, or a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof, said patient having one or A variety of diseases or symptoms, and the therapeutic agents are known to have a therapeutic effect on these symptoms.
  • a therapeutic agent is administered in a treated patient or population in an amount effective to alleviate one or more diseases or symptoms to induce the deterioration of such symptoms or to inhibit the development of such symptoms to any clinically measurable degree.
  • the amount of therapeutic agent (also referred to as a "therapeutically effective amount") effective to alleviate any particular disease or condition can vary depending on a variety of factors, such as the patient's disease state, age and weight, and the ability of the drug to produce the desired therapeutic effect in the patient. Evaluate whether the symptoms of the disease have been alleviated by any clinical test method that a doctor or other health care professional usually uses to assess the severity or progression of the symptoms.
  • any statistical test method known in the art such as Student's test, Chi-square test, Mann and Whitney's The U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determined that it should reduce symptoms of the target disease in a statistically significant number of subjects.
  • preventing cancer refers to delaying, inhibiting or preventing the onset of cancer in mammals where the onset of carcinogenesis or tumorigenesis has not been confirmed but has been identified by, for example, genetic screening or other methods Has cancer susceptibility.
  • the term also includes treating a mammal with a precancerous condition to terminate the progression of the precancerous condition to a malignancy or cause its regression.
  • the following embodiments are used to further describe the present disclosure, but these embodiments do not limit the scope of the present disclosure.
  • the experimental methods without specific conditions in the examples of the present disclosure are generally in accordance with conventional conditions, such as the manual of antibody technology experiments in Cold Spring Harbor and the molecular cloning manual; or in accordance with the conditions recommended by the raw material or commodity manufacturers.
  • the reagents without specific sources are conventional reagents purchased on the market.
  • Anti-human OX40 monoclonal antibody libraries were generated by immunizing mice.
  • the mice of the BalB / C and A / J strains (Yangzhou University Comparative Medical Center, Animal Production License Number; SCXK (Su) 2017-007) were used for experiments, females, 10 weeks of age.
  • Immune antigen was human OX40 recombinant protein with Fc tag (OX40-Fc: OX40, Leu, 29-Ala, 216 (Accession # NP_003318) and Fc fusion), purchased from Acro Biosystems, article number # OX40-H5255, and expressed using HEK293 as usual Method purification.
  • OX40-Fc OX40, Leu, 29-Ala, 216 (Accession # NP_003318) and Fc fusion
  • OX40-Fc was emulsified with Freund's adjuvant: Freund's complete adjuvant (sigma-aldrich, F5881-10ML) was used for the first immunization, and Freund's incomplete adjuvant (S5ma-10d) was used for the rest of the immunization.
  • the ratio of antigen to adjuvant was 1: 1, and 25 ⁇ g protein / 200 ⁇ l / mouse was injected per immunization. See details below:
  • mice serum was detected by the ELISA method in Example 2 to determine the antibody titer and the neutralizing activity of blocking OX40 / OX40L binding.
  • Mice with high serum titer, affinity and ability to block ligand binding were selected to kill mice after a final immunization.
  • Take spleen cells and SP2 / 0 myeloma cells ( CRL-1581 (TM )) were plated after fusion to obtain hybridomas.
  • the target hybridomas were selected by the indirect ELISA, capture ELISA, and cell-based functional screening of Example 2, and monoclonal antibodies were constructed by the limiting dilution method.
  • OX40 mouse monoclonal antibodies Nineteen OX40 mouse monoclonal antibodies have been established for serum-free production, and purified mouse monoclonal antibodies were obtained using protein A affinity chromatography. Hybridoma cells secreting activated anti-OX40 antibodies were screened by indirect ELISA, capture ELISA and cell functional activity. The brief steps for functional screening are as follows: Culture GS-H2 / OX40 stable cell line (purchased from genscript, cat # M00608). The diluted test antibody and OX40L (Sino Biological) (13127-H04H) solution were prepared and added to the GS-H2 / OX40 cells in the logarithmic growth phase.
  • OX40L Ino Biological
  • the cell supernatant was collected after the culture, and the IL-8 content in the supernatant was measured ( Human (IL-8kit, cisbio, cat # 62IL8PEB) was used.
  • Human IL-8kit, cisbio, cat # 62IL8PEB
  • antibody GPX4 prepared in patent 1A7.gr.1 in patent WO2015153513 was used as a positive reference
  • hIgG laboratory preparation
  • RNA of the cells can be obtained by conventional RNA extraction technology, and then the PCR product of the variable region of the monoclonal antibody can be obtained by reverse transcription polymerase chain reaction (RT-PCR).
  • RT-PCR reverse transcription polymerase chain reaction
  • the PCR product was separated and recovered by agarose gel, then cloned into a gene vector and transformed into E. coli.
  • Several transformed colonies were randomly selected, and the monoclonal antibody variable region was amplified by PCR for gene sequencing.
  • the corresponding sequences of the obtained exemplary murine monoclonal antibodies are shown below.
  • the heavy and light chain variable region sequences of the murine monoclonal antibody m4B5 are as follows:
  • the heavy and light chain variable region sequences of the murine monoclonal antibody m2G3 are as follows:
  • 20 ⁇ coating buffer was diluted to 1 ⁇ with deionized water, and the antigen human OX40-His (Acro biosytems, OXL-H52Q8) was prepared with 1 ⁇ coating solution (carbonate buffer solution) to a final concentration of 2 ⁇ g. / mL, add 100 ⁇ L per well, and incubate at 4 ° C overnight or 37 ° C for 2h. Wash the plate once with PBST, add 200 ⁇ L of blocking solution (PBST containing 5% skim milk) to each well, incubate at 37 ° C for 2 h, and wash the plate 4 times.
  • 1 ⁇ coating solution carbonate buffer solution
  • the murine antibody m2G3 and m4B5 heavy chain variable region (VH) plus human immunoglobulin heavy chain constant region, light chain variable region (VL) plus human immunoglobulin Kappa light chain constant region were cloned into Murine-human chimeric antibodies were produced on eukaryotic expression vectors by transfecting cells.
  • the heavy chain vector was designed as follows: signal peptide + heavy chain variable region sequence + human IgG1 constant region sequence.
  • the light chain vector is designed as follows: signal peptide + light chain variable region sequence + human Kappa constant region sequence.
  • the above sequences were inserted into the pCEP4 vector (Thermofisher, V04450), respectively. After obtaining the vector plasmid, extract the plasmid and send the plasmid for sequencing verification.
  • the validated plasmid was transfected into human 293F cells with PEI and continuously cultured. The 293F cells were cultured in serum-free medium (Shanghai Opmai Bio, OPM-293CD03) to logarithmic growth phase for cell transfection.
  • m2G3-NC stands for negative control
  • Ch2G3-NC, m4B5-NC, Ch4B5-NC are negative controls, which are the same constant regions used by m2G3, Ch2G3, m4B5, and Ch4B5, but the variable region is not Identify OX40.
  • humanized mouse antibodies were engineered.
  • the heavy chain variable region (VH) and light chain variable region (VL) of the chimeric antibody were subjected to site-directed amino acid mutations in the FR (framework region) region, respectively. According to different combinations of amino acid mutations, different humanized antibody heavy and light chains are designed. Cells transfected with plasmids encoding different light / heavy chain combinations can produce humanized antibodies. Briefly described as follows:
  • the heavy chain vector was designed as follows: signal peptide + mutated heavy chain variable region sequence + human IgG1 constant region sequence.
  • the light chain vector was designed as follows: signal peptide + mutated light chain variable region sequence + human Kappa constant region sequence.
  • the above sequences were inserted into the pCEP4 vector (Thermofisher, V04450), respectively.
  • a third-party gene synthesis company is requested to synthesize an expression vector according to the above design.
  • After obtaining the vector plasmid extract the plasmid and send the plasmid for sequencing verification.
  • the validated plasmid was transfected into human 293F cells with PEI and continuously cultured.
  • the 293F cells were cultured in serum-free medium (Shanghai Opmai Bio, OPM-293CD03) to logarithmic growth phase for cell transfection.
  • Cell culture conditions 5% CO 2 , 37 ° C, 125 rpm / min. During the culture period, feeds were added on days 1 and 3 until the cell viability was below 70%. The cell supernatant was collected and centrifuged. Load the centrifuged cell culture solution onto an antibody purification affinity column, wash the column with phosphate buffer, elute with glycine hydrochloric acid buffer (pH 2.7 0.1M Gly-HCl), neutralize with 1M Tris hydrochloric acid, pH 9.0, And dialysis with phosphate buffer, and finally obtain the purified humanized antibody.
  • glycine hydrochloric acid buffer pH 2.7 0.1M Gly-HCl
  • the humanized variable region sequence is as follows:
  • the sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, FR sequences in italics, and CDR sequences underlined.
  • variable regions of the designed humanized antibody are combined into different antibodies according to the following table:
  • 2G3-hu1 indicates that the humanized antibody 2G3-hu1 has a heavy chain variable region of hu2G3 and VH1, a light chain variable region of hu2G3 and VL1, and so on.
  • the humanized variable region sequence of m4B5 is as follows:
  • variable regions of the designed humanized antibody are combined into different antibodies according to the following table:
  • Hu4B5 and V1 indicate that the humanized antibody hu4B5 and V1 have the heavy chain variable region of hu4B5 and VH0, the light chain variable region of hu4B5 and VL0, and so on.
  • the HCDR2 sequence obtained after the mutation is as follows:
  • the heavy chain variable regions hu2G3, VH1.1, hu2G3, VH1.2 and the light chain variable region hu2G3, VL were combined to form a new optimized humanized antibody, as shown in Table 7.
  • the above-mentioned humanized antibody was subjected to affinity evaluation (refer to the capture ELISA in Example 2).
  • the experimental results show that the humanized molecule can bind to OX40.
  • the light chain variable region and the light chain constant region sequence in the above sequence are combined to form the final light chain sequence, and the heavy chain variable region and the heavy chain constant region in the above sequence are combined to form the final heavy chain sequence.
  • the specific light and heavy chain constant regions are not limited to the constant regions of antibodies of the present disclosure, and other light and heavy chain constant regions and mutants thereof known in the art may be used to increase the performance of the antibody.
  • IgG1 heavy chain constant region 1 + IgG1 heavy chain constant region:
  • Example 7 In vitro binding affinity and kinetic tests of humanized antibodies
  • the Biacore method is a recognized method for objectively detecting the affinity and kinetics of proteins.
  • the kit provided by Biacore was used to covalently link the test recombinant anti-OX40 antibody of the present invention to a CM5 (GE) chip using the NHS standard amino coupling method. Then, a series of concentration gradients of human OX40-His protein (Yiqiao Shenzhou # 10481-H08H) diluted in the same buffer solution were injected in each cycle before and after, the flow rate was 30 ⁇ L / min. After the injection, they were regenerated in the kit Reagent regeneration. The antigen-antibody binding kinetics were followed for 3 minutes and the dissociation kinetics were followed for 10 minutes.
  • the wells were coated with the ligand OX40 ligand (Acrobiosystem, OXL-H52Q8), and after blocking, a gradient dilution of the test antibody (antibody diluted in human Bio-OX40-FC (Acrobiosystem, OX40-H5255, labeled biotin) was added. Pre-incubate the solution for 40 min before adding the plate), and after 40 min incubation, wash the plate. SA-HRP (Jackson Immunoresearch, 016-030-084) was added, and after incubating for 40 minutes, a coloring solution and a stop solution were added, and the OD values were measured. The results are shown in the table below.
  • CD4 + memory T cells were isolated, added to the test antibody in a 96-well plate coated with anti-CD3 antibody (Chempartner, A05-001), and incubated at 37 ° C for 72h. The supernatant was taken to detect IFN- ⁇ .
  • the results are shown in Figure 2 and Table 13 shown. The results show that GPX4, 4B5 and 2G3 can significantly enhance the release of IFN- ⁇ , and 2G3 can reach the maximum stimulation effect at 10ng / mL.
  • Example 11 Inhibition of tumor cell growth by anti-OX40 antibodies
  • B-hTNFRSF4 (OX40) humanized mouse B-hTNFRSF4 (OX40) humanized mouse, Biosato Jiangsu Gene Biotechnology Co., Ltd.
  • female 17 to 20 g, 6 to 7 weeks.
  • MC38 tumor cells 7 in logarithmic growth phase (purchased from Nanjing Yinhe Biomedical Co., Ltd.) were collected, the cell concentration was adjusted to 5 ⁇ 10 6 / mL with PBS buffer, and 0.1 mL of cell suspension was inoculated into the flank of OX40 mice. Observe the mice after inoculation and monitor the tumor growth. On the 7th day after the inoculation, the average tumor volume of the flank of tumor-bearing mice reached 102.5 mm 3. Grouping and drug observation were performed according to the tumor volume. The grouping information is shown in Table 14.
  • Dosage (mg / kg) Route of administration Dosing frequency Control (IgG1) 3 i.p. Q3D ⁇ 6 GPX4 3 i.p. Q3D ⁇ 6 2G3 0.3 i.p. Q3D ⁇ 6 2G3 1 i.p. Q3D ⁇ 6 2G3 3 i.p. Q3D ⁇ 6
  • Measurement of tumor volume and body weight of tumor-bearing mice Measured twice a week using a vernier caliper.
  • the average tumor volume of the IgG1 control group reached 1732.593 mm 3
  • the average tumor volume of mice in the low-dose administration group (0.3 mg / kg) of the test compound 2 reached 930.37 mm 3
  • the average tumor volume of the tumor-bearing mice in the drug group (2G3, 1mg / kg) and the high-dose administration group (2G, 3mg / kg) was 303.49mm 3 and 155.79mm 3 , respectively. It was obvious (** P ⁇ 0.01) and showed a preliminary dose-dependent relationship, and its tumor growth inhibition rates reached 49%, 88%, and 97.0%, respectively.
  • the average tumor volume in the 3mg / kg GPX4 group was 362.47mm 3 , which was significantly different from the control group, and also showed a significant inhibitory effect on tumor growth (* P ⁇ 0.05), and their tumor growth inhibition rates reached 84%.

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Abstract

本申请提供抗OX40抗体、其抗原结合片段及其医药用途。进一步地，本申请提供一种嵌合抗体、人源化抗体，其包含来自所述抗OX40抗体及其抗原结合片段的CDR区，以及还提供包含所述抗OX40抗体及其抗原结合片段的药物组合物，及其作为治疗癌症的药物的用途。

Description

抗OX40抗体、其抗原结合片段及其医药用途
本申请要求2018年09月26日提交的201811128669.3和2018年11月26日提交的201811417666.1的优先权。
技术领域
本发明属于生物医药领域，涉及一种抗OX40抗体、其抗原结合片段，包含所述抗OX40抗体CDR区的嵌合抗体、人源化抗体，以及包含人抗OX40抗体及其抗原结合片段的药物组合物，以及其作为抗癌药物的用途。
背景技术
癌症是当今人类社会长期面临的严峻健康挑战。对于已扩散的实体肿瘤，传统的手术、化疗和放疗等疗法往往收效甚微。
肿瘤免疫治疗是肿瘤治疗领域一个持续热点。最近研究已证明，增强抗肿瘤T细胞功能可以用于抵抗癌症。有大量证据表明，肿瘤细胞通过诱导主要由调节T淋巴细胞(Treg；Quezda et al.Immunol Rev 2011；241:104-118)介导的主动免疫耐受，而“逃避”免疫系统。因此，效应T淋巴细胞(Teff)与耐受原性(tolerogenic)Treg之间的平衡，对于有效抗肿瘤免疫治疗至关重要。因此，可以通过增强肿瘤特异性Teff的效应功能和/或通过减弱肿瘤特异性Treg的抑制功能，获得有效的抗肿瘤免疫应答。
CD134(OX40)受体已被证明是介导这些应答的一种关键受体(Sugamura,K,Ishii,N,Weinberg,A.Therapeutic targeting of the effector T-cell co-stimulatory molecule OX40.Nature Rev Imm 2004；4:420-431)。
OX40是肿瘤坏死因子受体(TNFR)超家族的成员之一，是一种在细胞表面表达的分子量约50kDa的糖蛋白。OX40的胞外配体结合结构域由4个富含半胱氨酸的结构域(CRD)组成。OX40的天然配体是OX40L(CD252)，两者形成OX40-OX40L复合物。
OX40主要表达在激活的T细胞上，OX40是次级共刺激分子，在激活后24-72小时后表达。OX40的配体OX40L主要表达在在激活的抗原递呈细胞上。表达OX40的T淋巴细胞已被证实存在于各种人恶性肿瘤及癌症患者的引流淋巴结中。在严重联合免疫缺陷(SCID)小鼠模型中，OX40与OX40L结合结构域相互作用可增强抗肿瘤免疫，导致各种人恶性肿瘤细胞系，如淋巴瘤、前列腺癌、结肠癌及乳腺癌的肿瘤生长抑制。
目前，有多家国际制药公司在研发针对OX40的单克隆抗体。OX40抗体通过特异性刺激，激活免疫，提高患者自身对肿瘤的免疫系统反应，达到对肿瘤细胞进行杀伤的目的。相关专利如WO2013038191、WO2015153513、WO2016179517、 WO2017096182、CN110078825A、WO2016196228等。迄今为止，阿斯利康、BMS等公司各自开发的抗OX40抗体已在II期临床试验之中，而基因泰克、GSK等公司的相关产品也已处在临床试验阶段。
但是，上述药物在治疗效果方面仍有可提高的空间。因此，有必要继续开发具有高选择性、高亲和力和良好药效的抗OX40抗体。
发明内容
本公开提供一种抗OX40抗体或其抗原结合片段，所述抗体与人OX40特异性结合，并包如下所示的CDR：
(i)分别如SEQ ID NO：3、4、5所示的重链HCDR1、HCDR2、HCDR3或与SEQ ID NO：3、4、5所示的HCDR1、HCDR2、HCDR3分别具有3、2或1个氨基酸差异的HCDR变体；和/或，
分别如SEQ ID NO：6、7、8所示的轻链LCDR1、LCDR2、LCDR3或与SEQ ID NO：6、7、8所示的LCDR1、LCDR2、LCDR3分别具有3、2或1个氨基酸差异的LCDR变体；
具体地，分别如SEQ ID NO：3、4、5所示的重链HCDR1、HCDR2、HCDR3和分别如SEQ ID NO：6、7、8所示的轻链LCDR1、LCDR2、LCDR3；或
(ii)分别如SEQ ID NO：11、12、13所示的重链HCDR1、HCDR2、HCDR3或与SEQ ID NO：11、12、13所示的HCDR1、HCDR2、HCDR3分别具有3、2或1个氨基酸差异的HCDR变体；和/或，
分别如SEQ ID NO：14、15、16所示的轻链LCDR1、LCDR2、LCDR3或与SEQ ID NO：14、15、16所示的LCDR1、LCDR2、LCDR3分别具有3、2或1个氨基酸差异的LCDR变体；
具体地，分别如SEQ ID NO：11、12、13所示的重链HCDR1、HCDR2、HCDR3和分别如SEQ ID NO：14、15、16所示的轻链LCDR1、LCDR2、LCDR3；或
(iii)分别如SEQ ID NO：11、33、13所示的重链HCDR1、HCDR2、HCDR3或与SEQ ID NO：11、33、13所示的HCDR1、HCDR2、HCDR3分别具有3、2或1个氨基酸差异的HCDR变体；和/或，
分别如SEQ ID NO：14、15、16所示的轻链LCDR1、LCDR2、LCDR3或与SEQ ID NO：14、15、16所示的LCDR1、LCDR2、LCDR3分别具有3、2或1个氨基酸差异的LCDR变体；
具体地，分别如SEQ ID NO：11、33、13所示的重链HCDR1、HCDR2、HCDR3和分别如SEQ ID NO：14、15、16所示的轻链LCDR1、LCDR2、LCDR3；或
(vi)分别如SEQ ID NO：11、34、13所示的重链HCDR1、HCDR2、HCDR3或与SEQ ID NO：11、34、13所示的HCDR1、HCDR2、HCDR3分别具有3、2或1个氨基酸差异的HCDR变体；和/或，
分别如SEQ ID NO：14、15、16所示的轻链LCDR1、LCDR2、LCDR3或与SEQ ID NO：14、15、16所示的LCDR1、LCDR2、LCDR3分别具有3、2或1个氨基酸差异的LCDR变体；
具体地，分别如SEQ ID NO：11、34、13所示的重链HCDR1、HCDR2、HCDR3和分别如SEQ ID NO：14、15、16所示的轻链LCDR1、LCDR2、LCDR3。
在一些实施方案中，所述抗OX40抗体或其抗原结合片段为鼠源抗体、嵌合抗体、人源化抗体、人抗体或其片段，具体地，人源化抗体。
在一些实施方案中，所述单克隆抗体或抗原结合片段的CDR(包括3个重链CDR和3个轻链CDR)允许包含3、2或1个氨基酸差异(即CDR变体)，CDR变体是通过亲和力成熟方法筛选获得的。
在一些实施方案中，所述单克隆抗体或抗原结合片段与OX40的亲和力(KD)小于10 -8M、小于10 -9M或小于10 -10M。
在一些实施方案中，其中所述的鼠源抗体包含如下所示的重链可变区和轻链可变区：
(i)重链可变区氨基酸序列如SEQ ID NO：1所示；或与SEQ ID NO：1具有至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％序列同一性；和/或，
轻链可变区氨基酸序列如SEQ ID NO：2所示；或与SEQ ID NO：2具有至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％序列同一性；
(ii)重链可变区氨基酸序列如SEQ ID NO：9所示；或与SEQ ID NO：9具有至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％序列同一性；和/或，
轻链可变区氨基酸序列如SEQ ID NO：10所示；或与SEQ ID NO：10具有至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％序列同一性。
在一些实施方案中，其中所述的人源化抗体包含来源于人种系的FR区或其突变序列。
在一些实施方案中，其中所述的人源化抗体包含选自：
(i)重链可变区，其是SEQ ID NO：17、18、31、32中任一项所示；或与SEQ ID NO：17、18、31、32具有至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、 至少97％、至少98％、至少99％序列同一性；或
(ii)重链可变区，其是SEQ ID NO：26、27、28、29、30中任一项所示；或与SEQ ID NO：26、27、28、29、30具有至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％序列同一性。
在一种实施方案中，其中所述的抗OX40抗体或其抗原结合片段包含：
(i)轻链可变区，其是SEQ ID NO：19或20所示；或与SEQ ID NO：19或20具有至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％序列同一性；或
(ii)轻链可变区，其是SEQ ID NO：21、22、23、24、25中任一项所示；或与SEQ ID NO：21、22、23、24、25具有至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％序列同一性。
在一些具体实施方案中，抗OX40抗体或其抗原结合片段包含：
(i)如SEQ ID NO：17、18、31、32中任一项所示的重链可变区和SEQ ID NO：19或20所示的轻链可变区；或
(ii)如SEQ ID NO：26、27、28、29、30中任一项所示的重链可变区和SEQ ID NO：21、22、23、24、25中任一项所示的轻链可变区。
在一些具体实施方案中，抗OX40抗体或其抗原结合片包含如下所示的可变区：
(1)重链可变区如SEQ ID NO：17所示，轻链可变区如SEQ ID NO：19所示；
(2)重链可变区如SEQ ID NO：18所示，轻链可变区如SEQ ID NO：19所示；
(3)重链可变区如SEQ ID NO：31所示，轻链可变区如SEQ ID NO：19所示；
(4)重链可变区如SEQ ID NO：32所示，轻链可变区如SEQ ID NO：19所示；
(5)重链可变区如SEQ ID NO：17所示，轻链可变区如SEQ ID NO：20所示；
(6)重链可变区如SEQ ID NO：18所示，轻链可变区如SEQ ID NO：20所示；
(7)重链可变区如SEQ ID NO：31所示，轻链可变区如SEQ ID NO：20所示；和
(8)重链可变区如SEQ ID NO：32所示，轻链可变区如SEQ ID NO：20所示。
在一些实施方案中，抗OX40抗体或其抗原结合片包含如下所示的可变区：
(1)重链可变区如SEQ ID NO：26所示，轻链可变区如SEQ ID NO：21所示；
(2)重链可变区如SEQ ID NO：26所示，轻链可变区如SEQ ID NO：22所示；
(3)重链可变区如SEQ ID NO：26所示，轻链可变区如SEQ ID NO：23所示；
(4)重链可变区如SEQ ID NO：26所示，轻链可变区如SEQ ID NO：24所示；
(5)重链可变区如SEQ ID NO：26所示，轻链可变区如SEQ ID NO：25所示；
(6)重链可变区如SEQ ID NO：27所示，轻链可变区如SEQ ID NO：21所示；
(7)重链可变区如SEQ ID NO：27所示，轻链可变区如SEQ ID NO：22所示；
(8)重链可变区如SEQ ID NO：27所示，轻链可变区如SEQ ID NO：23所示；
(9)重链可变区如SEQ ID NO：27所示，轻链可变区如SEQ ID NO：24所示；
(10)重链可变区如SEQ ID NO：27所示，轻链可变区如SEQ ID NO：25所示；
(11)重链可变区如SEQ ID NO：28所示，轻链可变区如SEQ ID NO：21所示；
(12)重链可变区如SEQ ID NO：28所示，轻链可变区如SEQ ID NO：22所示；
(13)重链可变区如SEQ ID NO：28所示，轻链可变区如SEQ ID NO：23所示；
(14)重链可变区如SEQ ID NO：28所示，轻链可变区如SEQ ID NO：24所示；
(15)重链可变区如SEQ ID NO：28所示，轻链可变区如SEQ ID NO：25所示；
(16)重链可变区如SEQ ID NO：29所示，轻链可变区如SEQ ID NO：21所示；
(17)重链可变区如SEQ ID NO：29所示，轻链可变区如SEQ ID NO：22所示；
(18)重链可变区如SEQ ID NO：29所示，轻链可变区如SEQ ID NO：23所示；
(19)重链可变区如SEQ ID NO：29所示，轻链可变区如SEQ ID NO：24所示；
(20)重链可变区如SEQ ID NO：29所示，轻链可变区如SEQ ID NO：25所示；
(21)重链可变区如SEQ ID NO：30所示，轻链可变区如SEQ ID NO：21所示；
(22)重链可变区如SEQ ID NO：30所示，轻链可变区如SEQ ID NO：22所示；
(23)重链可变区如SEQ ID NO：30所示，轻链可变区如SEQ ID NO：23所示；
(24)重链可变区如SEQ ID NO：30所示，轻链可变区如SEQ ID NO：24所示；和
(25)重链可变区如SEQ ID NO：30所示，轻链可变区如SEQ ID NO：25所示。
在一些实施方案中，其中所述的抗OX40抗体包含恒定区；在一些具体实施方 案中，所述抗体为嵌合抗体或人源化抗体，所述抗体的重链恒定区源自人IgG1、IgG2、IgG3或IgG4或其突变序列，轻链恒定区源自人κ、λ链或其突变序列；在另一些具体实施方案中，所述重链恒定区的氨基酸序列如SEQ ID NO：35所示或与SEQ ID NO：35具有至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％序列同一性，所述轻链恒定区的氨基酸序列如SEQ ID NO：36所示或与SEQ ID NO：36具有至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％序列同一性。
在一些实施方案中，抗OX40抗体重链氨基酸序列如SEQ ID NO：39或37所示或与SEQ ID NO：39或37具有至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％序列同一性，和/或，所述抗OX40抗体轻链氨基酸序列如SEQ ID NO：40或38所示或与SEQ ID NO：40或38具有至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％序列同一性。
在一种实施方案中，其中所述抗OX40抗体包含：
(i)
重链，其氨基酸序列如SEQ ID NO：37所示或与SEQ ID NO：37具有至少85％序列同一性，和
轻链，其氨基酸序列如SEQ ID NO：38所示或与SEQ ID NO：38具有至少85％序列同一性；或
(ii)
重链，其氨基酸序列如SEQ ID NO：39所示与SEQ ID NO：39具有至少85％序列同一性，和
轻链，其氨基酸序列如SEQ ID NO：40或与SEQ ID NO：40具有至少85％序列同一性。
本公开还提供一种抗OX40抗体或其抗原结合片段，所述抗OX40抗体或其抗原结合片段与前述任一项所述的抗体或其抗原结合片段竞争性结合人OX40，或与前述任一项所述的抗体或其抗原结合片段结合相同的OX40抗原表位。
本公开还提供一种药物组合物，其含有：
-治疗/预防有效量的如上所述的抗OX40抗体或其抗原结合片段，以及
-一种或多种药学上可接受的载体、稀释剂、缓冲剂或赋形剂。
在一些具体的实施方案中，所述药物组合物可以制备成单位剂量形式，其单位剂量中可含有0.01至99重量％的抗OX40抗体或其抗原结合片段；或药物组合物单位剂量中含单克隆抗体或其抗原结合片段的量为0.1-2000mg，在一些具体实 施方案中，单克隆抗体或其抗原结合片段的量为1-1000mg。
在一些具体的实施方案中，单克隆抗体或其抗原结合片段可以是组合物中的唯一活性成分；在另一些具体的实施方案中，单克隆抗体或其抗原结合片段和其它活性成分组合使用。
本公开还提供一种分离的核酸分子，其编码如上所述的抗OX40抗体或其抗原结合片段。在此处的上下文中，这种核酸分子的互补序列，也包括在本申请范畴内。
本公开还提供一种载体，其包含上述的核酸分子，所述载体可以是真核表达载体、原核表达载体或病毒载体。
本公开还提供一种用如上所述的载体转化的宿主细胞，所述宿主细胞选自原核细胞和真核细胞。
在一些具体实施方案中，宿主细胞为真核细胞。在一些具体实施方案中，为哺乳动物细胞，其中所述的哺乳动物细胞包括但不限于CHO、HEK293、NSO。应当理解，本公开的宿主细胞不涉及任何能够发育成人类的细胞。
本公开还提供一种制备如上所述的抗OX40抗体或其抗原结合片段的方法，所述方法包括：
-培养上述宿主细胞，
-然后回收抗体或其抗原结合片段；
-任选地，对抗体或其抗原结合片段进行纯化。
本公开还提供用于检测或测定人OX40的方法，所述方法包括：
-使用上述抗OX40抗体或其抗原结合片段与样本(例如来源于人)相接触；
-任选地，确定样本中OX40的量、或确定是否存在OX40。
本公开还提供用于检测或测定人OX40的试剂，所述试剂包含上述任一项所述的抗OX40抗体或其抗原结合片段。
本公开还提供用于与人OX40相关的疾病的诊断剂，所述诊断剂包含根据上述的抗OX40抗体或其抗原结合片段。
本公开还提供用于诊断与人OX40相关的疾病的方法，所述方法包括使用上述抗OX40抗体或其抗原结合片段检测或测定人OX40或OX40阳性细胞。
本公开还提供上述抗OX40抗体或其抗原结合片段在制备与人OX40相关的疾病的诊断剂中的应用。
本公开还提供治疗疾病或病症的方法，包括向受试者施用有效量的上述抗OX40抗体或其抗原结合片段，或包含上述OX40抗体或其片段的药物组合物。
在一些实施方案中，所述疾病或病症可以为癌症或细胞增殖性疾病。在一些具体实施方案中，所述癌症为肺癌、前列腺癌、乳腺癌、头颈部癌、食管癌、胃癌、结肠癌、结直肠癌、膀胱癌、宫颈癌、子宫癌、卵巢癌、肝癌、黑色素瘤、 肾癌、鳞状细胞癌、血液系统癌症或者任何特征在于不受控细胞生长的其它疾病或病症。
在一些具体实施方案中，所述的血液系统癌症包括但不限于急性及慢性骨髓性白血病、急性淋巴细胞性白血病、慢性淋巴细胞性白血病、骨髓组织增殖性疾病、多发性骨髓瘤、何杰金氏(Hodgkin)疾病、非何杰金氏淋巴瘤、B细胞淋巴瘤、T细胞淋巴瘤、滤泡中心细胞淋巴瘤、慢性粒细胞白血病。
本公开还提供如上所述抗OX40抗体或其抗原结合片段，或包含上述OX40抗体或其片段的药物组合物在制备用于增强人类对象中的免疫应答的药物中的用途。
本公开还提供如上所述抗OX40抗体或其抗原结合片段，或包含上述OX40抗体或其片段的药物组合物在制备用于治疗/预防癌症的药物中的用途。
本公开还提供如上所述抗OX40抗体或其抗原结合片段，或包含上述OX40抗体或其片段的药物组合物在制备试剂盒中的用途。
在一些实施方案中，所述增强的免疫应答包括T效应细胞的免疫刺激/效应功能的增加，和/或T调节细胞的免疫抑制功能的下调。其中，所述增加可以是细胞增殖的结果，所述下调可以是细胞数量不增加(或细胞数量降低)的结果。
本公开中提供所述抗人OX40抗体或片段用于下述一项或多项：抑制Treg功能(例如抑制Treg的遏制性功能)、杀死表达OX40的细胞(例如表达高水平OX40的细胞)、提高效应T细胞功能和/或提高记忆T细胞功能、降低肿瘤免疫、增强T细胞功能和/或消减表达OX40的细胞。
本公开提供所述抗OX40抗体或片段用于以下的用途：治疗癌症、刺激受试者中的免疫反应、刺激抗原特异性T细胞反应、激活或共刺激T细胞、增加T细胞中的细胞因子(例如IL-2及/或IFN-γ)产生、和/或T细胞的增殖、减少或耗竭肿瘤中T调控性细胞的数目、和/或抑制肿瘤细胞生长。
本公开亦提供所述抗OX40抗体制备药剂的用途，所述药剂用于：刺激受试者中的免疫反应、刺激抗原特异性T细胞反应、激活或共刺激T细胞、增加T细胞中的IL-2和/或IFN-γ产生和/或T细胞的增殖、减少或耗竭肿瘤中T调控性细胞的数目和/或抑制肿瘤细胞生长。
附图说明
图1A至图1B：鼠源抗体和嵌合抗体与人OX40的亲和力ELISA检测结果。其中，图1A是鼠源抗体m2G3和嵌合抗体ch2G3的亲和力结果，m2G3-NC和ch2G3-NC为阴性对照；图1B是鼠源抗体m4B5和嵌合抗体ch4B5的亲和力结果，m4B5-NC和ch4B5-NC为阴性对照。
图2：抗OX40抗体刺激T细胞分泌IFN-γ的实验。结果显示，待测OX40抗体在10ng/mL抗体浓度时即可达到最大刺激效果。
图3A至图3B：抗OX40抗体小鼠体内抑瘤效果，图3A显示给药后不同小鼠体内肿瘤体积的变化；图3B显示给药后，不同抗OX40抗体对小鼠体内肿瘤质量的影响。结果显示给药治疗后第20天，在给药量为3mg/kg时，2G3抗体的肿瘤抑制率高达97％。
术语
为了更容易理解本公开，以下具体定义了某些技术和科学术语。除显而易见在本公开中的它处另有明确定义，否则本公开使用的所有其它技术和科学术语都具有本发明所属领域的一般技术人员通常理解的含义。
术语“增强T细胞功能”意指诱导、引起或刺激效应或记忆T细胞，使其具有更新、持续或放大的生物学功能。增强T细胞功能的例子包括：相对于干预前的水平，来自CD8 +效应T细胞的γ-干扰素分泌升高、来自CD4 +记忆和/或效应T细胞的γ-干扰素分泌升高、CD4 +效应和/或记忆T细胞增殖升高、CD8 +效应T细胞增殖升高、抗原响应性(例如清除)增强。
术语“增强免疫应答”是指刺激、激起、增加、改善或增强哺乳动物免疫系统的应答。免疫应答可以是细胞应答(即细胞介导的，如细胞毒性T淋巴细胞介导的)或者体液应答(即抗体介导的应答)，并且可以是初次或再次免疫应答。增强免疫应答的例子包括增加的CD4 +辅助T细胞活性及产生细胞毒性T细胞。免疫应答的增强可以用本领域技术人员已知的一些体外或体内测量进行评估，包括但不限于细胞毒性T淋巴细胞测定、细胞因子释放(例如IL-2的产生)、肿瘤消退、携带肿瘤动物的存活、抗体产生、免疫细胞增殖、细胞表面标记表达及细胞毒性。在一个实施方案中，所述方法增强细胞免疫应答，特别是细胞毒性T细胞应答。
“肿瘤免疫”指肿瘤逃避免疫识别和清除的过程。作为治疗概念，肿瘤免疫在其逃避免疫识别和清除的能力被减弱时，肿瘤被免疫系统识别并攻击，患者得到治疗。肿瘤识别的例子包括肿瘤结合、肿瘤收缩和肿瘤清除。
“T效应细胞”(“Teff”)，指具有细胞溶解活性的T细胞(例如，CD4 +及CD8 +T细胞)以及T辅助(Th)细胞，Teff分泌细胞因子、且激活并引导其他免疫细胞，但不包括调控性T细胞(Treg细胞)。本公开所述抗OX40抗体可激活Teff细胞，例如CD4+及CD8+Teff细胞。
“调节性T细胞”或“Treg细胞”意指专门化类型的CD4+T细胞，其能阻抑其它T细胞的应答。Treg细胞特征在于表达CD4、IL-2受体的α亚基(CD25)、和转录因子forkhead box P3(FOXP3)(Sakaguchi,Annu Rev Immunol 22,531-62(2004))，且在诱导和维持外周自体耐受中发挥至关重要作用，所述耐受针对肿瘤表达的抗原。
“OX40”是指一种结合OX40配体(OX40-L)的受体，它是TNF-受体超家族的成员。OX40亦称作肿瘤坏死因子受体超家族成员4(TNFRSF4)、ACT35、IMD16、 TXGP1L及CD 134。术语“OX40”包括由细胞天然表达的任何OX40变体或同种型。因此，本公开所述OX40抗体或片段可与来自除人类外的物种的OX40(例如，食蟹猴OX40)交叉反应。或者，所述OX40抗体或片段可对人OX40具有特异性，且不展现与其他物种的交叉反应性。
OX40或其变体及同种型从其天然表达的细胞或组织中分离，或使用本领域熟知的技术和/或本公开所述的技术以重组方式产生。除非另有说明，否则“OX40”可以来自任何脊椎动物来源，包括哺乳动物诸如灵长类(例如人)和啮齿类(例如小鼠和大鼠)的天然OX40。该术语涵盖“全长”，未加工的OX40以及因细胞中的加工所致的任何形式的OX40。该术语还涵盖OX40的天然发生变体，例如剪接变体或等位变体。
“OX40激活”指OX40受体的激活。通常，OX40激活导致信号转导。
“抗OX40抗体”或“结合OX40的抗体”指能够以足够亲和力结合OX40，使得该抗体可作为诊断剂和/或治疗剂用于靶向OX40的抗体。
术语“消减表达OX40的细胞”，是指抗OX40抗体或其片段杀死或消减表达OX40的细胞。消减表达OX40的细胞可以通过多种机制来实现，诸如抗体依赖性细胞(ADCC)介导的细胞毒性和/或吞噬作用。
术语“细胞因子”是由一类蛋白质的通称，其由细胞群释放，并作为细胞间介质作用于另一细胞。此类细胞因子的例子有淋巴因子、单核因子；白介素(IL)，诸如IL-1、IL-1α、IL-2、IL-3、IL-4、IL-5、IL-6、IL-7、IL-8、IL-9、IL-11、IL-12、IL-15；肿瘤坏死因子，诸如TNF-α或TNF-β；及其它多肽因子，包括LIF和kit配体(KL)和γ-干扰素。如本公开中使用的，术语细胞因子包括来自天然来源或来自重组细胞培养物的蛋白质及其生物学活性等效物，包括通过人工合成产生的小分子实体，及其药剂学可接受的衍生物和盐。
本公开所用氨基酸三字母代码和单字母代码如J.Biol.Chem,243,p3558(1968)中所述。
术语“抗体”不受任何特定的产生抗体的方法限制。例如，其包括，重组抗体、单克隆抗体和多克隆抗体。抗体可以是不同同种型的抗体，例如，IgG(例如，IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或IgM抗体。
抗体重链和轻链中靠近N端的约110个氨基酸的序列变化很大，为可变区(V区)；靠近C端的其余氨基酸序列相对稳定，为恒定区(C区)。可变区包括3个高变区(HVR)和4个序列相对保守的骨架区(FR)。3个高变区决定抗体的特异性，又称为互补性决定区(CDR)。每条轻链可变区(VL)和重链可变区(VH)由3个CDR区4个FR区组成，从氨基端到羧基端依次排列的顺序为：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。轻链的3个CDR区指LCDR1、LCDR2、和LCDR3；重链的3个CDR区指HCDR1、HCDR2和HCDR3。
本公开所述的抗体或抗原结合片段的LCVR区和HCVR区的CDR氨基酸残 基在数量和位置符合已知的Kabat编号规则(LCDR1-3，HCDR1-3)。
术语“重组人抗体”包括通过重组方法制备、表达、创建或分离的人抗体，所涉及的技术和方法在本领域中是熟知的，诸如：
(1)从人免疫球蛋白基因的转基因、转染色体动物(例如小鼠)或由其制备的杂交瘤中分离的抗体；
(2)从经转化以表达抗体的宿主细胞(如转染瘤)中分离的抗体；
(3)从重组组合人抗体文库中分离的抗体；以及
(4)通过将人免疫球蛋白基因序列剪接到其他DNA序列等方法制备、表达、创建或分离的抗体。此类重组人抗体包含可变区和恒定区，这些区域利用特定的由种系基因编码的人种系免疫球蛋白序列，但也包括随后诸如在抗体成熟过程中发生的重排和突变。
本公开的抗体包括鼠源抗体、嵌合抗体、人源化抗体、人抗体。
一些实施方案中为人源化抗体。
术语“鼠源抗体”为根据本领域知识和技能制备的针对人OX40的单克隆抗体。制备时用OX40抗原注射试验对象，然后分离表达具有所需序列或功能特性的抗体的脾细胞(B淋巴细胞)，再将B淋巴细胞与骨髓瘤细胞融合，获得相应杂交瘤细胞。
在一些实施方案中，所述的鼠源OX40抗体或其抗原结合片段，可进一步包含鼠源κ、λ链或其变体的轻链恒定区，或进一步包含鼠源IgG1,IgG2,IgG3或IgG4或其变体的重链恒定区。
术语“人抗体”包括具有人种系免疫球蛋白序列的可变和恒定区的抗体。本公开的人抗体可包括不由人种系免疫球蛋白序列编码的氨基酸残基(如通过体外随机或位点特异性诱变或通过体内体细胞突变所引入的突变)。然而，术语“人抗体”不包括这样的抗体，即其中已将衍生自另一种哺乳动物物种(诸如小鼠)种系的CDR序列移植到人骨架序列上(即“人源化抗体”)。
术语“人源化抗体(humanized antibody)”，是指将非人物种CDR序列移植到人的抗体可变区框架，即不同类型的人种系抗体框架序列中产生的抗体。可以克服由于嵌合抗体携带大量异源蛋白成分，从而诱导的异源性反应。此类构架序列可以从包括种系抗体基因序列的公共DNA数据库或公开的参考文献获得。如人重链和轻链可变区基因的种系DNA序列可以在“VBase”人种系序列数据库(www.mrccpe.com.ac.uk/vbase)中获得，以及在Kabat，E.A.等人，1991 Sequences of Proteins of Immunological Interest，第5版中找到。为避免免疫原性下降的同时，引起的活性下降，可对所述的人抗体可变区框架序列进行最少反向突变或回复突变，以保持活性。本公开的人源化抗体也包括进一步由噬菌体展示或酵母菌展示对CDR进行亲和力成熟后获得的人源化抗体。
在涉及CDR移植的情况中，由于与抗原接触的构架残基的变化而导致产生的 OX40抗体(或其抗原结合片段)对抗原的亲和力减弱。此类相互作用可以是体细胞高度突变的结果。因此，仍然需要将此类供体构架氨基酸移植至人源化抗体的构架。来自非人OX40抗体或其抗原结合片段的参与抗原结合的氨基酸残基，可通过检查非人单克隆抗体可变区序列和结构来鉴定。CDR供体构架中与种系不同的的各残基可被认为是相关的。如果不能确定最接近的种系，那么可将序列与亚型共有序列或具有高相似性百分数的鼠序列的共有序列相比较。稀有构架残基被认为可能是体细胞高度突变的结果，从而在结合中起着重要作用。在本公开一些实施方案中，所述的OX40人源化抗体的抗体轻链进一步包含人源κ、λ链或其变体的轻链恒定区。所述的OX40人源化抗体的抗体重链进一步包含人源IgG1、IgG2、IgG3、IgG4或其变体的重链恒定区，具体地，包含人源IgG1重链恒定区。
术语“回复突变”是指将人抗体来源的FR区氨基酸残基突变成原始来源抗体对应位置的氨基酸残基，通常是为了避人源化抗体引起的免疫原性下降的同时，引起的活性下降，对所述的人源化抗体可变区可进行最少的回复突变，以保持抗体的活性。
术语“嵌合抗体(chimeric antibody)”，是将非人抗体的可变区与人抗体的恒定区融合而成的抗体，可以减轻非人抗体诱发的免疫应答反应。例如，建立嵌合抗体，要选建立分泌鼠源性特异性单抗的杂交瘤，然后从小鼠杂交瘤细胞中克隆可变区基因，再要据需要克隆人抗体的恒定区基因，将小鼠可变区基因与人恒定区基因连接成嵌合基因后插入人载体中，最后在真核工业系统或原核工业系统中表达嵌合抗体分子。人抗体的恒定区可选自人源IgG1,IgG2,IgG3或IgG4或其变体的重链恒定区，具体地，包含人源IgG1重链恒定区。
术语抗体的“抗原结合片段”或“功能片段”是指抗体中的一个或多个片段，其保持特异性结合抗原(例如，OX40)的能力。已显示可利用全长抗体的片段来实现抗体的抗原结合功能。术语抗体的“抗原结合片段”中包含的结合片段的实例包括：
(i)Fab片段，由VL、VH、CL和CH1结构域组成的单价片段；
(ii)F(ab') 2片段，通过铰链区上的二硫桥连接两个Fab片段形成的二价片段，
(iii)由VH和CH1结构域组成的Fd片段；
(iv)由抗体的单臂的VH和VL结构域组成的Fv片段；
(v)单结构域或dAb片段(Ward等人，(1989)Nature，341：544-546)，其由VH结构域组成；和
(vi)分离的互补决定区(CDR)或
(vii)可任选地，通过合成的接头连接的两个或更多个分离的CDR的组合。
此外，虽然Fv片段的两个结构域VL和VH由单独的基因编码，但可使用重组方法，通过合成的接头连接它们，从而使得其能够产生单个蛋白质链(称为单链Fv(scFv)，在其中VL和VH区配对形成单价分子；参见，例如，Bird等 人(1988)Science 242:423-426；和Huston等人(1988)Proc.Natl.Acad.Sci USA85:5879-5883)。此类单链抗体也意欲包括在术语抗体的“抗原结合片段”中。使用本领域技术人员已知的常规技术获得此类抗体片段，并且以与对于完整抗体的方式相同的方式就功用性能筛选片段。可通过重组DNA技术或通过酶促或化学断裂完整免疫球蛋白来产生抗原结合部分。抗体可以是不同同种型的抗体，例如，IgG(例如，IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或IgM抗体。
一些实施方案中，“抗原结合片段”指具有抗原结合活性的Fab、Fv、sFv、F(ab’)2、线性抗体、单链抗体、scFv、sdAb、sdFv、纳米抗体、肽抗体(peptibody)、结构域抗体和多特异性抗体(双特异性抗体、双抗体(diabody)、三抗(triabody)和四抗(tetrabody)、串联二-scFv、串联三-scFv)。
“Fab”是通过用蛋白酶木瓜蛋白酶(切割H链的224位的氨基酸残基)处理IgG抗体分子所获得的片段中具有约50,000的分子量并具有抗原结合活性的抗体片段，其中H链N端侧的约一半和整个L链通过二硫键结合在一起。本公开的Fab可以通过用木瓜蛋白酶处理本公开的特异性识别人OX40并与胞外区的氨基酸序列或其三维结构结合的单克隆抗体来生产。此外，可以通过将编码所述抗体的Fab的DNA插入到原核生物表达载体或真核生物表达载体中并将载体导入到原核生物或真核生物中以表达Fab来生产所述Fab。
“F(ab')2”是通过用胃蛋白酶消化IgG铰链区中两个二硫键的下游部分，而获得的抗体片段，其分子量为约100,000，并具有抗原结合活性，并包含铰链位置相连的两个Fab区。
本公开的F(ab')2可以通过用胃蛋白酶处理本公开的单克隆抗体来生产。此外，可以通过用硫醚键或二硫键连接下面描述的Fab'来生产所述F(ab')2。
“Fab'”是通过切割上述F(ab')2的铰链区的二硫键而获得的分子量为约50,000并具有抗原结合活性的抗体片段。本公开的Fab'可以通过用还原剂例如二硫苏糖醇处理本公开的的F(ab')2来生产。
此外，可以通过将编码抗体的Fab'片段的DNA插入到原核生物表达载体或真核生物表达载体中，并将载体导入到原核生物或真核生物中，以表达Fab'来生产所述Fab'。
术语“单链抗体”、“单链Fv”或“scFv”意指包含通过接头连接的抗体重链可变结构域(或区域；VH)和抗体轻链可变结构域(或区域；VL)的分子。此类scFv分子可具有一般结构：NH 2-VL-接头-VH-COOH或NH 2-VH-接头-VL-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成，例如使用1-4个重复的变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA90:6444-6448)。可用于本公开的其他接头由Alfthan等人(1995)，Protein Eng.8:725-731，Choi等人(2001)，Eur.J.Immuno l.31:94-106，Hu等人(1996)，Cancer Res.56:3055-3061， Kipriyanov等人(1999)，J.Mol.Biol.293:41-56和Roovers等人(2001)，Cancer Immunol.描述。
本公开的scFv可以通过以下步骤来生产：获得编码本公开单克隆抗体VH和VL的cDNA，构建编码scFv的DNA，将所述DNA插入到原核生物表达载体或真核生物表达载体中，然后将所述表达载体导入到原核生物或真核生物中以表达scFv。
“双抗体”是其中scFv被二聚体化的抗体片段，是具有二价抗原结合活性的抗体片段。在二价抗原结合活性中，两个抗原可以是相同或不同的。
本公开的双抗体可以通过以下步骤来生产：获得编码本公开单克隆抗体的VH和VL的cDNA，构建编码scFv的DNA以使肽接头的氨基酸序列长度为8个残基或更少，将所述DNA插入到原核生物表达载体或真核生物表达载体中，然后将所述表达载体导入到原核生物或真核生物中以表达双抗体。
“dsFv”是通过将其中每个VH和VL中的一个氨基酸残基被半胱氨酸残基取代的多肽经由半胱氨酸残基之间的二硫键相连而获得的。可以按照已知方法(Protein Engineering,7,697(1994))基于抗体的三维结构预测来选择被半胱氨酸残基取代的氨基酸残基。
本公开的dsFv可以通过以下步骤来生产：获得编码本公开单克隆抗体的VH和VL的cDNA，构建编码dsFv的DNA，将所述DNA插入到原核生物表达载体或真核生物表达载体中，然后将所述表达载体导入到原核生物或真核生物中以表达dsFv。
“包含CDR的肽”是通过包含VH或VL的CDR中的一个或多个区域而构成的。包含多个CDR的肽可以被直接相连或经由适合的肽接头相连。
本公开的包含CDR的肽可以通过以下步骤来生产：构建编码本公开单克隆抗体的VH和VL的CDR的DNA，将所述DNA插入到原核生物表达载体或真核生物表达载体中，然后将所述表达载体导入到原核生物或真核生物中以表达所述肽。也可以通过化学合成方法例如Fmoc方法或tBoc方法来生产所述包含CDR的肽。
本公开中使用的术语“抗体框架(FR)”，是指可变结构域VL或VH的一部分，其用作该可变结构域的抗原结合环(CDR)的支架。从本质上讲，其是不具有CDR的可变结构域。
术语“氨基酸差异”是指多肽与其变体之间，在多肽片段上某个或某些氨基酸位点之间的差异，其中变体可以由多肽上某个或某些位点经替换、插入或缺失氨基酸获得。
术语“表位”是指抗原上与免疫球蛋白或抗体特异性结合的位点。表位可以由相邻的氨基酸、或通过蛋白质的三级折叠而并列的不相邻的氨基酸形成。由相邻的氨基酸形成的表位通常在暴露于变性溶剂后保持，而通过三级折叠形成的表位通常在变性溶剂处理后丧失。表位通常以独特的空间构象包括至少3-15个氨基酸。确定表位的方法在本领域中是熟知的，包括免疫印迹和免疫沉淀检测分析等。确定表位的空间构象的方法包括本领域中的技术和本公开所述的技术，例如X射线 晶体分析法和二维核磁共振等。
本发明所用的术语“特异性结合”是指抗体与预定的抗原上的表位结合。通常，当使用重组人OX40作为分析物并使用抗体作为配体，在仪器中通过表面等离子体共振(SPR)技术测定时，抗体以大约低于10 -7M或甚至更小的平衡解离常数(K D)与预定的抗原结合，并且其与预定抗原结合的亲和力是其与预定抗原(或紧密相关的抗原)之外的非特异性抗原(如BSA等)结合的亲和力的至少两倍。术语“识别抗原的抗体”在本公开中可以与术语“特异性结合的抗体”互换使用。
术语“KD”是指特定抗体-抗原相互作用的解离平衡常数。通常，本公开的抗体以小于大约10 -7M，例如小于大约10 -8M、10 -9M或10 -10M或更小的解离平衡常数(KD)结合OX40，例如，如使用表面等离子体共振(SPR)技术在BIACORE仪中测定的。
当术语“竞争”用于竞争相同表位的抗原结合蛋白(例如中和抗原结合蛋白或中和抗体)的情况中时，意指在抗原结合蛋白之间竞争，其通过以下测定法来测定：待检测的抗原结合蛋白(例如抗体或其功能片段)防止或抑制(例如降低)参考抗原结合蛋白(例如配体或参考抗体)与共同抗原(例如OX40抗原或其片段)的特异性结合。众多类型的竞争性结合测定可用于确定一种抗原结合蛋白是否与另一种竞争，这些测定例如：固相直接或间接放射免疫测定(RIA)、固相直接或间接酶免疫测定(EIA)、夹心竞争测定(参见例如Stahli等，1983，Methodsin Enzymology 9：242-253)；固相直接生物素-亲和素EIA(参见例如Kirkland等，1986，J.Immunol.137：3614-3619)、固相直接标记测定、固相直接标记夹心测定(参见例如Harlow和Lane，1988，Antibodies，A Laboratory Manual(抗体，实验室手册)，Cold Spring Harbor Press)；用I-125标记物的固相直接标记RIA(参见例如Morel等，1988，Molec.Immunol.25：7-15)；和直接标记的RIA(Moldenhauer等，1990，Scand.J.Immunol.32：77-82)。通常所述测定法涉及使用纯化抗原，其能与带有未标记的检测抗原结合蛋白及标记的参考抗原结合蛋白结合(所述抗原在固态表面或细胞表面上)。在待测抗原结合蛋白存在下，测量结合于固态表面或细胞表面的标记的量，来测量竞争性抑制。通常，待测抗原结合蛋白是过量存在的。由竞争性测定(竞争性抗原结合蛋白)鉴定的抗原结合蛋白包括：与参考抗原结合蛋白相同的表位发生结合的抗原结合蛋白；以及，与参考抗原结合蛋白结合的表位所充分接近的表位发生结合的抗原结合蛋白，所述两个表位在空间上互相妨碍结合的发生。在本公开实施例中提供关于用于测定竞争性结合的方法的其它详细资料。通常当竞争性抗原结合蛋白过量存在时，其将抑制(例如降低)至少40-45％、45-50％、50-55％、55-60％、60-65％、65-70％、70-75％或更多参考抗原结合蛋白与共同抗原的特异性结合。在某些情况下，结合被抑制至少80-85％、85-90％、90-95％、95-97％或更多。
术语“交叉反应”是指本发明的抗体与来自不同物种的OX40结合的能力。例如， 结合人OX40的本发明的抗体也可以结合另一物种的OX40。交叉反应性是通过在结合测定(例如SPR和ELISA)中检测与纯化抗原的特异性反应性，或与生理表达OX40的细胞的结合或功能性相互作用来测量。确定交叉反应性的方法包括如本公开所述的标准结合测定，例如表面等离子体共振(SPR)分析，或流式细胞术。
术语“抑制”或“阻断”可互换使用，并涵盖部分和完全抑制/阻断这两者。
术语“抑制生长”(例如涉及细胞)旨在包括细胞生长任何可测量的降低。
术语“诱导免疫应答”和“增强免疫应答”可互换使用，并指免疫应答对特定抗原的刺激(即，被动或适应性的)。针对诱导CDC或ADCC的术语“诱导”是指刺激特定的直接细胞杀伤机制。
“抗体依赖性细胞介导的细胞毒性”或“ADCC”指其中结合到某些细胞毒性细胞(例如NK细胞，嗜中性粒细胞和巨噬细胞)上存在的Fc受体(FcR)上的分泌型免疫球蛋白使得这些细胞毒性效应细胞能够特异性结合携带抗原的靶细胞，随后用细胞毒素杀死靶细胞的细胞毒性形式。介导ADCC的主要细胞(NK细胞)只表达FcγRIII，而单核细胞表达FcγRI、FcγRII和FcγRIII。Ravetch和Kinet,Annu.Rev.Immunol.9:457-92(1991)第464页表3总结了造血细胞上的FcR表达。为了评估目的分子的ADCC活性，可进行体外ADCC测定法，诸如美国专利No.5,500,362或5,821,337或美国专利No.6,737,056(Presta)中所记载的。可用于此类测定法的效应细胞包括PBMC和NK细胞。或者，可在体内评估目的分子的ADCC活性，例如在动物模型中，诸如Clynes等人PNAS(USA)95:652-656(1998)中所披露的。此外，可通过对IgG上Fc段的修饰，降低或消除抗体的ADCC效应功能。所述的修饰指在抗体的重链恒定区进行突变，如选自IgG1的N297A、L234A、L235A；IgG2/4嵌合体，IgG4的F235E、或L234A/E235A突变。
术语“补体依赖性细胞毒性”或“CDC”指存在补体时对靶细胞的溶解。经典补体途径的激活是由补体系统第一组分(C1q)结合抗体(适宜亚类的)起始的，该抗体已结合至其关联抗原。为了评估补体激活，可进行CDC测定法，例如Gazzano-Santoro等人,J.Immunol.Methods 202:163(1996)中所记载的。具有更改的Fc区氨基酸序列(具有变异Fc区的多肽)及提高或降低的C1q结合能力的多肽变体记载于例如美国专利No.6,194,551B1和WO 1999/51642。还可参见例如Idusogie等人,J.Immunol.164:4178-4184(2000)。
本公开中使用的术语“核酸分子”是指DNA分子和RNA分子。核酸分子可以是单链或双链的，但具体地，是双链DNA。当将核酸与另一个核酸序列置于功能关系中时，核酸是“有效连接的”。例如，如果启动子或增强子影响编码序列的转录，那么启动子或增强子有效地连接至所述编码序列。
术语“载体”是指能够运输与其连接的另一个核酸的核酸分子。在一个实施方案中，载体是“质粒”，其是指可将另外的DNA区段连接至其中的环状双链DNA环。在另一个实施方案中，载体是病毒载体，其中可将另外的DNA区段连接至病毒基 因组中。本公开中的载体能够在已引入它们的宿主细胞中自主复制(例如，具有细菌的复制起点的细菌载体和附加型哺乳动物载体)或可在引入宿主细胞后整合入宿主细胞的基因组，从而随宿主基因组一起复制(例如，非附加型哺乳动物载体)。
现有技术中熟知生产和纯化抗体和抗原结合片段的方法，如冷泉港的抗体实验技术指南，5-8章和15章。例如，鼠可以用人OX40或其片段免疫，所得到的抗体能被复性、纯化，并且可以用常规的方法进行氨基酸测序。抗原结合片段同样可以用常规方法制备。发明所述的抗体或抗原结合片段用基因工程方法在非人源的CDR区加上一个或多个人源FR区。人FR种系序列可以通过比对IMGT人类抗体可变区种系基因数据库和MOE软件，从ImMunoGeneTics(IMGT)的网站http://imgt.cines.fr得到，或者从免疫球蛋白杂志，2001ISBN012441351上获得。
本公开的抗体或抗原结合片段可用常规方法制备和纯化。比如，将编码重链和轻链的cDNA序列，可以克隆并重组至GS表达载体。重组的免疫球蛋白表达载体可以稳定地转染CHO细胞。作为一种更推荐的现有技术，哺乳动物类表达系统会导致抗体的糖基化，特别是在FC区的高度保守N端。通过表达与人源抗原特异性结合的抗体得到稳定的克隆。阳性的克隆在生物反应器的无血清培养基中扩大培养以生产抗体。分泌了抗体的培养液可以用常规技术纯化、收集。抗体可用常规方法进行过滤浓缩。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛，离子交换。得到的产物需立即冷冻，如-70℃，或者冻干。
“单克隆抗体”(mAb)，指由单一的克隆细胞株得到的抗体，所述的细胞株不限于真核的，原核的或噬菌体的克隆细胞株。单克隆抗体或抗原结合片段可以用如杂交瘤技术、重组技术、噬菌体展示技术，合成技术(如CDR-grafting)，或其它现有技术进行重组得到。
术语“宿主细胞”是指已向其中引入了表达载体的细胞。宿主细胞可包括微生物(例如细菌)、植物或动物细胞。易于转化的细菌包括肠杆菌科(enterobacteriaceae)的成员，例如大肠杆菌(Escherichia coli)或沙门氏菌(Salmonella)的菌株；芽孢杆菌科(Bacillaceae)例如枯草芽孢杆菌(Bacillus subtilis)；肺炎球菌(Pneumococcus)；链球菌(Streptococcus)和流感嗜血菌(Haemophilus influenzae)。适当的微生物包括酿酒酵母(Saccharomyces cerevisiae)和毕赤酵母(Pichia pastoris)。适当的动物宿主细胞系包括CHO(中国仓鼠卵巢细胞系)、NS0细胞、293细胞。
“保守修饰”或“保守置换或取代”是指具有类似特征(例如电荷、侧链大小、疏水性/亲水性、主链构象和刚性等)的其它氨基酸置换蛋白中的氨基酸，使得可频繁进行改变而不改变蛋白的生物学活性。本领域技术人员知晓，一般而言，多肽的非必需区域中的单个氨基酸置换基本上不改变生物学活性(参见例如Watson等(1987)Molecular Biology of the Gene,The Benjamin/Cummings Pub.Co.,第224页， (第4版))。另外，结构或功能类似的氨基酸的置换不大可能破环生物学活性。
“有效量”包含足以改善或预防医学疾病的症状或病症的量。有效量还意指足以允许或促进诊断的量。用于特定患者或兽医学受试者的有效量可依据以下因素而变化：例如，待治疗的病症、患者的总体健康情况、给药的方法途径和剂量以及副作用严重性。有效量可以是避免显著副作用或毒性作用的最大剂量或给药方案。
“外源性”指根据情况在生物、细胞或人体外产生的物质。
“内源性”指根据情况在生物、细胞或人体内产生的物质。
“突变序列”是指对本公开的核苷酸序列和氨基酸序列进行适当的替换、插入或缺失等突变修饰情况下，得到的与本公开的核苷酸序列和氨基酸序列具有不同百分比序列同一性程度的核苷酸序列和氨基酸序列。所述的序列同一性可以至少为85％、90％或95％，非限制性实施例包括85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、100％。两个序列之间的序列比较和同一性百分比测定可以通过National Center For Biotechnology Institute网站上可得的BLASTN/BLASTP算法的默认设置来进行。
本公开中“同源性”、“同一性”是指两个多核苷酸序列之间或两个多肽之间的序列相似性。当两个比较序列中的位置均被相同碱基或氨基酸单体亚基占据时，例如如果两个DNA分子的每一个位置都被腺嘌呤占据时，那么所述分子在该位置是同源的。两个序列之间的同源性百分率是两个序列共有的匹配或同源位置数除以比较的全部位置数×100的函数。例如，在序列最佳比对时，如果两个序列中的10个位置有6个匹配或同源，那么两个序列为60％同源；如果两个序列中的100个位置有95个匹配或同源，那么两个序列为95％同源。一般而言，当比对两个序列而得到最大的同源性百分率时进行比较。
本公开使用的表述“细胞”、“细胞系”和“细胞培养物”可互换使用，并且所有这类名称都包括后代。因此，“转化体”和“转化细胞”包括原代受试细胞和由其衍生的培养物，而不考虑转移数目。还应当理解的是，由于故意或非有意的突变，所有后代在DNA含量方面不可能精确相同。包括具有与最初转化细胞中筛选的相同的功能或生物学活性的突变后代。在意指不同名称的情况下，其由上下文清楚可见。
本公开使用的“聚合酶链式反应”或“PCR”是指其中微量的特定部分的核酸、RNA和/或DNA如在例如美国专利号4,683,195中所述扩增的程序或技术。一般来说，需要获得来自目标区域末端或之外的序列信息，使得可以设计寡核苷酸引物；这些引物在序列方面与待扩增模板的对应链相同或相似。2个引物的5’末端核苷酸可以与待扩增材料的末端一致。PCR可用于扩增特定的RNA序列、来自总基因组的特定DNA序列和由总细胞RNA转录的cDNA、噬菌体或质粒序列等。一般参见Mullis等(1987)Cold Spring Harbor Symp.Ouant.Biol.51:263；Erlich编辑，(1989)PCR TECHNOLOGY(Stockton Press,N.Y.)。本公开使用的PCR被视为用于扩增核酸测试样品的核酸聚合酶反应法的一个实例，但不是唯一的实例，所述方法 包括使用作为引物的已知核酸和核酸聚合酶，以扩增或产生核酸的特定部分。
“任选”或“任选地”意味着随后所描述地事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生的场合。例如，“任选包含1-3个抗体重链可变区”意味着特定序列的抗体重链可变区可以但不必须存在。
“药物组合物”表示含有一种或多种本公开的抗体或其抗原结合片段与其他化学组分的混合物，所述其他组分例如生理学/可药用的载体或赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
术语“癌症”指向或描述哺乳动物中特征为细胞生长不受调节的生理疾患。癌症的例子包括但不限于癌，淋巴瘤、母细胞瘤、肉瘤和白血病或淋巴样恶性肿瘤。此类癌症的更具体例子包括但不限于鳞状细胞癌(例如上皮鳞状细胞癌)、肺癌(包括小细胞肺癌、非小细胞肺癌、腺癌、和肺的鳞癌)、腹膜癌、肝细胞癌、胃癌(包括胃肠癌和胃肠基质癌)、胰腺癌、成胶质细胞瘤、宫颈癌、卵巢癌、肝癌、膀胱癌、尿道癌、肝瘤、乳腺癌、结肠癌、直肠癌、结肠直肠癌、子宫内膜癌或子宫癌、唾液腺癌、肾癌、前列腺癌、外阴癌、甲状腺癌、肛门癌、阴茎癌、黑素瘤、浅表扩散性黑素瘤、恶性雀斑样痣黑素瘤、肢端黑素瘤、结节性黑素瘤、多发性骨髓瘤和B细胞淋巴瘤、慢性淋巴细胞性白血病(CLL)、急性成淋巴细胞性白血病(ALL)、毛细胞性白血病、慢性成髓细胞性白血病、和移植后淋巴增殖性病症(PTLD)、以及与瘢痣病(phakomatoses)、水肿(诸如与脑瘤有关的)和梅格斯氏(Meigs)综合征有关的异常血管增殖、脑瘤和脑癌、以及头颈癌、及相关转移。
在某些实施方案中，适合于通过本公开的OX40抗体或片段来治疗的癌症包括乳腺癌、结肠直肠癌、直肠癌、非小细胞肺癌、成胶质细胞瘤、非何杰金氏淋巴瘤(NHL)、肾细胞癌、前列腺癌、肝癌、胰腺癌、软组织肉瘤、卡波西(Kaposi)氏肉瘤、类癌癌(carcinoid carcinoma)、头颈癌、卵巢癌、间皮瘤、和多发性骨髓瘤。在一些实施方案中，癌症选自：非小细胞肺癌、成胶质细胞瘤、成神经细胞瘤、黑素瘤、乳腺癌(例如三重阴性乳腺癌)、胃癌、结肠直肠癌(CRC)、和肝细胞癌。还有，在一些实施方案中，癌症选自：非小细胞肺癌、结肠直肠癌、成胶质细胞瘤和乳腺癌(例如三重阴性乳腺癌)，包括那些癌症的转移性形式。
术语“细胞增殖性病症”和“增殖性病症”指与一定程度的异常细胞增殖有关的病症。在一个实施方案中，细胞增殖性病症指癌症。
术语“肿瘤”指所有赘生性(neoplastic)细胞生长和增殖，无论是恶性的还是良性的，及所有癌前(pre-cancerous)和癌性细胞和组织。
术语“癌症”，“癌性”，“细胞增殖性病症”，“增殖性病症”和“肿瘤”在本公开中提到时并不互相排斥。
“给予”、“施用”和“处理”当应用于动物、人、受试者、细胞、组织、器官或生物流体时，是指外源性药物、治疗剂、诊断剂或组合物与动物、人、受试者、细 胞、组织、器官或生物流体的接触。“给予”、“施用”和“处理”可以指例如治疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触，以及试剂与流体的接触，其中所述流体与细胞接触。“给予”、“施用”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞体外和离体处理细胞。“处理”当应用于人、兽医学或研究受试者时，是指治疗处理、预防或预防性措施，研究和诊断应用。
“治疗”意指给予患者内用或外用治疗剂，例如包含本公开的任一种抗体或其抗原结合片段的组合物或编码抗体或其抗原结合片段的核酸分子，所述患者具有一种或多种疾病或症状，而已知所述治疗剂对这些症状具有治疗作用。通常，在受治疗患者或群体中以有效缓解一种或多种疾病或症状的量给予治疗剂，以诱导这类症状退化或抑制这类症状发展到任何临床可测量的程度。有效缓解任何具体疾病或症状的治疗剂的量(也称作“治疗有效量”)可根据多种因素变化，例如患者的疾病状态、年龄和体重，以及药物在患者产生需要疗效的能力。通过医生或其它专业卫生保健人士通常用于评价该症状的严重性或进展状况的任何临床检测方法，可评价疾病症状是否已被减轻。尽管本公开的实施方案(例如治疗方法或制品)在缓解每个目标疾病症状方面可能无效，但是根据本领域已知的任何统计学检验方法如Student t检验、卡方检验、依据Mann和Whitney的U检验、Kruskal-Wallis检验(H检验)、Jonckheere-Terpstra检验和Wilcoxon检验确定，其在统计学显著数目的受试者中应当减轻目标疾病症状。
术语“预防癌症”是指在哺乳动物中延迟、抑制或防止癌症发作，所述哺乳动物中癌发生或肿瘤发生的起始尚未得到证实，但是通过例如遗传筛查或其它方法确定，已鉴定其具有癌症易感性。该术语还包括治疗具有癌变前病症的哺乳动物以终止所述癌变前病症向恶性肿瘤的进展或导致其消退。
具体实施方式
以下结合实施例用于进一步描述本公开，但这些实施例并非限制着本公开的范围。本公开实施例中未注明具体条件的实验方法，通常按照常规条件，如冷泉港的抗体技术实验手册，分子克隆手册；或按照原料或商品制造厂商所建议的条件。未注明具体来源的试剂，为市场购买的常规试剂。
实施例1：抗体的制备
抗人OX40单抗库通过免疫小鼠产生。实验用BalB/C和A/J品系小鼠(扬州大学比较医学中心，动物生产许可证号；SCXK(苏)2017-007)，雌性，10周龄。
免疫抗原为带Fc标签的人OX40重组蛋白(OX40-Fc：OX40 Leu 29-Ala 216(Accession#NP_003318)与Fc融合)，购自Acro Biosystems公司，货号#OX40-H5255，使用HEK293表达后按常规方法纯化。
将OX40-Fc用弗氏佐剂乳化：首次免疫用弗氏完全佐剂(sigma-aldrich，F5881-10ML)，其余加强免疫用弗氏不完全佐剂(sigma-aldrich，F5506-10ML)。抗原与佐剂比例为1:1，每次免疫注射25μg蛋白/200μl/只小鼠。详细见如下：
Figure PCTCN2019107787-appb-000001
用实施例2中的ELISA方法检测小鼠血清，确定小鼠血清的抗体滴度和阻断OX40/OX40L结合的中和活性。选择血清效价、亲和力和配体结合阻断能力强的小鼠进行一次终免疫后处死小鼠。取脾细胞和SP2/0骨髓瘤细胞(
Figure PCTCN2019107787-appb-000002
CRL-1581 TM)融合后铺板获得杂交瘤，通过实施例2的间接ELISA，捕获ELISA以及基于细胞的功能性筛选选择目标杂交瘤，并通过有限稀释法建株单克隆抗体。
把已建株的19株OX40小鼠单抗进行无血清表达生产，并用protein A亲和层析技术得到纯化的小鼠单抗。通过间接ELISA，捕获ELISA及细胞功能学活性筛选其中的分泌激活型抗OX40抗体的杂交瘤细胞。功能性筛选的简要步骤如下：培养GS-H2/OX40稳定细胞系(购自genscript，cat#M00608)。制备稀释的待测抗体及OX40L(Sino Biological，13127-H04H)溶液添加到处于对数生长期的GS-H2/OX40细胞中，培养后收集细胞上清，测定上清中的IL-8含量(使用human IL-8kit，cisbio，cat#62IL8PEB)。实验中用抗体GPX4(参照专利WO2015153513专利中1A7.gr.1制备)做阳性参照，hIgG(实验室制备)做阴性参照。
根据细胞功能性活性，选择活性高的10株进行基因克隆和测序。通过常规RNA抽提技术即可获得细胞总RNA，然后通过逆转录聚合酶链式反应(RT-PCR)获得单克隆抗体可变区的PCR产物。PCR产物经琼脂糖凝胶分离回收，然后克隆到基因载体，转化大肠杆菌。随机挑选数个转化菌落，PCR扩增单克隆抗体可变区，用于基因测序。获得的示例性的鼠源单克隆抗体的相应序列如下所示。
鼠单克隆抗体m4B5的重链和轻链可变区序列如下：
m4B5重链可变区：
Figure PCTCN2019107787-appb-000003
m4B5轻链可变区：
Figure PCTCN2019107787-appb-000004
鼠单克隆抗体m4B5的CDR区序列如表1：
表1.鼠单克隆抗体m4B5的CDR区序列
名称 序列 编号
HCDR1 SYGLH SEQ ID NO：3
HCDR2 VIWSGGSTDYNAAFIS SEQ ID NO：4
HCDR3 EEYDV SEQ ID NO：5
LCDR1 RASQDISNYLN SEQ ID NO：6
LCDR2 YTSRLHS SEQ ID NO：7
LCDR3 QQGNTLPWT SEQ ID NO：8
鼠单克隆抗体m2G3的重链和轻链可变区序列如下：
m2G3重链可变区：
Figure PCTCN2019107787-appb-000005
m2G3轻链可变区：
Figure PCTCN2019107787-appb-000006
鼠单克隆抗体m2G3的CDR区序列如表2：
表2.鼠单克隆抗体m2G3的CDR区序列
名称 序列 编号
HCDR1 RYSVH SEQ ID NO：11
HCDR2 MIWDGGNTDYNSALKS SEQ ID NO：12
HCDR3 NPLYFSYAMDY SEQ ID NO：13
LCDR1 RASQDISNYLN SEQ ID NO：14
LCDR2 YTSRLQS SEQ ID NO：15
LCDR3 QQVNTFPFT SEQ ID NO：16
实施例2：抗体的ELISA鉴定与筛选方法
间接ELISA法：
将20×包被缓冲液用去离子水稀释至1×，用1×包被液(碳酸盐缓冲液)配制抗原人OX40-His(Acro biosytems,OXL-H52Q8)，使其终浓度为2μg/mL，每孔加液100μL，4℃过夜或37℃孵育2h。PBST洗板1次，每孔加入200μL封闭液(含5％脱脂牛奶的PBST)，37℃孵育2h，PBST洗板4次。每孔加入稀释好的一抗100μL(待测抗体浓度从10000ng/ml起5倍倍稀，7个梯度，即10000ng/ml，2000ng/ml，400ng/ml，80ng/ml，16ng/ml，3.2ng/ml，0.64ng/ml，空白孔为纯稀释液即2.5％脱脂牛奶的PBST)，37℃孵育40min。PBST洗板4次，用PBST缓冲液稀释酶标二抗(HRP标记山羊抗小鼠IgG，购自Jackson Immunoresearch,Cat#115036071或HRP标记山羊抗人IgG，购自Jackson Immunoresearch,Cat#109036098)，每孔加100μL，37℃孵育40min。PBST洗板4次，每孔加100μL TMB显色液，室温避光孵育3-15分钟，每孔加入50μl终止 液(1M硫酸)。设置酶标仪参数，在450-630nm处读取OD值，保存实验数据。
捕获ELISA：
将20×PBS缓冲液用去离子水稀释至1×，用1×PBS配制GAM二抗(Jackson Immunoresearch,115-006-071)，使其终浓度为2μg/mL，每孔加入100μL，4℃过夜或37℃孵育2h；PBST洗板1次，每孔加入200μL封闭液(含5％脱脂牛奶的PBST)，37℃孵育2h，PBST洗板4次。每孔加入稀释好的一抗100μL(待测抗体浓度从10000ng/ml起5倍倍稀，7个梯度，即10000ng/ml，2000ng/ml，400ng/ml，80ng/ml，16ng/ml，3.2ng/ml，0.64ng/ml，空白孔为纯稀释液即2.5％脱脂牛奶的PBST)，37℃孵育40min。PBST洗板4次，用2.5％脱脂牛奶的PBST稀释人OX40-FC-生物素(Acro biosystem，OX0-H5255，标记生物素)，每孔加100μl，37℃孵育40min，PBST洗板4次。每孔加100μL TMB显色液，室温避光孵育3-15分钟，每孔加入50μl终止液(1M硫酸)，设置酶标仪参数，在450-630nm处读取OD值，保存实验数据。
配体结合阻断ELISA：
将20×包被缓冲液用去离子水稀释至1×，用1×包被液(碳酸盐缓冲液)配置抗原OX40L-His(Acro biosytems,OXL-H52Q8)，使其终浓度为2μg/mL，每孔加入100μL，4℃过夜或37℃孵育2h；PBST洗板1次；每孔加入200μL封闭液(含5％脱脂牛奶的PBST)，37℃孵育2h；PBST洗板4次；用预先配制好的200ng/ml的人OX40-Fc溶液(配制在2.5％的脱脂牛奶中)梯度稀释小鼠血清/抗体，而后室温预孵育40min后，加到已封闭好的OX40L板子上，100μL/孔，孵育40min；PBST洗板4次；用PBST缓冲液稀释HRP标记的羊抗人二抗(GAH-HRP，Jackson Immunoresearch,109-035-006)，每孔加100μl，37℃孵育40min；PBST洗板4次；每孔加100μL TMB显色液，室温避光孵育3-15分钟；每孔加入50μl终止液(1M硫酸)，设置酶标仪参数，450-630nm处读取OD值，保存实验数据。
实施例3：抗OX40重组嵌合抗体的构建表达
将鼠源抗体m2G3和m4B5的重链可变区(VH)加上人免疫球蛋白重链恒定区，轻链可变区(VL)加上人免疫球蛋白Kappa轻链恒定区，分别克隆到真核表达载体上，通过转染细胞生产出鼠-人嵌合抗体。
重链载体设计如下：信号肽+重链可变区序列+人的IgG1恒定区序列。
轻链载体设计如下：信号肽+轻链可变区序列+人的Kappa恒定区序列。
分别将上述序列插入pCEP4载体(Thermofisher，V04450)。得到载体质粒后，提取质粒，将质粒送测序验证。将验证合格的质粒用PEI转染至人293F细胞中，连续培养，将293F细胞用无血清培养液(上海奥浦迈生物，OPM-293CD03)培养至对数生长期用于细胞转染。将21.4μg嵌合抗体轻链质粒和23.6μg嵌合抗体重链质粒溶解在10ml
Figure PCTCN2019107787-appb-000007
I Reduced Serum Medium(GIBCO，31985-070)中混匀，然后加入200μg PEI，混匀，室温孵育15min，加入50mL细胞中。细胞培养条件： 5％CO 2，37℃，125rpm/min。培养期间，第1天和第3天加补料，直到细胞活率低于70％，收取细胞上清，离心过滤。将离心过滤后的细胞培养液上样到抗体纯化亲和柱，经磷酸缓冲液洗柱、甘氨酸盐酸缓冲液(pH2.7 0.1M Gly-HCl)洗脱、1M Tris盐酸pH 9.0中和、以及磷酸缓冲液透析，最终获得纯化的嵌合抗体Ch2G3和Ch4B5。
实施例4：体外结合亲和力和动力学实验
对鼠源抗体和嵌合抗体进行人OX40的亲和力进行检测(方法步骤同实施例2)，结果见图1A和图1B。其中，m2G3-NC(-NC表示negative control)、Ch2G3-NC、m4B5-NC、Ch4B5-NC为阴性对照，其分别与m2G3、Ch2G3、m4B5、Ch4B5使用的相同的恒定区，但可变区不识别OX40。
结果显示，嵌合抗体Ch2G3和Ch4B5与人OX40具有很高的亲和力。
实施例5：抗OX40抗体的体外细胞报告基因实验
用抗CD3抗体(Chempartner,A05-001)包被孔板，4度放置过夜，PBS洗3次；收获Jurkat-NF-Kb luc-hOX40细胞(ATCC,TIB-152稳定细胞系由上海睿智化学构建)和Raji细胞(ATCC,CCL-86)，重悬然后混合两种细胞。添加50μl/孔稀释的待测抗体和50μl/孔已混合的两种细胞到细胞板中，细胞板放在37度5％CO 2孵育箱中孵育5小时。添加100μl one-Glo TM luciferase reagent(Promega,Cat#E6120)到每一孔中，室温孵育3min以上。上机检测发光信号，记录RLU读值，结果见表3。
表3.抗OX40抗体的体外激活报告基因结果
抗体 EC 50(nM)
Ch2G3 0.6371
Ch4B5 0.5589
结果显示，嵌合抗体Ch2G3和Ch4B5能有效的激活报告基因。
实施例6：小鼠抗体人源化实验
为了降低可能的免疫原性，将鼠源抗体进行人源化改造。将嵌合抗体的重链可变区(VH)和轻链可变区(VL)分别在FR(framework region)区域进行定点氨基酸突变。根据不同的氨基酸突变组合，设计不同的人源化抗体重链和轻链，将编码不同轻/重链组合的质粒转染细胞可以生产出人源化抗体。简述如下：
首先设计表达载体：
重链载体设计如下：信号肽+突变的重链可变区序列+人的IgG1恒定区序列。
轻链载体设计如下：信号肽+突变的轻链可变区序列+人的Kappa恒定区序列。
分别将上述序列插入pCEP4载体(Thermofisher，V04450)。请第三方基因合成公司按照上述设计合成表达载体，得到载体质粒后，提取质粒，将质粒送测序验证。将验证合格的质粒用PEI转染至人293F细胞中，连续培养，将293F细胞用无 血清培养液(上海奥浦迈生物，OPM-293CD03)培养至对数生长期用于细胞转染。将21.4μg编码人源化抗体轻链的质粒和23.6μl编码人源化抗体重链的质粒溶解在10ml
Figure PCTCN2019107787-appb-000008
I Reduced Serum Medium(GIBCO,31985-070)中混匀，然后加入200μg PEI，混匀，室温孵育15min，加入50mL细胞中。
细胞培养条件：5％CO 2，37℃，125rpm/min。培养期间，第1天和第3天加补料，直到细胞活率低于70％，收取细胞上清，离心过滤。将离心过滤后的细胞培养液上样到抗体纯化亲和柱，经磷酸缓冲液洗柱、甘氨酸盐酸缓冲液(pH2.7 0.1M Gly-HCl)洗脱、1M Tris盐酸pH9.0中和、以及磷酸缓冲液透析，最终获得纯化的人源化抗体。
人源化可变区序列如下：
>hu2G3 VH1
Figure PCTCN2019107787-appb-000009
>hu2G3 VH2
Figure PCTCN2019107787-appb-000010
>hu2G3 VL1
Figure PCTCN2019107787-appb-000011
>hu2G3 VL2
Figure PCTCN2019107787-appb-000012
注：顺序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，序列中斜体为FR序列，下划线为CDR序列。
将设计的人源化抗体的可变区按下表组合成不同的抗体：
表4.m2G3人源化抗体及其重轻链可变区
抗体 VH VL
2G3-hu1 hu2G3 VH1 hu2G3 VL1
2G3-hu2 hu2G3 VH2 hu2G3 VL1
2G3-hu3 hu2G3 VH1 hu2G3 VL2
2G3-hu4 hu2G3 VH2 hu2G3 VL2
注：2G3-hu1表示，人源化的抗体2G3-hu1的重链可变区为hu2G3 VH1，轻链可变区为hu2G3 VL1，其他类推。
m4B5的人源化可变区序列如下：
hu4B5 VL0：
Figure PCTCN2019107787-appb-000013
hu4B5 VL1：
Figure PCTCN2019107787-appb-000014
hu4B5 VL2：
Figure PCTCN2019107787-appb-000015
hu4B5 VL3：
Figure PCTCN2019107787-appb-000016
hu4B5 VL4：
Figure PCTCN2019107787-appb-000017
hu4B5 VH0：
Figure PCTCN2019107787-appb-000018
hu4B5 VH1：
Figure PCTCN2019107787-appb-000019
hu4B5 VH2：
Figure PCTCN2019107787-appb-000020
hu4B5 VH3：
Figure PCTCN2019107787-appb-000021
hu4B5 VH4：
Figure PCTCN2019107787-appb-000022
将设计的人源化抗体的可变区按下表组合成不同的抗体：
表5.m4B5人源化抗体及其重轻链可变区的对应关系
Figure PCTCN2019107787-appb-000023
注：Hu4B5 V1表示，人源化的抗体hu4B5 V1的重链可变区为hu4B5 VH0，轻链可变区为hu4B5 VL0，其他类推。
为了获得更好的m2G3的人源化改造抗体，对hu2G3 VH1的氨基酸序列进行突变，突变后的重链可变区序列如下：
hu2G3 VH1.1
Figure PCTCN2019107787-appb-000024
>hu2G3 VH1.2
Figure PCTCN2019107787-appb-000025
突变后获得的HCDR2序列如下：
表6.突变后获得的HCDR2序列
名称 序列 编号
HCDR2 V1 MIWDGGNTDYNAALKS SEQ ID NO：33
HCDR2 V2 MIWDGGNTDYAAPVKG SEQ ID NO：34
将重链可变区hu2G3 VH1.1、hu2G3 VH1.2与轻链可变区hu2G3 VL组合形成新的优化的人源化抗体，见表7。
表7.突变后，获得的新的人源化hu2G3抗体的轻/重链可变区
抗体 VH VL
2G3 hu2G3 VH1.1 hu2G3 VL1
2G3-hu5 hu2G3 VH1.1 hu2G3 VL2
2G3-hu6 hu2G3 VH1.2 hu2G3 VL1
2G3-hu7 hu2G3 VH1.2 hu2G3 VL2
将上述人源化抗体进行亲和力测评(参照实施例2中的捕获ELISA)，实验结果表明人源化后的分子可同OX40结合。
示例性m2G3及其人源化抗体亲和力测评(捕获ELISA)结果如表8所示：
表8.抗体亲和力
Figure PCTCN2019107787-appb-000026
示例性的m4B5及其改造抗体的捕获ELISA检测结果如表9所示：
表9.抗体亲和力
Figure PCTCN2019107787-appb-000027
将上述序列中的轻链可变区与轻链恒定区序列组合形成最终的轻链序列，将上述序列中的重链可变区与重链恒定区组合形成最终的重链序列。具体轻、重链恒定区并非作为本公开抗体恒定区的限制，也可选用本领域其它已知的轻、重链恒定区及其突变体来增加抗体的性能。
示例性的恒定区如下所示：
IgG1重链恒定区：
Figure PCTCN2019107787-appb-000028
kappa轻链恒定区：
Figure PCTCN2019107787-appb-000029
示例性的2G3、hu4B5-V7(又称4B5)抗体全长氨基酸序列如下：
2G3重链：
Figure PCTCN2019107787-appb-000030
2G3轻链：
Figure PCTCN2019107787-appb-000031
4B5重链：
Figure PCTCN2019107787-appb-000032
4B5轻链：
Figure PCTCN2019107787-appb-000033
阳性参照GPX4的制备参照专利WO2015153513专利中1A7.gr.1制备，其重轻链氨基酸序列如下所示：
GPX4重链：
Figure PCTCN2019107787-appb-000034
Figure PCTCN2019107787-appb-000035
GPX4轻链：
Figure PCTCN2019107787-appb-000036
实施例7：人源化抗体体外结合亲和力和动力学试验
Biacore方法是公认的客观检测蛋白相互间亲和力和动力学的检测方法。我们通过Biacore T200(GE)分析本发明待测OX40抗体表征亲和力及结合动力学。
利用由Biacore提供的试剂盒，采用NHS标准氨基偶联法将本发明待测重组抗OX40抗体共价连接至CM5(GE)芯片上。然后将稀释于同样缓冲液中的一系列浓度梯度的人OX40-His蛋白(义翘神州#10481-H08H)于前后各个循环进样，流速30μL/min，进样后均以试剂盒内配再生试剂再生。追踪抗原-抗体结合动力学3分钟并追踪解离动力学10分钟。使用GE的BIAevaluation软件以1:1(Langmuir)结合模型分析所得数据，以此法测定的嵌合抗体ka(k on)、kd(k off)和K D值显示于下表。
表10.人源化抗体的体外结合亲和力结果
待测抗体 ka(1/Ms) kd(1/s) K D(M)
2G3 4.371E+05 2.023E-03 4.628E-09
4B5 1.314E+05 3.694E-04 2.812E-09
结果显示，2G3和4B5可有效结合OX40。
实施例8：抗体阻断OX40与OX40L结合ELISA试验
用配体OX40配体(Acrobiosystem，OXL-H52Q8)包被孔板，封闭后，加入梯度稀释待测抗体(抗体稀释在含人Bio-OX40-FC(Acrobiosystem，OX40-H5255，标记生物素)的溶液中预孵育40min后加板)，孵育40min后，洗板。再加入SA-HRP(Jackson Immunoresearch,016-030-084)，孵育40min后，加入显色液和终止液，测OD值，结果见下表。
表11.抗体阻断OX40与OX40L结合实验结果
抗体 2G3 4B5
IC 50(nM) 1.88 0.725
结果显示，2G3和4B5均可阻断OX40同OX40L的结合。
实施例9:人源化抗体活性数据
用抗CD3抗体(Chempartner,A05-001)包被孔板，4℃放置过夜，PBS洗3次；收获Jurkat-NF-Kb luc-hOX40细胞(ATCC,TIB-152(稳定细胞系由睿智化学公司构建)和Raji细胞(ATCC,CCL-86)，重悬然后混合两种细胞。添加50μl/孔稀释的待测抗体，和50μl/孔已混合的两种细胞到孔板，细胞板放在37℃5％CO 2孵育箱中孵育5小时。添加one-Glo TM荧光素酶试剂(Promega,Cat#E6120)到每一孔中，室温孵育3min以上。上机检测发光信号，记录RLU读值。结果见表12，结果表明2G3和4B5可有效激活报告基因。
表12.人源化抗体激活报告基因实验结果
抗体 2G3 4B5
EC 50(nM) 2.294 0.3411
实施例10：体外细胞功能试验
分离CD4+记忆T细胞，同待测抗体加入到抗CD3抗体(Chempartner,A05-001)包被的96孔板中，37℃共孵育72h，取上清检测IFN-γ，结果如图2和表13所示。结果显示，GPX4，4B5及2G3可显著增强IFN-γ的释放，其中2G3在10ng/mL即可达到最大刺激效果。
表13.不同抗体对应的IFNγ(pg/mL)数值
Figure PCTCN2019107787-appb-000037
注：数值计算方法是：均值+/-SEM(平均值+/-平均值标准误差)，N＝3(三次重复)。
实施例11:抗OX40抗体对肿瘤细胞生长的抑制
B-hTNFRSF4(OX40)人源化小鼠(B-hTNFRSF4(OX40)人源化小鼠，百奥赛图江苏基因生物技术有限公司)，雌性，17至20g，6至7周。
收集对数生长期MC38肿瘤细胞7(购买于南京银河生物医药有限公司)，用PBS缓冲液调整细胞浓度为5×10 6/mL，接种0.1mL细胞悬液至OX40小鼠胁腹部。观察接种后小鼠并监测肿瘤的生长，在接种后第7天时荷瘤小鼠胁腹部平均肿瘤体积达到102.5mm 3，按照肿瘤体积大小进行分组和给药观察，分组信息见表14。
表14.小鼠分组及给药剂量表
给药组 给药剂量(mg/kg) 给药途径 给药频率
对照(IgG1) 3 i.p. Q3D×6
GPX4 3 i.p. Q3D×6
2G3 0.3 i.p. Q3D×6
2G3 1 i.p. Q3D×6
2G3 3 i.p. Q3D×6
检测OX40人源化抗体对小鼠MC38结肠癌细胞移植瘤的生长抑制作用。
肿瘤体积和荷瘤鼠体重测量：使用游标卡尺每周两次测量，肿瘤体积计算公式为V＝0.5a×b 2，a，b分别代表肿瘤的长径和宽径；肿瘤生长移植瘤TGI(％)＝[1-T/C]×100。所有荷瘤鼠体重每周测量两次。
给药治疗后第20天，IgG1对照组的平均肿瘤体积达到了1732.593mm 3，受试化合物2G3低剂量给药组(0.3mg/kg)小鼠平均肿瘤体积达到了930.37mm 3，中剂量给药组(2G3，1mg/kg)和高剂量给药组(2G，3mg/kg)组内荷瘤鼠平均肿瘤体积分别为303.49mm 3和155.79mm 3，中高剂量组抑制肿瘤生长与对照组差异明显(**P<0.01)，并表现出初步的剂量依赖关系，其肿瘤生长抑制率分别达到了49％、88％和97.0％。3mg/kg GPX4组中平均肿瘤体积分别为362.47mm 3，与对照组比较差异显著，也表现有明显的抑制肿瘤生长的作用(*P<0.05)，其肿瘤生长抑制率分别达到了84％。
给药后第20天结束实验，结果见表15和图3A和图3B。所有治疗小鼠被安乐死处理，剥离荷瘤鼠皮下移植瘤块并进行称量。其中对照组平均肿瘤块重量为1.568g,受试化合物2G3在低剂量组(0.3mg/kg)、中剂量组(1mg/kg)和高剂量组(3mg/kg)，平均肿瘤重量分别为0.926g、0.251g和0.181g，其中中高剂量组与对照组比较差异显著，抑制肿瘤生长作用明显(**P<0.01)。在同一时间，给药组GPX4 3mg/kg中，平均肿瘤重量为0.372g，与对照组比较差异明显，也表现出明显的抑制MC38肿瘤细胞生长的作用(**P<0.01)。
在实验过程中，所有治疗荷瘤小鼠体重变化无明显异常，同时在药物治疗过程中未见明显异常行为和其他表现。
表15.小鼠体内抑瘤效果
Figure PCTCN2019107787-appb-000038

Claims (18)

  1. 一种抗OX40抗体或其抗原结合片段，其中：
    重链可变区，包含：
    (I)分别如SEQ ID NO：3、4、5所示的HCDR1、HCDR2、HCDR3；或与SEQ ID NO：3、4、5所示的HCDR1、HCDR2、HCDR3分别具有3、2或1个氨基酸差异的HCDR变体；
    (II)分别如SEQ ID NO：11、12、13所示的HCDR1、HCDR2、HCDR3；或与SEQ ID NO：11、12、13所示的HCDR1、HCDR2、HCDR3分别具有3、2或1个氨基酸差异的HCDR变体；
    (III)分别如SEQ ID NO：11、33、13所示的HCDR1、HCDR2、HCDR3；或与SEQ ID NO：11、33、13所示的HCDR1、HCDR2、HCDR3分别具有3、2或1个氨基酸差异的HCDR变体；或
    (IV)分别如SEQ ID NO：11、34、13所示的HCDR1、HCDR2、HCDR3；或与SEQ ID NO：11、34、13所示的HCDR1、HCDR2、HCDR3分别具有3、2或1个氨基酸差异的HCDR变体；
    和/或，轻链可变区，包含：
    (I)分别如SEQ ID NO：6、7、8所示的LCDR1、LCDR2、LCDR3；或与SEQ ID NO：6、7、8所示的LCDR1、LCDR2、LCDR3分别具有3、2或1个氨基酸差异的LCDR变体；或
    (II)分别如SEQ ID NO：14、15、16所示的LCDR1、LCDR2、LCDR3；或与SEQ ID NO：14、15、16所示的LCDR1、LCDR2、LCDR3分别具有3、2或1个氨基酸差异的LCDR变体；
    优选地，所述抗OX40抗体或其抗原结合片段包含：
    (i)分别如SEQ ID NO：3、4、5所示的HCDR1、HCDR2、HCDR3，和分别如SEQ ID NO：6、7、8所示的LCDR1、LCDR2、LCDR3；
    (ii)分别如SEQ ID NO：11、12、13所示的HCDR1、HCDR2、HCDR3，和分别如SEQ ID NO：14、15、16所示的轻链LCDR1、LCDR2、LCDR3；
    (iii)分别如SEQ ID NO：11、33、13所示的HCDR1、HCDR2、HCDR3，和分别如SEQ ID NO：14、15、16所示的轻链LCDR1、LCDR2、LCDR3；或
    (iv)分别如SEQ ID NO：11、34、13所示的HCDR1、HCDR2、HCDR3和分别如SEQ ID NO：14、15、16所示的轻链LCDR1、LCDR2、LCDR3。
  2. 根据权利要求1所述的抗OX40抗体或其抗原结合片段，其为鼠源抗体、嵌合抗体、人抗体、人源化抗体或其片段。
  3. 根据权利要求1或2所述的抗OX40抗体或其抗原结合片段，其中：
    所述抗原结合片段选自：Fab、Fab’、Fv、scFv、F(ab')2片段；
    所述抗体的重链可变区还包含重链框架区，所述重链框架区源自人IgG1、IgG2、IgG3或IgG4或其变体。
  4. 根据权利要求1或2所述的抗OX40抗体或其抗原结合片段，其中所述的抗体包含恒定区；
    优选地，重链恒定区源自人IgG1、IgG2、IgG3或IgG4或其突变序列；
    优选地，轻链恒定区源自人κ、λ链或其突变序列；
    更优选地，所述重链恒定区的氨基酸序列如SEQ ID NO：35所示或与SEQ IDNO：35具有至少90％的序列同一性，
    更优选地，所述轻链恒定区的氨基酸序列如SEQ ID NO：36所示或与SEQ IDNO：36具有至少90％的序列同一性。
  5. 根据权利要求1或2所述的抗OX40抗体或其抗原结合片段，其中：
    重链可变区，包含：
    (I)如SEQ ID NO：1所示或与SEQ ID NO：1具有至少95％序列同一性的氨基酸序列；
    (II)如SEQ ID NO：9所示或与SEQ ID NO：9具有至少95％序列同一性的氨基酸序列；
    (III)如SEQ ID NO：17、18、31、32任一所示；或与SEQ ID NO：17、18、31、32具有至少95％序列同一性的氨基酸序列；或
    (IV)如SEQ ID NO：26、27、28、29、30任一所示；或与SEQ ID NO：26、27、28、29、30具有至少95％序列同一性的氨基酸序列；
    和/或，轻链可变区，包含：
    (I)如SEQ ID NO：2所示或与SEQ ID NO：2具有至少95％序列同一性的氨基酸序列；
    (II)如SEQ ID NO：10所示或与SEQ ID NO：10具有至少95％序列同一性的氨基酸序列；
    (III)如SEQ ID NO：19、20任一所示或与SEQ ID NO：19、20具有至少95％序列同一性的氨基酸序列；
    (IV)如SEQ ID NO：21、22、23、24、25任一所示或与SEQ ID NO：21、22、23、24、25具有至少95％序列同一性的氨基酸序列；
    优选地，所述抗OX40抗体或其抗原结合片段包含：
    (i)如SEQ ID NO：1所示的重链可变区，和如SEQ ID NO：2所示的轻链可变区；
    (ii)如SEQ ID NO：9所示的重链可变区，和如SEQ ID NO：10所示的轻链可变区；
    (iii)如SEQ ID NO：17或18所示的重链可变区，和如SEQ ID NO：19所示的轻链可变区；
    (iv)如SEQ ID NO：17或18所示的重链可变区，和如SEQ ID NO：20所示的轻链可变区；
    (v)如SEQ ID NO：26、27、28、29、30任一所示的重链可变区，和如SEQID NO：21、22、23、24、25任一所示的轻链可变区。
  6. 根据权利要求1或2所述的抗OX40抗体或其抗原结合片段，包含：
    (i)
    如SEQ ID NO：37所示或与SEQ ID NO：37具有至少85％、至少90％、至少95％、至少98％、至少99％序列同一性的重链，和/或，
    如SEQ ID NO：38所示或与SEQ ID NO：38具有至少85％、至少90％、至少95％、至少98％、至少99％序列同一性的轻链；或
    (ii)
    如SEQ ID NO：39所示或与SEQ ID NO：39具有至少85％、至少90％、至少95％、至少98％、至少99％序列同一性的重链，和/或，
    如SEQ ID NO：40所示或与SEQ ID NO：40具有至少85％、至少90％、至少95％、至少98％、至少99％序列同一性的轻链。
  7. 一种抗OX40抗体或其抗原结合片段，其与权利要求1至6中任一项所述抗OX40抗体或其抗原结合片段竞争性结合人OX40。
  8. 一种多核苷酸，其编码权利要求1至7任一项所述的抗OX40抗体或其抗原结合片段。
  9. 一种载体，其包含权利要求8所述多核苷酸，优选地，所述载体为真核表达载体、原核表达载体或病毒载体。
  10. 一种宿主细胞，其包含权利要求9所述的载体，
    优选地，所述宿主细胞选自：细菌、酵母、哺乳动物细胞；
    更优选地，所述宿主细胞选自：大肠杆菌、毕赤酵母、中国仓鼠卵巢细胞、人胚肾293细胞。
  11. 一种制备权利要求1至7任一项所述抗OX40抗体或其抗原结合片段的方法，所述方法包括：
    培养权利要求10所述的宿主细胞；
    回收所述抗OX40抗体或其抗原结合片段；
    任选地，纯化所述抗OX40抗体或其抗原结合片段。
  12. 一种检测或测定OX40的方法，包括：
    使权利要求1至7任一项所述的抗OX40抗体或其抗原结合片段与样本相接触的步骤。
  13. 一种试剂，包含权利要求1至7任一项所述的抗OX40抗体或其抗原结合片段。
  14. 一种药物组合物，其含有：
    治疗有效量或预防治疗有效量的权利要求1至7任一项所述的抗OX40抗体或其抗原结合片段；以及
    一种或多种药学上可接受的载体、稀释剂、缓冲剂或赋形剂。
  15. 一种治疗或预防疾病或病症的方法，包括：
    向受试者施用治疗有效量或预防治疗有效量的权利要求1至7中任一项的抗OX40抗体或其抗原结合片段，或权利要求14所述的药物组合物；
    优选地，所述疾病或病症为癌症；
    更优选地，所述癌症选自：肺癌、前列腺癌、乳腺癌、头颈部癌、食管癌、胃癌、结肠癌、结直肠癌、膀胱癌、宫颈癌、子宫癌、卵巢癌、肝癌、黑色素瘤、肾癌、鳞状细胞癌、血液系统癌症。
  16. 一种增强人受试者中的免疫应答的方法，包括：
    向受试者施用治疗有效量或预防治疗有效量的权利要求1至7中任一项的抗OX40抗体或其抗原结合片段，或权利要求14所述的药物组合物；
    优选地，所述增强的免疫应答包括T效应细胞的免疫刺激/效应功能的增加，和/或T调节细胞的免疫抑制功能的下调。
  17. 选自以下的任一项在制备药物中的用途：
    -权利要求1至7中任一项所述的抗OX40抗体或其抗原结合片段、
    -权利要求8所述的多核苷酸、
    -权利要求9所述的载体、
    -权利要求14所述的药物组合物；
    所述药物用于治疗或预防癌症，或用于增强人受试者中的免疫应答；
    优选地，所述癌症选自：肺癌、前列腺癌、乳腺癌、头颈部癌、食管癌、胃 癌、结肠癌、结直肠癌、膀胱癌、宫颈癌、子宫癌、卵巢癌、肝癌、黑色素瘤、肾癌、鳞状细胞癌、血液系统癌症。
  18. 权利要求1至7中任一项所述的抗OX40抗体或其抗原结合在制备试剂盒中的用途，所述试剂盒用于检测或诊断与OX40表达相关的疾病或病症；
    优选地，所述疾病或病症为癌症；
    更优选地，所述癌症选自：肺癌、前列腺癌、乳腺癌、头颈部癌、食管癌、胃癌、结肠癌、结直肠癌、膀胱癌、宫颈癌、子宫癌、卵巢癌、肝癌、黑色素瘤、肾癌、鳞状细胞癌、血液系统癌症。
PCT/CN2019/107787 2018-09-26 2019-09-25 抗ox40抗体、其抗原结合片段及其医药用途 WO2020063660A1 (zh)

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JP2021517014A JP2022502417A (ja) 2018-09-26 2019-09-25 抗ｏｘ４０抗体、その抗原結合フラグメント、および医薬用途
KR1020217011102A KR20210069058A (ko) 2018-09-26 2019-09-25 항-ox40 항체, 이의 항원-결합 단편 및 약학적 용도
AU2019347934A AU2019347934A1 (en) 2018-09-26 2019-09-25 An anti-OX40 antibody, antigen-binding fragment thereof, and the pharmaceutical use
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EP19867097.8A EP3858857A1 (en) 2018-09-26 2019-09-25 An anti-ox40 antibody, antigen-binding fragment thereof, and the pharmaceutical use
US17/278,259 US20210355229A1 (en) 2018-09-26 2019-09-25 An anti-ox40 antibody, antigen-binding fragment thereof, and the pharmaceutical use
MX2021003168A MX2021003168A (es) 2018-09-26 2019-09-25 Anticuerpo anti-ox40, fragmento de union al antigeno del mismo y uso farmaceutico.
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