WO2021129874A1 - Anti-ox40 antibody and use thereof - Google Patents

Anti-ox40 antibody and use thereof Download PDF

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WO2021129874A1
WO2021129874A1 PCT/CN2020/140259 CN2020140259W WO2021129874A1 WO 2021129874 A1 WO2021129874 A1 WO 2021129874A1 CN 2020140259 W CN2020140259 W CN 2020140259W WO 2021129874 A1 WO2021129874 A1 WO 2021129874A1
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antibody
cancer
seq
antigen
human
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PCT/CN2020/140259
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French (fr)
Chinese (zh)
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张宏恺
晋瑞娜
王媛
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南开大学
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Priority to CN202080073615.0A priority Critical patent/CN114630839A/en
Publication of WO2021129874A1 publication Critical patent/WO2021129874A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39566—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against immunoglobulins, e.g. anti-idiotypic antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—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]

Definitions

  • the present invention relates in particular to a novel anti-OX40 antibody, a composition comprising the anti-OX40 antibody, a nucleic acid encoding the anti-OX40 antibody, a method for preparing the anti-OX40 antibody, and the use of the anti-OX40 antibody.
  • nivolumab nivolumab
  • pembrolizumab nivolumab
  • nivolumab nivolumab
  • pembrolizumab nivolumab
  • diseases such as unresectable or metastatic melanoma and metastatic non-small cell lung cancer.
  • Patients treated with these drugs have produced an anti-tumor response measured by improvement in progression-free survival and/or overall survival.
  • the field still needs more cancer treatment products and methods to supplement the existing standard care.
  • PD-1 and CTLA-4 play an immunosuppressive effect in the process of T cell activation, thereby inhibiting the immune killing function of T cells on tumor cells; therefore, blocking monoclonal antibodies against these two targets can relieve this immunosuppression , To restore the anti-tumor immune function of T cells.
  • activated immune checkpoint molecules are gradually becoming new targets for drug development.
  • Activated immune checkpoint molecules mainly refer to the costimulatory signal molecules of T cell activation-T cell costimulatory receptors, belonging to the tumor necrosis factor receptor (TNFR) family, used to regulate the proliferation and activation of T cells And differentiation, including OX40, CD40, 4-1BB and GITR.
  • TNFR tumor necrosis factor receptor
  • the OX40 receptor also known as CD134 and TNFRSF4 (Tumor Necrosis Factor Receptor Superfamily Member 4), is a member of the TNFR superfamily receptor, which is not constitutively expressed on resting naive T cells, unlike CD28.
  • OX40 is a secondary costimulatory immune checkpoint molecule that is expressed 24 to 72 hours after activation; its ligand OX40L (also known as CD252 and TNFSF4) is not expressed on resting antigen presenting cells, but is expressed after activation. The expression of OX40 depends on the complete activation of T cells.
  • OX40 and its ligand OX40L combine to deliver costimulatory signals.
  • the interaction of OX40 and OX40L can recruit TNFR-related (TRAFs) molecules in the intracellular region of OX40 to form a signaling complex containing IKK ⁇ and IKK ⁇ as well as PI3k and PKB (Akt);
  • TCR TNFR-related
  • OX40 also cooperates with TCR signaling through unknown The mechanism enhances intracellular Ca 2+ , thereby enhancing NFAT into the nucleus.
  • OX40 can activate the classic NF- ⁇ B1 pathway or the non-canonical NF- ⁇ B2 pathway, PI3k/PKB and NFAT pathways, thereby regulating the genes that control T cell division and survival, as well as promoting the transcription of cytokine genes and the expression of cytokine receptors , Is essential for cell survival.
  • OX40 signaling can cause down-regulation including CTLA-4 and Foxp3.
  • OX40 When OX40 is combined with its ligand OX40L, it helps to improve the response of the immune system: 1. Increase the survival and expansion of effector T cells and memory T cells, and increase cytokines (such as IL-2, IL-4, IL-5) , IFN- ⁇ ) secretion; 2. Reduce the immunosuppressive activity of regulatory T cells, and further amplify the effect of T cell activation. In the tumor microenvironment, immune activation can lead to the expression of OX40. It can enhance the activation and proliferation of effector T cells, and inhibit regulatory T cells, resulting in a complex anti-tumor immune response. At present, a number of clinical trials of anti-OX40 antibodies for cancer treatment can be retrieved on the Clinical Trials website.
  • the present invention meets the above needs by providing a novel anti-OX40 antibody that specifically binds and activates OX40.
  • the present invention provides an isolated anti-OX40 antibody or antigen-binding fragment thereof, comprising: a heavy chain CDR1 domain shown in SEQ ID NO: 1, and a heavy chain CDR2 domain shown in SEQ ID NO: 2. , And the heavy chain variable region of the heavy chain CDR3 domain shown in SEQ ID NO: 3; and having the light chain CDR1 domain shown in SEQ ID NO: 9, and the light chain CDR2 domain shown in SEQ ID NO: 10, and The light chain variable region of the light chain CDR3 domain shown in SEQ ID NO: 11.
  • the present invention provides an isolated anti-OX40 antibody or antigen-binding fragment thereof, comprising: a heavy chain CDR1 domain shown in SEQ ID NO: 17, and a heavy chain CDR2 domain shown in SEQ ID NO: 18 , And the heavy chain variable region of the heavy chain CDR3 domain shown in SEQ ID NO: 19; and having the light chain CDR1 domain shown in SEQ ID NO: 25, and the light chain CDR2 domain shown in SEQ ID NO: 26, and The light chain variable region of the light chain CDR3 domain shown in SEQ ID NO: 27.
  • the present invention provides an isolated anti-OX40 antibody or antigen-binding fragment thereof, comprising: a heavy chain CDR1 domain shown in SEQ ID NO: 33, and a heavy chain CDR2 domain shown in SEQ ID NO: 34 , And the heavy chain variable region of the heavy chain CDR3 domain shown in SEQ ID NO: 35; and having the light chain CDR1 domain shown in SEQ ID NO: 41, and the light chain CDR2 domain shown in SEQ ID NO: 42, and The light chain variable region of the light chain CDR3 domain shown in SEQ ID NO: 43.
  • the present invention provides an antibody-drug conjugate comprising the OX40 antibody or antigen-binding fragment thereof described herein and another therapeutic agent; preferably, the anti-OX40 antibody or antigen-binding fragment thereof is combined with The additional therapeutic agent is connected by a linker.
  • the present invention provides a nucleic acid that encodes the anti-OX40 antibody or antigen-binding fragment thereof described herein.
  • the present invention provides an expression vector comprising the nucleic acid described herein.
  • the present invention provides a host cell comprising the nucleic acid described herein or the expression vector described herein.
  • the present invention provides a method for producing the anti-OX40 antibody or antigen-binding fragment thereof described herein, which comprises culturing the host cell described herein under conditions suitable for the expression of the antibody or antigen-binding fragment thereof , And recover the expressed antibody or its antigen-binding fragment from the culture medium.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the anti-OX40 antibody or antigen-binding fragment thereof described herein, or the antibody-drug conjugate described herein, or the nucleic acid described herein, or Said expression vector, and pharmaceutically acceptable carrier.
  • the present invention provides the anti-OX40 antibody or antigen-binding fragment thereof described herein, or the antibody-drug conjugate described herein, or the pharmaceutical composition described herein, for use in the treatment of cancer.
  • the present invention provides a method for treating cancer, which comprises administering to a subject in need a therapeutically effective amount of the anti-OX40 antibody or antigen-binding fragment thereof described herein, or the antibody described herein- A drug conjugate, or a pharmaceutical composition as described herein, to treat the cancer.
  • the present invention provides the anti-OX40 antibody or antigen-binding fragment thereof described herein, or the antibody-drug conjugate described herein, or the pharmaceutical composition described herein, in the preparation of a drug for the treatment of cancer In the use.
  • the present invention provides the anti-OX40 antibody or antigen-binding fragment thereof described herein, or the antibody-drug conjugate described herein, or the pharmaceutical composition described herein, which is used in one of the following or Multiple: Inhibition of Treg function (for example, suppression of Treg suppressive function), killing of OX40-expressing cells (for example, cells expressing high levels of OX40), improvement of effector T cell function and/or improvement of memory T cell function, reduction of tumor immunity, Enhance T cell function and/or deplete OX40-expressing cells.
  • Treg function for example, suppression of Treg suppressive function
  • OX40-expressing cells for example, cells expressing high levels of OX40
  • improvement of effector T cell function and/or improvement of memory T cell function reduction of tumor immunity
  • Enhance T cell function and/or deplete OX40-expressing cells Enhance T cell function and/or deplete OX40-expressing cells.
  • the present invention provides an anti-OX40 antibody or antigen-binding fragment thereof as described herein, or an antibody-drug conjugate as described herein, or a pharmaceutical composition as described herein, in preparation for the treatment of the following: Use of one or more drugs: inhibiting Treg function (for example, inhibiting the suppressive function of Treg), killing OX40-expressing cells (for example, cells expressing high levels of OX40), improving effector T cell function and/or improving memory T Cell function, reduce tumor immunity, enhance T cell function and/or reduce cells expressing OX40.
  • Treg function for example, inhibiting the suppressive function of Treg
  • OX40-expressing cells for example, cells expressing high levels of OX40
  • improving effector T cell function and/or improving memory T Cell function reduce tumor immunity, enhance T cell function and/or reduce cells expressing OX40.
  • the present invention provides a pharmaceutical combination comprising the anti-OX40 antibody or antigen-binding fragment thereof described herein, or the antibody-drug conjugate described herein, or the pharmaceutical composition described herein, and One or more additional therapeutic agents.
  • the present invention provides a kit comprising the anti-OX40 antibody or antigen-binding fragment thereof described herein, or the antibody-drug conjugate described herein, or the pharmaceutical composition described herein, preferably It further includes a drug delivery device.
  • Figure 1 500nM Fc-ILZ-OX40L stimulated sorting of Jurkat/NF-KappaB-GFP+hOX40 reporter cell line monoclonal, flow cytometry analysis of the positive rate of GFP expression in cells after stimulation.
  • Phage ELISA method detects the binding of phage monoclonal to OX40 recombinant protein, and calculates the ratio of phage to OX40 and phage to BSA OD450nm.
  • FIG. 3 HEK293FT cell culture supernatants transfected with 4 scFv-Fc expression plasmids with or without cross-linking secondary antibodies stimulated Jurkat/NF- ⁇ B-GFP+hOX40 reporter cells, and flow cytometry analyzed the GFP positive rate of cells.
  • Figure 4 Analysis of aggregation of anti-OX40 antibodies.
  • Figure 5 Anti-tumor efficacy of anti-OX40 antibodies in vivo.
  • the anti-OX40 antibody significantly inhibited tumor growth compared to the control.
  • Figure 6 T cell regulation effect of anti-OX40 antibody. Compared with the control, the anti-OX40 antibody significantly upregulated helper CD4+T cells and cytotoxic CD8+T cells in the spleen and tumors.
  • Figure 7 Dose-dependent effects of cross-linked and soluble anti-OX40 antibodies.
  • the present invention provides an isolated anti-OX40 antibody or antigen-binding fragment thereof, comprising: a heavy chain CDR1 domain shown in SEQ ID NO: 1, and a heavy chain CDR2 domain shown in SEQ ID NO: 2. , And the heavy chain variable region of the heavy chain CDR3 domain shown in SEQ ID NO: 3; and having the light chain CDR1 domain shown in SEQ ID NO: 9, and the light chain CDR2 domain shown in SEQ ID NO: 10, and The light chain variable region of the light chain CDR3 domain shown in SEQ ID NO: 11.
  • the present invention provides an isolated anti-OX40 antibody or antigen-binding fragment thereof, comprising: a heavy chain CDR1 domain shown in SEQ ID NO: 17, and a heavy chain CDR2 domain shown in SEQ ID NO: 18 , And the heavy chain variable region of the heavy chain CDR3 domain shown in SEQ ID NO: 19; and having the light chain CDR1 domain shown in SEQ ID NO: 25, and the light chain CDR2 domain shown in SEQ ID NO: 26, and The light chain variable region of the light chain CDR3 domain shown in SEQ ID NO: 27.
  • the present invention provides an isolated anti-OX40 antibody or antigen-binding fragment thereof, comprising: a heavy chain CDR1 domain shown in SEQ ID NO: 33, and a heavy chain CDR2 domain shown in SEQ ID NO: 34 , And the heavy chain variable region of the heavy chain CDR3 domain shown in SEQ ID NO: 35; and having the light chain CDR1 domain shown in SEQ ID NO: 41, and the light chain CDR2 domain shown in SEQ ID NO: 42, and The light chain variable region of the light chain CDR3 domain shown in SEQ ID NO: 43.
  • the anti-OX40 antibody or antigen-binding fragment thereof described herein comprises the heavy chain variable region shown in SEQ ID NO: 4, or has at least 80%, 81%, and the sequence shown in SEQ ID NO: 4 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , 99% or 100% identical heavy chain variable region; and the light chain variable region shown in SEQ ID NO: 12, or at least 80%, 81%, 82%, and the sequence shown in SEQ ID NO: 12 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% Or a light chain variable region with 100% identity.
  • the anti-OX40 antibody or antigen-binding fragment thereof described herein comprises the heavy chain variable region shown in SEQ ID NO: 20, or has at least 80%, 81%, and the sequence shown in SEQ ID NO: 20. 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , 99% or 100% identical heavy chain variable region; and the light chain variable region shown in SEQ ID NO: 28, or at least 80%, 81%, 82%, and the sequence shown in SEQ ID NO: 28 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% Or a light chain variable region with 100% identity.
  • the anti-OX40 antibody or antigen-binding fragment thereof described herein comprises the heavy chain variable region shown in SEQ ID NO: 36, or has at least 80%, 81%, and the sequence shown in SEQ ID NO: 36. 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , 99% or 100% identical heavy chain variable region; and the light chain variable region shown in SEQ ID NO: 44, or at least 80%, 81%, 82%, and the sequence shown in SEQ ID NO: 44 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% Or a light chain variable region with 100% identity.
  • the anti-OX40 antibody or antigen-binding fragment thereof described herein further comprises a heavy chain constant region and a light chain constant region; preferably, the heavy chain constant region is the heavy chain shown in SEQ ID NO: 5 or 21
  • the constant region or the sequence shown in SEQ ID NO: 5 or 21 has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical heavy chain constant region; and/or preferably, the light chain constant region is The light chain constant region shown in SEQ ID NO: 13, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% with the sequence shown in SEQ ID NO: 13 , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical light chain constant region.
  • the anti-OX40 antibody or antigen-binding fragment thereof described herein further comprises a heavy chain signal peptide connected to the heavy chain variable region and/or a heavy chain signal connected to the light chain variable region
  • the heavy chain signal peptide is the heavy chain signal peptide shown in SEQ ID NO: 6, or has at least 80%, 81%, 82%, 83%, 84% with the sequence shown in SEQ ID NO: 6 , 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity
  • the light chain signal peptide is the light chain signal peptide shown in SEQ ID NO: 14, or is at least 80%, 81%, 82% with the sequence shown in SEQ ID NO: 14 %, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 9
  • the anti-OX40 antibody or antigen-binding fragment thereof described herein is an IgG antibody or an antigen-binding fragment thereof, preferably an IgG1 antibody or an antigen-binding fragment thereof.
  • the anti-OX40 antibody or antigen-binding fragment thereof described herein is a monoclonal antibody or antigen-binding fragment thereof.
  • the anti-OX40 antigen-binding fragment described herein is Fab, Fab', F(ab')2, Fv, scFv, or sdAb.
  • the present invention provides an antibody-drug conjugate comprising the anti-OX40 antibody or antigen-binding fragment thereof as described herein and another therapeutic agent; preferably, the anti-OX40 antibody or antigen-binding fragment thereof The fragment and the additional therapeutic agent are connected by a linker.
  • the present invention provides a nucleic acid that encodes the anti-OX40 antibody or antigen-binding fragment thereof described herein.
  • nucleic acid described herein comprises:
  • the nucleic acid further comprises the heavy chain constant region nucleotide coding sequence shown in SEQ ID NO: 50 or 54 and/or the light chain constant region nucleotide coding sequence shown in SEQ ID NO: 52.
  • the present invention provides an expression vector comprising the nucleic acid described herein.
  • the present invention provides a host cell comprising the nucleic acid or expression vector described herein.
  • a method for producing the anti-OX40 antibody or antigen-binding fragment thereof described herein which comprises culturing the host cell described herein under conditions suitable for the expression of the antibody or antigen-binding fragment thereof, And recover the expressed antibody or its antigen-binding fragment from the culture medium.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the anti-OX40 antibody or antigen-binding fragment thereof described herein, or the antibody-drug conjugate described herein, or the nucleic acid described herein, or Said expression vector, and pharmaceutically acceptable carrier.
  • the anti-OX40 antibody or antigen-binding fragment thereof described herein, or the antibody-drug conjugate described herein, or the pharmaceutical composition described herein is used for the treatment of cancer.
  • the cancer is selected from squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), peritoneal cancer , Hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urethral 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, liver cancer, anal cancer, penile cancer, melanoma, superficial spreading mel
  • the present invention provides a method for treating cancer, which comprises administering to a subject in need a therapeutically effective amount of the anti-OX40 antibody or antigen-binding fragment thereof described herein, or the antibody described herein- A drug conjugate, or a pharmaceutical composition as described herein, to treat the cancer.
  • the cancer is selected from squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), peritoneal cancer , Hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urethral 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, liver cancer, anal cancer, penile cancer, melanoma, superficial spreading melanoma, Malignant freckle melanoma, acral melanoma, nodular melanoma, multiple myeloma and B-cell lymphoma, chronic
  • the present invention provides the anti-OX40 antibody or antigen-binding fragment thereof described herein, or the antibody-drug conjugate described herein, or the pharmaceutical composition described herein, in the preparation of a drug for the treatment of cancer
  • the cancer is selected from squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), peritoneal cancer , Hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urethral 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, liver cancer, anal cancer, pen
  • squamous cell carcinoma
  • Inhibit Treg function e.g., inhibit the suppressive function of Treg
  • kill OX40-expressing cells e.g., cells expressing high levels of OX40
  • improve effector T cell function and/or improve memory T cell function reduce tumor immunity
  • enhance T cells Function and/or deplete cells expressing OX40 e.g., deplete cells expressing OX40.
  • the anti-OX40 antibody or antigen-binding fragment thereof described herein, or the antibody-drug conjugate described herein, or the pharmaceutical composition described herein is prepared for the treatment of one of the following or Uses in a variety of drugs: inhibit Treg function (e.g., inhibit the suppressive function of Treg), kill OX40-expressing cells (e.g., 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 e.g., inhibit the suppressive function of Treg
  • kill OX40-expressing cells e.g., cells expressing high levels of OX40
  • improve effector T cell function and/or improve memory T cell function e.g., reduce tumor immunity, enhance T cell function and/or reduce cells expressing OX40.
  • the present invention provides a pharmaceutical combination comprising the anti-OX40 antibody or antigen-binding fragment thereof described herein, or the antibody-drug conjugate described herein, or the pharmaceutical composition described herein, and One or more additional therapeutic agents.
  • the present invention provides a kit comprising the anti-OX40 antibody or antigen-binding fragment thereof described herein, or the antibody-drug conjugate described herein, or the pharmaceutical composition described herein, preferably It further includes a drug delivery device.
  • comparison window refers to a segment having at least about 20 (usually 30 to about 75 or 40 to about 50) contiguous positions, where the two sequences can be compared with those having Compare the reference sequences of the same number of consecutive positions.
  • the optimized alignment of the sequences used for comparison can be performed using a program in a suite of bioinformatics software (Inc., Madison, WI), using default parameters.
  • This program implements several comparison schemes described in the following references: Dayhoff, MO, 1978, A model of evolutionary change in proteins-Matrices for detecting distant relationships.
  • the "percentage of sequence identity” is determined by comparing two optimized aligned sequences on a comparison window with at least 20 positions, where the polynucleotide or polypeptide sequence in the comparison window Part of the reference sequence (which does not contain additions or deletions) compared to the optimized alignment of the two sequences may contain 20% or less, usually 5% to 15% or 10% to 12% of additions or deletions (ie, gaps). ). The percentage is calculated by determining the number of positions where the same nucleic acid base or amino acid residue appears in both sequences to generate the number of matching positions, and dividing the number of matching positions by the total number of positions in the reference sequence (Ie, window size) and multiply the result by 100 to produce a percentage of sequence identity.
  • the variant may also be substantially homologous to the natural gene or part or complement thereof.
  • These polynucleotide variants can hybridize to naturally-occurring DNA sequences encoding natural antibodies (or complementary sequences) under moderately stringent conditions.
  • Suitable “moderately stringent conditions” include pre-washing in a solution of 5X SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridization at 50°C to 65°C, 5X SSC overnight; then at 65°C Wash twice, lasting 20 minutes, and use 2X, 0.5X and 0.2X SSC containing 0.1% SDS for each wash.
  • highly stringent conditions or “highly stringent conditions” are those of the following: (1) Use low ionic strength and high temperature for cleaning, such as 0.015M sodium chloride/0.0015M citric acid at 50°C Sodium/0.1% sodium lauryl sulfate; (2) Use denaturants such as formamide during hybridization, for example, with 0.1% bovine serum albumin/0.1% sucrose/0.1% polyvinylpyrrole at pH 6.5 Pyridone/50mM sodium phosphate buffer and 50% (v/v) formamide of 750mM sodium chloride and 75mM sodium citrate at 42°C; or (3) 50% formamide, 5X SSC at 42°C (0.75M NaCl, 0.075M sodium citrate), 50mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5X Denhardt hybridization solution, sonicated salmon sperm DNA (50 ⁇ g/mL), 0.1% SDS and 10% Dextran sulfate, and washed
  • nucleotide sequences encoding the polypeptides as described herein. Some of these polynucleotides have minimal homology with the nucleotide sequence of any natural gene. However, the present invention specifically contemplates polynucleotides that are altered due to differences in codon usage. In addition, alleles of genes comprising the polynucleotide sequences provided herein are within the scope of the present invention. Alleles are endogenous genes that change due to one or more mutations of nucleotides, such as deletions, additions, and/or substitutions. The resulting mRNA and protein may (but need not) have altered structure or function. Alleles can be identified using standard techniques such as hybridization, amplification, and/or database sequence comparison.
  • the polynucleotide of the present invention can be obtained using chemical synthesis, recombinant methods or PCR.
  • the method of chemical polynucleotide synthesis is well known in the art and need not be described in detail herein. Those skilled in the art can use the sequences provided herein and commercially available DNA synthesizers to generate the desired DNA sequence.
  • the polynucleotides containing the desired sequence can be inserted into a suitable vector, and the vector can be further introduced into a suitable host cell for replication and amplification, as described herein further discussion.
  • the polynucleotide can be inserted into the host cell by any means known in the art. Cells are transformed by direct uptake, endocytosis, transfection, F-hybridization or electroporation to introduce exogenous polynucleotides. Once introduced, the exogenous polynucleotide can be maintained in the cell as a non-integrating vector (such as a plasmid) or integrated into the host cell gene.
  • the polynucleotide thus amplified can be isolated from the host cell by methods well known in the art. See, for example, Sambrook et al., 1989.
  • PCR allows for the replication of DNA sequences. .
  • RNA can be obtained by using isolated DNA in a suitable vector and inserting it into a suitable host cell. When the cell replicates and the DNA is transcribed into RNA, the RNA can then be isolated using methods known to those skilled in the art.
  • Suitable cloning and expression vectors can include various components such as promoters, enhancers, and other transcriptional regulatory sequences.
  • the vector can also be constructed to allow subsequent cloning of antibody variable domains into different vectors.
  • Suitable cloning vectors can be constructed according to standard techniques or can be selected from a large number of cloning vectors available in the art. Although the selected cloning vector can vary depending on the host cell to be used, useful cloning vectors will generally have the ability to self-replicate, can have a single target for specific restriction endonucleases, and/or can carry targets that can be used for selection. The gene containing the marker in the clone of the vector.
  • Suitable examples include plasmids and bacterial viruses, for example, pUC18, pUC19, Bluescript (e.g., pBS SK+) and derivatives thereof, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA and shuttle vectors (such as pSA3 and pAT28).
  • Bluescript e.g., pBS SK+
  • shuttle vectors such as pSA3 and pAT28.
  • the expression vector is usually a replicable polynucleotide construct, which contains the polynucleotide according to the present invention. It implies that the expression vector must be replicable in the host cell, in the form of an episomal gene or as an integral part of chromosomal DNA.
  • Suitable expression vectors include (but are not limited to) plasmids and viral vectors, including adenovirus, adeno-associated virus, retrovirus, cosmid and the expression vector disclosed in PCT Publication No. WO 87/04462.
  • Vector components may generally include (but are not limited to) one or more of the following: a signal sequence; an origin of replication; one or more marker genes; suitable transcription control components (such as promoters, enhancers, and terminator).
  • suitable transcription control components such as promoters, enhancers, and terminator.
  • transcription control components such as ribosome binding sites, translation initiation sites, and termination codons, are also usually required.
  • the vector containing the polynucleotide of interest and/or the polynucleotide itself can be introduced into the host cell by any of a number of appropriate methods, including electroporation, the use of calcium chloride, rubidium chloride, calcium phosphate, Transfection of DEAE-dextran or other substances; microprojectile bombardment; lipofection; and infection (for example, where the vector is an infectious agent, such as a pox virus).
  • electroporation the use of calcium chloride, rubidium chloride, calcium phosphate, Transfection of DEAE-dextran or other substances; microprojectile bombardment; lipofection; and infection (for example, where the vector is an infectious agent, such as a pox virus).
  • the choice of introducing a vector or polynucleotide will generally depend on the characteristics of the host cell.
  • the antibodies of the present invention include human antibodies prepared, expressed, produced or isolated by recombinant methods, such as antibodies expressed using recombinant expression vectors transfected into host cells (further described in the following section II), isolated from recombinant combinatorial human antibody libraries Antibodies (further described in section III below), antibodies isolated from human immunoglobulin gene transgenic animals (e.g. mice) (see e.g. (Taylor, LD et al. (1992) Nucl. Acids Res. 20: 6287-6295) Or antibodies prepared, expressed, produced or isolated by any other method involving the splicing of human immunoglobulin gene sequences into other DNA sequences.
  • Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences ( See Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242).
  • the antibody or antibody portion of the present invention can be prepared by recombinantly expressing immunoglobulin light chain genes and heavy chain genes in host cells.
  • a host cell is transfected with one or more recombinant expression vectors carrying DNA fragments encoding the immunoglobulin light and heavy chains of the antibody, so that the light and heavy chains are expressed in the host cell, Furthermore, it is preferably secreted into a medium in which the host cell is cultured, from which the antibody can be recovered. Standard recombinant DNA methodology is used to obtain antibody heavy chain genes and antibody light chain genes, introduce these genes into a recombinant expression vector, and then introduce the vector into host cells.
  • Antibodies or antigen-binding fragments thereof can be recombinantly produced using suitable host cells.
  • the nucleic acid encoding the antibody or antigen-binding fragment thereof can be cloned into an expression vector, which can then be introduced into host cells such as E. coli cells, yeast cells, insect cells, apes COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells , Wherein the cell does not additionally produce immunoglobulin to obtain antibody synthesis in the recombinant host cell.
  • host cells include E. coli cells, yeast cells, insect cells, apes COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells , Wherein the cell does not additionally produce immunoglobulin to obtain antibody synthesis in the recombinant host cell.
  • preferred host cells include CHO cells, human embryonic kidney (HEK) 293 cells, or Sp2.0 cells.
  • Antibody fragments can be produced by proteolysis or other degradation of full-length antibodies by recombinant methods or by chemical synthesis.
  • Polypeptide fragments of antibodies (especially shorter polypeptides of up to about 50 amino acids) can be conveniently prepared by chemical synthesis. Methods for the chemical synthesis of proteins and peptides are known in the art and are commercially available.
  • the antibody or antigen-binding fragment thereof of the present invention can be affinity matured.
  • affinity matured antibodies can be obtained by procedures known in the art (Marks et al., 1992, Bio/Technology, 10:779-783; Barbas et al., 1994, Proc Nat. Acad. Sci, USA 91: 3809- 3813; Schier et al., 1995, Gene, 169:147-155; Yelton et al., 1995, J. Immunol., 155:1994-2004; Jackson et al., 1995, J. Immunol., 154(7): 3310 -9; Hawkins et al., 1992, J. Mol. Biol., 226:889-896; and WO2004/058184).
  • amino acid sequence variants of the antibodies provided herein are encompassed.
  • Amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions, and/or insertions and/or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletion, insertion, and substitution can be made to obtain the final construct, as long as the final construct possesses the desired characteristics, for example, antigen binding.
  • antibody variants with one or more amino acid substitutions are provided.
  • Sites of interest for alternative mutagenesis include HVR and FR.
  • Conservative substitutions are shown in Table A under the heading of "preferred substitutions”. More substantial changes are provided in Table A under the heading of "exemplary substitutions” and are described further below with reference to amino acid side chain categories.
  • Amino acid substitutions can be introduced into the antibody of interest, and the product screened for the desired activity, such as retained/improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.
  • amino acids can be grouped as follows:
  • Non-conservative substitutions would require replacing members of one of these categories with members of another category.
  • substitution variant involves replacing one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody).
  • a parent antibody e.g., a humanized or human antibody.
  • the resulting variants selected for further research will have certain biological changes (e.g. improvement) (e.g. increased affinity, decreased immunogenicity) relative to the parent antibody and/or will substantially retain the parent antibody Some of the biological characteristics.
  • An exemplary alternative variant is an affinity matured antibody, which can be conveniently generated, for example, using phage display-based affinity maturation techniques such as those described herein.
  • one or more HVR residues are mutated, and the variant antibodies are displayed on phage and screened for specific biological activities (such as binding affinity).
  • HVR Changes (e.g., substitutions) can be made to HVR, for example to improve antibody affinity.
  • HVR "hot spots", residues encoded by codons that undergo mutations at a high frequency during the somatic maturation process (see, for example, Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and/or Such changes are made to residues in contact with the antigen, wherein the resulting variant VH or VL is tested for binding affinity.
  • Affinity maturation through the construction and reselection of secondary libraries has been described in, for example, Hoogenboom et al., in Methods in Molecular Biology 178: 1-37 (edited by O'Brien et al., Human Press, Totowa, NJ, (2001)).
  • diversity is introduced as a variable gene for maturation selection by multiple methods (e.g., error-prone PCR, strand shuffling, or oligonucleotide-directed mutagenesis). Then, create a secondary library. Then, the library is screened to identify any antibody variants with the desired affinity.
  • Another method of introducing diversity involves an HVR-directed method, in which several HVR residues (eg, 4-6 residues at a time) are randomized. Alanine scanning mutagenesis or modeling can be used, for example, to specifically identify HVR residues involved in antigen binding. In particular, CDR-H3 and CDR-L3 are often targeted.
  • substitutions, insertions, or deletions can occur within one or more HVRs, as long as such changes do not substantially reduce the ability of the antibody to bind antigen.
  • conservative changes e.g., conservative substitutions, as provided herein
  • HVR that do not substantially reduce binding affinity.
  • such changes can be outside of the antigen contact residues in the HVR.
  • each HVR is unchanged or contains no more than 1, 2, or 3 amino acid substitutions.
  • alanine scanning mutagenesis One method that can be used to identify residues or regions in antibodies that can be targeted for mutagenesis is called “alanine scanning mutagenesis", as described by Cunningham and Wells (1989) Science, 244:1081-1085.
  • residues or groups of target residues for example, charged residues such as arg, asp, his, lys, and glu
  • neutral or negatively charged amino acids for example, alanine Acid or polyalanine
  • Further substitutions can be introduced at amino acid positions that indicate functional sensitivity to the initial substitution.
  • the crystal structure of the antigen-antibody complex is used to identify the contact points between the antibody and the antigen.
  • contact residues and neighboring residues can be targeted or eliminated.
  • the variants can be screened to determine whether they contain the desired properties.
  • Amino acid sequence insertions include amino and/or carboxyl terminal fusions ranging from one residue to a polypeptide containing 100 or more residues in length, and intra-sequence insertions of single or multiple amino acid residues.
  • terminal insertions include antibodies with N-terminal methionyl residues.
  • Other insertional variants of antibody molecules include fusions of the N- or C-terminus of the antibody with an enzyme (for example, for ADEPT) or a polypeptide that extends the serum half-life of the antibody.
  • the antibodies provided herein are modified to increase or decrease the degree of glycosylation of the antibody.
  • the addition or deletion of glycosylation sites of the antibody can be conveniently achieved by changing the amino acid sequence so that one or more glycosylation sites are created or eliminated.
  • the carbohydrate to which it is attached can be changed.
  • Natural antibodies produced by mammalian cells usually contain branched, biantennary oligosaccharides, which are generally attached to Asn297 of the CH2 domain of the Fc region through an N linkage. See, for example, Wright et al., TIBTECH 15:26-32 (1997).
  • Oligosaccharides may include various carbohydrates, for example, mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, and fucose attached to GlcNAc in the "stem" of the biantennary oligosaccharide structure.
  • the oligosaccharides in the antibodies of the invention can be modified to create antibody variants with certain improved properties.
  • antibody variants are provided that have a carbohydrate structure lacking fucose attached (directly or indirectly) to the Fc region.
  • the amount of fucose in such antibodies can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%.
  • Determine the amount of fucose by calculating the average amount of fucose in the sugar chain at Asn297 relative to the sum of all sugar structures (for example, complex, hybrid, and high-mannose structures) attached to Asn297, such as by Measured by MALDI-TOF mass spectrometry, for example, as described in WO 2008/077546.
  • Asn297 refers to the asparagine residue located at about position 297 in the Fc region (Eu numbering of residues in the Fc region); however, Asn297 can also be located at about ⁇ 2% upstream or downstream of position 297 due to minor sequence variations in the antibody. 3 amino acids, that is, between the 294th and 300th positions. Such fucosylation variants may have improved ADCC function. See, for example, US Patent Publication No. US 2003/0157108 (Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd).
  • Examples of publications involving "defucosylated” or “fucose deficient” antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002 /0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; TO 2005/035778; TO2005/ 053742; TO2002/031140; Okazaki et al., J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al., Biotech. Bioeng.
  • Examples of cell lines capable of producing defucosylated antibodies include LecI3CHO cells deficient in protein fucosylation (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application No. US 2003 /0157108A1, Presta, L; and WO 2004/056312A1, Adams, etc.), and knockout cell lines, such as ⁇ -1,6-fucosyltransferase gene FUT8 knockout CHO cells (see, for example, Yamane-Ohnuki, etc., Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4): 680-688 (2006); and WO2003/085107).
  • antibody variants having bipartite oligosaccharides for example, the biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc.
  • Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described in, for example, WO2003/011878 (Jean-Mairet et al.); US Patent No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umana et al.).
  • antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region Such antibody variants may have improved CDC function.
  • Such antibody variants are described in, for example, WO 1997/30087 (Patel et al.); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).
  • one or more amino acid modifications can be introduced into the Fc region of the antibodies provided herein, thereby generating Fc region variants.
  • the Fc region variant may comprise a human Fc region sequence (e.g., a human IgGl, IgG2, IgG3, or IgG4 Fc region) that contains amino acid modifications (e.g., substitutions) at one or more amino acid positions.
  • the present invention encompasses antibody variants possessing some but not all effector functions that make them desirable candidates for applications where the in vivo half-life of the antibody is important, and some Effector functions (such as complement and ADCC) are unnecessary or harmful.
  • In vitro and/or in vivo cytotoxicity assays can be performed to confirm the reduction/decrease of CDC and/or ADCC activity.
  • an Fc receptor (FcR) binding assay can be performed to ensure that the antibody lacks Fc ⁇ R binding (and therefore may lack ADCC activity), but retains FcRn binding ability.
  • the main cells that mediate ADCC, NK cells only express FcyRIII, while monocytes express FcyRI, FcyRII and FcyRIII.
  • FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991).
  • a non-limiting example of an in vitro assay for assessing the ADCC activity of a molecule of interest is described in U.S. Patent No. 5,500,362 (see, for example, Hellstrom, I. et al., Proc. Nat'I Acad. Sci USA 83:7059-7063 (1986)) And Hellstrom, I etc., Proc. Nat'I Acad. Sci. USA 82: 1499-1502 (1985); 5, 821, 337 (see Bruggemann, M. et al., J. Exp. Med.
  • a non-radioactive assay method can be used (see, for example, the ACT I TM non-radioactive cytotoxicity assay for flow cytometry (Cell Technology, Inc. Mountain View, CA; and the CytoTox96 non-radioactive cytotoxicity assay (Promega, Madison) , WI)). Effector cells useful for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells.
  • PBMC peripheral blood mononuclear cells
  • NK natural killer cells.
  • the ADCC activity of the molecule of interest can be assessed in vivo, for example in animal models Such as those disclosed in Clynes et al., Proc Nat'I Acad Sci USA 95:652-656 (1998).
  • Clq binding assays can also be performed to confirm that antibodies cannot bind to Clq and therefore lack CDC activity. See, for example, WO 2006/ 029879 and the Clq and C3c binding ELISA in WO 2005/100402.
  • CDC assays can be implemented (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS. et al., Blood 101:1045-1052 (2003); and Cragg, M ⁇ S ⁇ and MJ Glennie, Blood 103:2738-2743 (2004)).
  • Antibodies with reduced effector function include those with substitutions of one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (US Patent No. 6,737,056).
  • Fc mutants include Fc mutants having substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including so-called "DANA" in which residues 265 and 297 are substituted with alanine.
  • Fc mutant U.S. Patent No. 7,332,581.
  • antibody variants comprise an Fc region with one or more amino acid substitutions that improve ADCC, such as substitutions at positions 298, 333, and/or 334 (EU numbering of residues) in the Fc region.
  • changes are made to the Fc region that result in altered (ie, improved or reduced) Clq binding and/or complement dependent cytotoxicity (CDC), for example, as described in U.S. Patent No. 6,194,551 , WO 99/51642, and Idusogie et al., J.Tmmunol.164:4178-4184 (2000).
  • CDC complement dependent cytotoxicity
  • FcRn neonatal Fc receptor
  • the neonatal Fc receptor (FcRn) is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J Immunol. 117:587 (1976) and Kim et al. J. Immunol. 24:249 (1994)).
  • Those antibodies comprise an Fc region with one or more substitutions therein that improve the binding of the Fc region to FcRn.
  • Such Fc variants include those that have substitutions at one or more of Fc region residues 238,256, 265,272,286,303,305,307,311,312,317,340,356,360,362,376,378,380,382,413,424 or 434, for example, substitution of Fc region residue 434 (US Patent No. 7,371,826).
  • cysteine for example, "thioMAb” in which one or more residues of the antibody are replaced with cysteine residues.
  • the substituted residue is present in an accessible site of the antibody.
  • the reactive thiol group is thus positioned at the accessible site of the antibody, and can be used to conjugate the antibody with other modules, such as drug modules or linker-drug modules, to Create immunoconjugates as described further herein.
  • cysteine can be substituted for any one or more of the following residues: V205 of the light chain (Kabat numbering); A118 of the heavy chain (EU numbering); and the Fc region of the heavy chain S400 (EU numbering method).
  • Cysteine engineered antibodies can be generated as described in, for example, US Patent No. 7,521,541.
  • the antibodies provided herein can be further modified to contain additional non-proteinaceous modules known in the art and readily available.
  • Modules suitable for antibody derivatization include, but are not limited to, water-soluble polymers.
  • water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymer, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1 , 3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyamino acid (homopolymer or random copolymer), and dextran or poly( ⁇ -ethylene Pyrrolidone) polyethylene glycol, propylene glycol homopolymer, propylene oxide/ethylene oxide copolymer, polyoxyethylated polyol (such as glycerin), polyvinyl alcohol and mixtures thereof.
  • PEG polyethylene glycol
  • polyethylene glycol propionaldehyde Due to its stability in water, polyethylene glycol propionaldehyde may have advantages in production.
  • the polymer can be of any molecular weight and can be branched or unbranched.
  • the number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. Generally speaking, the number and/or type of polymers used for derivatization can be determined based on the following considerations, including but not limited to the specific properties or functions of the antibody to be improved, whether the antibody derivative will be used for treatment under specified conditions, etc. .
  • conjugates of antibodies and non-proteinaceous moieties that can be selectively heated by exposure to radiation are provided.
  • the non-proteinaceous module is carbon nanotubes (Kam et al., Proc. Natl. Acad. Sci. USA 102:11600-11605 (2005)).
  • the radiation can be of any wavelength, and includes, but is not limited to, a wavelength that does not damage ordinary cells, but heats the non-proteinaceous module to a temperature at which cells near the antibody-non-proteinaceous module are killed.
  • anti-OX40 antibodies provided herein can be identified, screened, or characterized by their physical/chemical properties and/or biological activities by a variety of assays known in the art.
  • the antibody of the present invention is tested for its antigen binding activity, for example, by a known method such as ELISA, Western blot, and the like. Methods known in the art can be used to determine OX40 binding, and exemplary methods are disclosed herein.
  • radioimmunoassay is used to measure binding.
  • An exemplary radioimmunoassay is illustrated.
  • the OX40 antibody was iodinated, and a competition reaction mixture containing a fixed concentration of iodinated antibody and a decreasing concentration of serially diluted unlabeled OX40 antibody was prepared.
  • Cells expressing OX40 for example, BT474 cells stably transfected with human OX40 are added to the reaction mixture.
  • the cells are washed to separate the free iodinated OX40 antibody from the OX40 antibody bound to the cells.
  • the level of bound iodinated OX40 antibody is determined, for example, by counting the radioactivity associated with the cells, and using standard methods to determine the binding affinity.
  • flow cytometry is used to assess the ability of OX40 antibodies to bind surface-expressed OX40 (e.g., on a subset of T cells).
  • Obtain peripheral leukocytes for example from humans, cynomolgus monkeys, rats or mice), and block the cells with serum.
  • the labeled OX40 antibody was added in serial dilutions, and T cells were also stained to identify T cell subsets (using methods known in the art).
  • surface plasmon resonance can be used to analyze OX40 binding. An exemplary surface plasmon resonance method is illustrated.
  • a competition assay can be used to identify antibodies that compete with any of the anti-OX40 antibodies disclosed herein for binding to OX40.
  • such competitive antibodies bind to the same epitope (e.g., linear or conformational epitope) as bound by any of the anti-OX40 antibodies disclosed herein. See Morris (1996) “Epitope Mapping Protocols", Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ) for detailed exemplary methods for locating epitopes bound by antibodies.
  • a competitive assay is exemplified.
  • a first labeled antibody which binds to OX, such as mab 1A7.gr.1, mab 3C8.gr5
  • a second unlabeled antibody which are tested to compete with the first antibody
  • the ability to bind to OX40 incubate the immobilized OX40 in a solution.
  • the second antibody may be present in the supernatant of the hybridoma.
  • the immobilized OX40 was incubated in a solution containing the first labeled antibody but not the second unlabeled antibody.
  • an assay method for identifying anti-OX40 antibodies with biological activity can include, for example, binding to OX40 (for example, binding to human and/or cyno OX40), increasing OX40-mediated signal transduction (for example, increasing NFkB-mediated transcription), and reducing cells expressing human OX40 (for example, T cells) ,
  • OX40 for example, binding to human and/or cyno OX40
  • increasing OX40-mediated signal transduction for example, increasing NFkB-mediated transcription
  • reducing cells expressing human OX40 for example, T cells
  • T effector cell function e.g. CD4+ effector T cells
  • enhance T effector T cells e.g. by increasing effector T cell proliferation and/or increasing effector T cell cytokine production (e.g.
  • interferon gamma interferon gamma
  • enhance the function of memory T cells e.g. CD4+ memory T cells
  • cytokine production of memory T cells e.g. gamma interferon
  • inhibiting the function of regulatory T cells e.g. by Treg suppression that reduces effector T cell function (eg CD4+ effector T cell function)
  • binds to human effector cells e.g. CD4+ effector T cell function
  • the antibodies of the invention are tested for such biological activity.
  • T cells e.g., memory or effector T cells
  • peripheral white blood cells e.g., separated from human whole blood using Ficoll gradient centrifugation.
  • Methods known in the art can be used to isolate memory T cells (e.g., CD4+ memory T cells) or effector T cells (e.g., CD4+ Teff cells) from PBMC.
  • memory T cells e.g., CD4+ memory T cells
  • effector T cells e.g., CD4+ Teff cells
  • MiItenyi CD4+ Memory T Cell Isolation Kit or Miltenyi Naive CD4+ T Cell Isolation Kit can be used.
  • the isolated T cells are cultured in the presence of antigen-presenting cells (for example, irradiated L cells expressing CD32 and CD80), and activated by adding anti-CD3 antibodies in the presence or absence of OX40 agonistic antibodies.
  • antigen-presenting cells for example, irradiated L cells expressing CD32 and CD80
  • OX40 agonistic antibodies The effects of agonistic OX40 antibodies on T cell proliferation can be measured using methods known in the art. For example, you can use the CellTiterGlo kit (Promega), and read the results on a multi-label reader (PerkinElmer).
  • the effect of agonistic OX40 antibody on T cell function can also be determined by analyzing the cytokines produced by T cells.
  • interferon gamma production by CD4+ T cells is measured, for example, by measuring interferon gamma in the cell culture supernatant. Methods for measuring interferon gamma are well known in the art.
  • T cells are isolated from human whole blood using methods known in the art (e.g., memory T cells or naive T cells).
  • the purified CD4+ naive T cells are labeled (for example, with CFSE), and the purified Treg cells are labeled with different reagents.
  • the irradiated antigen-presenting cells for example, L cells expressing CD32 and CD80 are co-cultured with labeled and purified naive CD4+ T cells and purified Treg.
  • Co-cultures were activated with anti-CD3 antibodies and tested in the presence or absence of agonistic OMO antibodies. After a suitable time (for example, 6 days of co-cultivation), FACS analysis is used to track the level of CD4+ naive T cell proliferation by dye dilution in reduced marker staining (for example, reduced CFSE marker staining).
  • FACS analysis is used to track the level of CD4+ naive T cell proliferation by dye dilution in reduced marker staining (for example, reduced CFSE marker staining).
  • a transgenic cell expressing human OX40 and a reporter gene comprising an NFkB promoter fused to a reporter gene (such as ⁇ -luciferase)
  • a reporter gene such as ⁇ -luciferase
  • Phagocytosis can be measured, for example, by using monocyte-derived macrophages or U937 cells (a human histiocytic lymphoma cell line with the morphology and characteristics of mature macrophages).
  • Cells expressing OX40 are added to monocyte-derived macrophages or U937 cells in the presence or absence of anti-OX40 agonistic antibodies. After the cells are cultured for a suitable period of time, the percentage of cells double-stained with markers for 1) macrophages or U937 cells and 2) cells expressing OX40 is checked, and this is divided by the markers for cells expressing OX40 ( For example, the total number of GFP) cells is used to determine the percentage of phagocytosis. It can be analyzed by flow cytometry. In another embodiment, the analysis can be performed by fluorescence microscopy analysis.
  • ADCC can be measured, for example, using methods known in the art. Exemplary methods are described in the definition section.
  • the level of OX40 on OX40-expressing cells used for testing in the ADCC assay is characterized. The cells are stained with a detectably labeled anti-OX40 antibody (for example, PE-labeled), and then the fluorescence level is measured using flow cytometry, and the results are presented as median fluorescence intensity (MFI).
  • MFI median fluorescence intensity
  • ADCC can be analyzed by the CellTiter Glo assay kit, and cell viability/cytotoxicity can be determined by chemiluminescence.
  • the corresponding recombinant Fcy receptors can be used to measure the binding affinity of various antibodies to FcyRIA, FcyRIIA, FcyRIIB, and the two allotypes of FcyRIIIA (F158 and V158) in an ELISA-based ligand binding assay.
  • the purified human Fc ⁇ receptor was expressed as a fusion protein containing the extracellular domain of the receptor ⁇ chain linked to the C-terminal Gly/6xHis/glutathione S-transferase (GST) polypeptide tag.
  • GST Gly/6xHis/glutathione S-transferase
  • Fc ⁇ RIIA CD32A
  • Fc ⁇ RIIB CD32B
  • the two allotypes of Fe ⁇ RIIIA CD16
  • F-158 and V-158 you can use goat anti-human kappa chain F(ab ') 2 fragment (ICN Biomedical; Irvine, CA) cross-linked (approximate molar ratio 1:3 antibody: cross-linking F(ab')2) as a multimer test antibody.
  • the plate was coated with anti-GST antibody (Genentech) and blocked with bovine serum albumin (BSA).
  • Fc ⁇ receptor was added to the plate at 25ng/well and incubated at room temperature 1 hour. After washing the plate, a serial dilution of the test antibody is added as a polymer complex, and the plate is incubated at room temperature for 2 hours.
  • PBS phosphate buffered saline
  • ELx405 TM plate washer Biotek Instruments; Winooski, VT
  • HRP horseradish peroxidase
  • TMB tetramethylbenzidine
  • the plate is incubated at room temperature for 5-20 minutes to allow color development. Stop the reaction with IM H3PO4, and use the microplate reader ( 190, Molecular Devices; Sunnyvale, CA) measured absorbance at 450 nm.
  • a dose-response binding curve was generated by plotting the mean absorbance values from duplicate antibody dilutions against antibody concentration. The binding curve was fitted with a four-parameter equation using SoftMax 190 (Molecular Devices) to determine the effective antibody concentration (EC50) at which 50% of the maximum response from the bound Fc ⁇ receptor was detected.
  • SoftMax 190 Molecular Devices
  • the loss of membrane integrity shown by, for example, propidium iodide (PI), trypan blue or 7AAD uptake can be assessed relative to a control.
  • the PI uptake assay can be performed in the absence of complement and immune effector cells.
  • the cells expressing OX40 are incubated in a separate medium or a medium containing a suitable monoclonal antibody at a concentration of, for example, about 10 ⁇ g/ml.
  • the cells are incubated for a certain period of time (for example, 1 or 3 days). After each treatment, the cells were washed and aliquoted.
  • the cells are aliquoted into 35mm strainer-capped 12x75 tubes (1 ml per tube, 3 tubes per treatment group) to remove cell clumps. Then PI (10 ⁇ g/ml) was added to the tube. The samples can be analyzed using a FACSCAN TM flow cytometer and FACSCONVERT TM CellQuest software (Becton Dickinson).
  • Cells for use in any of the above in vitro assays include cells or cell lines that naturally express OX40 or have been engineered to express OX40. Such cells include activated T cells that naturally express OX40, Treg cells, and activated memory T cells. Such cells also include cell lines that express OX40 and cell lines that do not normally express OX40 but have been transfected with nucleic acid encoding OX40. Exemplary cell lines provided herein for use in any of the above in vitro assays include transgenic BT474 cells expressing human OX40 (a human breast cancer cell line).
  • immunoconjugates of the invention can be used to replace or supplement the anti-OX40 antibody to perform any of the aforementioned assays.
  • anti-OX40 antibodies and other therapeutic agents can be used to perform any of the aforementioned assays.
  • the antibody or antigen-binding fragment thereof of the present invention can be formulated into a pharmaceutical composition.
  • the pharmaceutical composition may further comprise pharmaceutically acceptable carriers, excipients and/or stabilizers (Remington: The Science and practice of Pharmacy, 20th Edition, 2000, Lippincott Williams and Wilkins, Ed. KE Hoover), and lyophilized Formulation or aqueous solution.
  • Acceptable carriers, excipients or stabilizers are non-toxic to the recipient at the dose and concentration, and may contain buffers such as phosphoric acid, citric acid and other organic acids; antioxidants, including ascorbic acid and methionine; Preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexahydroxy quaternary ammonium chloride; algaecide; benzonine chloride; phenolic alcohol, butanol or benzyl alcohol; alkyl p-hydroxybenzoate , Such as methyl p-hydroxybenzoate or propyl p-hydroxybenzoate; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) Polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulin; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such
  • the antibody or antigen-binding fragment thereof of the present invention can be used for various therapeutic or diagnostic purposes.
  • the antibody or antigen-binding fragment thereof of the present invention can be used as an affinity purification agent (for example, for in vitro purification); as a diagnostic agent (for example, for detecting expression in specific cells, tissues, or serum).
  • Exemplary therapeutic uses of the antibodies of the present invention or antigen-binding fragments thereof include the treatment of cancer.
  • the antibody or antigen-binding fragment thereof of the present invention can also be used for prophylactic treatment.
  • the antibodies or antigen-binding fragments of the present invention can be administered to mammals, especially humans, by conventional techniques, such as intravenous (as a bolus injection or by continuous infusion over time), intramuscular, Intra-abdominal, intracerebral, subcutaneous, intra-articular, intra-synovial, intrathecal, oral, topical or inhalation.
  • the antibody or antigen-binding fragment thereof of the present invention can also be appropriately administered via intratumor, peritumor, intralesional, or perilesional routes.
  • the antibodies of the invention or antigen-binding fragments thereof are administered subcutaneously. In certain embodiments, the antibodies of the invention or antigen-binding fragments thereof are administered intravenously.
  • the pharmaceutical composition can be administered to subjects in need at a frequency that can vary with the severity of the disease.
  • the frequency may vary depending on the subject's disease susceptibility or tendency.
  • composition can be administered as a bolus injection or by continuous infusion to patients in need.
  • bolus administration of antibodies presented as Fab fragments can be administered from an amount of 0.0025 to 100 mg/kg body weight, 0.025 to 0.25 mg/kg, 0.010 to 0.10 mg/kg, or 0.10 to 0.50 mg/kg.
  • antibodies presented as Fab fragments can be 0.001 to 100 mg/kg body weight/min, 0.0125 to 1.25 mg/kg/min, 0.010 to 0.75 mg/kg/min, 0.010 to 1.0 mg/kg/min, or It is administered in an amount of 0.10 to 0.50 mg/kg/min for 1 to 24 hours, 1 to 12 hours, 2 to 12 hours, 6 to 12 hours, 2 to 8 hours, or 1 to 2 hours.
  • the dose can be from about 1 mg/kg to about 10 mg/kg, from about 2 mg/kg to about 10 mg/kg, from about 3 mg/kg to About 10 mg/kg, from about 4 mg/kg to about 10 mg/kg, from about 5 mg/kg to about 10 mg/kg, from about 1 mg/kg to about 20 mg/kg, from about 2 mg/kg to about 20 mg/kg, from About 3mg/kg to about 20mg/kg, from about 4mg/kg to about 20mg/kg, from about 5mg/kg to about 20mg/kg, about 1mg/kg or more, about 2mg/kg or more, about 3mg /kg or more, about 4mg/kg or more, about 5mg/kg or more, about 6mg/kg or more, about 7mg/kg or more, about 8mg/kg or more, about 9mg/kg Or more, about 10 mg/kg or more, about 11 mg
  • the composition can be administered to the patient via subcutaneous injection.
  • the dose of 1 to 100 mg of anti-OX40 antibody can be injected subcutaneously or intravenously at twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, every It is administered to the patient at a frequency of once every seven weeks, once every eight weeks, once every nine weeks, once every ten weeks, twice a month, once a month, once every two months, or once every three months.
  • the half-life of an anti-OX40 antibody in humans is about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days. Days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 22 days, about 23 days, about 24 days, about 25 days, About 26 days, about 27 days, about 28 days, about 29 days, about 30 days, from about 5 days to about 40 days, from about 5 days to about 35 days, from about 5 days to about 30 days, from about 5 days From about 10 days to about 40 days, from about 10 days to about 35 days, from about 10 days to about 30 days, from about 10 days to about 25 days, from about 15 days to about 40 days , From about 15 days to about 35 days, from about 15 days to about 40 days , From about 15 days to about 35 days, from about 15 days to about 30 days, or from about 15 days to about 25 days.
  • the pharmaceutical composition is administered subcutaneously or intravenously every 2 to 6 weeks at the following dose: from about 0.1 mg/kg to about 10 mg/kg, from about 0.5 mg/kg to about 10 mg/kg kg, from about 1 mg/kg to about 10 mg/kg, from about 1.5 mg/kg to about 10 mg/kg, from about 2 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 8 mg/kg, from about 0.5mg/kg to about 8mg/kg, from about 1mg/kg to about 8mg/kg, from about 1.5mg/kg to about 8mg/kg, from about 2mg/kg to about 8mg/kg, from about 0.1mg/kg To about 5mg/kg, from about 0.5mg/kg to about 5mg/kg, from about 1mg/kg to about 5mg/kg, from about 1.5mg/kg to about 5mg/kg, from about 2mg/kg to about 5mg/kg kg, about 0.5mg/kg
  • the pharmaceutical composition is administered subcutaneously or intravenously at a dose of about 2.0 mg/kg every 2 to 6 weeks. In certain embodiments, the pharmaceutical composition is administered subcutaneously or intravenously every 2 to 6 weeks at a dose of from about 2.0 mg/kg to about 10.0 mg/kg.
  • the pharmaceutical composition is administered subcutaneously every 2 weeks.
  • the antibodies or antigen-binding fragments thereof of the present invention can be used as monotherapy or in combination with other therapies to treat cancer.
  • an "antigen-binding fragment" of an antibody refers to a fragment of a full-length antibody that retains the ability to specifically bind to an antigen (preferably, has substantially the same binding affinity).
  • antigen-binding fragments include (i) Fab fragments, which are monovalent fragments composed of VL, VH, CL and CH1 domains; (ii) F(ab')2 fragments, which are contained in the hinge region connected by disulfide bonds (Iii) Fd fragment composed of VH and CH1 domains; (iv) Fv fragment composed of VL and VH domains of one arm of an antibody; (v) dAb fragment (Ward et al.
  • VL and VH isolated complementarity determining regions
  • dsFv disulfide-linked Fvs
  • anti- Id antibodies and intracellular antibodies.
  • VL and VH isolated complementarity determining regions
  • the synthetic linker allows it to be made into a single protein chain, in which the VL and VH regions Pair to form a monovalent molecule (called single-chain Fv (scFv)); see, for example, Bird et al.
  • Diabodies are bivalent bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but the use of a linker that is too short to allow pairing between two chains on the same chain is used to force all The domain is paired with the complementary domain of the other chain and creates two antigen-binding sites (see, for example, Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993); Poljak et al., 1994, Structure 2:1121-1123).
  • variable domain refers to the variable region of the antibody light chain (VL) or the variable region of the antibody heavy chain (VH), alone or in combination.
  • VL variable region of the antibody light chain
  • VH variable region of the antibody heavy chain
  • CDR complementarity determining regions
  • FR framework regions
  • the residues in the variable domains are numbered according to Kabat, which is the numbering system of the heavy chain variable domain or the light chain variable domain used for the compilation of antibodies. See Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids, which correspond to the shortening or insertion of the FR or CDR of the variable domain.
  • the Kabat numbering of residues can be determined by aligning the antibody's sequence to regions of homology with the "standard" Kabat numbering sequence.
  • Various algorithms for assigning Kabat numbers are available. Unless otherwise stated in this article, this article uses the algorithm implemented in Abysis (www.abysis.org) released in 2012 to assign Kabat numbers to variable regions.
  • CDR complementarity determining region
  • the "complementarity determining region” can be identified according to the definition of the aggregation, AbM, contact and/or configuration of both Kabat, Chothia, Kabat and Chothia well known in the art or any method of CDR determination. See, for example, Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th edition (highly variable regions); Chothia et al., 1989, Nature 342:877-883 (structural loop structure).
  • the AbM definition of CDR is a compromise between Kabat and Chothia and the use of Oxford Molecular's AbM antibody modeling software.
  • the "contact" definition of CDR is based on the definition described in MacCallum et al., 1996, J. Mol.
  • CDR may refer to a CDR defined by any method (including a combination of methods) known in the art.
  • Antigenic determinant refers to the range or region in the antigen (Ag) where the antibody specifically binds, for example, the range or region containing the amino acid residues that interact with the antibody (Ab).
  • the epitope can be linear or non-linear (e.g., conformational).
  • the antibody or antigen-binding fragment thereof When the binding of corresponding antibodies or antigen-binding fragments thereof is mutually exclusive, the antibody or antigen-binding fragment thereof basically binds to the same epitope as another antibody or antigen-binding fragment thereof. That is, the binding of one antibody or antigen-binding fragment thereof excludes simultaneous or continuous binding of other antibodies or antigen-binding fragments thereof. If the antigen can be adapted to the simultaneous binding of two corresponding antibodies or antigen-binding fragments thereof, the epitope is considered to be unique or not substantially the same.
  • paratope is derived from the above definition of "antigenic determinant” by twisting the angle, and refers to a range or region involved in antigen binding in an antibody molecule, for example, a range or region containing residues that interact with the antigen .
  • Paratopes can be linear or conformational (such as discrete residues in CDRs).
  • the epitope/paratope of a given antibody/antigen binding pair can be defined and characterized at different levels of detail using various experimental and computational epitope positioning methods. Experimental methods include mutagenesis, X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, hydrogen/deuterium exchange mass spectrometry (HX-MS) and various competitive binding methods. Because each method relies on the principle of uniqueness, the description of the epitope is closely related to the method by which the epitope has been determined. Therefore, the epitope/paratope of a given antibody/antigen pair will be defined differently depending on the localization method employed.
  • the epitope/paratope used for the interaction between the antibody (Ab) and the antigen (Ag) can be defined by the spatial coordinates of the atomic contact existing in the Ag-Ab interaction and the binding of the pair. The relative contribution of thermodynamics is defined by the information.
  • epitope/paratope residues can be characterized by defining the spatial coordinates of the atomic contact between Ag and Ab.
  • the epitope/paratopic residues can be defined by specific criteria, such as the distance between atoms in Ab and Ag (e.g., from a heavy atom of a homologous antibody and a heavy atom of the antigen equal to or less than about approx.
  • epitope/paratope residues are characterized by participating in hydrogen bonds with homologous antibodies/antigens, or with water molecules that are also hydrogen-bonded to homologous antibodies/antibodies (via water-mediated hydrogen bonding) Interaction.
  • epitope/paratopic residues are characterized by forming salt bridges with homologous antibody/antigen residues.
  • epitope/paratopic residues can be characterized by Residues that have non-zero changes in the masked surface area (BSA) due to the interaction with the homologous antibody/antigen.
  • BSA masked surface area
  • epitopes/paratopes can be characterized by functions, for example, by Competitive binding with other Abs.
  • the epitope/paratope can also be more generally defined as containing amino acid residues, where the substitution of another amino acid will change the characteristics of the interaction between Ab and Ag (for example, C Amino acid scan).
  • epitopes Since the description and definition of epitopes depend on the epitope mapping method used and the facts obtained at different levels of detail, it is inferred that the comparison of epitopes of different Abs on the same Ag can be similar at different levels of detail To proceed. For example, if it is described at the amino acid level, such as an epitope determined from X-ray structure, if it contains the same amino acid residue group, it is considered to be the same. If the binding of the corresponding antibodies is mutually exclusive, that is, the binding of one antibody excludes the simultaneous or sequential binding of other antibodies, then the epitopes characterized by competitive binding are considered to be overlapping; and if the antigen can accommodate two corresponding If antibodies bind at the same time, it is considered that the epitopes are distinct (unique).
  • the epitope and paratope of a given antibody/antigen pair can be identified by routine methods. For example, the general location of the epitope can be determined by assessing the ability of the antibody to bind to different fragments or variant polypeptides, as described more fully previously herein.
  • the specific residues in OX40 that can be contacted with the specific residues in the antibody can also be determined using routine methods.
  • the antibody/antigen complex can be crystallized. The crystal structure can be determined and used to identify specific sites of interaction between the antibody and the antigen.
  • specific binding is a term well known in the art, and methods for determining these specific bindings are also well known in the art. If a molecule reacts or binds to a specific cell or substance more frequently, faster, has a longer duration, and/or has greater affinity than the molecule reacts or binds to a replacement cell or substance, it is considered that the molecule exhibits "specific binding". If the antibody or antigen-binding fragment thereof binds to the target with greater affinity, binding, easier and/or longer duration than other substances, the antibody or antigen-binding fragment thereof "specifically binds" to the target.
  • an antibody or antigen-binding fragment thereof that specifically binds to OX40 is that the antibody binds to its homologous antigen with greater affinity, binding, easier and/or longer duration than binding to other antigens.
  • an anti-OX40 antibody can specifically bind to human OX40 in a sample, but does not substantially recognize or bind to other molecules in the sample.
  • an antibody or antigen-binding fragment thereof that specifically binds to a first target may or may not specifically bind to a second target. Therefore, "specific binding” does not necessarily require (although it may include) exclusive binding. Usually, but not necessarily, the reference to "binding" means specific binding.
  • Various analysis modes can be used to select antibodies or antigen-binding fragments thereof that specifically bind to the molecule of interest.
  • solid-phase ELISA immunoassay, immunoprecipitation, Biacore TM (GE Healthcare), KinExA, fluorescence activated cell sorting (FACS), Octet TM (FortéBio, Inc.) and Western blot analysis can be used Recognize antibodies or antigen-binding fragments thereof that specifically bind to an antigen.
  • the specific binding will be at least twice the background signal or noise, more usually at least 10 times the background, at least 50 times the background, at least 100 times the background, at least 500 times the background, at least 1000 times the background, or background At least 10,000 times of that.
  • Antibody binding specificities can be K D K D value and comparing the value with K D values to a control antibody known not to bind to OX40 evaluated by measuring the specific binding between the antibody and OX40.
  • K D is about ⁇ 10 -5 M or less, it is considered that the antibody "specifically" binds to the antigen.
  • the antibody or its antigen-binding fragment When compared to an antibody or its antigen-binding fragment that binds to other antigens, the antibody or its antigen-binding fragment does not bind to an antigen with greater affinity, binding, easier and/or longer duration, the antibody or its antigen-binding fragment
  • the antigen-binding fragment "substantially does not bind" to the antigen.
  • the combination will not be more than twice the background signal or noise.
  • it combines with the K D of 1 ⁇ 10 -4 M or more, 1 ⁇ 10 -3 M or more, 1 ⁇ 10 -2 M or more, or 1 ⁇ 10 -1 M or more. antigen.
  • competitive as used herein with respect to antibodies means that the binding of a first antibody or antigen-binding portion thereof to an antigen reduces subsequent binding of a second antibody or antigen-binding portion thereof to the same antigen.
  • the binding of the first antibody produces steric hindrance, conformational change, or binding to a common epitope (or part thereof), so that the binding of the second antibody to the same antigen is reduced.
  • Standard competitive binding analysis can be used to determine whether two antibodies compete with each other.
  • Biacore technology which may use surface plasmon resonance (SPR) technology, usually using a biosensor system (such as a system) to measure the degree of interaction.
  • SPR can be used in an in vitro competitive binding inhibition assay to determine the ability of an antibody to inhibit the binding of a second antibody.
  • Another analysis for measuring antibody competition uses an ELISA-based method.
  • a high-throughput method for "grading" antibodies based on antibody competition is described in WO2003/48731. If one antibody or antigen-binding fragment thereof reduces the binding of another antibody or antigen-binding fragment to OX40, there is competition. For example, sequential binding competition analysis can be used, and different antibodies can be added sequentially.
  • the first antibody can be added to achieve near-saturated binding. Then, the second antibody is added. If the binding of the second antibody to OX40 cannot be detected or compared to the parallel analysis in the absence of the first antibody (where the value can be set to 100%), it is significantly reduced (e.g., at least about 10%, at least about 10%). 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% reduction), then the two antibodies are considered to be competing with each other .
  • K i Concentration at which 50% inhibition occurs is referred to as K i.
  • this K i is equal to K D.
  • the binding affinities associated with different molecular interactions can be compared by comparing the K D values for individual antibody/antigen complexes.
  • the K D value for antibodies or other binding partners can be determined using methods established in the art.
  • Fc fusion protein is a protein in which one or more polypeptides are operably linked to an Fc polypeptide.
  • Fc fusion combines the Fc region of an immunoglobulin and a fusion partner.
  • the "Fc region” can be a native sequence Fc region or a variant Fc region.
  • the human IgG heavy chain Fc region is usually defined as an amino acid residue at position Cys226 or extending from Pro230 to its carboxyl terminus.
  • the numbering of residues in the Fc region is the numbering with the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, Md., 1991.
  • the Fc region of immunoglobulin usually contains two constant domains (CH 2 and CH 3 ). As known in the art, the Fc region can exist in dimer or monomer form.
  • terapéuticaally effective amount means an amount of an anti-OX40 antibody or antigen-binding fragment thereof, or a combination comprising the antibody or antigen-binding fragment thereof, sufficient to achieve the intended purpose.
  • the precise amount will depend on many factors, including (but not limited to) the components and physical characteristics of the therapeutic composition, the expected patient population, individual patient precautions, etc., and can be determined by those skilled in the art.
  • treatment includes prophylactic and/or therapeutic treatment. If administered before the clinical manifestation of the disease, disorder, or condition, the treatment is considered prophylactic.
  • Therapeutic treatment includes, for example, reducing or attenuating the severity of or shortening the length of the disease, disorder, or condition.
  • any of the anti-human OX40 antibodies provided herein can be used in treatment methods.
  • an anti-human OX40 agonistic antibody is provided for use as a medicine.
  • anti-human OX40 agonistic antibodies are provided for use in the treatment of cancer.
  • anti-human OX40 agonistic antibodies are provided for use in methods of treatment.
  • an anti-human OX40 agonist antibody is provided for use in a method of treating an individual with cancer, comprising administering an effective amount of the anti-human OX40 agonist antibody to the individual. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, such as described below.
  • an anti-human OX40 agonistic antibody for use in enhancing immune function (for example, by up-regulating a cell-mediated immune response) in an individual with cancer, including administering an effective amount of the anti-human OX40 to the individual Agonistic antibodies.
  • an anti-human OX40 agonistic antibody for enhancing T cell function in an individual with cancer including administering an effective amount of the anti-human OX40 agonistic antibody to the individual.
  • an anti-human OX40 agonistic antibody which is used to deplete human OX40-expressing cells (for example, OX40-expressing T cells, such as OX40-expressing Treg), including administering an effective amount of the anti-human OX40 to the individual Agonistic antibodies.
  • the abatement is performed by ADCC.
  • depletion is by phagocytosis.
  • an anti-human OX40 agonistic antibody which is used to treat individuals with tumor immunity.
  • anti-human OX40 agonistic antibodies are provided for use in the treatment of infections (eg, bacterial or viral or other pathogen infections).
  • infections eg, bacterial or viral or other pathogen infections.
  • the present invention provides an anti-human OX40 agonist antibody for use in a method of treating an individual with an infection, comprising administering an effective amount of the anti-human OX40 agonist antibody to the individual.
  • the infection is a viral and/or bacterial infection.
  • the infection is a pathogen infection.
  • the present invention provides the use of anti-OX40 antibody to manufacture or prepare medicine.
  • the drug is used to treat cancer.
  • the medicament is used in a method of treating cancer, which comprises administering an effective amount of the medicament to an individual with cancer. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, such as described below.
  • the drug is used to enhance immune function in an individual with cancer (for example, by up-regulating a cell-mediated immune response), which includes administering an effective amount of the drug to the individual.
  • the drug is used to enhance T cell function in an individual with cancer, which includes administering an effective amount of the drug to the individual.
  • the T cell dysfunctional disorder is cancer.
  • the drug is used to deplete human OX40 expressing cells (eg, high OX40 expressing cells, such as OX40 expressing T cells), which includes administering an effective amount of the drug to the individual.
  • the abatement is performed by ADCC.
  • depletion is by phagocytosis.
  • the drug is used to treat individuals with tumor immunity.
  • medicaments are provided for use in the treatment of infections (e.g., bacterial or viral or other pathogen infections).
  • the method of treating an individual with an infection by the medicament comprises administering an effective amount of the medicament to the individual.
  • the infection is a viral and/or bacterial infection.
  • the infection is a pathogen infection.
  • the present invention provides methods for treating cancer.
  • the method includes administering an effective amount of an anti-OX40 antibody to an individual with such cancer.
  • the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, such as described below.
  • the "individual" according to any of the above embodiments may be a human.
  • a method for enhancing T cell function in an individual with cancer comprising administering to the individual an effective amount of the anti-human OX40 agonist antibody.
  • a method for depleting cells expressing human OX40 for example, cells expressing high levels of OX40, such as T cells expressing OX40
  • the abatement is performed by ADCC.
  • depletion is by phagocytosis.
  • an anti-human OX40 agonistic antibody which is used to treat individuals with tumor immunity.
  • examples of cancer further include, but are not limited to, B-cell lymphoma (including low-grade/follicular non-Hodgkin’s lymphoma (NHL), small lymphocytic (SL) NHU intermediate/follicular NHL , Intermediate diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small nonnucleoblastic NHL, bulky disease NHL, mantle cell lymphoma, AIDS-related lymphoma, and Val Denstrom's (Waldenstrom) macroglobulinemia), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and post-transplant lymph Proliferative disorders (PTLD), and abnormal blood vessel proliferation associated with phakomatoses, edema (such as those associated with brain tumors), B cell proliferative disorders, and Meigs syndrome.
  • B-cell lymphoma including low-grade/follicular non
  • More specific examples include, but are not limited to, relapsed or refractory NHL, front-line low-grade NHL, stage III/IV NHL, chemotherapy-resistant NHL, precursor B lymphoblastic leukemia and/or lymphoma, small lymphoma Cellular lymphoma, B-cell chronic lymphocytic leukemia and/or prolymphocytic leukemia and/or small lymphocytic lymphoma, B-cell prelymphocytic lymphoma, immunocytoma and/or lymphoplasmacytic ( lymphoplasmacytic lymphoma, lymphoplasmacytic lymphoma, marginal zone B-cell lymphoma, splenic marginal zone lymphoma, extranodal marginal zone_MALT lymphoma, nodal marginal zone lymphoma, hair Cellular Leukemia, Cytocytoma and/or Plasma Cell Myeloma, Low Grade/Follicular Lymphoma, Intermediate/Follicular NHL, Mantle Cell Lymphoma, Folli
  • examples of cancer further include, but are not limited to, B-cell proliferative disorders, which further include, but are not limited to, lymphoma (eg, B-cell non-Hodgkin's lymphoma (NHL)) and lymphocytic leukemia.
  • lymphoma eg, B-cell non-Hodgkin's lymphoma (NHL)
  • NHL lymphocytic leukemia
  • lymphomas and lymphocytic leukemias include, for example, a) follicular lymphoma, b) small Non-Cleaved Cell Lymphoma/Burkitt’s lymphoma (including endemic Burkitt’s lymphoma, sporadic Burkitt’s lymphoma and non-Burkitt’s lymphoma), c) marginal zone lymphoma (including extranodal marginal zone B-cell lymphoma (mucosa-associated lymphoid tissue lymphoma, MALT) ), nodal marginal zone B-cell lymphoma and splenic marginal zone lymphoma), d) mantle cell lymphoma (MCL), e) large cell lymphoma (including B-cell diffuse large cell lymphoma (DLCL), diffuse mixed Cell lymphoma, immunoblastic lymphoma, primary mediastinal B-cell lymphoma, angiocentric lymphoma-pulmonary B-cell lympho
  • the cancer is a B cell proliferative disorder.
  • the B-cell proliferative disorder is lymphoma, non-Hodgkin's lymphoma (NHL), aggressive NHL, recurrent aggressive NHL, recurrent painless NHL, refractory NHL , Refractory painless NHL, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL), or mantle cell lymphoma.
  • NHL such as painless NHL and/or aggressive NHL.
  • the B-cell proliferative disorder is painless follicular lymphoma or diffuse large B-cell lymphoma.
  • the present invention provides a pharmaceutical formulation comprising any anti-OX40 antibody provided herein, for example for use in any of the above-mentioned treatment methods.
  • the pharmaceutical formulation comprises any anti-OX40 antibody provided herein and a pharmaceutically acceptable carrier.
  • the pharmaceutical formulation comprises any anti-OX40 antibody provided herein and at least one additional therapeutic agent, such as described below.
  • the anti-human OX40 agonist antibody kills OX40-expressing cells (for example, cells expressing high levels of OX40) by inhibiting Treg function (for example, inhibiting the suppressive function of Treg), and increasing Effector T cell function and/or improve memory T cell function to suppress tumor immunity.
  • the anti-human OX40 agonist antibody kills OX40-expressing cells (for example, cells expressing high levels of OX40) by inhibiting Treg function (for example, inhibiting the suppressive function of Treg), and increasing Effector T cell function and/or improve memory T cell function to treat cancer.
  • the anti-human OX40 agonist antibody kills OX40-expressing cells (for example, cells expressing high levels of OX40) by inhibiting Treg function (for example, inhibiting the suppressive function of Treg), and increasing Effector T cell function and/or improve memory T cell function to enhance immune function.
  • the anti-human OX40 agonist antibody kills OX40-expressing cells (for example, cells expressing high levels of OX40) by inhibiting Treg function (for example, inhibiting the suppressive function of Treg), and increasing Effector T cell function and/or improve memory T cell function to enhance T cell function.
  • the anti-human OX40 agonistic antibody is a subtractive anti-human OX40 agonistic antibody.
  • the anti-human OX40 agonist antibody treatment results in cell depletion (e.g., depletion of cells expressing OX40, such as depletion of cells expressing high levels of OX40).
  • the abatement is performed by ADCC.
  • depletion is by phagocytosis.
  • the anti-human OX40 agonist antibody may, for example, inhibit effector and/or memory T cell function (in some embodiments, effector T cell function) relative to the Treg function prior to administration of the OX40 agonist antibody. And/or memory T cell proliferation and/or cytokine secretion) Treg suppression to inhibit Treg function.
  • the anti-human OX40 agonist antibody increases effector T cell proliferation relative to effector T cell proliferation prior to administration of the OX40 agonist antibody.
  • the anti-human OX40 agonist antibody increases memory T cell proliferation relative to the memory T cell proliferation prior to administration of the OX40 agonist antibody.
  • the anti-human OX40 agonist antibody increases effector T cell cytokine production (eg, gamma-interferon production) relative to effector T cell cytokine production prior to administration of the OX40 agonist antibody. In some embodiments of any method, the anti-human OX40 agonist antibody increases memory T cell cytokine production (eg, ⁇ -interferon production) relative to memory T cell cytokine production prior to administration of the OX40 agonist antibody.
  • the anti-human OX40 agonist antibody increases CD4+ effector T cell proliferation and/or CD8+ relative to CD4+ effector T cell proliferation and/or CD8+ effector T cell proliferation prior to administration of the OX40 agonist antibody Effector T cell proliferation. In some embodiments of any method, the anti-human OX40 agonist antibody increases memory T cell proliferation (e.g., CD4+ memory T cell proliferation) relative to memory T cell proliferation prior to administration of the OX40 agonist antibody.
  • memory T cell proliferation e.g., CD4+ memory T cell proliferation
  • the CD4+ effector T cells in the individual have enhanced proliferation, cytokine secretion and/or lysis. Cell activity.
  • the number of CD4+ effector T cells is increased relative to before administration of the anti-human OX40 agonist antibody. In some embodiments, CD4+ effector T cell cytokine secretion is increased relative to before administration of the anti-human OX40 agonist antibody. In some embodiments of any method, the CD8+ effector T cells in the individual have enhanced proliferation, cytokine secretion, and/or lytic activity relative to prior to administration of the anti-human OX40 agonistic antibody. In some embodiments, the number of CD8+ effector T cells is increased relative to before administration of the anti-human OX40 agonistic antibody. In some embodiments, CD8+ effector T cell cytokine secretion is increased relative to before administration of the anti-human OX40 agonist antibody.
  • the anti-human OX40 agonist antibody binds to human effector cells, for example, to Fc ⁇ R expressed by human effector cells.
  • the human effector cell performs ADCC effector function.
  • the human effector cell performs phagocytic effector function.
  • the anti-human OX40 agonist antibody comprising a variant IgG1Fc polypeptide (which contains a mutation that eliminates binding to human effector cells, such as the DANA or N297G mutation) has a portion relative to the IgG1Fc portion containing the native sequence
  • the anti-human OX40 agonistic antibody reduces the activity (eg CD4+ effector T cell function, such as proliferation).
  • an anti-human OX40 agonist antibody comprising a variant IgG1Fc polypeptide (which contains a mutation that eliminates binding to human effector cells, such as the DANA or N297G mutation) does not possess substantial activity (e.g., CD4+ effector T cell function, Such as proliferation).
  • the anti-human OX40 agonistic antibody function requires antibody cross-linking.
  • the function is to stimulate the proliferation of CD4+ effector T cells.
  • antibody cross-linking is determined by providing an anti-human OX40 agonistic antibody adhered to a solid surface (e.g., cell culture plate).
  • antibody cross-linking is determined by introducing mutations (such as DANA or N297S mutations) in the IgG1 Fc portion of the antibody and testing the function of the mutant antibody.
  • the memory T cells in the individual have enhanced proliferation and/or cytokine secretion relative to prior to administration of the anti-human OX40 agonistic antibody.
  • the number of memory T cells is increased relative to before administration of the anti-human OX40 agonist antibody.
  • memory T cell cytokine secretion (level) is increased relative to before administration of the anti-human OX40 agonist antibody.
  • Tregs in the individual have reduced effector T cell function (e.g., proliferation and/or cytokine secretion) inhibition relative to prior to administration of the anti-human OX40 agonistic antibody.
  • the number of effector T cells is increased relative to before administration of the anti-human OX40 agonistic antibody. In some embodiments, effector T cell cytokine secretion (level) is increased relative to before administration of the anti-human OX40 agonist antibody.
  • the number of (infiltrating) CD4+ effector T cells in the tumor is relative to the administration of the anti-human OX40 Agonistic antibodies were previously elevated.
  • the number of (infiltrating) CD4+ effector T cells in the tumor expressing interferon-gamma e.g., total CD4+ cells expressing interferon-gamma, or, for example, total CD4+ cells
  • the percentage of CD4+ cells expressing ⁇ -interferon was increased relative to before administration of anti-human OX40 agonistic antibody.
  • the number of (infiltrating) CD8+ effector T cells in the tumor is relative to administration of anti-human OX40 agonism The antibody was previously elevated.
  • the number of (infiltrating) CD8+ effector T cells in a tumor expressing interferon-gamma is relative to Elevated before administration of anti-human OX40 agonistic antibody.
  • the number of (infiltrating) Tregs within the tumor is reduced relative to before administration of the anti-human OX40 agonistic antibody.
  • the administration of the anti-human OX40 agonistic antibody is combined with the administration of the tumor antigen.
  • the tumor antigen comprises a protein.
  • the tumor antigen comprises nucleic acid.
  • the tumor antigen is a tumor cell.
  • the cancer displays human effector cells (e.g., is infiltrated by human effector cells).
  • the methods used to detect human effector cells are well known in the art and include, for example, by IHC.
  • the cancer displays high levels of human effector cells.
  • the human effector cells are one or more of NK cells, macrophages, and monocytes.
  • the cancer is any cancer described herein.
  • the cancer is non-small cell lung cancer (NSCLC), glioblastoma, neuroblastoma, melanoma, breast cancer (e.g. triple negative breast cancer), gastric cancer, colorectal cancer (CRC), Or hepatocellular carcinoma.
  • the cancer displays FcR-expressing cells (e.g., is infiltrated by FcR-expressing cells).
  • Methods for detecting FcR are well known in the art and include, for example, by IHC.
  • the cancer displays high levels of FcR-expressing cells.
  • FcR is FcyR.
  • the FcR is an activating Fc ⁇ R.
  • the cancer is non-small cell lung cancer (NSCLC), glioblastoma, neuroblastoma, melanoma, breast cancer (e.g. triple negative breast cancer), gastric cancer, colorectal cancer (CRC), Or hepatocellular carcinoma.
  • NSCLC non-small cell lung cancer
  • glioblastoma glioblastoma
  • neuroblastoma melanoma
  • breast cancer e.g. triple negative breast cancer
  • CRC colorectal cancer
  • the "individual” according to any of the above embodiments is preferably a human.
  • the antibodies of the present invention can be used alone or in combination with other agents in therapy.
  • the antibody of the invention can be co-administered with at least one other therapeutic agent.
  • Such combination therapies documented above encompass combined administration (wherein two or more therapeutic agents are contained in the same formulation or separate formulations), and separate administration, in which case the administration of another therapeutic agent And/or the administration of the antibody of the present invention occurs before, at the same time, and/or after the agent.
  • the administration of the anti-OX40 antibody and the administration of the other therapeutic agent are within about one month, or within about one, two or three weeks, or within about 1, 2, 3, 4, 5, or 6 days of each other occur.
  • the antibodies of the present invention can also be used in combination with radiation therapy.
  • the anti-human OX40 agonist antibody can be administered in combination with chemotherapy or chemotherapeutic agents. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with radiotherapy or radiotherapy agents. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with a targeted therapy or a targeted therapeutic agent. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with immunotherapy or immunotherapeutic agents, such as monoclonal antibodies.
  • the anti-human OX40 agonist antibody can be combined with PARP inhibitors (eg Olaparanib, Rucaparib, Niraparib, Cediranib, BMN673, Veliparib), Trabectedin, nab-paclitaxel (albumin-bound paclitaxel, ABRAXANE), Trebananib , Pazopanib, Cediranib, Palbociclib, everolimus, fluorouracil (e.g.
  • the anti-human OX40 agonist antibody can be administered in combination with a PD-1 axis binding antagonist.
  • PD-1 axis binding antagonists include but are not limited to PD-1 binding antagonists, PD-L1 binding antagonists and PD-L2 binding antagonists.
  • Alternative names for "PD-1” include CD279 and SLEB2.
  • Alternative names for "PD-L1” include B7-H1, B7-4, CD274 and B7-H.
  • Alternative names for "PD-L2" include B7-DC, Btdc, and CD273.
  • PD-1, PD-L1, and PD-L2 are human PD-1, PD-L1 and PD-L2.
  • the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partner.
  • the PD-1 ligand binding partner is PD-L1 and/or PD-L2.
  • the PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding partner.
  • the PD-L1 binding partner is PD-1 and/or B7-1.
  • the PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to its binding partner.
  • the PD-L2 binding partner is PD-1.
  • the antagonist can be an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide.
  • the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody).
  • the anti-PD-1 antibody is selected from the group consisting of MDX-1106 (nivolumab, OPDIVO), Merck 3475 (MK-3475, pembrolizumab, KEYTRUDAWPCT-011 (Pidilizumab).
  • PD-1 The binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region (e.g., the Fc region of an immunoglobulin sequence)).
  • the PD-1 binding antagonist is AMP-224.
  • the PD-L1 binding antagonist is an anti-PD-L1 antibody.
  • the anti-PD-L1 binding antagonist is selected From the following group: YW243.55.S70, MPDL3280A, MEDI4736 and MDX-1105.
  • MDX-1105 also known as BMS-936559
  • Antibody YW243.55.S70 is WO Anti-PD-L1 described in 2010/077634 A1.
  • MDX-1106 also known as MDX-1106-04, ONO-4538, BMS-936558 or nivolumab
  • Merck 3475 is also known as MK-3475, SCH-900475 or pembrolizumab, is the anti-PD-1 antibody described in WO2009/114335.
  • CT-011 also known as hBAT, hBAT-1 or pidil izumab
  • AMP-224 also known as B7-DCIg
  • the anti-PD-1 antibody is MDX-1106.
  • Alternative names for "MDX-1106” include MDX-1106-04, ON0-4538, BMS-936558 or nivoIumab.
  • the anti-PD-1 antibody is nivolumab (CAS Registry Number: 946414- 94-4).
  • the anti-human OX40 agonist antibody can be administered in combination with an agonist directed against an activating costimulatory molecule.
  • the activating costimulatory molecule may include CD40, CD226, CD28, GITR, CD137, CD27, HVEM, or CD127.
  • the agonist against the activating costimulatory molecule is an agonist antibody that binds CD40, CD226, CD28, OX40, GITR, CD137, CD27, HVEM, or CD127.
  • the anti-human OX40 agonist antibody can be administered in combination with an antagonist against an inhibitory costimulatory molecule.
  • inhibitory costimulatory molecules may include CTLA-4 (also known as CD152), PD-1,'I'IM-3, BTLA, VISTA, LAG-3, B7-H3, B7-H4, IDO, TIGIT, MICA/B, or arginase.
  • the antagonist for inhibitory costimulatory molecules binds CTLA-4, PD-1,'I'IM-3, BTLA, VISTA, LAG-3 (e.g., LAG-3-IgG fusion protein (IMP321 )), B7-H3, B7-H4, IDO, TIGIT, MICA/B, or arginase antagonist antibody.
  • the anti-human OX40 agonist antibody can be administered in combination with an antagonist against CTLA-4 (also known as CD152), such as a blocking antibody.
  • CTLA-4 also known as CD152
  • the anti-human OX40 agonist antibody can be combined with ipilimumab (also known as MDX-010, MDX-101, or ) Combined application.
  • the anti-human OX40 agonistic antibody can be administered in combination with tremelimumab (also known as ticilimumab or CP-675,206).
  • the anti-human OX40 agonist antibody can be administered in combination with an antagonist against B7-H3 (also known as CD276), such as a blocking antibody.
  • the anti-human OX40 agonistic antibody can be administered in combination with MGA271. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an antagonist against TGFP, such as metelimumaM (also known as CAT-192), fresolimumab (also known as GC1008), or LY2157299.
  • an antagonist against TGFP such as metelimumaM (also known as CAT-192), fresolimumab (also known as GC1008), or LY2157299.
  • the anti-human OX40 agonist antibody can be administered in combination with a treatment comprising adoptive transfer of T cells expressing chimeric antigen receptor (CAR) (eg, cytotoxic T cells or CTL).
  • CAR chimeric antigen receptor
  • the anti-human OX40 agonistic antibody can be administered in combination with UCART19.
  • the anti-human OX40 agonistic antibody can be administered in combination with WT128z.
  • the anti-human OX40 agonistic antibody can be administered in combination with KTE-C19 (Kite).
  • the anti-human OX40 agonistic antibody can be administered in combination with CTL019 (Novartis).
  • the anti-human OX40 agonist antibody can be administered in combination with a treatment comprising adoptive transfer of T cells containing a dominant negative TGFI3 receptor, for example, a dominant negative TGFWI type receptor.
  • the anti-human OX40 agonistic antibody can be administered in combination with treatments that include the HERCREEM regimen (see, for example, ClinicalTrials.gov Identifier NCT00889954).
  • the anti-human OX40 agonist antibody can be administered in combination with an antagonist against CD19. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with MOR00208. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an antagonist against CD38. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with daratumumab.
  • the anti-human OX40 agonist antibody can be administered in combination with an agonist against CD137 (also known as TNFRSF9, 4-1BB, or ILA), such as an activating antibody.
  • the anti-human OX40 agonistic antibody can be administered in combination with urelumab (also known as BMS-663513).
  • the anti-human OX40 agonist antibody can be administered in combination with an agonist against CD40, such as an activating antibody.
  • the anti-human OX40 agonistic antibody can be administered in combination with CP-870893.
  • an anti-human OX40 agonist antibody can be administered in combination with an agonist against OX40 (also known as CD134), such as an activating antibody.
  • anti-human OX40 agonistic antibodies can be administered in combination with different anti-OX40 antibodies (eg, AgonOX).
  • the anti-human OX40 agonist antibody can be administered in combination with an agonist against CD27, such as an activating antibody.
  • the anti-human OX40 agonistic antibody can be administered in combination with CDX-1127.
  • the anti-human OX40 agonist antibody can be administered in combination with an antagonist against indoleamine-2,3-dioxygenase (IDO).
  • the IDO antagonist is 1-methyl-D-tryptophan (also known as I-D-MT).
  • the anti-human OX40 agonist antibody can be administered in combination with an agonist against CD137 (also known as TNFRSF9, 4-1BB, or ILA), such as an activating antibody.
  • the anti-human OX40 agonistic antibody can be administered in combination with urelumab (also known as BMS-663513).
  • the anti-human OX40 agonist antibody can be administered in combination with an agonist against CD40, such as an activating antibody.
  • the anti-human OX40 agonistic antibody can be administered in combination with CP-870893 or R07009789.
  • an anti-human OX40 agonist antibody can be administered in combination with an agonist against OX40 (also known as CD134), such as an activating antibody.
  • the anti-human OX40 agonist antibody can be administered in combination with an agonist against CD27, such as an activating antibody.
  • the anti-human OX40 agonistic antibody can be administered in combination with CDX-1127 (also known as varlilumab).
  • the anti-human OX40 agonist antibody can be administered in combination with an antagonist against indoleamine-2,3-dioxygenase (IDO).
  • the IDO antagonist is 1-methyl-D-tryptophan (also known as 1-D-MT).
  • the IDO antagonist is the IDO antagonist shown in WO2010/005958 (the content is clearly included through the record here).
  • the IDO antagonist is 4-( ⁇ 2-[(aminosulfonyl)amino]ethyl ⁇ amino)-N_(3-bromo-4-fluorophenyl)-N'-hydroxy-1, 2,5-oxadiazole-3-carboxamidine (e.g. as described in Example 23 of WO2010/005958).
  • the IDO antagonist is 4-( ⁇ 2-[(aminosulfonyl)amino]ethyl ⁇ amino)-N_(3-bromo-4-fluorophenyl)-N'-hydroxy-1, 2,5-oxadiazole-3-carboxamidine (e.g. as described in Example 23 of WO2010/005958).
  • the IDO antagonist is
  • the IDO antagonist is INCB24360. In some embodiments, the IDO antagonist is Indoximod (the D isomer of 1-methyl-tryptophan).
  • the anti-human OX40 agonist antibody can be administered in combination with an antibody-drug conjugate. In some embodiments, the antibody-drug conjugate comprises mertansine or monomethyl auristatin E (MMAE). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an anti-NaPi2b antibody-MMAE conjugate (also known as DNIB0600A, RG7599 or Iifastuzumab vedotin).
  • the anti-human OX40 agonistic antibody can be combined with trastuzumab emtansine (also known as T-DM1, ado_trastuzumab emtansine, or KADCYL. Genentech) co-administered.
  • the anti-human OX40 agonist antibody can be administered in combination with the anti-MUC16 antibody-MMAE conjugate, DMUC5754A.
  • the anti-human OX40 agonist antibody can be administered in combination with the anti-MUC16 antibody-MMAE conjugate, DMUC4064A.
  • the anti-human OX40 agonist antibody can be administered in combination with an antibody-drug conjugate that targets the endothelin B receptor (EDNBR), such as an antibody against EDNBR conjugated with MMAE.
  • EDNBR endothelin B receptor
  • the anti-human OX40 agonistic antibody can be combined with an antibody-drug conjugate targeting lymphocyte antigen 6 complex, locus E (Ly6E), such as an antibody against Ly6E (also called Ly6E) conjugated with MMAE As DLYE5953A) combined administration.
  • the anti-human OX40 agonistic antibody can be administered in combination with polatuzumab vedotin.
  • the anti-human OX40 agonist antibody can be administered in combination with an antibody-drug conjugate that targets CD30.
  • the anti-human OX40 agonistic antibody can be administered in combination with ADCETRIS (also known as brentuximab vedotin).
  • the anti-human OX40 agonistic antibody can be administered in combination with polatuzumab vedotin.
  • the anti-human OX40 agonist antibody can be administered in combination with an angiogenesis inhibitor.
  • anti-human OX40 agonistic antibodies can be administered in combination with antibodies directed against VEGF, such as VEGF-A.
  • the anti-human OX40 agonist antibody can be combined with bevacizumab (also known as Genentech) co-administered.
  • the anti-human OX40 agonistic antibody can be administered in combination with an antibody against Angiopoietin 2 (also known as Ang2).
  • the anti-human OX40 agonistic antibody can be administered in combination with MEDI3617.
  • an anti-human OX40 agonist antibody can be administered in combination with an antibody against VEGFR2. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with ramucirumab. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with a VEGF receptor fusion protein. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with aflibercept. In some embodiments, the anti-human OX40 agonistic antibody can interact with ziv-aflibercept (also known as VEGF trap or ) Combined application. In some embodiments, anti-human OX40 agonistic antibodies can be administered in combination with bispecific antibodies directed against VEGF and Ang2.
  • the anti-human OX40 agonistic antibody can be administered in combination with RG7221 (also known as vanucizumab).
  • an anti-human OX40 agonist antibody can be combined with an angiogenesis inhibitor and a PD-1 axis binding antagonist (for example, a PD-1 binding antagonist such as an anti-PD-1 antibody, a PD-L1 binding antagonist such as Anti-PD-L1 antibody, and PD-L2 binding antagonist (such as anti-PD-L2 antibody) are administered in combination.
  • a PD-1 binding antagonist such as an anti-PD-1 antibody
  • a PD-L1 binding antagonist such as Anti-PD-L1 antibody
  • PD-L2 binding antagonist such as anti-PD-L2 antibody
  • the anti-human OX40 agonist antibody can be combined with bevacizumab and PD-1 axis binding antagonist (for example, PD-1 binding antagonist such as anti-PD-1 antibody, PD-L1 binding antagonist such as anti-PD-1 PD-L1 antibody, and PD-L2 binding antagonist such as anti-PD-L2 antibody) are administered in combination.
  • PD-1 axis binding antagonist for example, PD-1 binding antagonist such as anti-PD-1 antibody, PD-L1 binding antagonist such as anti-PD-1 PD-L1 antibody, and PD-L2 binding antagonist such as anti-PD-L2 antibody
  • the anti-human OX40 agonistic antibody can be administered in combination with bevacizumab and MDX-1106 (nivolumab, OPDIVO).
  • the anti-human OX40 agonistic antibody can be administered in combination with bevacizumab and Merck 3475 (MK-3475, pembrolizumab, KEYTRUDA). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with bevacizumab and CT-011 (Pidilizumab). In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with bevacizumab and YW243.55.S70. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with bevacizumab and MPDL3280A.
  • the anti-human OX40 agonist antibody can be administered in combination with bevacizumab and MEDI4736. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with bevacizumab and MDX-1105.
  • the anti-human OX40 agonist antibody can be administered in combination with an anti-tumor agent. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an agent that targets CSF-1R (also known as M-CSFR or CD115). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an anti-CSF-1R antibody (also known as IMC-CS4 or LY3022855). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with the anti-CSF-1R antibody, RG7155 (also known as R05509554 or emactuzumab).
  • anti-human OX40 agonistic antibodies can be administered in combination with interferons, such as interferon- ⁇ or interferon- ⁇ .
  • the anti-human OX40 agonistic antibody can be administered in combination with Roferon-A (also known as recombinant interferon a-2a).
  • the anti-human OX40 agonistic antibody can be combined with GM-CSF (also known as recombinant human granulocyte macrophage colony stimulating factor, rhu GM-CSF, sargramostim, or ) Combined application.
  • the anti-human OX40 agonistic antibody can interact with IL-2 (also known as aldesleukin or ) Combined application.
  • the anti-human OX40 agonistic antibody can be administered in combination with IL-12.
  • the anti-human OX40 agonistic antibody can be administered in combination with IL27.
  • the anti-human OX40 agonistic antibody can be administered in combination with IL-15.
  • the anti-human OX40 agonistic antibody can be administered in combination with ALT-803.
  • anti-human OX40 agonistic antibodies can be administered in combination with antibodies that target CD20.
  • the CD20-targeting antibody is onuzumab (also known as GAlO 1 or ) Or rituximab.
  • anti-human OX40 agonistic antibodies can be administered in combination with antibodies that target GITR.
  • the antibody that targets GITR is TRX518.
  • the antibody targeting GITR is MK04166 (Merck).
  • the anti-human OX40 agonist antibody can be administered in combination with an inhibitor of Bruton's tyrosine kinase (BTK). In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with ibrutinib. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an inhibitor of vaccine dehydrogenase I (IDH1) and/or vaccine dehydrogenase 2 (IDH2). In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with AG-120 (Agios).
  • IDH1 vaccine dehydrogenase I
  • IDH2 vaccine dehydrogenase 2
  • the anti-human OX40 agonistic antibody can be administered in combination with AG-120 (Agios).
  • the anti-human OX40 agonist antibody can be combined with onuzumab and PD-1 axis binding antagonist (for example, PD-1 binding antagonist such as anti-PD-1 antibody, PD-L1 binding antagonist such as anti-PD-1 PD-L1 antibody, and PD-L2 binding antagonist such as anti-PD-L2 antibody) are administered in combination.
  • PD-1 axis binding antagonist for example, PD-1 binding antagonist such as anti-PD-1 antibody, PD-L1 binding antagonist such as anti-PD-1 PD-L1 antibody, and PD-L2 binding antagonist such as anti-PD-L2 antibody
  • anti-human OX40 agonistic antibodies can be administered in combination with cancer vaccines.
  • the cancer vaccine is a peptide cancer vaccine, which in some embodiments is a personalized peptide vaccine.
  • the peptide cancer vaccine is a multivalent long peptide, a multipeptide, a peptide mixture, a hybrid peptide, or a peptide-pulsed dendritic cell vaccine (see, for example, Yamada et al., Cancer Sci, 104:14- 21, 2013).
  • the anti-human OX40 agonist antibody can be administered in combination with an adjuvant.
  • the anti-human OX40 agonist antibody can be combined with a TLR agonist, such as Poly-ICLC (also known as ), LPS, MPL, or CpG ODN treatments are administered in combination.
  • TLR agonist such as Poly-ICLC (also known as ), LPS, MPL, or CpG ODN treatments are administered in combination.
  • the anti-human OX40 agonist antibody can be administered in combination with tumor necrosis factor (TNF) a.
  • TNF tumor necrosis factor
  • the anti-human OX40 agonistic antibody can be administered in combination with IL-1.
  • the anti-human OX40 agonistic antibody can be administered in combination with HMGB1.
  • the anti-human OX40 agonist antibody can be administered in combination with an IL-10 antagonist.
  • the anti-human OX40 agonist antibody can be administered in combination with an IL-4 antagonist. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an IL-13 antagonist. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an IL-17 antagonist. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an HVEM antagonist. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an ICOS agonist (for example, by administering ICOS-L, or an agonistic antibody against ICOS). In some embodiments, anti-human OX40 agonistic antibodies can be administered in combination with treatments that target CX3CL1.
  • anti-human OX40 agonistic antibodies can be administered in combination with treatments that target CXCL9. In some embodiments, anti-human OX40 agonistic antibodies can be administered in combination with treatments that target CXCL10. In some embodiments, anti-human OX40 agonistic antibodies can be administered in combination with treatments that target CCL5. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an LFA-I or ICAM1 agonist. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with a selectin agonist.
  • the anti-human OX40 agonist antibody can be administered in combination with an inhibitor of B-Raf. In some embodiments, the anti-human OX40 agonistic antibody can be combined with Verofenib (also known as ) Combined application. In some embodiments, the anti-human OX40 agonistic antibody can be combined with dabrafenib (also known as ) Combined application. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with encorafenib (LGX818).
  • the anti-human OX40 agonist antibody can be administered in combination with an EGFR inhibitor. In some embodiments, the anti-human OX40 agonistic antibody can be combined with erlotinib (also known as ) Combined application. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an inhibitor of EGFR-T790M. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with gefitinib. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with afatinib. In some embodiments, the anti-human OX40 agonist antibody can be combined with cetuximab (also known as ) Combined application.
  • the anti-human OX40 agonist antibody can be combined with panitumumab (also known as ) Combined application. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with rociletinib. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with AZD9291. In some embodiments, anti-human OX40 agonistic antibodies can be administered in combination with MEK, such as MEK1 (also known as MAP2K1) and/or MEK2 (also known as MAP2K2) inhibitors.
  • MEK such as MEK1 (also known as MAP2K1) and/or MEK2 (also known as MAP2K2) inhibitors.
  • the anti-human OX40 agonistic antibody can be administered in combination with cobimetinib (also known as CDC-0973 or XL-518). In some embodiments, the anti-human OX40 agonistic antibody can be combined with trametinib (also known as ) Combined application. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with binimetinib.
  • the anti-human OX40 agonist antibody can be administered in combination with inhibitors of B-Raf (e.g., verofenib or dabrafenib) and inhibitors of MEK (e.g., MEK1 and/or MEK2) (e.g., cobimetinib or trametinib) .
  • the anti-human OX40 agonist antibody can be administered in combination with an inhibitor of ERK (eg, ERK1/2).
  • the anti-human OX40 agonistic antibody can be administered in combination with GDC-0994.
  • the anti-human OX40 agonist antibody can be administered in combination with an inhibitor of B-Raf, an inhibitor of MEK, and an inhibitor of ERK1/2. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an inhibitor of EGFR, an inhibitor of MEK, and an inhibitor of ERK1/2. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with one or more MAP kinase pathway inhibitors. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with CK127. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an inhibitor of K-Ras.
  • the anti-human OX40 agonist antibody can be administered in combination with an inhibitor of c-Met. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with onartuzumab (also known as MetMAb). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an inhibitor of anapatic lymphoma kinase (ALK). In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with AF802 (also known as CH5424802 or alectinib). In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with crizotinib.
  • ALK anapatic lymphoma kinase
  • AF802 also known as CH5424802 or alectinib
  • the anti-human OX40 agonistic antibody can be administered in combination with crizotinib.
  • the anti-human OX40 agonistic antibody can be administered in combination with ceritinib. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an inhibitor of phosphatidylinositol 3-kinase (PI3K). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with buparlisib (BKM-120). In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with pictilisib (also known as GDC-0941). In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with buparlisib (also known as BKM-120).
  • PI3K phosphatidylinositol 3-kinase
  • the anti-human OX40 agonist antibody can be administered in combination with perifosine (also known as KRX-0401). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with a delta selective inhibitor of phosphatidylinositol 3-kinase (PI3K). In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with idelalisib (also known as GS-1101 or CAL-101). In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with taselisib (also known as GDC-0032).
  • perifosine also known as KRX-0401
  • PI3K delta selective inhibitor of phosphatidylinositol 3-kinase
  • the anti-human OX40 agonistic antibody can be administered in combination with idelalisib (also known as GS-1101 or CAL-101). In some embodiments, the anti-human OX40
  • the anti-human OX40 agonistic antibody can be administered in combination with BYL-719. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an inhibitor of Akt. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with MK2206. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with GSK690693. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with ipatasertib (also known as CDC-0068). In some embodiments, anti-human OX40 agonistic antibodies can be administered in combination with inhibitors of mTOR.
  • the anti-human OX40 agonistic antibody can be administered in combination with sirolimus (also known as rapamycin). In some embodiments, the anti-human OX40 agonistic antibody can interact with temsirolimus (also known as CCI-779 or ) Combined application. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with everolimus (also known as RADOO1). In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with ridaforolimus (also known as AP-23573, MK_8669, or deforolimus). In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with OSI-027.
  • sirolimus also known as rapamycin
  • the anti-human OX40 agonistic antibody can interact with temsirolimus (also known as CCI-779 or ) Combined application.
  • the anti-human OX40 agonistic antibody can be administered in combination with everolimus
  • the anti-human OX40 agonistic antibody can be administered in combination with AZD8055. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with INK128. In some embodiments, anti-human OX40 agonistic antibodies can be administered in combination with dual PI3K/mTOR inhibitors. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with XL765. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with GDC-0980. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with BEZ235 (also known as NVP-BEZ235).
  • BEZ235 also known as NVP-BEZ235
  • the anti-human OX40 agonistic antibody can be administered in combination with BGT226. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with GSK2126458. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with PF-04691502. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with PF-05212384 (also known as PKI-587).
  • the anti-human OX40 agonistic antibody can be administered in combination with an agent that selectively degrades the estrogen receptor. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with GDC-0927. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an inhibitor of HER3. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with duligotuzumab. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an inhibitor of LSD1. In some embodiments, anti-human OX40 agonistic antibodies can be administered in combination with inhibitors of MDM2.
  • anti-human OX40 agonistic antibodies can be administered in combination with inhibitors of BCL2. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with venetoclax. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an inhibitor of CHK1. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with CDC-0575. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an inhibitor of the activated hedgehog signaling pathway. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with ERIVEDGE.
  • anti-human OX40 agonistic antibodies can be administered in combination with radiation therapy. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with gemcitabine. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with nab-pacIitaxel (ABRAXANE). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with trastuzumab. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with TVEC. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with IL27.
  • the anti-human OX40 agonist antibody can be administered in combination with cyclophosphamide. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with an agent that recruits T cells to the tumor. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with lirilumab (IPH2102/BMS-986015). In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with Idelalisib. In some embodiments, anti-human OX40 agonistic antibodies can be administered in combination with antibodies that target CD3 and CD20. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with REGN1979. In some embodiments, anti-human OX40 agonistic antibodies can be administered in combination with antibodies that target CD3 and CD19. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with blinatumomab.
  • the anti-human OX40 agonistic antibody can be administered in combination with an oncolytic virus. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with carboplatin and nab-paclitaxel. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with carboplatin and paclitaxel. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with cisplatin and pemetrexed. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with cisplatin and gemcitabine. In some embodiments, anti-human OX40 agonistic antibodies can be administered in combination with FOLFOX. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with FOLFIRI.
  • Such combination therapies documented above encompass combined administration (wherein two or more therapeutic agents are contained in the same formulation or separate formulations), and separate administration, in which case the administration of another therapeutic agent And/or the adjuvant before, at the same time, and/or after the administration of the antibody of the invention occurs.
  • the antibodies of the present invention can also be used in combination with radiation therapy.
  • the antibodies of the present invention can be administered by any suitable means (including parenteral, intrapulmonary, and intranasal, and if desired for local treatment, intralesional administration).
  • Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.
  • the dosing can be carried out by any suitable route (for example, by injection, such as intravenous or subcutaneous injection).
  • Various dosing schedules are encompassed herein, including but not limited to single administration or multiple administrations at multiple time points, bolus administration, and pulse infusion.
  • the antibody of the present invention will be formulated, dosed and administered in a manner consistent with excellent medical practice. Factors considered in this context include the specific condition being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the condition, the location of drug delivery, the method of administration, the schedule of administration, and other factors known to the medical practitioner.
  • the antibody need not be but is optionally formulated with one or more agents currently used to prevent or treat the condition in question.
  • the effective amount of such other drugs depends on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors mentioned above. These are generally used at the same dosage and route of administration as described herein, or at about 1-99% of the dosage described herein, or at any dosage and any route determined empirically/clinically to be suitable.
  • the appropriate dose of the antibody of the present invention (when used alone or in combination with one or more other therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity of the disease and The course of the disease, whether the antibody is administered for prevention or treatment, previous therapies, the patient's clinical history and response to the antibody, and the consideration of the attending physician.
  • the antibody is suitable for administration to a patient in one or a series of treatments. Depending on the type and severity of the disease, about 1 ⁇ g/kg to 40 mg/kg of antibody can be administered to the patient as an initial candidate dose, whether for example by one or more divided administrations or by continuous infusion.
  • a typical daily dose may be in the range of about 1 ⁇ g/kg to 100 mg/kg or more.
  • the treatment will usually continue until the desired suppression of disease symptoms occurs.
  • Such doses may be administered intermittently, for example every week or every three weeks (for example, so that the patient receives about 2 to about 20 doses, or for example about 6 doses of antibody).
  • a higher initial loading agent can be applied, followed by one or more lower doses.
  • other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.
  • immunoconjugates of the present invention can be used in place of or in addition to anti-OX40 antibodies to implement any of the aforementioned formulations or therapeutic methods.
  • NF- ⁇ B transcription factor binding elements in the promoter of the NF- ⁇ B-GFP reporter gene lentiviral system (QIAGEN, CCS-013G), which can drive the expression of GFP.
  • QIAGEN NF- ⁇ B transcription factor binding elements in the promoter of the NF- ⁇ B-GFP reporter gene lentiviral system
  • Jurkat cells ATCC, TIB-152 were infected with NF- ⁇ B-GFP reporter lentivirus, and Jurkat cells infected with lentivirus were screened with puromycin (InvivoGen, ant-pr-1).
  • TNF ⁇ protein 10ng/ml TNF ⁇ protein (Sino Biological, 10602-HNAE) was added to stimulate the cells selected with puromycin, and 24 hours later, the cell subsets that showed GFP fluorescence were sorted by flow cytometer (BD, FACSAria III) to obtain Jurkat/ NF- ⁇ B-GFP cells.
  • the DNA sequences of the positive controls OX40L and HEL used in this article were synthesized by Jinweizhi Company.
  • OX40L and HEL purification insert the DNA sequence into the eukaryotic expression vector pFUSE (InvivoGen, pfuse-hg1fc1), and use standard procedures to transfect 293F cells with PEI (Thermo Fisher Scientific, R79007); use Freestyle medium (Lifetechnologies, 12338026) ) Culture the cells on a shaker at 37°C. After 7 days of culture, the supernatant was collected and purified on the AKTA system (GE) using a Superdex TM 200 Increase prepacked column (GE, 28-9909-44).
  • OX40 signal transduction can activate the NF- ⁇ B pathway. If OX40 is successfully expressed on the surface of the Jurkat/NF- ⁇ B-GFP cell membrane, OX40L stimulation will induce the expression of GFP.
  • the constructed Jurkat/NF- ⁇ B-GFP cells sorted as described above were infected with the human OX40 lentiviral supernatant obtained above. After 16 hours, 100nM of the OX40L protein obtained above (as a positive control) and HEL (as a negative control) were added. Control) was stimulated, and 24 hours later, the cells with the strongest GFP fluorescence were sorted into a 96-well plate using a flow cytometer for single cell sorting.
  • the cells in the above 96-well plate were cultured in RPMI 1640 medium (Lifetechnologies, C11875500CP) containing 10% fetal bovine serum (Biological Industries, 04-001-1A) for 2 weeks, and 100 nM of the above was added after the single cells grew up.
  • RPMI 1640 medium Lifetechnologies, C11875500CP
  • HEL human serum
  • NF- ⁇ B-GFP+hOX40 select OX40L strongly positive and HEL negative monoclonal as Jurkat/NF- ⁇ B-GFP+hOX40 (referred to as NF- ⁇ B-GFP+hOX40) cells
  • NF- ⁇ B-GFP+hOX40 select OX40L strongly positive and HEL negative monoclonal as Jurkat/NF- ⁇ B-GFP+hOX40 cells
  • NF- ⁇ B-GFP+hOX40 select OX40L strongly positive and HEL negative monoclonal as Jurkat/NF- ⁇ B-GFP+hOX40 cells
  • the peripheral blood of 30 healthy adults was purchased from Tianjin Blood Center and Shanghai Miaotong Biological Company, and PBMCs were obtained by centrifugation with ficoll separation solution (Haoyang, LDS1075). Centrifugation conditions: 20°C, 2000rpm, up 5 down 0 mode for 30 minutes.
  • the PBMC obtained as described above was used to construct a human natural antibody library, and the library was constructed by a conventional method (phage display, Tim Clackson and Henry B. Lowman).
  • the obtained antibody heavy chain and light chain variable regions are randomly combined and displayed on the N-terminus of phage capsid protein pIII in the form of single-chain antibody scFv to obtain a phage display antibody library with a storage capacity of 10 10 .
  • Phage screening is a conventional technology (Phage Display: A Laboratory Manual, Carlos F Barbas III).
  • the biotinylated OX40 protein (acrobiosystems) was incubated with the phage display antibody library obtained as described above for 2 hours at room temperature. After the incubation, add 150ul streptavidin magnetic beads Dynabeads M280 (Lifetechnologies, 11205D) directly, and incubate on a mixer at room temperature for 30 min.
  • PBS-tween PBS: Lifetechnologies, 70011044; Tween: Sigma-Aldrich, 9005-64-5
  • PBS Lifetechnologies, 70011044; Tween: Sigma-Aldrich, 9005-64-5
  • PBS Lifetechnologies, 70011044; Tween: Sigma-Aldrich, 9005-64-5
  • PH2.2 glycine-hydrochloric acid
  • Bacteriophage conjugated The eluted phage was used to infect E. coli XL1-blue (Agilent, 200236), and the helper phage VCSM13 (Agilent, 200251) was added for amplification and used in the next round of screening. A total of three rounds of screening were carried out. After three rounds, the antibody phages that bind to CD40 were enriched, and the screening results are shown in Table 1.
  • An ELISA plate (Corning, 3690) was coated with 1 ⁇ g/ml of human OX40 (Acrobiosystems, OX0-H5224), and incubated overnight at 4°C. The overnight induced phage supernatant was added to the ELISA plate after overnight incubation, incubated for 1 h at room temperature, and washed 8 times with 0.05% PBST.
  • BSA Solarbio, A8020-100
  • HRP-conjugated anti-M13 GE, 27-9421-01, M13 is phage capsid protein
  • a positive clone was defined as the OX40 binding signal more than 3 times that of the BSA control. The results are shown in Figure 2. The positive rates of the second and third rounds of screening were 4.5% and 40.9%, respectively.
  • lentiviral core plasmid and helper plasmids pPACKH1-GAG, pPACKH1-REV, and pVSVG were co-transfected into 293FT cells at a ratio of 1:1:1:1.
  • the medium was changed and cultured at 37°C containing 10% fetal bovine serum ( In the DMEM medium of Biological Industries, 04-001-1A), continue to culture for 48 hours to collect the supernatant containing the anti-OX40 antibody lentiviral library; infect HEK293 cells with 50,000 pg of the anti-OX40 antibody lentiviral library, and after 16 hours of infection, Centrifuge to remove the medium containing the lentivirus, add fresh medium, and sort the infected HEK293 cells into a 96-well plate using flow sorting technology, so that each well contains only one infected HEK293 cell, and culture the cells for 3 weeks.
  • 10% fetal bovine serum In the DMEM medium of Biological Industries, 04-001-1A
  • the supernatant was added with 3 ⁇ 10 5 Jurkat/NF- ⁇ B-GFP+hOX40 reporter cells obtained as described above, and 2.5 ⁇ g/ml goat anti-human Fc (SouthernBiotech, SBA-2048-01) secondary antibody was added at the same time.
  • the pFUSE vector was sequenced, and the sequences of the positive antibodies 11-1, 2-1 and 2-2-1 were obtained.
  • the heavy chain and light chain coding nucleotide sequences of the anti-OX40 antibody were synthesized (see SEQ ID NO:49-60 for the respective constant region and variable region coding nucleotide sequences) (gold Weizhi Company), cloned them into the vector pFUSE, and transiently co-transfected the plasmids containing the heavy chain and the light chain into 293F suspension cells with PEI at a ratio of 1:1 to express the full-length antibody.
  • the AKTA system was used for purification with Superdex TM 200 Increase pre-packed column.
  • Biacore T200 (GE Healthcare) is used to detect the affinity of anti-OX40 antibodies.
  • the antibody was flowed through the Protein A chip at 10 ⁇ L/min and captured on the chip.
  • Use Running buffer HBS-EP+, GE
  • the concentration is set to 2 ⁇ M, 1 ⁇ M, 500nM, 250nM, 125nM, 62.5nM, 31.25nM.
  • ELISA was used to evaluate the effect of anti-OX40 antibody on the binding of OX40L and OX40.
  • the tested anti-OX40 antibodies significantly inhibited tumor growth relative to the IgG1 control.
  • the spleen and tumor of the mouse were stripped, and the spleen was directly ground and filtered with a 200-mesh nylon mesh to prepare a single cell suspension.
  • the tumor tissue was cut into small pieces and placed in 2ml enzyme mixture (each ml enzyme contains 1500U collagenase, hyaluronic acid) Acidase 1000U and DNase 2.5ul), shake in an incubator at 150rpm and 37°C for 1h. Filter and grind the incompletely digested tissue pieces with a 200 mesh nylon mesh to prepare a single cell suspension, centrifuge at 350g for 7 minutes, resuspend the cells with 2ml of red blood cell lysate, incubate on ice for 15 minutes, wash twice with PBS and count.
  • CD45+CD3+CD4+ is defined as CD4 + T cells
  • CD45+CD3+CD8+ is defined as CD8 + T cells.
  • the tested anti-OX40 antibodies significantly up-regulated helper CD4+ T cells and cytotoxic CD8+ T cells in the spleen and tumor relative to the control.
  • the spleen and tumor of the mouse were stripped and a single cell suspension was prepared.
  • the CD45, CD3, CD4 and CD8 antibodies were stained, and the ratio of CD4+ and CD8+ T cells was analyzed by flow cytometry.
  • CD45+CD3+CD4+ is defined as CD4+ T cells.
  • CD45+CD3+CD8+ is defined as CD8+ T cells.
  • Anti-OX40 antibodies can be divided into two types according to their different ways of agonizing.
  • the first type of OX40 agonistic activity does not depend on the crosslinking of Fc receptors; the other type requires Fc receptor crosslinking to have CD40 agonistic activity.
  • Fc receptor crosslinking There are more tumor-associated inflammatory cells in tumor tissues and surrounding draining lymph nodes, and Fc receptor Fc ⁇ R2b gathers more around tumor cells. Therefore, the "cross-linked antibody” agonist has higher tissue selectivity.
  • the antibody can produce obvious agonistic effects in the tumor microenvironment, while in the normal tissues of the body, the ability to act will be kept at a low level, so that it can be Improve the safety window of treatment.

Abstract

A novel anti-OX40 antibody, a composition comprising the anti-OX40 antibody, a nucleic acid encoding the anti-OX40 antibody, a method for preparing the anti-OX40 antibody, and use of the anti-OX40 antibody.

Description

抗OX40抗体及其用途Anti-OX40 antibody and its use 技术领域Technical field
本发明涉及尤其是新型抗OX40抗体，包含所述抗OX40抗体的组合物，编码所述抗OX40抗体的核酸，制备所述抗OX40抗体的方法，和所述抗OX40抗体的用途。The present invention relates in particular to a novel anti-OX40 antibody, a composition comprising the anti-OX40 antibody, a nucleic acid encoding the anti-OX40 antibody, a method for preparing the anti-OX40 antibody, and the use of the anti-OX40 antibody.
背景技术Background technique
具有实体肿瘤的患者中的抗肿瘤免疫应答已经通过使用生物制剂进行治疗而增强。例如，两种抗PD-1单克隆抗体：纳武单抗(nivolumab)
Figure PCTCN2020140259-appb-000001
和派姆单抗(pembrolizumab)
Figure PCTCN2020140259-appb-000002
已在美国和欧盟批准用于治疗不可切除或转移性黑色素瘤和转移性非小细胞肺癌等疾病。使用这些药物治疗患者已经产生通过无进展存活期和/或总体存活期的改善而衡量的抗肿瘤应答。本领域仍然需要更多的癌症治疗产品和方法以补充现有标准护理。 The anti-tumor immune response in patients with solid tumors has been enhanced by treatment with biological agents. For example, two anti-PD-1 monoclonal antibodies: nivolumab (nivolumab)
Figure PCTCN2020140259-appb-000001
And pembrolizumab
Figure PCTCN2020140259-appb-000002
It has been approved in the United States and the European Union for the treatment of diseases such as unresectable or metastatic melanoma and metastatic non-small cell lung cancer. Patients treated with these drugs have produced an anti-tumor response measured by improvement in progression-free survival and/or overall survival. The field still needs more cancer treatment products and methods to supplement the existing standard care.
PD-1和CTLA-4在T细胞激活过程中发挥免疫抑制作用，从而抑制T细胞对肿瘤细胞的免疫杀伤功能；因而针对这两个靶点的阻断性单克隆抗体可以解除这种免疫抑制，恢复T细胞抗肿瘤免疫功能。除了这样的抑制型免疫检查点分子之外，激活型免疫检查点分子正逐渐成为药物研发的新靶标。PD-1 and CTLA-4 play an immunosuppressive effect in the process of T cell activation, thereby inhibiting the immune killing function of T cells on tumor cells; therefore, blocking monoclonal antibodies against these two targets can relieve this immunosuppression , To restore the anti-tumor immune function of T cells. In addition to such inhibitory immune checkpoint molecules, activated immune checkpoint molecules are gradually becoming new targets for drug development.
激活型免疫检查点分子，主要指T细胞活化的共刺激信号分子-T细胞共刺激受体，属于肿瘤坏死因子受体(tumor necrosis factor receptor，TNFR)家族，用于调节T细胞的增殖、激活和分化，包括OX40、CD40、4-1BB和GITR等。Activated immune checkpoint molecules mainly refer to the costimulatory signal molecules of T cell activation-T cell costimulatory receptors, belonging to the tumor necrosis factor receptor (TNFR) family, used to regulate the proliferation and activation of T cells And differentiation, including OX40, CD40, 4-1BB and GITR.
OX40受体，也称为CD134和TNFRSF4(肿瘤坏死因子受体超家族成员4)，是TNFR超家族受体的成员，其不在静息初始T细胞上组成型表达，与CD28不同。OX40是次级共刺激免疫检查点分子，在激活后24至72小时表达；其配体OX40L(也成为CD252、TNFSF4)也不在静息抗原递呈细胞上表达，而是在其激活后表达。OX40的表达依赖于T细胞的完全激活。The OX40 receptor, also known as CD134 and TNFRSF4 (Tumor Necrosis Factor Receptor Superfamily Member 4), is a member of the TNFR superfamily receptor, which is not constitutively expressed on resting naive T cells, unlike CD28. OX40 is a secondary costimulatory immune checkpoint molecule that is expressed 24 to 72 hours after activation; its ligand OX40L (also known as CD252 and TNFSF4) is not expressed on resting antigen presenting cells, but is expressed after activation. The expression of OX40 depends on the complete activation of T cells.
OX40与其配体OX40L结合传递共刺激信号。OX40和OX40L的相互作用能够在OX40的胞内区域内招募TNFR相关(TRAFs)分子，形成包含IKKα和IKKβ以及PI3k和PKB(Akt)的信号传导复合物；OX40还与TCR信号协同作用，通过未知机制增强细胞内Ca 2+，从而增强NFAT入核。 OX40可激活经典的NF-κB1途径或非经典的NF-κB2途径、PI3k/PKB和NFAT途径，进而调控制T细胞***和存活的基因，以及促进细胞因子基因的转录以及细胞因子受体的表达，对于细胞存活至关重要。OX40信号传导会引起包括CTLA-4和Foxp3的下调。 OX40 and its ligand OX40L combine to deliver costimulatory signals. The interaction of OX40 and OX40L can recruit TNFR-related (TRAFs) molecules in the intracellular region of OX40 to form a signaling complex containing IKKα and IKKβ as well as PI3k and PKB (Akt); OX40 also cooperates with TCR signaling through unknown The mechanism enhances intracellular Ca 2+ , thereby enhancing NFAT into the nucleus. OX40 can activate the classic NF-κB1 pathway or the non-canonical NF-κB2 pathway, PI3k/PKB and NFAT pathways, thereby regulating the genes that control T cell division and survival, as well as promoting the transcription of cytokine genes and the expression of cytokine receptors , Is essential for cell survival. OX40 signaling can cause down-regulation including CTLA-4 and Foxp3.
当OX40与其配体OX40L结合时，有助于提高免疫***应答能力：1.增加效应T细胞和记忆T细胞的存活和扩增，增加细胞因子(例如IL-2、IL-4、IL-5、IFN-γ)的分泌；2.降低调节性T细胞的免疫抑制活性，进一步放大T细胞活化效应。在肿瘤微环境中，免疫激活可导致OX40表达。可增强效应T细胞的活化和增殖，并抑制调节性T细胞，从而导致复杂的抗肿瘤免疫反应。目前在Clinical Trials网站上已经可以检索到若干抗OX40抗体治疗癌症的临床项目。When OX40 is combined with its ligand OX40L, it helps to improve the response of the immune system: 1. Increase the survival and expansion of effector T cells and memory T cells, and increase cytokines (such as IL-2, IL-4, IL-5) , IFN-γ) secretion; 2. Reduce the immunosuppressive activity of regulatory T cells, and further amplify the effect of T cell activation. In the tumor microenvironment, immune activation can lead to the expression of OX40. It can enhance the activation and proliferation of effector T cells, and inhibit regulatory T cells, resulting in a complex anti-tumor immune response. At present, a number of clinical trials of anti-OX40 antibodies for cancer treatment can be retrieved on the Clinical Trials website.
本领域需要更多的新型抗OX40抗体以提供新的癌症治疗选择。The art needs more new anti-OX40 antibodies to provide new cancer treatment options.
发明内容Summary of the invention
本发明通过提供特异性结合和激活OX40的新型抗OX40抗体而满足了上述需要。The present invention meets the above needs by providing a novel anti-OX40 antibody that specifically binds and activates OX40.
在一个方面，本发明提供了一种分离的抗OX40抗体或其抗原结合片段，其包含：具有SEQ ID NO:1所示重链CDR1结构域，SEQ ID NO:2所示重链CDR2结构域，和SEQ ID NO:3所示重链CDR3结构域的重链可变区；和具有SEQ ID NO:9所示轻链CDR1结构域，SEQ ID NO:10所示轻链CDR2结构域，和SEQ ID NO:11所示轻链CDR3结构域的轻链可变区。In one aspect, the present invention provides an isolated anti-OX40 antibody or antigen-binding fragment thereof, comprising: a heavy chain CDR1 domain shown in SEQ ID NO: 1, and a heavy chain CDR2 domain shown in SEQ ID NO: 2. , And the heavy chain variable region of the heavy chain CDR3 domain shown in SEQ ID NO: 3; and having the light chain CDR1 domain shown in SEQ ID NO: 9, and the light chain CDR2 domain shown in SEQ ID NO: 10, and The light chain variable region of the light chain CDR3 domain shown in SEQ ID NO: 11.
在一个方面，本发明提供了一种分离的抗OX40抗体或其抗原结合片段，其包含：具有SEQ ID NO:17所示重链CDR1结构域，SEQ ID NO:18所示重链CDR2结构域，和SEQ ID NO:19所示重链CDR3结构域的重链可变区；和具有SEQ ID NO:25所示轻链CDR1结构域，SEQ ID NO:26所示轻链CDR2结构域，和SEQ ID NO:27所示轻链CDR3结构域的轻链可变区。In one aspect, the present invention provides an isolated anti-OX40 antibody or antigen-binding fragment thereof, comprising: a heavy chain CDR1 domain shown in SEQ ID NO: 17, and a heavy chain CDR2 domain shown in SEQ ID NO: 18 , And the heavy chain variable region of the heavy chain CDR3 domain shown in SEQ ID NO: 19; and having the light chain CDR1 domain shown in SEQ ID NO: 25, and the light chain CDR2 domain shown in SEQ ID NO: 26, and The light chain variable region of the light chain CDR3 domain shown in SEQ ID NO: 27.
在一个方面，本发明提供了一种分离的抗OX40抗体或其抗原结合片段，其包含：具有SEQ ID NO:33所示重链CDR1结构域，SEQ ID NO:34所示重链CDR2结构域，和SEQ ID NO:35所示重链CDR3结构域的重链可变区；和具有SEQ ID NO:41所示轻链CDR1结构域，SEQ ID NO:42所示轻链CDR2结构域，和SEQ ID NO:43所示轻链CDR3结构域的轻链可变区。In one aspect, the present invention provides an isolated anti-OX40 antibody or antigen-binding fragment thereof, comprising: a heavy chain CDR1 domain shown in SEQ ID NO: 33, and a heavy chain CDR2 domain shown in SEQ ID NO: 34 , And the heavy chain variable region of the heavy chain CDR3 domain shown in SEQ ID NO: 35; and having the light chain CDR1 domain shown in SEQ ID NO: 41, and the light chain CDR2 domain shown in SEQ ID NO: 42, and The light chain variable region of the light chain CDR3 domain shown in SEQ ID NO: 43.
在一个方面，本发明提供了一种抗体-药物缀合物，其包含本文所述的OX40抗体或其抗原结合片段和另外的治疗剂；优选地，所述抗OX40抗体 或其抗原结合片段与所述另外的治疗剂通过接头连接。In one aspect, the present invention provides an antibody-drug conjugate comprising the OX40 antibody or antigen-binding fragment thereof described herein and another therapeutic agent; preferably, the anti-OX40 antibody or antigen-binding fragment thereof is combined with The additional therapeutic agent is connected by a linker.
在一个方面，本发明提供了一种核酸，其编码本文所述的抗OX40抗体或其抗原结合片段。In one aspect, the present invention provides a nucleic acid that encodes the anti-OX40 antibody or antigen-binding fragment thereof described herein.
在一个方面，本发明提供了一种表达载体，其包含本文所述的核酸。In one aspect, the present invention provides an expression vector comprising the nucleic acid described herein.
在一个方面，本发明提供了一种宿主细胞，其包含本文所述的核酸或本文所述的表达载体。In one aspect, the present invention provides a host cell comprising the nucleic acid described herein or the expression vector described herein.
在一个方面，本发明提供了一种用于产生本文所述的抗OX40抗体或其抗原结合片段的方法，其包括在适合于抗体或其抗原结合片段表达的条件下培养本文所述的宿主细胞，和从培养基中回收表达的抗体或其抗原结合片段。In one aspect, the present invention provides a method for producing the anti-OX40 antibody or antigen-binding fragment thereof described herein, which comprises culturing the host cell described herein under conditions suitable for the expression of the antibody or antigen-binding fragment thereof , And recover the expressed antibody or its antigen-binding fragment from the culture medium.
在一个方面，本发明提供了一种药物组合物，其包含本文所述的抗OX40抗体或其抗原结合片段，或本文所述的抗体-药物缀合物，或本文所述的核酸，或本文所述的表达载体，及药学上可接受的载体。In one aspect, the present invention provides a pharmaceutical composition comprising the anti-OX40 antibody or antigen-binding fragment thereof described herein, or the antibody-drug conjugate described herein, or the nucleic acid described herein, or Said expression vector, and pharmaceutically acceptable carrier.
在一个方面，本发明提供了本文所述的抗OX40抗体或其抗原结合片段，或本文所述的抗体-药物缀合物，或本文所述的药物组合物，其用于治疗癌症。In one aspect, the present invention provides the anti-OX40 antibody or antigen-binding fragment thereof described herein, or the antibody-drug conjugate described herein, or the pharmaceutical composition described herein, for use in the treatment of cancer.
在一个方面，本发明提供了一种用于治疗癌症的方法，其包括向需要的受试者施用治疗有效量的本文所述的抗OX40抗体或其抗原结合片段，或本文所述的抗体-药物缀合物，或本文所述的药物组合物，从而治疗所述癌症。In one aspect, the present invention provides a method for treating cancer, which comprises administering to a subject in need a therapeutically effective amount of the anti-OX40 antibody or antigen-binding fragment thereof described herein, or the antibody described herein- A drug conjugate, or a pharmaceutical composition as described herein, to treat the cancer.
在一个方面，本发明提供了本文所述的抗OX40抗体或其抗原结合片段，或本文所述的抗体-药物缀合物，或本文所述的药物组合物，在制备用于治疗癌症的药物中的用途。In one aspect, the present invention provides the anti-OX40 antibody or antigen-binding fragment thereof described herein, or the antibody-drug conjugate described herein, or the pharmaceutical composition described herein, in the preparation of a drug for the treatment of cancer In the use.
在一个方面，本发明提供了本文所述的抗OX40抗体或其抗原结合片段，或本文所述的抗体-药物缀合物，或本文所述的药物组合物，其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。In one aspect, the present invention provides the anti-OX40 antibody or antigen-binding fragment thereof described herein, or the antibody-drug conjugate described herein, or the pharmaceutical composition described herein, which is used in one of the following or Multiple: Inhibition of Treg function (for example, suppression of Treg suppressive function), killing of OX40-expressing cells (for example, cells expressing high levels of OX40), improvement of effector T cell function and/or improvement of memory T cell function, reduction of tumor immunity, Enhance T cell function and/or deplete OX40-expressing cells.
在一个方面，本发明提供了本文所述的抗OX40抗体或其抗原结合片段，或本文所述的抗体-药物缀合物，或本文所述的药物组合物，在制备用于治疗下述一项或多项的药物中的用途：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。In one aspect, the present invention provides an anti-OX40 antibody or antigen-binding fragment thereof as described herein, or an antibody-drug conjugate as described herein, or a pharmaceutical composition as described herein, in preparation for the treatment of the following: Use of one or more drugs: inhibiting Treg function (for example, inhibiting the suppressive function of Treg), killing OX40-expressing cells (for example, cells expressing high levels of OX40), improving effector T cell function and/or improving memory T Cell function, reduce tumor immunity, enhance T cell function and/or reduce cells expressing OX40.
在一个方面，本发明提供了一种药物组合，其包含本文所述的抗OX40抗体或其抗原结合片段，或本文所述的抗体-药物缀合物，或本文所述的药物组合物，以及一种或多种另外的治疗剂。In one aspect, the present invention provides a pharmaceutical combination comprising the anti-OX40 antibody or antigen-binding fragment thereof described herein, or the antibody-drug conjugate described herein, or the pharmaceutical composition described herein, and One or more additional therapeutic agents.
在一个方面，本发明提供了一种试剂盒，其包括本文所述的抗OX40抗体或其抗原结合片段，或本文所述的抗体-药物缀合物，或本文所述的药物组合物，优选其进一步包括给药装置。In one aspect, the present invention provides a kit comprising the anti-OX40 antibody or antigen-binding fragment thereof described herein, or the antibody-drug conjugate described herein, or the pharmaceutical composition described herein, preferably It further includes a drug delivery device.
附图说明Description of the drawings
图1：500nM Fc-ILZ-OX40L刺激分选的Jurkat/NF-KappaB-GFP+hOX40报告细胞系单克隆，流式细胞分析刺激后细胞GFP表达阳性率。Figure 1: 500nM Fc-ILZ-OX40L stimulated sorting of Jurkat/NF-KappaB-GFP+hOX40 reporter cell line monoclonal, flow cytometry analysis of the positive rate of GFP expression in cells after stimulation.
图2：Phage ELISA法检测噬菌体单克隆与OX40重组蛋白的结合，计算噬菌体对OX40和噬菌体对BSA的OD450nm的比值。Figure 2: Phage ELISA method detects the binding of phage monoclonal to OX40 recombinant protein, and calculates the ratio of phage to OX40 and phage to BSA OD450nm.
图3：转染4种scFv-Fc表达质粒的HEK293FT细胞培养上清加入或不加入交联二抗，刺激Jurkat/NF-κB-GFP+hOX40报告细胞，流式细胞分析细胞GFP阳性率。Figure 3: HEK293FT cell culture supernatants transfected with 4 scFv-Fc expression plasmids with or without cross-linking secondary antibodies stimulated Jurkat/NF-κB-GFP+hOX40 reporter cells, and flow cytometry analyzed the GFP positive rate of cells.
图4：抗OX40抗体的聚集的分析。Figure 4: Analysis of aggregation of anti-OX40 antibodies.
图5：抗OX40抗体的体内抗肿瘤效力。抗OX40抗体相对于对照显著抑制肿瘤生长。Figure 5: Anti-tumor efficacy of anti-OX40 antibodies in vivo. The anti-OX40 antibody significantly inhibited tumor growth compared to the control.
图6：抗OX40抗体的T细胞调节效果。抗OX40抗体相对于对照在脾和肿瘤中明显上调辅助CD4+T细胞和细胞毒性CD8+T细胞。Figure 6: T cell regulation effect of anti-OX40 antibody. Compared with the control, the anti-OX40 antibody significantly upregulated helper CD4+T cells and cytotoxic CD8+T cells in the spleen and tumors.
图7：交联和可溶性抗OX40抗体的剂量依赖性作用。Figure 7: Dose-dependent effects of cross-linked and soluble anti-OX40 antibodies.
具体实施方式Detailed ways
在一个方面，本发明提供了一种分离的抗OX40抗体或其抗原结合片段，其包含：具有SEQ ID NO:1所示重链CDR1结构域，SEQ ID NO:2所示重链CDR2结构域，和SEQ ID NO:3所示重链CDR3结构域的重链可变区；和具有SEQ ID NO:9所示轻链CDR1结构域，SEQ ID NO:10所示轻链CDR2结构域，和SEQ ID NO:11所示轻链CDR3结构域的轻链可变区。In one aspect, the present invention provides an isolated anti-OX40 antibody or antigen-binding fragment thereof, comprising: a heavy chain CDR1 domain shown in SEQ ID NO: 1, and a heavy chain CDR2 domain shown in SEQ ID NO: 2. , And the heavy chain variable region of the heavy chain CDR3 domain shown in SEQ ID NO: 3; and having the light chain CDR1 domain shown in SEQ ID NO: 9, and the light chain CDR2 domain shown in SEQ ID NO: 10, and The light chain variable region of the light chain CDR3 domain shown in SEQ ID NO: 11.
在一个方面，本发明提供了一种分离的抗OX40抗体或其抗原结合片段，其包含：具有SEQ ID NO:17所示重链CDR1结构域，SEQ ID NO:18所示重链CDR2结构域，和SEQ ID NO:19所示重链CDR3结构域的重链可变区；和具有SEQ ID NO:25所示轻链CDR1结构域，SEQ ID NO:26所示轻链CDR2结构域，和SEQ ID NO:27所示轻链CDR3结构域的轻链可变区。In one aspect, the present invention provides an isolated anti-OX40 antibody or antigen-binding fragment thereof, comprising: a heavy chain CDR1 domain shown in SEQ ID NO: 17, and a heavy chain CDR2 domain shown in SEQ ID NO: 18 , And the heavy chain variable region of the heavy chain CDR3 domain shown in SEQ ID NO: 19; and having the light chain CDR1 domain shown in SEQ ID NO: 25, and the light chain CDR2 domain shown in SEQ ID NO: 26, and The light chain variable region of the light chain CDR3 domain shown in SEQ ID NO: 27.
在一个方面，本发明提供了一种分离的抗OX40抗体或其抗原结合片 段，其包含：具有SEQ ID NO:33所示重链CDR1结构域，SEQ ID NO:34所示重链CDR2结构域，和SEQ ID NO:35所示重链CDR3结构域的重链可变区；和具有SEQ ID NO:41所示轻链CDR1结构域，SEQ ID NO:42所示轻链CDR2结构域，和SEQ ID NO:43所示轻链CDR3结构域的轻链可变区。In one aspect, the present invention provides an isolated anti-OX40 antibody or antigen-binding fragment thereof, comprising: a heavy chain CDR1 domain shown in SEQ ID NO: 33, and a heavy chain CDR2 domain shown in SEQ ID NO: 34 , And the heavy chain variable region of the heavy chain CDR3 domain shown in SEQ ID NO: 35; and having the light chain CDR1 domain shown in SEQ ID NO: 41, and the light chain CDR2 domain shown in SEQ ID NO: 42, and The light chain variable region of the light chain CDR3 domain shown in SEQ ID NO: 43.
在一个实施方式中，本文所述的抗OX40抗体或其抗原结合片段包含SEQ ID NO:4所示重链可变区，或与SEQ ID NO:4所示序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的重链可变区；和SEQ ID NO:12所示轻链可变区，或与SEQ ID NO:12所示序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的轻链可变区。In one embodiment, the anti-OX40 antibody or antigen-binding fragment thereof described herein comprises the heavy chain variable region shown in SEQ ID NO: 4, or has at least 80%, 81%, and the sequence shown in SEQ ID NO: 4 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , 99% or 100% identical heavy chain variable region; and the light chain variable region shown in SEQ ID NO: 12, or at least 80%, 81%, 82%, and the sequence shown in SEQ ID NO: 12 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% Or a light chain variable region with 100% identity.
在一个实施方式中，本文所述的抗OX40抗体或其抗原结合片段包含SEQ ID NO:20所示重链可变区，或与SEQ ID NO:20所示序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的重链可变区；和SEQ ID NO:28所示轻链可变区，或与SEQ ID NO:28所示序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的轻链可变区。In one embodiment, the anti-OX40 antibody or antigen-binding fragment thereof described herein comprises the heavy chain variable region shown in SEQ ID NO: 20, or has at least 80%, 81%, and the sequence shown in SEQ ID NO: 20. 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , 99% or 100% identical heavy chain variable region; and the light chain variable region shown in SEQ ID NO: 28, or at least 80%, 81%, 82%, and the sequence shown in SEQ ID NO: 28 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% Or a light chain variable region with 100% identity.
在一个实施方式中，本文所述的抗OX40抗体或其抗原结合片段包含SEQ ID NO:36所示重链可变区，或与SEQ ID NO:36所示序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的重链可变区；和SEQ ID NO:44所示轻链可变区，或与SEQ ID NO:44所示序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的轻链可变区。In one embodiment, the anti-OX40 antibody or antigen-binding fragment thereof described herein comprises the heavy chain variable region shown in SEQ ID NO: 36, or has at least 80%, 81%, and the sequence shown in SEQ ID NO: 36. 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , 99% or 100% identical heavy chain variable region; and the light chain variable region shown in SEQ ID NO: 44, or at least 80%, 81%, 82%, and the sequence shown in SEQ ID NO: 44 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% Or a light chain variable region with 100% identity.
在一个实施方式，本文所述的抗OX40抗体或其抗原结合片段进一步包含重链恒定区和轻链恒定区；优选地，所述重链恒定区为SEQ ID NO:5或21所示重链恒定区，或与SEQ ID NO:5或21所示序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的重链恒定区；和/或优选地，所述轻链恒定区为SEQ ID NO:13所示轻链恒定区，或与SEQ  ID NO:13所示序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的轻链恒定区。In one embodiment, the anti-OX40 antibody or antigen-binding fragment thereof described herein further comprises a heavy chain constant region and a light chain constant region; preferably, the heavy chain constant region is the heavy chain shown in SEQ ID NO: 5 or 21 The constant region or the sequence shown in SEQ ID NO: 5 or 21 has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical heavy chain constant region; and/or preferably, the light chain constant region is The light chain constant region shown in SEQ ID NO: 13, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% with the sequence shown in SEQ ID NO: 13 , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical light chain constant region.
在一个实施方式中，本文所述的抗OX40抗体或其抗原结合片段进一步包含连接到所述重链可变区的重链信号肽和/或连接到所述轻链可变区的重链信号肽；优选地，所述重链信号肽为SEQ ID NO:6所示重链信号肽，或与SEQ ID NO:6所示序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的重链信号肽；和/或优选地，所述轻链信号肽为SEQ ID NO:14所示轻链信号肽，或与SEQ ID NO:14所示序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的轻链信号肽。In one embodiment, the anti-OX40 antibody or antigen-binding fragment thereof described herein further comprises a heavy chain signal peptide connected to the heavy chain variable region and/or a heavy chain signal connected to the light chain variable region Preferably, the heavy chain signal peptide is the heavy chain signal peptide shown in SEQ ID NO: 6, or has at least 80%, 81%, 82%, 83%, 84% with the sequence shown in SEQ ID NO: 6 , 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity And/or preferably, the light chain signal peptide is the light chain signal peptide shown in SEQ ID NO: 14, or is at least 80%, 81%, 82% with the sequence shown in SEQ ID NO: 14 %, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, A light chain signal peptide with 99% or 100% identity.
在一个实施方式中，本文所述的抗OX40抗体或其抗原结合片段为IgG抗体或其抗原结合片段，优选为IgG1抗体或其抗原结合片段。In one embodiment, the anti-OX40 antibody or antigen-binding fragment thereof described herein is an IgG antibody or an antigen-binding fragment thereof, preferably an IgG1 antibody or an antigen-binding fragment thereof.
在一个实施方式中，本文所述的抗OX40抗体或其抗原结合片段为单克隆抗体或其抗原结合片段。In one embodiment, the anti-OX40 antibody or antigen-binding fragment thereof described herein is a monoclonal antibody or antigen-binding fragment thereof.
在一个实施方式中，本文所述的抗OX40抗原结合片段为Fab、Fab'、F(ab')2、Fv、scFv或sdAb。In one embodiment, the anti-OX40 antigen-binding fragment described herein is Fab, Fab', F(ab')2, Fv, scFv, or sdAb.
在一个方面，本发明提供了一种抗体-药物缀合物，其包含根据本文所述的抗OX40抗体或其抗原结合片段和另外的治疗剂；优选地，所述抗OX40抗体或其抗原结合片段与所述另外的治疗剂通过接头连接。In one aspect, the present invention provides an antibody-drug conjugate comprising the anti-OX40 antibody or antigen-binding fragment thereof as described herein and another therapeutic agent; preferably, the anti-OX40 antibody or antigen-binding fragment thereof The fragment and the additional therapeutic agent are connected by a linker.
在一个方面，本发明提供了一种核酸，其编码本文所述的抗OX40抗体或其抗原结合片段。In one aspect, the present invention provides a nucleic acid that encodes the anti-OX40 antibody or antigen-binding fragment thereof described herein.
在一个实施方式中，本文所述的核酸包含：In one embodiment, the nucleic acid described herein comprises:
(a)SEQ ID NO:49所示重链可变区核苷酸编码序列和/或SEQ ID NO:51所示轻链可变区核苷酸编码序列；(a) The nucleotide coding sequence of the heavy chain variable region shown in SEQ ID NO: 49 and/or the nucleotide coding sequence of the light chain variable region shown in SEQ ID NO: 51;
(b)SEQ ID NO:53所示重链可变区核苷酸编码序列和/或SEQ ID NO:55所示轻链可变区核苷酸编码序列；或(b) The nucleotide coding sequence of the heavy chain variable region shown in SEQ ID NO: 53 and/or the nucleotide coding sequence of the light chain variable region shown in SEQ ID NO: 55; or
(c)SEQ ID NO:57所示重链可变区核苷酸编码序列和/或SEQ ID NO:59所示轻链可变区核苷酸编码序列；(c) The nucleotide coding sequence of the heavy chain variable region shown in SEQ ID NO: 57 and/or the nucleotide coding sequence of the light chain variable region shown in SEQ ID NO: 59;
优选地，所述核酸进一步包含SEQ ID NO:50或54所示重链恒定区核苷酸编码序列和/或SEQ ID NO:52所示轻链恒定区核苷酸编码序列。Preferably, the nucleic acid further comprises the heavy chain constant region nucleotide coding sequence shown in SEQ ID NO: 50 or 54 and/or the light chain constant region nucleotide coding sequence shown in SEQ ID NO: 52.
在一个方面，本发明提供了一种表达载体，其包含本文所述的核酸。In one aspect, the present invention provides an expression vector comprising the nucleic acid described herein.
在一个方面，本发明提供了一种宿主细胞，其包含本文所述的核酸或 表达载体。In one aspect, the present invention provides a host cell comprising the nucleic acid or expression vector described herein.
在一个方面，本文提供了一种用于产生本文所述的抗OX40抗体或其抗原结合片段的方法，其包括在适合于抗体或其抗原结合片段表达的条件下培养本文所述的宿主细胞，和从培养基中回收表达的抗体或其抗原结合片段。In one aspect, provided herein is a method for producing the anti-OX40 antibody or antigen-binding fragment thereof described herein, which comprises culturing the host cell described herein under conditions suitable for the expression of the antibody or antigen-binding fragment thereof, And recover the expressed antibody or its antigen-binding fragment from the culture medium.
在一个方面，本发明提供了一种药物组合物，其包含本文所述的抗OX40抗体或其抗原结合片段，或本文所述的抗体-药物缀合物，或本文所述的核酸，或本文所述的表达载体，及药学上可接受的载体。In one aspect, the present invention provides a pharmaceutical composition comprising the anti-OX40 antibody or antigen-binding fragment thereof described herein, or the antibody-drug conjugate described herein, or the nucleic acid described herein, or Said expression vector, and pharmaceutically acceptable carrier.
在一个实施方式中，本文所述的抗OX40抗体或其抗原结合片段，或本文所述的抗体-药物缀合物，或本文所述的药物组合物用于治疗癌症。在一个实施方式中，所述癌症选自鳞状细胞癌(例如上皮鳞状细胞癌)、肺癌(包括小细胞肺癌、非小细胞肺癌、肺的腺癌、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、***、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、***癌、外阴癌、甲状腺癌、肝癌，***癌、***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、水肿(例如与脑瘤有关的)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。In one embodiment, the anti-OX40 antibody or antigen-binding fragment thereof described herein, or the antibody-drug conjugate described herein, or the pharmaceutical composition described herein is used for the treatment of cancer. In one embodiment, the cancer is selected from squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), peritoneal cancer , Hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urethral 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, liver cancer, anal cancer, penile cancer, melanoma, superficial spreading melanoma, Malignant freckle melanoma, acral melanoma, nodular melanoma, multiple myeloma and B-cell lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), Hairy cell leukemia, chronic myeloblastic leukemia, and post-transplant lymphoproliferative disorder (PTLD), as well as those related to keloidosis, edema (e.g., related to brain tumors), and Meigs syndrome Abnormal blood vessel proliferation, brain tumors and brain cancers, and head and neck cancers, and related metastases.
在一个方面，本发明提供了一种用于治疗癌症的方法，其包括向需要的受试者施用治疗有效量的本文所述的抗OX40抗体或其抗原结合片段，或本文所述的抗体-药物缀合物，或本文所述的药物组合物，从而治疗所述癌症。在一个实施方式中，所述癌症选自鳞状细胞癌(例如上皮鳞状细胞癌)、肺癌(包括小细胞肺癌、非小细胞肺癌、肺的腺癌、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、***、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、***癌、外阴癌、甲状腺癌、肝癌，***癌、***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、水肿(例如与脑瘤有关的)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。In one aspect, the present invention provides a method for treating cancer, which comprises administering to a subject in need a therapeutically effective amount of the anti-OX40 antibody or antigen-binding fragment thereof described herein, or the antibody described herein- A drug conjugate, or a pharmaceutical composition as described herein, to treat the cancer. In one embodiment, the cancer is selected from squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), peritoneal cancer , Hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urethral 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, liver cancer, anal cancer, penile cancer, melanoma, superficial spreading melanoma, Malignant freckle melanoma, acral melanoma, nodular melanoma, multiple myeloma and B-cell lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), Hairy cell leukemia, chronic myeloblastic leukemia, and post-transplant lymphoproliferative disorder (PTLD), as well as those related to keloidosis, edema (e.g., related to brain tumors), and Meigs syndrome Abnormal blood vessel proliferation, brain tumors and brain cancers, and head and neck cancers, and related metastases.
在一个方面，本发明提供了本文所述的抗OX40抗体或其抗原结合片段，或本文所述的抗体-药物缀合物，或本文所述的药物组合物，在制备用于治疗癌症的药物中的用途。在一个实施方式中，所述癌症选自鳞状细胞癌(例如上皮鳞状细胞癌)、肺癌(包括小细胞肺癌、非小细胞肺癌、肺的腺癌、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、***、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、***癌、外阴癌、甲状腺癌、肝癌，***癌、***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、水肿(例如与脑瘤有关的)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。In one aspect, the present invention provides the anti-OX40 antibody or antigen-binding fragment thereof described herein, or the antibody-drug conjugate described herein, or the pharmaceutical composition described herein, in the preparation of a drug for the treatment of cancer In the use. In one embodiment, the cancer is selected from squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), peritoneal cancer , Hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urethral 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, liver cancer, anal cancer, penile cancer, melanoma, superficial spreading melanoma, Malignant freckle melanoma, acral melanoma, nodular melanoma, multiple myeloma and B-cell lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), Hairy cell leukemia, chronic myeloblastic leukemia, and post-transplant lymphoproliferative disorder (PTLD), as well as those related to keloidosis, edema (e.g., related to brain tumors), and Meigs syndrome Abnormal blood vessel proliferation, brain tumors and brain cancers, and head and neck cancers, and related metastases.
在一个方面，本文提供了本文所述的抗OX40抗体或其抗原结合片段，或本文所述的抗体-药物缀合物，或本文所述的药物组合物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。In one aspect, provided herein is the anti-OX40 antibody or antigen-binding fragment thereof described herein, or the antibody-drug conjugate described herein, or the pharmaceutical composition described herein for use in one or more of the following: Inhibit Treg function (e.g., inhibit the suppressive function of Treg), kill OX40-expressing cells (e.g., cells expressing high levels of OX40), improve effector T cell function and/or improve memory T cell function, reduce tumor immunity, and enhance T cells Function and/or deplete cells expressing OX40.
在一个方面，本文提供了本文所述的抗OX40抗体或其抗原结合片段，或本文所述的抗体-药物缀合物，或本文所述的药物组合物在制备用于治疗下述一项或多项的药物中的用途：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。In one aspect, provided herein is the anti-OX40 antibody or antigen-binding fragment thereof described herein, or the antibody-drug conjugate described herein, or the pharmaceutical composition described herein is prepared for the treatment of one of the following or Uses in a variety of drugs: inhibit Treg function (e.g., inhibit the suppressive function of Treg), kill OX40-expressing cells (e.g., 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.
在一个方面，本发明提供了一种药物组合，其包含本文所述的抗OX40抗体或其抗原结合片段，或本文所述的抗体-药物缀合物，或本文所述的药物组合物，以及一种或多种另外的治疗剂。In one aspect, the present invention provides a pharmaceutical combination comprising the anti-OX40 antibody or antigen-binding fragment thereof described herein, or the antibody-drug conjugate described herein, or the pharmaceutical composition described herein, and One or more additional therapeutic agents.
在一个方面，本发明提供了一种试剂盒，其包括本文所述的抗OX40抗体或其抗原结合片段，或本文所述的抗体-药物缀合物，或本文所述的药物组合物，优选其进一步包括给药装置。In one aspect, the present invention provides a kit comprising the anti-OX40 antibody or antigen-binding fragment thereof described herein, or the antibody-drug conjugate described herein, or the pharmaceutical composition described herein, preferably It further includes a drug delivery device.
要理解，可以组合本文所述各个实施方式的一个、一些、或所有特性以形成本发明的其它实施方式。本发明的这些和其它方面对本领域技术人员会变得显而易见。通过下面的详述进一步描述本发明的这些和其它实施 方式。It is understood that one, some, or all of the characteristics of the various embodiments described herein can be combined to form other embodiments of the present invention. These and other aspects of the invention will become apparent to those skilled in the art. These and other embodiments of the present invention are further described by the following detailed description.
若当描述针对最大对映性比对时，两个序列中的核苷酸或氨基酸的序列是相同的，则两个多核苷酸或多肽序列被称为是“相同的”。两个序列之间的比较是通常通过于比较窗上比较所述序列而进行以识别及比较具有序列相似性的局部区域。如本文使用的“比较窗”是指具有至少约20个(通常30至约75或40至约50个)连续位置的区段，其中在两个序列经优化比对后，可将序列与具有相同数量连续位置的参考序列进行比较。If the sequence of nucleotides or amino acids in two sequences is the same when the description is directed to a maximum enantiomeric alignment, then the two polynucleotide or polypeptide sequences are said to be "identical." The comparison between two sequences is usually performed by comparing the sequences on a comparison window to identify and compare local regions of sequence similarity. As used herein, "comparison window" refers to a segment having at least about 20 (usually 30 to about 75 or 40 to about 50) contiguous positions, where the two sequences can be compared with those having Compare the reference sequences of the same number of consecutive positions.
用于比较的序列的优化比对可使用生物信息学软件(Inc.,Madison,WI)的套件中的程序，使用默认参数进行。此程序实施下列参考文献中描述的数种比对方案：Dayhoff,M.O.,1978,A model of evolutionarychange in proteins-Matrices for detecting distant relationships.In Dayhoff,M.O.(编)Atlas of Protein Sequence and Structure,National Biomedical ResearchFoundation,Washington DC，第5卷，增刊3，第345至358页；Hein J.,1990,UnifiedApproach to Alignment and Phylogenes，第626至645页，Methods in Enzymology，第183卷，Academic Press,Inc.,San Diego,CA；Higgins,D.G.及Sharp,P.M.,1989,CABIOS 5:151-153；Myers,E.W.及Muller W.,1988,CABIOS 4:11-17；Robinson,E.D.,1971,Comb.Theor.11:105；Santou,N.,Nes,M.,1987,Mol.Biol.Evol.4:406-425；Sneath,P.H.A.及Sokal,R.R.,1973,Numerical Taxonomy the Principles and Practice of NumericalTaxonomy,Freeman Press,San Francisco,CA；Wilbur,W.J.及Lipman,D.J.,1983,Proc.Natl.Acad.Sci.USA 80:726-730。The optimized alignment of the sequences used for comparison can be performed using a program in a suite of bioinformatics software (Inc., Madison, WI), using default parameters. This program implements several comparison schemes described in the following references: Dayhoff, MO, 1978, A model of evolutionary change in proteins-Matrices for detecting distant relationships. In Dayhoff, MO (Editor) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC, Volume 5, Supplement 3, Pages 345 to 358; Hein J., 1990, Unified Approach to Alignment and Phylogenes, Pages 626 to 645, Methods in Enzymology, Volume 183, Academic Press, Inc., San Diego, CA; Higgins, DG and Sharp, PM, 1989, CABIOS 5: 151-153; Myers, EW and Muller W., 1988, CABIOS 4: 11-17; Robinson, ED, 1971, Comb. Theor.11 :105;Santou,N.,Nes,M.,1987,Mol.Biol.Evol.4:406-425;Sneath,PHA and Sokal,RR,1973, Numerical Taxonomy the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, CA; Wilbur, WJ and Lipman, DJ, 1983, Proc. Natl. Acad. Sci. USA 80:726-730.
在一些实施方式中，“序列同一性的百分比”是通过在具有至少20个位置的比较的窗上比较两个经优化比对的序列进行测定，其中比较窗中的多核苷酸或多肽序列的部分相较于针对两个序列的优化比对的参考序列(其不包含添加或删除)可包含20％或以下、通常5％至15％或10％至12％的添加或删除(即，间隙)。该百分比是通过以下计算：通过测定其中两个序列中均出现相同核酸碱基或氨基酸残基的位置数量以产生具有匹配位置的数量，将匹配位置的数量除以参考序列中的位置的总数量(即，窗尺寸)及使所述结果乘以100以产生序列同一性的百分比。In some embodiments, the "percentage of sequence identity" is determined by comparing two optimized aligned sequences on a comparison window with at least 20 positions, where the polynucleotide or polypeptide sequence in the comparison window Part of the reference sequence (which does not contain additions or deletions) compared to the optimized alignment of the two sequences may contain 20% or less, usually 5% to 15% or 10% to 12% of additions or deletions (ie, gaps). ). The percentage is calculated by determining the number of positions where the same nucleic acid base or amino acid residue appears in both sequences to generate the number of matching positions, and dividing the number of matching positions by the total number of positions in the reference sequence (Ie, window size) and multiply the result by 100 to produce a percentage of sequence identity.
或者，变体亦可与天然基因或其的部分或补体基本上同源。这些多核苷酸变体可在中度严格的条件下与编码天然抗体(或互补序列)的天然生成的DNA序列杂交。Alternatively, the variant may also be substantially homologous to the natural gene or part or complement thereof. These polynucleotide variants can hybridize to naturally-occurring DNA sequences encoding natural antibodies (or complementary sequences) under moderately stringent conditions.
合适的“中度严格的条件”包括在5X SSC，0.5％SDS，1.0mM EDTA(pH 8.0)的溶液中预洗；在50℃至65℃，5X SSC下杂交整夜；接着在65℃下清洗两次，历时20分钟，及每次清洗使用含有0.1％SDS的2X、0.5X及0.2X  SSC。Suitable "moderately stringent conditions" include pre-washing in a solution of 5X SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridization at 50°C to 65°C, 5X SSC overnight; then at 65°C Wash twice, lasting 20 minutes, and use 2X, 0.5X and 0.2X SSC containing 0.1% SDS for each wash.
如本文使用，“高度严格的条件”或“高严格性条件”是那些下列者：(1)针对清洗采用低离子强度及高温，例如在50℃下用0.015M氯化钠/0.0015M柠檬酸钠/0.1％十二烷基硫酸钠；(2)在杂交期间采用变性剂，诸如甲酰胺，例如，具有在pH 6.5下的0.1％牛血清白蛋白/0.1％聚蔗糖/0.1％聚乙烯吡咯啶酮/50mM磷酸钠缓冲剂及在42℃下的750mM氯化钠、75mM柠檬酸钠的50％(v/v)甲酰胺；或(3)在42℃下采用50％甲酰胺、5X SSC(0.75M NaCl、0.075M柠檬酸钠)、50mM磷酸钠(pH 6.8)、0.1％焦磷酸钠、5X Denhardt杂交溶液、经声处理的鲑鱼***DNA(50μg/mL)、0.1％SDS及10％硫酸葡聚糖，及在42℃下于0.2X SSC(氯化钠/柠檬酸钠)及在55℃下于50％甲酰胺中清洗，接着在55℃下于由含有EDTA的0.1X SSC组成的高严格性清洗液中进行清洗。本领域技术人员将知晓为适应诸如探针长度及类似物的因素，如何任选地调节温度、离子强度等。As used herein, "highly stringent conditions" or "highly stringent conditions" are those of the following: (1) Use low ionic strength and high temperature for cleaning, such as 0.015M sodium chloride/0.0015M citric acid at 50°C Sodium/0.1% sodium lauryl sulfate; (2) Use denaturants such as formamide during hybridization, for example, with 0.1% bovine serum albumin/0.1% sucrose/0.1% polyvinylpyrrole at pH 6.5 Pyridone/50mM sodium phosphate buffer and 50% (v/v) formamide of 750mM sodium chloride and 75mM sodium citrate at 42℃; or (3) 50% formamide, 5X SSC at 42℃ (0.75M NaCl, 0.075M sodium citrate), 50mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5X Denhardt hybridization solution, sonicated salmon sperm DNA (50μg/mL), 0.1% SDS and 10% Dextran sulfate, and washed in 0.2X SSC (sodium chloride/sodium citrate) at 42°C and in 50% formamide at 55°C, then washed at 55°C with 0.1X SSC containing EDTA It is cleaned in the high-stringency cleaning solution. Those skilled in the art will know how to optionally adjust temperature, ionic strength, etc. to accommodate factors such as probe length and the like.
本领域普通技术人员将知晓由于遗传密码子的简并性，因此存在许多编码如本文描述的多肽的核苷酸序列。这些多核苷酸中的一些与具有任何天然基因的核苷酸序列具有最小同源性。然而，本发明具体预期因密码子使用中的差异而改变的多核苷酸。此外，包含本文提供的多核苷酸序列的基因的等位基因是于本发明的范围内。等位基因是由于核苷酸的一个或更多个突变诸如删除、添加及/或取代而变化的内源性基因。所得mRNA及蛋白质可(但无需)具有经改变的结构或功能。等位基因可使用标准技术(诸如杂交、扩增及/或数据库序列比较)进行识别。Those of ordinary skill in the art will know that due to the degeneracy of the genetic code, there are many nucleotide sequences encoding the polypeptides as described herein. Some of these polynucleotides have minimal homology with the nucleotide sequence of any natural gene. However, the present invention specifically contemplates polynucleotides that are altered due to differences in codon usage. In addition, alleles of genes comprising the polynucleotide sequences provided herein are within the scope of the present invention. Alleles are endogenous genes that change due to one or more mutations of nucleotides, such as deletions, additions, and/or substitutions. The resulting mRNA and protein may (but need not) have altered structure or function. Alleles can be identified using standard techniques such as hybridization, amplification, and/or database sequence comparison.
本发明的多核苷酸可使用化学合成、重组方法或PCR获得。化学多核苷酸合成的方法是本领域中熟知及本文中无需详细描述。本领域技术人员可使用本文提供的序列及市售DNA合成剂以产生所需DNA序列。The polynucleotide of the present invention can be obtained using chemical synthesis, recombinant methods or PCR. The method of chemical polynucleotide synthesis is well known in the art and need not be described in detail herein. Those skilled in the art can use the sequences provided herein and commercially available DNA synthesizers to generate the desired DNA sequence.
就使用重组方法制备多核苷酸而言，可将包含所需序列的多核苷酸***合适的载体内，及可将该载体进一步引入合适的宿主细胞内，以用于复制及扩增，如本文进一步讨论。多核苷酸可通过本领域中已知的任何方式***宿主细胞内。细胞是通过直接摄取、内吞作用、转染、F-杂交或电穿孔以引入外源性多核苷酸而进行转化。一经引入，外源性多核苷酸可作为非整合性载体(诸如质粒)维持于细胞内或整合于宿主细胞基因体内。经如此扩增的多核苷酸可通过本领域内熟知的方法自宿主细胞分离。参见，例如，Sambrook等人，1989。For the preparation of polynucleotides using recombinant methods, the polynucleotides containing the desired sequence can be inserted into a suitable vector, and the vector can be further introduced into a suitable host cell for replication and amplification, as described herein further discussion. The polynucleotide can be inserted into the host cell by any means known in the art. Cells are transformed by direct uptake, endocytosis, transfection, F-hybridization or electroporation to introduce exogenous polynucleotides. Once introduced, the exogenous polynucleotide can be maintained in the cell as a non-integrating vector (such as a plasmid) or integrated into the host cell gene. The polynucleotide thus amplified can be isolated from the host cell by methods well known in the art. See, for example, Sambrook et al., 1989.
或者，PCR容许DNA序列的复制。。Alternatively, PCR allows for the replication of DNA sequences. .
RNA可通过在合适的载体中使用经分离的DNA及将其***于合适的宿主细胞内获得。当细胞复制及将DNA转录成RNA时，该RNA可然后使 用本领域技术人员公知的方法进行分离。RNA can be obtained by using isolated DNA in a suitable vector and inserting it into a suitable host cell. When the cell replicates and the DNA is transcribed into RNA, the RNA can then be isolated using methods known to those skilled in the art.
合适的克隆及表达载体可包括各种组件，诸如启动子、增强子及其他转录调节序列。该载体亦可经构建以容许接着将抗体可变结构域克隆至不同载体内。Suitable cloning and expression vectors can include various components such as promoters, enhancers, and other transcriptional regulatory sequences. The vector can also be constructed to allow subsequent cloning of antibody variable domains into different vectors.
合适的克隆载体可根据标准技术进行构建或可选择自本领域中可获得的大量克隆载体。尽管所选克隆载体可根据预期使用的宿主细胞而改变，但有用的克隆载体将通常具有自我复制的能力，可具有针对特定限制核酸内切酶的单一靶及/或可携载针对可用于选择含有该载体的克隆中的标记物的基因。合适的实例包括质粒及细菌病毒，例如，pUC18、pUC19、Bluescript(例如，pBS SK+)及其衍生物，mp18、mp19、pBR322、pMB9、ColE1、pCR1、RP4、噬菌体DNA及穿梭载体(诸如pSA3及pAT28)。这些及许多其他克隆载体是可获得自商业供货商(诸如BioRad、Strategene及Invitrogen)。Suitable cloning vectors can be constructed according to standard techniques or can be selected from a large number of cloning vectors available in the art. Although the selected cloning vector can vary depending on the host cell to be used, useful cloning vectors will generally have the ability to self-replicate, can have a single target for specific restriction endonucleases, and/or can carry targets that can be used for selection. The gene containing the marker in the clone of the vector. Suitable examples include plasmids and bacterial viruses, for example, pUC18, pUC19, Bluescript (e.g., pBS SK+) and derivatives thereof, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA and shuttle vectors (such as pSA3 and pAT28). These and many other cloning vectors are available from commercial suppliers (such as BioRad, Strategene, and Invitrogen).
进一步提供表达载体。表达载体通常是可复制多核苷酸构建体，其含有根据本发明的多核苷酸。其暗示表达载体必须于宿主细胞中可复制，以游离基因的形式或以染色体DNA的整体部分的形式。合适的表达载体包括(但不限于)质粒、病毒载体，其包括腺病毒、腺相关病毒、逆转录病毒、黏质粒及揭示于PCT公开第WO 87/04462号中的表达载体。载体组件可通常包括(但不限于)下列中的一或更多者：信号序列；复制起点；一个或更多个标记基因；合适的转录控制组件(诸如启动子、增强子及终止子)。就表达(即，翻译)而言，亦通常需一个或更多个转录控制组件，诸如核糖体结合位点、翻译起始位点及终止密码子。An expression vector is further provided. The expression vector is usually a replicable polynucleotide construct, which contains the polynucleotide according to the present invention. It implies that the expression vector must be replicable in the host cell, in the form of an episomal gene or as an integral part of chromosomal DNA. Suitable expression vectors include (but are not limited to) plasmids and viral vectors, including adenovirus, adeno-associated virus, retrovirus, cosmid and the expression vector disclosed in PCT Publication No. WO 87/04462. Vector components may generally include (but are not limited to) one or more of the following: a signal sequence; an origin of replication; one or more marker genes; suitable transcription control components (such as promoters, enhancers, and terminator). In terms of expression (ie, translation), one or more transcription control components, such as ribosome binding sites, translation initiation sites, and termination codons, are also usually required.
含有目的多核苷酸的载体及/或所述多核苷酸本身可通过许多适当方式中的任何一者引入宿主细胞内，所述方式包括电穿孔、采用氯化钙、氯化铷、磷酸钙、DEAE-葡聚糖或其他物质的转染；微粒轰击(microprojectile bombardment)；脂质转染；及感染(例如，其中该载体是感染剂，诸如痘疮病毒)。引入载体或多核苷酸的选择将通常取决于该宿主细胞的特征。The vector containing the polynucleotide of interest and/or the polynucleotide itself can be introduced into the host cell by any of a number of appropriate methods, including electroporation, the use of calcium chloride, rubidium chloride, calcium phosphate, Transfection of DEAE-dextran or other substances; microprojectile bombardment; lipofection; and infection (for example, where the vector is an infectious agent, such as a pox virus). The choice of introducing a vector or polynucleotide will generally depend on the characteristics of the host cell.
本发明的抗体包括通过重组方法制备、表达、产生或分离的人抗体，例如利用转染入宿主细胞的重组表达载体表达的抗体(在以下部分II中进一步介绍)、从重组组合人抗体文库分离的抗体(在以下部分III中进一步介绍)、从人免疫球蛋白基因转基因动物(例如小鼠)分离的抗体(参见例如(Taylor，L.D.等(1992)Nucl.Acids Res.20：6287-6295)或者涉及将人免疫球蛋白基因序列剪接为其他DNA序列的任何其他方法制备、表达、产生或分离的抗体。这样的重组人抗体具有人种系免疫球蛋白序列来源的可变区和恒定区(参见Kabat，E.A.，et al.(1991)Sequences of Proteins of Immunological Interest，Fifth  Edition，U.S.Department of Health and Human Services，NIH Publication No.91-3242)。The antibodies of the present invention include human antibodies prepared, expressed, produced or isolated by recombinant methods, such as antibodies expressed using recombinant expression vectors transfected into host cells (further described in the following section II), isolated from recombinant combinatorial human antibody libraries Antibodies (further described in section III below), antibodies isolated from human immunoglobulin gene transgenic animals (e.g. mice) (see e.g. (Taylor, LD et al. (1992) Nucl. Acids Res. 20: 6287-6295) Or antibodies prepared, expressed, produced or isolated by any other method involving the splicing of human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences ( See Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242).
可通过在宿主细胞中重组表达免疫球蛋白轻链基因和重链基因，制备本发明的抗体或抗体部分。为了重组表达抗体，用一个或多个携带编码所述抗体的免疫球蛋白轻链和重链的DNA片段的重组表达载体转染宿主细胞，使得所述轻链和重链在宿主细胞中表达，而且优选分泌入培养所述宿主细胞的培养基中，从该培养基中可回收所述抗体。标准重组DNA方法学用来获得抗体重链基因和抗体轻链基因，使这些基因导入重组表达载体中，然后使所述载体导入宿主细胞中，所述标准方法例如为以下文献介绍的方法：Sambrook，Fritsch和Maniatis(编辑)，Molecular Cloning；A Laboratory Manual，第二版，Cold Spring Harbor，N.Y.，(1989)，Ausubel，F.M.等(编辑)CurrentProtocols in Molecuar Biology，Greene Publishing Associates，(1989)和Boss等的美国专利第4,816,397号。The antibody or antibody portion of the present invention can be prepared by recombinantly expressing immunoglobulin light chain genes and heavy chain genes in host cells. In order to express the antibody recombinantly, a host cell is transfected with one or more recombinant expression vectors carrying DNA fragments encoding the immunoglobulin light and heavy chains of the antibody, so that the light and heavy chains are expressed in the host cell, Furthermore, it is preferably secreted into a medium in which the host cell is cultured, from which the antibody can be recovered. Standard recombinant DNA methodology is used to obtain antibody heavy chain genes and antibody light chain genes, introduce these genes into a recombinant expression vector, and then introduce the vector into host cells. The standard method is, for example, the method described in the following literature: Sambrook , Fritsch and Maniatis (Editor), Molecular Cloning; A Laboratory Manual, Second Edition, Cold Spring Harbor, NY, (1989), Ausubel, FM, etc. (Editor) Current Protocols in Molecular Biology, Greene Publishing Associates, (1989) and Boss U.S. Patent No. 4,816,397
抗体或其抗原结合片段可使用合适的宿主细胞重组制造。编码该抗体或其抗原结合片段的核酸可克隆至表达载体内，其可然后引入宿主细胞诸如大肠杆菌细胞、酵母细胞、昆虫细胞、类人猿COS细胞、中国仓鼠卵巢(CHO)细胞或骨髓瘤细胞内，其中该细胞不另外产生免疫球蛋白，以于重组宿主细胞中获得抗体的合成。在本领域熟知的许多细胞中，优选的宿主细胞包括CHO细胞、人类胚胎肾(HEK)293细胞或Sp2.0细胞。Antibodies or antigen-binding fragments thereof can be recombinantly produced using suitable host cells. The nucleic acid encoding the antibody or antigen-binding fragment thereof can be cloned into an expression vector, which can then be introduced into host cells such as E. coli cells, yeast cells, insect cells, apes COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells , Wherein the cell does not additionally produce immunoglobulin to obtain antibody synthesis in the recombinant host cell. Among the many cells well known in the art, preferred host cells include CHO cells, human embryonic kidney (HEK) 293 cells, or Sp2.0 cells.
抗体片段可通过重组方法或通过化学合成由全长抗体的蛋白质水解或其他降解产生。抗体的多肽片段(尤其多达约50个氨基酸的较短多肽)可通过化学合成便利地制成。用于蛋白质及肽的化学合成的方法是本领域中已知及是可购买获得。Antibody fragments can be produced by proteolysis or other degradation of full-length antibodies by recombinant methods or by chemical synthesis. Polypeptide fragments of antibodies (especially shorter polypeptides of up to about 50 amino acids) can be conveniently prepared by chemical synthesis. Methods for the chemical synthesis of proteins and peptides are known in the art and are commercially available.
本发明的抗体或其抗原结合片段可经亲和力成熟。例如，经亲和力成熟的抗体可由本领域中已知的程序(Marks等人，1992,Bio/Technology,10:779-783；Barbas等人，1994,Proc Nat.Acad.Sci,USA 91:3809-3813；Schier等人，1995,Gene,169:147-155；Yelton等人，1995,J.Immunol.,155:1994-2004；Jackson等人，1995,J.Immunol.,154(7):3310-9；Hawkins等人，1992,J.Mol.Biol.,226:889-896；及WO2004/058184)产生。The antibody or antigen-binding fragment thereof of the present invention can be affinity matured. For example, affinity matured antibodies can be obtained by procedures known in the art (Marks et al., 1992, Bio/Technology, 10:779-783; Barbas et al., 1994, Proc Nat. Acad. Sci, USA 91: 3809- 3813; Schier et al., 1995, Gene, 169:147-155; Yelton et al., 1995, J. Immunol., 155:1994-2004; Jackson et al., 1995, J. Immunol., 154(7): 3310 -9; Hawkins et al., 1992, J. Mol. Biol., 226:889-896; and WO2004/058184).
抗体变体Antibody variants
在某些实施方式中，涵盖本文中提供的抗体的氨基酸序列变体。例如，可以期望改善抗体的结合亲和力和/或其它生物学特性。可以通过将合适的修饰引入编码抗体的核苷酸序列中，或者通过肽合成来制备抗体的氨基酸序列变体。此类修饰包括例如对抗体的氨基酸序列内的残基的删除，和/或 ***和/或替代。可以进行删除，***，和替代的任何组合以得到最终的构建体，只要最终的构建体拥有期望的特征，例如，抗原结合。In certain embodiments, amino acid sequence variants of the antibodies provided herein are encompassed. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions, and/or insertions and/or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletion, insertion, and substitution can be made to obtain the final construct, as long as the final construct possesses the desired characteristics, for example, antigen binding.
a)替代，***，和删除变体a) Replace, insert, and delete variants
在某些实施方式中，提供了具有一处或多处氨基酸替代的抗体变体。替代诱变感兴趣的位点包括HVR和FR。保守替代在表A中在″优选的替代″的标题下显示。更实质的变化在表A中在″例示性替代″的标题下提供，并且如下文参照氨基酸侧链类别进一步描述的。可以将氨基酸替代引入感兴趣的抗体中，并且对产物筛选期望的活性，例如保留/改善的抗原结合，降低的免疫原性，或改善的ADCC或CDC。In certain embodiments, antibody variants with one or more amino acid substitutions are provided. Sites of interest for alternative mutagenesis include HVR and FR. Conservative substitutions are shown in Table A under the heading of "preferred substitutions". More substantial changes are provided in Table A under the heading of "exemplary substitutions" and are described further below with reference to amino acid side chain categories. Amino acid substitutions can be introduced into the antibody of interest, and the product screened for the desired activity, such as retained/improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.
表ATable A
初始残基Initial residue 例示性替代Illustrative substitution 优选的替代Preferred alternative
Ala(A)Ala(A) Val；Leu；IleVal; Leu; Ile ValVal
Arg(R)Arg(R) Lys；Gln；AsnLys; Gln; Asn LysLys
Asn(N)Asn(N) Gln；His；Asp，Lys；ArgGln; His; Asp, Lys; Arg GlnGln
Asp(D)Asp(D) Glu；AsnGlu; Asn GluGlu
Cys(C)Cys(C) Ser；AlaSer; Ala SerSer
Gln(Q)Gln(Q) Asn；GluAsn; Glu AsnAsn
Glu(E)Glu(E) Asp；GlnAsp; Gln AspAsp
Gly(G)Gly(G) AlaAla AlaAla
His(H)His(H) Asn；Gln；Lys；ArgAsn; Gln; Lys; Arg ArgArg
Ile(I)Ile(I) Leu；Val；Met；Ala；Phe；正亮氨酸Leu; Val; Met; Ala; Phe; Norleucine LeuLeu
Leu(L)Leu(L) 正亮氨酸；Ile；Val；Met；Ala；PheNorleucine; Ile; Val; Met; Ala; Phe IleIle
Lys(K)Lys(K) Arg；Gln；AsnArg; Gln; Asn ArgArg
Met(M)Met(M) Leu；Phe；IleLeu; Phe; Ile LeuLeu
Phe(F)Phe(F) Trp；Leu；Val；Ile；Ala；TyrTrp; Leu; Val; Ile; Ala; Tyr TyrTyr
Pro(P)Pro(P) AlaAla AlaAla
Ser(S)Ser(S) ThrThr ThrThr
Thr(T)Thr(T) Val；SerVal; Ser SerSer
Trp(W)Trp(W) Tyr；PheTyr; Phe TyrTyr
Tyr(Y)Tyr(Y) Trp；Phe；Thr；SerTrp; Phe; Thr; Ser PhePhe
Val(V)Val(V) Ile；Leu；Met；Phe；Ala；正亮氨酸Ile; Leu; Met; Phe; Ala; Norleucine LeuLeu
依照共同的侧链特性，氨基酸可以如下分组：According to the common side chain characteristics, amino acids can be grouped as follows:
(1)疏水性的：正亮氨酸，Met，Ala，Val，Leu，IIe；(1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, IIe;
(2)中性，亲水性的：Cys,Ser,Thr,Asn,Gin；(2) Neutral and hydrophilic: Cys, Ser, Thr, Asn, Gin;
(3)酸性的：Asp，Glu；(3) Acidic: Asp, Glu;
(4)碱性的：His，Lys，Arg；(4) Basic: His, Lys, Arg;
(5)影响链取向的残基:Gly，Pro；(5) Residues that affect chain orientation: Gly, Pro;
(6)芳香族的：Trp，Tyr,Phe。(6) Aromatic: Trp, Tyr, Phe.
非保守替代会需要用这些类别之一的成员替换另一个类别的。Non-conservative substitutions would require replacing members of one of these categories with members of another category.
一类替代变体牵涉替代亲本抗体(例如人源化或人抗体)的一个或多个高变区残基。一般地，为进一步研究选择的所得变体相对于亲本抗体会具有某些生物学特性的改变(例如改善)(例如升高的亲和力，降低的免疫原性)和/或会基本上保留亲本抗体的某些生物学特性。例示性的替代变体是亲和力成熟的抗体，其可以例如使用基于噬菌体展示的亲和力成熟技术诸如本文中所描述的那些技术来方便地生成。简言之，将一个或多个HVR残基突变，并将变体抗体在噬菌体上展示，并对其筛选特定的生物学活性(例如结合亲和力)。One type of substitution variant involves replacing one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variants selected for further research will have certain biological changes (e.g. improvement) (e.g. increased affinity, decreased immunogenicity) relative to the parent antibody and/or will substantially retain the parent antibody Some of the biological characteristics. An exemplary alternative variant is an affinity matured antibody, which can be conveniently generated, for example, using phage display-based affinity maturation techniques such as those described herein. In short, one or more HVR residues are mutated, and the variant antibodies are displayed on phage and screened for specific biological activities (such as binding affinity).
可以对HVR做出变化(例如，替代)，例如以改善抗体亲和力。可以对HVR"热点"，即由在体细胞成熟过程期间以高频率经历突变的密码子编码的残基(见例如Chowdhury，Methods Mol.Biol.207:179-196(2008))，和/或接触抗原的残基做出此类变化，其中对所得的变体VH或VL测试结合亲和力。通过次级文库的构建和再选择进行的亲和力成熟已经记载于例如Hoogenboom等，于Methods in Molecular Biology 178:l_37(0'Brien等编，Human Press，Totowa，NJ，(2001))。在亲和力成熟的一些实施方式中，通过多种方法(例如，易错PCR，链改组，或寡核苷酸指导的诱变)将多样性引入为成熟选择的可变基因。然后，创建次级文库。然后，筛选文库以鉴定具有期望的亲和力的任何抗体变体。另一种引入多样性的方法牵涉HVR指导的方法，其中将几个HVR残基(例如，一次4-6个残基)随机化。可以例如使用丙氨酸扫描诱变或建模来特异性鉴定牵涉抗原结合的HVR残基。特别地，经常靶向CDR-H3和CDR-L3。Changes (e.g., substitutions) can be made to HVR, for example to improve antibody affinity. HVR "hot spots", residues encoded by codons that undergo mutations at a high frequency during the somatic maturation process (see, for example, Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and/or Such changes are made to residues in contact with the antigen, wherein the resulting variant VH or VL is tested for binding affinity. Affinity maturation through the construction and reselection of secondary libraries has been described in, for example, Hoogenboom et al., in Methods in Molecular Biology 178: 1-37 (edited by O'Brien et al., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced as a variable gene for maturation selection by multiple methods (e.g., error-prone PCR, strand shuffling, or oligonucleotide-directed mutagenesis). Then, create a secondary library. Then, the library is screened to identify any antibody variants with the desired affinity. Another method of introducing diversity involves an HVR-directed method, in which several HVR residues (eg, 4-6 residues at a time) are randomized. Alanine scanning mutagenesis or modeling can be used, for example, to specifically identify HVR residues involved in antigen binding. In particular, CDR-H3 and CDR-L3 are often targeted.
在某些实施方式中，可以在一个或多个HVR内发生替代，***，或删除，只要此类变化不实质性降低抗体结合抗原的能力。例如，可以对HVR做出保守变化(例如，保守替代，如本文中提供的)，其不实质性降低结合亲和力。例如，此类变化可以在HVR中的抗原接触残基以外。在上文提供的变体VH和VL序列的某些实施方式中，每个HVR是未改变的，或者含有不超过1，2或3处氨基酸替代。In certain embodiments, substitutions, insertions, or deletions can occur within one or more HVRs, as long as such changes do not substantially reduce the ability of the antibody to bind antigen. For example, conservative changes (e.g., conservative substitutions, as provided herein) can be made to HVR that do not substantially reduce binding affinity. For example, such changes can be outside of the antigen contact residues in the HVR. In certain embodiments of the variant VH and VL sequences provided above, each HVR is unchanged or contains no more than 1, 2, or 3 amino acid substitutions.
一种可用于鉴定抗体中可以作为诱变靶位的残基或区域的方法称作" 丙氨酸扫描诱变"，如由Cunningham和Wells(1989)Science,244:1081-1085所描述的。在此方法中，将残基或靶残基的组(例如，带电荷的残基诸如arg，asp，his，lys，和glu)鉴定，并用中性或带负电荷的氨基酸(例如，丙氨酸或多丙氨酸)替换以测定抗体与抗原的相互作用是否受到影响。可以在对初始替代表明功能敏感性的氨基酸位置引入进一步的替代。或者/另外，利用抗原-抗体复合物的晶体结构来鉴定抗体与抗原间的接触点。作为替代的候选，可以靶向或消除此类接触残基和邻近残基。可以筛选变体以确定它们是否含有期望的特性。One method that can be used to identify residues or regions in antibodies that can be targeted for mutagenesis is called "alanine scanning mutagenesis", as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, residues or groups of target residues (for example, charged residues such as arg, asp, his, lys, and glu) are identified, and neutral or negatively charged amino acids (for example, alanine Acid or polyalanine) to determine whether the interaction between the antibody and the antigen is affected. Further substitutions can be introduced at amino acid positions that indicate functional sensitivity to the initial substitution. Alternatively or additionally, the crystal structure of the antigen-antibody complex is used to identify the contact points between the antibody and the antigen. As an alternative candidate, such contact residues and neighboring residues can be targeted or eliminated. The variants can be screened to determine whether they contain the desired properties.
氨基酸序列***包括长度范围为1个残基至含有100或更多个残基的多肽的氨基和/或羧基端融合，及单个或多个氨基酸残基的序列内***。末端***的例子包括具有N端甲硫氨酰基残基的抗体。抗体分子的其它***变体包括抗体的N或C端与酶(例如对于ADEPT)或延长抗体的血清半衰期的多肽的融合物。Amino acid sequence insertions include amino and/or carboxyl terminal fusions ranging from one residue to a polypeptide containing 100 or more residues in length, and intra-sequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with N-terminal methionyl residues. Other insertional variants of antibody molecules include fusions of the N- or C-terminus of the antibody with an enzyme (for example, for ADEPT) or a polypeptide that extends the serum half-life of the antibody.
b)糖基化变体b) Glycosylation variants
在某些实施方式中，改变本文中提供的抗体以提高或降低抗体糖基化的程度。可以通过改变氨基酸序列，使得创建或消除一个或多个糖基化位点来方便地实现对抗体的糖基化位点的添加或删除。In certain embodiments, the antibodies provided herein are modified to increase or decrease the degree of glycosylation of the antibody. The addition or deletion of glycosylation sites of the antibody can be conveniently achieved by changing the amino acid sequence so that one or more glycosylation sites are created or eliminated.
在抗体包含Fc区的情况中，可以改变其附着的碳水化合物。由哺乳动物细胞生成的天然抗体通常包含分支的，双触角寡糖，其一般通过N连接附着于Fc区的CH2域的Asn297。见例如Wright等，TIBTECH15:26-32(1997)。寡糖可以包括各种碳水化合物，例如，甘露糖，N-乙酰葡糖胺(GlcNAc)，半乳糖，和唾液酸，以及附着于双触角寡糖结构"主干"中的GIcNAc的岩藻糖。在一些实施方式中，可以对本发明抗体中的寡糖进行修饰以创建具有某些改善的特性的抗体变体。In the case where the antibody contains an Fc region, the carbohydrate to which it is attached can be changed. Natural antibodies produced by mammalian cells usually contain branched, biantennary oligosaccharides, which are generally attached to Asn297 of the CH2 domain of the Fc region through an N linkage. See, for example, Wright et al., TIBTECH 15:26-32 (1997). Oligosaccharides may include various carbohydrates, for example, mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, and fucose attached to GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharides in the antibodies of the invention can be modified to create antibody variants with certain improved properties.
在一个实施方式中，提供了抗体变体，其具有缺乏附着(直接或间接)于Fc区的岩藻糖的碳水化合物结构。例如，此类抗体中的岩藻糖量可以是1％至80％，1％至65％，5％至65％或20％至40％。通过相对于附着于Asn297的所有糖结构(例如，复合的，杂合的和高甘露糖的结构)的总和，计算Asn297处糖链内岩藻糖的平均量来测定岩藻糖量，如通过MALDI-TOF质谱术测量的，例如如记载于WO 2008/077546的。Asn297指位于Fc区中的约第297位(Fe区残基的Eu编号方式)的天冬酰胺残基；然而，Asn297也可以由于抗体中的微小序列变异而位于第297位上游或下游约±3个氨基酸，即在第294位和第300位之间。此类岩藻糖基化变体可以具有改善的ADCC功能。见例如美国专利公开文本No.US 2003/0157108(Presta，L.)；US 2004/0093621(Kyowa Hakko Kogyo Co.,Ltd)。涉及"脱岩藻糖基化的"或"岩 藻糖缺乏的"抗体变体的出版物的例子包括:US 2003/0157108；W0 2000/61739；W0 2001/29246；US 2003/0115614；US 2002/0164328；US 2004/0093621；US 2004/0132140；US 2004/0110704；US 2004/0110282；US 2004/0109865；W0 2003/085119；W0 2003/084570；W0 2005/035586；TO 2005/035778；TO2005/053742；TO2002/031140；0kazaki等，J.Mol.Biol.336:1239-1249(2004)；Yamane-Ohnuki等,Biotech.Bioeng.87:614(2004)。能够生成脱岩藻糖基化抗体的细胞系的例子包括蛋白质岩藻糖基化缺陷的LecI3CHO细胞(Ripka等，Arch.Biochem.Biophys.249:533-545(1986)；美国专利申请No US 2003/0157108A1，Presta，L；及WO 2004/056312A1，Adams等)，和敲除细胞系，诸如α-l，6-岩藻糖基转移酶基因FUT8敲除CHO细胞(见例如Yamane-Ohnuki等，Biotech.Bioeng·87:614(2004)；Kanda，Y.等，Biotechnol.Bioeng.,94(4):680-688(2006)；及W02003/085107)。In one embodiment, antibody variants are provided that have a carbohydrate structure lacking fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. Determine the amount of fucose by calculating the average amount of fucose in the sugar chain at Asn297 relative to the sum of all sugar structures (for example, complex, hybrid, and high-mannose structures) attached to Asn297, such as by Measured by MALDI-TOF mass spectrometry, for example, as described in WO 2008/077546. Asn297 refers to the asparagine residue located at about position 297 in the Fc region (Eu numbering of residues in the Fc region); however, Asn297 can also be located at about ±±2% upstream or downstream of position 297 due to minor sequence variations in the antibody. 3 amino acids, that is, between the 294th and 300th positions. Such fucosylation variants may have improved ADCC function. See, for example, US Patent Publication No. US 2003/0157108 (Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications involving "defucosylated" or "fucose deficient" antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002 /0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; TO 2005/035778; TO2005/ 053742; TO2002/031140; Okazaki et al., J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004). Examples of cell lines capable of producing defucosylated antibodies include LecI3CHO cells deficient in protein fucosylation (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application No. US 2003 /0157108A1, Presta, L; and WO 2004/056312A1, Adams, etc.), and knockout cell lines, such as α-1,6-fucosyltransferase gene FUT8 knockout CHO cells (see, for example, Yamane-Ohnuki, etc., Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4): 680-688 (2006); and WO2003/085107).
进一步提供了具有两分型寡糖的抗体变体，例如其中附着于抗体Fc区的双触角寡糖是通过GIcNAc两分的。此类抗体变体可以具有降低的岩藻糖基化和/或改善的ADCC功能。此类抗体变体的例子记载于例如WO2003/011878(Jean-Mairet等)；美国专利No.6，602，684(Umana等)；及US 2005/0123546(Umana等)。还提供了在附着于Fc区的寡糖中具有至少一个半乳糖残基的抗体变体。此类抗体变体可以具有改善的CDC功能。此类抗体变体记载于例如WO 1997/30087(Patel等)；WO 1998/58964(Raju，S.)；及WO 1999/22764(Raju，S·)。Further provided are antibody variants having bipartite oligosaccharides, for example, the biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described in, for example, WO2003/011878 (Jean-Mairet et al.); US Patent No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umana et al.). Also provided are antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region. Such antibody variants may have improved CDC function. Such antibody variants are described in, for example, WO 1997/30087 (Patel et al.); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).
c)Fc区变体c) Fc region variants
在某些实施方式中，可以将一处或多处氨基酸修饰引入本文中提供的抗体的Fc区中，由此生成Fc区变体。Fc区变体可以包含在一个或多个氨基酸位置包含氨基酸修饰(例如替代)的人Fc区序列(例如，人IgGl，IgG2，IgG3或IgG4Fc区)。In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of the antibodies provided herein, thereby generating Fc region variants. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgGl, IgG2, IgG3, or IgG4 Fc region) that contains amino acid modifications (e.g., substitutions) at one or more amino acid positions.
在某些实施方式中，本发明涵盖拥有一些但不是所有效应器功能的抗体变体，所述效应器功能使其成为如下应用的期望候选物，其中抗体的体内半衰期是重要的，而某些效应器功能(诸如补体和ADCC)是不必要的或有害的。可以进行体外和/或体内细胞毒性测定法以确认CDC和/或ADCC活性的降低/消减。例如，可以进行Fc受体(FcR)结合测定法以确保抗体缺乏FcγR结合(因此有可能缺乏ADCC活性)，但是保留FcRn结合能力。介导ADCC的主要细胞NK细胞仅表达FcγRIII，而单核细胞表达FcγRI，FcγRII和FcγRIII。在Ravetch和Kinet,Annu.Rev.Tmmunol·9:457-492(1991)的第464页上的表3中汇总了造血细胞上的FcR表达。评估感兴趣分子的ADCC活性的体外测定法的非限制性例子记载于美国专利No.5,500,362(见例如 Hellstrom,I·等，Proc.Nat'I Acad.Sci USA 83:7059-7063(1986))和Hellstrom，I等，Proc.Nat'I Acad.Sci.USA 82:1499-1502(1985)；5，821，337(见Bruggemann，Μ·等，J.Exp.Med.166:1351-1361(1987))。或者，可以采用非放射性测定方法(见例如用于流式细胞术的ACT I TM非放射性细胞毒性测定法(Cell Technology，Inc.Mountain View，CA；和CytoTox96非放射性细胞毒性测定法(Promega,Madison，WI))。对于此类测定法有用的效应细胞包括外周血单个核细胞(PBMC)和天然杀伤(NK)细胞。或者/另外，可以在体内评估感兴趣分子的ADCC活性，例如在动物模型中，诸如披露于Clynes等，Proc Nat'I Acad Sci USA 95:652-656(1998)的。也可以实施Clq结合测定法以确认抗体不能结合Clq，并且因此缺乏CDC活性。见例如WO 2006/029879和WO 2005/100402中的Clq和C3c结合ELISA。为了评估补体激活，可以实施CDC测定法(见例如Gazzano-Santoro等，J·Immunol.Methods 202:163(1996)；Cragg，ΜS.等，Blood 101:1045-1052(2003)；及Cragg，Μ·S·和M.J.Glennie,Blood 103:2738-2743(2004))。也可以使用本领域中已知的方法来实施FcRn结合和体内清除/半衰期测定(见例如Petkova，S.B.等，Int'I.Immunol.18(12):1759-1769(2006))。 In certain embodiments, the present invention encompasses antibody variants possessing some but not all effector functions that make them desirable candidates for applications where the in vivo half-life of the antibody is important, and some Effector functions (such as complement and ADCC) are unnecessary or harmful. In vitro and/or in vivo cytotoxicity assays can be performed to confirm the reduction/decrease of CDC and/or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that the antibody lacks FcγR binding (and therefore may lack ADCC activity), but retains FcRn binding ability. The main cells that mediate ADCC, NK cells, only express FcyRIII, while monocytes express FcyRI, FcyRII and FcyRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). A non-limiting example of an in vitro assay for assessing the ADCC activity of a molecule of interest is described in U.S. Patent No. 5,500,362 (see, for example, Hellstrom, I. et al., Proc. Nat'I Acad. Sci USA 83:7059-7063 (1986)) And Hellstrom, I etc., Proc. Nat'I Acad. Sci. USA 82: 1499-1502 (1985); 5, 821, 337 (see Bruggemann, M. et al., J. Exp. Med. 166: 1351-1361 ( 1987)). Alternatively, a non-radioactive assay method can be used (see, for example, the ACT I TM non-radioactive cytotoxicity assay for flow cytometry (Cell Technology, Inc. Mountain View, CA; and the CytoTox96 non-radioactive cytotoxicity assay (Promega, Madison) , WI)). Effector cells useful for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively/in addition, the ADCC activity of the molecule of interest can be assessed in vivo, for example in animal models Such as those disclosed in Clynes et al., Proc Nat'I Acad Sci USA 95:652-656 (1998). Clq binding assays can also be performed to confirm that antibodies cannot bind to Clq and therefore lack CDC activity. See, for example, WO 2006/ 029879 and the Clq and C3c binding ELISA in WO 2005/100402. To assess complement activation, CDC assays can be implemented (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS. et al., Blood 101:1045-1052 (2003); and Cragg, M·S· and MJ Glennie, Blood 103:2738-2743 (2004)). Methods known in the art can also be used to implement FcRn binding and in vivo clearance/half-life Determination (see, for example, Petkova, SB, etc., Int'I. Immunol. 18(12): 1759-1769 (2006)).
具有降低的效应器功能的抗体包括那些具有Fc区残基238，265，269，270，297，327和329中的一个或多个的替代的(美国专利No.6,737,056)。此类Fc突变体包括在氨基酸位置265，269，270，297和327中的两处或更多处具有替代的Fc突变体，包括残基265和297替代成丙氨酸的所谓的"DANA"Fc突变体(美国专利No.7，332，581)。Antibodies with reduced effector function include those with substitutions of one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (US Patent No. 6,737,056). Such Fc mutants include Fc mutants having substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including so-called "DANA" in which residues 265 and 297 are substituted with alanine. Fc mutant (U.S. Patent No. 7,332,581).
描述了具有改善的或降低的对FcR的结合的某些抗体变体(见例如美国专利No.6,737,056；WO 2004/056312,及Shields等，J.Biol.Chem.9(2):6591-6604(2001))。Certain antibody variants with improved or reduced binding to FcR are described (see, for example, U.S. Patent No. 6,737,056; WO 2004/056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001)).
在某些实施方式中，抗体变体包含具有改善ADCC的一处或多处氨基酸替代，例如Fc区的位置298,333,和/或334(残基的EU编号方式)的替代的Fc区。In certain embodiments, antibody variants comprise an Fc region with one or more amino acid substitutions that improve ADCC, such as substitutions at positions 298, 333, and/or 334 (EU numbering of residues) in the Fc region.
在一些实施方式中，对Fc区做出改变，其导致改变的(即，改善的或降低的)Clq结合和/或补体依赖性细胞毒性(CDC)，例如，如记载于美国专利No.6,194,551，WO 99/51642，及Idusogie等，J.Tmmunol·164:4178-4184(2000)的。In some embodiments, changes are made to the Fc region that result in altered (ie, improved or reduced) Clq binding and/or complement dependent cytotoxicity (CDC), for example, as described in U.S. Patent No. 6,194,551 , WO 99/51642, and Idusogie et al., J.Tmmunol.164:4178-4184 (2000).
具有延长的半衰期和改善的对新生儿Fc受体(FcRn)的结合的抗体记载于US2005/0014934A1(Hinton等)，新生儿Fc受体(FcRn)负责将母体IgG转移至胎儿(Guyer等，J.Immunol·117:587(1976)及Kim等J.Immunol·24:249(1994))。那些抗体包含其中具有改善Fc区对FcRn结合的 一处或多处替代的Fc区。此类Fc变体包括那些在Fc区残基238,256，265,272,286,303,305,307,311,312,317,340,356,360,362,376,378,380,382,413,424或434中的一处或多处具有替代，例如，Fc区残基434的替代的(美国专利No.7,371,826)。Antibodies with extended half-life and improved binding to the neonatal Fc receptor (FcRn) are described in US2005/0014934A1 (Hinton et al.). The neonatal Fc receptor (FcRn) is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J Immunol. 117:587 (1976) and Kim et al. J. Immunol. 24:249 (1994)). Those antibodies comprise an Fc region with one or more substitutions therein that improve the binding of the Fc region to FcRn. Such Fc variants include those that have substitutions at one or more of Fc region residues 238,256, 265,272,286,303,305,307,311,312,317,340,356,360,362,376,378,380,382,413,424 or 434, for example, substitution of Fc region residue 434 (US Patent No. 7,371,826).
还可见Duncan和Winter，Nature 322:738-40(1988)；美国专利No 5,648,260；美国专利No.5,624,821；及WO 94/29351，其关注Fc区变体的其它例子。See also Duncan and Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94/29351, which focus on other examples of Fc region variants.
d)经半胱氨酸工程化改造的抗体变体d) Antibody variants engineered by cysteine
在某些实施方式中，可以期望创建经半胱氨酸工程化改造的抗体，例如，"thioMAb"，其中抗体的一个或多个残基用半胱氨酸残基替代。在具体的实施方式中，替代的残基存在于抗体的可接近位点。通过用半胱氨酸替代那些残基，反应性硫醇基团由此定位于抗体的可接近位点，并且可以用于将抗体与其它模块，诸如药物模块或接头-药物模块缀合，以创建免疫缀合物，如本文中进一步描述的。在某些实施方式中，可以用半胱氨酸替代下列残基之任一个或多个:轻链的V205(Kabat编号方式)；重链的A118(EU编号方式)；和重链Fc区的S400(EU编号方式)。可以如例如美国专利No.7,521,541所述生成经半胱氨酸工程化改造的抗体。In certain embodiments, it may be desirable to create antibodies engineered with cysteine, for example, "thioMAb" in which one or more residues of the antibody are replaced with cysteine residues. In a specific embodiment, the substituted residue is present in an accessible site of the antibody. By substituting cysteine for those residues, the reactive thiol group is thus positioned at the accessible site of the antibody, and can be used to conjugate the antibody with other modules, such as drug modules or linker-drug modules, to Create immunoconjugates as described further herein. In certain embodiments, cysteine can be substituted for any one or more of the following residues: V205 of the light chain (Kabat numbering); A118 of the heavy chain (EU numbering); and the Fc region of the heavy chain S400 (EU numbering method). Cysteine engineered antibodies can be generated as described in, for example, US Patent No. 7,521,541.
e)抗体衍生物e) Antibody derivatives
在某些实施方式中，可以进一步修饰本文中提供的抗体以含有本领域知道的且易于获得的额外非蛋白质性质模块。适合于抗体衍生化的模块包括但不限于水溶性聚合物。水溶性聚合物的非限制性例子包括但不限于聚乙二醇(PEG)，乙二醇/丙二醇共聚物，羧甲基纤维素，右旋糖苷，聚乙烯醇，聚乙烯吡咯烷酮，聚-1，3-二氧戊环，聚-1，3，6-三口恶烷，乙烯/马来酸酐共聚物，聚氨基酸(均聚物或随机共聚物)，和右旋糖苷或聚(η-乙烯吡咯烷酮)聚乙二醇，丙二醇均聚物，环氧丙烷/环氧乙烷共聚物，聚氧乙烯化多元醇(例如甘油)，聚乙烯醇及其混合物。由于其在水中的稳定性，聚乙二醇丙醛在生产中可能具有优势。聚合物可以是任何分子量，而且可以是分支的或不分支的。附着到抗体上的聚合物数目可以变化，而且如果附着了超过一个聚合物，那么它们可以是相同或不同的分子。一般而言，可根据下列考虑来确定用于衍生化的聚合物的数目和/或类型，包括但不限于抗体要改进的具体特性或功能，抗体衍生物是否将用于指定条件下的治疗等。In certain embodiments, the antibodies provided herein can be further modified to contain additional non-proteinaceous modules known in the art and readily available. Modules suitable for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymer, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1 , 3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyamino acid (homopolymer or random copolymer), and dextran or poly(η-ethylene Pyrrolidone) polyethylene glycol, propylene glycol homopolymer, propylene oxide/ethylene oxide copolymer, polyoxyethylated polyol (such as glycerin), polyvinyl alcohol and mixtures thereof. Due to its stability in water, polyethylene glycol propionaldehyde may have advantages in production. The polymer can be of any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. Generally speaking, the number and/or type of polymers used for derivatization can be determined based on the following considerations, including but not limited to the specific properties or functions of the antibody to be improved, whether the antibody derivative will be used for treatment under specified conditions, etc. .
在另一个实施方式中，提供了抗体和可以通过暴露于辐射选择性加热的非蛋白质性质模块的缀合物。在一个实施方式中，非蛋白质性质模块是碳纳米管(Kam等，Proc.Natl.Acad.Sci.USA 102:11600-11605(2005))。辐射可以是任何波长的，并且包括但不限于对普通细胞没有损害，但是将非 蛋白质性质模块加热至抗体-非蛋白质性质模块附近的细胞被杀死的温度的波长。In another embodiment, conjugates of antibodies and non-proteinaceous moieties that can be selectively heated by exposure to radiation are provided. In one embodiment, the non-proteinaceous module is carbon nanotubes (Kam et al., Proc. Natl. Acad. Sci. USA 102:11600-11605 (2005)). The radiation can be of any wavelength, and includes, but is not limited to, a wavelength that does not damage ordinary cells, but heats the non-proteinaceous module to a temperature at which cells near the antibody-non-proteinaceous module are killed.
测定法Assay
可以通过本领域中已知的多种测定法对本文中提供的抗OX40抗体鉴定，筛选，或表征其物理/化学特性和/或生物学活性。The anti-OX40 antibodies provided herein can be identified, screened, or characterized by their physical/chemical properties and/or biological activities by a variety of assays known in the art.
1.结合测定法和其它测定法1. Combining assays and other assays
一方面，对本发明的抗体测试其抗原结合活性，例如通过已知的方法诸如ELISA，Western印迹，等来进行。可使用本领域已知方法来测定OX40结合，本文中公开了例示性方法。在一个实施方式中，使用放射免疫测定法测量结合。例示了一种例示性放射免疫测定法。将OX40抗体碘化，并制备含有固定浓度的碘化抗体和递减浓度的连续稀释的未标记OX40抗体的竞争反应混合物。将表达OX40的细胞(例如经人OX40稳定转染的BT474细胞)添加至反应混合物。温育后，清洗细胞将游离的碘化OX40抗体与结合至细胞的OX40抗体分开。测定结合的碘化OX40抗体的水平，例如通过对与细胞联合的放射性计数来进行，并使用标准方法测定结合亲和力。在另一个实施方式中，使用流式细胞术评估OX40抗体结合表面表达的OX40(例如在T细胞子集上)的能力。获得外周白血球(例如来自人，食蟹猴，大鼠或小鼠)，并用血清封闭细胞。以连续稀释液添加经标记的OX40抗体，还对T细胞染色以鉴定T细胞子集(使用本领域已知方法)。样品温育和清洗后，使用流式细胞仪分选细胞，并使用本领域公知方法分析数据。在另一个实施方式中，可使用表面等离振子共振来分析OX40结合。例示了一种例示性表面等离振子共振方法。On the one hand, the antibody of the present invention is tested for its antigen binding activity, for example, by a known method such as ELISA, Western blot, and the like. Methods known in the art can be used to determine OX40 binding, and exemplary methods are disclosed herein. In one embodiment, radioimmunoassay is used to measure binding. An exemplary radioimmunoassay is illustrated. The OX40 antibody was iodinated, and a competition reaction mixture containing a fixed concentration of iodinated antibody and a decreasing concentration of serially diluted unlabeled OX40 antibody was prepared. Cells expressing OX40 (for example, BT474 cells stably transfected with human OX40) are added to the reaction mixture. After incubation, the cells are washed to separate the free iodinated OX40 antibody from the OX40 antibody bound to the cells. The level of bound iodinated OX40 antibody is determined, for example, by counting the radioactivity associated with the cells, and using standard methods to determine the binding affinity. In another embodiment, flow cytometry is used to assess the ability of OX40 antibodies to bind surface-expressed OX40 (e.g., on a subset of T cells). Obtain peripheral leukocytes (for example from humans, cynomolgus monkeys, rats or mice), and block the cells with serum. The labeled OX40 antibody was added in serial dilutions, and T cells were also stained to identify T cell subsets (using methods known in the art). After the sample is incubated and washed, the cells are sorted using a flow cytometer, and the data is analyzed using methods known in the art. In another embodiment, surface plasmon resonance can be used to analyze OX40 binding. An exemplary surface plasmon resonance method is illustrated.
另一方面，可使用竞争测定法来鉴定与本文中公开的任何抗OX40抗体竞争对OX40的结合的抗体。在某些实施方式中，此类竞争性抗体结合与本文中公开的任何抗OX40抗体所结合表位相同的表位(例如线性或构象表位)。用于定位抗体所结合表位的详细例示性方法见Morris(1996)“Epitope Mapping Protocols”，Methods in Molecular Biology vol.66(Humana Press，Totowa，NJ)。例示了一种竞争测定法。On the other hand, a competition assay can be used to identify antibodies that compete with any of the anti-OX40 antibodies disclosed herein for binding to OX40. In certain embodiments, such competitive antibodies bind to the same epitope (e.g., linear or conformational epitope) as bound by any of the anti-OX40 antibodies disclosed herein. See Morris (1996) "Epitope Mapping Protocols", Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ) for detailed exemplary methods for locating epitopes bound by antibodies. A competitive assay is exemplified.
在一种例示性竞争测定法中，在包含第一经标记抗体(其结合OX，例如mab 1A7.gr.1，mab 3C8.gr5)和第二未标记抗体(其要测试与第一抗体竞争对OX40的结合的能力)的溶液中温育固定化OX40。第二抗体可存在于杂交瘤上清液中。作为对照，在包含第一经标记抗体但不包含第二未标记抗体的溶液中温育固定化OX40。在允许第一抗体结合OX40的条件下温育后，除去过量的未结合抗体，并测量与固定化OX40联合的标记物的量。如果测试样品中与固定化OX40联合的标记物的量与对照样品相比实质性 降低，那么这指示第二抗体与第一抗体竞争对OX40的结合。参见Harlow and Lane(1988)Antibodies:A Laboratory Manual ch.14(Cold Spring Harbor Laboratory,Cold Spring Harbor,NY)。In an exemplary competition assay, a first labeled antibody (which binds to OX, such as mab 1A7.gr.1, mab 3C8.gr5) and a second unlabeled antibody (which are tested to compete with the first antibody) are included in an exemplary competition assay. The ability to bind to OX40) incubate the immobilized OX40 in a solution. The second antibody may be present in the supernatant of the hybridoma. As a control, the immobilized OX40 was incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow the first antibody to bind to OX40, the excess unbound antibody is removed, and the amount of the label combined with the immobilized OX40 is measured. If the amount of the label associated with immobilized OX40 in the test sample is substantially reduced compared to the control sample, this indicates that the second antibody competes with the first antibody for binding to OX40. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).
2.活性测定法2. Activity assay
一方面，提供用于鉴定具有生物学活性的抗OX40抗体的测定法。生物学活性可以包括例如结合OX40(例如结合人和/或食蟹猴OX40)，提高OX40介导的信号转导(例如提高NFkB介导的转录)，消减表达人OX40的细胞(例如T细胞)，通过ADCC和/或吞噬消减表达人OX40的细胞，增强T效应细胞功能(例如CD4+效应T细胞)(例如通过提高效应T细胞增殖和/或提高效应T细胞的细胞因子生成(例如γ干扰素))，增强记忆T细胞功能(例如CD4+记忆T细胞)(例如通过提高记忆T细胞增殖和/或提高记忆T细胞的细胞因子生成(例如γ干扰素))，抑制调节T细胞功能(例如通过降低效应T细胞功能(例如CD4+效应T细胞功能))的Treg遏制)，结合人效应细胞。还提供在体内和/或在体外具有此类生物学活性的抗体。In one aspect, an assay method for identifying anti-OX40 antibodies with biological activity is provided. Biological activities can include, for example, binding to OX40 (for example, binding to human and/or cyno OX40), increasing OX40-mediated signal transduction (for example, increasing NFkB-mediated transcription), and reducing cells expressing human OX40 (for example, T cells) , By ADCC and/or phagocytosis to deplete the cells expressing human OX40, enhance T effector cell function (e.g. CD4+ effector T cells) (e.g. by increasing effector T cell proliferation and/or increasing effector T cell cytokine production (e.g. interferon gamma) )), enhance the function of memory T cells (e.g. CD4+ memory T cells) (e.g. by increasing the proliferation of memory T cells and/or increasing the cytokine production of memory T cells (e.g. gamma interferon)), inhibiting the function of regulatory T cells (e.g. by Treg suppression that reduces effector T cell function (eg CD4+ effector T cell function), binds to human effector cells. An antibody having such biological activity in vivo and/or in vitro is also provided.
在某些实施方式中，对本发明的抗体测试此类生物学活性。In certain embodiments, the antibodies of the invention are tested for such biological activity.
可以使用本领域已知的方法来测定T细胞共刺激，而且本文中公开了例示性方法。例如，可以自外周白血球获得T细胞(例如记忆或效应T细胞)(例如使用Ficoll梯度离心自人全血分离)。可以使用本领域已知的方法自PBMC分离记忆T细胞(例如CD4+记忆T细胞)或效应T细胞(例如CD4+Teff细胞)。例如，可以使用MiItenyiCD4+记忆T细胞分离试剂盒或Miltenyi幼稚CD4+T细胞分离试剂盒。在抗原呈递细胞(例如经过照射的表达CD32和CD80的L细胞)存在下培养分离的T细胞，并通过在OX40激动性抗体存在或缺失下添加抗CD3抗体来活化。可以使用本领域公知的方法来测量激动性OX40抗体对T细胞增殖的影响。例如，可以使用Cell Titer Glo试剂盒(Promega)，并在多标记物读数仪(Perkin Elmer)上读取结果。还可以通过分析由T细胞生成的细胞因子来测定激动性OX40抗体对T细胞功能的影响。在一个实施方式中，测定CD4+T细胞的干扰素γ生成，例如通过测量细胞培养物上清液中的干扰素γ。用于测量干扰素γ的方法是本领域公知的。Methods known in the art can be used to determine T cell costimulation, and exemplary methods are disclosed herein. For example, T cells (e.g., memory or effector T cells) can be obtained from peripheral white blood cells (e.g., separated from human whole blood using Ficoll gradient centrifugation). Methods known in the art can be used to isolate memory T cells (e.g., CD4+ memory T cells) or effector T cells (e.g., CD4+ Teff cells) from PBMC. For example, MiItenyi CD4+ Memory T Cell Isolation Kit or Miltenyi Naive CD4+ T Cell Isolation Kit can be used. The isolated T cells are cultured in the presence of antigen-presenting cells (for example, irradiated L cells expressing CD32 and CD80), and activated by adding anti-CD3 antibodies in the presence or absence of OX40 agonistic antibodies. The effects of agonistic OX40 antibodies on T cell proliferation can be measured using methods known in the art. For example, you can use the CellTiterGlo kit (Promega), and read the results on a multi-label reader (PerkinElmer). The effect of agonistic OX40 antibody on T cell function can also be determined by analyzing the cytokines produced by T cells. In one embodiment, interferon gamma production by CD4+ T cells is measured, for example, by measuring interferon gamma in the cell culture supernatant. Methods for measuring interferon gamma are well known in the art.
可以使用本领域已知的方法来测定Treg细胞功能，而且本文中公开了例示性方法。在一个例子中，测定Treg遏制效应T细胞增殖的能力。使用本领域已知的方法自人全血分离T细胞(例如分离记忆T细胞或幼稚T细胞)。标记纯化后的CD4+幼稚T细胞(例如用CFSE)，并用不同试剂标记纯化后的Treg细胞。将经过照射的抗原呈递细胞(例如表达CD32和CD80 的L细胞)与经过标记的纯化后的幼稚CD4+Τ细胞和纯化后的Treg共培养。使用抗CD3抗体活化共培养物，并在激动性OMO抗体存在或缺失下测试。合适时间(例如共培养6天)后，使用FACS分析通过降低的标记物染色(例如降低的CFSE标记物染色)中的染料稀释来跟踪CD4+幼稚T细胞增殖的水平。Methods known in the art can be used to determine Treg cell function, and exemplary methods are disclosed herein. In one example, the ability of Tregs to suppress the proliferation of effector T cells is determined. T cells are isolated from human whole blood using methods known in the art (e.g., memory T cells or naive T cells). The purified CD4+ naive T cells are labeled (for example, with CFSE), and the purified Treg cells are labeled with different reagents. The irradiated antigen-presenting cells (for example, L cells expressing CD32 and CD80) are co-cultured with labeled and purified naive CD4+ T cells and purified Treg. Co-cultures were activated with anti-CD3 antibodies and tested in the presence or absence of agonistic OMO antibodies. After a suitable time (for example, 6 days of co-cultivation), FACS analysis is used to track the level of CD4+ naive T cell proliferation by dye dilution in reduced marker staining (for example, reduced CFSE marker staining).
可以使用本领域公知的方法来测定OX40信号传导，而且本文中公开了例示性方法。在一个实施方式中，生成表达人OX40和报告基因(包含融合至报告基因(例如β萤光素酶)的NFkB启动子)的转基因细胞。对细胞添加OX40激动性抗体导致NFkB转录升高，这使用针对报告基因的测定法来检测。Methods known in the art can be used to measure OX40 signaling, and exemplary methods are disclosed herein. In one embodiment, a transgenic cell expressing human OX40 and a reporter gene (comprising an NFkB promoter fused to a reporter gene (such as β-luciferase)) is generated. Adding an OX40 agonistic antibody to the cells resulted in an increase in NFkB transcription, which was detected using an assay for reporter genes.
可以例如通过使用单核细胞衍生的巨噬细胞或U937细胞(一种具有成熟巨噬细胞的形态和特征的人组织细胞性淋巴瘤细胞系)来测定吞噬作用。在抗OX40激动性抗体存在或缺失下将表达OX40的细胞添加至单核细胞衍生的巨噬细胞或U937细胞。将细胞培养合适时间段后，通过检查针对1)巨噬细胞或U937细胞和2)表达OX40的细胞的标志物双重染色的细胞的百分比，并将此除以显示表达OX40的细胞的标志物(例如GFP)的细胞的总数来测定吞噬百分比。可以通过流式细胞术来进行分析。在另一个实施方式中，可以通过荧光显微术分析来进行分析。Phagocytosis can be measured, for example, by using monocyte-derived macrophages or U937 cells (a human histiocytic lymphoma cell line with the morphology and characteristics of mature macrophages). Cells expressing OX40 are added to monocyte-derived macrophages or U937 cells in the presence or absence of anti-OX40 agonistic antibodies. After the cells are cultured for a suitable period of time, the percentage of cells double-stained with markers for 1) macrophages or U937 cells and 2) cells expressing OX40 is checked, and this is divided by the markers for cells expressing OX40 ( For example, the total number of GFP) cells is used to determine the percentage of phagocytosis. It can be analyzed by flow cytometry. In another embodiment, the analysis can be performed by fluorescence microscopy analysis.
可以例如使用本领域公知的方法测定ADCC。定义部分中描述了例示性方法。在一些实施方式中，表征在ADCC测定法中用于测试的表达OX40的细胞上的OX40水平。将细胞用可检测标记的抗OX40抗体(例如PE标记的)染色，然后使用流式细胞术测定荧光水平，并以中值荧光强度(MFI)呈现结果。在另一个实施方式中，可以通过CellTiter Glo测定法试剂盒来分析ADCC，而且可以通过化学发光来测定细胞存活力/细胞毒性。ADCC can be measured, for example, using methods known in the art. Exemplary methods are described in the definition section. In some embodiments, the level of OX40 on OX40-expressing cells used for testing in the ADCC assay is characterized. The cells are stained with a detectably labeled anti-OX40 antibody (for example, PE-labeled), and then the fluorescence level is measured using flow cytometry, and the results are presented as median fluorescence intensity (MFI). In another embodiment, ADCC can be analyzed by the CellTiter Glo assay kit, and cell viability/cytotoxicity can be determined by chemiluminescence.
可以使用相应重组Fcγ受体在基于ELISA的配体结合测定法中测量各种抗体对FcγRIA，FcγRIIA，FcγRIIB，和FcγRIIIA的两种同种异型(F158和V158)的结合亲和力。以含有连接至C端Gly/6xHis/谷胱甘肽S-转移酶(GST)多肽标签的受体γ链胞外域的融合蛋白表达纯化后的人Fcγ受体。如下测定抗体对那些人Fcγ受体的结合亲和力。对于低亲和力受体，即FcγRIIA(CD32A)，FcγRIIB(CD32B)，和FeγRIIIA(CD16)的两种同种异型，F-158和V-158,可以通过用山羊抗人卡帕链的F(ab')2片段(ICN Biomedical；Irvine，CA)交联(以近似摩尔比1:3抗体：交联用F(ab')2)作为多聚体测试抗体。将板用抗GST抗体(Genentech)包被，并用牛血清清蛋白(BSA)封闭。用含有0.05％Tween-20的磷酸盐缓冲盐水(PBS)及ELx405 TM洗板仪(Biotek Instruments；Winooski，VT)清洗后，以25ng/孔将Fcγ受体添加至板，并于 室温温育1小时。清洗板后，作为多聚体复合物添加测试抗体的系列稀释液，并将板于室温温育2小时。清洗板以去除未结合的抗体后，用辣根过氧化物酶(HRP)缀合的山羊抗人F(ab')2的F(ab')2片段(Jackson ImmunoResearch Laboratories；West Grove，PA)检测结合至Fcγ受体的抗体，接着添加底物，四甲基联苯胺(TMB)(Kirkegaard and Perry Laboratories；Gaithersburg，MD)。取决于所测试的Fcγ受体，将板于室温温育5-20分钟以容许显色。用IM H3PO4终止反应，并用微量板读数仪(
Figure PCTCN2020140259-appb-000003
190，Molecular Devices；Sunnyvale，CA)测量450nm处的吸光度。通过将来自一式两份抗体稀释液的均值吸光值针对抗体浓度绘图，生成剂量-响应结合曲线。使用SoftMax 190(Molecular Devices)用四参数方程拟合结合曲线后确定检测到来自结合Fcγ受体的最大响应50％时的有效抗体浓度的值(EC50)。 The corresponding recombinant Fcy receptors can be used to measure the binding affinity of various antibodies to FcyRIA, FcyRIIA, FcyRIIB, and the two allotypes of FcyRIIIA (F158 and V158) in an ELISA-based ligand binding assay. The purified human Fcγ receptor was expressed as a fusion protein containing the extracellular domain of the receptor γ chain linked to the C-terminal Gly/6xHis/glutathione S-transferase (GST) polypeptide tag. The binding affinity of antibodies to those human Fcγ receptors was determined as follows. For low-affinity receptors, namely FcγRIIA (CD32A), FcγRIIB (CD32B), and the two allotypes of FeγRIIIA (CD16), F-158 and V-158, you can use goat anti-human kappa chain F(ab ') 2 fragment (ICN Biomedical; Irvine, CA) cross-linked (approximate molar ratio 1:3 antibody: cross-linking F(ab')2) as a multimer test antibody. The plate was coated with anti-GST antibody (Genentech) and blocked with bovine serum albumin (BSA). After washing with phosphate buffered saline (PBS) containing 0.05% Tween-20 and ELx405 TM plate washer (Biotek Instruments; Winooski, VT), Fcγ receptor was added to the plate at 25ng/well and incubated at room temperature 1 hour. After washing the plate, a serial dilution of the test antibody is added as a polymer complex, and the plate is incubated at room temperature for 2 hours. After washing the plate to remove unbound antibody, horseradish peroxidase (HRP) conjugated goat anti-human F(ab')2 F(ab')2 fragment (Jackson ImmunoResearch Laboratories; West Grove, PA) The antibody bound to the Fcγ receptor is detected, followed by the addition of the substrate, tetramethylbenzidine (TMB) (Kirkegaard and Perry Laboratories; Gaithersburg, MD). Depending on the Fcy receptor tested, the plate is incubated at room temperature for 5-20 minutes to allow color development. Stop the reaction with IM H3PO4, and use the microplate reader (
Figure PCTCN2020140259-appb-000003
190, Molecular Devices; Sunnyvale, CA) measured absorbance at 450 nm. A dose-response binding curve was generated by plotting the mean absorbance values from duplicate antibody dilutions against antibody concentration. The binding curve was fitted with a four-parameter equation using SoftMax 190 (Molecular Devices) to determine the effective antibody concentration (EC50) at which 50% of the maximum response from the bound Fcγ receptor was detected.
为了选择诱导细胞死亡的抗体，可相对于对照评估通过例如碘化丙啶(PI)，锥虫蓝或7AAD摄取显示的膜完整性丧失。PI摄取测定法可在补体和免疫效应细胞缺失下实施。在单独的培养基或含有浓度为例如约10μg/ml的适宜单克隆抗体的培养基中温育表达OX40的细胞。将细胞温育某个时段(例如1或3天)。每次处理后，将细胞清洗并等分。在一些实施方式中，将细胞等分到35mm盖有滤网(strainer-capped)的12x75管中(每管1ml，每个处理组3管)以除去细胞团块。然后向管中加入PI(10μg/ml)。可以使用FACSCAN TM流式细胞仪和FACSCONVERT TMCellQuest软件(Becton Dickinson)分析样品。 In order to select antibodies that induce cell death, the loss of membrane integrity shown by, for example, propidium iodide (PI), trypan blue or 7AAD uptake can be assessed relative to a control. The PI uptake assay can be performed in the absence of complement and immune effector cells. The cells expressing OX40 are incubated in a separate medium or a medium containing a suitable monoclonal antibody at a concentration of, for example, about 10 μg/ml. The cells are incubated for a certain period of time (for example, 1 or 3 days). After each treatment, the cells were washed and aliquoted. In some embodiments, the cells are aliquoted into 35mm strainer-capped 12x75 tubes (1 ml per tube, 3 tubes per treatment group) to remove cell clumps. Then PI (10 μg/ml) was added to the tube. The samples can be analyzed using a FACSCAN ™ flow cytometer and FACSCONVERT ™ CellQuest software (Becton Dickinson).
供任何上述体外测定法使用的细胞包括天然表达OX40或经改造而表达OX40的细胞或细胞系。此类细胞包括天然表达OX40的活化后的T细胞，Treg细胞和活化后的记忆T细胞。此类细胞还包括表达OX40的细胞系和并非正常情况下表达OX40但已经用编码OX40的核酸转染的细胞系。本文中提供的供任何上述体外测定法使用的例示性细胞系包括表达人OX40的转基因BT474细胞(一种人乳腺癌细胞系)。Cells for use in any of the above in vitro assays include cells or cell lines that naturally express OX40 or have been engineered to express OX40. Such cells include activated T cells that naturally express OX40, Treg cells, and activated memory T cells. Such cells also include cell lines that express OX40 and cell lines that do not normally express OX40 but have been transfected with nucleic acid encoding OX40. Exemplary cell lines provided herein for use in any of the above in vitro assays include transgenic BT474 cells expressing human OX40 (a human breast cancer cell line).
理解的是，可以使用本发明的免疫缀合物替换或补充抗OX40抗体来进行任何上述测定法。It is understood that the immunoconjugates of the invention can be used to replace or supplement the anti-OX40 antibody to perform any of the aforementioned assays.
理解的是，可以使用抗OX40抗体和别的治疗剂来进行任何上述测定法。It is understood that anti-OX40 antibodies and other therapeutic agents can be used to perform any of the aforementioned assays.
配方及用途Formulation and use
本发明的抗体或其抗原结合片段可调配成药物组合物。该药物组合物可进一步包含药学上可接受的载体、赋形剂及/或稳定剂(Remington:The Science and practice ofPharmacy，第20版，2000,Lippincott Williams and  Wilkins,Ed.K.E.Hoover)，以冻干配方或水溶液的形式。可接受的载体、赋形剂或稳定剂在剂量及浓度下对接受者是无毒的，及可包含缓冲剂诸如磷酸、柠檬酸及其他有机酸；抗氧化剂，包括抗坏血酸及甲硫氨酸；防腐剂(诸如十八烷基二甲基苄基氯化铵；氯化六羟季铵；杀藻胺；氯化本索宁；酚醇、丁醇或苄醇；对羟基苯甲酸烷基酯，诸如对羟基苯甲酸甲酯或对羟基苯甲酸丙酯；儿茶酚；间苯二酚；环己醇；3-戊醇；及间甲酚)；低分子量(小于约10个残基)多肽；蛋白质，诸如血清白蛋白、明胶或免疫球蛋白；亲水性聚合物，诸如聚乙烯吡咯啶酮；氨基酸，诸如甘氨酸、谷氨酰胺、天冬酰氨酸、组氨酸、精氨酸或赖氨酸；单醣、二醣及其他醣，包括葡萄糖、甘露糖或葡聚糖；螯合剂，诸如EDTA；糖，诸如蔗糖、甘露醇、海藻糖或山梨醇；成盐相反离子，诸如钠；金属络合物(例如，Zn-蛋白质络合物)；及/或非离子型表面活性剂，诸如TWEEN TM、PLURONICS TM或聚乙二醇(PEG)。本文进一步描述药学上可接受的赋形剂。 The antibody or antigen-binding fragment thereof of the present invention can be formulated into a pharmaceutical composition. The pharmaceutical composition may further comprise pharmaceutically acceptable carriers, excipients and/or stabilizers (Remington: The Science and practice of Pharmacy, 20th Edition, 2000, Lippincott Williams and Wilkins, Ed. KE Hoover), and lyophilized Formulation or aqueous solution. Acceptable carriers, excipients or stabilizers are non-toxic to the recipient at the dose and concentration, and may contain buffers such as phosphoric acid, citric acid and other organic acids; antioxidants, including ascorbic acid and methionine; Preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexahydroxy quaternary ammonium chloride; algaecide; benzonine chloride; phenolic alcohol, butanol or benzyl alcohol; alkyl p-hydroxybenzoate , Such as methyl p-hydroxybenzoate or propyl p-hydroxybenzoate; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) Polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulin; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine Or lysine; monosaccharides, disaccharides, and other sugars, including glucose, mannose, or dextran; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as Sodium; metal complexes (for example, Zn-protein complexes); and/or non-ionic surfactants such as TWEEN ™ , PLURONICS ™ or polyethylene glycol (PEG). The pharmaceutically acceptable excipients are further described herein.
本发明的抗体或其抗原结合片段可用于各种治疗或诊断目的。例如，本发明的抗体或其抗原结合片段可用作亲和力纯化剂(例如，用于活体外纯化)；用作诊断剂(例如，用于检测特异性细胞、组织或血清中的表达)。The antibody or antigen-binding fragment thereof of the present invention can be used for various therapeutic or diagnostic purposes. For example, the antibody or antigen-binding fragment thereof of the present invention can be used as an affinity purification agent (for example, for in vitro purification); as a diagnostic agent (for example, for detecting expression in specific cells, tissues, or serum).
本发明的抗体或其抗原结合片段的例示性治疗用途包括治疗癌症。本发明的抗体或其抗原结合片段亦可用于预防性治疗。Exemplary therapeutic uses of the antibodies of the present invention or antigen-binding fragments thereof include the treatment of cancer. The antibody or antigen-binding fragment thereof of the present invention can also be used for prophylactic treatment.
就治疗应用而言，本发明的抗体或其抗原结合片段可通过常规技术向哺乳动物、尤其人类施用，所述技术诸如静脉内(作为推注或通过经时的连续输注)、肌内、腹腔内、脑内、皮下、关节内、滑膜内、鞘内、经口、局部或通过吸入。本发明的抗体或其抗原结合片段亦可适当通过肿瘤内、肿瘤周围、病灶内或病灶周围途径施用。For therapeutic applications, the antibodies or antigen-binding fragments of the present invention can be administered to mammals, especially humans, by conventional techniques, such as intravenous (as a bolus injection or by continuous infusion over time), intramuscular, Intra-abdominal, intracerebral, subcutaneous, intra-articular, intra-synovial, intrathecal, oral, topical or inhalation. The antibody or antigen-binding fragment thereof of the present invention can also be appropriately administered via intratumor, peritumor, intralesional, or perilesional routes.
在某些实施方式中，本发明的抗体或其抗原结合片段是经皮下施用。在某些实施方式中，本发明的抗体或其抗原结合片段是经静脉内施用。In certain embodiments, the antibodies of the invention or antigen-binding fragments thereof are administered subcutaneously. In certain embodiments, the antibodies of the invention or antigen-binding fragments thereof are administered intravenously.
药物组合物可以可随疾病的严重性变化的频率向有此需要的受试者施用。在预防治疗的情况下，该频率可取决于该受试者疾病的易发性或倾向而变化。The pharmaceutical composition can be administered to subjects in need at a frequency that can vary with the severity of the disease. In the case of prophylactic treatment, the frequency may vary depending on the subject's disease susceptibility or tendency.
所述组合物可作为推注或通过连续输注向有此需要的患者施用。例如，以Fab片段呈现的抗体的推注施用可以自0.0025至100mg/kg体重、0.025至0.25mg/kg、0.010至0.10mg/kg或0.10至0.50mg/kg的量进行施用。就连续输注而言，以Fab片段呈现的抗体可以0.001至100mg/kg体重/分钟、0.0125至1.25mg/kg/min、0.010至0.75mg/kg/min、0.010至1.0mg/kg/min或0.10至0.50mg/kg/min的量施用，持续1至24小时、1至12小时、2至12小时、6至12小时、2至8小时或1至2小时。The composition can be administered as a bolus injection or by continuous infusion to patients in need. For example, bolus administration of antibodies presented as Fab fragments can be administered from an amount of 0.0025 to 100 mg/kg body weight, 0.025 to 0.25 mg/kg, 0.010 to 0.10 mg/kg, or 0.10 to 0.50 mg/kg. For continuous infusion, antibodies presented as Fab fragments can be 0.001 to 100 mg/kg body weight/min, 0.0125 to 1.25 mg/kg/min, 0.010 to 0.75 mg/kg/min, 0.010 to 1.0 mg/kg/min, or It is administered in an amount of 0.10 to 0.50 mg/kg/min for 1 to 24 hours, 1 to 12 hours, 2 to 12 hours, 6 to 12 hours, 2 to 8 hours, or 1 to 2 hours.
就以全长抗体(具有完整恒定区)呈现的抗体的施用而言，剂量可为自约1mg/kg至约10mg/kg、自约2mg/kg至约10mg/kg、自约3mg/kg至约10mg/kg、自约4mg/kg至约10mg/kg、自约5mg/kg至约10mg/kg、自约1mg/kg至约20mg/kg、自约2mg/kg至约20mg/kg、自约3mg/kg至约20mg/kg、自约4mg/kg至约20mg/kg、自约5mg/kg至约20mg/kg、约1mg/kg或更多、约2mg/kg或更多、约3mg/kg或更多、约4mg/kg或更多、约5mg/kg或更多、约6mg/kg或更多、约7mg/kg或更多、约8mg/kg或更多、约9mg/kg或更多、约10mg/kg或更多、约11mg/kg或更多、约12mg/kg或更多、约13mg/kg或更多、约14mg/kg或更多、约15mg/kg或更多、约16mg/kg或更多、约17mg/kg或更多、约19mg/kg或更多或约20mg/kg或更多。施用的频率将取决于病症的严重性。频率可在自每周三次至每两周或三周一次的范围内变化。For the administration of the antibody presented as a full-length antibody (with a complete constant region), the dose can be from about 1 mg/kg to about 10 mg/kg, from about 2 mg/kg to about 10 mg/kg, from about 3 mg/kg to About 10 mg/kg, from about 4 mg/kg to about 10 mg/kg, from about 5 mg/kg to about 10 mg/kg, from about 1 mg/kg to about 20 mg/kg, from about 2 mg/kg to about 20 mg/kg, from About 3mg/kg to about 20mg/kg, from about 4mg/kg to about 20mg/kg, from about 5mg/kg to about 20mg/kg, about 1mg/kg or more, about 2mg/kg or more, about 3mg /kg or more, about 4mg/kg or more, about 5mg/kg or more, about 6mg/kg or more, about 7mg/kg or more, about 8mg/kg or more, about 9mg/kg Or more, about 10 mg/kg or more, about 11 mg/kg or more, about 12 mg/kg or more, about 13 mg/kg or more, about 14 mg/kg or more, about 15 mg/kg or more More, about 16 mg/kg or more, about 17 mg/kg or more, about 19 mg/kg or more, or about 20 mg/kg or more. The frequency of administration will depend on the severity of the condition. The frequency can vary from three times a week to once every two or three weeks.
另外，所述组合物可经由皮下注射向患者施用。例如，1至100mg抗OX40抗体的剂量可经由皮下或静脉内注射以一周两次、一周一次、每两周一次、每三周一次、每四周一次、每五周一次、每六周一次、每七周一次、每八周一次、每九周一次、每十周一次、一个月两次、一个月一次、每两个月一次或每三个月一次的频率向患者施用。In addition, the composition can be administered to the patient via subcutaneous injection. For example, the dose of 1 to 100 mg of anti-OX40 antibody can be injected subcutaneously or intravenously at twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, every It is administered to the patient at a frequency of once every seven weeks, once every eight weeks, once every nine weeks, once every ten weeks, twice a month, once a month, once every two months, or once every three months.
在某些实施方式中，抗OX40抗体于人类中的半衰期是约5天、约6天、约7天、约8天、约9天、约10天、约11天、约12天、约13天、约14天、约15天、约16天、约17天、约18天、约19天、约20天、约21天、约22天、约23天、约24天、约25天、约26天、约27天、约28天、约29天、约30天、自约5天至约40天、自约5天至约35天、自约5天至约30天、自约5天至约25天、自约10天至约40天、自约10天至约35天、自约10天至约30天、自约10天至约25天、自约15天至约40天、自约15天至约35天、自约15天至约30天、或自约15天至约25天。In certain embodiments, the half-life of an anti-OX40 antibody in humans is about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days. Days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 22 days, about 23 days, about 24 days, about 25 days, About 26 days, about 27 days, about 28 days, about 29 days, about 30 days, from about 5 days to about 40 days, from about 5 days to about 35 days, from about 5 days to about 30 days, from about 5 days From about 10 days to about 40 days, from about 10 days to about 35 days, from about 10 days to about 30 days, from about 10 days to about 25 days, from about 15 days to about 40 days , From about 15 days to about 35 days, from about 15 days to about 30 days, or from about 15 days to about 25 days.
在某些实施方式中，该药物组合物是以以下的剂量每2至6周进行皮下或静脉内施用：自约0.1mg/kg至约10mg/kg、自约0.5mg/kg至约10mg/kg、自约1mg/kg至约10mg/kg、自约1.5mg/kg至约10mg/kg、自约2mg/kg至约10mg/kg、自约0.1mg/kg至约8mg/kg、自约0.5mg/kg至约8mg/kg、自约1mg/kg至约8mg/kg、自约1.5mg/kg至约8mg/kg、自约2mg/kg至约8mg/kg、自约0.1mg/kg至约5mg/kg、自约0.5mg/kg至约5mg/kg、自约1mg/kg至约5mg/kg、自约1.5mg/kg至约5mg/kg、自约2mg/kg至约5mg/kg、约0.5mg/kg、约1.0mg/kg、约1.5mg/kg、约2.0mg/kg、约2.5mg/kg、约3.0mg/kg、约3.5mg/kg、约4.0mg/kg、约4.5mg/kg、约5.0mg/kg、约5.5mg/kg、约6.0mg/kg、约6.5mg/kg、约7.0mg/kg、约7.5mg/kg、约8.0mg/kg、约8.5mg/kg、约9.0mg/kg、约9.5mg/kg或约10.0mg/kg。In certain embodiments, the pharmaceutical composition is administered subcutaneously or intravenously every 2 to 6 weeks at the following dose: from about 0.1 mg/kg to about 10 mg/kg, from about 0.5 mg/kg to about 10 mg/kg kg, from about 1 mg/kg to about 10 mg/kg, from about 1.5 mg/kg to about 10 mg/kg, from about 2 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 8 mg/kg, from about 0.5mg/kg to about 8mg/kg, from about 1mg/kg to about 8mg/kg, from about 1.5mg/kg to about 8mg/kg, from about 2mg/kg to about 8mg/kg, from about 0.1mg/kg To about 5mg/kg, from about 0.5mg/kg to about 5mg/kg, from about 1mg/kg to about 5mg/kg, from about 1.5mg/kg to about 5mg/kg, from about 2mg/kg to about 5mg/kg kg, about 0.5mg/kg, about 1.0mg/kg, about 1.5mg/kg, about 2.0mg/kg, about 2.5mg/kg, about 3.0mg/kg, about 3.5mg/kg, about 4.0mg/kg, About 4.5 mg/kg, about 5.0 mg/kg, about 5.5 mg/kg, about 6.0 mg/kg, about 6.5 mg/kg, about 7.0 mg/kg, about 7.5 mg/kg, about 8.0 mg/kg, about 8.5 mg/kg, about 9.0 mg/kg, about 9.5 mg/kg, or about 10.0 mg/kg.
在某些实施方式中，该药物组合物是以约2.0mg/kg的剂量每2至6周进行皮下注射或静脉内施用。在某些实施方式中，该药物组合物是以自约2.0mg/kg至约10.0mg/kg的剂量每2至6周进行皮下注射或静脉内施用。In certain embodiments, the pharmaceutical composition is administered subcutaneously or intravenously at a dose of about 2.0 mg/kg every 2 to 6 weeks. In certain embodiments, the pharmaceutical composition is administered subcutaneously or intravenously every 2 to 6 weeks at a dose of from about 2.0 mg/kg to about 10.0 mg/kg.
在一个例示性实施方式中，药物组合物是每2周皮下施用。In an exemplary embodiment, the pharmaceutical composition is administered subcutaneously every 2 weeks.
本发明的抗体或其抗原结合片段可用作单药疗法或与其他疗法组合以治疗癌症。The antibodies or antigen-binding fragments thereof of the present invention can be used as monotherapy or in combination with other therapies to treat cancer.
定义definition
除非本文另有定义，否则结合本发明使用的科学及技术术语应具有本领域普通技术人员通常所了解的含义。此外，除非上下文另有要求，否则单数术语应包括复数及复数术语应包括单数。通常，结合本文描述的细胞及组织培养物、分子生物学、免疫学、微生物学、遗传学及蛋白质及核酸化学及杂交使用的术语表及技术是那些本领域中熟知及常用者。Unless otherwise defined herein, the scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. In addition, unless the context requires otherwise, singular terms shall include pluralities and plural terms shall include the singular. Generally, the glossary and techniques used in conjunction with cell and tissue culture, molecular biology, immunology, microbiology, genetics, protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art.
抗体的“抗原结合片段”是指全长抗体的保留特异性结合至抗原的能力的片段(优选地，具有基本上相同的结合亲和力)。抗原结合片段的实例包括(i)Fab片段，其为由VL、VH、CL及CH1域组成的单价片段；(ii)F(ab')2片段，其为包含于铰链区通过双硫键连接的两个Fab片段的二价片段；(iii)由VH及CH1域组成的Fd片段；(iv)由抗体的单臂的VL及VH域组成的Fv片段；(v)dAb片段(Ward等人，(1989)Nature 341:544-546)，其由VH域组成；及(vi)经分离的互补决定区(CDR)、经双硫键连接的Fvs(dsFv)及抗-特应(抗-Id)抗体及胞内抗体。此外，尽管Fv片段的两个域(VL及VH)是由不同基因编码，但其可使用重组方法通过合成连接体连接，该合成连接体使得其被制成单一蛋白链，其中VL及VH区配对以形成单价分子(称为单链Fv(scFv))；参见例如，Bird等人Science 242:423-426(1988)及Huston等人，Proc.Natl.Acad.Sci.USA 85:5879-5883(1988))。单链抗体的其他形式(诸如双抗体)亦包含于本发明中。双抗体是二价双特异性抗体，其中VH及VL域是表达于单一多肽链上，但使用太短以致于无法容许在相同链上的两个链之间配对的连接体，藉此迫使所述域与另一链的互补域配对，及产生两个抗原-结合位点(参见例如，Holliger等人，Proc.Natl.Acad.Sci.USA 90:6444-6448(1993)；Poljak等人，1994,Structure 2:1121-1123)。An "antigen-binding fragment" of an antibody refers to a fragment of a full-length antibody that retains the ability to specifically bind to an antigen (preferably, has substantially the same binding affinity). Examples of antigen-binding fragments include (i) Fab fragments, which are monovalent fragments composed of VL, VH, CL and CH1 domains; (ii) F(ab')2 fragments, which are contained in the hinge region connected by disulfide bonds (Iii) Fd fragment composed of VH and CH1 domains; (iv) Fv fragment composed of VL and VH domains of one arm of an antibody; (v) dAb fragment (Ward et al. , (1989) Nature 341:544-546), which consists of VH domains; and (vi) isolated complementarity determining regions (CDR), disulfide-linked Fvs (dsFv) and anti-atopic (anti- Id) Antibodies and intracellular antibodies. In addition, although the two domains (VL and VH) of the Fv fragment are encoded by different genes, they can be connected by a synthetic linker using a recombination method. The synthetic linker allows it to be made into a single protein chain, in which the VL and VH regions Pair to form a monovalent molecule (called single-chain Fv (scFv)); see, for example, Bird et al. Science 242:423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988)). Other forms of single chain antibodies (such as diabodies) are also included in the present invention. Diabodies are bivalent bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but the use of a linker that is too short to allow pairing between two chains on the same chain is used to force all The domain is paired with the complementary domain of the other chain and creates two antigen-binding sites (see, for example, Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993); Poljak et al., 1994, Structure 2:1121-1123).
抗体“可变域”是指抗体轻链(VL)的可变区或抗体重链(VH)的可变区，单独或组合。如本领域中已知，重链及轻链的可变区各由三个互补决定区(CDR)组成，及由四个框架区(FR)连接，及有助于抗体的抗原结合位点的形成。An antibody "variable domain" refers to the variable region of the antibody light chain (VL) or the variable region of the antibody heavy chain (VH), alone or in combination. As known in the art, the variable regions of the heavy chain and the light chain are each composed of three complementarity determining regions (CDR), and are connected by four framework regions (FR), and contribute to the antigen binding site of the antibody form.
可变域中的残基是根据Kabat编号，Kabat是用于抗体的编译的重链可 变域或轻链可变域的编号***。参见Kabat等人，Sequences of Proteins of ImmunologicalInterest，第5版，Public Health Service,National Institutes of Health,Bethesda,MD.(1991))。使用此编号***，实际直链氨基酸序列可含有较少或额外的氨基酸，其对应于可变域的FR或CDR的缩短或***。对于给定抗体，残基的Kabat编号可通过于该抗体的序列与“标准”Kabat编号的序列具有同源性的区域的比对来测定。用于分配Kabat编号的各种算法是可获得的。除非本文另有说明，否则本文使用2012年发布的Abysis(www.abysis.org)中执行的算法将Kabat编号分配至可变区。The residues in the variable domains are numbered according to Kabat, which is the numbering system of the heavy chain variable domain or the light chain variable domain used for the compilation of antibodies. See Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids, which correspond to the shortening or insertion of the FR or CDR of the variable domain. For a given antibody, the Kabat numbering of residues can be determined by aligning the antibody's sequence to regions of homology with the "standard" Kabat numbering sequence. Various algorithms for assigning Kabat numbers are available. Unless otherwise stated in this article, this article uses the algorithm implemented in Abysis (www.abysis.org) released in 2012 to assign Kabat numbers to variable regions.
抗体中的具体氨基酸残基位置(诸如互补位残基)是亦根据Kabat编号。Specific amino acid residue positions in antibodies (such as paratope residues) are also numbered according to Kabat.
“互补决定区”(CDR)可根据本领域中熟知的Kabat、Chothia、Kabat及Chothia两者的聚集、AbM、接触(contact)及/或构形定义或CDR测定的任何方法的定义加以识别。参见例如Kabat等人，1991,Sequences of Proteins of Immunological Interest，第5版(高度可变区)；Chothia等人，1989,Nature 342:877-883(结构性环结构)。CDR的AbM定义是在Kabat与Chothia之间折衷及使用Oxford Molecular的AbM抗体建模软件CDR的“接触”定义是基于阐述于MacCallum等人，1996,J.Mol.Biol.,262:732-745中的可见抗原接触。CDR的“构象”定义是基于制造有助于抗原结合的焓的残基(参见，例如，Makabe等人，2008,Journal of Biological Chemistry,283:1156-1166)。又其他CDR边界定义可非严格遵守上文方法中的一者，但虽然如此仍将与Kabat CDR的至少一部分重叠，然而其可根据特定残基或残基组或甚至全部CDR不显著影响抗原结合的预测或实验发现而经缩短或延长。如本文使用，CDR可是指通过本领域中已知的任何方法(包括方法的组合)定义的CDR。The "complementarity determining region" (CDR) can be identified according to the definition of the aggregation, AbM, contact and/or configuration of both Kabat, Chothia, Kabat and Chothia well known in the art or any method of CDR determination. See, for example, Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th edition (highly variable regions); Chothia et al., 1989, Nature 342:877-883 (structural loop structure). The AbM definition of CDR is a compromise between Kabat and Chothia and the use of Oxford Molecular's AbM antibody modeling software. The "contact" definition of CDR is based on the definition described in MacCallum et al., 1996, J. Mol. Biol., 262: 732-745 Visible antigen contact in the. The "conformational" definition of CDR is based on making residues that contribute to the enthalpy of antigen binding (see, for example, Makabe et al., 2008, Journal of Biological Chemistry, 283:1156-1166). Still other CDR boundary definitions may not strictly comply with one of the above methods, but even so will still overlap with at least a part of Kabat CDR, but it may not significantly affect antigen binding according to specific residues or residue groups or even all CDRs The predictions or experimental findings have been shortened or lengthened. As used herein, CDR may refer to a CDR defined by any method (including a combination of methods) known in the art.
“抗原决定基”是指抗原(Ag)中抗体特异性结合的范围或区域，例如，包含与该抗体(Ab)相互作用的氨基酸残基的范围或区域。抗原决定基可为直链或非直链(例如，构象)的。"Antigenic determinant" refers to the range or region in the antigen (Ag) where the antibody specifically binds, for example, the range or region containing the amino acid residues that interact with the antibody (Ab). The epitope can be linear or non-linear (e.g., conformational).
当相应的抗体或其抗原结合片段的结合呈互相排外性时，抗体或其抗原结合片段基本上结合与另一抗体或其抗原结合片段相同的抗原决定基。即，一种抗体或其抗原结合片段的结合排除其他抗体或其抗原结合片段的同时或连续结合。若该抗原可适应两种相应的抗体或其抗原结合片段的同时结合，则认为抗原决定基是独一无二的或不是实质性地相同。When the binding of corresponding antibodies or antigen-binding fragments thereof is mutually exclusive, the antibody or antigen-binding fragment thereof basically binds to the same epitope as another antibody or antigen-binding fragment thereof. That is, the binding of one antibody or antigen-binding fragment thereof excludes simultaneous or continuous binding of other antibodies or antigen-binding fragments thereof. If the antigen can be adapted to the simultaneous binding of two corresponding antibodies or antigen-binding fragments thereof, the epitope is considered to be unique or not substantially the same.
术语“互补位”是通过扭转角度衍生自“抗原决定基”的上文定义，及是指抗体分子中涉及抗原结合的范围或区域，例如，包含与该抗原相互作用的残基的范围或区域。互补位可为直链或构象的(诸如CDR中的不连续残基)。The term "paratope" is derived from the above definition of "antigenic determinant" by twisting the angle, and refers to a range or region involved in antigen binding in an antibody molecule, for example, a range or region containing residues that interact with the antigen . Paratopes can be linear or conformational (such as discrete residues in CDRs).
给定抗体/抗原结合对的抗原决定基/互补位可使用各种实验及计算抗原决定基定位方法在不同细节层次下经定义及特征分析。实验方法包括诱 变、X射线晶体学、核磁共振(NMR)光谱学、氢/氘交换质谱法(HX-MS)及各种竞争结合方法。因为各方法依赖于独一无二的原则，所以抗原决定基的描述是与已测定该抗原决定基的方法紧密联系。因此，给定抗体/抗原对的抗原决定基/互补位将取决于采用的定位方法而不同地定义。The epitope/paratope of a given antibody/antigen binding pair can be defined and characterized at different levels of detail using various experimental and computational epitope positioning methods. Experimental methods include mutagenesis, X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, hydrogen/deuterium exchange mass spectrometry (HX-MS) and various competitive binding methods. Because each method relies on the principle of uniqueness, the description of the epitope is closely related to the method by which the epitope has been determined. Therefore, the epitope/paratope of a given antibody/antigen pair will be defined differently depending on the localization method employed.
在其最细节层次下，用于抗体(Ab)与抗原(Ag)间相互作用的抗原决定基/互补位可通过界定存在于Ag-Ab相互作用中的原子接触的空间坐标及有关其对结合热力学的相对贡献的信息进行定义。在一种程度下，抗原决定基/互补位残基可通过界定Ag与Ab间原子接触的空间坐标进行特征分析。在一个方面，该抗原决定基/互补位残基可通过特定标准，例如Ab及Ag中原子间的距离(例如，距同源抗体的重原子及该抗原的重原子等于或小于约界定。在另一方面，抗原决定基/互补位残基的特征为参与与同源抗体/抗原，或与亦经氢结合至同源抗体/抗体(经水介导的氢结合)的水分子的氢键相互作用。在另一方面，抗原决定基/互补位残基的特征为与同源抗体/抗原的残基形成盐桥。在又另一方面，抗原决定基/互补位残基的特征可为因与同源抗体/抗原的相互作用而于掩蔽表面积(BSA)中具有非零变化的残基。在较为不详细的程度下，抗原决定基/互补位可通过函数进行特征描述，例如，通过与其他Ab的竞争结合。该抗原决定基/互补位亦可更一般地定义为包含氨基酸残基，其中通过另一氨基酸的取代将变更于Ab与Ag之间的相互作用的特征(例如，丙氨酸扫描)。At its most detailed level, the epitope/paratope used for the interaction between the antibody (Ab) and the antigen (Ag) can be defined by the spatial coordinates of the atomic contact existing in the Ag-Ab interaction and the binding of the pair. The relative contribution of thermodynamics is defined by the information. To a certain extent, epitope/paratope residues can be characterized by defining the spatial coordinates of the atomic contact between Ag and Ab. In one aspect, the epitope/paratopic residues can be defined by specific criteria, such as the distance between atoms in Ab and Ag (e.g., from a heavy atom of a homologous antibody and a heavy atom of the antigen equal to or less than about approx. On the other hand, epitope/paratope residues are characterized by participating in hydrogen bonds with homologous antibodies/antigens, or with water molecules that are also hydrogen-bonded to homologous antibodies/antibodies (via water-mediated hydrogen bonding) Interaction. On the other hand, epitope/paratopic residues are characterized by forming salt bridges with homologous antibody/antigen residues. In yet another aspect, epitope/paratopic residues can be characterized by Residues that have non-zero changes in the masked surface area (BSA) due to the interaction with the homologous antibody/antigen. To a less detailed level, epitopes/paratopes can be characterized by functions, for example, by Competitive binding with other Abs. The epitope/paratope can also be more generally defined as containing amino acid residues, where the substitution of another amino acid will change the characteristics of the interaction between Ab and Ag (for example, C Amino acid scan).
自抗原决定基的描述及定义依赖于所用抗原决定基作图(mapping)方法及在不同细节层次下获得的事实，推断于相同Ag上不同Ab的抗原决定基的比较可在不同细节层次下类似地进行。例如，于氨基酸层面描述，例如自X射线结构测定的抗原决定基，若其含有相同氨基酸残基组，则认为其是相同的。若相应抗体的结合是相互排他性的，即，一种抗体的结合排除其他抗体的同时或连续结合，则认为以竞争结合为特征的抗原决定基为重叠的；及若该抗原可适应两个相应抗体同时结合，则认为抗原决定基是各别(独特)的。Since the description and definition of epitopes depend on the epitope mapping method used and the facts obtained at different levels of detail, it is inferred that the comparison of epitopes of different Abs on the same Ag can be similar at different levels of detail To proceed. For example, if it is described at the amino acid level, such as an epitope determined from X-ray structure, if it contains the same amino acid residue group, it is considered to be the same. If the binding of the corresponding antibodies is mutually exclusive, that is, the binding of one antibody excludes the simultaneous or sequential binding of other antibodies, then the epitopes characterized by competitive binding are considered to be overlapping; and if the antigen can accommodate two corresponding If antibodies bind at the same time, it is considered that the epitopes are distinct (unique).
给定抗体/抗原对的抗原决定基及互补位可通过例行方法鉴别。例如，抗原决定基的一般位置可通过评估抗体结合至不同片段或变体多肽的能力测定，如本文先前更充分地描述。可与抗体内的特异性残基接触的OX40内的特异性残基亦可使用例行方法测定。例如，抗体/抗原复合体可经结晶。该晶体结构可经测定及用以鉴别在抗体与抗原之间相互作用的特异性位点。The epitope and paratope of a given antibody/antigen pair can be identified by routine methods. For example, the general location of the epitope can be determined by assessing the ability of the antibody to bind to different fragments or variant polypeptides, as described more fully previously herein. The specific residues in OX40 that can be contacted with the specific residues in the antibody can also be determined using routine methods. For example, the antibody/antigen complex can be crystallized. The crystal structure can be determined and used to identify specific sites of interaction between the antibody and the antigen.
术语“特异性结合”是本领域中熟知的术语，且本领域中亦熟知用以测定这些特异性结合的方法。若分子与特定细胞或物质反应或结合比该分子与 替代细胞或物质反应或结合更频繁、更快速、更久持续时间及/或更大亲和力，则认为该分子显示“特异性结合”。若抗体或其抗原结合片段结合靶相较于结合其他物质以更大亲和力、结合性、更容易及/或更久持续时间，则该抗体或其抗原结合片段“特异性结合”至靶。The term "specific binding" is a term well known in the art, and methods for determining these specific bindings are also well known in the art. If a molecule reacts or binds to a specific cell or substance more frequently, faster, has a longer duration, and/or has greater affinity than the molecule reacts or binds to a replacement cell or substance, it is considered that the molecule exhibits "specific binding". If the antibody or antigen-binding fragment thereof binds to the target with greater affinity, binding, easier and/or longer duration than other substances, the antibody or antigen-binding fragment thereof "specifically binds" to the target.
例如，特异性结合OX40的抗体或其抗原结合片段是抗体结合其同源抗原相较于结合其他抗原，以更大亲和力、结合性、更容易及/或更久持续时间。例如，在标准结合分析条件下，抗OX40抗体可特异性结合样本中的人类OX40，但基本上不识别或结合该样本中的其他分子。亦了解特异性结合第一靶的抗体或其抗原结合片段可或可不特异性结合至第二靶。因此，“特异性结合”不一定要求(虽然其可包括)排他性结合。通常，但未必，“结合”的提及意谓特异性结合。For example, an antibody or antigen-binding fragment thereof that specifically binds to OX40 is that the antibody binds to its homologous antigen with greater affinity, binding, easier and/or longer duration than binding to other antigens. For example, under standard binding analysis conditions, an anti-OX40 antibody can specifically bind to human OX40 in a sample, but does not substantially recognize or bind to other molecules in the sample. It is also understood that an antibody or antigen-binding fragment thereof that specifically binds to a first target may or may not specifically bind to a second target. Therefore, "specific binding" does not necessarily require (although it may include) exclusive binding. Usually, but not necessarily, the reference to "binding" means specific binding.
可使用各种分析模式以选择特异性结合目的分子的抗体或其抗原结合片段。例如，在许多测定中，固相ELISA免疫测定、免疫沈淀、Biacore TM(GE Healthcare)、KinExA、荧光活化的细胞分选(FACS)、Octet TM(FortéBio,Inc.)及Western印迹分析可用以识别特异性结合抗原的抗体或其抗原结合片段。通常，特异性结合将是背景信号或噪音的至少两倍，更通常是背景的至少10倍、背景的至少50倍、背景的至少100倍、背景的至少500倍、背景的至少1000倍或背景的至少10,000倍。 Various analysis modes can be used to select antibodies or antigen-binding fragments thereof that specifically bind to the molecule of interest. For example, in many assays, solid-phase ELISA immunoassay, immunoprecipitation, Biacore TM (GE Healthcare), KinExA, fluorescence activated cell sorting (FACS), Octet TM (FortéBio, Inc.) and Western blot analysis can be used Recognize antibodies or antigen-binding fragments thereof that specifically bind to an antigen. Generally, the specific binding will be at least twice the background signal or noise, more usually at least 10 times the background, at least 50 times the background, at least 100 times the background, at least 500 times the background, at least 1000 times the background, or background At least 10,000 times of that.
抗体结合的特异性可通过测定抗体与OX40之间的特异性结合的K D值及比较该K D值与已知不结合至OX40的对照抗体的K D值来评估。一般而言，当该K D是约×10 -5M或更小时，认为抗体”、“特异性”结合抗原。 Antibody binding specificities can be K D K D value and comparing the value with K D values to a control antibody known not to bind to OX40 evaluated by measuring the specific binding between the antibody and OX40. Generally speaking, when the K D is about ×10 -5 M or less, it is considered that the antibody "specifically" binds to the antigen.
当相较于抗体或其抗原结合片段结合其他抗原，该抗体或其抗原结合片段不以更大亲和力、结合性、更容易及/或以更久持续时间结合至一抗原时，该抗体或其抗原结合片段“基本上不结合”至该抗原。通常，该结合将不大于背景信号或噪音的两倍。一般而言，其以1×10 -4M或更大、1×10 -3M或更大、1×10 -2M或更大或1×10 -1M或更大的K D结合该抗原。 When compared to an antibody or its antigen-binding fragment that binds to other antigens, the antibody or its antigen-binding fragment does not bind to an antigen with greater affinity, binding, easier and/or longer duration, the antibody or its antigen-binding fragment The antigen-binding fragment "substantially does not bind" to the antigen. Generally, the combination will not be more than twice the background signal or noise. Generally speaking, it combines with the K D of 1×10 -4 M or more, 1×10 -3 M or more, 1×10 -2 M or more, or 1×10 -1 M or more. antigen.
如本文关于抗体使用的术语“竞争”意谓第一抗体或其抗原-结合部分结合至抗原减少后续第二抗体或其抗原-结合部分对相同抗原的结合。一般而言，第一抗体的结合产生空间位阻、构象变化或结合至共同抗原决定基(或其部分)，使得第二抗体对相同抗原的结合是减少。可使用标准竞争性结合分析以判定两种抗体是否相互竞争。The term "competitive" as used herein with respect to antibodies means that the binding of a first antibody or antigen-binding portion thereof to an antigen reduces subsequent binding of a second antibody or antigen-binding portion thereof to the same antigen. Generally speaking, the binding of the first antibody produces steric hindrance, conformational change, or binding to a common epitope (or part thereof), so that the binding of the second antibody to the same antigen is reduced. Standard competitive binding analysis can be used to determine whether two antibodies compete with each other.
用于抗体竞争的一种合适的分析涉及使用Biacore技术，该技术可使用表面等离子共振(SPR)技术，通常使用生物传感器***(诸如***)测量相互作用的程度。例如，SPR可用于活体外竞争性结合抑制分析中以测定一种抗体抑制第二抗体的结合的能力。用于测量抗体竞争的另一分析使用基于 ELISA的方法。此外，一种基于抗体的竞争来“分级”抗体的高通量方法是描述于WO2003/48731中。若一种抗体或其抗原结合片段减少另一抗体或其抗原结合片段对OX40的结合，则存在竞争。例如，可使用顺序结合竞争分析，及顺序添加不同抗体。可添加第一抗体以达成接近饱和的结合。然后，添加第二抗体。若第二抗体对OX40的结合无法检测到或相较于在缺乏该第一抗体的情况下(其中值可设定为100％)的平行分析是显著减少(例如，至少约10％、至少约20％、至少约30％、至少约40％、至少约50％、至少约60％、至少约70％、至少约80％或至少约90％减少)，则所述两个抗体视为互相竞争。One suitable analysis for antibody competition involves the use of Biacore technology, which may use surface plasmon resonance (SPR) technology, usually using a biosensor system (such as a system) to measure the degree of interaction. For example, SPR can be used in an in vitro competitive binding inhibition assay to determine the ability of an antibody to inhibit the binding of a second antibody. Another analysis for measuring antibody competition uses an ELISA-based method. In addition, a high-throughput method for "grading" antibodies based on antibody competition is described in WO2003/48731. If one antibody or antigen-binding fragment thereof reduces the binding of another antibody or antigen-binding fragment to OX40, there is competition. For example, sequential binding competition analysis can be used, and different antibodies can be added sequentially. The first antibody can be added to achieve near-saturated binding. Then, the second antibody is added. If the binding of the second antibody to OX40 cannot be detected or compared to the parallel analysis in the absence of the first antibody (where the value can be set to 100%), it is significantly reduced (e.g., at least about 10%, at least about 10%). 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% reduction), then the two antibodies are considered to be competing with each other .
亦可进行竞争性结合分析，其中抗体对抗原的结合是相较于靶对具该靶的另一结合配偶体(诸如原本结合该靶的另一抗体或可溶受体)的结合。50％抑制发生时的浓度被称为K i。在理想条件下，该K i是等于K D。因此，一般而言，K i的测量可便利地经取代以提供K D的上限值。与不同分子相互作用相关联的结合亲和力(例如，不同抗体针对给定抗原的结合亲和力的比较)可通过针对个别抗体/抗原复合体的K D值的比较而进行比较。针对抗体或其他结合配偶体的K D值可使用本领域中业已建立的方法进行测定。 Competitive binding analysis can also be performed, in which the binding of the antibody to the antigen is compared to the binding of the target to another binding partner with the target (such as another antibody or a soluble receptor that originally binds to the target). Concentration at which 50% inhibition occurs is referred to as K i. Under ideal conditions, this K i is equal to K D. Thus, in general, the measurement of K i can conveniently be substituted to provide an upper limit value of the K D. The binding affinities associated with different molecular interactions (for example, the comparison of the binding affinities of different antibodies for a given antigen) can be compared by comparing the K D values for individual antibody/antigen complexes. The K D value for antibodies or other binding partners can be determined using methods established in the art.
“Fc融合”蛋白是其中一个或更多个多肽以可操作方式连接至Fc多肽的蛋白质。Fc融合组合免疫球蛋白的Fc区及融合配偶体。该“Fc区”可为天然序列Fc区或变体Fc区。尽管免疫球蛋白重链的Fc区的边界可改变，该人类IgG重链Fc区通常被定义为自位置Cys226的氨基酸残基或自Pro230延伸至其羧基端。Fc区中的残基的编号是具有EU指数的编号，如描述于Kabat等人，Sequences of Proteins of Immunological Interest，第5版，PublicHealth Service,National Institutes of Health,Bethesda,Md.,1991中。免疫球蛋白的Fc区通常包含两个恒定域(CH 2及CH 3)。如本领域中已知，Fc区可以二聚物或单体形式存在。 An "Fc fusion" protein is a protein in which one or more polypeptides are operably linked to an Fc polypeptide. Fc fusion combines the Fc region of an immunoglobulin and a fusion partner. The "Fc region" can be a native sequence Fc region or a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain can be changed, the human IgG heavy chain Fc region is usually defined as an amino acid residue at position Cys226 or extending from Pro230 to its carboxyl terminus. The numbering of residues in the Fc region is the numbering with the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, Md., 1991. The Fc region of immunoglobulin usually contains two constant domains (CH 2 and CH 3 ). As known in the art, the Fc region can exist in dimer or monomer form.
术语“治疗有效量”意谓抗OX40抗体或其抗原结合片段或包含此抗体或其抗原结合片段的组合足以达成预期目的的量。精确量将取决于许多因素，包括(但不限于)治疗组合物的组分及物理特征、预期患者群体、个别患者注意事项等，及可由本领域技术人员判定。The term "therapeutically effective amount" means an amount of an anti-OX40 antibody or antigen-binding fragment thereof, or a combination comprising the antibody or antigen-binding fragment thereof, sufficient to achieve the intended purpose. The precise amount will depend on many factors, including (but not limited to) the components and physical characteristics of the therapeutic composition, the expected patient population, individual patient precautions, etc., and can be determined by those skilled in the art.
术语“治疗”包括预防性及/或治疗性治疗。若在疾病、失调症或病症的临床表现前施用，则认为该治疗是预防性的。治疗性治疗包括(例如)减轻或减弱疾病、失调症或病症的严重性或缩短疾病、失调症或病症的长度。The term "treatment" includes prophylactic and/or therapeutic treatment. If administered before the clinical manifestation of the disease, disorder, or condition, the treatment is considered prophylactic. Therapeutic treatment includes, for example, reducing or attenuating the severity of or shortening the length of the disease, disorder, or condition.
如本文使用，术语“约”是指值的+/-10％。As used herein, the term "about" means +/- 10% of the value.
治疗性方法和组合物Therapeutic methods and compositions
本文中提供的任何抗人OX40抗体可以在治疗方法中使用。Any of the anti-human OX40 antibodies provided herein can be used in treatment methods.
在一个方面，提供抗人OX40激动性抗体，其用作药物。在又一些方面，提供抗人OX40激动性抗体，其用于治疗癌症。在某些实施方式中，提供抗人OX40激动性抗体，其用于治疗方法。在某些实施方式中，提供抗人OX40激动性抗体，其用于治疗具有癌症的个体的方法，包括对该个体施用有效量的该抗人OX40激动性抗体。在一个此类实施方式中，该方法进一步包括对该个体施用有效量的至少一种别的治疗剂，例如下文所述。In one aspect, an anti-human OX40 agonistic antibody is provided for use as a medicine. In still other aspects, anti-human OX40 agonistic antibodies are provided for use in the treatment of cancer. In certain embodiments, anti-human OX40 agonistic antibodies are provided for use in methods of treatment. In some embodiments, an anti-human OX40 agonist antibody is provided for use in a method of treating an individual with cancer, comprising administering an effective amount of the anti-human OX40 agonist antibody to the individual. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, such as described below.
在一个方面，提供的是抗人OX40激动性抗体，其用于在具有癌症的个体中增强免疫功能(例如通过上调细胞介导的免疫应答)，包括对该个体施用有效量的该抗人OX40激动性抗体。在一个方面，提供的是抗人OX40激动性抗体，其用于在具有癌症的个体中增强T细胞功能，包括对该个体施用有效量的该抗人OX40激动性抗体。在一个方面，提供的是抗人OX40激动性抗体，其用于消减表达人OX40的细胞(例如表达OX40的T细胞，例如表达OX40的Treg)，包括对该个体施用有效量的该抗人OX40激动性抗体。在一些实施方式中，消减是通过ADCC进行的。在一些实施方式中，消减是通过吞噬进行的。提供的是抗人OX40激动性抗体，其用于治疗具有肿瘤免疫的个体。In one aspect, provided is an anti-human OX40 agonistic antibody for use in enhancing immune function (for example, by up-regulating a cell-mediated immune response) in an individual with cancer, including administering an effective amount of the anti-human OX40 to the individual Agonistic antibodies. In one aspect, provided is an anti-human OX40 agonistic antibody for enhancing T cell function in an individual with cancer, including administering an effective amount of the anti-human OX40 agonistic antibody to the individual. In one aspect, provided is an anti-human OX40 agonistic antibody, which is used to deplete human OX40-expressing cells (for example, OX40-expressing T cells, such as OX40-expressing Treg), including administering an effective amount of the anti-human OX40 to the individual Agonistic antibodies. In some embodiments, the abatement is performed by ADCC. In some embodiments, depletion is by phagocytosis. Provided is an anti-human OX40 agonistic antibody, which is used to treat individuals with tumor immunity.
在又一些方面，提供抗人OX40激动性抗体，其用于治疗感染(例如细菌或病毒或其它病原体感染)。在某些实施方式中，本发明提供抗人OX40激动性抗体，其用于治疗具有感染的个体的方法，包括对该个体施用有效量的该抗人OX40激动性抗体。在一些实施方式中，感染是病毒和/或细菌感染。在一些实施方式中，感染是病原体感染。In still other aspects, anti-human OX40 agonistic antibodies are provided for use in the treatment of infections (eg, bacterial or viral or other pathogen infections). In certain embodiments, the present invention provides an anti-human OX40 agonist antibody for use in a method of treating an individual with an infection, comprising administering an effective amount of the anti-human OX40 agonist antibody to the individual. In some embodiments, the infection is a viral and/or bacterial infection. In some embodiments, the infection is a pathogen infection.
在又一个方面，本发明提供抗OX40抗体制造或制备药物的用途。在一个实施方式中，该药物用于治疗癌症。在又一个实施方式中，该药物用于治疗癌症的方法，其包括对具有癌症的个体施用有效量的该药物。在一个此类实施方式中，该方法进一步包括对该个体施用有效量的至少一种别的治疗剂，例如下文所述。In yet another aspect, the present invention provides the use of anti-OX40 antibody to manufacture or prepare medicine. In one embodiment, the drug is used to treat cancer. In yet another embodiment, the medicament is used in a method of treating cancer, which comprises administering an effective amount of the medicament to an individual with cancer. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, such as described below.
在一个方面，该药物用于在具有癌症的个体中增强免疫功能(例如通过上调细胞介导的免疫应答)，其包括对该个体施用有效量的该药物。在一个方面，该药物用于在具有癌症的个体中增强T细胞功能，其包括对该个体施用有效量的该药物。在一些实施方式中，该T细胞功能障碍性病症是癌症。在一个方面，该药物用于消减表达人OX40的细胞(例如表达高OX40的细胞，例如表达OX40的T细胞)，其包括对该个体施用有效量的该药物。在一些实施方式中，消减是通过ADCC进行的。在一些实施方式中，消减是通过吞噬进行的。在一个方面，该药物用于治疗具有肿瘤免疫 的个体。In one aspect, the drug is used to enhance immune function in an individual with cancer (for example, by up-regulating a cell-mediated immune response), which includes administering an effective amount of the drug to the individual. In one aspect, the drug is used to enhance T cell function in an individual with cancer, which includes administering an effective amount of the drug to the individual. In some embodiments, the T cell dysfunctional disorder is cancer. In one aspect, the drug is used to deplete human OX40 expressing cells (eg, high OX40 expressing cells, such as OX40 expressing T cells), which includes administering an effective amount of the drug to the individual. In some embodiments, the abatement is performed by ADCC. In some embodiments, depletion is by phagocytosis. In one aspect, the drug is used to treat individuals with tumor immunity.
在又一些方面，提供药物，其用于治疗感染(例如细菌或病毒或其它病原体感染)。在某些实施方式中，该药物用于治疗具有感染的个体的方法，包括对该个体施用有效量的该药物。在一些实施方式中，感染是病毒和/或细菌感染。在一些实施方式中，感染是病原体感染。In still other aspects, medicaments are provided for use in the treatment of infections (e.g., bacterial or viral or other pathogen infections). In certain embodiments, the method of treating an individual with an infection by the medicament comprises administering an effective amount of the medicament to the individual. In some embodiments, the infection is a viral and/or bacterial infection. In some embodiments, the infection is a pathogen infection.
在又一个方面，本发明提供用于治疗癌症的方法。在一个实施方式中，该方法包括对具有此类癌症的个体施用有效量的抗OX40抗体。在一个此类实施方式中，该方法进一步包括对该个体施用有效量的至少一种别的治疗剂，例如下文所述。依照任何上述实施方式的“个体”可以是人。In yet another aspect, the present invention provides methods for treating cancer. In one embodiment, the method includes administering an effective amount of an anti-OX40 antibody to an individual with such cancer. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, such as described below. The "individual" according to any of the above embodiments may be a human.
在一个方面，提供的是用于在具有癌症的个体中增强免疫功能(例如通过上调细胞介导的免疫应答)的方法，包括对该个体施用有效量的该抗人OX40激动性抗体。在一个方面，提供的是用于在具有癌症的个体中增强T细胞功能的方法，包括对该个体施用有效量的该抗人OX40激动性抗体。在一个方面，提供的是用于消减表达人OX40的细胞(例如表达高水平OX40的细胞，例如表达OX40的T细胞)的方法，包括对该个体施用有效量的该抗人OX40激动性抗体。在一些实施方式中，消减是通过ADCC进行的。在一些实施方式中，消减是通过吞噬进行的。提供的是抗人OX40激动性抗体，其用于治疗具有肿瘤免疫的个体。In one aspect, provided is a method for enhancing immune function (for example, by up-regulating a cell-mediated immune response) in an individual with cancer, comprising administering to the individual an effective amount of the anti-human OX40 agonist antibody. In one aspect, provided is a method for enhancing T cell function in an individual with cancer, comprising administering to the individual an effective amount of the anti-human OX40 agonist antibody. In one aspect, provided is a method for depleting cells expressing human OX40 (for example, cells expressing high levels of OX40, such as T cells expressing OX40), comprising administering an effective amount of the anti-human OX40 agonistic antibody to the individual. In some embodiments, the abatement is performed by ADCC. In some embodiments, depletion is by phagocytosis. Provided is an anti-human OX40 agonistic antibody, which is used to treat individuals with tumor immunity.
在一些实施方式中，癌症的例子进一步包括但不限于B细胞淋巴瘤(包括低级/滤泡性非霍奇金氏淋巴瘤(NHL)，小淋巴细胞性(SL)NHU中级/滤泡性NHL，中级弥漫性NHL，高级成免疫细胞性NHL，高级成淋巴细胞性NHL，高级小无核裂细胞性NHL，贮积病(bulky disease)NHL，套细胞淋巴瘤，AIDS相关淋巴瘤，和瓦尔登斯特伦氏(Waldenstrom)巨球蛋白血症)，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)，B细胞增殖性病症，和梅格斯氏(Meigs)综合征有关的异常血管增殖。更具体例子包括但不限于复发性或顽固性NHL，前线(front line)低级NHL，阶段III/IV NHL，化疗耐受性NHL，前体B成淋巴细胞性白血病和/或淋巴瘤，小淋巴细胞性淋巴瘤，B细胞慢性淋巴细胞性白血病和/或前淋巴细胞性白血病和/或小淋巴细胞性淋巴瘤，B细胞前淋巴细胞性淋巴瘤，免疫细胞瘤和/或淋巴浆细胞性(lymphoplasmacytic)淋巴瘤，淋巴浆细胞性淋巴瘤，边缘区B细胞淋巴瘤，脾边缘区淋巴瘤，节外边缘区(extranodal marginal zone)_MALT淋巴瘤，节边缘区(nodal marginal zone)淋巴瘤，毛细胞性白血病，衆细胞瘤和/或浆细胞骨髓瘤，低级/滤泡淋巴瘤，中级/滤泡NHL，套细胞淋巴瘤，滤泡中心 淋巴瘤(滤泡的)，中级弥漫性NHL，弥漫性大B细胞淋巴瘤，攻击性(agressive)NHL(包括攻击性前线NHL和攻击性复发性NHL)，自体干细胞移植后复发性或顽固性NHL，原发性纵隔大B细胞淋巴瘤，原发性渗出性淋巴瘤，高级成免疫细胞NHL，高级成淋巴细胞NHL，高级小无核裂细胞NHL，贮积病(bulky disease)NHL，伯基特氏(Burkitt)淋巴瘤，前体(外周)大粒状淋巴细胞白血病，蕈样肉芽肿病和/或塞扎里(Sezary)综合征，皮肤淋巴瘤，间变性大细胞淋巴瘤，血管中心性淋巴瘤。In some embodiments, examples of cancer further include, but are not limited to, B-cell lymphoma (including low-grade/follicular non-Hodgkin’s lymphoma (NHL), small lymphocytic (SL) NHU intermediate/follicular NHL , Intermediate diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small nonnucleoblastic NHL, bulky disease NHL, mantle cell lymphoma, AIDS-related lymphoma, and Val Denstrom's (Waldenstrom) macroglobulinemia), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and post-transplant lymph Proliferative disorders (PTLD), and abnormal blood vessel proliferation associated with phakomatoses, edema (such as those associated with brain tumors), B cell proliferative disorders, and Meigs syndrome. More specific examples include, but are not limited to, relapsed or refractory NHL, front-line low-grade NHL, stage III/IV NHL, chemotherapy-resistant NHL, precursor B lymphoblastic leukemia and/or lymphoma, small lymphoma Cellular lymphoma, B-cell chronic lymphocytic leukemia and/or prolymphocytic leukemia and/or small lymphocytic lymphoma, B-cell prelymphocytic lymphoma, immunocytoma and/or lymphoplasmacytic ( lymphoplasmacytic lymphoma, lymphoplasmacytic lymphoma, marginal zone B-cell lymphoma, splenic marginal zone lymphoma, extranodal marginal zone_MALT lymphoma, nodal marginal zone lymphoma, hair Cellular Leukemia, Cytocytoma and/or Plasma Cell Myeloma, Low Grade/Follicular Lymphoma, Intermediate/Follicular NHL, Mantle Cell Lymphoma, Follicular Center Lymphoma (Follicular), Intermediate Diffuse NHL, Diffuse Large B-cell lymphoma, aggressive NHL (including aggressive frontline NHL and aggressive recurrent NHL), recurrent or refractory NHL after autologous stem cell transplantation, primary mediastinal large B-cell lymphoma, primary Exudative lymphoma, high-grade immune cell NHL, high-grade lymphoblast NHL, high-grade small nonnuclear cleft cell NHL, bulky disease NHL, Burkitt’s lymphoma, precursor (peripheral) ) Large granular lymphocytic leukemia, mycosis fungoides and/or Sezary syndrome, skin lymphoma, anaplastic large cell lymphoma, angiocentric lymphoma.
在一些实施方式中，癌症的例子进一步包括但不限于B细胞增殖性病症，其进一步包括但不限于淋巴瘤(例如B细胞非霍奇金氏淋巴瘤(NHL))和淋巴细胞性白血病。此类淋巴瘤和淋巴细胞性白血病包括例如a)滤泡性淋巴瘤，b)小无核裂细胞淋巴瘤(small Non-Cleaved Cell Lymphoma)/伯基特(Burkitt)氏淋巴瘤(包括地方性伯基特氏淋巴瘤，散发性伯基特氏淋巴瘤和非伯基特氏淋巴瘤)，c)边缘区淋巴瘤(包括结外边缘区B细胞淋巴瘤(粘膜相关淋巴组织淋巴瘤，MALT)，结边缘区B细胞淋巴瘤和脾边缘区淋巴瘤)，d)套细胞淋巴瘤(MCL)，e)大细胞淋巴瘤(包括B细胞弥漫性大细胞淋巴瘤(DLCL)，弥漫性混合细胞淋巴瘤，免疫母细胞性淋巴瘤，原发性纵隔B细胞淋巴瘤，血管中心性淋巴瘤-肺B细胞淋巴瘤)，f)毛细胞白血病，g)淋巴细胞性淋巴瘤，瓦尔登斯特伦(waldenstrom)氏巨球蛋白血症，h)急性淋巴细胞性白血病(ALL)，慢性淋巴细胞性白血病(CLL)/小淋巴细胞性淋巴瘤(SLL)，B细胞幼淋巴细胞白血病，i)浆细胞赘生物，浆细胞骨髓瘤，多发性骨髓瘤，浆细胞瘤，和/或j)霍奇金氏病。In some embodiments, examples of cancer further include, but are not limited to, B-cell proliferative disorders, which further include, but are not limited to, lymphoma (eg, B-cell non-Hodgkin's lymphoma (NHL)) and lymphocytic leukemia. Such lymphomas and lymphocytic leukemias include, for example, a) follicular lymphoma, b) small Non-Cleaved Cell Lymphoma/Burkitt’s lymphoma (including endemic Burkitt’s lymphoma, sporadic Burkitt’s lymphoma and non-Burkitt’s lymphoma), c) marginal zone lymphoma (including extranodal marginal zone B-cell lymphoma (mucosa-associated lymphoid tissue lymphoma, MALT) ), nodal marginal zone B-cell lymphoma and splenic marginal zone lymphoma), d) mantle cell lymphoma (MCL), e) large cell lymphoma (including B-cell diffuse large cell lymphoma (DLCL), diffuse mixed Cell lymphoma, immunoblastic lymphoma, primary mediastinal B-cell lymphoma, angiocentric lymphoma-pulmonary B-cell lymphoma), f) hairy cell leukemia, g) lymphocytic lymphoma, Waldens Waldenstrom's macroglobulinemia, h) acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL), B-cell young lymphocytic leukemia, i ) Plasma cell neoplasms, plasma cell myeloma, multiple myeloma, plasma cell tumor, and/or j) Hodgkin’s disease.
在任何方法的一些实施方式中，该癌症是B细胞增殖性病症。在一些实施方式中，该B细胞增殖性病症是淋巴瘤，非霍奇金(Hodgkin)氏淋巴瘤(NHL)，攻击性NHL，复发性攻击性NHL，复发性无痛性NHL，顽固性NHL，顽固性无痛性NHL，慢性淋巴细胞性白血病(CLL)，小淋巴细胞淋巴瘤，白血病，毛细胞白血病(HCL)，急性淋巴细胞性白血病(ALL)，或套细胞淋巴瘤。在一些实施方式中，该B细胞增殖性病症是NHL，诸如无痛性NHL和/或攻击性NHL。在一些实施方式中，该B细胞增殖性病症是无痛性滤泡性淋巴瘤或弥漫性大B细胞淋巴瘤。In some embodiments of any method, the cancer is a B cell proliferative disorder. In some embodiments, the B-cell proliferative disorder is lymphoma, non-Hodgkin's lymphoma (NHL), aggressive NHL, recurrent aggressive NHL, recurrent painless NHL, refractory NHL , Refractory painless NHL, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphocytic leukemia (ALL), or mantle cell lymphoma. In some embodiments, the B cell proliferative disorder is NHL, such as painless NHL and/or aggressive NHL. In some embodiments, the B-cell proliferative disorder is painless follicular lymphoma or diffuse large B-cell lymphoma.
在又一个方面，本发明提供药物配制剂，其包含本文中提供的任何抗OX40抗体，例如用于任何上述治疗方法。在一个实施方式中，药物配制剂包含本文中提供的任何抗OX40抗体和药学可接受载剂。在另一个实施方式中，药物配制剂包含本文中提供的任何抗OX40抗体和至少一种别的治疗剂，例如下文所述。In yet another aspect, the present invention provides a pharmaceutical formulation comprising any anti-OX40 antibody provided herein, for example for use in any of the above-mentioned treatment methods. In one embodiment, the pharmaceutical formulation comprises any anti-OX40 antibody provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical formulation comprises any anti-OX40 antibody provided herein and at least one additional therapeutic agent, such as described below.
在本发明的任何方法的一些实施方式中，该抗人OX40激动性抗体通 过抑制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细胞功能来抑制肿瘤免疫。在本发明的任何方法的一些实施方式中，该抗人OX40激动性抗体通过抑制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细胞功能来治疗癌症。在本发明的任何方法的一些实施方式中，该抗人OX40激动性抗体通过抑制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细胞功能来增强免疫功能。在本发明的任何方法的一些实施方式中，该抗人OX40激动性抗体通过抑制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细胞功能来增强T细胞功能。In some embodiments of any of the methods of the present invention, the anti-human OX40 agonist antibody kills OX40-expressing cells (for example, cells expressing high levels of OX40) by inhibiting Treg function (for example, inhibiting the suppressive function of Treg), and increasing Effector T cell function and/or improve memory T cell function to suppress tumor immunity. In some embodiments of any of the methods of the present invention, the anti-human OX40 agonist antibody kills OX40-expressing cells (for example, cells expressing high levels of OX40) by inhibiting Treg function (for example, inhibiting the suppressive function of Treg), and increasing Effector T cell function and/or improve memory T cell function to treat cancer. In some embodiments of any of the methods of the present invention, the anti-human OX40 agonist antibody kills OX40-expressing cells (for example, cells expressing high levels of OX40) by inhibiting Treg function (for example, inhibiting the suppressive function of Treg), and increasing Effector T cell function and/or improve memory T cell function to enhance immune function. In some embodiments of any of the methods of the present invention, the anti-human OX40 agonist antibody kills OX40-expressing cells (for example, cells expressing high levels of OX40) by inhibiting Treg function (for example, inhibiting the suppressive function of Treg), and increasing Effector T cell function and/or improve memory T cell function to enhance T cell function.
在任何方法的一些实施方式中，该抗人OX40激动性抗体是消减性抗人OX40激动性抗体。在一些实施方式中，该抗人OX40激动性抗体处理导致细胞消减(例如消减表达OX40的细胞，例如消减表达高水平OX40的细胞)。在一些实施方式中，消减是通过ADCC进行的。在一些实施方式中，消减是通过吞噬进行的。In some embodiments of any of the methods, the anti-human OX40 agonistic antibody is a subtractive anti-human OX40 agonistic antibody. In some embodiments, the anti-human OX40 agonist antibody treatment results in cell depletion (e.g., depletion of cells expressing OX40, such as depletion of cells expressing high levels of OX40). In some embodiments, the abatement is performed by ADCC. In some embodiments, depletion is by phagocytosis.
在任何方法的一些实施方式中，相对于施用该OX40激动性抗体之前的Treg功能，该抗人OX40激动性抗体例如通过抑制效应和/或记忆T细胞功能(在一些实施方式中，效应T细胞和/或记忆T细胞增殖和/或细胞因子分泌)的Treg遏制来抑制Treg功能。在任何方法的一些实施方式中，相对于施用该OX40激动性抗体之前的效应T细胞增殖，该抗人OX40激动性抗体提高效应T细胞增殖。在任何方法的一些实施方式中，相对于施用该OX40激动性抗体之前的记忆T细胞增殖，该抗人OX40激动性抗体提高记忆T细胞增殖。在任何方法的一些实施方式中，相对于施用该OX40激动性抗体之前的效应T细胞细胞因子生成，该抗人OX40激动性抗体提高效应T细胞细胞因子生成(例如γ-干扰素生成)。在任何方法的一些实施方式中，相对于施用该OX40激动性抗体之前的记忆T细胞细胞因子生成，该抗人OX40激动性抗体提高记忆T细胞细胞因子生成(例如γ-干扰素生成)。在任何方法的一些实施方式中，相对于施用该OX40激动性抗体之前的CD4+效应T细胞增殖和/或CD8+效应T细胞增殖，该抗人OX40激动性抗体提高CD4+效应T细胞增殖和/或CD8+效应T细胞增殖。在任何方法的一些实施方式中，相对于施用该OX40激动性抗体之前的记忆T细胞增殖，该抗人OX40激动性抗体提高记忆T细胞增殖(例如CD4+记忆T细 胞增殖)。在一些实施方式中，相对于施用该抗人OX40激动性抗体的之前的增殖，细胞因子分泌和/或溶胞活性，个体中的CD4+效应T细胞具有增强的增殖，细胞因子分泌和/或溶胞活性。In some embodiments of any of the methods, the anti-human OX40 agonist antibody may, for example, inhibit effector and/or memory T cell function (in some embodiments, effector T cell function) relative to the Treg function prior to administration of the OX40 agonist antibody. And/or memory T cell proliferation and/or cytokine secretion) Treg suppression to inhibit Treg function. In some embodiments of any method, the anti-human OX40 agonist antibody increases effector T cell proliferation relative to effector T cell proliferation prior to administration of the OX40 agonist antibody. In some embodiments of any method, the anti-human OX40 agonist antibody increases memory T cell proliferation relative to the memory T cell proliferation prior to administration of the OX40 agonist antibody. In some embodiments of any method, the anti-human OX40 agonist antibody increases effector T cell cytokine production (eg, gamma-interferon production) relative to effector T cell cytokine production prior to administration of the OX40 agonist antibody. In some embodiments of any method, the anti-human OX40 agonist antibody increases memory T cell cytokine production (eg, γ-interferon production) relative to memory T cell cytokine production prior to administration of the OX40 agonist antibody. In some embodiments of any method, the anti-human OX40 agonist antibody increases CD4+ effector T cell proliferation and/or CD8+ relative to CD4+ effector T cell proliferation and/or CD8+ effector T cell proliferation prior to administration of the OX40 agonist antibody Effector T cell proliferation. In some embodiments of any method, the anti-human OX40 agonist antibody increases memory T cell proliferation (e.g., CD4+ memory T cell proliferation) relative to memory T cell proliferation prior to administration of the OX40 agonist antibody. In some embodiments, relative to the proliferation, cytokine secretion and/or lysis activity prior to administration of the anti-human OX40 agonistic antibody, the CD4+ effector T cells in the individual have enhanced proliferation, cytokine secretion and/or lysis. Cell activity.
在本发明的任何方法的一些实施方式中，CD4+效应T细胞的数目相对于施用该抗人OX40激动性抗体之前升高。在一些实施方式中，CD4+效应T细胞细胞因子分泌相对于施用该抗人OX40激动性抗体之前升高。在任何方法的一些实施方式中，个体中的CD8+效应T细胞具有相对于施用该抗人OX40激动性抗体之前增强的增殖，细胞因子分泌和/或溶胞活性。在一些实施方式中，CD8+效应T细胞的数目相对于施用该抗人OX40激动性抗体之前升高。在一些实施方式中，CD8+效应T细胞细胞因子分泌相对于施用该抗人OX40激动性抗体之前升高。In some embodiments of any of the methods of the invention, the number of CD4+ effector T cells is increased relative to before administration of the anti-human OX40 agonist antibody. In some embodiments, CD4+ effector T cell cytokine secretion is increased relative to before administration of the anti-human OX40 agonist antibody. In some embodiments of any method, the CD8+ effector T cells in the individual have enhanced proliferation, cytokine secretion, and/or lytic activity relative to prior to administration of the anti-human OX40 agonistic antibody. In some embodiments, the number of CD8+ effector T cells is increased relative to before administration of the anti-human OX40 agonistic antibody. In some embodiments, CD8+ effector T cell cytokine secretion is increased relative to before administration of the anti-human OX40 agonist antibody.
在本发明的任何方法的一些实施方式中，该抗人OX40激动性抗体结合人效应细胞，例如结合由人效应细胞表达的FcγR。在一些实施方式中，该人效应细胞实施ADCC效应器功能。在一些实施方式中，该人效应细胞实施吞噬效应器功能。In some embodiments of any of the methods of the invention, the anti-human OX40 agonist antibody binds to human effector cells, for example, to FcγR expressed by human effector cells. In some embodiments, the human effector cell performs ADCC effector function. In some embodiments, the human effector cell performs phagocytic effector function.
在本发明的任何方法的一些实施方式中，包含变异IgG1Fc多肽(其包含消除对人效应细胞的结合的突变，例如DANA或N297G突变)的抗人OX40激动性抗体具有相对于包含天然序列IgG1Fc部分的抗人OX40激动性抗体降低的活性(例如CD4+效应T细胞功能，例如增殖)。在一些实施方式中，包含变异IgG1Fc多肽(其包含消除对人效应细胞的结合的突变，例如DANA或N297G突变)的抗人OX40激动性抗体并不拥有实质性活性(例如CD4+效应T细胞功能，例如增殖)。In some embodiments of any of the methods of the present invention, the anti-human OX40 agonist antibody comprising a variant IgG1Fc polypeptide (which contains a mutation that eliminates binding to human effector cells, such as the DANA or N297G mutation) has a portion relative to the IgG1Fc portion containing the native sequence The anti-human OX40 agonistic antibody reduces the activity (eg CD4+ effector T cell function, such as proliferation). In some embodiments, an anti-human OX40 agonist antibody comprising a variant IgG1Fc polypeptide (which contains a mutation that eliminates binding to human effector cells, such as the DANA or N297G mutation) does not possess substantial activity (e.g., CD4+ effector T cell function, Such as proliferation).
在本发明的任何方法的一些实施方式中，抗人OX40激动性抗体功能需要抗体交联。在一些实施方式中，功能是刺激CD4+效应T细胞增殖。在一些实施方式中，抗体交联是通过提供粘附至固体表面(例如细胞培养板)的抗人OX40激动性抗体而测定的。在一些实施方式中，抗体交联是通过在该抗体的IgG1Fc部分中引入突变(例如DANA或N297S突变)并测试突变体抗体的功能而测定的。In some embodiments of any of the methods of the invention, the anti-human OX40 agonistic antibody function requires antibody cross-linking. In some embodiments, the function is to stimulate the proliferation of CD4+ effector T cells. In some embodiments, antibody cross-linking is determined by providing an anti-human OX40 agonistic antibody adhered to a solid surface (e.g., cell culture plate). In some embodiments, antibody cross-linking is determined by introducing mutations (such as DANA or N297S mutations) in the IgG1 Fc portion of the antibody and testing the function of the mutant antibody.
在任何方法的一些实施方式中，个体中的记忆T细胞具有相对于施用该抗人OX40激动性抗体之前增强的增殖和/或细胞因子分泌。在一些实施方式中，记忆T细胞的数目相对于施用该抗人OX40激动性抗体之前升高。在一些实施方式中，记忆T细胞细胞因子分泌(水平)相对于施用该抗人OX40激动性抗体之前升高。在任何方法的一些实施方式中，个体中的Treg具有相对于施用该抗人OX40激动性抗体之前降低的效应T细胞功能(例如增殖和/或细胞因子分泌)抑制。在一些实施方式中，效应T细胞的数目相对 于施用该抗人OX40激动性抗体之前升高。在一些实施方式中，效应T细胞细胞因子分泌(水平)相对于施用该抗人OX40激动性抗体之前升高。In some embodiments of any method, the memory T cells in the individual have enhanced proliferation and/or cytokine secretion relative to prior to administration of the anti-human OX40 agonistic antibody. In some embodiments, the number of memory T cells is increased relative to before administration of the anti-human OX40 agonist antibody. In some embodiments, memory T cell cytokine secretion (level) is increased relative to before administration of the anti-human OX40 agonist antibody. In some embodiments of any method, Tregs in the individual have reduced effector T cell function (e.g., proliferation and/or cytokine secretion) inhibition relative to prior to administration of the anti-human OX40 agonistic antibody. In some embodiments, the number of effector T cells is increased relative to before administration of the anti-human OX40 agonistic antibody. In some embodiments, effector T cell cytokine secretion (level) is increased relative to before administration of the anti-human OX40 agonist antibody.
在本发明的任何方法的一些实施方式中，肿瘤内(浸润性)CD4+效应T细胞的数目(例如CD4+效应T细胞的总数，或例如CD45+细胞中CD4+细胞的百分比)相对于施用该抗人OX40激动性抗体之前升高。在本发明的任何方法的一些实施方式中，表达γ-干扰素的肿瘤内(浸润性)CD4+效应T细胞的数目(例如总的表达γ-干扰素的CD4+细胞，或例如总的CD4+细胞中表达γ-干扰素的CD4+细胞的百分比)相对于施用抗人OX40激动性抗体之前升高。In some embodiments of any of the methods of the invention, the number of (infiltrating) CD4+ effector T cells in the tumor (e.g., the total number of CD4+ effector T cells, or e.g., the percentage of CD4+ cells in CD45+ cells) is relative to the administration of the anti-human OX40 Agonistic antibodies were previously elevated. In some embodiments of any of the methods of the present invention, the number of (infiltrating) CD4+ effector T cells in the tumor expressing interferon-gamma (e.g., total CD4+ cells expressing interferon-gamma, or, for example, total CD4+ cells The percentage of CD4+ cells expressing γ-interferon) was increased relative to before administration of anti-human OX40 agonistic antibody.
在本发明的任何方法的一些实施方式中，肿瘤内(浸润性)CD8+效应T细胞的数目(例如CD8+效应T细胞的总数，或例如CD45+细胞中CD8+的百分比)相对于施用抗人OX40激动性抗体之前升高。在本发明的任何方法的一些实施方式中，表达γ-干扰素的肿瘤内(浸润性)CD8+效应T细胞的数目(例如总的CD8+细胞中表达γ-干扰素的CD8+细胞的百分比)相对于施用抗人OX40激动性抗体之前升高。In some embodiments of any of the methods of the present invention, the number of (infiltrating) CD8+ effector T cells in the tumor (e.g., the total number of CD8+ effector T cells, or e.g., the percentage of CD8+ in CD45+ cells) is relative to administration of anti-human OX40 agonism The antibody was previously elevated. In some embodiments of any of the methods of the present invention, the number of (infiltrating) CD8+ effector T cells in a tumor expressing interferon-gamma (e.g., the percentage of CD8+ cells expressing interferon-gamma in the total CD8+ cells) is relative to Elevated before administration of anti-human OX40 agonistic antibody.
在本发明的任何方法的一些实施方式中，肿瘤内(浸润性)Treg的数目(例如Treg的总数或例如CD4+细胞中Fox3p+细胞的百分比)相对于施用抗人OX40激动性抗体之前降低。In some embodiments of any of the methods of the invention, the number of (infiltrating) Tregs within the tumor (e.g., the total number of Tregs or e.g. the percentage of Fox3p+ cells in CD4+ cells) is reduced relative to before administration of the anti-human OX40 agonistic antibody.
在本发明的任何方法的一些实施方式中，抗人OX40激动性抗体的施用与肿瘤抗原的施用组合。在一些实施方式中，该肿瘤抗原包含蛋白质。在一些实施方式中，该肿瘤抗原包含核酸。在一些实施方式中，该肿瘤抗原是肿瘤细胞。In some embodiments of any of the methods of the invention, the administration of the anti-human OX40 agonistic antibody is combined with the administration of the tumor antigen. In some embodiments, the tumor antigen comprises a protein. In some embodiments, the tumor antigen comprises nucleic acid. In some embodiments, the tumor antigen is a tumor cell.
在本发明的任何方法的一些实施方式中，该癌症展示人效应细胞(例如受到人效应细胞浸润)。用于检测人效应细胞的是方法本领域公知的，包括例如通过IHC。在一些实施方式中，该癌症展示高水平的人效应细胞。在一些实施方式中，人效应细胞是NK细胞，巨噬细胞，单核细胞中的一项或多项。在一些实施方式中，该癌症是本文中描述的任何癌症。在一些实施方式中，该癌症是非小细胞肺癌(NSCLC)，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，结直肠癌(CRC)，或肝细胞癌。In some embodiments of any of the methods of the invention, the cancer displays human effector cells (e.g., is infiltrated by human effector cells). The methods used to detect human effector cells are well known in the art and include, for example, by IHC. In some embodiments, the cancer displays high levels of human effector cells. In some embodiments, the human effector cells are one or more of NK cells, macrophages, and monocytes. In some embodiments, the cancer is any cancer described herein. In some embodiments, the cancer is non-small cell lung cancer (NSCLC), glioblastoma, neuroblastoma, melanoma, breast cancer (e.g. triple negative breast cancer), gastric cancer, colorectal cancer (CRC), Or hepatocellular carcinoma.
在本发明的任何方法的一些实施方式中，该癌症展示表达FcR的细胞(例如受到表达FcR的细胞浸润)。用于检测FcR的方法是本领域公知的，包括例如通过IHC。在一些实施方式中，该癌症展示高水平的表达FcR的细胞。在一些实施方式中，FcR是FcγR。在一些实施方式中，FcR是活化性FcγR。在一些实施方式中，该癌症是非小细胞肺癌(NSCLC)，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，结直肠癌(CRC)，或肝细胞癌。In some embodiments of any of the methods of the invention, the cancer displays FcR-expressing cells (e.g., is infiltrated by FcR-expressing cells). Methods for detecting FcR are well known in the art and include, for example, by IHC. In some embodiments, the cancer displays high levels of FcR-expressing cells. In some embodiments, FcR is FcyR. In some embodiments, the FcR is an activating FcγR. In some embodiments, the cancer is non-small cell lung cancer (NSCLC), glioblastoma, neuroblastoma, melanoma, breast cancer (e.g. triple negative breast cancer), gastric cancer, colorectal cancer (CRC), Or hepatocellular carcinoma.
依照任何上述实施方式的"个体"优选是人。The "individual" according to any of the above embodiments is preferably a human.
可以单独或与疗法中的其它药剂组合使用本发明的抗体。例如，可以与至少一种别的治疗剂共施用本发明的抗体。The antibodies of the present invention can be used alone or in combination with other agents in therapy. For example, the antibody of the invention can be co-administered with at least one other therapeutic agent.
上文记录的此类组合疗法涵盖组合施用(其中两种或更多种治疗剂包含在同一配制剂或分开的配制剂中)，和分开施用，在该情况中，可以在施用别的治疗剂和/或药剂之前，同时，和/或之后发生本发明的抗体的施用。在一个实施方式中，抗OX40抗体的施用和别的治疗剂的施用彼此在约一个月内，或约一，两或三周内，或约1，2，3，4，5，或6天内发生。也可以与放射疗法组合使用本发明的抗体。Such combination therapies documented above encompass combined administration (wherein two or more therapeutic agents are contained in the same formulation or separate formulations), and separate administration, in which case the administration of another therapeutic agent And/or the administration of the antibody of the present invention occurs before, at the same time, and/or after the agent. In one embodiment, the administration of the anti-OX40 antibody and the administration of the other therapeutic agent are within about one month, or within about one, two or three weeks, or within about 1, 2, 3, 4, 5, or 6 days of each other occur. The antibodies of the present invention can also be used in combination with radiation therapy.
在一些实施方式中，抗人OX40激动性抗体可以与化疗或化疗剂联合施用。在一些实施方式中，抗人OX40激动性抗体可以与放疗或放疗剂联合施用。在一些实施方式中，抗人OX40激动性抗体可以与靶向疗法或靶向治疗剂联合施用。在一些实施方式中，抗人OX40激动性抗体可以与免疫疗法或免疫治疗剂，例如单克隆抗体联合施用。In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with chemotherapy or chemotherapeutic agents. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with radiotherapy or radiotherapy agents. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with a targeted therapy or a targeted therapeutic agent. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with immunotherapy or immunotherapeutic agents, such as monoclonal antibodies.
在一些实施方式中，抗人OX40激动性抗体可以与PARP抑制剂(例如Olaparanib，Rucaparib，Niraparib，Cediranib，BMN673，Veliparib)，Trabectedin，nab-paclitaxel(清蛋白结合的帕利他赛，ABRAXANE)，Trebananib，Pazopanib，Cediranib，Palbociclib，everolimus，氟尿嘧啶(例如FOLFOX，F0LFIRI)，IFL，regorafenib，Reolysin，Alimta，Zykadia，Sutent，Torisel(temsirolimus)，Inlyta(axitinib，Pfizer)，Afinitor(everolimus，Novartis)，Nexavar(sorafenib，Onyx/Bayer)，Votrient，Pazopanib，axitinib，IMA-901，AGS_003，cabozantinib，Vinflunine，Hsp90抑制剂(例如apatorsin)，Ad-GM-CSF(CT-0070)，Temazolomide，IL-2，IFNa，vinblastine，Thalomid，dacarbazine，cyclophosphamide，lenalidomide，azacytidine，lenalidomide，bortezomid(VELCADE)，amrubicine，carfilzomib，pralatrexate，和/或enzastaurin联合施用。In some embodiments, the anti-human OX40 agonist antibody can be combined with PARP inhibitors (eg Olaparanib, Rucaparib, Niraparib, Cediranib, BMN673, Veliparib), Trabectedin, nab-paclitaxel (albumin-bound paclitaxel, ABRAXANE), Trebananib , Pazopanib, Cediranib, Palbociclib, everolimus, fluorouracil (e.g. FOLFOX, F0LFIRI), IFL, regorafenib, Reolysin, Alimta, Zykadia, Sutent, Torresel (temsirolimus), Inlyta (axitinex, Pfizer (Pfizer), Afinitor (ever sorafenib, Onyx/Bayer), Votrient, Pazopanib, axitinib, IMA-901, AGS_003, cabozantinib, Vinflunine, Hsp90 inhibitors (such as apatorsin), Ad-GM-CSF (CT-0070), Temazolomide, IL-2, IFNa, Vinblastine, Thalomid, dacarbazine, cyclophosphamide, lenalidomide, azacytidine, lenalidomide, bortezomid (VELCADE), amrubicine, carfilzomib, palatrexate, and/or enzastaurin are administered in combination.
在一些实施方式中，抗人OX40激动性抗体可以与PD-1轴结合拮抗剂联合施用。PD-1轴结合拮抗剂包括但不限于PD-1结合拮抗剂，PD-L1结合拮抗剂和PD-L2结合拮抗剂。“PD-1”的备选名称包括CD279和SLEB2。“PD-L1”的备选名称包括B7-Hl，B7-4，CD274和B7-H。“PD-L2”的备选名称包括B7-DC，Btdc，和CD273。在一些实施方式中，PD-l，PD-Ll，和PD-L2是人PD-1，PD-L1和PD-L2。在一些实施方式中，PD-1结合拮抗剂是抑制PD-1结合其配体结合配偶的分子。在一个具体方面，PD-1配体结合配偶是 PD-Ll和/或PD-L2。在另一个实施方式中，PD-Ll结合拮抗剂是抑制PD-Ll结合其结合配偶的分子。在一个具体方面，PD-Ll结合配偶是PD-1和/或B7-1。在另一个实施方式中，PD-L2结合拮抗剂是抑制PD-L2结合其结合配偶的分子。在一个具体方面，PD-L2结合配偶是PD-1。拮抗剂可以是抗体，其抗原结合片段，免疫粘附素，融合蛋白，或寡肽。在一些实施方式中，PD-1结合拮抗剂是抗PD-1抗体(例如人抗体，人源化抗体，或嵌合抗体)。在一些实施方式中，抗PD-1抗体选自下组：MDX-1106(nivolumab，0PDIV0)，Merck 3475(MK-3475，pembrolizumab，KEYTRUDAWPCT-011(Pidilizumab)。在一些实施方式中，PD-1结合拮抗剂是免疫粘附素(例如包含融合至恒定区(例如免疫球蛋白序列的Fc区)的，PD-L1或PD-L2的胞外或PD-1结合部分的免疫粘附素)。在一些实施方式中，PD-1结合拮抗剂是AMP-224。在一些实施方式中，PD-Ll结合拮抗剂是抗PD-Ll抗体。在一些实施方式中，抗PD-Ll结合拮抗剂选自下组:YW243.55.S70，MPDL3280A，MEDI4736和MDX-1105。MDX-1105，也称作BMS-936559，是WO2007/005874中记载的抗PD-Ll抗体。抗体YW243.55.S70是WO 2010/077634 A1中记载的抗PD-Ll。MDX-1106，也称作MDX-1106-04，0N0-4538，BMS-936558或nivolumab，是WO2006/121168中记载的抗PD-1抗体。Merck 3475，也称作MK-3475，SCH-900475或pembrolizumab，是WO2009/114335中记载的抗PD-1抗体。CT-011，也称作hBAT，hBAT-1或pidil izumab，是WO2009/101611中记载的抗PD-1抗体。AMP-224，也称作B7-DCIg，是WO2010/027827和WO2011/066342中记载的PD-L2-Fc融合可溶性受体。在一些实施方式中，抗PD-1抗体是MDX-1106。“MDX-1106”的备选名称包括MDX-1106-04，0N0-4538，BMS-936558或nivoIumab。在一些实施方式中，抗PD-1抗体是nivolumab(CAS注册号：946414-94-4)。In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with a PD-1 axis binding antagonist. PD-1 axis binding antagonists include but are not limited to PD-1 binding antagonists, PD-L1 binding antagonists and PD-L2 binding antagonists. Alternative names for "PD-1" include CD279 and SLEB2. Alternative names for "PD-L1" include B7-H1, B7-4, CD274 and B7-H. Alternative names for "PD-L2" include B7-DC, Btdc, and CD273. In some embodiments, PD-1, PD-L1, and PD-L2 are human PD-1, PD-L1 and PD-L2. In some embodiments, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partner. In a specific aspect, the PD-1 ligand binding partner is PD-L1 and/or PD-L2. In another embodiment, the PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding partner. In a specific aspect, the PD-L1 binding partner is PD-1 and/or B7-1. In another embodiment, the PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to its binding partner. In a specific aspect, the PD-L2 binding partner is PD-1. The antagonist can be an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of MDX-1106 (nivolumab, OPDIVO), Merck 3475 (MK-3475, pembrolizumab, KEYTRUDAWPCT-011 (Pidilizumab). In some embodiments, PD-1 The binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region (e.g., the Fc region of an immunoglobulin sequence)). In some embodiments, the PD-1 binding antagonist is AMP-224. In some embodiments, the PD-L1 binding antagonist is an anti-PD-L1 antibody. In some embodiments, the anti-PD-L1 binding antagonist is selected From the following group: YW243.55.S70, MPDL3280A, MEDI4736 and MDX-1105. MDX-1105, also known as BMS-936559, is the anti-PD-L1 antibody described in WO2007/005874. Antibody YW243.55.S70 is WO Anti-PD-L1 described in 2010/077634 A1. MDX-1106, also known as MDX-1106-04, ONO-4538, BMS-936558 or nivolumab, is an anti-PD-1 antibody described in WO2006/121168. Merck 3475 , Also known as MK-3475, SCH-900475 or pembrolizumab, is the anti-PD-1 antibody described in WO2009/114335. CT-011, also known as hBAT, hBAT-1 or pidil izumab, is described in WO2009/101611 Anti-PD-1 antibody. AMP-224, also known as B7-DCIg, is the PD-L2-Fc fusion soluble receptor described in WO2010/027827 and WO2011/066342. In some embodiments, the anti-PD-1 antibody is MDX-1106. Alternative names for "MDX-1106" include MDX-1106-04, ON0-4538, BMS-936558 or nivoIumab. In some embodiments, the anti-PD-1 antibody is nivolumab (CAS Registry Number: 946414- 94-4).
在一些实施方式中，抗人OX40激动性抗体可以与针对活化性共刺激分子的激动剂联合施用。在一些实施方式中，活化性共刺激分子可包括CD40，CD226，CD28，GITR，CD137，CD27,HVEM，或CD127。在一些实施方式中，针对活化性共刺激分子的激动剂是结合CD40，CD226，CD28，OX40，GITR，CDl37，CD27，HVEM，或CDl27的激动性抗体。在一些实施方式中，抗人OX40激动性抗体可以与针对抑制性共刺激分子的拮抗剂联合施用。在一些实施方式中，抑制性共刺激分子可包括CTLA-4(也称作CD152)，PD-1，′I′IΜ-3，BTLA，VISTA，LAG-3，B7-H3，B7-H4，IDO，TIGIT，MICA/B，或精氨酸酶。在一些实施方式中，针对抑制性共刺激分子的拮抗剂是结合CTLA-4，PD-1，′I′IΜ-3，BTLA，VISTA，LAG-3(例如 LAG-3-IgG融合蛋白(IMP321))，B7-H3，B7-H4，IDO，TIGIT，MICA/B，或精氨酸酶的拮抗性抗体。In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an agonist directed against an activating costimulatory molecule. In some embodiments, the activating costimulatory molecule may include CD40, CD226, CD28, GITR, CD137, CD27, HVEM, or CD127. In some embodiments, the agonist against the activating costimulatory molecule is an agonist antibody that binds CD40, CD226, CD28, OX40, GITR, CD137, CD27, HVEM, or CD127. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an antagonist against an inhibitory costimulatory molecule. In some embodiments, inhibitory costimulatory molecules may include CTLA-4 (also known as CD152), PD-1,'I'IM-3, BTLA, VISTA, LAG-3, B7-H3, B7-H4, IDO, TIGIT, MICA/B, or arginase. In some embodiments, the antagonist for inhibitory costimulatory molecules binds CTLA-4, PD-1,'I'IM-3, BTLA, VISTA, LAG-3 (e.g., LAG-3-IgG fusion protein (IMP321 )), B7-H3, B7-H4, IDO, TIGIT, MICA/B, or arginase antagonist antibody.
在一些实施方式中，抗人OX40激动性抗体可以与针对CTLA_4(也称作CD152)的拮抗剂，例如阻断性抗体联合施用。在一些实施方式中，抗人OX40激动性抗体可以与ipiIimumab(也称作MDX-010，MDX-101，或
Figure PCTCN2020140259-appb-000004
)联合施用。在一些实施方式中，抗人OX40激动性抗体可以与tremelimumab(也称作ticilimumab或CP-675，206)联合施用。在一些实施方式中，抗人OX40激动性抗体可以与针对B7-H3(也称作CD276)的拮抗剂，例如阻断性抗体联合施用。在一些实施方式中，抗人OX40激动性抗体可以与MGA271联合施用。在一些实施方式中，抗人OX40激动性抗体可以与针对TGFP的拮抗剂，例如metelimumaM也称作CAT-192)，fresoIimumab(也称作GC1008)，或LY2157299联合施用。 In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an antagonist against CTLA-4 (also known as CD152), such as a blocking antibody. In some embodiments, the anti-human OX40 agonist antibody can be combined with ipilimumab (also known as MDX-010, MDX-101, or
Figure PCTCN2020140259-appb-000004
) Combined application. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with tremelimumab (also known as ticilimumab or CP-675,206). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an antagonist against B7-H3 (also known as CD276), such as a blocking antibody. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with MGA271. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an antagonist against TGFP, such as metelimumaM (also known as CAT-192), fresolimumab (also known as GC1008), or LY2157299.
在一些实施方式中，抗人OX40激动性抗体可以与包含过继转移表达嵌合抗原受体(CAR)的T细胞(例如细胞毒性T细胞或CTL)的治疗联合施用。在一些实施方式中，抗人OX40激动性抗体可以与UCART19联合施用。在一些实施方式中，抗人OX40激动性抗体可以与WT128z联合施用。在一些实施方式中，抗人OX40激动性抗体可以与KTE-C19(Kite)联合施用。在一些实施方式中，抗人OX40激动性抗体可以与CTL019(Novartis)联合施用。在一些实施方式中，抗人OX40激动性抗体可以与包含过继转移包含显性阴性TGFI3受体，例如，显性阴性TGFWI型受体的T细胞的治疗联合施用。在一些实施方式中，抗人OX40激动性抗体可以与包含HERCREEM方案的治疗(参见例如ClinicalTrials.gov Identifier NCT00889954)联合施用。In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with a treatment comprising adoptive transfer of T cells expressing chimeric antigen receptor (CAR) (eg, cytotoxic T cells or CTL). In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with UCART19. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with WT128z. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with KTE-C19 (Kite). In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with CTL019 (Novartis). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with a treatment comprising adoptive transfer of T cells containing a dominant negative TGFI3 receptor, for example, a dominant negative TGFWI type receptor. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with treatments that include the HERCREEM regimen (see, for example, ClinicalTrials.gov Identifier NCT00889954).
在一些实施方式中，抗人OX40激动性抗体可以与针对CD19的拮抗剂联合施用。在一些实施方式中，抗人OX40激动性抗体可以与M0R00208联合施用。在一些实施方式中，抗人OX40激动性抗体可以与针对CD38的拮抗剂联合施用。在一些实施方式中，抗人OX40激动性抗体可以与daratumumab联合施用。In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an antagonist against CD19. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with MOR00208. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an antagonist against CD38. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with daratumumab.
在一些实施方式中，抗人OX40激动性抗体可以与针对CD137(也称作TNFRSF9，4-1BB，或ILA)的激动剂，例如活化性抗体联合施用。在一些实施方式中，抗人OX40激动性抗体可以与urelumab(也称作BMS-663513)联合施用。在一些实施方式中，抗人OX40激动性抗体可以与针对CD40的激动剂，例如活化性抗体联合施用。在一些实施方式中，抗人OX40激动性抗体可以与CP-870893联合施用。在一些实施方式中，抗人OX40激动性抗体可以与针对OX40(也称作CDl34)的激动剂，例如活化性抗体联合施用。在一些实施方式中，抗人OX40激动性抗体可以与不同的抗OX40 抗体(例如AgonOX)联合施用。在一些实施方式中，抗人OX40激动性抗体可以与针对CD27的激动剂，例如活化性抗体联合施用。在一些实施方式中，抗人OX40激动性抗体可以与CDX-1127联合施用。在一些实施方式中，抗人OX40激动性抗体可以与针对吲哚胺-2，3_双加氧酶(IDO)的拮抗剂联合施用。在一些实施方式中，该IDO拮抗剂是1-甲基-D-色氨酸(也称作I-D-MT)。In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an agonist against CD137 (also known as TNFRSF9, 4-1BB, or ILA), such as an activating antibody. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with urelumab (also known as BMS-663513). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an agonist against CD40, such as an activating antibody. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with CP-870893. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with an agonist against OX40 (also known as CD134), such as an activating antibody. In some embodiments, anti-human OX40 agonistic antibodies can be administered in combination with different anti-OX40 antibodies (eg, AgonOX). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an agonist against CD27, such as an activating antibody. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with CDX-1127. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an antagonist against indoleamine-2,3-dioxygenase (IDO). In some embodiments, the IDO antagonist is 1-methyl-D-tryptophan (also known as I-D-MT).
在一些实施方式中，抗人OX40激动性抗体可以与针对CD137(也称作TNFRSF9，4-1BB，或ILA)的激动剂，例如活化性抗体联合施用。在一些实施方式中，抗人OX40激动性抗体可以与urelumab(也称作BMS-663513)联合施用。在一些实施方式中，抗人OX40激动性抗体可以与针对CD40的激动剂，例如活化性抗体联合施用。在一些实施方式中，抗人OX40激动性抗体可以与CP-870893或R07009789联合施用。在一些实施方式中，抗人OX40激动性抗体可以与针对OX40(也称作CD134)的激动剂，例如活化性抗体联合施用。在一些实施方式中，抗人OX40激动性抗体可以与针对CD27的激动剂，例如活化性抗体联合施用。在一些实施方式中，抗人OX40激动性抗体可以与CDX-1127(也称作varlilumab)联合施用。在一些实施方式中，抗人OX40激动性抗体可以与针对吲哚胺-2，3-双加氧酶(IDO)的拮抗剂联合施用。在一些实施方式中，该IDO拮抗剂是1-甲基-D-色氨酸(也称作1-D-MT)。在一些实施方式中，该IDO拮抗剂是W02010/005958(通过此处记录明确收录其内容)中所示IDO拮抗剂。在一些实施方案中，该IDO诘抗剂是4_({2_[(氨基磺酰基)氨基]乙基}氨基)-N_(3-溴-4-氟苯基)-Ν'-羟基-1，2，5-口恶二唑-3-甲脒(例如如W02010/005958实施例23中记载的)。在一些实施方式实施方式中，该IDO拮抗剂是In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an agonist against CD137 (also known as TNFRSF9, 4-1BB, or ILA), such as an activating antibody. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with urelumab (also known as BMS-663513). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an agonist against CD40, such as an activating antibody. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with CP-870893 or R07009789. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with an agonist against OX40 (also known as CD134), such as an activating antibody. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an agonist against CD27, such as an activating antibody. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with CDX-1127 (also known as varlilumab). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an antagonist against indoleamine-2,3-dioxygenase (IDO). In some embodiments, the IDO antagonist is 1-methyl-D-tryptophan (also known as 1-D-MT). In some embodiments, the IDO antagonist is the IDO antagonist shown in WO2010/005958 (the content is clearly included through the record here). In some embodiments, the IDO antagonist is 4-({2-[(aminosulfonyl)amino]ethyl}amino)-N_(3-bromo-4-fluorophenyl)-N'-hydroxy-1, 2,5-oxadiazole-3-carboxamidine (e.g. as described in Example 23 of WO2010/005958). In some embodiments, the IDO antagonist is
Figure PCTCN2020140259-appb-000005
Figure PCTCN2020140259-appb-000005
在一些实施方式中，该IDO拮抗剂是INCB24360。在一些实施方式中，该IDO拮抗剂是Indoximod(l-甲基-色氨酸的D异构体)。在一些实施方式中，抗人OX40激动性抗体可以与抗体-药物缀合物联合施用。在一些实施方式中，该抗体-药物缀合物包含mertansine或单甲基奥瑞司他汀E(MMAE)。在一些实施方式中，抗人OX40激动性抗体可以与抗NaPi2b 抗体-MMAE缀合物(也称作DNIB0600A，RG7599或Iifastuzumab vedotin)联合施用。在一些实施方式中，抗人OX40激动性抗体可以与trastuzumab emtansine(也称作T-DM1，ado_trastuzumab emtansine，或KADCYL.
Figure PCTCN2020140259-appb-000006
Genentech)联合施用。在一些实施方式中，抗人OX40激动性抗体可以与抗MUC16抗体-MMAE缀合物，DMUC5754A联合施用。在一些实施方式中，抗人OX40激动性抗体可以与抗MUC16抗体-MMAE缀合物，DMUC4064A联合施用。在一些实施方式中，抗人OX40激动性抗体可以与靶向内皮缩血管肽B受体(EDNBR)的抗体-药物缀合物，例如缀合有MMAE的针对EDNBR的抗体联合施用。在一些实施方式中，抗人OX40激动性抗体可以与靶向淋巴细胞抗原6复合物，基因座E(Ly6E)的抗体-药物缀合物，例如缀合有MMAE的针对Ly6E的抗体(也称作DLYE5953A)联合施用。在一些实施方式中，抗人OX40激动性抗体可以与polatuzumab vedotin联合施用。在一些实施方式中，抗人OX40激动性抗体可以与靶向CD30的抗体-药物缀合物联合施用。在一些实施方式中，抗人OX40激动性抗体可以与ADCETRIS(也称作brentuximab vedotin)联合施用。在一些实施方式中，抗人OX40激动性抗体可以与polatuzumab vedotin联合施用。 In some embodiments, the IDO antagonist is INCB24360. In some embodiments, the IDO antagonist is Indoximod (the D isomer of 1-methyl-tryptophan). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an antibody-drug conjugate. In some embodiments, the antibody-drug conjugate comprises mertansine or monomethyl auristatin E (MMAE). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an anti-NaPi2b antibody-MMAE conjugate (also known as DNIB0600A, RG7599 or Iifastuzumab vedotin). In some embodiments, the anti-human OX40 agonistic antibody can be combined with trastuzumab emtansine (also known as T-DM1, ado_trastuzumab emtansine, or KADCYL.
Figure PCTCN2020140259-appb-000006
Genentech) co-administered. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with the anti-MUC16 antibody-MMAE conjugate, DMUC5754A. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with the anti-MUC16 antibody-MMAE conjugate, DMUC4064A. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an antibody-drug conjugate that targets the endothelin B receptor (EDNBR), such as an antibody against EDNBR conjugated with MMAE. In some embodiments, the anti-human OX40 agonistic antibody can be combined with an antibody-drug conjugate targeting lymphocyte antigen 6 complex, locus E (Ly6E), such as an antibody against Ly6E (also called Ly6E) conjugated with MMAE As DLYE5953A) combined administration. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with polatuzumab vedotin. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an antibody-drug conjugate that targets CD30. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with ADCETRIS (also known as brentuximab vedotin). In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with polatuzumab vedotin.
在一些实施方式中，抗人OX40激动性抗体可以与血管发生抑制剂联合施用。在一些实施方式中，抗人OX40激动性抗体可以与针对VEGF，例如VEGF-A的抗体联合施用。在一些实施方式中，抗人OX40激动性抗体可以与贝伐珠单抗(也称作
Figure PCTCN2020140259-appb-000007
Genentech)联合施用。在一些实施方式中，抗人OX40激动性抗体可以与针对血管生成素2(也称作Ang2)的抗体联合施用。在一些实施方式中，抗人OX40激动性抗体可以与MEDI3617联合施用。在一些实施方式中，抗人OX40激动性抗体可以与针对VEGFR2的抗体联合施用。在一些实施方式中，抗人OX40激动性抗体可以与ramucirumab联合施用。在一些实施方式中，抗人OX40激动性抗体可以与VEGF受体融合蛋白联合施用。在一些实施方式中，抗人OX40激动性抗体可以与aflibercept联合施用。在一些实施方式中，抗人OX40激动性抗体可以与ziv-aflibercept(也称作VEGF陷阱或
Figure PCTCN2020140259-appb-000008
)联合施用。在一些实施方式中，抗人OX40激动性抗体可以与针对VEGF和Ang2的双特异性抗体联合施用。在一些实施方式中，抗人OX40激动性抗体可以与RG7221(也称作vanucizumab)联合施用。在一些实施方式中，抗人OX40激动性抗体可以与血管发生抑制剂联合和与PD-1轴结合拮抗剂(例如PD-1结合拮抗剂诸如抗PD-1抗体，PD-Ll结合拮抗剂诸如抗PD-Ll抗体，和PD-L2结合拮抗剂诸如抗PD-L2抗体)联合施用。在一些实施方式中，抗人OX40激动性抗体可以与贝伐珠单抗和PD-1轴结合拮抗剂(例如PD-1结合拮抗剂诸如抗 PD-1抗体，PD-L1结合拮抗剂诸如抗PD-Ll抗体，和PD-L2结合拮抗剂诸如抗PD-L2抗体)联合施用。在一些实施方式中，抗人OX40激动性抗体可以与贝伐珠单抗和MDX-1106(nivolumab，OPDIVO)联合施用。在一些实施方式中，抗人OX40激动性抗体可以与贝伐珠单抗和Merck 3475(MK-3475，pembrolizumab，KEYTRUDA)联合施用。在一些实施方式中，抗人OX40激动性抗体可以与贝伐珠单抗和CT-011(Pidilizumab)联合施用。在一些实施方式中，抗人OX40激动性抗体可以与贝伐珠单抗和YW243.55.S70联合施用。在一些实施方式中，抗人OX40激动性抗体可以与贝伐珠单抗和MPDL3280A联合施用。在一些实施方式中，抗人OX40激动性抗体可以与贝伐珠单抗和MEDI4736联合施用。在一些实施方式中，抗人OX40激动性抗体可以与贝伐珠单抗和MDX-1105联合施用。 In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an angiogenesis inhibitor. In some embodiments, anti-human OX40 agonistic antibodies can be administered in combination with antibodies directed against VEGF, such as VEGF-A. In some embodiments, the anti-human OX40 agonist antibody can be combined with bevacizumab (also known as
Figure PCTCN2020140259-appb-000007
Genentech) co-administered. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with an antibody against Angiopoietin 2 (also known as Ang2). In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with MEDI3617. In some embodiments, an anti-human OX40 agonist antibody can be administered in combination with an antibody against VEGFR2. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with ramucirumab. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with a VEGF receptor fusion protein. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with aflibercept. In some embodiments, the anti-human OX40 agonistic antibody can interact with ziv-aflibercept (also known as VEGF trap or
Figure PCTCN2020140259-appb-000008
) Combined application. In some embodiments, anti-human OX40 agonistic antibodies can be administered in combination with bispecific antibodies directed against VEGF and Ang2. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with RG7221 (also known as vanucizumab). In some embodiments, an anti-human OX40 agonist antibody can be combined with an angiogenesis inhibitor and a PD-1 axis binding antagonist (for example, a PD-1 binding antagonist such as an anti-PD-1 antibody, a PD-L1 binding antagonist such as Anti-PD-L1 antibody, and PD-L2 binding antagonist (such as anti-PD-L2 antibody) are administered in combination. In some embodiments, the anti-human OX40 agonist antibody can be combined with bevacizumab and PD-1 axis binding antagonist (for example, PD-1 binding antagonist such as anti-PD-1 antibody, PD-L1 binding antagonist such as anti-PD-1 PD-L1 antibody, and PD-L2 binding antagonist such as anti-PD-L2 antibody) are administered in combination. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with bevacizumab and MDX-1106 (nivolumab, OPDIVO). In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with bevacizumab and Merck 3475 (MK-3475, pembrolizumab, KEYTRUDA). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with bevacizumab and CT-011 (Pidilizumab). In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with bevacizumab and YW243.55.S70. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with bevacizumab and MPDL3280A. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with bevacizumab and MEDI4736. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with bevacizumab and MDX-1105.
在一些实施方式中，抗人OX40激动性抗体可以与抗肿瘤剂联合施用。在一些实施方式中，抗人OX40激动性抗体可以与靶向CSF-1R(也称作M-CSFR或CD115)的药剂联合施用。在一些实施方式中，抗人OX40激动性抗体可以与抗CSF-1R抗体(也称作IMC-CS4或LY3022855)联合施用。在一些实施方式中，抗人OX40激动性抗体可以与抗CSF-1R抗体，RG7155(也称作R05509554或emactuzumab)联合施用。在一些实施方式中，抗人OX40激动性抗体可以与干扰素，例如干扰素-α或干扰素-γ联合施用。在一些实施方式中，抗人OX40激动性抗体可以与Roferon-A(也称作重组干扰素a-2a)联合施用。在一些实施方式中，抗人OX40激动性抗体可以与GM-CSF(也称作重组人粒细胞巨噬细胞集落刺激因子，rhu GM-CSF，sargramostim，或
Figure PCTCN2020140259-appb-000009
)联合施用。在一些实施方式中，抗人OX40激动性抗体可以与IL-2(也称作aldesleukin或
Figure PCTCN2020140259-appb-000010
)联合施用。在一些实施方式中，抗人OX40激动性抗体可以与IL-12联合施用。在一些实施方式中，抗人OX40激动性抗体可以与IL27联合施用。在一些实施方式中，抗人OX40激动性抗体可以与IL-15联合施用。在一些实施方式中，抗人OX40激动性抗体可以与ALT-803联合施用。在一些实施方式中，抗人OX40激动性抗体可以与靶向CD20的抗体联合施用。在一些实施方式中，靶向CD20的抗体是奥奴珠单抗(也称作GAlO 1或
Figure PCTCN2020140259-appb-000011
)或利妥昔单抗。在一些实施方式中，抗人OX40激动性抗体可以与靶向GITR的抗体联合施用。在一些实施方式中，靶向GITR的抗体是TRX518。在一些实施方式中，靶向GITR的抗体是MK04166(Merck)。 In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an anti-tumor agent. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an agent that targets CSF-1R (also known as M-CSFR or CD115). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an anti-CSF-1R antibody (also known as IMC-CS4 or LY3022855). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with the anti-CSF-1R antibody, RG7155 (also known as R05509554 or emactuzumab). In some embodiments, anti-human OX40 agonistic antibodies can be administered in combination with interferons, such as interferon-α or interferon-γ. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with Roferon-A (also known as recombinant interferon a-2a). In some embodiments, the anti-human OX40 agonistic antibody can be combined with GM-CSF (also known as recombinant human granulocyte macrophage colony stimulating factor, rhu GM-CSF, sargramostim, or
Figure PCTCN2020140259-appb-000009
) Combined application. In some embodiments, the anti-human OX40 agonistic antibody can interact with IL-2 (also known as aldesleukin or
Figure PCTCN2020140259-appb-000010
) Combined application. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with IL-12. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with IL27. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with IL-15. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with ALT-803. In some embodiments, anti-human OX40 agonistic antibodies can be administered in combination with antibodies that target CD20. In some embodiments, the CD20-targeting antibody is onuzumab (also known as GAlO 1 or
Figure PCTCN2020140259-appb-000011
) Or rituximab. In some embodiments, anti-human OX40 agonistic antibodies can be administered in combination with antibodies that target GITR. In some embodiments, the antibody that targets GITR is TRX518. In some embodiments, the antibody targeting GITR is MK04166 (Merck).
在一些实施方式中，抗人OX40激动性抗体可以与Bruton氏酪氨酸激酶(BTK)的抑制剂联合施用。在一些实施方式中，抗人OX40激动性抗体可以与ibrutinib联合施用。在一些实施方式中，抗人OX40激动性抗体可以与 疫苗脱氢酶I(IDH1)和/或疫苗脱氢酶2(IDH2)的抑制剂联合施用。在一些实施方式中，抗人OX40激动性抗体可以与AG-120(Agios)联合施用。In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an inhibitor of Bruton's tyrosine kinase (BTK). In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with ibrutinib. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an inhibitor of vaccine dehydrogenase I (IDH1) and/or vaccine dehydrogenase 2 (IDH2). In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with AG-120 (Agios).
在一些实施方式中，抗人OX40激动性抗体可以与奥奴珠单抗和PD-1轴结合拮抗剂(例如PD-1结合拮抗剂诸如抗PD-1抗体，PD-Ll结合拮抗剂诸如抗PD-Ll抗体，和PD-L2结合拮抗剂诸如抗PD-L2抗体)联合施用。In some embodiments, the anti-human OX40 agonist antibody can be combined with onuzumab and PD-1 axis binding antagonist (for example, PD-1 binding antagonist such as anti-PD-1 antibody, PD-L1 binding antagonist such as anti-PD-1 PD-L1 antibody, and PD-L2 binding antagonist such as anti-PD-L2 antibody) are administered in combination.
在一些实施方式中，抗人OX40激动性抗体可以与癌症疫苗联合施用。在一些实施方式中，该癌症疫苗是肽癌症疫苗，其在一些实施方式中是个性化肽疫苗。在一些实施方式中，该肽癌症疫苗是多价长肽，多重肽，肽混合物，杂合肽，或经肽脉冲的树突细胞疫苗(参见例如Yamada et al.，Cancer Sci，104:14-21,2013)。在一些实施方式中，抗人OX40激动性抗体可以与佐剂联合施用。在一些实施方式中，抗人OX40激动性抗体可以与包含TLR激动剂，例如Poly-ICLC(也称作
Figure PCTCN2020140259-appb-000012
)，LPS，MPL，或CpG ODN的治疗联合施用。在一些实施方式中，抗人OX40激动性抗体可以与肿瘤坏死因子(TNF)a联合施用。在一些实施方式中，抗人OX40激动性抗体可以与IL-1联合施用。在一些实施方式中，抗人OX40激动性抗体可以与HMGBl联合施用。在一些实施方式中，抗人OX40激动性抗体可以与IL-10拮抗剂联合施用。在一些实施方式中，抗人OX40激动性抗体可以与IL-4拮抗剂联合施用。在一些实施方式中，抗人OX40激动性抗体可以与IL-13拮抗剂联合施用。在一些实施方式中，抗人OX40激动性抗体可以与IL-17拮抗剂联合施用。在一些实施方式中，抗人OX40激动性抗体可以与HVEM拮抗剂联合施用。在一些实施方式中，抗人OX40激动性抗体可以与ICOS激动剂(例如通过施用ICOS-L，或针对ICOS的激动性抗体)联合施用。在一些实施方式中，抗人OX40激动性抗体可以与靶向CX3CL1的治疗联合施用。在一些实施方式中，抗人OX40激动性抗体可以与靶向CXCL9的治疗联合施用。在一些实施方式中，抗人OX40激动性抗体可以与靶向CXCLl0的治疗联合施用。在一些实施方式中，抗人OX40激动性抗体可以与靶向CCL5的治疗联合施用。在一些实施方式中，抗人OX40激动性抗体可以与LFA-I或ICAM1激动剂联合施用。在一些实施方式中，抗人OX40激动性抗体可以与选择蛋白激动剂联合施用。 In some embodiments, anti-human OX40 agonistic antibodies can be administered in combination with cancer vaccines. In some embodiments, the cancer vaccine is a peptide cancer vaccine, which in some embodiments is a personalized peptide vaccine. In some embodiments, the peptide cancer vaccine is a multivalent long peptide, a multipeptide, a peptide mixture, a hybrid peptide, or a peptide-pulsed dendritic cell vaccine (see, for example, Yamada et al., Cancer Sci, 104:14- 21, 2013). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an adjuvant. In some embodiments, the anti-human OX40 agonist antibody can be combined with a TLR agonist, such as Poly-ICLC (also known as
Figure PCTCN2020140259-appb-000012
), LPS, MPL, or CpG ODN treatments are administered in combination. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with tumor necrosis factor (TNF) a. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with IL-1. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with HMGB1. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an IL-10 antagonist. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an IL-4 antagonist. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an IL-13 antagonist. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an IL-17 antagonist. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an HVEM antagonist. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an ICOS agonist (for example, by administering ICOS-L, or an agonistic antibody against ICOS). In some embodiments, anti-human OX40 agonistic antibodies can be administered in combination with treatments that target CX3CL1. In some embodiments, anti-human OX40 agonistic antibodies can be administered in combination with treatments that target CXCL9. In some embodiments, anti-human OX40 agonistic antibodies can be administered in combination with treatments that target CXCL10. In some embodiments, anti-human OX40 agonistic antibodies can be administered in combination with treatments that target CCL5. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an LFA-I or ICAM1 agonist. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with a selectin agonist.
在一些实施方式中，抗人OX40激动性抗体可以与B-Raf的抑制剂联合施用。在一些实施方式中，抗人OX40激动性抗体可以与维罗非尼(也称作
Figure PCTCN2020140259-appb-000013
)联合施用。在一些实施方式中，抗人OX40激动性抗体可以与dabrafenib(也称作
Figure PCTCN2020140259-appb-000014
)联合施用。在一些实施方式中，抗人OX40激动性抗体可以与encorafenib(LGX818)联合施用。 In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an inhibitor of B-Raf. In some embodiments, the anti-human OX40 agonistic antibody can be combined with Verofenib (also known as
Figure PCTCN2020140259-appb-000013
) Combined application. In some embodiments, the anti-human OX40 agonistic antibody can be combined with dabrafenib (also known as
Figure PCTCN2020140259-appb-000014
) Combined application. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with encorafenib (LGX818).
在一些实施方式中，抗人OX40激动性抗体可以与EGFR抑制剂联合 施用。在一些实施方式中，抗人OX40激动性抗体可以与erlotinib(也称作
Figure PCTCN2020140259-appb-000015
)联合施用。在一些实施方式中，抗人OX40激动性抗体可以与EGFR-T790M的抑制剂联合施用。在一些实施方式中，抗人OX40激动性抗体可以与gefitinib联合施用。在一些实施方式中，抗人OX40激动性抗体可以与afatinib联合施用。在一些实施方式中，抗人OX40激动性抗体可以与西妥昔单抗(也称作
Figure PCTCN2020140259-appb-000016
)联合施用。在一些实施方式中，抗人OX40激动性抗体可以与panitumumab(也称作
Figure PCTCN2020140259-appb-000017
)联合施用。在一些实施方式中，抗人OX40激动性抗体可以与rociletinib联合施用。在一些实施方式中，抗人OX40激动性抗体可以与AZD9291联合施用。在一些实施方式中，抗人OX40激动性抗体可以与MEK，诸如MEK1(也称作MAP2K1)和/或MEK2(也称作MAP2K2)的抑制剂联合施用。在一些实施方式中，抗人OX40激动性抗体可以与cobimetinib(也称作CDC-0973或XL-518)联合施用。在一些实施方式中，抗人OX40激动性抗体可以与trametinib(也称作
Figure PCTCN2020140259-appb-000018
)联合施用。在一些实施方式中，抗人OX40激动性抗体可以与binimetinib联合施用。 In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an EGFR inhibitor. In some embodiments, the anti-human OX40 agonistic antibody can be combined with erlotinib (also known as
Figure PCTCN2020140259-appb-000015
) Combined application. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an inhibitor of EGFR-T790M. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with gefitinib. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with afatinib. In some embodiments, the anti-human OX40 agonist antibody can be combined with cetuximab (also known as
Figure PCTCN2020140259-appb-000016
) Combined application. In some embodiments, the anti-human OX40 agonist antibody can be combined with panitumumab (also known as
Figure PCTCN2020140259-appb-000017
) Combined application. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with rociletinib. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with AZD9291. In some embodiments, anti-human OX40 agonistic antibodies can be administered in combination with MEK, such as MEK1 (also known as MAP2K1) and/or MEK2 (also known as MAP2K2) inhibitors. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with cobimetinib (also known as CDC-0973 or XL-518). In some embodiments, the anti-human OX40 agonistic antibody can be combined with trametinib (also known as
Figure PCTCN2020140259-appb-000018
) Combined application. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with binimetinib.
在一些实施方式中，抗人OX40激动性抗体可以与B-Raf的抑制剂(例如维罗非尼或dabrafenib)和MEK(例如MEKl和/或MEK2)的抑制剂(例如cobimetinib或trametinib)联合施用。在一些实施方式中，抗人OX40激动性抗体可以与ERK(例如ERK1/2)的抑制剂联合施用。在一些实施方式中，抗人OX40激动性抗体可以与GDC-0994联合施用。在一些实施方式中，抗人OX40激动性抗体可以与B-Raf的抑制剂，MEK的抑制剂，和ERK1/2的抑制剂联合施用。在一些实施方式中，抗人OX40激动性抗体可以与EGFR的抑制剂，MEK的抑制剂，和ERK1/2的抑制剂联合施用。在一些实施方式中，抗人OX40激动性抗体可以与一种或多种MAP激酶途径抑制剂联合施用。在一些实施方式中，抗人OX40激动性抗体可以与CK127联合施用。在一些实施方式中，抗人OX40激动性抗体可以与K-Ras的抑制剂联合施用。In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with inhibitors of B-Raf (e.g., verofenib or dabrafenib) and inhibitors of MEK (e.g., MEK1 and/or MEK2) (e.g., cobimetinib or trametinib) . In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an inhibitor of ERK (eg, ERK1/2). In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with GDC-0994. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an inhibitor of B-Raf, an inhibitor of MEK, and an inhibitor of ERK1/2. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an inhibitor of EGFR, an inhibitor of MEK, and an inhibitor of ERK1/2. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with one or more MAP kinase pathway inhibitors. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with CK127. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an inhibitor of K-Ras.
在一些实施方式中，抗人OX40激动性抗体可以与c-Met的抑制剂联合施用。在一些实施方式中，抗人OX40激动性抗体可以与onartuzumab(也称作MetMAb)联合施用。在一些实施方式中，抗人OX40激动性抗体可以与anaplatic淋巴瘤激酶(ALK)的抑制剂联合施用。在一些实施方式中，抗人OX40激动性抗体可以与AF802(也称作CH5424802或alectinib)联合施用。在一些实施方式中，抗人OX40激动性抗体可以与crizotinib联合施用。在一些实施方式中，抗人OX40激动性抗体可以与ceritinib联合施用。在一些实施方式中，抗人OX40激动性抗体可以与磷脂酰肌醇3-激酶(PI3K)的抑制 剂联合施用。在一些实施方式中，抗人OX40激动性抗体可以与buparlisib(BKM-120)联合施用。在一些实施方式中，抗人OX40激动性抗体可以与pictilisib(也称作GDC-0941)联合施用。在一些实施方式中，抗人OX40激动性抗体可以与buparlisib(也称作BKM-120)联合施用。在一些实施方式中，抗人OX40激动性抗体可以与perifosine(也称作KRX-0401)联合施用。在一些实施方式中，抗人OX40激动性抗体可以与磷脂酰肌醇3-激酶(PI3K)的δ选择性抑制剂联合施用。在一些实施方式中，抗人OX40激动性抗体可以与idelalisib(也称作GS-1101或CAL-101)联合施用。在一些实施方式中，抗人OX40激动性抗体可以与taselisib(也称作GDC-0032)联合施用。在一些实施方式中，抗人OX40激动性抗体可以与BYL-719联合施用。在一些实施方式中，抗人OX40激动性抗体可以与Akt的抑制剂联合施用。在一些实施方式中，抗人OX40激动性抗体可以与MK2206联合施用。在一些实施方式中，抗人OX40激动性抗体可以与GSK690693联合施用。在一些实施方式中，抗人OX40激动性抗体可以与ipatasertib(也称作CDC-0068)联合施用。在一些实施方式中，抗人OX40激动性抗体可以与mTOR的抑制剂联合施用。在一些实施方式中，抗人OX40激动性抗体可以与sirolimus(也称作rapamycin)联合施用。在一些实施方式中，抗人OX40激动性抗体可以与temsirolimus(也称作CCI-779或
Figure PCTCN2020140259-appb-000019
)联合施用。在一些实施方式中，抗人OX40激动性抗体可以与everolimus(也称作RADOO1)联合施用。在一些实施方式中，抗人OX40激动性抗体可以与ridaforoIimus(也称作AP-23573，MK_8669，或deforolimus)联合施用。在一些实施方式中，抗人OX40激动性抗体可以与0SI-027联合施用。在一些实施方式中，抗人OX40激动性抗体可以与AZD8055联合施用。在一些实施方式中，抗人OX40激动性抗体可以与INK128联合施用。在一些实施方式中，抗人OX40激动性抗体可以与双重PI3K/mT0R抑制剂联合施用。在一些实施方式中，抗人OX40激动性抗体可以与XL765联合施用。在一些实施方式中，抗人OX40激动性抗体可以与GDC-0980联合施用。在一些实施方式中，抗人OX40激动性抗体可以与BEZ235(也称作NVP-BEZ235)联合施用。在一些实施方式中，抗人OX40激动性抗体可以与BGT226联合施用。在一些实施方式中，抗人OX40激动性抗体可以与GSK2126458联合施用。在一些实施方式中，抗人OX40激动性抗体可以与PF-04691502联合施用。在一些实施方式中，抗人OX40激动性抗体可以与PF-05212384(也称作PKI-587)联合施用。 In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an inhibitor of c-Met. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with onartuzumab (also known as MetMAb). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an inhibitor of anapatic lymphoma kinase (ALK). In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with AF802 (also known as CH5424802 or alectinib). In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with crizotinib. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with ceritinib. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an inhibitor of phosphatidylinositol 3-kinase (PI3K). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with buparlisib (BKM-120). In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with pictilisib (also known as GDC-0941). In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with buparlisib (also known as BKM-120). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with perifosine (also known as KRX-0401). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with a delta selective inhibitor of phosphatidylinositol 3-kinase (PI3K). In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with idelalisib (also known as GS-1101 or CAL-101). In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with taselisib (also known as GDC-0032). In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with BYL-719. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an inhibitor of Akt. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with MK2206. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with GSK690693. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with ipatasertib (also known as CDC-0068). In some embodiments, anti-human OX40 agonistic antibodies can be administered in combination with inhibitors of mTOR. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with sirolimus (also known as rapamycin). In some embodiments, the anti-human OX40 agonistic antibody can interact with temsirolimus (also known as CCI-779 or
Figure PCTCN2020140259-appb-000019
) Combined application. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with everolimus (also known as RADOO1). In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with ridaforolimus (also known as AP-23573, MK_8669, or deforolimus). In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with OSI-027. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with AZD8055. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with INK128. In some embodiments, anti-human OX40 agonistic antibodies can be administered in combination with dual PI3K/mTOR inhibitors. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with XL765. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with GDC-0980. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with BEZ235 (also known as NVP-BEZ235). In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with BGT226. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with GSK2126458. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with PF-04691502. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with PF-05212384 (also known as PKI-587).
在一些实施方式中，抗人OX40激动性抗体可以与选择性降解***受体的药剂联合施用。在一些实施方式中，抗人OX40激动性抗体可以与 GDC-0927联合施用。在一些实施方式中，抗人OX40激动性抗体可以与HER3的抑制剂联合施用。在一些实施方式中，抗人OX40激动性抗体可以与duligotuzumab联合施用。在一些实施方式中，抗人OX40激动性抗体可以与LSDl的抑制剂联合施用。在一些实施方式中，抗人OX40激动性抗体可以与MDM2的抑制剂联合施用。在一些实施方式中，抗人OX40激动性抗体可以与BCL2的抑制剂联合施用。在一些实施方式中，抗人OX40激动性抗体可以与venetoclax联合施用。在一些实施方式中，抗人OX40激动性抗体可以与CHKl的抑制剂联合施用。在一些实施方式中，抗人OX40激动性抗体可以与CDC-0575联合施用。在一些实施方式中，抗人OX40激动性抗体可以与激活的hedgehog信号传导途径的抑制剂联合施用。在一些实施方式中，抗人OX40激动性抗体可以与ERIVEDGE联合施用。In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with an agent that selectively degrades the estrogen receptor. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with GDC-0927. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an inhibitor of HER3. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with duligotuzumab. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an inhibitor of LSD1. In some embodiments, anti-human OX40 agonistic antibodies can be administered in combination with inhibitors of MDM2. In some embodiments, anti-human OX40 agonistic antibodies can be administered in combination with inhibitors of BCL2. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with venetoclax. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an inhibitor of CHK1. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with CDC-0575. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with an inhibitor of the activated hedgehog signaling pathway. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with ERIVEDGE.
在一些实施方式中，抗人OX40激动性抗体可以与放射疗法联合施用。在一些实施方式中，抗人OX40激动性抗体可以与吉西他滨联合施用。在一些实施方式中，抗人OX40激动性抗体可以与nab-pacIitaxeI(ABRAXANE)联合施用。在一些实施方式中，抗人OX40激动性抗体可以与曲妥珠单抗联合施用。在一些实施方式中，抗人OX40激动性抗体可以与TVEC联合施用。在一些实施方式中，抗人OX40激动性抗体可以与IL27联合施用。在一些实施方式中，抗人OX40激动性抗体可以与环磷酰胺联合施用。在一些实施方式中，抗人OX40激动性抗体可以与募集T细胞至肿瘤的药剂联合施用。在一些实施方式中，抗人OX40激动性抗体可以与lirilumab(IPH2102/BMS-986015)联合施用。在一些实施方式中，抗人OX40激动性抗体可以与Idelalisib联合施用。在一些实施方式中，抗人OX40激动性抗体可以与靶向CD3和CD20的抗体联合施用。在一些实施方式中，抗人OX40激动性抗体可以与REGN1979联合施用。在一些实施方式中，抗人OX40激动性抗体可以与靶向CD3和CD19的抗体联合施用。在一些实施方式中，抗人OX40激动性抗体可以与blinatumomab联合施用。In some embodiments, anti-human OX40 agonistic antibodies can be administered in combination with radiation therapy. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with gemcitabine. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with nab-pacIitaxel (ABRAXANE). In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with trastuzumab. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with TVEC. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with IL27. In some embodiments, the anti-human OX40 agonist antibody can be administered in combination with cyclophosphamide. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with an agent that recruits T cells to the tumor. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with lirilumab (IPH2102/BMS-986015). In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with Idelalisib. In some embodiments, anti-human OX40 agonistic antibodies can be administered in combination with antibodies that target CD3 and CD20. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with REGN1979. In some embodiments, anti-human OX40 agonistic antibodies can be administered in combination with antibodies that target CD3 and CD19. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with blinatumomab.
在一些实施方式中，抗人OX40激动性抗体可以与溶瘤病毒联合施用。在一些实施方式中，抗人OX40激动性抗体可以与卡钼和nab-paclitaxel联合施用。在一些实施方式中，抗人OX40激动性抗体可以与卡铂和帕利他赛联合施用。在一些实施方式中，抗人OX40激动性抗体可以与顺铂和培美曲塞联合施用。在一些实施方式中，抗人OX40激动性抗体可以与顺铂和吉西他滨联合施用。在一些实施方式中，抗人OX40激动性抗体可以与FOLFOX联合施用。在一些实施方式中，抗人OX40激动性抗体可以与F0LFIRI联合施用。In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with an oncolytic virus. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with carboplatin and nab-paclitaxel. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with carboplatin and paclitaxel. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with cisplatin and pemetrexed. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with cisplatin and gemcitabine. In some embodiments, anti-human OX40 agonistic antibodies can be administered in combination with FOLFOX. In some embodiments, the anti-human OX40 agonistic antibody can be administered in combination with FOLFIRI.
上文记录的此类组合疗法涵盖组合施用(其中两种或更多种治疗剂包 含在同一配制剂或分开的配制剂中)，和分开施用，在该情况中，可以在施用别的治疗剂和/或佐剂之前，同时，和/或之后发生本发明抗体的施用。也可以与放射疗法组合使用本发明抗体。Such combination therapies documented above encompass combined administration (wherein two or more therapeutic agents are contained in the same formulation or separate formulations), and separate administration, in which case the administration of another therapeutic agent And/or the adjuvant before, at the same time, and/or after the administration of the antibody of the invention occurs. The antibodies of the present invention can also be used in combination with radiation therapy.
可以通过任何合适的手段(包括胃肠外，肺内，和鼻内，及若期望用于局部治疗的话，损伤内施用)来施用本发明抗体(和任何别的治疗剂)。胃肠外输注包括肌肉内，静脉内，动脉内，腹膜内，或皮下施用。部分根据施用是短暂的还是长期的，剂量给药可以通过任何合适的路径(例如通过注射，诸如静脉内或皮下注射)来进行。本文中涵盖各种剂量给药日程表，包括但不限于单次施用或在多个时间点里的多次施用，推注施用，和脉冲输注。The antibodies of the present invention (and any other therapeutic agents) can be administered by any suitable means (including parenteral, intrapulmonary, and intranasal, and if desired for local treatment, intralesional administration). Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Depending in part on whether the administration is short-lived or long-term, the dosing can be carried out by any suitable route (for example, by injection, such as intravenous or subcutaneous injection). Various dosing schedules are encompassed herein, including but not limited to single administration or multiple administrations at multiple time points, bolus administration, and pulse infusion.
本发明抗体会以一种符合优秀的医学实践的方式配制，定剂量和施用。在此背景中考虑的因素包括所治疗的特定病症，所治疗的特定哺乳动物，患者个体的临床状况，病症的起因，药剂投递部位，施用方法，施用日程表及医学从业人员知道的其它因素。抗体无需但任选与一种或多种目前用于预防或治疗所讨论病症的药剂一起配制。此类其它药物的有效量取决于配制剂中存在的抗体的量，病症或治疗的类型，及上文所述其它因素。这些通常以本文所述相同的剂量和施用途径使用，或以约1-99％的本文所述剂量使用，或以凭经验/临床上确定为适宜的任何剂量和任何途径使用。The antibody of the present invention will be formulated, dosed and administered in a manner consistent with excellent medical practice. Factors considered in this context include the specific condition being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the condition, the location of drug delivery, the method of administration, the schedule of administration, and other factors known to the medical practitioner. The antibody need not be but is optionally formulated with one or more agents currently used to prevent or treat the condition in question. The effective amount of such other drugs depends on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors mentioned above. These are generally used at the same dosage and route of administration as described herein, or at about 1-99% of the dosage described herein, or at any dosage and any route determined empirically/clinically to be suitable.
为了预防或治疗疾病，本发明抗体(当单独或与一种或多种其它别的治疗剂组合使用时)的适宜剂量会取决于要治疗的疾病的类型，抗体的类型，疾病的严重性和病程，施用抗体是预防还是治疗目的，之前的疗法，患者的临床史和对抗体的响应，及主治医师的斟酌。抗体适合于在一次或一系列的治疗中施用于患者。根据疾病的类型和严重性，约1μg/kg至40mg/kg的抗体可以作为初始候选剂量施用于患者，无论是例如通过一次或多次分开的施用或者是通过连续输注。根据上文所述因素，一种典型的日剂量可以在约lμg/kg至100mg/kg或更多的范围中。对于几天或更长时间上的重复施用，根据状况，治疗通常会持续直至疾病症状发生期望的阻抑。此类剂量可间歇施用，例如每周或每三周(例如使得患者接受约2至约20剂，或例如约6剂抗体)。可以施用较高的初始加载剂，接着是较低的一或多剂。然而，其它剂量方案可能是有用的。此疗法的进展易于通过常规技术和测定法来监测。In order to prevent or treat diseases, the appropriate dose of the antibody of the present invention (when used alone or in combination with one or more other therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity of the disease and The course of the disease, whether the antibody is administered for prevention or treatment, previous therapies, the patient's clinical history and response to the antibody, and the consideration of the attending physician. The antibody is suitable for administration to a patient in one or a series of treatments. Depending on the type and severity of the disease, about 1 μg/kg to 40 mg/kg of antibody can be administered to the patient as an initial candidate dose, whether for example by one or more divided administrations or by continuous infusion. Based on the factors mentioned above, a typical daily dose may be in the range of about 1 μg/kg to 100 mg/kg or more. For repeated administrations over several days or longer, depending on the condition, the treatment will usually continue until the desired suppression of disease symptoms occurs. Such doses may be administered intermittently, for example every week or every three weeks (for example, so that the patient receives about 2 to about 20 doses, or for example about 6 doses of antibody). A higher initial loading agent can be applied, followed by one or more lower doses. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.
应当理解，可以使用本发明的免疫缀合物代替或补充抗OX40抗体来实施任何上述配制剂或治疗性方法。It should be understood that the immunoconjugates of the present invention can be used in place of or in addition to anti-OX40 antibodies to implement any of the aforementioned formulations or therapeutic methods.
实施例Example
实施例1.抗OX40抗体的制备和筛选Example 1. Preparation and screening of anti-OX40 antibodies
实施例1.1构建报告细胞系Jurkat/NF-κB-GFP+hOX40Example 1.1 Construction of reporter cell line Jurkat/NF-κB-GFP+hOX40
NF-κB-GFP报告基因慢病毒***(QIAGEN，CCS-013G)的启动子中有多个NF-κB转录因子结合元件，该启动子可以驱动GFP表达。根据试剂盒的说明书，用NF-κB-GFP报告基因慢病毒感染Jurkat细胞(ATCC，TIB-152)，并用嘌呤霉素(InvivoGen，ant-pr-1)筛选感染慢病毒的Jurkat细胞。加入10ng/ml TNFα蛋白(Sino Biological，10602-HNAE)刺激利用嘌呤霉素筛选到的细胞，24h后用流式细胞仪(BD，FACSAria III)分选表现GFP荧光的细胞亚群，获得Jurkat/NF-κB-GFP细胞。There are multiple NF-κB transcription factor binding elements in the promoter of the NF-κB-GFP reporter gene lentiviral system (QIAGEN, CCS-013G), which can drive the expression of GFP. According to the instructions of the kit, Jurkat cells (ATCC, TIB-152) were infected with NF-κB-GFP reporter lentivirus, and Jurkat cells infected with lentivirus were screened with puromycin (InvivoGen, ant-pr-1). 10ng/ml TNFα protein (Sino Biological, 10602-HNAE) was added to stimulate the cells selected with puromycin, and 24 hours later, the cell subsets that showed GFP fluorescence were sorted by flow cytometer (BD, FACSAria III) to obtain Jurkat/ NF-κB-GFP cells.
将人OX40 CDS(Sino Biological，HG10481-UT)构建至慢病毒核心质粒pCDH(System Biosciences)中，使用标准步骤用PEI(polyscience，24885-2)向293FT细胞(Thermo Fisher Scientific，R70007)中按1：1：1：1共转染所获得的慢病毒核心质粒和辅助质粒pPACKH1-GAG、pPACKH1-REV、pVSVG(均来自System Biosciences)，转染6h后换液，37℃培养于含10％胎牛血清(Biological Industries，04-001-1A)的DMEM培养基(Lifetechnologies，C11995500CP)中，继续培养48h收集含有人OX40慢病毒的上清。Construct human OX40 CDS (Sino Biological, HG10481-UT) into the lentiviral core plasmid pCDH (System Biosciences), and use standard procedures to transfer PEI (polyscience, 24885-2) to 293FT cells (Thermo Fisher Scientific, R70007) and press 1 :1:1:1: The lentiviral core plasmids and helper plasmids pPACKH1-GAG, pPACKH1-REV, and pVSVG (all from System Biosciences) obtained by co-transfection, were transfected for 6 hours and then the medium was changed, and cultured at 37°C containing 10% fetus In DMEM medium (Lifetechnologies, C11995500CP) of bovine serum (Biological Industries, 04-001-1A), the culture was continued for 48 hours to collect the supernatant containing human OX40 lentivirus.
本文所用阳性对照OX40L和HEL的DNA序列在金唯智公司进行全基因合成。对于OX40L和HEL纯化，将DNA序列***真核表达载体pFUSE(InvivoGen，pfuse-hg1fc1)中，使用标准步骤用PEI转染至293F细胞(Thermo Fisher Scientific，R79007)；利用Freestyle培养基(Lifetechnologies，12338026)在37℃摇床培养细胞。培养7天后收集上清液，并在AKTA***(GE)上使用Superdex TM200 Increase预装柱(GE，28-9909-44)纯化。 The DNA sequences of the positive controls OX40L and HEL used in this article were synthesized by Jinweizhi Company. For OX40L and HEL purification, insert the DNA sequence into the eukaryotic expression vector pFUSE (InvivoGen, pfuse-hg1fc1), and use standard procedures to transfect 293F cells with PEI (Thermo Fisher Scientific, R79007); use Freestyle medium (Lifetechnologies, 12338026) ) Culture the cells on a shaker at 37°C. After 7 days of culture, the supernatant was collected and purified on the AKTA system (GE) using a Superdex TM 200 Increase prepacked column (GE, 28-9909-44).
OX40信号转导可以激活NF-κB通路，如果OX40成功表达于Jurkat/NF-κB-GFP细胞膜表面，OX40L刺激将会诱导GFP的表达。将如上所述分选获得的构建好的Jurkat/NF-κB-GFP细胞用上述获得的人OX40慢病毒上清感染，16h后加入100nM上述获得的OX40L蛋白(作为阳性对照)和HEL(作为阴性对照)进行刺激，24h后用流式细胞仪单细胞分选GFP荧光最强的细胞至96孔板中。OX40 signal transduction can activate the NF-κB pathway. If OX40 is successfully expressed on the surface of the Jurkat/NF-κB-GFP cell membrane, OX40L stimulation will induce the expression of GFP. The constructed Jurkat/NF-κB-GFP cells sorted as described above were infected with the human OX40 lentiviral supernatant obtained above. After 16 hours, 100nM of the OX40L protein obtained above (as a positive control) and HEL (as a negative control) were added. Control) was stimulated, and 24 hours later, the cells with the strongest GFP fluorescence were sorted into a 96-well plate using a flow cytometer for single cell sorting.
将上述96孔板中的细胞在含有10％胎牛血清(Biological Industries，04-001-1A)的RPMI 1640培养基(Lifetechnologies，C11875500CP)中培养2周，待单细胞长起来后分别加入100nM上述获得三聚化OX40L蛋白(作为阳性对照)和HEL(作为阴性对照)，挑选OX40L强阳性、HEL阴性的单克隆作为Jurkat/NF-κB-GFP+hOX40(简称NF-κB-GFP+hOX40)细胞，用于后续实验。结果如图1，OX40L刺激后Jurkat/NF-κB-GFP+hOX40单克隆GFP阳性率为70.7％，HEL刺激无激活。The cells in the above 96-well plate were cultured in RPMI 1640 medium (Lifetechnologies, C11875500CP) containing 10% fetal bovine serum (Biological Industries, 04-001-1A) for 2 weeks, and 100 nM of the above was added after the single cells grew up. Obtain trimerized OX40L protein (as a positive control) and HEL (as a negative control), and select OX40L strongly positive and HEL negative monoclonal as Jurkat/NF-κB-GFP+hOX40 (referred to as NF-κB-GFP+hOX40) cells For follow-up experiments. The results are shown in Figure 1. After OX40L stimulation, the positive rate of Jurkat/NF-κB-GFP+hOX40 monoclonal GFP was 70.7%, and there was no activation after HEL stimulation.
实施例1.2从噬菌体展示抗体库中筛选抗OX40抗体Example 1.2 Screening of anti-OX40 antibodies from the phage display antibody library
从天津市血液中心及上海秒通生物公司购买30例健康成人外周血，利用ficoll分离液(灏洋，LDS1075)离心获取PBMC，离心条件：20℃，2000rpm，升5降0模式30分钟。使用如上所述获得的PBMC构建人天然抗体库，库的构建为常规方法(phage display，Tim Clackson and Henry B.Lowman)。将获得抗体重链和轻链可变区随机组合以单链抗体scFv的形式展示于噬菌体衣壳蛋白pIII的N端，获得噬菌体展示抗体库，库容达到10 10。 The peripheral blood of 30 healthy adults was purchased from Tianjin Blood Center and Shanghai Miaotong Biological Company, and PBMCs were obtained by centrifugation with ficoll separation solution (Haoyang, LDS1075). Centrifugation conditions: 20°C, 2000rpm, up 5 down 0 mode for 30 minutes. The PBMC obtained as described above was used to construct a human natural antibody library, and the library was constructed by a conventional method (phage display, Tim Clackson and Henry B. Lowman). The obtained antibody heavy chain and light chain variable regions are randomly combined and displayed on the N-terminus of phage capsid protein pIII in the form of single-chain antibody scFv to obtain a phage display antibody library with a storage capacity of 10 10 .
噬菌体筛选为常规技术(Phage Display:A Laboratory Manual，Carlos F Barbas III)。首先，将生物素化OX40蛋白(acrobiosystems)与如上所述获得的噬菌体展示抗体库室温孵育2h。孵育结束后直接加入150ul链霉亲和素磁珠Dynabeads M280(Lifetechnologies，11205D)，在室温混匀仪上孵育30min。用0.05％PBS-tween(PBS：Lifetechnologies，70011044；Tween：Sigma-Aldrich，9005-64-5)洗去不与抗原结合的噬菌体，最后用0.2M甘氨酸-盐酸(PH2.2)洗脱与抗原结合的噬菌体。应用洗脱的噬菌体感染大肠杆菌XL1-blue(Agilent，200236)，加入辅助噬菌体VCSM13(Agilent，200251)扩增后用于下一轮筛选。共进行三轮筛选。三轮后，富集与CD40结合的抗体噬菌体，筛选结果如表1。Phage screening is a conventional technology (Phage Display: A Laboratory Manual, Carlos F Barbas III). First, the biotinylated OX40 protein (acrobiosystems) was incubated with the phage display antibody library obtained as described above for 2 hours at room temperature. After the incubation, add 150ul streptavidin magnetic beads Dynabeads M280 (Lifetechnologies, 11205D) directly, and incubate on a mixer at room temperature for 30 min. Use 0.05% PBS-tween (PBS: Lifetechnologies, 70011044; Tween: Sigma-Aldrich, 9005-64-5) to wash away the phage that does not bind to the antigen, and finally elution with the antigen with 0.2M glycine-hydrochloric acid (PH2.2) Bacteriophage conjugated. The eluted phage was used to infect E. coli XL1-blue (Agilent, 200236), and the helper phage VCSM13 (Agilent, 200251) was added for amplification and used in the next round of screening. A total of three rounds of screening were carried out. After three rounds, the antibody phages that bind to CD40 were enriched, and the screening results are shown in Table 1.
表1利用噬菌体展示技术从人天然抗体库中筛选抗OX40抗体Table 1 Screening of anti-OX40 antibodies from human natural antibody library using phage display technology
Figure PCTCN2020140259-appb-000020
Figure PCTCN2020140259-appb-000020
为了初步评估筛选到的抗OX40抗体的阳性率，我们分别挑取第二、三轮22个噬菌体单克隆，进行噬菌体ELISA分析。噬菌体ELISA为常规技术，具体如下：从深孔板中培养的噬菌体单克隆中各挑取第二、三轮22个噬菌体单克隆，在37℃300rpm下振摇培养至OD＝0.5～0.8，加入辅助噬菌体VCSM13扩增后30℃过夜诱导噬菌体，用于抗体表达。在ELISA板(Corning，3690)上包被1μg/ml的人源OX40(Acrobiosystems，OX0-H5224)，4℃过夜孵育。向过夜孵育后的ELISA板上加入过夜诱导的噬菌体上清，室温孵育1h，0.05％PBST洗8次。利用BSA(Solarbio，A8020-100)作为阴性对照。加入HRP偶联的抗-M13(GE，27-9421-01，M13为噬菌体衣壳蛋白)检测结合的抗体噬菌体。将OX40结合信号是BSA对照的3倍以上定义为阳性克隆。结果如图2，其中第二轮筛选、第三轮筛 选的阳性率分别为4.5％、40.9％。In order to preliminarily evaluate the positive rate of the anti-OX40 antibodies screened, we picked 22 phage monoclonals in the second and third rounds respectively, and performed phage ELISA analysis. Phage ELISA is a conventional technique, and the details are as follows: pick the second and third rounds of 22 phage single clones from the phage single clones cultured in the deep-well plate, shake culture at 37℃ 300rpm to OD=0.5～0.8, add The helper phage VCSM13 was amplified at 30°C overnight to induce phage for antibody expression. An ELISA plate (Corning, 3690) was coated with 1 μg/ml of human OX40 (Acrobiosystems, OX0-H5224), and incubated overnight at 4°C. The overnight induced phage supernatant was added to the ELISA plate after overnight incubation, incubated for 1 h at room temperature, and washed 8 times with 0.05% PBST. BSA (Solarbio, A8020-100) was used as a negative control. HRP-conjugated anti-M13 (GE, 27-9421-01, M13 is phage capsid protein) was added to detect the bound antibody phage. A positive clone was defined as the OX40 binding signal more than 3 times that of the BSA control. The results are shown in Figure 2. The positive rates of the second and third rounds of screening were 4.5% and 40.9%, respectively.
实施例1.3共刺激受体OX40的激动剂抗体的筛选Example 1.3 Screening of agonist antibodies for costimulatory receptor OX40
首先，我们将从第三轮噬菌体展示富集到的抗体亚克隆到分泌型慢病毒载体pCDH中，同时将N27 ScFv克隆到分泌型慢病毒载体pCDH中作为阴性对照。向293FT细胞中按1：1：1：1共转染慢病毒核心质粒和辅助质粒pPACKH1-GAG、pPACKH1-REV、pVSVG，转染6h后换液，37℃培养于含10％胎牛血清(Biological Industries，04-001-1A)的DMEM培养基中，继续培养48h收集含有抗OX40抗体慢病毒文库的上清；用50000pg所获得的抗OX40抗体的慢病毒文库感染HEK293细胞，感染16h后，离心去除含有慢病毒的培养基，添加新鲜培养基，利用流式分选技术将感染的HEK293细胞分选到96孔板中，使每个孔仅含有1个感染HEK293细胞，培养细胞3周，待细胞达到一定数量后取上清。上清中加入如上所述获得的Jurkat/NF-κB-GFP+hOX40报告细胞3×10 5个，同时加入2.5μg/ml山羊抗人Fc(SouthernBiotech，SBA-2048-01)的二抗，交联细胞分泌的抗体以增强激动剂抗体的激活强度；培养24h后测试细胞上清的活性，阳性抗体的检测结果如图3；从细胞上清活性为阳性的HEK293细胞中提取抗体基因，构建至pFUSE载体进行测序，获得阳性抗体11-1、2-1和2-2-1的序列。 First, we subcloned the antibodies enriched from the third round of phage display into the secreted lentiviral vector pCDH, and cloned N27 ScFv into the secreted lentiviral vector pCDH as a negative control. The lentiviral core plasmid and helper plasmids pPACKH1-GAG, pPACKH1-REV, and pVSVG were co-transfected into 293FT cells at a ratio of 1:1:1:1. After 6 hours of transfection, the medium was changed and cultured at 37°C containing 10% fetal bovine serum ( In the DMEM medium of Biological Industries, 04-001-1A), continue to culture for 48 hours to collect the supernatant containing the anti-OX40 antibody lentiviral library; infect HEK293 cells with 50,000 pg of the anti-OX40 antibody lentiviral library, and after 16 hours of infection, Centrifuge to remove the medium containing the lentivirus, add fresh medium, and sort the infected HEK293 cells into a 96-well plate using flow sorting technology, so that each well contains only one infected HEK293 cell, and culture the cells for 3 weeks. After the cells reach a certain number, take the supernatant. The supernatant was added with 3×10 5 Jurkat/NF-κB-GFP+hOX40 reporter cells obtained as described above, and 2.5μg/ml goat anti-human Fc (SouthernBiotech, SBA-2048-01) secondary antibody was added at the same time. Combine the antibodies secreted by the cells to enhance the activation intensity of the agonist antibody; test the activity of the cell supernatant after 24 hours of culture, and the detection result of the positive antibody is shown in Figure 3. The pFUSE vector was sequenced, and the sequences of the positive antibodies 11-1, 2-1 and 2-2-1 were obtained.
实施例1.4抗OX40抗体的表达和纯化Example 1.4 Expression and purification of anti-OX40 antibody
根据筛选出的抗OX40抗体的序列，合成抗OX40抗体的重链和轻链编码核苷酸序列(各自的恒定区和可变区编码核苷酸序列参见SEQ ID NO:49-60)(金唯智公司)，将其分别克隆至载体pFUSE中，用PEI将分别含有重链和轻链的质粒二者按1：1瞬时共转染至293F悬浮细胞，以表达全长抗体。培养1周后在AKTA***上使用Superdex TM200 Increase预装柱纯化。 According to the sequence of the anti-OX40 antibody screened out, the heavy chain and light chain coding nucleotide sequences of the anti-OX40 antibody were synthesized (see SEQ ID NO:49-60 for the respective constant region and variable region coding nucleotide sequences) (gold Weizhi Company), cloned them into the vector pFUSE, and transiently co-transfected the plasmids containing the heavy chain and the light chain into 293F suspension cells with PEI at a ratio of 1:1 to express the full-length antibody. After 1 week of cultivation, the AKTA system was used for purification with Superdex TM 200 Increase pre-packed column.
SDS-PAGE法鉴定纯化抗体，应用分子排阻预装柱Superdex 200分析抗体的聚集。结果如图4，其中2-2-1的洗脱峰形对称且洗脱时的保留时间与单个分子量的单克隆抗体保持一致，同时抗体中存在少量聚集体。SDS-PAGE method was used to identify and purified antibodies, and the molecular exclusion prepacked column Superdex 200 was used to analyze the aggregation of antibodies. The result is shown in Figure 4, where the elution peak shape of 2-2-1 is symmetrical and the retention time during elution is consistent with that of a single molecular weight monoclonal antibody, and there are a small amount of aggregates in the antibody.
实施例2.抗OX40抗体的结合性质Example 2. Binding properties of anti-OX40 antibodies
实施例2.1抗OX40抗体与OX40蛋白的结合动力学Example 2.1 The binding kinetics of anti-OX40 antibody and OX40 protein
Biacore T200(GE Healthcare)用于检测抗OX40抗体的亲和力。将抗体以10μL/min流过Protein A芯片，捕获在芯片上。用Running buffer(HBS-EP+，GE)梯度稀释待测抗原人OX40重组蛋白(Acrobiosystems，CD0-H5228)，浓度设置为2μM、1μM、500nM、250nM、125nM、62.5nM、31.25nM。将上述不同浓度OX40按30μL/min流速流过捕获有抗体 的芯片，结合120s，然后芯片以30μL/min的流速流过Running buffer，240s，抗原逐渐从捕获抗体的芯片上解离。数据处理使用Biacore T200仪器配套软件BIAevaluation software S200进行，计算结合常数(Ka)、解离常数(Kd)和平衡解离常数(KD)。结果如表2所示。Biacore T200 (GE Healthcare) is used to detect the affinity of anti-OX40 antibodies. The antibody was flowed through the Protein A chip at 10 μL/min and captured on the chip. Use Running buffer (HBS-EP+, GE) to dilute the human OX40 recombinant protein (Acrobiosystems, CD0-H5228) of the antigen to be tested. The concentration is set to 2μM, 1μM, 500nM, 250nM, 125nM, 62.5nM, 31.25nM. The above-mentioned different concentrations of OX40 were flowed through the antibody-captured chip at a flow rate of 30μL/min and bound for 120s, and then the chip was flowed through the Running buffer at a flow rate of 30μL/min for 240s, and the antigen gradually dissociated from the antibody-captured chip. Data processing was performed using BIAevaluation software S200, the supporting software of the Biacore T200 instrument, and the binding constant (Ka), dissociation constant (Kd) and equilibrium dissociation constant (KD) were calculated. The results are shown in Table 2.
表2应用表面等离子共振技术平台测量抗OX40抗体与人OX40的亲和力Table 2 Application of surface plasmon resonance technology platform to measure the affinity of anti-OX40 antibody and human OX40
Figure PCTCN2020140259-appb-000021
Figure PCTCN2020140259-appb-000021
实施例2.2抗OX40抗体阻断OX40L蛋白与OX40蛋白结合Example 2.2 Anti-OX40 antibody blocks the binding of OX40L protein to OX40 protein
用ELISA评价抗OX40抗体对OX40L与OX40结合的作用。首先，使用蛋白生物素化试剂盒(GeneCopoeia，BI001)，根据说明书生物素化OX40L。在ELISA板上包被1μg/ml的人源OX40，4℃过夜孵育。加入如实施例1.4获得的PBS稀释至不同浓度的抗OX40抗体，HEL为对照抗体，室温孵育1h后，加入2.5μg/ml生物素化的OX40L，室温孵育30min后，0.05％PBS-Tween洗8次。加入Streptavidin-HRP使用酶标仪(SpectraMax i3x)读取OD405数值，检测结合OX40L与OX40的结合量。结果，所测试的抗OX40抗体阻断OX40与OX40L的结合。ELISA was used to evaluate the effect of anti-OX40 antibody on the binding of OX40L and OX40. First, use the protein biotinylation kit (GeneCopoeia, BI001) to biotinylate OX40L according to the instructions. Coated 1μg/ml human OX40 on the ELISA plate and incubate at 4°C overnight. Add the anti-OX40 antibody diluted to different concentrations with PBS as obtained in Example 1.4. HEL is the control antibody. After incubating for 1 hour at room temperature, add 2.5μg/ml biotinylated OX40L. After incubating at room temperature for 30 minutes, wash with 0.05% PBS-Tween for 8 Times. Add Streptavidin-HRP and use a microplate reader (SpectraMax i3x) to read the OD405 value to detect the binding amount of OX40L and OX40. As a result, the tested anti-OX40 antibody blocked the binding of OX40 to OX40L.
实施例3.抗OX40抗体的体内抗肿瘤效力Example 3. In vivo anti-tumor efficacy of anti-OX40 antibodies
5只人源化OX40敲入C57BL/6小鼠购自上海南方模式生物研究中心。隔离5天后，用100μl PBS中的8×10 5个MC38肿瘤细胞(由南开大学张宏恺教授提供)背部皮下接种每只小鼠。当肿瘤大小达到70-100mm 3时，通过以100μl PBS中10mg/kg和q3dx3(每三天治疗一次，共治疗3次)向小鼠腹膜内施用抗OX40抗体(n＝5)而开始抗OX40抗体治疗，或者IgG1对照(n＝5)。每周两次测量肿瘤尺寸和小鼠体重，使用卡尺在两个方向上测量肿瘤尺寸，并且使用以下公式以mm3表述体积：V＝0.5a×b 2，其中a 和b分别是肿瘤的长直径和短直径。当对照组的肿瘤大小达到1000mm 3时，处死小鼠。 Five humanized OX40 knock-in C57BL/6 mice were purchased from Shanghai Southern Model Biology Research Center. After 5 days of isolation, each mouse was inoculated subcutaneously on the back with 8×10 5 MC38 tumor cells (provided by Professor Zhang Hongkai of Nankai University) in 100 μl PBS. When the tumor size reached 70-100mm 3 , anti-OX40 was started by intraperitoneal administration of anti-OX40 antibody (n=5) to mice at 10mg/kg and q3dx3 in 100μl PBS (one treatment every three days for a total of three treatments) Antibody treatment, or IgG1 control (n=5). Measure the tumor size and mouse body weight twice a week, use a caliper to measure the tumor size in two directions, and use the following formula to express the volume in mm3: V=0.5a×b 2 , where a and b are the long diameters of the tumor, respectively And short diameter. When the tumor size of the control group reached 1000 mm 3 , the mice were sacrificed.
如图5所示，所测试的抗OX40抗体相对于IgG1对照显著抑制肿瘤生长。OX40激动剂抗体在OX40人源化小鼠结肠癌模型中抑制肿瘤生长。将0.8×10 5小鼠结肠癌细胞MC38移植到OX40人源化C57BL/6小鼠背部皮下，待肿瘤体积到70-100mm 3时腹腔注射10mg/kg OX40激动剂抗体2-2-1，11-1和IgG对照抗体(n＝5)，每三天治疗一次，共治疗3次。 As shown in Figure 5, the tested anti-OX40 antibodies significantly inhibited tumor growth relative to the IgG1 control. The OX40 agonist antibody inhibited tumor growth in the OX40 humanized mouse colon cancer model. Transplant 0.8×10 5 mouse colon cancer cell MC38 into the back of OX40 humanized C57BL/6 mouse subcutaneously, and when the tumor volume reaches 70-100mm 3 , 10mg/kg OX40 agonist antibody 2-2-1, 11 is injected intraperitoneally -1 and IgG control antibody (n=5), treated once every three days for a total of 3 treatments.
实施例4.抗OX40抗体的T细胞调节效果Example 4. T cell regulation effect of anti-OX40 antibody
剥离小鼠的脾脏和肿瘤，脾脏直接通过研磨，200目尼龙网过滤制备成单细胞悬液，肿瘤组织切成小块后置于2ml酶混合液中(每ml酶含胶原蛋白酶1500U,透明质酸酶1000U和DNA酶2.5ul)，在培养箱中150rpm，37℃振荡1h。用200目尼龙网过滤并研磨没有消化完全的组织块制备成单细胞悬液，350g离心7分钟，用2ml红细胞裂解液重悬细胞，冰上孵育15分钟，用PBS洗2遍并计数。The spleen and tumor of the mouse were stripped, and the spleen was directly ground and filtered with a 200-mesh nylon mesh to prepare a single cell suspension. The tumor tissue was cut into small pieces and placed in 2ml enzyme mixture (each ml enzyme contains 1500U collagenase, hyaluronic acid) Acidase 1000U and DNase 2.5ul), shake in an incubator at 150rpm and 37°C for 1h. Filter and grind the incompletely digested tissue pieces with a 200 mesh nylon mesh to prepare a single cell suspension, centrifuge at 350g for 7 minutes, resuspend the cells with 2ml of red blood cell lysate, incubate on ice for 15 minutes, wash twice with PBS and count.
取1×10 7细胞，用兔抗鼠CD16/CD32抗体冰上封闭15分钟，PBS洗一遍。取1×10 6细胞重悬在100ul PBS中，分别加入CD45，CD3，CD4和CD45，CD3，CD8抗体各1ul，4℃避光染色30分钟，PBS洗两遍后流式细胞仪分析CD4 +T细胞和CD8 +T细胞比例。CD45+CD3+CD4+定义为CD4 +T细胞，CD45+CD3+CD8+定义为CD8 +T细胞。 Take 1×10 7 cells, block them on ice with rabbit anti-mouse CD16/CD32 antibody for 15 minutes, and wash them with PBS. Resuspend 1×10 6 cells in 100ul PBS, add 1ul each of CD45, CD3, CD4 and CD45, CD3 and CD8 antibodies, stain for 30 minutes at 4℃ in the dark, wash twice with PBS, and analyze CD4 + by flow cytometry The ratio of T cells to CD8 + T cells. CD45+CD3+CD4+ is defined as CD4 + T cells, and CD45+CD3+CD8+ is defined as CD8 + T cells.
如图6所示，所测试的抗OX40抗体相对于对照在脾和肿瘤中明显上调辅助CD4+T细胞和细胞毒性CD8+T细胞。剥离小鼠的脾脏和肿瘤并制备单细胞悬液，CD45、CD3、CD4和CD8抗体染色，流式细胞仪分析CD4+和CD8+T细胞的比例，CD45+CD3+CD4+定义为CD4+T细胞，CD45+CD3+CD8+定义为CD8+T细胞。As shown in Figure 6, the tested anti-OX40 antibodies significantly up-regulated helper CD4+ T cells and cytotoxic CD8+ T cells in the spleen and tumor relative to the control. The spleen and tumor of the mouse were stripped and a single cell suspension was prepared. The CD45, CD3, CD4 and CD8 antibodies were stained, and the ratio of CD4+ and CD8+ T cells was analyzed by flow cytometry. CD45+CD3+CD4+ is defined as CD4+ T cells. CD45+CD3+CD8+ is defined as CD8+ T cells.
实施例5.交联和可溶性抗OX40抗体的剂量依赖性作用Example 5. Dose-dependent effects of cross-linking and soluble anti-OX40 antibodies
抗OX40抗体按照其激动方式不同可分两类，第一类OX40激动活性不依赖Fc受体的交联；另一类需要Fc受体交联才具有CD40激动活性。肿瘤组织及周围引流***有更多肿瘤相关炎性细胞的存在,Fc受体FcγR2b在肿瘤细胞周围有更多聚集。因此，“交联抗体”激动剂就具有了更高的组织选择性,在肿瘤微环境中抗体才能产生明显的激动作用,而在身体正常组织部位,作用能力会保持在低水平,这样就能提高治疗的安全窗。Anti-OX40 antibodies can be divided into two types according to their different ways of agonizing. The first type of OX40 agonistic activity does not depend on the crosslinking of Fc receptors; the other type requires Fc receptor crosslinking to have CD40 agonistic activity. There are more tumor-associated inflammatory cells in tumor tissues and surrounding draining lymph nodes, and Fc receptor FcγR2b gathers more around tumor cells. Therefore, the "cross-linked antibody" agonist has higher tissue selectivity. The antibody can produce obvious agonistic effects in the tumor microenvironment, while in the normal tissues of the body, the ability to act will be kept at a low level, so that it can be Improve the safety window of treatment.
分别取3×10 5个/管如实施例1.1中获得的NF-κB-GFP+hOX40报告细胞，分别加入如实施例1.4中获得的稀释至不同浓度的抗OX抗体，利用 HEL抗体做阴性对照；为了交联，各组同时加入2.5μg/ml浓度的二抗，即山羊抗人Fc。在含有10％胎牛血清(Biological Industries，04-001-1A)的RPMI 1640培养基(Lifetechnologies，C11875500CP)中37℃共同培养24h后，PBS洗三次，应用流式细胞仪进行分析。获得的数据使用GraphPad Prism 6.0拟合曲线并计算EC50。结果如图7所示，其中2-2-1对OX40的激活依赖二抗交联，以交联形式激活NF-κB-GFP+hOX40报告细胞系(图7)。 Take 3×10 5 cells/tube of the NF-κB-GFP+hOX40 reporter cells obtained in Example 1.1, respectively, add the anti-OX antibodies diluted to different concentrations as obtained in Example 1.4, and use the HEL antibody as a negative control ; For cross-linking, each group added a 2.5μg/ml secondary antibody at the same time, namely goat anti-human Fc. After co-cultivation in RPMI 1640 medium (Lifetechnologies, C11875500CP) containing 10% fetal bovine serum (Biological Industries, 04-001-1A) at 37°C for 24 hours, the cells were washed three times with PBS and analyzed by flow cytometry. The obtained data was fitted with GraphPad Prism 6.0 and the EC50 was calculated. The results are shown in Figure 7, where the activation of OX40 by 2-2-1 depends on the cross-linking of the secondary antibody, which activates the NF-κB-GFP+hOX40 reporter cell line in a cross-linked form (Figure 7).
虽然上文已经结合优选实施方式描述了本发明的原理，但应当清楚地理解，该描述仅通过示例的方式进行，而非作为对本发明的范围的限制。Although the principle of the present invention has been described above in conjunction with the preferred embodiments, it should be clearly understood that the description is only made by way of example and not as a limitation to the scope of the present invention.
序列表Sequence Listing
Figure PCTCN2020140259-appb-000022
Figure PCTCN2020140259-appb-000022
Figure PCTCN2020140259-appb-000023
Figure PCTCN2020140259-appb-000023
Figure PCTCN2020140259-appb-000024
Figure PCTCN2020140259-appb-000024
Figure PCTCN2020140259-appb-000025
Figure PCTCN2020140259-appb-000025
Figure PCTCN2020140259-appb-000026
Figure PCTCN2020140259-appb-000026
Figure PCTCN2020140259-appb-000027
Figure PCTCN2020140259-appb-000027
Figure PCTCN2020140259-appb-000028
Figure PCTCN2020140259-appb-000028
Figure PCTCN2020140259-appb-000029
Figure PCTCN2020140259-appb-000029
Figure PCTCN2020140259-appb-000030
Figure PCTCN2020140259-appb-000030
Figure PCTCN2020140259-appb-000031
Figure PCTCN2020140259-appb-000031

Claims (24)

  1. 一种分离的抗OX40抗体或其抗原结合片段，其包含：An isolated anti-OX40 antibody or antigen-binding fragment thereof, which comprises:
    (a)具有SEQ ID NO:1所示重链CDR1结构域，SEQ ID NO:2所示重链CDR2结构域，和SEQ ID NO:3所示重链CDR3结构域的重链可变区；和具有SEQ ID NO:9所示轻链CDR1结构域，SEQ ID NO:10所示轻链CDR2结构域，和SEQ ID NO:11所示轻链CDR3结构域的轻链可变区；(a) A heavy chain variable region having a heavy chain CDR1 domain shown in SEQ ID NO: 1, a heavy chain CDR2 domain shown in SEQ ID NO: 2, and a heavy chain CDR3 domain shown in SEQ ID NO: 3; And a light chain variable region having a light chain CDR1 domain shown in SEQ ID NO: 9, a light chain CDR2 domain shown in SEQ ID NO: 10, and a light chain CDR3 domain shown in SEQ ID NO: 11;
    (b)具有SEQ ID NO:17所示重链CDR1结构域，SEQ ID NO:18所示重链CDR2结构域，和SEQ ID NO:19所示重链CDR3结构域的重链可变区；和具有SEQ ID NO:25所示轻链CDR1结构域，SEQ ID NO:26所示轻链CDR2结构域，和SEQ ID NO:27所示轻链CDR3结构域的轻链可变区；或(b) A heavy chain variable region having a heavy chain CDR1 domain shown in SEQ ID NO: 17, a heavy chain CDR2 domain shown in SEQ ID NO: 18, and a heavy chain CDR3 domain shown in SEQ ID NO: 19; And the light chain CDR1 domain shown in SEQ ID NO: 25, the light chain CDR2 domain shown in SEQ ID NO: 26, and the light chain variable region of the light chain CDR3 domain shown in SEQ ID NO: 27; or
    (c)具有SEQ ID NO:33所示重链CDR1结构域，SEQ ID NO:34所示重链CDR2结构域，和SEQ ID NO:35所示重链CDR3结构域的重链可变区；和具有SEQ ID NO:41所示轻链CDR1结构域，SEQ ID NO:42所示轻链CDR2结构域，和SEQ ID NO:43所示轻链CDR3结构域的轻链可变区。(c) A heavy chain variable region having a heavy chain CDR1 domain shown in SEQ ID NO: 33, a heavy chain CDR2 domain shown in SEQ ID NO: 34, and a heavy chain CDR3 domain shown in SEQ ID NO: 35; And a light chain variable region having a light chain CDR1 domain shown in SEQ ID NO: 41, a light chain CDR2 domain shown in SEQ ID NO: 42, and a light chain CDR3 domain shown in SEQ ID NO: 43.
  2. 根据权利要求1所述的抗OX40抗体或其抗原结合片段，其包含：The anti-OX40 antibody or antigen-binding fragment thereof according to claim 1, which comprises:
    (a)SEQ ID NO:4所示重链可变区，或与SEQ ID NO:4所示序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的重链可变区；和SEQ ID NO:12所示轻链可变区，或与SEQ ID NO:12所示序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的轻链可变区；(a) The heavy chain variable region shown in SEQ ID NO: 4, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% with the sequence shown in SEQ ID NO: 4 %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical heavy chain variable regions; and The light chain variable region shown in SEQ ID NO: 12, or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88 with the sequence shown in SEQ ID NO: 12 %, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical light chain variable regions;
    (b)SEQ ID NO:20所示重链可变区，或与SEQ ID NO:20所示序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的重链可变区；和SEQ ID NO:28所示轻链可变区，或与SEQ ID NO:28所示序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的轻链可变区；或(b) The heavy chain variable region shown in SEQ ID NO: 20, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% with the sequence shown in SEQ ID NO: 20 %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical heavy chain variable regions; and The light chain variable region shown in SEQ ID NO: 28, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88 with the sequence shown in SEQ ID NO: 28 %, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical light chain variable regions; or
    (c)SEQ ID NO:36所示重链可变区，或与SEQ ID NO:36所示序列 具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的重链可变区；和SEQ ID NO:44所示轻链可变区，或与SEQ ID NO:44所示序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的轻链可变区。(c) The heavy chain variable region shown in SEQ ID NO: 36, or the sequence shown in SEQ ID NO: 36 has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87 %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical heavy chain variable regions; and The light chain variable region shown in SEQ ID NO: 44, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88 with the sequence shown in SEQ ID NO: 44 %, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical light chain variable regions.
  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，其进一步包含重链恒定区和轻链恒定区；The anti-OX40 antibody or antigen-binding fragment thereof according to claim 1 or 2, which further comprises a heavy chain constant region and a light chain constant region;
    优选地，所述重链恒定区为SEQ ID NO:5或21所示重链恒定区，或与SEQ ID NO:5所示序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的重链恒定区；和/或Preferably, the heavy chain constant region is the heavy chain constant region shown in SEQ ID NO: 5 or 21, or is at least 80%, 81%, 82%, 83%, 84% with the sequence shown in SEQ ID NO: 5 , 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity Constant region of the heavy chain; and/or
    优选地，所述轻链恒定区为SEQ ID NO:13所示轻链恒定区，或与SEQ ID NO:13所示序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的轻链恒定区。Preferably, the light chain constant region is the light chain constant region shown in SEQ ID NO: 13, or is at least 80%, 81%, 82%, 83%, 84%, 85% with the sequence shown in SEQ ID NO: 13 %, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% Chain constant region.
  4. 根据权利要求1-3中任一项所述的抗OX40抗体或其抗原结合片段，其进一步包含连接到所述重链可变区的重链信号肽和/或连接到所述轻链可变区的重链信号肽；The anti-OX40 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, which further comprises a heavy chain signal peptide connected to the heavy chain variable region and/or connected to the light chain variable region. Heavy chain signal peptide in the region;
    优选地，所述重链信号肽为SEQ ID NO:6所示重链信号肽，或与SEQ ID NO:6所示序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的重链信号肽；和/或Preferably, the heavy chain signal peptide is the heavy chain signal peptide shown in SEQ ID NO: 6, or is at least 80%, 81%, 82%, 83%, 84%, 85% with the sequence shown in SEQ ID NO: 6 %, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity weight Chain signal peptide; and/or
    优选地，所述轻链信号肽为SEQ ID NO:14所示轻链信号肽，或与SEQ ID NO:14所示序列具有至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的轻链信号肽。Preferably, the light chain signal peptide is the light chain signal peptide shown in SEQ ID NO: 14, or has at least 80%, 81%, 82%, 83%, 84%, 85% with the sequence shown in SEQ ID NO: 14. %, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% Chain signal peptide.
  5. 根据权利要求1-4中任一项所述的抗OX40抗体或其抗原结合片段，其为IgG抗体或其抗原结合片段，优选为IgG1抗体或其抗原结合片段。The anti-OX40 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, which is an IgG antibody or an antigen-binding fragment thereof, preferably an IgG1 antibody or an antigen-binding fragment thereof.
  6. 根据权利要求1-5中任一项所述的抗OX40抗体或其抗原结合片段，其为单克隆抗体或其抗原结合片段。The anti-OX40 antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, which is a monoclonal antibody or antigen-binding fragment thereof.
  7. 根据权利要求1-6中任一项所述的抗OX40抗体或其抗原结合片段，其中所述抗原结合片段为Fab、Fab'、F(ab')2、Fv、scFv或sdAb。The anti-OX40 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the antigen-binding fragment is Fab, Fab', F(ab')2, Fv, scFv or sdAb.
  8. 一种抗体-药物缀合物，其包含根据权利要求1-7中任一项所述的抗OX40抗体或其抗原结合片段和另外的治疗剂；优选地，所述抗OX40抗体 或其抗原结合片段与所述另外的治疗剂通过接头连接。An antibody-drug conjugate comprising the anti-OX40 antibody or antigen-binding fragment thereof according to any one of claims 1-7 and another therapeutic agent; preferably, the anti-OX40 antibody or antigen-binding fragment thereof The fragment and the additional therapeutic agent are connected by a linker.
  9. 一种核酸，其编码根据权利要求1-7中任一项所述的抗OX40抗体或其抗原结合片段。A nucleic acid encoding the anti-OX40 antibody or antigen-binding fragment thereof according to any one of claims 1-7.
  10. 根据权利要求9所述的核酸，其包含：The nucleic acid of claim 9 comprising:
    (a)SEQ ID NO:49所示重链可变区核苷酸编码序列和/或SEQ ID NO:51所示轻链可变区核苷酸编码序列；(a) The nucleotide coding sequence of the heavy chain variable region shown in SEQ ID NO: 49 and/or the nucleotide coding sequence of the light chain variable region shown in SEQ ID NO: 51;
    (b)SEQ ID NO:53所示重链可变区核苷酸编码序列和/或SEQ ID NO:55所示轻链可变区核苷酸编码序列；或(b) The nucleotide coding sequence of the heavy chain variable region shown in SEQ ID NO: 53 and/or the nucleotide coding sequence of the light chain variable region shown in SEQ ID NO: 55; or
    (c)SEQ ID NO:57所示重链可变区核苷酸编码序列和/或SEQ ID NO:59所示轻链可变区核苷酸编码序列；(c) The nucleotide coding sequence of the heavy chain variable region shown in SEQ ID NO: 57 and/or the nucleotide coding sequence of the light chain variable region shown in SEQ ID NO: 59;
    优选地，所述核酸进一步包含SEQ ID NO:50或54所示重链恒定区核苷酸编码序列和/或SEQ ID NO:52所示轻链恒定区核苷酸编码序列。Preferably, the nucleic acid further comprises the heavy chain constant region nucleotide coding sequence shown in SEQ ID NO: 50 or 54 and/or the light chain constant region nucleotide coding sequence shown in SEQ ID NO: 52.
  11. 一种表达载体，其包含根据权利要求9或10所述的核酸。An expression vector comprising the nucleic acid according to claim 9 or 10.
  12. 一种宿主细胞，其包含根据权利要求9或10所述的核酸或根据权利要求11所述的表达载体。A host cell comprising the nucleic acid according to claim 9 or 10 or the expression vector according to claim 11.
  13. 一种用于产生根据权利要求1-7中任一项所述的抗OX40抗体或其抗原结合片段的方法，其包括在适合于抗体或其抗原结合片段表达的条件下培养根据权利要求12所述的宿主细胞，和从培养基中回收表达的抗体或其抗原结合片段。A method for producing the anti-OX40 antibody or antigen-binding fragment thereof according to any one of claims 1-7, which comprises culturing the antibody or antigen-binding fragment thereof under conditions suitable for expression of the antibody or antigen-binding fragment thereof according to claim 12. The host cell described above, and the expressed antibody or antigen-binding fragment thereof recovered from the culture medium.
  14. 一种药物组合物，其包含根据权利要求1-7中任一项所述的抗OX40抗体或其抗原结合片段，或根据权利要求8所述的抗体-药物缀合物，或根据权利要求9或10所述的核酸，或根据权利要求11所述的表达载体，及药学上可接受的载体。A pharmaceutical composition comprising the anti-OX40 antibody or antigen-binding fragment thereof according to any one of claims 1-7, or the antibody-drug conjugate according to claim 8, or according to claim 9. Or the nucleic acid of 10, or the expression vector of claim 11, and a pharmaceutically acceptable carrier.
  15. 根据权利要求1-7中任一项所述的抗OX40抗体或其抗原结合片段，或根据权利要求8所述的抗体-药物缀合物，或根据权利要求14所述的药物组合物，其用于治疗癌症。The anti-OX40 antibody or antigen-binding fragment thereof according to any one of claims 1-7, or the antibody-drug conjugate according to claim 8, or the pharmaceutical composition according to claim 14, which Used to treat cancer.
  16. 根据权利要求15所述的抗OX40抗体或其抗原结合片段或者抗体-药物缀合物或者药物组合物，其中所述癌症选自鳞状细胞癌(例如上皮鳞状细胞癌)、肺癌(包括小细胞肺癌、非小细胞肺癌、肺的腺癌、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、***、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、***癌、外阴癌、甲状腺癌、肝癌，***癌、***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、水肿(例如与脑瘤有关的)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。The anti-OX40 antibody or antigen-binding fragment thereof or antibody-drug conjugate or pharmaceutical composition according to claim 15, wherein the cancer is selected from the group consisting of squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small Cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, glioblastoma, cervical cancer Cancer, ovarian cancer, liver cancer, bladder cancer, urethral cancer, liver tumor, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid Cancer, liver cancer, anal cancer, penile cancer, melanoma, superficial spreading melanoma, malignant nevus melanoma, acral melanoma, nodular melanoma, multiple myeloma, and B cells Lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and post-transplant lymphoproliferative disorder (PTLD), and keloid , Edema (for example, related to brain tumors) and abnormal vascular proliferation related to Meigs syndrome, brain tumors and brain cancers, and head and neck cancers, and related metastases.
  17. 一种用于治疗癌症的方法，其包括向需要的受试者施用治疗有效量的根据权利要求1-7中任一项所述的抗OX40抗体或其抗原结合片段，或根据权利要求8所述的抗体-药物缀合物，或根据权利要求14所述的药物组合物，从而治疗所述癌症。A method for treating cancer, which comprises administering to a subject in need a therapeutically effective amount of the anti-OX40 antibody or antigen-binding fragment thereof according to any one of claims 1-7, or according to claim 8 The antibody-drug conjugate, or the pharmaceutical composition according to claim 14, thereby treating the cancer.
  18. 根据权利要求17所述的方法，其中所述癌症选自鳞状细胞癌(例如上皮鳞状细胞癌)、肺癌(包括小细胞肺癌、非小细胞肺癌、肺的腺癌、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、***、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、***癌、外阴癌、甲状腺癌、肝癌，***癌、***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、水肿(例如与脑瘤有关的)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。The method of claim 17, wherein the cancer is selected from the group consisting of squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung) ), peritoneal cancer, hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urethral cancer, liver tumor, breast cancer , Colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, melanoma, superficial spreading Melanoma, lentigines melanoma, acral melanoma, nodular melanoma, multiple myeloma and B-cell lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and post-transplant lymphoproliferative disorder (PTLD), as well as keloidosis, edema (for example, related to brain tumors), and Meigs Syndrome-related abnormal blood vessel proliferation, brain tumors and brain cancer, and head and neck cancer, and related metastases.
  19. 根据权利要求1-7中任一项所述的抗OX40抗体或其抗原结合片段，或根据权利要求8所述的抗体-药物缀合物，或根据权利要求14所述的药物组合物，在制备用于治疗癌症的药物中的用途。The anti-OX40 antibody or antigen-binding fragment thereof according to any one of claims 1-7, or the antibody-drug conjugate according to claim 8, or the pharmaceutical composition according to claim 14, in Use in the preparation of medicines for the treatment of cancer.
  20. 根据权利要求19所述的用途，其中所述癌症选自鳞状细胞癌(例如上皮鳞状细胞癌)、肺癌(包括小细胞肺癌、非小细胞肺癌、肺的腺癌、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、***、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、***癌、外阴癌、甲状腺癌、肝癌，***癌、***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、水肿(例如与脑瘤有关的)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。The use according to claim 19, wherein the cancer is selected from the group consisting of squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung) ), peritoneal cancer, hepatocellular carcinoma, gastric cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urethral cancer, liver tumor, breast cancer , Colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, melanoma, superficial spreading Melanoma, lentigines melanoma, acral melanoma, nodular melanoma, multiple myeloma and B-cell lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and post-transplant lymphoproliferative disorder (PTLD), as well as keloidosis, edema (for example, related to brain tumors), and Meigs Syndrome-related abnormal blood vessel proliferation, brain tumors and brain cancer, and head and neck cancer, and related metastases.
  21. 根据权利要求1-7中任一项所述的抗OX40抗体或其抗原结合片段，或根据权利要求8所述的抗体-药物缀合物，或根据权利要求14所述的药物组合物，其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。The anti-OX40 antibody or antigen-binding fragment thereof according to any one of claims 1-7, or the antibody-drug conjugate according to claim 8, or the pharmaceutical composition according to claim 14, which Used for one or more of the following: inhibiting Treg function (for example, inhibiting the suppressive function of Treg), killing OX40-expressing cells (for example, cells expressing high levels of OX40), improving effector T cell function and/or improving memory T Cell function, reduce tumor immunity, enhance T cell function and/or reduce cells expressing OX40.
  22. 根据权利要求1-7中任一项所述的抗OX40抗体或其抗原结合片段，或根据权利要求8所述的抗体-药物缀合物，或根据权利要求14所述的药物组合物，在制备用于治疗下述一项或多项的药物中的用途：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。The anti-OX40 antibody or antigen-binding fragment thereof according to any one of claims 1-7, or the antibody-drug conjugate according to claim 8, or the pharmaceutical composition according to claim 14, in Use in the preparation of drugs for treating one or more of the following: inhibiting Treg function (for example, inhibiting the suppressive function of Treg), killing OX40-expressing cells (for example, cells expressing high levels of OX40), and increasing effector T cells Function and/or improve memory T cell function, reduce tumor immunity, enhance T cell function and/or reduce cells expressing OX40.
  23. 一种药物组合，其包含根据权利要求1-7中任一项所述的抗OX40抗体或其抗原结合片段，或根据权利要求8所述的抗体-药物缀合物，或根据权利要求14所述的药物组合物，以及一种或多种另外的治疗剂。A pharmaceutical combination comprising the anti-OX40 antibody or antigen-binding fragment thereof according to any one of claims 1-7, or the antibody-drug conjugate according to claim 8, or the antibody-drug conjugate according to claim 14 The pharmaceutical composition described above, and one or more additional therapeutic agents.
  24. 一种试剂盒，其包括根据权利要求1-7中任一项所述的抗OX40抗体或其抗原结合片段，或根据权利要求8所述的抗体-药物缀合物，或根据权利要求14所述的药物组合物，优选其进一步包括给药装置。A kit comprising the anti-OX40 antibody or antigen-binding fragment thereof according to any one of claims 1-7, or the antibody-drug conjugate according to claim 8, or the antibody-drug conjugate according to claim 14 The aforementioned pharmaceutical composition preferably further comprises a drug delivery device.
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