WO2021228218A1 - 抗cd25抗体、其抗原结合片段及其医药用途 - Google Patents

抗cd25抗体、其抗原结合片段及其医药用途 Download PDF

Info

Publication number
WO2021228218A1
WO2021228218A1 PCT/CN2021/093791 CN2021093791W WO2021228218A1 WO 2021228218 A1 WO2021228218 A1 WO 2021228218A1 CN 2021093791 W CN2021093791 W CN 2021093791W WO 2021228218 A1 WO2021228218 A1 WO 2021228218A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
antibody
antigen
cancer
binding fragment
Prior art date
Application number
PCT/CN2021/093791
Other languages
English (en)
French (fr)
Inventor
陈思萌
张伟
姜福伟
廖成
Original Assignee
江苏恒瑞医药股份有限公司
上海盛迪医药有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 江苏恒瑞医药股份有限公司, 上海盛迪医药有限公司 filed Critical 江苏恒瑞医药股份有限公司
Priority to CN202180028356.4A priority Critical patent/CN115397860A/zh
Priority to EP21804918.7A priority patent/EP4151655A1/en
Priority to US17/998,418 priority patent/US20230174670A1/en
Priority to CA3182362A priority patent/CA3182362A1/en
Priority to JP2022567574A priority patent/JP2023525060A/ja
Publication of WO2021228218A1 publication Critical patent/WO2021228218A1/zh

Links

Images

Classifications

    • 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
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • 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
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • 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
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • 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
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present disclosure belongs to the field of biomedicine, especially the field of cancer immunotherapy, including methods for treating cancer (such as solid tumors), and involves the use of anti-CD25 antibodies or antigen-binding fragments thereof.
  • Tumor immunotherapy is a hot spot in the field of tumor therapy. At present, it mainly improves the number and activity of CD4 + and CD8 + T cells in tumors or suppresses the number and activity of suppressor immune cells in tumors, for example, suppressing regulatory T cells (Treg), bone marrow-derived Suppressor cells MDSC or tumor-associated macrophages.
  • Treg suppressing regulatory T cells
  • MDSC bone marrow-derived Suppressor cells
  • tumor-associated macrophages for example, suppressing regulatory T cells (Treg), bone marrow-derived Suppressor cells MDSC or tumor-associated macrophages.
  • the therapeutic strategy of reducing or eliminating Treg cells in tumors has been clinically validated.
  • CD25 is a type I membrane protein composed of 272 amino acids. Its expression on T cells is regulated by TCR signals, so it is often used as a sign of T cell activation.
  • IL-2 has an immune activating effect and is an immunomodulator for tumor treatment. It acts through the IL-2 receptor (IL-2R) on the cell surface.
  • IL-2R includes three subunits, IL-2R ⁇ (ie CD25), IL-2R ⁇ (ie CD122) and IL-2R ⁇ (ie CD132). Three subunits can form three receptor forms: the high binding capacity receptor contains all three subunits IL-2R ⁇ / ⁇ / ⁇ , the medium binding capacity receptor contains IL-2R ⁇ / ⁇ , and the low binding capacity receptor is IL -2R ⁇ .
  • IL-2R ⁇ (ie, CD25) alone has a low affinity for IL-2 (Kd is 10 -8 M), and does not transmit intracellular signals.
  • IL-2R ⁇ and IL-2R ⁇ are necessary for IL-2 to activate downstream signaling pathways.
  • the two receptor subunits form a heterodimer.
  • IL-2R ⁇ ie CD25
  • IL-2R ⁇ and IL-2R ⁇ form high-affinity receptors (Kd of 10 -11 M)
  • it can activate downstream JAK-STAT, PI3K-AKT and MAPK signaling pathways, thereby promoting T cells Proliferation and activity of lymphocytes such as NK cells.
  • Some existing anti-CD25 antibodies block or inhibit the binding of IL-2 to CD25, for example, see WO2004/045512, WO2006/108670, WO1993/011238, WO1990/007861 and WO2017/174331.
  • Basiliximab (basiliximab) and daclizumab (daclizumab, DAC) are IgG1 anti-human CD25 antibodies that inhibit the binding of IL-2 to CD25. Based on the CD25-mediated immune activation function of IL-2, both are Developed to reduce the activation of effector T cells (Teff).
  • Basiliximab is a chimeric anti-CD25 antibody currently approved for graft-versus-host disease (GVHD), while daclizumab is a humanized anti-CD25 antibody approved for the treatment of multiple sclerosis .
  • CD25 can also be used as a Treg-specific marker for the development of antibodies through antibody-mediated cytotoxicity (ADCC) and antibody-mediated phagocytosis (ADCP) to remove the Treg cells inside the tumor and relieve the inhibition of Treg on the T cells inside the tumor, thereby promoting anti-tumor immunity.
  • ADCC antibody-mediated cytotoxicity
  • ADCP antibody-mediated phagocytosis
  • CD25 also mediates the activation of effector T cells by IL-2. If CD25 antibody inhibits the IL-2 signaling pathway, it will inhibit Teff and antagonize its anti-tumor activity.
  • CD25 antibodies developed preclinically include 7D4 (rat anti-mouse CD25, see, for example, Malek et al. PNAS, 1983 Sep; 80(18): 5694-8; Onizuka Se et al., Cancer Res.
  • WO2019/175216 provides the CD25-targeting antibody RG6292 jointly developed by Roche and TUSK, which has entered clinical phase I to test its safety and efficacy in solid tumors.
  • WO2018167104 also provides anti-CD25 antibodies that do not affect the binding of IL-2 to CD25.
  • anti-CD25 antibodies that are more effective in inhibiting tumors, safer, does not inhibit the binding of IL-2 and CD25, and can eliminate Treg through ADCC and ADCP effects while avoiding inhibition of Teff activity is still urgently needed in the art .
  • the present disclosure provides anti-CD25 antibodies, antigen-binding fragments thereof, and nucleic acids encoding them, vectors, host cells, pharmaceutical compositions, methods for treating cancer (especially solid tumors), and pharmaceutical applications.
  • the present disclosure provides an anti-CD25 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region (VH) and/or a light chain variable region (VL), wherein:
  • VH contains HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 1
  • VL contains LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 2;
  • VH contains HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 3
  • VL contains LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 4;
  • VH contains HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 5
  • VL contains LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 6;
  • VH contains HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 7
  • VL contains LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 8;
  • VH contains HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 9
  • VL contains LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 10;
  • VH contains HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 11
  • VL contains LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 12;
  • VH contains HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 13
  • VL contains LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 14;
  • VH contains HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 15, and the VL contains LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 16;
  • VH contains HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 17
  • VL contains LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 18;
  • VH contains HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 19
  • VL contains LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 20;
  • VH contains HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 21, and the VL contains LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 22;
  • VH contains HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 23
  • VL contains LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 24;
  • the VH contains HCDR1, HCDR2 and HCDR3 in SEQ ID NO: 25, and the VL contains LCDR1, LCDR2 and LCDR3 in SEQ ID NO: 26; or
  • VH contains HCDR1, HCDR2, and HCDR3 in SEQ ID NO:59
  • VL contains LCDR1, LCDR2, and LCDR3 in SEQ ID NO:60.
  • CDRs are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system; in some specific embodiments, the CDRs are defined according to the Kabat numbering system.
  • antibodies or antigen-binding fragments thereof comprising VH and VL as shown in 1), 2), 3), 5) or 6) block or partially block the binding of IL-2 to CD25; others
  • antibodies or antigen-binding fragments thereof comprising VH and VL as shown in 4), 7), 8), 9), 10), 11), 12) or 13) do not block, do not inhibit or almost Does not block or inhibit the binding of IL-2 to CD25.
  • an anti-CD25 antibody or antigen-binding fragment thereof which comprises VH and/or VL, wherein:
  • the VH includes HCDR1 selected from SEQ ID NO: 27, 33, 39, or 45, and/or HCDR2 selected from SEQ ID NO: 28, 34, 40, or 46, and/or HCDR2 selected from SEQ ID NO: 29, HCDR3 of 35, 41, or 47;
  • the VL includes LCDR1 selected from SEQ ID NO: 30, 36, 42 or 48, and/or LCDR2 selected from SEQ ID NO: 31, 37, 43, or 49, and/or An LCDR3 selected from SEQ ID NO: 32, 38, 44, or 50.
  • an anti-CD25 antibody or antigen-binding fragment thereof comprising VH and/or VL, wherein:
  • the VH includes HCDR1 as shown in SEQ ID NO: 27 or with at most 3, 2 or 1 amino acid mutations, as shown in SEQ ID NO: 28 or at most 3, 2, or 1 with it HCDR2 with amino acid mutations as shown in SEQ ID NO: 29 or HCDR3 with at most 3, 2 or 1 amino acid mutations as shown in SEQ ID NO: 29; and/or the VL includes HCDR3 as shown in SEQ ID NO: 30 or with HCDR3 LCDR1 with at most 3, 2 or 1 amino acid mutations, as shown in SEQ ID NO: 31 or with LCDR2 with at most 3, 2 or 1 amino acid mutations, and as shown in SEQ ID NO: 32 with or with LCDR2 LCDR3 with at most 3, 2 or 1 amino acid mutations;
  • the VH includes HCDR1 as shown in SEQ ID NO: 33 or with at most 3, 2 or 1 amino acid mutations, as shown in SEQ ID NO: 34 or at most 3, 2, or 1 with it HCDR2 with amino acid mutations as shown in SEQ ID NO: 35 or HCDR3 with at most 3, 2 or 1 amino acid mutations as shown in SEQ ID NO: 35; and/or the VL includes HCDR3 as shown in SEQ ID NO: 36 or with HCDR3 LCDR1 with at most 3, 2 or 1 amino acid mutations, as shown in SEQ ID NO: 37 or with LCDR2 with at most 3, 2 or 1 amino acid mutations, and as shown in SEQ ID NO: 38 with or with LCDR2 LCDR3 with at most 3, 2 or 1 amino acid mutations;
  • the VH includes HCDR1 as shown in SEQ ID NO: 39 or with at most 3, 2 or 1 amino acid mutations, as shown in SEQ ID NO: 40 or at most 3, 2, or 1 with it HCDR2 with amino acid mutations as shown in SEQ ID NO: 41 or HCDR3 with at most 3, 2 or 1 amino acid mutations as shown in SEQ ID NO: 41; and/or the VL includes HCDR3 as shown in SEQ ID NO: 42 or with HCDR3 LCDR1 with at most 3, 2 or 1 amino acid mutations, as shown in SEQ ID NO: 43 or with LCDR2 with at most 3, 2 or 1 amino acid mutations, and as shown in SEQ ID NO: 44 with or with LCDR2 LCDR3 with at most 3, 2 or 1 amino acid mutations; or
  • the VH includes HCDR1 as shown in SEQ ID NO: 45 or at most 3, 2 or 1 amino acid mutations, as shown in SEQ ID NO: 46 or at most 3, 2, or 1 amino acid mutations therefrom.
  • the aforementioned anti-CD25 antibody or antigen-binding fragment thereof with amino acid mutations and the parent antibody or antigen-binding fragment have the same or substantially the same affinity and/or function (such as ADCC, ADCP, anti-tumor) that bind to human CD25 active).
  • the aforementioned anti-CD25 antibody or antigen-binding fragment thereof of the present disclosure binds to human CD25 with a dissociation equilibrium constant equal to or less than 10-7M. In some embodiments, it binds to human CD25 with a dissociation equilibrium constant equal to or less than 10-8M, 10-9M, 10-10M, or 10-11M.
  • an anti-CD25 antibody or antigen-binding fragment thereof comprising VH and/or VL, wherein:
  • the VH includes HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NOs: 27, 28, and 29, and the VL includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 30, 31, and 32, respectively;
  • the VH includes HCDR1, HCDR2, and HCDR3 shown in SEQ ID NOs: 33, 34, and 35
  • the VL includes LCDR1, LCDR2, and LCDR3 shown in SEQ ID NOs: 36, 37, and 38, respectively;
  • the VH includes HCDR1, HCDR2, and HCDR3 shown in SEQ ID NOs: 39, 40, and 41
  • the VL includes LCDR1, LCDR2, and LCDR3 shown in SEQ ID NOs: 42, 43, and 44, respectively; or
  • the VH includes HCDR1, HCDR2, and HCDR3 shown in SEQ ID NOs: 45, 46, and 47, respectively, and the VL includes LCDR1, LCDR2, and LCDR3 shown in SEQ ID NOs: 48, 49, and 50, respectively.
  • the anti-CD25 antibody or antigen-binding fragment thereof comprises VH and VL, wherein:
  • VH is shown in SEQ ID NO: 1
  • VL is shown in SEQ ID NO: 2;
  • VH is shown in SEQ ID NO: 3
  • VL is shown in SEQ ID NO: 4;
  • VH is shown in SEQ ID NO: 5
  • VL is shown in SEQ ID NO: 6
  • VH is shown in SEQ ID NO: 7
  • VL is shown in SEQ ID NO: 8;
  • VH is shown in SEQ ID NO: 9 and the VL is shown in SEQ ID NO: 10;
  • VH is shown in SEQ ID NO: 11
  • VL is shown in SEQ ID NO: 12;
  • VH is shown in SEQ ID NO: 13
  • VL is shown in SEQ ID NO: 14;
  • VH is shown in SEQ ID NO: 15
  • VL is shown in SEQ ID NO: 16;
  • the VH is shown in SEQ ID NO: 17, and the VL is shown in SEQ ID NO: 18; the antibody is a murine antibody or a fragment thereof.
  • an anti-CD25 antibody or antigen-binding fragment variant thereof which comprises VH and/or VL, and the VH and/or VL are respectively the same as the VH and/or VL of the aforementioned anti-CD25 antibody or antigen-binding fragment thereof.
  • VH and/or VL are respectively the same as the VH and/or VL of the aforementioned anti-CD25 antibody or antigen-binding fragment thereof.
  • the anti-CD25 antibody or antigen-binding fragment thereof is a murine antibody, a chimeric antibody, a human antibody, a humanized antibody or a fragment thereof, for example, a humanized antibody or a fragment thereof.
  • chimeric antibodies are prepared based on murine anti-CD25 antibodies or antigen-binding fragments thereof, humanized, and then backmutated.
  • the methods of humanization in US20030040606 and US7494647 are introduced here in full.
  • the anti-CD25 antibody or antigen-binding fragment thereof comprises VH and VL, wherein:
  • the VH includes FR1 to FR3 selected from IGHV1-46*01 and FR4 selected from IGHJ1*01, and the VL includes FR1 to FR3 selected from IGKV4-1*01 and FR4 selected from IGKJ4*01;
  • the VH includes FR1 to FR2 selected from IGHV1-18*01, FR3 selected from IGHV1-69*02, and FR4 selected from hIGHJ6*01_14, and the VL includes FR1 selected from IGKV3-11*01. FR2 from IGKV5-2*01, FR3 from IGKV6-21*01, and FR4 from hIGKJ4*01_12;
  • the VH includes FR1 selected from IGHV1-18*01, FR1 selected from IGHV4-31*01, FR3 selected from IGHV1-3*01, and FR4 selected from hIGHJ6*01, and the VL includes FR4 selected from IGKV4 -1*01 FR1 to FR3, and FR4 selected from hIGKJ2*01; or
  • the VH includes FR1 to FR3 selected from IGHV3-23*04 and FR4 selected from IGHJ1*01, and the VL includes FR1 to FR3 selected from IGKV2-28*01 and FR4 selected from IGKJ4*01.
  • the anti-CD25 antibody or antigen-binding fragment thereof comprises VH and/or VL, wherein,
  • the VH includes HCDR1, HCDR2, and HCDR3 shown in SEQ ID NOs: 27, 28, and 29, and FR1 to FR3 selected from IGHV1-46*01, and FR4 selected from IGHJ1*01;
  • the VL includes LCDR1, LCDR2, and LCDR3 shown in SEQ ID NOs: 30, 31, and 32, and FR1 to FR3 selected from IGKV4-1*01, and FR4 selected from IGKJ4*01;
  • the VH comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOs: 33, 34 and 35, and FR1 to FR2 selected from IGHV1-18*01, FR3 selected from IGHV1-69*02, and FR3 selected from IGHV1-69*02, respectively FR4 of hIGHJ6*01_14;
  • the VL includes LCDR1, LCDR2, and LCDR3 shown in SEQ ID NOs: 36, 37, and 38, and FR1 selected from IGKV3-11*01, FR2 selected from IGKV5-2*01 , FR3 selected from IGKV6-21*01, and FR4 selected from hIGKJ4*01_12;
  • the VH includes HCDR1, HCDR2 and HCDR3 shown in SEQ ID NOs: 39, 40 and 41, and FR1 selected from IGHV1-18*01, FR1 selected from IGHV4-31*012, selected from IGHV1-3 *01 FR3 and FR4 selected from hIGHJ6*01;
  • the VL includes LCDR1, LCDR2 and LCDR3 shown in SEQ ID NOs: 42, 43 and 44, and FR1 to FR3 selected from IGKV4-1*01 , And FR4 selected from hIGKJ2*01; or
  • the VH includes HCDR1, HCDR2, and HCDR3 shown in SEQ ID NOs: 45, 46, and 47, and FR1 to FR3 selected from IGHV3-23*04, and FR4 selected from IGHJ1*01;
  • the VL includes The LCDR1, LCDR2, and LCDR3 shown in SEQ ID NOs: 48, 49, and 50, and FR1 to FR3 selected from IGKV2-28*01, and FR4 selected from IGKJ4*01, respectively.
  • the above FR can be backmutated to maintain antibody activity.
  • the anti-CD25 antibody or antigen-binding fragment thereof comprises VH and/or VL, wherein:
  • VH is shown in SEQ ID NO: 19
  • VL is shown in SEQ ID NO: 20;
  • VH is shown in SEQ ID NO: 21, and the VL is shown in SEQ ID NO: 22;
  • VH is shown in SEQ ID NO: 23
  • VL is shown in SEQ ID NO: 24;
  • VH is shown in SEQ ID NO: 25
  • VL is shown in SEQ ID NO: 26;
  • the VH is shown in SEQ ID NO: 59, and the VL is shown in SEQ ID NO: 60; the antibody is a humanized antibody or a fragment thereof.
  • an anti-CD25 antibody or antigen-binding fragment variant thereof which comprises VH and/or VL, and the VH and/or VL are respectively the same as the VH and/or VL of the aforementioned anti-CD25 antibody or antigen-binding fragment thereof.
  • VH and/or VL are respectively the same as the VH and/or VL of the aforementioned anti-CD25 antibody or antigen-binding fragment thereof.
  • the anti-CD25 antibody or antigen-binding fragment thereof further comprises the heavy chain constant region and/or light chain constant region of the antibody, such as human IgG1, IgG2, IgG3, and IgG4 heavy chain constant regions and conventional variants thereof, so
  • the light chain constant region of the antibody is selected from human antibody ⁇ and ⁇ chain constant regions and conventional variants thereof; another example is murine IgG, such as murine IgG2a.
  • the antigen-binding fragment is selected from Fab, Fab', F(ab')2, single-chain antibody (scFv), dimerized V region (diabody), disulfide bond stabilized V region (dsFv) and other antigen-binding fragments of CDR-containing peptides.
  • an isolated monoclonal antibody or antigen-binding fragment thereof which competes with the anti-CD25 antibody or antigen-binding fragment thereof described in any one of the foregoing for binding to human CD25, or for binding to the same epitope.
  • an anti-CD25 antibody or antigen-binding fragment thereof which comprises VH and/or VL, wherein:
  • VH is shown in SEQ ID NO: 1
  • VL is shown in SEQ ID NO: 2;
  • VH is shown in SEQ ID NO: 3
  • VL is shown in SEQ ID NO: 4;
  • VH is shown in SEQ ID NO: 5
  • VL is shown in SEQ ID NO: 6;
  • VH is shown in SEQ ID NO: 9 and the VL is shown in SEQ ID NO: 10; or
  • the VH is shown in SEQ ID NO: 11, and the VL is shown in SEQ ID NO: 12; the antibody or antigen-binding fragment thereof blocks or partially blocks the binding of IL-2 and CD25.
  • an anti-CD25 antibody or antigen-binding fragment thereof which comprises VH and/or VL, wherein:
  • VH is shown in SEQ ID NO: 7
  • VL is shown in SEQ ID NO: 8;
  • VH is shown in SEQ ID NO: 13
  • VL is shown in SEQ ID NO: 14;
  • VH is shown in SEQ ID NO: 15
  • VL is shown in SEQ ID NO: 16;
  • VH is shown in SEQ ID NO: 17
  • VL is shown in SEQ ID NO: 18;
  • VH is shown in SEQ ID NO: 19
  • VL is shown in SEQ ID NO: 20;
  • VH is shown in SEQ ID NO: 21, and the VL is shown in SEQ ID NO: 22;
  • VH is shown in SEQ ID NO: 23
  • VL is shown in SEQ ID NO: 24;
  • VH is shown in SEQ ID NO: 25
  • VL is shown in SEQ ID NO: 26;
  • the VH is shown in SEQ ID NO: 59
  • the VL is shown in SEQ ID NO: 60
  • the antibody or antigen-binding fragment thereof does not block, does not inhibit, hardly blocks, does not inhibit, or is low
  • a degree of blocking and inhibiting the binding of IL-2 and CD25 compared to IL-2 signaling in the absence of antibodies, the anti-CD25 antibody or antigen-binding fragment blocks less than about 50%, about 40%, about 35%, about 30%, about 25%, About 20%, about 15%, about 10% of IL-2 signaling, for example, less than about 25% of IL-2 signaling.
  • an anti-CD25 antibody or antigen-binding fragment variant thereof which comprises VH and/or VL, and the VH and/or VL and the aforementioned anti-CD25 antibody or antigen-binding fragment thereof have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity.
  • an anti-CD25 antibody or antigen-binding fragment variant thereof which comprises VH and/or VL, and the VH and/or VL is combined with the heavy chain variable region VH of the aforementioned anti-CD25 antibody or antigen-binding fragment thereof.
  • VL contains 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid changes, respectively.
  • the amino acid changes may be conservative substitutions of amino acid residues in the variable region.
  • the aforementioned antibody or antigen-binding fragment containing amino acid changes has the same or substantially the same binding affinity and/or function (e.g. ADCC, ADCP, anti-tumor activity) to human CD25 as the parent antibody or antigen-binding fragment.
  • the anti-CD25 antibody or antigen-binding fragment thereof comprises a heavy chain and/or light chain, wherein,
  • the heavy chain is shown in SEQ ID NO: 51, and the light chain is shown in SEQ ID NO: 52;
  • the heavy chain is shown in SEQ ID NO: 53, and the light chain is shown in SEQ ID NO: 54;
  • the heavy chain is shown in SEQ ID NO: 55, and the light chain is shown in SEQ ID NO: 56;
  • the heavy chain is shown in SEQ ID NO: 57, and the light chain is shown in SEQ ID NO: 58; or
  • the heavy chain is shown in SEQ ID NO: 61, and the light chain is shown in SEQ ID NO: 62.
  • an anti-CD25 antibody or antigen-binding fragment thereof which comprises a heavy chain and/or light chain, and the heavy chain and/or light chain are combined with the heavy chain and/or of the aforementioned anti-CD25 antibody or antigen-binding fragment thereof
  • the light chains have at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity, respectively.
  • the anti-CD25 antibody or antigen-binding fragment thereof of the present disclosure is of the IgG1 type, the Fc region has a defucosylation site, and has an enhanced Fc ⁇ RIIIa binding ability.
  • the above-mentioned anti-CD25 antibody or its antigen-binding fragment can increase the ADCC effect on Treg cells and enhance its anti-tumor activity.
  • the anti-CD25 antibody or antigen-binding fragment thereof of the present disclosure is of the IgG1 type, and the Fc region has a defucosylation site (for example, the A330I mutation), and has reduced Fc ⁇ RIIb binding ability.
  • the anti-CD25 antibody or its antigen-binding fragment can reduce the ADCC/ADCP inhibitory signal mediated by the Fc ⁇ RIIb receptor, so as to enhance the ADCC effect on Treg cells and enhance its anti-tumor activity.
  • the anti-CD25 antibody or antigen-binding fragment of the present disclosure has at least one of the following characteristics:
  • the CD25 binding protein or anti-CD25 antibody of the present disclosure can bind to CD25 with a KD value of less than 1 ⁇ 10-7M, less than 1 ⁇ 10-8M, less than 1 ⁇ 10-9M, or less than 1 ⁇ 10-10M.
  • the anti-CD25 antibodies or antigen-binding fragments thereof of the present disclosure can inhibit tumor growth by at least about 10%, at least about 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%.
  • the anti-CD25 antibodies or antigen-binding fragments of the present disclosure bind to Fc ⁇ R with high affinity, for example, bind to activated receptors with high affinity.
  • the antibodies of the present disclosure bind to FcyRI and/or FcyRIIA and/or FcyRIIIA with high affinity.
  • the antibody binds to at least one activating Fcy receptor with a dissociation constant of less than about 10-6M, 10-7M, 10-8M, 10-9M, or 10-10M.
  • the anti-CD25 antibody or antigen-binding fragment of the present disclosure is an IgG1 antibody capable of binding to at least one Fc-activated receptor.
  • the antibody can bind to one or more receptors selected from FcyRI, FcyRIIa, FcyRIIc, FcyRIIIa, and FcyRIIIb.
  • the antibodies of the present disclosure are capable of binding to FcyRIIIA.
  • the antibodies of the present disclosure are capable of binding to FcyRIIIA and FcyRIIA and optionally FcyRI.
  • the antibody is capable of binding to these receptors with high affinity, for example with a dissociation constant of less than about 10-7M, 10-8M, 10-9M, or 10-10M.
  • the anti-CD25 antibodies or antigen-binding fragments of the present disclosure bind to the inhibitory receptor FcyRIIb with low affinity.
  • the antibody binds to FcyRIIb with a dissociation constant greater than about 10-7M, greater than about 10-6M, or greater than about 10-5M.
  • the anti-CD25 antibodies or antigen-binding fragments of the present disclosure are from the IgG1 subclass, and preferably have ADCC and/or ADCP activity. In other embodiments, the anti-CD25 antibodies of the present disclosure are from the IgG2 subclass.
  • the anti-CD25 antibodies or antigen-binding fragments of the present disclosure inhibit or block less than 50% of IL-2 signaling through CD25.
  • an anti-CD25 antibody or antigen-binding fragment inhibits or blocks less than about 40%, 35%, 30%, and preferably less than about 25% of IL-2 signaling Conduction.
  • the present disclosure provides isolated polynucleotides that encode the anti-CD25 antibodies or antigen-binding fragments thereof of the present disclosure.
  • the polynucleotide may be DNA or RNA.
  • the present disclosure provides an expression vector containing the polynucleotide as described above.
  • the expression vector can be a eukaryotic expression vector, a prokaryotic expression vector, a viral vector, such as a plasmid, a cosmid, or a phage.
  • the present disclosure provides a host cell transformed with an expression vector as described above, which may be a eukaryotic cell or a prokaryotic cell.
  • the host cell is a bacterium, yeast, or mammalian cell.
  • the host cell is E. coli, Pichia pastoris, Chinese hamster ovary (CHO) cell or human embryonic kidney (HEK) 293 cell.
  • the present disclosure provides a method for preparing an anti-CD25 antibody or antigen-binding fragment thereof, comprising: expressing the antibody or antigen-binding fragment thereof in a host cell as described above, and isolating the antibody or antigen-binding fragment from the host cell Combine fragments.
  • it can also include a purification step, for example, using a Sepharose FF column with adjusted buffer A or G to wash out non-specifically bound components, and then use a pH gradient method to elute the bound antibody. SDS-PAGE detection and collection.
  • it is filtered and concentrated by a conventional method. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange. The resulting product needs to be frozen immediately, such as -70°C, or lyophilized.
  • the methods for producing and purifying antibodies and antigen-binding fragments are well known and can be found in the prior art, such as Cold Spring Harbor's Antibody Experiment Technique Guide (chapters 5-8 and 15).
  • human FcRn or its fragments can be used to immunize mice, and the obtained antibody can be renatured, purified, and amino acid sequencing can be performed by conventional methods.
  • Antigen-binding fragments can also be prepared by conventional methods.
  • the engineered antibodies or antigen-binding fragments of the present disclosure can be prepared and purified by conventional methods.
  • the cDNA sequences encoding the heavy and light chains can be cloned and recombined into an expression vector.
  • the recombinant immunoglobulin expression vector can be stably transfected into CHO cells.
  • Mammalian expression systems can lead to glycosylation of antibodies, especially at the highly conserved N-terminus of the Fc region.
  • Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are expanded in the serum-free medium of the bioreactor to produce antibodies.
  • the antibody-secreted culture medium can be purified and collected by conventional techniques.
  • the antibody can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange.
  • the present disclosure provides a composition, such as a pharmaceutical composition, which contains a therapeutically effective amount of the anti-CD25 antibody or antigen-binding fragment thereof as described above and a pharmaceutically acceptable excipient, dilution or carrier.
  • the pharmaceutical composition may contain 0.01 to 99% by weight of the anti-CD25 antibody or its antigen-binding fragment in the unit dose, or the amount of the anti-CD25 antibody or its antigen-binding fragment in the unit dose of the pharmaceutical composition. It is 0.1-2000 mg, and in some embodiments, it is 1-1000 mg.
  • the present disclosure provides any one or any combination of anti-CD25 antibodies or antigen-binding fragments thereof, pharmaceutical compositions containing the anti-CD25 antibodies or antigen-binding fragments, encoding polynucleotides, for diagnosis, treatment, and prevention of diseases
  • the method and the use of preparing drugs and pharmaceutical compositions for example, for treating or preventing proliferative disorders (such as cancer or tumors) or delaying the progress of related disorders.
  • a method for preventing, treating or alleviating a condition of a subject comprising administering to the subject an anti-CD25 antibody or antigen-binding fragment thereof, a compound containing the anti-CD25 antibody or antigen-binding fragment thereof Pharmaceutical composition and/or encoding polynucleotide.
  • the subject's condition is a proliferative disease, such as a tumor or cancer.
  • the aforementioned subject has an established tumor, such as a solid tumor.
  • a method is provided to reduce the number of cells in a tumor or tumor-invasive Treg in a subject; in some embodiments, a method is provided to eliminate or inhibit the cellular activity of Tregs in a tumor or tumor-invasive in a subject, Both include administering to the subject the anti-CD25 antibody or antigen-binding fragment thereof, a pharmaceutical composition containing the anti-CD25 antibody or antigen-binding fragment thereof, and/or the encoding polynucleotide.
  • a method for increasing the ratio of Teff/Treg within a tumor of a subject comprising administering to the subject an anti-CD25 antibody or antigen-binding fragment thereof, containing the anti-CD25 antibody or antigen-binding
  • the pharmaceutical composition of the fragment and/or the encoding polynucleotide increases to more than 5, 10, 15, 20, 40, or 80.
  • a method for enhancing CDC, ADCC and/or ADCP against tumor cells in a subject comprising administering to the subject the anti-CD25 antibody or antigen-binding fragment thereof of the present disclosure, containing the anti-CD25 Pharmaceutical compositions of antibodies or antigen-binding fragments thereof and/or encoding polynucleotides.
  • the ADCC and/or ADCP effect on tumor cells in the subject is enhanced.
  • the ADCC effect on tumor cells in the subject is enhanced.
  • an anti-CD25 antibody or antigen-binding fragment thereof of the present disclosure a pharmaceutical composition containing the anti-CD25 antibody or an antigen-binding fragment thereof, and/or encoding polynucleotide are provided for preparing prevention, treatment, or alleviation of a subject
  • the pharmaceutical use of the disease for the preparation of a pharmaceutical use for reducing the number of cells in a subject's tumor or tumor-invasive Treg, and for the preparation of a medicine for eliminating or inhibiting the cell activity of the subject's tumor or tumor-invasive Treg
  • the pharmaceutical use of is used for the preparation of a pharmaceutical use for increasing the ratio of Teff/Treg in a subject's tumor, and for the preparation of a pharmaceutical use for enhancing the CDC, ADCC and/or ADCP of tumor cells in the subject.
  • the condition of the subject described above is a proliferative disorder (e.g., cancer or tumor) or suffers from a proliferative disorder (e.g., cancer or tumor).
  • a proliferative disorder e.g., cancer or tumor
  • Such tumors include, but are not limited to, cancer, lymphoma, leukemia, blastoma, and sarcoma.
  • cancers include squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer, glioma, hepatocellular carcinoma (HCC), Hodgkin's lymphoma, non-Hodgkin's lymphoma , Acute myeloid leukemia (AML), multiple myeloma, gastrointestinal (tract) cancer, kidney cancer, ovarian cancer, liver cancer, lymphoblastic leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, kidney cancer , Prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, brain cancer, stomach cancer, bladder cancer, liver cancer, breast cancer, colon cancer and head and neck cancer cancer.
  • the cancer or tumor may be a solid tumor, including but not limited to sarcoma (including mesenchymal tissues (such as cancellous bone, cartilage, fat, muscle, blood vessels, hematopoietic cells, or fibrous connective tissue)). Cytoplasmic-derived transformed cells), carcinomas (including tumors derived from epithelial cells), mesothelioma, neuroblastoma, retinoblastoma, etc.
  • sarcoma including mesenchymal tissues (such as cancellous bone, cartilage, fat, muscle, blood vessels, hematopoietic cells, or fibrous connective tissue)
  • Cytoplasmic-derived transformed cells including carcinomas (including tumors derived from epithelial cells), mesothelioma, neuroblastoma, retinoblastoma, etc.
  • Cancers involving solid tumors include, but are not limited to, brain cancer, lung cancer, stomach cancer, duodenal cancer, esophageal cancer, breast cancer, colon and rectal cancer, kidney cancer, bladder cancer, kidney cancer, pancreatic cancer, prostate cancer, ovarian cancer Cancer, melanoma, oral cancer, sarcoma, eye cancer, thyroid cancer, urethral cancer, vagina cancer, neck cancer, lymphoma, etc.
  • the cancer involves CD25-expressing tumors, including but not limited to lymphomas, such as Hodgkin's lymphoma and lymphocytic leukemia, such as chronic lymphocytic leukemia (CLL).
  • lymphomas such as Hodgkin's lymphoma
  • lymphocytic leukemia such as chronic lymphocytic leukemia (CLL).
  • CLL chronic lymphocytic leukemia
  • the present disclosure provides detection applications of anti-CD25 antibodies or antigen-binding fragments thereof.
  • the present disclosure provides reagents for detecting CD25, the reagents comprising anti-CD25 antibodies or antigen-binding fragments thereof.
  • the present disclosure also provides methods, systems or devices for detecting CD25 in vivo or in vitro, which include the use of anti-CD25 antibodies or antigen-binding fragments thereof.
  • the in vitro detection method, system or device may, for example, include (1) contacting the sample with an anti-CD25 antibody or antigen-binding fragment thereof; (2) detecting the formation between the anti-CD25 antibody or its antigen-binding fragment and the sample Complex; and/or (3) contacting a reference sample (eg, control sample) with the antibody; and (4) determining the degree of complex formation between the antibody and the sample by comparing with the reference sample.
  • a change in complex formation in the sample or subject indicates the presence of CD25 in the sample.
  • the CD25 binding protein or anti-CD25 antibody of the present disclosure may be labeled with a fluorophore and a chromophore.
  • kits which includes a CD25 binding protein or an anti-CD25 antibody, and may also include diagnostic instructions.
  • the kit may also contain at least one additional reagent, such as a marker or additional diagnostic agent.
  • the antibody can be formulated as a pharmaceutical composition.
  • the anti-CD25 antibodies or antigen-binding fragments thereof provided in the embodiments of the present disclosure have the characteristics of high specificity, high affinity, and low immunogenicity.
  • the antibody of the present disclosure has a good inhibitory effect on Treg, does not affect Teff, enhances ADCC, ADCP and/or CDC in the subject, and inhibits tumor occurrence and development.
  • Figure 1A and Figure 1B ELISA to identify recombinant protein activity.
  • Figure 1A is the activity detection result of recombinant human CD25 protein
  • Figure 1B is the activity detection result of recombinant monkey and recombinant mouse CD25 protein.
  • Figure 2 FACS determination of the cell line 2F8 stably expressing human CD25 protein, in which the white peak is the CD25 positive peak, the gray peak is the control peak, and the primary antibody is the human IgG1 negative control antibody.
  • Figure 3A to Figure 3C FACS assay to detect antibody function.
  • Figure 3A shows the FACS determination of the binding results of antibodies DAC, 7G7B6, Tab06 and CHO-K1 cells
  • Figure 3B shows the FACS determination of the binding results of antibodies DAC, 7G7B6, Tab06 and CHO-K1-CD25 cells stably expressing human CD25 protein
  • Figure 3C For FACS determination of the binding results of antibodies DAC, 7G7B6, Tab06 and Su-DHL-1, mouse IgG or human IgG isotype was used as a negative control.
  • Figure 4A to Figure 4C ELISA assay detects antibody binding to CD25.
  • Figure 4A is the binding result of 7G7B6, Tab06 and recombinant human CD25 protein
  • Figure 4B is the binding result of 7G7B6, Tab06 and recombinant monkey CD25 protein
  • Figure 4C is the binding result of 7D4 and recombinant mouse CD25 protein
  • the negative control used is Mouse IgG and human IgG.
  • Figure 5A to Figure 5C FACS detects the binding of chimeric anti-CD25 antibody to SU-DHL-1 cells.
  • Figure 5A shows the detection results of cAb001, cAb002, cAb004, and cAb006.
  • Figure 5B shows the detection results of cAb028, cAb029, and cAb037.
  • Figure 5C shows the detection results of cAb042 and cAb046.
  • the positive controls used are Tab06 and DAC, and the negative control is human IgG. (Ie hIgG).
  • Figure 6A to Figure 6G FACS detection of chimeric anti-CD25 antibody binding to CD25 antigen on Treg cells, activated CD4 + and CD8 + effector T cells.
  • Figure 6A is the result of cAb006
  • Figure 6B is the result of cAb037
  • Figure 6C is the result of cAb042
  • Figure 6D is the result of cAb046
  • Figure 6E is the result of the positive control 7G7B
  • Figure 6F is the result of the positive control DAC
  • Figure 6G is the positive Compare the results of Tab06.
  • Fig. 7A to Fig. 7B FACS detects the effect of chimeric anti-CD25 antibody on the binding ability of IL-2 and receptor CD25.
  • Figure 7A is a graph of the detection results of cAb001, cAb002, cAb028, and cAb029
  • Figure 7B is a graph of the detection results of cAb006, cAb037, cAb042, and cAb046.
  • the anti-CD25 antibody control used to block the binding of IL-2 and CD25 is DAC, which does not block
  • the anti-CD25 antibody control for IL-2 and CD25 is Tab06, and the negative control is human IgG (ie hIgG-1).
  • Figure 8 FACS detects the binding of humanized anti-CD25 antibody to SU-DHL-1 cells.
  • Figure 9 FACS detection of the effect of humanized anti-CD25 antibody on the pStat5 signaling pathway of human peripheral blood T lymphocytes.
  • Figure 10 Detect the ADCC effect on SU-DHL-1 cells mediated by humanized anti-CD25 antibody by detecting the fluorescence activity of ADCC reporter gene in cells.
  • Fig. 11A and Fig. 11B Humanized anti-CD25 antibody-mediated anti-tumor activity results on MC38 xenografted tumors of hCD25 mice, wherein Fig. 11A is a graph of tumor suppression effect, and Fig. 11B is a graph of corresponding mouse body weight.
  • Figure 12A and Figure 12B Humanized anti-CD25 antibody-mediated intratumoral lymphocyte analysis results of MC38 xenograft tumors in hCD25 mice.
  • Figure 12A shows the results of the antibody killing Treg, and
  • Figure 12B shows the antibody killing CD3. Up.
  • CD25 or CD25 protein or “CD25 polypeptide” may optionally include any such protein or variants, conjugates or fragments thereof, including but not limited to known or wild-type CD25 as described herein, And any naturally occurring splice variants, amino acid variants or isoforms.
  • the complete human CD25 sequence can be found under Uniprot accession number P01589, and its 22nd to 240th amino acids correspond to the extracellular domain of mature human CD25.
  • CD25 Binding to CD25 refers to the ability to interact with CD25 or its epitope, and the CD25 or its epitope may be of human origin.
  • Antigen-binding site refers to a discrete three-dimensional site on an antigen that is recognized by the antibody or antigen-binding fragment of the present disclosure.
  • Antibody refers to immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds. The amino acid composition and sequence of the constant region of the immunoglobulin heavy chain are different, so their antigenicity is also different. According to this, immunoglobulins can be divided into five categories, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE. The corresponding heavy chains are ⁇ chain, ⁇ chain, and ⁇ chain. , ⁇ chain and ⁇ chain.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • the light chain is divided into a kappa chain or a lambda chain by the difference of the constant region.
  • Each of the five types of Ig can have a kappa chain or a lambda chain.
  • the sequence of about 110 amino acids near the N-terminus of the antibody heavy and light chains varies greatly and is the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable and are the constant region (C region).
  • the variable region includes 3 hypervariable regions (HVR) and 4 framework regions (FR) with relatively conserved sequences. Three hypervariable regions determine the specificity of the antibody, also known as complementarity determining regions (CDR). Each light chain variable region (VL) and heavy chain variable region (VH) consists of 3 CDR regions and 4 FR regions. The sequence from the amino terminal to the carboxy terminal is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the 3 CDR regions of the heavy chain refer to HCDR1, HCDR2 and HCDR3. In some embodiments, the antibodies of the present disclosure specifically or substantially specifically bind to CD25.
  • CD25 binding antibody refers to an antibody capable of binding to the CD25 subunit of the IL-2 receptor. This subunit is also called the alpha subunit of the IL-2 receptor. Such antibodies are also referred to herein as “anti-CD25 antibodies”.
  • the number and position of the CDR amino acid residues of the VL region and VH region of the antibody or antigen-binding fragment of the present disclosure conform to the known Kabat numbering system.
  • the EU numbering in Kabat is also generally used for constant domains and/or Fc domains.
  • CDR the deterministic description of CDR and the identification of residues containing the binding site of the antibody can be completed by resolving the structure of the antibody and/or resolving the structure of the antibody-ligand complex. This can be achieved by any of various techniques known to those skilled in the art, such as X-ray crystallography. A variety of analysis methods can be used to identify CDRs, including but not limited to Kabat numbering system, Chothia numbering system, AbM numbering system, IMGT numbering system, contact definition, conformational definition.
  • the Kabat numbering system is a standard for numbering residues in antibodies and is commonly used to identify CDR regions (see, for example, Johnson & Wu, 2000, Nucleic Acids Res., 28:214-8).
  • the Chothia numbering system is similar to the Kabat numbering system, but the Chothia numbering system takes into account the location of certain structural loop regions. (See, for example, Chothia et al., 1986, J. Mol. Biol., 196:901-17; Chothia et al., 1989, Nature, 342:877-83).
  • the AbM numbering system uses a computer program integration suite produced by Oxford MolecuLar Group for modeling antibody structures (see, for example, Martin et al., 1989, Proc Natl Acad Sci (USA), 86: 9268-9272; "AbMTM, A Computer Program for Modeling Variable Regions of Antibodies, "Oxford, UK; Oxford MolecuLar, Ltd).
  • the AbM numbering system uses a combination of knowledge databases and ab initio methods to model the tertiary structure of antibodies from basic sequences (see Samudrala et al., 1999, in PROTEINS, Structure, Function and Genetics Suppl., 3:194-198, "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach).
  • the contact definition is based on the analysis of available complex crystal structures (see, for example, MacCallum et al., 1996, J. Mol. Biol., 5:732-45).
  • the position of the CDRs can be identified as residues that make enthalpy contributions to antigen binding (see, for example, Makabe et al., 2008, Journal of Biological Chemistry, 283:1156-1166).
  • other CDR boundary definitions may not strictly follow one of the above methods, but still overlap with at least a part of Kabat CDR, although they can be shortened or lengthened according to specific residues or residue groups that do not significantly affect the prediction of antigen binding or experimental results. .
  • CDR may refer to a CDR defined by any method (including a combination of methods) known in the art.
  • the methods used herein can utilize CDRs defined according to any of these methods.
  • the kabat numbering rules are used to define CDRs, but those skilled in the art can understand that CDRs can also be redefined according to any of Chothia, extended, AbM, IMGT, contact, and/or conformational definitions.
  • the Fc region of IgG antibodies interacts with several cellular Fc ⁇ receptors (Fc ⁇ R) to stimulate and regulate downstream effector mechanisms.
  • Fc ⁇ R Fc ⁇ receptors
  • the communication between the IgG antibody and the immune system is controlled and mediated by Fc ⁇ R.
  • Fc ⁇ R transmits the information sensed and collected by the antibody to the immune system, thereby providing the link between the innate and adaptive immune systems, especially in the context of biological therapy (Hayes J et al., 2016.
  • IgG subclasses have different abilities to bind Fc ⁇ R, and this differential combination determines their ability to trigger a series of functional reactions.
  • Fc ⁇ RIIIa is the main receptor involved in antibody-dependent cell-mediated cytotoxicity (ADCC) activation.
  • IgG1 followed by IgG3 showed the highest affinity for this receptor, reflecting their ability to effectively induce ADCC.
  • IgG2 has weak binding to this receptor, but it has been found that anti-CD25 antibodies with human IgG2 isotype can also effectively deplete Treg.
  • Treg refers to the CD4 + T lymphocyte lineage that specifically controls autoimmunity, allergy, and infection. Generally, they regulate the activity of T cell populations, but they can also affect certain innate immune system cell types. Treg can be identified by the expression of biomarkers CD4, CD25 and Foxp3. Naturally occurring Treg cells usually account for about 5 to 10% of peripheral CD4+ T lymphocytes. However, within the tumor microenvironment (ie, tumor-infiltrating Treg cells), they can account for 20 to 30% of the total CD4 + T lymphocyte population.
  • Treg cells can directly kill target cells through perforation factor or granzyme B-dependent pathways, such as Teff and APC (antigen presenting cells); cytotoxic T lymphocyte-associated antigen 4 (CTLA4 + ) Treg cells can be used by APC Indole amine 2,3-dioxygenase (IDO) expression, which in turn inhibits T cell activation by reducing tryptophan; Treg cells can release interleukin-10 (IL-10) and transforming growth factor in the body (TGF ⁇ ), thereby directly inhibiting T cell activation and inhibiting APC function by inhibiting the expression of MHC molecules, CD80, CD86 and IL-12. Treg cells can also suppress immunity by expressing high levels of CTLA4, which can bind to CD80 and CD86 on antigen presenting cells and prevent the correct activation of effector T cells.
  • CTLA4 + cytotoxic T lymphocyte-associated antigen 4
  • IDO interleukin-10
  • TGF ⁇ transforming growth factor in the body
  • Treg cells can also suppress
  • Immunoeffector cells herein refer to immune cells involved in the effector phase of an immune response.
  • exemplary immune cells include myeloid or lymphoid cells, such as lymphocytes (e.g., B cells and T cells including cytolytic T cells (CTL)), killer cells, natural killer cells, macrophages, monocytes, Eosinophils, neutrophils, polymorphonuclear cells, granulocytes, mast cells and basophils.
  • Inhibition of the binding of IL-2 to CD25 includes where the anti-CD25 antibody does not block or inhibit the signal transduction of IL-2 through CD25, especially does not inhibit CD25-expressing cells Interleukin-2 signal transduction in. That is, compared to IL-2 signaling in the absence of the antibody, the anti-CD25 antibody of the present disclosure inhibits less than 50% of IL-2 signaling through CD25. Preferably, the anti-CD25 antibody inhibits less than about 40%, 35%, 30%, and preferably less than about 25% of IL-2 signaling compared to IL-2 signaling in the absence of the antibody.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • FcR Fc receptors
  • NK natural killer cells
  • FcR Fc receptors
  • ADCP antibody-dependent cell-mediated phagocytosis
  • CDC complement-dependent cytotoxicity
  • ADCC can be increased by eliminating the fucose moiety from the glycan of the antibody, for example, by producing the antibody in the YB2/0 cell line, or by introducing specific mutations in the Fc portion of human IgG1 (e.g., S298A/E333A/K334A, S239D/I332E/A330L, G236A/S239D/A330L/I332E) (Lazar et al. (2006) Proc Natl Acad Sci USA 103, 2005-2010; Smith et al. (2012) Proc Nat 25Acad Sci USA 109,6181-6) .
  • ADCP can also be increased by introducing specific mutations on the Fc part of human IgG1 (Richards et al. (2008) Mol Cancer Ther 7, 2517-27).
  • a "murine antibody” in the present disclosure is a monoclonal antibody against human CD25 or its epitope prepared according to the knowledge and skills in the art. During preparation, the test subject is injected with CD25 antigen, and then hybridomas expressing antibodies with the desired sequence or functional properties are isolated.
  • the murine anti-human CD25 antibody or antigen-binding fragment thereof may further comprise the light chain constant region of murine kappa, lambda chain or a variant thereof, or further comprise murine IgG1 , IgG2, IgG3 or IgG4 or variants of the heavy chain constant region.
  • a "chimeric antibody” is an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can reduce the immune response induced by the murine antibody.
  • To establish a chimeric antibody it is necessary to select a hybridoma that secretes a murine-specific monoclonal antibody, and then clone the variable region gene from the mouse hybridoma cell, and then clone the constant region gene of the human antibody as needed, and change the mouse variable region gene.
  • the region gene and the human constant region gene are connected to form a chimeric gene and then inserted into a human vector, and finally the chimeric antibody molecule is expressed in a eukaryotic industrial system or a prokaryotic industrial system.
  • the constant region of a human antibody can be selected from the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or its variants, preferably comprising human IgG2 or IgG4 heavy chain constant region, or using amino acid mutations without ADCC (antibody-dependent cell-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity) toxic IgG1.
  • ADCC antibody-dependent cell-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity
  • Humanized antibody also known as CDR-grafted antibody, refers to an antibody produced by grafting mouse CDR sequences into a human antibody variable region framework. It can overcome the strong immune response induced by the chimeric antibody carrying a large amount of mouse protein components. In order to avoid the decrease of immunogenicity and the decrease of activity at the same time, the variable region of the human antibody can be subjected to minimal reverse mutation to maintain activity.
  • the antibody of the present disclosure may be an affinity matured humanized antibody, and the CDR of the parent sequence (including all sequences) of the affinity matured antibody is at least 80% identical, such as 90% identical.
  • An affinity matured antibody is an antibody that has one or more changed amino acids in one or more CDRs, which results in an improved affinity for CD25 of the antibody compared to a parent antibody that does not have the changed amino acid.
  • Human antibodies include antibodies having variable and constant regions of human germline immunoglobulin sequences.
  • the human antibodies of the present disclosure may include amino acid residues that are not encoded by human germline immunoglobulin sequences (such as mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).
  • “human antibodies” do not include antibodies in which CDR sequences derived from the germline of another mammalian species (such as a mouse) have been grafted onto human framework sequences (ie, "humanized antibodies”).
  • Antigen-binding fragments include single-chain antibodies (ie, full-length heavy and light chains); Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, Fab-Fv, Fab-dsFv , Single domain antibody (e.g. VH or VL or VHH), scFv, bivalent or trivalent or tetravalent antibody, Bis-scFv, diabody, tribody, triabody, tetrabody and epitope binding fragments of any of the above (see e.g. Holliger and Hudson, 2005, Nature Biotech. 23(9): 1126-1136; Adair and Lawson, 2005, Drug Design Reviews-Online 2(3), 209-217).
  • the antigen-binding fragments of the present disclosure also include Fab and Fab' fragments described in WO2005/003169, WO2005/003170 and WO2005/003171.
  • Multivalent antibodies may comprise multispecific such as bispecific or may be monospecific (see e.g. WO92/22583 and WO05/113605), an example of the latter is the Tri-Fab described in WO 92/22583 (or TFM).
  • the anti-CD25 antibodies of the present disclosure encompass bi- and multispecific antibodies.
  • Epitope refers to a site on an antigen that specifically binds to an immunoglobulin or antibody.
  • Epitopes can be formed by adjacent amino acids or non-adjacent amino acids that are juxtaposed by tertiary folding of the protein. Epitopes formed by adjacent amino acids are usually maintained after exposure to a denaturing solvent, while epitopes formed by tertiary folding are usually lost after treatment with the denaturing solvent.
  • Epitopes usually include at least 3-15 amino acids in a unique spatial conformation. Methods to determine what epitope is bound by a given antibody are well known in the art, including immunoblotting and immunoprecipitation detection analysis. Methods for determining the spatial conformation of an epitope include the techniques in the art and the techniques described herein, such as X-ray crystal analysis and two-dimensional nuclear magnetic resonance.
  • Specific binding and “selective binding” refer to the binding of an antibody to an epitope on a predetermined antigen.
  • an antibody when measured by surface plasmon resonance (SPR) technology in the instrument, the antibody balances approximately below 10-7M or even less.
  • the dissociation constant (KD) binds to a predetermined antigen or its epitope, and its binding affinity to the predetermined antigen or its epitope is a non-specific antigen other than the predetermined antigen (or its epitope) or closely related antigens (Such as BSA, etc.) at least twice the binding affinity.
  • KD dissociation constant
  • Antibody that recognizes antigen can be used interchangeably with “antibody that specifically binds” herein.
  • Binding affinity refers to the apparent association constant or Ka.
  • Ka is the reciprocal of the dissociation constant (Kd).
  • Kd dissociation constant
  • binding to a specific protein target molecules may have at least 10 -5, 10 -6 binding affinity, 10-7, 10-8, 10-9, 10-10, and 10 -11 M's.
  • the higher affinity binding of the binding ligand to the first target can be achieved by a higher binding Ka (or Kd) of the first target than the Ka (or a lower Kd) of the second target. Value) to display.
  • the binding protein has specificity for the first target (for example, the protein in the first conformation or its analogue) .
  • the difference in binding affinity is at least 1.5, 2, 3, 4, 5, 10, 15, 20, 50, 70, 80, 100, 500, 1000, or 105 times.
  • Constant substitution refers to substitution with another amino acid residue that has similar properties to the original amino acid residue.
  • lysine, arginine, and histidine have similar properties in that they have basic side chains
  • aspartic acid and glutamic acid have similar properties in that they have acidic side chains.
  • glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine and tryptophan have similar properties in that they have uncharged polar side chains
  • alanine , Valine, leucine, threonine, isoleucine, proline, phenylalanine and methionine have similar properties in that they have non-polar side chains.
  • tyrosine, phenylalanine, tryptophan and histidine have similar properties in that they have aromatic side chains. Therefore, it will be obvious to a person skilled in the art that even when an amino acid residue in a group showing similar properties as described above is substituted, it will not show a specific change in properties.
  • Sequence homology or “sequence identity” refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When the positions in the two comparison sequences are occupied by the same base or amino acid monomer subunit, for example, if each position of the two DNA molecules is occupied by adenine, then the molecules are homologous at that position .
  • the percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared ⁇ 100%. For example, in the optimal sequence alignment, if there are 6 matches or homology in 10 positions in the two sequences, then the two sequences are 60% homologous. Generally speaking, the comparison is made when two sequences are aligned to obtain the greatest percentage of homology.
  • Cross-reactivity refers to the ability of the antibodies of the present disclosure to bind to CD25 from different species.
  • an antibody of the present disclosure that binds to human CD25 can also bind to CD25 of another species.
  • Cross-reactivity is measured by detecting specific reactivity with purified antigen in binding assays such as SPR and ELISA, or binding or functional interaction with cells that physiologically express CD25. Methods of determining cross-reactivity include standard binding assays as described herein, such as surface plasmon resonance analysis, or flow cytometry.
  • Inhibition or blocking are used interchangeably and encompasses both partial and complete inhibition/blocking. Inhibition/blocking of CD25 preferably reduces or alters the normal level or type of activity that occurs when CD25 binding occurs without inhibition or blocking. Inhibition and blocking are also intended to include any measurable reduction in binding affinity for CD25 when contacted with anti-CD25 antibody compared to CD25 not contacted with anti-CD25 antibody.
  • Inhibiting growth (e.g., referring to cells) is intended to include any measurable decrease in cell growth.
  • the methods for producing and purifying antibodies and antigen-binding fragments are well known and can be found in the prior art, such as Cold Spring Harbor's Antibody Experiment Technique Guide (chapters 5-8 and 15).
  • human CD25 or its fragments can be used to immunize mice, and the obtained antibodies can be renatured and purified, and amino acid sequencing can be performed by conventional methods.
  • Antigen-binding fragments can also be prepared by conventional methods.
  • the antibodies or antigen-binding fragments of the invention are genetically engineered to add one or more human FR regions to the non-human CDR regions.
  • the human FR germline sequence can be obtained from the website of ImmunoGeneTics (IMGT) http://imgt.cines.fr, or from the Journal of Immunoglobulin, 2001ISBN012441351.
  • the antibodies of the present disclosure may be polyclonal, monoclonal, heterologous, allogeneic, syngeneic, or modified forms thereof, and monoclonal antibodies are particularly suitable for use in various embodiments.
  • the antibodies of the present disclosure are recombinant antibodies.
  • "recombinant” generally refers to products such as cells or nucleic acids, proteins, or vectors, meaning that the cells, nucleic acids, proteins, or vectors have been modified by introducing heterologous nucleic acids or proteins or altering natural nucleic acids or proteins, or The cells are derived from cells so modified.
  • recombinant cells express genes that are not present in the natural (non-recombinant) cell form or express natural genes that are originally abnormally expressed, underexpressed, or not expressed at all.
  • Monoclonal antibody or “monoclonal antibody” refers to antibodies obtained from a single cloned cell line, and the cell line is not limited to eukaryotic, prokaryotic or phage cloned cell lines. Monoclonal antibodies or antigen-binding fragments can be obtained by recombination using, for example, hybridoma technology, recombination technology, phage display technology, synthesis technology (such as CDR-grafting), or other existing technologies.
  • the antibodies or antigen-binding fragments of the present disclosure can be prepared and purified by conventional methods.
  • the cDNA sequences encoding the heavy and light chains can be cloned and recombined into an expression vector.
  • the recombinant immunoglobulin expression vector can be stably transfected into CHO cells.
  • Mammalian expression systems can lead to glycosylation of antibodies, especially at the highly conserved N-terminus of the Fc region.
  • Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are expanded in the serum-free medium of the bioreactor to produce antibodies.
  • the antibody-secreted culture medium can be purified and collected by conventional techniques.
  • the antibody can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange.
  • the resulting product needs to be frozen immediately, such as -70°C, or lyophilized.
  • administering when applied to animals, humans, experimental subjects, cells, tissues, organs or biological fluids refer to exogenous drugs, therapeutic agents, diagnostic agents or compositions and animals , Human, subject, cell, tissue, organ or biological fluid contact.
  • administering can refer to, for example, treatment, pharmacokinetics, diagnosis, research, and experimental methods.
  • the treatment of cells includes contact of reagents with cells, and contact of reagents with fluids, where the fluids are in contact with cells.
  • administering “administration” and “treatment” also mean the treatment of, for example, cells by reagents, diagnostics, binding compositions, or by another cell in vitro and ex vivo.
  • Treatment when applied to human, veterinary or research subjects, refers to therapeutic treatment, preventive or preventive measures, research and diagnostic applications.
  • Treatment means administering an internal or external therapeutic agent to a subject, such as a composition comprising any one of the antibodies or antigen-binding fragments or conjugates thereof of the present disclosure, the subject has, or is suspected of being suffering from Yes, tend to suffer from one or more diseases or their symptoms, and the therapeutic agent is known to have a therapeutic effect on these symptoms.
  • the therapeutic agent is administered in the subject or population to be treated in an amount effective to alleviate one or more symptoms of the disease, whether by inducing the regression of such symptoms or inhibiting the development of such symptoms to any clinically measured extent.
  • the amount of the therapeutic agent effective to alleviate the symptoms of any particular disease can vary depending on a variety of factors, such as the subject’s disease state, age and weight, and the amount of the drug that produces the desired therapeutic effect in the subject. ability. Through any clinical testing methods commonly used by doctors or other professional health care professionals to evaluate the severity or progression of the symptoms, it can be evaluated whether the symptoms of the disease have been alleviated.
  • the embodiments of the present disclosure may be ineffective in alleviating the symptoms of the target disease in a subject, according to any statistical test methods known in the art such as Student's t test, chi-square test, and basis Mann and Whitney's U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determined that it should reduce the symptoms of the target disease in a statistically significant number of subjects.
  • any statistical test methods known in the art such as Student's t test, chi-square test, and basis Mann and Whitney's U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determined that it should reduce the symptoms of the target disease in a statistically significant number of subjects.
  • treatment of cancer includes (a) inhibiting cancer, that is, preventing its development, including but not limited to blocking or delaying the progression of cancer, blocking or delaying the metastasis of cancer; and/or (b) alleviating cancer , That is, causing cancer to regress, including but not limited to reducing or alleviating one or more symptoms related to the cancer, alleviating or alleviating metastatic cancer, and/or reducing or eliminating tumors.
  • Prevention herein refers to delaying or preventing the onset of cancer symptoms. Prevention may be absolute (therefore no disease will occur) or only effective in certain individuals or for a limited time.
  • Effective amount includes an amount sufficient to improve or prevent the symptoms or conditions of medical conditions.
  • An effective amount also means an amount sufficient to allow or facilitate diagnosis.
  • the effective amount for a particular subject or veterinary subject can vary depending on factors such as the condition to be treated, the subject's general health, the method of administration and dosage, and the severity of side effects.
  • the effective amount can be the maximum dose or dosing schedule that avoids significant side effects or toxic effects.
  • Cell Cell
  • cell line cell line
  • cell culture all such names include their progeny. It should also be understood that due to deliberate or unintentional mutations, all offspring cannot be exactly the same in terms of DNA content. Including mutant progeny with the same function or biological activity as screened in the original transformed cell.
  • “Pharmaceutical composition” means a mixture containing one or more antibodies or antigen-binding fragments or physiologically/pharmaceutically acceptable salts or prodrugs and other chemical components of the antibodies or antigen-binding fragments described herein, as well as other components such as physiological/pharmacological Medicinal carriers and excipients.
  • the purpose of the pharmaceutical composition is to promote the administration to the organism, which is beneficial to the absorption of the active ingredient and thus the biological activity.
  • Tumor applies to subjects who have been diagnosed with or suspected of having tumors. Cancer refers to malignant or potentially malignant neoplasms or tissue masses of any size, and includes primary tumors and secondary neoplasms. "Cancer”, “malignant tumor”, “neoplastic”, “tumor” and “cancer” are also used interchangeably herein, and refer to tumors and tumors that exhibit relatively abnormal, uncontrolled, and/or autonomous growth Cells, therefore they exhibit an abnormal growth phenotype characterized by a significant loss of control over cell proliferation. Generally, cells of interest for detection or treatment include precancerous (eg benign), malignant, pre-metastatic, metastatic and non-metastatic cells.
  • a "solid tumor” is an abnormal growth or mass of tissue, which usually does not contain cysts or fluid areas, especially tumors and/or metastases (regardless of where they are located) other than leukemia or non-solid lymphoma.
  • Solid tumors can be benign or malignant. Different types of solid tumors are named after the cell type that formed them and/or the tissue or organ in which they are located.
  • solid tumors include, but are not limited to, sarcomas (including cancers derived from transformed cells of mesenchymal origin in tissues such as cancellous bone, cartilage, fat, muscle, blood vessels, hematopoietic cells, or fibrous connective tissue), cancers (including Tumors from epithelial cells), melanoma, lymphoma, mesothelioma, neuroblastoma and retinoblastoma.
  • sarcomas including cancers derived from transformed cells of mesenchymal origin in tissues such as cancellous bone, cartilage, fat, muscle, blood vessels, hematopoietic cells, or fibrous connective tissue
  • cancers including Tumors from epithelial cells
  • melanoma lymphoma
  • mesothelioma mesothelioma
  • neuroblastoma neuroblastoma
  • retinoblastoma retinoblastoma
  • the articles "one” (a) and “one” (an) used herein refer to one or more than one (ie at least one/kind) of the grammatical objects of the article.
  • an element refers to one or more than one element.
  • Recombinant human CD25-Fc fusion protein (purchased from ACRO Biosystems) is a fusion of human IgG1Fc tag protein to the C-terminus of human CD25 protein (GenBank, accession number: NP_000408.1).
  • Recombinant human CD25-His protein (purchased from Beijing Yiqiao Shenzhou) is a polyhistidine tag fused to the C-terminus of human CD25 protein (GenBank, accession number: NP_000408.1) Met1-Cys213.
  • the recombinant monkey CD25-His protein (purchased from Beijing Yiqiao Shenzhou) is a His tag fused to the C-terminus of the monkey CD25 protein (GenBank, accession number: NP_001270633.1) Met1-Arg213.
  • Recombinant mouse CD25-His protein (purchased from Beijing Yiqiao Shenzhou) is a His tag fused to the C-terminus of mouse CD25 protein (GenBank, accession number: NP_032393.3) Met1-Lys236.
  • Use the anti-CD25 antibody DAC (Efalizumab, daclizumab) to perform a series of quality control tests on the purchased recombinant protein, such as activity verification.
  • ELISA enzyme-linked immunosorbent assay
  • HRP horseradish peroxidase labeled
  • Example 2 Establishment of a stable cell line expressing recombinant human CD25 protein
  • a cell line stably expressing recombinant human CD25 protein was obtained through screening.
  • the nucleotide sequence encoding human CD25 was cloned and ligated into the pCDNA3.4 vector (purchased from Invitrogen) and a plasmid was prepared.
  • the CHO-K1 cell line (all purchased from Invitrogen) was transfected with Lipofectamine 2000, and the CD-CHO medium containing G418 was selectively cultured for 2 weeks.
  • the limiting dilution method was used for subcloning in a 96-well culture plate and placed Culture in an incubator containing 5% CO 2 (v/v) at 37°C. After 2 weeks, select some monoclonal wells to expand into 24-well plates, and then expand into 6-well plates for cultivation. The amplified clones were detected and screened by flow analysis.
  • Figure 2 shows that the selected cell line 2F8 can stably express human CD25 protein.
  • DAC, 7G7B6, Tab06, and 7D4 are all anti-CD25 antibodies.
  • the sequence of the DAC is listed in US5530101, which is incorporated into the present disclosure by reference.
  • 7G7B6 has been suggested as a targeting part for targeting radionuclides to CD25-expressing lymphomas (Zhang et al, 2009, Cancer Biother Radiopharm 24(3), 303-309).
  • Tab06 is an anti-CD25 antibody that does not inhibit the binding of IL-2 to CD25.
  • the sequence is listed in WO2018167104 (sequences 27 and 29 of the patent), which is incorporated into the present disclosure by reference.
  • 7D4 is a rat IgM anti-mouse CD25 antibody.
  • 7D4 has been widely used to detect CD25 positive cells (Malek, 1983, Immunology, Vol. 80) ,pp.5694-5698; Onizuka S et al., 1999. Canc Res. 59, 3128-3133).
  • the functional verification of the detection antibodies DAC, 7G7B6, Tab06, and 7D4 was performed by FACS. Specifically, the logarithmic growth phase SU-DHL-1 cells, CHO-K1 cells and CHO-K1 cells stably expressing human CD25 (CHO-K1-CD25 cells) were collected by centrifugation, washed with PBS and centrifuged at 200g for 5 minutes. The plate was plated with 100 ⁇ L of culture medium containing 2 ⁇ 10 5 cells, and the detection antibody (100 nM, gradient dilution) was added after centrifugation at 400 g for 5 minutes. The control antibody was of human IgG isotype.
  • ELISA for functional verification of 7G7B6 and Tab06. Specifically: Dilute recombinant human CD25-His protein, recombinant monkey CD25-His protein and recombinant mouse CD25-his protein to 1 ⁇ g/mL with PBS, add 100 ⁇ L per well, and incubate overnight at 4°C; add ELISA Blocking solution (containing 1% BSA, pH 7.4 PBS phosphate buffer), after blocking at 37°C for 2 hours, successively add successively diluted detection antibodies and incubate at 37°C for 1 hour; wash the plate 2-3 with plate washing solution Second; add horseradish peroxidase labeled (HRP) secondary antibody, incubate at 37°C for 1 hour, and wash the plate 2-3 times with a plate washing solution. Add 100 ⁇ L of TMB substrate to each well. After incubating for 15 minutes at room temperature, add 50 ⁇ L of stop solution 2M HCl to each well, and read the OD450nm value.
  • HRP horseradish peroxid
  • 7G7B6 and Tab06 can bind to human CD25 protein and monkey CD25 protein, respectively, indicating that 7G7B6 and Tab06 have human monkey cross-reactivity; 7D4 can bind to mouse CD25 protein, indicating that 7D4 has mouse CD25 protein The binding activity.
  • Recombinant human CD25-Fc was used as an immunogen to immunize 6-8 weeks old Balb/c and SJL/J mice, and recombinant human CD25-His was used as an immunogen to immunize SJL/J mice.
  • the initial dose of immunization is 50 ⁇ g per mouse.
  • the immunization was boosted, and the immunization dose was 25 ⁇ g per mouse.
  • each booster immunization interval is 3 weeks. Serum samples were collected one week after each boost, and the antibody activity in mouse serum was detected by ELISA.
  • the plate was coated with 1ug/mL recombinant human CD25-His, overnight at 4°C, blocked with PBST buffer containing 1% BSA for 1 hour, and the plate was washed 3 times. Start with 1:100 in blocking buffer, 10-fold dilution of mouse serum, incubate at 37°C for 1 hour, wash the plate 3 times, and incubate with anti-mouse IgG-Fc-HRP secondary antibody for 1 hour. Wash with PBST 3 times, add 100 ⁇ L of TMB substrate to each well, and stop the reaction with 2M HCl after 15 minutes. Use a microplate reader to read the absorbance at 450nm. Mouse sera immunogenic recombinant human CD25-Fc immunized with different degrees of binding to the immunogen, the highest serum dilution at 5 to 1:10, still exhibits antigen-antibody reaction.
  • mice were sacrificed and the spleens were taken, and the spleen cells were collected by grinding.
  • NH 4 OH with a final concentration of 1% (w/w) was added to lyse the mixed red blood cells in the spleen cells to obtain a spleen cell suspension, and the cells were washed three times by centrifugation at 1000 rpm.
  • the mouse spleen cells and mouse myeloma cells SP2/0 were mixed at a ratio of 5:1 in the number of living cells, and the cells were fused using a high-efficiency electrofusion method.
  • ELISA was used to screen the fused cells, and the positive clones with OD450nm>1.0 were amplified to a 24-well cell plate.
  • a recombinant chimeric antibody is obtained by replacing the constant region of the mouse monoclonal antibody, and then the nucleotide sequence encoding the variable region of the mouse monoclonal antibody is cloned to contain the constant regions encoding the human heavy and light chains
  • the (Human IgG1, kappa) protein sequence pTT5 vector is transfected into HEK293 cells, that is, the variable regions of the heavy and light chains of the chimeric antibody obtained are the same as those of the murine antibody. .
  • the mouse anti-CDR is chimerized into the appropriate human GermLine framework (Bioinformation.2014; 10(4): 180-186; Methods Mol Biol .2019;1904:213-230), and then introduce back mutations at sites that may affect antibody-antigen binding, and finally clone the nucleotide sequence encoding the variable region of the humanized monoclonal antibody into the human body.
  • the light chain constant region (Human IgG1, kappa) protein sequence pTT5 vector was transfected into HEK293 cells. After 5 days, the cells were removed by centrifugation and the cell culture solution was filtered.
  • the harvested cell culture supernatant was loaded onto a protein A column (MabSelect SuRe, GE), the bound antibody was eluted with glycine, and the eluate was neutralized with 1M Tris.
  • a protein A column MobSelect SuRe, GE
  • the antibody variable region framework selection is shown in Table 2, the antibody variable region sequence is shown in Table 3, the heavy chain CDR (HCDR1 ⁇ HCDR2 ⁇ HCDR3) and light chain CDR (LCDR1 ⁇ LCDR2 ⁇ LCDR3) are shown in Table 4, and the full length of the antibody amino acid sequence is shown in Table 4. table 5.
  • PR006 is a humanized antibody of cAb006
  • PR071 is a humanized antibody of cAb037
  • PR031 is a humanized antibody of cAb042
  • PR058 and PR157 are humanized antibodies of cAb046.
  • ELISA method was used to detect the binding activity of chimeric anti-CD25 antibody and CD25 recombinant protein.
  • Antibody number EC 50 (nM) Antibody number EC 50 (nM)
  • Antibody number EC 50 (nM) cAb001 0.3991 cAb029 0.1145 cAb028 0.2295 cAb002 0.5244 cAb037 0.1467 DAC 0.3973 cAb004 0.3106 cAb042 0.3486 Tab06 0.6144 cAb006 0.2338 cAb046 5.569 To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To
  • Antibody number EC 50 (nM) Antibody number EC 50 (nM)
  • Antibody number EC 50 (nM) cAb001 0.2284 cAb028 0.3444 cAb046 0.3547 cAb002 0.2738 cAb029 0.1661 DAC 0.2319 cAb004 0.2552 cAb037 0.1653 Tab06 0.4032 cAb006 0.2004 cAb042 0.224 To To To To To To To To To To To To To To To To To To To To To To To To To To To To To
  • the lymphoma cell line SU-DHL-1 highly expresses CD25
  • flow cytometry to detect the binding of chimeric anti-CD25 antibody to SU-DHL-1 cells.
  • the SU-DHL-1 cells in the logarithmic growth phase were selected, the cells were collected, washed with PBS and centrifuged. Each well was plated with 100 ⁇ l 2 ⁇ 10 5 cells, and centrifuged at 400 g for 5 minutes. Add different concentrations of antibodies to be tested, incubate on ice for 1 hour, wash with PBS, and centrifuge at 400g for 5 minutes. Add the goat anti-human secondary antibody Alexa Fluor 488 with fluorophore, stain in ice bath for 1 hour, wash with PBS and test on the machine. As shown in Figures 5A-5C, the tested chimeric anti-CD25 antibodies of the present disclosure can all bind to SU-DHL-1 cells. Table 8 shows the combined EC 50 values.
  • Antibody number EC 50 (nM) Antibody number EC 50 (nM)
  • Antibody number EC 50 (nM) cAb001 0.1799 cAb028 0.1319 cAb046 0.3455 cAb002 0.1001 cAb029 0.1193 DAC 0.3407 cAb004 0.1681 cAb037 0.1502 Tab06 0.8323 cAb006 0.3286 cAb042 0.501 To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To To
  • Example 8 Binding of chimeric anti-CD25 antibody to Treg cells/activated CD4 + T cells/CD8 + T cells
  • CD25 is expressed in both activated Treg and activated effector T cells
  • the anti-CD25 antibody of the present disclosure has a binding difference to CD25 expressed on Treg and CD4 + and CD8 + effector T cells
  • FACS Fluorescence Activated Cell Sorting
  • PBMC peripheral blood mononuclear cells
  • Fresh PBMC were enriched with CD4 + cell negative screening and enrichment kit cells and CD8 + T cell screening kit respectively, and high-purity CD4 + and CD8 + effector T cells were isolated, and the cell purity was greater than 90%. And adding magnetic beads coated with anti-CD3/CD28 antibody to activate and expand the cells. After 3 days, CD25 highly expressed effector CD4 + T cells and effector CD8 + T cells were obtained.
  • Treg cells induced in vitro and the CD4 + and CD8 + effector T cells activated in vitro were added to the antibody to be tested in different dilutions and incubated on ice for 1 h, and then washed with PBS. Then add goat anti-human antibody Alexa Fluor488 ice bath for staining for 1 hour, wash with PBS, add 200 ⁇ L flow cytometry solution to each well under the same conditions for simultaneous detection.
  • Example 9 The effect of chimeric anti-CD25 antibody on IL-2 and receptor binding ability
  • Inhibition rate % 100 ⁇ (maximum binding fluorescence intensity when no antibody is added-fluorescence intensity after antibody binding)/binding intensity when no antibody is added.
  • Example 7 The flow cytometry method in Example 7 was used to detect the binding of the chimeric anti-CD25 antibody to SU-DHL-1 cells.
  • Figure 8 shows that the humanized anti-CD25 antibodies RP006, PR031, PR058, and PR071 of the present disclosure all have good SU-DHL-1 binding activity.
  • Example 11 The effect of humanized anti-CD25 antibody on the pStat5 signaling pathway of human peripheral blood T lymphocytes
  • CD25 forms a high-affinity receptor with ⁇ and ⁇ chains. After binding to IL-2, it activates the downstream JAK-STAT, PI3K-AKT and MAPK signaling pathways. The synergistic antibody up-regulates the effect of IL-2 on phosphorylated STAT-5 in PBMC.
  • DAC can significantly inhibit the phosphorylation of Stat5 in CD3 cells induced by IL-2, while Tab06 cannot inhibit the phosphorylation of Stat5 induced by IL-2.
  • PR006 can weakly inhibit the phosphorylation of Stat5 at 300 nM and 100 nM, and PR031, PR058, and PR071 do not inhibit the phosphorylation of Stat5 mediated by IL-2.
  • ADCC Antibody-mediated cytotoxicity
  • ADCC reporter cells Promega G7010 ADCC Bioassay Eector Cells
  • SU-DHL-1 cells SU-DHL-1 cells at a 1:1 ratio
  • PR006, PR031, PR058, and PR071 all have good ADCC activities, among which the activities of PR058 and PR071 are significantly stronger than those of antibody 686.
  • sequence of antibody 686 please refer to sequence 5 and sequence 9 of US20190284287A.
  • the anti-tumor activity of humanized anti-CD25 antibodies was evaluated in the MC38 colon cancer mouse model of B-hIL2RA humanized mice.
  • the aforementioned variable regions of PR058 and 686 were linked to mouse IgG2a to construct PR058 mIgG2a, 686 mIgG2a antibodies for animal experiments.
  • MC38 colon cancer cells resuspended in PBS were inoculated subcutaneously on the right side of B-hIL2RA humanized mice at a concentration of 5 ⁇ 10 5 cells/0.1 mL and a volume of 0.1 mL/head.
  • the average tumor volume reaches about 113mm 3
  • the route of administration in all groups was intraperitoneal injection, once a week, three times in a row, and the experiment ended 7 days after the last administration.
  • the tumor volume and body weight were measured twice a week, and the mouse body weight and tumor volume were recorded.
  • the animals were euthanized, the tumors were stripped and weighed, photographed, and the relative tumor inhibition rate (TGI%) was calculated.
  • PR058 has the best inhibitory effect on the growth of MC38 subcutaneous xenograft tumors at a dose level of 3 mg/kg, which is better than 686.
  • Figure 11B shows that the experimental animals are in good activity and eating state during the administration period, and the weight of the experimental animals has increased to a certain extent, indicating that the test drug has no obvious toxic effect on the experimental animals, and the safety is good.
  • the results of intratumor lymphocyte analysis shown in Figure 12A and Figure 12B show that PR058 can more strongly mediate Treg killing and up-regulate the number of CD3.
  • * means p ⁇ 0.05, which is statistically different compared with mIgG2a group; ** means p ⁇ 0.01, which is statistically significant compared with mIgG2a group; *** means p ⁇ 0.001, which is statistically different compared with mIgG2a group Its significant statistical difference; ns means no statistical difference compared with the mIgG2a group.

Abstract

提供了抗CD25抗体、其抗原结合片段及其医药用途。具体地，提供抗CD25抗体及其制备抗肿瘤药物的用途。

Description

抗CD25抗体、其抗原结合片段及其医药用途
本申请要求2020年05月14日提交的中国专利申请(申请号202010408502.3)的优先权。本申请引用上述中国专利申请的全文。
技术领域
本公开属于生物医药领域，特别是癌症免疫治疗领域，包括治疗癌症(例如实体瘤)的方法，涉及抗CD25抗体或其抗原结合片段的使用。
背景技术
肿瘤免疫治疗是肿瘤治疗领域的热点，目前主要通过提高肿瘤内部CD4 +和CD8 +T细胞或者抑制肿瘤内部抑制型免疫细胞的数目和活性，例如，抑制调节性T细胞(Treg)、骨髓来源的抑制性细胞MDSC或肿瘤相关巨噬细胞。通过降低或消除肿瘤内部Treg细胞的治疗策略已经得到临床验证。
CD25是由272个氨基酸组成的I型膜蛋白，其在T细胞上的表达受TCR信号的调控，因此常作为T细胞激活的标志。IL-2具有免疫激活作用，是肿瘤治疗的免疫调节剂，其通过细胞表面的IL-2受体(IL-2R)发生作用。IL-2R包括三个亚基，IL-2Rα(即CD25)、IL-2Rβ(即CD122)和IL-2Rγ(即CD132)。三个亚基可以形成三种受体形式：高结合力受体包含所有三个亚基IL-2Rα/β/γ，中结合力受体包含IL-2Rβ/γ，低结合力受体为IL-2Rα。IL-2Rα(即CD25)单独与IL-2的亲和力较低(Kd为10 -8M)，并且不传导细胞内信号。IL-2Rβ和IL-2Rγ是IL-2激活下游信号通路所必需的，当IL-2同时结合IL-2Rβ和IL-2Rγ时，两个受体亚基形成异源二聚体。当IL-2Rα(即CD25)与IL-2Rβ和IL-2Rγ形成高亲和力受体(Kd为10 -11M)时，能激活下游JAK-STAT，PI3K-AKT和MAPK信号通路，从而促进T细胞、NK细胞等淋巴细胞的增殖与活性。
部分已有的抗CD25抗体阻断或抑制IL-2与CD25结合，例如参见WO2004/045512、WO2006/108670、WO1993/011238、WO1990/007861和WO2017/174331。巴利昔单抗(basiliximab)和达利珠单抗(daclizumab，DAC)均是抑制IL-2与CD25结合的IgG1型抗人CD25抗体，基于CD25介导IL-2的免疫激活功能，均被开发用于减少效应T细胞(Teff)的活化。巴利昔单抗是目前被批准用于移植物抗宿主疾病(GVHD)的嵌和型抗CD25抗体，而达利珠单抗是被批准用于治疗多发性硬化症的人源化抗CD25抗体。
由于CD25在Treg上的表达丰度远高于其它免疫细胞，因此CD25也可以作为Treg特异性标记物用于开发抗体，通过抗体介导的细胞毒作用(ADCC)和抗体介导的细胞吞噬作用(ADCP)来去除肿瘤内部的Treg细胞，解除Treg对于肿瘤内部T细胞的抑制，从而促进抗肿瘤免疫。但CD25同时也介导IL-2对效应T细胞的激活，如果CD25抗体抑制了IL-2信号通路，将会抑制Teff，拮抗其抗肿 瘤活性。一些文献提到抗CD25单独或组合用于癌症或与Treg消耗有关的用途(WO2004/074437、WO 2006/108670、WO 2006/050172、WO 2011/077245、WO2016/021720、WO 2004/045512)，因此需要允许IL-2与CD25结合的抗CD25抗体。临床前研发的CD25抗体包括7D4(大鼠抗小鼠CD25，例如参见Malek et al.PNAS，1983Sep；80(18):5694-8；Onizuka S et al.,Cancer Res.1999Jul 1；59(13):3128-33)、7G7B6(抗人CD25，例如参见Zhang et al.，Cancer Biother Radiopharm.2009Jun；24(3):303-309)、PC61(大鼠抗小鼠CD25，例如参见Yulius Y Setiady et al.，Eur J Immunol.2010Mar；40(3):780-6)，在小鼠癌症模型中进行测试时，PC61由于其对CD25和IL2结合抑制作用，未能显示出抗肿瘤活性，而7D4由于其不影响IL2对CD25的结合，显示出优于PC61的抗肿瘤活性。此外，WO2019/175216中提供了罗氏公司和TUSK公司联合开发的靶向CD25的抗体RG6292，已经进入临床I期，以检测其在实体瘤中的安全性及药效。WO2018167104中也提供了不影响IL-2与CD25结合的抗CD25抗体。
开发抑制肿瘤效果更优的、安全性更高的、不抑制IL-2与CD25的结合的、能够通过ADCC和ADCP效应消除Treg，同时避免抑制Teff活性的抗CD25抗体仍然是本领域亟需的。
发明内容
本公开提供抗CD25抗体、其抗原结合片段，及其编码核酸、载体、宿主细胞、药物组合物、用于治疗癌症(特别是实体瘤)的方法和制药用途。
抗CD25抗体、其抗原结合片段
本公开提供抗CD25抗体或其抗原结合片段，包含重链可变区(VH)和/或轻链可变区(VL)，其中：
1)所述VH含有SEQ ID NO:1中的HCDR1、HCDR2和HCDR3，所述VL含有SEQ ID NO:2中的LCDR1、LCDR2和LCDR3；
2)所述VH含有SEQ ID NO：3中的HCDR1、HCDR2和HCDR3，所述VL含有SEQ ID NO:4中的LCDR1、LCDR2和LCDR3；
3)所述VH含有SEQ ID NO:5中的HCDR1、HCDR2和HCDR3，所述VL含有SEQ ID NO:6中的LCDR1、LCDR2和LCDR3；
4)所述VH含有SEQ ID NO:7中的HCDR1、HCDR2和HCDR3，所述VL含有SEQ ID NO:8中的LCDR1、LCDR2和LCDR3；
5)所述VH含有SEQ ID NO:9中的HCDR1、HCDR2和HCDR3，所述VL含有SEQ ID NO:10中的LCDR1、LCDR2和LCDR3；
6)所述VH含有SEQ ID NO:11中的HCDR1、HCDR2和HCDR3，所述VL含有SEQ ID NO:12中的LCDR1、LCDR2和LCDR3；
7)所述VH含有SEQ ID NO:13中的HCDR1、HCDR2和HCDR3，所述VL 含有SEQ ID NO:14中的LCDR1、LCDR2、LCDR3；
8)所述VH含有SEQ ID NO:15中的HCDR1、HCDR2和HCDR3，所述VL含有SEQ ID NO:16中的LCDR1、LCDR2和LCDR3；
9)所述VH含有SEQ ID NO:17中的HCDR1、HCDR2和HCDR3，所述VL含有SEQ ID NO:18中的LCDR1、LCDR2和LCDR3；
10)所述VH含有SEQ ID NO:19中的HCDR1、HCDR2和HCDR3，所述VL含有SEQ ID NO:20中的LCDR1、LCDR2和LCDR3；
11)所述VH含有SEQ ID NO:21中的HCDR1、HCDR2和HCDR3，所述VL含有SEQ ID NO:22中的LCDR1、LCDR2和LCDR3；
12)所述VH含有SEQ ID NO:23中的HCDR1、HCDR2和HCDR3，所述VL含有SEQ ID NO:24中的LCDR1、LCDR2和LCDR3；
13)所述VH含有SEQ ID NO:25中的HCDR1、HCDR2和HCDR3，所述VL含有SEQ ID NO:26中的LCDR1、LCDR2和LCDR3；或
14)所述VH含有SEQ ID NO:59中的HCDR1、HCDR2和HCDR3，所述VL含有SEQ ID NO:60中的LCDR1、LCDR2和LCDR3。
上述CDR是根据Kabat、IMGT、Chothia、AbM或Contact编号系统定义的；一些具体实施方案中，CDR是根据Kabat编号系统定义的。
一些实施方案中，包含如1)、2)、3)、5)或6)所示的VH和VL的抗体或其抗原结合片段阻断或部分阻断IL-2与CD25的结合；另一些实施方案中，包含如4)、7)、8)、9)、10)、11)、12)或13)所示的VH和VL的抗体或其抗原结合片段不阻断、不抑制或几乎不阻断、不抑制IL-2与CD25的结合。
一些实施方案中，提供抗CD25抗体或其抗原结合片段，其包含VH和/或VL，其中：
所述VH包含选自SEQ ID NO：27、33、39或45的HCDR1，和/或选自SEQ ID NO：28、34、40或46的HCDR2，和/或选自SEQ ID NO：29、35、41或47的HCDR3；所述VL包含选自SEQ ID NO：30、36、42或48的LCDR1，和/或选自SEQ ID NO：31、37、43或49的LCDR2，和/或选自SEQ ID NO：32、38、44或50的LCDR3。
一些实施方案中，提供抗CD25抗体或其抗原结合片段，包含VH和/或VL，其中：
i)所述VH包含如SEQ ID NO：27所示或与其具有至多3个、2个或1个氨基酸突变的HCDR1，如SEQ ID NO：28所示或与其具有至多3个、2个或1个氨基酸突变的HCDR2，和如SEQ ID NO：29所示或与其具有至多3个、2个或1个氨基酸突变的HCDR3；和/或所述VL包含如SEQ ID NO：30所示或与其具有至多3个、2个或1个氨基酸突变的LCDR1，如SEQ ID NO：31所示或与其具有至多3个、2个或1个氨基酸突变的LCDR2，和如SEQ ID NO：32所示或与其具有 至多3个、2个或1个氨基酸突变的LCDR3；
ii)所述VH包含如SEQ ID NO：33所示或与其具有至多3个、2个或1个氨基酸突变的HCDR1，如SEQ ID NO：34所示或与其具有至多3个、2个或1个氨基酸突变的HCDR2，和如SEQ ID NO：35所示或与其具有至多3个、2个或1个氨基酸突变的HCDR3；和/或所述VL包含如SEQ ID NO：36所示或与其具有至多3个、2个或1个氨基酸突变的LCDR1，如SEQ ID NO：37所示或与其具有至多3个、2个或1个氨基酸突变的LCDR2，和如SEQ ID NO：38所示或与其具有至多3个、2个或1个氨基酸突变的LCDR3；
iii)所述VH包含如SEQ ID NO：39所示或与其具有至多3个、2个或1个氨基酸突变的HCDR1，如SEQ ID NO：40所示或与其具有至多3个、2个或1个氨基酸突变的HCDR2，和如SEQ ID NO：41所示或与其具有至多3个、2个或1个氨基酸突变的HCDR3；和/或所述VL包含如SEQ ID NO：42所示或与其具有至多3个、2个或1个氨基酸突变的LCDR1，如SEQ ID NO：43所示或与其具有至多3个、2个或1个氨基酸突变的LCDR2，和如SEQ ID NO：44所示或与其具有至多3个、2个或1个氨基酸突变的LCDR3；或
iv)所述VH包含如SEQ ID NO：45所示或与其具有至多3个、2个或1个氨基酸突变的HCDR1，如SEQ ID NO：46所示或与其具有至多3个、2个或1个氨基酸突变的HCDR2，和如SEQ ID NO：47所示或与其具有至多3个、2个或1个氨基酸突变的HCDR3；和/或所述VL包含如SEQ ID NO：48所示或与其具有至多3个、2个或1个氨基酸突变的LCDR1，如SEQ ID NO：49所示或与其具有至多3个、2个或1个氨基酸突变的LCDR2，和如SEQ ID NO：50所示或与其具有至多3个、2个或1个氨基酸突变的LCDR3。
一些具体实施方案中，上述具有氨基酸突变的抗CD25抗体或其抗原结合片段与亲本抗体或抗原结合片段具有相同或基本相同的与人CD25结合的亲和力和\或功能(例如ADCC、ADCP、抗肿瘤活性)。
一些实施方案中，本公开的前述抗CD25抗体或其抗原结合片段以等于或小于10-7M解离平衡常数与人CD25结合。在一些实施方案中，以等于或小于10-8M、10-9M、10-10M或10-11M解离平衡常数与人CD25结合。
一些实施方案中，提供抗CD25抗体或其抗原结合片段，包含VH和/或VL，其中：
所述VH包含分别如SEQ ID NO：27、28和29所示的HCDR1、HCDR2的HCDR3，所述VL包含分别如SEQ ID NO：30、31和32所示的LCDR1、LCDR2和LCDR3；
所述VH包含分别如SEQ ID NO：33、34和35所示的HCDR1、HCDR2和HCDR3，所述VL包含分别如SEQ ID NO：36、37和38所示的LCDR1、LCDR2和LCDR3；
所述VH包含分别如SEQ ID NO：39、40和41所示的HCDR1、HCDR2和HCDR3，所述VL包含分别如SEQ ID NO：42、43和44所示的LCDR1、LCDR2和LCDR3；或
所述VH包含分别如SEQ ID NO：45、46和47所示的HCDR1、HCDR2和HCDR3，所述VL包含分别如SEQ ID NO：48、49和50所示的LCDR1、LCDR2和LCDR3。
一些实施方案中，抗CD25抗体或其抗原结合片段包含VH和VL，其中：
所述VH如SEQ ID NO：1所示，所述VL如SEQ ID NO：2所示；
所述VH如SEQ ID NO：3所示，所述VL如SEQ ID NO：4所示；
所述VH如SEQ ID NO：5所示，所述VL如SEQ ID NO：6所示
所述VH如SEQ ID NO：7所示，所述VL如SEQ ID NO：8所示；
所述VH如SEQ ID NO：9所示，所述VL如SEQ ID NO：10所示；
所述VH如SEQ ID NO：11所示，所述VL如SEQ ID NO：12所示；
所述VH如SEQ ID NO：13所示，所述VL如SEQ ID NO：14所示；
所述VH如SEQ ID NO：15所示，所述VL如SEQ ID NO：16所示；或
所述VH如SEQ ID NO：17所示，所述VL如SEQ ID NO：18所示；所述抗体为鼠源抗体或其片段。
一些具体实施方案中，提供抗CD25抗体或其抗原结合片段变体，其包含VH和/或VL，所述VH和/或VL与前述抗CD25抗体或其抗原结合片段的VH和/或VL分别具有至少80％、85％、90％、95％、96％、97％、98％或99％的序列同一性。
一些实施方案中，抗CD25抗体或其抗原结合片段为鼠源抗体、嵌合抗体、人抗体、人源化抗体或其片段，例如，为人源化抗体或其片段。
一些实施方案中，基于鼠源抗CD25抗体或其抗原结合片段制备嵌和抗体，进行人源化，后进行回复突变。此处全文引入US20030040606和US7494647中人源化的方法。
一些实施方案中，抗CD25抗体或其抗原结合片段包含VH和VL，其中：
所述VH包含选自IGHV1-46*01的FR1至FR3、和选自IGHJ1*01的FR4，所述VL包含选自IGKV4-1*01的FR1至FR3、和选自IGKJ4*01的FR4；
所述VH包含选自IGHV1-18*01的FR1至FR2、选自IGHV1-69*02的FR3、和选自hIGHJ6*01_14的FR4，所述VL包含选自IGKV3-11*01的FR1、选自IGKV5-2*01的FR2、选自IGKV6-21*01的FR3、和选自hIGKJ4*01_12的FR4；
所述VH包含选自IGHV1-18*01的FR1、选自IGHV4-31*01的FR2、选自IGHV1-3*01的FR3、和选自hIGHJ6*01的FR4，所述VL包含选自IGKV4-1*01的FR1至FR3、和选自hIGKJ2*01的FR4；或
所述VH包含选自IGHV3-23*04的FR1至FR3、和选自IGHJ1*01的FR4，所述VL包含选自IGKV2-28*01的FR1至FR3、和选自IGKJ4*01的FR4。
一些具体实施方案中，抗CD25抗体或其抗原结合片段包含VH和/或VL，其中，
所述VH包含分别如SEQ ID NO：27、28和29所示的HCDR1、HCDR2和HCDR3，和选自IGHV1-46*01的FR1至FR3、和选自IGHJ1*01的FR4；所述VL包含分别如SEQ ID NO：30、31和32所示的LCDR1、LCDR2和LCDR3，和选自IGKV4-1*01的FR1至FR3、和选自IGKJ4*01的FR4；
所述VH包含分别如SEQ ID NO：33、34和35所示的HCDR1、HCDR2和HCDR3，和选自IGHV1-18*01的FR1至FR2、选自IGHV1-69*02的FR3、和选自hIGHJ6*01_14的FR4；所述VL包含分别如SEQ ID NO：36、37和38所示的LCDR1、LCDR2和LCDR3，和选自IGKV3-11*01的FR1、选自IGKV5-2*01的FR2、选自IGKV6-21*01的FR3、和选自hIGKJ4*01_12的FR4；
所述VH包含分别如SEQ ID NO：39、40和41所示的HCDR1、HCDR2和HCDR3，和选自IGHV1-18*01的FR1、选自IGHV4-31*01的FR2、选自IGHV1-3*01的FR3、和选自hIGHJ6*01的FR4；所述VL包含分别如SEQ ID NO：42、43和44所示的LCDR1、LCDR2和LCDR3，和选自IGKV4-1*01的FR1至FR3、和选自hIGKJ2*01的FR4；或
所述VH包含分别如SEQ ID NO：45、46和47所示的HCDR1、HCDR2和HCDR3，和选自IGHV3-23*04的FR1至FR3、和选自IGHJ1*01的FR4；所述VL包含分别如SEQ ID NO：48、49和50所示的LCDR1、LCDR2和LCDR3，和选自IGKV2-28*01的FR1至FR3、和选自IGKJ4*01的FR4。可以对上述FR进行回复突变，以保持抗体活性。
一些实施方案中，抗CD25抗体或其抗原结合片段包含VH和/或VL，其中：
所述VH如SEQ ID NO：19所示，所述VL如SEQ ID NO：20所示；
所述VH如SEQ ID NO：21所示，所述VL如SEQ ID NO：22所示；
所述VH如SEQ ID NO：23所示，所述VL如SEQ ID NO：24所示；
所述VH如SEQ ID NO：25所示，所述VL如SEQ ID NO：26所示；或
所述VH如SEQ ID NO：59所示，所述VL如SEQ ID NO：60所示；所述抗体为人源化抗体或其片段。
一些具体实施方案中，提供抗CD25抗体或其抗原结合片段变体，其包含VH和/或VL，所述VH和/或VL与前述抗CD25抗体或其抗原结合片段的VH和/或VL分别具有至少80％、85％、90％、95％、96％、97％、98％或99％的序列同一性。
一些实施方案中，抗CD25抗体或其抗原结合片段进一步包含抗体的重链恒定区和/或轻链恒定区，例如人IgG1、IgG2、IgG3和IgG4的重链恒定区及其常规变体，所述抗体的轻链恒定区选自人抗体κ和λ链恒定区及其常规变体；又例如鼠的IgG，例如鼠IgG2a。
一些实施方案中，所述抗原结合片段选自Fab、Fab'、F(ab')2、单链抗体(scFv)、 二聚化的V区(双抗体)、二硫键稳定化的V区(dsFv)和其他包含CDR的肽的抗原结合片段。
一些实施方案中，还提供一种分离的单克隆抗体或其抗原结合片段，其与前述任一项所述抗CD25抗体或其抗原结合片段竞争结合人CD25，或竞争结合相同的表位。
一些实施方案中，提供抗CD25抗体或其抗原结合片段，其包含VH和/或VL，其中：
所述VH如SEQ ID NO：1所示，所述VL如SEQ ID NO：2所示；
所述VH如SEQ ID NO：3所示，所述VL如SEQ ID NO：4所示；
所述VH如SEQ ID NO：5所示，所述VL如SEQ ID NO：6所示；
所述VH如SEQ ID NO：9所示，所述VL如SEQ ID NO：10所示；或
所述VH如SEQ ID NO：11所示，所述VL如SEQ ID NO：12所示；所述抗体或其抗原结合片段阻断或部分阻断IL-2与CD25的结合。
另一些实施方案中，提供抗CD25抗体或其抗原结合片段，其包含VH和/或VL，其中：
所述VH如SEQ ID NO：7所示，所述VL如SEQ ID NO：8所示；
所述VH如SEQ ID NO：13所示，所述VL如SEQ ID NO：14所示；
所述VH如SEQ ID NO：15所示，所述VL如SEQ ID NO：16所示；
所述VH如SEQ ID NO：17所示，所述VL如SEQ ID NO：18所示；
所述VH如SEQ ID NO：19所示，所述VL如SEQ ID NO：20所示；
所述VH如SEQ ID NO：21所示，所述VL如SEQ ID NO：22所示；
所述VH如SEQ ID NO：23所示，所述VL如SEQ ID NO：24所示；
所述VH如SEQ ID NO：25所示，所述VL如SEQ ID NO：26所示；或
所述VH如SEQ ID NO：59所示，所述VL如SEQ ID NO：60所示；所述抗体或其抗原结合片段不阻断、不抑制，几乎不阻断、不抑制，或较低程度的阻断、抑制IL-2与CD25的结合。例如，与不存在抗体的情况下的IL-2信号传导相比，所述抗CD25抗体或抗原结合片段阻断小于约50％、约40％、约35％、约30％、约25％、约20％、约15％、约10％的IL-2信号传导，例如小于约25％的IL-2信号传导。
一些实施方案中，提供抗CD25抗体或其抗原结合片段变体，其包含VH和/或VL，所述VH和/或VL与前述抗CD25抗体或其抗原结合片段的VH和/或VL具有至少80％、85％、90％、95％、96％、97％、98％或99％的序列同一性。
一些实施方案中，提供抗CD25抗体或其抗原结合片段变体，其包含VH和/或VL，所述VH和/或VL与前述抗CD25抗体或其抗原结合片段的重链可变区VH和/或VL相比，分别包含0、1、2、3、4、5、6、7、8、9或10个氨基酸变化。所述氨基酸变化可以是可变区中的氨基酸残基保守性置换。一些实施方案中，上 述包含氨基酸变化的抗体或抗原结合片段与亲本抗体或抗原结合片段具有相同或基本相同的与人CD25结合的亲和力和\或功能(例如ADCC、ADCP、抗肿瘤活性)。
一些实施方案中，抗CD25抗体或其抗原结合片段包含重链和/或轻链，其中，
所述重链如SEQ ID NO：51所示，所述轻链如SEQ ID NO：52所示；
所述重链如SEQ ID NO：53所示，所述轻链如SEQ ID NO：54所示；
所述重链如SEQ ID NO：55所示，所述轻链如SEQ ID NO：56所示；
所述重链如SEQ ID NO：57所示，所述轻链如SEQ ID NO：58所示；或
所述重链如SEQ ID NO：61所示，所述轻链如SEQ ID NO：62所示。
一些实施方案中，提供抗CD25抗体或其抗原结合片段，其包含重链和/或轻链，所述重链和/或轻链与前述抗CD25抗体或其抗原结合片段的重链和/或轻链分别具有至少70％、80％、85％、90％、95％、96％、97％、98％或99％的序列同一性。
一些实施方案中，本公开的抗CD25抗体或其抗原结合片段是IgG1型，Fc区具有去岩藻糖化位点，具有增强的FcγRIIIa结合能力。上述抗CD25抗体或其抗原结合片段能增加对Treg细胞的ADCC作用，增强其抗肿瘤活性。
一些实施方案中，本公开的抗CD25抗体或其抗原结合片段是IgG1型，Fc区具有去岩藻糖化位点(例如A330I突变)，具有降低的FcγRIIb结合能力。上述抗CD25抗体或其抗原结合片段能降低FcγRIIb受体介导的对ADCC/ADCP抑制性信号，以增强对Treg细胞的ADCC作用，增强其抗肿瘤活性。
一些实施方案中，本公开的抗CD25抗体或抗原结合片段具有下述特征中的至少一个：
(a)结合人CD25的KD值小于1×10-7M；
(b)不抑制(或基本上不抑制)IL-2与CD25的结合；
(c)消耗肿瘤浸润性Treg，不影响(或基本上不影响)Teff的功能；
(d)以高于1的活化抑制率(A/I)结合Fcγ受体；
(e)以比结合FcγRI、FcγRIIc和/或FcγRIIb更高的亲和力结合FcγRIIIa，以比结合FcγRIIb更高的亲和力结合FcγRIIIa；
(f)抑制多种肿瘤生长。
本公开的CD25结合蛋白或抗CD25抗体结合CD25的KD值可以小于1×10-7M，小于1×10-8M，小于1×10-9M，或小于1×10-10M。
本公开的抗CD25抗体或其抗原结合片段能够抑制肿瘤生长至少约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％。
一些实施方案中，本公开的抗CD25抗体或抗原结合片段以高亲和力结合FcγR，例如，以高亲和力结合活化受体。一些具体实施方案中，本公开的抗体以高亲和力结合FcγRI和/或FcγRIIA和/或FcγRIIIA。在具体的实施方式中，抗体 与至少一种活化性Fcγ受体以小于约10-6M、10-7M、10-8M、10-9M或10-10M的解离常数结合。
一些实施方案中，本公开的抗CD25抗体或抗原结合片段是IgG1抗体，能够与至少一种Fc活化受体结合。例如，该抗体可与选自FcγRI、FcγRIIa、FcγRIIc、FcγRIIIa和FcγRIIIb中的一种或多种受体结合。在一些具体实施方案中，本公开的抗体能够与FcγRIIIA结合。一些实施方案中，本公开的抗体能够与FcγRIIIA和FcγRIIA和任选的FcγRI结合。在一个方面，抗体能够以高亲和力与这些受体结合，例如以小于约10-7M、10-8M、10-9M或10-10M的解离常数结合。
一些实施方案中，本公开的抗CD25抗体或抗原结合片段以低亲和力结合抑制性受体FcγRIIb。在一个方面，抗体以高于约10-7M、高于约10-6M或高于约10-5M的解离常数结合FcγRIIb。
一些实施方案中，本公开的抗CD25抗体或抗原结合片段来自IgG1亚类，优选具有ADCC和/或ADCP活性。在另一些实施方案中，本公开的抗CD25抗体来自IgG2亚类。
一些实施方案中，本公开的抗CD25抗体或抗原结合片段抑制或阻断少于50％的通过CD25的IL-2信号传导。例如，与不存在抗体的情况下的IL-2信号传导相比，抗CD25抗体或抗原结合片段抑制或阻断小于约40％、35％、30％，优选小于约25％的IL-2信号传导。
多核苷酸和载体
本公开提供经分离的多核苷酸，其编码本公开的抗CD25抗体或其抗原结合片段。所述多核苷酸可以是DNA或RNA。
本公开提供含有如上所述的多核苷酸的表达载体，表达载体可以是真核表达载体、原核表达载体、病毒载体，例如质粒、粘粒、噬菌体。
宿主细胞
本公开提供用如上所述的表达载体转化的宿主细胞，其可以是真核细胞、原核细胞。
一些实施方案中，所述宿主细胞为细菌、酵母菌、哺乳动物细胞。一些具体实施方案中，所述宿主细胞为大肠杆菌、毕赤酵母、中国仓鼠卵巢(CHO)细胞或人胚肾(HEK)293细胞。
制备方法
本公开提供一种用于制备抗CD25抗体或其抗原结合片段的方法包括：在如前所述的宿主细胞中表达该抗体或其抗原结合片段，并自该宿主细胞中分离该抗体或其抗原结合片段。
可选地，还可以包含纯化步骤，例如，用含调整过的缓冲液的A或G Sepharose FF柱进行纯化，洗去非特异性结合的组分，再用PH梯度法洗脱结合的抗体，用SDS-PAGE检测，收集。可选地，用常规方法进行过滤浓缩。可溶的混合物和多 聚体，也可以用常规方法去除，比如分子筛、离子交换。得到的产物需立即冷冻，如-70℃，或者冻干。
生产和纯化抗体和抗原结合片段的方法在现有技术中熟知和能找到，如冷泉港的抗体实验技术指南(5-8章和15章)。如，可以用人FcRn或其片段免疫小鼠，所得到的抗体能被复性、纯化，并且可以用常规的方法进行氨基酸测序。抗原结合片段同样可以用常规方法制备。
本公开工程化的抗体或抗原结合片段可用常规方法制备和纯化。比如，编码重链和轻链的cDNA序列，可以克隆并重组至表达载体。重组的免疫球蛋白表达载体可以稳定地转染CHO细胞。哺乳动物类表达系统会导致抗体的糖基化，特别是在Fc区的高度保守N端。通过表达与人源抗原特异性结合的抗体得到稳定的克隆。阳性的克隆在生物反应器的无血清培养基中扩大培养以生产抗体。分泌了抗体的培养液可以用常规技术纯化、收集。抗体可用常规方法进行过滤浓缩。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛，离子交换。
组合物
本公开提供一种组合物，例如药物组合物，其含有治疗有效量的如上所述的抗CD25抗体或其抗原结合片段和可药用的赋形剂、稀释或载体。在一些具体实施方式中，所述药物组合物单位计量中可含有0.01至99重量％的抗CD25抗体或其抗原结合片段，或药物组合物单位剂量中含抗CD25抗体或其抗原结合片段的量为0.1-2000mg，在一些具体实施方式中为1-1000mg。
治疗方法和制药用途
本公开提供抗CD25抗体或其抗原结合片段、含有所述抗CD25抗体或其抗原结合片段的药物组合物、编码多核苷酸中的任一项或其任意组合，用于诊断、治疗、预防疾病的方法和制备药物、药物组合物的用途(例如，用于治疗或预防增殖性病症(例如癌症或肿瘤)或延缓相关病症进展。
一些实施方案中，提供预防、治疗或缓解受试者病症的方法，包含向所述受试者施用本公开的抗CD25抗体或其抗原结合片段、含有所述抗CD25抗体或其抗原结合片段的药物组合物和/或编码多核苷酸。一些具体实施方案中，所述受试者病症是增殖性疾病，例如肿瘤或癌症。一些实施方案中，上述受试者患有已形成的肿瘤，例如实体瘤。
一些实施方案中，提供减少受试者肿瘤内部或肿瘤侵润性Treg的细胞数量方法；一些实施方案中，提供消除或抑制受试者肿瘤内部或肿瘤侵润性的Treg的细胞活性的方法，均包括向所述受试者施用本公开的抗CD25抗体或其抗原结合片段、含有所述抗CD25抗体或其抗原结合片段的药物组合物和/或编码多核苷酸。
一些实施方案中，提供增加受试者肿瘤内部Teff/Treg的比例的方法，包括向所述受试者施用本公开的抗CD25抗体或其抗原结合片段、含有所述抗CD25抗体或其抗原结合片段的药物组合物和/或编码多核苷酸。在一些具体实施方式中，肿 瘤中效应T细胞与调节性T细胞的比例(Teff/Treg)增加至超过5、10、15、20、40或80。
一些实施方案中，提供增强受试者体内针对肿瘤细胞的CDC、ADCC和/或ADCP的方法，包括向所述受试者施用本公开的抗CD25抗体或其抗原结合片段、含有所述抗CD25抗体或其抗原结合片段的药物组合物和/或编码多核苷酸。一些具体实施方案中，受试者体内针对肿瘤细胞的ADCC和/或ADCP效应增强。一些更具体实施方案中，受试者体内针对肿瘤细胞的ADCC效应增强。
一些实施方案中，提供本公开抗CD25抗体或其抗原结合片段、含有所述抗CD25抗体或其抗原结合片段的药物组合物和/或编码多核苷酸用于制备预防、治疗或缓解受试者病症的制药用途，用于制备减少受试者肿瘤内部或肿瘤侵润性Treg的细胞数量的制药用途，用于制备消除或抑制受试者肿瘤内部或肿瘤侵润性的Treg的细胞活性的药物的制药用途，用于制备增加受试者肿瘤内部Teff/Treg的比例的制药用途，用于制备增强受试者体内针对肿瘤细胞的CDC、ADCC和/或ADCP的制药用途。
一些具体实施方案中，上述受试者的病症为增殖性病症(例如癌症或肿瘤)或患有增殖性病症(例如癌症或肿瘤)。所述肿瘤包括但不限于癌、淋巴瘤、白血病、胚细胞瘤和肉瘤。这类癌症的更具体的实例包括鳞状细胞癌、骨髓瘤、小细胞肺癌、非小细胞肺癌、神经胶质瘤、肝细胞癌(HCC)、霍奇金淋巴瘤、非霍奇金淋巴瘤、急性髓性白血病(AML)、多种骨髓瘤、胃肠(道)癌、肾癌、卵巢癌、肝脏癌、淋巴母细胞白血病、淋巴细胞白血病、结直肠癌、子宫内膜癌、肾癌、前列腺癌、甲状腺癌、黑色素瘤、软骨肉瘤、神经母细胞瘤、胰腺癌、多形性胶质母细胞瘤、宫颈癌、脑癌、胃癌、膀胱癌、肝癌、乳腺癌、结肠癌和头颈癌。
一些具体实施方案中，所述癌症或肿瘤可以是实体瘤，包括但不限于肉瘤(包括由组织(例如松质骨、软骨、脂肪、肌肉、血管、造血细胞或纤维结缔组织)中的间充质来源的转化细胞产生的癌症)、癌(包括由上皮细胞产生的肿瘤)、间皮瘤、神经母细胞瘤、视网膜母细胞瘤等。涉及实体瘤的癌症包括但不限于，脑癌、肺癌、胃癌、十二指肠癌、食道癌、乳腺癌、结肠和直肠癌、肾癌、膀胱癌、肾脏癌、胰腺癌、前列腺癌、卵巢癌、黑色素瘤、口腔癌、肉瘤、眼癌、甲状腺癌、尿道癌、阴道癌、颈癌、淋巴瘤等。
一些具体实施方案中，所述癌症涉及表达CD25的肿瘤，包括但不限于淋巴瘤，例如霍奇金淋巴瘤和淋巴细胞性白血病，例如慢性淋巴细胞白血病(CLL)。
检测用途
本公开提供抗CD25抗体或其抗原结合片段的检测用途。
本公开提供检测CD25的试剂，所述试剂包含抗CD25抗体或其抗原结合片段。本公开还提供用于体内或体外检测CD25的方法、系统或装置，其包括使用抗CD25抗体或其抗原结合片段。
一些实施方案中，体外检测方法、系统或装置可能例如包括(1)使样品与抗CD25抗体或其抗原结合片段接触；(2)检测在抗CD25抗体或其抗原结合片段和样品之间形成的复合物；和/或(3)使参比样品(例如，对照样品)与抗体接触；和(4)通过与参比样品比较，确定抗体和样品之间复合物形成的程度。如与对照样品或受试者中相比，样品或受试者中复合物形成的变化(例如，统计学上的显著变化)表示样品中存在CD25。
一些实施方案中，出于检测目的，本公开的CD25结合蛋白或抗CD25抗体可以用荧光团和发色团标记。
一些实施方案中，还提供试剂盒，所述试剂盒包含与CD25结合蛋白或抗CD25抗体，还可以包含诊断使用说明。试剂盒还可以含有至少一种额外的试剂，如标记物或额外的诊断剂。对于体内使用，抗体可以配制为药物组合物。
本公开实施例提供的抗CD25抗体或其抗原结合片段具有高特异性、高亲和力、低免疫原性的特点。同时，本公开的抗体具有良好的抑制Treg，不影响Teff，增强受试者体内ADCC、ADCP和\或CDC，抑制肿瘤发生发展的效果。
附图说明：
图1A和图1B：ELISA鉴定重组蛋白活性。其中，图1A为重组人CD25蛋白的活性检测结果，图1B为重组猴和重组小鼠CD25蛋白的活性检测结果。
图2：FACS测定稳定表达人CD25蛋白的细胞株2F8，其中，白色峰为CD25阳性峰，灰色峰为对照峰，一抗为人IgG1阴性对照抗体。
图3A至图3C：FACS测定检测抗体功能。图3A为FACS测定抗体DAC、7G7B6、Tab06与CHO-K1细胞的结合结果，图3B为FACS测定抗体DAC、7G7B6、Tab06与稳定表达人CD25蛋白的CHO-K1-CD25细胞的结合结果，图3C为FACS测定抗体DAC、7G7B6、Tab06与Su-DHL-1的结合结果，其中，使用小鼠IgG或人IgG同种型作为阴性对照。
图4A至图4C：ELISA测定检测抗体与CD25结合。图4A为7G7B6、Tab06与重组人CD25蛋白的结合结果，图4B为7G7B6、Tab06与重组猴CD25蛋白的结合结果，图4C为7D4与重组小鼠CD25蛋白的结合结果，所使用的阴性对照为小鼠IgG和人IgG。
图5A至图5C：FACS检测嵌合型抗CD25抗体与SU-DHL-1细胞结合。图5A为cAb001、cAb002、cAb004、cAb006的检测结果，图5B为cAb028、cAb029、cAb037的检测结果，图5C为cAb042、cAb046的检测结果，所使用的阳性对照为Tab06和DAC，阴性对照为人IgG(即hIgG)。
图6A至图6G：FACS检测嵌合型抗CD25抗体与Treg细胞、激活的CD4 +和CD8 +效应T细胞上的CD25抗原结合。图6A为cAb006的结果，图6B为cAb037的结果，图6C为cAb042的结果，图6D为cAb046的结果，图6E为阳性对照7G7B6 的结果，图6F为阳性对照DAC的结果，图6G为阳性对照Tab06的结果。
图7A至图7B：FACS检测嵌合型抗CD25抗体对于IL-2和受体CD25的结合能力的影响。图7A为cAb001、cAb002、cAb028、cAb029的检测结果图，图7B为cAb006、cAb037、cAb042、cAb046的检测结果图，所使用的阻碍IL-2和CD25结合的抗CD25抗体对照为DAC，不阻碍IL-2和CD25结合的抗CD25抗体对照为Tab06，阴性对照为人IgG(即hIgG-1)。
图8：FACS检测人源化抗CD25抗体与SU-DHL-1细胞的结合。
图9：FACS检测人源化抗CD25抗体对人外周血T淋巴细胞pStat5信号通路的影响。
图10：通过检测ADCC报告基因在细胞中的荧光活性，检测人源化抗CD25抗体介导的对SU-DHL-1细胞的ADCC作用。
图11A和图11B：人源化抗CD25抗体介导的对hCD25小鼠的MC38移植瘤的抗肿瘤活性结果，其中，图11A肿瘤抑制效果图，图11B为对应的小鼠体重图。
图12A和图12B：人源化抗CD25抗体介导的对hCD25小鼠的MC38移植瘤的肿瘤内淋巴细胞分析结果，其中，图12A为抗体对Treg的杀伤结果，图12B为抗体对CD3的上调。
具体实施方式
发明详述
为了更容易理解本公开，以下具体定义了某些技术和科学。除显而易见在本文件中的它处另有明确定义，否则本文使用的所有其它技术和科学都具有本公开所属领域的一般技术人员通常理解的含义。
本公开所用氨基酸三字母代码和单字母代码如J.Biol.Chem，243，p3558(1968)中所述。
“CD25”或“CD25蛋白质”或“CD25多肽”可以任选地包括任何这类蛋白质或其变异体、结合物或片段，包括(但不限于)如本文所述的已知或野生型CD25，以及任何天然产生的剪接变异体、氨基酸变异体或同工型。完整的人类CD25序列可以在Uniprot登录号P01589找到，其第22至240个氨基酸对应成熟人CD25的胞外域。
“与CD25结合”，指能与CD25或其表位相互作用，所述CD25或其表位可以是人源的。“抗原结合位点”指抗原上不连续的，由本公开抗体或抗原结合片段识别的三维空间位点。
“抗体”指免疫球蛋白，是由两条相同的重链和两条相同的轻链通过链间二硫键连接而成的四肽链结构。免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将免疫球蛋白分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、 α链和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4。轻链通过恒定区的不同分为κ链或λ链。五类Ig中第每类Ig都可以有κ链或λ链。抗体重链和轻链靠近N端的约110个氨基酸的序列变化很大，为可变区(V区)；靠近C端的其余氨基酸序列相对稳定，为恒定区(C区)。可变区包括3个高变区(HVR)和4个序列相对保守的骨架区(FR)。3个高变区决定抗体的特异性，又称为互补性决定区(CDR)。每条轻链可变区(VL)和重链可变区(VH)由3个CDR区4个FR区组成，从氨基端到羧基端依次排列的顺序为：FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。轻链的3个CDR区指LCDR1，LCDR2，和LCDR3；重链的3个CDR区指HCDR1，HCDR2和HCDR3。在一些实施例中，本公开的抗体特异性地或基本上特异性地结合到CD25。
“结合CD25的抗体”是指能够结合IL-2受体的CD25亚基的抗体。该亚基也称为IL-2受体的α亚基。这种抗体在本文中也称为“抗CD25抗体”。
本公开的抗体或抗原结合片段的VL区和VH区的CDR氨基酸残基在数量和位置符合已知的Kabat编号系统。Kabat中的EU编号一般也用于恒定结构域和/或Fc结构域。
对于“CDR”的确定或定义，能够通过分辨抗体的结构和/或分辨抗体-配体复合物的结构来完成CDR的确定性描绘和包含抗体的结合位点的残基的鉴定。这可通过本领域技术人员已知的各种技术中的任一种，例如X射线晶体学来实现。多种分析方法可用于鉴定CDR，包括但不限于Kabat编号系统、Chothia编号系统、AbM编号系统、IMGT编号系统、接触定义、构象定义。Kabat编号系统是用于编号抗体中残基的标准并且通常用于鉴定CDR区域(参见例如Johnson&Wu，2000，Nucleic Acids Res.，28：214-8)。Chothia编号系统与Kabat编号系统类似，但Chothia编号系统考虑了某些结构环区域的位置。(参见例如Chothia等，1986，J.Mol.Biol.，196：901-17；Chothia等人，1989，Nature，342：877-83)。AbM编号系统使用建模抗体结构的由Oxford MolecuLar Group生产的计算机程序集成套件(参见例如Martin等，1989，ProcNatl Acad Sci(USA)，86：9268-9272；“AbMTM，A Computer Program for ModelingVariable Regions of Antibodies，”Oxford，UK；Oxford MolecuLar，Ltd)。AbM编号系统使用知识数据库和从头开始方法的组合，从基本序列建模抗体的三级结构(参见Samudrala等，1999，在PROTEINS，Structure，Function and Genetics Suppl.，3：194-198中的“Ab Initio Protein Structure Prediction Using a Combined HierarchicalApproach”描述的那些)。接触定义基于可用复杂晶体结构的分析(参见例如MacCallum等，1996，J.Mol.Biol.，5：732-45)。构象定义中，CDR的位置可鉴定为对抗原结合做出焓贡献的残基(参见例如Makabe等，2008，Journal ofBiological Chemistry，283：1156-1166)。另外其它的CDR边界定义可能不严格遵循上述方法之一，但仍然与Kabat CDR的至少一部分重叠，尽管根据特定残基 或残基组不显著影响抗原结合的预测或实验结果，它们可缩短或延长。如本文使用的，CDR可指通过本领域已知的任何方法(包括方法的组合)定义的CDR。本文使用的方法可利用根据这些方法中的任一种定义的CDR。本公开的实施例中采用kabat编号规则定义CDR，但本领域技术人员能够理解的是，也可以根据Chothia、延伸的、AbM、IMGT、接触和/或构象定义中的任一个来重新定义CDR。
IgG抗体的Fc区与几种细胞Fcγ受体(FcγR)相互作用以刺激和调节下游效应子机制。有五种活化受体，即FcγRI(CD64)、FcγRIIa(CD32a)、FcγRIIc(CD32c)、FcγRIIIa(CD16a)和FcγRIIIb(CD16b)，以及一种抑制性受体FcγRIIb(CD32b)。IgG抗体与免疫系统的通讯由FcγR控制和介导，FcγR将抗体感知和收集的信息传递给免疫系统，从而提供先天和适应性免疫系统之间的联系，尤其是在生物治疗的背景下(Hayes J et al.,2016.JInflamm Res 9:209–219)。IgG亚类结合FcγR的能力不同，这种差异结合决定了它们引发一系列功能性反应的能力。例如，在人类中，FcγRIIIa是参与抗体依赖性细胞介导的细胞毒作用(ADCC)活化的主要受体，IgG1紧接着IgG3显示出对该受体的最高亲和力，反映出它们有效诱导ADCC的能力，IgG2对该受体具有弱结合，但已发现具有人IgG2同种型的抗CD25抗体也能有效地消耗Treg。
“调节性T细胞”、“Treg”或“Treg细胞”是指专门控制自身免疫、变态反应和感染的CD4 +T淋巴细胞谱系。通常，它们调节T细胞群的活性，但它们也可以影响某些先天免疫系统细胞类型。可通过生物标志物CD4、CD25和Foxp3的表达来鉴定Treg。天然存在的Treg细胞通常占外周CD4 +T淋巴细胞的约5至10％。然而，在肿瘤微环境(即肿瘤浸润性Treg细胞)内，它们可占总CD4 +T淋巴细胞群的20至30％。活化的人Treg细胞可通过穿孔因子或颗粒酶B依赖性途径直接杀死靶细胞，如Teff和APC(抗原递呈细胞)；细胞毒性T淋巴细胞相关抗原4(CTLA4 +)Treg细胞通过APC来诱导吲哚胺2，3-双加氧酶(IDO)表达，这些转而通过减少色氨酸抑制T细胞活化；Treg细胞可以在体内释放白细胞介素-10(IL-10)和转化生长因子(TGFβ)，从而通过抑制MHC分子、CD80、CD86和IL-12的表达来直接抑制T细胞活化并抑制APC功能。Treg细胞还可以通过表达高水平的CTLA4来抑制免疫，CTLA4可以与抗原呈递细胞上的CD80和CD86结合并阻止效应T细胞的正确活化。
本文的“免疫效应细胞”是指参与免疫应答的效应阶段的免疫细胞。示例性免疫细胞包括髓样或淋巴样细胞，例如淋巴细胞(例如、B细胞和包括细胞溶解性T细胞(CTL)的T细胞)、杀伤细胞、天然杀伤细胞、巨噬细胞、单核细胞、嗜酸性粒细胞、嗜中性粒细胞、多形核细胞、粒细胞、肥大细胞和嗜碱性粒细胞。
本文的“不抑制”、“不阻断”、“非阻断性”、“非IL-2阻断性”、“无阻断”和类似(关于在抗CD25抗体存在下不阻断或不抑制IL-2与CD25的结合，例如“不抑制IL-2与CD25的结合”)包括其中抗CD25抗体不阻断或不抑制IL-2通过CD25 进行信号传导，特别是不抑制表达CD25的细胞中的白细胞介素-2信号传导。也就是说，与不存在抗体的情况下的IL-2信号传导相比，本公开的抗CD25抗体抑制少于50％的通过CD25的IL-2信号传导。优选地，与不存在抗体的情况下的IL-2信号传导相比，抗CD25抗体抑制小于约40％、35％、30％，优选小于约25％的IL-2信号传导。
“抗体依赖性细胞介导的细胞毒作用”(ADCC)是指一种细胞介导的反应，其中表达Fc受体(FcR)的非特异性细胞毒性细胞(例如自然杀伤(NK)细胞、嗜中性粒细胞和巨噬细胞)识别靶细胞上结合的抗体从而导致靶细胞裂解。“抗体依赖性细胞介导的吞噬作用”(ADCP)是指一种细胞介导的反应，其中表达Fc受体(FcR)的吞噬细胞(例如巨噬细胞)识别靶细胞上结合的抗体从而导致对靶细胞的吞噬作用。“补体依赖性细胞毒作用”(CDC)是指在补体存在下，抗体对表达抗原的细胞的裂解。CDC、ADCC和ADCP可以使用本领域已知并可用的测定方法(Clynes et al.(1998)Proc Natl Acad Sci USA95,652-6)来测量，抗体的恒定区在抗体固定补体和介导细胞依赖性细胞毒作用和吞噬作用的能力中是重要的。因此，可以基于抗体是否需要介导ADCC、ADCP来选择抗体的同种型。
ADCC可以通过从抗体聚糖中消除岩藻糖部分的方法来增加，例如通过在YB2/0细胞系中产生抗体，或者通过在人IgG1的Fc部分上引入特定突变(例如S298A/E333A/K334A、S239D/I332E/A330L、G236A/S239D/A330L/I332E)(Lazar et al.(2006)Proc Natl AcadSci USA 103,2005-2010；Smith et al.(2012)Proc Natl 25Acad Sci USA 109,6181-6)。ADCP也可以通过在人IgG1的Fc部分上引入特定突变来增加(Richards et al.(2008)MolCancer Ther 7,2517-27)。
“鼠源抗体”在本公开中为根据本领域知识和技能制备的针对人CD25或其表位的单克隆抗体。制备时用CD25抗原注射试验对象，然后分离表达具有所需序列或功能特性的抗体的杂交瘤。在本公开一个具体的实施方案中，所述的鼠源抗人CD25抗体或其抗原结合片段，可进一步包含鼠源κ、λ链或其变体的轻链恒定区，或进一步包含鼠源IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区。
“嵌合抗体(chimeric antibody)”，是将鼠源性抗体的可变区与人抗体的恒定区融合而成的抗体，可以减轻鼠源性抗体诱发的免疫应答反应。建立嵌合抗体，要选建立分泌鼠源性特异性单抗的杂交瘤，然后从小鼠杂交瘤细胞中克隆可变区基因，再要据需要克隆人抗体的恒定区基因，将小鼠可变区基因与人恒定区基因连接成嵌合基因后插入人载体中，最后在真核工业系统或原核工业系统中表达嵌合抗体分子。人抗体的恒定区可选自人源IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区，优选包含人源IgG2或IgG4重链恒定区，或者使用氨基酸突变后无ADCC(antibody-dependent cell-mediated cytotoxicity，抗体依赖的细胞介导的细胞毒作用)毒性的IgG1。
“人源化抗体(humanized antibody)”，也称为CDR移植抗体(CDR-grafted  antibody)，是指将小鼠的CDR序列移植到人的抗体可变区框架中产生的抗体。可以克服嵌合抗体由于携带大量小鼠蛋白成分，从而诱导的强烈的免疫应答反应。为避免在免疫原性下降的同时引起活性的下降，可对所述的人抗体可变区可进行最少反向突变，以保持活性。本公开的抗体可以是亲和力成熟的人源化抗体，亲和力成熟的抗体亲本序列(包含所有序列)的CDR至少80％相同，例如90％相同。亲和力成熟的抗体是在一个或多个CDR中具有一个或多个改变的氨基酸的抗体，其导致抗体与不具有改变的氨基酸的亲本抗体相比对CD25的亲和力改善。
“人抗体”包括具有人种系免疫球蛋白序列的可变和恒定区的抗体。本公开的人抗体可包括不由人种系免疫球蛋白序列编码的氨基酸残基(如通过体外随机或位点特异性诱变或通过体内体细胞突变所引入的突变)。然而，“人抗体”不包括这样的抗体，即其中已将衍生自另一种哺乳动物物种(诸如小鼠)种系的CDR序列移植到人骨架序列上(即“人源化抗体”)。
“抗原结合片段”包括单链抗体(即全长重链和轻链)；Fab、修饰的Fab、Fab’、修饰的Fab’、F(ab’)2、Fv、Fab-Fv、Fab-dsFv、单结构域抗体(例如VH或VL或VHH)、scFv、二价或三价或四价抗体、Bis-scFv、diabody、tribody、triabody、tetrabody和上述任意一种的表位结合片段(参见例如Holliger and Hudson,2005,Nature Biotech.23(9):1126-1136；Adair and Lawson,2005,Drug Design Reviews-Online 2(3),209-217)。产生和制备这些抗体片段的方法在本领域是公知的(参见例如Verma等人,1998,Journal ofImmunological Methods,216,165-181)。Fab-Fv形式首先公开于WO2009/040562，其二硫键稳定化形式Fab-dsFv首先公开于WO2010/035012。本公开的抗原结合片段还包括描述于WO2005/003169、WO2005/003170和WO2005/003171中的Fab和Fab’片段。多价抗体可包含多特异性例如双特异性或可以是单特异性的(参见例如WO92/22583和WO05/113605)，后者的一个实例是描述于WO 92/22583中的Tri-Fab(或TFM)。在一些具体实施方式中，本公开的抗CD25抗体涵盖双、多特异性抗体。
“表位”是指抗原上与免疫球蛋白或抗体特异性结合的位点。表位可以由相邻的氨基酸、或通过蛋白质的三级折叠而并列的不相邻的氨基酸形成。由相邻的氨基酸形成的表位通常在暴露于变性溶剂后保持，而通过三级折叠形成的表位通常在变性溶剂处理后丧失。表位通常以独特的空间构象包括至少3-15个氨基酸。确定什么表位由给定的抗体结合的方法在本领域中是熟知的，包括免疫印迹和免疫沉淀检测分析等。确定表位的空间构象的方法包括本领域中的技术和本文所述的技术，例如X射线晶体分析法和二维核磁共振等。
“特异性结合”、“选择性结合”是指抗体与预定的抗原上的表位结合。通常，当使用重组人CD25或其表位作为分析物并使用抗体作为配体，在仪器中通过表面等离子体共振(SPR)技术测定时，抗体以大约低于10-7M或甚至更小的平衡解离常数(KD)与预定的抗原或其表位结合，并且其与预定抗原或其表位结合的亲 和力是其与预定抗原(或其表位)或紧密相关的抗原之外的非特异性抗原(如BSA等)结合的亲和力的至少两倍。“识别抗原的抗体”在本文中可以与“特异性结合的抗体”互换使用。
“结合亲和力”是指表观缔合常数或Ka。Ka是解离常数(Kd)的倒数。例如结合蛋白对特定靶分子可具有至少10 -5、10 -6、10 -7、10 -8、10 -9、10 -10和10 -11M的结合亲和力。相对于第二靶标，结合配体与第一靶标的更高的亲和结合可通过比结合第二靶标的Ka(或更小数值的Kd)更高的结合第一靶标的Ka(或Kd的数值)来显示。在这些情况下，相对于第二靶标(例如第二构象或其类似物的相同蛋白；或第二蛋白)，结合蛋白对第一靶标有特异性(例如第一构象或其类似物的蛋白)。结合亲和力的差异(例如对特异性或其它比较)至少是1.5、2、3、4、5、10、15、20、50、70、80、100、500、1000或105倍。
“保守性置换”指置换为具有与原始氨基酸残基相似的特性的另一个氨基酸残基。例如，赖氨酸、精氨酸和组氨酸具有相似的特性，在于它们具有碱性侧链，并且天冬氨酸和谷氨酸具有相似的特性，在于它们具有酸性侧链。此外，甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸和色氨酸具有相似的特性，在于它们具有不带电荷极性侧链，并且丙氨酸、缬氨酸、亮氨酸、苏氨酸、异亮氨酸、脯氨酸、苯丙氨酸和甲硫氨酸具有相似的特性，在于它们具有非极性侧链。另外，酪氨酸、苯丙氨酸、色氨酸和组氨酸具有相似的特性，在于它们具有芳族侧链。因此，本领域技术人员将显而易见，甚至当置换如上文所述的显示相似特性的组中的氨基酸残基时，它将不显示特性的特定变化。
“序列同源性”或“序列同一性”是指两个多核苷酸序列之间或两个多肽之间的序列相似性。当两个比较序列中的位置均被相同碱基或氨基酸单体亚基占据时，例如如果两个DNA分子的每一个位置都被腺嘌呤占据时，那么所述分子在该位置是同源的。两个序列之间的同源性百分率是两个序列共有的匹配或同源位置数除以比较的位置数×100％的函数。例如，在序列最佳比对时，如果两个序列中的10个位置有6个匹配或同源，那么两个序列为60％同源。一般而言，当比对两个序列而得到最大的同源性百分率时进行比较。
“交叉反应”是指本公开的抗体与来自不同物种的CD25结合的能力。例如，结合人CD25的本公开的抗体也可以结合另一物种的CD25。交叉反应性是通过在结合测定(例如SPR和ELISA)中检测与纯化抗原的特异性反应性，或与生理表达CD25的细胞的结合或功能性相互作用来测量。确定交叉反应性的方法包括如本文所述的标准结合测定，例如表面等离子体共振分析，或流式细胞术。
“抑制”或“阻断”可互换使用，并涵盖部分和完全抑制/阻断这两者。对CD25的抑制/阻断优选地降低或改变无抑制或阻断的情况下发生CD25结合时出现活性的正常水平或类型。抑制和阻断也旨在包括与抗CD25抗体接触时，与未与抗CD25抗体接触的CD25相比，任何可测量的CD25结合亲和力降低。
“抑制生长”(例如涉及细胞)旨在包括细胞生长任何可测量的降低。
生产和纯化抗体和抗原结合片段的方法在现有技术中熟知和能找到，如冷泉港的抗体实验技术指南(5-8章和15章)。如，可以用人CD25或其片段免疫小鼠，所得到的抗体能被复性、纯化，并且可以用常规的方法进行氨基酸测序。抗原结合片段同样可以用常规方法制备。发明所述的抗体或抗原结合片段用基因工程方法在非人源的CDR区加上一个或多个人FR区。人FR种系序列可以从ImMunoGeneTics(IMGT)的网站http：//imgt.cines.fr得到，或者从免疫球蛋白杂志，2001ISBN012441351上获得。
本公开的抗体可以是多克隆的、单克隆的、异种的、同种异体的、同基因的或其经过修饰的形式，其中单克隆抗体尤其适用于多个实施例中。一般来说，本公开的抗体是重组抗体。如本文所用的“重组”泛指例如细胞或核酸、蛋白质或载体等产品，表示所述细胞、核酸、蛋白质或载体已经通过引入异源核酸或蛋白质或改变天然核酸或蛋白质而加以修饰，或所述细胞来源于如此修饰的细胞。例如，重组细胞表达天然(非重组)细胞形式内不存在的基因或表达原本异常表达、低表达或完全不表达的天然基因。
“单克隆抗体”或“单抗”指由单一的克隆细胞株得到的抗体，所述的细胞株不限于真核的、原核的或噬菌体的克隆细胞株。单克隆抗体或抗原结合片段可以用如杂交瘤技术、重组技术、噬菌体展示技术，合成技术(如CDR-嫁接)，或其它现有技术进行重组得到。
本公开的抗体或抗原结合片段可用常规方法制备和纯化。比如，编码重链和轻链的cDNA序列，可以克隆并重组至表达载体。重组的免疫球蛋白表达载体可以稳定地转染CHO细胞。哺乳动物类表达系统会导致抗体的糖基化，特别是在Fc区的高度保守N端。通过表达与人源抗原特异性结合的抗体得到稳定的克隆。阳性的克隆在生物反应器的无血清培养基中扩大培养以生产抗体。分泌了抗体的培养液可以用常规技术纯化、收集。抗体可用常规方法进行过滤浓缩。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛，离子交换。得到的产物需立即冷冻，如-70℃，或者冻干。
可使用本领域技术人员已知的常规技术，就与相同表位的结合竞争性筛选抗体。例如，可进行竞争和交叉竞争研究，以获得彼此竞争或交叉竞争与抗原结合的抗体。基于它们的交叉竞争来获得结合相同表位的抗体的高通量方法描述于国际专利公开WO03/48731中。因此，可使用本领域技术人员已知的常规技术，获得与本公开的抗体分子竞争结合CD25上的相同表位的抗体及其抗原结合片段。此处全文引入WO2018167104中检测抗体竞争结合的方法。
“给予”、“施用”和“处理”当应用于动物、人、实验受试者、细胞、组织、器官或生物流体时，是指外源性药物、治疗剂、诊断剂或组合物与动物、人、受试者、细胞、组织、器官或生物流体的接触。“给予”、“施用”和“处理”可以指 例如治疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触，以及试剂与流体的接触，其中所述流体与细胞接触。“给予”、“施用”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞体外和离体处理例如细胞。“处理”当应用于人、兽医学或研究受试者时，是指治疗处理、预防或预防性措施，研究和诊断应用。
“治疗”意指给予受试者内用或外用治疗剂，诸如包含本公开的任一种抗体或其抗原结合片段或其偶联物的组合物，所述受试者已经患有、疑似患有、倾向于患有一种或多种疾病或其症状，而已知所述治疗剂对这些症状具有治疗作用。通常，在受治疗受试者或群体中以有效缓解一种或多种疾病症状的量给予治疗剂，无论是通过诱导这类症状退化还是抑制这类症状发展到任何临床右测量的程度。有效缓解任何具体疾病症状的治疗剂的量(也称作“治疗有效量”)可根据多种因素变化，例如受试者的疾病状态、年龄和体重，以及药物在受试者产生需要疗效的能力。通过医生或其它专业卫生保健人士通常用于评价该症状的严重性或进展状况的任何临床检测方法，可评价疾病症状是否已被减轻。尽本公开的实施方案(例如治疗方法或制品)在缓解某个受试者中目标疾病症状方面可能无效，但是根据本领域已知的任何统计学检验方法如Student t检验、卡方检验、依据Mann和Whitney的U检验、Kruskal-Wallis检验(H检验)、Jonckheere-Terpstra检验和Wilcoxon检验确定，其在统计学显著数目的受试者中应当减轻目标疾病症状。
在本文中使用的术语“治疗癌症”包括(a)抑制癌症，即阻止其发展，包括但不限于阻断或延缓癌症的进展、阻断或延缓癌症的转移；和/或(b)缓解癌症，即引起癌症消退，包括但不限于减轻或缓解所述癌症相关的一种或多种症状、减轻或缓解转移的癌症、和/或使肿瘤减小或消除。
本文的“预防”是指延迟或预防癌症症状的发作。预防可能是绝对的(由此不会发生疾病)或仅在某些个体中或有限的时间内有效。
“有效量”包含足以改善或预防医字病症的症状或病症的量。有效量还意指足以允许或促进诊断的量。用于特定受试者或兽医学受试者的有效量可依据以下因素而变化：如待治疗的病症、受试者的总体健康情况、给药的方法途径和剂量以及副作用严重性。有效量可以是避免显著副作用或毒性作用的最大剂量或给药方案。
“细胞”、“细胞系”和“细胞培养物”可互换使用，并且所有这类名称都包括其后代。还应当理解的是，由于故意或非有意的突变，所有后代在DNA含量方面不可能精确相同。包括具有与最初转化细胞中筛选的相同的功能或生物学活性的突变后代。
“任选”或“任选地”意味着随后所描述地事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生地场合。例如，“任选包含1-3个抗体重链可变区”意味着特定序列的抗体重链可变区可以但不必须存在。
“药物组合物”表示含有一种或多种本文所述抗体或抗原结合片段或其生理学上/可药用的盐或前体药物与其他化学组分的混合物，以及其他组分例如生理学/可药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
“肿瘤”适用于被诊断出或被怀疑患有肿瘤的受试者，癌症是指任何大小的恶性或潜在恶性赘生物或组织肿块，并且包括原发性肿瘤和继发性赘生物。“癌症”、“恶性肿瘤”、“赘生物”、“肿瘤”和“癌症”在本文中也可互换使用，指的是表现出相对异常、不受控制和/或自主生长的肿瘤和肿瘤细胞，因此它们表现出以显著丧失对细胞增殖的控制为特征的异常生长表型。通常，用于检测或治疗的目的细胞包括癌前(例如良性)、恶性、转移前、转移和非转移细胞。“实体瘤”是组织的异常生长或肿块，其通常不包含囊肿或液体区域，特别是除白血病或非实体淋巴癌之外的肿瘤和/或转移(无论位于何处)。实体瘤可以是良性或恶性的。不同类型的实体瘤以形成它们的细胞类型和/或它们所在的组织或器官命名。实体瘤的实例包括但不限于肉瘤(包括从组织，例如松质骨、软骨、脂肪、肌肉、血管、造血细胞或纤维结缔组织中的间充质来源的转化细胞产生的癌症)、癌(包括来自上皮细胞的肿瘤)、黑色素瘤、淋巴瘤、间皮瘤、神经母细胞瘤和视网膜母细胞瘤。
如无具体说明，在本文使用的冠词“一种”(a)和“一”(an)是指一个或多于一个(即至少一个/种)所述冠词的语法对象。举例来说，“一种元素”是指一个或多于一个元素。
具体实施方式
以下结合实施例用于进一步描述，但这些实施例并非限制的范围。
实施例或测试例中未注明具体条件的实验方法，通常按照常规条件，或按照原料或商品制造厂商所建议的条件。参见Sambrook等，分子克隆，实验室手册，冷泉港实验室；当代分子生物学方法，Ausubel等著，Greene出版协会，Wiley Interscience，NY。未注明具体来源的试剂，为市场购买的常规试剂。
实施例1.重组CD25蛋白的活性检测
重组人CD25-Fc融合蛋白(购于ACRO Biosystems)是将人IgG1Fc标签蛋白融合在人CD25蛋白(GenBank，登录号：NP_000408.1)的C端。重组人CD25-His蛋白(购于北京义翘神州)是将多组氨酸标签融合在人CD25蛋白(GenBank，登录号：NP_000408.1)Met1-Cys213的C端。重组猴CD25-His蛋白(购于北京义翘神州)是将His标签融合在猴CD25蛋白(GenBank，登录号：NP_001270633.1)Met1-Arg213的C端。重组小鼠CD25-His蛋白(购于北京义翘神州)是将His标签融合在小鼠CD25蛋白(GenBank，登录号：NP_032393.3)Met1-Lys236的C端。使用抗CD25抗体DAC(Efalizumab，达利珠单抗)对购入的重组蛋白进行一 系列的质控检测，如活性验证。
通过ELISA(酶联免疫吸附实验)验证重组蛋白活性。具体为：用PBS将重组人CD25-Fc融合蛋白、人CD25-His蛋白、猴CD25-His蛋白、鼠CD25-His蛋白分别稀释至1μg/mL，加入ELISA微孔板，每孔各100μL，4℃孵育过夜；加入ELISA封闭液(含1％BSA(w/v),pH7.4的PBS磷酸盐缓冲液)，37℃封闭2小时后，依次加入梯度稀释的检测抗体DAC和7D4(抗小鼠CD25抗体)，37℃温育1小时，用洗板液洗板2-3次；加入辣根过氧化物酶标记(HRP)的二抗，37℃温育1小时，用洗板液洗板2-3次。加入TMB底物每孔100μL，室温孵育15分钟后，每孔加入50μL终止液(2M HCl)。用ELISA板机(SpectraMax M5e)读取OD450nm值，结果如图1A和1B所示。
结果显示，两种重组人CD25蛋白、重组猴CD25-His蛋白均能够与DAC结合，且检测信号随检测抗体的浓度变化而变化；重组小鼠CD25-His蛋白不能够与DAC结合，但可以与7D4结合，证明上述重组CD25蛋白均具有活性。
实施例2.建立表达重组人CD25蛋白的稳定细胞株
用编码重组人CD25蛋白的质粒转染CHO-K1细胞后，通过筛选获得稳定表达重组人CD25蛋白的细胞株。编码人CD25的核苷酸序列被克隆连接到pCDNA3.4载体(购自Invitrogen)并制备质粒。用Lipofectamine 2000对CHO-K1细胞系(均购自Invitrogen)进行转染，使用含G418的CD-CHO培养基选择性培养2周，有限稀释法在96孔培养板中进行亚克隆，并置于37℃含5％CO 2(v/v)培养箱内培养，2周后选择部分单克隆孔扩增至24孔板内，随后扩增至6孔板中培养。对扩增后的克隆通过流式分析法进行检测筛选。图2结果显示筛选出的细胞株2F8能够稳定表达人CD25蛋白。
实施例3.对检测抗体进行功能验证
DAC、7G7B6、Tab06、7D4均为抗CD25抗体。DAC的序列在US5530101中列出，该专利通过引用结合至本公开。7G7B6已被建议作为使放射性核素靶向表达CD25的淋巴瘤的靶向部分(Zhang et al，2009，Cancer Biother Radiopharm 24(3),303-309)。Tab06是一种不抑制IL-2与CD25的结合的抗CD25抗体，序列在WO2018167104中列出(该专利的序列27和29)，该专利通过引用结合至本公开。7D4是大鼠IgM抗小鼠CD25抗体，在PC61存在下或用PC61治疗后或具有相似结合特性的抗体存在下，7D4已被广泛用于检测CD25阳性细胞(Malek,1983,Immunology,Vol.80,pp.5694-5698；Onizuka S et al.,1999.Canc Res.59,3128-3133)。
通过FACS对检测抗体DAC、7G7B6、Tab06、7D4进行功能验证。具体为：离心收集对数生长期的SU-DHL-1细胞、CHO-K1细胞和稳定表达人CD25的CHO-K1 细胞(CHO-K1-CD25细胞)，PBS清洗后200g离心5分钟。用100μL含2x 10 5细胞数的培养液铺板，400g离心5分钟后加入检测抗体(100nM，梯度稀释)，其中对照组抗体为人IgG同种型。冰上孵育1h，用PBS洗去多余的抗体，400g离心5分钟。加入二抗Alexa Fluor 488羊抗人(Fc)抗体(购自Life Technology，货号A11013)冰浴染色1小时，PBS洗去多余的二抗，400g离心5分钟。向每个孔加入200μL流式检测液后，上机检测。
如图3A-3C所示，在与Su-DHL-1细胞和稳定表达人CD25的CHO-K1细胞(CHO-K1-CD25细胞)(即单克隆2F8)的结合实验中，DAC的信号最强，Tab06次之，7G7B6的信号最弱；3种抗体均与能Su-DHL-1细胞和稳定表达人CD25的CHO-K1细胞结合，都不与CHO-K1细胞结合。
对7G7B6和Tab06使用ELISA进行功能验证。具体为：用PBS将重组人CD25-His蛋白、重组猴CD25-His蛋白和重组小鼠CD25-his蛋白稀释至1μg/mL，加入ELISA微孔板，每孔100μL，4℃孵育过夜；加入ELISA封闭液(含1％BSA，pH7.4的PBS磷酸缓冲液)，37℃封闭2小时后，依次分别加入梯度稀释的检测抗体，37℃温育1小时；用洗板液洗板2-3次；加入辣根过氧化物酶标记(HRP)的二抗，37℃温育1小时，用洗板液洗板2-3次。每孔加入TMB底物100μL，室温孵育15分钟后，每孔加入50μL终止液2M HCl，读取OD450nm值。
如图4A-4C所示，7G7B6与Tab06分别可以与人CD25蛋白和猴CD25蛋白结合，表明7G7B6与Tab06具有人猴交叉反应活性；7D4可以与小鼠CD25蛋白结合，表明7D4具有小鼠CD25蛋白的结合活性。
实施例4.重组CD25蛋白免疫小鼠获得鼠源抗CD25单克隆抗体
使用重组人CD25-Fc作为免疫原免疫6-8周龄的Balb/c和SJL/J小鼠，使用重组人CD25-His作为免疫原免疫SJL/J小鼠。初次免疫剂量为每只小鼠50μg。初次免疫2周后，加强免疫，免疫剂量为每只小鼠25μg。以后每次加强免疫间隔3周。每次加强免疫一周后采集血清样品，用ELISA检测小鼠血清中抗体活性。用1ug/mL重组人CD25-His包板，4℃过夜，用含1％BSA的PBST缓冲液封闭1小时，洗板3次。在封闭缓冲液中以1：100开始，10倍梯度稀释小鼠血清，37℃孵育1小时，洗板3次，与抗小鼠IgG-Fc-HRP的二抗温育1小时。PBST洗涤3次，每孔加入100μL TMB底物，15分钟后用2M HCl终止反应。使用酶标仪读取450nm处的吸光度。经免疫原重组人CD25-Fc免疫的小鼠血清对免疫原有不同程度的结合，血清的最高稀释倍数为在1:10 5时，仍能呈现抗原抗体反应。
最后一次免疫腹腔注射100μg重组人CD25-Fc与重组人CD25-His，5天后处死小鼠后取脾脏，碾磨收集脾细胞。加入终浓度1％(w/w)的NH 4OH裂解脾细胞中混杂的红细胞，获得脾细胞悬液，1000转离心清洗细胞3次。按活细胞数目5:1比率将小鼠脾脏细胞与小鼠骨髓瘤细胞SP2/0混合，使用高效电融合方法进行细胞融合。 使用20％胎牛血清、含1×HAT(w/w)的DMEM培养基稀释融合后的细胞至96孔细胞培养板中，200μL每孔共计1x 10 5个细胞，置于5％(v/v)CO 2的37℃培养箱中。14天后使用ELISA筛选融合后的细胞，将OD450nm>1.0的阳性克隆扩增至24孔细胞板，在37℃,5％(v/v)CO 2条件下，使用含10％(w/w)HT胎牛血清的DMEM培养基继续扩大培养。3天后取24孔细胞板培养液离心收集上清，用ELISA、FACS确定对重组人CD25蛋白和CD25阳性细胞的结合活性。两轮单克隆化，并使用上述ELISA检测方法与流式检测方法筛选出优异单克隆株。抗体经测序的氨基酸序列示于表1，下划线为互补决定区CDR序列(使用Kabat编号系统)。
表1.鼠源抗CD25抗体序列
Figure PCTCN2021093791-appb-000001
Figure PCTCN2021093791-appb-000002
实施例5.制备重组嵌合型抗CD25抗体以及人源化过程
对于嵌合抗体，通过替换鼠源单克隆抗体的恒定区，获得重组嵌合型抗体，随后将编码鼠源单克隆抗体可变区的核苷酸序列克隆到含有编码人重、轻链恒定区(Human IgG1,kappa)蛋白序列的pTT5载体上后转染HEK293细胞，即，所获得的嵌合抗体的重链、轻链可变区分别与鼠源抗体的重链、轻链可变区相同。对于人源化抗体：通过同源建模预测鼠源单抗的结构后，将鼠抗CDR嵌合到合适的人GermLine框架上(Bioinformation.2014；10(4):180–186；Methods Mol Biol.2019；1904:213-230)，随后在有可能影响抗体-抗原结合的位点引入回复突变，最后将人源化单克隆抗体编码可变区的核苷酸序列克隆到含有编码人重、轻链恒定区(Human IgG1,kappa)蛋白序列的pTT5载体上后转染HEK293细胞。5天后离心去除细胞收集并过滤细胞培养液。pH调节至7.0后，将收获的细胞培养液上清液上样至蛋白A柱(MabSelect SuRe，GE)，使用甘氨酸洗脱结合的抗体，1M Tris中和洗脱液。
抗体可变区框架选择见表2，抗体可变区序列参见表3，重链CDR(HCDR1\HCDR2\HCDR3)和轻链CDR(LCDR1\LCDR2\LCDR3)见表4，抗体氨基酸序列全长见表5。其中，PR006是cAb006的人源化的抗体，PR071是cAb037的人源化抗体，PR031是cAb042的人源化抗体，PR058、PR157是cAb046的人源化抗体。
表2.抗体可变区框架选择
Figure PCTCN2021093791-appb-000003
Figure PCTCN2021093791-appb-000004
表3.部分不阻断IL-2功能的人源化抗CD25抗体的可变区序列
Figure PCTCN2021093791-appb-000005
表4.部分不阻断IL-2功能的人源化抗CD25抗体的可变区序列(kabat编号系统)
Figure PCTCN2021093791-appb-000006
Figure PCTCN2021093791-appb-000007
表5.人源化抗体的重轻链全长氨基酸序列
Figure PCTCN2021093791-appb-000008
Figure PCTCN2021093791-appb-000009
Figure PCTCN2021093791-appb-000010
实施例6.嵌合型抗CD25抗体对人(human)和食蟹猴(cyno)CD25蛋白亲和力检测
用ELISA方法检测嵌合型抗CD25抗体与CD25重组蛋白的结合活性。以1g/mL人CD25-His/猴CD25-His浓度，每孔50uL，包被96孔板。用清洗液将未结合抗原清洗掉(清洗液：1×PBST)。清洗后以1x PBST配置的1％BSA封闭液37℃封闭1小时。清洗液洗板3次后，加入不同稀释浓度的待测嵌合抗体，37℃温箱孵育1小时。清洗液洗板3次后，加入100μl 1:5000稀释的羊抗人IgG二抗，37℃温箱孵育0.5小时洗板后，取TMB显色液A液和B液1：1比例混合后显色。15分钟用1M盐酸终止显色反应，检测450nm的荧光值。结果显示，所检测的嵌合型抗CD25抗体均可以和人以及猴的CD25蛋白结合，ELISA检测的半数有效浓度(EC 50)见表6和表7。
表6.嵌合型抗CD25抗体与猴CD25结合的EC 50(ELISA)
抗体编号 EC 50(nM) 抗体编号 EC 50(nM) 抗体编号 EC 50(nM)
cAb001 0.3991 cAb029 0.1145 cAb028 0.2295
cAb002 0.5244 cAb037 0.1467 DAC 0.3973
cAb004 0.3106 cAb042 0.3486 Tab06 0.6144
cAb006 0.2338 cAb046 5.569    
表7.嵌合型抗CD25抗体与人CD25结合的EC 50(ELISA)
抗体编号 EC 50(nM) 抗体编号 EC 50(nM) 抗体编号 EC 50(nM)
cAb001 0.2284 cAb028 0.3444 cAb046 0.3547
cAb002 0.2738 cAb029 0.1661 DAC 0.2319
cAb004 0.2552 cAb037 0.1653 Tab06 0.4032
cAb006 0.2004 cAb042 0.224    
实施例7.嵌合型抗CD25抗体与SU-DHL-1细胞的结合
由于淋巴瘤细胞系SU-DHL-1高表达CD25，因此我们用流式细胞术来检测嵌合型抗CD25抗体与SU-DHL-1细胞的结合。选取对数生长期的SU-DHL-1细胞，收集细胞，PBS清洗后离心。每孔用100μl 2×10 5细胞铺板，400g离心5分钟。加入不同浓度的待检抗体，冰上孵育1h后，PBS洗涤，400g离心5min。加入带有荧光基团的羊抗人二抗Alexa Fluor 488，冰浴染色1h，PBS清洗后上机检测。如图5A-5C所示，所检测的本公开的嵌合型抗CD25抗体均可和SU-DHL-1细胞结 合。表8为结合的EC 50值。
表8.嵌合型抗CD25抗体与SU-DHL-1细胞结合的EC 50
抗体编号 EC 50(nM) 抗体编号 EC 50(nM) 抗体编号 EC 50(nM)
cAb001 0.1799 cAb028 0.1319 cAb046 0.3455
cAb002 0.1001 cAb029 0.1193 DAC 0.3407
cAb004 0.1681 cAb037 0.1502 Tab06 0.8323
cAb006 0.3286 cAb042 0.501    
实施例8.嵌合型抗CD25抗体与Treg细胞/激活的CD4 +T细胞/CD8 +T细胞的结合
由于CD25在激活的Treg和激活的效应T细胞中都有表达，为了检测本公开的抗CD25抗体对于表达在Treg和CD4 +和CD8 +效应T细胞上的CD25是否存在结合差别，以下用FACS的方法比较了抗体对Treg和激活后的效应T细胞上的结合能力。
取人外周血单核细胞(PBMC)用磁珠法分离提取天然CD4 +细胞，并加入诱导Treg的培养基((AIM V+10％FBS+human IL-2(400U/mL)+Rapamycin(0.1g/mL)或TGFβ(20ng/mL)+atRA(200nM))，同时加入包被了抗CD3/CD28抗体的磁珠。培养期间保持细胞密度为2×10 6/mL，37℃，5％CO 2条件下培养。培养7天后用Dynabeads TM ReguLatory CD4 +/CD25 +T Cell Kit(Thermo Fisher,11363D)分离细胞得到激活型的CD4 +CD25 +Treg细胞，得到FOXP3阳性率大于90％的Treg细胞。
新鲜PBMC中分别用CD4 +细胞负性筛选富集试剂盒细胞和CD8 +T细胞筛选试剂盒富集，分离得到高纯度的CD4 +和CD8 +效应T细胞，细胞纯度大于90％。并加入包被了抗CD3/CD28抗体的磁珠使细胞激活和扩增。3天后得到CD25高表达的效应CD4 +T细胞和效应CD8 +T细胞。
将体外诱导的Treg细胞和体外激活的CD4 +和CD8 +效应T细胞分别加入不同稀释浓度的待检抗体冰上孵育1h后，用PBS洗涤。再加入羊抗人抗体Alexa Fluor488冰浴染色1h，用PBS洗涤，每孔加入200μL流式检测液在同样条件下同时上机检测。
结果如图6A-6G所示，所有检测嵌合抗体均可和Treg细胞、激活的CD4 +和CD8 +效应T细胞上的CD25抗原结合。但从结合程度而言，与Treg的结合力要远远大于和CD4 +T细胞的结合，和CD4 +T细胞的结合略大于和CD8 +T细胞的结合。
实施例9.嵌合型抗CD25抗体对于IL-2和受体结合能力的影响
收集对数生长期的SU-DHL-1细胞与200nM IL-2-biotin以及不同浓度的待测嵌合型抗CD25抗体，4℃孵育1h后，PBS洗一次细胞。加入Alexa Fluor 488  Streptavidin(1:1000)4℃孵育1h，用PBS去多余的二抗，用FACS检测荧光细胞。如果抗体影响了IL-2和CD25的结合，荧光强度变弱。
使用如下公式计算抑制率：
抑制率％＝100×(未加抗体时最大结合荧光强度-抗体结合后荧光强度)/未加抗体时的结合强度。
结果如图7A-7B所示，DAC、cAb001、cAb002、cAb028、cAb029不同程度地抑制IL-2与SU-DHL-1的结合，提示以上嵌合型抗体对于对IL-2信号通路有抑制，而cAb006、cAb037、cAb042、cAb046与Tab006一样不抑制IL-2与SU-DHL-1的结合，提示以上嵌合型抗体属于非拮抗型抗体。
实施例10.人源化抗CD25抗体对SUDH-L1的细胞结合
用实施例7中的流式细胞术方法来检测嵌合型抗CD25抗体与SU-DHL-1细胞的结合。图8显示本公开人源化抗CD25抗体RP006、PR031、PR058以及PR071均具有良好的SU-DHL-1结合活性。
实施例11.人源化抗CD25抗体对人外周血T淋巴细胞pStat5信号通路的影响
CD25作为IL-2细胞因子受体的α链，与β和γ链形成高亲和力受体后，与IL-2结合后，激活下游JAK-STAT，PI3K-AKT和MAPK信号通路，因此进一步检测嵌合型抗体对于IL-2在PBMC中上调磷酸化STAT-5的作用。
复苏冻存PBMC，将人源化抗CD25抗体和PBMC共孵育(37℃，5％CO 2)。30分钟后分别就加入100U/mL的IL-2诱导10分钟。将激活后的PBMC固定每孔加入200μL染色液(含2％FBS的PBS)加入5μL的V450小鼠抗人CD3标记T细胞，将未结合抗体洗去后，加入300μl预冷破膜剂Perm Buffer III，涡旋混匀，冰浴30分钟。再用Alexa
Figure PCTCN2021093791-appb-000011
647小鼠抗Stat5染色60分钟。去除多余的抗体，用流式细胞仪检测。
如图9所示，DAC能明显抑制IL-2诱导的CD3细胞内Stat5的磷酸化，而Tab06不能抑制IL-2诱导的Stat5的磷酸化。PR006在300nM和100nM能微弱地抑制Stat5的磷酸化，PR031、PR058、PR071不抑制IL-2介导的Stat5的磷酸化。
实施例12.人源化抗CD25抗体介导的对SU-DHL-1细胞的ADCC作用
抗体介导细胞细胞毒杀伤(ADCC)是抗体治疗的一个重要功能，通过ADCC的活性杀伤CD25高表达的Treg细胞达到激活肿瘤免疫的作用是我们期望的，以下检测了本公开的人源化抗CD25抗体介导ADCC的活性。
使用ADCC报告基因细胞(Promega公司G7010 ADCC Bioassay E ector Cells)与SU-DHL-1细胞1:1比例混合，加入不同浓度的抗体孵育6小时后，检测细胞荧 光活性，从图10和表9可以看出，PR006、PR031、PR058以及PR071均具有良好的ADCC活性，其中，PR058、PR071活性明显强于抗体686。
抗体686序列参见US20190284287A的序列5、序列9。
表9.人源化抗体ADCC活性
Figure PCTCN2021093791-appb-000012
实施例13.人源化抗CD25抗体介导对hCD25小鼠的MC38移植瘤模型的抗肿瘤活性
在B-hIL2RA人源化小鼠的MC38结肠癌小鼠模型中评估人源化抗CD25抗体的抗肿瘤活性。将前述PR058、686的可变区连接到小鼠IgG2a上构建PR058 mIgG2a、686 mIgG2a抗体，用于动物实验。
将PBS重悬的MC38结肠癌细胞以5×10 5个/0.1mL浓度，0.1mL/只的体积接种于B-hIL2RA人源化小鼠的右侧皮下。当平均肿瘤体积达到大约113mm 3时，挑选个体肿瘤体积合适的48只小鼠随机分组，每组8只，共6组，分别为：G1同种型mIgG2a(3mg/kg)对照组、G2 PR058 mIgG2a(3mg/kg)组、G3 686 mIgG2a(3mg/kg)组。所有组给药途径均为腹腔注射，每周给药1次，连续给药3次，末次给药7天后结束实验。每周测量肿瘤体积及体重2次，记录小鼠体重和肿瘤体积。实验结束时，动物安乐死，剥取肿瘤称重、拍照，计算相对肿瘤抑制率(TGI％)。
研究结果如图11A所示，PR058在3mg/kg剂量水平下，对MC38皮下移植瘤的生长具有最优的抑制作用，优于686。图11B显示实验动物在给药期间活动和进食状态良好，实验动物体重均有一定程度的上升，表明受试药物未对实验动物产生明显毒性作用，安全性较好。如图12A、图12B的肿瘤内淋巴细胞分析结果显示，PR058能更强地介导Treg杀伤，以及CD3数目上调。其中，*表示p<0.05，与mIgG2a组相比有统计学差异；**表示p<0.01，与mIgG2a组相比有显著统计学差异；***表示p<0.001，与mIgG2a组相比有及其显著统计学差异；ns表示与mIgG2a组相比无统计学差异。
虽然以上描述了本公开的具体实施方式，但是本领域的技术人员应当理解，这些仅是举例说明，在不背离本发明的原理和实质的前提下，可以对这些实施方式做出多种变更或修改。因此，本公开的保护范围由所附权利要求书限定。

Claims (23)

  1. 一种抗CD25抗体或其抗原结合片段，包含重链可变区(VH)和轻链可变区(VL)，其中：
    1)所述VH含有SEQ ID NO：17中的HCDR1、HCDR2和HCDR3，和所述VL含有SEQ ID NO：18中的LCDR1、LCDR2和LCDR3；
    2)所述VH含有SEQ ID NO：1中的HCDR1、HCDR2和HCDR3，和所述VL含有SEQ ID NO：2中的LCDR1、LCDR2和LCDR3；
    3)所述VH含有SEQ ID NO：3中的HCDR1、HCDR2和HCDR3，和所述VL含有SEQ ID NO：4中的LCDR1、LCDR2和LCDR3；
    4)所述VH含有SEQ ID NO：5中的HCDR1、HCDR2和HCDR3，和所述VL含有SEQ ID NO：6中的LCDR1、LCDR2和LCDR3；
    5)所述VH含有SEQ ID NO：7中的HCDR1、HCDR2和HCDR3，和所述VL含有SEQ ID NO：8中的LCDR1、LCDR2和LCDR3；
    6)所述VH含有SEQ ID NO：9中的HCDR1、HCDR2和HCDR3，和所述VL含有SEQ ID NO：10中的LCDR1、LCDR2和LCDR3；
    7)所述VH含有SEQ ID NO：11中的HCDR1、HCDR2和HCDR3，和所述VL含有SEQ ID NO：12中的LCDR1、LCDR2和LCDR3；
    8)所述VH含有SEQ ID NO：13中的HCDR1、HCDR2和HCDR3，和所述VL含有SEQ ID NO：14中的LCDR1、LCDR2和LCDR3；或
    9)所述VH含有SEQ ID NO：15中的HCDR1、HCDR2和HCDR3，和所述VL含有SEQ ID NO：16中的LCDR1、LCDR2和LCDR3；
    所述CDR是根据Kabat、IMGT、Chothia、AbM或Contact编号系统定义的；优选地，所述CDR是根据Kabat编号系统定义的。
  2. 一种抗CD25抗体或其抗原结合片段，包含VH和VL，其中：
    1)所述VH包含分别如SEQ ID NO：45、46、47所示的HCDR1、HCDR2、HCDR3，和所述VL包含分别如SEQ ID NO：48、49、50所示的LCDR1、LCDR2、LCDR3；
    2)所述VH包含分别如SEQ ID NO：27、28、29所示的HCDR1、HCDR2、HCDR3，和所述VL包含分别如SEQ ID NO：30、31、32所示的LCDR1、LCDR2、LCDR3；
    3)所述VH包含分别如SEQ ID NO：33、34、35所示的HCDR1、HCDR2、HCDR3，和所述VL包含分别如SEQ ID NO：36、37、38所示的LCDR1、LCDR2、LCDR3；或
    4)所述VH包含分别如SEQ ID NO：39、40、41所示的HCDR1、HCDR2、 HCDR3，和所述VL包含分别如SEQ ID NO：42、43、44所示的LCDR1、LCDR2、LCDR3。
  3. 如权利要求1-2任一项所述的抗CD25抗体或其抗原结合片段，其中所述抗CD25抗体为鼠源抗体、嵌合抗体、人抗体或人源化抗体；
    优选地，所述抗CD25抗体为人源化抗体。
  4. 如权利要求1-2任一项所述的抗CD25抗体或其抗原结合片段，其中：
    1)所述VH包含选自IGHV1-46*01的FR1至FR3、和选自IGHJ1*01的FR4，和所述VL包含选自IGKV4-1*01的FR1至FR3、和选自IGKJ4*01的FR4；
    2)所述VH包含选自IGHV1-18*01的FR1至FR2、选自IGHV1-69*02的FR3、和选自hIGHJ6*01_14的FR4，和所述VL包含选自IGKV3-11*01的FR1、选自IGKV5-2*01的FR2、选自IGKV6-21*01的FR3、和选自hIGKJ4*01_12的FR4；
    3)所述VH包含选自IGHV1-18*01的FR1、选自IGHV4-31*01的FR2、选自IGHV1-3*01的FR3、和选自hIGHJ6*01的FR4，和所述VL包含选自IGKV4-1*01的FR1至FR3、和选自hIGKJ2*01的FR4；或
    4)所述VH包含选自IGHV3-23*04的FR1至FR3、选自IGHJ1*01的FR4，和所述VL包含选自IGKV2-28*01的FR1至FR3、和选自IGKJ4*01的FR4。
  5. 如权利要求1-4中任一项所述的抗CD25抗体或其抗原结合片段，其中：
    所述VH如SEQ ID NO：25所示或与之具有至少90％、至少95％序列同一性，和所述VL如SEQ ID NO：26所示或与之具有至少90％、至少95％序列同一性；
    所述VH如SEQ ID NO：59所示或与之具有至少90％、至少95％序列同一性，和所述VL如SEQ ID NO：60所示或与之具有至少90％、至少95％序列同一性；
    所述VH如SEQ ID NO：1所示或与之具有至少90％、至少95％序列同一性，和所述VL如SEQ ID NO：2所示或与之具有至少90％、至少95％序列同一性；
    所述VH如SEQ ID NO：3所示或与之具有至少90％、至少95％序列同一性，和所述VL如SEQ ID NO：4所示或与之具有至少90％、至少95％序列同一性；
    所述VH如SEQ ID NO：5所示或与之具有至少90％、至少95％序列同一性，和所述VL如SEQ ID NO：6所示或与之具有至少90％、至少95％序列同一性；
    所述VH如SEQ ID NO：7所示或与之具有至少90％、至少95％序列同一性，和所述VL如SEQ ID NO：8所示或与之具有至少90％、至少95％序列同一性；
    所述VH如SEQ ID NO：9所示或与之具有至少90％、至少95％序列同一性，和所述VL如SEQ ID NO：10所示或与之具有至少90％、至少95％序列同一性；
    所述VH如SEQ ID NO：11所示或与之具有至少90％、至少95％序列同一性，和所述VL如SEQ ID NO：12所示或与之具有至少90％、至少95％序列同一性；
    所述VH如SEQ ID NO：13所示或与之具有至少90％、至少95％序列同一性，和所述VL如SEQ ID NO：14所示或与之具有至少90％、至少95％序列同一性；
    所述VH如SEQ ID NO：15所示或与之具有至少90％、至少95％序列同一性，和所述VL如SEQ ID NO：16所示或与之具有至少90％、至少95％序列同一性；
    所述VH如SEQ ID NO：17所示或与之具有至少90％、至少95％序列同一性，和所述VL如SEQ ID NO：18所示或与之具有至少90％、至少95％序列同一性；
    所述VH如SEQ ID NO：19所示或与之具有至少90％、至少95％序列同一性，和所述VL如SEQ ID NO：20所示；
    所述VH如SEQ ID NO：21所示或与之具有至少90％、至少95％序列同一性，和所述VL如SEQ ID NO：22所示或与之具有至少90％、至少95％序列同一性；或
    所述VH如SEQ ID NO：23所示或与之具有至少90％、至少95％序列同一性，和所述VL如SEQ ID NO：24所示或与之具有至少90％、至少95％序列同一性。
  6. 如权利要求1-5中任一项所述的抗CD25抗体或其抗原结合片段，其中所述抗原结合片段为scFv、Fv、Fab或Fab’片段。
  7. 如权利要求1-5中任一项所述的抗CD25抗体或其抗原结合片段，其中所述抗CD25抗体为IgG抗体，优选为IgG1、IgG2、IgG4；
    优选地，所述抗CD25抗体在Fc区具有去岩藻糖化位点，以增强与FcγRIIIa的结合能力，和/或降低与FcγRIIb的结合能力；
    更优选地，所述去岩藻糖化位点为297位。
  8. 如权利要求1-7中任一项所述的抗CD25抗体或其抗原结合片段，包含重链和轻链，其中：
    1)所述重链如SEQ ID NO：51所示或与之具有至少80％、至少90％、至少95％序列同一性，和所述轻链如SEQ ID NO：52所示或与之具有至少80％、至少90％、至少95％序列同一性；
    2)所述重链如SEQ ID NO：53所示或与之具有至少80％、至少90％、至少95％序列同一性，和所述轻链如SEQ ID NO：54所示或与之具有至少80％同一性；
    3)所述重链如SEQ ID NO：55所示或与之具有至少80％、至少90％、至少95％序列同一性，和所述轻链如SEQ ID NO：56所示或与之具有至少80％、至少90％、至少95％序列同一性；
    4)所述重链如SEQ ID NO：57所示或与之具有至少80％、至少90％、至少95％序列同一性，和所述轻链如SEQ ID NO：58所示或与之具有至少80％、至少90％、至少95％序列同一性；或
    5)所述重链如SEQ ID NO：61所示或与之具有至少80％、至少90％、至少95％序列同一性，和所述轻链如SEQ ID NO：62所示或与之具有至少80％、至少90％、至少95％序列同一性。
  9. 如权利要求1-8中任一项所述的抗CD25抗体或其抗原结合片段，其中，所述抗CD25抗体或其抗原结合片段具有下述特征中的至少一项：
    (a)结合人CD25的KD值小于1×10 -7M；
    (b)不抑制或基本上不抑制IL-2与CD25的结合；
    (c)消耗肿瘤浸润性Treg，不影响或基本上不影响Teff的功能；
    (d)以高于1的活化抑制率(A/I)结合Fcγ受体；
    (e)以比结合FcγRI、FcγRIIc和/或FcγRIIb更高的亲和力结合FcγRIIIa，优选地，以比结合FcγRIIb更高的亲和力结合FcγRIIIa；
    (f)抑制肿瘤生长；和
    (g)引发增强的CDC、ADCC和/或ADCP反应；优选地，引发增加的ADCC反应。
  10. 一种抗CD25抗体或其抗原结合片段，其与权利要求1-9中任一项所述的抗CD25抗体或其抗原结合片段竞争性结合人CD25，或竞争性结合、或结合相同的人CD25表位。
  11. 一种多核苷酸，其编码权利要求1-10中任一项所述的抗CD25抗体或其抗原结合片段。
  12. 一种载体，其含有如权利要求11所述的多核苷酸。
  13. 一种宿主细胞，其包含权利要求12所述的载体，
    优选地，所述宿主细胞为细菌、酵母或哺乳动物细胞；
    更优选地，所述宿主细胞为大肠杆菌、毕赤酵母、中国仓鼠卵巢细胞或人胚肾293细胞。
  14. 一种制备抗CD25抗体或其抗原结合片段的方法，包括：
    在权利要求13所述的宿主细胞中表达权利要求1-10任一项所述的抗CD25抗体或其抗原结合片段，以及
    从所述宿主细胞中分离所述抗CD25抗体或其抗原结合片段。
  15. 一种药物组合物，其含有：
    权利要求1-10中任一项所述的抗CD25抗体或其抗原结合片段；
    以及，一种或多种可药用的赋形剂、稀释剂或载体。
  16. 选自以下的任一项或其任意组合在制备药物或药物组合物中的用途：
    权利要求1-10中任一项所述的抗CD25抗体或其抗原结合片段，权利要求11所述的多核苷酸和权利要求15所述的药物组合物，其中所述药物或药物组合物用于治疗癌症。
  17. 一种治疗患有癌症的受试者的方法，包括：
    向受试者施用治疗有效量的权利要求1-10中任一项所述的抗CD25抗体或抗原结合片段、权利要求11所述的多核苷酸、权利要求15所述的药物组合物或其任意组合。
  18. 一种减少受试者肿瘤内部或肿瘤侵润性Treg细胞数量和/或抑制其活性的方法，包括：
    向受试者施用治疗有效量的权利要求1-10任一项所述的抗CD25抗体或抗原结合片段、权利要求11所述的多核苷酸、权利要求15所述的药物组合物或其任意组合。
  19. 一种增加受试者肿瘤内部Teff/Treg的比例的方法，包括：
    向受试者施用治疗有效量的权利要求1-10任一项所述的抗CD25抗体或抗原结合片段、权利要求11所述的多核苷酸、权利要求15所述的药物组合物或其任意组合。
  20. 一种增强受试者体内针对肿瘤细胞的CDC、ADCC和/或ADCP的方法，包括：
    向受试者施用治疗有效量的权利要求1-10任一项所述的抗CD25抗体或抗原结合片段、权利要求11所述的多核苷酸、权利要求15所述的药物组合物或其任意组合。
  21. 如权利要求16的用途和权利要求17-20任一项所述的方法，其中所述受试者患有癌症或肿瘤，
    优选地，所述癌症或肿瘤选自鳞状细胞癌、骨髓瘤、小细胞肺癌、非小细胞肺癌、神经胶质瘤、肝细胞癌(HCC)、霍奇金淋巴瘤、非霍奇金淋巴瘤、急性髓性白血病(AML)、多种骨髓瘤、胃肠(道)癌、肾癌、卵巢癌、肝脏癌、淋巴母细胞白血病、淋巴细胞白血病、结直肠癌、子宫内膜癌、肾癌、前列腺癌、甲状腺癌、黑色素瘤、软骨肉瘤、神经母细胞瘤、胰腺癌、多形性胶质母细胞瘤、宫颈癌、脑癌、胃癌、膀胱癌、肝癌、乳腺癌、结肠癌和头颈癌。
  22. 一种在体内或体外检测CD25的方法，所述方法包括使用权利要求1-10中任一项所述的抗CD25抗体或抗原结合片段、和/或权利要求11所述的多核苷酸，
    优选地，如果CD25存在，检测所述抗CD25抗体或抗原结合片段、多核苷酸和所述CD25之间的相互作用。
  23. 一种用于检测CD25的试剂盒，其包含权利要求1-10中任一项所述的抗CD25抗体或抗原结合片段和\或权利要求11所述的多核苷酸。
PCT/CN2021/093791 2020-05-14 2021-05-14 抗cd25抗体、其抗原结合片段及其医药用途 WO2021228218A1 (zh)

Priority Applications (5)

Application Number Priority Date Filing Date Title
CN202180028356.4A CN115397860A (zh) 2020-05-14 2021-05-14 抗cd25抗体、其抗原结合片段及其医药用途
EP21804918.7A EP4151655A1 (en) 2020-05-14 2021-05-14 Anti-cd25 antibodies, antigen-binding fragments thereof, and medical uses thereof
US17/998,418 US20230174670A1 (en) 2020-05-14 2021-05-14 Anti-cd25 antibodies, antigen-binding fragments thereof, and medical uses thereof
CA3182362A CA3182362A1 (en) 2020-05-14 2021-05-14 Anti-cd25 antibodies, antigen-binding fragments thereof, and medical uses thereof
JP2022567574A JP2023525060A (ja) 2020-05-14 2021-05-14 抗ｃｄ２５抗体、その抗原結合断片及びその医薬用途

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010408502 2020-05-14
CN202010408502.3 2020-05-14

Publications (1)

Publication Number Publication Date
WO2021228218A1 true WO2021228218A1 (zh) 2021-11-18

Family

ID=78525333

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/093791 WO2021228218A1 (zh) 2020-05-14 2021-05-14 抗cd25抗体、其抗原结合片段及其医药用途

Country Status (7)

Country Link
US (1) US20230174670A1 (zh)
EP (1) EP4151655A1 (zh)
JP (1) JP2023525060A (zh)
CN (1) CN115397860A (zh)
CA (1) CA3182362A1 (zh)
TW (1) TW202144429A (zh)
WO (1) WO2021228218A1 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024040194A1 (en) 2022-08-17 2024-02-22 Capstan Therapeutics, Inc. Conditioning for in vivo immune cell engineering

Citations (24)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990007861A1 (en) 1988-12-28 1990-07-26 Protein Design Labs, Inc. CHIMERIC IMMUNOGLOBULINS SPECIFIC FOR p55 TAC PROTEIN OF THE IL-2 RECEPTOR
WO1992022583A2 (en) 1991-06-11 1992-12-23 Celltech Limited Tri- and tetra-valent monospecific antigen-binding proteins
WO1993011238A1 (en) 1991-12-06 1993-06-10 Sumitomo Pharmaceuticals Company, Limited Humanized b-b10, an anti-il2 receptor antibody
US5530101A (en) 1988-12-28 1996-06-25 Protein Design Labs, Inc. Humanized immunoglobulins
US20030040606A1 (en) 2001-06-27 2003-02-27 Leung Shawn Shui-On Reducing immunogenicities of immunoglobulins by framework-patching
WO2003048731A2 (en) 2001-12-03 2003-06-12 Abgenix, Inc. Antibody categorization based on binding characteristics
WO2004045512A2 (en) 2002-11-15 2004-06-03 Genmab A/S Human monoclonal antibodies against cd25
WO2004074437A2 (en) 2003-02-14 2004-09-02 University Of Southern California Compositions and methods for cancer immunotherapy
WO2005003169A2 (en) 2003-07-01 2005-01-13 Celltech R & D Limited Modified antibody fab fragments
WO2005003170A2 (en) 2003-07-01 2005-01-13 Celltech R & D Limited Modified antibody fragments
WO2005003171A2 (en) 2003-07-01 2005-01-13 Celltech R & D Limited Modified antibody fragments
WO2005113605A1 (en) 2004-05-19 2005-12-01 Celltech R & D Limited Cross-linked antibodies
WO2006050172A2 (en) 2004-10-29 2006-05-11 University Of Southern California Combination cancer immunotherapy with co-stimulatory molecules
WO2006108670A2 (en) 2005-04-15 2006-10-19 Novartis Ag Use of cd25 antibodies in immunotherapy
US7494647B2 (en) 1998-04-03 2009-02-24 Chugai Seiyaku Kabushiki Kaisha Humanized antibody against human tissue factor (TF) and process of production of the humanized antibody
WO2009040562A1 (en) 2007-09-26 2009-04-02 Ucb Pharma S.A. Dual specificity antibody fusions
WO2010035012A1 (en) 2008-09-26 2010-04-01 Ucb Pharma S.A. Biological products
WO2011077245A2 (en) 2009-12-23 2011-06-30 Fondazione Centro San Raffaele Del Monte Tabor Compositions
WO2016021720A1 (ja) 2014-08-07 2016-02-11 学校法人兵庫医科大学 Ｉｌ－１８と分子標的抗体とを併用する癌治療薬
CN105377892A (zh) * 2013-03-15 2016-03-02 艾伯维生物技术有限公司 抗cd25抗体及其用途
WO2017174331A1 (en) 2016-04-07 2017-10-12 Cancer Research Technology Limited Anti cd25 fc gamma receptor bispecific antibodies for tumor specific cell depletion
WO2018167104A1 (en) 2017-03-17 2018-09-20 Tusk Therapeutics Ltd Fc-optimized anti-cd25 for tumour specific cell depletion
WO2019008386A1 (en) * 2017-07-06 2019-01-10 Tusk Therapeutics Ltd COMPOUNDS AND METHODS FOR DEPLOYING SPECIFIC CELLS FROM A TUMOR
WO2019175215A1 (en) * 2018-03-13 2019-09-19 Tusk Therapeutics Ltd Anti-cd25 for tumour specific cell depletion

Patent Citations (29)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5530101A (en) 1988-12-28 1996-06-25 Protein Design Labs, Inc. Humanized immunoglobulins
WO1990007861A1 (en) 1988-12-28 1990-07-26 Protein Design Labs, Inc. CHIMERIC IMMUNOGLOBULINS SPECIFIC FOR p55 TAC PROTEIN OF THE IL-2 RECEPTOR
WO1992022583A2 (en) 1991-06-11 1992-12-23 Celltech Limited Tri- and tetra-valent monospecific antigen-binding proteins
WO1993011238A1 (en) 1991-12-06 1993-06-10 Sumitomo Pharmaceuticals Company, Limited Humanized b-b10, an anti-il2 receptor antibody
US7494647B2 (en) 1998-04-03 2009-02-24 Chugai Seiyaku Kabushiki Kaisha Humanized antibody against human tissue factor (TF) and process of production of the humanized antibody
US20030040606A1 (en) 2001-06-27 2003-02-27 Leung Shawn Shui-On Reducing immunogenicities of immunoglobulins by framework-patching
WO2003048731A2 (en) 2001-12-03 2003-06-12 Abgenix, Inc. Antibody categorization based on binding characteristics
CN101124244A (zh) * 2002-11-15 2008-02-13 根马布股份公司 抗cd25的人单克隆抗体
WO2004045512A2 (en) 2002-11-15 2004-06-03 Genmab A/S Human monoclonal antibodies against cd25
WO2004074437A2 (en) 2003-02-14 2004-09-02 University Of Southern California Compositions and methods for cancer immunotherapy
WO2005003171A2 (en) 2003-07-01 2005-01-13 Celltech R & D Limited Modified antibody fragments
WO2005003170A2 (en) 2003-07-01 2005-01-13 Celltech R & D Limited Modified antibody fragments
WO2005003169A2 (en) 2003-07-01 2005-01-13 Celltech R & D Limited Modified antibody fab fragments
WO2005113605A1 (en) 2004-05-19 2005-12-01 Celltech R & D Limited Cross-linked antibodies
WO2006050172A2 (en) 2004-10-29 2006-05-11 University Of Southern California Combination cancer immunotherapy with co-stimulatory molecules
WO2006108670A2 (en) 2005-04-15 2006-10-19 Novartis Ag Use of cd25 antibodies in immunotherapy
WO2009040562A1 (en) 2007-09-26 2009-04-02 Ucb Pharma S.A. Dual specificity antibody fusions
WO2010035012A1 (en) 2008-09-26 2010-04-01 Ucb Pharma S.A. Biological products
WO2011077245A2 (en) 2009-12-23 2011-06-30 Fondazione Centro San Raffaele Del Monte Tabor Compositions
CN105377892A (zh) * 2013-03-15 2016-03-02 艾伯维生物技术有限公司 抗cd25抗体及其用途
WO2016021720A1 (ja) 2014-08-07 2016-02-11 学校法人兵庫医科大学 Ｉｌ－１８と分子標的抗体とを併用する癌治療薬
WO2017174331A1 (en) 2016-04-07 2017-10-12 Cancer Research Technology Limited Anti cd25 fc gamma receptor bispecific antibodies for tumor specific cell depletion
WO2018167104A1 (en) 2017-03-17 2018-09-20 Tusk Therapeutics Ltd Fc-optimized anti-cd25 for tumour specific cell depletion
CN110869388A (zh) * 2017-03-17 2020-03-06 塔斯克疗法有限公司 用于肿瘤特异性细胞消耗的Fc优化的抗CD25
WO2019008386A1 (en) * 2017-07-06 2019-01-10 Tusk Therapeutics Ltd COMPOUNDS AND METHODS FOR DEPLOYING SPECIFIC CELLS FROM A TUMOR
CN111094346A (zh) * 2017-07-06 2020-05-01 塔斯克疗法有限公司 用于肿瘤特异性细胞消耗的化合物和方法
WO2019175215A1 (en) * 2018-03-13 2019-09-19 Tusk Therapeutics Ltd Anti-cd25 for tumour specific cell depletion
US20190284287A1 (en) 2018-03-13 2019-09-19 Tusk Therapeutics Ltd. Anti-cd25 antibody agents
WO2019175216A1 (en) 2018-03-13 2019-09-19 Tusk Therapeutics Ltd Anti-cd25 for tumour specific cell depletion

Non-Patent Citations (26)

* Cited by examiner, † Cited by third party
Title
ADAIRLAWSON, DRUG DESIGN REVIEWS-ONLINE, vol. 2, no. 3, 2005, pages 209 - 217
ANNE GOUBIER; SOLOMON ISABELLE; VARGAS FREDERICK ARCE; ZERVAS DIMITRIOS; QING CHEN; SALIMU JOSEPHINE; BROWN MARK; MERCHIERS PASCAL: "Non-IL-2 Blocking Anti-CD25-Targeting Antibodies: Depletion of Regulatory T-cells Driving Optimaleffector Response for Rejection of Established Tumors", CANCER IMMUNOLOGY RESEARCH; FOURTH CRI-CIMT-EATI-AACR INTERNATIONAL CANCER IMMUNOTHERAPY. TRANSLATING SCIENCE INTO SURVIVAL; SEPTEMBER 30 - OCTOBER 3, 2018; NEW YORK, NY,20190201; 20180930 - 20181003AMERICAN ASSOCIATION FOR CANCER RESEARCH, US, vol. 7, no. 2, Suppl, 28 February 2019 (2019-02-28), pages 1 - 4, XP055637738, ISSN: 2326-6066, DOI: 10.1158/2326-6074.CRICIMTEATIAACR18-A192 *
BIOINFORMATION, vol. 10, no. 4, 2014, pages 180 - 186
CHOTHIA ET AL., J. MOL. BIOL., vol. 196, 1986, pages 901 - 17
CHOTHIA ET AL., NATURE, vol. 342, 1989, pages 877 - 83
CLYNES ET AL., PROC NATL ACAD SCI USA, vol. 95, 1998, pages 652 - 6
HAYES J ET AL., JINFLAMM RES, vol. 9, 2016, pages 209 - 219
HOLLIGERHUDSON, NATURE BIOTECH., vol. 23, no. 9, 2005, pages 1126 - 1136
JOHNSONWU, NUCLEIC ACIDS RES., vol. 28, 2000, pages 214 - 8
LAZAR ET AL., PROC NATL ACAD SCI USA, vol. 103, 2006, pages 2005 - 2010
MACCALLUM ET AL., J. MOL. BIOL., vol. 5, 1996, pages 732 - 45
MAKABE ET AL., JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 283, 2008, pages 1156 - 1166
MALEK ET AL., PNAS, vol. 80, no. 18, September 1983 (1983-09-01), pages 5694 - 5698
MALEK, IMMUNOLOGY, vol. 80, 1983, pages 5694 - 5698
MARTIN ET AL., PROC NATL ACAD SCI (USA, vol. 86, 1989, pages 9268 - 9272
METHODS MOLBIOL, vol. 1904, 2019, pages 213 - 230
MICHAEL J. FLYNN; JOHN A. HARTLEY: "The emerging role of anti‐CD25 directed therapies as both immune modulators and targeted agents in cancer", BRITISH JOURNAL OF HAEMATOLOGY, JOHN WILEY, HOBOKEN, USA, vol. 179, no. 1, 30 May 2017 (2017-05-30), Hoboken, USA, pages 20 - 35, XP071122009, ISSN: 0007-1048, DOI: 10.1111/bjh.14770 *
ONIZUKA S ET AL., CANC RES, vol. 59, 1999, pages 3128 - 3133
ONIZUKA S ET AL., CANCERRES., vol. 59, no. 13, 1 July 1999 (1999-07-01), pages 3128 - 3133
RICHARDS ET AL., MOL CANCER THER, vol. 7, 2008, pages 2517 - 2527
SAMUDRALA ET AL.: "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach", PROTEINS, STRUCTURE, FUNCTION AND GENETICS, 1999, pages 194 - 198, XP001146416
SMITH ET AL., PROC NATL ACAD SCI USA, vol. 109, 2012, pages 6181 - 6186
VERMA ET AL., JOURNAL OF IMMUNOLOGICAL METHODS, vol. 216, 1998, pages 165 - 181
YULIUS Y SETIADY ET AL., EUR J IMMUNOL., vol. 40, no. 3, March 2010 (2010-03-01), pages 780 - 6
ZHANG ET AL., CANCER BIOTHER RADIOPHARM, vol. 24, no. 3, 2009, pages 303 - 309
ZHANG ET AL., CANCER BIOTHER RADIOPHARM., vol. 24, no. 3, June 2009 (2009-06-01), pages 303 - 309

Also Published As

Publication number Publication date
CN115397860A (zh) 2022-11-25
CA3182362A1 (en) 2021-11-18
EP4151655A1 (en) 2023-03-22
TW202144429A (zh) 2021-12-01
JP2023525060A (ja) 2023-06-14
US20230174670A1 (en) 2023-06-08

Similar Documents

Publication Publication Date Title
WO2017148424A1 (zh) 一种pdl-1抗体、其药物组合物及其用途
CN110366560B (zh) 抗b7-h4抗体、其抗原结合片段及其医药用途
US11525005B2 (en) Anti-CD40 antibody, antigen binding fragment thereof and medical use thereof
KR20210143192A (ko) 변형된 Fc 단편, 이를 포함하는 항체 및 이의 응용
JP7053479B2 (ja) ＩＬ７受容体細胞外ドメインα鎖に対する非拮抗性抗体及び癌治療におけるそれらの使用
WO2019141268A1 (zh) 抗4-1bb抗体、其抗原结合片段及其医药用途
CN112969714B (zh) 抗cd40抗体、其抗原结合片段及其医药用途
WO2020063660A1 (zh) 抗ox40抗体、其抗原结合片段及其医药用途
TW202304997A (zh) 新型抗ｃｄ４抗體
WO2021228218A1 (zh) 抗cd25抗体、其抗原结合片段及其医药用途
TWI770619B (zh) 對lif具有專一性的結合分子及其用途
CN114907479A (zh) 抗cd112r抗体及其用途
JP2022553927A (ja) Ｉｌｔ－２阻害剤での癌の処置
CN116135883A (zh) 抗cd25抗体、其抗原结合片段及其医药用途
WO2021209066A1 (zh) 特异性抗原结合分子，其制备方法及医药用途
WO2022262749A1 (zh) 靶向pd1和/或ox40的特异性结合蛋白
WO2022258015A1 (en) Antibodies and bispecific binding proteins that bind ox40 and/or pd-l1
RU2779128C2 (ru) Антитело к cd40, его антигенсвязывающий фрагмент и его медицинское применение
TW202221038A (zh) 抗ｔｒｏｐ－２抗體、其抗原結合片段或其突變體、及其醫藥用途

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21804918

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 3182362

Country of ref document: CA

ENP Entry into the national phase

Ref document number: 2022567574

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2021804918

Country of ref document: EP

Effective date: 20221214