WO2021164722A1 - 抗il-2抗体、其抗原结合片段及其医药用途 - Google Patents

抗il-2抗体、其抗原结合片段及其医药用途 Download PDF

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WO2021164722A1
WO2021164722A1 PCT/CN2021/076806 CN2021076806W WO2021164722A1 WO 2021164722 A1 WO2021164722 A1 WO 2021164722A1 CN 2021076806 W CN2021076806 W CN 2021076806W WO 2021164722 A1 WO2021164722 A1 WO 2021164722A1
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seq
antibody
antigen
lcdr1
hcdr1
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PCT/CN2021/076806
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English (en)
French (fr)
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林�源
朱福香
廖成
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江苏恒瑞医药股份有限公司
上海盛迪医药有限公司
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Priority to AU2021223063A priority Critical patent/AU2021223063A1/en
Priority to KR1020227031261A priority patent/KR20220143869A/ko
Priority to BR112022016326A priority patent/BR112022016326A2/pt
Priority to US17/904,517 priority patent/US20230089620A1/en
Priority to JP2022549830A priority patent/JP2023515480A/ja
Priority to CN202180010561.8A priority patent/CN115210257A/zh
Priority to MX2022010218A priority patent/MX2022010218A/es
Priority to CA3169980A priority patent/CA3169980A1/en
Priority to EP21756707.2A priority patent/EP4108683A4/en
Publication of WO2021164722A1 publication Critical patent/WO2021164722A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/244—Interleukins [IL]
    • C07K16/246—IL-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/55—IL-2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/577—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 tolerising response
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • 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
    • C07K2317/70—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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies

Definitions

  • the present disclosure relates to antibodies, such as antibodies that specifically bind to interleukin-2 (IL-2) and antigen-binding fragments thereof.
  • IL-2 interleukin-2
  • the present disclosure relates to the anti-IL-2 antibody, its antigen-binding fragment and IL-2 complex (including fusion protein), and its use as a medicine for the treatment of immune-related (such as autoimmune diseases, inflammatory disorders).
  • IL-2 Human Interleukin-2
  • TCGF T cell growth factor
  • the gene is located on chromosome 4 (4q27), including a total sequence of 7 kb, consisting of 133 amino acids, with a molecular weight of approximately 15kD.
  • TCGF T cell growth factor
  • the stimulating factor in the culture medium was purified and identified as a single protein, namely IL-2.
  • T cells can secrete IL-2 and express IL-2 receptor (IL-2R) on the cell surface after being activated by TCR and CD28.
  • IL-2R IL-2 receptor
  • This model makes IL-2 a molecule that plays a central role in the T cell immune response.
  • subsequent in vivo experiments found that after knocking out IL-2 or its receptor, the animals developed autoimmunity. Subsequent experiments showed that IL-2 can not only activate effector cells such as T cells and NK cells, but also activate regulatory T cells (Treg), thereby suppressing excessive immunity against itself.
  • IL-2 acts through 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 ⁇ and IL-2R ⁇ are necessary for IL-2 to activate downstream signaling pathways.
  • IL-2 When IL-2 binds to IL-2R ⁇ and IL-2R ⁇ at the same time, the two receptor subunits form a heterodimer, phosphoric acid STAT5 in the cell can enter the nucleus to cause the corresponding gene transcription and expression; IL-2R ⁇ is not necessary for signal transduction, but it can enhance the binding of IL-2 to IL-2R ⁇ and IL-2R ⁇ .
  • Tregs continue to express the high binding and force receptor IL-2R ⁇ / ⁇ / ⁇ , while the inactivated CD8+ T cells and NK cells only express the medium binding force receptor IL-2R ⁇ / ⁇ . Therefore, low-dose IL-2 preferentially binds and activates Treg, while high-dose IL-2 can also activate immune effector cells such as CD8+ T cells and NK cells.
  • low-dose IL-2 has been reported to be used in a series of immune-related diseases, such as the treatment of autoimmune diseases and inflammatory diseases, such as system Systemic lupus erythematosus (SLE), type I diabetes (T1D), chronic graft versus host disease (cGvHD), bone marrow transplantation (HSCT), alopecia areata (alopecia areata), hepatitis C virus-induced vasculitis, chronic GvHD, asthma, dermatitis, alopecia areata, acute lung injury, chronic beryllium dust, sepsis, etc.
  • SLE Systemic lupus erythematosus
  • T1D type I diabetes
  • cGvHD chronic graft versus host disease
  • HSCT bone marrow transplantation
  • alopecia areata hepatitis C virus-induced vasculitis
  • chronic GvHD asthma
  • dermatitis alopecia areata
  • acute lung injury chronic be
  • IL-2 has a short half-life and requires frequent administration; on the other hand, IL-2 has a smaller administration window. If the dose is slightly higher, it may activate immune effector cells and aggravate autoimmune diseases. The nature or shortcomings of these IL-2 itself make it difficult to maintain a sustained low dose of IL-2 in patients, limiting its further application.
  • Anti-IL-2 antibodies can change the binding of IL-2 to IL-2R.
  • WO2017/070561 and WO2015/109212 provide anti-IL-2 antibodies.
  • the present disclosure satisfies these needs.
  • the present disclosure provides anti-IL-2 antibodies and antigen-binding fragments thereof, and complexes comprising the antibodies, antigen-binding fragments, encoding nucleic acids, vectors, host cells, pharmaceutical compositions, and methods for treating or delaying immune-related diseases , And its pharmaceutical uses.
  • an antibody comprising IL-2 or an antigen-binding fragment thereof is provided, and the K D value of its affinity with IL-2 is ⁇ 5 nM.
  • IL-2 antibodies or antigen-binding fragments thereof are provided, and the affinity K D value of IL-2 with IL-2 is> 5 nM or ⁇ 10 nM, and ⁇ 200 nM or ⁇ 150 nM or ⁇ 100 nM or ⁇ 80 nM or ⁇ 60 nM.
  • the IL-2 antibody or antigen-binding fragment thereof has a K D value of the affinity of IL-2> 5 nM and ⁇ 60 nM,> 5 nM and ⁇ 50 nM, ⁇ 10 nM and ⁇ 50 nM, ⁇ 10 nM and ⁇ 60 nM , >5nM and ⁇ 80nM, ⁇ 10nM and ⁇ 80nM, >5nM and ⁇ 100nM, ⁇ 10nM and ⁇ 100nM, >5nM and ⁇ 150nM, or ⁇ 10nM and ⁇ 150nM.
  • the IL-2 antibody or antigen-binding fragment thereof of the present disclosure contains a heavy chain variable region (VH) and a light chain variable region (VL), the VH has HCDR1, HCDR2, HCDR3, and VL has LCDR1, LCDR2, LCDR3.
  • VH heavy chain variable region
  • VL light chain variable region
  • the IL-2 antibody or antigen-binding fragment thereof contains VH and VL:
  • HCDR1, HCDR2, and HCDR3 of the VH correspond to HCDR1, HCDR2, and HCDR3 in SEQ ID NO:1, respectively
  • LCDR1, LCDR2, and LCDR3 of the VL respectively correspond to LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 2;
  • HCDR1, HCDR2, and HCDR3 of the VH correspond to HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 3, respectively, and LCDR1, LCDR2, and LCDR3 of the VL respectively correspond to LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 4;
  • HCDR1, HCDR2, and HCDR3 of the VH respectively correspond to HCDR1, HCDR2, HCDR3 in SEQ ID NO: 5
  • LCDR1, LCDR2, and LCDR3 of the VL respectively correspond to LCDR1, LCDR2, LCDR3 in SEQ ID NO: 6;
  • HCDR1, HCDR2, and HCDR3 of the VH respectively correspond to HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 7
  • LCDR1, LCDR2, and LCDR3 of the VL respectively correspond to LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 8;
  • HCDR1, HCDR2, and HCDR3 of the VH correspond to HCDR1, HCDR2, HCDR3 in SEQ ID NO: 9 respectively
  • LCDR1, LCDR2, and LCDR3 of the VL respectively correspond to LCDR1, LCDR2, LCDR3 in SEQ ID NO: 10; or
  • HCDR1, HCDR2, and HCDR3 of the VH correspond to HCDR1, HCDR2, and HCDR3 in SEQ ID NO: 11, respectively
  • LCDR1, LCDR2, and LCDR3 of the VL correspond to LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 12, respectively.
  • the above-mentioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering system.
  • the anti-IL-2 antibody or antigen-binding fragment thereof comprises:
  • SEQ ID NO: HCDR2 shown in 14, 20, 26, 32, 38 or 44, and/or
  • SEQ ID NO: HCDR3 shown in 15, 21, 27, 33, 39 or 45, and/or
  • SEQ ID NO: LCDR1 shown in 16, 22, 28, 34, 40 or 46, and/or
  • SEQ ID NO: LCDR2 shown in 17, 23, 29, 35, 41 or 47, and/or
  • SEQ ID NO: LCDR3 shown in 18, 24, 30, 36, 42 or 48.
  • the described embodiment uses the Kabat numbering system.
  • the anti-IL-2 antibody or antigen-binding fragment thereof comprises:
  • SEQ ID NO: HCDR1 shown in 49, 55, 61, 67, 73 or 79, and/or
  • SEQ ID NO: HCDR2 shown in 50, 56, 62, 68, 74 or 80, and/or
  • SEQ ID NO: LCDR1 shown in 52, 58, 64, 70, 76 or 82, and/or
  • SEQ ID NO: LCDR2 shown in 53, 59, 65, 71, 77 or 83, and/or
  • SEQ ID NO: LCDR3 shown in 54, 60, 66, 72, 78 or 84.
  • the described embodiment uses the Chothia numbering system.
  • the anti-IL-2 antibody or antigen-binding fragment thereof comprises:
  • SEQ ID NO: HCDR2 shown in 86, 92, 98, 104, 110 or 116, and/or
  • SEQ ID NO: HCDR3 shown in 87, 93, 99, 105, 111 or 117, and/or
  • SEQ ID NO: LCDR1 shown in 88, 94, 100, 106, 112, or 118, and/or
  • SEQ ID NO: LCDR2 shown in 89, 95, 101, 107, 113 or 119, and/or
  • SEQ ID NO: LCDR3 shown in 90, 96, 102, 108, 114, or 120.
  • the described embodiment adopts the IMGT numbering system.
  • the anti-IL-2 antibody or antigen-binding fragment thereof comprises:
  • SEQ ID NO: HCDR1 shown in 121, 127, 133, 139, 145 or 151, and/or
  • SEQ ID NO: HCDR2 shown in 122, 128, 134, 140, 146 or 152, and/or
  • SEQ ID NO: HCDR3 shown in 123, 129, 135, 141, 147 or 153, and/or
  • SEQ ID NO: LCDR1 shown in 124, 130, 136, 142, 148 or 154, and/or
  • SEQ ID NO: LCDR2 shown in 125, 131, 137, 143, 149 or 155, and/or
  • SEQ ID NO: LCDR3 shown in 126, 132, 138, 144, 150 or 156.
  • the described embodiment adopts the AbM numbering system.
  • the above anti-IL-2 antibodies or their antigen-binding fragments are divided into two groups, the first group includes:
  • SEQ ID NO: LCDR2 shown in 17, 29 or 41, and/or
  • SEQ ID NO: LCDR2 shown in 53, 65 or 77, and/or
  • SEQ ID NO: LCDR1 shown in 88, 100 or 112, and/or
  • SEQ ID NO: LCDR2 shown in 89, 101 or 113, and/or
  • SEQ ID NO: HCDR3 shown in 123, 135 or 147, and/or
  • SEQ ID NO: LCDR2 shown in 125, 137 or 149, and/or
  • SEQ ID NO: LCDR3 shown in 126, 138 or 150.
  • the second group contains:
  • SEQ ID NO: LCDR3 shown in 24, 36 or 48;
  • SEQ ID NO: LCDR3 shown in 60, 72 or 84;
  • SEQ ID NO: LCDR2 shown in 95, 107 or 119, and/or
  • SEQ ID NO: LCDR1 shown in 130, 142 or 154, and/or
  • SEQ ID NO: LCDR2 shown in 131, 143 or 155, and/or
  • SEQ ID NO: LCDR3 shown in 132, 144 or 156.
  • the K D value of the affinity of the IL-2 antibody or antigen-binding fragment thereof to IL-2 of the first group is> 5 nM or ⁇ 10 nM, and ⁇ 200 nM or ⁇ 150 nM or ⁇ 100 nM or ⁇ 80 nM or ⁇ 60nM.
  • the IL-2 antibody or antigen-binding fragment thereof has a K D value of the affinity of IL-2> 5 nM and ⁇ 60 nM,> 5 nM and ⁇ 50 nM, ⁇ 10 nM and ⁇ 60 nM, ⁇ 10 nM and ⁇ 50 nM , >5nM and ⁇ 80nM, ⁇ 10nM and ⁇ 80nM, >5nM and ⁇ 100nM, ⁇ 10nM and ⁇ 100nM, >5nM and ⁇ 150nM, or ⁇ 10nM and ⁇ 150nM.
  • one or more amino acid mutations may also exist in the CDR regions.
  • the mutated amino acids can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.
  • the K D value of its affinity for IL-2 is ⁇ 5 nM.
  • an IL-2 antibody or antigen-binding fragment thereof which binds to the same antigen or epitope as the aforementioned anti-IL-2 antibody or antigen-binding fragment thereof, or which binds to the aforementioned anti-IL-2 antibody or antigen-binding fragment thereof Competing for binding to the same antigen or epitope, or for binding to the aforementioned anti-IL-2 antibody or antigen-binding fragment thereof, or for binding to IL-2.
  • the aforementioned anti-IL-2 antibody or antigen-binding fragment thereof reduces or blocks the binding of IL-2 to IL-2R ⁇ , for example, blocks the binding of IL-2 to IL-2R ⁇ .
  • the aforementioned anti-IL-2 antibody or antigen-binding fragment thereof reduces the binding of IL-2 to IL-2R ⁇ .
  • the aforementioned anti-IL-2 antibody or antigen-binding fragment thereof is a fully human antibody or antigen-binding fragment thereof, or a humanized antibody or antigen-binding fragment thereof.
  • the aforementioned anti-IL-2 antibody or antigen-binding fragment thereof is an IgG antibody or antigen-binding fragment thereof, for example, an IgG1, IgG4 antibody or an antigen-binding fragment thereof.
  • the aforementioned anti-IL-2 antibody or antigen-binding fragment thereof is an IgG1 antibody with reduced effector function, such as an IgG1 antibody with reduced or missing ADCC function (for example, an IgG1 antibody with an Fc with a LALA mutation).
  • the aforementioned anti-IL-2 antibody or antigen-binding fragment thereof comprises HCDR1-3 and/or LCDR1-3 selected from the following group:
  • HCDR1-3 shown in SEQ ID NO: 151-153 and/or LCDR1-3 shown in SEQ ID NO: 154-156.
  • an anti-IL-2 antibody or antigen-binding fragment thereof which comprises VH and VL:
  • VH includes HCDR1-3 shown in SEQ ID NO: 37-39, and its VL includes LCDR1-3 shown in SEQ ID NO: 40-42;
  • VH includes HCDR1-3 shown in SEQ ID NO: 13-15, and its VL includes LCDR1-3 shown in SEQ ID NO: 16-18;
  • VH includes HCDR1-3 shown in SEQ ID NO: 25-27, and its VL includes LCDR1-3 shown in SEQ ID NO: 28-30;
  • VH includes HCDR1-3 shown in SEQ ID NO: 19-21, and its VL includes LCDR1-3 shown in SEQ ID NO: 22-24;
  • VH includes the HCDR1-3 shown in SEQ ID NO: 31-33, and its VL includes the LCDR1-3 shown in SEQ ID NO: 34-36; or
  • VH includes the HCDR1-3 shown in SEQ ID NO: 43-45, and its VL includes the LCDR1-3 shown in SEQ ID NO: 46-48;
  • VH includes the HCDR1-3 shown in SEQ ID NO: 73-75, and its VL includes the LCDR1-3 shown in SEQ ID NO: 76-78;
  • VH includes the HCDR1-3 shown in SEQ ID NO: 49-51, and its VL includes the LCDR1-3 shown in SEQ ID NO: 52-54;
  • VH includes the HCDR1-3 shown in SEQ ID NO: 61-63, and its VL includes the LCDR1-3 shown in SEQ ID NO: 64-66;
  • VH includes HCDR1-3 shown in SEQ ID NO: 55-57, and its VL includes LCDR1-3 shown in SEQ ID NO: 58-60;
  • VH includes the HCDR1-3 shown in SEQ ID NO: 67-69, and its VL includes the LCDR1-3 shown in SEQ ID NO: 70-72;
  • VH includes the HCDR1-3 shown in SEQ ID NO: 79-81, and its VL includes the LCDR1-3 shown in SEQ ID NO: 82-84;
  • VH includes the HCDR1-3 shown in SEQ ID NO: 109-111, and its VL includes the LCDR1-3 shown in SEQ ID NO: 112-114;
  • VH includes the HCDR1-3 shown in SEQ ID NO: 85-87, and its VL includes the LCDR1-3 shown in SEQ ID NO: 88-90;
  • VH includes the HCDR1-3 shown in SEQ ID NO: 97-99, and its VL includes the LCDR1-3 shown in SEQ ID NO: 100-102;
  • VH includes the HCDR1-3 shown in SEQ ID NO: 91-93, and its VL includes the LCDR1-3 shown in SEQ ID NO: 94-96;
  • VH includes the HCDR1-3 shown in SEQ ID NO: 103-105, and its VL includes the LCDR1-3 shown in SEQ ID NO: 106-108; or
  • VH includes the HCDR1-3 shown in SEQ ID NO: 115-117, and its VL includes the LCDR1-3 shown in SEQ ID NO: 118-120;
  • VH includes the HCDR1-3 shown in SEQ ID NO: 145-147, and its VL includes the LCDR1-3 shown in SEQ ID NO: 148-150;
  • VH includes the HCDR1-3 shown in SEQ ID NO: 121-123, and its VL includes the LCDR1-3 shown in SEQ ID NO: 124-126;
  • VH includes the HCDR1-3 shown in SEQ ID NO: 133-135, and its VL includes the LCDR1-3 shown in SEQ ID NO: 136-138;
  • VH includes the HCDR1-3 shown in SEQ ID NO: 127-129, and its VL includes the LCDR1-3 shown in SEQ ID NO: 130-132;
  • VH includes the HCDR1-3 shown in SEQ ID NO: 139-141, and its VL includes the LCDR1-3 shown in SEQ ID NO: 142-144; or
  • VH includes the HCDR1-3 shown in SEQ ID NO: 151-153
  • VL includes the LCDR1-3 shown in SEQ ID NO: 154-156.
  • the aforementioned anti-IL-2 antibody or antigen-binding fragment thereof comprises:
  • VH as shown in one of SEQ ID NO: 1, 3, 5, 7, 9, 11 or at least 90%, 95%, 98%, 99% identical to it;
  • the aforementioned anti-IL-2 antibody or antigen-binding fragment thereof comprises VH and/or VL selected from the following group:
  • the heavy chain variable region (VH) of the aforementioned anti-IL-2 antibody or antigen-binding fragment thereof is connected to the human IgG1 heavy chain constant region, and the light chain variable region (VL) is connected to the kappa or lamda chain constant region.
  • the aforementioned anti-IL-2 antibody comprises:
  • HC that is shown in one of SEQ ID NO: 157, 159, 161, 163, 165, 167 or has at least 80%, 90%, 95%, 98%, 99% identity with it;
  • An LC that is shown in one of SEQ ID NO: 158, 160, 162, 164, 166, 168 or has at least 80%, 90%, 95%, 98%, 99% identity with it.
  • the aforementioned anti-IL-2 antibody or antigen-binding fragment thereof comprises HC and LC selected from the following group:
  • HC Full-length heavy chain
  • LC Full-length light chain
  • HC as shown in SEQ ID NO: 159 or at least 80%, 90%, 95%, 98%, 99% identical to it, as shown in SEQ ID NO: 160 or at least 80%, 90% with it , 95%, 98%, 99% identity LC;
  • HC as shown in SEQ ID NO: 167 or at least 80%, 90%, 95%, 98%, 99% identical to it, as shown in SEQ ID NO: 168 or at least 80%, 90% , 95%, 98%, 99% identity LC.
  • the antigen-binding fragment of the anti-IL-2 antibody is Fab, Fv, sFv, Fab', F(ab') 2 , linear antibody, single chain antibody, scFv, sdAb, sdFv, nanobody, peptide antibody peptibody , Domain antibodies and multispecific antibodies (bispecific antibodies, diabody, triabody and tetrabody, tandem two-scFv, tandem three-scFv), for example specifically scFv, Fv, Fab or Fab' fragments.
  • the present disclosure provides a complex comprising IL-2 antibody or antigen-binding fragment thereof and IL-2.
  • the IL-2 antibody or its antigen-binding fragment and IL-2 in the complex bind non-covalently, and the K D value of the affinity of the IL-2 antibody or its antigen-binding fragment with IL-2 is ⁇ 5nM.
  • the IL-2 antibody or its antigen-binding fragment and IL-2 in the complex bind covalently (for example, to form a fusion protein), wherein the IL-2 antibody or its antigen-binding fragment and IL-2
  • the K D value of the affinity is >5nM or ⁇ 10nM, and ⁇ 200nM or ⁇ 150nM or ⁇ 100nM or ⁇ 80nM or ⁇ 60nM.
  • the K D value of the affinity is> 5 nM and ⁇ 60 nM,> 5 nM and ⁇ 50 nM, ⁇ 10 nM and ⁇ 60 nM, ⁇ 10 nM and ⁇ 50 nM,> 5 nM and ⁇ 80 nM, ⁇ 10 nM and ⁇ 80 nM , >5nM and ⁇ 100nM, ⁇ 10nM and ⁇ 100nM, >5nM and ⁇ 150nM, or ⁇ 10nM and ⁇ 150nM.
  • a fusion protein which comprises an anti-IL-2 antibody or an antigen fragment thereof and IL-2, wherein the affinity of the anti-IL-2 antibody or an antigen fragment thereof to IL-2 has a K D value> 5 nM or ⁇ 10 nM , And ⁇ 50nM or ⁇ 60nM or ⁇ 80nM.
  • the K D value of the affinity of the anti-IL-2 antibody or its antigen fragment to IL-2 is greater than ⁇ 10 nM and ⁇ 60 nM.
  • the anti-IL-2 antibody or antigen fragment thereof and IL-2 are bound by covalent or non-covalent forces.
  • the anti-IL-2 antibody or antigenic fragment thereof forms a fusion protein with IL-2.
  • IL-2 is connected to the light chain variable region or heavy chain variable region of the anti-IL-2 antibody or its antigen fragment, for example, IL-2 is connected to the light chain variable region or heavy chain of the anti-IL-2 antibody or its antigen fragment.
  • the N-terminus of the chain variable region is connected.
  • IL-2 is linked to the N-terminus of the light chain variable region.
  • the IL-2 in the aforementioned fusion protein and the anti-IL-2 antibody or antigen fragment thereof are connected via a linker.
  • the linker is selected from the group consisting of: (G m S n ) x or (GGNGT) x or (YGNGT) x amino acid sequences, wherein m and n are each independently selected from an integer of 1-8 (e.g., 1, 2, 3, 4, 5, 6, 7 or 8), x is independently selected from an integer of 1-20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20).
  • the linker has an amino acid sequence as shown in (G 4 S)x, and x is independently selected from an integer of 1-6 (for example, x is 4 or 5).
  • the linker may also be other linkers with similar flexibility and length to the amino acid sequence shown in (G m S n ) x or (GGNGT) x or (YGNGT) x; the linker may also be selected from AKTTPKLEEGEFSEAR , AKTTPKLEEGEFSEARV, AKTTPKLGG, SAKTTPKLGG, AKTTPKLEEGEFSEARV, SAKTTP, SAKTTPKLGG, RADAAP, RADAAPTVS, RADAAAAGGPGS, RADAAAA (G 4 S) 4 , SAKTTP, SAKTTPKLEEGE, SAKTTPKLGG, SAKTTPKLEAP, SAKTTPKLGG, SAKTA, VTAA, VTAAPK, VTKA, VTAAPT, SAKTTPK, VTPA,
  • the above-mentioned complex selectively enhances the activity or promotes the proliferation of cells that highly express IL-2Ra, such as regulatory T (Treg) cells.
  • the complex is a fusion protein formed by an anti-IL-2 antibody or an antigen fragment thereof and IL-2.
  • IL-2 in the aforementioned fusion protein has an amino acid substitution or N-terminal deletion of the third amino acid (as an example, T3).
  • the third amino acid substitution of IL-2 is Ala, Gln or Glu (as an example, T3A, T3Q, T3E), and the N-terminal deletion is the deletion of the first 3 or 7 amino acids of the N-terminal.
  • the aforementioned fusion protein comprises HC and LC selected from the following group:
  • the aforementioned complex of the present disclosure selectively enhances the effect of IL-2 on Treg activity (e.g., pro-proliferation activity, pro-STAT5 phosphorylation activity), and/or selectively increases IL-2 pro-Treg cells The ability to express one or more of FOXP3, CD25 and Icos.
  • Treg activity e.g., pro-proliferation activity, pro-STAT5 phosphorylation activity
  • IL-2 pro-Treg cells The ability to express one or more of FOXP3, CD25 and Icos.
  • the compound of the present disclosure further has one or more of (i)-(iii):
  • Treg cells Compared with CD8+ T cells, Treg cells promote STAT5 phosphorylation to a higher degree;
  • the Fc region of the anti-IL-2 antibodies or antigen-binding fragments thereof of the present disclosure comprises one or more amino acid substitutions, and the one or more amino acid substitutions reduce the binding of the Fc region to Fc receptors, for example, the Fc region It binds to Fc ⁇ receptors and reduces or eliminates effector functions.
  • the anti-IL-2 antibodies or antigen-binding fragments thereof of the present disclosure have reduced or eliminated ADCC function.
  • the anti-IL-2 antibodies or antigen-binding fragments thereof of the present disclosure have a modified Fc region, and the modified Fc region has a 50%, 80% reduction in binding affinity to the Fc receptor compared with the natural Fc region. %, 90% or more than 95%.
  • the Fc receptor is an Fc ⁇ receptor.
  • the Fc receptor is a human Fc ⁇ receptor, such as Fc ⁇ RI, Fc ⁇ RIIa, Fc ⁇ RIIB, Fc ⁇ RIIIa.
  • the engineered Fc region also has a reduced binding affinity for complement (such as C1q) compared to the natural Fc region.
  • the engineered Fc region has no reduced binding affinity for neonatal Fc receptor (FcRn) compared to the natural Fc region.
  • the modified Fc region has reduced effector functions, and the reduced effector functions may include, but are not limited to, one or more of the following: reduced complement dependent cytotoxicity (CDC), reduced Antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent cellular phagocytosis (ADCP), reduced cytokine secretion, reduced immune complex-mediated antigen uptake by antigen-presenting cells, reduced interaction with NK cells Binding, decreased binding to macrophages, decreased binding to monocytes, decreased binding to polymorphonuclear cells, decreased direct signaling induced apoptosis, decreased dendritic cell maturation or reduction The T cells are triggered.
  • CDC reduced complement dependent cytotoxicity
  • ADCC Antibody-dependent cell-mediated cytotoxicity
  • ADCP reduced antibody-dependent cellular phagocytosis
  • cytokine secretion reduced immune complex-mediated anti
  • substitution of amino acid residues at positions 238, 265, 269, 270, 297, 327, and 329 can reduce effector functions.
  • the Fc region is a human IgG 1 Fc region, and the amino acid residues at positions 234 and 235 are A, and the numbering is based on the EU index.
  • substitution of amino acid residues at positions such as 228 can reduce effector functions.
  • Amino acid substitutions that change effector functions are selected from one or more of the following: S298A/E333A/K334A; S239D/I332E/A330L; S239D/I332E/G236A; G236A/S239D/A330L/I332E; F243L/R292P/Y300L /V305I/P396L; K326A/E333A; K326W/E333S; K326M/E333S; C221D/D222C; S267E/H268F/S324T; E345R; S298A/E333A/K334A/N434A; E294 is missing/T307P/N434Y; T256N/A378V/S383N/S383N N434Y; T252L/T253S/T254F; M252Y/S254T/T256E; M428L/
  • the present disclosure provides an anti-IL-2 antibody or antigen-binding fragment thereof having a moderate affinity for IL-2, where the intermediate affinity is the affinity of the antibody and IL-2 with a K D value> 5 nM or ⁇ 10 nM, and ⁇ 200 nM or ⁇ 150nM or ⁇ 100nM or ⁇ 80nM or ⁇ 60nM (for example, ⁇ 10nM and ⁇ 50nM).
  • the antibody and IL-2 form a fusion protein
  • the antibody and IL-2 are connected through a linker.
  • the antibody and IL-2 are also bound by non-covalent bonds, but the degree of non-covalent bond binding (ie, affinity) is not higher than that of IL-2R ⁇ and IL-2.
  • the fusion protein when the fusion protein contacts IL-2R ⁇ on the cell surface, the non-covalent bond between the anti-IL-2 antibody and IL-2 is dissociated, and IL-2 binds to IL-2R ⁇ , thereby activating the cell.
  • Tregs continue to express the high binding and force receptor IL-2R ⁇ / ⁇ / ⁇ , while the inactivated CD8+ T cells and NK cells only express the medium binding force receptor IL-2R ⁇ / ⁇ . Therefore, the fusion protein of this type of antibody and IL-2 can selectively or preferentially activate Treg.
  • Such antibodies provided in the present disclosure are, for example, A2-22, B2-15, D2-60, and antibodies with the same HCDR1-3 and LCDR1-3.
  • the present disclosure also provides an anti-IL-2 antibody or antigen-binding fragment thereof with high affinity to IL-2, where the high affinity is that the affinity K D value of the antibody to IL-2 is ⁇ 5 nM.
  • the antibody and IL-2 can be bound by a non-covalent bond, and the degree of non-covalent bond binding (ie, affinity) is similar to or slightly higher than the avidity of IL-2R ⁇ and IL-2 or slightly higher than that of IL-2R ⁇ and IL-2. 2.
  • the complex (non-covalent bond) of the antibody and IL-2 contacts the IL-2R ⁇ on the cell surface, the non-covalent bond between the anti-IL-2 antibody and IL-2 is dissociated Or partially dissociated, IL-2 binds to IL-2R ⁇ , thereby activating the cell. Therefore, the complex (non-covalent bond) of this type of antibody and IL-2 can also selectively or preferentially activate Treg.
  • the present disclosure does not exclude the fusion protein form of this type of antibody and IL-2.
  • Such antibodies provided in the present disclosure are, for example, C2-53, D3-68, and antibodies with the same HCDR1-3 and LCDR1-3.
  • the affinity of anti-IL-2 antibodies or antigen-binding fragments thereof to IL-2 is tested by conventional methods in the art.
  • the affinity is expressed by the K D value, for example, Biacore (see Example 6 for the specific detection method) is used for detection.
  • the affinity values expressed as 50 EC for example, using ELISA assay (see Example 5 Detection particular embodiment).
  • some of the anti-IL-2 antibodies or antigen-binding fragments thereof of the present disclosure can reduce the binding of IL-2 and IL-2R ⁇ , which is detected by ELISA test, see Example 3 for details; or by Octet test For detection, see Example 4 for details.
  • an antibody having HCDR1-3 and LCDR1-3 in the sequence described in SEQ ID NO: 9 and 10 (such as D2-60) has an HCDR1- in the sequence described in SEQ ID NO: 5 and 6
  • Antibodies against 3 and LCDR1-3 (for example, B2-15), and antibodies against HCDR1-3 and LCDR1-3 in the sequence described in SEQ ID NO:1 and 3 (for example, A2-22) cannot block IL-2 and IL
  • the binding of -2R ⁇ can only reduce the binding of IL-2 and IL-2R ⁇ .
  • Example 3 can be understood that at the high antibody concentration of Example 3 (for example, 10 ⁇ g/mL), a meaningful IC 50 value cannot be obtained by fitting the curve.
  • Increase can be understood as being able to affect the binding of IL-2 and IL-2R ⁇ to a certain extent under the conditions of Example 3 (for example, Figure 1A), or under the experimental conditions of Example 4, having greater than 0.600 (for example, 0.700, 0.754, 0.848) Octet level of IL-2R ⁇ blocking value.
  • some of the anti-IL-2 antibodies or antigen-binding fragments thereof of the present disclosure can block the binding of IL-2 and IL-2R ⁇ , which is detected by ELISA experiments, see Example 3 for details; or by Octet For experimental detection, see Example 4 for details.
  • an antibody having HCDR1-3 and LCDR1-3 in the sequence described in SEQ ID NO: 3 and 4 has HCDR1- in the sequence described in SEQ ID NO: 7 and 8.
  • 3 and LCDR1-3 antibodies for example, C2-53
  • antibodies with HCDR1-3 and LCDR1-3 in the sequence described in SEQ ID NO: 11 and 12 for example, D3-68) can block IL-2 and IL -2R ⁇ binding.
  • Blocking the binding of IL-2 and IL-2R ⁇ can be understood to be able to obtain an effective IC 50 value for blocking the binding of IL-2 and IL-2R ⁇ under the conditions of Example 3 (for example, 0.2-5 ⁇ g/mL, more for example 0.5-2 ⁇ g/mL), or under the experimental conditions of Example 4, the blocking value of IL-2R ⁇ with Octet levels of less than 0.600 (for example, 0.410, 0.428, 0.473).
  • some of the anti-IL-2 antibodies or antigen-binding fragments thereof of the present disclosure can block the binding of IL-2 and IL-2R ⁇ , which is detected by ELISA experiment, see Example 3 for details; or Detected by the Octet experiment, see Example 4 for details.
  • an antibody having HCDR1-3 and LCDR1-3 in the sequence described in SEQ ID NO: 9 and 10 (such as D2-60) has an HCDR1- in the sequence described in SEQ ID NO: 5 and 6 3 and LCDR1-3 antibodies (for example, B2-15), having HCDR1-3 and LCDR1-3 antibodies (for example, A2-22) in the sequence described in SEQ ID NO:1 and 3, having SEQ ID NO:1 2.
  • Antibodies against HCDR1-3 and LCDR1-3 in the sequence e.g. A3-21
  • antibodies having HCDR1-3 and LCDR1-3 in the sequence described in SEQ ID NO: 7 and 8 e.g., C2-53
  • Antibodies with HCDR1-3 and LCDR1-3 in the sequences described in SEQ ID NOs: 11 and 12 can block the binding of IL-2 to IL-2R ⁇ .
  • blocking the binding of IL-2 and IL-2R ⁇ can be understood as obtaining an effective IC 50 value for blocking the binding of IL-2 and IL-2R ⁇ under the conditions of Example 3 (for example, 0.1-9 ⁇ g/mL, For example, 1-9 ⁇ g/mL), or under the conditions of Example 3, having an Octet level of less than 0.300 (for example, less than 0.2, more for example, less than 0.1), the blocking value of IL-2R ⁇ .
  • Both IL-2 or the complex of the present disclosure can activate T cells (e.g., Treg and CD8+ T cells).
  • T cells e.g., Treg and CD8+ T cells.
  • "Selectively enhance the effect of IL-2 on Treg activity” is to set the effect of IL-2 on Treg activity as A, and the effect on CD8+ T cell activity as B; the compound of the present disclosure has an effect on Treg activity.
  • the effect is A'
  • the effect on CD8+T cell activity is B', then the ratio of A'/B' is higher than the ratio of A/B (to any degree).
  • the "active" obtained under the same test conditions including, but not limited to, the analyte (e.g. IL-2, or a complex of the present disclosure) promoting cell proliferation EC 50, promoting cell STAT5 phosphorylation EC 50 (as described in Example 10 method of detection).
  • the expression “the degree of activation of Treg activity is higher than the degree of activation of CD8+ T cells” and the expression “the degree of inhibition of Treg activity is lower than the degree of inhibition of CD8+ T cells” are equivalent.
  • Means that compared with CD8+ T cells, the anti-IL-2 antibodies, antigen-binding fragments or complexes of the present disclosure activate Treg activity to a higher degree (to any degree).
  • the meaning of "lower degree of inhibition of cytotoxicity” is equivalent and means that the anti-IL-2 antibody, antigen-binding fragment or complex thereof of the present disclosure phosphorylates STAT5 in Treg cells compared to CD8+ T cells.
  • the degree of promotion is higher (to any degree). Refer to Example 7 for the detection method of STAT5 phosphorylation.
  • the expression "compared to the proliferation of CD8+ T cells, CD4+ T cells or NK cells, promotes the proliferation of Treg cells to a higher degree” and the expression "compared to CD8+ T cells, CD4+ T cells or The meaning of NK cell proliferation has a lower degree of inhibition of Treg cell proliferation" is equivalent, and it means that compared to the proliferation of CD8+ T cells (or CD4+ T cells, or NK cells), the anti-IL of the present disclosure -2 antibodies, their antigen-binding fragments or complexes promote the proliferation of Treg cells to a higher degree (to any degree).
  • Increase the expression of one or more of FOXP3, CD25 and Icos in Treg cells encompasses an increase in the expression of one or more of FOXP3, CD25 and Icos of Treg by the complex of the present disclosure to any degree; or Compared with CD8+ T cells, CD4+ T cells, or NK cells, the anti-IL-2 antibodies, antigen-binding fragments or complexes thereof of the present disclosure inhibit one or more of FOXP3, CD25, and Icos to a lower degree.
  • the present disclosure provides isolated polynucleotides that encode the aforementioned anti-IL-2 antibodies, antigen-binding fragments thereof, or the aforementioned complexes (for example, fusion proteins).
  • 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. In some specific embodiments, the host cell is Escherichia coli, Pichia pastoris, Chinese hamster ovary (CHO) cells or human embryonic kidney (HEK) 293 cells.
  • the present disclosure provides a method for preparing an anti-IL-2 antibody, an antigen-binding fragment thereof, an anti-IL-2 antibody, or a complex thereof (for example, a fusion protein), including:
  • the preparation method of the present disclosure may further include a purification step.
  • a purification step For example, use A or G Sepharose FF column containing adjusted buffer for purification, wash away non-specifically bound components, and then use pH gradient method to elute the bound antibody, detect by SDS-PAGE, and collect.
  • 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 IL-2 or its fragments can be used to immunize mice, and the obtained antibody 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 ImmunoGeneTics (IMGT) database, or from the Journal of Immunoglobulin, 2001ISBN012441351.
  • 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.
  • composition such as a pharmaceutical composition, which contains a therapeutically effective amount of an anti-IL-2 antibody, an antigen-binding fragment thereof, or a complex (such as a fusion protein) as described above, and a pharmaceutically acceptable excipient, Dilution or carrier.
  • the unit dose of the pharmaceutical composition may contain 0.01 to 99% by weight of an anti-IL-2 antibody, an antigen-binding fragment thereof or an anti-IL-2 antibody, a complex thereof (such as a fusion protein), or
  • the unit dose of the pharmaceutical composition contains an anti-IL-2 antibody, an antigen-binding fragment thereof, an anti-IL-2 antibody, or a complex (such as a fusion protein) in an amount of 0.1-2000 mg, and in some embodiments, 1-1000 mg.
  • the present disclosure provides the use of any one or a combination selected from the following in the preparation of a medicine or a pharmaceutical composition: the aforementioned anti-IL-2 antibody, its antigen-binding fragment, a complex (such as a fusion protein), and an encoding polynucleotide.
  • the above-mentioned drugs or pharmaceutical compositions are used to treat or prevent immune-related disorders (for example, autoimmune diseases, inflammatory diseases) or delay the progress of immune-related disorders.
  • immune-related disorders for example, autoimmune diseases, inflammatory diseases
  • a method for treating or preventing an autoimmune disease or delaying the progression of an autoimmune disease comprising administering to a subject an effective amount of an anti-IL-2 antibody or an antigen-binding fragment thereof according to the present disclosure to treat or delay the disease, Complexes (e.g. fusion proteins) or pharmaceutical compositions according to the present disclosure.
  • the aforementioned autoimmune diseases and inflammatory diseases are selected from any one of the following: inflammatory skin diseases, including psoriasis and dermatitis (such as atopic dermatitis); dermatomyositis; systemic scleroderma and sclerosis ; Conditions associated with inflammatory bowel disease (such as Crohn's disease and ulcerative colitis); colitis; gastritis; respiratory distress syndrome (including adult respiratory distress syndrome and ARDS); dermatitis; meningitis; encephalitis ; Uveitis; glomerulonephritis; allergic conditions such as eczema and asthma and other conditions involving T cell infiltration and chronic inflammatory response; atherosclerosis; leukocyte adhesion defects; rheumatoid arthritis; systemic lupus erythematosus (SLE); diabetes (such as type I diabetes); multiple sclerosis; Raynaud’s syndrome; autoimmune thyroiditis; allergic encephalomyelitis; S
  • autoimmune diseases and inflammatory diseases are selected from: lupus, graft-versus-host disease, hepatitis C-induced vasculitis, type I diabetes, multiple sclerosis, atopic disease or inflammatory bowel disease.
  • a method for increasing the ratio of Tregs to non-Tregs in a T cell population comprising combining the T cell population with an effective amount of the aforementioned anti-IL-2 antibody, antigen-binding fragment or complex thereof (E.g. fusion protein) contact.
  • the ratio of CD3 + FoxP3 + cells to CD3 + FoxP3 - is increased.
  • the ratio of CD3 + FoxP3 + cells to CD3 + FoxP3- is increased by at least 50%.
  • a method for increasing the ratio of Treg to non-Treg in the peripheral blood of a subject comprising combining a population of T cells with an effective amount of the anti-IL-2 antibody or antigen-binding fragment thereof. Or contact with a complex (such as a fusion protein).
  • a complex such as a fusion protein.
  • the ratio of CD3 + FoxP3 + cells to CD3 + FoxP3 - is increased.
  • the ratio of CD3 + FoxP3 + cells to CD3 + FoxP3- is increased by at least 50%.
  • a method for any one selected from the group consisting of: increasing the ratio of Treg to natural killer (NK) cells in a T cell population, or increasing the number of Tregs, or increasing the activity of Tregs; the method comprises An effective amount of the above-mentioned anti-IL-2 antibody, antigen-binding fragment or complex thereof (for example, fusion protein) is administered.
  • the ratio of CD3 + FoxP3 + cells to CD3- CD19 - lymphocytes expressing CD56 and/or CD16 is increased.
  • the ratio of CD3 + FoxP3 + cells to CD3- CD19 - lymphocytes expressing CD56 and/or CD16 is increased by at least 50%.
  • a method for any one selected from the group consisting of: increasing the ratio of Treg to natural killer (NK) cells in the peripheral blood of a subject, or increasing the number of Tregs, or increasing the activity of Tregs includes administering an effective amount of the above-mentioned anti-IL-2 antibody, antigen-binding fragment or complex thereof (for example, a fusion protein).
  • the ratio of CD3 + FoxP3 + cells to CD3- CD19 - lymphocytes expressing CD56 and/or CD16 is increased.
  • the ratio of CD3 + FoxP3 + cells to CD3- CD19 - lymphocytes expressing CD56 and/or CD16 is increased by at least 50%.
  • a method for monitoring the response of a subject to the aforementioned anti-IL-2 antibody, antigen-binding fragment or complex thereof comprising detecting changes in the subject, so The change is selected from any one or a combination of the following: an increase in body temperature, an increase in CRP in the peripheral blood of the subject, a decrease in platelets in the peripheral blood of the subject, and the subject A decrease in neutrophils in the peripheral blood of the subject, or a decrease in albumin in the peripheral blood of the subject.
  • the treatment is terminated, suspended, the frequency of dosing is reduced, or the amount of dosing is reduced.
  • the change comprises any one or a combination selected from: an increase in body temperature of at least 0.5° C., an increase in CRP of the subject’s peripheral blood of at least 0.2 mg/mL, and at least 0.8 A reduction of platelets in the peripheral blood of at least 0.8 times, a reduction of neutrophils in the peripheral blood of the subject at least 0.8 times, or a reduction of albumin in the peripheral blood of the subject at least 0.4 times .
  • the aforementioned anti-IL-2 antibodies, antigen-binding fragments or complexes thereof can specifically bind IL-2 and reduce the binding of IL-2 to IL-2R ⁇ and/or IL-2R ⁇ .
  • These antibodies, antigen-binding fragments or complexes inhibit the proliferation of non-Treg cells (including effector CD8 + , non-Treg CD4 + and NK cells) more than the inhibition of Treg cell proliferation; and/or compared with isotype control antibodies , Increase Treg proliferation; and/or increase the ratio of Treg cells/non-Treg cells; or maintain Treg markers.
  • Figure 1A and Figure 1B are the results of an ELISA experiment in which an anti-IL-2 antibody blocks the binding of IL-2 to IL-2R ⁇ and IL-2R ⁇ , respectively.
  • Figure 2 shows the results of detecting the binding ability of anti-IL-2 antibodies to IL-2 through ELISA experiments.
  • Figures 3A to 3H show the experimental results of the non-covalent complex of anti-IL-2 antibody and IL-2 stimulating the phosphorylation of STAT5 in human peripheral blood Treg and CD8 + T cells.
  • Figures 4A to 4D are experimental results of the fusion protein stimulating the phosphorylation activity of STAT5 in human peripheral blood regulatory T cells Treg and CD8 + T cells.
  • Figures 5A to 5F are the experimental results of the effect of the fusion protein on the proliferation of lymphocytes in the peripheral blood of Balb/c mice.
  • Figure 6 shows the experimental results of the effect of the fusion protein on the proliferation of lymphocytes in the spleen of mice immunized with OVA.
  • Figure 7 shows the results of the efficacy of the fusion protein in a mouse model of delayed allergic reaction.
  • Figures 8A to 8B show the pharmacodynamic results of the fusion protein in a mouse arthritis model.
  • Figures 9A to 9F are experimental results of fusion proteins formed by anti-IL-2 antibodies and different IL-2 variants stimulating the phosphorylation activity of STAT5 in human peripheral blood regulatory T cells and CD8 + T cells.
  • Figure 11A and Figure 11B are the experimental results of the effect of D2-60-b-T3E on the content of dsDNA IgG and IgM in the serum of MRL/lpr mice (**P ⁇ 0.01 vs. model control group).
  • Interleukin-2 or "IL-2” is intended to be interpreted broadly, including any IL-2 related products. Including but not limited to human and non-human IL-2 homologs, fragments or truncations, fusion proteins (such as fusion with signal peptide or other active or inactive ingredients, active ingredients such as antibodies or antigen-binding fragments), modifications Forms (such as PEGylation, glycosylation, albumin conjugation/fusion, Fc conjugation and/fusion, hydroxyethylation, etc.), and conservatively modified proteins.
  • the term encompasses any natural IL-2 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats).
  • the term also encompasses unprocessed IL-2 and any form of IL-2 derived from processing in cells, naturally occurring IL-2 variants, such as splice variants or allelic variants, and IL-2 Conservatively modified variants of 2.
  • IL-2R ⁇ refers to any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats)
  • Any natural CD25 including "full-length” unprocessed CD25 and any form of CD25 derived from processing in cells, also includes naturally-occurring CD25 variants, such as splice variants or allelic variants.
  • CD25 is human CD25, and an exemplary sequence is shown in SEQ ID NO. 37.
  • High-affinity IL-2 receptor refers to the heterotrimeric form of IL-2 receptor, which consists of receptor gamma subunit (also known as universal cytokine receptor gamma subunit, gammac or CD132), receptor The ⁇ subunit (also called CD122, p70 or IL-2R ⁇ ) and the receptor ⁇ subunit (also called CD25, p55, IL-2R ⁇ ) are composed.
  • a "medium affinity IL-2 receptor” refers to an IL-2 receptor that contains only gamma and beta subunits without alpha subunits (see, for example, Olejniczak and Kasprzak, Med Sci Monit 14, RA179-189 (2008). )).
  • Constant modifications apply to amino acid and nucleotide sequences.
  • conservative modification refers to those nucleic acids that encode the same or substantially the same amino acid sequence, or in the case where the nucleotide does not encode the amino acid sequence, to the substantially same nucleotide sequence.
  • conservative modification refers to the replacement of amino acids in proteins with other amino acids with similar characteristics (such as charge, side chain size, hydrophobicity/hydrophilicity, main chain conformation and rigidity, etc.) so that frequent changes can be made. Does not change the biological activity of the protein.
  • amino acid mutations include amino acid substitutions, deletions, insertions, modifications and any combination thereof to realize the final construct so that the final construct possesses the desired characteristics, such as enhanced stability and increased activity.
  • Amino acid sequence deletions and insertions include amino and/or carboxy terminal deletions and amino acid insertions.
  • Preferred amino acid mutations are amino acid substitutions.
  • a non-conservative amino acid can be substituted, that is, one amino acid can be replaced with another amino acid having a different structure and/or chemical properties.
  • Preferred amino acid substitutions include the substitution of hydrophilic amino acids for hydrophobic amino acids.
  • Amino acid substitutions include non-naturally occurring amino acids or naturally-occurring amino acid derivatives of 20 standard amino acids (e.g. 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine Amino acid) substitution.
  • Amino acid mutations can be generated using genetic or chemical methods known in the art, including methods such as site-directed mutagenesis, PCR, gene synthesis, and chemical modification.
  • Antibody is used in the broadest sense and covers various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (such as bispecific antibodies), full-length antibodies and antibody fragments ( Or antigen-binding fragments, or antigen-binding portions) as long as they exhibit the desired antigen-binding activity.
  • Antibody can refer to immunoglobulin, which is a tetrapeptide chain structure composed of two heavy chains and two 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 antigen-specificity is also different.
  • 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.
  • the same type of Ig can be divided into different subclasses according to the amino acid composition of the hinge region and the number and position of heavy chain disulfide bonds.
  • 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 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 (CDR) and 4 framework regions (FR) with relatively conservative 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) is composed 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.
  • CDR the deterministic description of CDR can be completed by resolving the structure of the antibody and/or the structure of the antibody-ligand complex, and identifying the residues involved in the antigen binding site. 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 de-novo 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).
  • 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. For any given embodiment containing more than one CDR, the CDR can be defined according to any of Kabat, Chothia, extended, AbM, IMGT, contact, and/or conformational definitions.
  • Monoclonal antibody or “monoclonal antibody” refers to antibodies obtained from a substantially homogeneous antibody population, that is, each antibody contained in the population is the same except for possible naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific and are directed against a single antigenic site. In addition, unlike polyclonal antibodies, each monoclonal antibody is directed against a single determinant on the antigen.
  • the modifier "monoclonal” indicates the characteristics of an antibody as obtained from a substantially homogeneous antibody population, and is not construed as requiring the production of the antibody by any specific method.
  • monoclonal antibodies used in accordance with the present disclosure can be prepared by the hybridoma method first described by Kohler and Milstein, 1975, Nature 256:495, or can be prepared by, for example, the recombinant DNA method described in U.S. Patent No. 4,816,567.
  • monoclonal antibodies can also be isolated from the generated phage library using the technique described in McCafferty et al., 1990, Nature 348:552-554.
  • 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”).
  • Human antibody or “recombinant human antibody” includes human antibodies prepared, expressed, created or isolated by recombinant methods, and the techniques and methods involved are well known in the art, such as:
  • Antibodies prepared, expressed, created or isolated by methods such as splicing human immunoglobulin gene sequences to other DNA sequences.
  • Such recombinant human antibodies contain variable and constant regions, which utilize specific human germline immunoglobulin sequences encoded by germline genes, but also include subsequent rearrangements and mutations such as those that occur during antibody maturation.
  • Antigen (Ag) refers to a molecule used to immunize immunocompetent vertebrates to produce antibodies (Ab) that recognize Ag; or to screen in expression libraries (e.g., phage, yeast, or ribosome display libraries) The molecule or mimetic used.
  • Ag is defined more broadly and is generally expected to include target molecules recognized by Ab. Therefore, Ag includes molecules used in the immunization process for the production of Abs or library screening for selection of Abs, or parts or mimetics thereof.
  • full-length IL-2 from mammalian species such as human, monkey, mouse, and rat IL-2
  • monomers and multimers such as Dimers, trimers, etc.
  • truncated variants and other variants of IL-2 are all called antigens.
  • 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 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, for example, 3 to 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.
  • 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 Fab-Fv format was first disclosed in WO2009/040562, and the disulfide bond stabilized Fab-dsFv format was first disclosed in WO2010/035012.
  • 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).
  • Binding to IL-2 refers to the ability to interact with IL-2 or its epitope, and the IL-2 or its epitope may be of human origin.
  • Antigen binding site refers to a continuous or discontinuous three-dimensional site on an antigen that is recognized by the antibody or antigen-binding fragment of the present disclosure.
  • Treg means a specialized CD4+ T cell type that can suppress the response of other T cells.
  • Tregs are characterized by expressing IL-2 receptor alpha subunit (CD25) and transcription factor forkhead box P3 (FOXP3), and in inducing and maintaining peripheral autotolerance to antigens (including those expressed by tumors) Plays a key role in.
  • Treg requires IL-2 to achieve its function and development and the induction of its inhibitory characteristics.
  • Antibody effector functions refer to those biological activities that can be attributed to the Fc region of an antibody (natural sequence Fc region or amino acid sequence variant Fc region) and vary with antibody isotype.
  • Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; cell surface receptors (such as B cell receptors) Body) down-regulation; and B cell activation.
  • ADCC means that cells expressing Fc receptors directly kill target cells coated with antibodies by recognizing the Fc segment of antibodies. The ADCC effect function of the antibody can be reduced or eliminated by modifying the Fc section of IgG.
  • the modification refers to mutations in the constant region of the heavy chain of the antibody, such as N297A, L234A, L235A, P329G selected from IgG1; IgG2/4chimera, and F234A/L235A mutations of IgG4.
  • CDC refers to the cytotoxic form of the complement cascade that activates the complement cascade by binding the complement component C1q to the antibody Fc. Methods for detecting ADCC and CDC activities of antibodies are known in the art. For example, CDC can be evaluated by measuring the binding activity between the antibody to be tested and the Fc receptor (for example, C1q).
  • Fc receptor or “FcR” describes a receptor that binds to the Fc region of an antibody.
  • the preferred FcR is human FcR.
  • a preferred FcR is an FcR ( ⁇ receptor) that binds to an IgG antibody, and includes receptors of the Fc ⁇ RI, Fc ⁇ RII, and Fc ⁇ RIII subclasses, including allelic variants and alternatively spliced forms of these receptors.
  • FcyRII receptors include FcyRIIA ("activating receptor”) and FcyRIIB ("inhibiting receptor”), which have similar amino acid sequences that differ mainly in their cytoplasmic domains.
  • the activation receptor Fc ⁇ RIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain.
  • the inhibitory receptor Fc ⁇ RIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain (see review M. Daeron, Annu. Rev. Immunol. 15:203-234 (1997)).
  • FcR is reviewed in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J.Lab.Clin.Med.126: 330-41 (1995).
  • FcR herein encompasses other FcRs, including those to be identified in the future.
  • the term also includes the neonatal receptor FcRn responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)).
  • Polynucleotide or “nucleic acid” refers to a chain of nucleotides of any length, including DNA and RNA.
  • the nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and/or their analogs, or any substrate that can be incorporated into the chain by DNA or RNA polymerase.
  • 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 is approximately lower than 10 -7 M or even more.
  • the small equilibrium dissociation constant (K D ) binds to the predetermined antigen or its epitope, and its binding affinity to the predetermined antigen or its epitope is that of the predetermined antigen (or its epitope) or closely related antigens.
  • the binding affinity of foreign non-specific antigens is at least twice.
  • the term "antibody that recognizes an antigen” can be used interchangeably with the term “antibody that specifically binds” herein.
  • Bindity is used herein as a measure of the strength of the non-covalent interaction between two molecules (eg, an antibody or antigen-binding fragment thereof and an antigen).
  • affinity is used to describe a monovalent interaction (intrinsic activity).
  • the binding affinity between two molecules can be quantified by determining the dissociation constant (KD). KD can be determined by measuring the kinetics of complex formation and dissociation using, for example, a surface plasmon resonance (SPR) method (Biacore).
  • SPR surface plasmon resonance
  • the rate constants corresponding to the association and dissociation of the monovalent complex are called the association rate constant ka (or kon) and the dissociation rate constant kd (or koff), respectively.
  • the value of the dissociation constant can be directly determined by well-known methods, and can be calculated by methods such as those described in Caceci et al. (1984, Byte 9:340-362) even for complex mixtures.
  • a double filter nitrocellulose filter binding assay such as Wong & Lohman: that disclosed in (1993, Proc.Natl.Acad.Sci.USA 90 5428-5432) to determine the K D.
  • binding kinetics and binding affinity of antibodies can also be determined by standards known in the art, such as surface plasmon resonance (SPR), for example, by using the Biacore TM system or KinExA.
  • SPR surface plasmon resonance
  • the K D value of each antibody/antigen complex can be compared to compare the binding affinities associated with different molecular interactions, for example, the comparison of the binding affinities of different antibodies for a given antigen.
  • the specificity of the interaction can be determined and compared by determining and comparing the K D value of the target interaction (for example, the specific interaction between the antibody and the antigen) and the non-target interaction (for example, a control antibody that is not known to bind IL-2). ) K D value for evaluation.
  • the affinity of the IL-2 antibody or antigen-binding fragment of the present disclosure to bind to its target is at least 2-fold, 10-fold, 50-fold greater than its binding affinity to another non-IL-2 molecule. 100 times, 200 times, 500 times, 1,000 times or 10,000 times, this is not a restrictive definition.
  • the "complex" of an anti-IL-2 antibody or antigen-binding fragment thereof and IL-2 refers to a complex comprising at least one anti-IL-2 antibody or antigen-binding fragment thereof of the present disclosure, which specifically binds to IL-2 or expresses IL-2 cells.
  • the complex comprises an anti-IL-2 antibody or antigen-binding fragment thereof and an IL-2 molecule bound by covalent force, non-covalent force or any other force.
  • the anti-IL-2 antibody or antigen-binding fragment thereof forms a fusion protein with IL-2.
  • the anti-IL-2 antibody or antigen-binding fragment thereof binds to IL-2 non-covalently.
  • the anti-IL-2 antibody or antigen-binding fragment thereof and IL-2 can still be maintained as a complex. It should be understood that, among other variables, the anti-IL-2 antibody or antigen-binding fragment thereof and IL-2 will form complexes with different binding abilities based on the different K D values of the binding interaction between them.
  • Inhibition or “blocking” are used interchangeably and encompasses both partial and complete inhibition/blocking. Inhibition and blocking are also intended to include any measurable binding affinity for IL-2 that promotes cell proliferation (e.g., T Cell) activity is reduced.
  • an antibody that "competes for binding" with a reference antibody refers to an antibody that blocks the binding of the reference antibody to the antigen by 50% or more, or the binding to the antigen is blocked by the reference antibody by 50% or more in a competition assay.
  • Antibody The antigen binding activity of the anti-IL-2 antibody of the present disclosure can be tested by known methods such as ELISA, Western blotting, etc. For example, the use of a competition assay can be used to identify antibodies that compete with IL-2 for binding.
  • the competitive antibody binds to the same epitope (e.g., linear or conformational epitope) as the antigen binding molecule or anti-IL-2 antibody.
  • the method described in International Patent Publication WO03/48731 Therefore, conventional techniques known to those skilled in the art can be used to obtain antibodies and antigen-binding fragments thereof that compete with the antibody molecules of the present disclosure for binding to the same epitope on IL-2.
  • 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 IL-2 antibody or antigen-binding fragment thereof of the present disclosure or a complex with IL-2 as a therapeutic agent, so The subject has, is suspected of having, or is prone to suffer from one or more immune-related diseases or symptoms thereof, and the therapeutic agent is known to have a therapeutic effect on these symptoms.
  • the therapeutic agent is administered to 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 measurable degree.
  • the amount of the therapeutic agent (also referred to as "therapeutically effective amount") that is effective to alleviate the symptoms of any particular disease can vary according to various 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.
  • an “effective amount” includes an amount sufficient to ameliorate or prevent the symptoms or conditions of the medical condition.
  • 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.
  • “Homology” or “identity” refers to the sequence similarity between two polynucleotide sequences or between two polypeptides.
  • 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.
  • “Host cell” includes individual cells or cell cultures, which may be or have been recipients of vectors for incorporation of polynucleotide inserts.
  • a host cell includes the progeny of a single host cell, and due to natural, accidental or deliberate mutations, the progeny may not necessarily be exactly the same as the original parent cell (in morphology or genomic DNA complement).
  • Host cells include cells transfected and/or transformed in vivo with polynucleotides of the present disclosure.
  • Cell Cell line
  • “cell culture” are used interchangeably, and 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.
  • Vector means a construct capable of being delivered in a host cell and in some embodiments expressing one or more genes or sequences of interest.
  • examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors combined with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes , And certain eukaryotic cells such as producer cells.
  • “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.
  • “Pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” includes any material that, when combined with an active ingredient, allows the ingredient to retain biological activity and does not react with the subject's immune system. Examples include, but are not limited to, any standard pharmaceutical carrier, such as phosphate buffered saline solution, water, emulsions such as oil/water emulsions, and various types of wetting agents.
  • the diluent for aerosol or parenteral administration is phosphate buffered saline (PBS) or physiological (0.9%) saline.
  • PBS phosphate buffered saline
  • Compositions containing such carriers are formulated by well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, editor, Mack Publishing Co., Easton, PA, 1990; and R Remington, The Science and Practice of Pharmacy 20th Edition Mack Publishing, 2000).
  • the range of "1 to 10" should be regarded as including any and all sub-ranges (and including the end value) between the minimum value 1 and the maximum value 10; that is, starting with a minimum value of 1 or greater All sub-ranges of, for example, 1 to 6.1, and all sub-ranges ending with a maximum value of 10 or less, for example, 5.5 to 10.
  • the preserved natural library phage suspension was diluted and blocked with BSA, incubated with magnetic beads Dynabeads (M-280, invitrogen), and the phage after the negative sieve incubation was collected.
  • the magnetic beads were combined and washed according to the Kingfisher magnetic bead screening system method (Thermo Scientific), and 5% BSA was incubated with the magnetic beads.
  • the Dynabeads were coated and blocked with biotin-labeled IL-2 (Sanyou Biopharmaceutical, GenBank Accession No: P60568-1) with a human Fc tag, and the phage suspension collected after the negative sieve was incubated with the Dynabeads and followed by the Kingfisher magnetic beads The screening system method is combined and cleaned.
  • the phages are eluted with trypsin. After elution, the phage solution was thoroughly mixed with E. coli SS320 cells (Sanyou Biopharmaceutical) in the logarithmic growth phase, and then incubated at 37°C for 30 min. Spread E. coli SS320 on 2YT-Car + -Tet + plates and cultivate overnight in a 37°C incubator. Calculate phage input, output, etc., scrape the input phage and perform 3 rounds of screening.
  • the 2000 clones obtained in the third round of screening were selected and cultured overnight in a 96-well deep-well plate. Centrifuge to take the supernatant.
  • the anti-human IgG (STAR161, Bio-rad) was diluted with PBS to 2 ⁇ g/mL, and the ELISA plate was coated. 30 ⁇ L was added to each well at 4°C overnight.
  • the plate was washed 3 times with PBST (PBS, pH 7.4+0.1% Tween 20). Blocked with 1% BSA for 1 hour at room temperature, and washed the plate 3 times with PBST. 30 ⁇ L of supernatant after centrifugation was added, and the plate was washed 3 times with PBST for 2 hours at room temperature.
  • the biotin-labeled IL-2 with human Fc label was diluted with PBS to 2 ⁇ g/mL and added 30 ⁇ L per well for 1 hour at room temperature, and the plate was washed 3 times with PBST.
  • the above 364 clones were inoculated into a 50 mL volume of 2YT-Car + -Tet + medium at a ratio of 1:1000, and cultured overnight at 37°C. Transfer the bacterial solution to a 50mL centrifuge tube. Centrifuge at room temperature, discard the supernatant, and add 0.5 mL of protein lysis solution (10mM Tris-HCl pH 9.0, 1mM EDTA, 5mM MgCl 2 , 25U/mL Bezonase Nuclease (Merck)) pre-cooled at 4°C. Incubate for 1 hour on ice. Centrifuge at 4°C to collect the supernatant for later use.
  • protein lysis solution 10mM Tris-HCl pH 9.0, 1mM EDTA, 5mM MgCl 2 , 25U/mL Bezonase Nuclease (Merck)
  • IL-2R ⁇ -Fc (CJ82, Novoprotein) (GenBank Accession No: NP_000869.1) was diluted to 8 ⁇ g/mL with PBS, coated with ELISA plate, 30 ⁇ L was added to each well, 4°C overnight. Wash the plate 3 times with PBST. Blocked with 1% BSA for 1 hour at room temperature, and washed the plate 3 times with PBST. After incubating the obtained Fab lysate with biotin-labeled human Fc-labeled IL-2 (2.5 ⁇ g/mL) for 1 hour at room temperature, 30 ⁇ L was added to each well for 1 hour at room temperature, and the plate was washed 3 times with PBST.
  • the heavy chain and light chain variable regions of the six antibodies in Example 1 were connected with the human IgG1 heavy chain constant region and the kappa or lamda chain constant region to form a full-length fully human anti-IL-2 antibody.
  • the heavy chain constant region has L234A and L235A mutations (LALA mutations) to remove the possible ADCC function of the antibody.
  • the full-length sequences of the 6 antibodies are shown in Table 3.
  • HC is the constant region of human IgG1 heavy chain
  • LC is the constant region of kappa chain
  • the above sequence was synthesized, digested with BamHI and XhoI, and inserted into the pcDNA3.1 expression vector (Life Technologies Cat. No. V790-20) through the BamHI/XhoI restriction site.
  • the expression vector and the transfection reagent PEI (Polysciences, Inc. Cat. No. 23966) were transfected into HEK293 cells (Life Technologies Cat. No. 11625019) at a ratio of 1:2, and incubated in a CO 2 incubator 4- 5 days. After the expressed antibody is recovered by centrifugation, the antibody is purified according to a conventional method, and after identification, the full-length fully human antibody of the present disclosure is obtained.
  • Example 3 ELISA experiment where anti-IL-2 antibody can block or reduce the binding of IL-2 and IL-2R ⁇ , and can block the binding of IL-2 and IL-2R ⁇
  • Dilute IL-2R ⁇ -Fc (CJ78, Novoprotein) (Accession#NP_000408) with PBS to 0.5 ⁇ g/mL, coat the ELISA plate, add 30 ⁇ L to each well, overnight at 4°C. Wash the plate 3 times with PBST. Blocked with 1% BSA for 1 hour at room temperature, and washed the plate 3 times with PBST.
  • the anti-IL-2 antibody was diluted with PBS, pH 7.4 in a concentration gradient to 10 ⁇ g/mL, 5 ⁇ g/mL, 2.5 ⁇ g/mL, 1.25 ⁇ g/mL, 0.63 ⁇ g/mL, 0.31 ⁇ g/mL, 0.16 ⁇ g/mL, 0.08 ⁇ g/mL, respectively, with biotin-labeled human Fc-labeled IL-2 (final concentration 1 ⁇ g/mL) after 15 minutes of room temperature incubation, 30 ⁇ L was added to each well for 1 hour at room temperature, and the plate was washed 3 times with PBST. Add 30 ⁇ L of the secondary antibody NeutrAvidin-HRP diluted 1:6000, wash the plate 9 times with PBST for 1 hour at room temperature. Add 30 ⁇ L TMB to develop color at room temperature for 5-10 minutes, then add 30 ⁇ L 2M HCl or H 2 SO 4 to stop the reaction, and read the data with a microplate reader OD 450.
  • Antibody number IC 50 ( ⁇ g/mL) A2-22 N.A. A3-21 1.456 B2-15 N.A. C2-53 0.6591 D2-60 N.A. D3-68 1.196
  • IL-2R ⁇ -Fc Dilute IL-2R ⁇ -Fc (CJ82, Novoprotein) with PBS to 8 ⁇ g/mL, coat the ELISA plate, add 30 ⁇ L to each well, overnight at 4°C. Wash the plate 3 times with PBST. Blocked with 1% BSA for 1 hour at room temperature, and washed the plate 3 times with PBST.
  • Example 4 Anti-IL-2 antibody blocks or reduces the binding of IL-2 and IL-2R ⁇ , and the Octet experiment of blocking the binding of IL-2 and IL-2R ⁇
  • the Streptavidin biosensor (Fortebio, #18-5020) was soaked in 200 ⁇ L of KB buffer (PBS, pH 7.4, 0.05% tween-20, 0.1% BSA) for 60 seconds, and then wetted. Then, the biotinylated IL-2R ⁇ -Fc (CJ78, Novoprotein) was diluted to 10 ⁇ g/mL with KB buffer, and the sensor was placed in 200 ⁇ L of this solution for 150 seconds. Soak the sensor in KB buffer for 60 seconds to elute excess IL-2R ⁇ .
  • KB buffer PBS, pH 7.4, 0.05% tween-20, 0.1% BSA
  • IL-2-His (CX66, Novoprotein) was mixed with anti-IL-2 antibody, and diluted with KB buffer to a final concentration of 100 nM and 500 nM, respectively, and incubated at room temperature for 30 minutes. Place the sensor in the mixed solution and bind for 300 seconds. The stronger the anti-IL-2 antibody's ability to block or reduce the binding of IL-2 to IL-2R ⁇ , the lower the reading. Read the value at 230 seconds when the combination starts, as shown in Table 6.
  • the Streptavidin biosensor (Fortebio, #18-5020) was soaked in 200 ⁇ L of KB buffer (PBS, pH 7.4, 0.05% tween-20, 0.1% BSA) for 60 seconds, and then wetted. Then, the biotinylated IL-2-Fc (Sanyou Biopharmaceutical) was diluted to 10 ⁇ g/mL with KB buffer, and the sensor was placed in 200 ⁇ L of this solution for 300 seconds. Soak the sensor in KB buffer for 60 seconds to elute excess IL-2. The anti-IL-2 antibody was diluted with KB buffer to a final concentration of 500 nM, and the sensor was placed in the antibody solution to bind for 300 seconds.
  • KB buffer PBS, pH 7.4, 0.05% tween-20, 0.1% BSA
  • Embodiment 5 Anti-IL-2 antibody and IL-2 binding ELISA experiment
  • Biotin-labeled IL-2 (Sanyou Biopharmaceutical) with human Fc label was diluted with PBS to 2 ⁇ g/mL and 30 ⁇ L was added to each well for 1 hour at room temperature, and the plate was washed 3 times with PBST.
  • the results are shown in Figure 2 and Table 8. The results show that under this experimental method, all antibodies bind to IL-2, and C2-53 binds strongly.
  • Antibody number EC 50 ( ⁇ g/mL) A2-22 1.289 A3-21 1.350 B2-15 2.117 C2-53 0.7301 D2-60 1.428 D3-68 1.205
  • Example 6 Affinity and kinetics of anti-IL-2 antibody and IL-2 Biacore experiment
  • Protein A sensor chip GE, Cat#29127556 of the Biacore instrument (Biacore T200, GE) to capture the antibody, where the anti-IL-2 antibody is diluted with 1 ⁇ HBS-EP to 1 ⁇ g/mL, and continues at a flow rate of 10 ⁇ L/min. 30 seconds. Then, a series of concentration gradient IL-2 (C013, Novoprotein) flowed through the chip surface at a flow rate of 30 ⁇ L/min, and the binding lasted for 120 seconds. The dissociation flow rate is 30 ⁇ L/min for 300 seconds, the reaction signal is detected in real time, and the binding and dissociation curves are obtained.
  • Example 7 Determination of STAT5 phosphorylation activity in human peripheral blood PBMC by the non-covalent complex of anti-IL-2 antibody and IL-2
  • Basic medium RPMI 1640+10% fetal bovine serum.
  • Antibody mixture CD3 APC-Cy7 (BD 557832), CD4 BB515 (BD 564419), CD8 BB700 (BD 566452), CD25 BV421 (BD 564033), pSTAT5 AF647 (BD 562076).
  • Human PBMC STAT5 phosphorylation experiment adjust freshly isolated human PBMC cells to a density of 6.5 ⁇ 10 6 cells/mL with a basal medium, and place 80 ⁇ L in a 96-well plate. Premix different concentrations of anti-IL-2 antibodies and different concentrations of IL-2 for 30 minutes at room temperature, add 20 ⁇ L to 90 ⁇ L PBMC, and stimulate at 37°C for 20 minutes. Immediately afterwards, the cells were fixed with pre-warmed BD Cytofix buffer (BD, Cat No. 554655) at 37°C for 15 minutes, and then fixed on ice for 15 minutes. Centrifuge at 400g for 7 minutes at 4°C. Remove the supernatant and wash once with 150 ⁇ L PBS.
  • BD Cytofix buffer BD, Cat No. 554655
  • BD Phosflow Perm Buffer III (BD, Cat No. 558050) pre-cooled at -20°C to break the membrane at -20°C overnight. Centrifuge at 500g for 7 minutes at 4°C. The supernatant was removed, and 150 ⁇ L PBS was added, and washed twice at pH 7.4. Add 100 ⁇ L of FcR blocking reagent diluted 1:200 and incubate at 4°C for 20 minutes. Centrifuge at 500g for 7 minutes at 4°C. The supernatant was removed, 50 ⁇ L of antibody mixture was added, and the cells were stained at 4°C for 1 hour. Centrifuge at 500g for 7 minutes at 4°C.
  • BD Phosflow Perm Buffer III BD, Cat No. 558050
  • CD8 + T cells are defined as CD3 + CD4 - CD8 + cells
  • Treg are defined as CD3 + CD4 + CD8 + CD25 + cells.
  • A3-21 and C2-53 can inhibit or more reduce the activity of IL-2 on CD8 + T cells and other immune effector cells while not affecting or less reducing the activity of IL-2 on Treg.
  • D3-68 can also reduce the activity of IL-2 on CD8 + T cells more than Treg, although this ability may not be as significant as A3-21 and C2-53.
  • the non-covalent complex of anti-IL-2 antibody and IL-2 may be easily dissociated in vivo to generate free IL-2, causing toxicity.
  • the fusion protein For antibody numbering, if IL-2 is at the N-terminus of the antibody heavy chain, the fusion protein has the suffix a; if IL-2 is at the N-terminus of the antibody light chain, the fusion protein has the suffix b.
  • the fused IL-2 For the fused IL-2, it carries a mutation of T3A (that is, the amino acid at position 3 is mutated from Thr to Ala) to remove possible glycosylation.
  • Human IL-2 mature protein does not contain amino acid M at position 1, so the numbering starts from amino acid A at position 2.
  • the amino acid sequences of the 12 fusion proteins are as follows:
  • Antibody number Expression amount (mg/L) purity(%) A2-22-a 39 90.780 A2-22-b 104 96.308 A3-21-a 82 89.358 A3-21-b 2 82.095 B2-15-a 89 53.821 B2-15-b 70 99.839 C2-53-a N.A. 54.940 C2-53-b 6 26.598 D2-60-a 2.4 40.248 D2-60-b 129 97.540 D3-68-a 0.3 N.A. D3-68-b 106 100
  • IL-2 coupling to the N-terminus of the heavy chain or light chain will greatly affect the expression and purity of the fusion protein.
  • the expression level or purity is low, suggesting that in the fusion protein, IL-2 and the antibody may not form a normal intramolecular binding, but form an intermolecular binding, which leads to protein aggregation.
  • the fusion protein with higher expression (at least 20mg/L) and purity (at least 90%) is selected for subsequent identification.
  • the Protein A biosensor (Fortebio, #18-5010) was immersed in 200 ⁇ L of KB buffer (PBS, pH 7.4, 0.02% tween-20, 0.1% BSA) for 60 seconds, and then wetted. Then, use KB buffer to dilute the fusion protein to 10 ⁇ g/mL, place the sensor in 200 ⁇ L of the solution, and stop when the reading reaches 1.2 nm. Soak the sensor in KB buffer for 100 seconds to elute the excess antibody-IL-2 fusion protein.
  • IL-2R ⁇ (ILA-H52H9, Acrobiosystem) was diluted 2-fold with KB buffer to between 100nM-3.125nM. Place the sensor in the solution and bind for 60 seconds.
  • the fusion protein's STAT5 phosphorylation activity on Treg and CD8 + T cells in human peripheral blood (PBMC) was determined.
  • Figures 4A to 4D the experimental results show that compared with IL-2, all fusion proteins have reduced activity on Treg and CD8 + T cells. But for A2-22-a, A2-22-b, B2-15-b, D2-60-b, A3-21-b, the activity of these fusion proteins on CD8+ T cells is lower than that on Treg More, suggesting that IL-2 in these fusion proteins can be more biased to activate Treg.
  • Example 11 Determination of the effect of the fusion protein on the peripheral blood immune cells of Balb/c mice
  • BALB/c mice purchased from Beijing Charles River Experimental Animal Technology Co., Ltd.
  • Female, 6-8 weeks old, weighing 18-20 g were bred adaptively for 5 days before the formal experiment. All BALB/c mice were raised in an SPF animal room IVC constant temperature and pressure system, where the temperature was 20 to 26°C, the humidity was 40 to 70%, and the light cycle was 12 hours bright/12 hours dark. There are no more than 6 BALB/c mice in each cage.
  • the mice were grouped according to their body weight, and the administration was started after grouping. The type of administration, dosage and route of administration are shown in Table 16.
  • the model grouping day is the 0th day.
  • the mixed staining solution includes CD3 APC-Cy7 (Biolegend 100329), CD8 PE (Biolegend 100708), CD4 PE-Cy7 (eBioscience 25-0042-82), CD25 PerCP-Cy5.5 (BD 561112). Add 100 ⁇ L of mixed staining solution to each sample and incubate at 4°C for 30 minutes. Wash twice with PBS containing 1% FBS.
  • the membrane was fixed and broken with True-Nuclear TM Transcription Factor Buffer Set (Biolegend 424401) for 60 minutes, and 100 ⁇ L of anti-mouse Foxp3 antibody (Biolegend 126405) and anti-mouse Ki67 antibody (eBioscience, 25-5698-82) were incubated for 60 minutes at room temperature. Wash twice with PBS (pH 7.4), and finally resuspend with 500 ⁇ L PBS, pH 7.4 washing solution, and analyze on the machine.
  • CD8 + T cells are defined as CD3 + CD4 - CD8 + cells
  • Treg are defined as CD3 + CD4 + CD25 + Foxp3 + cells.
  • Example 12 Determination of the effect of fusion protein on spleen immune cells of Balb/c mice immunized with chicken ovalbumin OVA
  • OVA Chicken ovalbumin
  • CFA Freund's complete adjuvant
  • Male C57B16/J mice purchased from Shanghai Experimental Animal Center), 6-8 weeks old, weighing 18-20 g. Mice were grouped according to their body weights.
  • 8 mL of OVA solution was taken, mixed with 8 mL of CFA, and emulsified in a water-in-oil state.
  • Each animal was immunized by intraperitoneal injection of 200 ⁇ L.
  • mice in each group were injected subcutaneously (sc) with the fusion protein to be tested and the PBS control 10 mL/kg.
  • the type of administration, dosage and route of administration are shown in Table 17.
  • the method includes: taking out the mouse spleen under aseptic conditions after soaking in 75% ethanol for 5 minutes, rinsing the mouse spleen for 1-2 times with PBS, and then cutting the mouse spleen into pieces. Place the filter screen in a 50mL centrifuge tube, transfer the spleen tissue fragments to the screen, and grind, adding fresh PBS continuously during the process. Centrifuge at 1800 rpm for 3 minutes, and discard the supernatant.
  • the detection cells and their markers are as follows:
  • GEB Germinal Center B cells
  • Follicular T helper cells (Tfh, follicular T helper cells) (PD1 high, CXCR5 high, FOXP3-CD4+);
  • Follicular regulatory T cells (Tfr, follicular regulatory T cells) (PD1 high, CXCR5 high, FOXP3+CD4+);
  • A2-22-b, B2-15-b, D2-60-b can stimulate the proliferation of Treg and Tfr in the spleen, and inhibit the number of Tfh and GCB in the spleen, indicating that A2-22-b, B2-15-b, D2-60-b can suppress the immune system by activating Treg.
  • Example 13 Pharmacodynamic experiment of the fusion protein in a mouse model of delayed allergic reaction
  • Male ICR mice purchased from Shanghai Experimental Animal Center), 6 weeks old, weighing 18-20 g. The mice were grouped according to body weight. On the 0th day of the experiment, the mice were immunized by smearing 50 ⁇ L of 1% DNFB solution on their abdomen.
  • mice Male DBA/1 pure mice were purchased from Shanghai Slack Laboratory Animal Co., Ltd.
  • the arthritis model is induced by immunization with bovine type II collagen (Biolead, 20022).
  • bovine type II collagen Biolead, 20022
  • Each mouse was injected intracutaneously at the base of the tail, and a booster injection was given 3 weeks later.
  • the model animals were randomly divided into 6 groups, namely: normal control group, model group, IL-2 (C013, novoprotein) group, A2-22-b group, and B2-15-b group, with 8 animals in each group.
  • normal control animals were selected as the normal control group, and the groups are shown in Table 19.
  • mice will be weighed every 3 days and the severity of joint inflammation will be scored semi-quantitatively (clinical scores):
  • Each paw has a maximum score of 4 points, and each mouse has a maximum score of 16 points. The observation of arthritis condition continued until the end of the experiment.
  • Detection of serum anti-bovine type II collagen antibody concentration At the end of the experiment, blood is collected, serum is separated, and stored at -80°C. ELISA detection kit (Chondrex, 20322T) was used to detect the level of anti-bovine type II collagen antibody in serum. The specific steps are carried out in accordance with the instructions.
  • Example 15 The influence of different connecting arm lengths on the expression and purity of fusion protein
  • the (G 4 S) 5 connecting arm between IL-2 and the light chain of the anti-IL-2 antibody B2-15 in B2-15-b was shortened by the number of repetitions of G4S to 4, while the heavy chain remained unchanged to obtain B2
  • the light chain sequence of -15-b variant B2-15-b-(G 4 S) 4 is shown in Table 20.
  • Example 2 According to the method in Example 2, the above-mentioned B2-15-b variant was expressed and purified, and the expression amount could still reach 47 mg/L, and the SEC-HPLC purity was 99%. It is proved that different link arm lengths can realize the expression of fusion protein.
  • Example 16 Determination of STAT5 phosphorylation activity of human peripheral blood (PBMC) fusion protein between anti-IL-2 antibody and different IL-2 variants
  • Mutate IL-2 introduce T3E, or T3Q, or remove the first 3 amino acids at the N-terminus of IL-2 (B2-15-b-Del3), or remove the first 7 amino acids at the N-terminus of IL-2 (B2-15-b- Del7), or directly use wild-type IL-2, respectively conjugate with anti-IL-2 antibody.
  • the following fusion proteins were produced (the heavy chain sequence of each antibody remains unchanged), see Table 21.
  • the fusion protein of the above-mentioned anti-IL-2 antibody and IL-2 variant was tested for the phosphorylation activity of STAT5 in Treg and CD8 + T cells in human peripheral blood (PBMC).
  • PBMC peripheral blood
  • Example 17 Pharmacodynamic experiment of fusion protein in MRL/lpr spontaneous lupus erythematosus mouse model
  • Female BALB/c mice and female MRL/lpr mice were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. Taking female BALB/c mice as normal control, female MRL/lpr mice were randomly divided into 3 groups according to proteinuria level and body weight, namely: MRL/lpr control group (model control group), 1 and 3 mg/kg administration Group.
  • MRL/lpr control group model control group
  • D2-60-b-T3E is administered by subcutaneous injection, once every 3 days, at a dose of 1, 3 mg/kg; the administration starts at 8 weeks of age and continues for 8 weeks.
  • the urine protein content of the mice in each group was measured every two weeks; after 8 consecutive weeks of administration, the following tests were carried out: 1Determine the content of anti-double-stranded DNA IgM and IgG in the serum of each group of mice; 2Determine the contents of each group Serum creatinine (CRE) and blood urea nitrogen (BUN) levels in mice; 3Determine the levels of IL-6, IL-10 and IFN- ⁇ in the serum of mice in each group; 4Perform on the kidney tissue of each group of mice Pathological analysis and scoring.
  • CRE Serum creatinine
  • BUN blood urea nitrogen
  • the tail reflex method was used to collect mouse urine
  • the urine protein detection kit (CBB method, Nanjing Jiancheng Institute of Biological Engineering, #C035-2-1) was used to detect the urine protein concentration of mice.
  • CBB method Nanjing Jiancheng Institute of Biological Engineering, #C035-2-1
  • the urine protein detection kit was used to detect the urine protein concentration of mice.
  • the kidneys will be damaged due to the deposition of dsDNA antibody, which will lead to the decline of glomerular filtration function and reabsorption capacity, and the increase of urine protein concentration.
  • the level of urine protein in mice will gradually increase.
  • mice tested every two weeks showed that there was no significant difference in the urine protein content of each group of mice at the beginning.
  • the urine protein level of mice in the MRL/lpr control group gradually increased. It was significantly higher than the normal group, indicating that MRL/lpr mice had severe spontaneous kidney damage due to the deposition of autoantibodies.
  • the average urine protein content of mice in the 1 and 3 mg/kg D2-60-b-T3E administration groups was lower than the model control group and showed a dose-dependent manner, but due to the large individual differences in the data, there was no statistical difference compared with the model control group .
  • the data is shown in Figure 10.
  • ELISA method was used to detect the content of anti-double-stranded DNA (anti-dsDNA) IgG and IgM in serum.
  • anti-dsDNA anti-double-stranded DNA
  • the average value of anti-dsDNA IgG and IgM content in the serum of the 3mg/kg D2-60-b-T3E administration group was significantly lower than that of the model control group (P ⁇ 0.01), although the 1mg/kg group had IgG and IgM content It is lower than the trend of the model control group, but the difference from the model control group is not statistically significant.
  • the experimental results are shown in Figure 11A and Figure 11B.
  • This experiment uses the creatinine (CRE) determination kit (Nanjing Jiancheng Institute of Biological Engineering, #C011-2-1) and the urea nitrogen (BUN) test kit (Nanjing Jiancheng Institute of Biological Engineering, #C013-2-1) detection experiment At the end of the mouse serum creatinine and urea nitrogen content.
  • CRE creatinine
  • BUN urea nitrogen
  • Urea nitrogen and creatinine are the metabolites of human protein and muscle respectively.
  • the kidney is the final excretion organ. After renal function is impaired, blood urea nitrogen and creatinine cannot be effectively excreted.
  • Blood urea nitrogen (BUN) and blood creatinine (CRE) The concentration gradually increases due to retention. Therefore, the levels of BUN and CRE in serum are the main indicators of clinically reflecting renal function.
  • the serum levels of creatinine (CRE) and urea nitrogen (BUN) in the MRL/lpr control group (model control group) mice were significantly increased (P ⁇ 0.0001), suggesting that the MRL/lpr model group mice There is functional renal impairment.
  • the serum creatinine content of mice in the 1 and 3 mg/kg administration groups was significantly lower than the model control group (P ⁇ 0.01, P ⁇ 0.0001), which can improve the renal function of MRL/lpr mice to a certain extent, and 3mg/kg administration
  • the content of creatinine in the serum of mice in the drug group was significantly lower than that in the 1 mg/kg group.
  • the serum urea nitrogen content of the mice in the 3mg/kg administration group was also significantly lower than the model control group (P ⁇ 0.05), indicating that 3mg/kg D2-60-b-T3E can reverse the spontaneous kidney of MRL/lpr mice damage.
  • the results are shown in Figure 12A and Figure 12B.
  • ELISA method was used to detect the levels of cytokines IFN- ⁇ , IL-6 and IL-10 in serum.
  • the left kidney of the mouse was fixed with 4% paraformaldehyde, and the sections were embedded in paraffin and stained with H&E. The sections were observed under a microscope and pathological scores were performed. Pathological damages to glomeruli, renal interstitium, and blood vessels were performed. Carry out grading and scoring, see Table 24 for scoring standards.
  • MRL/lpr control group mice showed severe glomerular, renal interstitial and vascular pathological damage, glomerular sclerosis, some inward depression, a large number of cells infiltrated in the perivascular and interstitial area, kidney Severe swelling and hyperplasia of the pellet appeared. Compared with normal mice, the kidney pathological score of MRL/lpr control group (model control group) mice was significantly increased (P ⁇ 0.001), suggesting that the kidneys of model control group mice have obvious lesions.
  • the pathological damage of glomerulus, renal interstitium and blood vessel of mice in the administration group was reduced to a certain extent.
  • the kidney pathology scores of mice in the 1 and 3 mg/kg administration groups were significantly lower than the model control group (P ⁇ 0.05, P ⁇ 0.0001), indicating that D2-60-b-T3E can significantly reduce MRL/lpr spontaneous renal disease.
  • the results are shown in Figure 15.

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Abstract

抗IL-2抗体、其抗原结合片段及其医药用途。进一步地，所述抗IL-2抗体、其抗原结合片段与IL-2的复合物（包括融合蛋白），及其作为药物用于治疗自身免疫疾病、炎性病症的用途。

Description

抗IL-2抗体、其抗原结合片段及其医药用途
本申请要求2020年02月21日提交的中国专利申请(申请号202010107662.4)的优先权。
技术领域
本公开涉及抗体，例如特异性结合白细胞介素-2(IL-2)的抗体及其抗原结合片段。本公开涉及所述抗IL-2抗体、其抗原结合片段与IL-2的复合物(包括融合蛋白)，及其作为药物用于治疗免疫相关(如自身免疫疾病、炎性病症)的用途。
背景技术
人白细胞介素-2(Interleukin-2，IL-2)，也称为T细胞生长因子(TCGF)，基因位于4号染色体(4q27)，包括共7kb的序列，由133个氨基酸组成，分子量约15kD。1976年和1977年，Doris Morgan，Francis Ruscetti，Robert Gallo和Steven Gillis，Kendal Smith等分别发现活化后的T细胞培养液可以促进T细胞增殖。之后培养液中的刺激因子被纯化并被鉴定为单一的蛋白质即IL-2。
最初的体外细胞实验表明T细胞在通过TCR和CD28活化之后，可以分泌IL-2并在细胞表面表达IL-2受体(IL-2R)。IL-2和其受体的结合可以引起T细胞的增殖和使T细胞产生效应。这一模型使得IL-2成为T细胞免疫反应中起到中心作用的一个分子。不过随后的体内实验发现在敲除IL-2或其受体后，动物产生了自身免疫。随后的实验表明IL-2不仅可以激活如T细胞和NK细胞的效应细胞，还可以激活调节性T细胞(Treg)，从而抑制过量的针对自身的免疫。
IL-2通过细胞表面的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。其中，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，磷酸化细胞内的STAT5，进入细胞核导致相应的基因转录和表达；IL-2Rα并非信号传导所必需，但可以增强IL-2与IL-2Rβ和IL-2Rγ的结合。Treg持续表达高结和力受体IL-2Rα/β/γ，而未被激活的CD8+T细胞和NK细胞等只表达中结合力受体IL-2Rβ/γ。因此，低剂量IL-2优先结合并激活Treg，而高剂量IL-2则也可以激活CD8+T细胞和NK细胞等免疫效应细胞。
由于Treg具有免疫抑制作用，而低剂量IL-2具有优先激活Treg的性质，因此低剂量IL-2被报道可以用于一系列免疫相关，如自身免疫疾病和炎性疾病的治疗，、例如系统性红斑狼疮(systemic lupus erythematosus，SLE)、I型糖尿病(type I diabetes，T1D)、慢性移植物对抗宿主疾病(chronic Graft versus Host Disease，cGvHD)、骨髓移植(hematopoietic stem cell transplantation，HSCT)、斑秃(alopecia areata)、丙型肝炎病毒诱导的血管炎(hepatitis C virus-induced vasculitis)、慢性GvHD、哮喘、皮炎、斑秃、急性肺损伤、慢性铍尘病、败血症等。在很多接受低剂量IL-2处理的患者中，Treg与效应T细胞的比例均有上升，症状也有所缓解。 然而一方面，IL-2半衰期较短，需要频繁给药；另一方面，IL-2的给药窗口较小，如果剂量略高，则可能激活免疫效应细胞，加重自身免疫疾病。这些IL-2本身的性质或缺点使得IL-2难以在患者体内保持持续的低剂量，限制了其进一步应用。
抗IL-2抗体可以改变IL-2与IL-2R的结合。WO2017/070561和WO2015/109212提供了抗IL-2抗体。但本领域中对于治疗IL-2介导的疾病、病症和状况的新型疗法仍然存在长期未满足的需求。本公开满足了这些需求。
发明内容
本公开提供抗IL-2抗体及其抗原结合片段，和包含所述抗体、抗原结合片段的复合物、编码核酸、载体、宿主细胞、药物组合物、其用于治疗或延缓免疫相关疾病的方法，及其制药用途。
抗IL-2抗体或其抗原结合片段
一些实施方案中，提供包含IL-2抗体或其抗原结合片段，其与IL-2的亲合力的K D值≤5nM。
另一些实施方案中，提供IL-2抗体或其抗原结合片段，其与IL-2的亲合力K D值＞5nM或≥10nM，且≤200nM或≤150nM或≤100nM或≤80nM或≤60nM。在一些具体实施方案中，IL-2抗体或其抗原结合片段与IL-2的亲合力的K D值＞5nM且≤60nM，＞5nM且≤50nM，≥10nM且≤50nM，≥10nM且≤60nM，＞5nM且≤80nM，≥10nM且≤80nM，＞5nM且≤100nM，≥10nM且≤100nM，＞5nM且≤150nM，或≥10nM且≤150nM。
一些实施方案中，本公开的IL-2抗体或其抗原结合片段，其含有重链可变区(VH)和轻链可变区(VL)，所述VH具有HCDR1、HCDR2、HCDR3，所述VL具有LCDR1、LCDR2、LCDR3。
一些实施方案中，所述IL-2抗体或其抗原结合片段含有VH和VL：
所述VH的HCDR1、HCDR2、HCDR3分别对应于SEQ ID NO：1中的HCDR1、HCDR2、HCDR3，所述VL的LCDR1、LCDR2、LCDR3分别对应于SEQ ID NO：2中的LCDR1、LCDR2、LCDR3；
所述VH的HCDR1、HCDR2、HCDR3分别对应于SEQ ID NO：3中的HCDR1、HCDR2、HCDR3，所述VL的LCDR1、LCDR2、LCDR3分别对应于SEQ ID NO：4中的LCDR1、LCDR2、LCDR3；
所述VH的HCDR1、HCDR2、HCDR3分别对应于SEQ ID NO：5中的HCDR1、HCDR2、HCDR3，所述VL的LCDR1、LCDR2、LCDR3分别对应于SEQ ID NO：6中的LCDR1、LCDR2、LCDR3；
所述VH的HCDR1、HCDR2、HCDR3分别对应于SEQ ID NO：7中的HCDR1、HCDR2、HCDR3，所述VL的LCDR1、LCDR2、LCDR3分别对应于SEQ ID NO：8中的LCDR1、LCDR2、LCDR3；
所述VH的HCDR1、HCDR2、HCDR3分别对应于SEQ ID NO：9中的HCDR1、HCDR2、HCDR3，所述VL的LCDR1、LCDR2、LCDR3分别对应于SEQ ID NO：10中的LCDR1、LCDR2、LCDR3；或
所述VH的HCDR1、HCDR2、HCDR3分别对应于SEQ ID NO：11中的HCDR1、HCDR2、HCDR3，所述VL的LCDR1、LCDR2、LCDR3分别对应于SEQ ID NO：12中的LCDR1、LCDR2、LCDR3。上述HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3是根据Kabat、IMGT、Chothia、AbM或Contact编号系统定义的。
一些实施方案中，抗IL-2抗体或其抗原结合片段包含：
SEQ ID NO：13、19、25、31、37或43所示的HCDR1，和/或
SEQ ID NO：14、20、26、32、38或44所示的HCDR2，和/或
SEQ ID NO：15、21、27、33、39或45所示的HCDR3，和/或
SEQ ID NO：16、22、28、34、40或46所示的LCDR1，和/或
SEQ ID NO：17、23、29、35、41或47所示的LCDR2，和/或
SEQ ID NO：18、24、30、36、42或48所示的LCDR3。所述实施方案采用Kabat编号系统。
另一些实施方案中，抗IL-2抗体或其抗原结合片段包含：
SEQ ID NO：49、55、61、67、73或79所示的HCDR1，和/或
SEQ ID NO：50、56、62、68、74或80所示的HCDR2，和/或
SEQ ID NO：51、57、63、69、75或81所示的HCDR3，和/或
SEQ ID NO：52、58、64、70、76或82所示的LCDR1，和/或
SEQ ID NO：53、59、65、71、77或83所示的LCDR2，和/或
SEQ ID NO：54、60、66、72、78或84所示的LCDR3。所述实施方案采用Chothia编号系统。
另一些实施方案中，抗IL-2抗体或其抗原结合片段包含：
SEQ ID NO：85、91、97、103、109或115所示的HCDR1，和/或
SEQ ID NO：86、92、98、104、110或116所示的HCDR2，和/或
SEQ ID NO：87、93、99、105、111或117所示的HCDR3，和/或
SEQ ID NO：88、94、100、106、112或118所示的LCDR1，和/或
SEQ ID NO：89、95、101、107、113或119所示的LCDR2，和/或
SEQ ID NO：90、96、102、108、114或120所示的LCDR3。所述实施方案采用IMGT编号系统。
另一些实施方案中，抗IL-2抗体或其抗原结合片段包含：
SEQ ID NO：121、127、133、139、145或151所示的HCDR1，和/或
SEQ ID NO：122、128、134、140、146或152所示的HCDR2，和/或
SEQ ID NO：123、129、135、141、147或153所示的HCDR3，和/或
SEQ ID NO：124、130、136、142、148或154所示的LCDR1，和/或
SEQ ID NO：125、131、137、143、149或155所示的LCDR2，和/或
SEQ ID NO：126、132、138、144、150或156所示的LCDR3。所述实施方案采用AbM编号系统。
以上抗IL-2抗体或其抗原结合片段分为两组，第一组包含：
SEQ ID NO：13、25或37所示的HCDR1，和/或
SEQ ID NO：14、26或38所示的HCDR2，和/或
SEQ ID NO：15、27或39所示的HCDR3，和/或
SEQ ID NO：16、28或40所示的LCDR1，和/或
SEQ ID NO：17、29或41所示的LCDR2，和/或
SEQ ID NO：18、30或42所示的LCDR3；
SEQ ID NO：49、61或73所示的HCDR1，和/或
SEQ ID NO：50、62或74所示的HCDR2，和/或
SEQ ID NO：51、63或75所示的HCDR3，和/或
SEQ ID NO：52、64或76所示的LCDR1，和/或
SEQ ID NO：53、65或77所示的LCDR2，和/或
SEQ ID NO：54、66或78所示的LCDR3；
SEQ ID NO：85、97或109所示的HCDR1，和/或
SEQ ID NO：86、98或110所示的HCDR2，和/或
SEQ ID NO：87、99或111所示的HCDR3，和/或
SEQ ID NO：88、100或112所示的LCDR1，和/或
SEQ ID NO：89、101或113所示的LCDR2，和/或
SEQ ID NO：90、102或114所示的LCDR3；或
SEQ ID NO：121、133或145所示的HCDR1，和/或
SEQ ID NO：122、134或146所示的HCDR2，和/或
SEQ ID NO：123、135或147所示的HCDR3，和/或
SEQ ID NO：124、136或148所示的LCDR1，和/或
SEQ ID NO：125、137或149所示的LCDR2，和/或
SEQ ID NO：126、138或150所示的LCDR3。
第二组包含：
SEQ ID NO：19、31或43所示的HCDR1，和/或
SEQ ID NO：20、32或44所示的HCDR2，和/或
SEQ ID NO：21、33或45所示的HCDR3，和/或
SEQ ID NO：22、34或46所示的LCDR1，和/或
SEQ ID NO：23、35或47所示的LCDR2，和/或
SEQ ID NO：24、36或48所示的LCDR3；
SEQ ID NO：55、67或79所示的HCDR1，和/或
SEQ ID NO：56、68或80所示的HCDR2，和/或
SEQ ID NO：57、69或81所示的HCDR3，和/或
SEQ ID NO：58、70或82所示的LCDR1，和/或
SEQ ID NO：59、71或83所示的LCDR2，和/或
SEQ ID NO：60、72或84所示的LCDR3；
SEQ ID NO：91、103或115所示的HCDR1，和/或
SEQ ID NO：92、104或116所示的HCDR2，和/或
SEQ ID NO：93、105或117所示的HCDR3，和/或
SEQ ID NO：94、106或118所示的LCDR1，和/或
SEQ ID NO：95、107或119所示的LCDR2，和/或
SEQ ID NO：96、108或120所示的LCDR3；或
SEQ ID NO：127、139或151所示的HCDR1，和/或
SEQ ID NO：128、140或152所示的HCDR2，和/或
SEQ ID NO：129、141或153所示的HCDR3，和/或
SEQ ID NO：130、142或154所示的LCDR1，和/或
SEQ ID NO：131、143或155所示的LCDR2，和/或
SEQ ID NO：132、144或156所示的LCDR3。
第一组的抗IL-2抗体或其抗原结合片段，其CDR区还可以存在一个或多个氨基酸突变。突变的氨基酸可以为1、2、3、4、5、6、7、8、9、10、11、12、13、14、15个。在一些实施方案中，第一组的IL-2抗体或其抗原结合片段与IL-2的亲合力的K D值>5nM或≥10nM，且≤200nM或≤150nM或≤100nM或≤80nM或≤60nM。在一些具体实施方案中，IL-2抗体或其抗原结合片段与IL-2的亲合力的K D值>5nM且≤60nM，>5nM且≤50nM，≥10nM且≤60nM，≥10nM且≤50nM，>5nM且≤80nM，≥10nM且≤80nM，>5nM且≤100nM，≥10nM且≤100nM，>5nM且≤150nM，或≥10nM且≤150nM。
第二组的抗IL-2抗体或其抗原结合片段，其CDR区还可以存在一个或多个氨基酸突变。突变的氨基酸可以为1、2、3、4、5、6、7、8、9、10、11、12、13、14、15个。在一些实施方案中，其与IL-2的亲合力的K D值≤5nM。
一些实施方案中，提供IL-2抗体或其抗原结合片段，其结合与前述抗IL-2抗体或其抗原结合片段相同的抗原或表位，或与前述抗IL-2抗体或其抗原结合片段竞争结合相同的抗原或表位，或与前述抗IL-2抗体或其抗原结合片段结合或竞争结合IL-2。
一些实施方案中，前述抗IL-2抗体或其抗原结合片段降低或阻断IL-2与IL-2Rβ的结合，例如，阻断IL-2与IL-2Rβ的结合。
一些实施方案中，前述抗IL-2抗体或其抗原结合片段降低IL-2与IL-2Rα的结合。
一些实施方案中，前述抗IL-2抗体或其抗原结合片段为全人抗体或其抗原结合片段，或人源化抗体或其抗原结合片段。
一些实施方案中，前述抗IL-2抗体或其抗原结合片段为IgG抗体或其抗原结合片段，例如，为IgG1、IgG4抗体或其抗原结合片段。一些具体实施方案中，前述抗IL-2抗体或其抗原结合片段为降低的效应子功能的IgG1抗体，例如ADCC功能降低或消失的IgG1抗体(例如具有LALA突变的Fc的IgG1抗体)。
一些实施方案中，前述抗IL-2抗体或其抗原结合片段，包含选自以下组的HCDR1-3和/或LCDR1-3：
SEQ ID NO：13-15所示的HCDR1-3，和/或SEQ ID NO：16-18所示的LCDR1-3；
SEQ ID NO：19-21所示的HCDR1-3，和/或SEQ ID NO：22-24所示的LCDR1-3；
SEQ ID NO：25-27所示的HCDR1-3，和/或SEQ ID NO：28-30所示的LCDR1-3；
SEQ ID NO：31-33所示的HCDR1-3，和/或SEQ ID NO：34-36所示的LCDR1-3；
SEQ ID NO：37-39所示的HCDR1-3，和/或SEQ ID NO：40-42所示的LCDR1-3；
SEQ ID NO：43-45所示的HCDR1-3，和/或SEQ ID NO：46-48所示的LCDR1-3；
SEQ ID NO：49-51所示的HCDR1-3，和/或SEQ ID NO：52-54所示的LCDR1-3；
SEQ ID NO：55-57所示的HCDR1-3，和/或SEQ ID NO：58-60所示的LCDR1-3；
SEQ ID NO：61-63所示的HCDR1-3，和/或SEQ ID NO：64-66所示的LCDR1-3；
SEQ ID NO：67-69所示的HCDR1-3，和/或SEQ ID NO：70-72所示的LCDR1-3；
SEQ ID NO：73-75所示的HCDR1-3，和/或SEQ ID NO：76-78所示的LCDR1-3；
SEQ ID NO：79-81所示的HCDR1-3，和/或SEQ ID NO：82-84所示的LCDR1-3；
SEQ ID NO：85-87所示的HCDR1-3，和/或SEQ ID NO：88-90所示的LCDR1-3；
SEQ ID NO：91-93所示的HCDR1-3，和/或SEQ ID NO：94-96所示的LCDR1-3；
SEQ ID NO：97-99所示的HCDR1-3，和/或SEQ ID NO：100-102所示的LCDR1-3；
SEQ ID NO：103-105所示的HCDR1-3，和/或SEQ ID NO：106-108所示的LCDR1-3；
SEQ ID NO：109-111所示的HCDR1-3，和/或SEQ ID NO：112-114所示的LCDR1-3；
SEQ ID NO：115-117所示的HCDR1-3，和/或SEQ ID NO：118-120所示的LCDR1-3；
SEQ ID NO：121-123所示的HCDR1-3，和/或SEQ ID NO：124-126所示的LCDR1-3；
SEQ ID NO：127-129所示的HCDR1-3，和/或SEQ ID NO：130-132所示的LCDR1-3；
SEQ ID NO：133-135所示的HCDR1-3，和/或SEQ ID NO：136-138所示的LCDR1-3；
SEQ ID NO：139-141所示的HCDR1-3，和/或SEQ ID NO：142-144所示的LCDR1-3；
SEQ ID NO：145-147所示的HCDR1-3，和/或SEQ ID NO：148-150所示的LCDR1-3；或
SEQ ID NO：151-153所示的HCDR1-3，和/或SEQ ID NO：154-156所示的LCDR1-3。
一些实施方案中，提供抗IL-2抗体或其抗原结合片段，其包含VH和VL:
(i)根据Kabat编号系统定义的，
其VH包含SEQ ID NO：37-39所示的HCDR1-3，其VL包含SEQ ID NO：40-42所示的LCDR1-3；
其VH包含SEQ ID NO：13-15所示的HCDR1-3，其VL包含SEQ ID NO：16-18所示的LCDR1-3；
其VH包含SEQ ID NO：25-27所示的HCDR1-3，其VL包含SEQ ID NO：28-30所示的LCDR1-3；
其VH包含SEQ ID NO：19-21所示的HCDR1-3，其VL包含SEQ ID NO：22-24所示的LCDR1-3；
其VH包含SEQ ID NO：31-33所示的HCDR1-3，其VL包含SEQ ID NO：34-36所示的LCDR1-3；或
其VH包含SEQ ID NO：43-45所示的HCDR1-3，其VL包含SEQ ID NO：46-48所示的LCDR1-3；
(ii)根据Chothia编号系统定义的，
其VH包含SEQ ID NO：73-75所示的HCDR1-3，其VL包含SEQ ID NO：76-78所示的LCDR1-3；
其VH包含SEQ ID NO：49-51所示的HCDR1-3，其VL包含SEQ ID NO：52-54所示的LCDR1-3；
其VH包含SEQ ID NO：61-63所示的HCDR1-3，其VL包含SEQ ID NO：64-66所示的LCDR1-3；
其VH包含SEQ ID NO：55-57所示的HCDR1-3，其VL包含SEQ ID NO：58-60所示的LCDR1-3；
其VH包含SEQ ID NO：67-69所示的HCDR1-3，其VL包含SEQ ID NO：70-72所示的LCDR1-3；
其VH包含SEQ ID NO：79-81所示的HCDR1-3，其VL包含SEQ ID NO：82-84所示的LCDR1-3；
(iii)根据IMGT编号系统定义的，
其VH包含SEQ ID NO：109-111所示的HCDR1-3，其VL包含SEQ ID NO：112-114所示的LCDR1-3；
其VH包含SEQ ID NO：85-87所示的HCDR1-3，其VL包含SEQ ID NO：88-90所示的LCDR1-3；
其VH包含SEQ ID NO：97-99所示的HCDR1-3，其VL包含SEQ ID NO：100-102所示的LCDR1-3；
其VH包含SEQ ID NO：91-93所示的HCDR1-3，其VL包含SEQ ID NO：94-96所示的LCDR1-3；
其VH包含SEQ ID NO：103-105所示的HCDR1-3，其VL包含SEQ ID NO：106-108所示的LCDR1-3；或
其VH包含SEQ ID NO：115-117所示的HCDR1-3，其VL包含SEQ ID NO：118-120所示的LCDR1-3；
(iv)根据AbM编号系统定义的，
其VH包含SEQ ID NO：145-147所示的HCDR1-3，其VL包含SEQ ID NO：148-150所示的LCDR1-3；
其VH包含SEQ ID NO：121-123所示的HCDR1-3，其VL包含SEQ ID NO：124-126所示的LCDR1-3；
其VH包含SEQ ID NO：133-135所示的HCDR1-3，其VL包含SEQ ID NO：136-138所示的LCDR1-3；
其VH包含SEQ ID NO：127-129所示的HCDR1-3，其VL包含SEQ ID NO：130-132所示的LCDR1-3；
其VH包含SEQ ID NO：139-141所示的HCDR1-3，其VL包含SEQ ID NO：142-144所示的LCDR1-3；或
其VH包含SEQ ID NO：151-153所示的HCDR1-3，其VL包含SEQ ID NO：154-156所示的LCDR1-3。
一些实施方案中，前述的抗IL-2抗体或其抗原结合片段，其包含：
如SEQ ID NO：1、3、5、7、9、11之一所示或与之具有至少90％、95％、98％、99％同一性的VH；和/或
如SEQ ID NO：2、4、6、8、10、12之一所示或与之具有至少90％、95％、98％、99％同一性的VH。
一些实施方案中，前述的抗IL-2抗体或其抗原结合片段，其包含选自下列组的VH和/或VL：
如SEQ ID NO：1所示或与之具有至少90％同一性的VH，如SEQ ID NO：2所示或与之具有至少90％同一性的VL；
如SEQ ID NO：3所示或与之具有至少90％同一性的VH，如SEQ ID NO：4所示或与之具有至少90％同一性的VL；
如SEQ ID NO：5所示或与之具有至少90％同一性的VH，如SEQ ID NO：6所示或与之具有至少90％同一性的VL；
如SEQ ID NO：7所示或与之具有至少90％同一性的VH，如SEQ ID NO：8所示或与之具有至少90％同一性的VL；
如SEQ ID NO：9所示或与之具有至少90％同一性的VH，如SEQ ID NO：10所示或与之具有至少90％同一性的VL；或
如SEQ ID NO：11所示或与之具有至少90％同一性的VH，如SEQ ID NO：12所示或与之具有至少90％同一性的VL。
一些实施方案中，前述抗IL-2抗体或其抗原结合片段重链可变区(VH)与人IgG1重链恒定区连接，轻链可变区(VL)与kappa或lamda链恒定区连接。
一些实施方案中，前述抗IL-2抗体包含：
如SEQ ID NO：157、159、161、163、165、167之一所示或与之具有至少80％、90％、95％、98％、99％同一性的HC；和/或
如SEQ ID NO：158、160、162、164、166、168之一所示或与之具有至少80％、90％、95％、98％、99％同一性的LC。
一些实施方案中，前述抗IL-2抗体或其抗原结合片段包含选自以下组的HC和LC：
如SEQ ID NO：157所示或与之具有至少80％、90％、95％、98％、99％同一性的全长重链(HC)，如SEQ ID NO：158所示或与之具有至少80％、90％、95％、98％、99％同一性的全长轻链(LC)；
如SEQ ID NO：159所示或与之具有至少80％、90％、95％、98％、99％同一性的HC，如SEQ ID NO：160所示或与之具有至少80％、90％、95％、98％、99％ 同一性的LC；
如SEQ ID NO：161所示或与之具有至少80％、90％、95％、98％、99％同一性的HC，如SEQ ID NO：162所示或与之具有至少80％、90％、95％、98％、99％同一性的LC；
如SEQ ID NO：163所示或与之具有至少90％同一性的HC，如SEQ ID NO：164所示或与之具有至少80％、90％、95％、98％、99％同一性的LC；
如SEQ ID NO：165所示或与之具有至少90％同一性的HC，如SEQ ID NO：166所示或与之具有至少80％、90％、95％、98％、99％同一性的LC；或
如SEQ ID NO：167所示或与之具有至少80％、90％、95％、98％、99％同一性的HC，如SEQ ID NO：168所示或与之具有至少80％、90％、95％、98％、99％同一性的LC。
一些实施方案中，抗IL-2抗体的抗原结合片段为Fab、Fv、sFv、Fab’、F(ab’) 2、线性抗体、单链抗体、scFv、sdAb、sdFv、纳米抗体、肽抗体peptibody、结构域抗体和多特异性抗体(双特异性抗体、diabody、triabody和tetrabody、串联二-scFv、串联三-scFv)，例如具体为scFv、Fv、Fab或Fab’片段。
复合物
本公开提供包含IL-2抗体或其抗原结合片段和IL-2的复合物。
一些具体实施方案中，复合物中IL-2抗体或其抗原结合片段和IL-2以非共价力结合，IL-2抗体或其抗原结合片段与IL-2的亲合力的K D值≤5nM。
另一些具体实施方案中，复合物中IL-2抗体或其抗原结合片段和IL-2以共价力结合(例如形成融合蛋白)，其中，IL-2抗体或其抗原结合片段与IL-2的亲合力的K D值>5nM或≥10nM，且≤200nM或≤150nM或≤100nM或≤80nM或≤60nM。在一些具体实施方案中，所述亲合力的K D值>5nM且≤60nM，>5nM且≤50nM，≥10nM且≤60nM，≥10nM且≤50nM，>5nM且≤80nM，≥10nM且≤80nM，>5nM且≤100nM，≥10nM且≤100nM，>5nM且≤150nM，或≥10nM且≤150nM。
一些实施方案中，提供融合蛋白，其包含抗IL-2抗体或其抗原片段与IL-2，其中抗IL-2抗体或其抗原片段与IL-2亲合力的K D值>5nM或≥10nM，且≤50nM或≤60nM或≤80nM。一些具体方案中，抗IL-2抗体或其抗原片段与IL-2亲合力的K D值大于≥10nM，且≤60nM。
一些实施方案中，抗IL-2抗体或其抗原片段与IL-2之间通过共价或非共价力结合。一些实施方案中，抗IL-2抗体或其抗原片段与IL-2形成融合蛋白。IL-2与抗IL-2抗体或其抗原片段的轻链可变区或重链可变区连接，例如，IL-2与抗IL-2抗体或其抗原片段的轻链可变区或重链可变区的N端连接。一些具体实施方案中，IL-2与轻链可变区的N端连接。
一些实施方案中，上述融合蛋白中的IL-2与抗IL-2抗体或其抗原片段通过连接子连接。所述连接子选自：如(G mS n) x或(GGNGT) x或(YGNGT) x所示的氨基酸序列，其中m、n各自独立地选自1-8的整数(例如，1、2、3、4、5、6、7或8)，x独立地选自1-20的整数(例如，1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20)。在一些具体实施方案中，所述连接子 具有如(G 4S)x所示的氨基酸序列，x独立地选自1-6的整数(例如，x为4或5)。所述连接子还可以为与上述(G mS n) x或(GGNGT) x或(YGNGT) x所示的氨基酸序列具有类似柔性和长度的其他连接子；所述连接子还可以选自AKTTPKLEEGEFSEAR、AKTTPKLEEGEFSEARV、AKTTPKLGG、SAKTTPKLGG、AKTTPKLEEGEFSEARV、SAKTTP、SAKTTPKLGG、RADAAP、RADAAPTVS、RADAAAAGGPGS、RADAAAA(G 4S) 4、SAKTTP、SAKTTPKLGG、SAKTTPKLEEGEFSEARV、ADAAP、ADAAPTVSIFPP、TVAAP、TVAAPSVFIFPP、QPKAAP、QPKAAPSVTLFPP、AKTTPP、AKTTPPSVTPLAP、AKTTAP、AKTTAPSVYPLAP、ASTKGP、ASTKGPSVFPLAP、GENKVEYAPALMALS、GPAKELTPLKEAKVS、GHEAAAVMQVQYPAS。
一些实施方案中，上述复合物选择性增强高表达IL-2Ra的细胞的活性或促进其增殖，所述高表达IL-2Ra的细胞例如为调节性T(Treg)细胞。一些具体实施方案中，复合物为抗IL-2抗体或其抗原片段与IL-2形成的融合蛋白。
一些实施方案中，上述融合蛋白中的IL-2存在第三位氨基酸(作为一个示例，T3)的氨基酸取代或N端缺失。一些具体实施方案中，IL-2的第三位氨基酸取代为Ala、Gln或Glu(作为一个示例，T3A、T3Q、T3E)，N端缺失为N端前3个或前7个氨基酸缺失。
一些实施方案中，上述融合蛋白包含选自以下组的HC和LC：
SEQ ID No：169所示的HC，SEQ ID No：158所示的LC；
SEQ ID No：171所示的HC，SEQ ID No：160所示的LC；
SEQ ID No：159所示的HC，SEQ ID No：172所示的LC；
SEQ ID No：173所示的HC，SEQ ID No：162所示的LC；
SEQ ID No：175所示的HC，SEQ ID No：164所示的LC；
SEQ ID No：163所示的HC，SEQ ID No：176所示的LC；
SEQ ID No：177所示的HC，SEQ ID No：166所示的LC；
SEQ ID No：179所示的HC，SEQ ID No：168所示的LC；
SEQ ID No：167所示的HC，SEQ ID No：180所示的LC；
SEQ ID No：161所示的HC，SEQ ID No：174、181-186任一所示的LC；
SEQ ID No：157所示的HC，SEQ ID No：170、187所示的LC；
SEQ ID No：165所示的HC，SEQ ID No：178、188、189任一所示的LC；
或与上述HC、LC具有至少80％、90％、95％、98％、99％同一性的序列。
一些实施方案中，前述本公开的复合物选择性的增强了IL-2对Treg活性的作用(例如促增殖活性、促STAT5磷酸化活性)，和/或选择性地提高IL-2促Treg细胞中FOXP3、CD25和Icos中的一种或多种表达的能力。
一些实施方案中，本公开的复合物还具有(i)-(iii)中的一项或多项：
(i)对Treg的活性激活程度高于对CD8+T细胞的活性激活程度；
(ii)相较于在CD8+T细胞中，在Treg细胞中对STAT5磷酸化的促进程度更高；
(iii)相较于CD8+T细胞、CD4+T细胞或NK细胞的增殖，对Treg细胞的 增殖促进程度更高。
一些实施方案中，本公开的抗IL-2抗体或其抗原结合片段的Fc区包含一个或多个氨基酸取代，所述一个或多个氨基酸取代减少Fc区与Fc受体的结合，例如Fc区与Fcγ受体的结合，并且降低或消除效应子功能。一些具体实施方案中，本公开的抗IL-2抗体或其抗原结合片段具有降低或消除的ADCC功能。
一些实施方案中，本公开的抗IL-2抗体或其抗原结合片段具有改造的Fc区，所述改造的Fc区与天然Fc区相比，对Fc受体的结合亲合力下降50％、80％、90％或95％以上。在一些实施方案中，所述的Fc受体是Fcγ受体。在一些实施方案中，所述Fc受体是人Fcγ受体，例如FcγRI、FcγRIIa、FcγRIIB、FcγRIIIa。在一些实施方案中，改造的Fc区与天然Fc区相比，对补体(如C1q)的结合亲合力也降低。在一些实施方案中，改造的Fc区与天然Fc区相比，对新生儿Fc受体(FcRn)的结合亲合力不降低。在一些实施方案中，改造的Fc区具有降低的效应子功能，所述降低的效应子功能可以包括但不限于以下中的一个或多个：降低的补体依赖性细胞毒性(CDC)、降低的抗体依赖性细胞介导的细胞毒性(ADCC)、降低的抗体依赖性细胞吞噬(ADCP)、减少的细胞因子分泌、减少的免疫复合物介导的抗原呈递细胞的抗原摄取、减少的与NK细胞的结合、减少的与巨噬细胞的结合、减少的与单核细胞的结合、减少的与多形核细胞的结合、减少的直接信号传导诱导性细胞凋亡、降低的树突细胞成熟或减少的T细胞引发。
一些实施方案中，对于IgG 1Fc区，在238、265、269、270、297、327和329等位置的氨基酸残基取代可降低的效应子功能。在一些实施方案中，所述Fc区是人IgG 1Fc区，并且在234和235位置的氨基酸残基为A，编号依据为EU索引。对于IgG 4Fc区，在228等位置的氨基酸残基取代可降低的效应子功能。改变效应子功能的氨基酸取代选自以下中的一组或更多组：S298A/E333A/K334A；S239D/I332E/A330L；S239D/I332E/G236A；G236A/S239D/A330L/I332E；F243L/R292P/Y300L/V305I/P396L；K326A/E333A；K326W/E333S；K326M/E333S；C221D/D222C；S267E/H268F/S324T；E345R；S298A/E333A/K334A/N434A；E294缺失/T307P/N434Y；T256N/A378V/S383N/N434Y；T252L/T253S/T254F；M252Y/S254T/T256E；M428L/N434S；L234A/L235A；S228P/L235E；L234A/L235A/P331S；L234A/L235A/P329G；D265A/E233P；H268Q/V309L/A330S/P331S；V234A/G237A/P238S/H268A/V309L/A300S/P331S；L234A/L235A/G237A/P238S/H268A/V309L/A300S/P331S；S228P/F234A/L235A；D270A/P329A；L234F/L235E；L234F/L235E/P331S；F241A/V264A/D265A；N297G/D265A；和L234Y/G236W/S298A。
本公开提供与IL-2具有中等亲合力的抗IL-2抗体或其抗原结合片段，所述中等亲合力为抗体与IL-2的亲合力K D值>5nM或≥10nM，且≤200nM或≤150nM或≤100nM或≤80nM或≤60nM(例如≥10nM且≤50nM)。当所述抗体与IL-2形成融合 蛋白时，所述抗体与IL-2通过连接子连接。此外，所述抗体与IL-2还通过非共价键结合，但所述非共价键结合程度(即亲合力)不高于IL-2Rα与IL-2的亲合力。因此，当所述融合蛋白与细胞表面的IL-2Rα接触时，抗IL-2抗体与IL-2间的非共价键解离，IL-2与IL-2Rα结合，从而激活细胞。Treg持续表达高结和力受体IL-2Rα/β/γ，而未被激活的CD8+T细胞和NK细胞等只表达中结合力受体IL-2Rβ/γ。因此，这类抗体与IL-2的融合蛋白能够选择性或优先激活Treg。本公开提供的这类抗体例如A2-22、B2-15、D2-60及与之具有相同HCDR1-3、LCDR1-3的抗体。
本公开还提供与IL-2具有高亲合力的抗IL-2抗体或其抗原结合片段，所述高亲合力为抗体与IL-2的亲合力K D值≤5nM。所述抗体与IL-2可以通过非共价键结合，所述非共价键结合程度(即亲合力)与IL-2Rα与IL-2的亲合力相似或略高于IL-2Rα与IL-2的亲合力，因此，所述抗体与IL-2的复合物(非共价键)与细胞表面的IL-2Rα接触时，抗IL-2抗体与IL-2间的非共价键解离或部分解离，IL-2与IL-2Rα结合，从而激活细胞。因此，这类抗体与IL-2的复合物(非共价键)也能够选择性或优先激活Treg。本公开并不排除这类抗体与IL-2的融合蛋白形式。本公开提供的这类抗体例如C2-53、D3-68及与之具有相同HCDR1-3、LCDR1-3的抗体。
本公开中，抗IL-2抗体或其抗原结合片段与IL-2的亲合力采用本领域的常规方法检测。当亲合力用K D值表示时，采用例如Biacore(具体检测方法参见实施例6)检测。当亲合力用EC 50值表示时，采用例如ELISA(具体检测方法参见实施例5)检测。
一些实施方案中，本公开的部分抗IL-2抗体或其抗原结合片段，能降低IL-2与IL-2Rα的结合，这是通过ELISA实验检测的，具体参见实施例3；或通过Octet实验检测的，具体参见实施例4。这部分实施方案中，具有SEQ ID NO：9、10所述序列中的HCDR1-3和LCDR1-3的抗体(例如D2-60)，具有SEQ ID NO：5、6所述序列中的HCDR1-3和LCDR1-3的抗体(例如B2-15)，具有SEQ ID NO：1、3所述序列中的HCDR1-3和LCDR1-3的抗体(例如A2-22)不能阻断IL-2与IL-2Rα的结合，仅能降低IL-2与IL-2Rα的结合。此处的“不能阻断IL-2与IL-2Rα的结合”可以理解在实施例3的高抗体浓度下(例如10μg/mL)仍不能通过拟合曲线获得有意义的IC 50值。“降低”可以理解为在实施例3条件下仍能一定程度影响IL-2与IL-2Rα的结合(例如图1A)，或在实施例4的实验条件下，具有大于0.600(例如为0.700、0.754、0.848)的Octet水平对IL-2Rα的阻断数值。
一些实施方案中，本公开的部分抗IL-2抗体或其抗原结合片段，能阻断IL-2与IL-2Rα的结合，这是通过ELISA实验检测的，具体参见实施例3；或通过Octet实验检测的，具体参见实施例4。这部分实施方案中，具有SEQ ID NO：3、4所述序列中的HCDR1-3和LCDR1-3的抗体(例如A3-21)，具有SEQ ID NO：7、8所述序列中的HCDR1-3和LCDR1-3的抗体(例如C2-53)，具有SEQ ID NO：11、12所述序列中的HCDR1-3和LCDR1-3的抗体(例如D3-68)能阻断IL-2与IL-2Rα结合。“阻断IL-2与IL-2Rα的结合”可以理解为能在实施例3条件下获得有效的 阻断IL-2与IL-2Rα结合的IC 50值(例如0.2-5μg/mL，更例如0.5-2μg/mL)，或在实施例4的实验条件下，具有小于0.600(例如为0.410、0.428、0.473)的Octet水平对IL-2Rα的阻断数值。
一些实施方案中，本公开的部分抗IL-2抗体或其抗原结合片段，均能阻断IL-2与IL-2Rβ的结合，这是通过ELISA实验检测的，具体参见实施例3；或通过Octet实验检测的，具体参见实施例4。这部分实施方案中，具有SEQ ID NO：9、10所述序列中的HCDR1-3和LCDR1-3的抗体(例如D2-60)，具有SEQ ID NO：5、6所述序列中的HCDR1-3和LCDR1-3的抗体(例如B2-15)，具有SEQ ID NO：1、3所述序列中的HCDR1-3和LCDR1-3的抗体(例如A2-22)，具有SEQ ID NO：1、2所述序列中的HCDR1-3和LCDR1-3的抗体(例如A3-21)，具有SEQ ID NO：7、8所述序列中的HCDR1-3和LCDR1-3的抗体(例如C2-53)，具有SEQ ID NO：11、12所述序列中的HCDR1-3和LCDR1-3的抗体(例如D3-68)均能阻断IL-2与IL-2Rβ结合。此处的“阻断IL-2与IL-2Rβ的结合”可以理解为在实施例3条件下获得有效的阻断IL-2与IL-2Rβ结合的IC 50值(例如0.1-9μg/mL，更例如1-9μg/mL)，或在实施例3条件下，具有小于0.300(例如小于0.2，更例如小于0.1)的Octet水平对IL-2Rβ的阻断数值。
IL-2或本公开的复合物均能够激活T细胞(例如，Treg和CD8+T细胞)。“选择性地增强了IL-2对Treg活性的作用”，是设定IL-2对Treg活性的作用为A，对CD8+T细胞活性的作用为B；本公开的复合物对Treg活性的作用为A’，对CD8+T细胞活性的作用为B’，则A’/B’的比值高于A/B的比值(任意程度上)。所述“活性”在同等检测条件下获得，包括但不限于待测物(例如IL-2或本公开的复合物)促细胞增殖的EC 50、促细胞STAT5磷酸化的EC 50(如实施例10的检测方法)。
本公开中，表述“对Treg的活性激活程度高于对CD8+T细胞的活性激活程度”和表述“对Treg的活性抑制程度低于对CD8+T细胞的活性抑制程度”的含义是等同的，是指相较于CD8+T细胞，本公开的抗IL-2抗体、其抗原结合片段或复合物对Treg的活性激活程度更高(任意程度上)。
本公开中，表述“相较于在CD8+T细胞中，在Treg细胞中对STAT5磷酸化的促进程度更高”和表述“相较于在CD8+T细胞中，在Treg细胞中对STAT5磷酸化的抑制程度更低”的含义是等同的，是指相较于在CD8+T细胞中，本公开的抗IL-2抗体、其抗原结合片段或复合物在Treg细胞中对STAT5磷酸化的促进程度更高(任意程度上)。所述STAT5磷酸化的检测方法参见实施例7。
本公开中，表述“相较于CD8+T细胞、CD4+T细胞或NK细胞的增殖，对Treg细胞的增殖促进程度更高”和表述“相较于CD8+T细胞、CD4+T细胞或NK细胞的增殖，对Treg细胞的增殖抑制程度更低”的含义是等同的，是指相较于对CD8+T细胞(或CD4+T细胞、或NK细胞)的增殖，本公开的抗IL-2抗体、其抗原结合片段或复合物对Treg细胞的增殖促进程度更高(任意程度上)。
“增加Treg细胞中FOXP3、CD25和Icos中的一种或多种的表达”涵盖本公开的复合物对Treg的FOXP3、CD25和Icos的一种或多种的表达在任意程度上的 增加；或较之CD8+T细胞、CD4+T细胞、或NK细胞，本公开的抗IL-2抗体、其抗原结合片段或复合物在任意程度更低地抑制FOXP3、CD25和Icos中一种或多种的表达。
多核苷酸
本公开提供经分离的多核苷酸，其编码前述抗IL-2抗体、其抗原结合片段，或编码前述复合物(例如融合蛋白)。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，其可以是真核细胞或原核细胞。
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IL-2抗体、其抗原结合片段或抗IL-2抗体、其复合物(例如融合蛋白)的方法，包括：
-在如前所述的宿主细胞中表达该抗体、其抗原结合片段或复合物(例如融合蛋白)，和
-自该宿主细胞中分离该抗体、其抗原结合片段或复合物(例如融合蛋白)。
可选地，本公开的制备方法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IL-2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)数据库得到，或者从免疫球蛋白杂志，2001ISBN012441351上获得。
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IL-2抗体、其抗原结合片段、或复合物(例如融合蛋白)，以及可药用的赋形剂、稀释或载体。在一些具体实施方式中，所述药物组合物单位剂量中可含有0.01至99重量％的抗IL-2抗体、其抗原结合片段或抗IL-2抗体、其复合物(例如融合蛋白)，或药物组合物单位剂量中含抗IL-2抗体、其抗原结合片段或抗IL-2抗体、复合物(例如融合蛋白)的量为0.1-2000mg，在一些具体实施方式中为1-1000mg。
方法和用途
本公开提供选自以下的任一项或组合在制备药物或药物组合物中的用途：前述抗IL-2抗体、其抗原结合片段，复合物(例如融合蛋白)，编码多核苷酸。
一些实施方案中，上述药物或药物组合物用于治疗或预防免疫相关病症(例如自身免疫疾病、炎性疾病)或延缓免疫相关病症进展。
一些实施方案中，提供治疗或预防自身免疫疾病或延缓自身免疫疾病进展的方法，该方法包括给予受试者治疗或延缓疾病有效量的根据本公开的抗IL-2抗体、其抗原结合片段，复合物(例如融合蛋白)或根据本公开的药物组合物。
一些实施方案中，上述自身免疫疾病、炎性疾病选自以下的任一项：炎性皮肤病，包括牛皮癣和皮炎(例如特应性皮炎)；皮肌炎；系统性硬皮病和硬化症；与炎性肠病相关的状况(例如克罗恩氏病和溃疡性结肠炎)；结肠炎；胃炎；呼吸窘迫综合征(包括成人呼吸窘迫综合征和ARDS)；皮炎；脑膜炎；脑炎；葡萄膜炎；肾小球肾炎；过敏性状况如湿疹和哮喘以及涉及T细胞浸润和慢性炎症应答的其它状况；动脉粥样硬化；白细胞粘附缺陷；类风湿性关节炎；系统性红斑狼疮(SLE)；糖尿病(例如I型糖尿病)；多发性硬化症；雷诺氏综合征；自身免疫性甲状腺炎；变态反应性脑脊髓炎；干燥综合征；青少年型糖尿病；以及通常在结核、肉瘤样病、多肌炎、肉芽肿病和血管炎中发现的由细胞因子和T淋巴细胞介导的与急性和迟发性超敏反应相关的免疫应答；韦格纳氏病；恶性贫血(阿狄森氏病)；涉及白细胞渗出的疾病；中枢神经系统(CNS)炎性病症；多器官损伤综合征；溶血性贫血(包括但不限于冷球蛋白血症或库姆氏阳性贫血)；重症肌无力；抗原-抗体复合物介导的疾病；抗肾小球基底膜病；抗磷脂综合征；过敏性神经炎；格雷夫斯病；朗-爱二氏肌无力综合征；类天疱疮；天疱疮；自身免疫性多内分泌腺疾病；白癜风；赖特氏病；僵人综合征；白塞病；巨细胞动脉炎；免疫复合物肾炎；IgA肾病；IgM多发性神经病；免疫性血小板减少性紫癜(ITP)或自身免疫性血小板减少症和自身免疫性溶血性疾病；桥本氏甲状腺炎；自身免疫性肝炎；自身免疫性血友病；自身免疫性淋巴细胞增生综合征(ALPS)；自身免疫性葡萄膜视网膜炎；格-巴二氏综合征；古德帕斯丘综合征；混合性结缔组织病；自身免疫相关性不孕症；结节性多动脉炎；斑秃；特发性粘液性水肿；移植物抗宿主病；和肌营养不良(杜兴氏肌营养不良、贝克型肌营养不良、强直性肌营养不良、肢带型肌营养不良、面肩肱型肌营养不良、先天性肌营养不良、眼咽型肌营养不良、远端型营养不良和埃德型营养不良)、妊娠的自发消失、特应性疾病或炎性肠病。
一些具体方案中，自身免疫疾病、炎性疾病选自：狼疮、移植物抗宿主病、丙型肝炎诱导的脉管炎、I型糖尿病、多发性硬化、特应性疾病或炎性肠病。
一些实施方案中，提供一种用于增加T细胞群体内Treg对非Treg的比率的方法，所述方法包括将T细胞群体与有效量的上述抗IL-2抗体、其抗原结合片段或复合物(例如融合蛋白)接触。在一个实施方案中，CD3 +FoxP3 +细胞对CD3 +FoxP3 -的比率增加。在另一个实施方案中，CD3 +FoxP3 +细胞对CD3+FoxP3-的比率增加至少50％。
一些实施方案中，提供一种用于增加受试者的外周血中Treg对非Treg的比率的方法，所述方法包括将T细胞群体与有效量的上述抗IL-2抗体、其抗原结合片段或复合物(例如融合蛋白)接触。在一个实施方案中，CD3 +FoxP3 +细胞对CD3 +FoxP3 -的比率增加。在另一个实施方案中，CD3 +FoxP3 +细胞对CD3+FoxP3-的比率增加至少50％。
一些实施方案中，提供一种用于选自以下任一项的方法：增加T细胞群体内的Treg对天然杀伤(NK)细胞的比率、或增加Treg数量、或提高Treg活性；所述方法包括施用有效量的上述抗IL-2抗体、其抗原结合片段或复合物(例如融合蛋白)。在一个实施方案中，CD3 +FoxP3 +细胞对表达CD56和/或CD16的CD3 -CD19 -淋巴细胞的比率增加。在另一个实施方案中，CD3 +FoxP3 +细胞对表达CD56和/或CD16的CD3 -CD19 -淋巴细胞的比率增加至少50％。
一些实施方案中，提供一种用于选自以下任一项的方法：增加受试者的外周血中Treg对天然杀伤(NK)细胞的比率、或增加Treg数量、或提高Treg活性，所述方法包括施用有效量的上述抗IL-2抗体、其抗原结合片段或复合物(例如融合蛋白)。在一个实施方案中，CD3 +FoxP3 +细胞对表达CD56和/或CD16的CD3 -CD19 -淋巴细胞的比率增加。在另一个实施方案中，CD3 +FoxP3 +细胞对表达CD56和/或CD16的CD3 -CD19 -淋巴细胞的比率增加至少50％。
一些实施方案中，提供监测受试者对上述抗IL-2抗体、其抗原结合片段或复合物(例如融合蛋白)的应答的方法，所述方法包括检测所述受试者中的变化，所述变化选自以下的任一项或组合：体温的升高、所述受试者的外周血中的CRP的增加、所述受试者的外周血中的血小板的减少、所述受试者的外周血中的嗜中性粒细胞的减少、或所述受试者的外周血中的白蛋白的减少。在检测到所述变化后，终止、暂停所述治疗、减少给药频率或减少给药量。在一个实施方案中，所述变化包括选自以下的任一项或组合：至少0.5℃的体温的升高、至少0.2mg/mL的所述受试者的外周血的CRP的增加、至少0.8倍的所述外周血中的血小板的减少、至少0.8倍的所述受试者的外周血中的嗜中性粒细胞的减少或至少0.4倍的受试者的外周血中的白蛋白的减少。
一些实施方案中，前述抗-IL-2抗体、其抗原结合片段或复合物(例如融合蛋白)能特异性结合IL-2，并且降低IL-2与IL-2Rα和/或IL-2Rβ的结合。这些抗体、抗原结合片段或复合物对非Treg细胞(包括效应CD8 +、非Treg CD4 +和NK细胞)的增殖抑制，超过对Treg细胞增殖的抑制；和/或与同种型对照抗体相比，增加Treg增殖；和/或增加Treg细胞/非Treg细胞的比率；或维持Treg标记物。
附图说明：
图1A和图1B分别为抗IL-2抗体阻断IL-2与IL-2Rα和IL-2Rβ结合的ELISA实验结果。
图2为通过ELISA实验检测抗IL-2抗体与IL-2的结合能力结果。
图3A至图3H为抗IL-2抗体与IL-2的非共价复合体刺激人外周血Treg和CD8 +T细胞STAT5磷酸化的实验结果。
图4A至图4D为融合蛋白刺激人外周血调节性T细胞Treg和CD8 +T细胞中STAT5磷酸化活性的实验结果。
图5A至图5F为融合蛋白对Balb/c小鼠体内外周血中淋巴细胞增殖影响的实验结果。
图6为融合蛋白对经过OVA免疫的小鼠脾脏中淋巴细胞增殖影响的实验结果。
图7为融合蛋白在小鼠延迟性过敏反应模型中的药效结果。
图8A至图8B为融合蛋白在小鼠关节炎模型中的药效结果。
图9A至图9F为抗IL-2抗体与不同IL-2变体形成的融合蛋白刺激人外周血调节性T细胞Treg和CD8 +T细胞中STAT5磷酸化活性的实验结果。
图10为D2-60-b-T3E对MRL/lpr小鼠尿中蛋白含量影响的实验结果(n＝7至10)。
图11A和图11B分别为D2-60-b-T3E对MRL/lpr小鼠血清中dsDNA IgG、IgM含量影响的实验结果(**P<0.01vs.模型对照组)。
图12A和图12B分别为D2-60-b-T3E对MRL/lpr小鼠肾功能影响的实验结果。*P<0.05，**P<0.01，****P<0.0001vs.模型对照组，n＝9至10。
图13A和图13B分别为D2-60-b-T3E对MRL/lpr小鼠血清IL-6、INF-γ含量影响的实验结果。*P<0.05，**P<0.01，****P<0.0001vs.模型对照组，n＝9至10。
图14为D2-60-b-T3E对MRL/lpr小鼠血清IL-10含量影响的实验结果。*P<0.05，**P<0.01，****P<0.0001vs.模型对照组，n＝9至10。
图15为D2-60-b-T3E对MRL/lpr小鼠病理评分的影响。*P<0.05，**P<0.01，****P<0.0001vs.模型对照组，n＝9至10。
具体实施方式
术语
为了更容易理解本公开，以下具体定义了某些技术和科学术语。除显而易见在本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术语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。
“白介素-2”或“IL-2”旨在被广义地解释，包括任意IL-2相关的产品。包括 但不限于人和非人的IL-2同系物、片段或截短体、融合蛋白(如与信号肽融合或其他活性、非活性成份融合，活性成份例如抗体或其抗原结合片段)、修饰形式(如PEG化、糖基化、白蛋白缀合/融合、Fc缀和/融合、羟乙基化等)、和保守修饰的蛋白等。该术语涵盖来自任何脊椎动物来源，包括哺乳动物如灵长类(例如人)和啮齿动物(例如小鼠和大鼠)的任何天然的IL-2。该术语还涵盖未加工的IL-2以及源自细胞中的加工的任何形式的IL-2，还涵盖天然存在的IL-2变体，例如剪接变体或等位变体，还涵盖IL-2的保守修饰变体。
“IL-2Rα”、“CD25”或“IL-2受体的α亚基”指来自任何脊椎动物来源，包括哺乳动物如灵长类(例如人)和啮齿动物(例如小鼠和大鼠)的任何天然CD25，包括“全长”的未加工的CD25以及源自细胞中的加工的任何形式的CD25，还包括天然存在的CD25变体，例如剪接变体或等位变体。在某些实施方案中，CD25是人CD25，例示性序列如SEQ ID NO.37所示。
“高亲合力IL-2受体”指IL-2受体的异型三聚体形式，其由受体γ亚基(也称为通用细胞因子受体γ亚基、γc或CD132)、受体β亚基(也称为CD122、p70或IL-2Rβ)和受体α亚基(也称为CD25、p55、IL-2Rα)组成。比较而言，“中等亲合力IL-2受体”指仅包含γ亚基和β亚基而无α亚基的IL-2受体(参见例如Olejniczak和Kasprzak,MedSci Monit14,RA179-189(2008))。
“保守修饰”适用于氨基酸和核苷酸序列。对于特定的核苷酸序列，保守改性是指编码相同或基本相同的氨基酸序列的那些核酸，或在核苷酸不编码氨基酸序列的情况下，是指基本上相同的核苷酸序列。对于氨基酸序列，“保守修饰”是指具有类似特征(例如电荷、侧链大小、疏水性/亲水性、主链构象和刚性等)的其它氨基酸置换蛋白中的氨基酸，使得可频繁进行改变而不改变蛋白的生物学活性。本领域技术人员知晓，一般而言，多肽的非必需区域中的单个氨基酸置换基本上不改变生物学活性(参见例如Watson等(1987)Molecμlar Biology of the Gene，The Benjamin/Cummings Pub.Co.，第224页，(第4版))。
“氨基酸突变”包括氨基酸取代、缺失、插入、修饰及其任意组合，以实现最终构建体，使得最终构建体拥有期望的特性，例如增强的稳定性、提高的活性。氨基酸序列缺失和插入包括氨基和/或羧基端缺失和氨基酸插入。优选的氨基酸突变是氨基酸取代。为了改变例如抗IL-2抗体或其抗原结合片段的结合特性，可以将非保守性的氨基酸取代，即将一个氨基酸用具有不同结构和/或化学特性的另一种氨基酸替换。优选的氨基酸取代包括用亲水性氨基酸替换疏水性氨基酸。氨基酸取代包括由非天然存在的氨基酸或由20种标准氨基酸的天然存在的氨基酸衍生物(例如4-羟脯氨酸、3-甲基组氨酸、鸟氨酸、高丝氨酸、5-羟赖氨酸)替换。可以使用本领域中公知的遗传或化学方法生成氨基酸突变，包括定点诱变、PCR、基因合成、化学修饰等方法。
“抗体”以最广义使用，涵盖各种抗体结构，包括但不限于单克隆抗体，多 克隆抗体；单特异性抗体，多特异性抗体(例如双特异性抗体)，全长抗体和抗体片段(或抗原结合片段，或抗原结合部分)，只要它们展现出期望的抗原结合活性。抗体可以指免疫球蛋白，是由两条重链和两条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-特异性也不同。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(CDR)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。
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，并鉴定参与抗原结合位点的残基。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de-novo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)。
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。对于包含超过一个CDR的任何给定实施例，可根据Kabat、Chothia、延伸的、AbM、IMGT、接触和/或构象定义中的任一个来定义CDR。
“单克隆抗体”或“单抗”指从基本上同质的抗体群体获得的抗体，即除了可能以少量存在的可能天然存在的突变之外，群体包含的各个抗体是相同的。单克隆抗体是高度特异性的，针对单个抗原位点。此外，与多克隆抗体不同，每种单克隆抗体针对抗原上的单个决定簇。修饰语“单克隆”指示如从基本上同质的抗体群体获得的抗体的特征，并且不被解释为需要通过任何特定方法产生抗体。例如，根据本公开使用的单克隆抗体可通过首先由Kohler和Milstein，1975，Nature256：495描述的杂交瘤方法来制备，或者可通过例如美国专利号4,816,567中所述的重组DNA方法来制备。例如，单克隆抗体也可从使用McCafferty等，1990，Nature348：552-554中描述的技术，从所生成的噬菌体文库中分离。
“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”)。
“人抗体”或“重组人抗体”包括通过重组方法制备、表达、创建或分离的人抗体，所涉及的技术和方法在本领域中是熟知的，诸如：
(1)从人免疫球蛋白基因的转基因、转染色体动物(例如小鼠)或由其制备的杂交瘤中分离的抗体；
(2)从经转化以表达抗体的宿主细胞如转染瘤中分离的抗体；
(3)从重组组合人抗体文库中分离的抗体；以及
(4)通过将人免疫球蛋白基因序列剪接到其他DNA序列等方法制备、表达、创建或分离的抗体。
此类重组人抗体包含可变区和恒定区，这些区域利用特定的由种系基因编码的人种系免疫球蛋白序列，但也包括随后诸如在抗体成熟过程中发生的重排和突变。
“抗原(Ag)”指用于免疫接种免疫活性的脊椎动物的分子，以产生识别Ag的抗体(Ab)；或在表达文库(例如尤其是噬菌体、酵母或核糖体展示文库)中进行筛选所使用的分子或模拟物。在本文中，Ag被更广义地定义，并且一般预期包括被Ab识别的靶分子。因此，Ag包括用于产生Ab的免疫接种过程或用于选择Ab的文库筛选中使用的分子、或其部分或模拟物。因此，对于本公开的与IL-2结合的抗体，来自哺乳动物物种的全长IL-2(例如人、猴、小鼠和大鼠IL-2)，包括其单体和多聚体，例如二聚体、三聚体等，以及IL-2的截短变体和其它变体均被称为抗原。
“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例如3至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。
“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)。
“与IL-2结合”，指能与IL-2或其表位相互作用，所述IL-2或其表位可以是人源的。
“抗原结合位点”指抗原上连续或不连续的，由本公开抗体或抗原结合片段识别的三维空间位点。
“调节性T细胞”或“Treg”意指一种能抑制其它T细胞的应答的特殊化CD4+T细胞类型。Treg的特征在于表达IL-2受体的α亚基(CD25)和转录因子叉头框P3(FOXP3)，并在诱导和维持对抗原(包括那些由肿瘤表达的抗原)的外周自体耐受性中起着关键作用。Treg需要IL-2来实现其功能和发育以及其抑制性特征的诱导。
“效应子功能”指那些可归于抗体Fc区(天然序列Fc区或氨基酸序列变体Fc区)且随抗体同种型而变化的生物学活性。抗体效应子功能的例子包括：C1q结合和补体依赖性细胞毒性(CDC)；Fc受体结合；抗体依赖性细胞介导的细胞毒性(ADCC)；吞噬作用；细胞表面受体(例如B细胞受体)下调；和B细胞活化。“ADCC”是指表达Fc受体的细胞通过识别抗体的Fc段直接杀伤被抗体包被的靶细胞。可通过对IgG上Fc段的修饰，降低或消除抗体的ADCC效应功能。所述的修饰指在抗体的重链恒定区进行突变，如选自IgG1的N297A、L234A、L235A、P329G；IgG2/4chimera，IgG4的F234A/L235A突变。“CDC”是指通过使补体成分C1q与抗体Fc结合来激活补体级联的细胞毒性形式。检测抗体的ADCC、CDC活性的方法是本领域已知的，例如可通过测定待测抗体与Fc受体(例如C1q)之间的结合活性来评价CDC。
“Fc受体”或“FcR”描述结合抗体Fc区的受体。优选的FcR是人FcR。此外，优选的FcR是结合IgG抗体的FcR(γ受体)，且包括FcγRI、FcγRII和FcγRIII亚类的受体，包括这些受体的等位基因变体和选择性剪接形式。FcγRII受体包括FcγRIIA(“激活受体”)和FcγRIIB(“抑制受体”)，它们具有主要在其胞质结构域中不同的相似氨基酸序列。激活受体FcγRIIA在其胞质结构域中包含基于免疫受体酪氨酸的激活基序(ITAM)。抑制受体FcγRIIB在其胞质结构域中包含基于免疫受体酪氨酸的抑制基序(ITIM)(参见综述M.Daeron,Annu.Rev.Immunol.15:203-234(1997))。FcR综述于Ravetch和Kinet,Annu.Rev.Immunol.9:457-92(1991)；Capel等,Immunomethods 4:25-34(1994)；及de Haas等,J.Lab.Clin.Med.126:330-41(1995)中。本文的术语“FcR”涵盖了其他FcR，包括有待在将来鉴定的那些。该术语还包括负责将母体IgG转移至胎儿的新生儿受体FcRn(Guyer等，J.Immunol.117:587(1976)和Kim等,J.Immunol.24:249(1994))。
“多核苷酸”或“核酸”指任何长度的核苷酸链，包括DNA和RNA。核苷酸可为脱氧核糖核苷酸、核糖核苷酸、经修饰的核苷酸或碱基和/或其类似物、或者可通过DNA或RNA聚合酶掺入链内的任何底物。
“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IL-2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(K D)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合力是其与预定抗原(或其表位)或紧密相关的抗原之外的非特异性抗原(如BSA等)结合的亲合力的至少两倍。术语“识别抗原的抗体”在本文中可以与术语“特异性结合的抗体”互换使用。
“亲合力(avidity)”在本文中用作两个分子(例如抗体或其抗原结合片段与抗原)之间的非共价相互作用的强度量度。术语“亲合力”用于描述单价相互作用(固有活性)。两个分子之间的结合亲合力可通过确定解离常数(KD)来量化。可通过使用例如表面等离子共振(SPR)方法(Biacore)测量复合物形成和解离的动力学来确定KD。对应于单价复合物的结合和解离的速率常数分别被称为结合速率常数ka(或kon)和解离速率常数kd(或koff)。K D通过方程K D＝kd/ka与ka和kd有关。解离常数的值可通过众所周知的方法直接确定，并且可通过方法例如Caceci等人(1984，Byte 9：340-362)中所述的那些甚至对于复杂混合物进行计算。例如，可使用双重过滤硝化纤维素滤器结合测定如Wong&Lohman(1993，Proc.Natl.Acad.Sci.USA 90：5428-5432)中公开的那种来确定K D。评估抗体针对靶抗原的结合能力的其它标准测定是本领域已知的，包括例如ELISA、蛋白质印迹、RIA和流式细胞术分析、以及本文其它地方例举的其它测定。抗体的结合动力学和结合亲合力也可通过本领域已知的标准测定，例如表面等离子共振(SPR)，例如通过使用Biacore TM系统或KinExA来评价。可通过比较各个抗体/抗原复合物的K D值来比较与不同分子相互作用相关的结合亲合力，例如，不同抗体对于给定 抗原的结合亲合力的比较。类似地，相互作用的特异性可通过确定和比较目的相互作用(例如抗体和抗原之间的特异性相互作用)的K D值与非目的相互作用(例如已知不结合IL-2的对照抗体)的K D值进行评价。在一些实施方案中，本公开的IL-2抗体或抗原结合片段能够与其靶结合的亲合力比它与另一种非IL-2分子结合的亲合力大至少2倍、10倍、50倍、100倍、200倍、500倍、1,000倍或10,000倍，这里不属于限制性定义。
抗IL-2抗体或其抗原结合片段与IL-2的“复合物”指包含本公开的至少一种抗IL-2抗体或其抗原结合片段的复合物，其特异性结合IL-2或表达IL-2的细胞。该复合物包含通过共价力、非共价力或任何其它力结合的抗IL-2抗体或其抗原结合片段和IL-2分子。在一些实施方案中，抗IL-2抗体或其抗原结合片段与IL-2形成融合蛋白。在另一些实施方案中，抗IL-2抗体或其抗原结合片段与IL-2通过非共价力结合。在一些实施方案中，即使在施用复合物后，抗IL-2抗体或其抗原结合片段和IL-2仍可作为复合物的形式维持。应理解，在其它变量中，抗IL-2抗体或其抗原结合片段和IL-2将基于它们之间的结合相互作用的不同的K D值而形成结合能力不同的复合物。
“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。抑制和阻断也旨在包括与抗IL-2抗体接触时，与未与抗IL-2抗体接触的IL-2相比，任何可测量的IL-2结合亲合力、促细胞增殖(例如T细胞)活性降低。
与参照抗体“竞争结合的抗体”指在竞争测定法中，将参照抗体对抗原的结合阻断50％或更多的抗体，或其对抗原的结合被参照抗体阻断50％或更多的抗体。可以通过已知方法如ELISA、蛋白印迹法等，测试本公开的抗IL-2抗体的抗原结合活性。例如，采用竞争测定法可用于鉴定与IL-2竞争结合的抗体。在某些实施方案中，所述竞争性抗体与抗原结合分子或抗IL-2抗体结合相同的表位(例如线性或构象表位)。例如，国际专利公开WO03/48731中描述的方法。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IL-2上的相同表位的抗体及其抗原结合片段。
“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IL-2抗体或其抗原结合片段或其与IL-2的复合物的组合物作为治疗剂，所述受试者已经患有、疑似患有、倾向于患有一种或多种免疫相关的疾病或其症状，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能测量的程度。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。尽管本公开的实施方案(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。
“有效量”包含足以改善或预防医学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。
“同源性”或“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。
“宿主细胞”包括各个细胞或细胞培养物，其可为或已是用于掺入多核苷酸插入片段的载体的受体。宿主细胞包括单个宿主细胞的子代，并且由于天然、偶然或有意的突变，子代可不一定与原始亲本细胞完全相同(在形态学或基因组DNA互补体中)。宿主细胞包括用本公开的多核苷酸在体内转染和/或转化的细胞。“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。
“载体”意指能够在宿主细胞中递送并且在一些实施例中表达一种或多种目的基因或序列的构建体。载体的例子包括但不限于病毒载体、裸露DNA或RNA表达载体、质粒、粘粒或噬菌体载体、与阳离子缩合剂结合的DNA或RNA表达载体、包封在脂质体中的DNA或RNA表达载体、以及某些真核细胞如生产细胞。
“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。
“药学可接受的载体”或“药学可接受的赋形剂”包括当与活性成分组合时，允许该成分保留生物学活性并且不与受试者的免疫系统反应的任何材料。例子包括但不限于任何标准药物载体，例如磷酸盐缓冲盐水溶液、水、乳剂如油/水乳剂、和各种类型的润湿剂。在一些实施例中，用于气雾剂或肠胃外施用的稀释剂是磷酸盐缓冲盐水(PBS)或生理(0.9％)盐水。包含此类载体的组合物通过众所周知的常规方法配制(参见例如Remington′s Pharmaceutical Sciences，第18版，A.Gennaro，编辑，Mack PublishingCo.，Easton，PA，1990；以及R Remington，The Science and Practice of Pharmacy第20版Mack Publishing，2000)。
本文中，当“约”应用于值或参数时，包括(且描述)涉及该值或参数本身的实施方案。例如，提及“约X”的描述包括“X”的描述。数字范围包括定义该范围的数字在内。尽管阐述本公开的广泛范围的数值范围和参数是近似值，但在具体实施例中阐述的数值尽可能精确地报道。然而，任何数值固有地包含必然来源于它们各自测试测量中发现的标准差的某些误差。此外，本文公开的所有范围都应理解为涵盖其中包含的任何和所有子范围。例如，“1至10”的所述范围应该视为包括最小值1和最大值10之间的任何和所有子范围(且包括端值在内)；即以最小值1或更大的值开始的所有子范围，例如1至6.1，并且以最大值10或更小的值结束的所有子范围，例如5.5至10。
实施例
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.抗IL-2抗体的筛选
A)人源天然噬菌体Fab库的筛选
将保存的天然库噬菌体悬液用BSA稀释封闭，与磁珠Dynabeads(M-280，invitrogen)共孵育，收集负筛孵育后的噬菌体。按照Kingfisher磁珠筛选系统方法(Thermo Scientific)对磁珠进行结合和清洗，将5％BSA与磁珠孵育。用生物素标记的带有人Fc标签的IL-2(Sanyou Biopharmaceutical，GenBank Accession No：P60568-1)包被封闭Dynabeads，将负筛后收集的噬菌体悬液与所述Dynabeads孵育，并按照Kingfisher磁珠筛选系统方法进行结合和清洗。用胰酶洗脱噬菌体。洗脱后噬菌体溶液与生长对数期的大肠杆菌SS320细胞(Sanyou Biopharmaceutical)充分混合后，37℃静置孵育30min。将大肠杆菌SS320涂布在2YT-Car +-Tet +平板上，37℃培养箱过夜培养。计算噬菌体输入、输出等，刮板制备输入的噬菌体并进行3轮筛选。
B)ELISA初筛能结合IL-2的表达了抗IL-2的Fab的单克隆噬菌体
挑选第3轮筛选获得的2000个克隆在96孔深孔板中培养过夜。离心取上清。将抗人IgG(STAR161，Bio-rad)用PBS稀释到2μg/mL，包被ELISA板，每孔 加入30μL，4℃过夜。PBST(PBS，pH 7.4+0.1％Tween 20)洗板3次。1％BSA室温封闭1小时，PBST洗板3次。加入离心后的上清30μL，室温2小时，PBST洗板3次。将用生物素标记的带有人Fc标签的IL-2用PBS稀释到2μg/mL每孔加入30μL，室温1小时，PBST洗板3次。加入1:8000稀释的二抗NeutrAvidin-HRP30μL，室温1小时，PBST洗板9次加入30μL TMB室温显色5-10分钟，之后加入30μL 2M HCl或者H 2SO 4终止反应，酶标仪OD 450读取数据。最终获得364株具有特异序列的克隆。
C)ELISA初筛能够阻断IL-2与IL-2Rβ结合的抗IL-2的Fab
将上述364株克隆按照1:1000的比例接种到50mL体积的2YT-Car +-Tet +培养基中，37℃培养过夜。将菌液转移至50mL离心管。常温条件下离心处理，弃掉上清，向其中加入4℃预冷的蛋白裂解液(10mM Tris-HCl pH 9.0，1mM EDTA，5mM MgCl 2，25U/mL Bezonase核酸酶(Merck))0.5mL，冰上静置孵育1小时。4℃条件下离心处理收集上清备用。
将IL-2Rβ-Fc(CJ82，Novoprotein)(GenBank登录No：NP_000869.1)用PBS稀释到8μg/mL，包被ELISA板，每孔加入30μL，4℃过夜。PBST洗板3次。1％BSA室温封闭1小时，PBST洗板3次。将获得的Fab裂解液与用生物素标记的带有人Fc标签的IL-2(2.5μg/mL)室温温育1小时后，每孔加入30μL，室温1小时，PBST洗板3次。加入1:8000稀释的二抗NeutrAvidin-HRP 30μL，室温1小时，PBST洗板9次加入30μL TMB室温显色5-10分钟，之后加入30μL 2M HCl或者H 2SO 4终止反应，酶标仪OD 450读取数据。
获得具有较强的阻断IL-2与IL-2Rβ结合能力的6个Fab。这6个全人源抗体的重链和轻链可变区序列如表1所示，不同编号系统的互补决定区(CDR)如表2所示。
表1.全人源抗IL-2抗体重链(HCVR)和轻链(LCVR)可变区序列
Figure PCTCN2021076806-appb-000001
Figure PCTCN2021076806-appb-000002
表2.全人源抗IL-2抗体的重链和轻链互补决定区(CDR)序列
Figure PCTCN2021076806-appb-000003
Figure PCTCN2021076806-appb-000004
Figure PCTCN2021076806-appb-000005
实施例2:全长全人源抗IL-2抗体的制备
将实施例1中6个抗体的重链和轻链可变区与人IgG1重链恒定区和kappa或lamda链恒定区连接，构造形成全长全人源抗IL-2抗体。其中重链恒定区带有L234A和L235A突变(LALA突变)，用以去除抗体可能的ADCC功能。6个抗体的全长序列如表3所示。
表3.全人源抗IL-2抗体的重轻链全长序列
Figure PCTCN2021076806-appb-000006
Figure PCTCN2021076806-appb-000007
Figure PCTCN2021076806-appb-000008
Figure PCTCN2021076806-appb-000009
(注：HC的下划线部分为人IgG1重链恒定区，LC的下划线部分为kappa链恒定区)
合成上述序列，用BamHI和XhoI消化后，通过BamHI/XhoI酶切位点插入到pcDNA3.1表达载体(Life Technologies Cat.No.V790-20)中。将表达载体和转染试剂PEI(Polysciences，Inc.Cat.No.23966)以1:2的比例转染HEK293细胞(Life Technologies Cat.No.11625019)，并置于CO 2孵育箱中孵育4-5天。表达的抗体通过离心回收后，按常规方法进行抗体纯化，经鉴定，得到本公开的全长全人源抗体。
实施例3:抗IL-2抗体能阻断或降低IL-2与IL-2Rα结合，能阻断IL-2和IL-2Rβ结合的ELISA实验
A)抗体阻断或降低IL-2与IL-2Rα结合的ELISA实验
将IL-2Rα-Fc(CJ78，Novoprotein)(Accession#NP_000408)用PBS稀释到0.5μg/mL，包被ELISA板，每孔加入30μL，4℃过夜。PBST洗板3次。1％BSA室温封闭1小时，PBST洗板3次。将抗IL-2抗体用PBS，pH7.4做浓度梯度稀释至10μg/mL，5μg/mL，2.5μg/mL，1.25μg/mL，0.63μg/mL，0.31μg/mL，0.16μg/mL，0.08μg/mL，分别与用生物素标记的带有人Fc标签的IL-2(终浓度1μg/mL)室温温育15分钟后，每孔加入30μL，室温1小时，PBST洗板3次。加入1:6000稀释的二抗NeutrAvidin-HRP30μL，室温1小时，PBST洗板9次。加入30μL TMB室温显色5-10分钟，之后加入30μL 2M HCl或者H 2SO 4终止反应，酶标仪OD 450读取数据。
结果如图1A所示。结果显示，C2-53、D3-68和A3-21可以完全阻断IL-2与IL-2Rα的结合，其余克隆在10μg/mL高浓度下可能仅有微弱的阻断(即降低)IL-2与IL-2Rα结合的能力。各个抗体的IC 50如表4。
表4.抗IL-2抗体阻断IL-2与IL-2Rα的结合的IC 50
抗体编号 IC 50(μg/mL)
A2-22 N.A.
A3-21 1.456
B2-15 N.A.
C2-53 0.6591
D2-60 N.A.
D3-68 1.196
(注：N.A.：由于阻断效果较弱，无法拟合曲线获得有意义的IC50。)
B)抗体阻断IL-2与IL-2Rβ结合的ELISA实验
将IL-2Rβ-Fc(CJ82，Novoprotein)用PBS稀释到8μg/mL,包被ELISA板，每孔加入30μL，4℃过夜。PBST洗板3次。1％BSA室温封闭1小时，PBST洗板3次。将抗IL-2抗体用PBS，pH7.4做浓度梯度稀释至10μg/mL，5μg/mL，2.5μg/mL，1.25μg/mL，0.63μg/mL，0.31μg/mL，0.16μg/mL，0.08μg/mL，分别与用生物素标记的带有人Fc标签的IL-2(终浓度5μg/mL)室温温育15分钟后，每孔加入30μL，室温1小时，PBST洗板3次。加入1:6000稀释的二抗NeutrAvidin-HRP 30μL，室温1小时，PBST洗板9次。加入30μL TMB室温显色5-10分钟，之后加入30μL 2M HCl或者H 2SO 4终止反应，酶标仪OD 450读取数据。
结果如图1B所示。其中，所有抗体都可以完全阻断IL-2与IL-2Rβ的结合。各个抗体的IC 50如表5。
表5.抗IL-2抗体阻断IL-2与IL-2Rβ的结合的IC 50
抗体编号 IC 50(μg/mL)
A2-22 2.728
A3-21 3.475
B2-15 3.045
C2-53 1.441
D2-60 8.272
D3-68 2.134
实施例4:抗IL-2抗体阻断或降低IL-2与IL-2Rα的结合，阻断IL-2和IL-2Rβ结合的Octet实验
A)抗体阻断或降低IL-2与IL-2Rα结合的Octet实验
将Streptavidin生物传感器(Fortebio，#18-5020)浸泡在200μL的KB缓冲液(PBS，pH 7.4，0.05％tween-20，0.1％BSA)中60秒，进行湿润处理。然后，用KB缓冲液将生物素化的IL-2Rα-Fc(CJ78，Novoprotein)稀释到10μg/mL，将传感器置于200μL该溶液中150秒。将传感器浸泡于KB缓冲液中60秒，以洗脱多余的IL-2Rα。将IL-2-His(CX66，Novoprotein)与抗IL-2抗体混合，并以KB缓冲液稀释至终浓度分别为100nM和500nM，室温温育30分钟。将传感器置于该混合溶液中结合300秒。抗IL-2抗体阻断或降低IL-2与IL-2Rα结合的能力越强，则读数越低。读取开始结合230秒时的数值，如表6所示。
表6.抗IL-2抗体在Octet实验中阻断或降低IL-2与IL-2Rα的结合
抗体编号 Octet水平对IL-2Rα阻断数值
C2-53 0.410
A3-21 0.428
D3-68 0.473
B2-15 0.700
D2-60 0.754
A2-22 0.848
B)抗体阻断IL-2与IL-2Rβ结合的Octet实验
将Streptavidin生物传感器(Fortebio，#18-5020)浸泡在200μL的KB缓冲液(PBS，pH 7.4，0.05％tween-20，0.1％BSA)中60秒，进行湿润处理。然后，用KB缓冲液将生物素化的IL-2-Fc(Sanyou Biopharmaceutical)稀释到10μg/mL，将传感器置于200μL该溶液中300秒。将传感器浸泡于KB缓冲液中60秒，以洗脱多余的IL-2。将抗IL-2抗体以KB缓冲液稀释至终浓度500nM，将传感器置于抗体溶液中结合300秒。将传感器浸泡于KB缓冲液中60秒，以洗脱多余的抗IL-2抗体。将IL-2Rβ-Fc(CJ82，Novoprotein)以KB缓冲液稀释至终浓度8000nM，将传感器置于抗体溶液中结合300秒。抗IL-2抗体阻断IL-2与IL-2Rβ结合的能力越强，则读数越低。读取在IL-2Rβ-Fc开始结合230秒时的数值，如表7所示。
表7.抗IL-2抗体在Octet实验中阻断IL-2与IL-2Rβ的结合
抗体编号 Octet水平对IL-2Rβ阻断数值
A3-21 0.003
C2-53 0.017
D3-68 0.027
A2-22 0.042
B2-15 0.056
D2-60 0.086
实施例5:抗IL-2抗体与IL-2的结合ELISA实验
将抗人-IgG(STAR161，Bio-rad)用PBS稀释到2μg/mL，包被ELISA板，每孔加入30μL，4℃过夜。PBST洗板3次。1％BSA室温封闭1小时，PBST洗板3次。将抗IL-2抗体用PBS梯度稀释到20μg/mL，10μg/mL，5μg/mL，2.5μg/mL，1.25μg/mL，0.625μg/mL，0.3125μg/mL，0.15625μg/mL，加30μL抗体到ELISA板中，室温1小时，PBST洗板3次。将用生物素标记的带有人Fc标签的IL-2(Sanyou Biopharmaceutical)用PBS稀释到2μg/mL每孔加入30μL，室温1小时，PBST洗板3次。加入1:6000稀释的二抗NeutrAvidin-HRP 30μL，室温1小时，PBST洗板9次加入30μL TMB室温显色5-10分钟，之后加入30μL 2M HCl或者H 2SO 4终止反应，酶标仪OD 450读取数据。结果如图2和表8所示。结果显示，在该实验方法下，所有抗体均与IL-2有结合，其中C2-53结合较强。
表8.抗IL-2抗体与IL-2结合的EC 50
抗体编号 EC 50(μg/mL)
A2-22 1.289
A3-21 1.350
B2-15 2.117
C2-53 0.7301
D2-60 1.428
D3-68 1.205
实施例6：抗IL-2抗体与IL-2的亲合力和动力学Biacore实验
用Biacore仪器(Biacore T200，GE)的Protein A传感芯片(GE，Cat#29127556)捕获抗体，其中抗IL-2抗体用1×HBS-EP稀释至1μg/mL，以10μL/min的流速持续30秒。然后，在芯片表面以30μL/min的流速流经一系列浓度梯度的IL-2(C013，Novoprotein)，结合持续120秒。解离流速30μL/min，持续300秒，实时检测反应信号，获得结合和解离曲线。在每个循环解离完成后，用10mM Gly-HCl pH 2.0将芯片洗净再生。实验得到的数据以1:1结合模型进行拟合，得出抗IL-2抗体与IL-2的结合力数值，见表9。结果显示，所有抗体均与IL-2有结合，亲合力跨度从C2-53的0.267nM到A2-22的39.8nM不等。
表9.抗IL-2抗体与IL-2的亲合力
抗体编号 Kon(1/Ms) Koff(1/s) KD(M)
C2-53 1.64E+07 4.37E-03 2.67E-10
D3-68 1.16E+07 5.46E-02 4.72E-09
D2-60 5.09E+05 6.51E-03 1.28E-08
B2-15 1.46E+06 4.48E-02 3.06E-08
A2-22 1.33E+06 5.27E-02 3.98E-08
实施例7：抗IL-2抗体与IL-2的非共价复合体对人外周血PBMC中STAT5磷酸化活性的测定
依据在不同浓度的抗IL-2抗体与IL-2非共价复合体处理下，人外周血中各细胞群体(包括Treg，CD4 +T细胞，CD8 +T细胞)中STAT5磷酸化水平，检测抗体对IL-2活性的调节能力。
基础培养基：RPMI 1640+10％胎牛血清。
抗体混合物：CD3 APC-Cy7(BD 557832)，CD4 BB515(BD 564419)，CD8 BB700(BD 566452)，CD25 BV421(BD 564033)，pSTAT5 AF647(BD 562076)。
人PBMC STAT5磷酸化实验：将新鲜分离的人PBMC细胞用基础培养基调整至6.5×10 6个细胞/mL的密度，取80μL置于96孔板中。将不同浓度的抗IL-2抗体与不同浓度的IL-2室温预混30分钟，取20μL加入90μL PBMC中，37℃刺激20分钟。之后立即用预热的BD Cytofix缓冲液(BD，Cat No.554655)在37℃固定细胞15分钟，然后在冰上继续固定15分钟。400g 4℃离心7分钟。去除上清液，用150μL PBS洗涤一次。加入150μL在-20℃预冷的BD Phosflow Perm Buffer III(BD，Cat No.558050)-20℃破膜过夜。500g 4℃离心7分钟。去除上清液，加入150μL PBS，pH 7.4洗涤两次。加入100μL按1:200稀释的FcR封闭剂，在4℃下温育20分钟。500g 4℃离心7分钟。去除上清液，加入50μL抗体混合物，对细胞在4℃染色1小时。500g 4℃离心7分钟。去除上清液，加入200μL PBS，pH 7.4洗涤一次，用150μL PBS，pH 7.4重悬细胞，用流式细胞仪(BD FACSCelesta)检测。CD8 +T细胞定义为 CD3 +CD4 -CD8 +的细胞，Treg定义为CD3 +CD4 +CD8 +CD25 +的细胞。
对上述两个细胞群的pSTAT5荧光数值(MFI)进行统计，计算每个浓度下的数值与完全激活状态下的最大pSTAT5 MFI数值的百分比，采用计算机程序或四参数回归计算法进行拟合。结果如图3A至图3H所示。
结果表明，随着抗体浓度增加，IgG1同种型并不会对IL-2激活Treg和CD8 +T细胞的pSTAT5产生任何影响。相反，A3-21，C2-53在很大的浓度范围内，基本不影响IL-2对Treg的活性，却在很大程度上降低了IL-2对CD8 +T细胞的活性。只有在高浓度下，比如100nM A3-21和33.3nM C2-53，IL-2对Treg的活性有一定下降，而在该条件下，IL-2对CD8 +T细胞完全没有活性。也即，A3-21和C2-53可以在不影响或较少的降低IL-2对Treg的活性的同时，抑制或更多的降低IL-2对CD8 +T细胞等免疫效应细胞的活性。类似的，D3-68也可以相对于Treg更多的降低IL-2对CD8 +T细胞的活性，虽然这种能力可能不如A3-21和C2-53显著。
实施例8：融合蛋白的制备
抗IL-2抗体与IL-2的非共价复合体体内可能容易解离，生成游离的IL-2，造成毒性。我们通过(G 4S) 5连接臂(即，GGGGSGGGGSGGGGSGGGGSGGGGS)将人IL-2偶联至抗IL-2抗体重链或轻链的N端，得到12个抗IL-2抗体与IL-2的融合蛋白。
对于抗体编号，若IL-2在抗体重链的N端，则该融合蛋白带有后缀a；若IL-2在抗体轻链的N端，则该融合蛋白带有后缀b。
对于所融合的IL-2，其带有T3A(即第3位氨基酸由Thr突变为Ala)的突变，以去除可能的糖基化。人IL-2成熟蛋白不含有第1位的氨基酸M，因此编号从第2位的氨基酸A开始计数。12个融合蛋白氨基酸序列如下：
表10.融合蛋白的全长序列
Figure PCTCN2021076806-appb-000010
Figure PCTCN2021076806-appb-000011
Figure PCTCN2021076806-appb-000012
Figure PCTCN2021076806-appb-000013
Figure PCTCN2021076806-appb-000014
Figure PCTCN2021076806-appb-000015
依照实施例2中的方法对上述12个融合蛋白进行表达和纯化，表达量和在SEC-HPLC中的纯度如下表11。
表11.融合蛋白的表达量和纯度
抗体编号 表达量(mg/L) 纯度(％)
A2-22-a 39 90.780
A2-22-b 104 96.308
A3-21-a 82 89.358
A3-21-b 2 82.095
B2-15-a 89 53.821
B2-15-b 70 99.839
C2-53-a N.A. 54.940
C2-53-b 6 26.598
D2-60-a 2.4 40.248
D2-60-b 129 97.540
D3-68-a 0.3 N.A.
D3-68-b 106 100
结果显示，对于不同抗IL-2抗体，IL-2偶联在重链或轻链的N端，会极大的影响融合蛋白的表达量和纯度。表达量或纯度低，提示在融合蛋白中IL-2与抗体可能无法形成正常的分子内结合，而是形成分子间结合，从而引起蛋白质的聚集。挑选表达量(至少20mg/L)和纯度(至少90％)较高的融合蛋白进行后续鉴定。
实施例9：融合蛋白与IL-2Rα和IL-2Rβ结合的Octet实验
将Protein A生物传感器(Fortebio，#18-5010)浸泡在200μL的KB缓冲液(PBS，pH 7.4，0.02％tween-20，0.1％BSA)中60秒，进行湿润处理。然后，用KB缓冲液将融合蛋白稀释到10μg/mL，将传感器置于200μL该溶液中，待读数为1.2nm时停止。将传感器浸泡于KB缓冲液中100秒，以洗脱多余的抗体-IL-2融合蛋白。将IL-2Rα(ILA-H52H9，Acrobiosystem)用KB缓冲液以2倍梯度稀释至100nM-3.125nM之间。将传感器置于该溶液中结合60秒。将传感器置于KB缓冲液中解离60秒。采用动态1：1结合方式拟合，则融合蛋白与IL-2Rα的亲合力如表12所示。结果显示，与IL-2相比，偶联了抗IL-2抗体的IL-2与IL-2Rα的结合均有减弱，其中A3-21-a， D3-68-b完全不结合IL-2Rα。
表12.融合蛋白与IL-2Rα的亲合力
待测物 Kon(1/Ms) Koff(1/s) KD(M)
IL-2 1.40E+06 2.51E-02 1.80E-08
A2-22-a 9.22E+05 3.75E-02 4.06E-08
A2-22-b 1.63E+06 4.13E-02 2.53E-08
A3-21-a N.A. N.A. 不结合
B2-15-b 1.23E+06 3.30E-02 2.68E-08
D2-60-b 1.24E+06 5.61E-02 4.51E-08
D3-68-b N.A. N.A. 不结合
(注：N.A.：不结合，所以无法拟合得到数值。其中IL-2与IL-2Rα的亲合力实验，IL-2Rα作为固定相，IL-2作为流动相)。
与上述方法类似，用Octet采用稳态模式拟合数据，测得抗体-IL-2融合蛋白与IL-2Rβ(CD2-H5221，Acrobiosystem)的亲合力如表13所示。其中，抗体融合蛋白作为固定相，IL-2Rβ作为流动相。结果显示，与IL-2相比，偶联了抗IL-2抗体的IL-2绝大多数完全不结合，个别抗体-IL2融合蛋白，如D3-68-b与IL-2Rβ的结合减弱，但仍然有一定程度的结合。其中IL-2与IL-2Rβ的亲合力实验，IL-2Rβ作为固定相，IL-2作为流动相。
表13.抗IL-2抗体与IL-2融合蛋白与IL-2Rβ的亲合力
待测物 KD(nM)
IL-2 4.6E-07
A2-22-a 不结合
A2-22-b 不结合
A3-21-a 不结合
B2-15-b 不结合
D2-60-b 不结合
D3-68-b 1.1E-06
实施例10：融合蛋白对人外周血(PBMC)STAT5磷酸化活性的测定
依照实施例7中的方法，测定融合蛋白对人外周血(PBMC)中Treg和CD8 +T细胞中的STAT5磷酸化活性。如图4A至图4D所示，实验结果显示，相比IL-2，所有融合蛋白对Treg和CD8 +T细胞的活性均有所降低。但是对于A2-22-a，A2-22-b，B2-15-b，D2-60-b，A3-21-b，这些融合蛋白对CD8+T细胞的活性降低相比对Treg的活性降低更多，提示了这些融合蛋白中的IL-2可以更偏向于激活Treg。
而对于D3-68-b，其对Treg的活性降低相比对CD8 +T细胞的活性降低更多(图4C和图4D所示)。融合蛋白激活Treg和CD8 +T细胞中pSTAT5的EC50如表14和表15所示。
表14.融合蛋白激活Treg细胞中pSTAT5的EC 50
待测物 EC 50(nM)
IL-2 0.0003714
A2-22-a 0.04439
A2-22-b 0.05677
B2-15-b 0.01072
D2-60-b 0.1940
D3-68-b 0.1731
A3-21-a 0.1923
表15.融合蛋白激活CD8 +T细胞中pSTAT5的EC 50
待测物 EC 50(nM)
IL-2 0.4343
A2-22-a N.A.
A2-22-b N.A.
B2-15-b N.A.
D2-60-b N.A.
D3-68-b 31.06
A3-21-a N.A.
(注：N.A.：在最高测试浓度下数值依然低，无法拟合获得准确EC 50)。
实施例11：融合蛋白对Balb/c小鼠外周血免疫细胞影响的测定
BALB/c小鼠(购自北京Charles River实验动物技术有限公司)，雌性，6-8周龄，体重18-20g，在正式实验前，适应性饲养5天。所有的BALB/c小鼠饲养于SPF级动物房IVC恒温恒压系统中，其中温度20至26℃，湿度40至70％，光照周期12小时明/12小时暗。每个笼盒内饲养不多于6只BALB/c小鼠。小鼠按照体重进行分组，分组后开始给药，给药种类、给药剂量和给药途径见表16。模型分组当天为第0天。
表16.小鼠给药方案
Figure PCTCN2021076806-appb-000016
每个时间点新鲜采集的抗凝血用红细胞裂解液裂解红细胞，PBS洗一次。用含有1％FBS的PBS配制混合染色液。混合染色液包括CD3 APC-Cy7(Biolegend 100329)、CD8 PE(Biolegend 100708)、CD4 PE-Cy7(eBioscience 25-0042-82)、CD25 PerCP-Cy5.5(BD 561112)。每个样本加入100μL混合染色液，4度温育30分钟。用含有1％FBS的PBS洗两次。用True-Nuclear TM Transcription Factor Buffer  Set(Biolegend 424401)进行固定破膜60分钟，100μL抗小鼠Foxp3抗体(Biolegend 126405)和抗小鼠Ki67抗体(eBioscience，25-5698-82)室温孵育60分钟。用PBS(pH 7.4)洗两次，最后用500μLPBS，pH 7.4洗液重悬，上机分析。其中，CD8 +T细胞定义为CD3 +CD4 -CD8 +的细胞，Treg定义为CD3 +CD4 +CD25 +Foxp3 +的细胞。
实验结果如图5A至图5F所示。对于A2-22-b，B2-15-b，D2-60-b，与第0天相比，小鼠全血中Treg占CD4 +T细胞的百分比从第2天开始上升，Treg中Ki67+的百分比也从第2天开始显著上升，表明A2-22-b，B2-15-b，D2-60-b可以强烈刺激Treg的增殖。相反，对于CD4 +CD25 -T细胞和CD8+T细胞，Ki67+的百分比则几乎没有变化或变化较小，CD8 +T细胞占CD3 +T细胞的百分比也几乎未变，说明A2-22-b，B2-15-b，D2-60-b几乎不活化CD8 +T细胞。
实施例12：融合蛋白对接受鸡卵清蛋白OVA免疫的Balb/c小鼠的脾脏免疫细胞的影响的测定
鸡卵清蛋白(OVA)，Sigma A5503，使用PBS溶解，配制成0.5mg/mL。弗氏完全佐剂(CFA，含灭活结核分枝杆菌1mg/mL)，Sigma，F5881。雄性C57B16/J小鼠(购自上海实验动物中心)，6-8周龄，体重18-20g。小鼠按照体重进行分组，实验第1天，取OVA溶液8mL，与8mL CFA混匀、乳化呈油包水状态。每只动物腹腔注射200μL进行免疫。分别于第3天和第8天，各组小鼠皮下注射(sc)待测融合蛋白和PBS对照10mL/kg。给药种类、给药剂量和给药途径见表17。
表17.小鼠免疫方案
Figure PCTCN2021076806-appb-000017
第10天时，小鼠安乐死，取脾脏处理后流式细胞仪检测相关免疫细胞。方法包括：75％乙醇浸泡5分钟后在无菌条件下取出小鼠脾脏，PBS漂洗1-2次后，将小鼠脾脏剪碎。将过滤网置于50mL离心管中，将脾脏组织碎片转移到筛网上，研磨，过程中不断加入新鲜的PBS。1800转/分钟离心3分钟，弃上清。加入10mL红细胞裂解液冰上静置5分钟后加入PBS终止裂解，1800转/分钟离心3分钟两次。根据BioLegend流式抗体染色步骤进行染色后，将细胞用PBS重悬离心两次，过筛，进行流式细胞检测。
检测细胞及其标记物如下：
脾脏生殖中心B细胞(GCB，Germinal Center B cells)(Fas+GL-7+B220+)；
卵泡辅助性T细胞(Tfh，follicular T helper cells)(PD1高，CXCR5高，FOXP3-CD4+)；
卵泡调节性T细胞(Tfr，follicular regulatory T cells)(PD1高，CXCR5高，FOXP3+CD4+)；
调节性T细胞Treg细胞(FOXP3+CD25+CD4+CXCR5低)；
实验结果如图6所示。A2-22-b，B2-15-b，D2-60-b均可以刺激脾脏中的Treg和Tfr增殖，抑制脾脏中的Tfh和GCB数量，表明A2-22-b，B2-15-b，D2-60-b具有通过激活Treg抑制免疫系统的作用。
实施例13：融合蛋白在小鼠延迟性过敏反应模型中的药效实验
实验方法：取2，4二硝基氟苯(DNFB，Sigma，42085-50G)，用丙酮：橄榄油＝4：1溶解，配制成1％(1g/100mL)溶液备用，另稀释成0.5％(0.5g/100mL)溶液备用。雄性ICR小鼠(购自上海实验动物中心)，6周周龄，体重18-20g。小鼠按照体重进行分组。实验第0天，小鼠腹部涂抹1％的DNFB溶液50μL免疫，实验第5天时，小鼠右耳内外侧分别涂抹0.5％的DNFB溶液10μL(共20μL)进行激发，分别于实验第6天使用8mm直径打孔器取下左侧和右侧耳朵组织称重，并计算左、右两侧耳朵组织重量的差值。待测药从实验开始前2天(第-2天)开始，每5天皮下给药1次。
给药种类、给药剂量和给药途径见表18。
表18.小鼠免疫方案
Figure PCTCN2021076806-appb-000018
实验结果如图7所示。A2-22-b，B2-15-b，D2-60-b均可以降低耳朵肿胀程度，并呈现剂量效应，表明A2-22-b，B2-15-b，D2-60-b具有抑制免疫系统的作用。
实施例14：融合蛋白在小鼠关节炎模型中的药效实验
雄性DBA/1纯系小鼠购自上海斯莱克实验动物有限公司。关节炎模型由牛II型胶原(Biolead，20022)免疫诱发。实验第0天，取2.5mL牛II胶原/冰乙酸(2mg/mL)中，再与2.5mL完全弗氏佐剂充分混悬，成油包水乳状，取0.1毫升在乙醚轻麻醉下于每个小鼠的尾根部皮内注射，3周后加强注射一次。造模动物随机分为6组，分别为：正常对照组，造模型组，IL-2(C013，novoprotein)组、A2-22-b组、B2-15-b组，每组各8只。同时选用正常对照动物作为正常对照组，分组见表19。
表19.小鼠免疫方案
Figure PCTCN2021076806-appb-000019
(注：Q5D，每五天一次；Q2D，每两天一次；i.p.，腹腔给药；s.c.，皮下给药。)
关节炎症评价：小鼠从二次免疫开始，每3天进行称重和关节炎病情严重程度半定量计分(clinical scores)：
0分：无红肿；
1分：在爪上有一处红肿炎症；
2分：整爪上有轻微炎症或者有两处及以上红肿；
3分：整爪上有中度红肿；
4分：整个爪上有严重红肿甚至关节僵硬、畸形及活动障碍。
每只爪最高得分4分，每只鼠最高得分16分。关节炎病情观察一直持续到实验结束。
血清抗牛II型胶原抗体浓度检测：实验结束时，收集血，分离血清，-80℃保存。采用ELISA检测试剂盒(Chondrex，20322T)检测血清中抗牛II型胶原抗体的水平。具体步骤按说明书进行。
实验结果如图8A和图8B所示。和正常对照组相比，模型对照组小鼠体重明显下降，关节炎评分显著升高(p<0.001)；和模型对照组相比，B2-15-b可显著抑制关节炎造模小鼠体重减轻，可显著降低关节炎评分(p<0.05)。和正常对照组相比，模型对照组血清抗牛II型胶原IgG2a抗体的浓度显著增加(p<0.001)；和模型对照组相比，B2-15-b给药可显著下调关节炎小鼠血清抗牛II型胶原IgG2a抗体的分泌(p<0.01)。
实施例15：不同连接臂长度对融合蛋白表达量和纯度的影响
将B2-15-b中IL-2与抗IL-2抗体B2-15轻链之间的(G 4S) 5连接臂缩短G4S的重复次数至4次，而重链保持不变，获得B2-15-b的变体B2-15-b-(G 4S) 4，其轻链序列如表20。
表20.融合蛋白中抗体的全长轻链序列
Figure PCTCN2021076806-appb-000020
按照实施例2中的方法对上述B2-15-b的变体进行表达和纯化，仍可以达到表达量47mg/L，SEC-HPLC纯度为99％。证明不同连接臂长度能实现融合蛋白的表达。
实施例16：抗IL-2抗体与不同IL-2变体之间的融合蛋白对人外周血(PBMC)STAT5磷酸化活性的测定
将IL-2进行突变，引入T3E，或T3Q，或去除IL-2N端前3个氨基酸(B2-15-b-Del3)，或去除IL-2N端前7个氨基酸(B2-15-b-Del7)，或直接使用野生型IL-2，分别与抗IL-2抗体进行偶联。产生如下融合蛋白(每一个抗体的重链序列保持不变)，见表21。
表21.融合蛋白中抗体的轻链全长序列
Figure PCTCN2021076806-appb-000021
Figure PCTCN2021076806-appb-000022
Figure PCTCN2021076806-appb-000023
依照实施例7中的方法，测定上述抗IL-2抗体与IL-2变体的融合蛋白对人外周血(PBMC)中Treg和CD8 +T细胞中的STAT5磷酸化活性。如图9A至图9F、 表22所示，实验结果显示，这些IL-2变体基本不影响抗体融合蛋白对Treg和CD8+T细胞的活性。这些融合蛋白依然更偏向于激活Treg，而不是CD8 +T细胞。
表22.抗IL-2抗体与不同IL-2变体的融合蛋白激活Treg细胞中pSTAT5的EC 50
融合蛋白编号 EC 50(nM)
A2-22-b 0.168
A2-22-b-Del3 0.144
B2-15-b 0.016
B2-15-b-T3E 0.031
B2-15-b-WT 0.041
B2-15-b-T3Q 0.029
B2-15-b-Del3 0.038
B2-15-b-Del7 0.055
D2-60-b 0.114
D2-60-b-Del3 0.229
D2-60-b-T3E 0.168
实施例17：融合蛋白在MRL/lpr自发性红斑狼疮小鼠模型中的药效实验
雌性BALB/c小鼠和雌性MRL/lpr小鼠购自上海斯莱克实验动物有限公司(Shanghai SLAC Laboratory Animal Co.,Ltd)。以雌性BALB/c小鼠为正常对照，雌性MRL/lpr小鼠按蛋白尿水平及体重随机分为3组，分别为：MRL/lpr对照组(模型对照组)，1和3mg/kg给药组。D2-60-b-T3E经皮下注射给药，每3天给药一次，剂量为1、3mg/kg；8周龄时开始给药，连续给药8周。
表23.实验设计与分组
Figure PCTCN2021076806-appb-000024
给药期间，每两周测定各组小鼠尿蛋白含量；连续8周给药结束后进行如下检测：①测定各组小鼠血清中抗双链DNA的IgM和IgG的含量；②测定各组小鼠血清中血肌酐(CRE)和血尿素氮(BUN)含量；③测定各组小鼠血清中IL-6、IL-10和IFN-γ的含量；④对各组小鼠的肾脏组织进行病理分析和评分。
1)小鼠尿蛋白含量的检测
本实验使用提尾反射法收集小鼠尿液，采用尿蛋白检测试剂盒(CBB法，南京建成生物工程研究所，#C035-2-1)检测小鼠尿蛋白浓度。MRL/lpr小鼠红斑狼疮发病的过程中，肾脏会因dsDNA抗体沉积而发生损伤，进而导致肾小球滤过功能和重吸收能力下降，尿蛋白浓度升高。随着MRL/lpr小鼠狼疮肾炎的发展，小鼠尿蛋白水平会逐渐升高。每两周进行的小鼠尿蛋白水平检测显示，最初每组小 鼠尿蛋白含量无明显差异，随着实验的进行，MRL/lpr对照组(模型对照组)小鼠尿蛋白水平逐渐升高并明显高于正常组，说明由于自身抗体的沉积，MRL/lpr小鼠出现严重的自发性肾损伤。1和3mg/kg D2-60-b-T3E给药组小鼠平均尿蛋白含量低于模型对照组并呈现剂量依赖性，但由于数据个体差异较大，和模型对照组相比无统计学差异。数据见图10。
2)血清中抗双链DNA的IgM和IgG的含量检测
采用ELISA法检测血清中抗双链DNA(抗-dsDNA)IgG、IgM的含量。具体方法参见ELISA试剂盒(Alpha Diagnostic，#5120和#5130)说明书。3mg/kg的D2-60-b-T3E给药组小鼠血清中抗-dsDNA IgG和IgM含量的平均值显著低于模型对照组(P<0.01)，1mg/kg组虽然有IgG和IgM含量低于模型对照组的趋势，但和模型对照组的差异无统计学意义。实验结果见图11A和图11B。
3)D2-60-b-T3E对MRL/lpr小鼠肾功能的影响
本实验使用肌酐(CRE)测定试剂盒(南京建成生物工程研究所，#C011-2-1)和尿素氮(BUN)测试盒(南京建成生物工程研究所，#C013-2-1)检测实验结束时小鼠血清中血肌酐和尿素氮的含量。
尿素氮和肌酐分别是人体蛋白质和肌肉的代谢产物，肾脏是其终排泄器官，肾功能受损后，血中尿素氮和肌酐无法有效排出，血尿素氮(BUN)和血肌酐(CRE)的浓度因潴留而逐渐升高。因此血清中BUN和CRE水平是临床上反映肾功能的主要指标。与正常小鼠相比，MRL/lpr对照组(模型对照组)小鼠血清中肌酐(CRE)和尿素氮(BUN)含量均显著升高(P<0.0001)，提示MRL/lpr模型组小鼠出现功能性肾受损。
1和3mg/kg给药组小鼠血清中肌酐的含量显著低于模型对照组(P<0.01，P<0.0001)，可以一定程度上改善MRL/lpr小鼠的肾功能，并且3mg/kg给药组小鼠血清中肌酐的含量显著低于1mg/kg给药组。3mg/kg给药组小鼠血清中尿素氮的含量同样显著低于模型对照组(P<0.05)，说明3mg/kg的D2-60-b-T3E可以逆转MRL/lpr小鼠的自发性肾损伤。结果见图12A和图12B。
4)D2-60-b-T3E对MRL/lpr小鼠血清IL-6，IL10和INF-γ含量的影响
采用ELISA方法检测血清中细胞因子IFN-γ、IL-6和IL-10的含量。
与正常小鼠相比，MRL/lpr对照组(模型组)小鼠血清中IL-6和IFN-γ的含量显著升高(P<0.0001)。3mg/kg给药组小鼠血清中IL-6的含量显著低于模型对照组与1mg/kg给药组(P<0.0001)，呈现出剂量依赖关系。3mg/kg给药组小鼠血清中IFN-γ的含量显著低于模型对照组(P<0.01)。结果见图13A和图13B。
与正常小鼠相比，MRL/lpr对照组(模型对照组)小鼠血清中IL-10的含量显著下降(P<0.0001)。3mg/kg D2-60-b-T3E给药组小鼠血清中IL-10的含量显著高于模型对照组(P<0.001)。结果见图14。
5)D2-60-b-T3E对MRL/lpr小鼠肾脏病变的影响
实验结束时，取小鼠左肾用4％多聚甲醛中固定，石蜡包埋切片后H&E染色，在显微镜下观察切片，并进行病理评分，对肾小球、肾间质以及血管等病理损伤进行分级评分，评分标准见表24。
表24.肾脏病理评分标准
Figure PCTCN2021076806-appb-000025
MRL/lpr对照组(模型对照组)小鼠肾脏出现严重的肾小球、肾间质及血管病理损伤，肾小球硬化，部分向内凹陷，大量细胞浸润在血管周及间质区域，肾小球出现严重的肿胀与增生。与正常小鼠相比，MRL/lpr对照组(模型对照组)小鼠肾脏病理评分显著升高(P<0.001)，提示模型对照组小鼠的肾脏出现明显的病变。
给药组小鼠的肾小球、肾间质及血管病理损伤有一定程度的减轻。1和3mg/kg给药组小鼠肾脏病理评分显著低于模型对照组(P<0.05，P<0.0001)，表明D2-60-b-T3E能显著减轻MRL/lpr自发性肾脏病变。结果见图15。

Claims (27)

  1. 抗IL-2抗体或其抗原结合片段，其包含重链可变区VH和轻链可变区VL，其中：
    所述VH含有SEQ ID NO：9中的HCDR1、HCDR2、HCDR3，所述VL含有SEQ ID NO：10中的LCDR1、LCDR2、LCDR3；
    所述VH含有SEQ ID NO：1中的HCDR1、HCDR2、HCDR3，所述VL含有SEQ ID NO：2中的LCDR1、LCDR2、LCDR3；
    所述VH含有SEQ ID NO：5中的HCDR1、HCDR2、HCDR3，所述VL含有SEQ ID NO：6中的LCDR1、LCDR2、LCDR3；
    所述VH含有SEQ ID NO：3中的HCDR1、HCDR2、HCDR3，所述VL含有SEQ ID NO：4中的LCDR1、LCDR2、LCDR3；
    所述VH含有SEQ ID NO：7中的HCDR1、HCDR2、HCDR3，所述VL含有SEQ ID NO：8中的LCDR1、LCDR2、LCDR3；或
    所述VH含有SEQ ID NO：11中的HCDR1、HCDR2、HCDR3，所述VL含有SEQ ID NO：12中的LCDR1、LCDR2、LCDR3；
    上述CDR是根据Kabat、IMGT、Chothia、AbM或Contact编号系统定义的；
    优选地，根据Kabat编号系统定义的，
    所述VH包含SEQ ID NO：37-39所示的HCDR1-3，所述VL包含SEQ ID NO：40-42所示的LCDR1-3；
    所述VH包含SEQ ID NO：13-15所示的HCDR1-3，所述VL包含SEQ ID NO：16-18所示的LCDR1-3；
    所述VH包含SEQ ID NO：25-27所示的HCDR1-3，所述VL包含SEQ ID NO：28-30所示的LCDR1-3；
    所述VH包含SEQ ID NO：19-21所示的HCDR1-3，所述VL包含SEQ ID NO：22-24所示的LCDR1-3；
    所述VH包含SEQ ID NO：31-33所示的HCDR1-3，所述VL包含SEQ ID NO：34-36所示的LCDR1-3；或
    所述VH包含SEQ ID NO：43-45所示的HCDR1-3，所述VL包含SEQ ID NO：46-48所示的LCDR1-3；
    优选地，根据Chothia编号系统定义的，
    所述VH包含SEQ ID NO：73-75所示的HCDR1-3，所述VL包含SEQ ID NO：76-78所示的LCDR1-3；
    所述VH包含SEQ ID NO：49-51所示的HCDR1-3，所述VL包含SEQ ID NO：52-54所示的LCDR1-3；
    所述VH包含SEQ ID NO：61-63所示的HCDR1-3，所述VL包含SEQ ID NO：64-66所示的LCDR1-3；
    所述VH包含SEQ ID NO：55-57所示的HCDR1-3，所述VL包含SEQ ID NO：58-60所示的LCDR1-3；
    所述VH包含SEQ ID NO：67-69所示的HCDR1-3，所述VL包含SEQ ID NO：70-72所示的LCDR1-3；或
    所述VH包含SEQ ID NO：79-81所示的HCDR1-3，所述VL包含SEQ ID NO：82-84所示的LCDR1-3；
    优选地，根据IMGT编号系统定义的，
    所述VH包含SEQ ID NO：109-111所示的HCDR1-3，所述VL包含SEQ ID NO：112-114所示的LCDR1-3；
    所述VH包含SEQ ID NO：85-87所示的HCDR1-3，所述VL包含SEQ ID NO：88-90所示的LCDR1-3；
    所述VH包含SEQ ID NO：97-99所示的HCDR1-3，所述VL包含SEQ ID NO：100-102所示的LCDR1-3；
    所述VH包含SEQ ID NO：91-93所示的HCDR1-3，所述VL包含SEQ ID NO：94-96所示的LCDR1-3；
    所述VH包含SEQ ID NO：103-105所示的HCDR1-3，所述VL包含SEQ ID NO：106-108所示的LCDR1-3；或
    所述VH包含SEQ ID NO：115-117所示的HCDR1-3，所述VL包含SEQ ID NO：118-120所示的LCDR1-3；
    优选地，根据AbM编号系统定义的，
    所述VH包含SEQ ID NO：145-147所示的HCDR1-3，所述VL包含SEQ ID NO：148-150所示的LCDR1-3；
    所述VH包含SEQ ID NO：121-123所示的HCDR1-3，所述VL包含SEQ ID NO：124-126所示的LCDR1-3；
    所述VH包含SEQ ID NO：133-135所示的HCDR1-3，所述VL包含SEQ ID NO：136-138所示的LCDR1-3；
    所述VH包含SEQ ID NO：127-129所示的HCDR1-3，所述VL包含SEQ ID NO：130-132所示的LCDR1-3；
    所述VH包含SEQ ID NO：139-141所示的HCDR1-3，所述VL包含SEQ ID NO：142-144所示的LCDR1-3；或
    所述VH包含SEQ ID NO：151-153所示的HCDR1-3，所述VL包含SEQ ID NO：154-156所示的LCDR1-3。
  2. 如权利要求1所述的抗IL-2抗体或其抗原结合片段，其包含：
    如SEQ ID NO：9、1、5、3、7、11之一所示或与之具有至少90％、至少95％同一性的VH；和
    如SEQ ID NO：10、2、6、4、8、12之一所示或与之具有至少95％同一性的VL；
    优选地，所述抗IL-2抗体或其抗原结合片段包含：
    如SEQ ID NO：9所示或与之具有至少95％同一性的VH，
    如SEQ ID NO：10所示或与之具有至少95％同一性的VL；或
    如SEQ ID NO：1所示或与之具有至少95％同一性的VH，
    如SEQ ID NO：2所示或与之具有至少95％同一性的VL；或
    如SEQ ID NO：5所示或与之具有至少95％同一性的VH，
    如SEQ ID NO：6所示或与之具有至少95％同一性的VL；或
    如SEQ ID NO：3所示或与之具有至少95％同一性的VH，
    如SEQ ID NO：4所示或与之具有至少95％同一性的VL；或
    如SEQ ID NO：7所示或与之具有至少95％同一性的VH，
    如SEQ ID NO：8所示或与之具有至少95％同一性的VL；或
    如SEQ ID NO：11所示或与之具有至少95％同一性的VH，
    如SEQ ID NO：12所示或与之具有至少95％同一性的VL。
  3. 如权利要求1或2所述的抗IL-2抗体或其抗原结合片段，其包含：
    如SEQ ID NO：165、157、161、159、163、167之一所示或与之具有至少80％、90％、95％同一性的HC；和
    如SEQ ID NO：166、158、162、160、164、168之一所示或与之具有至少80％、90％、95％同一性的LC；
    优选地，所述抗IL-2抗体包含：
    如SEQ ID NO：165所示或与之具有至少90％同一性的HC，
    如SEQ ID NO：166所示或与之具有至少90％同一性的LC；或
    如SEQ ID NO：157所示或与之具有至少90％同一性的HC，
    如SEQ ID NO：158所示或与之具有至少90％同一性的LC；或
    如SEQ ID NO：161所示或与之具有至少90％同一性的HC，
    如SEQ ID NO：162所示或与之具有至少90％同一性的LC；或
    如SEQ ID NO：159所示或与之具有至少90％同一性的HC，
    如SEQ ID NO：160所示或与之具有至少90％同一性的LC；或
    如SEQ ID NO：163所示或与之具有至少90％同一性的HC，
    如SEQ ID NO：164所示或与之具有至少90％同一性的LC；或
    如SEQ ID NO：167所示或与之具有至少90％同一性的HC，
    如SEQ ID NO：168所示或与之具有至少90％同一性的LC。
  4. 如前述权利要求任一项所述的抗IL-2抗体或其抗原结合片段，其为全人抗体或其抗原结合片段。
  5. 如前述权利要求任一项所述的抗IL-2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。
  6. 如前述权利要求任一项所述的抗IL-2抗体或其抗原结合片段，其为IgG抗体或其抗原结合片段，优选为IgG1、IgG4抗体或其抗原结合片段。
  7. 一种复合物，其包含：
    权利要求1-6任一项所述的抗IL-2抗体或其抗原片段与IL-2，和
    IL-2。
  8. 如权利要求7所述的复合物，其选择性增强高表达IL-2Ra的细胞的活性或促进其增殖，优选地，所述高表达IL-2Ra的细胞为调节性T细胞(Treg)。
  9. 如权利要求7或8所述的复合物，所述抗IL-2抗体或其抗原片段与IL-2之间通过共价或非共价作用力结合。
  10. 如权利要求7-9任一项所述的复合物，其为抗IL-2抗体或其抗原片段与IL-2形成的融合蛋白。
  11. 如权利要求10所述的复合物，所述融合蛋白中，IL-2连接至抗IL-2抗体或其抗原片段的轻链(LC)或重链(HC)；
    优选地，IL-2连接至抗IL-2抗体或其抗原片段的LC或HC的N端；
    更优选地，IL-2连接至抗IL-2抗体或其抗原片段的LC的N端。
  12. 如权利要求10或11所述的复合物，所述融合蛋白中，IL-2通过连接子连接至抗IL-2抗体或其抗原片段，
    优选地，所述连接子包含如(G 4S) x所示的氨基酸序列，其中，x独立地选自1-20的整数；
    更优选地，连接子为(G 4S) 5所示的氨基酸序列。
  13. 如权利要求7-12任一项所述的复合物，其中，IL-2存在第三位氨基酸取代或N端缺失，
    优选地，所述第三位氨基酸取代选自以下的任一项：3A、3Q、3E；
    所述N端缺失为N端前3个或前7个氨基酸的缺失。
  14. 如权利要求10-13任一项所述的复合物，其中所述融合蛋白包含选自以下任一组的HC和LC：
    SEQ ID No：169所示或与之具有至少90％序列同一性的HC，和
    SEQ ID No：158所示或与之具有至少90％序列同一性的LC；
    SEQ ID No：171所示或与之具有至少90％序列同一性的HC，和
    SEQ ID No：160所示或与之具有至少90％序列同一性的LC；
    SEQ ID No：159所示或与之具有至少90％序列同一性的HC，和
    SEQ ID No：172所示或与之具有至少90％序列同一性的LC；
    SEQ ID No：173所示或与之具有至少90％序列同一性的HC，和
    SEQ ID No：162所示或与之具有至少90％序列同一性的LC；
    SEQ ID No：175所示或与之具有至少90％序列同一性的HC，和
    SEQ ID No：164所示或与之具有至少90％序列同一性的LC；
    SEQ ID No：163所示或与之具有至少90％序列同一性的HC，和
    SEQ ID No：176所示或与之具有至少90％序列同一性的LC；
    SEQ ID No：177所示或与之具有至少90％序列同一性的HC，和
    SEQ ID No：166所示或与之具有至少90％序列同一性的LC；
    SEQ ID No：179所示或与之具有至少90％序列同一性的HC，和
    SEQ ID No：168所示或与之具有至少90％序列同一性的LC；
    SEQ ID No：167所示或与之具有至少90％序列同一性的HC，和
    SEQ ID No：180所示或与之具有至少90％序列同一性的LC；
    SEQ ID No：161所示或与之具有至少90％序列同一性的HC，和
    SEQ ID No：174、181-186任一所示或与之具有至少90％序列同一性的LC；
    SEQ ID No：157所示或与之具有至少90％序列同一性的HC，和
    SEQ ID No：170、187任一所示或与之具有至少90％序列同一性的LC；或
    SEQ ID No：165所示或与之具有至少90％序列同一性的HC，和
    SEQ ID No：178、188、189任一所示或与之具有至少90％序列同一性的LC。
  15. 如权利要求7-14任一项所述的复合物，所述复合物选择性地增强了IL-2对Treg活性的作用。
  16. 抗IL-2抗体或其抗原结合片段，其降低IL-2与IL-2Rα结合，并且阻断IL-2与IL-2Rβ的结合，所述抗IL-2抗体或其抗原结合片段与人IL-2的亲合力K D值＞5nM，优选为＞5nM且≤100nM，更优选为≥10nM且≤60nM。
  17. 如权利要求16所述的抗IL-2抗体或其抗原结合片段，其含有VH和VL，
    所述VH含有SEQ ID NO：9中的HCDR1、HCDR2、HCDR3，所述VL含有SEQ ID NO：10中的LCDR1、LCDR2、LCDR3；
    所述VH含有SEQ ID NO：1中的HCDR1、HCDR2、HCDR3，所述VL含有SEQ ID NO：2中的LCDR1、LCDR2、LCDR3；或
    所述VH含有SEQ ID NO：5中的HCDR1、HCDR2、HCDR3，所述VL含有SEQ ID NO：6中的LCDR1、LCDR2、LCDR3；
    上述CDR是根据Kabat、IMGT、Chothia、AbM或Contact编号系统定义的。
  18. 抗IL-2抗体或其抗原结合片段，其阻断IL-2与IL-2Rα结合，并且阻断IL-2与IL-2Rβ的结合，所述抗IL-2抗体或其抗原结合片段与人IL-2的亲合力K D值≤5nM。
  19. 如权利要求18所述的抗IL-2抗体或其抗原结合片段，其含有VH和VL，
    所述VH含有SEQ ID NO：3中的HCDR1、HCDR2、HCDR3，所述VL含有SEQ ID NO：4中的LCDR1、LCDR2、LCDR3；
    所述VH含有SEQ ID NO：7中的HCDR1、HCDR2、HCDR3，所述VL含有SEQ ID NO：8中的LCDR1、LCDR2、LCDR3；或
    所述VH含有SEQ ID NO：11中的HCDR1、HCDR2、HCDR3，所述VL含有SEQ ID NO：12中的LCDR1、LCDR2、LCDR3；
    上述CDR是根据Kabat、IMGT、Chothia、AbM或Contact编号系统定义的。
  20. 抗IL-2抗体或其抗原结合片段，其与权利要求1-6、16-19中抗IL-2抗体或其抗原结合片段竞争结合人IL-2；优选地，竞争结合相同的人IL-2表位。
  21. 药物组合物，其含有：
    权利要求1-6、16-19任一项所述的抗IL-2抗体或其抗原结合片段、或权利要求7-15任一项所述的复合物，以及
    一种或多种可药用的赋形剂、稀释剂或载体。
  22. 经分离的多核苷酸，其编码权利要求1-6、16-19任一项所述的抗IL-2抗体或其抗原结合片段或权利要求7-15任一项所述的复合物。
  23. 宿主细胞，其转入或包含权利要求22所述的多核苷酸。
  24. 制备抗IL-2抗体或其抗原结合片段、或其复合物的方法，包括：
    在权利要求23所述的宿主细胞中，表达抗IL-2抗体或其抗原结合片段、或其复合物，以及
    从所述宿主细胞中分离所述抗IL-2抗体或其抗原结合片段、或其复合物。
  25. 治疗自身免疫疾病或延缓自身免疫疾病进展的方法，所述方法包括：
    向受试者施用治疗或延缓疾病有效量的权利要求1-6、16-19任一项所述的抗IL-2抗体或其抗原结合片段，权利要求7-15任一项所述的复合物，权利要求21所述的药物组合物、或权利要求22所述的多核苷酸，
    优选地，所述自身免疫疾病选自以下的任一项或组合：狼疮、移植物抗宿主病、丙型肝炎诱导的脉管炎、I型糖尿病、多发性硬化、炎性肠病、慢性GvHD、哮喘、皮炎、斑秃、急性肺损伤、败血症、反应性关节炎、类风湿性关节炎。
  26. 增加T细胞群体中的Treg对非Treg的比率、或增加Treg数量、或提高Treg活性的方法，其包括：将所述T细胞群体与有效量的权利要求1-6或16-19任一项所述的抗IL-2抗体或其抗原结合片段、权利要求7-15任一项所述的复合物、或权利要求21所述的药物组合物接触。
  27. 增加受试者外周血中的Treg对非Treg的比率、或增加Treg数量、或提高Treg活性的方法，其包括：
    向所述受试者施用有效量的权利要求1-6或16-19任一项所述的抗IL-2抗体或其抗原结合片段、权利要求7-15任一项所述的复合物、或权利要求21所述的药物组合物。
PCT/CN2021/076806 2020-02-21 2021-02-19 抗il-2抗体、其抗原结合片段及其医药用途 WO2021164722A1 (zh)

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BR112022016326A BR112022016326A2 (pt) 2020-02-21 2021-02-19 Anticorpo anti-il-2, e fragmento de ligação ao antígeno do mesmo e uso médico do mesmo
US17/904,517 US20230089620A1 (en) 2020-02-21 2021-02-19 Anti-IL-2 Antibody, and Antigen-Binding Fragment Thereof and Medical Use Thereof
JP2022549830A JP2023515480A (ja) 2020-02-21 2021-02-19 抗ｉｌ－２抗体、その抗原結合断片及びその医薬用途
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MX2022010218A MX2022010218A (es) 2020-02-21 2021-02-19 Anticuerpo anti-il-2 y fragmento de union a antigeno del mismo y su uso medico.
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