WO2022267926A1 - 一种抗tnfr2抗体及其制备方法和应用 - Google Patents

一种抗tnfr2抗体及其制备方法和应用 Download PDF

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Publication number
WO2022267926A1
WO2022267926A1 PCT/CN2022/098506 CN2022098506W WO2022267926A1 WO 2022267926 A1 WO2022267926 A1 WO 2022267926A1 CN 2022098506 W CN2022098506 W CN 2022098506W WO 2022267926 A1 WO2022267926 A1 WO 2022267926A1
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seq
amino acid
acid sequence
variable region
chain variable
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PCT/CN2022/098506
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English (en)
French (fr)
Inventor
姜晓玲
殷刘松
吴崇兵
周金花
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盛禾(中国)生物制药有限公司
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Priority to CN202280052089.9A priority Critical patent/CN117836325A/zh
Publication of WO2022267926A1 publication Critical patent/WO2022267926A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Definitions

  • the invention belongs to the field of biomedicine, and in particular relates to an anti-TNFR2 antibody and its preparation method and application.
  • enhancing the body's own ability to resist disease by modulating the immune response is an effective alternative and/or supplement to traditional treatments.
  • enhancing activity against T lymphocytes to target and treat various diseases eg, cancer or autoimmune diseases
  • the immune response against disease can be enhanced by inhibiting the ability of Tregs to suppress the activity of T lymphocytes or by enhancing the ability of effector T cells to suppress tumors.
  • Tumor necrosis factor receptor 2 also known as TNFRSF1B and CD120b, is a costimulatory member of the tumor necrosis factor receptor superfamily (TNFRSF), which includes such genes as GITR, OX40, CD27, CD40, and 4-IBB (CD137) of protein.
  • TNFR2 is a cell surface receptor expressed on T cells that has been shown to enhance the activation of effector T cells and reduce Treg-mediated suppression. By regulating TRAF2/3 and NF-kB signaling, TNFR2 can mediate the transcription of genes that promote cell survival and proliferation.
  • TNFR2 can be expressed on cancer cells, tumor-infiltrating Tregs, and effector T cells.
  • the invention provides an antibody directed at the target of TNFR2, which is used to enhance the immune ability against tumors.
  • the first object of the present invention is to provide an anti-TNFR2 antibody or an antigen-binding fragment thereof.
  • the second object of the present invention is to provide the gene encoding the above-mentioned anti-TNFR2 antibody or its antigen-binding fragment.
  • the third object of the present invention is to provide a vector comprising the gene encoding the above-mentioned anti-TNFR2 antibody or an antigen-binding fragment thereof.
  • the fourth object of the present invention is to provide a host cell comprising the gene vector encoding the above-mentioned anti-TNFR2 antibody or an antigen-binding fragment thereof.
  • the fifth object of the present invention is to provide a method for expressing the above-mentioned anti-TNFR2 antibody or antigen-binding fragment thereof.
  • the sixth object of the present invention is to provide drug conjugates comprising the above-mentioned anti-TNFR2 antibodies or antigen-binding fragments thereof.
  • the seventh object of the present invention is to provide the use of the combination comprising the above-mentioned anti-TNFR2 antibody or its antigen-binding fragment and chemotherapy in the manufacture of drugs for treating cancer or autoimmune diseases.
  • the eighth object of the present invention is to provide applications comprising the above-mentioned anti-TNFR2 antibody or antigen-binding fragment thereof.
  • the ninth object of the present invention is to provide a pharmaceutical composition comprising an anti-TNFR2 antibody or an antigen-binding fragment thereof.
  • the CDR1, CDR2 and CDR3 of the heavy chain variable region are composed of the amino acid sequences SEQ ID NO: 18, SEQ ID NO: 58 and SEQ ID NO: 20, respectively, and the CDR1, CDR2 and CDR3 of the light chain variable region CDR2 and CDR3 consist of SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, respectively; or
  • CDR1, CDR2 and CDR3 of the heavy chain variable region are composed of amino acid sequences SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, respectively, and the CDR1, CDR2 and CDR3 of the light chain variable region CDR2 and CDR3 consist of SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively; or
  • the CDR1, CDR2 and CDR3 of the heavy chain variable region are composed of the amino acid sequences SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively, and the CDR1, CDR3 of the light chain variable region CDR2 and CDR3 consist of SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively; or
  • the CDR1, CDR2 and CDR3 of the heavy chain variable region are composed of amino acid sequences SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, respectively, and the CDR1, CDR2 and CDR3 of the light chain variable region CDR2 and CDR3 consist of SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, respectively; or
  • the CDR1, CDR2 and CDR3 of the heavy chain variable region are composed of amino acid sequences SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively, and the CDR1, CDR2 and CDR3 of the light chain variable region CDR2 and CDR3 consist of SEQ ID NO: 27, SEQ ID NO: 15 and SEQ ID NO: 16, respectively; or
  • the CDR1, CDR2 and CDR3 of the heavy chain variable region are composed of amino acid sequences SEQ ID NO: 18, SEQ ID NO: 55 and SEQ ID NO: 20, respectively, and the CDR1, CDR2 and CDR3 of the light chain variable region CDR2 and CDR3 consist of SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, respectively; or
  • the CDR1, CDR2 and CDR3 of the heavy chain variable region are composed of amino acid sequences SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, respectively, and the CDR1, CDR2 and CDR3 of the light chain variable region CDR2 and CDR3 consist of SEQ ID NO: 28, SEQ ID NO: 7 and SEQ ID NO: 8, respectively; or
  • CDR1, CDR2 and CDR3 of the heavy chain variable region are composed of amino acid sequences SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 respectively, and the CDR1, CDR2 and CDR3 of the light chain variable region CDR2 and CDR3 consist of SEQ ID NO: 33, SEQ ID NO: 7 and SEQ ID NO: 8, respectively; or
  • the CDR1, CDR2 and CDR3 of the heavy chain variable region are composed of amino acid sequences SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 36, respectively, and the CDR1, CDR2 and CDR3 of the light chain variable region CDR2 and CDR3 consist of SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively; or
  • CDR1, CDR2 and CDR3 of the heavy chain variable region are composed of amino acid sequences SEQ ID NO: 18, SEQ ID NO: 55 and SEQ ID NO: 61, respectively, and the CDR1, CDR1, and CDR3 of the light chain variable region CDR2 and CDR3 consist of SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, respectively; or
  • CDR1, CDR2 and CDR3 of the heavy chain variable region are composed of amino acid sequences SEQ ID NO: 18, SEQ ID NO: 55 and SEQ ID NO: 64, respectively, and the CDR1, CDR1, and CDR3 of the light chain variable region CDR2 and CDR3 consist of SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, respectively; or
  • CDR1, CDR2 and CDR3 of the heavy chain variable region are composed of amino acid sequences SEQ ID NO: 18, SEQ ID NO: 58 and SEQ ID NO: 61, respectively, and the CDR1, CDR1, and CDR3 of the light chain variable region CDR2 and CDR3 consist of SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, respectively; or
  • the CDR1, CDR2 and CDR3 of the heavy chain variable region are composed of amino acid sequences SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 36 respectively, and the CDR1, CDR2 and CDR3 of the light chain variable region CDR2 and CDR3 consist of SEQ ID NO: 28, SEQ ID NO: 7 and SEQ ID NO: 8, respectively; or
  • CDR1, CDR2 and CDR3 of the heavy chain variable region are composed of amino acid sequences SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 45, respectively, and the CDR1, CDR2 and CDR3 of the light chain variable region CDR2 and CDR3 consist of SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively; or
  • CDR1, CDR2 and CDR3 of the heavy chain variable region are composed of amino acid sequences SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 45, respectively, and the CDR1, CDR1, and CDR3 of the light chain variable region CDR2 and CDR3 consist of SEQ ID NO: 28, SEQ ID NO: 7 and SEQ ID NO: 48, respectively; or
  • the CDR1, CDR2 and CDR3 of the heavy chain variable region are composed of amino acid sequences SEQ ID NO: 18, SEQ ID NO: 58 and SEQ ID NO: 64 respectively, and the CDR1, CDR2 and CDR3 of the light chain variable region CDR2 and CDR3 consist of SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, respectively.
  • the heavy chain variable region has the amino acid sequence given by SEQ ID NO: 56, or has at least 80%, 85%, 90%, 91%, 92% of the amino acid sequence given by SEQ ID NO: 56 , 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical sequences, or have one or more (preferably 1, 2) compared with the amino acid sequence of SEQ ID NO: 56 , 3, 4, 5, 6, 7, 8, 9, 10) amino acid sequences of conservative amino acid mutations (preferably substitutions, insertions or deletions),
  • the light chain variable region has the amino acid sequence given by SEQ ID NO: 57, or has at least 80%, 85%, 90%, 91%, 92%, 93% of the amino acid sequence given by SEQ ID NO: 57 , 94%, 95%, 96%, 97%, 98%, 99% or more identical sequences, or have one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid sequences with conservative amino acid mutations (preferably substitutions, insertions or deletions); or
  • the heavy chain variable region has the amino acid sequence given by SEQ ID NO: 1, or has at least 80%, 85%, 90%, 91%, 92% of the amino acid sequence given by SEQ ID NO: 1 , 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical sequences, or have one or more (preferably 1, 2) compared with the amino acid sequence of SEQ ID NO: 1 , 3, 4, 5, 6, 7, 8, 9, 10) amino acid sequences of conservative amino acid mutations (preferably substitutions, insertions or deletions),
  • the light chain variable region has the amino acid sequence given by SEQ ID NO: 5, or has at least 80%, 85%, 90%, 91%, 92%, 93% of the amino acid sequence given by SEQ ID NO: 5 , 94%, 95%, 96%, 97%, 98%, 99% or more identical sequences, or have one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid sequences with conservative amino acid mutations (preferably substitutions, insertions or deletions); or
  • the heavy chain variable region has the amino acid sequence given by SEQ ID NO: 9, or has at least 80%, 85%, 90%, 91%, 92% of the amino acid sequence given by SEQ ID NO: 9 , 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical sequences, or have one or more (preferably 1, 2) compared with the amino acid sequence of SEQ ID NO: 9 , 3, 4, 5, 6, 7, 8, 9, 10) amino acid sequences of conservative amino acid mutations (preferably substitutions, insertions or deletions),
  • the light chain variable region has the amino acid sequence given by SEQ ID NO: 13, or has at least 80%, 85%, 90%, 91%, 92%, 93% of the amino acid sequence given by SEQ ID NO: 13 , 94%, 95%, 96%, 97%, 98%, 99% or more identical sequences, or have one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid sequences with conservative amino acid mutations (preferably substitutions, insertions or deletions); or
  • the heavy chain variable region has the amino acid sequence given by SEQ ID NO: 17, or has at least 80%, 85%, 90%, 91%, 92% of the amino acid sequence given by SEQ ID NO: 17 , 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical sequences, or have one or more (preferably 1, 2) compared with the amino acid sequence of SEQ ID NO: 17 , 3, 4, 5, 6, 7, 8, 9, 10) amino acid sequences of conservative amino acid mutations (preferably substitutions, insertions or deletions),
  • the light chain variable region has the amino acid sequence given by SEQ ID NO: 21, or has at least 80%, 85%, 90%, 91%, 92%, 93% of the amino acid sequence given by SEQ ID NO: 21 , 94%, 95%, 96%, 97%, 98%, 99% or more identical sequences, or have one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid sequences with conservative amino acid mutations (preferably substitutions, insertions or deletions); or
  • the heavy chain variable region has the amino acid sequence given by SEQ ID NO: 49, or has at least 80%, 85%, 90%, 91%, 92% of the amino acid sequence given by SEQ ID NO: 49 , 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical sequences, or have one or more (preferably 1, 2) compared with the amino acid sequence of SEQ ID NO: 49 , 3, 4, 5, 6, 7, 8, 9, 10) amino acid sequences of conservative amino acid mutations (preferably substitutions, insertions or deletions),
  • the light chain variable region has the amino acid sequence given by SEQ ID NO: 50, or has at least 80%, 85%, 90%, 91%, 92%, 93% of the amino acid sequence given by SEQ ID NO: 50 , 94%, 95%, 96%, 97%, 98%, 99% or more identical sequences, or have one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid sequences with conservative amino acid mutations (preferably substitutions, insertions or deletions); or
  • the heavy chain variable region has the amino acid sequence given by SEQ ID NO: 51, or has at least 80%, 85%, 90%, 91%, 92% of the amino acid sequence given by SEQ ID NO: 51 , 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical sequences, or have one or more (preferably 1, 2) compared with the amino acid sequence of SEQ ID NO: 51 , 3, 4, 5, 6, 7, 8, 9, 10) amino acid sequences of conservative amino acid mutations (preferably substitutions, insertions or deletions),
  • the light chain variable region has the amino acid sequence given by SEQ ID NO: 52, or has at least 80%, 85%, 90%, 91%, 92%, 93% of the amino acid sequence given by SEQ ID NO: 52 , 94%, 95%, 96%, 97%, 98%, 99% or more identical sequences, or have one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid sequences with conservative amino acid mutations (preferably substitutions, insertions or deletions); or
  • the heavy chain variable region has the amino acid sequence given by SEQ ID NO: 53, or has at least 80%, 85%, 90%, 91%, 92% of the amino acid sequence given by SEQ ID NO: 53 , 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical sequences, or have one or more (preferably 1, 2) compared with the amino acid sequence of SEQ ID NO: 53 , 3, 4, 5, 6, 7, 8, 9, 10) amino acid sequences of conservative amino acid mutations (preferably substitutions, insertions or deletions),
  • the light chain variable region has the amino acid sequence given by SEQ ID NO: 54, or has at least 80%, 85%, 90%, 91%, 92%, 93% of the amino acid sequence given by SEQ ID NO: 54 , 94%, 95%, 96%, 97%, 98%, 99% or more identical sequences, or have one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid sequences with conservative amino acid mutations (preferably substitutions, insertions or deletions); or
  • the heavy chain variable region has the amino acid sequence given by SEQ ID NO: 25, or has at least 80%, 85%, 90%, 91%, 92% of the amino acid sequence given by SEQ ID NO: 25 , 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical sequences, or have one or more (preferably 1, 2) compared with the amino acid sequence of SEQ ID NO: 25 , 3, 4, 5, 6, 7, 8, 9, 10) amino acid sequences of conservative amino acid mutations (preferably substitutions, insertions or deletions),
  • the light chain variable region has the amino acid sequence given by SEQ ID NO: 26, or has at least 80%, 85%, 90%, 91%, 92%, 93% of the amino acid sequence given by SEQ ID NO: 26 , 94%, 95%, 96%, 97%, 98%, 99% or more identical sequences, or have one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid sequences with conservative amino acid mutations (preferably substitutions, insertions or deletions); or
  • the heavy chain variable region has the amino acid sequence given by SEQ ID NO: 29, or has at least 80%, 85%, 90%, 91%, 92% of the amino acid sequence given by SEQ ID NO: 29 , 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical sequences, or have one or more (preferably 1, 2) compared with the amino acid sequence of SEQ ID NO: 29 , 3, 4, 5, 6, 7, 8, 9, 10) amino acid sequences of conservative amino acid mutations (preferably substitutions, insertions or deletions),
  • the light chain variable region has the amino acid sequence given by SEQ ID NO: 30, or has at least 80%, 85%, 90%, 91%, 92%, 93% of the amino acid sequence given by SEQ ID NO: 30 , 94%, 95%, 96%, 97%, 98%, 99% or more identical sequences, or have one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid sequences with conservative amino acid mutations (preferably substitutions, insertions or deletions); or
  • the heavy chain variable region has the amino acid sequence given by SEQ ID NO: 31, or has at least 80%, 85%, 90%, 91%, 92% of the amino acid sequence given by SEQ ID NO: 31 , 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical sequences, or have one or more (preferably 1, 2) compared with the amino acid sequence of SEQ ID NO: 31 , 3, 4, 5, 6, 7, 8, 9, 10) amino acid sequences of conservative amino acid mutations (preferably substitutions, insertions or deletions),
  • the light chain variable region has the amino acid sequence given by SEQ ID NO: 32, or has at least 80%, 85%, 90%, 91%, 92%, 93% of the amino acid sequence given by SEQ ID NO: 32 , 94%, 95%, 96%, 97%, 98%, 99% or more identical sequences, or have one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid sequences with conservative amino acid mutations (preferably substitutions, insertions or deletions); or
  • the heavy chain variable region has the amino acid sequence given by SEQ ID NO: 34, or has at least 80%, 85%, 90%, 91%, 92% of the amino acid sequence given by SEQ ID NO: 34 , 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical sequences, or have one or more (preferably 1, 2) compared with the amino acid sequence of SEQ ID NO: 34 , 3, 4, 5, 6, 7, 8, 9, 10) amino acid sequences of conservative amino acid mutations (preferably substitutions, insertions or deletions),
  • the light chain variable region has the amino acid sequence given by SEQ ID NO: 35, or has at least 80%, 85%, 90%, 91%, 92%, 93% of the amino acid sequence given by SEQ ID NO: 35 , 94%, 95%, 96%, 97%, 98%, 99% or more identical sequences, or have one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid sequences with conservative amino acid mutations (preferably substitutions, insertions or deletions); or
  • the heavy chain variable region has the amino acid sequence given by SEQ ID NO: 59, or has at least 80%, 85%, 90%, 91%, 92% of the amino acid sequence given by SEQ ID NO: 59 , 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical sequences, or have one or more (preferably 1, 2) compared with the amino acid sequence of SEQ ID NO: 59 , 3, 4, 5, 6, 7, 8, 9, 10) amino acid sequences of conservative amino acid mutations (preferably substitutions, insertions or deletions),
  • the light chain variable region has the amino acid sequence given by SEQ ID NO: 60, or has at least 80%, 85%, 90%, 91%, 92%, 93% of the amino acid sequence given by SEQ ID NO: 60 , 94%, 95%, 96%, 97%, 98%, 99% or more identical sequences, or have one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid sequences with conservative amino acid mutations (preferably substitutions, insertions or deletions); or
  • the heavy chain variable region has the amino acid sequence given by SEQ ID NO: 62, or has at least 80%, 85%, 90%, 91%, 92% of the amino acid sequence given by SEQ ID NO: 62 , 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical sequences, or have one or more (preferably 1, 2) compared with the amino acid sequence of SEQ ID NO: 62 , 3, 4, 5, 6, 7, 8, 9, 10) amino acid sequences of conservative amino acid mutations (preferably substitutions, insertions or deletions),
  • the light chain variable region has the amino acid sequence given by SEQ ID NO: 63, or has at least 80%, 85%, 90%, 91%, 92%, 93% of the amino acid sequence given by SEQ ID NO: 63 , 94%, 95%, 96%, 97%, 98%, 99% or more identical sequences, or have one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid sequences with conservative amino acid mutations (preferably substitutions, insertions or deletions); or
  • the heavy chain variable region has the amino acid sequence given by SEQ ID NO: 65, or has at least 80%, 85%, 90%, 91%, 92% of the amino acid sequence given by SEQ ID NO: 65 , 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical sequences, or have one or more (preferably 1, 2) compared with the amino acid sequence of SEQ ID NO: 65 , 3, 4, 5, 6, 7, 8, 9, 10) amino acid sequences of conservative amino acid mutations (preferably substitutions, insertions or deletions),
  • the light chain variable region has the amino acid sequence given by SEQ ID NO: 66, or has at least 80%, 85%, 90%, 91%, 92%, 93% of the amino acid sequence given by SEQ ID NO: 66 , 94%, 95%, 96%, 97%, 98%, 99% or more identical sequences, or have one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid sequences with conservative amino acid mutations (preferably substitutions, insertions or deletions); or
  • the heavy chain variable region has the amino acid sequence given by SEQ ID NO: 37, or has at least 80%, 85%, 90%, 91%, 92% of the amino acid sequence given by SEQ ID NO: 37 , 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical sequences, or have one or more (preferably 1, 2) compared with the amino acid sequence of SEQ ID NO: 37 , 3, 4, 5, 6, 7, 8, 9, 10) amino acid sequences of conservative amino acid mutations (preferably substitutions, insertions or deletions),
  • the light chain variable region has the amino acid sequence given by SEQ ID NO: 38, or has at least 80%, 85%, 90%, 91%, 92%, 93% of the amino acid sequence given by SEQ ID NO: 38 , 94%, 95%, 96%, 97%, 98%, 99% or more identical sequences, or have one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid sequences with conservative amino acid mutations (preferably substitutions, insertions or deletions); or
  • the heavy chain variable region has the amino acid sequence given by SEQ ID NO: 39, or has at least 80%, 85%, 90%, 91%, 92% of the amino acid sequence given by SEQ ID NO: 39 , 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical sequences, or have one or more (preferably 1, 2) compared with the amino acid sequence of SEQ ID NO: 39 , 3, 4, 5, 6, 7, 8, 9, 10) amino acid sequences of conservative amino acid mutations (preferably substitutions, insertions or deletions),
  • the light chain variable region has the amino acid sequence given by SEQ ID NO: 40, or has at least 80%, 85%, 90%, 91%, 92%, 93% of the amino acid sequence given by SEQ ID NO: 40 , 94%, 95%, 96%, 97%, 98%, 99% or more identical sequences, or have one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid sequences with conservative amino acid mutations (preferably substitutions, insertions or deletions); or
  • the heavy chain variable region has the amino acid sequence given by SEQ ID NO: 41, or has at least 80%, 85%, 90%, 91%, 92% of the amino acid sequence given by SEQ ID NO: 41 , 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical sequences, or have one or more (preferably 1, 2) compared with the amino acid sequence of SEQ ID NO: 41 , 3, 4, 5, 6, 7, 8, 9, 10) amino acid sequences of conservative amino acid mutations (preferably substitutions, insertions or deletions),
  • the light chain variable region has the amino acid sequence given by SEQ ID NO: 42, or has at least 80%, 85%, 90%, 91%, 92%, 93% of the amino acid sequence given by SEQ ID NO: 42 , 94%, 95%, 96%, 97%, 98%, 99% or more identical sequences, or have one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid sequences with conservative amino acid mutations (preferably substitutions, insertions or deletions); or
  • the heavy chain variable region has the amino acid sequence given by SEQ ID NO: 43, or has at least 80%, 85%, 90%, 91%, 92% of the amino acid sequence given by SEQ ID NO: 43 , 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical sequences, or have one or more (preferably 1, 2) compared with the amino acid sequence of SEQ ID NO: 43 , 3, 4, 5, 6, 7, 8, 9, 10) amino acid sequences of conservative amino acid mutations (preferably substitutions, insertions or deletions),
  • the light chain variable region has the amino acid sequence given by SEQ ID NO: 44, or has at least 80%, 85%, 90%, 91%, 92%, 93% of the amino acid sequence given by SEQ ID NO: 44 , 94%, 95%, 96%, 97%, 98%, 99% or more identical sequences, or have one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid sequences with conservative amino acid mutations (preferably substitutions, insertions or deletions); or
  • the heavy chain variable region has the amino acid sequence given by SEQ ID NO: 46, or has at least 80%, 85%, 90%, 91%, 92% of the amino acid sequence given by SEQ ID NO: 46 , 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical sequences, or have one or more (preferably 1, 2) compared with the amino acid sequence of SEQ ID NO: 46 , 3, 4, 5, 6, 7, 8, 9, 10) amino acid sequences of conservative amino acid mutations (preferably substitutions, insertions or deletions),
  • the light chain variable region has the amino acid sequence given by SEQ ID NO: 47, or has at least 80%, 85%, 90%, 91%, 92%, 93% of the amino acid sequence given by SEQ ID NO: 47 , 94%, 95%, 96%, 97%, 98%, 99% or more identical sequences, or have one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid sequences with conservative amino acid mutations (preferably substitutions, insertions or deletions); or
  • the heavy chain variable region has the amino acid sequence given by SEQ ID NO: 67, or has at least 80%, 85%, 90%, 91%, 92% of the amino acid sequence given by SEQ ID NO: 67 , 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical sequences, or have one or more (preferably 1, 2) compared with the amino acid sequence of SEQ ID NO: 67 , 3, 4, 5, 6, 7, 8, 9, 10) amino acid sequences of conservative amino acid mutations (preferably substitutions, insertions or deletions),
  • the light chain variable region has the amino acid sequence given by SEQ ID NO: 68, or has at least 80%, 85%, 90%, 91%, 92%, 93% of the amino acid sequence given by SEQ ID NO: 68 , 94%, 95%, 96%, 97%, 98%, 99% or more identical sequences, or have one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid sequences of conservative amino acid mutations (preferably substitutions, insertions or deletions).
  • a polynucleotide characterized in that it encodes the antibody or antigen-binding fragment thereof according to any one of schemes 1-2.
  • a recombinant vector, transgenic cell line, phage, recombinant bacteria or viral vector characterized in that it contains the polynucleotide according to Scheme 8.
  • An isolated host cell characterized in that it contains the recombinant vector, transgenic cell line, phage, recombinant bacteria or viral vector according to Scheme 9.
  • An antibody expression method characterized in that, using the recombinant vector, transgenic cell line, phage, recombinant bacteria or viral vector according to the scheme 9 to express in the host cell according to any one of the schemes 11-15 antibody protein.
  • antibody or antigen-binding fragment thereof is selected from the group consisting of monoclonal antibody or antigen-binding fragment thereof, polyclonal antibody or antigen-binding fragment thereof, humanized antibody or antigen-binding fragment thereof, primatized antibody or antigen-binding fragment thereof, bispecific antibody or antigen-binding fragment thereof, multispecific antibody or antigen-binding fragment thereof, dual variable Immunoglobulin domains, monovalent antibodies or antigen-binding fragments thereof, chimeric antibodies or antigen-binding fragments thereof, single-chain Fv molecules (scFv), bispecific antibodies, trispecific antibodies, nanobodies, antibody-like protein scaffolds, structures Domain antibodies, Fv fragments, Fab fragments, F(ab')2 molecules, and tandem scFv.
  • scFv single-chain Fv molecules
  • An antibody-drug conjugate characterized in that the antibody or antigen-binding fragment thereof according to any one of schemes 1-2 is optionally coupled to a therapeutic agent through a linker.
  • a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of schemes 1-2 and a pharmaceutically acceptable carrier, diluent or excipient.
  • composition according to scheme 23 wherein said composition further comprises an additional therapeutic agent.
  • the tumor is selected from the group consisting of ovarian cancer, melanoma, prostate cancer, intestinal cancer, gastric cancer, esophageal cancer, breast cancer, lung cancer, kidney cancer, pancreatic cancer, and uterine cancer , liver cancer, bladder cancer, cervical cancer, oral cancer, brain cancer, testicular cancer, skin cancer, colorectal cancer, malignant glioma, thyroid cancer.
  • the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to TNFR2 with high affinity.
  • the antibody can mediate the killing activity on Treg cells, mediate the proliferation and activation of CD8+ T cells, and have excellent anti- Tumor efficacy.
  • Figure 1 is a schematic diagram of the FACS binding of a rat antibody to hTNFR2;
  • Figure 2 is a schematic diagram of ligand blocking ELISA detection of rat antibody binding to hTNFR2;
  • Figure 3 is a schematic diagram of the killing activity of Treg cells mediated by anti-TNFR2 antibodies
  • Figure 4 is a schematic diagram of anti-TNFR2 antibody-mediated proliferation of CD8+ T cells
  • Figure 5 is a schematic diagram of the activation of CD8+ T cells mediated by anti-TNFR2 antibodies
  • Figure 6 is a schematic diagram of the in vivo anti-tumor efficacy evaluation of chimeric antibodies
  • FIG. 8 In vivo drug efficacy experiment of mouse MC38 transplanted tumor mouse model - body weight change diagram
  • FIG. 9 In vivo drug efficacy experiment in mouse B16-F10 transplanted tumor mouse model - graph of tumor volume change.
  • isolated antibodies including murine antibodies, chimeric antibodies, human antibodies, that specifically bind a particular epitope on TNFR2 (eg, human TNFR2).
  • the BMK in this paper includes BMK2 and BMK6.
  • the sequences are respectively from: SEQ ID NO:3 and SEQ ID NO:4 in WO2017083525A1 patent; SEQ ID NO:150 and SEQ ID NO:151 in WO2020061210A1 patent.
  • TNFR2 and TNFR1 jointly mediate the activity of TNF ⁇ .
  • TNFR1 is a membrane-bound protein of 55 kD
  • TNFR2 is a membrane-bound protein of 75 kD.
  • TNFR2 can regulate the binding of TNF ⁇ to TNFR1 and thus regulate the level of TNF ⁇ necessary to stimulate the action of NF-kB.
  • TNFR2 can also be cleaved (or undergo alternative splicing) by metalloproteases to generate a soluble receptor that maintains affinity for TNF ⁇ .
  • the present invention also provides a composition. It contains the antibody of the present invention or its active fragment, and a pharmaceutically acceptable carrier.
  • the materials are formulated in a nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, the pH of which can vary with the nature of the material to be formulated and the condition to be treated.
  • the prepared pharmaceutical composition can be administered through conventional routes, including but not limited to: intratumoral, intraperitoneal, intravenous, or topical administration.
  • the pharmaceutical composition of the invention can be directly used for binding TNFR2 protein molecules, and thus can be used for treating tumors.
  • other therapeutic agents may also be used concomitantly.
  • the pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99.999wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the above-mentioned antibody of the present invention or its binding fragment (or its conjugate) and pharmaceutical acceptable carrier or excipient.
  • Such carriers include, but are not limited to: saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical formulation should match the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of injection, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably produced under sterile conditions.
  • the amount of active ingredient administered is a therapeutically effective amount.
  • the polypeptides of the invention can also be used with other therapeutic agents.
  • the antibodies of the invention can be used alone, or combined or conjugated with a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or a combination of any of these.
  • Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or substances capable of producing a detectable product. enzyme.
  • Therapeutic agents that can be combined or coupled with the antibody of the present invention include, but are not limited to: 1. Radionuclide; 2. Biotoxin; 3. Cytokines such as IL-2, etc.; 4. Gold nanoparticles/nanorods; 5. Viruses 6.
  • Liposomes 7. Nanomagnetic particles; 8. Prodrug-activating enzymes (eg, DT-diaphorase (DTD) or biphenylhydrolase-like protein (BPHL)); 9. Chemotherapeutic agents (eg, , cisplatin) or any form of nanoparticles, etc.
  • DTD DT-diaphorase
  • BPHL biphenylhydrolase-like protein
  • an "antibody” refers to a natural immunoglobulin or an immunoglobulin produced by partial or complete synthesis.
  • Antibodies can be isolated from natural sources such as plasma or serum in which the antibodies are naturally present, or culture supernatants of hybridoma cells producing the antibodies. Alternatively, it can be partially or completely synthesized by using a technique of gene recombination or the like.
  • Preferred antibodies include, for example, antibodies to immunoglobulin isotypes or subclasses of these isotypes.
  • Known human immunoglobulins include nine classes (isotypes) of IgG1, IgG2, IgG3, IgG4, IgAl, IgA2, IgD, IgE, and IgM. Among these isotypes, antibodies of the invention may include IgGl, IgG2, IgG3, IgG4.
  • antibody or “immunoglobulin” used interchangeably herein include whole antibodies and any antigen-binding fragment (antigen-binding portion) or single chains thereof.
  • An “antibody” comprises at least one heavy (H) chain and one light (L) chain.
  • these heavy and light chains are interconnected by disulfide bonds, and there are two pairs of heavy and light chains in pairs. These two are also interconnected by disulfide bonds, and each heavy chain consists of a heavy chain variable region (abbreviated here as VH) and a heavy chain constant region, which consists of three domains CH1, CH2 and CH3 .
  • VH heavy chain variable region
  • CH1 heavy chain constant region
  • the light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region, which consists of one domain, CL, and the VH and VL regions can be further subdivided into hypervariable regions called complementary decision area.
  • Regions (CDRs) interspersed with more conserved regions called framework regions (FR) or joining regions (J) (JH or JL in heavy and light chains, respectively)
  • each VH and VL consists of three CDRs , three FRs and one J domain, which are arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, J.
  • the variable regions of the heavy and light chains bind antigen.
  • the constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) or humoral factors, such as the first part (CIq) of the classical complement system. Fragments of full-length antibodies have been shown to perform the antigen-binding function of the antibody. Examples of binding fragments expressed as antigen-binding portions or antibody fragments include (i) Fab fragments, monovalent fragments consisting of: (ii) F(ab') fragments, comprising two disulfide bonds linked by a hinge region.
  • the two domains VL and VH of the Fv fragment are encoded as separate genes, they can be linked using recombinant methods through a synthetic linker that makes them a single protein chain in which the VL and VH regions pair to form a monovalent molecule
  • Single chain homologues of such immunoglobulin fragments are such single chain antibodies and are also intended to be encompassed within the term "antibody”.
  • Antibody fragments are obtained using conventional techniques known to those skilled in the art and screened for the presence of such fragments in the antibody. The utility in.
  • Antigen-binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins. The numbering of amino acid positions (such as amino acid residues in the Fc region) and target regions in the antibodies described herein, such as CDR, using the Kabat system.
  • isotype refers to the antibody class (eg, IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD, and IgE antibodies) encoded by the heavy chain constant region genes.
  • recombinant antibody refers to an antibody prepared, expressed, produced or isolated by recombinant means, for example (a) from an animal (eg, mouse) that is transgenic or transchromosomal for an immunoglobulin gene (eg, human immunoglobulin) Antibody genes or hybridomas made therefrom, (b) antibodies isolated from host cells transformed to express the antibodies, e.g., from transfectomas, (c) using phage display from a combined combinatorial antibody library, , comprising human antibody sequences) isolated antibodies, (d) antibodies prepared, expressed, produced or isolated by any other means of splicing immunoglobulin gene sequences (eg, human immunoglobulin genes) to other DNA sequences.
  • a from an animal (eg, mouse) that is transgenic or transchromosomal for an immunoglobulin gene (eg, human immunoglobulin) Antibody genes or hybridomas made therefrom
  • antibodies isolated from host cells transformed to express the antibodies e.g., from transfectomas
  • Such recombinant antibodies may have variable and constant regions derived from human germline immunoglobulin sequences.
  • such recombinant human antibodies may be subjected to in vitro mutagenesis; the VH and VL regions of the recombinant antibodies are sequences derived from and related to human germline VH and VL sequences, but in vivo the human antibody species May not occur naturally in the library.
  • chimeric immunoglobulin refers to an immunoglobulin or antibody whose variable regions are derived from a first species and whose constant regions are derived from a second species. Chimeric immunoglobulins or antibodies can be constructed, eg, by genetic engineering, from immunoglobulin gene segments belonging to different species.
  • humanized antibody refers to an antibody that includes at least one humanized antibody chain (ie, at least one humanized light or heavy chain).
  • humanized antibody chain i.e., “humanized immunoglobulin chain” refers to an antibody chain (i.e., a light chain or a heavy chain, respectively) having a variable region comprising substantially all of the human antibody Variable framework regions and complementarity determination. Regions (CDRs) derived substantially from a non-human antibody (eg, at least one CDR, two CDRs or three CDRs).
  • the humanized antibody chain also includes constant regions (eg, one constant region or portion thereof in the case of a light chain, preferably three constant regions in the case of a heavy chain).
  • human antibody is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human immunoglobulin sequences. Furthermore, if the antibody comprises a constant region, the constant region is also derived from human germline immunoglobulin sequences. Human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (eg, mutations introduced by random site-specific mutagenesis in vitro or somatic mutation in vivo).
  • Human antibodies may have at least one or more amino acids substituted with amino acid residues, eg, activity-enhancing amino acid residues not encoded by human immunoglobulin sequences. Typically, human antibodies can have as many as twenty positions substituted with amino acid residues that are not part of the human germline immunoglobulin sequence. In specific embodiments, these substitutions are within CDR regions. In some other embodiments, the substitutions are within the framework regions.
  • multispecific or “multifunctional antibody” is an artificial hybrid antibody having multiple different binding sites; sites.
  • Bispecific antibodies can be produced by a variety of methods, including fusion of hybridomas or linking of Fab' fragments.
  • isolated is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities.
  • isolated antibodies typically are substantially free of other cellular material and/or chemicals.
  • Fc region refers to the C-terminal region of an antibody heavy chain.
  • the Fc region comprises the constant region of the antibody, but does not include the first constant region immunoglobulin domain (eg, CH1 or CL).
  • the term "antigen" is an entity (eg, a protein entity or a peptide) to which an antibody binds, eg, TNFR2.
  • the terms “specifically binds” and “selectively binds” mean that an antibody exhibits significant affinity for a particular antigen or epitope, and generally does not exhibit significant cross-reactivity with other antigens or epitopes.
  • "Appreciable” or preferred binding includes binding with a KD of 10" 7 , 10" 8 , 10" 9 or 10" 10 M or more preferably.
  • the KD (affinity constant) of the antibody-antigen interaction represents the antibody concentration at which 50% of the antibody and antigen molecules bind together.
  • 50% of the higher affinity (i.e., stronger) antibody binds the antigen at a lower antibody concentration than would be required to achieve the same percentage binding with a lower affinity antibody molecular.
  • a lower KD value indicates a higher (stronger) affinity.
  • a "better" affinity is stronger and numerically lower than its affinity, which has a lower numerical value for its KD of 10 ⁇ 7 M and therefore better affinity compared to a KD of 10 ⁇ 6 M. It is generally preferred to have a KD value of less than 10 ⁇ 7 M , thus preferably greater than 10 ⁇ 8 M, intermediate values as described herein are also contemplated, and preferred binding affinities can be expressed as a range of affinities, e.g. for the antibodies disclosed herein
  • the TNFR2 antibody is 10 -7 to 10 -12 M, more preferably 10 -8 to 10 -12 M.
  • an antibody that "exhibits no appreciable cross-reactivity” or “does not bind with a physiologically relevant affinity” is an antibody that does not significantly bind the antibody.
  • Off-target antigens eg, non-TNFR2 proteins
  • Specific or selective binding can be determined according to any technique in the art. Recognized methods for determining such binding include, for example, based on Scatchard analysis, biomacromolecular interaction assays, biofilm layer interferometry and/or competitive (competition) binding assays.
  • epitope means an antigenic determinant in an antigen, and refers to an antigenic site to which a domain of an antigen-binding molecule comprising an antibody variable region disclosed in this specification binds.
  • an epitope can be defined in terms of its structure.
  • the epitope can also be defined based on the antigen-binding activity of the antigen-binding molecule that recognizes the epitope.
  • the antigen is a peptide or polypeptide
  • the epitope can be specified by the amino acid residues forming the epitope.
  • the epitope is a sugar chain, the epitope can be identified by its specific sugar chain structure.
  • a linear epitope is an epitope whose primary amino acid sequence is recognized. Such linear epitopes usually contain at least three, most often at least five, eg about 8 to 10 or 6 to 20 amino acids in their specified sequence.
  • “conformational epitopes” are epitopes in which the primary amino acid sequence containing the epitope is not the only determinant of the recognized epitope (e.g., the primary amino acid sequence of a conformational epitope is not necessarily defined by the epitope antibody recognition).
  • a conformational epitope may contain a greater number of amino acids than a linear epitope.
  • Conformational epitope recognizing antibodies recognize the three-dimensional structure of a peptide or protein. For example, when a protein molecule folds and forms a three-dimensional structure, the amino acids and/or polypeptide backbones that form a conformational epitope become aligned and the epitope can be recognized by antibodies.
  • Methods for determining epitope conformation include, for example, but are not limited to, X-ray crystallography, two-dimensional nuclear magnetic resonance, site-specific spin labeling, and electron paramagnetic resonance.
  • vector is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double-stranded DNA loop. Other DNA fragments can be ligated into it.
  • viral vector a type of vector
  • certain vectors are capable of autonomous replication in the host cells into which they are introduced (eg, bacterial vectors and episomal mammalian vectors of bacterial origin with replication).
  • Other vectors eg, non-exogenous mammalian vectors
  • vectors are capable of directing the expression of the genes they express. These vectors are referred to herein as “recombinant expression vectors” (or simply “expression vectors”).
  • expression vectors useful in recombinant DNA techniques are usually in the form of plasmids.
  • plasmid and vector
  • other forms of expression vectors such as viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions, are also contemplated.
  • inhibitor refers to any statistically significant reduction in biological activity, including partial and complete blocking of that activity.
  • “inhibition” can mean a statistically significant decrease of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97% , 98%, 99% or 100% biological activity.
  • activation refers to any statistically significant biological activity that activates cells, for example, “activation” can mean a statistically significant increase in biological activity, about 1%, 10%, 20%, 30% , 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, 1000%, 10000% and above biological activity.
  • the phrase "inhibits the binding of a TNFR2 ligand to TNFR2” refers to the ability of the antibody to statistically significantly reduce the binding of a TNFR2 ligand (eg, TNF ⁇ ) to TNFR2 relative to the absence of the TNFR2 antibody. In other words, in the presence of antibody, there is a statistically significant decrease in the amount of TNFR2 ligand bound to TNFR2 relative to the control (no antibody).
  • a TNFR2 ligand eg, TNF ⁇
  • the amount of TNFR2 ligand bound to TNFR2 can be reduced by at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or relative to the absence
  • the amount of antibody (control) is at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or about 100%.
  • the reduction in TNFR2 ligand binding can be measured using art-recognized techniques that measure the binding of labeled TNFR2 ligand (e.g., radiolabeled TNF ⁇ ) to cells expressing TNFR2 in the presence or absence of (control) antibodies. combined level.
  • the term "inhibiting tumor growth” includes any measurable reduction in tumor growth, eg, inhibition of tumor growth by at least about 10%, eg, at least about 20%, at least about 50%. 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 99%, or about 100%.
  • treatment refers to the treatment or prophylaxis described herein.
  • the method of “treating” is for administering to a subject or subject predisposed to a tumor or cancer.
  • an anti-TNFR2 antibody e.g., an anti-human TNFR2 antibody
  • an anti-TNFR2 antibody is the disease or disorder, or to prolong Survival of the subject such that it would prolong survival without this treatment.
  • variable fragment refers to the smallest unit of an antibody-derived antigen-binding domain, which consists of a pair of antibody light chain variable region (VL) and antibody heavy chain variable region (VH) .
  • VL antibody light chain variable region
  • VH antibody heavy chain variable region
  • Fv preferably includes, for example, a pair of Fv as an antigen-binding molecule or the like comprising: scFv, single-chain antibody and sc(Fv)2.
  • scFv single chain antibody
  • sc(Fv)2 all refer to antibody fragments of a single polypeptide chain, containing variable regions derived from heavy and light chains, but no constant regions.
  • single chain antibodies also contain a polypeptide linker between the VH and VL domains, which enables formation of the desired structure believed to allow antigen binding.
  • CHOK1-hTNFR2 refers to a cell constructed by a certain technique.
  • the specific construction methods include the following methods, but are not limited to the following methods. Specific steps: (1) Gene synthesis and molecular construction: Jinweizhi optimized the codon and synthesized the sequence of human TNFR2 protein. The synthetic gene was further subcloned into the modified pSBbi-GB vector. (2) Transient transfection: One day before transfection, prepare 1.0 ⁇ 10 ⁇ 6 CHOK1 cells with viability higher than 95% in a T25 culture flask, and start transfection at 70%-90% confluence.
  • the concentration of blasticidin was 10ug/mL. Change the medium every 2-3 days. After recovery from 2 to 3 weeks of antibiotic selection, a stable pool of cells will result. Stable single cell lines were further generated by BD FACS Melody sorting. Cells are first counted and their viability measured. After incubation with primary and secondary antibodies, single cells were sorted into 96-well plates and grown in an incubator until colonies were visualized. After FACS detection, high-expression single clones were amplified and incorporated into the library. (4) FACS detection: Transiently transfected cells were transferred to a 96-well U-bottom plate (Corning-3799) at a density of 2x10 ⁇ 5 cells/well.
  • Anti-TNFR2 antibody was diluted at 2ug/mL in 2% FBS/1XPBS, 100 ⁇ L per well, and then incubated at 4°C for 1 hour. Cells were washed twice and resuspended in 100 ⁇ L 2% BSA/1XPBS. The secondary antibody (goat anti-human IgG Fc-Alexa647) was diluted 1:500 in 2% FBS/1XPBS, 100 ⁇ L per well, and then incubated at 4°C for 30 minutes. Cells were washed twice and resuspended in 100 ⁇ L 2% FBS/1XPBS. Fluorescence was measured by flow cytometry (BD Canto II) and analyzed by FlowJo. (5) Stability test: the cell bank or cell line was passaged for more than 3 weeks after P1 for stability test. FACS was used for stability confirmation.
  • Treg Treg cells
  • suppressor T cells which are characterized by the expression of the biomarkers CD4, FOXP3 and CD25, representing the regulation of the immune system, maintenance of Antigen tolerance and protection of T cell subsets in autoimmune disease.
  • Tregs are immunosuppressive and generally suppress or downregulate the induction and proliferation of effector T (Teff) cells.
  • Tregs can develop in the thymus (so-called CD4 + Foxp3 + "native" Tregs) or differentiate from naive CD4 + T cells in the periphery, for example, after exposure to TGF ⁇ or retinoic acid.
  • Teff Teff cell
  • effector T cell is a cell formed by proliferation and differentiation of T cells after being stimulated by an antigen. Effector T cells have the function of releasing lymphokines, and in this process, a small part of T cells become memory T cells. Effector T cells contact with target cells to stimulate granule exocytosis, and the released perforin forms pores on the surface of target cells through polymerization, thereby mediating the killing effect. The process of target cell death is similar to apoptosis. At the same time, effector T cells can also release immune active substances - lymphokines, such as interleukin, interferon and so on.
  • immune active substances - lymphokines such as interleukin, interferon and so on.
  • Recombinant human TNFR2-His Tag protein (Sino, Cat: 10417-H08H) was used as the immunogen to immunize SD rats. In order to avoid poor response or apoptosis during the immunization process, 3-4 rats could be immunized at the same time. Negative serum was taken 1 day in advance, and for the first immunization, 60 ⁇ g recombinant human TNFR2-His Tag protein fully emulsified in Freund's complete adjuvant was injected at multiple points by subcutaneous immunization, intraperitoneal immunization and plantar immunization, and subcutaneous immunization and foot immunization were performed on the 23rd day.
  • the basic immunization method was to inject 40 ⁇ g recombinant human TNFR2-His Tag protein fully emulsified with Freund’s complete adjuvant in multiple points for the second immunization, and to inject 40 ⁇ g immunogen in the same way as the second immunization every two weeks for the third and fourth times.
  • the first, fifth, sixth and seventh immunizations After 30 days, antiserum titers were assessed by testing sera collected from tail bleeds in recombinant human TNFR2-His Tag protein-coated ELISA plates at various dilutions from 1:100 to 1:1,000,000. When the titer results meet the requirements and the anti-human TNFR2 antibody is detected at a dilution >1:1,000,000, the rat spleen and lymph nodes can be harvested for cell fusion.
  • Myeloma cells SP2/0 (ATCC) were subcultured one day before fusion, so that the cells were in the logarithmic growth phase during the experiment. Before fusion, the cells were collected in a centrifuge tube, centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded And add 10mL DMEM complete medium to mix the cells for later use.
  • the spleen cells and lymph node cells used in the experiment were obtained from SD rats immunized seven times with recombinant human TNFR2-His Tag protein. The cell suspension was centrifuged at 2000 rpm for 8 minutes, the supernatant was discarded and DMEM complete medium was added to mix the cells for further use.
  • the feeder cells used in the fusion selection culture were obtained from macrophages in the peritoneal cavity of unimmunized animals. Before fusion, the collected macrophage suspension was centrifuged at 100 rpm for 8 minutes, the supernatant was discarded and 25 mL of HAT was added. The selection medium was mixed and divided into two 24-well culture plates to assist the growth of new hybrid B lymphocyte hybridomas.
  • B lymphocytes and myeloma cells were mixed at a ratio of 1:2, and the suspension was centrifuged at 1000 rpm for 8 minutes, and the precipitate was collected and washed twice with electrofusion solution. Then take the precipitation plus electrofusion solution to 1.2mL, and inject 0.4mL of cell suspension into each of the three multi-electrode cuvettes.
  • the AC electric field sinusoidal signal frequency of dielectric electrophoresis is 1MHz, the amplitude is 250V/cm, and it is applied for 30 seconds; then immediately add a perforating RC electric pulse with an amplitude of 5kV/cm and a pulse width of 20 ⁇ s , the number of pulses is 3, and the time interval is 1 second. Let stand at room temperature for 10 minutes, then wash out the fusion with a total of 5 mL of PFM solution, and incubate at 37° C. for 30 minutes.
  • centrifuge at 100 rpm for 8 minutes, take the precipitate, add 500mL HAT to mix and dispense into 96-well culture plate, the number of cells is 1 ⁇ 104/well (the existing feeder cells are 1.2* 104 /well) , placed in a CO 2 incubator at 37°C.
  • Example 3 Indirect ELISA method for screening positive clones
  • Positive cell clones were screened by indirect ELISA. Square array test was used to determine the coating concentration for detection of recombinant human TNFR2-His Tag protein (Sino; Cat: 10417-H08H).
  • the detection antigen coating buffer was diluted horizontally, 50 ⁇ L per well to coat the ELISA plate, overnight at 4°C; washed 3 times with PBST, and 200 ⁇ L of blocking solution was added to each well, overnight at 4°C; the serum of the immune mice was longitudinally diluted, 50 ⁇ L per well, The same multiple dilution of normal rat serum was used as a negative control, and incubated at 37°C for 2 hours; washed with PBST for the third time, added enzyme-labeled secondary antibody at a working concentration, 50 ⁇ L per well, incubated at 37°C for 1.5 hours, after washing with PBST, TMB developed color, The enzyme-linked detector measures the value of OD450 to determine the optimal coating concentration of the detection antigen.
  • the established indirect ELISA method was used to detect the antibody secreted by the hybridoma cells.
  • the specific method is as follows: add the hybridoma cell culture supernatant to the ELISA plate pre-coated with recombinant human TNFR2-His Tag protein (Sino; Cat: 10417-H08H), 50 ⁇ L/well, and use the SP2/0 cell supernatant as negative Control, immune polyantiserum as a positive control, 37 ° C water bath for 2 h; PBST washed 3 times; add the working concentration of HRP-labeled goat anti-mouse IgG and IgM antibodies, 50 ⁇ L / well, 37 ° C water bath for 1.5 h; after washing, TMB showed Color for 10 minutes, and display the OD450 reading of the microplate reader after the termination. The OD450 reading of the tested well is more than two times greater than that of the negative control, which is judged as positive.
  • Embodiment 4 FACS method further screens positive clone
  • CHOK1 and Flpin-T-Rex293F cell lines were transfected with lentiviral vectors carrying human TNFR2, monkey TNFR2 or mouse TNFR2.
  • Cells were sorted to establish human TNFR2, monkey TNFR2 and mouse TNFR2 stable expression cell lines, CHOK1-hTNFR2, CHOK1-cTNFR2 and Flpin-T-Rex293F-mTNFR2.
  • Monoclonalization of hybridoma cells was carried out by two rounds of limiting dilution method, and the binding activity of antibodies produced by subcloned cells to human and monkey TNFR2 proteins was determined by ELISA and FACS detection methods.
  • Embodiment 5 the preparation of antibody
  • the monoclonal antibody is mainly obtained by in vitro culture. Expand the cell line to a T75 culture flask, culture until the cell coverage rate is 80-90%, discard the cell supernatant, add 30mL hybridoma production medium (hybridoma-SFM, Gibco), 37 ° C, 5% CO2 culture. After 2-3 days of culture, add hybridoma production medium (hybridoma-SFM, Gibco), if the cell viability is lower than 30%, fresh living cells can be added. After culturing for 6-7 days, when the cell survival rate is lower than 20%, the culture supernatant is collected after low-speed centrifugation, and stored at 4°C for future use.
  • hybridoma production medium hybridoma production medium
  • Example 6 FACS binding activity of anti-TNFR2 antibodies to human TNFR2
  • CHOK1-hTNFR2 cells were plated at 1 ⁇ 10 5 cells/well (containing 1% BSA). Add 100 uL of isotype control and candidate antibody at a concentration of 100 nm, and dilute 11 gradients (including 1% BSA) by 4 times. Cells were incubated at 4°C for 1 hour and then washed twice with excess FACS buffer. The cells were resuspended in 100 ⁇ L of FACS buffer, added goat anti-rat IgG Fc-AF647 (1:500), 100 ul in 1% BSA, incubated at 4° C. in the dark for 30 minutes and washed twice with excess FACS buffer. Cells were fixed in fixation buffer and then analyzed by flow cytometry. Candidate antibodies with high specific binding activity to human TNFR2 were screened by FACS method. As shown in Table 1 and Figure 1, 11 sequences showed comparable binding ability to hTNFR2 compared to BMK2.
  • Antibody EC50(nM) maximum mean fluorescence parental cells Antibody 01 0.045 921 92.9 Antibody 02 0.067 1239 65 Antibody 03 0.045 383 64 Antibody 04 0.025 1322 61.3 Antibody 05 0.038 949 64.2 Antibody 06 0.077 1342 74.5 Antibody 07 0.050 1392 64.7 Antibody 08 0.081 1481 71 Antibody 09 0.065 1370 68.9 Antibody 10 0.077 907 64.1 Antibody 11 0.493 1007 76 BMK2 0.051 1610 135 uIgG1 isotype control >100 97 134
  • Coat anti-His antibody (1ug/mL), 50ul/well, wash 3 times with PBST after overnight incubation at 4°C.
  • Add recombinant human TNFR2-His Tag protein (Sino; Cat: 10417-H08H), 0.25ug/mL (containing 2% BSA), 50ul/well, incubate at room temperature for 1 hour, and wash 3 times with PBST.
  • the TMB was developed for 10 minutes, and the OD450 reading of the microplate reader was displayed after the termination. According to the experimental results, it is judged whether the candidate antibody has ligand blocking activity. As shown in Table 2 and Figure 2, 2 sequences showed better ligand blocking ability than BMK2; 6 sequences showed ligand blocking ability comparable to BMK2.
  • Antibody IC50(nM) Inhibition rate(%) Antibody 01 2.72 93.2 Antibody 02 0.962 96.2 Antibody 03 10.4 91.6 Antibody 04 0.584 96.0 Antibody 05 1.27 95.9 Antibody 06 1.79 96.4 Antibody 07 >100 15.4 Antibody 08 1.40 96.1 Antibody 09 1.18 96.1 Antibody 10 >100 12.2 Antibody 11 >100 22.4 BMK2 1.54 95.3 uIgG1 isotype control >100 11.7
  • Embodiment 8 Affinity detection of anti-TNFR2 antibody and human TNFR2
  • Activator preparation Prepare by mixing 400mM EDC and 100mM NHS(GE) immediately before use.
  • CM5 sensor chip for 420 s at a flow rate of 10 ⁇ L/min.
  • the channel was then injected with 30 ⁇ g/mL anti-rat Fc IgG in 10 mM NaAc (pH 4.5) at a flow rate of 10 ⁇ L/min.
  • Chip 420s was inactivated with 1M ethanolamine-hydrochloric acid (GE) at a flow rate of 10 ⁇ L/min.
  • GE ethanolamine-hydrochloric acid
  • Candidate antibodies in running buffer 1 ⁇ HBS-EP+(10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% Tween 20, pH7.4) were captured onto Fc2 with anti-rat Fc IgG at a flow rate of 10 ⁇ L/min.
  • 10nm recombinant human TNFR2-His Tag protein (Sino; Cat: 10417-H08H) and running buffer were sequentially injected into Fc1-Fc2 at a flow rate of 30min, combined for 180s, and then dissociated for 3600s.
  • 10 mM glycine (pH 1.5) was injected as regeneration buffer after each dissociation.
  • Chips were regenerated with 10 mM glycine (pH 1.5).
  • CD4+ T cells were isolated from peripheral blood mononuclear cells (PBMC) of healthy volunteers.
  • PBMC peripheral blood mononuclear cells
  • PE-anti-CD25 (1:50) and Alexa Fluor 488-anti-Foxp3 (1:50) were used for staining, and BD FACSC anto II flow cytometer was used for counting and analysis.
  • Table 4 and Figure 3 the six antibody sequences all showed excellent killing activity on Treg cells.
  • Coating candidate antibody (containing 5ug/mL OKT3), 20ug/mL, 4-fold dilution in 3 gradients, incubated overnight at 4°C.
  • Counting analysis was performed using a BD FACS Canto II flow cytometer. The results are shown in Figure 4, the six antibody sequences all showed excellent effects on promoting the proliferation of CD8+ T cells.
  • Coating candidate antibody (containing 5ug/mL OKT3), 20ug/mL, 4-fold dilution in 3 gradients, incubated overnight at 4°C.
  • the release of IFN ⁇ and IL-2 were detected by ELISA, respectively.
  • the results are shown in Figure 5, all of the six antibody sequences showed excellent effects on promoting the activation of CD8+ T cells.
  • Antibody 04 VH SEQ ID NO:01
  • Antibody 04 VL SEQ ID NO:05
  • Antibody 10 VH SEQ ID NO:09
  • Antibody 10 VL SEQ ID NO:13
  • Antibody 11 VH SEQ ID NO:17
  • DNA fragment is light chain DNA, gene synthesis of heavy chain variable region and human IgG1 heavy chain constant region (SEQ ID NO:70)
  • the DNA fragment is heavy chain DNA.
  • the pcDNA3.1-G418 vector contains the promoter CMV Promoter, the eukaryotic screening marker G418 tag and the prokaryotic screening tag Ampicillin.
  • the recombinant plasmids containing the above-mentioned genes of interest were transformed into Escherichia coli competent cells DH5 ⁇ , and the transformed bacteria were coated on a medium containing 100 ⁇ g/mL ampicillin.
  • Example 14 In vivo anti-tumor efficacy evaluation of chimeric antibodies
  • Antibody 04-xIgG1K, antibody 10-xIgG1K, and antibody 11-xIgG1K were evaluated for their antitumor efficacy in the mouse colon cancer cell line MC38 transplanted tumor C57BL/6-hTNFR2 humanized mouse model.
  • Mouse colon cancer MC38 cells were cultured in a single layer in vitro, and the culture conditions were RPMI1640 medium plus 10% fetal bovine serum, 2mm glutamine, and cultured at 37°C and 5% CO 2 . Routine digestion with trypsin-EDTA was performed twice a week for passaging. When the cell saturation is 80%-90%, collect the cells, count and inoculate. 0.1 mL (5 ⁇ 10 5 cells) of MC38 cells were subcutaneously inoculated on the right back of each mouse, and the administration began when the average tumor volume reached 60 mm3.
  • mice intraperitoneal injections of antibody 04-xIgG1K, antibody 10-xIgG1K, and antibody 11-xIgG1K, twice a week, 2.0 mg/kg each time, and give the same dose of human IgG as a control, and co-administer 5 times.
  • Routine inspections include observation of tumor growth and the impact of drug treatment on the daily behavior of the animals, such as behavioral activities, food and water intake (visual inspection only), and body weight changes (measure body weight twice a week) , appearance signs or other abnormal conditions. Based on the number of animals in each group, the number of apoptotic animals and side effects in each group were recorded.
  • the experimental index is to investigate whether tumor growth is inhibited, delayed or cured.
  • Tumor diameters were measured with vernier calipers three times a week.
  • the antitumor efficacy of compounds was evaluated by TGI (%) or relative tumor proliferation rate T/C (%).
  • TGI (%) reflects tumor growth inhibition rate.
  • TGI (%) [(1-(Average tumor volume at the end of administration of a certain treatment group-Average tumor volume at the beginning of administration of this treatment group))/(Average tumor volume at the end of treatment of the solvent control group Volume - average tumor volume at the beginning of treatment in the solvent control group)] ⁇ 100%.
  • Relative tumor proliferation rate T/C (%): the calculation formula is as follows: T/C% TRTV/CRTV ⁇ 100% (TRTV: RTV of the treatment group; CRTV: RTV of the negative control group).
  • the average tumor volume of TRTV and CRTV take the data on the same day.
  • the mouse colon cancer cell line MC38 xenograft tumor model tumor-bearing mice were given human IgG control respectively, and the tumor growth curves of antibody 04-xIgG1K, antibody 10-xIgG1K, and antibody 11-xIgG1K are as shown in Figure 6, where the abscissa indicates that after the start of treatment days, the vertical axis represents the tumor volume.
  • antibody 04-xIgG1K, antibody 10-xIgG1K, antibody 11-xIgG1K and BMK6 have equivalent or better anti-tumor efficacy in vivo, and the in vivo tumor efficacy of antibody 11-xIgG1K is significantly better than BMK6.
  • VH and VL sequences were compared to the library of known human germline sequences on the NCBI website (https://www.ncbi.nlm.nih.gov/igblast/).
  • the databases used were IMGT human VH gene and IMGT human VL ⁇ gene.
  • human germline IGVH1-2 was selected as the acceptor sequence
  • the human light chain IGKJ4 (allele 1) junction region (J gene) was selected from the international ImMunoGeneTics information Human junction region sequences compiled at http://www.imgt.org.
  • human germline IGKV1-16 was selected as the acceptor sequence
  • human light chain IGKJ2 (allele 1) junction region (J gene) was selected from the international ImMunoGeneTics information Human junction region sequences compiled at http://www.imgt.org.
  • human germline IGVH3-15 was selected as the acceptor sequence
  • human light chain IGKJ4 (allele 1) junction region (J gene) was selected from the international ImMunoGeneTics information Human junction region sequences compiled at http://www.imgt.org.
  • human germline IGKV1-27 was selected as the acceptor sequence
  • human light chain IGKJ2 allele 1 junction region (J gene) was selected from the the international ImMunoGeneTics information Human junction region sequences compiled at http://www.imgt.org.
  • human germline IGVH1-69 was selected as the acceptor sequence
  • human light chain IGKJ4 allele 1 junction region (J gene) was selected from the international ImMunoGeneTics information Human junction region sequences compiled at http://www.imgt.org.
  • human germline IGKV3D-15 was selected as the acceptor sequence, and the human light chain IGKJ2 (allele 1) junction region (J gene) was selected from the international ImMunoGeneTics information Human junction region sequences compiled at http://www.imgt.org. CDRs were determined according to the AbM definition. Altering the human germline framework positions to the corresponding parental murine sequences optimizes binding of the humanized antibodies. Through the above methods, we obtained the following multiple humanized antibodies.
  • Antibody serial number Antibody 12 VH:SEQ ID NO:25; VL:SEQ ID NO:26 Antibody 13 VH: SEQ ID NO: 31; VL: SEQ ID NO: 32 Antibody 14 VH: SEQ ID NO: 34; VL: SEQ ID NO: 35 Antibody 15 VH: SEQ ID NO: 37; VL: SEQ ID NO: 38 Antibody 16 VH: SEQ ID NO: 41; VL: SEQ ID NO: 42 Antibody 17 VH: SEQ ID NO: 43; VL: SEQ ID NO: 44 Antibody 18 VH:SEQ ID NO:29; VL:SEQ ID NO:30 Antibody 19 VH: SEQ ID NO: 39; VL: SEQ ID NO: 40 Antibody 20 VH: SEQ ID NO: 46; VL: SEQ ID NO: 47 Antibody 21 VH: SEQ ID NO: 49; VL: SEQ ID NO: 50 antibody 22 VH: SEQ ID NO: 51; VL: SEQ ID NO: 52 Antibody 23 VH: SEQ ID
  • Blocking block with 3% skimmed milk powder, 300 ⁇ L/well, incubate at 37°C for 1 hour, discard the blocking solution, wash 4 times with a plate washer, and pat dry on flat paper.
  • sample dilution the reference product and the test product are diluted to 10 ⁇ g/mL with 3% skimmed milk powder, and the initial concentration is used as the initial concentration for 3-fold dilution, and a total of 11 gradients are diluted, and a blank well is set up, and only the dilution is added.
  • liquid 100 ⁇ L/well, incubate at 37°C for 1h. Discard the liquid in the wells, wash 4 times with a plate washer, and pat dry on flat paper.
  • Color development Add TMB color development solution, 100 ⁇ L/well, wrap it with aluminum foil, and develop color at 37°C in the dark for 8 minutes.
  • Stop color development add stop solution 1M HCl to stop color reaction, 100 ⁇ L/well.
  • the humanized antibody has comparable or better binding activity to the corresponding chimeric antibody, indicating that the antibody maintains high binding activity after humanization.
  • Sensor preparation soak the AHC sensor with 0.02% PBST (0.02% Tween 20, pH 7.4, 1*PBS) as a buffer solution for 600s before use, and remove the sucrose covered on the sensor surface.
  • PBST 0.02% Tween 20, pH 7.4, 1*PBS
  • the AHC sensor was equilibrated with 0.02% PBST (0.02% Tween 20, pH7.4, 1*PBS) as a buffer for 60s, the TNFR2 antibody in the sample plate was immobilized for 300s, and the secondary equilibrated buffer was 180s.
  • 100nm human W3785-hPro1.ECD.His (Human-TNFR2-His) protein was bound to TNFR2 antibody for 300s, and then dissociated for 600s. After dissociation, 10mM glycine (pH2.0) was used as regeneration buffer for 30s.
  • the sensor was regenerated with 10 mM glycine (pH 2.0).
  • Example 18 Drug efficacy in vivo in mouse colon cancer cell line MC38 transplanted tumor C57BL/6hTNFR2 mouse model
  • Mouse colon cancer MC38 cells were cultured in a single layer in vitro, and the culture conditions were DMEM medium plus 10% fetal bovine serum, 2mm glutamine, and cultured at 37°C and 5% CO 2 . Routine digestion with trypsin-EDTA was performed twice a week for passaging. When the cell saturation is 80%-90%, collect the cells, count and inoculate.
  • MC38 cells 0.1 mL (5x10 5 cells) were subcutaneously inoculated on the right back of each mouse, and the administration began when the average tumor volume reached 54 mm 3 .
  • the experimental index is to investigate whether tumor growth is inhibited, delayed or cured. Tumor diameters were measured three times a week.
  • the curative effect of tumor inhibition was evaluated by TGI (%). TGI (%) reflects tumor growth inhibition rate.
  • TGI (%) [(1-(Average tumor volume at the end of administration of a certain treatment group-Average tumor volume at the beginning of administration of this treatment group))/(Average tumor volume at the end of treatment of the solvent control group Volume - average tumor volume at the beginning of treatment in the solvent control group)] ⁇ 100%.
  • Example 19 In vivo drug efficacy of mouse melanoma cell line B16-F10 transplanted tumor C57BL/6hTNFR2 mouse model

Abstract

提供了以高亲和力与TNFR2特异性结合的抗体或其抗原结合片段。还提供了编码所述抗体或其抗原结合片段的核酸分子，用于表达所述抗体或其抗原结合片段的表达载体和宿主细胞，以及所述抗体或其抗原结合片段的生产方法。还提供了包含所述抗体或其抗原结合片段的抗体药物偶联物、药物组合物以及与其它治疗剂的联合疗法，以及使用所述抗体或其抗原结合片段制备治疗多种疾病的药物。

Description

一种抗TNFR2抗体及其制备方法和应用 技术领域
本发明属于生物医药领域，具体涉及一种抗TNFR2抗体及其制备方法和应用。
背景技术
最近的研究表明，通过调节免疫应答来增强人体自身抵抗疾病的能力是对传统治疗方法的一种有效的替代和/或补充。例如，研究表明，增强针对T淋巴细胞的活性以靶向和治疗各种疾病(例如，癌症或自身免疫性疾病)在治疗上是有效的。可以通过抑制Treg抑制T淋巴细胞活性的能力或者通过增强效应T细胞抑制肿瘤的能力来增强针对疾病的免疫应答。
肿瘤坏死因子受体2(TNFR2)，也称为TNFRSF1B和CD120b，是肿瘤坏死因子受体超家族(TNFRSF)的共刺激成员，其中包括诸如GITR，OX40，CD27，CD40和4-IBB(CD137)的蛋白质。TNFR2是在T细胞上表达的细胞表面受体，已被证明能增强效应T细胞的激活并减少Treg介导的抑制作用。通过调节TRAF2/3和NF-kB信号传导，TNFR2可以介导促进细胞存活和增殖的基因的转录。TNFR2可以在癌细胞，浸润肿瘤的Treg和效应T细胞上表达。本发明提供一种针对TNFR2靶点的抗体，用于增强针对肿瘤的免疫能力。
发明内容
针对现有问题的不足，本发明的第一个目的是提供一种抗TNFR2抗体或其抗原结合片段。
本发明的第二个目的是提供上述抗TNFR2抗体或其抗原结合片段的编码基因。
本发明的第三个目的是提供包含上述抗TNFR2抗体或其抗原结合片段的编码基因的载体。
本发明的第四个目的是提供包含上述抗TNFR2抗体或其抗原结合片段的编码基因载体的宿主细胞。
本发明的第五个目的是提供表达包含上述抗TNFR2抗体或其抗原结合片段的方法。
本发明的第六个目的是提供包含上述抗TNFR2抗体或其抗原结合片段的药物偶联物。
本发明的第七个目的是提供包含上述抗TNFR2抗体或其抗原结合片段和化学疗法的组合在制造用于治疗癌症或自身免疫性疾病药物方面的用途。
本发明的第八个目的是提供包含上述抗TNFR2抗体或其抗原结合片段的应用。
本发明的第九个目的是提供一种包含抗TNFR2抗体或其抗原结合片段的药物组合物。
本发明解决其技术问题采用的技术方案是：
1.一种能够特异性地结合人肿瘤坏死因子受体2(TNFR2)的抗体或其抗原结合片段，其中，包含重链和轻链，其特征在于，所述重链可变区包含重链互补决定区CDR1、CDR2和CDR3，所述轻链可变区包含轻链互补决定区CDR1、CDR2和CDR3，其中，
(01)所述重链可变区的CDR1、CDR2和CDR3分别由氨基酸序列SEQ ID NO：18、SEQ ID NO：58和SEQ ID NO：20组成，且所述轻链可变区的CDR1、CDR2和CDR3分别由SEQ ID NO：22、SEQ ID NO：23和SEQ ID NO：24组成；或
(02)所述重链可变区的CDR1、CDR2和CDR3分别由氨基酸序列SEQ ID NO：2、SEQ ID NO：3和SEQ ID NO：4组成，且所述轻链可变区的CDR1、CDR2和CDR3分别由SEQ ID NO：6、SEQ ID NO：7和SEQ ID NO：8组成；或
(03)所述重链可变区的CDR1、CDR2和CDR3分别由氨基酸序列SEQ ID NO：10、SEQ ID NO：11和SEQ ID NO：12组成，且所述轻链可变区的CDR1、CDR2和CDR3分别由SEQ ID NO：14、SEQ ID NO：15和SEQ ID NO：16组成；或
(04)所述重链可变区的CDR1、CDR2和CDR3分别由氨基酸序列SEQ ID NO：18、SEQ ID NO：19和SEQ ID NO：20组成，且所述轻链可变区的CDR1、CDR2和CDR3分别由SEQ ID NO：22、SEQ ID NO：23和SEQ ID NO：24组成；或
(05)所述重链可变区的CDR1、CDR2和CDR3分别由氨基酸序列SEQ ID NO：10、SEQ ID NO：11和SEQ ID NO：12组成，且所述轻链可变区的CDR1、CDR2和CDR3分别由SEQ ID NO：27、SEQ ID NO：15和SEQ ID NO：16组成；或
(06)所述重链可变区的CDR1、CDR2和CDR3分别由氨基酸序列SEQ ID NO：18、SEQ ID NO：55和SEQ ID NO：20组成，且所述轻链可变区的CDR1、CDR2和CDR3分别由SEQ ID NO：22、SEQ ID NO：23和SEQ ID NO：24组成；或
(07)所述重链可变区的CDR1、CDR2和CDR3分别由氨基酸序列SEQ ID NO：2、SEQ ID NO：3和SEQ ID NO：4组成，且所述轻链可变区的CDR1、CDR2和CDR3分别由SEQ ID NO：28、SEQ ID NO：7和SEQ ID NO：8组成；或
(08)所述重链可变区的CDR1、CDR2和CDR3分别由氨基酸序列SEQ ID NO：2、SEQ ID NO：3和SEQ ID NO：4组成，且所述轻链可变区的CDR1、 CDR2和CDR3分别由SEQ ID NO：33、SEQ ID NO：7和SEQ ID NO：8组成；或
(09)所述重链可变区的CDR1、CDR2和CDR3分别由氨基酸序列SEQ ID NO：2、SEQ ID NO：3和SEQ ID NO：36组成，且所述轻链可变区的CDR1、CDR2和CDR3分别由SEQ ID NO：6、SEQ ID NO：7和SEQ ID NO：8组成；或
(10)所述重链可变区的CDR1、CDR2和CDR3分别由氨基酸序列SEQ ID NO：18、SEQ ID NO：55和SEQ ID NO：61组成，且所述轻链可变区的CDR1、CDR2和CDR3分别由SEQ ID NO：22、SEQ ID NO：23和SEQ ID NO：24组成；或
(11)所述重链可变区的CDR1、CDR2和CDR3分别由氨基酸序列SEQ ID NO：18、SEQ ID NO：55和SEQ ID NO：64组成，且所述轻链可变区的CDR1、CDR2和CDR3分别由SEQ ID NO：22、SEQ ID NO：23和SEQ ID NO：24组成；或
(12)所述重链可变区的CDR1、CDR2和CDR3分别由氨基酸序列SEQ ID NO：18、SEQ ID NO：58和SEQ ID NO：61组成，且所述轻链可变区的CDR1、CDR2和CDR3分别由SEQ ID NO：22、SEQ ID NO：23和SEQ ID NO：24组成；或
(13)所述重链可变区的CDR1、CDR2和CDR3分别由氨基酸序列SEQ ID NO：2、SEQ ID NO：3和SEQ ID NO：36组成，且所述轻链可变区的CDR1、CDR2和CDR3分别由SEQ ID NO：28、SEQ ID NO：7和SEQ ID NO：8组成；或
(14)所述重链可变区的CDR1、CDR2和CDR3分别由氨基酸序列SEQ ID NO：2、SEQ ID NO：3和SEQ ID NO：45组成，且所述轻链可变区的CDR1、CDR2和CDR3分别由SEQ ID NO：6、SEQ ID NO：7和SEQ ID NO：8组成；或
(15)所述重链可变区的CDR1、CDR2和CDR3分别由氨基酸序列SEQ ID NO：2、SEQ ID NO：3和SEQ ID NO：45组成，且所述轻链可变区的CDR1、CDR2和CDR3分别由SEQ ID NO：28、SEQ ID NO：7和SEQ ID NO：48组成；或
(16)所述重链可变区的CDR1、CDR2和CDR3分别由氨基酸序列SEQ ID NO：18、SEQ ID NO：58和SEQ ID NO：64组成，且所述轻链可变区的CDR1、CDR2和CDR3分别由SEQ ID NO：22、SEQ ID NO：23和SEQ ID NO：24组成。
2.根据权利要求1中所述的抗体或其抗原结合片段，其特征在于，其中：
(01)所述重链可变区具有SEQ ID NO：56给出的氨基酸序列，或与SEQ ID NO：56给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、 94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：56的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列，
所述轻链可变区具有SEQ ID NO：57给出的氨基酸序列，或与SEQ ID NO：57给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：57的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列；或
(02)所述重链可变区具有SEQ ID NO：1给出的氨基酸序列，或与SEQ ID NO：1给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：1的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列，
所述轻链可变区具有SEQ ID NO：5给出的氨基酸序列，或与SEQ ID NO：5给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：5的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列；或
(03)所述重链可变区具有SEQ ID NO：9给出的氨基酸序列，或与SEQ ID NO：9给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：9的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列，
所述轻链可变区具有SEQ ID NO：13给出的氨基酸序列，或与SEQ ID NO：13给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：13的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列；或
(04)所述重链可变区具有SEQ ID NO：17给出的氨基酸序列，或与SEQ ID NO：17给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：17的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列，
所述轻链可变区具有SEQ ID NO：21给出的氨基酸序列，或与SEQ ID NO：21给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：21的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10 个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列；或
(05)所述重链可变区具有SEQ ID NO：49给出的氨基酸序列，或与SEQ ID NO：49给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：49的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列，
所述轻链可变区具有SEQ ID NO：50给出的氨基酸序列，或与SEQ ID NO：50给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：50的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列；或
(06)所述重链可变区具有SEQ ID NO：51给出的氨基酸序列，或与SEQ ID NO：51给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：51的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列，
所述轻链可变区具有SEQ ID NO：52给出的氨基酸序列，或与SEQ ID NO：52给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：52的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列；或
(07)所述重链可变区具有SEQ ID NO：53给出的氨基酸序列，或与SEQ ID NO：53给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：53的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列，
所述轻链可变区具有SEQ ID NO：54给出的氨基酸序列，或与SEQ ID NO：54给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：54的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列；或
(08)所述重链可变区具有SEQ ID NO：25给出的氨基酸序列，或与SEQ ID NO：25给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：25的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列，
所述轻链可变区具有SEQ ID NO：26给出的氨基酸序列，或与SEQ ID  NO：26给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：26的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列；或
(09)所述重链可变区具有SEQ ID NO：29给出的氨基酸序列，或与SEQ ID NO：29给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：29的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列，
所述轻链可变区具有SEQ ID NO：30给出的氨基酸序列，或与SEQ ID NO：30给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：30的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列；或
(10)所述重链可变区具有SEQ ID NO：31给出的氨基酸序列，或与SEQ ID NO：31给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：31的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列，
所述轻链可变区具有SEQ ID NO：32给出的氨基酸序列，或与SEQ ID NO：32给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：32的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列；或
(11)所述重链可变区具有SEQ ID NO：34给出的氨基酸序列，或与SEQ ID NO：34给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：34的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列，
所述轻链可变区具有SEQ ID NO：35给出的氨基酸序列，或与SEQ ID NO：35给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：35的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列；或
(12)所述重链可变区具有SEQ ID NO：59给出的氨基酸序列，或与SEQ ID NO：59给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO： 59的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列，
所述轻链可变区具有SEQ ID NO：60给出的氨基酸序列，或与SEQ ID NO：60给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：60的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列；或
(13)所述重链可变区具有SEQ ID NO：62给出的氨基酸序列，或与SEQ ID NO：62给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：62的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列，
所述轻链可变区具有SEQ ID NO：63给出的氨基酸序列，或与SEQ ID NO：63给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：63的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列；或
(14)所述重链可变区具有SEQ ID NO：65给出的氨基酸序列，或与SEQ ID NO：65给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：65的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列，
所述轻链可变区具有SEQ ID NO：66给出的氨基酸序列，或与SEQ ID NO：66给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：66的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列；或
(15)所述重链可变区具有SEQ ID NO：37给出的氨基酸序列，或与SEQ ID NO：37给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：37的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列，
所述轻链可变区具有SEQ ID NO：38给出的氨基酸序列，或与SEQ ID NO：38给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：38的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列；或
(16)所述重链可变区具有SEQ ID NO：39给出的氨基酸序列，或与SEQ ID NO：39给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：39的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列，
所述轻链可变区具有SEQ ID NO：40给出的氨基酸序列，或与SEQ ID NO：40给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：40的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列；或
(17)所述重链可变区具有SEQ ID NO：41给出的氨基酸序列，或与SEQ ID NO：41给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：41的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列，
所述轻链可变区具有SEQ ID NO：42给出的氨基酸序列，或与SEQ ID NO：42给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：42的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列；或
(18)所述重链可变区具有SEQ ID NO：43给出的氨基酸序列，或与SEQ ID NO：43给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：43的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列，
所述轻链可变区具有SEQ ID NO：44给出的氨基酸序列，或与SEQ ID NO：44给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：44的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列；或
(19)所述重链可变区具有SEQ ID NO：46给出的氨基酸序列，或与SEQ ID NO：46给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：46的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列，
所述轻链可变区具有SEQ ID NO：47给出的氨基酸序列，或与SEQ ID NO：47给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、 94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：47的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列；或
(20)所述重链可变区具有SEQ ID NO：67给出的氨基酸序列，或与SEQ ID NO：67给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：67的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列，
所述轻链可变区具有SEQ ID NO：68给出的氨基酸序列，或与SEQ ID NO：68给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：68的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列。
3.根据方案1-2中任一项所述的抗体或其抗原结合片段，其结合Treg的TNFR2，并能杀伤Treg，具有杀伤率至少10％、20％、30％、40％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、96％、97％、98％、99％或100％。
4.根据方案1-2中任一项所述的抗体或其抗原结合片段，其结合Teff的TNFR2，促进Teff细胞增殖分化。
5.根据方案1-2中任一项所述的抗体或其抗原结合片段，其结合Teff的TNFR2，促进Teff生物活性。
6.根据方案1-2中任一项所述的抗体或其抗原结合片段，其与TNFR2之间的解离常数KD小于10nM。
7.根据方案1-2中任一项所述的抗体或其抗原结合片段，其与TNFR2之间的解离常数KD小于1nM。
8.一种多核苷酸，其特征在于，其编码根据方案1-2中任一项所述的抗体或其抗原结合片段。
9.一种重组载体、转基因细胞系、噬菌体、重组菌或病毒载体，其特征在于，其含有根据方案8所述的多核苷酸。
10.根据方案9所述的重组载体、转基因细胞系、噬菌体、重组菌或病毒载体在制备抗TNFR2抗体中的应用。
11.一种分离的宿主细胞，其特征在于，其含有根据方案9所述的重组载体、转基因细胞系、噬菌体、重组菌或病毒载体。
12.根据方案11所述的宿主细胞，其中所述宿主细胞是原核细胞。
13.根据方案11所述的宿主细胞，其中所述宿主细胞是真核细胞。
14.根据方案13所述的宿主细胞，其中所述真核细胞是哺乳动物细胞。
15.根据方案14所述的宿主细胞，其中所述哺乳动物细胞是CHO细胞。
16.一种抗体表达方法，其特征在于，用根据方案9所述的重组载体、转基因细胞系、噬菌体、重组菌或病毒载体在根据方案11-15中任一项所述的宿主细胞中表达抗体蛋白。
17.根据方案1-2中任一项所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段包括鼠源的、人鼠嵌合的、人源化的或全人源的。
18.根据方案1-2中任一项所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段选自由以下组成的组：单克隆抗体或其抗原结合片段、多克隆抗体或其抗原结合片段、人源化抗体或其抗原结合片段、灵长类化抗体或其抗原结合片段、双特异性抗体或其抗原结合片段、多特异性抗体或其抗原结合片段、双重可变免疫球蛋白结构域、单价抗体或其抗原结合片段、嵌合抗体或其抗原结合片段、单链Fv分子(scFv)、双特异性抗体、三特异性抗体、纳米抗体、抗体样蛋白支架、结构域抗体、Fv片段、Fab片段、F(ab’)2分子以及串联scFv。
19.根据方案1-2中任一项所述的抗体或其抗原结合片段，其中，所述抗体为单克隆抗体。
20.根据方案1-2中任一项所述的抗体或其抗原结合片段在制备药物中的应用，所述药物含有所述抗体或其抗原结合片段和化学疗法，用于在人类患者中治疗肿瘤，其中将所述抗体和所述化学疗法配制成能提供比所述试剂各自的效果之和更大的治疗效果。
21.一种抗体药物偶联物，其特征在于，根据方案1-2中任一项所述的抗体或其抗原结合片段任选通过连接子与治疗剂偶联。
22.根据方案21所述的抗体药物偶联物，其中所述治疗剂是细胞毒性剂。
23.一种药物组合物，其包含根据方案1-2中任一项所述的抗体或其抗原结合片段和药学上可接受的载体、稀释剂或赋形剂。
24.根据方案23所述的药物组合物，其中所述组合物还包含额外治疗剂。
25.根据方案24所述的药物组合物，其中所述额外治疗剂是免疫治疗剂。
26.根据方案1-2中任一项所述的抗体或其抗原结合片段在如下(a)、(b)、(c)、(d)或(e)中的应用：
(a)制备诱导Treg细胞凋亡的药物中的应用；
(b)制备靶向肿瘤细胞杀伤的药物中的应用；
(c)制备阻断TNF-TNFR2肿瘤信号通路的药物中的应用；
(d)制备促进Teff细胞增殖分化和/或生物活性的药物中的应用；
(e)制备治疗自身免疫性疾病的药物中的应用。
27.根据方案26所述的应用，其特征在于，所述肿瘤选自：卵巢癌、黑色素瘤、前列腺癌、肠癌、胃癌、食管癌、乳腺癌、肺癌、肾癌、胰腺癌、子宫癌、肝癌、膀胱癌、子宫颈癌、口腔癌、脑癌、睾丸癌、皮肤癌、结肠直肠癌、恶性胶质瘤、甲状腺癌。
本发明提供了以高亲和力与TNFR2特异性结合的抗体或其抗原结合片段，所述抗体可以介导对Treg细胞的杀伤活性，介导对CD8+T细胞的增殖和活化作用，具有优异的抗肿瘤药效。
附图说明
图1为大鼠抗体与hTNFR2的FACS结合示意图；
图2为大鼠抗体与hTNFR2结合的配体阻断ELISA检测示意图；
图3为抗TNFR2抗体介导的对Treg细胞的杀伤活性示意图；
图4为抗TNFR2抗体介导的对CD8+T细胞的增殖作用示意图；
图5为抗TNFR2抗体介导的对CD8+T细胞的活化作用示意图；
[根据细则91更正 01.07.2022]　
图6为嵌合抗体的体内抗肿瘤药效评价示意图；
[根据细则91更正 01.07.2022]　
图7小鼠MC38移植肿瘤小鼠模型体内药效实验-肿瘤体积变化图；
[根据细则91更正 01.07.2022]　
图8小鼠MC38移植肿瘤小鼠模型体内药效实验-体重变化图；
[根据细则91更正 01.07.2022]　
图9小鼠B16-F10移植肿瘤小鼠模型体内药效实验-肿瘤体积变化图。
具体实施方式
提供以下定义以帮助理解本文中阐述的发明。
本文提供了分离的抗体，包括鼠抗体、嵌合抗体、人抗体，其特异性结合TNFR2(例如人TNFR2)上的特定表位。本文还提供了制备抗体的方法、包含本发明抗体或其抗原结合片段的药物偶联物、药物组合物以及与其它治疗剂的联合疗法，以及使用本发明的抗体或其抗原结合片段制备治疗多种疾病的药物。
本文的BMK包含BMK2、BMK6。序列分别来自：WO2017083525A1专利中的SEQ ID NO:3和SEQ ID NO:4；WO2020061210A1专利中的SEQ ID NO:150和SEQ ID NO:151。
术语“肿瘤坏死因子受体2”，“TNFR2”，“CD120b”，“p75”，“p75TNFR”，“p80TNF-α受体”，“TBPII”，“TNFBR”，“TNFR1B”，“TNF-R75”本文中可互换使用的“和”TNFR80包括所有家族成员、突变体、等位基因、片段和物种。TNFR2与TNFR1共同介导TNFα的活性。TNFR1是55kD的膜结合蛋白，而TNFR2是75kD的膜结合蛋白。TNFR2可以调节TNFα与TNFR1的结合，因此可以调节刺激NF-kB作用所必需的TNFα水平。TNFR2也可以被金属蛋白酶切割(或进行选择性剪接)，从而生成可溶受体，从而维持对TNFα的亲和力。
本发明还提供了一种组合物。它含有本发明的抗体或其活性片段，以及药学上可接受的载体。通常，可将这些物质配制于无毒的、惰性的和药学上可接受的水性载体介质中，pH值可随被配制物质的性质以及待治疗的病症而有所变化。配制好的药物组合物可以通过常规途径进行给药，其中包括但不限于：瘤内、腹膜内、静脉内、或局部给药。
本发明的药物组合物可直接用于结合TNFR2蛋白分子，因而可用于治疗肿瘤。此外，还可同时使用其他治疗剂。
本发明的药物组合物含有安全有效量(如0.001-99.999wt％，优选0.01-90wt％，更优选0.1-80wt％)的本发明上述的抗体或其结合片段(或其偶联物)以及药 学上可接受的载体或赋形剂。这类载体包括但不限于：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。药物制剂应与给药方式相匹配。本发明的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。药物组合物如针剂、溶液宜在无菌条件下制造。活性成分的给药量是治疗有效量。此外，本发明的多肽还可与其他治疗剂一起使用。
本发明的抗体可以单独使用，也可与可检测标记物(为诊断目的)、治疗剂、PK(蛋白激酶)修饰部分或任何以上这些物质的组合结合或偶联。用于诊断目的可检测标记物包括但不限于：荧光或发光标记物、放射性标记物、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂、或能够产生可检测产物的酶。可与本发明抗体结合或偶联的治疗剂包括但不限于：1.放射性核素；2.生物毒素；3.细胞因子如IL-2等；4.金纳米颗粒/纳米棒；5.病毒颗粒；6.脂质体；7.纳米磁粒；8.前药激活酶(例如，DT-心肌黄酶(DTD)或联苯基水解酶样蛋白质(BPHL))；9.化疗剂(例如，顺铂)或任何形式的纳米颗粒等。
在本说明书中，“抗体”是指天然的免疫球蛋白或者通过部分或完全合成而制备的免疫球蛋白。抗体可从天然存在该抗体的血浆或血清等的天然资源、或者产生抗体的杂交瘤细胞的培养上清中分离。备选地，可通过使用基因重组等的技术部分或完全地合成。优选的抗体包括，例如，免疫球蛋白的同种型或这些同种型的亚类的抗体。已知人免疫球蛋白包括IgG1、IgG2、IgG3、IgG4、IgAl、IgA2、IgD、IgE、IgM这9种类别(同种型)。在这些同种型中，本发明的抗体可以包括IgGl、IgG2、IgG3、IgG4。
本文可互换使用的术语“抗体”或“免疫球蛋白”包括整个抗体及其任何抗原结合片段(抗原结合部分)或其单链。“抗体”包括至少一条重(H)链和一条轻(L)链。例如，在天然存在的IgG中，这些重链和轻链通过二硫键相互连接，并且存在两对成对的重链和轻链。这两个也通过二硫键相互连接，每条重链由重链可变区(在此缩写为VH)和重链恒定区组成，重链恒定区由三个结构域CH1，CH2和CH3组成。轻链由轻链可变区(在本文中简称为VL)和轻链恒定区组成，轻链恒定区由一个结构域CL组成，VH和VL区可进一步细分为高变区，称为互补决定区。区域(CDR)，散布着更保守的区域，称为框架区域(FR)或连接区域(J)(分别在重链和轻链中为JH或JL)，每个VH和VL由三个CDR组成，三个FR和一个J结构域，其从氨基末端到羧基末端按以下顺序排列：FR1，CDR1，FR2，CDR2，FR3，CDR3，J。重链和轻链的可变区与抗原结合。抗体的恒定区可以介导免疫球蛋白与宿主组织或因子的结合，包括免疫系统的各种细胞(例如效应细胞)或体液因子，例如经典补码系统的第一部分(C1q)。已经显示出全长抗体的片段可以执行抗体的抗原结合功能。表示为抗原结合部分或抗体片段的结合片段的实例包括(i)Fab片段，由以下组成的单价片段：(ii)F(ab') 2片段，是包含两个通过铰链区的二硫键连接的Fab片段的二价片 段；(iii)由VH和CH1域组成的Fd片段；(iv)由抗体单臂的VL和VH结构域组成的Fv片段，(v)包括VH和VL结构域的dAb；(vi)dAb片段由VH结构域组成；(vii)由VH或VL结构域组成的dAb；(viii)分离的互补决定区(CDR)或(ix)两个或多个分离的CDR的组合，可以任选地通过合成接头连接。此外，尽管Fv片段的两个结构域VL和VH被编码对于单独的基因，它们可以使用重组方法通过合成接头连接起来，该接头使它们成为一条蛋白链，其中VL和VH区配对形成单价分子(这种免疫球蛋白片段的单链同源物是这种单链抗体也意图包含在术语“抗体”之内。使用本领域技术人员已知的常规技术获得抗体片段，并筛选该片段在该抗体中的效用。抗原结合部分可以通过重组DNA技术或完整的免疫球蛋白的酶促或化学裂解来产生。本文所述抗体中氨基酸位置的编号(例如Fc区的氨基酸残基)和目标区域，例如CDR，使用Kabat系统。
如本文所用，“同种型”是指由重链恒定区基因编码的抗体类别(例如，IgG1，IgG2，IgG3，IgG4，IgM，IgA1，IgA2，IgD和IgE抗体)。
术语“重组抗体”是指通过重组方式制备，表达，产生或分离的抗体，例如(a)从针对免疫球蛋白基因(例如人免疫球蛋白)转基因或转染色体的动物(例如，老鼠)分离的抗体基因或由其制备的杂交瘤，(b)从宿主细胞分离的抗体，该宿主细胞经转化表达抗体，例如从转染瘤中表达，(c)使用噬菌体展示从组合的组合抗体库(例如，包含人抗体序列)分离的抗体，(d)通过将免疫球蛋白基因序列(例如人免疫球蛋白基因)剪接至其他DNA序列的任何其他方式制备，表达，产生或分离的抗体。这类重组抗体可能具有源自人种系免疫球蛋白序列的可变区和恒定区。然而，在某些实施方案中，此类重组人抗体可进行体外诱变；重组抗体的VH和VL区是源自人种系VH和VL序列并与之相关的序列，但在体内人抗体种系库中可能不自然存在。
术语“嵌合免疫球蛋白”或“嵌合抗体”是指其可变区衍生自第一物种而其恒定区衍生自第二物种的免疫球蛋白或抗体。嵌合免疫球蛋白或抗体可以例如通过基因工程由属于不同物种的免疫球蛋白基因区段构建。
术语“人源化抗体”是指包括至少一条人源化抗体链(即，至少一条人源化轻链或重链)的抗体。术语“人源化抗体链”(即“人源化免疫球蛋白链”)是指具有可变区的抗体链(即分别为轻链或重链)，所述可变区包括人抗体实质性的可变框架区和互补性决定。基本上来自非人抗体的区域(CDR)(例如，至少一个CDR，两个CDR或三个CDR)。在一些实施方案中，人源化抗体链还包括恒定区(例如，在轻链的情况下，一个恒定区或其一部分，在重链的情况下，优选三个恒定区)。
如本文所用，术语“人抗体”旨在包括具有可变区的抗体，其中，构架区和CDR区均来自人免疫球蛋白序列。此外，如果抗体包含恒定区，则恒定区也是来源于人种系免疫球蛋白序列。人抗体可以包括人种系免疫球蛋白序列未编码的氨基酸残基(例如，体外通过随机位点特异性诱变或体内体细胞突变引入的突 变)。
人抗体可以具有至少一个或多个被氨基酸残基取代的氨基酸，所述氨基酸残基例如是不由人免疫球蛋白序列编码的增强活性的氨基酸残基。通常，人抗体可以具有多达二十个位置，该位置被不属于人种系免疫球蛋白序列一部分的氨基酸残基取代。在特定的实施方案中，这些替换在CDR区域内。在一些其它的实施方案中，这些替换在框架区域内。
如本文所用，术语“多特异性”或“多功能抗体”是具有多个不同的结合位；点的人工杂交抗体。双特异性抗体可以通过多种方法产生，包括融合杂交瘤或连接Fab'片段。
如本文所用，术语“分离的”旨在指基本上不含具有不同抗原特异性的其他抗体的抗体。此外，分离出的抗体通常基本上不含其他细胞物质和/或化学物质。
如本文所用，术语“Fc区”，“Fc结构域”或“Fc”是指抗体重链的C末端区域。因此，Fc区包含抗体的恒定区，但不包括第一恒定区免疫球蛋白结构域(例如，CH1或CL)。
如本文所用，术语“抗原”是抗体结合的实体(例如，蛋白质实体或肽)，例如TNFR2。
如本文所用，术语“特异性结合”和“选择性结合”是指抗体对特定抗原或表位表现出明显的亲和力，并且通常不与其他抗原或表位表现出明显的交叉反应性。“可观的”或优选的结合包括以10 -7，10 -8，10 -9或10 -10M或更优选的KD结合。抗体抗原相互作用的KD(亲和常数)表示50％的抗体和抗原分子结合在一起的抗体浓度。因此，在合适的固定抗原浓度下，亲和力更高(即更强)的抗体的50％与以较低亲和力抗体实现相同百分比结合所需的抗体浓度相比，其以较低的抗体浓度结合抗原分子。因此，较低的KD值表示较高(较强)的亲和力。如本文所用，“更好”的亲和力比其亲和力更强，并且数值较低，其10 -7M的KD的数值较低，因此与10 -6M的KD相比，亲和力更好。通常优选具有小于10 - 7M，因此优选大于10 -8M的KD值，也可以考虑在本文所述的中间值，并且可以将优选的结合亲和力表示为亲和力的范围，例如对于本文公开的抗TNFR2抗体为10 -7至10 -12M，更优选为10 -8至10 -12M。“不表现出明显的交叉反应性”或“不以生理学相关的亲和力结合”的抗体是不会明显结合抗体的抗体。脱靶抗原(例如非TNFR2蛋白)或表位。特异性或选择性结合可以根据任何本领域技术来确定。确定这种结合的公认方法包括，例如，根据斯卡查德分析，生物大分子相互作用分析，生物膜层干涉技术和/或竞争性(竞争)结合测定法。
如本文所用，术语“表位”意指抗原中的抗原决定簇，并且是指在本说明书中公开的包含抗体可变区的抗原结合分子的结构域所结合的抗原位点。因此，例如可以根据其结构来定义表位。另外，也可以根据识别该表位的抗原结合分子中的抗原结合活性来定义该表位。当抗原是肽或多肽时，表位可以由形成表 位的氨基酸残基指定。或者，当表位是糖链时，表位可以通过其特定的糖链结构来确定。
线性表位是含有其一级氨基酸序列被识别的表位。这种线性表位通常在其特定序列中含有至少三个，最常见的至少五个，例如约8至10或6至20个氨基酸。
与线性表位相反，“构象表位”是其中含有表位的一级氨基酸序列不是识别的表位的唯一决定簇的表位(例如，构象表位的一级氨基酸序列不一定被表位限定抗体识别)。与线性表位相比，构象表位可含有更多数量的氨基酸。构象表位识别抗体识别肽或蛋白质的三维结构。例如，当蛋白质分子折叠并形成三维结构时，形成构象表位的氨基酸和/或多肽主链变得对齐，并且表位可被抗体识别。用于确定表位构象的方法包括例如X射线晶体学，二维核磁共振，位点特异性自旋标记和电子顺磁共振，但不限于此。
如本文所用，术语“载体”旨在指能够转运已与其连接的另一核酸的核酸分子。一种类型的载体是“质粒”，其是指环状双链DNA环。其他DNA片段可以连接到其中。载体的另一种类型是病毒载体，其中可以将另外的DNA片段连接到病毒基因组中。某些载体能够在引入它们的宿主细胞中自主复制(例如，具有复制的细菌起源的细菌载体和游离型哺乳动物载体)。可以将其他载体(例如非外源哺乳动物载体)整合到宿主细胞的基因组中，然后导入宿主细胞中，从而与宿主基因组一起复制。此外，某些载体能够指导其表达的基因表达这些载体在本文中称为“重组表达载体”(或简称为“表达载体”)。通常，在重组DNA技术中有用的表达载体通常是质粒的形式。术语“质粒”和“载体”可以互换使用。然而，还考虑了具有等同功能的其他形式的表达载体，例如病毒载体(例如复制缺陷型逆转录病毒，腺病毒和腺相关病毒)。
如本文所用，术语“抑制”是指任何统计学上显著降低的生物活性，包括部分和完全阻断该活性。例如，“抑制”可以表示统计上的显著下降，约为10％，20％，30％，40％，50％，60％，70％，80％，90％，95％，96％，97％，98％，99％或100％的生物活性。
如本文所用，术语“激活”是指任何统计学上显著激活细胞的生物活性，例如，“激活”可以表示统计上的生物活性显著上升，约为提升1％，10％，20％，30％，40％，50％，60％，70％，80％，90％，100％，200％，500％，1000％，10000％及以上的生物活性。
如本文所用，短语“抑制TNFR2配体与TNFR2的结合”是指相对于不存在TNFR2抗体的情况，抗体在统计学上显著降低TNFR2配体(例如，TNFα)与TNFR2的结合的能力。换句话说，在抗体存在下，相对于对照(无抗体)，与TNFR2结合的TNFR2配体的量在统计学上显著减少。在本文公开的抗TNFR2抗体的存在下，与TNFR2结合的TNFR2配体的量可以减少至少约10％，或至少约20％，或至少约30％，或至少约40％，或相对于不存在抗体(对照)的量， 至少约50％，或至少约60％，或至少约70％，或至少约80％，或至少约90％，或约100％。可以使用本领域公认的技术来测量TNFR2配体结合的减少，该技术在存在或不存在(对照)抗体的情况下测量标记的TNFR2配体(例如，放射性标记的TNFα)与表达TNFR2的细胞的结合水平。
如本文所用，术语“抑制肿瘤生长”包括肿瘤生长的任何可测量的降低，例如，肿瘤生长的抑制至少约10％，例如至少约20％，至少约50％。30％，至少约40％，至少约50％，至少约60％，至少约70％，至少约80％，至少约90％，至少约99％或约100％。
如本文所用，术语“治疗”是指本文所述的治疗或预防措施。“治疗”的方法用于给予易患肿瘤或癌症的受试者或受试者。为了预防，治愈，延缓，减轻疾病或病症或复发性疾病或病症的一种或多种症状，本文所述的抗TNFR2抗体(例如，抗人TNFR2抗体)是这种疾病或病症，或者为了延长受试者的生存期，使其在没有这种治疗的情况下延长生存期。
如本文所用，术语“可变片段(Fv)”是指抗体衍生的抗原结合结构域的最小单位，其由一对抗体轻链可变区(VL)和抗体重链可变区(VH)构成。1988年，Skerra和Pluckthun发现，通过在细菌信号序列下游插入抗体基因并在大肠杆菌中诱导该基因的表达，可以从大肠杆菌周质部分制备同源和活性抗体。在由周质级分制备的Fv中，VH以与抗原结合的方式与VL结合。
在此，Fv优选包括，例如，一对作为抗原结合分子等的Fv，其包含：scFv，单链抗体和sc(Fv)2。
如本文所用，术语“scFv”、“单链抗体”和“sc(Fv)2”均指单个多肽链的抗体片段，其含有源自重链和轻链的可变区，但没有恒定区。通常，单链抗体还在VH和VL结构域之间含有多肽接头，这使得能够形成被认为允许抗原结合的所需结构。
如本文所用，术语“CHOK1-hTNFR2”是指一种通过一定的技术构建的细胞。具体构建方法包括以下的方法，但不限于以下的方法。具体步骤：(1)基因合成和分子构建：金唯智对人TNFR2蛋白的序列进行了密码子优化和合成。将合成的基因进一步亚克隆到修饰的pSBbi-GB载体中。(2)瞬时转染：在转染前一天，在T25培养瓶中准备具有高于95％活率的1.0x10^6个CHOK1细胞，70％-90％的汇合度时开始进行转染。目的基因与转座酶质粒pCMV(CAT)T7-SB100以9:1比例进行混合，然后加入
Figure PCTCN2022098506-appb-000001
DNA转染试剂混合，然后添加细胞培养基。将细胞保持在37℃的恒温箱中，同时将CO2浓度保持在8％。孵育48小时后，首先通过胰蛋白酶分离2x10^5细胞，然后在1,500rpm，4℃下离心4分钟，用于FACS检测TNFR2表达水平。(3)稳定细胞池和细胞系产生：在瞬时转染FACS确认TNFR2表达后，将细胞池进一步在含有杀稻瘟素的培养基中进行培养，以进行稳定细胞池选择。杀稻瘟素的浓度为10ug/mL。2-3天换一次培养基。从2到3周的抗生素选择中恢复后，将会产生 稳定的细胞池。通过BD FACS Melody分选进一步产生稳定的单细胞系。首先对细胞进行计数并测量其活力。用一抗和二抗孵育后，将单个细胞分选到96孔板中，在培养箱中培养至可观察到克隆。FACS检测后，将高表达单克隆扩增并入库。(4)FACS检测：将瞬时转染的细胞以2x10^5细胞/孔的密度转移到96孔U型底板(Corning-3799)。抗TNFR2抗体在2％FBS/1XPBS中以2ug/mL稀释，每孔100μL，然后在4℃下孵育1小时。将细胞洗涤两次并重新悬浮在100μL2％BSA/1XPBS中。二抗(山羊抗人IgGFc-Alexa647)在2％FBS/1XPBS中以1:500稀释，每孔100μL，然后在4℃下孵育30分钟。将细胞洗涤两次并重新悬浮在100μL 2％FBS/1XPBS中。通过流式细胞术(BD Canto II)测量荧光并通过FlowJo进行分析。(5)稳定性测试：细胞库或细胞系在P1后传代超过3周用于稳定性测试。FACS用于稳定性确认。
如本文所用，术语“Treg”、“Treg细胞”或“调节性T细胞”有时被称为抑制性T细胞，其特征在于表达生物标志物CD4、FOXP3和CD25，代表调节免疫系统、维持对自身抗原的耐受性并预防自身免疫疾病的T细胞亚群。Treg是免疫抑制性的并且通常抑制或下调效应T(Teff)细胞的诱导和增殖。Treg可以在胸腺中发展(所谓的CD4 +Foxp3 +“天然”Treg)或从外周的幼稚CD4 +T细胞中分化，例如，在暴露于TGFβ或视黄酸之后。
如本文所用，术语“Teff”、“Teff细胞”或“效应T细胞”是T细胞接受抗原刺激后，经过增殖，分化形成的细胞。效应T细胞具有释放淋巴因子的功能，在此过程中，有一小部分T细胞成为记忆T细胞。效应T细胞与靶细胞接触而激发颗粒胞吐，所释放的穿孔素通过聚合作用而在靶细胞表面形成小孔，从而介导杀伤作用，其靶细胞死亡过程类似于细胞凋亡。同时，效应T细胞还能释放出免疫活性物质-淋巴因子，如白细胞介素，干扰素等。
以下的实施例便于更好地理解本发明，但并不限定本发明。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。
实施例1：动物免疫
用重组人TNFR2-His Tag蛋白(Sino，Cat：10417-H08H)作为免疫原，免疫SD大鼠，为避免大鼠反应不佳或免疫过程中凋亡，可同时免疫3～4只。提前1天采取阴性血清，首次免疫，以皮下免疫、腹腔免疫和足底免疫方式多点注射60μg经弗氏完全佐剂充分乳化的重组人TNFR2-His Tag蛋白，第23天以皮下免疫和足底免疫方式多点注射40μg经弗氏完全佐剂充分乳化的重组人TNFR2-His Tag蛋白进行二次免疫，每隔两周以二次免疫相同的方式注射40μg免疫原进行第三次、第四次、第五次、第六次及第七次免疫。30天后，通过在1：100至1：1000000的不同稀释度下，在重组人TNFR2-His Tag蛋白包被的ELISA板中对从尾部放血收集的血清进行试验来评估抗血清滴度。当效价结果 满足要求，在>1：1000000的稀释度下检测到抗人TNFR2抗体时，可收获大鼠脾脏和淋巴结，进行细胞融合。
实施例2：细胞融合
骨髓瘤细胞SP2/0(ATCC)于融合前一天将细胞传代，使实验时细胞处于对数生长期，融合前将细胞收集于离心管中，经1000转/分离心5分钟，弃上清液并加入10mL DMEM完全培养基将细胞混匀备用。实验用脾细胞和淋巴结细胞取自经重组人TNFR2-His Tag蛋白七次免疫的SD大鼠，融合前将大鼠处死取出脾脏和淋巴结，加入10mL DMEM完全培养基将细胞压磨过网，得细胞悬液经2000转/分离心8分钟，弃上清液并加入DMEM完全培养基将细胞混匀备用。融合体选择培养中所用的饲养细胞取自未经免疫的动物腹腔中的巨噬细胞，融合前将收集的巨噬细胞悬液经100转/分离心8分钟，弃上清液并加入25mL HAT选择培养基混匀，分装于2块24孔培养板中，以辅助新的杂种B淋巴细胞杂交瘤生长。
将B淋巴细胞和骨髓瘤细胞按1:2混合，将其悬浮液以1000转/分离心8分钟，取沉淀，用电融合液洗两次。再取沉淀加电融合液至1.2mL，向三个多电极小池各注入0.4mL细胞悬浮液。对每个电极小池依次施加如下电场条件：电介质电泳之交流电场正弦信号频率为1MHz，振幅为250V/cm，施加30秒钟；然后立即加穿孔RC电脉冲幅度为5kV/cm，脉冲宽度为20μs，脉冲个数为3，时间间隔为l秒钟。室温下静置10分钟，再用总量为5mL的PFM液将融合物冲洗取出，于37℃温育30分钟。然后经100转/分离心8分钟，取沉淀，加入500mL HAT混匀并分注于96孔培养板，细胞数为1×10 4个/孔(己有饲养细胞1.2*10 4个/孔)，放入CO 2培养箱于37℃培养。
实施例3：间接ELISA方法筛选阳性克隆
采用间接ELISA方法筛选阳性细胞克隆。方阵试验确定检测重组人TNFR2-His Tag蛋白(Sino；Cat：10417-H08H)的包被浓度。检测抗原包被缓冲液横向梯度稀释，每孔50μL包被ELISA板，4℃过夜；PBST洗涤3次，每孔加入200μL封闭液，4℃过夜；免疫鼠血清纵向倍比稀释，每孔50μL，正常大鼠血清同样倍数稀释作为阴性对照，37℃孵育2h；用PBST洗第三次，加入工作浓度的酶标二抗，每孔50μL，37℃孵育1.5h，PBST洗涤后，TMB显色，酶联检测仪测定OD450的值，判定检测抗原的最佳包被浓度。
采用建好的间接ELISA方法检测杂交瘤细胞分泌的抗体情况。具体方法如下：将杂交瘤细胞培养上清加入预先包被好重组人TNFR2-His Tag蛋白(Sino；Cat：10417-H08H)的ELISA板中，50μL/孔，以SP2/0细胞上清作为阴性对照，免疫多抗血清作为阳性对照，37℃水浴2h；PBST洗涤3次；加入工作浓度的HRP标记的羊抗鼠IgG和IgM抗体，50μL/孔，37℃水浴1.5h；洗涤后， TMB显色10min，显示终止后酶标仪测定OD450读数。被测孔OD450读数大于阴性对照两倍以上判定为阳性。
实施例4：FACS方法进一步筛选阳性克隆
用携带人TNFR2、猴TNFR2或小鼠TNFR2的慢病毒载体转导入CHOK1和Flpin-T-Rex293F细胞系。分选细胞以建立人TNFR2、猴TNFR2和小鼠TNFR2稳定表达细胞系，CHOK1-hTNFR2、CHOK1-cTNFR2和Flpin-T-Rex293F-mTNFR2。分别将100μL FACS缓冲液中的1×10 5个稳定表达细胞等分至独立的管中，并添加100uL杂交瘤细胞培养上清。将细胞在4℃下孵育30分钟，然后用过量FACS缓冲液洗涤两次。将细胞重新悬浮于100μL FACS缓冲液中，并将0.005μg抗鼠IgG-PE二抗添加到样品中，孵育30分钟并用过量FACS缓冲液洗涤两次。将细胞在固定缓冲液中固定，随后通过流式细胞术进行分析。FACS方法筛选出与人TNFR2特异性结合，且与猴、鼠有种属交叉反应的抗体。
采用两轮有限稀释法进行杂交瘤细胞的单克隆化，通过ELISA和FACS检测方法确定亚克隆细胞产生抗体与人、猴TNFR2蛋白的结合活性。
实施例5：抗体的制备
获得稳定的杂交瘤细胞系后，主要采用体外培养法获取单抗。将细胞株扩增至在T75培养瓶内，培养至细胞覆盖率为80-90％，将细胞上清弃去，加入30mL杂交瘤生产培养基(hybridoma-SFM，Gibco)，37℃，5％CO 2培养。培养2-3天后添加杂交瘤生产培养基(hybridoma-SFM，Gibco)，细胞活率低于30％，则可添加新鲜的活细胞。培养6-7天，待细胞存活率低于20％，低速离心后收集培养上清，4℃储存备用。
用MabSelect SuRe-LX(GE公司)亲和层析柱纯化抗体：①装柱，将购买的Protein A填料适量装于重力层析柱中用平衡缓冲液(PBS，pH7.4)冲洗至平衡；②上样，将经过0.22μm滤膜过滤的杂交瘤细胞培养上清加入装好的层析柱中，控制流速1滴/秒；③平衡，上完样液后使用平衡缓冲液冲洗至平衡；④洗脱，加入洗脱缓冲液(50mM醋酸/醋酸钠，pH3.5)冲洗柱子并收集洗脱液；⑤再生，洗脱完成后加入2倍柱体积0.1M NaOH冲洗柱子，2倍柱体积的20％乙醇冲洗后置于4℃保存。最后采用SDS-PAGE法和SEC-HPLC法鉴定抗体纯度，紫外微量分光光度计法测定抗体浓度。
实施例6：抗TNFR2抗体与人TNFR2的FACS结合活性
将CHOK1-hTNFR2细胞以1×10 5个/孔进行铺板(包含1％BSA)。添加100uL同型对照及候选抗体，浓度为100nm，4倍稀释11个梯度(包含1％BSA)。将细胞在4℃下孵育1小时，然后用过量FACS缓冲液洗涤两次。将 细胞重新悬浮于100μL FACS缓冲液中，加入山羊抗大鼠IgG Fc-AF647(1:500)，100ul在1％BSA，避光4℃下孵育30分钟并用过量FACS缓冲液洗涤两次。将细胞在固定缓冲液中固定，随后通过流式细胞术进行分析。FACS方法筛选出与人TNFR2特异性结合活性高的候选抗体。如表1和图1所示，与BMK2相比，11条序列显示出与hTNFR2相当的结合能力。
表1
抗体 EC50(nM) 最大平均荧光值 亲代细胞
抗体01 0.045 921 92.9
抗体02 0.067 1239 65
抗体03 0.045 383 64
抗体04 0.025 1322 61.3
抗体05 0.038 949 64.2
抗体06 0.077 1342 74.5
抗体07 0.050 1392 64.7
抗体08 0.081 1481 71
抗体09 0.065 1370 68.9
抗体10 0.077 907 64.1
抗体11 0.493 1007 76
BMK2 0.051 1610 135
uIgG1同型对照 >100 97 134
实施例7：配体阻断实验
包被抗-His抗体，(1ug/mL)，50ul/孔，4℃过夜孵育后PBST洗涤3次。加入2％BSA，200ul/孔，室温孵育1小时，PBST洗涤3次。加入重组人TNFR2-His Tag蛋白(Sino；Cat：10417-H08H)，0.25ug/mL(包含2％BSA)，50ul/孔，室温孵育1小时，PBST洗涤3次。加入一抗(同型对照或候选抗体)，浓度为100nM，用含有1.6nM重组人TNF-alpha-Biotin蛋白(Sino；Cat：10602-HANE-B)及2％BSA溶液4倍稀释11个梯度，室温孵育2小时，PBST洗涤3次。加入二抗，SA-HRP(1：5000)，50ul/孔，包含2％BSA，室温孵育1小时，PBST洗涤3次。
洗涤后，TMB显色10min，显示终止后酶标仪测定OD450读数。根据实验结果判断候选抗体是否具有配体阻断活性。如表2和图2所示，2条序列显示出比BMK2更好的配体阻断能力；6条序列显示出与BMK2相当的配体阻断能力。
表2
抗体 IC50(nM) 抑制率(％)
抗体01 2.72 93.2
抗体02 0.962 96.2
抗体03 10.4 91.6
抗体04 0.584 96.0
抗体05 1.27 95.9
抗体06 1.79 96.4
抗体07 >100 15.4
抗体08 1.40 96.1
抗体09 1.18 96.1
抗体10 >100 12.2
抗体11 >100 22.4
BMK2 1.54 95.3
uIgG1同型对照 >100 11.7
实施例8：抗TNFR2抗体与人TNFR2的亲和力检测
设备：Biacore 8K(GE)。
传感器芯片：CM5芯片(GE)。
(1)固定：
活化剂准备：在使用前立即混合400mM EDC和100mM NHS(GE)制备。
以10μL/min的流速激活CM5传感器芯片420s。然后用30μg/mL的抗大鼠Fc IgG在10mM NaAc(pH4.5)中以10μL/min的流速注入通道。用1M乙醇胺-盐酸(GE)以10μL/min的流速灭活芯片420s。
(2)样品捕获：
运行缓冲液1×HBS-EP+(10mM HEPES，150mM NaCl，3mM EDTA，0.05％吐温20，pH7.4)中候选抗体，用抗大鼠Fc IgG以10μL/min的流速捕获到Fc2上。将10nm的重组人TNFR2-His Tag蛋白(Sino；Cat：10417-H08H)和运行缓冲液以30min的流速依次注入Fc1-Fc2，结合180s，然后解离3600s。每解离后注入10mM甘氨酸(pH1.5)作为再生缓冲液。
(3)再生：
用10mM甘氨酸(pH1.5)再生芯片。
(4)数据分析：
从测试结果图中减去参考通道Fc1和缓冲通道的结果图。实验数据符合1：1结合模型。用分子量26.6kDa计算重组人TNFR2-His Tag蛋白的摩尔浓度。
结果见表3，候选抗体与人TNFR2间SPR亲和力高，达到0.01nM-1nM。
表3.候选抗体与人TNFR2间SPR亲和力
抗体 ka(1/Ms) kd(1/s) KD(M)
抗体01 1.24E+06 1.05E-02 8.45E-09
抗体02 3.79E+05 1.05E-05 2.77E-11
抗体03 2.22E+05 4.13E-03 1.87E-08
抗体04 1.32E+06 <1.00E-05* <7.58E-12
抗体05 2.76E+05 2.81E-04 1.02E-09
抗体06 3.18E+05 3.33E-04 1.05E-09
抗体07 6.95E+05 2.61E-04 3.76E-10
抗体08 3.72E+05 <1.00E-05* <2.69E-11
抗体09 3.73E+05 1.37E-05 3.66E-11
抗体10 4.92E+05 7.40E-05 1.50E-10
抗体11 1.20E+05 1.69E-04 1.40E-09
BMK2 2.13E+05 1.59E-04 7.48E-10
*低于检测下限
实施例9：抗TNFR2抗体介导的对Treg细胞的杀伤活性
从健康志愿者外周血单个核细胞(PBMC)中分离得到CD4+T细胞。候选抗体，200nM，5倍稀释9个梯度，加入200U/mL IL2及20ng/mL重组人TNF-alpha。加入CD4+T细胞，2×10 5个/孔，37℃孵育72小时。PE-anti-CD25(1:50)和Alexa Fluor 488-anti-Foxp3(1:50)进行染色，使用BD FACSC anto II流式细胞仪计数分析。结果如表4和图3所示，6条抗体序列均显示出非常优异的对Treg细胞杀伤活性。
表4.抗TNFR2抗体介导的对Treg细胞的杀伤活性
Figure PCTCN2022098506-appb-000002
Figure PCTCN2022098506-appb-000003
实施例10：抗TNFR2抗体介导的对CD8+T细胞的增殖作用
包被候选抗体(含有5ug/mL OKT3)，20ug/mL，4倍稀释3个梯度，4℃过夜孵育。加入用CFSE进行标记的CD8+T细胞，2×10 5个/孔，加入1ug/mL anti-CD28，37℃孵育96小时。使用BD FACS Canto II流式细胞仪计数分析。结果如图4所示，6条抗体序列均显示出非常优异的促进CD8+T细胞增值的作用。
实施例11：抗TNFR2抗体介导的对CD8+T细胞的活化作用
包被候选抗体(含有5ug/mL OKT3)，20ug/mL，4倍稀释3个梯度，4℃过夜孵育。加入用CD8+T细胞，2×10 5个/孔，加入1ug/mL anti-CD28，37℃孵育16小时。通过ELISA分别检测IFNγ和IL-2的释放。结果如图5所示，6条抗体序列均显示出非常优异的促进CD8+T细胞活化的作用。
实施例12：单克隆抗体的测序
选取3个候选抗体进行测序，并最终获得了抗体04、抗体10和抗体11的氨基酸/核苷酸序列。轻链可变区序列和重链可变区序列与序列表的对应关系如表5。
表5.编号与序列对应关系
抗体04 VH SEQ ID NO:01
抗体04 VL SEQ ID NO:05
抗体10 VH SEQ ID NO:09
抗体10 VL SEQ ID NO:13
抗体11 VH SEQ ID NO:17
抗体11 VL SEQ ID NO:21
实施例13：嵌合抗体的重组表达制备
基因合成轻链可变区与人IgG1 kappa轻链恒定区(SEQ ID NO:69)DNA片段为轻链DNA，基因合成重链可变区与人IgG1重链恒定区(SEQ ID NO:70)的DNA片段为重链DNA。利用分子克隆技术，插入pcDNA3.1-G418载体中，构建成哺乳动物细胞表达质粒。pcDNA3.1-G418载体含有启动子CMV Promoter、真核筛选标记G418标签和原核筛选标签Ampicillin。基因合成得到 抗体04-xIgG1K、抗体10-xIgG1K、抗体11-xIgG1K抗体表达轻链和重链的核苷酸序列，用HindIII和XhoI对载体和目的片段进行双酶切，回收后通过DNA连接酶进行酶连，并转化大肠杆菌感受态细胞DH5α，挑选出阳性克隆并进行质粒提取和酶切验证，获得含抗体04-xIgG1K、抗体10-xIgG1K、抗体11-xIgG1K重链和轻链的重组质粒。
根据《分子克隆实验指南》(2002年，科学出版社)所述方法将含有上述各目的基因的重组质粒转化至大肠杆菌感受态细胞DH5α中，将转化细菌涂布在含100μg/mL氨苄青霉素的LB平板上培养，挑选质粒克隆至液体LB培养基中培养，260rpm摇菌14小时，由无内毒素质粒大抽试剂盒抽提质粒，用无菌水溶解并用核酸蛋白定量仪进行浓度测定。
在37℃、8％CO2、100rpm下培养ExpiCHO至细胞密度6×10 6cells/mL。使用脂质体分别将含抗体04-xIgG1K、抗体10-xIgG1K、抗体11-xIgG1K重链和轻链的重组质粒转染到上述细胞中，转染质粒浓度为1μg/mL，脂质体浓度参照ExpiCHO TM Expression System试剂盒确定，在32℃、5％CO2，100rpm下培养7-10天。转染18-22h之后和5-8天之间分别补料一次。4000rpm离心上述培养产物，0.22μm滤膜过滤并收集培养基上清液，采用Protein A纯化所得的抗体。
实施例14：嵌合抗体体内抗肿瘤药效评价
在小鼠结肠癌细胞系MC38移植肿瘤C57BL/6-hTNFR2人源化小鼠模型中，对抗体04-xIgG1K、抗体10-xIgG1K、抗体11-xIgG1K进行体内抗肿瘤药效评价。
小鼠结肠癌MC38细胞体外单层培养，培养条件为RPMI1640培养基中加10％胎牛血清，2mm谷氨酰胺，37℃ 5％CO 2培养。一周两次用胰酶-EDTA进行常规消化处理传代。当细胞饱和度为80％-90％时，收取细胞，计数，接种。将0.1mL(5x10 5cells)MC38细胞皮下接种于每只小鼠的右后背，肿瘤平均体积达到60mm3时开始分组给药。
给与荷瘤小鼠腹腔注射抗体04-xIgG1K、抗体10-xIgG1K、抗体11-xIgG1K，每周给药2次，每次2.0mg/kg，并给与相同剂量人IgG作为对照，共给药5次。
每天监测动物的健康状况及凋亡情况，例行检查包括观察肿瘤生长和药物治疗对动物日常行为表现的影响如行为活动，摄食摄水量(仅目测)，体重变化(每周测量两次体重)，外观体征或其它不正常情况。基于各组动物数量记录各组内动物凋亡数和副作用。
实验指标是考察肿瘤生长是否被抑制、延缓或治愈。每周三次用游标卡尺测量肿瘤直径。肿瘤体积的计算公式为：V＝0.5a×b2，a和b分别表示肿瘤的长径和短径。化合物的抑瘤疗效用TGI(％)或相对肿瘤增殖率T/C(％)评价。 TGI(％)，反映肿瘤生长抑制率。TGI(％)的计算：TGI(％)＝【(1-(某处理组给药结束时平均瘤体积－该处理组开始给药时平均瘤体积))/(溶剂对照组治疗结束时平均瘤体积－溶剂对照组开始治疗时平均瘤体积)】×100％。相对肿瘤增殖率T/C(％)：计算公式如下：T/C％＝TRTV/CRTV×100％(TRTV：治疗组RTV；CRTV：阴性对照组RTV)。根据肿瘤测量的结果计算出相对肿瘤体积(relative tumor volume，RTV)，计算公式为RTV＝Vt/V0，其中V0是分组给药时(即d0)测量所得平均肿瘤体积，Vt为某一次测量时的平均肿瘤体积，TRTV与CRTV取同一天数据。
小鼠结肠癌细胞系MC38移植瘤模型荷瘤鼠分别给予人IgG对照，抗体04-xIgG1K、抗体10-xIgG1K、抗体11-xIgG1K的肿瘤生长曲线如图6所述，其中横坐标表示开始治疗后的天数，纵坐标表示肿瘤体积。如图6，抗体04-xIgG1K、抗体10-xIgG1K、抗体11-xIgG1K和BMK6具有相当或更优的体内抗肿瘤药效，其中抗体11-xIgG1K的体内肿瘤药效显著优于BMK6。
实施例15：抗体的人源化改造
将VH和VL序列与NCBI网站上的已知人种系序列的文库进行比较(https://www.ncbi.nlm.nih.gov/igblast/)。所用数据库是IMGT人VH基因和IMGT人VLκ基因。对于抗体04VH，选择人种系IGVH1-2作为接受体序列，并且人轻链IGKJ4(等位基因1)接合区(J基因)选自在
Figure PCTCN2022098506-appb-000004
the international ImMunoGeneTics information
Figure PCTCN2022098506-appb-000005
http://www.imgt.org处汇编的人接合区序列。对于抗体04VL，选择人种系IGKV1-16作为接受体序列，并且人轻链IGKJ2(等位基因1)接合区(J基因)选自在
Figure PCTCN2022098506-appb-000006
the international ImMunoGeneTics information
Figure PCTCN2022098506-appb-000007
http://www.imgt.org处汇编的人接合区序列。对于抗体10VH，选择人种系IGVH3-15作为接受体序列，并且人轻链IGKJ4(等位基因1)接合区(J基因)选自在
Figure PCTCN2022098506-appb-000008
the international ImMunoGeneTics information
Figure PCTCN2022098506-appb-000009
http://www.imgt.org处汇编的人接合区序列。对于抗体10VL，选择人种系IGKV1-27作为接受体序列，并且人轻链IGKJ2(等位基因1)接合区(J基因)选自在
Figure PCTCN2022098506-appb-000010
the international ImMunoGeneTics information
Figure PCTCN2022098506-appb-000011
http://www.imgt.org处汇编的人接合区序列。对于抗体11VH，选择人种系IGVH1-69作为接受体序列，并且人轻链IGKJ4(等位基因1)接合区(J基因)选自在
Figure PCTCN2022098506-appb-000012
the international ImMunoGeneTics information
Figure PCTCN2022098506-appb-000013
http://www.imgt.org处汇编的人接合区序列。对于抗体11VL，选择人种系IGKV3D-15作为接受体序列，并且人轻链IGKJ2(等位基因1)接合区(J基因)选自在
Figure PCTCN2022098506-appb-000014
the international ImMunoGeneTics information
Figure PCTCN2022098506-appb-000015
http://www.imgt.org处汇编的人接合区序列。根据AbM定义确定CDR。使人种系框架位置改变成相应亲本鼠序列使人源化抗体的结合最优化。通过以上方法，我们得到了以下多个人源化抗体。
抗体 序列编号
抗体12 VH:SEQ ID NO:25；VL:SEQ ID NO:26
抗体13 VH:SEQ ID NO:31；VL:SEQ ID NO:32
抗体14 VH:SEQ ID NO:34；VL:SEQ ID NO:35
抗体15 VH:SEQ ID NO:37；VL:SEQ ID NO:38
抗体16 VH:SEQ ID NO:41；VL:SEQ ID NO:42
抗体17 VH:SEQ ID NO:43；VL:SEQ ID NO:44
抗体18 VH:SEQ ID NO:29；VL:SEQ ID NO:30
抗体19 VH:SEQ ID NO:39；VL:SEQ ID NO:40
抗体20 VH:SEQ ID NO:46；VL:SEQ ID NO:47
抗体21 VH:SEQ ID NO:49；VL:SEQ ID NO:50
抗体22 VH:SEQ ID NO:51；VL:SEQ ID NO:52
抗体23 VH:SEQ ID NO:53；VL:SEQ ID NO:54
抗体24 VH:SEQ ID NO:56；VL:SEQ ID NO:57
抗体25 VH:SEQ ID NO:59；VL:SEQ ID NO:60
抗体26 VH:SEQ ID NO:62；VL:SEQ ID NO:63
抗体27 VH:SEQ ID NO:65；VL:SEQ ID NO:66
抗体28 VH:SEQ ID NO:67；VL:SEQ ID NO:68
实施例16：人源化抗体的结合活性测定
(1)包被：用包被液(1×PBS，pH7.4)将Human-TNFR2-His稀释为0.5μg/mL，包被到96孔酶标板中，100μL/孔，4℃过夜。倒掉包被液，1×PBST洗板每孔300μL，用洗板机洗涤4次，并在平板纸上拍干。
(2)封闭：用3％脱脂奶粉封闭，300μL/孔，37℃孵育1h，倒掉封闭液，洗板机洗涤4次，平板纸上拍干。
(3)样品稀释：参比品和供试品用3％脱脂奶粉稀释为10μg/mL，以此为初始浓度进行3倍稀释，共稀释11个梯度，另设1个空白孔，只加稀释液。100μL/孔，37℃孵育1h。弃去孔中液体，洗板机洗涤4次，平板纸上拍干。
(4)加酶标二抗：用3％脱脂奶粉将Peroxidase-conjugated AffiniPure F(ab’)2 Fragment Goat Anti-Human IgG按1：20000稀释，100μL/孔，37℃孵育1h。洗板机洗涤6次，平板纸上拍干。
(5)显色：加入TMB显色液，100μL/孔，并用铝箔纸包好，37℃避光显色8min。
(6)终止显色：加入终止液1M HCl终止显色反应，100μL/孔。
(7)在酶标仪上450nm处读数。
(8)人源化抗体与对应的嵌合抗体具有相当或更优的结合活性，表明抗体人 源化后保持高结合活性。
表6
组合 EC50(ug/mL)
抗体04-xIgG1K(参照品) 0.005815
抗体12 0.01348
抗体13 0.004955
抗体14 0.00375
抗体15 0.003197
抗体16 0.02026
抗体17 0.01488
抗体10-xIgG1K(参照品) 0.01429
抗体21 0.01469
抗体22 0.01373
实施例17：人源化抗体的亲和力测定
设备：OCTET Red96e(Fortebio)。
传感器：AHC。
(1)实验设置：
传感器准备：在使用前以0.02％PBST(0.02％吐温20，pH7.4，1*PBS)作为缓冲液浸润AHC传感器600s，除去传感器表面覆盖的蔗糖。
(2)固化及捕获：
AHC传感器以0.02％PBST(0.02％吐温20，pH7.4，1*PBS)作为缓冲液平衡60s，固化样品板中的TNFR2抗体300s，二次平衡缓冲液180s。100nm的人W3785-hPro1.ECD.His(Human-TNFR2-His)蛋白与TNFR2抗体结合300s，然后解离600s。解离后以10mM甘氨酸(pH2.0)作为再生缓冲液，再生30s。
(3)再生：
用10mM甘氨酸(pH2.0)再生传感器。
(4)数据分析：
由表7可知，人源化抗体与对应的抗体相比，具有相当或更优的结合活性，说明人源化抗体能保持高亲和力。
表7.亲和力试验结果
Figure PCTCN2022098506-appb-000016
Figure PCTCN2022098506-appb-000017
实施例18：小鼠结肠癌细胞系MC38移植肿瘤C57BL/6hTNFR2小鼠模型体内药效
小鼠结肠癌MC38细胞体外单层培养，培养条件为DMEM培养基中加10％胎牛血清，2mm谷氨酰胺，37℃ 5％CO 2培养。一周两次用胰酶-EDTA进行常规消化处理传代。当细胞饱和度为80％-90％时，收取细胞，计数，接种。
将0.1mL(5x10 5cells)MC38细胞皮下接种于每只小鼠的右后背，肿瘤平均体积达到54mm 3时开始分组给药。实验指标是考察肿瘤生长是否被抑制、延缓或治愈。每周三次测量肿瘤直径。肿瘤体积的计算公式为：V＝0.5a×b 2，a和b分别表示肿瘤的长径和短径。抑瘤疗效用TGI(％)评价。TGI(％)，反映肿瘤生长抑制率。TGI(％)的计算：TGI(％)＝【(1-(某处理组给药结束时平均瘤体积－该处理组开始给药时平均瘤体积))/(溶剂对照组治疗结束时平均瘤体积－溶剂对照组开始治疗时平均瘤体积)】×100％。
表8.体内药效实验动物分组及给药方案
Figure PCTCN2022098506-appb-000018
Figure PCTCN2022098506-appb-000019
结果如图7所示，其中横坐标表示分组给药后的天数，纵坐标表示肿瘤体积。开始细胞接种6天后，达到54mm 3进行分笼与给药，给药14天后，生理盐水对照组的小鼠平均荷瘤体积达到2452±563mm 3；而构建抗体治疗组的小鼠荷瘤体积为1093±184mm 3，肿瘤生长受到显著抑制(TGI为56.65％)，同时生理盐水对照组和构建抗体治疗组小鼠体重均缓慢上长(如图8)，表明该构建抗体显示出良好的抗肿瘤活性并具有良好的安全性。
实施例19：小鼠黑色素瘤细胞系B16-F10移植肿瘤C57BL/6hTNFR2小鼠模型体内药效
通过将1×10 6个的小鼠黑色素瘤B16-F10细胞皮下注射至雌性裸鼠右后背以建立皮下移植肿瘤模型，肿瘤平均体积达到150mm 3时开始分组给药。10mpk构建抗体、10mpk同型对照或等体积PBS进行腹腔注射治疗，每3天给药一次，每周给药两次。实验指标是考察肿瘤生长是否被抑制、延缓或治愈。每周三次测量肿瘤直径。肿瘤体积的计算公式为：V＝0.5a×b 2，a和b分别表示肿瘤的长径和短径。抑瘤疗效用TGI(％)评价。TGI(％)，反映肿瘤生长抑制率。TGI(％)的计算：TGI(％)＝【(1-(某处理组给药结束时平均瘤体积－该处理组开始给药时平均瘤体积))/(溶剂对照组治疗结束时平均瘤体积－溶剂对照组开始治疗时平均瘤体积)】×100％。
结果如图9所示，其中横坐标表示分组给药后的天数，纵坐标表示肿瘤体积。开始细胞接种8天后，达到150mm 3进行分笼与给药，给药21天后，PBS对照与同型对照治疗组的小鼠平均荷瘤体积达到4273mm 3；而构建抗体治疗组的小鼠荷瘤体积仅为967mm 3，肿瘤生长受到显著抑制(TGI为80.3％)，该构建抗体显示出良好的抗肿瘤活性。

Claims (27)

  1. 一种能够特异性地结合人肿瘤坏死因子受体2(TNFR2)的抗体或其抗原结合片段，其中，包含重链和轻链，其特征在于，所述重链可变区包含重链互补决定区CDR1、CDR2和CDR3，所述轻链可变区包含轻链互补决定区CDR1、CDR2和CDR3，其中，
    (01)所述重链可变区的CDR1、CDR2和CDR3分别由氨基酸序列SEQ ID NO：18、SEQ ID NO：58和SEQ ID NO：20组成，且所述轻链可变区的CDR1、CDR2和CDR3分别由SEQ ID NO：22、SEQ ID NO：23和SEQ ID NO：24组成；或
    (02)所述重链可变区的CDR1、CDR2和CDR3分别由氨基酸序列SEQ ID NO：2、SEQ ID NO：3和SEQ ID NO：4组成，且所述轻链可变区的CDR1、CDR2和CDR3分别由SEQ ID NO：6、SEQ ID NO：7和SEQ ID NO：8组成；或
    (03)所述重链可变区的CDR1、CDR2和CDR3分别由氨基酸序列SEQ ID NO：10、SEQ ID NO：11和SEQ ID NO：12组成，且所述轻链可变区的CDR1、CDR2和CDR3分别由SEQ ID NO：14、SEQ ID NO：15和SEQ ID NO：16组成；或
    (04)所述重链可变区的CDR1、CDR2和CDR3分别由氨基酸序列SEQ ID NO：18、SEQ ID NO：19和SEQ ID NO：20组成，且所述轻链可变区的CDR1、CDR2和CDR3分别由SEQ ID NO：22、SEQ ID NO：23和SEQ ID NO：24组成；或
    (05)所述重链可变区的CDR1、CDR2和CDR3分别由氨基酸序列SEQ ID NO：10、SEQ ID NO：11和SEQ ID NO：12组成，且所述轻链可变区的CDR1、CDR2和CDR3分别由SEQ ID NO：27、SEQ ID NO：15和SEQ ID NO：16组成；或
    (06)所述重链可变区的CDR1、CDR2和CDR3分别由氨基酸序列SEQ ID NO：18、SEQ ID NO：55和SEQ ID NO：20组成，且所述轻链可变区的CDR1、CDR2和CDR3分别由SEQ ID NO：22、SEQ ID NO：23和SEQ ID NO：24组成；或
    (07)所述重链可变区的CDR1、CDR2和CDR3分别由氨基酸序列SEQ ID NO：2、SEQ ID NO：3和SEQ ID NO：4组成，且所述轻链可变区的CDR1、CDR2和CDR3分别由SEQ ID NO：28、SEQ ID NO：7和SEQ ID NO：8组成；或
    (08)所述重链可变区的CDR1、CDR2和CDR3分别由氨基酸序列SEQ ID NO：2、SEQ ID NO：3和SEQ ID NO：4组成，且所述轻链可变区的CDR1、CDR2和CDR3分别由SEQ ID NO：33、SEQ ID NO：7和SEQ ID NO：8组成；或
    (09)所述重链可变区的CDR1、CDR2和CDR3分别由氨基酸序列SEQ ID  NO：2、SEQ ID NO：3和SEQ ID NO：36组成，且所述轻链可变区的CDR1、CDR2和CDR3分别由SEQ ID NO：6、SEQ ID NO：7和SEQ ID NO：8组成；或
    (10)所述重链可变区的CDR1、CDR2和CDR3分别由氨基酸序列SEQ ID NO：18、SEQ ID NO：55和SEQ ID NO：61组成，且所述轻链可变区的CDR1、CDR2和CDR3分别由SEQ ID NO：22、SEQ ID NO：23和SEQ ID NO：24组成；或
    (11)所述重链可变区的CDR1、CDR2和CDR3分别由氨基酸序列SEQ ID NO：18、SEQ ID NO：55和SEQ ID NO：64组成，且所述轻链可变区的CDR1、CDR2和CDR3分别由SEQ ID NO：22、SEQ ID NO：23和SEQ ID NO：24组成；或
    (12)所述重链可变区的CDR1、CDR2和CDR3分别由氨基酸序列SEQ ID NO：18、SEQ ID NO：58和SEQ ID NO：61组成，且所述轻链可变区的CDR1、CDR2和CDR3分别由SEQ ID NO：22、SEQ ID NO：23和SEQ ID NO：24组成；或
    (13)所述重链可变区的CDR1、CDR2和CDR3分别由氨基酸序列SEQ ID NO：2、SEQ ID NO：3和SEQ ID NO：36组成，且所述轻链可变区的CDR1、CDR2和CDR3分别由SEQ ID NO：28、SEQ ID NO：7和SEQ ID NO：8组成；或
    (14)所述重链可变区的CDR1、CDR2和CDR3分别由氨基酸序列SEQ ID NO：2、SEQ ID NO：3和SEQ ID NO：45组成，且所述轻链可变区的CDR1、CDR2和CDR3分别由SEQ ID NO：6、SEQ ID NO：7和SEQ ID NO：8组成；或
    (15)所述重链可变区的CDR1、CDR2和CDR3分别由氨基酸序列SEQ ID NO：2、SEQ ID NO：3和SEQ ID NO：45组成，且所述轻链可变区的CDR1、CDR2和CDR3分别由SEQ ID NO：28、SEQ ID NO：7和SEQ ID NO：48组成；或
    (16)所述重链可变区的CDR1、CDR2和CDR3分别由氨基酸序列SEQ ID NO：18、SEQ ID NO：58和SEQ ID NO：64组成，且所述轻链可变区的CDR1、CDR2和CDR3分别由SEQ ID NO：22、SEQ ID NO：23和SEQ ID NO：24组成。
  2. 根据权利要求1中所述的抗体或其抗原结合片段，其特征在于，其中：
    (01)所述重链可变区具有SEQ ID NO：56给出的氨基酸序列，或与SEQ ID NO：56给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：56的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列，
    所述轻链可变区具有SEQ ID NO：57给出的氨基酸序列，或与SEQ ID NO：57给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：57的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列；或
    (02)所述重链可变区具有SEQ ID NO：1给出的氨基酸序列，或与SEQ ID NO：1给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：1的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列，
    所述轻链可变区具有SEQ ID NO：5给出的氨基酸序列，或与SEQ ID NO：5给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：5的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列；或
    (03)所述重链可变区具有SEQ ID NO：9给出的氨基酸序列，或与SEQ ID NO：9给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：9的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列，
    所述轻链可变区具有SEQ ID NO：13给出的氨基酸序列，或与SEQ ID NO：13给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：13的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列；或
    (04)所述重链可变区具有SEQ ID NO：17给出的氨基酸序列，或与SEQ ID NO：17给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：17的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列，
    所述轻链可变区具有SEQ ID NO：21给出的氨基酸序列，或与SEQ ID NO：21给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：21的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列；或
    (05)所述重链可变区具有SEQ ID NO：49给出的氨基酸序列，或与SEQ ID NO：49给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、 94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：49的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列，
    所述轻链可变区具有SEQ ID NO：50给出的氨基酸序列，或与SEQ ID NO：50给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：50的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列；或
    (06)所述重链可变区具有SEQ ID NO：51给出的氨基酸序列，或与SEQ ID NO：51给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：51的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列，
    所述轻链可变区具有SEQ ID NO：52给出的氨基酸序列，或与SEQ ID NO：52给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：52的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列；或
    (07)所述重链可变区具有SEQ ID NO：53给出的氨基酸序列，或与SEQ ID NO：53给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：53的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列，
    所述轻链可变区具有SEQ ID NO：54给出的氨基酸序列，或与SEQ ID NO：54给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：54的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列；或
    (08)所述重链可变区具有SEQ ID NO：25给出的氨基酸序列，或与SEQ ID NO：25给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：25的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列，
    所述轻链可变区具有SEQ ID NO：26给出的氨基酸序列，或与SEQ ID NO：26给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：26的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10 个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列；或
    (09)所述重链可变区具有SEQ ID NO：29给出的氨基酸序列，或与SEQ ID NO：29给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：29的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列，
    所述轻链可变区具有SEQ ID NO：30给出的氨基酸序列，或与SEQ ID NO：30给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：30的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列；或
    (10)所述重链可变区具有SEQ ID NO：31给出的氨基酸序列，或与SEQ ID NO：31给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：31的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列，
    所述轻链可变区具有SEQ ID NO：32给出的氨基酸序列，或与SEQ ID NO：32给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：32的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列；或
    (11)所述重链可变区具有SEQ ID NO：34给出的氨基酸序列，或与SEQ ID NO：34给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：34的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列，
    所述轻链可变区具有SEQ ID NO：35给出的氨基酸序列，或与SEQ ID NO：35给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：35的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列；或
    (12)所述重链可变区具有SEQ ID NO：59给出的氨基酸序列，或与SEQ ID NO：59给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：59的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列，
    所述轻链可变区具有SEQ ID NO：60给出的氨基酸序列，或与SEQ ID  NO：60给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：60的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列；或
    (13)所述重链可变区具有SEQ ID NO：62给出的氨基酸序列，或与SEQ ID NO：62给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：62的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列，
    所述轻链可变区具有SEQ ID NO：63给出的氨基酸序列，或与SEQ ID NO：63给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：63的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列；或
    (14)所述重链可变区具有SEQ ID NO：65给出的氨基酸序列，或与SEQ ID NO：65给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：65的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列，
    所述轻链可变区具有SEQ ID NO：66给出的氨基酸序列，或与SEQ ID NO：66给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：66的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列；或
    (15)所述重链可变区具有SEQ ID NO：37给出的氨基酸序列，或与SEQ ID NO：37给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：37的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列，
    所述轻链可变区具有SEQ ID NO：38给出的氨基酸序列，或与SEQ ID NO：38给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：38的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列；或
    (16)所述重链可变区具有SEQ ID NO：39给出的氨基酸序列，或与SEQ ID NO：39给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO： 39的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列，
    所述轻链可变区具有SEQ ID NO：40给出的氨基酸序列，或与SEQ ID NO：40给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：40的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列；或
    (17)所述重链可变区具有SEQ ID NO：41给出的氨基酸序列，或与SEQ ID NO：41给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：41的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列，
    所述轻链可变区具有SEQ ID NO：42给出的氨基酸序列，或与SEQ ID NO：42给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：42的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列；或
    (18)所述重链可变区具有SEQ ID NO：43给出的氨基酸序列，或与SEQ ID NO：43给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：43的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列，
    所述轻链可变区具有SEQ ID NO：44给出的氨基酸序列，或与SEQ ID NO：44给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：44的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列；或
    (19)所述重链可变区具有SEQ ID NO：46给出的氨基酸序列，或与SEQ ID NO：46给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：46的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列，
    所述轻链可变区具有SEQ ID NO：47给出的氨基酸序列，或与SEQ ID NO：47给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：47的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列；或
    (20)所述重链可变区具有SEQ ID NO：67给出的氨基酸序列，或与SEQ ID NO：67给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：67的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列，
    所述轻链可变区具有SEQ ID NO：68给出的氨基酸序列，或与SEQ ID NO：68给出的氨基酸序列具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或以上同一性的序列，或与SEQ ID NO：68的氨基酸序列相比具有一个或多个(优选1、2、3、4、5、6、7、8、9、10个)保守氨基酸突变(优选置换、插入或缺失)的氨基酸序列。
  3. 根据权利要求1-3中任一项所述的抗体或其抗原结合片段，其结合Treg的TNFR2，并能杀伤Treg，具有杀伤率至少10％、20％、30％、40％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、96％、97％、98％、99％或100％。
  4. 根据权利要求1-3中任一项所述的抗体或其抗原结合片段，其结合Teff的TNFR2，促进Teff细胞增殖分化。
  5. 根据权利要求1-3中任一项所述的抗体或其抗原结合片段，其结合Teff的TNFR2，促进Teff生物活性。
  6. 根据权利要求1-3中任一项所述的抗体或其抗原结合片段，其与TNFR2之间的解离常数KD小于10nM。
  7. 根据权利要求1-3中任一项所述的抗体或其抗原结合片段，其与TNFR2之间的解离常数KD小于1nM。
  8. 一种多核苷酸，其特征在于，其编码根据权利要求1-3中任一项所述的抗体或其抗原结合片段。
  9. 一种重组载体、转基因细胞系、噬菌体、重组菌或病毒载体，其特征在于，其含有根据权利要求9所述的多核苷酸。
  10. 根据权利要求10所述的重组载体、转基因细胞系、噬菌体、重组菌或病毒载体在制备抗TNFR2抗体中的应用。
  11. 一种分离的宿主细胞，其特征在于，其含有根据权利要求10所述的重组载体、转基因细胞系、噬菌体、重组菌或病毒载体。
  12. 根据权利要求12所述的宿主细胞，其中所述宿主细胞是原核细胞。
  13. 根据权利要求12所述的宿主细胞，其中所述宿主细胞是真核细胞。
  14. 根据权利要求14所述的宿主细胞，其中所述真核细胞是哺乳动物细胞。
  15. 根据权利要求15所述的宿主细胞，其中所述哺乳动物细胞是CHO细胞。
  16. 一种抗体表达方法，其特征在于，用根据权利要求10所述的重组载 体、转基因细胞系、噬菌体、重组菌或病毒载体在根据权利要求12-16中任一项所述的宿主细胞中表达抗体蛋白。
  17. 根据权利要求1-3中任一项所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段包括鼠源的、人鼠嵌合的、人源化的或全人源的。
  18. 根据权利要求1-3中任一项所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段选自由以下组成的组：单克隆抗体或其抗原结合片段、多克隆抗体或其抗原结合片段、人源化抗体或其抗原结合片段、灵长类化抗体或其抗原结合片段、双特异性抗体或其抗原结合片段、多特异性抗体或其抗原结合片段、双重可变免疫球蛋白结构域、单价抗体或其抗原结合片段、嵌合抗体或其抗原结合片段、单链Fv分子(scFv)、双特异性抗体、三特异性抗体、纳米抗体、抗体样蛋白支架、结构域抗体、Fv片段、Fab片段、F(ab’)2分子以及串联scFv。
  19. 根据权利要求1-3中任一项所述的抗体或其抗原结合片段，其中，所述抗体为单克隆抗体。
  20. 根据权利要求1-3中任一项所述的抗体或其抗原结合片段在制备药物中的应用，所述药物含有所述抗体或其抗原结合片段和化学疗法，用于在人类患者中治疗肿瘤，其中将所述抗体和所述化学疗法配制成能提供比所述试剂各自的效果之和更大的治疗效果。
  21. 一种抗体药物偶联物，其特征在于，根据权利要求1-3中任一项所述的抗体或其抗原结合片段任选通过连接子与治疗剂偶联。
  22. 根据权利要求22所述的抗体药物偶联物，其中所述治疗剂是细胞毒性剂。
  23. 一种药物组合物，其包含根据权利要求1-3中任一项所述的抗体或其抗原结合片段和药学上可接受的载体、稀释剂或赋形剂。
  24. 根据权利要求24所述的药物组合物，其中所述组合物还包含额外治疗剂。
  25. 根据权利要求25所述的药物组合物，其中所述额外治疗剂是免疫治疗剂。
  26. 根据权利要求1-3中任一项所述的抗体或其抗原结合片段在如下(a)、(b)、(c)、(d)或(e)中的应用：
    (a)制备诱导Treg细胞凋亡的药物中的应用；
    (b)制备靶向肿瘤细胞杀伤的药物中的应用；
    (c)制备阻断TNF-TNFR2肿瘤信号通路的药物中的应用；
    (d)制备促进Teff细胞增殖分化和/或生物活性的药物中的应用；
    (e)制备治疗自身免疫性疾病的药物中的应用。
  27. 根据权利要求27所述的应用，其特征在于，所述肿瘤选自：卵巢癌、黑色素瘤、前列腺癌、肠癌、胃癌、食管癌、乳腺癌、肺癌、肾癌、胰腺癌、 子宫癌、肝癌、膀胱癌、子宫颈癌、口腔癌、脑癌、睾丸癌、皮肤癌、结肠直肠癌、恶性胶质瘤、甲状腺癌。
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