WO2021093753A1 - 一种能够与人4-1bb结合的分子及其应用 - Google Patents

一种能够与人4-1bb结合的分子及其应用 Download PDF

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WO2021093753A1
WO2021093753A1 PCT/CN2020/127993 CN2020127993W WO2021093753A1 WO 2021093753 A1 WO2021093753 A1 WO 2021093753A1 CN 2020127993 W CN2020127993 W CN 2020127993W WO 2021093753 A1 WO2021093753 A1 WO 2021093753A1
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antibody
seq
consistency
positions
antigen
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PCT/CN2020/127993
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English (en)
French (fr)
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程联胜
刘雯婷
张大艳
曾小丽
王凤荣
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合肥瀚科迈博生物技术有限公司
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Priority claimed from CN201911105605.6A external-priority patent/CN112794904B/zh
Priority claimed from CN201911106016.XA external-priority patent/CN112794906B/zh
Priority claimed from CN201911105611.1A external-priority patent/CN112794905B/zh
Application filed by 合肥瀚科迈博生物技术有限公司 filed Critical 合肥瀚科迈博生物技术有限公司
Priority to US17/774,972 priority Critical patent/US20220411523A1/en
Priority to AU2020381735A priority patent/AU2020381735A1/en
Priority to CN202080058703.3A priority patent/CN114269788A/zh
Priority to KR1020227016283A priority patent/KR20220087479A/ko
Priority to EP20886450.4A priority patent/EP4059963A4/en
Priority to JP2022523656A priority patent/JP7432716B2/ja
Publication of WO2021093753A1 publication Critical patent/WO2021093753A1/zh

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Definitions

  • the present invention relates to the field of biotechnology, in particular to a molecule capable of binding to human 4-1BB and its application.
  • 4-1BB (CD137) is a member of the tumor necrosis factor (TNF) receptor family. It is encoded by the tumor necrosis factor receptor superfamily member 9 (TNFRSF9) gene. It is the process of Kwon and Weissman in the process of screening and differentiation of activated murine T cell clones. First found in. Schwarzh et al. isolated human 4-1BB with homology to murine 4-1BB from human activated T cells and named it CD137. 4-1BB is a type I membrane glycoprotein, which is different from PD-1/PD- The expression of L1, 4-1BB is activation-dependent, and it mediates the costimulatory signal of T cell activation. It is widely present in immune cells including activated NK cells, activated T cells, dendritic cells (DC), mast cells, The surface of monocytes, neutrophils and Treg cells.
  • TNF tumor necrosis factor
  • TNFRSF9 tumor necrosis factor receptor superfamily member 9
  • Human 4-1BB has 255 amino acids and is expressed in the form of monomers and dimers on the cell surface, and is easy to form trimers with ligands, thereby turning on signal transduction.
  • 4-1BB is an activating costimulatory molecule, and its signal pathway can promote the differentiation, proliferation and cytokine production of T cells, especially CD8+ T cells.
  • 4-1BB is mainly expressed on activated T cells and mainly acts on the middle and late stages of T cell immune response.
  • the 4-1BB signaling pathway can achieve anti-tumor effects by enhancing the function of tumor-specific CD8+ T cells. It can also enhance the anti-tumor immune response mediated by CD8+ T cells by enhancing the immune function of NK cells, DC and CD4+ T cells.
  • 4-1BB recruits TNFR-related factors TRAF1 and TRAF2 to form heterotrimers, through the c-Jun N-terminal kinase (JNK) pathway, extracellular signal-regulated kinase (ERK) pathway, and through ⁇ -catenin and AKT pathways to enhance signal transduction
  • JNK c-Jun N-terminal kinase
  • ERK extracellular signal-regulated kinase
  • AKT extracellular signal-regulated kinase
  • the main transcription factor NF- ⁇ B regulates the 4-1BB signal and promotes the production and secretion of cytokines.
  • the 4-1BB/4-1BBL interaction can improve the immune defense of T cell virus infection.
  • 4-1BB monotherapy and combination therapy tumor models have confirmed durable anti-tumor protective T cell memory response.
  • the purpose of the present invention is to provide a molecule capable of binding to human 4-1BB and its application.
  • the molecule provided by the present invention that can bind to human 4-1BB can be a humanized antibody against human 4-1BB or an antigen-binding fragment thereof (such as a Fab fragment, a ScFv fragment) or an antibody fusion protein (such as a complete humanized antibody). , ScFv, Fab fragments fused with other proteins to form antibody fusion proteins) or anti-human 4-1BB humanized antibodies coupled to ADC antibodies with biologically active small molecule drugs or fully humanized antibodies, ScFv, Fab fragments and other targets Double antibody formed by dot antibody.
  • the present invention claims an antibody.
  • the amino acid sequences of HCDR1, HCDR2 and HCDR3 in the variable region of the heavy chain are shown in SEQ ID No. 1, 31-35, 50-64, 98-106 from the N-terminus.
  • the amino acid sequences of LCDR1, LCDR2 and LHCDR3 in the light chain variable region are shown in SEQ ID No. 2 from the N-terminus to positions 24-34, 50-56, and 89-97.
  • amino acid sequence of the heavy chain variable region is any one of the following:
  • SEQ ID No.1 starts from the N-terminal position 1-117 (that is, SEQ ID No.16 starts from the N-terminal position 1-117), or with SEQ ID No.1 from the N-terminal position 1-117 Positions (ie SEQ ID No. 16 from the N-terminal position 1-117) have a consistency of 99% or more, 95% or more, 90% or more, 85% or more, 80% or more than 75% (the inconsistencies are preferably Framework area (FR));
  • amino acid sequence of the light chain variable region can be any of the following:
  • SEQ ID No. 2 starts from the N-terminal position 1-107 (that is, SEQ ID No. 16 starts from the N-terminal position 133-239), or with SEQ ID No. 2 starts from the N-terminal position 1-107 Positions (ie SEQ ID No. 16 from the 133-239th position from the N-terminus) have a consistency of 99% or more, 95% or more, 90% or more, 85% or more, 80% or more than 75%. Framework region (FR)).
  • SEQ ID No.20 has positions 133-239 from the N-terminus, or with SEQ ID No.20 positions 133-239 from the N-terminus with 99% or more, 95% or more, 90% or more, or 85% or more , 80% or more or 75% or more consistency (the inconsistency is preferably in the framework region (FR)).
  • SEQ ID No.21 has positions 133-239 from the N-terminus, or with SEQ ID No.21 positions 133-239 from the N-terminus with 99% or more, 95% or more, 90% or more, or 85% or more , 80% or more or 75% or more consistency (the inconsistency is preferably in the framework region (FR)).
  • SEQ ID No.23 has positions 133-239 from the N-terminus, or with SEQ ID No.23 positions 133-239 from the N-terminus with 99% or more, 95% or more, 90% or more, or 85% or more , 80% or more or 75% or more consistency (the inconsistency is preferably in the framework region (FR)).
  • the heavy chain constant region of the antibody is a human IgG4 constant region, and the specific amino acid sequence is shown at positions 118-444 from the N-terminus of SEQ ID No. 1.
  • the light chain constant region of the antibody is a human kappa light chain constant region, and the specific amino acid sequence is shown in SEQ ID No. 2 at positions 108-214 from the N-terminus.
  • amino acid sequence of the heavy chain is SEQ ID No. 1, or the amino acid sequence of SEQ ID No. 1 is 99% or more, 95% or more, 90% or more, 85% or more, 80% or more or 75% or more. Consistency (the inconsistency is preferably in the framework region (FR)).
  • the amino acid sequence of the light chain is SEQ ID No. 2, or has more than 99%, more than 95%, more than 90%, more than 85%, more than 80%, or more than 75% identity with SEQ ID No. 2 (inconsistent The location is preferably in the framework region (FR)).
  • the antibody of the present invention can be of any class, such as IgG, IgM, IgE, IgA or IgD.
  • the 4-1BB antibody is of the IgG class, such as IgG1, IgG2, IgG3 or IgG4 subtype, more preferably IgG4 subtype.
  • the light chain type of the antibody of the present invention can be a kappa chain or a lambda chain, preferably a Kappa chain.
  • the antibody is specifically an anti-human 4-1BB humanized antibody Hanke10F4.
  • the Hanke10F4 mainly binds to Domain 1 and Domain 2A of the extracellular domain of human 4-1BB.
  • IgG such as IgG1, IgG2, IgG3 or IgG4;
  • the antibody or its antigen-binding fragment of the present invention has agonist activity on human 4-1BB, can stimulate the proliferation of CD4 + T and CD8 + T cells, and can activate the downstream NF- ⁇ B signal pathway by binding to the cell surface 4-1BB molecule , Thereby significantly promoting the production and secretion of the cytokine IFN- ⁇ , and can significantly inhibit the occurrence and development of tumors in humanized mice with human 4-1BB gene knock-in, indicating to a certain extent that the antibody or its antigen-binding portion
  • the immune system can be regulated by regulating the activity of immune cells, and can be used as immune enhancers for anti-tumor or antiviral immune responses, or immune regulators for T cell-mediated autoimmune diseases.
  • the antibodies of the present invention can be prepared by methods known in the art, and can be prepared by expression in transfected cells such as immortalized eukaryotic cells such as myeloma or hybridoma cells. Antibodies can be converted from one class or subtype to another by methods known in the art. In addition, the antibodies provided by the present invention may be monoclonal antibodies or polyclonal antibodies, preferably monoclonal antibodies.
  • the antibodies of the present invention can be produced by techniques known in the art, including conventional monoclonal antibody techniques, such as standard somatic cell hybridization techniques, viral or oncogene transformation of B lymphocytes, or recombinant antibody techniques, as detailed below Narrated.
  • Hybridoma production is a commonly used method and is known in the art.
  • the amino acid sequence of the human 4-1BB antigen is shown in SEQ ID No. 5 (positions 24-184 are the extracellular region).
  • the immune antigen is isolated or purified human 4-1BB, which can be a fragment of human 4-1BB, such as Extracellular domain of human 4-1BB.
  • Animal immunization can be carried out by any method known in the art, immunizing non-human animals such as mice, rats, sheep and the like.
  • an antibody-producing immortalized cell line is prepared from the cells isolated from the immunized animal.
  • Hybridomas can be expanded in homologous animals, animals lacking an immune system, and nude mice, or expanded in cell culture in vitro. Methods of selecting, cloning and expanding hybridomas are well known to those skilled in the art.
  • the biphasic agar diffusion test was used to detect the immunoglobulin subtype secreted by hybridoma cells as IgG1.
  • the antibodies of the present invention can also be prepared by phage display methods. This phage display method for isolating human antibodies has been established in the art.
  • the antibody After the antibody is produced and expressed, it contains antibodies, dimers, single light and heavy chains, or other immunoglobulin forms, which can be purified according to standard methods in the art, such as ammonium sulfate precipitation, affinity column, column chromatography, gel Electrophoresis, etc.
  • the present invention claims the antigen-binding fragments of the aforementioned antibodies.
  • the antigen-binding fragment may comprise one or more of the following: (1) the light chain of the aforementioned antibody; (2) the heavy chain of the aforementioned antibody; (3) the light chain of the aforementioned antibody Variable region; (4) heavy chain variable region of the aforementioned antibody; (5) one or more CDR regions of the aforementioned antibody.
  • the antigen-binding fragment can be any of the following: (1) Fab fragment, which is a monovalent fragment composed of VL, VH, CL and CH1 domains; (2) F(ab')2 fragment, which is Bivalent fragment, including two Fab fragments linked by disulfide bonds in the hinge region; (3) Fd fragment, composed of VH and CH1 domains; (4) FV fragment, composed of VL and VH domains of one arm of an antibody (5) Isolated CDRs (such as CDRs from the light chain and/or CDRs from the heavy chain); (6) Single-chain antibodies, etc., where the single-chain antibody can have a scFv structure, and the single-chain antibody can have different tags, such as scFv-His, scFv-Fc, etc.
  • the antigen-binding fragment provided by the present invention is a tagged single-chain antibody (scFv-Fc).
  • a single chain antibody (scFv-Fc) can be composed of a single polypeptide chain comprising a VL domain connected to a VH domain connected to a human IgG4 Fc tag, where the VL domain and the VH domain are paired to form a monovalent molecule.
  • Single-chain antibodies can be prepared according to methods known in the art.
  • the present invention specifically claims an anti-4-1BB single-chain antibody.
  • the anti-4-1BB single-chain antibody claimed in the present invention is formed by connecting a heavy chain variable region and a light chain variable region; the amino acid sequences of HCDR1, HCDR2 and HCDR3 in the heavy chain variable region are as shown in SEQ ID No. 16 is shown at positions 31-35, 50-64, and 98-106 from the N-terminus; the amino acid sequences of LCDR1, LCDR2 and LHCDR3 in the light chain variable region are shown in SEQ ID No. 16 from the N end of the 156th to 166th, 182th to 188th, and 221-229th.
  • amino acid sequence of the heavy chain variable region of the single-chain antibody is any one of the following:
  • amino acid sequence of the light chain variable region of the single-chain antibody can be any of the following:
  • SEQ ID No.16 has positions 133-239 from the N-terminus, or with SEQ ID No.16 positions 133-239 from the N-terminus with 99% or more, 95% or more, 90% or more, or 85% or more , 80% or more or 75% or more consistency (the inconsistency is preferably in the framework region (FR)).
  • SEQ ID No.20 has positions 133-239 from the N-terminus, or with SEQ ID No.20 positions 133-239 from the N-terminus with 99% or more, 95% or more, 90% or more, or 85% or more , 80% or more or 75% or more consistency (the inconsistency is preferably in the framework region (FR)).
  • SEQ ID No.21 has positions 133-239 from the N-terminus, or with SEQ ID No.21 positions 133-239 from the N-terminus with 99% or more, 95% or more, 90% or more, or 85% or more , 80% or more or 75% or more consistency (the inconsistency is preferably in the framework region (FR)).
  • SEQ ID No.23 has positions 133-239 from the N-terminus, or with SEQ ID No.23 positions 133-239 from the N-terminus with 99% or more, 95% or more, 90% or more, or 85% or more , 80% or more or 75% or more consistency (the inconsistency is preferably in the framework region (FR)).
  • amino acid sequence of the single-chain antibody can be any of the following:
  • Positions 1-239 of SEQ ID No. 18, or with positions 1-239 of SEQ ID No. 18, have 99% or more, 95% or more, 90% or more, 85% or more, 80% or more, or 75 % Consistency (the inconsistency is preferably in the framework region (FR)).
  • Positions 1-239 of SEQ ID No. 19, or with positions 1-239 of SEQ ID No. 19 have 99% or more, 95% or more, 90% or more, 85% or more, 80% or more or 75 % Consistency (the inconsistency is preferably in the framework region (FR)).
  • Positions 1-239 of SEQ ID No. 20, or with positions 1-239 of SEQ ID No. 20 have 99% or more, 95% or more, 90% or more, 85% or more, 80% or more % Consistency (the inconsistency is preferably in the framework region (FR)).
  • Positions 1-239 of SEQ ID No. 21, or with positions 1-239 of SEQ ID No. 21 have 99% or more, 95% or more, 90% or more, 85% or more, 80% or more % Consistency (the inconsistency is preferably in the framework region (FR)).
  • Positions 1-239 of SEQ ID No. 22, or with positions 1-239 of SEQ ID No. 22 have 99% or more, 95% or more, 90% or more, 85% or more, 80% or more % Consistency (the inconsistency is preferably in the framework region (FR)).
  • Positions 1-239 of SEQ ID No. 23, or with positions 1-239 of SEQ ID No. 23 have 99% or more, 95% or more, 90% or more, 85% or more, 80% or more % Consistency (the inconsistency is preferably in the framework region (FR)).
  • Positions 1-239 of SEQ ID No. 24, or with positions 1-239 of SEQ ID No. 24 have 99% or more, 95% or more, 90% or more, 85% or more, 80% or more % Consistency (the inconsistency is preferably in the framework region (FR)).
  • SEQ ID No. 25, positions 1-239, or with SEQ ID No. 25, positions 1-239 have 99% or more, 95% or more, 90% or more, 85% or more, 80% or more, or 75 % Consistency (the inconsistency is preferably in the framework region (FR)).
  • SEQ ID No. 26, position 1-239, or with SEQ ID No. 26, position 1-239 has 99% or more, 95% or more, 90% or more, 85% or more, 80% or more % Consistency (the inconsistency is preferably in the framework region (FR)).
  • SEQ ID No. 27, position 1-239, or with SEQ ID No. 27, position 1-239 has 99% or more, 95% or more, 90% or more, 85% or more, 80% or more or 75 % Consistency (the inconsistency is preferably in the framework region (FR)).
  • SEQ ID No. 28 positions 1-239, or with positions 1-239 of SEQ ID No. 28, have 99% or more, 95% or more, 90% or more, 85% or more, 80% or more, or 75 % Consistency (the inconsistency is preferably in the framework region (FR)).
  • the present invention claims the antibody fusion protein formed by the aforementioned antibody or the aforementioned antigen-binding fragment and other proteins.
  • the other protein can be toxin, enzyme, cell membrane protein molecule, cytokine or receptor protein, etc.
  • the antibody fusion protein claimed in the present invention may be a single-chain antibody with a tag protein formed by the aforementioned single-chain antibody and a tag protein.
  • the tag protein is Fc.
  • the Fc may be derived from, for example, IgG, IgM, IgE, IgA or IgD.
  • the Fc is from the IgG class, such as IgG1, IgG2, IgG3 or IgG4 subtype, more preferably IgG4 subtype.
  • the Fc is preferably of human origin.
  • amino acid sequence of the Fc is as shown in SEQ ID No.16 at positions 240-472, or with SEQ ID No.16 at positions 240-472 from the N-terminus with more than 99%, more than 95%, Consistency of over 90%, over 85%, over 80%, or over 75%.
  • amino acid sequence of the single-chain antibody with the tagged protein may be any of the following:
  • (d1) As shown in SEQ ID No. 16, or with SEQ ID No. 16 with 99% or more, 95% or more, 90% or more, 85% or more, 80% or more than 75% consistency (the inconsistency is preferably in Framework region (FR)).
  • (d2) SEQ ID No. 18, or with SEQ ID No. 18 with 99% or more, 95% or more, 90% or more, 85% or more, 80% or more, or 75% or more consistency (the inconsistencies are preferably Framework region (FR)).
  • the present invention claims a double antibody formed by the aforementioned antibody or the aforementioned antigen-binding fragment or the aforementioned single-chain antibody with a tag protein and other target antibodies.
  • the present invention claims nucleic acid molecules.
  • the nucleic acid molecule claimed in the present invention encodes the aforementioned antibody or the aforementioned antigen-binding fragment or the aforementioned antibody fusion protein or the aforementioned double antibody.
  • the nucleic acid molecule of the present invention may be DNA or RNA, and may or may not contain intron sequences.
  • the nucleic acid molecule is a cDNA molecule.
  • nucleic acid molecule the nucleotide sequences encoding HCDR1, HCDR2, and HCDR3 in the heavy chain variable region of the antibody are as shown in SEQ ID No. 3 from the 5'end of positions 91 to 105, The nucleotide sequences of LCDR1, LCDR2, and LHCDR3 in the light chain variable region encoding the antibody are shown in SEQ ID No. 4 and 70-102 from the 5'end. Digits, 148-168th, and 265-291 are shown.
  • the nucleotide sequence encoding the variable region of the heavy chain of the antibody is SEQ ID No. 3 from the 5'end of positions 1-351 or the same as SEQ ID No. 3 3 From the 5'end, positions 1-351 have a consistency of 99% or more, 95% or more, 90% or more, 85% or more, 80% or more or 75% or more (the inconsistency is preferably in the framework region (FR)).
  • the nucleotide sequence of the variable region of the light chain encoding the antibody is SEQ ID No. 4 from the 5'end of positions 1-321 or with SEQ ID No. 4 from the 5'end of positions 1-321 Have a consistency of 99% or more, 95% or more, 90% or more, 85% or more, 80% or more or 75% or more (the inconsistency is preferably in the framework region (FR)).
  • nucleotide sequence encoding the variable region of the heavy chain constant region of the antibody is SEQ ID No. 3 at positions 352-1332 from the 5'end.
  • nucleotide sequence of the constant variable region of the light chain encoding the antibody is SEQ ID No. 4 from the 5'end of the 322-642th position.
  • the nucleotide sequence encoding the heavy chain of the antibody is SEQ ID No. 3, or is more than 99%, more than 95%, more than 90%, 85% with respect to SEQ ID No. 3. % Or more, 80% or more or 75% or more consistency (the inconsistency is preferably in the framework region (FR)).
  • the nucleotide sequence of the light chain that encodes the antibody is SEQ ID No. 4 or a sequence of 99% or more, 95% or more, 90% or more, 85% or more, 80% or more or 75% or more of SEQ ID No. 4 Consistency (the inconsistency is preferably in the framework region (FR)).
  • nucleic acid molecules of the present invention can be obtained using appropriate molecular biology techniques.
  • cDNA encoding the light chain and the heavy chain of the antibody prepared by the hybridoma can be obtained by PCR amplification or cDNA cloning technology.
  • the nucleic acid molecule of the present invention can encode the amino acid sequence of a light chain or a part of a heavy chain, a full-length light chain or a heavy chain, or an antibody derivative or an antigen-binding fragment thereof.
  • the isolated DNA encoding VH can be converted into a full-length heavy chain gene by ligating the DNA encoding VH to another DNA molecule encoding the heavy chain constant region (CH1, CH2, and CH3).
  • Human heavy chain constant region gene sequences are known in the art, and DNA fragments containing these regions can be obtained by standard PCR amplification.
  • the heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region. Preferred is the IgG4 constant region.
  • the isolated DNA encoding the VL region can be converted into a full-length light chain gene by ligating the DNA encoding VL to another DNA molecule encoding the light chain constant region CL.
  • Human light chain constant region gene sequences are known in the art, and DNA fragments containing these regions can be obtained by standard PCR amplification.
  • the light chain constant region can be a Kappa or Lambda constant region.
  • the present invention specifically claims nucleic acid molecules encoding the aforementioned single-chain antibody or the aforementioned single-chain antibody with a tag protein or the aforementioned fusion antibody.
  • nucleotide sequences encoding HCDR1, HCDR2, and HCDR3 in the heavy chain variable region are as shown in SEQ ID No. 17 from the 5'end of the 91-105th position and the 148th-position sequence from the 5'end.
  • the nucleotide sequences encoding LCDR1, LCDR2 and LHCDR3 in the light chain variable region are shown in SEQ ID No. 17, 466-498 and 544 from the 5'end in sequence. -564, 661-687.
  • the nucleotide sequence encoding the variable region of the heavy chain is SEQ ID No. 17 from the 5'end of positions 1-351 or the same as SEQ ID No. 17 from the 5'end. Positions 1-351 from the end have a consistency of 99% or more, 95% or more, 90% or more, 85% or more, 80% or more or 75% or more (the inconsistency is preferably in the framework region (FR)).
  • the nucleotide sequence encoding the variable region of the light chain is SEQ ID No. 17 from the 5'end of the 397 to 717 or more than 99% of SEQ ID No. 17 from the 397 to 717 of the 5'end , 95% or more, 90% or more, 85% or more, 80% or more or 75% or more consistency (the inconsistency is preferably in the framework region (FR)).
  • the nucleotide sequence encoding the single-chain antibody is SEQ ID No. 17 from the 5'end of positions 1-717 or the same as SEQ ID No. 17 from the 5'end of positions 1-717. % Or more, 95% or more, 90% or more, 85% or more, 80% or more or 75% or more consistency (the inconsistency is preferably in the framework region (FR)).
  • nucleotide sequence encoding the single-chain antibody with the tag protein is SEQ ID No. 17, or the nucleotide sequence of SEQ ID No. 17 is more than 99%, more than 95%, more than 90%, more than 85%, 80% or more or 75% or more consistency (the inconsistency is preferably in the framework region (FR))
  • the nucleic acid molecules of the present invention can be obtained using appropriate molecular biology techniques.
  • the present invention claims to protect expression cassettes, recombinant vectors, and recombinant cells containing the aforementioned nucleic acid molecules.
  • the vector is an expression vector that can be used for expression of binding molecules such as antibodies or antigen-binding fragments thereof (such as single-chain antibodies) or single-chain antibodies with tagged proteins (such as fusion of single-chain antibodies and Fc) or fusion antibodies.
  • binding molecules such as antibodies or antigen-binding fragments thereof (such as single-chain antibodies) or single-chain antibodies with tagged proteins (such as fusion of single-chain antibodies and Fc) or fusion antibodies.
  • DNA encoding partial or full-length light chain and heavy chain is inserted into an expression vector, and transcription and translation of the DNA molecule are started.
  • the antibody light chain gene and the antibody heavy chain gene can be inserted into a separate vector or inserted into the same expression vector.
  • the insertion method can be any suitable method known, such as linking complementary restriction sites on antibody gene fragments to the vector.
  • the light chain and heavy chain variable regions of the antibodies described in the present invention can be used to generate full-length antibody genes of any antibody type and subtype.
  • the present invention further provides a host cell (ie, the recombinant cell) containing the nucleic acid molecule provided by the present invention.
  • the host cell may be any cell available for expression vectors.
  • higher eukaryotic host cells such as mammalian cells, include, for example, Chinese Hamster Ovary (CHO) cells, lower eukaryotic host cells, such as yeast cells, and may be prokaryotic cells, such as bacterial cells, E. coli, and the like.
  • Transfection methods for introducing recombinant nucleic acid constructs into host cells include electroporation, calcium phosphate transfection, DEAE-dextran, lipofection, and phage infection.
  • the expression vector encoding the antibody gene can be introduced into a host cell by a known method, and the host cell can be cultured enough to allow the binding molecule to be expressed in the host cell, thereby producing the antibody.
  • the vector can be a plasmid, cosmid, phage or viral vector.
  • the plasmid can be pCDNA3.4 and pCDNA3.4-L or others.
  • nucleotide sequence of the anti-4-1BB antibody (or single-chain antibody or fusion antibody) of the present invention can easily use known methods, such as directed evolution and point mutation methods, to mutate the nucleotide sequence of the anti-4-1BB antibody (or single-chain antibody or fusion antibody) of the present invention.
  • Those artificially modified have the nucleotide sequence of the anti-4-1BB antibody (or single-chain antibody or fusion antibody) of the present invention with 99% or more, 95% or more, 90% or more, 85% or more, 80% Nucleotides with more than or more than 75% identity, as long as they encode the anti-4-1BB antibody and have the anti-4-1BB antibody activity, are derived from the nucleotide sequence of the present invention and are equivalent to those of the present invention sequence.
  • the DNA fragment shown in SEQ ID No. 3 (the coding gene of the antibody heavy chain) is cloned into the pcDNA3.4 vector between the restriction sites Xba I and Hand III to obtain the expression.
  • the recombinant expression vector of the heavy chain of the antibody (named pcDNA3.4-H); the DNA fragment shown in SEQ ID No. 4 (the coding gene of the antibody light chain) was cloned into the restriction site Xba I and the pcDNA3.4 vector After Hand III, a recombinant expression vector (named pcDNA3.4-L) expressing the light chain of the antibody was obtained.
  • a kozak co-recognition was also introduced upstream of the DNA fragments shown in SEQ ID No. 3 and SEQ ID No. 4 (the genes encoding the heavy and light chains of the antibody).
  • the sequence (5'-GCCACC-3') and the coding sequence of the signal peptide (5'-ATGGAGTTTGGGCTGAGTTGGGTCTTTCTGGTCGCAATTC TGCTGAAGGGAGTGCAGTGC-3'), and the coding sequence of the signal peptide is connected to the 5'ends of SEQ ID No. 3 and SEQ ID No. 4.
  • the recombinant cell is a recombinant cell obtained by co-transfecting the aforementioned two recombinant expression vectors (pcDNA3.4-H and pcDNA3.4-L) for expressing the antibody heavy chain and light chain respectively into HEK293 cells.
  • the recombinant cell can express the aforementioned antibody Hanke10F4.
  • the present invention claims ADC antibodies.
  • the ADC antibody claimed in the present invention is the aforementioned antibody or the aforementioned antigen-binding fragment or the aforementioned antibody fusion protein (such as a single-chain antibody with a tag protein) or the aforementioned double-antibody coupling with biological Formed after active small molecule drugs.
  • the present invention claims a pharmaceutical composition.
  • the pharmaceutical composition claimed in the present invention comprises: (A1) the aforementioned antibody or the aforementioned antigen-binding fragment (such as a single-chain antibody) or the aforementioned antibody fusion protein (such as the fusion of a single-chain antibody and Fc) or the aforementioned The double antibody or the ADC antibody described above; (A2) a pharmaceutically acceptable excipient, diluent or carrier.
  • the present invention claims the aforementioned antibody or the aforementioned antigen-binding fragment (such as a single-chain antibody) or the aforementioned antibody fusion protein (such as the fusion of a single-chain antibody with Fc) or the aforementioned double antibody or Application of the aforementioned nucleic acid molecule or expression cassette, recombinant vector or recombinant cell or the aforementioned ADC antibody or the aforementioned pharmaceutical composition in any of the following:
  • the antibody or antigen-binding fragment thereof can be used as a diagnostic tool to detect 4-1BB in the blood or tissue of patients with cancer, autoimmune or other diseases.
  • the 4-1BB can be human 4-1BB or monkey 4-1BB.
  • the cancer may be a cancer in which the expression of 4-1BB is unregulated.
  • the autoimmune disease may be an autoimmune disease in which the expression of 4-1BB is unregulated.
  • the inflammatory disease may be an inflammatory disease in which the expression of 4-1BB is imbalanced.
  • infectious disease may be an infectious disease in which the expression of 4-1BB is imbalanced.
  • the present invention claims any of the following methods:
  • (C1) A method for detecting 4-1BB, including the following steps: using the aforementioned antibody or the aforementioned antigen-binding fragment (such as single-chain antibody) or the aforementioned antibody fusion protein (such as single-chain antibody and Fc Fusion) or the aforementioned double antibody or the aforementioned nucleic acid molecule or expression cassette, recombinant vector or recombinant cell to detect the sample to be tested.
  • the aforementioned antibody or the aforementioned antigen-binding fragment such as single-chain antibody
  • the aforementioned antibody fusion protein such as single-chain antibody and Fc Fusion
  • the antibody or antigen-binding fragment thereof can be used as a diagnostic tool to detect 4-1BB in the blood or tissue of patients with cancer, autoimmune or other diseases.
  • the 4-1BB can be human 4-1BB or monkey 4-1BB.
  • (C2) A method for blocking the 4-1BB/4-1BBL signaling pathway, including the following steps: using the aforementioned antibody or the aforementioned antigen-binding fragment (such as a single-chain antibody) or the aforementioned antibody fusion protein (Single-chain antibody and Fc fusion) or the above-mentioned double antibody or the above-mentioned nucleic acid molecule or expression cassette, recombinant vector or recombinant cell or the above-mentioned ADC antibody or the above-mentioned pharmaceutical composition block 4-1BB/4 -1BBL signal pathway.
  • the aforementioned antibody or the aforementioned antigen-binding fragment such as a single-chain antibody
  • the aforementioned antibody fusion protein Single-chain antibody and Fc fusion
  • the above-mentioned double antibody or the above-mentioned nucleic acid molecule or expression cassette recombinant vector or recombinant cell or the above-mentioned ADC antibody or the above-mentioned pharmaceutical composition
  • (C3) A method for stimulating T cell activation, including the following steps: using the aforementioned antibody or the aforementioned antigen-binding fragment (such as single-chain antibody) or the aforementioned antibody fusion protein (such as single-chain antibody and Fc Fusion) or the above-mentioned double antibody or the above-mentioned nucleic acid molecule or expression cassette, recombinant vector or recombinant cell or the above-mentioned ADC antibody or the above-mentioned pharmaceutical composition stimulates T cell activation.
  • the aforementioned antigen-binding fragment such as single-chain antibody
  • the aforementioned antibody fusion protein such as single-chain antibody and Fc Fusion
  • the above-mentioned double antibody or the above-mentioned nucleic acid molecule or expression cassette recombinant vector or recombinant cell or the above-mentioned ADC antibody or the above-mentioned pharmaceutical composition stimulates T cell activation.
  • (C4) A method for promoting the secretion of IFN- ⁇ from T cells, including the following steps: using the aforementioned antibody or the aforementioned antigen-binding fragment (such as a single-chain antibody) or the aforementioned antibody fusion protein (such as a single-chain antibody) The antibody is fused with Fc) or the double antibody described above or the nucleic acid molecule or expression cassette described above, recombinant vector or recombinant cell or the ADC antibody described above or the pharmaceutical composition described above to promote the secretion of IFN- ⁇ from T cells.
  • the aforementioned antigen-binding fragment such as a single-chain antibody
  • the aforementioned antibody fusion protein such as a single-chain antibody
  • the antibody is fused with Fc) or the double antibody described above or the nucleic acid molecule or expression cassette described above, recombinant vector or recombinant cell or the ADC antibody described above or the pharmaceutical composition described above to promote the secretion of IFN- ⁇ from T cells.
  • (C5) A method for inhibiting the growth of colon cancer cells, comprising the following steps: using the aforementioned antibody or the aforementioned antigen-binding fragment (such as a single-chain antibody) or the aforementioned antibody fusion protein (such as a single-chain antibody and Fc fusion) or the aforementioned double antibody or the aforementioned nucleic acid molecule or expression cassette, recombinant vector or recombinant cell or the aforementioned ADC antibody or the aforementioned pharmaceutical composition inhibits the growth of colon cancer cells.
  • the aforementioned antibody or the aforementioned antigen-binding fragment such as a single-chain antibody
  • the aforementioned antibody fusion protein such as a single-chain antibody and Fc fusion
  • the aforementioned double antibody or the aforementioned nucleic acid molecule or expression cassette recombinant vector or recombinant cell or the aforementioned ADC antibody or the aforementioned pharmaceutical composition inhibits the growth of colon cancer cells.
  • (C6) A method for inhibiting the growth of colon cancer tumors, comprising the following steps: using the aforementioned antibody or the aforementioned antigen-binding fragment (such as a single-chain antibody) or the aforementioned antibody fusion protein (such as a single-chain antibody and Fc fusion) or the aforementioned double antibody or the aforementioned nucleic acid molecule or expression cassette, recombinant vector or recombinant cell or the aforementioned ADC antibody or the aforementioned pharmaceutical composition inhibits the growth of colon cancer tumors.
  • the aforementioned antibody or the aforementioned antigen-binding fragment such as a single-chain antibody
  • the aforementioned antibody fusion protein such as a single-chain antibody and Fc fusion
  • the aforementioned double antibody or the aforementioned nucleic acid molecule or expression cassette recombinant vector or recombinant cell or the aforementioned ADC antibody or the aforementioned pharmaceutical composition inhibits the growth of colon cancer tumors.
  • (C7) A method for the treatment and/or prevention of colon cancer, comprising the following steps: using the aforementioned antibody or the aforementioned antigen-binding fragment (such as a single-chain antibody) or the aforementioned antibody fusion protein (such as a single-chain antibody)
  • the antibody is fused with Fc) or the double antibody described above or the nucleic acid molecule or expression cassette, recombinant vector or recombinant cell described above or the ADC antibody described above or the pharmaceutical composition described above to treat and/or prevent colon cancer.
  • a method of preparing an immune enhancer comprising the following steps: using the aforementioned antibody or the aforementioned antigen-binding fragment (such as single-chain antibody) or the aforementioned antibody fusion protein (such as single-chain antibody and Fc Fusion) or the above-mentioned double antibody or the above-mentioned nucleic acid molecule or expression cassette, recombinant vector or recombinant cell, or the above-mentioned ADC antibody or the above-mentioned pharmaceutical composition as an active ingredient to prepare an immune enhancer.
  • the aforementioned antigen-binding fragment such as single-chain antibody
  • the aforementioned antibody fusion protein such as single-chain antibody and Fc Fusion
  • (C9) A method of preparing an immune modulator, comprising the following steps: using the aforementioned antibody or the aforementioned antigen-binding fragment (such as single-chain antibody) or the aforementioned antibody fusion protein (such as single-chain antibody and Fc Fusion) or the aforementioned double antibody or the aforementioned nucleic acid molecule or expression cassette, recombinant vector or recombinant cell, the aforementioned ADC antibody or the aforementioned pharmaceutical composition as an active ingredient to prepare an immunomodulator.
  • the aforementioned antibody or the aforementioned antigen-binding fragment such as single-chain antibody
  • the aforementioned antibody fusion protein such as single-chain antibody and Fc Fusion
  • (C10) A method for treating and/or preventing and/or diagnosing a disease characterized by 4-1BB expression imbalance, comprising the following steps: using the aforementioned antibody or the aforementioned antigen-binding fragment (such as a single-chain antibody) or The aforementioned antibody fusion protein (such as the fusion of a single-chain antibody and Fc) or the aforementioned double antibody or the aforementioned nucleic acid molecule or expression cassette, recombinant vector or recombinant cell or the aforementioned ADC antibody or the aforementioned pharmaceutical composition treatment And/or prevent and/or diagnose diseases characterized by 4-1BB expression imbalance.
  • the aforementioned antibody or the aforementioned antigen-binding fragment such as a single-chain antibody
  • the aforementioned antibody fusion protein such as the fusion of a single-chain antibody and Fc
  • the aforementioned double antibody or the aforementioned nucleic acid molecule or expression cassette recombinant vector or recombinant cell or the aforementioned ADC antibody or the
  • (C11) A method for treating and/or preventing and/or diagnosing cancer, comprising the steps of: using the aforementioned antibody or the aforementioned antigen-binding fragment (such as a single-chain antibody) or the aforementioned antibody fusion protein (such as single-chain antibody and Fc fusion) or the aforementioned double antibody or the aforementioned nucleic acid molecule or expression cassette, recombinant vector or recombinant cell or the aforementioned ADC antibody or the aforementioned pharmaceutical composition for treatment and/or prevention and/or Diagnose cancer.
  • the aforementioned antibody or the aforementioned antigen-binding fragment such as a single-chain antibody
  • the aforementioned antibody fusion protein Such as single-chain antibody and Fc fusion
  • the aforementioned double antibody or the aforementioned nucleic acid molecule or expression cassette recombinant vector or recombinant cell or the aforementioned ADC antibody or the aforementioned pharmaceutical composition for treatment and/or prevention and/or Diagnose cancer.
  • the cancer may be a cancer in which the expression of 4-1BB is unregulated.
  • (C12) A method of treating and/or preventing and/or diagnosing autoimmune diseases, comprising the following steps: using the aforementioned antibody or the aforementioned antigen-binding fragment (such as a single-chain antibody) or the aforementioned antibody fusion Protein (such as single-chain antibody and Fc fusion) or the aforementioned double antibody or the aforementioned nucleic acid molecule or expression cassette, recombinant vector or recombinant cell or the aforementioned ADC antibody or the aforementioned pharmaceutical composition for treatment and/or prevention and / Or diagnosis of autoimmune diseases.
  • the aforementioned antibody or the aforementioned antigen-binding fragment such as a single-chain antibody
  • the aforementioned antibody fusion Protein such as single-chain antibody and Fc fusion
  • the autoimmune disease may be an autoimmune disease in which the expression of 4-1BB is unregulated.
  • a method of treating and/or preventing and/or diagnosing inflammatory diseases comprising the following steps: using the aforementioned antibody or the aforementioned antigen-binding fragment (such as a single-chain antibody) or the aforementioned antibody fusion protein (Single-chain antibody and Fc fusion) or the aforementioned double antibody or the aforementioned nucleic acid molecule or expression cassette, recombinant vector or recombinant cell or the aforementioned ADC antibody or the aforementioned pharmaceutical composition for treatment and/or prevention and/ Or diagnose inflammatory diseases.
  • the aforementioned antibody or the aforementioned antigen-binding fragment such as a single-chain antibody
  • the aforementioned antibody fusion protein Single-chain antibody and Fc fusion
  • the aforementioned double antibody or the aforementioned nucleic acid molecule or expression cassette recombinant vector or recombinant cell or the aforementioned ADC antibody or the aforementioned pharmaceutical composition for treatment and/or prevention and/ Or diagnose inflammatory diseases.
  • the inflammatory disease may be an inflammatory disease in which the expression of 4-1BB is imbalanced.
  • (C14) A method of treating and/or preventing and/or diagnosing infectious diseases, comprising the following steps: using the aforementioned antibody or the aforementioned antigen-binding fragment (such as a single-chain antibody) or the aforementioned antibody fusion Protein (such as single-chain antibody and Fc fusion) or the aforementioned double antibody or the aforementioned nucleic acid molecule or expression cassette, recombinant vector or recombinant cell or the aforementioned ADC antibody or the aforementioned pharmaceutical composition for treatment and/or prevention and / Or diagnosis of infectious diseases.
  • the aforementioned antibody or the aforementioned antigen-binding fragment such as a single-chain antibody
  • the aforementioned antibody fusion Protein such as single-chain antibody and Fc fusion
  • double antibody or the aforementioned nucleic acid molecule or expression cassette recombinant vector or recombinant cell or the aforementioned ADC antibody or the aforementioned pharmaceutical composition for treatment and/or prevention and / Or diagnosis of infectious diseases.
  • infectious disease may be an infectious disease in which the expression of 4-1BB is imbalanced.
  • Figure 1 shows SDS-PAGE of hybridoma antibody.
  • Figure 2 shows the structure of the antibody.
  • Figure 3 shows that antibodies secreted by hybridomas recognize human 4-1BB.
  • Figure 4 shows that hybridomas secrete antibodies that recognize monkey 4-1BB.
  • Figure 5 shows that the antigen-binding fragment recognizes human 4-1BB.
  • Figure 6 shows that the antigen-binding fragment recognizes monkey 4-1BB.
  • Figure 7 shows FACS comparing the affinity of each antigen-binding fragment with human 4-1BB.
  • Figure 8 shows the affinity comparison of Hanke10F4 and HKB6.
  • Figure 9 shows that the antibody Hanke10F4 blocks the binding of 4-1BBL to 4-1BB.
  • Figure 10 shows the competition of the antibody Hanke10F4 with the control antibody epitope.
  • Figure 11 shows the affinity of FACS detection antibodies to antigens with different point mutations.
  • Figure 12 shows Hanke10F4 and HKB6 epitope competition.
  • Figure 13 shows the determination of luciferase activity.
  • A is the result of "3.5 Luciferase Reporter Gene Activity Test”.
  • B is the test result of "3.9 Stability (1) Accelerated Stability”.
  • Figure 14 shows the detection of the effect of antibody Hanke10F4 on CD4 + T cell activation.
  • the three bar graphs in each group are from left to right, respectively, the activation effect of CD4 + T cells under the condition of high, medium, and low antibody concentration (2 ⁇ g/ml, 0.4 ⁇ g/ml, 0.08 ⁇ g/ml).
  • Figure 15 shows the detection of the effect of antibody Hanke10F4 on CD8 + T cell activation.
  • the three bar graphs in each group are the activation effects of T cells under high, medium, and low antibody concentrations (3 ⁇ g/ml, 0.5 ⁇ g/ml, 0.08 ⁇ g/ml).
  • Figure 16 is an antibody inhibiting MC38 tumor model. Each line represents the tumor growth curve of a mouse.
  • Figure 17 shows the antibody Hanke10F4 challenge test.
  • Figure 18 shows the toxicological test of antibody Hanke10F4.
  • Figure 19 shows the Hanke10F4Tm detection.
  • the following examples facilitate a better understanding of the present invention, but do not limit the present invention.
  • the experimental methods in the following examples are conventional methods unless otherwise specified.
  • the test materials used in the following examples, unless otherwise specified, are all purchased from conventional biochemical reagent stores.
  • the quantitative experiments in the following examples are all set to repeat the experiment three times, and the results are averaged.
  • the positive control antibodies are Utomilumab from Pfizer (PF-05082566, patent number: US8337850B2) and Urelumab from Bristol Myers Squibb (BMS-663513, patent number: US7288638B2), both of which are anti-human antibodies.
  • 4-1BB antibody The negative control antibody is human IgG (Nanjing GenScript product).
  • the first position of each nucleotide sequence in the sequence list is the 5'terminal nucleotide of the corresponding DNA, and the last position is the 3'terminal nucleotide of the corresponding DNA.
  • the pCDNA3.4 vector (Invitrogen, Cat: A14697) in the following examples.
  • Paving buffer phosphate buffer with pH 7.0;
  • Washing solution Phosphate buffer with pH 7.0 containing only 0.05% by volume Tween 20;
  • Chromogenic substrate Tetramethylbenzidine
  • the solvent of the phosphate buffer with a pH of 7.0 is water, and the solutes are sodium chloride, potassium chloride, potassium dihydrogen phosphate, and disodium hydrogen phosphate;
  • the sodium chloride is used in the phosphoric acid with a pH of 7.0
  • the concentration in the salt buffer is 135 mM
  • the concentration of the potassium chloride in the pH 7.0 phosphate buffer is 2.7 mM
  • the potassium dihydrogen phosphate is in the pH 7.0 phosphate buffer
  • the concentration of the disodium hydrogen phosphate in the pH 7.0 phosphate buffer is 8 mM.
  • mice and three C57b1/6 mice aged 4 to 6 weeks were selected, and the extracellular segment of human 4-1BB (SEQ ID No. 5, positions 24-186) was used as the antigen to immunize the mice.
  • the immunization was once every 2 weeks for a total of 3 immunizations.
  • blood was collected from the tail vein, and the antibody production was detected by indirect ELISA.
  • the appropriate immunized mice were selected, and the eyes were removed and the neck was sacrificed.
  • the mouse spleen cells were aseptically harvested, and cell fusion was performed according to conventional methods to prepare antibodies.
  • Human 4-1BB monoclonal antibody hybridoma cell line was used as the antigen to immunize the mice.
  • the immunization was once every 2 weeks for a total of 3 immunizations.
  • blood was collected from the tail vein, and the antibody production was detected by indirect ELISA.
  • the appropriate immunized mice were selected, and the eyes were removed and the neck was sacrificed.
  • the anti-human 4-1BB monoclonal antibody hybridoma cells were screened by ELISA.
  • the ELISA plate was coated with 10 ⁇ g/ml human 4-1BB (SEQ ID No. 5), and blocked overnight at 4°C.
  • TMB tetramethylbenzidine
  • the positive cell clones were screened by the ELISA method and then repeatedly subcloned until all the hybridoma cell culture supernatants were detected as 100% positive.
  • the cloning of hybridoma cells uses the limiting dilution method.
  • the culture plate is placed in a 37°C, 5% CO 2 incubator for culture, and cell clones can be observed under the microscope after about 5 days. Change the medium at the right time, test, take the positive and good monoclonal cell line for expansion culture, then obtain the anti-human 4-1BB monoclonal antibody hybridoma cell line, and freeze the cell line in time.
  • the positive and growing anti-human 4-1BB antibody hybridoma cell lines obtained from the screening were gradually expanded and cultured, centrifuged (10,000 rpm for 10 minutes), the supernatant was taken, and the obtained supernatant was purified with a Protein G affinity chromatography column.
  • the specific operation is: first equilibrate the Protein G column (GE company) with PBS, then incubate the supernatant through the column, first use solution A (formulation: solvent is water, solute and concentration: 20mM sodium phosphate, 500mM NaCl, pH5.0) Pre-elute 5 column volumes, then use B solution (formulation: solvent is water, solute and concentration: 20mM sodium acetate, 150mM NaCl, pH3.5) to elute 5 column volumes, collect elution peaks, and then concentrate at 30KDa Concentrate in a centrifuge tube to obtain antibodies.
  • solution A formulation: solvent is water, solute and concentration: 20mM sodium phosphate, 500mM NaCl, pH5.0
  • Pre-elute 5 column volumes Pre-elute 5 column volumes
  • use B solution formulation: solvent is water, solute and concentration: 20mM sodium acetate, 150mM NaCl, pH3.5
  • the obtained anti-human 4-1BB antibody was sequenced, and the results showed that the obtained anti-human 4-1BB antibody had a molecular weight of 150 Kb, including heavy and light chains, and was a typical intact antibody.
  • the structure of the antibody is shown in Figure 2.
  • the affinity of the four hybridomas secreted antibodies to recognize human 4-1BB was evaluated by ELISA.
  • a 96-well plate (96-well ELISA plate, Nunc) was coated with human 4-1BB (SEQ ID No. 5) at a concentration of 1 ⁇ g/ml, 100 ⁇ l/well, and overnight at 4°C. Wash the plate three times, add 300 ⁇ l of blocking solution to each well (see above for the recipe), and block for 1 hour at 37°C. Wash the plate three times with the washing solution (see above for the recipe), dilute the antibody with the sample diluent (PBS-T with 1% bovine serum albumin) to 4000ng/ml, and then use 7 Ep tubes to dilute 4-fold into a gradient solution.
  • sample diluent PBS-T with 1% bovine serum albumin
  • the antibodies secreted by hybridomas can recognize and bind to human 4-1BB.
  • the OD450 value of the ELISA result is shown in Table 2, and the analysis diagram is shown in Fig. 3.
  • the ELISA method was used to evaluate the affinity of the hybridoma secreted antibody to recognize monkey 4-1BB.
  • the monkey 4-1BB (Cy4-1BB) (SEQ ID No. 6) antigen was diluted to 1 ⁇ g/ml with plating buffer (see above for the formula), and 100 ⁇ l was added to each well for plating, overnight at 4°C.
  • the secreted antibody 37G10F4 obtained from the panning as described in the present invention was selected. Analyze the antibody gene sequence, and then use the homology modeling and optimization of antibody Fab, surface scanning to determine the humanized mutation site, virtual mutation and molecular dynamics simulation, determine the key amino acid and a series of analysis and design reasonable humanized antibody. Through humanization, 12 candidate scFv-Fc (human IgG4) antigen-binding fragments (Table 4) were obtained for further identification as in this example.
  • the 12 candidate scFv-Fc (human IgG4) antigen-binding fragments in Table 4 have the same and different complementarity determining regions (CDRs) in the heavy chain variable region (VH) and light chain variable region (VL)
  • CDRs complementarity determining regions
  • FR framework regions
  • the amino acid sequences of HCDR1, HCDR2, and HCDR3 in the heavy chain variable region are shown in SEQ ID No. 16 at positions 31-35, positions 50-64, and positions 98-106 from the N-terminus; light chain variable region
  • the amino acid sequences of LCDR1, LCDR2 and LHCDR3 in SEQ ID No. 16 are shown in sequence at positions 156-166, 182-188, and 221-229 from the N-terminus.
  • amino acid sequence of scFvB60103 is shown in SEQ ID No. 16, positions 1-117 are the variable region of the heavy chain, positions 133-239 are the variable region of the light chain, and positions 240-472 are Fc.
  • the coding gene sequence of scFvB60103 is shown in SEQ ID No. 17, and positions 1-351 are the coding genes of the heavy chain variable region (positions 91-105, 148-192, and 292-318 respectively encode three HCDR), positions 397-717 are the coding genes of the light chain variable region (positions 466-498, 544-564, and 661-687 respectively encode three LCDRs), and positions 718-1416 are the coding genes of Fc.
  • the 12 candidate scFv-Fc molecules in Table 4 were expressed and purified. Take scFvB60103 as an example.
  • the gene encoding scFvB60103 antibody (SEQ ID No. 17) was artificially synthesized, then inserted into the pCDNA3.4 vector, and expressed by transient transduction. Transient operation specifically uses Expi293 expression system (Thermo Fisher) products, and operates according to the product manual. Add Expi Fectamine transfection reagent and DNA plasmid to OptiMEM to obtain solutions A and B, and then mix solution A and solution B to obtain solution C. Solution C was added to Expi293 cells (Thermo Fisher), and Expi293 cells were cultured for 5 days.
  • the specific operation is: first equilibrate the Protein G column (GE company) with PBS, then incubate the supernatant through the column, first use solution A (formulation: solvent is water, solute and concentration: 20mM sodium phosphate, 500mM NaCl, pH5.0) Pre-elute 5 column volumes, then use B solution (formulation: solvent is water, solute and concentration: 20mM sodium acetate, 150mM NaCl, pH3.5) to elute 5 column volumes, collect elution peaks, and then concentrate at 30KDa Concentrate in a centrifuge tube to obtain scFvB60103 target molecules.
  • ELISA was used to identify the affinity of scFv molecules with human and monkey 4-1BB molecules (see related steps in Example 1 for specific methods). As shown in Table 4, the results show that all 12 scFv-Fc molecules can recognize human and monkey 4-1BB, and scFvB60103 has the best recognition ability. The ELISA results are shown in Figure 5 and Figure 6.
  • the reagents used include:
  • the buffer is a solution obtained by adding BSA and sodium azide to PBS, the mass percentage concentration of BSA in the buffer is 2%, and the mass percentage concentration of sodium azide is 0.02%; goat anti-human FITC secondary antibody (Sigma, stored in the dark).
  • CHO-K1-hu4-1BB the FACS method was used to detect the difference in affinity between scFv molecules with better binding activity to human 4-1BB and human 4-1BB molecules in the ELISA test results.
  • the cells used in the experiment were CHO-K1 cells (CHO-K1-hu4-1BB) with high expression of human 4-1BB on the cell membrane constructed by Hefei Hanke Maibo and wild-type CHO-K1 without 4-1BB expression (Shanghai Cells of Chinese Academy of Sciences) Library).
  • the human 4-1BB gene sequence (SEQ ID No. 7) was artificially synthesized, and then inserted into the pCDNA3.4 vector according to common methods in the art, and then introduced into wild-type CHO-K1 cells with Lipofectamine 3000 transfection reagent (Invitrogen). Then G418 (purchased from Shenggong Bioengineering (Shanghai) Co., Ltd.) was added for pressure screening, and finally CHO-K1 cells (CHO-K1-hu4-1BB) with high expression of human 4-1BB on the cell membrane were obtained for use.
  • a centrifuge tube containing antibody diluent with an antibody concentration of 0 ⁇ g/ml was added as a blank control.
  • the exemplary antibody Hanke10F4 identified in this example was obtained from the humanized modification of the hybridoma secretory antibody 37G10F4, that is, it was transformed from scFvB60103 in Table 4.
  • Hanke10F4 will be expressed and purified in detail, and then identified in ELISA, FACS, luciferase reporter gene determination, and T cell activation system.
  • the full-length gene sequence was synthesized.
  • the heavy chain encoding gene of Hanke10F4 antibody is shown in SEQ ID No. 3, and the light chain encoding gene of Hanke 10F4 antibody is shown in SEQ ID No. 4.
  • Positions 1-351 of SEQ ID No. 3 are the coding genes of the heavy chain variable region VH, and the coding sequences of CDR1, CDR2, and CDR3 are as shown in SEQ ID No. 3, 91-105 and 148-192, respectively. , As shown in positions 292-318.
  • Positions 1-321 of SEQ ID No. 4 are the coding genes of the light chain variable region VL, and the coding sequences of CDR1, CDR2, and CDR3 are as shown at positions 70-102 and 148-168 of SEQ ID No. 4, respectively. , As shown in the 265-291 position.
  • the terminator (TGA) and the recognition sequence of HindIII (5'-AAGCTT-3') are added to the 3'end of the TGA in turn, and the TGA is connected to the 3'end of SEQ ID No. 3 and SEQ ID No. 4, and the resulting new
  • the DNA sequences of are recorded as heavy chain gene A and light chain gene A.
  • the heavy chain gene A was artificially synthesized, and the heavy chain gene A was digested with XbaI and HindIII and connected to the vector backbone obtained by digesting the pCDNA3.4 vector with XbaI and HindIII to obtain the recombinant vector.
  • the recombinant vector verified by sequencing was named as pCDNA3.4-H; the light chain gene A was digested with XbaI and HindIII and connected with the vector backbone obtained by digesting pCDNA3.4 vector with XbaI and HindIII to obtain a recombinant vector.
  • the recombinant vector verified by sequencing was named pCDNA3 .4-L.
  • pCDNA3.4-H and pCDNA3.4-L into the human embryonic kidney cell line HEK293F (ATCC American Cell Bank) to obtain recombinant cells, and then place the recombinant cells in a 37°C, 5% CO 2 shaking incubator for culture. The speed is 120 rpm.
  • the protein A affinity chromatography column was used to purify the antibody protein from the culture supernatant.
  • the specific operation is: first equilibrate the Protein A column (GE company) with PBS, and then incubate the supernatant through the column, first use A solution (formulation: solvent is water, solute and concentration: 20mM sodium phosphate, 500mM NaCl, pH5.0) Pre-elute 5 column volumes, then use B solution (formulation: solvent is water, solute and concentration: 20mM sodium acetate, 150mM NaCl, pH3.5) to elute 5 column volumes, collect elution peaks, and then concentrate at 30KDa
  • the anti-human 4-1BB antibody is obtained by concentrating in a centrifuge tube to obtain the antibody.
  • the antibody is composed of a heavy chain and a light chain.
  • the amino acid sequence of the heavy chain is shown in SEQ ID No. 1, and the amino acid sequence of the light chain is shown in SEQ ID No. 2.
  • the heavy chain type of Hanke10F4 antibody is IgG4, and the light chain type is kappa chain.
  • Positions 1-117 of SEQ ID No. 1 are the heavy chain variable region VH, and the sequences of CDR1, CDR2, and CDR3 are as shown in SEQ ID No. 1, positions 31-35, positions 50-64, and positions 98, respectively. 106 shows.
  • step 1.4 For the method, refer to step 1.4 in Example 1.
  • Human 4-1BB or monkey 4-1BB were directly coated on 96-well plates, antibody samples (Hanke10F4 and Utomilumab) were used as primary antibodies, and horseradish peroxidase-labeled goat anti-mouse was used as Secondary Antibodies.
  • Tetramethylbenzidine (TMB) is a color reagent, and sulfuric acid stops the color development. Measure the OD value at 450nm with BIO-TEK ELX-800 microplate reader.
  • Table 6 shows the ELISA results of the antibody Hanke10F4 in the form of IgG4, which can recognize both human 4-1BB and monkey 4-1BB.
  • the reagents used include:
  • the buffer is a solution obtained by adding BSA and sodium azide to PBS, the mass percentage concentration of BSA in the buffer is 2%, and the mass percentage concentration of sodium azide is 0.02%; goat anti-human FITC secondary antibody (Sigma, stored in the dark).
  • the cells used in the experiment were CHO-K1 cells (CHO-K1-hu4-1BB) with high expression of human 4-1BB on the cell membrane surface constructed by Hefei Hanke Maibo and CHO-K1 cells (CHO-K1 with high expression of monkey 4-1BB on the cell membrane surface).
  • CHO-K1-cy4-1BB CHO-K1 with high expression of monkey 4-1BB on the cell membrane surface
  • wild-type CHO-K1 without 4-1BB expression from Shanghai Cell Bank of Chinese Academy of Sciences.
  • CHO-K1-hu4-1BB was used to detect the binding activity of Hanke10F4 monoclonal antibody to human 4-1BB.
  • T75 flask cultures using the test cell to 80% of full scale, trypsinized, centrifuged at 1000 rpm for 5min, cells were collected (about 10 cells per bottle 6), with about 1ml buffer, resuspended, and centrifuged the cells and resuspended to give Cell suspension, the cell concentration is 1 ⁇ 10 7 cells/ml.
  • Prepare 25 ⁇ l of Hanke10F4 antibody diluent of each dilution gradient add 25 ⁇ l of cell suspension to each centrifuge tube with different concentrations of antibody and mix well. The final antibody concentration is shown in Table 7.
  • the centrifuge tube of the antibody dilution was used as a blank control. After incubating for 30 minutes, wash twice with 1ml FACS buffer, add goat anti-human FITC secondary antibody, pipette to resuspend, and incubate for 30 minutes in the dark. Wash twice with 1ml FACS buffer solution again, then add 500 ⁇ l FACS buffer solution to each tube to resuspend, place on ice and protect from light, and test on the machine.
  • the antibody Utomilumab (Pfizer Pharmaceutical Co., Ltd.) was used as a positive control.
  • the CHO-K1-hu4-1BB test was used to compare the binding activity of Hanke10F4 and HKB6 monoclonal antibodies with human 4-1BB.
  • T75 flask cultures using the test cell to 80% of full scale, trypsinized, centrifuged at 1000 rpm for 5min, cells were collected (about 10 cells per bottle 6), with about 1ml buffer, resuspended, and centrifuged the cells and resuspended to give Cell suspension, the cell concentration is 1 ⁇ 10 7 cells/ml.
  • HKB6 is the HKB6 antibody described in the patent application number 201811541545.8.
  • the h4-1BB-His (Acro company, article number: Q07011-1) antigen was used to detect the affinity of Utomilumab, Hanke10F4 or Urelumab by using the Fortebio octec instrument using biofilm layer interference (BLI) technology.
  • BBI biofilm layer interference
  • ka represents the generation rate of the binding product per unit time
  • kd represents the percentage of the binding product degraded into reactants per unit time
  • KD value represents the strength of the affinity
  • CHO-K1-hu4-1BB was used to compare the binding activity between Hanke10F4 antibody and human 4-1BBL.
  • the secondary antibody is SA-FITC (Biolegend), and other operations (except the following) and diluent are the same as the FACS detection in step 3.2.
  • the amino acid sequence of human 4-1BBL is shown in SEQ ID No.9.
  • the Hanke10F4 is labeled Bio-Hanke10F4 with biotin, and the labeling method is the same as the conventional protein labeling method in the art. Dilute the Bio-Hanke10F4 to 1.5 ⁇ g/ml solution A with the sample diluent. At the same time, prepare diluent B of human 4-1BBL-hIgG (or Hanke10F4), the concentration of fixed solution A remains unchanged, and the concentration of solution B is a certain multiple of A (see Table 9 for specific multiples). Mix Solution A and Solution B in equal proportions and incubate with CHO-K1-hu4-1BB.
  • CHO-K1-hu4-1BB was used to compare the epitope competition between Hanke10F4 antibody and control antibodies Utomilumab and Urelumab.
  • the secondary antibody is SA-FITC (Biolegend), and other operations (except the following) and diluent are the same as the FACS detection in step 3.2.
  • the Hanke10F4 was labeled Bio-Hanke10F4 with biotin, and the solution A with a concentration of 1.5 ⁇ g/ml was diluted with the sample diluent.
  • the concentration of fixation solution A remains unchanged, and the concentration of solution B is a certain multiple of A (see Table 10 for specific multiples).
  • the extracellular region of human 4-1BB antigen was replaced with the corresponding extracellular region of mouse 4-1BB to synthesize different target genes and inserted into pCDNA3.4 vector respectively to obtain DNA plasmids of different 4-1BB antigens.
  • the expression cells were obtained by transient transduction.
  • the cell lines with different 4-1BB antigens on the surface are shown in Table 12. It is used to analyze the binding activity of antibodies to different 4-1BB antigens by FACS, and then analyze the specific epitopes of the extracellular domain of human 4-1BB.
  • Transient operation specifically uses Expi293 expression system (Thermo Fisher) products, and operates according to the product manual. Add Expi Fectamine transfection reagent and DNA plasmid to OptiMEM to obtain solutions A and B, and then mix solution A and solution B to obtain solution C. Add Solution C to Expi293 cells (Thermo Fisher) and incubate overnight to obtain a cell line that highly expresses 4-1BB antigen on the cell surface.
  • Expi293 expression system Thermo Fisher
  • the full-length amino acid sequence of the human 4-1BB antigen is shown in SEQ ID No. 5 (the corresponding coding gene is shown in SEQ ID No. 7), and the extracellular region is at positions 24-186 (the corresponding coding gene is shown in SEQ ID No. .7 shown in bits 70-558).
  • the full-length amino acid sequence of the murine 4-1BB antigen is shown in SEQ ID No. 10 (the corresponding coding gene is shown in SEQ ID No. 11), and the extracellular region is at positions 24-187 (the corresponding coding gene is shown in SEQ ID No. 11. 70-561).
  • step 3.2 Using the cell line obtained by transient, follow the FACS detection procedure in step 3.2, and sequentially add antibody samples (Utomilumab, Hanke10F4 or Urelumab) as the primary antibody and incubate with the cells, then add goat anti-human FITC as the secondary antibody and incubate with FACS buffer again Wash twice, then add 500 ⁇ l FACS buffer to each tube to resuspend, protect from light, and test on the machine.
  • antibody samples Utomilumab, Hanke10F4 or Urelumab
  • Utomilumab mainly binds Domain 3 and Domain 4 of the extracellular domain of human 4-1BB
  • Urelumab mainly binds Domain 1 of the extracellular domain of human 4-1BB.
  • Hanke10F4 mainly binds to the Domain1 and Domain 2A regions of the extracellular domain of human 4-1BB, and it interacts with the control antibodies Utomilumab and Urelumab.
  • the antigen binding epitopes are all different (Table 12).
  • Hanke10F4 has a certain degree of competition with the positive control antibody, which may be caused by the spatial epitope of the antigen antibody.
  • the specific structure of the antigen in cell A refers to the total length of the human 4-1BB antigen shown in SEQ ID No.5
  • the amino acids at positions 24-86 are replaced with amino acids at positions 24-86 in the full length of the murine 4-1BB antigen shown in SEQ ID No. 10.
  • the specific structure of the antigen in cell B refers to the 64th in the full length of the human 4-1BB antigen shown in SEQ ID No. 5
  • the amino acid at position -118 is replaced with amino acid at position 64-118 of SEQ ID No. 10.
  • the specific structure of the antigen in cell C refers to the 87th in the full length of the human 4-1BB antigen shown in SEQ ID No. 5.
  • the amino acid at position -133 is replaced with amino acid at position 87-133 of SEQ ID No. 10.
  • the specific structure of the antigen in cell D refers to the 97th in the full length of the human 4-1BB antigen shown in SEQ ID No. 5
  • the amino acid at position -159 is replaced with amino acid at position 97-159 of SEQ ID No. 10.
  • the specific structure of the antigen in cell E refers to the 119th in the full length of the human 4-1BB antigen shown in SEQ ID No. 5
  • the amino acid at position -186 is replaced with amino acid at position 119-186 of SEQ ID No. 10.
  • the specific structure of the antigen in cell F refers to the full length of the human 4-1BB antigen shown in SEQ ID No. 5
  • the amino acids at positions 24-46 and 139-186 in SEQ ID No. 10 are replaced with amino acids at positions 24-46 and 139-186.
  • amino acids 101 and 132 of the human 4-1BB antigen are the key binding sites of Utomilulab, and position 42 is the key binding site of Urelumab.
  • the amino acids at the above positions are mutated one by one to synthesize different target genes to obtain DNA plasmids with different 4-1BB antigens, and transiently transduced to obtain cell lines expressing different 4-1BB antigens on the cell surface (see step (1) for related steps). As shown in Table 13.
  • step 3.2 Using the cell line obtained by transient, follow the FACS detection step in step 3.2, add antibody Hanke10F4 and incubate with the cells, then add goat anti-human FITC as the secondary antibody to incubate, wash twice with FACS buffer, and then add 500 ⁇ l FACS buffer to each tube Resuspend the solution, avoid light, and test on the machine to analyze the binding activity of antibodies to different 4-1BB antigens.
  • the result is shown in Figure 11.
  • CHO-K1-hu4-1BB was used to compare the antigen-binding epitope competition between Hanke10F4 antibody and HKB6.
  • the secondary antibody is SA-FITC (Biolegend), and other operations (except the following) and diluent are the same as the FACS detection in step 3.2.
  • the Hanke10F4 was labeled Bio-Hanke10F4 with biotin, and the solution A with a concentration of 1.5 ⁇ g/ml was diluted with the sample diluent.
  • the concentration of fixation solution A remains unchanged, and the concentration of solution B is a certain multiple of A (see Table 14 for specific multiples).
  • HEK-293/NF ⁇ B-Luci/41-BB cells are HEK293 cell lines constructed by Hefei Hanke Maibo Biotechnology Company that stably express human 4-1BB membrane protein and NF ⁇ B-Luciferase reporter gene.
  • the specific operation is as follows: insert the human 4-1BB sequence (SEQ ID No. 7) as the target gene into the pCDNA3.4 vector to obtain plasmid A.
  • the NF ⁇ B element sequence SEQ ID No. 12
  • the luciferase gene SEQ ID No. 12
  • plasmid B is used as the target gene and inserted into different positions in the pGL4.10 vector (Ubao Bio) according to the methods commonly used in the art to obtain plasmid B. Then use Lipofectamine 3000 transfection reagent (Invitrogen) to introduce the A and B plasmids into HEK293 cells (Shanghai Cell Bank, Chinese Academy of Sciences). Then add Puromycin (Gibco) and Hygromycin B (Shenggong Biological Engineering (Shanghai) Co., Ltd.) pressure screening, and finally get HEK-293/NF ⁇ B-Luci/41-BB.
  • Lipofectamine 3000 transfection reagent Invitrogen
  • Puromycin Gabco
  • Hygromycin B Shenggong Biological Engineering (Shanghai) Co., Ltd.
  • the antibody and the cross-linked antibody Fab' goat anti-human IgG Fc were mixed at a concentration ratio of 1:2, they were added to a 96-well plate, 50 ⁇ l/well. After standing for 18-24h in the incubator, add 100 ⁇ l of ONE-Glo Luciferase assay sysytem reagent (Promega) and incubate for 10min at room temperature to detect the chemiluminescence value.
  • Isolation of human CD4 + T cells Collect whole blood from healthy people, use lymphocyte separation fluid (Sigma), separate PBMC cells according to its instructions, and use human CD4 + T cell magnetic beads (BD Company, catalog number 557767), The human CD4 + T cells were isolated and purified according to the instructions for use.
  • CD4 + T cells to a 96-cell culture plate after incubation at 37°C, with a density of 1 ⁇ 10 5 cells/well, and then place the 96-well plate in a 37°C, 5% CO 2 incubator for 3 days , Collect the cell supernatant, and detect the secretion of cytokine INF- ⁇ by ELISA.
  • the anti-CD3 antibody was diluted with PBS to 0.5 ⁇ g/ml, added to a 96-well plate (Corning), 60 ⁇ l/well, and the 96-well plate was placed at 37° C. and incubated for 1 hour. Wash with PBS, and then add the stable CHO-K1 cell line (CHO-K1-CD32A or CHO-K1-CD32B or CHO-K1) to a 96-well plate, 100 ⁇ l/well, 1 ⁇ 10 4 cells/well. Place the 96-well plate in the cell incubator overnight.
  • the cells used in the experiment were CHO-K1 cells (CHO-K1-CD32A) with high expression of human Fc ⁇ R ⁇ A (CD32A) on the cell membrane constructed by Hefei Hanke Maibo and CHO- with high expression of human Fc ⁇ R ⁇ B (CD32B) on the cell membrane.
  • K1 cells CHO-K1-CD32B
  • CHO-K1 cells CHO-K1-CD32B
  • the human Fc ⁇ R ⁇ A coding gene sequence (SEQ ID No.14) and the human Fc ⁇ R ⁇ B coding gene sequence (SEQ ID No.15) were artificially synthesized, and then inserted into the pCDNA3.4 vector according to common methods in the art, and then used separately Lipofectamine 3000 transfection reagent (Invitrogen) was introduced into wild-type CHO-K1 cells.
  • CHO-K1 cells (CHO-K1-CD32A) with high expression of human Fc ⁇ R ⁇ A on the cell membrane and high expression of human Fc ⁇ R ⁇ on the cell membrane surface CHO-K1 cells of B (CHO-K1-CD32B).
  • Isolation of human CD8 + T cells Collect whole blood from healthy people, use lymphocyte separation medium (Sigma), separate PBMC cells according to its instructions, and use human CD8 + T cell magnetic beads (BD Company, catalog number 557766), The human CD8 + T cells were isolated and purified according to the instructions for use.
  • the cell supernatant was aspirated, and then 100 ⁇ l of human CD8 + T cells were added to each well, 1 ⁇ 10 5 cells/well. Then, the antibody was diluted with medium to the target concentration and added to a 96-well plate, 100 ⁇ l/well, the final concentrations were: 3 ⁇ g/ml, 0.5 ⁇ g/ml, 0.08 ⁇ g/ml, and 3 replicate wells were set for each concentration.
  • the 96-well plate was placed in a cell incubator and incubated for 3 days, and the secretion of cytokine INF- ⁇ in the cell culture supernatant was detected by ELISA.
  • B-hCD137(4-1BB) mice were selected to detect the anti-tumor efficacy of Hanke10F4 in vivo.
  • the B-hCD137(4-1BB) mouse model is a genetically engineered mouse, which is a human-derived h4-1BB gene that is chimeric in the genome of the C57BL/6 mouse with a genetic background, from Biocytogram.
  • the MC38 cell line was subcutaneously inoculated on the back (shaved) side of the tested mice (each mouse was inoculated with 2 ⁇ 10 6 cells, the cells were mixed with Matrigel at a volume ratio of 1:1, and then inoculated, 200 ⁇ l).
  • the mice were randomly divided into 5 groups according to the experimental design, with 8 mice in each group.
  • the day of group administration is defined as day 0.
  • the grouping situation and dosing schedule are shown in Table 15:
  • N is the number of animals in each group
  • Hanke10F4 In the Hanke10F4 administration group, after the tumor subsided, the observation was continued for 1 month, and the tumor did not recur. Mice with tumor regression were again inoculated with 4 ⁇ 10 6 MC38 cells. After 15 days, tumors did not form. As shown in Figure 17, Hanke10F4 can not only inhibit tumor growth, but also mobilize memory T cell function in mice. The tumor will not recur and has immune function to tumors of the same type.
  • B-hCD137(4-1BB) mice were selected to detect the anti-tumor efficacy of Hanke10F4 in vivo.
  • the B-hCD137(4-1BB) mouse model is a genetically engineered mouse, which is a human-derived h4-1BB gene that is chimeric in the genome of the C57BL/6 mouse with a genetic background, from Biocytogram.
  • test mice were randomly divided into 4 groups, and they were given physiological saline, Hanke10F4, Utomilumab, and Urelumab, respectively, twice a week for 3 weeks, and the concentration of each administration was 30 mg/kg. After the administration, the tail was removed and blood was collected to detect the contents of AST (aspartate aminotransferase) and ALT (alanine aminotransferase) in the peripheral blood of the mice.
  • AST aspartate aminotransferase
  • ALT alanine aminotransferase
  • the AST content of Urelumab was significantly higher, indicating that the antibody has a risk of liver toxicity, while the AST and ALT content of Hanke10F4 and Utomilumab were not significantly different from those of the normal saline group, indicating that Hanke10F4 and Utomilumab also have Low toxicity.
  • Hanke10F4 was administered to 40 SD rats (half female and half male) intravenously with a single intravenous injection, with three doses of high (30mg/kg), medium (10mg/kg), and low (3mg/kg), according to different times FOB test to evaluate the effect of Hanke10F 4 on the central nervous system of SD rats.
  • the 4 cynomolgus monkeys were randomly divided into two groups: a dose climbing group and a fixed-dose group according to their sex and weight. Each group consisted of 2 males and half males. They were administered by intravenous injection, as shown in Table 16.
  • the first digit of the animal number represents the group (1 and 2 respectively represent the Hanke10F4 dose climbing group and the Hanke10F4 high dose group); the second letter represents the gender (F is female, M is male).
  • the detection time point is once during the adaptation period, and once every 24h after administration on D8, D15, D22, and D29; once at the end of the administration period (D35).
  • the experimental results showed that during the administration period, the monkeys in each group had no abnormalities in general observation, body weight, food intake/food consumption, body temperature, respiration, electrocardiogram, blood pressure, blood coagulation index, etc.
  • all animal blood biochemical indexes and indexes (alanine aminotransferase ALT, aspartate aminotransferase AST) of Hanke10F4 in each dose group fluctuated within the normal range, and there was no obvious trend of change. See Table 17 for details.
  • the maximum tolerated dose (MTD) of Hanke10F4 in cynomolgus monkeys is ⁇ 200mg/kg.
  • T0 is the initial time
  • 3M is the end of 3 months.
  • the Tm value the temperature at which half of the Hanke10F4 protein unfolds.
  • the Tm1 value of Hanke10F4 is 64.4°C and the Tm2 value is 71.5°C (Tm1 and Tm2 are the two temperatures at which the fluorescence peak changes during protein unfolding), That is, the stability of the Hanke10F4 antibody is good ( Figure 19).
  • the antibody Hanke10F4 provided by the present invention can bind to human 4-1BB, exhibit high affinity to human 4-1BB and effectively enhance T cell response.
  • the antibody competitively binds to human 4-1BB to a certain extent with human 4-1BBL.
  • the antibody produces INF- ⁇ in the induction of T cell costimulation assay, which can be used in a mouse model. Obviously inhibit tumor growth, and the tumor will not recur.
  • the antibody of the present invention can be used to regulate the immune response mediated by T cells and antibodies, and can be used in anti-tumor immunotherapy.
  • the single-chain antibody fusion antibody provided by the present invention can bind to human and monkey 4-1BB.
  • the binding of the antibody of the present invention to human 4-1BB leads to an enhanced immune response.
  • the antibodies and antigen-binding fragments provided by the present invention can be used as immune enhancers or immune modulators for T cell-mediated autoimmune diseases.
  • the antibodies and antigen-binding fragments thereof can also be used as diagnostic tools to detect 4-1BB in the blood or tissues of patients with cancer, autoimmune or other diseases.
  • the antibody provided by the invention has good anti-tumor efficacy, strong safety and good stability.
  • the antibodies and antigen-binding fragments thereof provided by the present invention have a wide range of therapeutic applications as immune modulators, such as cancer, autoimmune diseases, inflammatory diseases, and infectious diseases.

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Abstract

提供一种能够与人4-1BB结合的分子及其应用。所提供的抗体的重链可变区中HCDR1、HCDR2和HCDR3的氨基酸序列依次如SEQ ID No.1自N端起第31-35位、第50-64位、第98-106位所示；轻链可变区中LCDR1、LCDR2和LHCDR3的氨基酸序列依次如SEQ ID No.2自N端起第24-34位、第50-56位、第89-97位所示。所提供的抗体能结合人和猴4-1BB，并对人4-1BB表现高亲和力以及有效增强T细胞反应，可用于调节T细胞和抗体介导的免疫反应，作为免疫调节物具有广泛的治疗用途，诸如癌症、自身免疫疾病、炎症疾病、和感染疾病等。

Description

一种能够与人4-1BB结合的分子及其应用 技术领域
本发明涉及生物技术领域，特别涉及一种能够与人4-1BB结合的分子及其应用。
背景技术
4-1BB(CD137)属肿瘤坏死因子(TNF)受体家族的成员，由肿瘤坏死因子受体超家族成员9(TNFRSF9)基因编码，是由Kwon和Weissman在激活的鼠T细胞克隆分化筛选过程中首先发现的。Schwarzh等从人活化T细胞中分离出与鼠4-1BB具有同源性的人4-1BB，并将其命名为CD137。4-1BB是Ⅰ型膜糖蛋白，不同于PD-1/PD-L1，4-1BB的表达具有激活依赖性，其介导T细胞活化的共刺激信号，广泛存在于免疫细胞包括激活的NK细胞、活化的T细胞、树突状细胞(DC)、肥大细胞、单核细胞、中性粒细胞以及Treg细胞表面。
人的4-1BB具有255个氨基酸，在细胞表面以单体和二聚体形式表达，并且易于与配体形成三聚体，进而开启信号转导。
研究表明，4-1BB是激活型共刺激分子，其信号通路可促进T细胞，特别是CD8+T细胞的分化、增殖以及细胞因子的产生。4-1BB主要在活化的T细胞上表达，主要作用于T细胞免疫应答的中晚期。4-1BB信号通路可以通过增强肿瘤特异性CD8+T细胞功能实现抗肿瘤作用，还可以通过增强NK细胞、DC以及CD4+T细胞的免疫功能提高CD8+T细胞介导的抗肿瘤免疫应答。4-1BB募集TNFR相关因子TRAF1和TRAF2形成异源三聚体，通过c-Jun N末端激酶(JNK)途径，细胞外信号调节激酶(ERK)途径，以及通过β-catenin和AKT途径加强信号转导，并通过主转录因子NF-κB调节4-1BB信号，促使细胞因子的产生和分泌。此外，4-1BB/4-1BBL相互作用能够提高T细胞病毒感染的免疫防御作用。进一步的，4-1BB单一治疗和组合治疗肿瘤模型已经确定持久的抗肿瘤保护性T细胞记忆应答。
目前，并无关于4-1BB的激动剂药物上市，最快的处在临床研发阶段，不能满足各种疾病包括癌症的对于免疫治疗方法的需求。针对4-1BB靶点，研制一种“加速器”药物，利用现有的癌症药物(如罗氏的Rituxan)帮助激活免疫系统，开发新型肿瘤免疫疗法，适应市场需求，避免PD-1/PD-L1的耐药劣势，具有很好的应用前景。
发明公开
本发明的目的是提供一种能够与人4-1BB结合的分子及其应用。
本发明所提供的能够与人4-1BB结合的分子可为抗人4-1BB的人源化抗体或其抗原结合片段(如Fab片段、ScFv片段)或抗体融合蛋白(如完整人源化抗体、ScFv、Fab片段与其他蛋白融合形成的抗体融合蛋白)或抗人4-1BB人源化抗体偶联具有生物活性小分子药物的ADC抗体或完整人源化抗体、ScFv、Fab片段与其他靶点抗体形成的双抗。
第一方面，本发明要求保护一种抗体。
本发明所要求保护的抗体，重链可变区中HCDR1、HCDR2和HCDR3的氨基酸序列依次如SEQ ID No.1自N端起第31-35位、第50-64位、第98-106位所示；轻链可变区中LCDR1、LCDR2和LHCDR3的氨基酸序列依次如SEQ ID No.2自N端起第24-34位、第50-56位、第89-97位所示。
进一步地，所述重链可变区的氨基酸序列为如下任一：
(a1)SEQ ID No.1自N端起第1-117位(即SEQ ID No.16自N端起第1-117位)，或者与SEQ ID No.1自N端起第1-117位(即SEQ ID No.16自N端起第1-117位)具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))；
(a2)SEQ ID No.18自N端起第1-117位，或者与SEQ ID No.18自N端起第1-117位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))；
(a3)SEQ ID No.19自N端起第1-117位，或者与SEQ ID No.19自N端起第1-117位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))；
(a4)SEQ ID No.20自N端起第1-117位，或者与SEQ ID No.20自N端起第1-117位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))；
(a5)SEQ ID No.21自N端起第1-117位，或者与SEQ ID No.21自N端起第1-117位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))；
(a6)SEQ ID No.22自N端起第1-117位，或者与SEQ ID No.22自N端起第1-117位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))；
(a7)SEQ ID No.23自N端起第1-117位，或者与SEQ ID No.23自N端起第1-117位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))；
(a8)SEQ ID No.24自N端起第1-117位，或者与SEQ ID No.24自N端起第1-117位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))；
(a9)SEQ ID No.25自N端起第1-117位，或者与SEQ ID No.25自N端起第1-117位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))；
(a10)SEQ ID No.26自N端起第1-117位，或者与SEQ ID No.26自N端起第1-117位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))；
(a11)SEQ ID No.27自N端起第1-117位，或者与SEQ ID No.27自N端起第 1-117位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))；
(a12)SEQ ID No.28自N端起第1-117位，或者与SEQ ID No.28自N端起第1-117位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))。
进一步地，所述轻链可变区的氨基酸序列可为如下任一：
(b1)SEQ ID No.2自N端起第1-107位(即SEQ ID No.16自N端起第133-239位)，或者与SEQ ID No.2自N端起第1-107位(即SEQ ID No.16自N端起第133-239位)具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))。
(b2)SEQ ID No.18自N端起第133-239位，或者与SEQ ID No.18自N端起第133-239位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))。
(b3)SEQ ID No.19自N端起第133-239位，或者与SEQ ID No.19自N端起第133-239位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))。
(b4)SEQ ID No.20自N端起第133-239位，或者与SEQ ID No.20自N端起第133-239位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))。
(b5)SEQ ID No.21自N端起第133-239位，或者与SEQ ID No.21自N端起第133-239位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))。
(b6)SEQ ID No.22自N端起第133-239位，或者与SEQ ID No.22自N端起第133-239位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))。
(b7)SEQ ID No.23自N端起第133-239位，或者与SEQ ID No.23自N端起第133-239位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))。
(b8)SEQ ID No.24自N端起第133-239位，或者与SEQ ID No.24自N端起第133-239位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))。
(b9)SEQ ID No.25自N端起第133-239位，或者与SEQ ID No.25自N端起第133-239位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))。
(b10)SEQ ID No.26自N端起第133-239位，或者与SEQ ID No.26自N端起第133-239位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))。
(b11)SEQ ID No.27自N端起第133-239位，或者与SEQ ID No.27自N端起第133-239位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))。
(b12)SEQ ID No.28自N端起第133-239位，或者与SEQ ID No.28自N端起第133-239位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))。
所述抗体的重链恒定区为人IgG4恒定区，具体氨基酸序列如SEQ ID No.1自N端起第118-444位所示。所述抗体的轻链恒定区为人kappa轻链恒定区，具体氨基酸序列如SEQ ID No.2自N端起第108-214位所示。
更进一步地，所述重链的氨基酸序列为SEQ ID No.1，或者与SEQ ID No.1具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))。所述轻链的氨基酸序列为SEQ ID No.2，或者与SEQ ID No.2具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))。
本发明所述抗体可以是任何类别，如IgG、IgM、IgE、IgA或IgD。优选4-1BB抗体是IgG类别，如IgG1、IgG2、IgG3或IgG4亚型，更优选IgG4亚型。
本发明所述抗体的轻链类型可以是κ链，也可以是λ链，优选为Kappa链。
在本发明的具体实施方式中，所述抗体具体为抗人4-1BB的人源化抗体Hanke10F4。所述Hanke10F4主要结合人4-1BB胞外区的Domain1和Domain 2A。
本发明所提供的抗体具有如下一或多种性质或特性：
1)特异性结合人4-1BB；
2)结合人和食蟹猴4-1BB；
3)结合人或食蟹猴4-1BB；
4)一定程度上，能阻断人4-1BBL(4-1BB配体)与人4-1BB的结合；
5)是IgG，如IgG1、IgG2、IgG3或IgG4；
6)是人抗体，或者人源化抗体。
本发明所述抗体或其抗原结合片段对人4-1BB具有激动剂活性，能刺激CD4 +T和CD8 +T细胞增殖，能够通过结合细胞表面4-1BB分子，引起下游NF-κB信号通路激活，从而显著促进细胞因子IFN-γ的产生与分泌，并且能显著抑制人4-1BB基因敲入的人源化小鼠体内肿瘤的发生与发展，一定程度上说明所述抗体或其抗原结合部分可以通过调节免疫细胞活性进而对免疫系统进行调控，能够运用于免疫增强物抗肿瘤或抗病毒免疫反应的免疫增强物，或T细胞介导的自身免疫疾病的免疫调节物。
本发明的抗体可通过本领域已知的方法制备，可通过在转染的细胞诸如永生化的真核细胞诸如骨髓瘤或杂交瘤细胞中表达来制备。抗体可以通过本领域已知的方法从一个类别或亚型转变为另一个类别或亚型。此外，本发明提供的抗体可以是单克隆抗体或者多克隆抗体，优选单克隆抗体。
本发明所述的抗体可以通过本领域已知的技术产生，包括常规的单克隆抗体技术，例如标准的体细胞杂交技术，B淋巴细胞的病毒或致癌基因转化，或重组抗体技术，如下文详述。
杂交瘤产生是常用的方法，为本领域已知。首先，瞬转表达人4-1BB蛋白，免疫Balb/c小鼠。人4-1BB抗原氨基酸序列如SEQ ID No.5所示(其中第24-184位为胞外区)，免疫抗原是分离的或者纯化的人4-1BB，可以是人4-1BB片段，如人4-1BB的胞外结构域。动物免疫，可以是本领域已知的任何方法进行，免疫非人动物如小鼠、大鼠、绵羊等。在用人4-1BB抗原免疫动物之后，从分离自免疫的动物的细胞中制备产生抗体的永生化细胞系。在免疫后，处死动物，获得永生化淋巴结和/或脾B细胞，用常规方法进行细胞融合，用ELISA法筛选阳性细胞克隆，即产生抗4-1BB抗体的细胞，再反复亚克隆，进一步筛选，选择生长状态良好、抗体产量高、培养上清100％阳性的杂交瘤。杂交瘤可以在同系动物、缺少免疫系统的动物及裸鼠体内扩增或者在体外细胞培养中扩增。选择、克隆和扩增杂交瘤的方法为本领域技术人员熟知。用双相琼脂扩散实验检测杂交瘤细胞分泌的免疫球蛋白亚型为IgG1。本发明抗体也可以用噬菌体展示方法制备。这种用于分离人抗体的噬菌体展示方法是本领域已经建立的。
所述抗体生产表达后，包含抗体、二聚体、单个轻、重链，或其他免疫球蛋白形式，可根据本领域标准方法纯化，诸如硫酸铵沉淀、亲和柱、柱层析，凝胶电泳等。
第二方面，本发明要求保护前文所述抗体的抗原结合片段。
其中，所述抗原结合片段可包含如下中的一种或多种：(1)前文所述抗体的轻链；(2)前文所述抗体的重链；(3)前文所述抗体的轻链可变区；(4)前文所述抗体的重链可变区；(5)前文所述抗体的一或多个CDR区。
进一步地，所述抗原结合片段可为如下任一：(1)Fab片段，其是有VL、VH、CL和CH1结构域组成的单价片段；(2)F(ab’)2片段，其是二价片段，包含铰链区由二硫键链接的两个Fab片段；(3)Fd片段，由VH和CH1结构域组成；(4)FV片段，由抗体的单臂的VL和VH结构域组成；(5)分离的CDR(如来自轻链的CDR和/或来自重链的CDR)；(6)单链抗体等，其中单链抗体可以为scFv结构，单链抗体可以带不同标签，如scFv-His，scFv-Fc等。
在本发明的具体实施方式中，本发明提供的抗原结合片段是带有标签的单链抗体(scFv-Fc)。单链抗体(scFv-Fc)可由包含连接与VH结构域的VL结构域的单个多肽链连接人的IgG4Fc标签组成，其中VL结构域和VH结构域被配对以形成单价分子。单链抗体可以根据本领域已知方法制备。
在该方面中，本发明特别的要求保护一种抗4-1BB的单链抗体。
本发明所要求保护的抗4-1BB的单链抗体，由重链可变区和轻链可变区连接而成；所述重链可变区中HCDR1、HCDR2和HCDR3的氨基酸序列依次如SEQ ID No.16自N端起第31-35位、第50-64位、第98-106位所示；所述轻链可变区中LCDR1、 LCDR2和LHCDR3的氨基酸序列依次如SEQ ID No.16自N端起第156-166位、第182-188位、第221-229位所示。
进一步地，所述单链抗体的重链可变区的氨基酸序列为如下任一：
(a1)SEQ ID No.16自N端起第1-117位，或者与SEQ ID No.16自N端起第1-117位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))；
(a2)SEQ ID No.18自N端起第1-117位，或者与SEQ ID No.18自N端起第1-117位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))；
(a3)SEQ ID No.19自N端起第1-117位，或者与SEQ ID No.19自N端起第1-117位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))；
(a4)SEQ ID No.20自N端起第1-117位，或者与SEQ ID No.20自N端起第1-117位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))；
(a5)SEQ ID No.21自N端起第1-117位，或者与SEQ ID No.21自N端起第1-117位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))；
(a6)SEQ ID No.22自N端起第1-117位，或者与SEQ ID No.22自N端起第1-117位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))；
(a7)SEQ ID No.23自N端起第1-117位，或者与SEQ ID No.23自N端起第1-117位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))；
(a8)SEQ ID No.24自N端起第1-117位，或者与SEQ ID No.24自N端起第1-117位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))；
(a9)SEQ ID No.25自N端起第1-117位，或者与SEQ ID No.25自N端起第1-117位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))；
(a10)SEQ ID No.26自N端起第1-117位，或者与SEQ ID No.26自N端起第1-117位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))；
(a11)SEQ ID No.27自N端起第1-117位，或者与SEQ ID No.27自N端起第1-117位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))；
(a12)SEQ ID No.28自N端起第1-117位，或者与SEQ ID No.28自N端起第 1-117位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))。
进一步地，所述单链抗体的轻链可变区的氨基酸序列可为如下任一：
(b1)SEQ ID No.16自N端起第133-239位，或者与SEQ ID No.16自N端起第133-239位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))。
(b2)SEQ ID No.18自N端起第133-239位，或者与SEQ ID No.18自N端起第133-239位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))。
(b3)SEQ ID No.19自N端起第133-239位，或者与SEQ ID No.19自N端起第133-239位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))。
(b4)SEQ ID No.20自N端起第133-239位，或者与SEQ ID No.20自N端起第133-239位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))。
(b5)SEQ ID No.21自N端起第133-239位，或者与SEQ ID No.21自N端起第133-239位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))。
(b6)SEQ ID No.22自N端起第133-239位，或者与SEQ ID No.22自N端起第133-239位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))。
(b7)SEQ ID No.23自N端起第133-239位，或者与SEQ ID No.23自N端起第133-239位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))。
(b8)SEQ ID No.24自N端起第133-239位，或者与SEQ ID No.24自N端起第133-239位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))。
(b9)SEQ ID No.25自N端起第133-239位，或者与SEQ ID No.25自N端起第133-239位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))。
(b10)SEQ ID No.26自N端起第133-239位，或者与SEQ ID No.26自N端起第133-239位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))。
(b11)SEQ ID No.27自N端起第133-239位，或者与SEQ ID No.27自N端起第133-239位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))。
(b12)SEQ ID No.28自N端起第133-239位，或者与SEQ ID No.28自N端起 第133-239位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))。
更进一步地，所述单链抗体的氨基酸序列可为如下任一：
(c1)SEQ ID No.16的第1-239位，或者与SEQ ID No.16的第1-239位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))。
(c2)SEQ ID No.18的第1-239位，或者与SEQ ID No.18的第1-239位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))。
(c3)SEQ ID No.19的第1-239位，或者与SEQ ID No.19的第1-239位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))。
(c4)SEQ ID No.20的第1-239位，或者与SEQ ID No.20的第1-239位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))。
(c5)SEQ ID No.21的第1-239位，或者与SEQ ID No.21的第1-239位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))。
(c6)SEQ ID No.22的第1-239位，或者与SEQ ID No.22的第1-239位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))。
(c7)SEQ ID No.23的第1-239位，或者与SEQ ID No.23的第1-239位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))。
(c8)SEQ ID No.24的第1-239位，或者与SEQ ID No.24的第1-239位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))。
(c9)SEQ ID No.25的第1-239位，或者与SEQ ID No.25的第1-239位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))。
(c10)SEQ ID No.26的第1-239位，或者与SEQ ID No.26的第1-239位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))。
(c11)SEQ ID No.27的第1-239位，或者与SEQ ID No.27的第1-239位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))。
(c12)SEQ ID No.28的第1-239位，或者与SEQ ID No.28的第1-239位具有 99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))。
第三方面，本发明要求保护前文所述的抗体或前文所述的抗原结合片段与其他蛋白形成的抗体融合蛋白。
其中，所述其他蛋白可为毒素、酶、细胞膜蛋白分子、细胞因子或受体蛋白等。
进一步地，本发明要求保护的所述抗体融合蛋白可为前文所述单链抗体与标签蛋白形成的带有标签蛋白的单链抗体。
在本发明的具体实施方式中，所述标签蛋白为Fc。
进一步地，所述Fc可来自如IgG、IgM、IgE、IgA或IgD。优选所述Fc来自IgG类别，如IgG1、IgG2、IgG3或IgG4亚型，更优选IgG4亚型。所述Fc优选为人源。
更进一步地，所述Fc的氨基酸序列如SEQ ID No.16的第240-472位所示，或者与SEQ ID No.16自N端起第240-472位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性。
更加具体地，所述带有标签蛋白的单链抗体的氨基酸序列可为如下任一：
(d1)SEQ ID No.16所示，或者与SEQ ID No.16具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))。
(d2)SEQ ID No.18所示，或者与SEQ ID No.18具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))。
(d3)SEQ ID No.19所示，或者与SEQ ID No.19具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))。
(d4)SEQ ID No.20所示，或者与SEQ ID No.20具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))。
(d5)SEQ ID No.21所示，或者与SEQ ID No.21具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))。
(d6)SEQ ID No.22所示，或者与SEQ ID No.22具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))。
(d7)SEQ ID No.23所示，或者与SEQ ID No.23具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))。
(d8)SEQ ID No.24所示，或者与SEQ ID No.24具有99％以上、95％以上、 90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))。
(d9)SEQ ID No.25所示，或者与SEQ ID No.25具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))。
(d10)SEQ ID No.26所示，或者与SEQ ID No.26具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))。
(d11)SEQ ID No.27所示，或者与SEQ ID No.27具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))。
(d12)SEQ ID No.28所示，或者与SEQ ID No.28具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))。
第四方面，本发明要求保护前文所述的抗体或前文所述抗原结合片段或前文所述带有标签蛋白的单链抗体与其他靶点抗体形成的双抗。
第五方面，本发明要求保护核酸分子。
本发明所要求保护的核酸分子编码前文所述的抗体或前文所述抗原结合片段或前文所述抗体融合蛋白或前文所述的双抗。
本发明的核酸分子可以是DNA或RNA，可以包含或不包含内含子序列。优选的，所述核酸分子是cDNA分子。
进一步地，在所述核酸分子中，编码所述抗体的重链可变区中HCDR1、HCDR2和HCDR3的核苷酸序列依次如SEQ ID No.3自5’端起第91-105位、第148-192位、第292-318位所示；编码所述抗体的轻链可变区中LCDR1、LCDR2和LHCDR3的核苷酸序列依次如SEQ ID No.4自5’端起第70-102位、第148-168位、第265-291位所示。
更进一步地，在所述核酸分子中，编码所述抗体的所述重链可变区的核苷酸序列为SEQ ID No.3自5’端起第1-351位或者与SEQ ID No.3自5’端起第1-351位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))。编码所述抗体的所述轻链可变区的核苷酸序列为SEQ ID No.4自5’端起第1-321位或者与SEQ ID No.4自5’端起第1-321位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))。
在所述核酸分子中，编码所述抗体的所述重链恒定区变区的核苷酸序列为SEQ ID No.3自5’端起第352-1332位。编码所述抗体的所述轻链恒变区的核苷酸序列为SEQ ID No.4自5’端起第322-642位。
更加具体地，在所述核酸分子中，编码所述抗体的重链的核苷酸序列为SEQ  ID No.3或者与SEQ ID No.3具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))。编码所述抗体的轻链的核苷酸序列为SEQ ID No.4或者与SEQ ID No.4具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))。
本发明的核酸分子可以用合适的分子生物学技术获得。对于由杂交瘤表达的抗体，可以通过PCR扩增或者cDNA克隆技术获得编码由所述杂交瘤制备的抗体轻链和重链的cDNA。
本发明的核酸分子可以编码轻链或重链的一部分、全长轻链或重链，或抗体衍生物或其抗原结合片段的氨基酸序列。
编码VH的分离的DNA可以通过将编码VH的DNA连接于编码重链恒定区(CH1、CH2和CH3)的另一个DNA分子而转化为全长重链基因。人类重链恒定区基因序列是本领域已知的，包含这些区域的DNA片段可以通过标准PCR扩增获得。重链恒定区可以是IgG1、IgG2、IgG3、IgG4、IgA、IgE、IgM或IgD恒定区。优选的是IgG4恒定区。
编码VL区的分离的DNA可以通过将编码的VL的DNA连接于编码轻链恒定区CL的另一个DNA分子而转化为全长轻链基因。人类轻链恒定区基因序列是本领域已知的，包含这些区域的DNA片段可以通过标准的PCR扩增获得。轻链恒定区可以是Kappa或Lambda恒定区。
在该方面中，本发明特别的要求保护编码前文所述的单链抗体或前文所述带有标签蛋白的单链抗体或前文所述融合抗体的核酸分子。
进一步地，在所述核酸分子中，编码所述重链可变区中HCDR1、HCDR2和HCDR3的核苷酸序列依次如SEQ ID No.17自5’端起第91-105位、第148-192位、第292-318位所示；编码所述轻链可变区中LCDR1、LCDR2和LHCDR3的核苷酸序列依次如SEQ ID No.17自5’端起第466-498位、第544-564位、第661-687位所示。
更进一步地，在所述核酸分子中，编码所述重链可变区的核苷酸序列为SEQ ID No.17自5’端起第1-351位或者与SEQ ID No.17自5’端起第1-351位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))。编码所述轻链可变区的核苷酸序列为SEQ ID No.17自5’端起第397-717位或者与SEQ ID No.17自5’端起第397-717位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))。
更加具体地，编码所述单链抗体的核苷酸序列为SEQ ID No.17自5’端起第1-717位或者与SEQ ID No.17自5’端起第1-717位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))。
更加具体地，编码所述带有标签蛋白的单链抗体的核苷酸序列为SEQ ID No.17或者与SEQ ID No.17具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))
本发明的核酸分子可以用合适的分子生物学技术获得。
第六方面，本发明要求保护含有前文所述核酸分子的表达盒、重组载体、重组细胞。
其中，所述载体是可用于结合分子如抗体或其抗原结合片段(如单链抗体)或带有标签蛋白的单链抗体(如单链抗体与Fc融合)或融合抗体表达的表达载体。
为表达本发明的抗体(或单链抗体或融合抗体)，编码部分或全长轻链和重链的DNA被插入表达载体，开始该DNA分子的转录和翻译。抗体轻链基因和抗体重链基因可以插入单独载体中，或者插入相同表达载体中。插入方法可以是已知的任何合适的方法，如抗体基因片段上的互补限制位点与载体的链接。本发明描述的抗体的轻链和重链可变区可用于产生任何抗体种型和亚型的全长抗体基因。
本发明进一步提供含本发明提供核酸分子的宿主细胞(即所述重组细胞)。所述宿主细胞可以是表达载体可利用的任何细胞。例如高等真核宿主细胞，如哺乳动物细胞包含例如中国仓鼠卵巢(CHO)细胞，低等真核宿主细胞，如酵母细胞，及可以是原核细胞，如细菌细胞、大肠杆菌等。重组核酸构建体导入宿主细胞中的转染方法如电穿孔、磷酸钙转染、DEAE-葡聚糖、脂质转染、噬菌体感染等。可通过已知方法将当编码抗体基因的表达载体导入宿主细胞中，通过将该宿主细胞培养足以使得结合分子在宿主细胞中表达，由此产生抗体。
所述载体可为质粒、黏粒、噬菌体或病毒载体。所述质粒可为pCDNA3.4和pCDNA3.4-L或其他。
本领域普通技术人员可以很容易地采用已知的方法，例如定向进化和点突变的方法，对本发明所述抗4-1BB抗体(或单链抗体或融合抗体)的核苷酸序列进行突变。那些经过人工修饰的，具有与本发明所述抗4-1BB抗体(或单链抗体或融合抗体)的核苷酸序列具有99％以上、95％以上、90％以上、85％以上、80％以上或75％以上的同一性的核苷酸，只要编码所述抗4-1BB抗体且具有所述抗4-1BB抗体活性，均是衍生于本发明的核苷酸序列并且等同于本发明的序列。
在本发明的具体实施方式中，将SEQ ID No.3所示DNA片段(抗体重链的编码基因)克隆到pcDNA3.4载体的酶切位点Xba I和Hand III之间后得到表达所述抗体的重链的重组表达载体(命名为pcDNA3.4-H)；将SEQ ID No.4所示DNA片段(抗体轻链的编码基因)克隆到pcDNA3.4载体的酶切位点Xba I和Hand III之间后得到表达所述抗体的轻链的重组表达载体(命名为pcDNA3.4-L)。为了更加有利于蛋白表达，构建所述重组表达载体时，在SEQ ID No.3和SEQ ID No.4所示DNA片段(抗体重链和轻链的编码基因)的上游还引入了kozak共识别序列(5’-GCCACC-3’)和信号肽的编码序列(5’-ATGGAGTTTGGGCTGAGTTGGGTCTTTCTGGTCGCAATTC TGCTGAAGGGAGTGCAGTGC-3’)，信号肽的编码序列与SEQ ID No.3和SEQ ID No.4的5’端相连。所述重组细胞 为将上述分别表达所述抗体重链和轻链的两个重组表达载体(pcDNA3.4-H和pcDNA3.4-L)共转染HEK293细胞后得到的重组细胞。所述重组细胞即可表达前文所述抗体Hanke10F4。
第七方面，本发明要求保护ADC抗体。
本发明要求保护的所述ADC抗体为前文所述的抗体或前文所述抗原结合片段或前文所述抗体融合蛋白(如带有标签蛋白的单链抗体)或前文所述双抗偶联具有生物活性小分子药物后形成。
第八方面，本发明要求保护药物组合物。
本发明所要求保护的药物组合物包含：(A1)前文所述的抗体或前文所述抗原结合片段(如单链抗体)或前文所述抗体融合蛋白(如单链抗体与Fc融合)或前文所述双抗或前文所述ADC抗体；(A2)药学可接受的赋形剂、稀释剂或载体。
第九方面，本发明要求保护前文所述的抗体或前文所述的抗原结合片段(如单链抗体)或前文所述抗体融合蛋白(如单链抗体与Fc融合)或前文所述双抗或前文所述核酸分子或表达盒、重组载体或重组细胞或前文所述ADC抗体或前文所述药物组合物在如下任一中的应用：
(B1)制备用于检测4-1BB的产品，或检测4-1BB；
在实际应用中，可将所述抗体或其抗原结合片段用作诊断工具检测癌症、自身免疫或其他疾病的患者的血液或组织中的4-1BB。
其中，所述4-1BB可为人4-1BB，也可为猴4-1BB。
(B2)制备用于阻断4-1BB/4-1BBL信号通路的产品，或阻断4-1BB/4-1BBL信号通路；
(B3)制备用于刺激T细胞活化的产品，或刺激T细胞活化；
(B4)制备用于促进T细胞分泌IFN-γ的产品，或促进T细胞分泌IFN-γ；
(B5)制备用于抑制结肠癌细胞生长的产品，或抑制结肠癌细胞生长；
(B6)制备用于抑制结肠癌肿瘤生长的产品，或抑制结肠癌肿瘤生长；
(B7)制备用于治疗和/或预防结肠癌的产品，或治疗和/或预防结肠癌；
(B8)作为免疫增强物，或制备免疫增强物；
(B9)作为免疫调节物，或制备免疫调节物；
(B10)制备用于治疗和/或预防和/或诊断具有4-1BB表达失调特征疾病的产品，或治疗和/或预防和/或诊断具有4-1BB表达失调特征疾病；
(B11)制备用于治疗和/或预防和/或诊断癌症的产品，或治疗和/或预防和/或诊断癌症；
进一步地，所述癌症可为4-1BB表达失调的癌症。
(B12)制备用于治疗和/或预防和/或诊断自身免疫疾病的产品，或治疗和/或预防和/或诊断自身免疫疾病；
进一步地，所述自身免疫疾病可为4-1BB表达失调的自身免疫疾病。
(B13)制备用于治疗和/或预防和/或诊断炎症疾病的产品，或治疗和/或预防和/或诊断炎症疾病；
进一步地，所述炎症疾病可为4-1BB表达失调的炎症疾病。
(B14)制备用于治疗和/或预防和/或诊断感染性疾病的产品，或治疗和/或预防和/或诊断感染性疾病。
进一步地，所述感染性疾病可为4-1BB表达失调的感染性疾病。
第九方面，本发明要求保护如下任一方法：
(C1)一种检测4-1BB的方法，包括如下步骤：采用前文所述的抗体或前文所述的抗原结合片段(如单链抗体)或前文所述抗体融合蛋白(如单链抗体与Fc融合)或前文所述双抗或前文所述的核酸分子或表达盒、重组载体或重组细胞对待测样本进行检测。
在实际应用中，可将所述抗体或其抗原结合片段用作诊断工具检测癌症、自身免疫或其他疾病的患者的血液或组织中的4-1BB。
其中，所述4-1BB可为人4-1BB，也可为猴4-1BB。
(C2)一种阻断4-1BB/4-1BBL信号通路的方法，包括如下步骤：采用前文所述的抗体或前文所述的抗原结合片段(如单链抗体)或前文所述抗体融合蛋白(如单链抗体与Fc融合)或前文所述双抗或前文所述核酸分子或表达盒、重组载体或重组细胞或前文所述ADC抗体或前文所述药物组合物阻断4-1BB/4-1BBL信号通路。
(C3)一种刺激T细胞活化的方法，包括如下步骤：采用前文所述的抗体或前文所述的抗原结合片段(如单链抗体)或前文所述抗体融合蛋白(如单链抗体与Fc融合)或前文所述双抗或前文所述核酸分子或表达盒、重组载体或重组细胞或前文所述ADC抗体或前文所述药物组合物刺激T细胞活化。
(C4)一种促进T细胞分泌IFN-γ的方法，包括如下步骤：采用前文所述的抗体或前文所述的抗原结合片段(如单链抗体)或前文所述抗体融合蛋白(如单链抗体与Fc融合)或前文所述双抗或前文所述核酸分子或表达盒、重组载体或重组细胞或前文所述ADC抗体或前文所述药物组合物促进T细胞分泌IFN-γ。
(C5)一种抑制结肠癌细胞生长的方法，包括如下步骤：采用前文所述的抗体或前文所述的抗原结合片段(如单链抗体)或前文所述抗体融合蛋白(如单链抗体与Fc融合)或前文所述双抗或前文所述核酸分子或表达盒、重组载体或重组细胞或前文所述ADC抗体或前文所述药物组合物抑制结肠癌细胞生长。
(C6)一种抑制结肠癌肿瘤生长的方法，包括如下步骤：采用前文所述的抗体或前文所述的抗原结合片段(如单链抗体)或前文所述抗体融合蛋白(如单链抗体与Fc融合)或前文所述双抗或前文所述核酸分子或表达盒、重组载体或重组细胞或前文所述ADC抗体或前文所述药物组合物抑制结肠癌肿瘤生长。
(C7)一种治疗和/或预防结肠癌的方法，包括如下步骤：采用前文所述的抗体或前文所述的抗原结合片段(如单链抗体)或前文所述抗体融合蛋白(如 单链抗体与Fc融合)或前文所述双抗或前文所述核酸分子或表达盒、重组载体或重组细胞或前文所述ADC抗体或前文所述药物组合物治疗和/或预防结肠癌。
(C8)一种制备免疫增强物的方法，包括如下步骤：采用前文所述的抗体或前文所述的抗原结合片段(如单链抗体)或前文所述抗体融合蛋白(如单链抗体与Fc融合)或前文所述双抗或前文所述核酸分子或表达盒、重组载体或重组细胞或前文所述ADC抗体或前文所述药物组合物作为活性成分制备免疫增强物。
(C9)一种制备免疫调节物的方法，包括如下步骤：采用前文所述的抗体或前文所述的抗原结合片段(如单链抗体)或前文所述抗体融合蛋白(如单链抗体与Fc融合)或前文所述双抗或前文所述核酸分子或表达盒、重组载体或重组细胞或前文所述ADC抗体或前文所述药物组合物作为活性成分制备免疫调节物。
(C10)一种治疗和/或预防和/或诊断具有4-1BB表达失调特征疾病的方法，包括如下步骤：采用前文所述的抗体或前文所述的抗原结合片段(如单链抗体)或前文所述抗体融合蛋白(如单链抗体与Fc融合)或前文所述双抗或前文所述核酸分子或表达盒、重组载体或重组细胞或前文所述ADC抗体或前文所述药物组合物治疗和/或预防和/或诊断具有4-1BB表达失调特征疾病。
(C11)一种治疗和/或预防和/或诊断癌症的方法，包括如下步骤：采用前文所述的抗体或前文所述的抗原结合片段(如单链抗体)或前文所述抗体融合蛋白(如单链抗体与Fc融合)或前文所述双抗或前文所述核酸分子或表达盒、重组载体或重组细胞或前文所述ADC抗体或前文所述药物组合物治疗和/或预防和/或诊断癌症。
进一步地，所述癌症可为4-1BB表达失调的癌症。
(C12)一种治疗和/或预防和/或诊断自身免疫疾病的方法，包括如下步骤：采用前文所述的抗体或前文所述的抗原结合片段(如单链抗体)或前文所述抗体融合蛋白(如单链抗体与Fc融合)或前文所述双抗或前文所述核酸分子或表达盒、重组载体或重组细胞或前文所述ADC抗体或前文所述药物组合物治疗和/或预防和/或诊断自身免疫疾病。
进一步地，所述自身免疫疾病可为4-1BB表达失调的自身免疫疾病。
(C13)一种治疗和/或预防和/或诊断炎症疾病的方法，包括如下步骤：采用前文所述的抗体或前文所述的抗原结合片段(如单链抗体)或前文所述抗体融合蛋白(如单链抗体与Fc融合)或前文所述双抗或前文所述核酸分子或表达盒、重组载体或重组细胞或前文所述ADC抗体或前文所述药物组合物治疗和/或预防和/或诊断炎症疾病。
进一步地，所述炎症疾病可为4-1BB表达失调的炎症疾病。
(C14)一种治疗和/或预防和/或诊断感染性疾病的方法，包括如下步骤：采用前文所述的抗体或前文所述的抗原结合片段(如单链抗体)或前文所述抗 体融合蛋白(如单链抗体与Fc融合)或前文所述双抗或前文所述核酸分子或表达盒、重组载体或重组细胞或前文所述ADC抗体或前文所述药物组合物治疗和/或预防和/或诊断感染性疾病。
进一步地，所述感染性疾病可为4-1BB表达失调的感染性疾病。
附图说明
图1为杂交瘤抗体SDS-PAGE。
图2为抗体结构图。
图3为杂交瘤分泌抗体识别人4-1BB。
图4为杂交瘤分泌抗体识别猴4-1BB。
图5为抗原结合片段识别人4-1BB。
图6为抗原结合片段识别猴4-1BB。
图7为FACS比较各抗原结合片段与人4-1BB亲和力。
图8为Hanke10F4和HKB6亲和力比较。
图9为抗体Hanke10F4阻断4-1BBL与4-1BB结合。
图10为抗体Hanke10F4与对照抗体表位竞争。
图11为FACS检测抗体与不同点突变抗原亲和。
图12为Hanke10F4和HKB6表位竞争。
图13为萤光素酶活性测定。A为“3.5荧光素酶报告基因活性检测”结果。B为“3.9稳定性(1)加速稳定性”检测结果。
图14为抗体Hanke10F4激活CD4 +T细胞效应检测。图中每组三个柱形图由左到右依次分别为抗体高、中、低(2μg/ml、0.4μg/ml、0.08μg/ml)浓度条件下CD4 +T细胞的激活效应。
图15为抗体Hanke10F4激活CD8 +T细胞效应检测。图中每组三个柱形图由左到右依次分别为抗体高、中、低(3μg/ml、0.5μg/ml、0.08μg/ml)浓度条件下T细胞的激活效应。
图16为抗体抑制MC38肿瘤模型。每个线条代表一只小鼠的肿瘤生长曲线。
图17为抗体Hanke10F4挑战试验。
图18为抗体Hanke10F4毒理检测。
图19为Hanke10F4Tm检测。
实施发明的最佳方式
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。以下实施例中的定量试验，均设置三次重复实验，结果取平均值。
下述实施例中，阳性对照抗体为Pfizer公司的Utomilumab(PF-05082566，专利号：US8337850B2)和Bristol Myers Squibb公司的Urelumab(BMS-663513，专利号：US7288638B2)，这两种抗体均为抗人4-1BB抗体。阴性对照抗体为 人IgG(南京金斯瑞产品)。
下述实施例中，如无特殊说明，序列表中各核苷酸序列的第1位均为相应DNA的5′末端核苷酸，末位均为相应DNA的3′末端核苷酸。
下述实施例中的pCDNA3.4载体(Invitrogen，Cat:A14697)。
常规器材和试剂如下：
1、96孔酶标板(Nunc公司)；
2、铺板缓冲液：pH为7.0的磷酸盐缓冲液；
3、洗涤液：仅含0.05％体积百分含量Tween 20的pH为7.0的磷酸盐缓冲液；
4、封闭液：仅含10g/L BSA的洗涤液。
5、辣根过氧化物酶标记亲和素；
6、显色底物：四甲基联苯胺；
7、终止液：1M硫酸。
其中，所述pH为7.0的磷酸盐缓冲液的溶剂为水，溶质为氯化钠、氯化钾、磷酸二氢钾、磷酸氢二钠；所述氯化钠在所述pH为7.0的磷酸盐缓冲液中的浓度为135mM，所述氯化钾在所述pH为7.0的磷酸盐缓冲液中的浓度为2.7mM，所述磷酸二氢钾在所述pH为7.0的磷酸盐缓冲液中的浓度1.5mM，所述磷酸氢二钠在所述pH为7.0的磷酸盐缓冲液中的浓度为8mM。
实施例1、杂交瘤抗体的制备
1.1小鼠免疫
选用4～6周龄的Balb/c小鼠3只和C57b1/6小鼠3只，以人4-1BB胞外段(SEQ ID No.5的第24-186位)为抗原免疫小鼠，每2周免疫1次，共免疫3次。第3次免疫后尾静脉采血，用间接ELISA检测抗体产生情况，选择合适的免疫的小鼠，摘眼球后脱颈处死，无菌摘取小鼠脾细胞，，按常规方法进行细胞融合制备抗人4-1BB单克隆抗体杂交瘤细胞株。
1.2杂交瘤筛选
用ELISA筛选抗人4-1BB单克隆抗体杂交瘤细胞。用10μg/ml人4-1BB(SEQ ID No.5)包被ELISA板，于4℃过夜并封闭。依次加入待测细胞培养上清液，于37℃孵育1小时，再用PBST洗板3次，加入4000倍稀释的辣根过氧化物酶标记的羊抗鼠(ThermoFisher公司)于37℃孵育45min，PBST洗板3次后，用四甲基联苯胺(tetramethylbenzidine，TMB)(ThermoFisher公司)底物显色后，于450nm波长测定OD值。
用ELISA法筛选阳性的细胞克隆再反复亚克隆，直到所有杂交瘤细胞培养上清检测为100％阳性。杂交瘤细胞的克隆化用有限稀释法。将培养板置于37℃、5％的CO 2孵箱中培养，约5天后在显微镜下可观察到细胞克隆。适时换液，检测，取阳性并且长势好的单克隆细胞株进行扩大培养，即得到抗人4-1BB单克隆抗体杂交瘤细胞株，及时冻存细胞株。
1.3杂交瘤抗体鉴定
将筛选所得阳性并且长势好的抗人4-1BB抗体杂交瘤细胞株逐级扩大培养，离心(10000rpm离心10分钟)，取上清，将所得上清用Protein G亲和层析柱纯化。具体操作是：先用PBS平衡Protein G柱(GE公司)，然后培养上清过柱，先采用A液(配方：溶剂为水，溶质及浓度为：20mM磷酸钠、500mM NaCl，pH5.0)预洗脱5个柱体积，再采用B液(配方：溶剂为水，溶质及浓度为：20mM醋酸钠、150mM NaCl，pH3.5)洗脱5个柱体积，收集洗脱峰，然后30KDa浓缩离心管浓缩获得抗体。
免疫得到15个杂交瘤抗体细胞株，通过筛选，最终得到6个候选杂交瘤母细胞株，通过亚型鉴定及初步筛选后，细胞株详情及分别对应抗体如表1所示。
表1、抗人4-1BB杂交瘤细胞株
序号 编号 抗体亚型 融合细胞
1 37G10F4 IgG1,κ Sp2/0
2 23C2E9 IgG1,κ Sp2/0
3 2C2F2 IgG1,κ Sp2/0
4 32C2G6 IgG2b,κ Sp2/0
5 13F8H8 IgM,κ Sp2/0
6 37G11E2B3 IgM,κ Sp2/0
淘汰13F8H8和37G11E2B3两个候选，收集细胞上清，分别纯化、浓缩，对样品进行SDS-PAGE电泳，电泳检测结果如图1所示。
对得到的抗人4-1BB抗体进行测序，结果显示，所得抗人4-1BB抗体为分子量大小为150Kb，包含重链和轻链，为典型的完整抗体。抗体结构如图2所示。
1.4杂交瘤分泌抗体识别人4-1BB
通过ELISA评估4种杂交瘤分泌抗体识别人4-1BB的亲和力。96孔板(96孔酶标板，Nunc公司)用人4-1BB(SEQ ID No.5)包被，浓度为1μg/ml，100μl/孔，4℃过夜。洗板三次，每孔加300μl封闭液(配方见上文)，37℃封闭1小时。用洗涤液(配方见上文)洗板三次，将抗体用样品稀释液(PBS-T加入1％牛血清白蛋白)稀释到4000ng/ml，再用7个Ep管4倍稀释成梯度溶液，每个浓度设置两个复孔，100μl/孔，孵育1小时。用洗涤液洗板三次，每孔加稀释8000倍的辣根过氧化物酶标记羊抗鼠(ThermoFisher公司)100μl，振荡0.5小时。洗板三次，每孔加四甲基联苯胺(tetramethylbenzidine，TMB)(ThermoFisher公司)100μl。避光显色，加1M的硫酸终止反应。用BIO-TEK ELX-800酶标仪在450nm下测OD值。
通过上述步骤，杂交瘤分泌抗体均能识别并结合人4-1BB，ELISA结果OD450值如表2所示，分析图如图3所示。
表2、杂交瘤分泌抗体识别人4-1BB
Figure PCTCN2020127993-appb-000001
Figure PCTCN2020127993-appb-000002
1.5杂交瘤分泌抗体识别猴4-1BB
采用ELISA方法评估杂交瘤分泌抗体识别猴4-1BB的亲和力。具体将猴4-1BB(Cy4-1BB)(SEQ ID No.6)抗原用铺板缓冲液(配方见上文)稀释到1μg/ml，每孔加100μl铺板，4℃过夜。将杂交瘤分泌抗体与阳性对照抗体Utomilumab用样品稀释液稀释到1000ng/ml，再用7个Ep管10倍稀释成梯度溶液，每个浓度设置两个复孔，每孔100μl加入板内，振荡1小时。再次洗板后，每孔加稀释8000倍的辣根过氧化物酶标记羊抗鼠(ThermoFisher公司)100μl，振荡0.5小时。清洗之后，加四甲基联苯胺(tetramethylbenzidine，TMB)(ThermoFisher公司)100μl显色。
通过上述步骤，实验证明杂交瘤分泌抗体均能识别Cy4-1BB分子，ELISA结果OD450值如表3所示，分析图如图4所示。
表3、杂交瘤分泌抗体识别猴4-1BB
Figure PCTCN2020127993-appb-000003
实施例2、抗体人源化及scFv-Fc抗原结合片段性质鉴定
选择得自如本发明所述淘选的分泌型抗体37G10F4。分析抗体基因序列，然后通过抗体Fab的同源建模与优化，表面扫描确定人源化突变位点，虚拟突变 与分子动力学模拟，确定关键氨基酸等一系列分析设计合理的人源化抗体。通过人源化改造，得到12个候选scFv-Fc(人IgG4)形式的抗原结合片段(表4)，用于如本实施例进一步鉴定。
表4中的12个候选scFv-Fc(人IgG4)形式的抗原结合片段，其重链可变区(VH)和轻链可变区(VL)中的互补决定区(CDR)均相同，不同之处仅在于VH和VL中的框架区(FR)有氨基酸突变；Fc区也相同。重链可变区中HCDR1、HCDR2和HCDR3的氨基酸序列依次如SEQ ID No.16自N端起第31-35位、第50-64位、第98-106位所示；轻链可变区中LCDR1、LCDR2和LHCDR3的氨基酸序列依次如SEQ ID No.16自N端起第156-166位、第182-188位、第221-229位所示。
其中，scFvB60103的氨基酸序列如SEQ ID No.16所示，第1-117位为重链可变区，第133-239位为轻链可变区，第240-472位为Fc。
对应于基因水平，scFvB60103的编码基因序列如SEQ ID No.17所示，第1-351位为重链可变区的编码基因(第91-105、148-192、292-318位分别编码三个HCDR)，第397-717位为轻链可变区的编码基因(第466-498、544-564、661-687位分别编码三个LCDR)，第718-1416位为Fc的编码基因。
表达和纯化表4中的12个候选scFv-Fc分子。以scFvB60103为例说明。人工合成scFvB60103抗体的编码基因(SEQ ID No.17)，然后将其插入pCDNA3.4载体，通过瞬转表达。瞬转操作具体使用Expi293表达系统(Thermo Fisher)产品，根据产品说明书操作流程。在OptiMEM中分别加入Expi Fectamine转染试剂、DNA质粒得到溶液A和B，然后将溶液A和溶液B混匀得到溶液C。将溶液C加入Expi293细胞(Thermo Fisher)中，培养Expi293细胞5天后。离心(10000rpm离心10分钟)，取上清，将所得上清用Protein G亲和层析柱纯化。具体操作是：先用PBS平衡Protein G柱(GE公司)，然后培养上清过柱，先采用A液(配方：溶剂为水，溶质及浓度为：20mM磷酸钠、500mM NaCl，pH5.0)预洗脱5个柱体积，再采用B液(配方：溶剂为水，溶质及浓度为：20mM醋酸钠、150mM NaCl，pH3.5)洗脱5个柱体积，收集洗脱峰，然后30KDa浓缩离心管浓缩获得scFvB60103目的分子。
(1)ELISA鉴定
ELISA鉴定scFv分子与人和猴4-1BB分子的亲和力(具体方法参见实施例1相关步骤)。具体如表4所示，结果显示，12种scFv-Fc分子均能识别人和猴4-1BB，其中scFvB60103识别能力最优。ELISA结果如图5和图6所示。
表4、scFv识别人和猴4-1BB
Figure PCTCN2020127993-appb-000004
Figure PCTCN2020127993-appb-000005
(2)FACS检测
所用试剂包括：
FACS缓冲液，该缓冲液为向PBS中添加BSA和叠氮化钠得到的溶液，该缓冲液中BSA的质量百分比浓度为2％，叠氮化钠的质量百分比浓度为0.02％；羊抗人FITC二抗(Sigma公司，避光保存)。
使用CHO-K1-hu4-1BB，用FACS方法检测ELISA检测结果中与人4-1BB结合活性较优的scFv分子与人4-1BB分子亲和力的差异。实验所用细胞为合肥瀚科迈博公司构建的细胞膜表面高表达人4-1BB的CHO-K1细胞(CHO-K1-hu4-1BB)和无4-1BB表达的野生型CHO-K1(中科院上海细胞库)。
人工合成人4-1BB基因序列(SEQ ID No.7)，然后按照本领域通用方法插入pCDNA3.4载体，然后用Lipofectamine 3000转染试剂(Invitrogen公司)导入到野生型CHO-K1细胞。再加入G418(购于生工生物工程(上海)股份有限公司)加压筛选，最终得到细胞膜高表达人4-1BB的CHO-K1细胞(CHO-K1-hu4-1BB，备用。
使用T75瓶培养待测细胞至80％满度，胰酶消化，1000rpm离心5min，收集细胞(每瓶细胞数在10 6个左右)，用约1ml缓冲液重悬清洗、离心并重悬细胞，得到细胞悬液，细胞浓度为1×10 7个细胞/ml。准备各个稀释梯度的各供试抗体(见表5)的稀释液25μl，向加有不同浓度抗体的各离心管中加入25μl的细胞悬液混匀，最终抗体浓度如表5中所示，将加入抗体浓度为0μg/ml的抗体稀释液的离心管作为空白对照。孵育30min后，用1ml FACS缓冲液清洗两次后，加入羊抗人FITC二抗，吹打重悬，避光孵育30min。再次用1ml FACS缓冲液清洗两次，然后每管加入500μl FACS缓冲液重悬，置于冰上避光，上机检测。将抗体Utomilumab(辉瑞制药有限公司)作为阳性对照。
通过上述步骤，比较不同scFv分子与人4-1BB的结合活性高低，如表5和图7所示，结果显示scFvB60103对应的EC50值最低，即其与人4-1BB亲和力最高，可以作为最优分子。
表5、FACS检测scFv与人4-1BB亲和力检测结果
Figure PCTCN2020127993-appb-000006
Figure PCTCN2020127993-appb-000007
实施例3、IgG人源化抗体
本实施例鉴别的实施例性抗体Hanke10F4，为从杂交瘤分泌型抗体37G10F4人源化改造得到，即从表4中的scFvB60103转变而来。本实施例将详述表达和纯化Hanke10F4，然后在ELISA、FACS、荧光素酶报告基因测定、T细胞激活体系中鉴定。
3.1抗体表达与纯化
根据测序得到的基因序列，合成全长基因序列，Hanke10F4抗体的重链编码基因如SEQ ID No.3所示，Hanke10F4抗体的轻链编码基因如SEQ ID No.4所示。
SEQ ID No.3的第1-351位为重链可变区VH的编码基因，其中CDR1、CDR2和CDR3的编码序列分别如SEQ ID No.3的第91-105位、第148-192位、第292-318位所示。
SEQ ID No.4的第1-321位为轻链可变区VL的编码基因，其中CDR1、CDR2和CDR3的编码序列分别如SEQ ID No.4的第70-102位、第148-168位、第265-291位所示。
分别在SEQ ID No.3和SEQ ID No.4的5’端依次加上XbaI的识别序列(5’-TCTAGA-3’)、kozak共识别序列(5’-GCCACC-3’)和信号肽的编码序列(5’-ATGGAGTTTGGGCTGAGTTGGGTCTTTCTGGTCGCAATTCTGCTGAAGGGAGTGCAGTGC-3’)，信号肽的编码序列与SEQ ID No.3和SEQ ID No.4的5’端相连，然后分别在SEQ ID No.3和SEQ ID No.4的3’末端依次加上终止子(TGA)和HindIII的识别序列(5’-AAGCTT-3’)，TGA与SEQ ID No.3和SEQ ID No.4的3’端相连，将得到的新的DNA序列分别记为重链基因甲和轻链基因甲。人工合成重链基因甲，将重链基因甲用XbaI和HindIII酶切后与经XbaI和HindIII酶切pCDNA3.4载体得到的载体骨架连接，得到重组载体，将经测序验证正确的重组载体命名为pCDNA3.4-H；将轻链基因甲用XbaI和HindIII酶切后与经XbaI和HindIII酶切pCDNA3.4载体得到的载体骨架连接，得到重组载体，将经测序验证正确的重组载体命名为pCDNA3.4-L。
将pCDNA3.4-H和pCDNA3.4-L导入到人胚胎肾细胞系HEK293F(ATCC美国细胞库)，得到重组细胞，然后将重组细胞放入37℃、5％CO 2振荡培养箱中培养， 转速120rpm。
利用Protein A亲和层析柱从培养上清中纯化抗体蛋白。具体操作是：先用PBS平衡Protein A柱(GE公司)，然后培养上清过柱，先采用A液(配方：溶剂为水，溶质及浓度为：20mM磷酸钠、500mM NaCl，pH5.0)预洗脱5个柱体积，再采用B液(配方：溶剂为水，溶质及浓度为：20mM醋酸钠、150mM NaCl，pH3.5)洗脱5个柱体积，收集洗脱峰，然后30KDa浓缩离心管浓缩获得所述抗体即获得抗人4-1BB抗体。
对抗人4-1BB抗体进行测序，结果显示，所得抗人4-1BB抗体为完整抗体，即该抗体为Hanke10F4。该抗体由重链和轻链组成，重链的氨基酸序列为序列如SEQ ID No.1所示，轻链的氨基酸序列如SEQ ID No.2所示。Hanke10F4抗体的重链类型为IgG4，轻链类型为κ链。
SEQ ID No.1的第1-117位为重链可变区VH，其中CDR1、CDR2和CDR3的序列分别如SEQ ID No.1的第31-35位、第50-64位、第98-106位所示。
SEQ ID No.2的第1-107位轻链可变区VL中CDR1、CDR2和CDR3的序列分别如SEQ ID No.2的第24-34位、第50-56位、第89-97位所示。
3.2对人或猴4-1BB亲和力鉴定
(1)ELISA检测
方法参见实施例1中步骤1.4，分别以人4-1BB或猴4-1BB直接包被96孔板，抗体样品(Hanke10F4和Utomilumab)为一抗，以辣根过氧化物酶标记羊抗鼠为二抗。四甲基联苯胺(tetramethylbenzidine，TMB)为显色试剂，硫酸终止显色。用BIO-TEK ELX-800酶标仪在450nm下测OD值。
表6显示IgG4形式的抗体Hanke10F4的ELISA结果，与人4-1BB和猴4-1BB均能识别。
表6、ELISA测定中Hanke10F4的结合
Figure PCTCN2020127993-appb-000008
(2)FACS检测
所用试剂包括：
FACS缓冲液，该缓冲液为向PBS中添加BSA和叠氮化钠得到的溶液，该缓冲液中BSA的质量百分比浓度为2％，叠氮化钠的质量百分比浓度为0.02％；羊抗人FITC二抗(Sigma公司，避光保存)。
实验所用细胞为合肥瀚科迈博公司构建的细胞膜表面高表达人4-1BB的CHO-K1细胞(CHO-K1-hu4-1BB)和细胞膜表面高表达猴4-1BB的CHO-K1细胞(CHO-K1-cy4-1BB)以及无4-1BB表达的野生型CHO-K1(来自于中科院上海细胞库)。
人工合成人4-1BB基因序列(SEQ ID No.7)和猴4-1BB基因序列(SEQ ID No.8)，然后按照本领域通用方法分别插入pCDNA3.4载体，然后分别用Lipofectamine 3000转染试剂(Invitrogen公司)导入到野生型CHO-K1细胞。再加入G418(购于生工生物工程(上海)股份有限公司)加压筛选，最终得到细胞膜高表达人4-1BB的CHO-K1细胞(CHO-K1-hu4-1BB)和细胞膜表面高表达猴4-1BB的CHO-K1细胞(CHO-K1-cy4-1BB)。
使用CHO-K1-hu4-1BB检测Hanke10F4单克隆抗体与人4-1BB结合活性。使用T75瓶培养待测细胞至80％满度，胰酶消化，1000rpm离心5min，收集细胞(每瓶细胞数在10 6个左右)，用约1ml缓冲液重悬清洗、离心并重悬细胞，得到细胞悬液，细胞浓度为1×10 7个细胞/ml。准备各个稀释梯度的Hanke10F4抗体稀释液25μl，向加有不同浓度抗体的各离心管中加入25μl的细胞悬液混匀，最终抗体浓度如表7中所示，将加入抗体浓度为0μg/ml的抗体稀释液的离心管作为空白对照。孵育30min后，用1ml FACS缓冲液清洗两次后，加入羊抗人FITC二抗，吹打重悬，避光孵育30min。再次用1ml FACS缓冲液清洗两次，然后每管加入500μl FACS缓冲液重悬，置于冰上避光，上机检测。将抗体Utomilumab(辉瑞制药有限公司)作为阳性对照。
按照上述步骤，使用CHO-K1-cy4-1BB检测Hanke10F4单克隆抗体与猴4-1BB结合活性。
通过上述步骤，实验证明Hanke10F4单克隆抗体与人4-1BB和猴4-1BB分子均具有很高的结合活性，如表7所示。
表7、FACS检测4-1BB抗体亲和力检测结果
Figure PCTCN2020127993-appb-000009
Figure PCTCN2020127993-appb-000010
(3)FACS比较抗体亲和力
使用CHO-K1-hu4-1BB检测比较Hanke10F4、HKB6单克隆抗体与人4-1BB结合活性。使用T75瓶培养待测细胞至80％满度，胰酶消化，1000rpm离心5min，收集细胞(每瓶细胞数在10 6个左右)，用约1ml缓冲液重悬清洗、离心并重悬细胞，得到细胞悬液，细胞浓度为1×10 7个细胞/ml。准备各个稀释梯度的Hanke10F4抗体稀释液25μl或者HKB6抗体稀释液25μl，向加有不同浓度抗体的各离心管中加入25μl的细胞悬液混匀，最终抗体浓度如表7中所示，将加入抗体浓度为0μg/ml的抗体稀释液的离心管作为空白对照。孵育30min后，用1ml FACS缓冲液清洗两次后，加入羊抗人FITC二抗，吹打重悬，避光孵育30min。再次用1ml FACS缓冲液清洗两次，然后每管加入500μl FACS缓冲液重悬，置于冰上避光，上机检测。
其中HKB6为专利申请号为201811541545.8中所述HKB6抗体。
通过上述步骤，如表8和图8所示，Hanke10F4结合人4-1BB的亲和力明显高于HKB6。
表8、Hankering10F4和HKB6亲和力比较
Figure PCTCN2020127993-appb-000011
(4)抗体亲和力测定
利用h4-1BB-His(Acro公司，货号：Q07011-1)抗原，通过Fortebio octec仪器采用生物膜层干涉(BLI)技术，检测Utomilumab、Hanke10F4或Urelumab的亲和力。
结果如表9所示，Hanke10F4具有较高的抗体亲和力，与Utomilumab亲和不相上下。
表9、抗体亲和力
样品 KD(M) ka(1/Ms) kd(1/s)
Hanke10F4 1.39E-08 1.73E+05 2.41E-03
Utomilumab 2.21E-08 2.43E+05 5.38E-03
Urelumab 5.76E-09 2.83E+05 1.63E-03
注：ka代表单位时间内结合产物的生成速率；kd代表单位时间内结合产物降解为反应物的百分比；KD值代表亲和作用的强弱。
3.3配体竞争
使用CHO-K1-hu4-1BB比较Hanke10F4抗体与人4-1BBL之间竞争结合活性。二抗为SA-FITC(Biolegend公司)，其他操作(除下述外)、稀释液同步骤3.2中FACS检测。人4-1BBL氨基酸序列如SEQ ID No.9所示。
将Hanke10F4用生物素标记为Bio-Hanke10F4，标记方法同本领域常规标记蛋白方法一致。用样品稀释液将Bio-Hanke10F4稀释浓度为1.5μg/ml的溶液A。同时配制人4-1BBL-hIgG(或Hanke10F4)稀释液B，固定溶液A浓度不变，溶液B浓度为A的一定倍数(具体倍数见表9)。将溶液A和溶液B等比例混匀，与CHO-K1-hu4-1BB孵育。
同样操作检测反向竞争，将人4-1BBL用生物素标记为Bio-4-1BBL，并用样品稀释液稀释浓度为2μg/ml的溶液A。同时配制Hanke10F4(或人4-1BBL-hIgG)稀释液B，固定溶液A浓度不变，溶液B浓度为A的一定倍数(具体倍数见表8)。将溶液A和B等比例混匀，与CHO-K1-hu4-1BB孵育。
通过上述步骤，实验证明Hanke10F4单克隆抗体与人4-1BBL的结合位点有竞争关系，具体结果见表10和图9所示，即该单克隆抗体在一定程度上随着药物剂量会阻断人4-1BB/4-1BBL信号通路。
表10、FACS检测配体阻断
Figure PCTCN2020127993-appb-000012
3.4表位竞争
3.4.1 Hanke10F4与阳性对照抗体抗原结合表位竞争
使用CHO-K1-hu4-1BB比较Hanke10F4抗体与对照抗体Utomilumab、Urelumab之间表位竞争关系。二抗为SA-FITC(Biolegend公司)，其他操作(除 下述外)、稀释液同步骤3.2中FACS检测。
将Hanke10F4用生物素标记为Bio-Hanke10F4，并用样品稀释液稀释浓度为1.5μg/ml的溶液A。同时配制Utomilumab、Urelumab、Hanke10F4抗体稀释液B，固定溶液A浓度不变，溶液B浓度为A的一定倍数(具体倍数见表10)。将溶液A和B等比例混匀，与CHO-K1-hu4-1BB孵育。
通过上述步骤，重复实验证明Hanke10F4单克隆抗体与Utomilumab和Urelumab的抗原结合位点有一定程度竞争关系，与Utomilumab的竞争强于与Urelumab的竞争。具体结果见表11和图10所示。
表11、FACS检测表位竞争
Figure PCTCN2020127993-appb-000013
3.4.2 Hanke10F4与对照抗体抗原结合表位分析
(1)方法一
将人4-1BB抗原胞外区分段用鼠4-1BB相应胞外区替代，合成不同的目的基因，分别插入pCDNA3.4载体，得到不同4-1BB抗原的DNA质粒，通过瞬转得到表达细胞表面不同4-1BB抗原的细胞株，具体见表12。用于FACS分析抗体与不同4-1BB抗原的结合活性，进而分析抗体结合人4-1BB胞外区的具体表位。
瞬转操作具体使用Expi293表达系统(Thermo Fisher)产品，根据产品说明书操作流程。在OptiMEM中分别加入Expi Fectamine转染试剂、DNA质粒得到溶液A和B，然后将溶液A和溶液B混匀得到溶液C。将溶液C加入Expi293细胞(Thermo Fisher)中，过夜孵育得到细胞表面高表达4-1BB抗原的细胞株。
其中，人4-1BB抗原全长氨基酸序列如SEQ ID No.5所示(对应编码基因如SEQ ID No.7所示)，胞外区为第24-186位(对应编码基因如SEQ ID No.7的第70-558位所示)。鼠4-1BB抗原全长氨基酸序列如SEQ ID No.10所示(对应编码基因如SEQ ID No.11所示)，胞外区为第24-187位(对应编码基因如SEQ ID No.11的第70-561位所示)。
利用瞬转得到的细胞株，按照步骤3.2中FACS检测步骤，依次加入抗体样品(Utomilumab、Hanke10F4或Urelumab)作为一抗与细胞孵育，然后加入羊抗人FITC作为二抗孵育，再次用FACS缓冲液清洗两次，然后每管加入500μl FACS缓冲液重悬，避光，上机检测。
根据专利及文献报道分析，Utomilumab主要结合人4-1BB胞外区的Domain 3和Domain 4，Urelumab主要结合人4-1BB胞外区的Domain1。通过上述操作，可以得知Urelumab的荧光强度趋势在细胞A和F两组降低，Utomilumab在细胞B、C和D三组降低，符合研究报道。而Hanke10F4荧光强度趋势在细胞A和F两组降低，且在细胞A组大幅度降低，降低程度远远低于Urelumab的降低程度，又因为Hanke10F4的荧光强度在细胞B组不降反增，因此说明Domain1和2A对于Hanke10F4与人4-1BB的亲和很关键，而Domain2B、3A、3B影响不大，Hanke10F4主要结合人4-1BB胞外区的Domain1和Domain 2A区域，与对照抗体Utomilumab和Urelumab的抗原结合表位均不相同(表12)。Hanke10F4与阳性对照抗体又具有一定程度的竞争关系，这种竞争关系可能由于抗原抗体的空间表位导致。
表12、抗体结合人4-1BB表位平均荧光强度分析
Figure PCTCN2020127993-appb-000014
注：细胞A中抗原的具体结构“人4-1BB+鼠4-1BB domain1,2A,2B(aa24-86)”指的是将SEQ ID No.5所示的人4-1BB抗原全长中的第24-86位的氨基酸替换为SEQ ID No.10所示的鼠4-1BB抗原全长中第24-86位的氨基酸。细胞B中抗原的具体结构“人4-1BB+鼠4-1BB domain2B,3A,3B(aa64-118)”指的是将SEQ ID No.5所示的人4-1BB抗原全长中的第64-118位的氨基酸替换为SEQ ID No.10的第64-118位氨基酸。细胞C中抗原的具体结构“人4-1BB+鼠4-1BB domain3A,3B,4A(aa87-133)”指的是将SEQ ID No.5所示的人4-1BB抗原全长中的第87-133位的氨基酸替换为SEQ ID No.10的第87-133位氨基酸。细胞D中抗原的具体结构“人4-1BB+鼠4-1BB domain3B,4A,4B(aa97-159)”指的是将SEQ ID No.5所示的人4-1BB抗原全长中的第97-159位的氨基酸替换为SEQ ID No.10的第97-159位氨基酸。细胞E中抗原的具体结构“人4-1BB+鼠4-1BB  domain4A,4B,NF(aa119-186)”指的是将SEQ ID No.5所示的人4-1BB抗原全长中的第119-186位的氨基酸替换为SEQ ID No.10的第119-186位氨基酸。细胞F中抗原的具体结构“人4-1BB+鼠4-1BB domain1,4B,NF(aa24-46,aa139-186)”指的是将SEQ ID No.5所示的人4-1BB抗原全长中的第24-46以及139-186位的氨基酸替换为SEQ ID No.10的第24-46以及139-186位氨基酸。
(2)方法二
根据文献分析，人4-1BB抗原氨基酸101、132位为Utomilulab关键结合位点，42位为Urelumab关键结合位点。将上述位点氨基酸逐个突变，合成不同的目的基因，得到不同4-1BB抗原的DNA质粒，瞬转得到表达细胞表面不同4-1BB抗原的细胞株(参见步骤(1)相关步骤进行)，具体如表13所示。
表13、人4-1BB点突变瞬转细胞
细胞序号 抗原信息
WT 人4-1BB全长
M101R 人4-1BB 101位氨基酸M突变为R
I132R 人4-1BB 132位氨基酸I突变为R
N42R 人4-1BB 42位氨基酸N突变为R
利用瞬转得到的细胞株，按照步骤3.2中FACS检测步骤，加入抗体Hanke10F4与细胞孵育，然后加入羊抗人FITC作为二抗孵育，再次用FACS缓冲液清洗两次，然后每管加入500μl FACS缓冲液重悬，避光，上机检测，分析抗体与不同4-1BB抗原的结合活性。结果如图11所示。Utomilumab与Urelumab均与关键氨基酸突变后的抗原不结合，而Hanke10F4与上述所有抗原结合能力无明显区别，也没有显著下降现象，即说明Hanke10F 4与抗原结合的关键氨基酸与Utomilumab、Urelumab抗体不一致，Hanke10F4具体独特的抗原结合位点。
3.4.3 Hanke10F4与HKB6抗原结合表位竞争
使用CHO-K1-hu4-1BB比较Hanke10F4抗体与HKB6之间抗原结合表位竞争关系。二抗为SA-FITC(Biolegend公司)，其他操作(除下述外)、稀释液同步骤3.2中FACS检测。
将Hanke10F4用生物素标记为Bio-Hanke10F4，并用样品稀释液稀释浓度为1.5μg/ml的溶液A。同时配制HKB6、Hanke10F4抗体稀释液B，固定溶液A浓度不变，溶液B浓度为A的一定倍数(具体倍数见表14)。将溶液A和B等比例混匀，与CHO-K1-hu4-1BB孵育。
通过上述步骤，实验证明Hanke10F4单克隆抗体与HKB6没有竞争关系，即两个抗体的抗原结合表位不同，具体结果见表14和图12所示。
表14、Hanke10F4和HKB6表位竞争
Figure PCTCN2020127993-appb-000015
Figure PCTCN2020127993-appb-000016
3.5荧光素酶报告基因活性检测
HEK-293/NFκB-Luci/41-BB细胞为合肥瀚科迈博生物技术公司构建的稳定表达人4-1BB膜蛋白以及NFκB-Luciferase报告基因的HEK293细胞株。具体操作如下：将人4-1BB序列(SEQ ID No.7)作为目的基因插入pCDNA3.4载体中，得到质粒A，同时将NFκB元件序列(SEQ ID No.12)以及荧光素酶基因(SEQ ID No.13)作为目的基因，按照本领域通用方法分别插入pGL4.10载体(优宝生物)中不同位点，得到质粒B。然后用Lipofectamine 3000转染试剂(Invitrogen公司)将A、B两种质粒一起导入到HEK293细胞(中科院上海细胞库)。再加入Puromycin(Gibco)和Hygromycin B(生工生物工程(上海)股份有限公司)加压筛选，最终得到HEK-293/NFκB-Luci/41-BB。
配制加压培养基(DMEM+10％FBS+1％P/S+1ug/ml Puromycin+250ug/ml Hygromycin B),于37℃、5％CO2静态培养箱内维持培养HEK-293/NFκB-Luci/41-BB细胞。取对数生长期的细胞，胰酶消化后1000RPM离心5min，重悬于完全培养基(DMEM+10％FBS+1％P/S)，将50μl细胞铺板于96孔板(Corning，3917)中，为3×10 4个/孔。将抗体与交联抗体Fab’羊抗人IgG Fc(Jackson，109-006-008)按1：2浓度比混匀后，加入96孔板，50μl/孔。在培养箱内静置18-24h后，加入100μl的ONE-Glo Luciferase assay sysytem试剂(Promega)室温孵育10min，检测化学发光值。
通过上述步骤，实验证明Hanke10F4单克隆抗体与Utomilumab均能激活报告基因表达信号，具体结果见图13中A所示。这说明Hanke10F4与Utomilumab均能激活T细胞活化信号通路，启动T细胞免疫功能，Hanke10F4的活化效应明显高于Utomilumab。
3.6体外药效
3.6.1 CD4 +T细胞激活效应检测
将anti-CD3抗体(Biolegend,货号317325)利用PBS稀释到1μg/ml，得到anti-CD3抗体溶液；将Hanke10F4抗体取高、中、低三个浓度，利用PBS进行稀释，分别得到Hanke10F4抗体浓度为4μg/ml、0.4μg/ml、0.04μg/ml的溶液。将anti-CD3抗体溶液分别与三种浓度的Hanke10F4抗体溶液按照1:1的体积比混合均匀后，加入96孔板包板，50μl/孔，设3个复孔，将96孔板静置于37℃孵育1小时。第二天使用前，用PBS清洗三次。设置阳性对照抗体为Utomilumab，阴性对照抗体为IgG(Biolegend,货号403601)。
人CD4 +T细胞分离：从健康人中抽取全血，使用淋巴细胞分离液(Sigma公司)，按照其说明书操作分离PBMC细胞，并使用人CD4 +T细胞磁珠(BD公司，货号557767)，根据说明书分离纯化得到人CD4 +T细胞，备用。
将CD4 +T细胞加入37℃孵育好包板后的96细胞培养板中，密度为1×10 5个/孔，再将96孔板放于37℃，5％CO 2培养箱中培养3天，收集细胞上清，ELISA检测细胞因子INF-γ的分泌量。
通过上述步骤，实验证明Hanke10F4抗体与Utomilumab类似，均能刺激CD4+T细胞活化并分泌细胞因子INF-γ。具体结果见图14所示。
3.6.2 CD8 +T细胞激活效应检测
将anti-CD3抗体利用PBS稀释到0.5μg/ml，加入96孔板(Corning)，60μl/孔，将96孔板静置于37℃孵育1小时。用PBS清洗，然后将CHO-K1稳转细胞株(CHO-K1-CD32A或CHO-K1-CD32B或CHO-K1)加入96孔板，100μl/孔，1×10 4个/孔。再将96孔板置于细胞培养箱过夜。
实验所用细胞为合肥瀚科迈博公司构建的细胞膜表面高表达人FcγR‖A(CD32A)的CHO-K1细胞(CHO-K1-CD32A)和细胞膜表面高表达人FcγR‖B(CD32B)的CHO-K1细胞(CHO-K1-CD32B)和CHO-K1细胞。
人工合成人FcγR‖A的编码基因序列(SEQ ID No.14)以及人FcγR‖B的编码基因序列(SEQ ID No.15)，然后分别按照本领域通用方法插入pCDNA3.4载体，然后分别用Lipofectamine 3000转染试剂(Invitrogen公司)导入到野生型CHO-K1细胞。再加入G418(购于生工生物工程(上海)股份有限公司)加压筛选，最终得到细胞膜高表达人FcγR‖A的CHO-K1细胞(CHO-K1-CD32A)和细胞膜表面高表达人FcγR‖B的CHO-K1细胞(CHO-K1-CD32B)。
人CD8 +T细胞分离：从健康人中抽取全血，使用淋巴细胞分离液(Sigma公司)，按照其说明书操作分离PBMC细胞，并使用人CD8 +T细胞磁珠(BD公司，货号557766)，根据说明书分离纯化得到人CD8 +T细胞，备用。
96孔板过夜孵育后，将细胞上清吸除，再每孔加入100μl人CD8 +T细胞，1×10 5个/孔。再将抗体用培养基稀释到目标浓度加入96孔板，100μl/孔，最终浓度分别为：3μg/ml、0.5μg/ml、0.08μg/ml，每个浓度设置3个复孔。将96孔板放入细胞培养箱孵育3天，用ELISA法检测细胞培养上清中细胞因子INF-γ的分泌量。
通过上述步骤，实验证明Hanke10F4抗体与Utomilumab、Urelumab类似，均能刺激T细胞活化，并且对于T细胞的活化依赖于FcγR‖A、FcγR‖B。具体结果见图15所示。
3.7体内药效
实验选择B-hCD137(4-1BB)小鼠检测Hanke10F4体内抗肿瘤药效。B-hCD137(4-1BB)小鼠模型是基因工程鼠，是在遗传背景C57BL/6小鼠的基因组嵌合有人源的h4-1BB基因，来自百奥赛图。
在受试小鼠后背(剃毛)一侧皮下接种MC38细胞系(每只小鼠接种2×10 6细胞，细胞与Matrigel以1:1体积比混匀后接种，200μl)。当荷瘤鼠平均瘤体积达到100mm 3时，将小鼠按实验设计随机分入5组，每组8只。分组给药当天定义为第0天。分组情况和给药方案如表15所示：
表15、分组和给药方案
Figure PCTCN2020127993-appb-000017
注：N为每组动物只数
在肿瘤接种后，每周两次检查动物生存和活动情况。包括：肿瘤生长情况，活动能力，饮食，体重和其他异常行为。
通过上述步骤，实验证明Hanke10F4抗体能够抑制MC38肿瘤生长，具有明显的剂量依赖性，且同等剂量条件下肿瘤抑制能力明显强于Utomilumab抗体的抗肿瘤药效几乎一致。具体结果见图16所示。
Hanke10F4给药组，肿瘤消退后，继续观察1个月，肿瘤没有复发。将肿瘤消退的小鼠再次接种4×10 6个MC38细胞，15天后，肿瘤并没有形成，如图17所示，说明Hanke10F4不仅能够抑制肿瘤生长，并且能够调动小鼠体内记忆T细胞功能，使得肿瘤不会复发，且对同型肿瘤具有免疫功能。
3.8安全性评估
3.8.1小鼠毒理实验
实验选择B-hCD137(4-1BB)小鼠检测Hanke10F4体内抗肿瘤药效。B-hCD137(4-1BB)小鼠模型是基因工程鼠，是在遗传背景C57BL/6小鼠的基因组嵌合有人源的h4-1BB基因，来自百奥赛图。
将受试小鼠随机分为4组，分别给生理盐水、Hanke10F4、Utomilumab及Urelumab，每周给药2次，给药3周，每次给药浓度为30mg/kg。给药结束后，断尾取血，检测小鼠外周血中AST(谷草转氨酶)与ALT(谷丙转氨酶)的含量。如图18，Urelumab相比于生理盐水组，AST含量显著升高，表示该抗体具有肝毒性风险，而Hanke10F4与Utomilumab的AST和ALT含量均与生理盐水组无明显区别，表明Hanke10F4与Utomilumab同样具有低毒性。
3.8.2 SD大鼠静脉注射给予Hanke10F4功能观察组合(FOB)试验
将40只SD大鼠(雌、雄各半)单次静脉注射给予Hanke10F4，设置高(30mg/kg)、中(10mg/kg)、低(3mg/kg)三个给药剂量，根据不同时间点的功能观察组合(FOB)试验，评估Hanke10F 4对SD大鼠中枢神经系统的影响。
实验期间，安慰剂对照组、Hanke10F4低剂量组、Hanke10F4中剂量组、Hanke10F4高剂量组，大鼠一般状态观察均未见明显异常。给药前和给药后0.5h、6h、24h、48h、168h，各组动物身体姿势、立毛等指标未见药物相关的异常变化。未见动物自残或攻击笼内其他动物，也未见动物死亡；各组动物出笼难易、徒手保定观察、流泪、流涎、分泌物、眼球突出、瞳孔、眼睑闭合、皮肤颜色、立毛、呼吸等指标未见异常变化；各组动物靠近反应、接触反应、惊恐反应、腹部紧张度、身体张力、耳廓反射、掐尾反射观察、平面翻正反射观察、空中翻正反射、瞳孔反射观察、眼睑反射、屈肌反应、伸肌反应等指标未见异常变化。综合结果，本实验条件下，SD大鼠单次静脉注射给予3、10和30mg/kg的Hanke10F4，对动物中枢神经系统功能无明显影响。
3.8.3食蟹猴毒理
将4只食蟹猴分性别按体重随机分为剂量爬坡组、固定剂量组共2个组，每组2只，雌雄各半,采用静脉注射途径给药，具体见表16。
表16、食蟹猴毒理试验分组以及各组给药方案
Figure PCTCN2020127993-appb-000018
注：动物编号的首位数字代表组别(1、2分别代表Hanke10F4剂量爬坡组、Hanke10F4高剂量组)；第二位字母代表性别(F为雌性，M为雄性)。
检测时间点为适应期1次，D8、D15、D22、D29给药后24h各测定1次；给药期结束(D35)测定1次。实验结果显示，给药期间，各组猴子一般观察、体重和摄食量/耗食量、体温、呼吸、心电图、血压、凝血指标等均未见异常。与给药前相比，Hanke10F4各剂量组所有动物血液生化指标各指标(谷丙转氨酶ALT、谷草转氨酶AST)均在正常范围内波动，未见明显变化趋势，具体见表17。剂量爬坡组，≥100mg/kg剂量时，雄性动物白细胞及中性粒细胞、单核细胞升高，细胞因子IL-6略有升高，再次给药又降回原水平，具体见表18。因此Hanke10F4在食蟹猴体内最大耐受剂量(MTD)≥200mg/kg。
表17、食蟹猴血液学个体数据
Figure PCTCN2020127993-appb-000019
表18、食蟹猴细胞因子个体数据
Figure PCTCN2020127993-appb-000020
Figure PCTCN2020127993-appb-000021
3.9稳定性
(1)加速稳定性
配制F01(pH5.2)和F02(pH5.5)两种缓冲液，将Hanke10F4设为20mg/ml，4℃放置3个月(3M)，检测抗体浓度、聚集降解等各项理化特性,具体包含：通过可变光程分光光度计测定Hanke10F4蛋白浓度，通过高效液相色谱仪检测Hanke10F4分子大小异质体纯度(SEC-HPLC)和电荷异质体纯度(CEX-HPLC)，通过毛细管电泳分析系统检测Hanke10F4非还原毛细管凝胶电泳蛋白纯度(NR-CE)。结果如表19所示，Hanke10F4在不同pH缓冲液中，4℃放置3个月后与最初时间的样本(Hanke10F4/T0)相比，未出现明显变化，Hanke10F4稳定性良好。
同时，取4℃放置1个月(1M)、2个月(2M)、3个月(3M)的Hanke10F4样本与实验开始时间点的样本(Hanke10F4/T0)，采用前文所述的报告基因法检测抗体生物学活性，最高浓度设为4ug/ml，并以2倍梯度稀释9个浓度点。实验结果如图13中B所示，Hanke10F4在4℃放置3个月后，抗体生物学活性良好。
表19、Hanke10F4加速稳定性检测
Figure PCTCN2020127993-appb-000022
Figure PCTCN2020127993-appb-000023
注：T0为初始时；3M为3个月末。
(2)Tm值检测
通过差示扫描荧光(differential scanning fluorimetry,DSF)检测抗体Hanke10F4在两种波长(350nm和330nm)下的荧光强度，计算荧光强度比值，检测蛋白去折叠过程中荧光强度和荧光峰值的变化，从而得出Hanke10F4一半蛋白去折叠时的温度，即Tm值，结果测得Hanke10F4的Tm1值为64.4℃，Tm2值为71.5℃(Tm1和Tm2为蛋白去折叠过程中荧光峰值发生变化的两个温度)，即Hanke10F4抗体的稳定性良好(图19)。
工业应用
本发明所提供的抗体Hanke10F4能结合人4-1BB，并对人4-1BB表现高亲和力以及有效增强T细胞反应。一方面所述抗体在一定程度上与人4-1BBL竞争性结合人4-1BB，另一方面，所述抗体在T细胞共刺激测定法诱导中INF-γ的生成，在小鼠模型中能明显抑制肿瘤生长，且肿瘤不会复发。本发明所述抗体，可用于调节T细胞和抗体介导的免疫反应，可用于抗肿瘤免疫治疗。本发明所提供的单链抗体的融合抗体能结合人和猴4-1BB。
本发明抗体与人4-1BB的结合导致免疫反应增强。本发明提供的抗体及其抗原结合片段可用作免疫增强物，或T细胞介导的自身免疫疾病的免疫调节物。所述抗体及其抗原结合片段也可用作诊断工具检测癌症、自身免疫或其他疾病的患者的血液或组织中的4-1BB。
本发明所提供的抗体抗肿瘤药效好，安全性强，稳定性好。
本发明提供的抗体及其抗原结合片段作为免疫调节物具有广泛的治疗用途，所述疾病诸如癌症、自身免疫疾病、炎症疾病、和感染疾病等。

Claims (44)

  1. 抗体，其特征在于：所述抗体的重链可变区中HCDR1、HCDR2和HCDR3的氨基酸序列依次如SEQ ID No.1自N端起第31-35位、第50-64位、第98-106位所示。
  2. 根据权利要求1所述的抗体，其特征在于：所述抗体的轻链可变区中LCDR1、LCDR2和LHCDR3的氨基酸序列依次如SEQ ID No.2自N端起第24-34位、第50-56位、第89-97位所示。
  3. 根据权利要求1所述的抗体，其特征在于：所述重链可变区的氨基酸序列为如下任一：
    (a1)SEQ ID No.1自N端起第1-117位，或者与SEQ ID No.1自N端起第1-117位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性；
    (a2)SEQ ID No.18自N端起第1-117位，或者与SEQ ID No.18自N端起第1-117位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性；
    (a3)SEQ ID No.19自N端起第1-117位，或者与SEQ ID No.19自N端起第1-117位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性；
    (a4)SEQ ID No.20自N端起第1-117位，或者与SEQ ID No.20自N端起第1-117位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性；
    (a5)SEQ ID No.21自N端起第1-117位，或者与SEQ ID No.21自N端起第1-117位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性；
    (a6)SEQ ID No.22自N端起第1-117位，或者与SEQ ID No.22自N端起第1-117位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性；
    (a7)SEQ ID No.23自N端起第1-117位，或者与SEQ ID No.23自N端起第1-117位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性；
    (a8)SEQ ID No.24自N端起第1-117位，或者与SEQ ID No.24自N端起第1-117位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性；
    (a9)SEQ ID No.25自N端起第1-117位，或者与SEQ ID No.25自N端起第1-117位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性；
    (a10)SEQ ID No.26自N端起第1-117位，或者与SEQ ID No.26自N端起第1-117位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性；
    (a11)SEQ ID No.27自N端起第1-117位，或者与SEQ ID No.27自N端起第1-117位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性；
    (a12)SEQ ID No.28自N端起第1-117位，或者与SEQ ID No.28自N端起第1-117位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性。
  4. 根据权利要求2所述的抗体，其特征在于：所述轻链可变区的氨基酸序列为如下任一：
    (b1)SEQ ID No.2自N端起第1-107位，或者与SEQ ID No.2自N端起第1-107位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性；
    (b2)SEQ ID No.18自N端起第133-239位，或者与SEQ ID No.18自N端起第133-239位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性；
    (b3)SEQ ID No.19自N端起第133-239位，或者与SEQ ID No.19自N端起第133-239位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性；
    (b4)SEQ ID No.20自N端起第133-239位，或者与SEQ ID No.20自N端起第133-239位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性。
    (b5)SEQ ID No.21自N端起第133-239位，或者与SEQ ID No.21自N端起第133-239位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性；
    (b6)SEQ ID No.22自N端起第133-239位，或者与SEQ ID No.22自N端起第133-239位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性；
    (b7)SEQ ID No.23自N端起第133-239位，或者与SEQ ID No.23自N端起第133-239位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性；
    (b8)SEQ ID No.24自N端起第133-239位，或者与SEQ ID No.24自N端起第133-239位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性；
    (b9)SEQ ID No.25自N端起第133-239位，或者与SEQ ID No.25自N端起第133-239位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性；
    (b10)SEQ ID No.26自N端起第133-239位，或者与SEQ ID No.26自N端起第133-239位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性；
    (b11)SEQ ID No.27自N端起第133-239位，或者与SEQ ID No.27自N端起第133-239位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性；
    (b12)SEQ ID No.28自N端起第133-239位，或者与SEQ ID No.28自N端起第133-239位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性。
  5. 根据权利要求1-4中任一所述的抗体，其特征在于：所述抗体为IgG、IgM、IgE、IgA或IgD。
  6. 根据权利要求5所述的抗体，其特征在于：所述IgG为IgG1、IgG2、IgG3或IgG4亚型。
  7. 根据权利要求1-6中任一所述的抗体，其特征在于：所述抗体的轻链类型为κ链或λ链。
  8. 根据权利要求6或7所述的抗体，其特征在于：所述抗体为IgG4亚型，所述抗体的轻链类型为Kappa链。
  9. 根据权利要求1-8中任一所述的抗体，其特征在于：所述抗体为人源化抗体。
  10. 根据权利要求1-9中任一所述的抗体，其特征在于：所述抗体的重链的氨基酸序列为SEQ ID No.1，或者与SEQ ID No.1具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性。
  11. 根据权利要求1-10中任一所述的抗体，其特征在于：所述抗体的轻链的氨基酸序列为SEQ ID No.2或者与SEQ ID No.2具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性。
  12. 权利要求1-11中任一所述抗体的抗原结合片段。
  13. 根据权利要求12所述的抗原结合片段，其特征在于：所述抗原结合片段包含如下中的一种或多种：所述抗体的轻链；所述抗体的重链；所述抗体的轻链可变区；所述抗体的重链可变区；所述抗体的一或多个CDR区。
  14. 根据权利要求13所述的抗原结合片段，其特征在于：所述抗原结合片段为如下任一：Fab片段；F(ab’)2片段；Fd片段；FV片段；分离的CDR；单链抗体。
  15. 根据权利要求14所述的抗原结合片段，其特征在于：所述抗原结合片段为单链抗体；所述单链抗体的氨基酸全序列为如下任一：
    (c1)SEQ ID No.16的第1-239位，或者与SEQ ID No.16的第1-239位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性；
    (c2)SEQ ID No.18的第1-239位，或者与SEQ ID No.18的第1-239位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性；
    (c3)SEQ ID No.19的第1-239位，或者与SEQ ID No.19的第1-239位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性；
    (c4)SEQ ID No.20的第1-239位，或者与SEQ ID No.20的第1-239位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性；
    (c5)SEQ ID No.21的第1-239位，或者与SEQ ID No.21的第1-239位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性；
    (c6)SEQ ID No.22的第1-239位，或者与SEQ ID No.22的第1-239位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性；
    (c7)SEQ ID No.23的第1-239位，或者与SEQ ID No.23的第1-239位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性；
    (c8)SEQ ID No.24的第1-239位，或者与SEQ ID No.24的第1-239位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性；
    (c9)SEQ ID No.25的第1-239位，或者与SEQ ID No.25的第1-239位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性；
    (c10)SEQ ID No.26的第1-239位，或者与SEQ ID No.26的第1-239位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性；
    (c11)SEQ ID No.27的第1-239位，或者与SEQ ID No.27的第1-239位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性；
    (c12)SEQ ID No.28的第1-239位，或者与SEQ ID No.28的第1-239位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性(不一致处优选在骨架区(FR))。
  16. 由权利要求1-11中任一所述的抗体或12-15中任一所述的抗原结合片段与其他蛋白形成的抗体融合蛋白。
  17. 根据权利要求16所述的抗体融合蛋白，其特征在于：所述抗体融合蛋白为由权利要求14或15中所述的单链抗体与标签蛋白形成的带有标签蛋白的单链抗体。
  18. 根据权利要求17所述的抗体融合蛋白，其特征在于：所述标签蛋白为Fc。
  19. 根据权利要求18所述的抗体融合蛋白，其特征在于：所述Fc为IgG Fc。
  20. 根据权利要求18所述的抗体融合蛋白，其特征在于：所述IgG为IgG4。
  21. 根据权利要求18-20中任一所述的抗体融合蛋白，其特征在于：所述Fc的氨基酸序列如SEQ ID No.16的第240-472位所示，或者与SEQ ID No.16自N端起第240-472位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性。
  22. 根据权利要求21所述的抗体融合蛋白，其特征在于：所述带有标签蛋白的单链抗体的氨基酸序列为如下任一：
    (d1)SEQ ID No.16所示，或者与SEQ ID No.16具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性；
    (d2)SEQ ID No.18所示，或者与SEQ ID No.18具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性；
    (d3)SEQ ID No.19所示，或者与SEQ ID No.19具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性；
    (d4)SEQ ID No.20所示，或者与SEQ ID No.20具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性；
    (d5)SEQ ID No.21所示，或者与SEQ ID No.21具有99％以上、95％以上、 90％以上、85％以上、80％以上或者75％以上的一致性；
    (d6)SEQ ID No.22所示，或者与SEQ ID No.22具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性；
    (d7)SEQ ID No.23所示，或者与SEQ ID No.23具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性；
    (d8)SEQ ID No.24所示，或者与SEQ ID No.24具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性；
    (d9)SEQ ID No.25所示，或者与SEQ ID No.25具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性；
    (d10)SEQ ID No.26所示，或者与SEQ ID No.26具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性；
    (d11)SEQ ID No.27所示，或者与SEQ ID No.27具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性；
    (d12)SEQ ID No.28所示，或者与SEQ ID No.28具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性。
  23. 由权利要求1-11中任一所述的抗体或权利要求12-15中任一所述抗原结合片段与其他靶点抗体形成的双抗。
  24. 核酸分子，其特征在于：所述核酸分子编码权利要求1-11中任一所述的抗体或权利要求12-15中任一所述抗原结合片段或权利要求16-22中任一所述抗体融合蛋白或权利要求23所述的双抗。
  25. 根据权利要求23所述的核酸分子，其特征在于：在所述核酸分子中，编码权利要求1-11中任一所述抗体的重链可变区中HCDR1、HCDR2和HCDR3的核苷酸序列依次如SEQ ID No.3自5’端起第91-105位、第148-192位、第292-318位所示。
  26. 根据权利要求23所述的核酸分子，其特征在于：在所述核酸分子中，编码权利要求1-11中任一所述抗体的轻链可变区中LCDR1、LCDR2和LHCDR3的核苷酸序列依次如SEQ ID No.4自5’端起第70-102位、第148-168位、第265-291位所示。
  27. 根据权利要求25所述的核酸分子，其特征在于：在所述核酸分子中，编码所述抗体的所述重链可变区的核苷酸序列为SEQ ID No.3自5’端起第1-351位或者与SEQ ID No.3自5’端起第1-351位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性。
  28. 根据权利要求26所述的核酸分子，其特征在于：编码所述抗体的所述轻链可变区的核苷酸序列为SEQ ID No.4自5’端起第1-321位或者与SEQ ID No.4自5’端起第1-321位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性。
  29. 根据权利要求27所述的核酸分子，其特征在于：在所述核酸分子中， 编码所述抗体的重链的核苷酸序列为SEQ ID No.3或者与SEQ ID No.3具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性。
  30. 根据权利要求28所述的核酸分子，其特征在于：在所述核酸分子中，编码所述抗体的轻链的核苷酸序列为SEQ ID No.4或者与SEQ ID No.4具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性。
  31. 根据权利要求27或28所述的核酸分子，其特征在于：编码所述单链抗体的核苷酸序列为SEQ ID No.17自5’端起第1-717位或者与SEQ ID No.17自5’端起第1-717位具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性。
  32. 根据权利要求31所述的核酸分子，其特征在于：编码所述带有标签蛋白的单链抗体的核苷酸序列为SEQ ID No.17或者与SEQ ID No.17具有99％以上、95％以上、90％以上、85％以上、80％以上或者75％以上的一致性。
  33. 含有权利要求24-32中任一所述核酸分子的表达盒、重组载体或重组细胞。
  34. 根据权利要求33所述的表达盒、重组载体或重组细胞，其特征在于：所述重组细胞为高等真核宿主细胞、低等真核宿主细胞、原核细胞。
  35. 根据权利要求34所述的表达盒、重组载体或重组细胞，其特征在于：所述高等真核宿主细胞为哺乳动物细胞；所述低等真核宿主细胞为酵母细胞；所述原核细胞为细菌细胞、大肠杆菌。
  36. 根据权利要求33所述的表达盒、重组载体或重组细胞，其特征在于：所述重组载体为质粒、黏粒、噬菌体或病毒载体。
  37. ADC抗体，其特征在于：所述ADC抗体为权利要求1-11中任一所述的抗体或权利要求12-15中任一所述抗原结合片段或权利要求16-22中任一所述抗体融合蛋白或权利要求23所述的双抗偶联具有生物活性小分子药物后形成。
  38. 药物组合物，其特征在于：所述药物组合物包含：
    (A1)权利要求1-11中任一所述的抗体或权利要求12-15中任一所述抗原结合片段或权利要求16-22中任一所述抗体融合蛋白或权利要求23所述的双抗或权利要求37所述ADC抗体；
    (A2)药学可接受的赋形剂、稀释剂或载体。
  39. 权利要求1-11中任一所述的抗体或权利要求12-15中任一所述的抗原结合片段或权利要求16-22中任一所述的抗体融合蛋白或权利要求23所述的双抗或权利要求24-32中任一所述的核酸分子或权利要求33-36中任一所述的表达盒、重组载体或重组细胞或权利要求37所述ADC抗体或权利要求38所述的药物组合物在如下任一中的应用：
    (B1)制备用于检测4-1BB的产品，或检测4-1BB；
    (B2)制备用于阻断4-1BB/4-1BBL信号通路的产品，或阻断4-1BB/4-1BBL信号通路；
    (B3)制备用于刺激T细胞活化的产品，或刺激T细胞活化；
    (B4)制备用于促进T细胞分泌IFN-γ的产品，或促进T细胞分泌IFN-γ；
    (B5)制备用于抑制结肠癌细胞生长的产品，或抑制结肠癌细胞生长；
    (B6)制备用于抑制结肠癌肿瘤生长的产品，或抑制结肠癌肿瘤生长；
    (B7)制备用于治疗和/或预防结肠癌的产品，或治疗和/或预防结肠癌；
    (B8)作为免疫增强物，或制备免疫增强物；
    (B9)作为免疫调节物，或制备免疫调节物；
    (B10)制备用于治疗和/或预防和/或诊断具有4-1BB表达失调特征疾病的产品，或治疗和/或预防和/或诊断具有4-1BB表达失调特征疾病；
    (B11)制备用于治疗和/或预防和/或诊断癌症的产品，或治疗和/或预防和/或诊断癌症；
    (B12)制备用于治疗和/或预防和/或诊断自身免疫疾病的产品，或治疗和/或预防和/或诊断自身免疫疾病；
    (B13)制备用于治疗和/或预防和/或诊断炎症疾病的产品，或治疗和/或预防和/或诊断炎症疾病；
    (B14)制备用于治疗和/或预防和/或诊断感染性疾病的产品，或治疗和/或预防和/或诊断感染性疾病。
  40. 根据权利要求39所述的应用，其特征在于：所述4-1BB为人4-1BB或猴4-1BB。
  41. 根据权利要求39所述的应用，其特征在于：所述癌症为4-1BB表达失调的癌症；所述自身免疫疾病为4-1BB表达失调的自身免疫疾病；所述炎症疾病为4-1BB表达失调的炎症疾病；所述感染性疾病为4-1BB表达失调的感染性疾病。
  42. 如下任一方法：
    (C1)一种检测4-1BB的方法，包括如下步骤：采用权利要求1-11中任一所述的抗体或权利要求12-15中任一所述的抗原结合片段或权利要求16-22中任一所述的抗体融合蛋白或权利要求23所述的双抗或权利要求24-32中任一所述的核酸分子或权利要求33-36中任一所述的表达盒、重组载体或重组细胞对待测样本进行检测；
    (C2)一种阻断4-1BB/4-1BBL信号通路的方法，包括如下步骤：采用权利要求1-11中任一所述的抗体或权利要求12-15中任一所述的抗原结合片段或权利要求16-22中任一所述的抗体融合蛋白或权利要求23所述的双抗或权利要求24-32中任一所述的核酸分子或权利要求33-36中任一所述的表达盒、重组载体或重组细胞或权利要求37所述ADC抗体或权利要求38所述的药物组合物阻断4-1BB/4-1BBL信号通路；
    (C3)一种刺激T细胞活化的方法，包括如下步骤：采用权利要求1-11中任一所述的抗体或权利要求12-15中任一所述的抗原结合片段或权利要求16-22 中任一所述的抗体融合蛋白或权利要求23所述的双抗或权利要求24-32中任一所述的核酸分子或权利要求33-36中任一所述的表达盒、重组载体或重组细胞或权利要求37所述ADC抗体或权利要求38所述的药物组合物刺激T细胞活化；
    (C4)一种促进T细胞分泌IFN-γ的方法，包括如下步骤：采用权利要求1-11中任一所述的抗体或权利要求12-15中任一所述的抗原结合片段或权利要求16-22中任一所述的抗体融合蛋白或权利要求23所述的双抗或权利要求24-32中任一所述的核酸分子或权利要求33-36中任一所述的表达盒、重组载体或重组细胞或权利要求37所述ADC抗体或权利要求38所述的药物组合物促进T细胞分泌IFN-γ；
    (C5)一种抑制结肠癌细胞生长的方法，包括如下步骤：采用权利要求1-11中任一所述的抗体或权利要求12-15中任一所述的抗原结合片段或权利要求16-22中任一所述的抗体融合蛋白或权利要求23所述的双抗或权利要求24-32中任一所述的核酸分子或权利要求33-36中任一所述的表达盒、重组载体或重组细胞或权利要求37所述ADC抗体或权利要求38所述的药物组合物抑制结肠癌细胞生长；
    (C6)一种抑制结肠癌肿瘤生长的方法，包括如下步骤：采用权利要求1-11中任一所述的抗体或权利要求12-15中任一所述的抗原结合片段或权利要求16-22中任一所述的抗体融合蛋白或权利要求23所述的双抗或权利要求24-32中任一所述的核酸分子或权利要求33-36中任一所述的表达盒、重组载体或重组细胞或权利要求37所述ADC抗体或权利要求38所述的药物组合物抑制结肠癌肿瘤生长；
    (C7)一种治疗和/或预防结肠癌的方法，包括如下步骤：采用权利要求1-11中任一所述的抗体或权利要求12-15中任一所述的抗原结合片段或权利要求16-22中任一所述的抗体融合蛋白或权利要求23所述的双抗或权利要求24-32中任一所述的核酸分子或权利要求33-36中任一所述的表达盒、重组载体或重组细胞或权利要求37所述ADC抗体或权利要求38所述的药物组合物治疗和/或预防结肠癌；
    (C8)一种制备免疫增强物的方法，包括如下步骤：采用权利要求1-11中任一所述的抗体或权利要求12-15中任一所述的抗原结合片段或权利要求16-22中任一所述的抗体融合蛋白或权利要求23所述的双抗或权利要求24-32中任一所述的核酸分子或权利要求33-36中任一所述的表达盒、重组载体或重组细胞或权利要求37所述ADC抗体或权利要求38所述的药物组合物作为活性成分制备免疫增强物；
    (C9)一种制备免疫调节物的方法，包括如下步骤：采用权利要求1-11中任一所述的抗体或权利要求12-15中任一所述的抗原结合片段或权利要求16-22中任一所述的抗体融合蛋白或权利要求23所述的双抗或权利要求24-32中任一所述的核酸分子或权利要求33-36中任一所述的表达盒、重组载体或重组细胞 或权利要求37所述ADC抗体或权利要求38所述的药物组合物作为活性成分制备免疫调节物；
    (C10)一种治疗和/或预防和/或诊断具有4-1BB表达失调特征疾病的方法，包括如下步骤：采用权利要求1-11中任一所述的抗体或权利要求12-15中任一所述的抗原结合片段或权利要求16-22中任一所述的抗体融合蛋白或权利要求23所述的双抗或权利要求24-32中任一所述的核酸分子或权利要求33-36中任一所述的表达盒、重组载体或重组细胞或权利要求37所述ADC抗体或权利要求38所述的药物组合物治疗和/或预防和/或诊断具有4-1BB表达失调特征疾病；
    (C11)一种治疗和/或预防和/或诊断癌症的方法，包括如下步骤：采用权利要求1-11中任一所述的抗体或权利要求12-15中任一所述的抗原结合片段或权利要求16-22中任一所述的抗体融合蛋白或权利要求23所述的双抗或权利要求24-32中任一所述的核酸分子或权利要求33-36中任一所述的表达盒、重组载体或重组细胞或权利要求37所述ADC抗体或权利要求38所述的药物组合物治疗和/或预防和/或诊断癌症；
    (C12)一种治疗和/或预防和/或诊断自身免疫疾病的方法，包括如下步骤：采用权利要求1-11中任一所述的抗体或权利要求12-15中任一所述的抗原结合片段或权利要求16-22中任一所述的抗体融合蛋白或权利要求23所述的双抗或权利要求24-32中任一所述的核酸分子或权利要求33-36中任一所述的表达盒、重组载体或重组细胞或权利要求37所述ADC抗体或权利要求38所述的药物组合物治疗和/或预防和/或诊断自身免疫疾病；
    (C13)一种治疗和/或预防和/或诊断炎症疾病的方法，包括如下步骤：采用权利要求1-11中任一所述的抗体或权利要求12-15中任一所述的抗原结合片段或权利要求16-22中任一所述的抗体融合蛋白或权利要求23所述的双抗或权利要求24-32中任一所述的核酸分子或权利要求33-36中任一所述的表达盒、重组载体或重组细胞或权利要求37所述ADC抗体或权利要求38所述的药物组合物治疗和/或预防和/或诊断炎症疾病；
    (C14)一种治疗和/或预防和/或诊断感染性疾病的方法，包括如下步骤：采用权利要求1-11中任一所述的抗体或权利要求12-15中任一所述的抗原结合片段或权利要求16-22中任一所述的抗体融合蛋白或权利要求23所述的双抗或权利要求24-32中任一所述的核酸分子或权利要求33-36中任一所述的表达盒、重组载体或重组细胞或权利要求37所述ADC抗体或权利要求38所述的药物组合物治疗和/或预防和/或诊断感染性疾病。
  43. 根据权利要求42所述的方法，其特征在于：所述4-1BB为人4-1BB或猴4-1BB。
  44. 根据权利要求42所述的方法，其特征在于：所述癌症为4-1BB表达失调的癌症；所述自身免疫疾病为4-1BB表达失调的自身免疫疾病；所述炎症疾病为4-1BB表达失调的炎症疾病；所述感染性疾病为4-1BB表达失调的感染性 疾病。
PCT/CN2020/127993 2019-11-13 2020-11-11 一种能够与人4-1bb结合的分子及其应用 WO2021093753A1 (zh)

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