WO2023274286A1 - Anti-crtam antibody and application thereof - Google Patents

Anti-crtam antibody and application thereof Download PDF

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WO2023274286A1
WO2023274286A1 PCT/CN2022/102253 CN2022102253W WO2023274286A1 WO 2023274286 A1 WO2023274286 A1 WO 2023274286A1 CN 2022102253 W CN2022102253 W CN 2022102253W WO 2023274286 A1 WO2023274286 A1 WO 2023274286A1
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seq
amino acid
acid sequence
variable region
chain variable
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Chinese (zh)
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白米雪
肖扬
蒋美玲
李婷婷
赵立文
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南京圣和药业股份有限公司
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    • C07—ORGANIC CHEMISTRY
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • C07—ORGANIC CHEMISTRY
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
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    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
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    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host

Definitions

  • the present invention relates to the technical field of antibody medicines, in particular to an anti-CRTAM antibody or its antigen-binding fragment, a pharmaceutical composition containing the anti-CRTAM antibody or its antigen-binding fragment and applications thereof.
  • CRTAM Class-I MHC-restricted T cell associated molecule
  • IgSF immunoglobulin superfamily
  • the ligand is Nectin-like 2 (Necl-2).
  • the expression of CRTAM is strictly regulated by TCR (T cell receptor) activation and is only expressed on activated immune cells.
  • TCR T cell receptor
  • PBMC Peripheral blood mononuclear cell
  • the promoter of CRTAM is regulated by AP-1 transcription factor like IL-2.
  • CRTAM activation will stimulate CD8+ T cells to secrete IFN- ⁇ and enhance the killing ability of CD8+ T cells and NK cells.
  • CRTAM can affect the polarization of CD4+ T cells, promote the further differentiation of CD4+ T cells after MHC and TCR combined activation, and increase the secretion of cytokines such as IFN- ⁇ , IL-17, and IL-22 ability.
  • Necl-2 of CRTAM also known as TSLC1 (The tumor suppressor in lung cancer-1) and CADM1
  • TSLC1 The tumor suppressor in lung cancer-1
  • CADM1 The tumor suppressor in lung cancer-1
  • nectin and necl are calcium ion-independent cell adhesion molecules
  • Necl-2 and CRTAM The combination can activate T cells and NK cells to produce killing effect. Necl-2 expression is gradually reduced to silence on tumor cells, which is thought to be another tumor escape mechanism.
  • Literature shows that the expression of Necl-2 in tumor cells can inhibit the growth and invasion of tumor cells; at the same time, the combination of Necl-2 and CRTAM can further activate CD8+ T cells and NK cells, and improve the killing ability of immune cells.
  • the Necl-2 and CRTAM pathways do not overlap with the PD1-PDL1 pathway and have complementary roles. Tumor cells will achieve the purpose of immune escape by down-regulating the expression of Necl-2 protein.
  • CRTAM is a new and safe immune co-stimulatory molecule with potential therapeutic ability for solid tumors, so research and development of CRTAM agonistic antibodies is of great significance.
  • an anti-CRTAM antibody or an antigen-binding fragment thereof which comprises a heavy chain variable region and/or a light chain variable region
  • the heavy chain variable region comprises a complementarity determining region of the heavy chain variable region 1 (HCDR1), the complementarity determining region 2 (HCDR2) of the heavy chain variable region, and/or the complementarity determining region 3 (HCDR3) of the heavy chain variable region comprising the complementarity of the light chain variable region
  • the invention provides an anti-CRTAM antibody or antigen-binding fragment thereof comprising a heavy chain variable region and/or a light chain variable region, wherein:
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 selected from the group consisting of:
  • the light chain variable region comprises LCDR1, LCDR2 and LCDR3 selected from the following group:
  • the present invention provides an anti-CRTAM antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region and a light chain variable region, wherein HCDR1, HCDR2 and HCDR3 of the heavy chain variable region SEQ ID NO: 1, 2 and 3 or an amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, 2 and 3, and LCDR1, LCDR2 of the light chain variable region and LCDR3 are respectively SEQ ID NO: 4, 5 and 6, SEQ ID NO: 7, 5 and 6 or have the amino acid sequence shown in SEQ ID NO: 4, 5 and 6 or SEQ ID NO: 7, 5 and 6 Amino acid sequences with at least 85% sequence identity; or
  • the HCDR1, HCDR2 and HCDR3 are respectively SEQ ID NO: 8, 9 and 10, SEQ ID NO: 8, 11 and 10 or with SEQ ID NO: 8, 9 and 10 or SEQ ID NO: 8, 11 and 10
  • An amino acid sequence having at least 85% sequence identity to the amino acid sequence shown and said LCDR1, LCDR2 and LCDR3 are SEQ ID NO: 12, 13 and 14, SEQ ID NO: 15, 13 and 14, respectively, or with SEQ ID NO: 12, 13 and 14 or an amino acid sequence having at least 85% sequence identity to the amino acid sequence shown in SEQ ID NO: 15, 13 and 14; or
  • the HCDR1, HCDR2 and HCDR3 are respectively SEQ ID NO: 16, 17 and 18 or an amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 16, 17 and 18, and the LCDR1, LCDR2 and LCDR3 are SEQ ID NO: 19, 20, and 21, respectively, or an amino acid sequence having at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 19, 20, and 21; or
  • the HCDR1, HCDR2 and HCDR3 are respectively SEQ ID NO: 22, 23 and 24, SEQ ID NO: 22, 25 and 24 or with SEQ ID NO: 22, 23 and 24 or SEQ ID NO: 22, 25 and 24
  • the amino acid sequence shown has an amino acid sequence of at least 85% sequence identity
  • said LCDR1, LCDR2 and LCDR3 are respectively SEQ ID NO:26, 27 and 28 or the amino acid sequence shown in SEQ ID NO:26, 27 and 28 Amino acid sequences having at least 85% sequence identity.
  • amino acid sequence of the heavy chain variable region is selected from the group consisting of SEQ ID NO:80, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86 and SEQ ID NO:87, SEQ ID NO:80, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86 or SEQ ID NO:87 are substituted, deleted or added with one or more Amino acid sequences obtained from amino acids and functionally identical to SEQ ID NO: 80, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86 or SEQ ID NO: 87, and functionally identical to SEQ ID NO: 80, SEQ ID NO: 80, SEQ ID NO: ID NO:84, SEQ ID NO:85, SEQ ID NO:86 or SEQ ID NO:87 has an amino acid sequence of at least 85% sequence identity, and the amino acid sequence of the light chain variable region is selected
  • the amino acid sequence of the heavy chain variable region is SEQ ID NO: 80
  • SEQ ID NO: 80 is substituted, deleted or added with one or An amino acid sequence obtained from multiple amino acids and having the same function as SEQ ID NO: 80 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 80
  • the amino acid sequence of the light chain variable region is SEQ ID NO :81, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids of SEQ ID NO:81 and having the same function as SEQ ID NO:81 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:81 .
  • amino acid sequence of the heavy chain variable region is selected from SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86 and SEQ ID NO: 87, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86 or SEQ ID NO: 87 obtained by substitution, deletion or addition of one or more amino acids and identical to SEQ ID NO: 84, An amino acid sequence that is functionally identical to SEQ ID NO:85, SEQ ID NO:86, or SEQ ID NO:87, and has at least The amino acid sequence of 85% sequence identity, and the aminoacid sequence of described light chain variable region is selected from SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90 and SEQ ID NO:91, SEQ ID NO: 88.
  • SEQ ID NO: 89, SEQ ID NO: 90 or SEQ ID NO: 91 obtained by substitution, deletion or addition of one or more amino acids and identical to SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90 or a functionally identical amino acid sequence of SEQ ID NO:91, and an amino acid sequence having at least 85% sequence identity to SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90 or SEQ ID NO:91.
  • the amino acid sequence of the heavy chain variable region is SEQ ID NO: 87
  • SEQ ID NO: 87 is substituted, deleted or added with one or An amino acid sequence obtained from multiple amino acids and having the same function as SEQ ID NO: 87 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 87
  • the amino acid sequence of the light chain variable region is SEQ ID NO :90
  • an amino acid sequence obtained by substituting, deleting or adding one or more amino acids of SEQ ID NO:90 and having the same function as SEQ ID NO:90 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:90 or
  • the amino acid sequence of the heavy chain variable region is SEQ ID NO: 87, the amino acid sequence obtained by replacing, deleting or adding one or more amino acids of SEQ ID NO: 87 and having the same function as SEQ ID NO: 87 or the amino acid sequence with SEQ ID NO: 87 ID NO:87 has an amino acid sequence of at least 85% sequence identity, and the amino acid sequence of the light chain variable region is SEQ ID NO:92, and SEQ ID NO:92 is obtained by substitution, deletion or addition of one or more amino acids An amino acid sequence that is functionally identical to SEQ ID NO:92 or an amino acid sequence that has at least 85% sequence identity to SEQ ID NO:92.
  • amino acid sequence of the heavy chain variable region is selected from SEQ ID NO:45, SEQ ID NO:106, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53 and SEQ ID NO:54, SEQ ID NO:45, SEQ ID NO:106, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO: 52, SEQ ID NO: 53 or SEQ ID NO: 54 obtained by substitution, deletion or addition of one or more amino acids and obtained with SEQ ID NO: 45, SEQ ID NO: 106, SEQ ID NO: 50, SEQ ID The functionally identical amino acid sequence of NO:51, SEQ ID NO:52, SEQ ID NO:53 or SEQ ID NO:54, and SEQ ID NO:45, SEQ ID NO:106, SEQ ID NO:50, SEQ ID NO :51, SEQ ID NO:52, SEQ ID NO:52, SEQ ID NO:SEQ ID NO:54, SEQ ID NO:45, SEQ ID NO:106, S
  • the amino acid sequence of the heavy chain variable region is SEQ ID NO: 45
  • SEQ ID NO: 45 is substituted, deleted or added with one or The amino acid sequence obtained by multiple amino acids and having the same function as SEQ ID NO: 45 or the amino acid sequence having at least 85% sequence identity with SEQ ID NO: 45
  • the amino acid sequence of the light chain variable region is SEQ ID NO :46, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids of SEQ ID NO:46 and having the same function as SEQ ID NO:46 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:46 .
  • the amino acid sequence of the heavy chain variable region is SEQ ID NO: 106
  • SEQ ID NO: 106 is substituted, deleted or added with one or The amino acid sequence obtained by multiple amino acids and having the same function as SEQ ID NO: 106 or the amino acid sequence having at least 85% sequence identity with SEQ ID NO: 106
  • the amino acid sequence of the light chain variable region is SEQ ID NO :46, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids of SEQ ID NO:46 and having the same function as SEQ ID NO:46 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:46 .
  • amino acid sequence of the heavy chain variable region is selected from SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53 and SEQ ID NO:54
  • SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53 or SEQ ID NO:54 are substituted, deleted or added with one or more Amino acid sequences obtained from amino acids and having the same function as SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53 or SEQ ID NO:54, as well as the same amino acid sequence as SEQ ID NO:50, SEQ ID NO:50, SEQ ID NO:52 ID NO:51, SEQ ID NO:52, SEQ ID NO:53 or SEQ ID NO:54 have an amino acid sequence of at least 85% sequence identity, and the amino acid sequence of the light chain variable region is selected from SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53 and S
  • SEQ ID NO:63 The functionally identical amino acid sequence of SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65 or SEQ ID NO:66, and SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57 , SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ An amino acid sequence having at least 85% sequence identity to ID NO:64, SEQ ID NO:65 or SEQ ID NO:66.
  • the amino acid sequence of the heavy chain variable region is SEQ ID NO: 51
  • SEQ ID NO: 51 is substituted, deleted or added with one or An amino acid sequence obtained from multiple amino acids and having the same function as SEQ ID NO:51 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:51
  • the amino acid sequence of the light chain variable region is SEQ ID NO :66, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids of SEQ ID NO:66 and having the same function as SEQ ID NO:66 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:66 ;or
  • the amino acid sequence of the heavy chain variable region is SEQ ID NO: 51, an amino acid sequence obtained by replacing, deleting or adding one or more amino acids to SEQ ID NO: 51 and having the same function as SEQ ID NO: 51 or with SEQ ID NO: 51 ID NO:51 has an amino acid sequence of at least 85% sequence identity, and the amino acid sequence of the light chain variable region is SEQ ID NO:67, and SEQ ID NO:67 is obtained by substitution, deletion or addition of one or more amino acids An amino acid sequence that is functionally identical to SEQ ID NO: 67 or an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 67.
  • amino acid sequence of the heavy chain variable region is selected from the group consisting of SEQ ID NO:68, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74 and SEQ ID NO:75, SEQ ID NO:68, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74 or SEQ ID NO:75 are substituted, deleted or added with one or more Amino acid sequences obtained from amino acids and functionally identical to SEQ ID NO: 68, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74 or SEQ ID NO: 75, and amino acid sequences identical to those of SEQ ID NO: 68, SEQ ID NO: ID NO:72, SEQ ID NO:73, SEQ ID NO:74 or SEQ ID NO:75 has an amino acid sequence of at least 85% sequence identity, and the amino acid sequence of the light chain variable region is selected from SEQ ID NO:68, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74 or SEQ
  • SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, and SEQ ID NO:79 SEQ ID NO:69, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, or SEQ ID NO:79 is obtained by substitution, deletion or addition of one or more amino acids and has the same function as SEQ ID NO:69, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78 or SEQ ID NO:79
  • the amino acid sequence of the heavy chain variable region is SEQ ID NO: 68
  • SEQ ID NO: 68 is substituted, deleted or added with one or An amino acid sequence obtained from multiple amino acids and having the same function as SEQ ID NO:68 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:68
  • the amino acid sequence of the light chain variable region is SEQ ID NO :69
  • an amino acid sequence obtained by substituting, deleting or adding one or more amino acids of SEQ ID NO:69 and having the same function as SEQ ID NO:69 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:69 is substituted, deleted or added with one or An amino acid sequence obtained from multiple amino acids and having the same function as SEQ ID NO:68 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:68
  • amino acid sequence of the heavy chain variable region is selected from SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74 and SEQ ID NO: 75, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74 or SEQ ID NO: 75 obtained by substitution, deletion or addition of one or more amino acids and identical to SEQ ID NO: 72, An amino acid sequence that is functionally identical to SEQ ID NO:73, SEQ ID NO:74, or SEQ ID NO:75, and has at least The amino acid sequence of 85% sequence identity, and the aminoacid sequence of described light chain variable region is selected from SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78 and SEQ ID NO:79, SEQ ID NO: 76.
  • SEQ ID NO: 77, SEQ ID NO: 78 or SEQ ID NO: 79 obtained by substitution, deletion or addition of one or more amino acids and identical to SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78 or an amino acid sequence that is functionally identical to SEQ ID NO: 79, and an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78 or SEQ ID NO: 79.
  • the amino acid sequence of the heavy chain variable region is SEQ ID NO: 75
  • SEQ ID NO: 75 is substituted, deleted or added with one or An amino acid sequence obtained from multiple amino acids and having the same function as SEQ ID NO:75 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:75
  • the amino acid sequence of the light chain variable region is SEQ ID NO :77, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids of SEQ ID NO:77 and having the same function as SEQ ID NO:77 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:77 .
  • amino acid sequence of the heavy chain variable region is selected from SEQ ID NO:45, SEQ ID NO:106, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53 and SEQ ID NO:54, SEQ ID NO:45, SEQ ID NO:106, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO: 52, SEQ ID NO: 53 or SEQ ID NO: 54 obtained by substitution, deletion or addition of one or more amino acids and obtained with SEQ ID NO: 45, SEQ ID NO: 106, SEQ ID NO: 50, SEQ ID The functionally identical amino acid sequence of NO:51, SEQ ID NO:52, SEQ ID NO:53 or SEQ ID NO:54, and SEQ ID NO:45, SEQ ID NO:106, SEQ ID NO:50, SEQ ID NO :51, SEQ ID NO:52, SEQ ID NO:52, SEQ ID NO:SEQ ID NO:54, SEQ ID NO:45, SEQ ID NO:106, S
  • SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO: 66 or SEQ ID NO: 67 obtained by substitution, deletion or addition of one or more amino acids and with SEQ ID NO: 46, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63 , SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66 or SEQ ID NO:67 functionally identical amino acid sequences, and SEQ ID NO:46, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO
  • the amino acid sequence of the heavy chain variable region is SEQ ID NO: 45
  • SEQ ID NO: 45 is substituted, deleted or added with one or The amino acid sequence obtained by multiple amino acids and having the same function as SEQ ID NO: 45 or the amino acid sequence having at least 85% sequence identity with SEQ ID NO: 45
  • the amino acid sequence of the light chain variable region is SEQ ID NO :46, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids of SEQ ID NO:46 and having the same function as SEQ ID NO:46 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:46 .
  • the amino acid sequence of the heavy chain variable region is SEQ ID NO: 106
  • SEQ ID NO: 106 is substituted, deleted or added with one or The amino acid sequence obtained by multiple amino acids and having the same function as SEQ ID NO: 106 or the amino acid sequence having at least 85% sequence identity with SEQ ID NO: 106
  • the amino acid sequence of the light chain variable region is SEQ ID NO :46, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids of SEQ ID NO:46 and having the same function as SEQ ID NO:46 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:46 .
  • amino acid sequence of the heavy chain variable region is selected from SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53 and SEQ ID NO:54
  • SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53 or SEQ ID NO:54 are substituted, deleted or added with one or more Amino acid sequence obtained from amino acids and identical to SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53 or SEQ ID NO:54, and SEQ ID NO:50, SEQ ID NO:50, SEQ ID NO:52 ID NO:51, SEQ ID NO:52, SEQ ID NO:53 or SEQ ID NO:54 have an amino acid sequence of at least 85% sequence identity, and the amino acid sequence of the light chain variable region is selected from SEQ ID NO: 55.
  • SEQ ID NO:63 The functionally identical amino acid sequence of SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65 or SEQ ID NO:66, and SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57 , SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ An amino acid sequence having at least 85% sequence identity to ID NO:64, SEQ ID NO:65 or SEQ ID NO:66.
  • the amino acid sequence of the heavy chain variable region is SEQ ID NO: 51
  • SEQ ID NO: 51 is substituted, deleted or added with one or An amino acid sequence obtained from multiple amino acids and having the same function as SEQ ID NO:51 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:51
  • the amino acid sequence of the light chain variable region is SEQ ID NO :66, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids of SEQ ID NO:66 and having the same function as SEQ ID NO:66 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:66 ;or
  • the amino acid sequence of the heavy chain variable region is SEQ ID NO: 51, an amino acid sequence obtained by replacing, deleting or adding one or more amino acids to SEQ ID NO: 51 and having the same function as SEQ ID NO: 51 or with SEQ ID NO: 51 ID NO:51 has an amino acid sequence of at least 85% sequence identity, and the amino acid sequence of the light chain variable region is SEQ ID NO:67, and SEQ ID NO:67 is obtained by substitution, deletion or addition of one or more amino acids An amino acid sequence that is functionally identical to SEQ ID NO: 67 or an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 67.
  • amino acid sequence of the heavy chain variable region is selected from the group consisting of SEQ ID NO:93, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100 and SEQ ID NO:101
  • SEQ ID NO:93, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100 or SEQ ID NO:101 is obtained by substitution, deletion or addition of one or more amino acids and is identical to SEQ ID NO:93, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100 or SEQ ID NO:101 functionally identical amino acid sequence, and at least 85% identical to SEQ ID NO:93, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100 or SEQ ID NO:101 the amino acid sequence of sequence identity, and the amino acid sequence of sequence identity, and the amino acid sequence of sequence identity, and the amino acid sequence of sequence identity, and the amino acid
  • the amino acid sequence of the heavy chain variable region is SEQ ID NO: 93
  • SEQ ID NO: 93 is substituted, deleted or added with one or An amino acid sequence obtained from multiple amino acids and having the same function as SEQ ID NO:93 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:93
  • the amino acid sequence of the light chain variable region is SEQ ID NO :94, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids of SEQ ID NO:94 and having the same function as SEQ ID NO:94 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:94 .
  • amino acid sequence of the heavy chain variable region is selected from SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99 and SEQ ID NO: 100, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99 or SEQ ID NO: 100 obtained by substitution, deletion or addition of one or more amino acids and identical to SEQ ID NO: 97, An amino acid sequence that is functionally identical to SEQ ID NO:98, SEQ ID NO:99, or SEQ ID NO:100, and has at least The amino acid sequence of 85% sequence identity, and the aminoacid sequence of described light chain variable region is selected from SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104 and SEQ ID NO:105, SEQ ID NO: 102.
  • SEQ ID NO: 103, SEQ ID NO: 104 or SEQ ID NO: 105 is obtained by substitution, deletion or addition of one or more amino acids and is identical to SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104 or a functionally identical amino acid sequence of SEQ ID NO: 105, and an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104 or SEQ ID NO: 105.
  • the amino acid sequence of the heavy chain variable region is SEQ ID NO: 99
  • SEQ ID NO: 99 is substituted, deleted or added with one or An amino acid sequence obtained from multiple amino acids and having the same function as SEQ ID NO:99 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:99
  • the amino acid sequence of the light chain variable region is SEQ ID NO :104, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids of SEQ ID NO:104 and having the same function as SEQ ID NO:104 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:104 ;or
  • the amino acid sequence of the heavy chain variable region is SEQ ID NO: 101, an amino acid sequence obtained by replacing, deleting or adding one or more amino acids to SEQ ID NO: 101 and having the same function as SEQ ID NO: 101 or identical to SEQ ID NO: 101.
  • ID NO: 101 has an amino acid sequence of at least 85% sequence identity
  • the amino acid sequence of the light chain variable region is SEQ ID NO: 104
  • SEQ ID NO: 104 is obtained by substitution, deletion or addition of one or more amino acids
  • the present invention provides an anti-CRTAM antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region and a light chain variable region, wherein HCDR1, HCDR2 and HCDR3 of the heavy chain variable region Respectively SEQ ID NO:1, 2 and 3, the LCDR1, LCDR2 and LCDR3 of the light chain variable region are respectively SEQ ID NO:4, 5 and 6, the amino acid sequence of the heavy chain variable region is SEQ ID NO:80, the amino acid sequence obtained by replacing, deleting or adding one or more amino acids of SEQ ID NO:80 and having the same function as SEQ ID NO:80 or having at least 85% sequence identity with SEQ ID NO:80 and all
  • the amino acid sequences of HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 1, 2 and 3, and the amino acid sequence of the light chain variable region is SEQ ID NO: 81, SEQ ID NO: 81 is substituted, deleted or Amino acid sequence obtained by adding one or more amino acids and having the same
  • the present invention provides an anti-CRTAM antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region and a light chain variable region, wherein HCDR1, HCDR2 and HCDR3 of the heavy chain variable region Respectively SEQ ID NO: 1, 2 and 3, LCDR1, LCDR2 and LCDR3 of the light chain variable region are respectively SEQ ID NO: 4, 5 and 6,
  • the amino acid sequence of the heavy chain variable region is selected from SEQ ID NO: ID NO:84, SEQ ID NO:85, SEQ ID NO:86 and SEQ ID NO:87, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86 or SEQ ID NO:87 substituted, deleted Or the amino acid sequence obtained by adding one or more amino acids and having the same function as SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86 or SEQ ID NO: 87, and the same amino acid sequence as SEQ ID NO: 84, SEQ ID NO:85, SEQ ID NO:86
  • the light chain can be The LCDR1, LCDR2 and LCDR3 of the variable region are SEQ ID NO: 4, 5 and 6 respectively, the amino acid sequence of the heavy chain variable region is SEQ ID NO: 87, and the amino acid sequence of the light chain variable region is SEQ ID NO: 87 ID NO:90.
  • the present invention provides an anti-CRTAM antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region and a light chain variable region, wherein HCDR1, HCDR2 and HCDR3 of the heavy chain variable region Respectively SEQ ID NO:1, 2 and 3, the LCDR1, LCDR2 and LCDR3 of the light chain variable region are respectively SEQ ID NO:7, 5 and 6, the amino acid sequence of the heavy chain variable region is SEQ ID NO:87, the amino acid sequence obtained by replacing, deleting or adding one or more amino acids of SEQ ID NO:87 and having the same function as SEQ ID NO:87 or having at least 85% sequence identity with SEQ ID NO:87 and all
  • the amino acid sequences of the HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 1, 2 and 3, and the amino acid sequence of the light chain variable region are SEQ ID NO: 92, and SEQ ID NO: 92 is substituted, deleted or Amino acid sequence obtained by adding one or more amino acids and having
  • the present invention provides an anti-CRTAM antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are respectively SEQ ID NO:8, 9 and 10, the LCDR1, LCDR2 and LCDR3 of the light chain variable region are SEQ ID NO:12, 13 and 14 respectively, the amino acid sequence of the heavy chain variable region is SEQ ID NO: 45.
  • An amino acid sequence obtained by substituting, deleting or adding one or more amino acids of SEQ ID NO: 45 and having the same function as SEQ ID NO: 45 or having at least 85% sequence identity with SEQ ID NO: 45 and the HCDR1 , HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NOs: 8, 9 and 10, and the amino acid sequence of the light chain variable region is SEQ ID NO: 46, and SEQ ID NO: 46 is substituted, deleted or added or a plurality of amino acids obtained and having the same amino acid sequence as SEQ ID NO:46 or having at least 85% sequence identity with SEQ ID NO:46 and said LCDR1, LCDR2 and LCDR3 such as SEQ ID NO:12, 13 and 14 Amino acid sequence shown.
  • the light chain can be The LCDR1, LCDR2 and LCDR3 of the variable region are SEQ ID NO: 12, 13 and 14 respectively, the amino acid sequence of the heavy chain variable region is SEQ ID NO: 106, and SEQ ID NO: 106 is substituted, deleted or added one or Amino acid sequence obtained from multiple amino acids and functionally identical to SEQ ID NO: 106 or having at least 85% sequence identity with SEQ ID NO: 106 and said HCDR1, HCDR2 and HCDR3 are as set forth in SEQ ID NO: 8, 11 and 10
  • the amino acid sequence shown, and the amino acid sequence of the light chain variable region is SEQ ID NO: 46, SEQ ID NO: 46 is obtained by substitution, deletion or addition of one or more amino acids and has the same function as SEQ ID NO: 46 or an
  • the light chain can be The LCDR1, LCDR2 and LCDR3 of the variable region are SEQ ID NO:12, 13 and 14 respectively, and the amino acid sequence of the heavy chain variable region is selected from SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53 and SEQ ID NO:54, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53 or SEQ ID NO:54 are substituted, deleted or added with one or more Amino acid sequences obtained from amino acids and having the same function as SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53 or SEQ ID NO:54, as well as the same amino acid sequence as SEQ ID NO:50,
  • SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65 and SEQ ID NO:66 SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO: 65 or SEQ ID NO: 66 obtained by substitution, deletion or addition of one or more amino acids and with SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO: 65 or SEQ ID NO: 66 function The same amino acid sequence, and with SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:
  • the light chain can be The LCDR1, LCDR2 and LCDR3 of the variable region are SEQ ID NO:12, 13 and 14 respectively, the amino acid sequence of the heavy chain variable region is SEQ ID NO:51, and the amino acid sequence of the light chain variable region is SEQ ID NO: ID NO:66.
  • the light chain can be The LCDR1, LCDR2 and LCDR3 of the variable region are SEQ ID NO: 15, 13 and 14 respectively, the amino acid sequence of the heavy chain variable region is SEQ ID NO: 51, and SEQ ID NO: 51 is substituted, deleted or added with one or Amino acid sequence obtained from multiple amino acids and functionally identical to SEQ ID NO: 51 or having at least 85% sequence identity to SEQ ID NO: 51 and said HCDR1, HCDR2 and HCDR3 are as set forth in SEQ ID NOs: 8, 11 and 10
  • the amino acid sequence shown, and the amino acid sequence of the light chain variable region is SEQ ID NO: 67, SEQ ID NO: 67 obtained through substitution, deletion or addition of one or more amino acids and has the same function as SEQ ID NO: 67 or an
  • the present invention provides an anti-CRTAM antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are respectively SEQ ID NO:16,17,18, the LCDR1, LCDR2 and LCDR3 of the light chain variable region are SEQ ID NO:19,20,21 respectively, the amino acid sequence of the heavy chain variable region is SEQ ID NO: 68.
  • An amino acid sequence obtained by replacing, deleting or adding one or more amino acids of SEQ ID NO:68 and having the same function as SEQ ID NO:68 or having at least 85% sequence identity with SEQ ID NO:68 and the HCDR1 , HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NO: 16, 17, 18, and the amino acid sequence of the light chain variable region is SEQ ID NO: 69, and SEQ ID NO: 69 is substituted, deleted or added or a plurality of amino acids obtained and having the same amino acid sequence as SEQ ID NO: 69 or having at least 85% sequence identity with SEQ ID NO: 69 and said LCDR1, LCDR2 and LCDR3 such as SEQ ID NO: 19, 20, 21 Amino acid sequence shown.
  • the anti-CRTAM antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are respectively SEQ ID NO:16,17,18, the LCDR1, LCDR2 and LCDR3 of the light chain variable region are SEQ ID NO:19,20,21 respectively, the amino acid sequence of the heavy chain variable region is selected from SEQ ID NO :72, SEQ ID NO:73, SEQ ID NO:74 and SEQ ID NO:75, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74 or SEQ ID NO:75 are substituted, deleted or added Amino acid sequences obtained by one or more amino acids and functionally identical to SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74 or SEQ ID NO:75, and sequences identical to SEQ ID NO:72, SEQ ID NO: 73.
  • SEQ ID NO: 74 or SEQ ID NO: 75 has at least 85% sequence identity and said HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NO: 16, 17, 18, and said light chain can be
  • the amino acid sequence of the variable region is selected from SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78 and SEQ ID NO:79, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78 or SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78 or SEQ ID NO:78 ID NO:79 is obtained by substitution, deletion or addition of one or more amino acids and has the same amino acid sequence as SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78 or SEQ ID NO:79, and SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78 or SEQ ID NO: 79 has at least 85% sequence identity and said LCDR1, LCDR2 and LCDR3 are shown
  • the anti-CRTAM antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are SEQ ID NO: ID NO: 16, 17, 18, the LCDR1, LCDR2 and LCDR3 of the light chain variable region are SEQ ID NO: 19, 20, 21 respectively, the amino acid sequence of the heavy chain variable region is SEQ ID NO: 75 , an amino acid sequence obtained by substituting, deleting or adding one or more amino acids of SEQ ID NO:75 and having the same function as SEQ ID NO:75 or having at least 85% sequence identity with SEQ ID NO:75 and said HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NO: 16, 17, 18, and the amino acid sequence of the light chain variable region is SEQ ID NO: 77, and SEQ ID NO: 77 is substituted, deleted or added with one or The amino acid sequence obtained by multiple amino acids and having the same function
  • the anti-CRTAM antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are SEQ ID NO: ID NO: 22, 23 and 24, the LCDR1, LCDR2 and LCDR3 of the light chain variable region are respectively SEQ ID NO: 26, 27 and 28, the amino acid sequence of the heavy chain variable region is SEQ ID NO: 93 , an amino acid sequence obtained by substituting, deleting or adding one or more amino acids of SEQ ID NO:93 and having the same function as SEQ ID NO:93 or having at least 85% sequence identity with SEQ ID NO:93 and said HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NO:22, 23 and 24, and the amino acid sequence of the light chain variable region is SEQ ID NO:94, and SEQ ID NO:94 is substituted, deleted or added with one or An amino acid sequence obtained from a plurality of amino
  • the anti-CRTAM antibody or antigen-binding fragment thereof according to the present invention comprises a heavy chain variable region and a light chain variable region, wherein HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are SEQ ID NO:22, 23 and 24, the LCDR1, LCDR2 and LCDR3 of the light chain variable region are respectively SEQ ID NO:26, 27 and 28, the amino acid sequence of the heavy chain variable region is selected from SEQ ID NO: 97.
  • SEQ ID NO: 98, SEQ ID NO: 99 and SEQ ID NO: 100, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99 or SEQ ID NO: 100 are substituted, missing or have one added or a plurality of amino acids obtained and with SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99 or SEQ ID NO: 100 functionally identical amino acid sequence, and with SEQ ID NO: 97, SEQ ID NO: 98 , SEQ ID NO:99 or SEQ ID NO:100 have at least 85% sequence identity and said HCDR1, HCDR2 and HCDR3 have amino acid sequences as shown in SEQ ID NO:22, 23 and 24, and said light chain is variable
  • the amino acid sequence of the region is selected from SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104 and SEQ ID NO: 105, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104 or S
  • the anti-CRTAM antibody or antigen-binding fragment thereof according to the present invention comprises a heavy chain variable region and a light chain variable region, wherein HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are SEQ ID NO: ID NOs: 22, 23 and 24, the LCDR1, LCDR2 and LCDR3 of the light chain variable region are SEQ ID NO: 26, 27 and 28 respectively, and the amino acid sequence of the heavy chain variable region is SEQ ID NO: 99 , an amino acid sequence obtained by substituting, deleting or adding one or more amino acids of SEQ ID NO:99 and having the same function as SEQ ID NO:99 or having at least 85% sequence identity with SEQ ID NO:99 and said HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NOs: 22, 23 and 24, and the amino acid sequence of the light chain variable region is SEQ ID NO: 104, and SEQ ID NO: 104 is substituted, deleted or added with one or An amino acid sequence obtained from multiple amino
  • the anti-CRTAM antibody or antigen-binding fragment thereof according to the present invention comprises a heavy chain variable region and a light chain variable region, wherein HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are SEQ ID NO: ID NOs: 22, 25 and 24, the LCDR1, LCDR2 and LCDR3 of the light chain variable region are SEQ ID NO: 26, 27 and 28 respectively, and the amino acid sequence of the heavy chain variable region is SEQ ID NO: 101 , an amino acid sequence obtained by substituting, deleting or adding one or more amino acids of SEQ ID NO:101 and having the same function as SEQ ID NO:101 or having at least 85% sequence identity with SEQ ID NO:101 and said HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NOs: 22, 25 and 24, and the amino acid sequence of the light chain variable region is SEQ ID NO: 104, and SEQ ID NO: 104 is substituted, deleted or added with one or An amino acid sequence obtained from multiple amino
  • the invention provides an anti-CRTAM antibody or antigen-binding fragment thereof, wherein said antibody is a monoclonal antibody. In some embodiments, the invention provides an anti-CRTAM antibody or antigen-binding fragment thereof, wherein the antibody is a murine monoclonal antibody, chimeric antibody, humanized antibody, bispecific antibody, or fully human antibody.
  • the anti-CRTAM antibody according to the present invention is a murine antibody, which also contains the heavy chain constant region of murine IgG1, IgG2a, IgG2b, IgG2c, IgG3 or variants thereof, and murine The light chain constant region of the kappa, lambda chain or variants thereof.
  • the antibody heavy chain of the anti-CRTAM chimeric antibody or its antigen-binding fragment further comprises a heavy chain constant region of murine IgG1, IgG2a, IgG2b, IgG2c, IgG3 or a mutant sequence thereof, preferably Contains human IgG1 or IgG2 heavy chain constant region, or IgG4 constant region that significantly reduces ADCC (antibody-dependent cell-mediated cytotoxicity) toxicity after amino acid mutation.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • the present invention provides an anti-CRTAM humanized antibody or an antigen-binding fragment thereof, wherein the heavy chain comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or a variant thereof, wherein The light chain comprises the light chain constant region of human kappa, lambda chain or variants thereof.
  • the anti-CRTAM humanized antibody or antigen-binding fragment thereof of the present invention further comprises a heavy chain constant region of human IgG1 or IgG2 or a variant thereof, and a light chain of a human ⁇ chain or a variant thereof. Chain constant region.
  • the anti-CRTAM antibody or antigen-binding fragment is an Fc-silenced engineered IgG1 antibody or antigen-binding fragment that has reduced binding to an Fc receptor or Does not bind to Fc receptors.
  • the antibody is an IgG4 antibody.
  • the anti-CRTAM antibody or antigen-binding fragment is a bispecific antibody or antigen-binding fragment that mediates T cell cytotoxicity and/or NK cell cytotoxicity.
  • the anti-CRTAM antibody or antigen-binding fragment is capable of inducing and/or enhancing activation of immune cells.
  • said immune cells are T cells.
  • said immune cells are NK cells.
  • the anti-CRTAM antibody or antigen-binding fragment is a bispecific or multispecific antibody or antigen-binding fragment that binds to a CRTAM protein and binds to one or more additional binding targets, preferably the Additional binding targets are one or more tumor antigens. In some specific embodiments, said one or more additional binding targets are immunomodulatory molecules.
  • the present invention provides an anti-CRTAM antibody or an antigen-binding fragment thereof, wherein the antigen-binding fragment is Fab, Fab', Fv, scFv, F(ab') 2 , F(ab) 2 , dAb or single domain antibodies.
  • Another aspect of the invention provides an isolated nucleic acid encoding an anti-CRTAM antibody or antigen-binding fragment thereof according to the invention.
  • the isolated nucleic acid of the present invention it comprises:
  • an isolated nucleic acid according to the invention comprising:
  • the isolated nucleic acid of the present invention it comprises:
  • an isolated nucleic acid according to the invention comprising:
  • the isolated nucleic acid of the present invention it comprises:
  • an isolated nucleic acid according to the invention comprising:
  • the isolated nucleic acid of the present invention it comprises:
  • an isolated nucleic acid according to the invention comprising:
  • Another aspect of the present invention provides an expression vector expressing the anti-CRTAM antibody or antigen-binding fragment thereof of the present invention.
  • An expression vector according to the present invention which comprises the isolated nucleic acid molecule of the present invention.
  • a chimeric antigen receptor (CAR) fusion protein comprising the anti-CRTAM antibody of the present invention or an antigen-binding fragment thereof.
  • the chimeric antigen receptor fusion protein comprises an anti-CRTAM antibody or antigen-binding fragment thereof of the invention, which is a single chain variable fragment (scFv) directed against the VH and VL of the CRTAM antigen.
  • the scFv against VH and VL of CRTAM antigen has HCDR1, HCDR2 and HCDR3 of the heavy chain variable region and LCDR1, LCDR2 and LCDR3 of the light chain variable region described in the above embodiments.
  • Another aspect of the present invention provides a host cell transformed with the above expression vector.
  • host cells according to the invention are selected from prokaryotic cells and eukaryotic cells.
  • the host cell is bacteria, preferably Escherichia coli.
  • said host cell is a mammalian cell.
  • Another aspect of the present invention provides a method for preparing the anti-CRTAM antibody or antigen-binding fragment thereof of the present invention, comprising the steps of expressing the antibody in the host cell and isolating the antibody from the host cell.
  • compositions which comprises the anti-CRTAM antibody or antigen-binding fragment thereof of the present invention and a pharmaceutically acceptable carrier.
  • the present invention provides a pharmaceutical composition, which comprises the anti-CRTAM antibody or antigen-binding fragment thereof of the present invention, and further comprises other active components, such as other antibodies, targeted drugs, and the like.
  • the pharmaceutically acceptable carrier is selected from antioxidants, polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, sugar alcohols, ions, and surfactants.
  • the pharmaceutically acceptable carrier is a buffered aqueous solution.
  • said pharmaceutically acceptable carrier is in the form of liposomes.
  • the anti-CRTAM humanized antibody or antigen-binding fragment thereof of the present invention can be mixed with pharmaceutically acceptable carriers, diluents or excipients to prepare pharmaceutical preparations suitable for oral or parenteral administration.
  • Methods of administration include, but are not limited to, oral, intradermal, intramuscular, intraperitoneal, intravenous, intracerebral, intraocular, intratracheal, subcutaneous, and intranasal routes.
  • the formulation can be administered by any route, for example, by infusion or bolus injection, or by absorption through the epithelium or mucocutaneous (eg, oral mucosa or rectum, etc.). Administration can be systemic or local.
  • the formulations can be prepared by methods known in the art, and contain carriers, diluents or excipients commonly used in the field of pharmaceutical formulations.
  • Another aspect of the invention provides a method of treating cancer comprising administering to an individual in need thereof an anti-CRTAM antibody or antigen-binding fragment thereof of the invention or a pharmaceutical composition of the invention.
  • the tumor is selected from lung cancer, leukemia, lymphoma, breast cancer, gastric cancer, intestinal cancer, esophageal cancer, ovarian cancer, testicular cancer, thyroid cancer, bone cancer, cervical cancer, uterine cancer, kidney cancer, Liver cancer, gallbladder cancer, bile duct cancer, bladder cancer, pancreatic cancer, glioma, skin cancer, prostate cancer, nasopharyngeal cancer, glioma, glioblastoma, sarcoma and neuroendocrine tumors.
  • the tumor is selected from small cell lung cancer, non-small cell lung cancer, melanoma, hepatocellular carcinoma, hematological malignancies, lymphoma, breast cancer, gastric cancer, bowel cancer, esophageal cancer, ovarian cancer, Cervical cancer, kidney cancer, bladder cancer, pancreatic cancer and glioma.
  • the anti-CRTAM antibody or antigen-binding fragment thereof provided by the present invention has significant anti-tumor effect, does not affect the combination of CRTAM and its ligand, does not affect its normal function, and at the same time, the immunogenicity of the humanized antibody is greatly reduced, effectively eliminating the The rejection reaction of the immune system to the exogenous monoclonal antibody can be used in the preparation of drugs for the treatment of various tumor diseases, and has broad market prospects.
  • the term "at least 80% sequence identity” means at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity.
  • the term “at least 85% sequence identity” means at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity.
  • sequence identity of the present invention can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% %. Sequence comparison and determination of percent identity between two sequences can be performed by the BLASTN/BLASTP algorithm on the website of the National Center For Biotechnology Institute.
  • the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged relative to each other in three-dimensional space to form an antigen-binding surface.
  • the antigen-binding surface is complementary to the three-dimensional surface of the bound antigen, and the three hypervariable regions of each heavy and light chain are referred to as "complementarity determining regions" or "CDRs.”
  • CDRs complementarity determining regions
  • the “antibody” of the present invention refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen.
  • Antibodies can be whole antibodies and any antigen-binding fragments or single chains thereof.
  • An “antibody” of the present invention includes any protein or peptide comprising an Ig molecule that has at least a portion of the biological activity of binding an antigen.
  • Examples of “antibodies” of the invention include, but are not limited to, the CDRs of a heavy or light chain or a ligand-binding portion thereof, a heavy or light chain variable region, a heavy or light chain constant region, a framework region, or any portion thereof.
  • the "antigen-binding fragment” in the present invention refers to Fab fragments, Fab' fragments, F(ab') 2 fragments, Fv fragments and scFv fragments that bind to human CRTAM with antigen-binding activity.
  • the Fv fragment contains the antibody heavy chain variable region and the light chain variable region, but has no constant region, and has the smallest antibody fragment with all antigen-binding sites.
  • Fv antibodies also contain a polypeptide linker between the VH and VL domains and are capable of forming the structures required for antigen binding. Different linkers can also be used to connect two antibody variable regions into one polypeptide chain, called single-chain antibody or single-chain Fv (scFv).
  • the anti-CRTAM antibody of the present invention may be a single-chain variable region fragment (scFv), which is derived from a single-chain polypeptide of an antibody and retains the ability to bind antigen.
  • scFv single-chain variable region fragment
  • Examples of scFv include antibody polypeptides formed by recombinant DNA techniques in which the Fv regions of immunoglobulin heavy chain (H chain) and light chain (L chain) fragments are linked via a spacer sequence.
  • H chain immunoglobulin heavy chain
  • L chain light chain
  • the antibody of the present invention refers to an immunoglobulin molecule or an immunologically active part thereof, that is, a molecule comprising an antigen-binding site that specifically binds to (immunoreacts with) an antigen.
  • "Specifically binds” means that an antibody reacts with one or more epitopes of an antigen but does not react with or binds other polypeptides with very low affinity (Kd > 10-6).
  • Antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, dAb (domain antibody), single chain, Fab, Fab' and F(ab')2 fragments, Fv, scFv, and Fab expression libraries.
  • Monoclonal antibodies are antibodies derived from a single clonal cell strain, not limited to eukaryotic, prokaryotic, or phage clonal cell strains. Monoclonal antibodies or antigen-binding fragments can be recombined using hybridoma technology, recombinant technology, phage display technology and synthetic technology such as CDR grafting or other existing technologies.
  • the "mouse antibody” described in the present invention is a monoclonal antibody against human CRTAM prepared according to the knowledge and skills in the art. In preparation, test subjects are injected with hCRTAM antigen, and hybridomas expressing antibodies having the desired sequence or functional properties are isolated.
  • the "chimeric antibody” of the present invention is an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can reduce the immune response induced by the murine antibody.
  • To establish a chimeric antibody it is necessary to first establish a hybridoma that secretes a mouse-derived specific monoclonal antibody, then clone the variable region gene from the mouse hybridoma cell, and then clone the constant region gene of the human antibody as required, and then clone the variable region gene of the mouse
  • the gene is connected with the human constant region gene to form a chimeric gene and then inserted into a human vector, and finally the chimeric antibody molecule is expressed in a eukaryotic industrial system or a prokaryotic industrial system.
  • the "humanized antibody” of the present invention is also called a CDR-grafted antibody, which is an antibody produced by grafting a mouse CDR sequence into a human antibody variable region framework (FR).
  • FR human antibody variable region framework
  • Such variable region framework sequences can be obtained from public DNA databases or published references, eg from the ImMunoGeneTics (IMGT) website http://imgt.cines.fr or from Immunoglobulin Journal, 2001 ISBN012441351.
  • Fig. 1 is the result of an anti-CRTAM humanized antibody blocking activity of human CRTAM binding to CADM1 (ELISA), wherein the abscissa is the antibody concentration (ng/mL), and the ordinate is the absorbance value at OD450.
  • Figure 2 is the experimental results (ELISA) of the competition between the anti-CRTAM antibody HuC68-17 and 5A11 for binding to human CRTAM, where the abscissa is the antibody concentration (ng/mL), and the ordinate is the absorbance at OD450.
  • Figure 3 is the experimental results (ELISA) of the competition between the anti-CRTAM antibody HuC94-31 and 5A11 for binding to human CRTAM, where the abscissa is the antibody concentration (ng/mL), and the ordinate is the absorbance at OD450.
  • Figure 4 is the experimental results (ELISA) of the competition between the anti-CRTAM antibody HuC110-34 and 5A11 for binding to human CRTAM, where the abscissa is the antibody concentration (ng/mL), and the ordinate is the absorbance at OD450.
  • Figure 5 is the experimental results (ELISA) of the competition between the anti-CRTAM antibody HuC124-42 and 5A11 for binding to human CRTAM, where the abscissa is the antibody concentration (ng/mL), and the ordinate is the absorbance at OD450.
  • a gene fragment encoding the full-length human CRTAM protein was synthesized, and the amino acid sequence was designed as shown in SEQ ID NO:29.
  • the nucleotide sequence was cloned into the eukaryotic expression plasmid pTargetT to obtain the expression plasmid pT-hCRTAM.
  • a gene fragment encoding the full-length CRTAM protein of monkey origin was synthesized, and the amino acid sequence design was shown in SEQ ID NO:33.
  • the nucleotide sequence was cloned into the eukaryotic expression plasmid pTargetT to obtain the expression plasmid pT-cCRTAM.
  • the amino acid sequence of the fused extracellular region of human CRTAM protein and the mIgG1-Fc tag is shown in SEQ ID NO:30. After codon optimization of the extracellular domain sequence of human CRTAM protein, the nucleotide sequence of hCRTAM-mFc with tag was synthesized and cloned into the eukaryotic expression plasmid pHR to obtain its expression plasmid pHR-hCRTAM-mFc.
  • the amino acid sequences of the fused human CRTAM protein extracellular region and hIgG1-Fc or His tag are shown in SEQ ID NO: 31 and SEQ ID NO: 32.
  • the tagged hCRTAM- The nucleotide sequences of hFc and hCRTAM-His were cloned into the eukaryotic expression plasmid pHR respectively to obtain the expression plasmids pHR-hCRTAM-hFc and pHR-hCRTAM-His.
  • the amino acid sequence of the fused extracellular region of the monkey-derived CRTAM protein and the mIgG1-Fc tag is shown in SEQ ID NO:34. After codon-optimizing the extracellular domain sequence of the monkey-derived CRTAM protein, the nucleotide sequence of cCRTAM-mFc with a tag was synthesized and cloned into the eukaryotic expression plasmid pHR to obtain its expression plasmid pHR-cCRTAM-mFc.
  • the amino acid sequences of the fused extracellular region of the monkey-derived CRTAM protein and the hIgG1-Fc or His tag are shown in SEQ ID NO:35 and SEQ ID NO:36.
  • the tagged cCRTAM- The nucleotide sequences of hFc and cCRTAM-His were cloned into the eukaryotic expression plasmid pHR respectively to obtain the expression plasmids pHR-cCRTAM-hFc and pHR-cCRTAM-His.
  • the amino acid sequence of the fused extracellular region of the mouse CRTAM protein and the mIgG1-Fc tag is shown in SEQ ID NO:37. After codon-optimizing the extracellular domain sequence of the mouse CRTAM protein, the nucleotide sequence of the tagged mCRTAM-mFc was synthesized and cloned into the eukaryotic expression plasmid pHR to obtain its expression plasmid pHR-mCRTAM-mFc.
  • amino acid sequences of the fused extracellular region of the mouse CRTAM protein and the hIgG1-Fc or His tag are shown in SEQ ID NO:38 and SEQ ID NO:39, and the codon optimization of the above amino acid sequence is performed to synthesize the tagged mCRTAM-
  • the nucleotide sequences of hFc and mCRTAM-His were cloned into the eukaryotic expression plasmid pHR respectively to obtain the expression plasmids pHR-mCRTAM-hFc and pHR-mCRTAM-His.
  • the amino acid sequence of the fused extracellular region of human CADM1 protein and the mIgG1-Fc tag is shown in SEQ ID NO: 40, and the nucleotide sequence of the tagged CADM1-mFc was synthesized after codon optimization of the above amino acid sequence. It was cloned into the eukaryotic expression plasmid pHR to obtain its expression plasmid pHR-CADM1-mFc.
  • the amino acid sequences of the fused human CADM1 protein extracellular region and hIgG1-Fc or His tag are shown in SEQ ID NO: 41 and SEQ ID NO: 42. After codon optimization of the above amino acid sequence, the tagged CADM1- The nucleotide sequences of hFc and CADM1-His were cloned into the eukaryotic expression plasmid pHR respectively to obtain the expression plasmids pHR-CADM1-hFc and pHR-CADM1-His.
  • the humanized antibody 5A11 (hereinafter referred to as 5A11) disclosed in PCT application WO2019/086878 was used as a positive control antibody. 5A11 was prepared according to the method disclosed in WO2019/086878. The amino acid sequence of 5A11 is as follows:
  • 5A11 heavy chain amino acid sequence SEQ ID NO: 43;
  • the codons of the amino acid sequence corresponding to the 5A11 antibody were artificially optimized, and the light and heavy chain gene fragments were respectively cloned into the eukaryotic expression plasmid pHR to obtain the heavy chain eukaryotic expression plasmid pHR-5A11-hG1m of 5A11, and the light chain expression Plasmid pHR-5A11-h ⁇ .
  • the eukaryotic expression plasmid pT-hCRTAM was electrotransfected into CHO-K1 cells (Shanghai Institute of Cell Biology, Chinese Academy of Sciences) at a voltage of 160V and a square pulse of 15msec . cultured in an incubator. After 24 hours, medium containing 1000 ⁇ g/mL G418 (Gibco, #10131-027) was used for pressurized culture.
  • the positive rate of the transfected pool was detected by flow cytometry, and the cells of the pool with a higher positive rate were plated (according to the cell density of 1 ⁇ 106 cells/mL, 100 ⁇ L/well, plated in a 96-well plate) , the cells were incubated with 5A11 antibody and Goat pAb to Hu IgG (PE) (Abcam, ab98596) antibody, the mean value at 392nm wavelength was detected by flow cytometry (ACEABIO, Novocyte 2060R), and the data was generated by GraphPad for data analysis.
  • the positive cell line was subcloned, and the cloned CHO-K1 cell line with high expression of hCRTAM protein was selected and named as CHO-K1-hCRTAM.
  • the eukaryotic expression plasmid pT-cCRTAM was transfected into CHO-K1 cells (Shanghai Institute of Cell Biology, Chinese Academy of Sciences) by electroporation under 160V voltage and 15msec square pulse, and placed in 37°C, 5% CO2 concentration cultured in an incubator. After 24 hours, medium containing 1000 ⁇ g/mL G418 (Gibco, #10131-027) was used for pressurized culture.
  • the positive rate of the transfected pool was detected by flow cytometry, and the cells of the pool with a higher positive rate were plated (according to the cell density of 1 ⁇ 106 cells/mL, 100 ⁇ L/well, plated in a 96-well plate) , the cells were incubated with 5A11 antibody and Goat pAb to Hu IgG (PE) (Abcam, ab98596) antibody, the mean value at 392nm wavelength was detected by flow cytometry (ACEABIO, Novocyte 2060R), and the data was generated by GraphPad for data analysis.
  • the positive cell lines were subcloned, and the cloned CHO-K1 cell line with high expression of cCRTAM protein was selected and named as CHO-K1-cCRTAM.
  • Inoculate 293E cells (derived from ATCC) at a density of 1 ⁇ 106/ml in a 1L cell culture flask, add fresh preheated FreeStyle293 medium to make the total volume after inoculation reach 250mL, and store at 37°C, 8% CO 2. Cultivate overnight at 100 rpm in a humidified cell shaker. Take 7.5mL FreeStyle293 medium, add 500 ⁇ L of 1mg/mL PEI solution, mix well, and let it stand for 5min.
  • the mixed solution of PEI and FreeStyle293 medium was added to the plasmid, mixed evenly, and allowed to stand at room temperature for 20 minutes, then added to the cell culture, and cultured at 37° C., 8% CO 2 , in a humidified cell shaker at 100 rpm.
  • the cells were fed, and 12.5 mL of OPM-CHO PFF05 (Shanghai OPM Biotechnology Co., Ltd., F81279) and 5 mL of glucose (the concentration of the mother solution was 180 g/L) were added to each bottle. ) and 2.5mL of glutamine (the concentration of the stock solution is 200mM).
  • the cell culture was centrifuged at 2000rpm for 5min to collect the supernatant, and then centrifuged at 6000rpm for 20min to collect the supernatant, filtered using 0.45 ⁇ m and 0.22 ⁇ m filter cups respectively, and the collected filtrate was stored in a refrigerator at 4°C until purification.
  • AKTA GE, AKTA pure-150
  • affinity chromatography column according to protein properties (see Table 1 for affinity chromatography columns adapted to different proteins), and the specific purification steps are as follows:
  • Sample loading load the cell expression supernatant with a retention time of 5 minutes;
  • On-line cleaning 0.1M NaOH for 30 minutes, retention time 5 minutes;
  • Embodiment 2 Preparation of anti-CRTAM monoclonal antibody
  • the hCRTAM antigen proteins with different labels and adjuvant are used to immunize experimental animals together, or CHO-K1-hCRTAM cell line is used for cellular immunization.
  • Experimental animals include Balb/c strain mice, SJL strain mice and SD rats.
  • Protein immunization For mouse immunization, a mouse was immunized with 50 ⁇ g antigen for the first immunization, and 25 ⁇ g/mouse of antigenic protein was used in the later stages; SD rat immunization was immunized with 100 ⁇ g antigen for the first immunization, and 50 ⁇ g/mouse of antigenic protein was used in the later stages.
  • the immune adjuvant can be Freund's adjuvant (Sigma) or Quick Antibody-Mouse5W (Q5W, Beijing Boaolong Immunotechnology Co., Ltd.). Freund's adjuvant was used to emulsify the antigen, and hCRTAM antigen protein samples with different labels were added dropwise to the adjuvant solution, and vortexed while dropping to mix thoroughly, and the dosage of the adjuvant was carried out according to the instructions. Mix well to form a water-in-oil emulsion and immunize. Using Quick Antibody-Mouse5W as an adjuvant, hCRTAM antigen protein samples with different labels were mixed with Quick Antibody-Mouse5W at a volume ratio of 1:1.
  • mice were immunized by intramuscular injection.
  • Cellular immunization CHO-K1-hCRTAM cells were resuspended in PBS, mice were immunized at 1 ⁇ 10 7 cells/mouse; SD rats were immunized at 2 ⁇ 10 7 cells/mouse.
  • the immunization scheme is shown in Table 2.
  • mice/rats after booster immunization were sacrificed and soaked in 75% alcohol, the spleen was dissected out, ground with a grinding rod, and filtered through a cell mesh to prepare a single cell suspension.
  • the spleen cell suspension was centrifuged at 2000 rpm for 5 min, and the supernatant was discarded.
  • the cell suspension was transferred to 15 mL RPMI 1640 complete medium containing 20% FBS, and left at room temperature for 20 min. Resuspend the confluent cells in RPMI 1640 medium containing 1 ⁇ HAT, 1 ⁇ BIOMYC3, and 20% FBS. Add the cell suspension to several 96-well cell culture plates at 100 ⁇ L/well to ensure that the cell volume per well is about 4 ⁇ 10 4 cells/well, and culture in a 37°C cell culture incubator. After 5 days, 100 ⁇ L/well RPMI 1640 complete medium (containing 20% FBS, 1 ⁇ HAT, 1 ⁇ BIOMYC-3) was added.
  • the cell culture supernatant of the hybridoma mother clone was taken, and the hybridoma mother clone binding to hCRTAM protein and cCRTAM protein was screened by ELISA, and the mother clone capable of binding to CHO-K1-hCRTAM cell line was further screened by flow cytometry .
  • the mother clones with strong binding ability were screened by ELISA and FACS, and the positive mother clones were subcloned by the limiting dilution method. After one week of culture, the binding activity of the supernatant of the subclones and hCRTAM protein was detected by ELISA, and then the secretory anti-hCRTAM antibody was obtained. monoclonal cell line. A number of better monoclonal cell lines were obtained, labeled as C68, C94, C110, and C124 respectively.
  • the monoclonal antibody cell line was determined according to the activity analysis results of the supernatant of the subclones, and expanded for culture.
  • the culture condition is 1640 medium containing 10% FBS, 1 ⁇ NAEE, 1 ⁇ sodium pyruvate, and 1% penicillin-streptomycin double antibody.
  • the confluence of the cells is greater than >80%, the cells are subcultured and expanded.
  • the culture reaches about 50 mL, the supernatant is collected and the antibody is purified. The purity of the obtained antibody was confirmed by SDS-PAGE gel electrophoresis.
  • the subcloned positive hybridoma cells were expanded and cultured, and an appropriate amount of cells was taken to extract total RNA according to the instructions of the RNeasy Plus Mini Kit (Qiagen, 74134) kit, and reverse transcription was performed using the Prime Script 1st strand cDNA Synthesis Kit (Takara, 6110A) The kit synthesizes the first strand of cDNA.
  • Design universal primers based on the conserved sequences at both ends of the variable region of the antibody (the 5' end contains the homologous arm sequence for homologous recombination with the eukaryotic expression vector), and use cDNA as a template to perform PCR amplification of the variable region gene of the antibody. Thereby obtaining the gene fragments of mouse anti-light chain and heavy chain variable domains respectively; Design primers (references: 1. Anke Krebber, Susanne Bornhauser, Jorg Burmester et al. a reengineered phage display system.
  • Embodiment 3 Construction of anti-CRTAM chimeric antibody
  • the purified mouse antibody heavy chain and light chain variable region gene fragments were respectively combined with the linearized eukaryotic expression plasmid pHR-hG1m/pHR containing the human antibody heavy chain or light chain constant region -h ⁇ co-transformed Escherichia coli DH5 ⁇ competent cells.
  • the mixture was evenly spread on the surface of the agar plate containing the ampicillin antibiotic, cultured upside down in a constant temperature incubator at 37°C overnight, and then several single colonies were picked for DNA sequencing.
  • the chimeric antibodies with correct sequencing were marked as C68CHI, C94CHI, C110CHI, and C124CHI, respectively.
  • the amino acid sequences of heavy chain variable regions and light chain variable regions of antibodies C68CHI, C94CHI, C110CHI, and C124CHI are identical to those of the corresponding murine antibodies C68, C94, C110, and C124.
  • the chimeric antibody heavy and light chain plasmids were co-transfected into HEK293E cells, expressed and purified to obtain the chimeric antibody, and then the purity test, activity analysis and affinity test were performed.
  • the chimeric antibody C68CHI was mutated to screen for better antibodies.
  • the 53rd N in the heavy chain variable region of C68CHI is mutated to S, which improves the stability of the antibody and is labeled as C68CHIm1.
  • the amino acid sequence of the heavy chain variable region of C68CHIm1 is SEQ ID NO: 106
  • the amino acid sequence of the light chain variable region is SEQ ID NO: 46
  • HCDR1, HCDR2 and HCDR3 are SEQ ID NO: 8
  • LCDR1, LCDR2 and LCDR3 are SEQ ID NO: 12, 13 and 14, respectively.
  • the chimeric antibody sequences are shown in Table 4.
  • multiple chimeric antibodies with good activity such as C68CHIm1, C94CHI, C110CHI, and C124CHI, were selected for humanized antibody transformation.
  • the first step is to compare the mouse antibody sequences in the Immune Gene Database (IMGT) to confirm the mouse germlines of the variable regions of the C68CHIm1, C94CHI, C110CHI, and C124CHI antibodies.
  • IMGT Immune Gene Database
  • the FR regions of the heavy chain variable region sequences of C68CHIm1, C94CHI, C110CHI, and C124CHI antibodies are respectively related to mouse antibody germline genes (IGHV3-2*02, IGHV5-9-1*01, IGHV1-85*01, IGHV1- 9*01) were most similar; the FR sequences of antibody light chain variable regions were most similar to mouse antibodies (IGKV1-110*01, IGKV10-96*01, IGKV1-110*01, IGKV5-48*01).
  • C68CHIm1, C94CHI, C110CHI, C124CHI antibody framework region sequence FR1-FR3 search for a fully human framework with similar 3D structure but low immunogenicity in the human framework region library to replace FR1-FR3 of C68CHIm1, C94CHI, C110CHI, C124CHI Sequence, heavy chain/light chain full-length sequence was used for 3D modeling and structural comparison analysis with the original antibody heavy chain/light chain sequence, considering antigenicity and 3D structural similarity, and finally selected 5 humanized heavy chains of C68CHIm1 Chain variable region (see SEQ ID NO:50 ⁇ 54) and 12 humanized light chain variable regions (see SEQ ID NO:55 ⁇ 59, 60 ⁇ 66), 4 humanized heavy chains of C94CHI can be Variable region (see SEQ ID NO:72 ⁇ 75) and 4 humanized light chain variable regions (see SEQ ID NO:76 ⁇ 79), 4 humanized heavy chain variable regions of C110CHI (see SEQ ID NO:84 ⁇ 87) and 5 humanized light chain variable regions (see SEQ ID
  • Humanized antibodies with good purity, activity and affinity were selected and labeled as HuC68-16, HuC94-31, HuC110-32 and HuC124-22 respectively. The sequences are shown in Table 5.
  • the humanized antibodies HuC68-16, HuC110-32, and HuC124-22 were mutated to screen for better antibodies.
  • the 34th G in the variable region of the light chain of HuC68-16 was mutated to K (see SEQ ID NO: 67), the stability of the antibody was improved, and it was labeled as HuC68-17.
  • the 34th G in the variable region of the light chain of HuC110-32 was mutated to K (see SEQ ID NO: 92), which improved the stability of the antibody and was labeled as HuC110-34.
  • the 62nd G in the variable region of the heavy chain of HuC124-22 is mutated to A (see SEQ ID NO: 101), the stability of the antibody is improved, and it is labeled as HuC124-42.
  • the optimized antibody sequences are shown in Table 6.
  • an appropriate antibody subtype framework is selected to match the optimized humanized antibody variable region to construct a complete humanized antibody.
  • the oligonucleotide fragments were annealed and ligated by Overlap PCR, and then the complete light chain was amplified using specific primers (the 5' end contains homology arm sequences for homologous recombination with eukaryotic expression vectors) and the heavy chain variable region nucleotide fragment; the purified light chain variable region nucleotide fragment and the linearized eukaryotic expression plasmid containing the human ⁇ light chain constant region were co-transformed into Escherichia coli DH5 ⁇ competent cells, and the The purified nucleotide fragment of the heavy chain variable region and
  • the positive clones with correct sequencing were inoculated in 2 ⁇ YT liquid medium containing ampicillin antibiotics, cultured with shaking at 37°C for more than 12 hours, and then the bacteria were collected for plasmid extraction to obtain humanized antibody light chain and heavy chain expression plasmids , Use a nucleic acid quantitative analyzer to detect the concentration and purity of the plasmid.
  • the plasmid was transfected into HEK293E cells, expressed and purified to obtain a large amount of antibodies, and the purity test, activity analysis and affinity test were performed.
  • the complete humanized antibody sequences are listed in Table 7.
  • hCRTAM-His protein (1.5 ⁇ g/mL, 50 ⁇ L/well, prepared in Example 1) was coated onto a 96-well microtiter plate, and incubated at 37° C. for 2 h. After washing 3 times with 1x PBST, block with PBST containing 5% skimmed milk at 37°C for 2h.
  • the anti-CRTAM antibody of the present invention was used as a primary antibody starting from 10 ⁇ g/mL, and added to a microtiter plate (50 ⁇ L/well) by 5-fold gradient dilution, with a total of 8 concentrations, the concentrations were 10000 ng/mL and 2000 ng respectively.
  • control antibody was 5A11 (prepared in Example 1); After washing with 1x PBST for 3 times, pat dry, use Anti-Human IgG HRP (Jackson, 109-035-003, 1:5000, 50 ⁇ L/well) as the secondary antibody, and incubate at 37°C for 1 hour.
  • Anti-Human IgG HRP Jackson, 109-035-003, 1:5000, 50 ⁇ L/well
  • Antibody binding activity was analyzed by ELISA.
  • the cCRTAM-His protein (2 ⁇ g/mL, 50 ⁇ L/well, prepared in Example 1) was coated onto a 96-well microtiter plate, and incubated at 37° C. for 2 h. After washing 3 times with 1x PBST, block with PBST containing 5% skimmed milk at 37°C for 2h.
  • the anti-CRTAM antibody of the present invention was used as a primary antibody starting from 10 ⁇ g/mL, and added to a microtiter plate (50 ⁇ L/well) by 5-fold gradient dilution, with a total of 8 concentrations, the concentrations were 10000 ng/mL and 2000 ng respectively.
  • control antibody was 5A11 (prepared in Example 1); After washing with 1x PBST for 3 times, pat dry, use Anti-Human IgG HRP (Jackson, 109-035-003, 1:5000, 50 ⁇ L/well) as the secondary antibody, and incubate at 37°C for 1 hour.
  • Anti-Human IgG HRP Jackson, 109-035-003, 1:5000, 50 ⁇ L/well
  • Example 7 Determination of the blocking activity of anti-CRTAM antibodies to CRTAM and its ligand CADM1 (ELISA)
  • hCRTAM-His protein (1.5 ⁇ g/mL, 50 ⁇ L/well, prepared in Example 1) was coated onto a 96-well microtiter plate, and incubated at 37° C. for 2 h. After washing 3 times with 1x PBST, block with PBST containing 5% skimmed milk at 37°C for 2h. At the same time, the ligand protein hCADM1-His was labeled with biotin-NHS ester (biovsion, 2347-50), and the labeled ligand was named hCADM1-His-biotin.
  • the hCADM1-His-biotin ligand solution with a concentration of 20 ⁇ g/mL was configured, and the primary antibody was diluted with the above ligand solution as a diluent, and the anti-CRTAM antibody of the present invention was used as a primary antibody.
  • the concentrations were 100000ng/mL, 33333ng/mL, 11111ng/mL, 3704ng/mL, 1235ng/mL, 412ng /mL, 137ng/mL, 46ng/mL, incubated at 37°C for 1.5h
  • the control antibody was 5A11 (prepared in Example 1); washed 3 times with 1x PBST and patted dry
  • the secondary antibody was Streptavidin-HRP (BD, 554066 , 1:10000, 50 ⁇ L/well), and incubated at 37°C for 1h.
  • Embodiment 8 Determination of binding activity of anti-CRTAM antibody to human CRTAM on cell surface (FACS)
  • the binding activity of the antibody to hCRTAM on the surface of CHO-K1-hCRTAM cells was analyzed by FACS. After the CHO-K1-hCRTAM cells were digested, they were resuspended with 2% FBS-PBS solution and counted. Spread the above cells on a cell plate in a manner of 1.5x105 cells per well.
  • the anti-CRTAM antibody of the present invention is used as a primary antibody starting from 20 ⁇ g/ml, and added to the cell plate in a 5-fold gradient dilution, with a total of 8 concentrations, and the concentration is 20000 ng/ml.
  • control antibody is 5A11; wash three times with PBS After cells, the secondary antibody was incubated with PE-Anti-Human IgG (Biolegend, Cat. No. 409303, 1.25 ⁇ l/well) at 4°C for 1 h; after washing with PBS three times, flow cytometry was used to detect the antibody produced by binding to the cell surface. Fluorescence intensity, the results are shown in Table 10.
  • the binding activity of the antibody to cCRTAM on the surface of CHO-K1-cCRTAM cells was analyzed by FACS. After the CHO-K1-cCRTAM cells were digested, they were resuspended with 2% FBS-PBS solution and counted. Spread the above cells on a cell plate in a manner of 1.5x105 cells per well.
  • the anti-CRTAM antibody of the present invention is used as a primary antibody starting from 20 ⁇ g/ml, and added to the cell plate in a 5-fold gradient dilution, with a total of 8 concentrations, and the concentration is 20000 ng/ml.
  • Antibody name HuC94-31 HuC110-34 HuC124-42 EC50(ng/mL) 35.02 50.69 90.38
  • the ELISA method was used to analyze the epitope competition activity of the anti-CRTAM antibody of the present invention and 5A11 on human CRTAM.
  • hCRTAM-His protein (2 ⁇ g/mL, 50 ⁇ L/well, prepared in Example 1) was coated onto a 96-well microtiter plate, and incubated at 37° C. for 2 h. After washing 3 times with 1x PBST, block with PBST containing 5% skimmed milk at 37°C for 2h.
  • the anti-CRTAM antibody of the present invention was labeled with Biotin-NHS ester TM (Biovsion Company, 2347-50), and the labeled antibody was named as HuC68-17-biotin, HuC94-31-biotin, HuC110-34-biotin, HuC124-biotin, respectively. 42-biotin.
  • the anti-CRTAM antibody of the present invention (competition positive control) and 5A11 are used as the primary antibody starting from 100 ⁇ g/mL, 5-fold serial dilution is added to the microplate (50 ⁇ L/well), a total of 8 concentrations, the concentration 100000ng/mL, 20000ng/mL, 4000ng/mL, 800ng/mL, 160ng/mL, 32ng/mL, 6.4ng/mL, 1.28ng/mL, incubate at 37°C for 1.5h; wash with 1x PBST for 3 times and shoot Dry, use Streptavidin-HRP (BD, 5540
  • the experimental results show that the humanized anti-CRTAM antibodies HuC68-17, HuC94-31, HuC110-34, HuC124-42 of the present invention have almost no epitope competition with 5A11, and do not block the binding of 5A11 and human CRTAM, indicating that the present invention
  • the humanized anti-CRTAM antibody has a completely different binding epitope to CRTAM than 5A11.
  • the binding affinity of the humanized anti-CRTAM antibody of the present invention to the antigen hCRTAM-His was determined by Fortebio Octet.
  • the anti-CRTAM antibody was diluted to a concentration of 10 ⁇ g/mL with SD buffer (PBS+0.02% Tween20+0.1% BSA), and the antigen hCRTAM-His was diluted with a 4-fold concentration gradient of SD buffer to make its concentration 12 ⁇ g/mL, 3 ⁇ g/mL mL, 0.75 ⁇ g/mL, 0 ⁇ g/mL, the AHC sensor was used to immobilize the antibody, and the affinity was determined according to the operating procedures of Fortebio Octet RED96. The specific parameters and experimental results are shown in Table 12.
  • SEB staphylococcal enterotoxin B was used to stimulate healthy human PBMC to detect the in vitro efficacy of anti-CRTAM antibody.
  • PBMCs Resuscitate PBMCs according to the required cell volume, add them to 8-9ml IMDM complete medium, centrifuge at 900g for 10min, discard the supernatant; Add the wells into a 96-well plate; use 4 times the concentration (40 ⁇ g/mL), 50 ⁇ L/well, prepare the antibody to be detected and Isotype with IMDM complete medium, mark it, and vortex; add the antibody solution into the 96-well plate, Add 50 ⁇ L/well of culture medium to the control group, place the 96-well plate in a 37°C incubator, and incubate the cells and antibodies for 1 h; Corresponding wells; 96-well plates were placed in a 37°C, 5% CO 2 incubator and incubated for 72 hours, and 150 ⁇ L of cell-free supernatant was collected, diluted in a certain proportion, and tested according to the hIFN- ⁇ ELISA detection kit (R&D ELISA kit, R&D system Cat: DY
  • Table 13 anti-CRTAM antibody promotes PBMC to release IFN- ⁇
  • Example 13 Anti-tumor experiment of HCC827 (del19) human non-small cell lung cancer cell PBMC reconstruction model 1. Experimental materials
  • HCC827 (del19) cells were purchased from the American Type Culture Collection (ATCC);
  • mice female, 6-8 weeks old, weighing 18-20 grams, were purchased from Biocytogen (Jiangsu) Gene Biotechnology Co., Ltd.;
  • control substance 5A11 was prepared in Example 1 and used as a positive control; the reference substance PBS was used as a negative control; before the test, the anti-CRTAM antibody of the present invention was prepared in PBS to a concentration of 1 mg/mL.
  • PBMCs come from the fresh blood of healthy blood donors, and are injected into the tail vein 1 hour before the antibody is given after grouping on DAY1 (10M PBMC cells are injected per mouse). The tail vein was administered twice a week, and the tumor diameter was measured twice a week with a vernier caliper, and the tumor volume was calculated.
  • the tumor volume measured for 22 days of continuous administration was recorded, and the tumor volume growth curve was drawn with GraphPad Prism. After the experiment, the tumor weight was detected, and the tumor inhibition rate (TGI)% of each group was calculated. The results are shown in Table 14.
  • Example 14 Anti-tumor experiment of NUGC-4 human gastric cancer cell PBMC reconstruction model
  • NUGC-4 human gastric cancer cells were purchased from the American Type Culture Collection (ATCC);
  • mice female, 6-8 weeks old, weighing 18-20 grams, were purchased from Biocytogen (Jiangsu) Gene Biotechnology Co., Ltd.;
  • the control substance is PBS, which is used as a negative control; before the test, the anti-CRTAM antibody of the present invention is prepared with PBS to 1 mg/mL.
  • mice Mix 100 ⁇ L PBS containing 5 ⁇ 10 6 NUGC-4 (del19) cells with 100 ⁇ L matrigel and inoculate them subcutaneously on the right hind limb of the mice. Grouping began when the tumors grew to an average volume of 110 mm 3 .
  • PBMCs come from the fresh blood of healthy blood donors, and are injected into the tail vein 1 hour before the antibody is given after grouping on DAY1 (10M PBMC cells are injected per mouse). The tail vein was administered twice a week, and the tumor diameter was measured twice a week with a vernier caliper, and the tumor volume was calculated.
  • the tumor volume measured on 21 days of continuous administration was recorded, and the tumor volume growth curve was drawn with GraphPad Prism. After the experiment, the tumor weight was detected, and the tumor inhibition rate (TGI)% of each group was calculated. The results are shown in Table 15.
  • anti-CRTAM antibodies provided by the present invention have significant anti-tumor effects and can significantly inhibit tumor growth, suggesting that these antibodies can be used in the preparation of anti-tumor drugs and have good market prospects.

Abstract

The present invention provides an anti-CRTAM antibody or an antigen-binding fragment thereof, a pharmaceutical composition comprising the anti-CRTAM antibody or the antigen-binding fragment thereof, and an application of the anti-CRTAM antibody or the antigen-binding fragment thereof in the preparation of an anti-tumor drug.

Description

抗CRTAM抗体及其应用Anti-CRTAM antibody and its application 技术领域technical field
本发明涉及抗体药物技术领域，尤其涉及一种抗CRTAM抗体或其抗原结合片段，包含抗CRTAM抗体或其抗原结合片段的药物组合物以及它们的应用。The present invention relates to the technical field of antibody medicines, in particular to an anti-CRTAM antibody or its antigen-binding fragment, a pharmaceutical composition containing the anti-CRTAM antibody or its antigen-binding fragment and applications thereof.
背景技术Background technique
CRTAM(Class-I MHC-restricted T cell associated molecule)为免疫球蛋白超家族(IgSF)的一员，是主要表达于激活后的CD8+T细胞和NK细胞上的共刺激性靶点，它的配体是Nectin-like 2(Necl-2)。CRTAM的表达严格受到TCR(T cell receptor)激活的调控，仅表达于激活的免疫细胞上。在健康人的PBMC(Peripheral blood mononuclear cell)中表达量很低，在哮喘病人的PBMC中高表达。CRTAM的启动子和IL-2一样受到AP-1转录因子的调控。CRTAM激活后会刺激CD8+T细胞分泌IFN-γ以及增强CD8+T细胞和NK细胞的杀伤能力。同时，有文献报道CRTAM能够影响CD4+T细胞的极化，在MHC和TCR结合激活后，促进CD4+T细胞的进一步分化，提高IFN-γ、IL-17、IL-22等细胞因子的分泌能力。CRTAM的配体Necl-2又被称为TSLC1(The tumor suppressor in lung cancer-1)和CADM1，是Necl家族的一员，nectin和necl是非钙离子依赖的细胞粘附分子，Necl-2与CRTAM结合能够激活T细胞、NK细胞产生杀伤作用。在肿瘤细胞上Necl-2的表达会逐渐减少至沉默，这被认为是另一条肿瘤逃逸的机制。CRTAM (Class-I MHC-restricted T cell associated molecule), a member of the immunoglobulin superfamily (IgSF), is a costimulatory target mainly expressed on activated CD8+ T cells and NK cells. The ligand is Nectin-like 2 (Necl-2). The expression of CRTAM is strictly regulated by TCR (T cell receptor) activation and is only expressed on activated immune cells. The expression level in PBMC (Peripheral blood mononuclear cell) of healthy people is very low, and it is highly expressed in PBMC of asthmatic patients. The promoter of CRTAM is regulated by AP-1 transcription factor like IL-2. CRTAM activation will stimulate CD8+ T cells to secrete IFN-γ and enhance the killing ability of CD8+ T cells and NK cells. At the same time, it has been reported that CRTAM can affect the polarization of CD4+ T cells, promote the further differentiation of CD4+ T cells after MHC and TCR combined activation, and increase the secretion of cytokines such as IFN-γ, IL-17, and IL-22 ability. The ligand Necl-2 of CRTAM, also known as TSLC1 (The tumor suppressor in lung cancer-1) and CADM1, is a member of the Necl family, nectin and necl are calcium ion-independent cell adhesion molecules, Necl-2 and CRTAM The combination can activate T cells and NK cells to produce killing effect. Necl-2 expression is gradually reduced to silence on tumor cells, which is thought to be another tumor escape mechanism.
文献表明，Necl-2在肿瘤细胞的表达会抑制肿瘤细胞的生长和侵袭；同时Necl-2与CRTAM结合能够进一步激活CD8+T细胞和NK细胞，提高免疫细胞的杀伤能力。Necl-2和CRTAM通路与PD1-PDL1通路不重合且具备互补的作用。肿瘤细胞会通过下调Necl-2蛋白的表达达到免疫逃逸的目的。Literature shows that the expression of Necl-2 in tumor cells can inhibit the growth and invasion of tumor cells; at the same time, the combination of Necl-2 and CRTAM can further activate CD8+ T cells and NK cells, and improve the killing ability of immune cells. The Necl-2 and CRTAM pathways do not overlap with the PD1-PDL1 pathway and have complementary roles. Tumor cells will achieve the purpose of immune escape by down-regulating the expression of Necl-2 protein.
CRTAM是一个全新、安全的免疫共刺激分子，具有潜在的实体瘤治疗能力，因此研究开发CRTAM激动性抗体具有重要的意义。CRTAM is a new and safe immune co-stimulatory molecule with potential therapeutic ability for solid tumors, so research and development of CRTAM agonistic antibodies is of great significance.
发明内容Contents of the invention
本发明一方面提供一种抗CRTAM抗体或其抗原结合片段，其包含重链可变区和/或轻链可变区，其中所述重链可变区包含重链可变区的互补决定区1(HCDR1)、重链可变区的互补决定区2(HCDR2)和/或重链可变区的互补决定区3(HCDR3)，所述轻链可变区包含轻链可变区的互补决定区1(LCDR1)、轻链可变区的互补决定区2(LCDR2)和/或轻链可变区的互补决定区3(LCDR3)。One aspect of the present invention provides an anti-CRTAM antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region and/or a light chain variable region, wherein the heavy chain variable region comprises a complementarity determining region of the heavy chain variable region 1 (HCDR1), the complementarity determining region 2 (HCDR2) of the heavy chain variable region, and/or the complementarity determining region 3 (HCDR3) of the heavy chain variable region comprising the complementarity of the light chain variable region The complementarity determining region 1 (LCDR1), the complementarity determining region 2 (LCDR2) of the light chain variable region and/or the complementarity determining region 3 (LCDR3) of the light chain variable region.
在一些实施方案中，本发明提供一种抗CRTAM抗体或其抗原结合片段，其包含重链可变区和/或轻链可变区，其中：In some embodiments, the invention provides an anti-CRTAM antibody or antigen-binding fragment thereof comprising a heavy chain variable region and/or a light chain variable region, wherein:
(1)所述重链可变区包含选自如下组的HCDR1、HCDR2和HCDR3：(1) The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 selected from the group consisting of:
(a1)如SEQ ID NO:1、2和3所示的氨基酸序列；(a1) an amino acid sequence as shown in SEQ ID NO: 1, 2 and 3;
(a2)如SEQ ID NO:8、9和10所示的氨基酸序列；(a2) amino acid sequences as shown in SEQ ID NO:8, 9 and 10;
(a3)如SEQ ID NO:8、11和10所示的氨基酸序列；(a3) amino acid sequences as shown in SEQ ID NO:8, 11 and 10;
(a4)如SEQ ID NO:16、17和18所示的氨基酸序列；(a4) amino acid sequences as shown in SEQ ID NO: 16, 17 and 18;
(a5)如SEQ ID NO:22、23和24所示的氨基酸序列；(a5) amino acid sequences as shown in SEQ ID NO:22, 23 and 24;
(a6)如SEQ ID NO:22、25和24所示的氨基酸序列；和(a6) amino acid sequences as shown in SEQ ID NO: 22, 25 and 24; and
(a7)与(a1)、(a2)、(a3)、(a4)、(a5)或(a6)所示的氨基酸序列具有至少85％序列同一性的氨基酸序列；和/或(a7) an amino acid sequence having at least 85% sequence identity to the amino acid sequence shown in (a1), (a2), (a3), (a4), (a5) or (a6); and/or
(2)所述轻链可变区包含选自如下组的LCDR1、LCDR2和LCDR3：(2) the light chain variable region comprises LCDR1, LCDR2 and LCDR3 selected from the following group:
(b1)如SEQ ID NO:4、5和6所示的氨基酸序列；(b1) an amino acid sequence as shown in SEQ ID NO: 4, 5 and 6;
(b2)如SEQ ID NO:7、5和6所示的氨基酸序列；(b2) amino acid sequences as shown in SEQ ID NO:7, 5 and 6;
(b3)如SEQ ID NO:12、13和14所示的氨基酸序列；(b3) amino acid sequences as shown in SEQ ID NO: 12, 13 and 14;
(b4)如SEQ ID NO:15、13和14所示的氨基酸序列；(b4) amino acid sequences as shown in SEQ ID NO: 15, 13 and 14;
(b5)如SEQ ID NO:19、20和21所示的氨基酸序列；(b5) amino acid sequences as shown in SEQ ID NO: 19, 20 and 21;
(b6)如SEQ ID NO:26、27和28所示的氨基酸序列；(b6) amino acid sequences as shown in SEQ ID NO:26, 27 and 28;
(b7)与(b1)、(b2)、(b3)、(b4)、(b5)或(b6)所示的氨基酸序列具有至少85％序列同一性的氨基酸序列。(b7) An amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in (b1), (b2), (b3), (b4), (b5) or (b6).
在一个具体的实施方案中，本发明提供一种抗CRTAM抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中所述重链可变区的HCDR1、HCDR2和HCDR3分别为SEQ ID NO:1、2和3或与SEQ ID NO:1、2和3所示的氨基酸序列具有至少85％序列同一性的氨基酸序列，和所述轻链可变区的LCDR1、 LCDR2和LCDR3分别为SEQ ID NO:4、5和6、SEQ ID NO:7、5和6或与SEQ ID NO:4、5和6或SEQ ID NO:7、5和6所示的氨基酸序列具有至少85％序列同一性的氨基酸序列；或In a specific embodiment, the present invention provides an anti-CRTAM antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region and a light chain variable region, wherein HCDR1, HCDR2 and HCDR3 of the heavy chain variable region SEQ ID NO: 1, 2 and 3 or an amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, 2 and 3, and LCDR1, LCDR2 of the light chain variable region and LCDR3 are respectively SEQ ID NO: 4, 5 and 6, SEQ ID NO: 7, 5 and 6 or have the amino acid sequence shown in SEQ ID NO: 4, 5 and 6 or SEQ ID NO: 7, 5 and 6 Amino acid sequences with at least 85% sequence identity; or
所述HCDR1、HCDR2和HCDR3分别为SEQ ID NO:8、9和10、SEQ ID NO:8、11和10或与SEQ ID NO:8、9和10或SEQ ID NO:8、11和10所示的氨基酸序列具有至少85％序列同一性的氨基酸序列，和所述LCDR1、LCDR2和LCDR3分别为SEQ ID NO:12、13和14、SEQ ID NO:15、13和14或与SEQ ID NO:12、13和14或SEQ ID NO:15、13和14所示的氨基酸序列具有至少85％序列同一性的氨基酸序列；或The HCDR1, HCDR2 and HCDR3 are respectively SEQ ID NO: 8, 9 and 10, SEQ ID NO: 8, 11 and 10 or with SEQ ID NO: 8, 9 and 10 or SEQ ID NO: 8, 11 and 10 An amino acid sequence having at least 85% sequence identity to the amino acid sequence shown, and said LCDR1, LCDR2 and LCDR3 are SEQ ID NO: 12, 13 and 14, SEQ ID NO: 15, 13 and 14, respectively, or with SEQ ID NO: 12, 13 and 14 or an amino acid sequence having at least 85% sequence identity to the amino acid sequence shown in SEQ ID NO: 15, 13 and 14; or
所述HCDR1、HCDR2和HCDR3分别为SEQ ID NO:16、17和18或与SEQ ID NO:16、17和18所示的氨基酸序列具有至少85％序列同一性的氨基酸序列，和所述LCDR1、LCDR2和LCDR3分别为SEQ ID NO:19、20和21或与SEQ ID NO:19、20和21所示的氨基酸序列具有至少85％序列同一性的氨基酸序列；或The HCDR1, HCDR2 and HCDR3 are respectively SEQ ID NO: 16, 17 and 18 or an amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 16, 17 and 18, and the LCDR1, LCDR2 and LCDR3 are SEQ ID NO: 19, 20, and 21, respectively, or an amino acid sequence having at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO: 19, 20, and 21; or
所述HCDR1、HCDR2和HCDR3分别为SEQ ID NO:22、23和24、SEQ ID NO:22、25和24或与SEQ ID NO:22、23和24或SEQ ID NO:22、25和24所示的氨基酸序列具有至少85％序列同一性的氨基酸序列，和所述LCDR1、LCDR2和LCDR3分别为SEQ ID NO:26、27和28或与SEQ ID NO:26、27和28所示的氨基酸序列具有至少85％序列同一性的氨基酸序列。在一些实施方案中，根据本发明的抗CRTAM抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列选自SEQ ID NO:80、SEQ ID NO:84、SEQ ID NO:85、SEQ ID NO:86和SEQ ID NO:87，SEQ ID NO:80、SEQ ID NO:84、SEQ ID NO:85、SEQ ID NO:86或SEQ ID NO:87经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:80、SEQ ID NO:84、SEQ ID NO:85、SEQ ID NO:86或SEQ ID NO:87功能相同的氨基酸序列，以及与SEQ ID NO:80、SEQ ID NO:84、SEQ ID NO:85、SEQ ID NO:86或SEQ ID NO:87具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列选自SEQ ID NO:81、SEQ ID NO:88、SEQ ID NO:89、SEQ ID NO:90、SEQ ID NO:91和SEQ ID NO:92，SEQ ID NO:81、SEQ ID NO:88、SEQ ID NO:89、SEQ ID NO:90、SEQ ID NO:91或SEQ ID NO:92经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:81、SEQ ID NO:88、SEQ ID NO:89、SEQ ID NO:90、SEQ ID NO:91或SEQ ID NO:92功能相同的氨基酸序 列，以及与SEQ ID NO:81、SEQ ID NO:88、SEQ ID NO:89、SEQ ID NO:90、SEQ ID NO:91或SEQ ID NO:92具有至少85％序列同一性的氨基酸序列。The HCDR1, HCDR2 and HCDR3 are respectively SEQ ID NO: 22, 23 and 24, SEQ ID NO: 22, 25 and 24 or with SEQ ID NO: 22, 23 and 24 or SEQ ID NO: 22, 25 and 24 The amino acid sequence shown has an amino acid sequence of at least 85% sequence identity, and said LCDR1, LCDR2 and LCDR3 are respectively SEQ ID NO:26, 27 and 28 or the amino acid sequence shown in SEQ ID NO:26, 27 and 28 Amino acid sequences having at least 85% sequence identity. In some embodiments, according to the anti-CRTAM antibody or antigen-binding fragment thereof of the present invention, wherein the amino acid sequence of the heavy chain variable region is selected from the group consisting of SEQ ID NO:80, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86 and SEQ ID NO:87, SEQ ID NO:80, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86 or SEQ ID NO:87 are substituted, deleted or added with one or more Amino acid sequences obtained from amino acids and functionally identical to SEQ ID NO: 80, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86 or SEQ ID NO: 87, and functionally identical to SEQ ID NO: 80, SEQ ID NO: 80, SEQ ID NO: ID NO:84, SEQ ID NO:85, SEQ ID NO:86 or SEQ ID NO:87 has an amino acid sequence of at least 85% sequence identity, and the amino acid sequence of the light chain variable region is selected from SEQ ID NO: 81. SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91 and SEQ ID NO:92, SEQ ID NO:81, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO: 90, SEQ ID NO: 91 or SEQ ID NO: 92 obtained by substitution, deletion or addition of one or more amino acids and obtained with SEQ ID NO: 81, SEQ ID NO: 88, SEQ ID NO: 89, Amino acid sequences functionally identical to SEQ ID NO:90, SEQ ID NO:91 or SEQ ID NO:92, and to SEQ ID NO:81, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:90, SEQ ID NO:92 An amino acid sequence having at least 85% sequence identity to ID NO:91 or SEQ ID NO:92.
在一些实施方案中，根据本发明的抗CRTAM抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列为SEQ ID NO:80，SEQ ID NO:80经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:80功能相同的氨基酸序列或与SEQ ID NO:80具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:81，SEQ ID NO:81经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:81功能相同的氨基酸序列或与SEQ ID NO:81具有至少85％序列同一性的氨基酸序列。In some embodiments, according to the anti-CRTAM antibody or antigen-binding fragment thereof of the present invention, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 80, SEQ ID NO: 80 is substituted, deleted or added with one or An amino acid sequence obtained from multiple amino acids and having the same function as SEQ ID NO: 80 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 80, and the amino acid sequence of the light chain variable region is SEQ ID NO :81, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids of SEQ ID NO:81 and having the same function as SEQ ID NO:81 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:81 .
在一些实施方案中，根据本发明的抗CRTAM抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列选自SEQ ID NO:84、SEQ ID NO:85、SEQ ID NO:86和SEQ ID NO:87，SEQ ID NO:84、SEQ ID NO:85、SEQ ID NO:86或SEQ ID NO:87经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:84、SEQ ID NO:85、SEQ ID NO:86或SEQ ID NO:87功能相同的氨基酸序列，以及与SEQ ID NO:84、SEQ ID NO:85、SEQ ID NO:86或SEQ ID NO:87具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列选自SEQ ID NO:88、SEQ ID NO:89、SEQ ID NO:90和SEQ ID NO:91，SEQ ID NO:88、SEQ ID NO:89、SEQ ID NO:90或SEQ ID NO:91经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:88、SEQ ID NO:89、SEQ ID NO:90或SEQ ID NO:91功能相同的氨基酸序列，以及与SEQ ID NO:88、SEQ ID NO:89、SEQ ID NO:90或SEQ ID NO:91具有至少85％序列同一性的氨基酸序列。In some embodiments, according to the anti-CRTAM antibody or antigen-binding fragment thereof of the present invention, wherein the amino acid sequence of the heavy chain variable region is selected from SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86 and SEQ ID NO: 87, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86 or SEQ ID NO: 87 obtained by substitution, deletion or addition of one or more amino acids and identical to SEQ ID NO: 84, An amino acid sequence that is functionally identical to SEQ ID NO:85, SEQ ID NO:86, or SEQ ID NO:87, and has at least The amino acid sequence of 85% sequence identity, and the aminoacid sequence of described light chain variable region is selected from SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90 and SEQ ID NO:91, SEQ ID NO: 88. SEQ ID NO: 89, SEQ ID NO: 90 or SEQ ID NO: 91 obtained by substitution, deletion or addition of one or more amino acids and identical to SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90 or a functionally identical amino acid sequence of SEQ ID NO:91, and an amino acid sequence having at least 85% sequence identity to SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90 or SEQ ID NO:91.
在一些实施方案中，根据本发明的抗CRTAM抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列为SEQ ID NO:87，SEQ ID NO:87经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:87功能相同的氨基酸序列或与SEQ ID NO:87具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:90，SEQ ID NO:90经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:90功能相同的氨基酸序列或与SEQ ID NO:90具有至少85％序列同一性的氨基酸序列；或者In some embodiments, according to the anti-CRTAM antibody or antigen-binding fragment thereof of the present invention, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 87, SEQ ID NO: 87 is substituted, deleted or added with one or An amino acid sequence obtained from multiple amino acids and having the same function as SEQ ID NO: 87 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 87, and the amino acid sequence of the light chain variable region is SEQ ID NO :90, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids of SEQ ID NO:90 and having the same function as SEQ ID NO:90 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:90 ;or
所述重链可变区的氨基酸序列为SEQ ID NO:87，SEQ ID NO:87经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:87功能相同的氨基酸序列或与SEQ ID NO:87具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的 氨基酸序列为SEQ ID NO:92，SEQ ID NO:92经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:92功能相同的氨基酸序列或与SEQ ID NO:92具有至少85％序列同一性的氨基酸序列。The amino acid sequence of the heavy chain variable region is SEQ ID NO: 87, the amino acid sequence obtained by replacing, deleting or adding one or more amino acids of SEQ ID NO: 87 and having the same function as SEQ ID NO: 87 or the amino acid sequence with SEQ ID NO: 87 ID NO:87 has an amino acid sequence of at least 85% sequence identity, and the amino acid sequence of the light chain variable region is SEQ ID NO:92, and SEQ ID NO:92 is obtained by substitution, deletion or addition of one or more amino acids An amino acid sequence that is functionally identical to SEQ ID NO:92 or an amino acid sequence that has at least 85% sequence identity to SEQ ID NO:92.
在一些实施方案中，根据本发明的抗CRTAM抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列选自SEQ ID NO:45、SEQ ID NO:106、SEQ ID NO:50、SEQ ID NO:51、SEQ ID NO:52，SEQ ID NO:53和SEQ ID NO:54，SEQ ID NO:45、SEQ ID NO:106、SEQ ID NO:50、SEQ ID NO:51、SEQ ID NO:52，SEQ ID NO:53或SEQ ID NO:54经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:45、SEQ ID NO:106、SEQ ID NO:50、SEQ ID NO:51、SEQ ID NO:52，SEQ ID NO:53或SEQ ID NO:54功能相同的氨基酸序列，以及与SEQ ID NO:45、SEQ ID NO:106、SEQ ID NO:50、SEQ ID NO:51、SEQ ID NO:52，SEQ ID NO:53或SEQ ID NO:54具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列选自SEQ ID NO:46、SEQ ID NO:55、SEQ ID NO:56、SEQ ID NO:57、SEQ ID NO:58、SEQ ID NO:59、SEQ ID NO:60、SEQ ID NO:61、SEQ ID NO:62、SEQ ID NO:63、SEQ ID NO:64、SEQ ID NO:65、SEQ ID NO:66和SEQ ID NO:67，SEQ ID NO:46、SEQ ID NO:55、SEQ ID NO:56、SEQ ID NO:57、SEQ ID NO:58、SEQ ID NO:59、SEQ ID NO:60、SEQ ID NO:61、SEQ ID NO:62、SEQ ID NO:63、SEQ ID NO:64、SEQ ID NO:65、SEQ ID NO:66或SEQ ID NO:67经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:46、SEQ ID NO:55、SEQ ID NO:56、SEQ ID NO:57、SEQ ID NO:58、SEQ ID NO:59、SEQ ID NO:60、SEQ ID NO:61、SEQ ID NO:62、SEQ ID NO:63、SEQ ID NO:64、SEQ ID NO:65、SEQ ID NO:66或SEQ ID NO:67功能相同的氨基酸序列，以及与SEQ ID NO:46、SEQ ID NO:55、SEQ ID NO:56、SEQ ID NO:57、SEQ ID NO:58、SEQ ID NO:59、SEQ ID NO:60、SEQ ID NO:61、SEQ ID NO:62、SEQ ID NO:63、SEQ ID NO:64、SEQ ID NO:65、SEQ ID NO:66或SEQ ID NO:67具有至少85％序列同一性的氨基酸序列。In some embodiments, according to the anti-CRTAM antibody or antigen-binding fragment thereof of the present invention, wherein the amino acid sequence of the heavy chain variable region is selected from SEQ ID NO:45, SEQ ID NO:106, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53 and SEQ ID NO:54, SEQ ID NO:45, SEQ ID NO:106, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO: 52, SEQ ID NO: 53 or SEQ ID NO: 54 obtained by substitution, deletion or addition of one or more amino acids and obtained with SEQ ID NO: 45, SEQ ID NO: 106, SEQ ID NO: 50, SEQ ID The functionally identical amino acid sequence of NO:51, SEQ ID NO:52, SEQ ID NO:53 or SEQ ID NO:54, and SEQ ID NO:45, SEQ ID NO:106, SEQ ID NO:50, SEQ ID NO :51, SEQ ID NO:52, SEQ ID NO:53 or SEQ ID NO:54 have an amino acid sequence of at least 85% sequence identity, and the amino acid sequence of the light chain variable region is selected from the group consisting of SEQ ID NO:46, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66 and SEQ ID NO:67, SEQ ID NO:46, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO: 57. SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO: 66 or SEQ ID NO: 67 obtained by substitution, deletion or addition of one or more amino acids and with SEQ ID NO: 46, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, Amino acid sequences functionally identical to SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66 or SEQ ID NO:67, and to SEQ ID NO:46, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO: ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO An amino acid sequence having at least 85% sequence identity to SEQ ID NO:65, SEQ ID NO:66 or SEQ ID NO:67.
在一些实施方案中，根据本发明的抗CRTAM抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列为SEQ ID NO:45，SEQ ID NO:45经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:45功能相同的氨基酸序列或与SEQ ID NO:45具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:46，SEQ ID NO:46经取代、缺失或添加一个或多个氨基 酸获得的且与SEQ ID NO:46功能相同的氨基酸序列或与SEQ ID NO:46具有至少85％序列同一性的氨基酸序列。In some embodiments, according to the anti-CRTAM antibody or antigen-binding fragment thereof of the present invention, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 45, SEQ ID NO: 45 is substituted, deleted or added with one or The amino acid sequence obtained by multiple amino acids and having the same function as SEQ ID NO: 45 or the amino acid sequence having at least 85% sequence identity with SEQ ID NO: 45, and the amino acid sequence of the light chain variable region is SEQ ID NO :46, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids of SEQ ID NO:46 and having the same function as SEQ ID NO:46 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:46 .
在一些实施方案中，根据本发明的抗CRTAM抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列为SEQ ID NO:106，SEQ ID NO:106经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:106功能相同的氨基酸序列或与SEQ ID NO:106具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:46，SEQ ID NO:46经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:46功能相同的氨基酸序列或与SEQ ID NO:46具有至少85％序列同一性的氨基酸序列。In some embodiments, according to the anti-CRTAM antibody or antigen-binding fragment thereof of the present invention, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 106, SEQ ID NO: 106 is substituted, deleted or added with one or The amino acid sequence obtained by multiple amino acids and having the same function as SEQ ID NO: 106 or the amino acid sequence having at least 85% sequence identity with SEQ ID NO: 106, and the amino acid sequence of the light chain variable region is SEQ ID NO :46, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids of SEQ ID NO:46 and having the same function as SEQ ID NO:46 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:46 .
在一些实施方案中，根据本发明的抗CRTAM抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列选自SEQ ID NO:50、SEQ ID NO:51、SEQ ID NO:52，SEQ ID NO:53和SEQ ID NO:54，SEQ ID NO:50、SEQ ID NO:51、SEQ ID NO:52，SEQ ID NO:53或SEQ ID NO:54经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:50、SEQ ID NO:51、SEQ ID NO:52，SEQ ID NO:53或SEQ ID NO:54功能相同的氨基酸序列，以及与SEQ ID NO:50、SEQ ID NO:51、SEQ ID NO:52，SEQ ID NO:53或SEQ ID NO:54具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列选自SEQ ID NO:55、SEQ ID NO:56、SEQ ID NO:57、SEQ ID NO:58、SEQ ID NO:59、SEQ ID NO:60、SEQ ID NO:61、SEQ ID NO:62、SEQ ID NO:63、SEQ ID NO:64、SEQ ID NO:65和SEQ ID NO:66，SEQ ID NO:55、SEQ ID NO:56、SEQ ID NO:57、SEQ ID NO:58、SEQ ID NO:59、SEQ ID NO:60、SEQ ID NO:61、SEQ ID NO:62、SEQ ID NO:63、SEQ ID NO:64、SEQ ID NO:65或SEQ ID NO:66经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:55、SEQ ID NO:56、SEQ ID NO:57、SEQ ID NO:58、SEQ ID NO:59、SEQ ID NO:60、SEQ ID NO:61、SEQ ID NO:62、SEQ ID NO:63、SEQ ID NO:64、SEQ ID NO:65或SEQ ID NO:66功能相同的氨基酸序列，以及与SEQ ID NO:55、SEQ ID NO:56、SEQ ID NO:57、SEQ ID NO:58、SEQ ID NO:59、SEQ ID NO:60、SEQ ID NO:61、SEQ ID NO:62、SEQ ID NO:63、SEQ ID NO:64、SEQ ID NO:65或SEQ ID NO:66具有至少85％序列同一性的氨基酸序列。In some embodiments, according to the anti-CRTAM antibody or antigen-binding fragment thereof of the present invention, wherein the amino acid sequence of the heavy chain variable region is selected from SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53 and SEQ ID NO:54, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53 or SEQ ID NO:54 are substituted, deleted or added with one or more Amino acid sequences obtained from amino acids and having the same function as SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53 or SEQ ID NO:54, as well as the same amino acid sequence as SEQ ID NO:50, SEQ ID NO:50, SEQ ID NO:52 ID NO:51, SEQ ID NO:52, SEQ ID NO:53 or SEQ ID NO:54 have an amino acid sequence of at least 85% sequence identity, and the amino acid sequence of the light chain variable region is selected from SEQ ID NO: 55. SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65 and SEQ ID NO:66, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65 or SEQ ID NO:66 substituted, deleted or added with one or more amino acids Obtained and with SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO: 62. The functionally identical amino acid sequence of SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65 or SEQ ID NO:66, and SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57 , SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ An amino acid sequence having at least 85% sequence identity to ID NO:64, SEQ ID NO:65 or SEQ ID NO:66.
在一些实施方案中，根据本发明的抗CRTAM抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列为SEQ ID NO:51，SEQ ID NO:51经取代、缺失或 添加一个或多个氨基酸获得的且与SEQ ID NO:51功能相同的氨基酸序列或与SEQ ID NO:51具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:66，SEQ ID NO:66经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:66功能相同的氨基酸序列或与SEQ ID NO:66具有至少85％序列同一性的氨基酸序列；或者In some embodiments, according to the anti-CRTAM antibody or antigen-binding fragment thereof of the present invention, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 51, SEQ ID NO: 51 is substituted, deleted or added with one or An amino acid sequence obtained from multiple amino acids and having the same function as SEQ ID NO:51 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:51, and the amino acid sequence of the light chain variable region is SEQ ID NO :66, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids of SEQ ID NO:66 and having the same function as SEQ ID NO:66 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:66 ;or
所述重链可变区的氨基酸序列为SEQ ID NO:51，SEQ ID NO:51经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:51功能相同的氨基酸序列或与SEQ ID NO:51具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:67，SEQ ID NO:67经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:67功能相同的氨基酸序列或与SEQ ID NO:67具有至少85％序列同一性的氨基酸序列。The amino acid sequence of the heavy chain variable region is SEQ ID NO: 51, an amino acid sequence obtained by replacing, deleting or adding one or more amino acids to SEQ ID NO: 51 and having the same function as SEQ ID NO: 51 or with SEQ ID NO: 51 ID NO:51 has an amino acid sequence of at least 85% sequence identity, and the amino acid sequence of the light chain variable region is SEQ ID NO:67, and SEQ ID NO:67 is obtained by substitution, deletion or addition of one or more amino acids An amino acid sequence that is functionally identical to SEQ ID NO: 67 or an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 67.
在一些实施方案中，根据本发明的抗CRTAM抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列选自SEQ ID NO:68、SEQ ID NO:72、SEQ ID NO:73、SEQ ID NO:74和SEQ ID NO:75，SEQ ID NO:68、SEQ ID NO:72、SEQ ID NO:73、SEQ ID NO:74或SEQ ID NO:75经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:68、SEQ ID NO:72、SEQ ID NO:73、SEQ ID NO:74或SEQ ID NO:75功能相同的氨基酸序列，以及与SEQ ID NO:68、SEQ ID NO:72、SEQ ID NO:73、SEQ ID NO:74或SEQ ID NO:75具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列选自SEQ ID NO:69、SEQ ID NO:76、SEQ ID NO:77、SEQ ID NO:78和SEQ ID NO:79，SEQ ID NO:69、SEQ ID NO:76、SEQ ID NO:77、SEQ ID NO:78或SEQ ID NO:79经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:69、SEQ ID NO:76、SEQ ID NO:77、SEQ ID NO:78或SEQ ID NO:79功能相同的氨基酸序列，以及与SEQ ID NO:69、SEQ ID NO:76、SEQ ID NO:77、SEQ ID NO:78或SEQ ID NO:79具有至少85％序列同一性的氨基酸序列。In some embodiments, according to the anti-CRTAM antibody or antigen-binding fragment thereof of the present invention, wherein the amino acid sequence of the heavy chain variable region is selected from the group consisting of SEQ ID NO:68, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74 and SEQ ID NO:75, SEQ ID NO:68, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74 or SEQ ID NO:75 are substituted, deleted or added with one or more Amino acid sequences obtained from amino acids and functionally identical to SEQ ID NO: 68, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74 or SEQ ID NO: 75, and amino acid sequences identical to those of SEQ ID NO: 68, SEQ ID NO: ID NO:72, SEQ ID NO:73, SEQ ID NO:74 or SEQ ID NO:75 has an amino acid sequence of at least 85% sequence identity, and the amino acid sequence of the light chain variable region is selected from SEQ ID NO: 69. SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, and SEQ ID NO:79, SEQ ID NO:69, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, or SEQ ID NO:79 is obtained by substitution, deletion or addition of one or more amino acids and has the same function as SEQ ID NO:69, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78 or SEQ ID NO:79 An identical amino acid sequence, and an amino acid sequence having at least 85% sequence identity to SEQ ID NO:69, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78 or SEQ ID NO:79.
在一些实施方案中，根据本发明的抗CRTAM抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列为SEQ ID NO:68，SEQ ID NO:68经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:68功能相同的氨基酸序列或与SEQ ID NO:68具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:69，SEQ ID NO:69经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:69功能相同的氨基酸序列或与SEQ ID NO:69具有至 少85％序列同一性的氨基酸序列。In some embodiments, according to the anti-CRTAM antibody or antigen-binding fragment thereof of the present invention, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 68, SEQ ID NO: 68 is substituted, deleted or added with one or An amino acid sequence obtained from multiple amino acids and having the same function as SEQ ID NO:68 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:68, and the amino acid sequence of the light chain variable region is SEQ ID NO :69, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids of SEQ ID NO:69 and having the same function as SEQ ID NO:69 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:69 .
在一些实施方案中，根据本发明的抗CRTAM抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列选自SEQ ID NO:72、SEQ ID NO:73、SEQ ID NO:74和SEQ ID NO:75，SEQ ID NO:72、SEQ ID NO:73、SEQ ID NO:74或SEQ ID NO:75经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:72、SEQ ID NO:73、SEQ ID NO:74或SEQ ID NO:75功能相同的氨基酸序列，以及与SEQ ID NO:72、SEQ ID NO:73、SEQ ID NO:74或SEQ ID NO:75具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列选自SEQ ID NO:76、SEQ ID NO:77、SEQ ID NO:78和SEQ ID NO:79，SEQ ID NO:76、SEQ ID NO:77、SEQ ID NO:78或SEQ ID NO:79经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:76、SEQ ID NO:77、SEQ ID NO:78或SEQ ID NO:79功能相同的氨基酸序列，以及与SEQ ID NO:76、SEQ ID NO:77、SEQ ID NO:78或SEQ ID NO:79具有至少85％序列同一性的氨基酸序列。In some embodiments, according to the anti-CRTAM antibody or antigen-binding fragment thereof of the present invention, wherein the amino acid sequence of the heavy chain variable region is selected from SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74 and SEQ ID NO: 75, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74 or SEQ ID NO: 75 obtained by substitution, deletion or addition of one or more amino acids and identical to SEQ ID NO: 72, An amino acid sequence that is functionally identical to SEQ ID NO:73, SEQ ID NO:74, or SEQ ID NO:75, and has at least The amino acid sequence of 85% sequence identity, and the aminoacid sequence of described light chain variable region is selected from SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78 and SEQ ID NO:79, SEQ ID NO: 76. SEQ ID NO: 77, SEQ ID NO: 78 or SEQ ID NO: 79 obtained by substitution, deletion or addition of one or more amino acids and identical to SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78 or an amino acid sequence that is functionally identical to SEQ ID NO: 79, and an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78 or SEQ ID NO: 79.
在一些实施方案中，根据本发明的抗CRTAM抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列为SEQ ID NO:75，SEQ ID NO:75经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:75功能相同的氨基酸序列或与SEQ ID NO:75具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:77，SEQ ID NO:77经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:77功能相同的氨基酸序列或与SEQ ID NO:77具有至少85％序列同一性的氨基酸序列。In some embodiments, according to the anti-CRTAM antibody or antigen-binding fragment thereof of the present invention, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 75, SEQ ID NO: 75 is substituted, deleted or added with one or An amino acid sequence obtained from multiple amino acids and having the same function as SEQ ID NO:75 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:75, and the amino acid sequence of the light chain variable region is SEQ ID NO :77, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids of SEQ ID NO:77 and having the same function as SEQ ID NO:77 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:77 .
在一些实施方案中，根据本发明的抗CRTAM抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列选自SEQ ID NO:45、SEQ ID NO:106、SEQ ID NO:50、SEQ ID NO:51、SEQ ID NO:52，SEQ ID NO:53和SEQ ID NO:54，SEQ ID NO:45、SEQ ID NO:106、SEQ ID NO:50、SEQ ID NO:51、SEQ ID NO:52，SEQ ID NO:53或SEQ ID NO:54经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:45、SEQ ID NO:106、SEQ ID NO:50、SEQ ID NO:51、SEQ ID NO:52，SEQ ID NO:53或SEQ ID NO:54功能相同的氨基酸序列，以及与SEQ ID NO:45、SEQ ID NO:106、SEQ ID NO:50、SEQ ID NO:51、SEQ ID NO:52，SEQ ID NO:53或SEQ ID NO:54具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列选自SEQ ID NO:46、SEQ ID NO:55、SEQ ID NO:56、SEQ ID NO:57、SEQ ID NO:58、SEQ ID NO:59、SEQ ID NO:60、SEQ ID NO: 61、SEQ ID NO:62、SEQ ID NO:63、SEQ ID NO:64、SEQ ID NO:65、SEQ ID NO:66和SEQ ID NO:67，SEQ ID NO:46、SEQ ID NO:55、SEQ ID NO:56、SEQ ID NO:57、SEQ ID NO:58、SEQ ID NO:59、SEQ ID NO:60、SEQ ID NO:61、SEQ ID NO:62、SEQ ID NO:63、SEQ ID NO:64、SEQ ID NO:65、SEQ ID NO:66或SEQ ID NO:67经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:46、SEQ ID NO:55、SEQ ID NO:56、SEQ ID NO:57、SEQ ID NO:58、SEQ ID NO:59、SEQ ID NO:60、SEQ ID NO:61、SEQ ID NO:62、SEQ ID NO:63、SEQ ID NO:64、SEQ ID NO:65、SEQ ID NO:66或SEQ ID NO:67功能相同的氨基酸序列，以及与SEQ ID NO:46、SEQ ID NO:55、SEQ ID NO:56、SEQ ID NO:57、SEQ ID NO:58、SEQ ID NO:59、SEQ ID NO:60、SEQ ID NO:61、SEQ ID NO:62、SEQ ID NO:63、SEQ ID NO:64、SEQ ID NO:65、SEQ ID NO:66或SEQ ID NO:67具有至少85％序列同一性的氨基酸序列。In some embodiments, according to the anti-CRTAM antibody or antigen-binding fragment thereof of the present invention, wherein the amino acid sequence of the heavy chain variable region is selected from SEQ ID NO:45, SEQ ID NO:106, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53 and SEQ ID NO:54, SEQ ID NO:45, SEQ ID NO:106, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO: 52, SEQ ID NO: 53 or SEQ ID NO: 54 obtained by substitution, deletion or addition of one or more amino acids and obtained with SEQ ID NO: 45, SEQ ID NO: 106, SEQ ID NO: 50, SEQ ID The functionally identical amino acid sequence of NO:51, SEQ ID NO:52, SEQ ID NO:53 or SEQ ID NO:54, and SEQ ID NO:45, SEQ ID NO:106, SEQ ID NO:50, SEQ ID NO :51, SEQ ID NO:52, SEQ ID NO:53 or SEQ ID NO:54 have an amino acid sequence of at least 85% sequence identity, and the amino acid sequence of the light chain variable region is selected from the group consisting of SEQ ID NO:46, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66 and SEQ ID NO:67, SEQ ID NO:46, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO: 57. SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO: 66 or SEQ ID NO: 67 obtained by substitution, deletion or addition of one or more amino acids and with SEQ ID NO: 46, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63 , SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66 or SEQ ID NO:67 functionally identical amino acid sequences, and SEQ ID NO:46, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID An amino acid sequence having at least 85% sequence identity to NO:65, SEQ ID NO:66 or SEQ ID NO:67.
在一些实施方案中，根据本发明的抗CRTAM抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列为SEQ ID NO:45，SEQ ID NO:45经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:45功能相同的氨基酸序列或与SEQ ID NO:45具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:46，SEQ ID NO:46经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:46功能相同的氨基酸序列或与SEQ ID NO:46具有至少85％序列同一性的氨基酸序列。In some embodiments, according to the anti-CRTAM antibody or antigen-binding fragment thereof of the present invention, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 45, SEQ ID NO: 45 is substituted, deleted or added with one or The amino acid sequence obtained by multiple amino acids and having the same function as SEQ ID NO: 45 or the amino acid sequence having at least 85% sequence identity with SEQ ID NO: 45, and the amino acid sequence of the light chain variable region is SEQ ID NO :46, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids of SEQ ID NO:46 and having the same function as SEQ ID NO:46 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:46 .
在一些实施方案中，根据本发明的抗CRTAM抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列为SEQ ID NO:106，SEQ ID NO:106经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:106功能相同的氨基酸序列或与SEQ ID NO:106具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:46，SEQ ID NO:46经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:46功能相同的氨基酸序列或与SEQ ID NO:46具有至少85％序列同一性的氨基酸序列。In some embodiments, according to the anti-CRTAM antibody or antigen-binding fragment thereof of the present invention, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 106, SEQ ID NO: 106 is substituted, deleted or added with one or The amino acid sequence obtained by multiple amino acids and having the same function as SEQ ID NO: 106 or the amino acid sequence having at least 85% sequence identity with SEQ ID NO: 106, and the amino acid sequence of the light chain variable region is SEQ ID NO :46, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids of SEQ ID NO:46 and having the same function as SEQ ID NO:46 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:46 .
在一些实施方案中，根据本发明的抗CRTAM抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列选自SEQ ID NO:50、SEQ ID NO:51、SEQ ID NO:52，SEQ ID NO:53和SEQ ID NO:54，SEQ ID NO:50、SEQ ID NO:51、SEQ ID NO:52，SEQ ID NO:53或SEQ ID NO:54经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:50、SEQ ID NO:51、SEQ ID NO:52，SEQ ID NO:53 或SEQ ID NO:54功能相同的氨基酸序列，以及与SEQ ID NO:50、SEQ ID NO:51、SEQ ID NO:52，SEQ ID NO:53或SEQ ID NO:54具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列选自SEQ ID NO:55、SEQ ID NO:56、SEQ ID NO:57、SEQ ID NO:58、SEQ ID NO:59、SEQ ID NO:60、SEQ ID NO:61、SEQ ID NO:62、SEQ ID NO:63、SEQ ID NO:64、SEQ ID NO:65和SEQ ID NO:66，SEQ ID NO:55、SEQ ID NO:56、SEQ ID NO:57、SEQ ID NO:58、SEQ ID NO:59、SEQ ID NO:60、SEQ ID NO:61、SEQ ID NO:62、SEQ ID NO:63、SEQ ID NO:64、SEQ ID NO:65或SEQ ID NO:66经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:55、SEQ ID NO:56、SEQ ID NO:57、SEQ ID NO:58、SEQ ID NO:59、SEQ ID NO:60、SEQ ID NO:61、SEQ ID NO:62、SEQ ID NO:63、SEQ ID NO:64、SEQ ID NO:65或SEQ ID NO:66功能相同的氨基酸序列，以及与SEQ ID NO:55、SEQ ID NO:56、SEQ ID NO:57、SEQ ID NO:58、SEQ ID NO:59、SEQ ID NO:60、SEQ ID NO:61、SEQ ID NO:62、SEQ ID NO:63、SEQ ID NO:64、SEQ ID NO:65或SEQ ID NO:66具有至少85％序列同一性的氨基酸序列。In some embodiments, according to the anti-CRTAM antibody or antigen-binding fragment thereof of the present invention, wherein the amino acid sequence of the heavy chain variable region is selected from SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53 and SEQ ID NO:54, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53 or SEQ ID NO:54 are substituted, deleted or added with one or more Amino acid sequence obtained from amino acids and identical to SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53 or SEQ ID NO:54, and SEQ ID NO:50, SEQ ID NO:50, SEQ ID NO:52 ID NO:51, SEQ ID NO:52, SEQ ID NO:53 or SEQ ID NO:54 have an amino acid sequence of at least 85% sequence identity, and the amino acid sequence of the light chain variable region is selected from SEQ ID NO: 55. SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65 and SEQ ID NO:66, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65 or SEQ ID NO:66 substituted, deleted or added with one or more amino acids Obtained and with SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO: 62. The functionally identical amino acid sequence of SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65 or SEQ ID NO:66, and SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57 , SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ An amino acid sequence having at least 85% sequence identity to ID NO:64, SEQ ID NO:65 or SEQ ID NO:66.
在一些实施方案中，根据本发明的抗CRTAM抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列为SEQ ID NO:51，SEQ ID NO:51经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:51功能相同的氨基酸序列或与SEQ ID NO:51具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:66，SEQ ID NO:66经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:66功能相同的氨基酸序列或与SEQ ID NO:66具有至少85％序列同一性的氨基酸序列；或者In some embodiments, according to the anti-CRTAM antibody or antigen-binding fragment thereof of the present invention, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 51, SEQ ID NO: 51 is substituted, deleted or added with one or An amino acid sequence obtained from multiple amino acids and having the same function as SEQ ID NO:51 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:51, and the amino acid sequence of the light chain variable region is SEQ ID NO :66, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids of SEQ ID NO:66 and having the same function as SEQ ID NO:66 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:66 ;or
所述重链可变区的氨基酸序列为SEQ ID NO:51，SEQ ID NO:51经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:51功能相同的氨基酸序列或与SEQ ID NO:51具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:67，SEQ ID NO:67经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:67功能相同的氨基酸序列或与SEQ ID NO:67具有至少85％序列同一性的氨基酸序列。The amino acid sequence of the heavy chain variable region is SEQ ID NO: 51, an amino acid sequence obtained by replacing, deleting or adding one or more amino acids to SEQ ID NO: 51 and having the same function as SEQ ID NO: 51 or with SEQ ID NO: 51 ID NO:51 has an amino acid sequence of at least 85% sequence identity, and the amino acid sequence of the light chain variable region is SEQ ID NO:67, and SEQ ID NO:67 is obtained by substitution, deletion or addition of one or more amino acids An amino acid sequence that is functionally identical to SEQ ID NO: 67 or an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 67.
在一些实施方案中，根据本发明的抗CRTAM抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列选自SEQ ID NO:93、SEQ ID NO:97、SEQ ID NO:98、SEQ ID NO:99、SEQ ID NO:100和SEQ ID NO:101，SEQ ID NO:93、SEQ  ID NO:97、SEQ ID NO:98、SEQ ID NO:99、SEQ ID NO:100或SEQ ID NO:101经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:93、SEQ ID NO:97、SEQ ID NO:98、SEQ ID NO:99、SEQ ID NO:100或SEQ ID NO:101功能相同的氨基酸序列，以及与SEQ ID NO:93、SEQ ID NO:97、SEQ ID NO:98、SEQ ID NO:99、SEQ ID NO:100或SEQ ID NO:101具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列选自SEQ ID NO:94、SEQ ID NO:102、SEQ ID NO:103、SEQ ID NO:104和SEQ ID NO:105，SEQ ID NO:94、SEQ ID NO:102、SEQ ID NO:103、SEQ ID NO:104或SEQ ID NO:105经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:94、SEQ ID NO:102、SEQ ID NO:103、SEQ ID NO:104或SEQ ID NO:105功能相同的氨基酸序列，以及与SEQ ID NO:94、SEQ ID NO:102、SEQ ID NO:103、SEQ ID NO:104或SEQ ID NO:105具有至少85％序列同一性的氨基酸序列。In some embodiments, according to the anti-CRTAM antibody or antigen-binding fragment thereof of the present invention, wherein the amino acid sequence of the heavy chain variable region is selected from the group consisting of SEQ ID NO:93, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100 and SEQ ID NO:101, SEQ ID NO:93, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100 or SEQ ID NO:101 is obtained by substitution, deletion or addition of one or more amino acids and is identical to SEQ ID NO:93, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100 or SEQ ID NO:101 functionally identical amino acid sequence, and at least 85% identical to SEQ ID NO:93, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100 or SEQ ID NO:101 the amino acid sequence of sequence identity, and the amino acid sequence of the light chain variable region is selected from SEQ ID NO:94, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104 and SEQ ID NO:105, SEQ ID NO: 94, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104 or SEQ ID NO: 105 obtained by substitution, deletion or addition of one or more amino acids and identical to SEQ ID NO: 94, Amino acid sequences functionally identical to SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104 or SEQ ID NO: 105, and SEQ ID NO: 94, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 103, SEQ ID NO: An amino acid sequence having at least 85% sequence identity to ID NO: 104 or SEQ ID NO: 105.
在一些实施方案中，根据本发明的抗CRTAM抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列为SEQ ID NO:93，SEQ ID NO:93经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:93功能相同的氨基酸序列或与SEQ ID NO:93具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:94，SEQ ID NO:94经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:94功能相同的氨基酸序列或与SEQ ID NO:94具有至少85％序列同一性的氨基酸序列。In some embodiments, according to the anti-CRTAM antibody or antigen-binding fragment thereof of the present invention, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 93, SEQ ID NO: 93 is substituted, deleted or added with one or An amino acid sequence obtained from multiple amino acids and having the same function as SEQ ID NO:93 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:93, and the amino acid sequence of the light chain variable region is SEQ ID NO :94, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids of SEQ ID NO:94 and having the same function as SEQ ID NO:94 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:94 .
在一些实施方案中，根据本发明的抗CRTAM抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列选自SEQ ID NO:97、SEQ ID NO:98、SEQ ID NO:99和SEQ ID NO:100，SEQ ID NO:97、SEQ ID NO:98、SEQ ID NO:99或SEQ ID NO:100经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:97、SEQ ID NO:98、SEQ ID NO:99或SEQ ID NO:100功能相同的氨基酸序列，以及与SEQ ID NO:97、SEQ ID NO:98、SEQ ID NO:99或SEQ ID NO:100具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列选自SEQ ID NO:102、SEQ ID NO:103、SEQ ID NO:104和SEQ ID NO:105，SEQ ID NO:102、SEQ ID NO:103、SEQ ID NO:104或SEQ ID NO:105经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:102、SEQ ID NO:103、SEQ ID NO:104或SEQ ID NO:105功能相同的氨基酸序列，以及与SEQ ID NO:102、SEQ ID NO:103、SEQ ID NO:104或SEQ ID NO:105具有至少85％序列同一性的氨基 酸序列。In some embodiments, according to the anti-CRTAM antibody or antigen-binding fragment thereof of the present invention, wherein the amino acid sequence of the heavy chain variable region is selected from SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99 and SEQ ID NO: 100, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99 or SEQ ID NO: 100 obtained by substitution, deletion or addition of one or more amino acids and identical to SEQ ID NO: 97, An amino acid sequence that is functionally identical to SEQ ID NO:98, SEQ ID NO:99, or SEQ ID NO:100, and has at least The amino acid sequence of 85% sequence identity, and the aminoacid sequence of described light chain variable region is selected from SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104 and SEQ ID NO:105, SEQ ID NO: 102. SEQ ID NO: 103, SEQ ID NO: 104 or SEQ ID NO: 105 is obtained by substitution, deletion or addition of one or more amino acids and is identical to SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104 or a functionally identical amino acid sequence of SEQ ID NO: 105, and an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104 or SEQ ID NO: 105.
在一些实施方案中，根据本发明的抗CRTAM抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列为SEQ ID NO:99，SEQ ID NO:99经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:99功能相同的氨基酸序列或与SEQ ID NO:99具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:104，SEQ ID NO:104经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:104功能相同的氨基酸序列或与SEQ ID NO:104具有至少85％序列同一性的氨基酸序列；或者In some embodiments, according to the anti-CRTAM antibody or antigen-binding fragment thereof of the present invention, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 99, SEQ ID NO: 99 is substituted, deleted or added with one or An amino acid sequence obtained from multiple amino acids and having the same function as SEQ ID NO:99 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:99, and the amino acid sequence of the light chain variable region is SEQ ID NO :104, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids of SEQ ID NO:104 and having the same function as SEQ ID NO:104 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:104 ;or
所述重链可变区的氨基酸序列为SEQ ID NO:101，SEQ ID NO:101经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:101功能相同的氨基酸序列或与SEQ ID NO:101具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:104，SEQ ID NO:104经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:104功能相同的氨基酸序列或与SEQ ID NO:104具有至少85％序列同一性的氨基酸序列。The amino acid sequence of the heavy chain variable region is SEQ ID NO: 101, an amino acid sequence obtained by replacing, deleting or adding one or more amino acids to SEQ ID NO: 101 and having the same function as SEQ ID NO: 101 or identical to SEQ ID NO: 101. ID NO: 101 has an amino acid sequence of at least 85% sequence identity, and the amino acid sequence of the light chain variable region is SEQ ID NO: 104, and SEQ ID NO: 104 is obtained by substitution, deletion or addition of one or more amino acids An amino acid sequence that is functionally identical to SEQ ID NO: 104 or an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 104.
在一些具体的实施方案中，本发明提供一种抗CRTAM抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中所述重链可变区的HCDR1、HCDR2和HCDR3分别为SEQ ID NO:1、2和3，所述轻链可变区的LCDR1、LCDR2和LCDR3分别为SEQ ID NO:4、5和6，所述重链可变区的氨基酸序列为SEQ ID NO:80，SEQ ID NO:80经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:80功能相同的氨基酸序列或与SEQ ID NO:80具有至少85％序列同一性且所述HCDR1、HCDR2和HCDR3如SEQ ID NO:1、2和3所示的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:81，SEQ ID NO:81经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:81功能相同的氨基酸序列或与SEQ ID NO:81具有至少85％序列同一性且所述LCDR1、LCDR2和LCDR3如SEQ ID NO:4、5和6所示的氨基酸序列。In some specific embodiments, the present invention provides an anti-CRTAM antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region and a light chain variable region, wherein HCDR1, HCDR2 and HCDR3 of the heavy chain variable region Respectively SEQ ID NO:1, 2 and 3, the LCDR1, LCDR2 and LCDR3 of the light chain variable region are respectively SEQ ID NO:4, 5 and 6, the amino acid sequence of the heavy chain variable region is SEQ ID NO:80, the amino acid sequence obtained by replacing, deleting or adding one or more amino acids of SEQ ID NO:80 and having the same function as SEQ ID NO:80 or having at least 85% sequence identity with SEQ ID NO:80 and all The amino acid sequences of HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 1, 2 and 3, and the amino acid sequence of the light chain variable region is SEQ ID NO: 81, SEQ ID NO: 81 is substituted, deleted or Amino acid sequence obtained by adding one or more amino acids and having the same function as SEQ ID NO: 81 or having at least 85% sequence identity with SEQ ID NO: 81 and said LCDR1, LCDR2 and LCDR3 such as SEQ ID NO: 4, 5 and the amino acid sequence shown in 6.
在一些具体的实施方案中，本发明提供一种抗CRTAM抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中所述重链可变区的HCDR1、HCDR2和HCDR3分别为SEQ ID NO:1、2和3，所述轻链可变区的LCDR1、LCDR2和LCDR3分别为SEQ ID NO:4、5和6，所述重链可变区的氨基酸序列选自SEQ ID NO:84、SEQ ID NO:85、SEQ ID NO:86和SEQ ID NO:87，SEQ ID NO:84、SEQ ID NO:85、SEQ ID NO:86或SEQ ID NO:87经取代、缺失或添加一个或多 个氨基酸获得的且与SEQ ID NO:84、SEQ ID NO:85、SEQ ID NO:86或SEQ ID NO:87功能相同的氨基酸序列，以及与SEQ ID NO:84、SEQ ID NO:85、SEQ ID NO:86或SEQ ID NO:87具有至少85％序列同一性且所述HCDR1、HCDR2和HCDR3如SEQ ID NO:1、2和3所示的氨基酸序列，且所述轻链可变区的氨基酸序列选自SEQ ID NO:88、SEQ ID NO:89、SEQ ID NO:90和SEQ ID NO:91，SEQ ID NO:88、SEQ ID NO:89、SEQ ID NO:90或SEQ ID NO:91经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:88、SEQ ID NO:89、SEQ ID NO:90或SEQ ID NO:91功能相同的氨基酸序列，以及与SEQ ID NO:88、SEQ ID NO:89、SEQ ID NO:90或SEQ ID NO:91具有至少85％序列同一性且所述LCDR1、LCDR2和LCDR3如SEQ ID NO:4、5和6所示的氨基酸序列。In some specific embodiments, the present invention provides an anti-CRTAM antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region and a light chain variable region, wherein HCDR1, HCDR2 and HCDR3 of the heavy chain variable region Respectively SEQ ID NO: 1, 2 and 3, LCDR1, LCDR2 and LCDR3 of the light chain variable region are respectively SEQ ID NO: 4, 5 and 6, the amino acid sequence of the heavy chain variable region is selected from SEQ ID NO: ID NO:84, SEQ ID NO:85, SEQ ID NO:86 and SEQ ID NO:87, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86 or SEQ ID NO:87 substituted, deleted Or the amino acid sequence obtained by adding one or more amino acids and having the same function as SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86 or SEQ ID NO: 87, and the same amino acid sequence as SEQ ID NO: 84, SEQ ID NO:85, SEQ ID NO:86 or SEQ ID NO:87 have at least 85% sequence identity and said HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NO:1, 2 and 3, and said light The amino acid sequence of the chain variable region is selected from the group consisting of SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90 and SEQ ID NO:91, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90 or an amino acid sequence obtained by substituting, deleting or adding one or more amino acids of SEQ ID NO:91 and having the same function as SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90 or SEQ ID NO:91, and having at least 85% sequence identity to SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90 or SEQ ID NO: 91 and said LCDR1, LCDR2 and LCDR3 such as SEQ ID NO: 4, 5 and 6 Amino acid sequence shown.
在一些实施方案中，根据本发明的抗CRTAM抗体或其抗原结合片段，其中所述重链可变区的HCDR1、HCDR2和HCDR3分别为SEQ ID NO:1、2和3，所述轻链可变区的LCDR1、LCDR2和LCDR3分别为SEQ ID NO:4、5和6，所述重链可变区的氨基酸序列为SEQ ID NO:87，且所述轻链可变区的氨基酸序列为SEQ ID NO:90。In some embodiments, according to the anti-CRTAM antibody or antigen-binding fragment thereof of the present invention, wherein the HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are SEQ ID NO: 1, 2 and 3 respectively, the light chain can be The LCDR1, LCDR2 and LCDR3 of the variable region are SEQ ID NO: 4, 5 and 6 respectively, the amino acid sequence of the heavy chain variable region is SEQ ID NO: 87, and the amino acid sequence of the light chain variable region is SEQ ID NO: 87 ID NO:90.
在一些具体的实施方案中，本发明提供一种抗CRTAM抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中所述重链可变区的HCDR1、HCDR2和HCDR3分别为SEQ ID NO:1、2和3，所述轻链可变区的LCDR1、LCDR2和LCDR3分别为SEQ ID NO:7、5和6，所述重链可变区的氨基酸序列为SEQ ID NO:87，SEQ ID NO:87经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:87功能相同的氨基酸序列或与SEQ ID NO:87具有至少85％序列同一性且所述HCDR1、HCDR2和HCDR3如SEQ ID NO:1、2和3所示的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:92，SEQ ID NO:92经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:92功能相同的氨基酸序列或与SEQ ID NO:92具有至少85％序列同一性且所述LCDR1、LCDR2和LCDR3如SEQ ID NO:7、5和6所示的氨基酸序列。In some specific embodiments, the present invention provides an anti-CRTAM antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region and a light chain variable region, wherein HCDR1, HCDR2 and HCDR3 of the heavy chain variable region Respectively SEQ ID NO:1, 2 and 3, the LCDR1, LCDR2 and LCDR3 of the light chain variable region are respectively SEQ ID NO:7, 5 and 6, the amino acid sequence of the heavy chain variable region is SEQ ID NO:87, the amino acid sequence obtained by replacing, deleting or adding one or more amino acids of SEQ ID NO:87 and having the same function as SEQ ID NO:87 or having at least 85% sequence identity with SEQ ID NO:87 and all The amino acid sequences of the HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 1, 2 and 3, and the amino acid sequence of the light chain variable region are SEQ ID NO: 92, and SEQ ID NO: 92 is substituted, deleted or Amino acid sequence obtained by adding one or more amino acids and having the same function as SEQ ID NO: 92 or having at least 85% sequence identity with SEQ ID NO: 92 and said LCDR1, LCDR2 and LCDR3 such as SEQ ID NO: 7, 5 and the amino acid sequence shown in 6.
在一些实施方案中，本发明提供一种抗CRTAM抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中所述重链可变区的HCDR1、HCDR2和HCDR3分别为SEQ ID NO:8、9和10，所述轻链可变区的LCDR1、LCDR2和LCDR3分别为SEQ ID NO:12、13和14，所述重链可变区的氨基酸序列为SEQ ID NO:45，SEQ ID NO:45经取代、缺失或添加一个或多个氨基酸获得的且与SEQ  ID NO:45功能相同的氨基酸序列或与SEQ ID NO:45具有至少85％序列同一性且所述HCDR1、HCDR2和HCDR3如SEQ ID NO:8、9和10所示的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:46，SEQ ID NO:46经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:46功能相同的氨基酸序列或与SEQ ID NO:46具有至少85％序列同一性且所述LCDR1、LCDR2和LCDR3如SEQ ID NO:12、13和14所示的氨基酸序列。In some embodiments, the present invention provides an anti-CRTAM antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are respectively SEQ ID NO:8, 9 and 10, the LCDR1, LCDR2 and LCDR3 of the light chain variable region are SEQ ID NO:12, 13 and 14 respectively, the amino acid sequence of the heavy chain variable region is SEQ ID NO: 45. An amino acid sequence obtained by substituting, deleting or adding one or more amino acids of SEQ ID NO: 45 and having the same function as SEQ ID NO: 45 or having at least 85% sequence identity with SEQ ID NO: 45 and the HCDR1 , HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NOs: 8, 9 and 10, and the amino acid sequence of the light chain variable region is SEQ ID NO: 46, and SEQ ID NO: 46 is substituted, deleted or added or a plurality of amino acids obtained and having the same amino acid sequence as SEQ ID NO:46 or having at least 85% sequence identity with SEQ ID NO:46 and said LCDR1, LCDR2 and LCDR3 such as SEQ ID NO:12, 13 and 14 Amino acid sequence shown.
在一些实施方案中，根据本发明的抗CRTAM抗体或其抗原结合片段，其中所述重链可变区的HCDR1、HCDR2和HCDR3分别为SEQ ID NO:8、11和10，所述轻链可变区的LCDR1、LCDR2和LCDR3分别为SEQ ID NO:12、13和14，所述重链可变区的氨基酸序列为SEQ ID NO:106，SEQ ID NO:106经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:106功能相同的氨基酸序列或与SEQ ID NO:106具有至少85％序列同一性且所述HCDR1、HCDR2和HCDR3如SEQ ID NO:8、11和10所示的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:46，SEQ ID NO:46经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:46功能相同的氨基酸序列或与SEQ ID NO:46具有至少85％序列同一性且所述LCDR1、LCDR2和LCDR3如SEQ ID NO:12、13和14所示的氨基酸序列。In some embodiments, according to the anti-CRTAM antibody or antigen-binding fragment thereof of the present invention, wherein the HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are SEQ ID NO: 8, 11 and 10, respectively, the light chain can be The LCDR1, LCDR2 and LCDR3 of the variable region are SEQ ID NO: 12, 13 and 14 respectively, the amino acid sequence of the heavy chain variable region is SEQ ID NO: 106, and SEQ ID NO: 106 is substituted, deleted or added one or Amino acid sequence obtained from multiple amino acids and functionally identical to SEQ ID NO: 106 or having at least 85% sequence identity with SEQ ID NO: 106 and said HCDR1, HCDR2 and HCDR3 are as set forth in SEQ ID NO: 8, 11 and 10 The amino acid sequence shown, and the amino acid sequence of the light chain variable region is SEQ ID NO: 46, SEQ ID NO: 46 is obtained by substitution, deletion or addition of one or more amino acids and has the same function as SEQ ID NO: 46 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 46 and the LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 12, 13 and 14.
在一些实施方案中，根据本发明的抗CRTAM抗体或其抗原结合片段，其中所述重链可变区的HCDR1、HCDR2和HCDR3分别为SEQ ID NO:8、11和10，所述轻链可变区的LCDR1、LCDR2和LCDR3分别为SEQ ID NO:12、13和14，所述重链可变区的氨基酸序列选自SEQ ID NO:50、SEQ ID NO:51、SEQ ID NO:52，SEQ ID NO:53和SEQ ID NO:54，SEQ ID NO:50、SEQ ID NO:51、SEQ ID NO:52，SEQ ID NO:53或SEQ ID NO:54经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:50、SEQ ID NO:51、SEQ ID NO:52，SEQ ID NO:53或SEQ ID NO:54功能相同的氨基酸序列，以及与SEQ ID NO:50、SEQ ID NO:51、SEQ ID NO:52，SEQ ID NO:53或SEQ ID NO:54具有至少85％序列同一性且所述HCDR1、HCDR2和HCDR3如SEQ ID NO:8、11和10所示的氨基酸序列，且所述轻链可变区的氨基酸序列选自SEQ ID NO:55、SEQ ID NO:56、SEQ ID NO:57、SEQ ID NO:58、SEQ ID NO:59、SEQ ID NO:60、SEQ ID NO:61、SEQ ID NO:62、SEQ ID NO:63、SEQ ID NO:64、SEQ ID NO:65和SEQ ID NO:66，SEQ ID NO:55、SEQ ID NO:56、SEQ ID NO:57、SEQ ID NO:58、SEQ ID  NO:59、SEQ ID NO:60、SEQ ID NO:61、SEQ ID NO:62、SEQ ID NO:63、SEQ ID NO:64、SEQ ID NO:65或SEQ ID NO:66经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:55、SEQ ID NO:56、SEQ ID NO:57、SEQ ID NO:58、SEQ ID NO:59、SEQ ID NO:60、SEQ ID NO:61、SEQ ID NO:62、SEQ ID NO:63、SEQ ID NO:64、SEQ ID NO:65或SEQ ID NO:66功能相同的氨基酸序列，以及与SEQ ID NO:55、SEQ ID NO:56、SEQ ID NO:57、SEQ ID NO:58、SEQ ID NO:59、SEQ ID NO:60、SEQ ID NO:61、SEQ ID NO:62、SEQ ID NO:63、SEQ ID NO:64、SEQ ID NO:65或SEQ ID NO:66具有至少85％序列同一性且所述LCDR1、LCDR2和LCDR3如SEQ ID NO:12、13和14所示的氨基酸序列。In some embodiments, according to the anti-CRTAM antibody or antigen-binding fragment thereof of the present invention, wherein the HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are SEQ ID NO: 8, 11 and 10, respectively, the light chain can be The LCDR1, LCDR2 and LCDR3 of the variable region are SEQ ID NO:12, 13 and 14 respectively, and the amino acid sequence of the heavy chain variable region is selected from SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53 and SEQ ID NO:54, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53 or SEQ ID NO:54 are substituted, deleted or added with one or more Amino acid sequences obtained from amino acids and having the same function as SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53 or SEQ ID NO:54, as well as the same amino acid sequence as SEQ ID NO:50, SEQ ID NO:50, SEQ ID NO:52 ID NO:51, SEQ ID NO:52, SEQ ID NO:53 or SEQ ID NO:54 have at least 85% sequence identity and the HCDR1, HCDR2 and HCDR3 are as shown in SEQ ID NO:8, 11 and 10 Amino acid sequence, and the amino acid sequence of the light chain variable region is selected from SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO: 60. SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65 and SEQ ID NO:66, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO: 65 or SEQ ID NO: 66 obtained by substitution, deletion or addition of one or more amino acids and with SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65 or SEQ ID NO:66 function The same amino acid sequence, and with SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65 or SEQ ID NO:66 has at least 85% sequence identity and said LCDR1, LCDR2 and LCDR3 are as in SEQ ID NO:12 , 13 and 14 shown amino acid sequences.
在一些实施方案中，根据本发明的抗CRTAM抗体或其抗原结合片段，其中所述重链可变区的HCDR1、HCDR2和HCDR3分别为SEQ ID NO:8、11和10，所述轻链可变区的LCDR1、LCDR2和LCDR3分别为SEQ ID NO:12、13和14，所述重链可变区的氨基酸序列为SEQ ID NO:51，且所述轻链可变区的氨基酸序列为SEQ ID NO:66。In some embodiments, according to the anti-CRTAM antibody or antigen-binding fragment thereof of the present invention, wherein the HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are SEQ ID NO: 8, 11 and 10, respectively, the light chain can be The LCDR1, LCDR2 and LCDR3 of the variable region are SEQ ID NO:12, 13 and 14 respectively, the amino acid sequence of the heavy chain variable region is SEQ ID NO:51, and the amino acid sequence of the light chain variable region is SEQ ID NO: ID NO:66.
在一些实施方案中，根据本发明的抗CRTAM抗体或其抗原结合片段，其中所述重链可变区的HCDR1、HCDR2和HCDR3分别为SEQ ID NO:8、11和10，所述轻链可变区的LCDR1、LCDR2和LCDR3分别为SEQ ID NO:15、13和14，所述重链可变区的氨基酸序列为SEQ ID NO:51，SEQ ID NO:51经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:51功能相同的氨基酸序列或与SEQ ID NO:51具有至少85％序列同一性且所述HCDR1、HCDR2和HCDR3如SEQ ID NO:8、11和10所示的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:67，SEQ ID NO:67经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:67功能相同的氨基酸序列或与SEQ ID NO:67具有至少85％序列同一性且所述LCDR1、LCDR2和LCDR3如SEQ ID NO:15、13和14所示的氨基酸序列。In some embodiments, according to the anti-CRTAM antibody or antigen-binding fragment thereof of the present invention, wherein the HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are SEQ ID NO: 8, 11 and 10, respectively, the light chain can be The LCDR1, LCDR2 and LCDR3 of the variable region are SEQ ID NO: 15, 13 and 14 respectively, the amino acid sequence of the heavy chain variable region is SEQ ID NO: 51, and SEQ ID NO: 51 is substituted, deleted or added with one or Amino acid sequence obtained from multiple amino acids and functionally identical to SEQ ID NO: 51 or having at least 85% sequence identity to SEQ ID NO: 51 and said HCDR1, HCDR2 and HCDR3 are as set forth in SEQ ID NOs: 8, 11 and 10 The amino acid sequence shown, and the amino acid sequence of the light chain variable region is SEQ ID NO: 67, SEQ ID NO: 67 obtained through substitution, deletion or addition of one or more amino acids and has the same function as SEQ ID NO: 67 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 67 and the LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 15, 13 and 14.
在一些实施方案中，本发明提供一种抗CRTAM抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中所述重链可变区的HCDR1、HCDR2和HCDR3分别为SEQ ID NO:16、17、18，所述轻链可变区的LCDR1、LCDR2和LCDR3分别为SEQ ID NO:19、20、21，所述重链可变区的氨基酸序列为SEQ ID NO:68，SEQ ID NO:68经取代、缺失或添加一个或多个氨基酸获得的且与SEQ  ID NO:68功能相同的氨基酸序列或与SEQ ID NO:68具有至少85％序列同一性且所述HCDR1、HCDR2和HCDR3如SEQ ID NO:16、17、18所示的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:69，SEQ ID NO:69经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:69功能相同的氨基酸序列或与SEQ ID NO:69具有至少85％序列同一性且所述LCDR1、LCDR2和LCDR3如SEQ ID NO:19、20、21所示的氨基酸序列。In some embodiments, the present invention provides an anti-CRTAM antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are respectively SEQ ID NO:16,17,18, the LCDR1, LCDR2 and LCDR3 of the light chain variable region are SEQ ID NO:19,20,21 respectively, the amino acid sequence of the heavy chain variable region is SEQ ID NO: 68. An amino acid sequence obtained by replacing, deleting or adding one or more amino acids of SEQ ID NO:68 and having the same function as SEQ ID NO:68 or having at least 85% sequence identity with SEQ ID NO:68 and the HCDR1 , HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NO: 16, 17, 18, and the amino acid sequence of the light chain variable region is SEQ ID NO: 69, and SEQ ID NO: 69 is substituted, deleted or added or a plurality of amino acids obtained and having the same amino acid sequence as SEQ ID NO: 69 or having at least 85% sequence identity with SEQ ID NO: 69 and said LCDR1, LCDR2 and LCDR3 such as SEQ ID NO: 19, 20, 21 Amino acid sequence shown.
在一些实施方案中，根据本发明的抗CRTAM抗体或其抗原结合片段，，其包含重链可变区和轻链可变区，其中所述重链可变区的HCDR1、HCDR2和HCDR3分别为SEQ ID NO:16、17、18，所述轻链可变区的LCDR1、LCDR2和LCDR3分别为SEQ ID NO:19、20、21，所述重链可变区的氨基酸序列选自SEQ ID NO:72、SEQ ID NO:73、SEQ ID NO:74和SEQ ID NO:75，SEQ ID NO:72、SEQ ID NO:73、SEQ ID NO:74或SEQ ID NO:75经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:72、SEQ ID NO:73、SEQ ID NO:74或SEQ ID NO:75功能相同的氨基酸序列，以及与SEQ ID NO:72、SEQ ID NO:73、SEQ ID NO:74或SEQ ID NO:75具有至少85％序列同一性且所述HCDR1、HCDR2和HCDR3如SEQ ID NO:16、17、18所示的氨基酸序列，且所述轻链可变区的氨基酸序列选自SEQ ID NO:76、SEQ ID NO:77、SEQ ID NO:78和SEQ ID NO:79，SEQ ID NO:76、SEQ ID NO:77、SEQ ID NO:78或SEQ ID NO:79经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:76、SEQ ID NO:77、SEQ ID NO:78或SEQ ID NO:79功能相同的氨基酸序列，以及与SEQ ID NO:76、SEQ ID NO:77、SEQ ID NO:78或SEQ ID NO:79具有至少85％序列同一性且所述LCDR1、LCDR2和LCDR3如SEQ ID NO:19、20、21所示的氨基酸序列。In some embodiments, the anti-CRTAM antibody or antigen-binding fragment thereof according to the present invention comprises a heavy chain variable region and a light chain variable region, wherein HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are respectively SEQ ID NO:16,17,18, the LCDR1, LCDR2 and LCDR3 of the light chain variable region are SEQ ID NO:19,20,21 respectively, the amino acid sequence of the heavy chain variable region is selected from SEQ ID NO :72, SEQ ID NO:73, SEQ ID NO:74 and SEQ ID NO:75, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74 or SEQ ID NO:75 are substituted, deleted or added Amino acid sequences obtained by one or more amino acids and functionally identical to SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74 or SEQ ID NO:75, and sequences identical to SEQ ID NO:72, SEQ ID NO: 73. SEQ ID NO: 74 or SEQ ID NO: 75 has at least 85% sequence identity and said HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NO: 16, 17, 18, and said light chain can be The amino acid sequence of the variable region is selected from SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78 and SEQ ID NO:79, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78 or SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78 or SEQ ID NO:78 ID NO:79 is obtained by substitution, deletion or addition of one or more amino acids and has the same amino acid sequence as SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78 or SEQ ID NO:79, and SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78 or SEQ ID NO: 79 has at least 85% sequence identity and said LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 19, 20, 21 amino acid sequence.
在一些实施方案中，根据本发明的抗CRTAM抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中所述重链可变区的HCDR1、HCDR2和HCDR3分别为SEQ ID NO:16、17、18，所述轻链可变区的LCDR1、LCDR2和LCDR3分别为SEQ ID NO:19、20、21，所述重链可变区的氨基酸序列为SEQ ID NO:75，SEQ ID NO:75经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:75功能相同的氨基酸序列或与SEQ ID NO:75具有至少85％序列同一性且所述HCDR1、HCDR2和HCDR3如SEQ ID NO:16、17、18所示的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:77，SEQ ID NO:77经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:77功能相同的氨基酸序列或与 SEQ ID NO:77具有至少85％序列同一性且所述LCDR1、LCDR2和LCDR3如SEQ ID NO:19、20、21所示的氨基酸序列。In some embodiments, the anti-CRTAM antibody or antigen-binding fragment thereof according to the present invention comprises a heavy chain variable region and a light chain variable region, wherein HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are SEQ ID NO: ID NO: 16, 17, 18, the LCDR1, LCDR2 and LCDR3 of the light chain variable region are SEQ ID NO: 19, 20, 21 respectively, the amino acid sequence of the heavy chain variable region is SEQ ID NO: 75 , an amino acid sequence obtained by substituting, deleting or adding one or more amino acids of SEQ ID NO:75 and having the same function as SEQ ID NO:75 or having at least 85% sequence identity with SEQ ID NO:75 and said HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NO: 16, 17, 18, and the amino acid sequence of the light chain variable region is SEQ ID NO: 77, and SEQ ID NO: 77 is substituted, deleted or added with one or The amino acid sequence obtained by multiple amino acids and having the same function as SEQ ID NO: 77 or having at least 85% sequence identity with SEQ ID NO: 77 and the LCDR1, LCDR2 and LCDR3 are as shown in SEQ ID NO: 19, 20, 21 The amino acid sequence shown.
在一些实施方案中，根据本发明的抗CRTAM抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中所述重链可变区的HCDR1、HCDR2和HCDR3分别为SEQ ID NO:22、23和24，所述轻链可变区的LCDR1、LCDR2和LCDR3分别为SEQ ID NO:26、27和28，所述重链可变区的氨基酸序列为SEQ ID NO:93，SEQ ID NO:93经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:93功能相同的氨基酸序列或与SEQ ID NO:93具有至少85％序列同一性且所述HCDR1、HCDR2和HCDR3如SEQ ID NO:22、23和24所示的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:94，SEQ ID NO:94经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:94功能相同的氨基酸序列或与SEQ ID NO:94具有至少85％序列同一性且所述LCDR1、LCDR2和LCDR3如SEQ ID NO:26、27和28所示的氨基酸序列。In some embodiments, the anti-CRTAM antibody or antigen-binding fragment thereof according to the present invention comprises a heavy chain variable region and a light chain variable region, wherein HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are SEQ ID NO: ID NO: 22, 23 and 24, the LCDR1, LCDR2 and LCDR3 of the light chain variable region are respectively SEQ ID NO: 26, 27 and 28, the amino acid sequence of the heavy chain variable region is SEQ ID NO: 93 , an amino acid sequence obtained by substituting, deleting or adding one or more amino acids of SEQ ID NO:93 and having the same function as SEQ ID NO:93 or having at least 85% sequence identity with SEQ ID NO:93 and said HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NO:22, 23 and 24, and the amino acid sequence of the light chain variable region is SEQ ID NO:94, and SEQ ID NO:94 is substituted, deleted or added with one or An amino acid sequence obtained from a plurality of amino acids and having the same function as SEQ ID NO: 94 or having at least 85% sequence identity with SEQ ID NO: 94 and said LCDR1, LCDR2 and LCDR3 are as set forth in SEQ ID NO: 26, 27 and 28 The amino acid sequence shown.
在一些实施方案中，根据本发明的抗CRTAM抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中所述重链可变区的HCDR1、HCDR2和HCDR3分别为SEQ ID NO:22、23和24，所述轻链可变区的LCDR1、LCDR2和LCDR3分别为SEQ ID NO:26、27和28，所述重链可变区的氨基酸序列选自SEQ ID NO:97、SEQ ID NO:98、SEQ ID NO:99和SEQ ID NO:100，SEQ ID NO:97、SEQ ID NO:98、SEQ ID NO:99或SEQ ID NO:100经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:97、SEQ ID NO:98、SEQ ID NO:99或SEQ ID NO:100功能相同的氨基酸序列，以及与SEQ ID NO:97、SEQ ID NO:98、SEQ ID NO:99或SEQ ID NO:100具有至少85％序列同一性且所述HCDR1、HCDR2和HCDR3如SEQ ID NO:22、23和24所示的氨基酸序列，且所述轻链可变区的氨基酸序列选自SEQ ID NO:102、SEQ ID NO:103、SEQ ID NO:104和SEQ ID NO:105，SEQ ID NO:102、SEQ ID NO:103、SEQ ID NO:104或SEQ ID NO:105经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:102、SEQ ID NO:103、SEQ ID NO:104或SEQ ID NO:105功能相同的氨基酸序列，以及与SEQ ID NO:102、SEQ ID NO:103、SEQ ID NO:104或SEQ ID NO:105具有至少85％序列同一性且所述LCDR1、LCDR2和LCDR3如SEQ ID NO:26、27和28所示的氨基酸序列。In some embodiments, the anti-CRTAM antibody or antigen-binding fragment thereof according to the present invention comprises a heavy chain variable region and a light chain variable region, wherein HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are SEQ ID NO: ID NO:22, 23 and 24, the LCDR1, LCDR2 and LCDR3 of the light chain variable region are respectively SEQ ID NO:26, 27 and 28, the amino acid sequence of the heavy chain variable region is selected from SEQ ID NO: 97. SEQ ID NO: 98, SEQ ID NO: 99 and SEQ ID NO: 100, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99 or SEQ ID NO: 100 are substituted, missing or have one added or a plurality of amino acids obtained and with SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99 or SEQ ID NO: 100 functionally identical amino acid sequence, and with SEQ ID NO: 97, SEQ ID NO: 98 , SEQ ID NO:99 or SEQ ID NO:100 have at least 85% sequence identity and said HCDR1, HCDR2 and HCDR3 have amino acid sequences as shown in SEQ ID NO:22, 23 and 24, and said light chain is variable The amino acid sequence of the region is selected from SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104 and SEQ ID NO: 105, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104 or SEQ ID NO:105 obtained by substituting, deleting or adding one or more amino acids and having the same amino acid sequence as SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104 or SEQ ID NO:105, and SEQ ID NO:105 ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104 or SEQ ID NO: 105 has at least 85% sequence identity and said LCDR1, LCDR2 and LCDR3 are as set forth in SEQ ID NOs: 26, 27 and 28 amino acid sequence.
在一些实施方案中，根据本发明的抗CRTAM抗体或其抗原结合片段，其包 含重链可变区和轻链可变区，其中所述重链可变区的HCDR1、HCDR2和HCDR3分别为SEQ ID NO:22、23和24，所述轻链可变区的LCDR1、LCDR2和LCDR3分别为SEQ ID NO:26、27和28，所述重链可变区的氨基酸序列为SEQ ID NO:99，SEQ ID NO:99经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:99功能相同的氨基酸序列或与SEQ ID NO:99具有至少85％序列同一性且所述HCDR1、HCDR2和HCDR3如SEQ ID NO:22、23和24所示的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:104，SEQ ID NO:104经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:104功能相同的氨基酸序列或与SEQ ID NO:104具有至少85％序列同一性且所述LCDR1、LCDR2和LCDR3如SEQ ID NO:26、27和28所示的氨基酸序列。In some embodiments, the anti-CRTAM antibody or antigen-binding fragment thereof according to the present invention comprises a heavy chain variable region and a light chain variable region, wherein HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are SEQ ID NO: ID NOs: 22, 23 and 24, the LCDR1, LCDR2 and LCDR3 of the light chain variable region are SEQ ID NO: 26, 27 and 28 respectively, and the amino acid sequence of the heavy chain variable region is SEQ ID NO: 99 , an amino acid sequence obtained by substituting, deleting or adding one or more amino acids of SEQ ID NO:99 and having the same function as SEQ ID NO:99 or having at least 85% sequence identity with SEQ ID NO:99 and said HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NOs: 22, 23 and 24, and the amino acid sequence of the light chain variable region is SEQ ID NO: 104, and SEQ ID NO: 104 is substituted, deleted or added with one or An amino acid sequence obtained from multiple amino acids and having the same function as SEQ ID NO: 104 or having at least 85% sequence identity with SEQ ID NO: 104 and said LCDR1, LCDR2 and LCDR3 are as set forth in SEQ ID NO: 26, 27 and 28 The amino acid sequence shown.
在一些实施方案中，根据本发明的抗CRTAM抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中所述重链可变区的HCDR1、HCDR2和HCDR3分别为SEQ ID NO:22、25和24，所述轻链可变区的LCDR1、LCDR2和LCDR3分别为SEQ ID NO:26、27和28，所述重链可变区的氨基酸序列为SEQ ID NO:101，SEQ ID NO:101经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:101功能相同的氨基酸序列或与SEQ ID NO:101具有至少85％序列同一性且所述HCDR1、HCDR2和HCDR3如SEQ ID NO:22、25和24所示的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:104，SEQ ID NO:104经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:104功能相同的氨基酸序列或与SEQ ID NO:104具有至少85％序列同一性且所述LCDR1、LCDR2和LCDR3如SEQ ID NO:26、27和28所示的氨基酸序列。In some embodiments, the anti-CRTAM antibody or antigen-binding fragment thereof according to the present invention comprises a heavy chain variable region and a light chain variable region, wherein HCDR1, HCDR2 and HCDR3 of the heavy chain variable region are SEQ ID NO: ID NOs: 22, 25 and 24, the LCDR1, LCDR2 and LCDR3 of the light chain variable region are SEQ ID NO: 26, 27 and 28 respectively, and the amino acid sequence of the heavy chain variable region is SEQ ID NO: 101 , an amino acid sequence obtained by substituting, deleting or adding one or more amino acids of SEQ ID NO:101 and having the same function as SEQ ID NO:101 or having at least 85% sequence identity with SEQ ID NO:101 and said HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NOs: 22, 25 and 24, and the amino acid sequence of the light chain variable region is SEQ ID NO: 104, and SEQ ID NO: 104 is substituted, deleted or added with one or An amino acid sequence obtained from multiple amino acids and having the same function as SEQ ID NO: 104 or having at least 85% sequence identity with SEQ ID NO: 104 and said LCDR1, LCDR2 and LCDR3 are as set forth in SEQ ID NO: 26, 27 and 28 The amino acid sequence shown.
在一些实施方案中，本发明提供抗CRTAM抗体或其抗原结合片段，其中所述抗体是单克隆抗体。在一些实施方案中，本发明提供抗CRTAM抗体或其抗原结合片段，其中所述抗体是鼠源单克隆抗体、嵌合抗体、人源化抗体、双特异性抗体或完全人抗体。In some embodiments, the invention provides an anti-CRTAM antibody or antigen-binding fragment thereof, wherein said antibody is a monoclonal antibody. In some embodiments, the invention provides an anti-CRTAM antibody or antigen-binding fragment thereof, wherein the antibody is a murine monoclonal antibody, chimeric antibody, humanized antibody, bispecific antibody, or fully human antibody.
在一些具体的实施方案中，根据本发明的抗CRTAM抗体为鼠源抗体，其还含有鼠源的IgG1、IgG2a、、IgG2b、IgG2c、IgG3或其变体的重链恒定区，和鼠源的κ、λ链或其变体的轻链恒定区。In some specific embodiments, the anti-CRTAM antibody according to the present invention is a murine antibody, which also contains the heavy chain constant region of murine IgG1, IgG2a, IgG2b, IgG2c, IgG3 or variants thereof, and murine The light chain constant region of the kappa, lambda chain or variants thereof.
在一些优选的实施方案中，所述的抗CRTAM嵌合抗体或其抗原结合片段的抗体重链进一步包含鼠源IgG1、IgG2a、、IgG2b、IgG2c、IgG3或其突变序列的重链恒定区，优选包含人源IgG1或IgG2重链恒定区，或者使用氨基酸突变后 显著降低ADCC(抗体依赖的细胞介导的细胞毒作用)毒性的IgG4恒定区。In some preferred embodiments, the antibody heavy chain of the anti-CRTAM chimeric antibody or its antigen-binding fragment further comprises a heavy chain constant region of murine IgG1, IgG2a, IgG2b, IgG2c, IgG3 or a mutant sequence thereof, preferably Contains human IgG1 or IgG2 heavy chain constant region, or IgG4 constant region that significantly reduces ADCC (antibody-dependent cell-mediated cytotoxicity) toxicity after amino acid mutation.
在一些实施方案中，本发明提供一种抗CRTAM人源化抗体或其抗原结合片段，其中所述重链包含人源的IgG1、IgG2、IgG3、IgG4或其变体的重链恒定区，所述轻链包含人源的κ、λ链或其变体的轻链恒定区。In some embodiments, the present invention provides an anti-CRTAM humanized antibody or an antigen-binding fragment thereof, wherein the heavy chain comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or a variant thereof, wherein The light chain comprises the light chain constant region of human kappa, lambda chain or variants thereof.
在一些优选的实施方案中，本发明的抗CRTAM人源化抗体或其抗原结合片段还包含人源IgG1或IgG2或其变体的重链恒定区，和人源κ链或其变体的轻链恒定区。In some preferred embodiments, the anti-CRTAM humanized antibody or antigen-binding fragment thereof of the present invention further comprises a heavy chain constant region of human IgG1 or IgG2 or a variant thereof, and a light chain of a human κ chain or a variant thereof. Chain constant region.
在一些实施方案中，所述抗CRTAM抗体或抗原结合片段是Fc沉默的工程化IgG1抗体或抗原结合片段，所述Fc沉默的工程化IgG1抗体或抗原结合片段具有降低的与Fc受体结合或者不与Fc受体结合。在另一个实施方案中，所述抗体是IgG4抗体。In some embodiments, the anti-CRTAM antibody or antigen-binding fragment is an Fc-silenced engineered IgG1 antibody or antigen-binding fragment that has reduced binding to an Fc receptor or Does not bind to Fc receptors. In another embodiment, the antibody is an IgG4 antibody.
在一些实施方案中，所述抗CRTAM抗体或抗原结合片段是介导T细胞细胞毒性和/或NK细胞细胞毒性的双特异性抗体或抗原结合片段。In some embodiments, the anti-CRTAM antibody or antigen-binding fragment is a bispecific antibody or antigen-binding fragment that mediates T cell cytotoxicity and/or NK cell cytotoxicity.
在一些实施方案中，所述抗CRTAM抗体或抗原结合片段能够诱导和/或增强免疫细胞的活化。在一个优选的实施方案中，所述免疫细胞是T细胞。在另一优选的实施方案中，所述免疫细胞是NK细胞。In some embodiments, the anti-CRTAM antibody or antigen-binding fragment is capable of inducing and/or enhancing activation of immune cells. In a preferred embodiment, said immune cells are T cells. In another preferred embodiment, said immune cells are NK cells.
在一些实施方案中，所述抗CRTAM抗体或抗原结合片段是双特异性或多特异性抗体或抗原结合片段，其与CRTAM蛋白结合并且与一个或多个另外的结合靶结合，优选地所述另外的结合靶是一种或多种肿瘤抗原。在一些具体的实施方案中，所述一个或多个另外的结合靶是免疫调节分子。In some embodiments, the anti-CRTAM antibody or antigen-binding fragment is a bispecific or multispecific antibody or antigen-binding fragment that binds to a CRTAM protein and binds to one or more additional binding targets, preferably the Additional binding targets are one or more tumor antigens. In some specific embodiments, said one or more additional binding targets are immunomodulatory molecules.
在一些实施方案中，本发明提供抗CRTAM抗体或其抗原结合片段，其中所述的抗原结合片段为Fab、、Fab'、Fv、scFv、F(ab') 2、F(ab) 2、dAb或单结构域抗体。 In some embodiments, the present invention provides an anti-CRTAM antibody or an antigen-binding fragment thereof, wherein the antigen-binding fragment is Fab, Fab', Fv, scFv, F(ab') 2 , F(ab) 2 , dAb or single domain antibodies.
本发明的另一方面提供一种分离的核酸，其编码根据本发明的抗CRTAM抗体或其抗原结合片段。Another aspect of the invention provides an isolated nucleic acid encoding an anti-CRTAM antibody or antigen-binding fragment thereof according to the invention.
在一些具体的实施方案中，根据本发明的分离的核酸，其包含：In some specific embodiments, according to the isolated nucleic acid of the present invention, it comprises:
编码重链可变区如SEQ ID NO:80、SEQ ID NO:84、SEQ ID NO:85、SEQ ID NO:86或SEQ ID NO:87的核苷酸序列；和编码轻链可变区如SEQ ID NO:81、SEQ ID NO:88、SEQ ID NO:89、SEQ ID NO:90、SEQ ID NO:91或SEQ ID NO:92的核苷酸序列。A nucleotide sequence encoding a heavy chain variable region such as SEQ ID NO:80, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86 or SEQ ID NO:87; and a light chain variable region such as The nucleotide sequence of SEQ ID NO:81, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91 or SEQ ID NO:92.
在一些优选的实施方案中，根据本发明的分离的核酸，其包含：In some preferred embodiments, an isolated nucleic acid according to the invention comprising:
编码重链可变区如SEQ ID NO:80的核苷酸序列；和编码轻链可变区如SEQ ID NO:81的核苷酸序列；或者A nucleotide sequence encoding a heavy chain variable region such as SEQ ID NO:80; and a nucleotide sequence encoding a light chain variable region such as SEQ ID NO:81; or
编码重链可变区如SEQ ID NO:87的核苷酸序列；和编码轻链可变区如SEQ ID NO:90或SEQ ID NO:92的核苷酸序列。A nucleotide sequence encoding a heavy chain variable region such as SEQ ID NO:87; and a nucleotide sequence encoding a light chain variable region such as SEQ ID NO:90 or SEQ ID NO:92.
在一些具体的实施方案中，根据本发明的分离的核酸，其包含：In some specific embodiments, according to the isolated nucleic acid of the present invention, it comprises:
编码重链可变区如SEQ ID NO:45、SEQ ID NO:106、SEQ ID NO:50、SEQ ID NO:51、SEQ ID NO:52，SEQ ID NO:53或SEQ ID NO:54的核苷酸序列；和编码轻链可变区如SEQ ID NO:46、SEQ ID NO:55、SEQ ID NO:56、SEQ ID NO:57、SEQ ID NO:58、SEQ ID NO:59、SEQ ID NO:60、SEQ ID NO:61、SEQ ID NO:62、SEQ ID NO:63、SEQ ID NO:64、SEQ ID NO:65、SEQ ID NO:66或SEQ ID NO:67的核苷酸序列。A nucleus encoding a heavy chain variable region such as SEQ ID NO:45, SEQ ID NO:106, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53 or SEQ ID NO:54 Nucleotide sequence; and coding light chain variable region such as SEQ ID NO:46, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID The nucleotide sequence of NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66 or SEQ ID NO:67 .
在一些优选的实施方案中，根据本发明的分离的核酸，其包含：In some preferred embodiments, an isolated nucleic acid according to the invention comprising:
编码重链可变区如SEQ ID NO:45或SEQ ID NO:106的核苷酸序列；和编码轻链可变区如SEQ ID NO:46的核苷酸序列；或者A nucleotide sequence encoding a heavy chain variable region such as SEQ ID NO:45 or SEQ ID NO:106; and a nucleotide sequence encoding a light chain variable region such as SEQ ID NO:46; or
编码重链可变区如SEQ ID NO:51的核苷酸序列；和编码轻链可变区如SEQ ID NO:66或SEQ ID NO:67的核苷酸序列。A nucleotide sequence encoding a heavy chain variable region such as SEQ ID NO:51; and a nucleotide sequence encoding a light chain variable region such as SEQ ID NO:66 or SEQ ID NO:67.
在一些具体的实施方案中，根据本发明的分离的核酸，其包含：In some specific embodiments, according to the isolated nucleic acid of the present invention, it comprises:
编码重链可变区如SEQ ID NO:68、SEQ ID NO:72、SEQ ID NO:73、SEQ ID NO:74或SEQ ID NO:75的核苷酸序列；和编码轻链可变区如SEQ ID NO:69、SEQ ID NO:76、SEQ ID NO:77、SEQ ID NO:78或SEQ ID NO:79的核苷酸序列。A nucleotide sequence encoding a heavy chain variable region such as SEQ ID NO:68, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74 or SEQ ID NO:75; and a light chain variable region such as The nucleotide sequence of SEQ ID NO:69, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78 or SEQ ID NO:79.
在一些优选的实施方案中，根据本发明的分离的核酸，其包含：In some preferred embodiments, an isolated nucleic acid according to the invention comprising:
编码重链可变区如SEQ ID NO:68的核苷酸序列；和编码轻链可变区如SEQ ID NO:69的核苷酸序列；或者A nucleotide sequence encoding a heavy chain variable region such as SEQ ID NO:68; and a nucleotide sequence encoding a light chain variable region such as SEQ ID NO:69; or
编码重链可变区如SEQ ID NO:75的核苷酸序列；和编码轻链可变区如SEQ ID NO:77的核苷酸序列。A nucleotide sequence encoding a heavy chain variable region such as SEQ ID NO:75; and a nucleotide sequence encoding a light chain variable region such as SEQ ID NO:77.
在一些具体的实施方案中，根据本发明的分离的核酸，其包含：In some specific embodiments, according to the isolated nucleic acid of the present invention, it comprises:
编码重链可变区如SEQ ID NO:93、SEQ ID NO:97、SEQ ID NO:98、SEQ ID NO:99、SEQ ID NO:100或SEQ ID NO:101的核苷酸序列；和编码轻链可变区如SEQ ID NO:94、SEQ ID NO:102、SEQ ID NO:103、SEQ ID NO:104或SEQ  ID NO:105的核苷酸序列。A nucleotide sequence encoding a heavy chain variable region such as SEQ ID NO:93, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100 or SEQ ID NO:101; and encoding A light chain variable region such as the nucleotide sequence of SEQ ID NO:94, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104 or SEQ ID NO:105.
在一些优选的实施方案中，根据本发明的分离的核酸，其包含：In some preferred embodiments, an isolated nucleic acid according to the invention comprising:
编码重链可变区如SEQ ID NO:93的核苷酸序列；和编码轻链可变区如SEQ ID NO:94的核苷酸序列；或者A nucleotide sequence encoding a heavy chain variable region such as SEQ ID NO:93; and a nucleotide sequence encoding a light chain variable region such as SEQ ID NO:94; or
编码重链可变区如SEQ ID NO:99或SEQ ID NO:101的核苷酸序列；和编码轻链可变区如SEQ ID NO:104的核苷酸序列。A nucleotide sequence encoding a heavy chain variable region such as SEQ ID NO:99 or SEQ ID NO:101; and a nucleotide sequence encoding a light chain variable region such as SEQ ID NO:104.
本发明的另一方面提供一种表达载体，其表达本发明的抗CRTAM抗体或其抗原结合片段。根据本发明的表达载体其包含本发明的分离的核酸分子。Another aspect of the present invention provides an expression vector expressing the anti-CRTAM antibody or antigen-binding fragment thereof of the present invention. An expression vector according to the present invention which comprises the isolated nucleic acid molecule of the present invention.
本发明的另一方面提供一种嵌合抗原受体(CAR)融合蛋白，其包含本发明的抗CRTAM抗体或其抗原结合片段。在一些实施方案中，所述嵌合抗原受体融合蛋白包含本发明的抗CRTAM抗体或其抗原结合片段，其为针对CRTAM抗原的VH和VL的单链可变片段(scFv)。所述针对CRTAM抗原的VH和VL的scFv具有以上实施方案中描述的重链可变区的HCDR1、HCDR2和HCDR3和轻链可变区的LCDR1、LCDR2和LCDR3。Another aspect of the present invention provides a chimeric antigen receptor (CAR) fusion protein comprising the anti-CRTAM antibody of the present invention or an antigen-binding fragment thereof. In some embodiments, the chimeric antigen receptor fusion protein comprises an anti-CRTAM antibody or antigen-binding fragment thereof of the invention, which is a single chain variable fragment (scFv) directed against the VH and VL of the CRTAM antigen. The scFv against VH and VL of CRTAM antigen has HCDR1, HCDR2 and HCDR3 of the heavy chain variable region and LCDR1, LCDR2 and LCDR3 of the light chain variable region described in the above embodiments.
本发明的另一方面提供一种如上所述的表达载体转化的宿主细胞。Another aspect of the present invention provides a host cell transformed with the above expression vector.
在一些实施方案中，根据本发明的宿主细胞选自原核细胞和真核细胞。在一些实施方案中，所述的宿主细胞为细菌，优选为大肠杆菌。在另一个优选的实施方案中，所述的宿主细胞为哺乳动物细胞。In some embodiments, host cells according to the invention are selected from prokaryotic cells and eukaryotic cells. In some embodiments, the host cell is bacteria, preferably Escherichia coli. In another preferred embodiment, said host cell is a mammalian cell.
本发明的另一方面提供制备本发明的抗CRTAM抗体或其抗原结合片段的方法，包括在所述宿主细胞中表达抗体以及从宿主细胞中分离所述抗体的步骤。Another aspect of the present invention provides a method for preparing the anti-CRTAM antibody or antigen-binding fragment thereof of the present invention, comprising the steps of expressing the antibody in the host cell and isolating the antibody from the host cell.
本发明的另一方面提供一种药物组合物，其包含本发明的抗CRTAM抗体或其抗原结合片段和药学可接受的载体。在一些实施方案中，本发明提供药物组合物，其包含本发明的抗CRTAM抗体或其抗原结合片段，还包含其他活性组分，如其他抗体、靶向药物等。在一些实施方案中，所述药学可接受的载体选自抗氧化剂、多肽、蛋白质、亲水性聚合物、氨基酸、糖、螯合剂、糖醇、离子和表面活性剂。在一个具体的实施方案中，所述药学可接受的载体为缓冲水溶液。在另一个具体的实施方案中，所述药学可接受的载体为脂质体的形式。Another aspect of the present invention provides a pharmaceutical composition, which comprises the anti-CRTAM antibody or antigen-binding fragment thereof of the present invention and a pharmaceutically acceptable carrier. In some embodiments, the present invention provides a pharmaceutical composition, which comprises the anti-CRTAM antibody or antigen-binding fragment thereof of the present invention, and further comprises other active components, such as other antibodies, targeted drugs, and the like. In some embodiments, the pharmaceutically acceptable carrier is selected from antioxidants, polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, sugar alcohols, ions, and surfactants. In a specific embodiment, the pharmaceutically acceptable carrier is a buffered aqueous solution. In another specific embodiment, said pharmaceutically acceptable carrier is in the form of liposomes.
可以将本发明的抗CRTAM人源化抗体或其抗原结合片段与药学上可接受的载体、稀释剂或赋形剂混合制备成药物制剂，以适合于经口或胃肠外给药。给药方法包括，但不限于经口、皮内、肌内、腹膜内、静脉内、脑内、眼内、气管内、皮下、鼻内途径。所述制剂可以通过任何途径施用，例如通过输注或推注， 通过经上皮或皮肤粘膜(例如口腔粘膜或直肠等)吸收的途径施用。给药可以是全身的或局部的。所述制剂可通过本领域已知的方法制备，且包含药物制剂领域常规使用的载体、稀释剂或赋形剂。The anti-CRTAM humanized antibody or antigen-binding fragment thereof of the present invention can be mixed with pharmaceutically acceptable carriers, diluents or excipients to prepare pharmaceutical preparations suitable for oral or parenteral administration. Methods of administration include, but are not limited to, oral, intradermal, intramuscular, intraperitoneal, intravenous, intracerebral, intraocular, intratracheal, subcutaneous, and intranasal routes. The formulation can be administered by any route, for example, by infusion or bolus injection, or by absorption through the epithelium or mucocutaneous (eg, oral mucosa or rectum, etc.). Administration can be systemic or local. The formulations can be prepared by methods known in the art, and contain carriers, diluents or excipients commonly used in the field of pharmaceutical formulations.
本发明的另一方面提供治疗癌症的方法，所述方法包括向有此需要的个体施用本发明的抗CRTAM抗体或其抗原结合片段或本发明的药物组合物。Another aspect of the invention provides a method of treating cancer comprising administering to an individual in need thereof an anti-CRTAM antibody or antigen-binding fragment thereof of the invention or a pharmaceutical composition of the invention.
本发明的另一方面提供本发明的抗CRTAM抗体或其抗原结合片段或本发明的药物组合物在制备预防和/或***的药物中的应用。在一些实施方案中，所述肿瘤选自肺癌、白血病、淋巴瘤、乳腺癌、胃癌、肠癌、食管癌、卵巢癌、睾丸癌、甲状腺癌、骨癌、***、子宫癌、肾癌、肝癌、胆囊癌、胆管癌、膀胱癌、胰腺癌、神经胶质瘤、皮肤癌、***癌、鼻咽癌、神经胶质瘤、胶质母细胞瘤、肉瘤和神经内分泌肿瘤。在一些实施方案中，所述肿瘤选自小细胞肺癌、非小细胞肺癌、黑素瘤、肝细胞癌、血液***恶性肿瘤、淋巴瘤、乳腺癌、胃癌、肠癌、食管癌、卵巢癌、***、肾癌、膀胱癌、胰腺癌和神经胶质瘤。Another aspect of the present invention provides the application of the anti-CRTAM antibody or antigen-binding fragment thereof of the present invention or the pharmaceutical composition of the present invention in the preparation of drugs for preventing and/or treating tumors. In some embodiments, the tumor is selected from lung cancer, leukemia, lymphoma, breast cancer, gastric cancer, intestinal cancer, esophageal cancer, ovarian cancer, testicular cancer, thyroid cancer, bone cancer, cervical cancer, uterine cancer, kidney cancer, Liver cancer, gallbladder cancer, bile duct cancer, bladder cancer, pancreatic cancer, glioma, skin cancer, prostate cancer, nasopharyngeal cancer, glioma, glioblastoma, sarcoma and neuroendocrine tumors. In some embodiments, the tumor is selected from small cell lung cancer, non-small cell lung cancer, melanoma, hepatocellular carcinoma, hematological malignancies, lymphoma, breast cancer, gastric cancer, bowel cancer, esophageal cancer, ovarian cancer, Cervical cancer, kidney cancer, bladder cancer, pancreatic cancer and glioma.
本发明提供的抗CRTAM抗体或其抗原结合片段具有显著的抗肿瘤作用，不影响CRTAM与其配体的结合，不影响其正常功能，同时人源化后的抗体免疫原性大大降低，有效消除人体免疫***对外源性单抗的排异反应，可在制备用于治疗各类肿瘤疾病的药物中应用，具有广阔的市场前景。The anti-CRTAM antibody or antigen-binding fragment thereof provided by the present invention has significant anti-tumor effect, does not affect the combination of CRTAM and its ligand, does not affect its normal function, and at the same time, the immunogenicity of the humanized antibody is greatly reduced, effectively eliminating the The rejection reaction of the immune system to the exogenous monoclonal antibody can be used in the preparation of drugs for the treatment of various tumor diseases, and has broad market prospects.
定义definition
除非另有定义，本文中使用的科学和技术术语的含义是本领域技术人员所通常理解的含义。本文中所述的细胞和组织培养、分子生物学以及蛋白质和寡或多核苷酸化学及杂交中使用的命名和技术是本领域公知且普遍使用的。对于重组DNA、寡核苷酸合成和组织培养与转化(如电穿孔、脂质转染)，使用了标准技术。酶促反应和纯化技术根据生产商的说明书或本领域普遍使用或本文所述的方法进行。前述技术和方法通常根据本领域公知且本说明书中引用和讨论的多部综合和较具体的文献中描述的那样使用。参见例如Sambrook等，Molecular Cloning:A Laboratory Manual)(第2版，Cold Spring Harbor Laboratory Press，纽约冷泉港(1989))。本文所述的分析化学、合成有机化学以及医学和药学化学中使用的命名以及实验室方法和技术是本领域公知且普遍使用的。Unless otherwise defined, the meanings of scientific and technical terms used herein are the meanings commonly understood by those skilled in the art. The nomenclature and techniques used in cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization described herein are those well known and commonly used in the art. For recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (eg, electroporation, lipofection), standard techniques are used. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or methods commonly used in the art or described herein. The foregoing techniques and methods are generally employed as described in various general and more specific documents that are well known in the art and that are cited and discussed throughout the present specification. See, eg, Sambrook et al., Molecular Cloning: A Laboratory Manual) (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989)). The nomenclature used in, and the laboratory methods and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art.
在本发明中，术语“至少80％序列同一性”是指至少80％，81％，82％，83％，84％，85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％， 97％，98％，99％，100％的序列同一性。在本发明中，术语“至少85％序列同一性”是指至少85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％，100％的序列同一性。在一些优选的实施方案中，本发明所述的序列同一性可以至少为90％，91％，92％，93％，94％，95％，96％，97％，98％，99％，100％。两个序列之间的序列比较和同一性百分比测定可以通过National Center For Biotechnology Instutute网站上的BLASTN/BLASTP算法来进行。In the present invention, the term "at least 80% sequence identity" means at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity. In the present invention, the term "at least 85% sequence identity" means at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity. In some preferred embodiments, the sequence identity of the present invention can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% %. Sequence comparison and determination of percent identity between two sequences can be performed by the BLASTN/BLASTP algorithm on the website of the National Center For Biotechnology Institute.
在抗体分子中，轻链的三个高变区和重链的三个高变区在三维空间中以相对彼此的位置排列以形成抗原结合表面。抗原结合表面与所结合抗原的三维表面互补，且每条重链和轻链的三个高变区均被称作“互补决定区”或“CDR”。氨基酸向每个结构域的分配是根据Kabat《免疫学感兴趣的蛋白质的序列》(国立卫生研究院，马里兰州贝塞斯达(1987和1991))或Chothia和Lesk，J.Mol.Biol.196:901-917(1987)，Chothia等，Nature 342:878-883(1989)定义。In an antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged relative to each other in three-dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of the bound antigen, and the three hypervariable regions of each heavy and light chain are referred to as "complementarity determining regions" or "CDRs." The assignment of amino acids to each domain was according to Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD (1987 and 1991)) or Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987), defined by Chothia et al., Nature 342:878-883 (1989).
本发明的“抗体”是指特异性地识别并结合抗原的多肽或多肽复合物。抗体可以是完整抗体和其任何抗原结合片段或单链。本发明的“抗体”包括含有Ig分子的具有结合抗原的生物活性的至少一部分的任何蛋白质或肽。本发明“抗体”的实例包括但不限于重链或轻链的CDR或其配体结合部分、重链或轻链可变区、重链或轻链恒定区、框架区或其任何部分。The "antibody" of the present invention refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. Antibodies can be whole antibodies and any antigen-binding fragments or single chains thereof. An "antibody" of the present invention includes any protein or peptide comprising an Ig molecule that has at least a portion of the biological activity of binding an antigen. Examples of "antibodies" of the invention include, but are not limited to, the CDRs of a heavy or light chain or a ligand-binding portion thereof, a heavy or light chain variable region, a heavy or light chain constant region, a framework region, or any portion thereof.
本发明所述的“抗原结合片段”是指具有抗原结合活性的Fab片段、Fab’片段、F(ab’) 2片段及与人CRTAM结合的Fv片段、scFv片段。Fv片段含有抗体重链可变区和轻链可变区，但没有恒定区，并具有全部抗原结合位点的最小抗体片段。一般地，Fv抗体还包含在VH和VL结构域之间的多肽接头，且能够形成抗原结合所需的结构。也可以用不同的连接物将两个抗体可变区连接成一条多肽链，称为单链抗体或单链Fv(scFv)。本发明的抗CRTAM抗体可以是单链可变区片段(scFv)，其源自抗体的单链多肽，保留了结合抗原的能力。scFv的实例包括通过重组DNA技术形成的抗体多肽，其中免疫球蛋白重链(H链)和轻链(L链)片段的Fv区经由间隔序列连接。制备scFv的各种方法是本领域技术人员所熟知的。 The "antigen-binding fragment" in the present invention refers to Fab fragments, Fab' fragments, F(ab') 2 fragments, Fv fragments and scFv fragments that bind to human CRTAM with antigen-binding activity. The Fv fragment contains the antibody heavy chain variable region and the light chain variable region, but has no constant region, and has the smallest antibody fragment with all antigen-binding sites. Typically, Fv antibodies also contain a polypeptide linker between the VH and VL domains and are capable of forming the structures required for antigen binding. Different linkers can also be used to connect two antibody variable regions into one polypeptide chain, called single-chain antibody or single-chain Fv (scFv). The anti-CRTAM antibody of the present invention may be a single-chain variable region fragment (scFv), which is derived from a single-chain polypeptide of an antibody and retains the ability to bind antigen. Examples of scFv include antibody polypeptides formed by recombinant DNA techniques in which the Fv regions of immunoglobulin heavy chain (H chain) and light chain (L chain) fragments are linked via a spacer sequence. Various methods of preparing scFv are well known to those skilled in the art.
本发明所述的抗体指免疫球蛋白分子或其免疫活性部分，即包含特异性结合抗原(与其免疫反应)的抗原结合位点的分子。“特异性结合”指抗体与抗原的一种或多种抗原决定簇反应而不与其他多肽反应或以很低的亲和性(Kd>10-6)结合 其他多肽。抗体包括但不限于多克隆、单克隆、嵌合、dAb(结构域抗体)、单链、Fab、Fab’和F(ab’)2片段、Fv、scFv及Fab表达文库。单克隆抗体(mAb)是由单一的克隆细胞株得到的抗体，所述的细胞株不限于真核的、原核的或噬菌体的克隆细胞株。单克隆抗体或抗原结合片段可以用如杂交瘤技术、重组技术、噬菌体展示技术及合成技术如CDR grafting或其它现有技术进行重组得到。The antibody of the present invention refers to an immunoglobulin molecule or an immunologically active part thereof, that is, a molecule comprising an antigen-binding site that specifically binds to (immunoreacts with) an antigen. "Specifically binds" means that an antibody reacts with one or more epitopes of an antigen but does not react with or binds other polypeptides with very low affinity (Kd > 10-6). Antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, dAb (domain antibody), single chain, Fab, Fab' and F(ab')2 fragments, Fv, scFv, and Fab expression libraries. Monoclonal antibodies (mAbs) are antibodies derived from a single clonal cell strain, not limited to eukaryotic, prokaryotic, or phage clonal cell strains. Monoclonal antibodies or antigen-binding fragments can be recombined using hybridoma technology, recombinant technology, phage display technology and synthetic technology such as CDR grafting or other existing technologies.
本发明所述的“鼠源抗体”为根据本领域知识和技能制备的对人CRTAM的单克隆抗体。制备时用hCRTAM抗原注射试验对象，然后分离表达具有所需序列或功能特性的抗体的杂交瘤。The "mouse antibody" described in the present invention is a monoclonal antibody against human CRTAM prepared according to the knowledge and skills in the art. In preparation, test subjects are injected with hCRTAM antigen, and hybridomas expressing antibodies having the desired sequence or functional properties are isolated.
本发明所述的“嵌合抗体”是将鼠源性抗体的可变区与人抗体的恒定区融合而成的抗体，可以减轻鼠源性抗体诱发的免疫应答反应。建立嵌合抗体，要先建立分泌鼠源性特异性单抗的杂交瘤，然后从小鼠杂交瘤细胞中克隆可变区基因，再根据需要克隆人抗体的恒定区基因，将小鼠可变区基因与人恒定区基因连接成嵌合基因后***人载体中，最后在真核工业***或原核工业***中表达嵌合抗体分子。The "chimeric antibody" of the present invention is an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can reduce the immune response induced by the murine antibody. To establish a chimeric antibody, it is necessary to first establish a hybridoma that secretes a mouse-derived specific monoclonal antibody, then clone the variable region gene from the mouse hybridoma cell, and then clone the constant region gene of the human antibody as required, and then clone the variable region gene of the mouse The gene is connected with the human constant region gene to form a chimeric gene and then inserted into a human vector, and finally the chimeric antibody molecule is expressed in a eukaryotic industrial system or a prokaryotic industrial system.
本发明所述的“人源化抗体”也称为CDR移植抗体，是将小鼠的CDR序列移植到人的抗体可变区框架(FR)中产生的抗体。此类可变区框架序列可以从公共的DNA数据库或公开的参考文献获得，例如从ImMunoGeneTics(IMGT)网站http://imgt.cines.fr得到或从免疫球蛋白杂志，2001ISBN012441351上获得。The "humanized antibody" of the present invention is also called a CDR-grafted antibody, which is an antibody produced by grafting a mouse CDR sequence into a human antibody variable region framework (FR). Such variable region framework sequences can be obtained from public DNA databases or published references, eg from the ImMunoGeneTics (IMGT) website http://imgt.cines.fr or from Immunoglobulin Journal, 2001 ISBN012441351.
附图说明Description of drawings
图1是抗CRTAM人源化抗体阻断人CRTAM结合CADM1活性实验结果(ELISA)，其中横坐标是抗体浓度(ng/mL)，纵坐标是OD450处的吸光值。Fig. 1 is the result of an anti-CRTAM humanized antibody blocking activity of human CRTAM binding to CADM1 (ELISA), wherein the abscissa is the antibody concentration (ng/mL), and the ordinate is the absorbance value at OD450.
图2是抗CRTAM抗体HuC68-17与5A11竞争结合人CRTAM的实验结果(ELISA)，其中横坐标是抗体浓度(ng/mL)，纵坐标是OD450处的吸光值。Figure 2 is the experimental results (ELISA) of the competition between the anti-CRTAM antibody HuC68-17 and 5A11 for binding to human CRTAM, where the abscissa is the antibody concentration (ng/mL), and the ordinate is the absorbance at OD450.
图3是抗CRTAM抗体HuC94-31与5A11竞争结合人CRTAM的实验结果(ELISA)，其中横坐标是抗体浓度(ng/mL)，纵坐标是OD450处的吸光值。Figure 3 is the experimental results (ELISA) of the competition between the anti-CRTAM antibody HuC94-31 and 5A11 for binding to human CRTAM, where the abscissa is the antibody concentration (ng/mL), and the ordinate is the absorbance at OD450.
图4是抗CRTAM抗体HuC110-34与5A11竞争结合人CRTAM的实验结果(ELISA)，其中横坐标是抗体浓度(ng/mL)，纵坐标是OD450处的吸光值。Figure 4 is the experimental results (ELISA) of the competition between the anti-CRTAM antibody HuC110-34 and 5A11 for binding to human CRTAM, where the abscissa is the antibody concentration (ng/mL), and the ordinate is the absorbance at OD450.
图5是抗CRTAM抗体HuC124-42与5A11竞争结合人CRTAM的实验结果 (ELISA)，其中横坐标是抗体浓度(ng/mL)，纵坐标是OD450处的吸光值。Figure 5 is the experimental results (ELISA) of the competition between the anti-CRTAM antibody HuC124-42 and 5A11 for binding to human CRTAM, where the abscissa is the antibody concentration (ng/mL), and the ordinate is the absorbance at OD450.
具体实施方式detailed description
下面代表性的实施例是为了更好地说明本发明，而非用于限制本发明的保护范围。以下实施例中未注明条件的实验方法通常按照常规条件，如冷泉港的抗体技术实验手册、分子克隆手册等，或按照原料或商品制造厂商所建议的条件进行。实施例中使用的材料、试剂如无特殊说明均为商购获得。The following representative examples are for better illustrating the present invention, but not for limiting the protection scope of the present invention. Experimental methods not indicated in the following examples are generally carried out under conventional conditions, such as Cold Spring Harbor Antibody Technology Experiment Manual, Molecular Cloning Manual, etc., or according to the conditions suggested by raw material or commodity manufacturers. The materials and reagents used in the examples are all commercially available unless otherwise specified.
实施例1：CRTAM抗体相关抗原蛋白及阳性对照抗体的制备Example 1: Preparation of CRTAM antibody-related antigen protein and positive control antibody
1、抗原蛋白及阳性对照抗体的表达载体构建1. Construction of expression vectors for antigenic proteins and positive control antibodies
(1)抗原蛋白的表达载体构建(1) Expression vector construction of antigenic protein
合成编码人源CRTAM蛋白全长的基因片段，氨基酸序列设计如SEQ ID NO:29所示。将其核苷酸序列克隆至真核表达质粒pTargetT上，获得其表达质粒pT-hCRTAM。A gene fragment encoding the full-length human CRTAM protein was synthesized, and the amino acid sequence was designed as shown in SEQ ID NO:29. The nucleotide sequence was cloned into the eukaryotic expression plasmid pTargetT to obtain the expression plasmid pT-hCRTAM.
合成编码猴源CRTAM蛋白全长的基因片段，氨基酸序列设计如SEQ ID NO:33所示。将其核苷酸序列克隆至真核表达质粒pTargetT上，获得其表达质粒pT-cCRTAM。A gene fragment encoding the full-length CRTAM protein of monkey origin was synthesized, and the amino acid sequence design was shown in SEQ ID NO:33. The nucleotide sequence was cloned into the eukaryotic expression plasmid pTargetT to obtain the expression plasmid pT-cCRTAM.
融合的人源CRTAM蛋白胞外区和mIgG1-Fc标签的氨基酸序列如SEQ ID NO:30所示。对人源CRTAM蛋白胞外区序列进行密码子优化后，合成带有标签的hCRTAM-mFc的核苷酸序列，并将克隆至真核表达质粒pHR上，获得其表达质粒pHR-hCRTAM-mFc。The amino acid sequence of the fused extracellular region of human CRTAM protein and the mIgG1-Fc tag is shown in SEQ ID NO:30. After codon optimization of the extracellular domain sequence of human CRTAM protein, the nucleotide sequence of hCRTAM-mFc with tag was synthesized and cloned into the eukaryotic expression plasmid pHR to obtain its expression plasmid pHR-hCRTAM-mFc.
融合的人源CRTAM蛋白胞外区和hIgG1-Fc或His标签的氨基酸序列如SEQ ID NO:31和SEQ ID NO:32所示，对上述氨基酸序列进行密码子优化后合成带有标签的hCRTAM-hFc和hCRTAM-His的核苷酸序列，并将其分别克隆至真核表达质粒pHR上，获得其表达质粒pHR-hCRTAM-hFc、pHR-hCRTAM-His。The amino acid sequences of the fused human CRTAM protein extracellular region and hIgG1-Fc or His tag are shown in SEQ ID NO: 31 and SEQ ID NO: 32. After codon optimization of the above amino acid sequence, the tagged hCRTAM- The nucleotide sequences of hFc and hCRTAM-His were cloned into the eukaryotic expression plasmid pHR respectively to obtain the expression plasmids pHR-hCRTAM-hFc and pHR-hCRTAM-His.
融合的猴源CRTAM蛋白胞外区和mIgG1-Fc标签的氨基酸序列如SEQ ID NO:34所示。对猴源CRTAM蛋白胞外区序列进行密码子优化后，合成带有标签的cCRTAM-mFc的核苷酸序列，并将克隆至真核表达质粒pHR上，获得其表达质粒pHR-cCRTAM-mFc。The amino acid sequence of the fused extracellular region of the monkey-derived CRTAM protein and the mIgG1-Fc tag is shown in SEQ ID NO:34. After codon-optimizing the extracellular domain sequence of the monkey-derived CRTAM protein, the nucleotide sequence of cCRTAM-mFc with a tag was synthesized and cloned into the eukaryotic expression plasmid pHR to obtain its expression plasmid pHR-cCRTAM-mFc.
融合的猴源CRTAM蛋白胞外区和hIgG1-Fc或His标签的氨基酸序列如SEQ  ID NO:35和SEQ ID NO:36所示，对上述氨基酸序列进行密码子优化后合成带有标签的cCRTAM-hFc和cCRTAM-His的核苷酸序列，并将其分别克隆至真核表达质粒pHR上，获得其表达质粒pHR-cCRTAM-hFc、pHR-cCRTAM-His。The amino acid sequences of the fused extracellular region of the monkey-derived CRTAM protein and the hIgG1-Fc or His tag are shown in SEQ ID NO:35 and SEQ ID NO:36. After codon optimization of the above amino acid sequence, the tagged cCRTAM- The nucleotide sequences of hFc and cCRTAM-His were cloned into the eukaryotic expression plasmid pHR respectively to obtain the expression plasmids pHR-cCRTAM-hFc and pHR-cCRTAM-His.
融合的鼠源CRTAM蛋白胞外区和mIgG1-Fc标签的氨基酸序列如SEQ ID NO:37所示。对鼠源CRTAM蛋白胞外区序列进行密码子优化后，合成带有标签的mCRTAM-mFc的核苷酸序列，并将克隆至真核表达质粒pHR上，获得其表达质粒pHR-mCRTAM-mFc。The amino acid sequence of the fused extracellular region of the mouse CRTAM protein and the mIgG1-Fc tag is shown in SEQ ID NO:37. After codon-optimizing the extracellular domain sequence of the mouse CRTAM protein, the nucleotide sequence of the tagged mCRTAM-mFc was synthesized and cloned into the eukaryotic expression plasmid pHR to obtain its expression plasmid pHR-mCRTAM-mFc.
融合的鼠源CRTAM蛋白胞外区和hIgG1-Fc或His标签的氨基酸序列如SEQ ID NO:38和SEQ ID NO:39所示，对上述氨基酸序列进行密码子优化后合成带有标签的mCRTAM-hFc和mCRTAM-His的核苷酸序列，并将其分别克隆至真核表达质粒pHR上，获得其表达质粒pHR-mCRTAM-hFc、pHR-mCRTAM-His。The amino acid sequences of the fused extracellular region of the mouse CRTAM protein and the hIgG1-Fc or His tag are shown in SEQ ID NO:38 and SEQ ID NO:39, and the codon optimization of the above amino acid sequence is performed to synthesize the tagged mCRTAM- The nucleotide sequences of hFc and mCRTAM-His were cloned into the eukaryotic expression plasmid pHR respectively to obtain the expression plasmids pHR-mCRTAM-hFc and pHR-mCRTAM-His.
(2)配体蛋白的表达载体构建(2) Expression vector construction of ligand protein
融合的人源CADM1蛋白胞外区和mIgG1-Fc标签的氨基酸序列如SEQ ID NO:40所示，对上述氨基酸序列进行密码子优化后合成带有标签的CADM1-mFc的核苷酸序列。将其克隆至真核表达质粒pHR上，获得其表达质粒pHR-CADM1-mFc。The amino acid sequence of the fused extracellular region of human CADM1 protein and the mIgG1-Fc tag is shown in SEQ ID NO: 40, and the nucleotide sequence of the tagged CADM1-mFc was synthesized after codon optimization of the above amino acid sequence. It was cloned into the eukaryotic expression plasmid pHR to obtain its expression plasmid pHR-CADM1-mFc.
融合的人源CADM1蛋白胞外区和hIgG1-Fc或His标签的氨基酸序列如SEQ ID NO:41和SEQ ID NO:42所示，对上述氨基酸序列进行密码子优化后合成带有标签的CADM1-hFc和CADM1-His的核苷酸序列，并将其分别克隆至真核表达质粒pHR上，获得其表达质粒pHR-CADM1-hFc、pHR-CADM1-His。The amino acid sequences of the fused human CADM1 protein extracellular region and hIgG1-Fc or His tag are shown in SEQ ID NO: 41 and SEQ ID NO: 42. After codon optimization of the above amino acid sequence, the tagged CADM1- The nucleotide sequences of hFc and CADM1-His were cloned into the eukaryotic expression plasmid pHR respectively to obtain the expression plasmids pHR-CADM1-hFc and pHR-CADM1-His.
(3)阳性对照抗体的表达载体构建(3) Expression vector construction of positive control antibody
使用PCT申请WO2019/086878中公开的人源化抗体5A11(本文简称5A11)作为阳性对照抗体。参照WO2019/086878中公开的方法制备5A11。5A11的氨基酸序列如下所示：The humanized antibody 5A11 (hereinafter referred to as 5A11) disclosed in PCT application WO2019/086878 was used as a positive control antibody. 5A11 was prepared according to the method disclosed in WO2019/086878. The amino acid sequence of 5A11 is as follows:
5A11重链氨基酸序列：SEQ ID NO:43；5A11 heavy chain amino acid sequence: SEQ ID NO: 43;
5A11轻链氨基酸序列：SEQ ID NO:44；5A11 light chain amino acid sequence: SEQ ID NO: 44;
对5A11抗体所对应的氨基酸序列进行密码子人工优化，将其轻、重链基因片段分别克隆到真核表达质粒pHR上，获得5A11的重链真核表达质粒pHR-5A11-hG1m，轻链表达质粒pHR-5A11-hλ。The codons of the amino acid sequence corresponding to the 5A11 antibody were artificially optimized, and the light and heavy chain gene fragments were respectively cloned into the eukaryotic expression plasmid pHR to obtain the heavy chain eukaryotic expression plasmid pHR-5A11-hG1m of 5A11, and the light chain expression Plasmid pHR-5A11-hλ.
2、抗原蛋白的表达与纯化2. Expression and purification of antigenic protein
(1)表达抗原蛋白的稳定转染细胞株构建(1) Construction of stable transfected cell lines expressing antigenic proteins
将真核表达质粒pT-hCRTAM在160V电压，15msec的方形脉冲下以电转的方式转染到CHO-K1细胞(中国科学院上海细胞生物学研究所)，置于37℃，5％CO 2浓度的培养箱中培养。24h后采用含1000μg/mL G418(Gibco，#10131-027)的培养基进行加压培养。转染16天后采用流式细胞术检测转染pool的阳性率，将阳性率较高的pool的细胞进行铺板(按照1×10 6个/mL的细胞密度，100μL/孔，铺96孔板)，采用5A11抗体和Goat pAb to Hu IgG(PE)(Abcam,ab98596)抗体与细胞孵育，以流式细胞仪(ACEABIO,Novocyte 2060R)检测392nm波长下mean值，使用GraphPad生成进行数据分析。将阳性细胞株进行亚克隆，挑选出高表达hCRTAM蛋白的克隆化的CHO-K1细胞株，命名为CHO-K1-hCRTAM。 The eukaryotic expression plasmid pT-hCRTAM was electrotransfected into CHO-K1 cells (Shanghai Institute of Cell Biology, Chinese Academy of Sciences) at a voltage of 160V and a square pulse of 15msec . cultured in an incubator. After 24 hours, medium containing 1000 μg/mL G418 (Gibco, #10131-027) was used for pressurized culture. 16 days after transfection, the positive rate of the transfected pool was detected by flow cytometry, and the cells of the pool with a higher positive rate were plated (according to the cell density of 1 ×106 cells/mL, 100 μL/well, plated in a 96-well plate) , the cells were incubated with 5A11 antibody and Goat pAb to Hu IgG (PE) (Abcam, ab98596) antibody, the mean value at 392nm wavelength was detected by flow cytometry (ACEABIO, Novocyte 2060R), and the data was generated by GraphPad for data analysis. The positive cell line was subcloned, and the cloned CHO-K1 cell line with high expression of hCRTAM protein was selected and named as CHO-K1-hCRTAM.
将真核表达质粒pT-cCRTAM在160V电压，15msec的方形脉冲下以电转的方式转染到CHO-K1细胞(中国科学院上海细胞生物学研究所)，置于37℃，5％CO 2浓度的培养箱中培养。24h后采用含1000μg/mL G418(Gibco，#10131-027)的培养基进行加压培养。转染16天后采用流式细胞术检测转染pool的阳性率，将阳性率较高的pool的细胞进行铺板(按照1×10 6个/mL的细胞密度，100μL/孔，铺96孔板)，采用5A11抗体和Goat pAb to Hu IgG(PE)(Abcam,ab98596)抗体与细胞孵育，以流式细胞仪(ACEABIO,Novocyte 2060R)检测392nm波长下mean值，使用GraphPad生成进行数据分析。将阳性细胞株进行亚克隆，挑选出高表达cCRTAM蛋白的克隆化的CHO-K1细胞株，命名为CHO-K1-cCRTAM。 The eukaryotic expression plasmid pT-cCRTAM was transfected into CHO-K1 cells (Shanghai Institute of Cell Biology, Chinese Academy of Sciences) by electroporation under 160V voltage and 15msec square pulse, and placed in 37°C, 5% CO2 concentration cultured in an incubator. After 24 hours, medium containing 1000 μg/mL G418 (Gibco, #10131-027) was used for pressurized culture. 16 days after transfection, the positive rate of the transfected pool was detected by flow cytometry, and the cells of the pool with a higher positive rate were plated (according to the cell density of 1 ×106 cells/mL, 100 μL/well, plated in a 96-well plate) , the cells were incubated with 5A11 antibody and Goat pAb to Hu IgG (PE) (Abcam, ab98596) antibody, the mean value at 392nm wavelength was detected by flow cytometry (ACEABIO, Novocyte 2060R), and the data was generated by GraphPad for data analysis. The positive cell lines were subcloned, and the cloned CHO-K1 cell line with high expression of cCRTAM protein was selected and named as CHO-K1-cCRTAM.
(2)标签抗原蛋白的表达(2) Expression of tagged antigen protein
在1L细胞培养瓶中接种密度为1×10 6个/ml的293E细胞(来源于ATCC)，加入新鲜的预热的FreeStyle293培养基，使接种后总体积达到250mL，置37℃，8％CO 2，加湿的细胞摇床中100rpm培养过夜。取7.5mL FreeStyle293培养基，加入1mg/mL的PEI溶液500μL，混合均匀，静置5min，同时取250μg待转染质粒加入8mL FreeStyle293培养基中，混合均匀，静置5min，其中标签抗原蛋白质粒pHR-hCRTAM-mFc、pHR-hCRTAM-hFc、pHR-hCRTAM-His、 pHR-cCRTAM-mFc、pHR-cCRTAM-hFc、pHR-cCRTAM-His、pHR-mCRTAM-mFc、pHR-mCRTAM-hFc、pHR-mCRTAM-His、pHR-CADM1-mFc、pHR-CADM1-hFc、pHR-CADM1-His分别转染，阳性对照抗体5A11重链质粒pHR-5A11-hG1m和轻链质粒pHR-5A11-hλ按照质量比1.1：1共同转染。将PEI与FreeStyle293培养基的混合溶液加入到质粒中，混合均匀，室温静置20min，然后加入细胞培养物中，置37℃，8％CO 2，加湿的细胞摇床中100rpm培养。在细胞转染后第1天和第3天对细胞进行补料，每瓶加入12.5mL的OPM-CHO PFF05(上海奥浦迈生物科技有限公司，F81279)、5mL的葡萄糖(母液浓度为180g/L)和2.5mL的谷氨酰胺(母液浓度为200mM)。当细胞活力降至75％时，收集细胞上清。将细胞培养物2000rpm离心5min，收集上清，再6000rpm离心20min，收集上清，分别使用0.45μm和0.22μm的滤杯过滤，收集滤液保存于4℃冰箱待纯化。 Inoculate 293E cells (derived from ATCC) at a density of 1 ×106/ml in a 1L cell culture flask, add fresh preheated FreeStyle293 medium to make the total volume after inoculation reach 250mL, and store at 37°C, 8% CO 2. Cultivate overnight at 100 rpm in a humidified cell shaker. Take 7.5mL FreeStyle293 medium, add 500μL of 1mg/mL PEI solution, mix well, and let it stand for 5min. - hCRTAM-mFc, pHR-hCRTAM-hFc, pHR-hCRTAM-His, pHR-cCRTAM-mFc, pHR-cCRTAM-hFc, pHR-cCRTAM-His, pHR-mCRTAM-mFc, pHR-mCRTAM-hFc, pHR-mCRTAM -His, pHR-CADM1-mFc, pHR-CADM1-hFc, pHR-CADM1-His were transfected respectively, the positive control antibody 5A11 heavy chain plasmid pHR-5A11-hG1m and light chain plasmid pHR-5A11-hλ according to the mass ratio of 1.1: 1 co-transfection. The mixed solution of PEI and FreeStyle293 medium was added to the plasmid, mixed evenly, and allowed to stand at room temperature for 20 minutes, then added to the cell culture, and cultured at 37° C., 8% CO 2 , in a humidified cell shaker at 100 rpm. On the 1st and 3rd day after cell transfection, the cells were fed, and 12.5 mL of OPM-CHO PFF05 (Shanghai OPM Biotechnology Co., Ltd., F81279) and 5 mL of glucose (the concentration of the mother solution was 180 g/L) were added to each bottle. ) and 2.5mL of glutamine (the concentration of the stock solution is 200mM). When the cell viability dropped to 75%, the cell supernatant was collected. The cell culture was centrifuged at 2000rpm for 5min to collect the supernatant, and then centrifuged at 6000rpm for 20min to collect the supernatant, filtered using 0.45μm and 0.22μm filter cups respectively, and the collected filtrate was stored in a refrigerator at 4°C until purification.
(3)亲和层析柱纯化(3) Affinity chromatography column purification
利用AKTA(GE，AKTA pure-150)根据蛋白性质采用亲和层析柱进行纯化(不同蛋白适配的亲和层析柱见表1)，具体纯化步骤如下：Use AKTA (GE, AKTA pure-150) to purify with affinity chromatography column according to protein properties (see Table 1 for affinity chromatography columns adapted to different proteins), and the specific purification steps are as follows:
表1不同蛋白适配的亲和层析柱Table 1 Affinity chromatography columns adapted to different proteins
Figure PCTCN2022102253-appb-000001
Figure PCTCN2022102253-appb-000001
清洗：超纯水清洗设备及管路2min，流速10mL/min，后用0.1M NaOH清洗层析***；Cleaning: clean the equipment and pipeline with ultrapure water for 2 minutes, flow rate 10mL/min, and then clean the chromatography system with 0.1M NaOH;
接柱：将层析柱接入层析设备，并用超纯水冲洗5min；后用0.1M NaOH冲洗30min，保留时间5min；Connect the column: Connect the chromatography column to the chromatography equipment, and rinse with ultrapure water for 5 minutes; then rinse with 0.1M NaOH for 30 minutes, and the retention time is 5 minutes;
平衡：20mM PB+0.15M NaCl，pH 7.2平衡5个CV(柱体积)；Balance: 20mM PB+0.15M NaCl, pH 7.2 balance 5 CV (column volume);
上样：将细胞表达上清上样，保留时间5min；Sample loading: load the cell expression supernatant with a retention time of 5 minutes;
后平衡：20mM PB+0.15M NaCl，pH 7.2平衡5个CV；After balance: 20mM PB+0.15M NaCl, pH 7.2 balance 5 CV;
洗脱：50mM醋酸，pH＝3.4洗脱，保留时间5min。UV280至50mAu左右时开始收集，降至50mAu左右时停止收集。用1M Tris-HCl，pH 9.0将样品 pH调节至7.0；Elution: 50mM acetic acid, pH=3.4 elution, retention time 5min. Start collecting when UV280 reaches about 50mAu, and stop collecting when it drops to about 50mAu. Adjust the sample pH to 7.0 with 1M Tris-HCl, pH 9.0;
再平衡：20mM PB+0.15M NaCl，pH 7.2平衡3个CV，保留时间5min；Rebalance: 20mM PB+0.15M NaCl, pH 7.2, balance 3 CV, retention time 5min;
在线清洗：0.1M NaOH清洗30min，保留时间5min；On-line cleaning: 0.1M NaOH for 30 minutes, retention time 5 minutes;
清洗保存：纯化水清洗10min，后20％乙醇2个CV。Cleaning and storage: wash with purified water for 10 minutes, then 2 CVs with 20% ethanol.
实施例2：抗CRTAM单克隆抗体的制备Embodiment 2: Preparation of anti-CRTAM monoclonal antibody
1、杂交瘤单克隆的制备1. Preparation of hybridoma monoclonal
(1)动物免疫(1) Animal immunity
采用不同标签的hCRTAM抗原蛋白与佐剂共同免疫实验动物，或采用CHO-K1-hCRTAM细胞株进行细胞免疫。实验动物包括Balb/c品系小鼠、SJL品系小鼠和SD大鼠。蛋白免疫：小鼠免疫按照首次免疫50μg抗原免疫一只小鼠，后期均使用抗原蛋白25μg/只；SD大鼠免疫按照首次免疫100μg抗原免疫一只大鼠，后期均使用抗原蛋白50μg/只。免疫佐剂可以是弗氏佐剂(Sigma)或Quick Antibody-Mouse5W(Q5W，北京博奥龙免疫技术有限公司)。采用弗氏佐剂乳化抗原，将不同标签的hCRTAM抗原蛋白样品逐滴加入到佐剂溶液中，边滴加边涡旋以充分混合，佐剂使用剂量参考说明书进行。混合均匀形成油包水的乳状后免疫。采用Quick Antibody-Mouse5W作为佐剂，将不同标签的hCRTAM抗原蛋白样品与Quick Antibody-Mouse5W按照1:1的体积比进行混合，混匀后即采用肌肉注射的方式，免疫SD大鼠。细胞免疫：CHO-K1-hCRTAM细胞使用PBS重悬，小鼠免疫按照1×10 7个细胞/只；SD大鼠免疫按照2×10 7个细胞/只。免疫方案如表2所示。 The hCRTAM antigen proteins with different labels and adjuvant are used to immunize experimental animals together, or CHO-K1-hCRTAM cell line is used for cellular immunization. Experimental animals include Balb/c strain mice, SJL strain mice and SD rats. Protein immunization: For mouse immunization, a mouse was immunized with 50 μg antigen for the first immunization, and 25 μg/mouse of antigenic protein was used in the later stages; SD rat immunization was immunized with 100 μg antigen for the first immunization, and 50 μg/mouse of antigenic protein was used in the later stages. The immune adjuvant can be Freund's adjuvant (Sigma) or Quick Antibody-Mouse5W (Q5W, Beijing Boaolong Immunotechnology Co., Ltd.). Freund's adjuvant was used to emulsify the antigen, and hCRTAM antigen protein samples with different labels were added dropwise to the adjuvant solution, and vortexed while dropping to mix thoroughly, and the dosage of the adjuvant was carried out according to the instructions. Mix well to form a water-in-oil emulsion and immunize. Using Quick Antibody-Mouse5W as an adjuvant, hCRTAM antigen protein samples with different labels were mixed with Quick Antibody-Mouse5W at a volume ratio of 1:1. After mixing, SD rats were immunized by intramuscular injection. Cellular immunization: CHO-K1-hCRTAM cells were resuspended in PBS, mice were immunized at 1×10 7 cells/mouse; SD rats were immunized at 2×10 7 cells/mouse. The immunization scheme is shown in Table 2.
表2动物免疫方案Table 2 Animal immunization scheme
Figure PCTCN2022102253-appb-000002
Figure PCTCN2022102253-appb-000002
Figure PCTCN2022102253-appb-000003
Figure PCTCN2022102253-appb-000003
*i.m.肌内注射；s.c.皮下注射；i.p.腹腔注射。*i.m. intramuscular injection; s.c. subcutaneous injection; i.p. intraperitoneal injection.
(2)杂交瘤融合(2) Hybridoma Fusion
脾细胞的获取和制备：将加强免疫后的小鼠/大鼠处死后浸泡75％的酒精中，解剖取出脾脏，用研磨棒研磨后，经细胞筛网过滤后制备成单细胞悬液。将脾细胞悬液2000rpm离心5min，弃上清。加入2mL红细胞裂解液，室温裂解红细胞2min，加入PBS至20mL，1500rpm离心7min，弃上清，重悬后进行活细胞计数。收集培养瓶中的Sp2/0细胞，1000rpm离心5min后弃上清，重悬后进行活细胞计数。按脾细胞：Sp2/0细胞＝1.6：1的比例混合细胞，1500rpm离心7min后弃上清。用20mL电转缓冲液重悬细胞，1500rpm离心7min。弃上清，重复一次。分别用适量电转缓冲液重悬细胞，保证细胞浓度2×10 7个细胞/mL左右。把细胞悬液加入9mL电转融合槽中融合。融合后将细胞悬液转入到含有20％FBS的15mL RPMI 1640完全培养基中，室温放置20min。用含1×HAT、1×BIOMYC3、20％FBS的RPMI 1640培养基重悬融合细胞。按100μL/孔将细胞悬液加到若干块96孔细胞培养板中，保证每孔细胞量约为4×10 4个细胞/孔，置于37℃细胞培养箱中培养。5天后补加100μL/孔RPMI 1640完全培养基(含20％FBS，1×HAT，1×BIOMYC-3)。 Acquisition and preparation of splenocytes: The mice/rats after booster immunization were sacrificed and soaked in 75% alcohol, the spleen was dissected out, ground with a grinding rod, and filtered through a cell mesh to prepare a single cell suspension. The spleen cell suspension was centrifuged at 2000 rpm for 5 min, and the supernatant was discarded. Add 2 mL of erythrocyte lysate, lyse the erythrocytes at room temperature for 2 minutes, add PBS to 20 mL, centrifuge at 1500 rpm for 7 minutes, discard the supernatant, resuspend and count viable cells. Collect the Sp2/0 cells in the culture flask, centrifuge at 1000rpm for 5min, discard the supernatant, resuspend and count live cells. The cells were mixed according to the ratio of splenocytes:Sp2/0 cells=1.6:1, and the supernatant was discarded after centrifugation at 1500rpm for 7min. The cells were resuspended with 20mL electroporation buffer and centrifuged at 1500rpm for 7min. Discard the supernatant and repeat once. Resuspend the cells with an appropriate amount of electroporation buffer to ensure a cell concentration of about 2×10 7 cells/mL. Add the cell suspension to a 9mL electroporation fusion tank for fusion. After fusion, the cell suspension was transferred to 15 mL RPMI 1640 complete medium containing 20% FBS, and left at room temperature for 20 min. Resuspend the confluent cells in RPMI 1640 medium containing 1×HAT, 1×BIOMYC3, and 20% FBS. Add the cell suspension to several 96-well cell culture plates at 100 μL/well to ensure that the cell volume per well is about 4×10 4 cells/well, and culture in a 37°C cell culture incubator. After 5 days, 100 μL/well RPMI 1640 complete medium (containing 20% FBS, 1×HAT, 1×BIOMYC-3) was added.
(3)杂交瘤及亚克隆筛选(3) Hybridoma and subclone screening
融合一周后，取杂交瘤母克隆的细胞培养上清，通过ELISA筛选结合hCRTAM蛋白和cCRTAM蛋白的杂交瘤母克隆，进一步通过流式细胞术筛选出能结合CHO-K1-hCRTAM细胞株的母克隆。One week after the fusion, the cell culture supernatant of the hybridoma mother clone was taken, and the hybridoma mother clone binding to hCRTAM protein and cCRTAM protein was screened by ELISA, and the mother clone capable of binding to CHO-K1-hCRTAM cell line was further screened by flow cytometry .
通过ELISA和FACS筛选出结合能力较强的母克隆，利用有限稀释法将阳性母克隆进行亚克隆，培养一周后利用ELISA检测亚克隆上清与hCRTAM蛋白的结合活性，进而获得分泌抗hCRTAM抗体的单克隆细胞株。获得多个较好的单克隆细胞株，分别标记为C68、C94、C110、C124。The mother clones with strong binding ability were screened by ELISA and FACS, and the positive mother clones were subcloned by the limiting dilution method. After one week of culture, the binding activity of the supernatant of the subclones and hCRTAM protein was detected by ELISA, and then the secretory anti-hCRTAM antibody was obtained. monoclonal cell line. A number of better monoclonal cell lines were obtained, labeled as C68, C94, C110, and C124 respectively.
2、单克隆抗体的制备2. Preparation of monoclonal antibodies
根据亚克隆上清活性分析结果确定单克隆抗体细胞株，将其扩大培养。培养条件是含有10％FBS、1×NAEE、1×丙酮酸钠、1％青链霉素双抗的1640培养基。待细胞汇合度大于>80％时，进将细胞传代扩培，待培养至约50mL时收集上清，纯化抗体。获得抗体经SDS-PAGE凝胶电泳确定纯度良好。The monoclonal antibody cell line was determined according to the activity analysis results of the supernatant of the subclones, and expanded for culture. The culture condition is 1640 medium containing 10% FBS, 1×NAEE, 1×sodium pyruvate, and 1% penicillin-streptomycin double antibody. When the confluence of the cells is greater than >80%, the cells are subcultured and expanded. When the culture reaches about 50 mL, the supernatant is collected and the antibody is purified. The purity of the obtained antibody was confirmed by SDS-PAGE gel electrophoresis.
3、单克隆抗体测序3. Monoclonal antibody sequencing
将经亚克隆操作的阳性杂交瘤细胞进行扩大培养，取适量细胞按RNeasy Plus Mini Kit(Qiagen，74134)试剂盒说明书提取总RNA，利用Prime Script 1st strand cDNA Synthesis Kit(Takara，6110A)反转录试剂盒合成cDNA第一条链。The subcloned positive hybridoma cells were expanded and cultured, and an appropriate amount of cells was taken to extract total RNA according to the instructions of the RNeasy Plus Mini Kit (Qiagen, 74134) kit, and reverse transcription was performed using the Prime Script 1st strand cDNA Synthesis Kit (Takara, 6110A) The kit synthesizes the first strand of cDNA.
根据抗体可变区两端保守序列设计通用引物(5’端含有用于与真核表达载体发生同源重组的同源臂序列)，以cDNA为模板进行抗体可变区基因的PCR扩增，从而分别获得鼠抗轻链与重链可变区的基因片段；设计引物(参考文献：1.Anke Krebber,Susanne Bornhauser,Jorg Burmester et al.Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system.Journal of Immunological Methods,1997,201:35–55；2.Simon
Figure PCTCN2022102253-appb-000004
Antibody variable-region sequencing as a method for hybridoma cell-line authentication,2008,78:1071–1078)，进行DNA测序获得序列。鼠抗测序结果见表3。 Design universal primers based on the conserved sequences at both ends of the variable region of the antibody (the 5' end contains the homologous arm sequence for homologous recombination with the eukaryotic expression vector), and use cDNA as a template to perform PCR amplification of the variable region gene of the antibody. Thereby obtaining the gene fragments of mouse anti-light chain and heavy chain variable domains respectively; Design primers (references: 1. Anke Krebber, Susanne Bornhauser, Jorg Burmester et al. a reengineered phage display system. Journal of Immunological Methods, 1997, 201:35–55; 2.Simon
Figure PCTCN2022102253-appb-000004
Antibody variable-region sequencing as a method for hybridoma cell-line authentication, 2008, 78:1071–1078), DNA sequencing was performed to obtain the sequence. The results of mouse anti-sequencing are shown in Table 3.
表3抗CRTAM鼠源单克隆抗体序列Table 3 Anti-CRTAM mouse monoclonal antibody sequence
Figure PCTCN2022102253-appb-000005
Figure PCTCN2022102253-appb-000005
实施例3：抗CRTAM嵌合抗体的构建Embodiment 3: Construction of anti-CRTAM chimeric antibody
将纯化后(纯化步骤见实施例1)的小鼠抗体重链与轻链可变区基因片段分别与线性化的含有人抗体重链或轻链恒定区的真核表达质粒pHR-hG1m/pHR-hλ共转化大肠杆菌DH5α感受态细胞。将混合液均匀涂布于含有氨苄抗生素的琼脂平板表面，于37℃恒温培养箱倒置过夜培养后分别挑取若干单菌落进行DNA测序。将测序正确的嵌合抗体分别标记为C68CHI、C94CHI、C110CHI、C124CHI。抗体C68CHI、C94CHI、C110CHI、C124CHI的重链可变区氨基酸序列和轻链可变区氨基酸序列与相应的鼠源抗体C68、C94、C110、C124相同。The purified mouse antibody heavy chain and light chain variable region gene fragments (see Example 1 for the purification steps) were respectively combined with the linearized eukaryotic expression plasmid pHR-hG1m/pHR containing the human antibody heavy chain or light chain constant region -hλ co-transformed Escherichia coli DH5α competent cells. The mixture was evenly spread on the surface of the agar plate containing the ampicillin antibiotic, cultured upside down in a constant temperature incubator at 37°C overnight, and then several single colonies were picked for DNA sequencing. The chimeric antibodies with correct sequencing were marked as C68CHI, C94CHI, C110CHI, and C124CHI, respectively. The amino acid sequences of heavy chain variable regions and light chain variable regions of antibodies C68CHI, C94CHI, C110CHI, and C124CHI are identical to those of the corresponding murine antibodies C68, C94, C110, and C124.
将嵌合抗体重轻链质粒共转染HEK293E细胞，表达纯化获得嵌合抗体，然后进行纯度检测、活性分析及亲和力测定。The chimeric antibody heavy and light chain plasmids were co-transfected into HEK293E cells, expressed and purified to obtain the chimeric antibody, and then the purity test, activity analysis and affinity test were performed.
利用定点突变的方法，将嵌合抗体C68CHI进行基因突变，以筛选更优的抗 体。C68CHI重链可变区第53位N突变为S，抗体稳定性提高，标记为C68CHIm1。C68CHIm1的重链可变区氨基酸序列为SEQ ID NO:106，轻链可变区氨基酸序列为SEQ ID NO:46，HCDR1、HCDR2和HCDR3分别为SEQ ID NO:8、11和10，LCDR1、LCDR2和LCDR3分别为SEQ ID NO:12、13和14。嵌合抗体序列见表4。Using site-directed mutagenesis, the chimeric antibody C68CHI was mutated to screen for better antibodies. The 53rd N in the heavy chain variable region of C68CHI is mutated to S, which improves the stability of the antibody and is labeled as C68CHIm1. The amino acid sequence of the heavy chain variable region of C68CHIm1 is SEQ ID NO: 106, the amino acid sequence of the light chain variable region is SEQ ID NO: 46, HCDR1, HCDR2 and HCDR3 are SEQ ID NO: 8, 11 and 10, LCDR1, LCDR2 and LCDR3 are SEQ ID NO: 12, 13 and 14, respectively. The chimeric antibody sequences are shown in Table 4.
表4抗CRTAM嵌合抗体序列表Table 4 anti-CRTAM chimeric antibody sequence list
Figure PCTCN2022102253-appb-000006
Figure PCTCN2022102253-appb-000006
实施例4：抗CRTAM人源化抗体的构建及生产Example 4: Construction and production of anti-CRTAM humanized antibody
根据嵌合抗体的免疫活性分析，选择C68CHIm1、C94CHI、C110CHI、C124CHI等多个活性好的嵌合抗体进行人源化抗体改造。According to the analysis of the immune activity of chimeric antibodies, multiple chimeric antibodies with good activity, such as C68CHIm1, C94CHI, C110CHI, and C124CHI, were selected for humanized antibody transformation.
抗体的人源化改造，首先是通过与免疫基因数据库(IMGT)中的小鼠抗体序列进行比对，确认C68CHIm1、C94CHI、C110CHI、C124CHI抗体可变区的鼠源种系，经过同源比对，C68CHIm1、C94CHI、C110CHI、C124CHI抗体的重链可变区序列的FR区分别与小鼠抗体种系基因(IGHV3-2*02、IGHV5-9-1*01、IGHV1-85*01、IGHV1-9*01)最为相似；抗体轻链可变区的FR序列则分别与小鼠抗体(IGKV1-110*01、IGKV10-96*01、IGKV1-110*01、IGKV5-48*01)最为相似。以C68CHIm1、C94CHI、C110CHI、C124CHI抗体框架区序列FR1-FR3作为模板，在人框架区库中寻找3D结构相似但是免疫原性较低的全人框架替代C68CHIm1、C94CHI、C110CHI、C124CHI的FR1-FR3序列，重链/轻链全长序列进行3D建模并和原抗体重链/轻链序列进行结构比对分析，综合考虑抗原性和3D结构相似度，最终选择C68CHIm1的5条人源化重链可变区(参见SEQ ID NO:50～54)和12条人源化轻链可变区(参见SEQ ID NO:55～59、60～66)、C94CHI的4条人源化重链可变区(参见SEQ ID NO:72～75)和4条人源化轻链可变区(参见SEQ ID NO:76～79)、C110CHI的4条人源化重链可变区(参见SEQ ID NO:84～87)和5条人源化轻链可变区(参见SEQ ID NO:88～91)以及C124CHI 的4条人源化重链可变区(参见SEQ ID NO:97～100)和4条人源化轻链可变区(参见SEQ ID NO:102～105)进行下一步优化。C68CHIm1、C94CHI、C110CHI、C124CHI的人源化抗体非CDR区序列均达到95％以上人源化。For the humanization of antibodies, the first step is to compare the mouse antibody sequences in the Immune Gene Database (IMGT) to confirm the mouse germlines of the variable regions of the C68CHIm1, C94CHI, C110CHI, and C124CHI antibodies. , the FR regions of the heavy chain variable region sequences of C68CHIm1, C94CHI, C110CHI, and C124CHI antibodies are respectively related to mouse antibody germline genes (IGHV3-2*02, IGHV5-9-1*01, IGHV1-85*01, IGHV1- 9*01) were most similar; the FR sequences of antibody light chain variable regions were most similar to mouse antibodies (IGKV1-110*01, IGKV10-96*01, IGKV1-110*01, IGKV5-48*01). Using C68CHIm1, C94CHI, C110CHI, C124CHI antibody framework region sequence FR1-FR3 as a template, search for a fully human framework with similar 3D structure but low immunogenicity in the human framework region library to replace FR1-FR3 of C68CHIm1, C94CHI, C110CHI, C124CHI Sequence, heavy chain/light chain full-length sequence was used for 3D modeling and structural comparison analysis with the original antibody heavy chain/light chain sequence, considering antigenicity and 3D structural similarity, and finally selected 5 humanized heavy chains of C68CHIm1 Chain variable region (see SEQ ID NO:50～54) and 12 humanized light chain variable regions (see SEQ ID NO:55～59, 60～66), 4 humanized heavy chains of C94CHI can be Variable region (see SEQ ID NO:72～75) and 4 humanized light chain variable regions (see SEQ ID NO:76～79), 4 humanized heavy chain variable regions of C110CHI (see SEQ ID NO:84～87) and 5 humanized light chain variable regions (see SEQ ID NO:88～91) and 4 humanized heavy chain variable regions of C124CHI (see SEQ ID NO:97～100) and 4 humanized light chain variable regions (see SEQ ID NO: 102-105) for further optimization. The non-CDR region sequences of the humanized antibodies of C68CHIm1, C94CHI, C110CHI, and C124CHI all reached more than 95% humanization.
挑选纯度、活性和亲和力均较好的人源化抗体，分别标记为HuC68-16、HuC94-31、HuC110-32、HuC124-22，序列见表5。Humanized antibodies with good purity, activity and affinity were selected and labeled as HuC68-16, HuC94-31, HuC110-32 and HuC124-22 respectively. The sequences are shown in Table 5.
表5抗CRTAM人源化抗体序列Table 5 Anti-CRTAM humanized antibody sequence
Figure PCTCN2022102253-appb-000007
Figure PCTCN2022102253-appb-000007
利用定点突变的方法，将人源化抗体HuC68-16、HuC110-32、HuC124-22进行基因突变，以筛选更优的抗体。HuC68-16轻链可变区第34位G突变为K(参见SEQ ID NO:67)，抗体稳定性提高，标记为HuC68-17。HuC110-32轻链可变区第34位G突变为K(参见SEQ ID NO:92)，抗体稳定性提高，标记为HuC110-34。HuC124-22重链可变区第62位G突变为A(参见SEQ ID NO:101)，抗体稳定性提高，标记为HuC124-42。优化后抗体序列见表6。Using site-directed mutagenesis, the humanized antibodies HuC68-16, HuC110-32, and HuC124-22 were mutated to screen for better antibodies. The 34th G in the variable region of the light chain of HuC68-16 was mutated to K (see SEQ ID NO: 67), the stability of the antibody was improved, and it was labeled as HuC68-17. The 34th G in the variable region of the light chain of HuC110-32 was mutated to K (see SEQ ID NO: 92), which improved the stability of the antibody and was labeled as HuC110-34. The 62nd G in the variable region of the heavy chain of HuC124-22 is mutated to A (see SEQ ID NO: 101), the stability of the antibody is improved, and it is labeled as HuC124-42. The optimized antibody sequences are shown in Table 6.
表6抗CRTAM人源化抗体优化后序列Table 6 Optimized sequence of anti-CRTAM humanized antibody
Figure PCTCN2022102253-appb-000008
Figure PCTCN2022102253-appb-000008
考虑到抗体的功能与稳定性，选择合适的抗体亚型骨架与优化后的人源化抗体可变区进行匹配，构建完整的人源化抗体。将人源化抗体轻链与重链可变区氨基酸序列反转录成相对应的核苷酸序列，根据表达宿主进行密码子优化，并生成相邻片段之间含有互补序列的寡核苷酸片段，通过Overlap PCR将寡核苷酸片段退火后连接起来，再利用特异性引物(5’端含有用于与真核表达载体发生同源重组的同源臂序列)扩增出完整的轻链与重链可变区核苷酸片段；将纯化后的轻链可变区核苷酸片段与线性化的含有人κ轻链恒定区的真核表达质粒共转化大 肠杆菌DH5α感受态细胞，将纯化后的重链可变区核苷酸片段与人IgG重链恒定区的真核表达质粒共转化大肠杆菌DH5α感受态细胞，分别将转化质粒的感受态细胞均匀涂布于含有氨苄抗生素的琼脂平板表面，于37℃恒温培养箱过夜培养后分别挑取若干单菌落进行DNA测序。Considering the function and stability of the antibody, an appropriate antibody subtype framework is selected to match the optimized humanized antibody variable region to construct a complete humanized antibody. Reverse transcribe the amino acid sequence of the humanized antibody light chain and heavy chain variable region into the corresponding nucleotide sequence, perform codon optimization according to the expression host, and generate oligonucleotides containing complementary sequences between adjacent fragments The oligonucleotide fragments were annealed and ligated by Overlap PCR, and then the complete light chain was amplified using specific primers (the 5' end contains homology arm sequences for homologous recombination with eukaryotic expression vectors) and the heavy chain variable region nucleotide fragment; the purified light chain variable region nucleotide fragment and the linearized eukaryotic expression plasmid containing the human κ light chain constant region were co-transformed into Escherichia coli DH5α competent cells, and the The purified nucleotide fragment of the heavy chain variable region and the eukaryotic expression plasmid of the human IgG heavy chain constant region were co-transformed into Escherichia coli DH5α competent cells, and the competent cells of the transformed plasmid were evenly spread on agar containing ampicillin antibiotics On the surface of the plate, after culturing overnight in a 37°C constant temperature incubator, several single colonies were picked for DNA sequencing.
将测序正确的阳性克隆接种于含有氨苄抗生素的2×YT液体培养基中，于37℃振荡培养12小时以上，然后收集菌体进行质粒提取，从而获得人源化抗体轻链与重链表达质粒，使用核酸定量分析仪检测质粒的浓度与纯度。The positive clones with correct sequencing were inoculated in 2×YT liquid medium containing ampicillin antibiotics, cultured with shaking at 37°C for more than 12 hours, and then the bacteria were collected for plasmid extraction to obtain humanized antibody light chain and heavy chain expression plasmids , Use a nucleic acid quantitative analyzer to detect the concentration and purity of the plasmid.
将质粒转染HEK293E细胞，表达纯化获得大量抗体，进行纯度检测、活性分析及亲和力的检测。完整的人源化抗体序列见表7。The plasmid was transfected into HEK293E cells, expressed and purified to obtain a large amount of antibodies, and the purity test, activity analysis and affinity test were performed. The complete humanized antibody sequences are listed in Table 7.
表7抗CRTAM人源化抗体完整序列Table 7 Complete sequence of anti-CRTAM humanized antibody
Figure PCTCN2022102253-appb-000009
Figure PCTCN2022102253-appb-000009
实施例5：抗CRTAM抗体与人CRTAM结合活性测定(ELISA)Example 5: Anti-CRTAM antibody binding activity assay (ELISA) to human CRTAM
采用ELISA分析抗体的结合活性。将hCRTAM-His蛋白(1.5μg/mL，50μL/孔，实施例1中制得)包被到96孔酶标板，于37℃孵育2h。用1x PBST清洗3次后用含5％脱脂牛奶的PBST 37℃封闭2h。用1x PBST清洗3次后，本发明的抗CRTAM抗体作为一抗从10μg/mL开始，5倍梯度稀释加入酶标板(50μL/孔)，共8个浓度，浓度分别为10000ng/mL、2000ng/mL、400ng/mL、80ng/mL、16ng/mL、3.2ng/mL、0.64ng/mL、0.128ng/mL，37℃孵育1.5h，对照抗体为5A11(实施例1中制得)；用1x PBST清洗3次后拍干，二抗使用Anti-Human IgG HRP(Jackson，109-035-003，1:5000，50μL/孔)，37℃孵育1h。用1xPBST清洗5次后拍干，加入单组分TMB显色液(索莱宝，PR1200，50μL/孔)，避光显色约5min，加入2M硫酸(50μL/孔)终止，使用酶标仪(thermo，Multiskan FC)读取OD450值。使用GraphPad生成EC50，结果如表8所示。Antibody binding activity was analyzed by ELISA. hCRTAM-His protein (1.5 μg/mL, 50 μL/well, prepared in Example 1) was coated onto a 96-well microtiter plate, and incubated at 37° C. for 2 h. After washing 3 times with 1x PBST, block with PBST containing 5% skimmed milk at 37°C for 2h. After washing 3 times with 1x PBST, the anti-CRTAM antibody of the present invention was used as a primary antibody starting from 10 μg/mL, and added to a microtiter plate (50 μL/well) by 5-fold gradient dilution, with a total of 8 concentrations, the concentrations were 10000 ng/mL and 2000 ng respectively. /mL, 400ng/mL, 80ng/mL, 16ng/mL, 3.2ng/mL, 0.64ng/mL, 0.128ng/mL, incubated at 37°C for 1.5h, the control antibody was 5A11 (prepared in Example 1); After washing with 1x PBST for 3 times, pat dry, use Anti-Human IgG HRP (Jackson, 109-035-003, 1:5000, 50 μL/well) as the secondary antibody, and incubate at 37°C for 1 hour. After washing with 1xPBST for 5 times, pat dry, add one-component TMB chromogenic solution (Soleibao, PR1200, 50 μL/well), and develop color in the dark for about 5 minutes, add 2M sulfuric acid (50 μL/well) to terminate, and use a microplate reader (thermo, Multiskan FC) read OD450 value. EC50 was generated using GraphPad, and the results are shown in Table 8.
表8抗CRTAM人源化抗体与人CRTAM结合活性Table 8 Anti-CRTAM humanized antibody binding activity to human CRTAM
抗体名称Antibody name HuC94-31HuC94-31 HuC110-34HuC110-34 HuC124-42HuC124-42 5A115A11
EC50(ng/mL)EC50(ng/mL) 11.4111.41 11.1711.17 9.919.91 38.1838.18
实验结果显示，本发明的抗CRTAM抗体具有较好的与人CRTAM结合的能力。Experimental results show that the anti-CRTAM antibody of the present invention has a better ability to bind to human CRTAM.
实施例6：抗CRTAM抗体与食蟹猴CRTAM结合活性测定(ELISA)Example 6: Determination of the Binding Activity of Anti-CRTAM Antibodies to CRTAM in Cynomolgus Monkeys (ELISA)
采用ELISA分析抗体的结合活性。将cCRTAM-His蛋白(2μg/mL，50μL/孔，实施例1中制得)包被到96孔酶标板，于37℃孵育2h。用1x PBST清洗3次后用含5％脱脂牛奶的PBST 37℃封闭2h。用1x PBST清洗3次后，本发明的抗CRTAM抗体作为一抗从10μg/mL开始，5倍梯度稀释加入酶标板(50μL/孔)，共8个浓度，浓度分别为10000ng/mL、2000ng/mL、400ng/mL、80ng/mL、16ng/mL、3.2ng/mL、0.64ng/mL、0.128ng/mL，37℃孵育1.5h，对照抗体为5A11(实施例1中制得)；用1x PBST清洗3次后拍干，二抗使用Anti-Human IgG HRP(Jackson，109-035-003，1:5000，50μL/孔)，37℃孵育1h。用1xPBST清洗5次后拍干，加入单组分TMB显色液(索莱宝，PR1200，50μL/孔)，避光显色约5min，加入2M硫酸(50μL/孔)终止，使用酶标仪(thermo，Multiskan FC)读取OD450值。使用GraphPad生成EC50，结果如表9所示。Antibody binding activity was analyzed by ELISA. The cCRTAM-His protein (2 μg/mL, 50 μL/well, prepared in Example 1) was coated onto a 96-well microtiter plate, and incubated at 37° C. for 2 h. After washing 3 times with 1x PBST, block with PBST containing 5% skimmed milk at 37°C for 2h. After washing 3 times with 1x PBST, the anti-CRTAM antibody of the present invention was used as a primary antibody starting from 10 μg/mL, and added to a microtiter plate (50 μL/well) by 5-fold gradient dilution, with a total of 8 concentrations, the concentrations were 10000 ng/mL and 2000 ng respectively. /mL, 400ng/mL, 80ng/mL, 16ng/mL, 3.2ng/mL, 0.64ng/mL, 0.128ng/mL, incubated at 37°C for 1.5h, the control antibody was 5A11 (prepared in Example 1); After washing with 1x PBST for 3 times, pat dry, use Anti-Human IgG HRP (Jackson, 109-035-003, 1:5000, 50 μL/well) as the secondary antibody, and incubate at 37°C for 1 hour. After washing with 1xPBST for 5 times, pat dry, add one-component TMB chromogenic solution (Soleibao, PR1200, 50 μL/well), and develop color in the dark for about 5 minutes, add 2M sulfuric acid (50 μL/well) to terminate, and use a microplate reader (thermo, Multiskan FC) read OD450 value. EC50 was generated using GraphPad, and the results are shown in Table 9.
表9抗CRTAM抗体与食蟹猴CRTAM结合活性Table 9 Anti-CRTAM antibody binding activity to cynomolgus monkey CRTAM
抗体名称Antibody name HuC94-31HuC94-31 HuC110-34HuC110-34 HuC124-42HuC124-42
EC50(ng/mL)EC50(ng/mL) 4.684.68 8.178.17 4.784.78
实验结果显示，本发明的抗CRTAM抗体具有较好的与食蟹猴CRTAM结合的能力。Experimental results show that the anti-CRTAM antibody of the present invention has a better ability to bind to CRTAM in cynomolgus monkeys.
实施例7：抗CRTAM抗体对CRTAM与其配体CADM1的阻断活性测定(ELISA)Example 7: Determination of the blocking activity of anti-CRTAM antibodies to CRTAM and its ligand CADM1 (ELISA)
采用ELISA法分析抗体对人CRTAM与其配体CADM1的阻断活性。将hCRTAM-His蛋白(1.5μg/mL，50μL/孔，实施例1中制得)包被到96孔酶标板，于37℃孵育2h。用1x PBST清洗3次后用含5％脱脂牛奶的PBST 37℃封闭2h。同时用biotin-NHS ester(biovsion，2347-50)标记配体蛋白hCADM1-His，标记好的配体命名为hCADM1-His-biotin。封闭好的酶标板用1x PBST清洗3次后，配置浓度为20μg/mL的hCADM1-His-biotin配体溶液，以上述配体溶液为稀释液稀释一抗，本发明的抗CRTAM抗体作为一抗从100μg/mL开始，3倍梯度稀释加入酶标板(50μL/孔)，共8个浓度，浓度分别为100000ng/mL、33333ng/mL、11111ng/mL、3704ng/mL、1235ng/mL、412ng/mL、137ng/mL、46ng/mL，37℃孵育1.5h，对照抗体为5A11(实施例1中制得)；用1x PBST清洗3次后 拍干，二抗使用Streptavidin-HRP(BD，554066，1:10000，50μL/孔)，37℃孵育1h。用1xPBST清洗5次后拍干，加入单组分TMB显色液(索莱宝，PR1200，50μL/孔)，避光显色约5min，加入2M硫酸(50μL/孔)终止，使用酶标仪(thermo，Multiskan FC)读取OD450值。使用GraphPad生成IC50，结果如图1所示。The blocking activity of the antibody on human CRTAM and its ligand CADM1 was analyzed by ELISA. hCRTAM-His protein (1.5 μg/mL, 50 μL/well, prepared in Example 1) was coated onto a 96-well microtiter plate, and incubated at 37° C. for 2 h. After washing 3 times with 1x PBST, block with PBST containing 5% skimmed milk at 37°C for 2h. At the same time, the ligand protein hCADM1-His was labeled with biotin-NHS ester (biovsion, 2347-50), and the labeled ligand was named hCADM1-His-biotin. After the sealed ELISA plate was washed 3 times with 1x PBST, the hCADM1-His-biotin ligand solution with a concentration of 20 μg/mL was configured, and the primary antibody was diluted with the above ligand solution as a diluent, and the anti-CRTAM antibody of the present invention was used as a primary antibody. Starting from 100μg/mL, 3-fold serial dilution was added to the microplate plate (50μL/well), a total of 8 concentrations, the concentrations were 100000ng/mL, 33333ng/mL, 11111ng/mL, 3704ng/mL, 1235ng/mL, 412ng /mL, 137ng/mL, 46ng/mL, incubated at 37°C for 1.5h, the control antibody was 5A11 (prepared in Example 1); washed 3 times with 1x PBST and patted dry, the secondary antibody was Streptavidin-HRP (BD, 554066 , 1:10000, 50 μL/well), and incubated at 37°C for 1h. After washing with 1xPBST for 5 times, pat dry, add one-component TMB chromogenic solution (Soleibao, PR1200, 50 μL/well), and develop color in the dark for about 5 minutes, add 2M sulfuric acid (50 μL/well) to terminate, and use a microplate reader (thermo, Multiskan FC) read OD450 value. IC50 was generated using GraphPad, and the results are shown in Figure 1.
实验结果显示，本发明的抗CRTAM抗体优于对照抗体5A11，几乎不阻断人CRTAM与CADM1的结合，不影响其正常功能。Experimental results show that the anti-CRTAM antibody of the present invention is better than the control antibody 5A11, hardly blocking the combination of human CRTAM and CADM1, and not affecting its normal function.
实施例8：抗CRTAM抗体与细胞表面人CRTAM结合活性测定(FACS)Embodiment 8: Determination of binding activity of anti-CRTAM antibody to human CRTAM on cell surface (FACS)
采用FACS分析抗体与CHO-K1-hCRTAM细胞表面的hCRTAM的结合活性。CHO-K1-hCRTAM细胞消化后，用2％FBS-PBS的溶液重悬，计数。将上述细胞按照每孔1.5x10 5个细胞的方式铺细胞板，本发明的抗CRTAM抗体作为一抗从20μg/ml开始，5倍梯度稀释加入细胞板，共8个浓度，浓度分别为20000ng/mL、4000ng/mL、800ng/mL、160ng/mL、32ng/mL、6.4ng/mL、1.28ng/mL、0.26ng/mL，4℃条件下孵育1.5h，对照抗体为5A11；使用PBS清洗三次细胞后，二抗使用PE-Anti-Human IgG(Biolegend，Cat.No.409303，1.25μl/孔)4℃条件下孵育1h；PBS三次洗涤后使用流式细胞仪检测抗体与细胞表面结合产生的荧光强度，结果如表10所示。 The binding activity of the antibody to hCRTAM on the surface of CHO-K1-hCRTAM cells was analyzed by FACS. After the CHO-K1-hCRTAM cells were digested, they were resuspended with 2% FBS-PBS solution and counted. Spread the above cells on a cell plate in a manner of 1.5x105 cells per well. The anti-CRTAM antibody of the present invention is used as a primary antibody starting from 20 μg/ml, and added to the cell plate in a 5-fold gradient dilution, with a total of 8 concentrations, and the concentration is 20000 ng/ml. mL, 4000ng/mL, 800ng/mL, 160ng/mL, 32ng/mL, 6.4ng/mL, 1.28ng/mL, 0.26ng/mL, incubate at 4°C for 1.5h, the control antibody is 5A11; wash three times with PBS After cells, the secondary antibody was incubated with PE-Anti-Human IgG (Biolegend, Cat. No. 409303, 1.25 μl/well) at 4°C for 1 h; after washing with PBS three times, flow cytometry was used to detect the antibody produced by binding to the cell surface. Fluorescence intensity, the results are shown in Table 10.
表10抗CRTAM抗体与细胞表面hCRTAM结合活性Table 10 Anti-CRTAM antibody binding activity to cell surface hCRTAM
抗体名称Antibody name HuC110-34HuC110-34 5A115A11
EC50(ng/mL)EC50(ng/mL) 41.7441.74 60.7060.70
实验结果显示，本发明的人源化抗CRTAM抗体HuC110-34具有较好的与细胞表面hCRTAM结合能力。Experimental results show that the humanized anti-CRTAM antibody HuC110-34 of the present invention has better binding ability to hCRTAM on the cell surface.
实施例9：抗CRTAM抗体与细胞表面食蟹猴CRTAM结合活性测定(FACS)Example 9: Determination of the Binding Activity of Anti-CRTAM Antibodies to Cell Surface Cynomolgus Monkey CRTAM (FACS)
采用FACS分析抗体与CHO-K1-cCRTAM细胞表面的cCRTAM的结合活性。CHO-K1-cCRTAM细胞消化后，用2％FBS-PBS的溶液重悬，计数。将上述细胞按照每孔1.5x10 5个细胞的方式铺细胞板，本发明的抗CRTAM抗体作为一抗从20μg/ml开始，5倍梯度稀释加入细胞板，共8个浓度，浓度分别为20000ng/mL、4000ng/mL、800ng/mL、160ng/mL、32ng/mL、6.4ng/mL、1.28ng/mL、0.26ng/mL，4℃条件下孵育1.5h；使用PBS清洗三次细胞后，二抗使用PE-Anti-Human IgG(Biolegend，Cat.No.409303，1.25μl/孔)4℃条件下孵育1h；PBS三次洗涤后使用流式细胞仪检测抗体与细胞表面结合产生的荧光强度，结果如表11所示。 The binding activity of the antibody to cCRTAM on the surface of CHO-K1-cCRTAM cells was analyzed by FACS. After the CHO-K1-cCRTAM cells were digested, they were resuspended with 2% FBS-PBS solution and counted. Spread the above cells on a cell plate in a manner of 1.5x105 cells per well. The anti-CRTAM antibody of the present invention is used as a primary antibody starting from 20 μg/ml, and added to the cell plate in a 5-fold gradient dilution, with a total of 8 concentrations, and the concentration is 20000 ng/ml. mL, 4000ng/mL, 800ng/mL, 160ng/mL, 32ng/mL, 6.4ng/mL, 1.28ng/mL, 0.26ng/mL, incubated at 4°C for 1.5h; after washing the cells three times with PBS, the secondary antibody Use PE-Anti-Human IgG (Biolegend, Cat. No. 409303, 1.25 μl/well) to incubate at 4°C for 1 h; after washing with PBS three times, use flow cytometry to detect the fluorescence intensity generated by the binding of the antibody to the cell surface, and the results are as follows: Table 11 shows.
表11抗CRTAM抗体与细胞表面食蟹猴CRTAM结合活性Table 11 Anti-CRTAM antibody binding activity to cell surface cynomolgus monkey CRTAM
抗体名称Antibody name HuC94-31HuC94-31 HuC110-34HuC110-34 HuC124-42HuC124-42
EC50(ng/mL)EC50(ng/mL) 35.0235.02 50.6950.69 90.3890.38
实验结果显示，本发明的人源化抗CRTAM抗体具有较好的与细胞表面cCRTAM结合能力。Experimental results show that the humanized anti-CRTAM antibody of the present invention has better ability to bind to cell surface cCRTAM.
实施例10：抗CRTAM抗体与5A11对人CRTAM的表位竞争活性测定(ELISA)Example 10: Anti-CRTAM antibody and 5A11 epitope competition activity assay (ELISA) for human CRTAM
采用ELISA法分析本发明的抗CRTAM抗体与5A11对人CRTAM的表位竞争活性。将hCRTAM-His蛋白(2μg/mL，50μL/孔，实施例1中制得)包被到96孔酶标板，于37℃孵育2h。用1x PBST清洗3次后用含5％脱脂牛奶的PBST37℃封闭2h。同时用Biotin-NHS ester TM(Biovsion公司，2347-50)标记本发明的抗CRTAM抗体，标记好的抗体分别命名为HuC68-17-biotin、HuC94-31-biotin、HuC110-34-biotin、HuC124-42-biotin。封闭好的酶标板用1x PBST清洗3次后，分别配置浓度为1μg/mL的HuC68-17-biotin、HuC94-31-biotin、HuC110-34-biotin、HuC124-42-biotin溶液，以上述溶液为稀释液分别稀释一抗，本发明的抗CRTAM抗体(竞争阳性对照)与5A11作为一抗从100μg/mL开始，5倍梯度稀释加入酶标板(50μL/孔)，共8个浓度，浓度分别为100000ng/mL、20000ng/mL、4000ng/mL、800ng/mL、160ng/mL、32ng/mL、6.4ng/mL、1.28ng/mL，37℃孵育1.5h；用1x PBST清洗3次后拍干，二抗使用Streptavidin-HRP(BD，554066，1:10000，50μL/孔)，37℃孵育1h。用1xPBST清洗5次后拍干，加入单组分TMB显色液(索莱宝，PR1200，50μL/孔)，避光显色约5min，加入2M硫酸(50μL/孔)终止，使用酶标仪(thermo，Multiskan FC)读取OD450值。使用GraphPad生成IC50，结果如图2～5所示。 The ELISA method was used to analyze the epitope competition activity of the anti-CRTAM antibody of the present invention and 5A11 on human CRTAM. hCRTAM-His protein (2 μg/mL, 50 μL/well, prepared in Example 1) was coated onto a 96-well microtiter plate, and incubated at 37° C. for 2 h. After washing 3 times with 1x PBST, block with PBST containing 5% skimmed milk at 37°C for 2h. At the same time, the anti-CRTAM antibody of the present invention was labeled with Biotin-NHS ester TM (Biovsion Company, 2347-50), and the labeled antibody was named as HuC68-17-biotin, HuC94-31-biotin, HuC110-34-biotin, HuC124-biotin, respectively. 42-biotin. After the sealed microtiter plate was washed 3 times with 1x PBST, HuC68-17-biotin, HuC94-31-biotin, HuC110-34-biotin, HuC124-42-biotin solutions with a concentration of 1 μg/mL were respectively prepared, and the above solutions To dilute the primary antibody for the diluent, the anti-CRTAM antibody of the present invention (competition positive control) and 5A11 are used as the primary antibody starting from 100 μg/mL, 5-fold serial dilution is added to the microplate (50 μL/well), a total of 8 concentrations, the concentration 100000ng/mL, 20000ng/mL, 4000ng/mL, 800ng/mL, 160ng/mL, 32ng/mL, 6.4ng/mL, 1.28ng/mL, incubate at 37°C for 1.5h; wash with 1x PBST for 3 times and shoot Dry, use Streptavidin-HRP (BD, 554066, 1:10000, 50 μL/well) as the secondary antibody, and incubate at 37°C for 1 hour. After washing with 1xPBST for 5 times, pat dry, add one-component TMB chromogenic solution (Soleibao, PR1200, 50 μL/well), and develop color in the dark for about 5 minutes, add 2M sulfuric acid (50 μL/well) to terminate, and use a microplate reader (thermo, Multiskan FC) read OD450 value. Use GraphPad to generate IC50, and the results are shown in Figures 2-5.
实验结果显示，本发明的人源化抗CRTAM抗体HuC68-17、HuC94-31、HuC110-34、HuC124-42与5A11几乎不存在表位竞争，不阻断5A11与人CRTAM的结合，表明本发明的人源化抗CRTAM抗体与5A11对CRTAM的结合表位完全不同。The experimental results show that the humanized anti-CRTAM antibodies HuC68-17, HuC94-31, HuC110-34, HuC124-42 of the present invention have almost no epitope competition with 5A11, and do not block the binding of 5A11 and human CRTAM, indicating that the present invention The humanized anti-CRTAM antibody has a completely different binding epitope to CRTAM than 5A11.
实施例11：抗CRTAM抗体与人CRTAM蛋白的亲和力测定Example 11: Affinity Determination of Anti-CRTAM Antibody and Human CRTAM Protein
利用Fortebio Octet对本发明的人源化抗CRTAM抗体结合抗原hCRTAM-His(实施例1中制得)的亲和力进行测定。将抗CRTAM抗体用SD缓冲液(PBS+0.02％Tween20+0.1％BSA)稀释到浓度10μg/mL，抗原hCRTAM-His用SD 缓冲液4倍浓度梯度稀释，使其浓度为12μg/mL、3μg/mL、0.75μg/mL、0μg/mL，选用AHC传感器固化抗体，按Fortebio Octet RED96的操作规程进行亲和力测定，具体参数及实验结果如表12所示。The binding affinity of the humanized anti-CRTAM antibody of the present invention to the antigen hCRTAM-His (prepared in Example 1) was determined by Fortebio Octet. The anti-CRTAM antibody was diluted to a concentration of 10 μg/mL with SD buffer (PBS+0.02% Tween20+0.1% BSA), and the antigen hCRTAM-His was diluted with a 4-fold concentration gradient of SD buffer to make its concentration 12 μg/mL, 3 μg/mL mL, 0.75 μg/mL, 0 μg/mL, the AHC sensor was used to immobilize the antibody, and the affinity was determined according to the operating procedures of Fortebio Octet RED96. The specific parameters and experimental results are shown in Table 12.
表12抗CRTAM抗体与人CRTAM蛋白的亲和力测定Table 12 Affinity determination of anti-CRTAM antibody and human CRTAM protein
Figure PCTCN2022102253-appb-000010
Figure PCTCN2022102253-appb-000010
实验结果显示，本发明的人源化抗CRTAM抗体的亲和力显著优于5A11，显示出更持久的与人CRTAM蛋白结合能力。Experimental results show that the affinity of the humanized anti-CRTAM antibody of the present invention is significantly better than that of 5A11, showing a longer-lasting ability to bind to human CRTAM protein.
实施例12：抗CRTAM抗体的体外药效检测Example 12: In Vitro Drug Efficacy Detection of Anti-CRTAM Antibody
采用SEB(葡萄球菌肠毒素B)刺激健康人PBMC的方法来检测抗CRTAM抗体的体外药效。SEB (staphylococcal enterotoxin B) was used to stimulate healthy human PBMC to detect the in vitro efficacy of anti-CRTAM antibody.
按所需细胞量复苏PBMC，加到8-9ml的IMDM完全培养基中，900g离心10min，弃上清；用适量培养基重悬，用血球计数板计数，并重悬为1M/mL，100μL/孔加入96孔板中；按4倍浓度(即40μg/mL)，50μL/孔，用IMDM完全培养基配制待检测抗体和Isotype，做好标记，涡旋；将抗体溶液加入96孔板中，对照组加入50μL/孔的培养基，96孔板置于37℃孵箱中，细胞和抗体孵育1h；按4倍浓度(即200ng/mL)用IMDM完全培养基配制SEB溶液，50μL/孔加入对应孔中；96孔板置于37℃，5％CO 2孵箱中孵育72h后，收集无细胞上清150μL，按一定比例稀释后，按照hIFN-γELISA检测试剂盒(R&D
Figure PCTCN2022102253-appb-000011
ELISA试剂盒，R&D system Cat：DY285B)说明书检测上清中IFN-γ的浓度，结果如表 Resuscitate PBMCs according to the required cell volume, add them to 8-9ml IMDM complete medium, centrifuge at 900g for 10min, discard the supernatant; Add the wells into a 96-well plate; use 4 times the concentration (40 μg/mL), 50 μL/well, prepare the antibody to be detected and Isotype with IMDM complete medium, mark it, and vortex; add the antibody solution into the 96-well plate, Add 50 μL/well of culture medium to the control group, place the 96-well plate in a 37°C incubator, and incubate the cells and antibodies for 1 h; Corresponding wells; 96-well plates were placed in a 37°C, 5% CO 2 incubator and incubated for 72 hours, and 150 μL of cell-free supernatant was collected, diluted in a certain proportion, and tested according to the hIFN-γELISA detection kit (R&D
Figure PCTCN2022102253-appb-000011
ELISA kit, R&D system Cat: DY285B) instructions to detect the concentration of IFN-γ in the supernatant, the results are shown in the table
13所示。表13抗CRTAM抗体促进PBMC释放IFN-γ13. Table 13 anti-CRTAM antibody promotes PBMC to release IFN-γ
Figure PCTCN2022102253-appb-000012
Figure PCTCN2022102253-appb-000012
实验结果显示，与5A11相比，本发明的抗CRTAM抗体具有更好地促IFN-γ 释放能力。Experimental results show that, compared with 5A11, the anti-CRTAM antibody of the present invention has a better ability to promote IFN-γ release.
实施例13：HCC827(del19)人非小细胞肺癌细胞PBMC重建模型抗肿瘤实验1、实验材料Example 13: Anti-tumor experiment of HCC827 (del19) human non-small cell lung cancer cell PBMC reconstruction model 1. Experimental materials
(1)实验细胞及动物(1) Experimental cells and animals
HCC827(del19)细胞购自美国典型培养物保藏中心(ATCC)；HCC827 (del19) cells were purchased from the American Type Culture Collection (ATCC);
NSG小鼠，雌性，6-8周龄，体重18-20克，购自百奥赛图(江苏)基因生物技术有限公司；NSG mice, female, 6-8 weeks old, weighing 18-20 grams, were purchased from Biocytogen (Jiangsu) Gene Biotechnology Co., Ltd.;
(2)供试品及对照品(2) Test product and reference product
对照品5A11由实施例1制得，用作阳性对照；对照品PBS用作阴性对照；试验前，将本发明的抗CRTAM抗体用PBS配制为1mg/mL。The control substance 5A11 was prepared in Example 1 and used as a positive control; the reference substance PBS was used as a negative control; before the test, the anti-CRTAM antibody of the present invention was prepared in PBS to a concentration of 1 mg/mL.
2、实验方法2. Experimental method
HCC827(del19)人非小细胞肺癌细胞PBMC重建模型PBMC reconstruction model of HCC827(del19) human non-small cell lung cancer cells
将含有1×10 7个HCC827(del19)细胞的50μL PBS与50μL基质胶混合后接种于小鼠的右后肢皮下，待肿瘤生长至平均体积达100mm 3时开始分组。PBMC来自健康献血者的新鲜血液，于DAY1分组后给抗体前1小时进行尾静脉注射(每只小鼠注射10M的PBMC细胞)。尾静脉给药每周2次，每周两次用游标卡尺测量肿瘤直径，计算肿瘤体积，肿瘤体积的计算公式为：V＝0.5a×b 2，a和b分别表示肿瘤的长径和短径，计算相对肿瘤增殖率(T/C)。肿瘤生长抑制率用下列公式计算:TGI(％)＝[1-(Ti-T0)/(Vi-V0)]×100,其中Ti为某一天某给药组的平均肿瘤体积，T0为此给药组在开始给药时的平均肿瘤体积；Vi为某一天(与Ti同一天)溶媒对照组的平均肿瘤体积，V0为溶媒对照组在给开始药时的平均肿瘤体积。记录连续给药22天测量的肿瘤体积，用GraphPad Prism绘制瘤体积生长曲线。在实验结束后检测肿瘤重量，并计算各组的肿瘤抑制率(TGI)％，结果如表14所示。 Mix 50 μL of PBS containing 1×10 7 HCC827 (del19) cells with 50 μL of Matrigel and inoculate subcutaneously on the right hind limb of the mouse, and start grouping when the tumor grows to an average volume of 100 mm 3 . PBMCs come from the fresh blood of healthy blood donors, and are injected into the tail vein 1 hour before the antibody is given after grouping on DAY1 (10M PBMC cells are injected per mouse). The tail vein was administered twice a week, and the tumor diameter was measured twice a week with a vernier caliper, and the tumor volume was calculated. The formula for calculating the tumor volume was: V=0.5a×b 2 , where a and b represented the long diameter and short diameter of the tumor, respectively , to calculate the relative tumor proliferation rate (T/C). The tumor growth inhibition rate is calculated with the following formula: TGI (%)=[1-(Ti-T0)/(Vi-V0)]×100, wherein Ti is the average tumor volume of a certain administration group on a certain day, and T0 is given for this purpose. The average tumor volume of the drug group at the beginning of administration; Vi is the average tumor volume of the vehicle control group on a certain day (the same day as Ti), and V0 is the average tumor volume of the vehicle control group at the beginning of drug administration. The tumor volume measured for 22 days of continuous administration was recorded, and the tumor volume growth curve was drawn with GraphPad Prism. After the experiment, the tumor weight was detected, and the tumor inhibition rate (TGI)% of each group was calculated. The results are shown in Table 14.
表14 HCC827(del19)人非小细胞肺癌细胞PBMC重建模型抗肿瘤结果Table 14 Anti-tumor results of HCC827(del19) human non-small cell lung cancer cell PBMC reconstruction model
Figure PCTCN2022102253-appb-000013
Figure PCTCN2022102253-appb-000013
实验结果显示，相比于5A11，本发明的抗CRTAM抗体能够更好地抑制HCC827(del19)人非小细胞肺癌肿瘤生长。Experimental results show that, compared with 5A11, the anti-CRTAM antibody of the present invention can better inhibit the growth of HCC827 (del19) human non-small cell lung cancer tumor.
实施例14：NUGC-4人胃癌细胞PBMC重建模型抗肿瘤实验Example 14: Anti-tumor experiment of NUGC-4 human gastric cancer cell PBMC reconstruction model
1、实验材料1. Experimental materials
(1)实验细胞及动物(1) Experimental cells and animals
NUGC-4人胃癌细胞购自美国典型培养物保藏中心(ATCC)；NUGC-4 human gastric cancer cells were purchased from the American Type Culture Collection (ATCC);
NSG小鼠，雌性，6-8周龄，体重18-20克，购自百奥赛图(江苏)基因生物技术有限公司；NSG mice, female, 6-8 weeks old, weighing 18-20 grams, were purchased from Biocytogen (Jiangsu) Gene Biotechnology Co., Ltd.;
(2)供试品及对照品(2) Test product and reference product
对照品为PBS，用作阴性对照；试验前，将本发明的抗CRTAM抗体用PBS配制为1mg/mL。The control substance is PBS, which is used as a negative control; before the test, the anti-CRTAM antibody of the present invention is prepared with PBS to 1 mg/mL.
2、实验方法2. Experimental method
NUGC-4人胃癌细胞PBMC重建模型NUGC-4 human gastric cancer cell PBMC reconstruction model
将含有5×10 6个NUGC-4(del19)细胞的100μL PBS与100μL基质胶混合后接种于小鼠的右后肢皮下，待肿瘤生长至平均体积达110mm 3时开始分组。PBMC来自健康献血者的新鲜血液，于DAY1分组后给抗体前1小时进行尾静脉注射(每只小鼠注射10M的PBMC细胞)。尾静脉给药每周2次，每周两次用游标卡尺测量肿瘤直径，计算肿瘤体积，肿瘤体积的计算公式为：V＝0.5a×b 2，a和b分别表示肿瘤的长径和短径，计算相对肿瘤增殖率(T/C)。肿瘤生长抑制率用下列公式计算:TGI(％)＝[1-(Ti-T0)/(Vi-V0)]×100,其中Ti为某一天某给药组的平均肿瘤体积，T0为此给药组在开始给药时的平均肿瘤体积；Vi为某一天(与Ti同一天)溶媒对照组的平均肿瘤体积，V0为溶媒对照组在给开始药时的平均肿瘤体积。记录连续给药21天测量的肿瘤体积，用GraphPad Prism绘制瘤体积生长曲线。在实验结束后检测肿瘤重量，并计算各组的肿瘤抑制率(TGI)％，结果如表15所示。 Mix 100 μL PBS containing 5×10 6 NUGC-4 (del19) cells with 100 μL matrigel and inoculate them subcutaneously on the right hind limb of the mice. Grouping began when the tumors grew to an average volume of 110 mm 3 . PBMCs come from the fresh blood of healthy blood donors, and are injected into the tail vein 1 hour before the antibody is given after grouping on DAY1 (10M PBMC cells are injected per mouse). The tail vein was administered twice a week, and the tumor diameter was measured twice a week with a vernier caliper, and the tumor volume was calculated. The formula for calculating the tumor volume was: V=0.5a×b 2 , where a and b represented the long diameter and short diameter of the tumor, respectively , to calculate the relative tumor proliferation rate (T/C). The tumor growth inhibition rate is calculated with the following formula: TGI (%)=[1-(Ti-T0)/(Vi-V0)]×100, wherein Ti is the average tumor volume of a certain administration group on a certain day, and T0 is given for this purpose. The average tumor volume of the drug group at the beginning of administration; Vi is the average tumor volume of the vehicle control group on a certain day (the same day as Ti), and V0 is the average tumor volume of the vehicle control group at the beginning of drug administration. The tumor volume measured on 21 days of continuous administration was recorded, and the tumor volume growth curve was drawn with GraphPad Prism. After the experiment, the tumor weight was detected, and the tumor inhibition rate (TGI)% of each group was calculated. The results are shown in Table 15.
表15 NUGC-4人胃癌细胞PBMC重建模型抗肿瘤结果Table 15 Anti-tumor results of NUGC-4 human gastric cancer cell PBMC reconstruction model
Figure PCTCN2022102253-appb-000014
Figure PCTCN2022102253-appb-000014
实验结果显示，本发明的抗CRTAM抗体能够较好地抑制NUGC-4人胃癌细胞生长。Experimental results show that the anti-CRTAM antibody of the present invention can better inhibit the growth of NUGC-4 human gastric cancer cells.
以上实施例证明本发明提供的抗CRTAM抗体具有显著的抗肿瘤作用，可明显抑制肿瘤增长，提示这些抗体可在制备抗肿瘤药物中应用，具有好的市场前景。The above examples prove that the anti-CRTAM antibodies provided by the present invention have significant anti-tumor effects and can significantly inhibit tumor growth, suggesting that these antibodies can be used in the preparation of anti-tumor drugs and have good market prospects.
尽管以上已经对本发明作了详细描述，但是本领域技术人员理解，在不偏离本发明的精神和范围的前提下可以对本发明进行各种修改和改变。本发明的权利范围并不限于上文所作的详细描述，而应归属于权利要求书。Although the present invention has been described in detail above, it will be understood by those skilled in the art that various modifications and changes can be made to the present invention without departing from the spirit and scope of the present invention. The scope of rights of the present invention is not limited to the detailed description above, but should be attributed to the claims.

Claims (12)

  1. 一种抗CRTAM抗体或其抗原结合片段，其包含重链可变区和轻链可变区，其中：An anti-CRTAM antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein:
    (1)所述重链可变区包含选自如下组的HCDR1、HCDR2和HCDR3：(1) The heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 selected from the group consisting of:
    (a1)如SEQ ID NO:1、2和3所示的氨基酸序列；(a1) an amino acid sequence as shown in SEQ ID NO: 1, 2 and 3;
    (a2)如SEQ ID NO:8、9和10所示的氨基酸序列；(a2) amino acid sequences as shown in SEQ ID NO:8, 9 and 10;
    (a3)如SEQ ID NO:8、11和10所示的氨基酸序列；(a3) amino acid sequences as shown in SEQ ID NO:8, 11 and 10;
    (a4)如SEQ ID NO:16、17和18所示的氨基酸序列；(a4) amino acid sequences as shown in SEQ ID NO: 16, 17 and 18;
    (a5)如SEQ ID NO:22、23和24所示的氨基酸序列；(a5) amino acid sequences as shown in SEQ ID NO:22, 23 and 24;
    (a6)如SEQ ID NO:22、25和24所示的氨基酸序列；和(a6) amino acid sequences as shown in SEQ ID NO: 22, 25 and 24; and
    (a7)与(a1)、(a2)、(a3)、(a4)、(a5)或(a6)所示的氨基酸序列具有至少85％序列同一性的氨基酸序列；和(a7) an amino acid sequence having at least 85% sequence identity to the amino acid sequence shown in (a1), (a2), (a3), (a4), (a5) or (a6); and
    (2)所述轻链可变区包含选自如下组的LCDR1、LCDR2和LCDR3：(2) the light chain variable region comprises LCDR1, LCDR2 and LCDR3 selected from the following group:
    (b1)如SEQ ID NO:4、5和6所示的氨基酸序列；(b1) an amino acid sequence as shown in SEQ ID NO: 4, 5 and 6;
    (b2)如SEQ ID NO:7、5和6所示的氨基酸序列；(b2) amino acid sequences as shown in SEQ ID NO:7, 5 and 6;
    (b3)如SEQ ID NO:12、13和14所示的氨基酸序列；(b3) amino acid sequences as shown in SEQ ID NO: 12, 13 and 14;
    (b4)如SEQ ID NO:15、13和14所示的氨基酸序列；(b4) amino acid sequences as shown in SEQ ID NO: 15, 13 and 14;
    (b5)如SEQ ID NO:19、20和21所示的氨基酸序列；(b5) amino acid sequences as shown in SEQ ID NO: 19, 20 and 21;
    (b6)如SEQ ID NO:26、27和28所示的氨基酸序列；(b6) amino acid sequences as shown in SEQ ID NO:26, 27 and 28;
    (b7)与(b1)、(b2)、(b3)、(b4)、(b5)或(b6)所示的氨基酸序列具有至少85％序列同一性的氨基酸序列。(b7) An amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in (b1), (b2), (b3), (b4), (b5) or (b6).
  2. 如权利要求1所述的抗CRTAM抗体或其抗原结合片段，其具有：The anti-CRTAM antibody or antigen-binding fragment thereof according to claim 1, which has:
    所述HCDR1、HCDR2和HCDR3分别为SEQ ID NO:1、2和3或与SEQ ID NO:1、2和3所示的氨基酸序列具有至少85％序列同一性的氨基酸序列，和所述LCDR1、LCDR2和LCDR3分别为SEQ ID NO:4、5和6、SEQ ID NO:7、5和6或与SEQ ID NO:4、5和6或SEQ ID NO:7、5和6所示的氨基酸序列具有至少85％序列同一性的氨基酸序列；或The HCDR1, HCDR2 and HCDR3 are respectively SEQ ID NO: 1, 2 and 3 or an amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, 2 and 3, and the LCDR1, LCDR2 and LCDR3 are respectively SEQ ID NO: 4, 5 and 6, SEQ ID NO: 7, 5 and 6 or the amino acid sequence shown in SEQ ID NO: 4, 5 and 6 or SEQ ID NO: 7, 5 and 6 Amino acid sequences having at least 85% sequence identity; or
    所述HCDR1、HCDR2和HCDR3分别为SEQ ID NO:8、9和10、SEQ ID NO:8、11和10或与SEQ ID NO:8、9和10或SEQ ID NO:8、11和10所示的氨基 酸序列具有至少85％序列同一性的氨基酸序列，和所述LCDR1、LCDR2和LCDR3分别为SEQ ID NO:12、13和14、SEQ ID NO:15、13和14或与SEQ ID NO:12、13和14或SEQ ID NO:15、13和14所示的氨基酸序列具有至少85％序列同一性的氨基酸序列；或The HCDR1, HCDR2 and HCDR3 are respectively SEQ ID NO: 8, 9 and 10, SEQ ID NO: 8, 11 and 10 or with SEQ ID NO: 8, 9 and 10 or SEQ ID NO: 8, 11 and 10 An amino acid sequence having at least 85% sequence identity to the amino acid sequence shown, and said LCDR1, LCDR2 and LCDR3 are SEQ ID NO: 12, 13 and 14, SEQ ID NO: 15, 13 and 14, respectively, or with SEQ ID NO: 12, 13 and 14 or an amino acid sequence having at least 85% sequence identity to the amino acid sequence shown in SEQ ID NO: 15, 13 and 14; or
    所述HCDR1、HCDR2和HCDR3分别为SEQ ID NO:16、17和18或与SEQ ID NO:16、17和18所示的氨基酸序列具有至少85％序列同一性的氨基酸序列，和所述LCDR1、LCDR2和LCDR3分别为SEQ ID NO:19、20和21或与SEQ ID NO:19、20和21所示的氨基酸序列具有至少85％序列同一性的氨基酸序列；或所述HCDR1、HCDR2和HCDR3分别为SEQ ID NO:22、23和24、SEQ ID NO:22、25和24或与SEQ ID NO:22、23和24或SEQ ID NO:22、25和24所示的氨基酸序列具有至少85％序列同一性的氨基酸序列，和所述LCDR1、LCDR2和LCDR3分别为SEQ ID NO:26、27和28或与SEQ ID NO:26、27和28所示的氨基酸序列具有至少85％序列同一性的氨基酸序列。The HCDR1, HCDR2 and HCDR3 are respectively SEQ ID NO: 16, 17 and 18 or an amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 16, 17 and 18, and the LCDR1, LCDR2 and LCDR3 are respectively SEQ ID NO: 19, 20 and 21 or an amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 19, 20 and 21; or the HCDR1, HCDR2 and HCDR3 are respectively is SEQ ID NO:22, 23 and 24, SEQ ID NO:22, 25 and 24 or at least 85% identical to the amino acid sequence shown in SEQ ID NO:22, 23 and 24 or SEQ ID NO:22, 25 and 24 An amino acid sequence with sequence identity, and said LCDR1, LCDR2 and LCDR3 are SEQ ID NO: 26, 27 and 28 respectively or have at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 26, 27 and 28 amino acid sequence.
  3. 如权利要求1或2所述的抗CRTAM抗体或其抗原结合片段，其中：The anti-CRTAM antibody or antigen-binding fragment thereof as claimed in claim 1 or 2, wherein:
    所述重链可变区的氨基酸序列选自SEQ ID NO:80、SEQ ID NO:84、SEQ ID NO:85、SEQ ID NO:86和SEQ ID NO:87，SEQ ID NO:80、SEQ ID NO:84、SEQ ID NO:85、SEQ ID NO:86或SEQ ID NO:87经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:80、SEQ ID NO:84、SEQ ID NO:85、SEQ ID NO:86或SEQ ID NO:87功能相同的氨基酸序列，以及与SEQ ID NO:80、SEQ ID NO:84、SEQ ID NO:85、SEQ ID NO:86或SEQ ID NO:87具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列选自SEQ ID NO:81、SEQ ID NO:88、SEQ ID NO:89、SEQ ID NO:90、SEQ ID NO:91和SEQ ID NO:92，SEQ ID NO:81、SEQ ID NO:88、SEQ ID NO:89、SEQ ID NO:90、SEQ ID NO:91或SEQ ID NO:92经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:81、SEQ ID NO:88、SEQ ID NO:89、SEQ ID NO:90、SEQ ID NO:91或SEQ ID NO:92功能相同的氨基酸序列，以及与SEQ ID NO:81、SEQ ID NO:88、SEQ ID NO:89、SEQ ID NO:90、SEQ ID NO:91或SEQ ID NO:92具有至少85％序列同一性的氨基酸序列；或者The amino acid sequence of the heavy chain variable region is selected from SEQ ID NO:80, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86 and SEQ ID NO:87, SEQ ID NO:80, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86 or SEQ ID NO:87 obtained by substitution, deletion or addition of one or more amino acids and with SEQ ID NO:80, SEQ ID NO:84, SEQ ID An amino acid sequence functionally identical to NO:85, SEQ ID NO:86 or SEQ ID NO:87, and an amino acid sequence identical to SEQ ID NO:80, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86 or SEQ ID NO :87 has an amino acid sequence of at least 85% sequence identity, and the amino acid sequence of the light chain variable region is selected from the group consisting of SEQ ID NO:81, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91 and SEQ ID NO:92, SEQ ID NO:81, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91 or SEQ ID NO:92 substituted, Obtained by deletion or addition of one or more amino acids and having the same function as SEQ ID NO:81, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91 or SEQ ID NO:92 Amino acid sequences, and amino acid sequences having at least 85% sequence identity to SEQ ID NO:81, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91 or SEQ ID NO:92 ;or
    所述重链可变区的氨基酸序列选自SEQ ID NO:45、SEQ ID NO:106、SEQ ID NO:50、SEQ ID NO:51、SEQ ID NO:52，SEQ ID NO:53和SEQ ID NO:54，SEQ ID NO:45、SEQ ID NO:106、SEQ ID NO:50、SEQ ID NO:51、SEQ ID NO: 52，SEQ ID NO:53或SEQ ID NO:54经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:45、SEQ ID NO:106、SEQ ID NO:50、SEQ ID NO:51、SEQ ID NO:52，SEQ ID NO:53或SEQ ID NO:54功能相同的氨基酸序列，以及与SEQ ID NO:45、SEQ ID NO:106、SEQ ID NO:50、SEQ ID NO:51、SEQ ID NO:52，SEQ ID NO:53或SEQ ID NO:54具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列选自SEQ ID NO:46、SEQ ID NO:55、SEQ ID NO:56、SEQ ID NO:57、SEQ ID NO:58、SEQ ID NO:59、SEQ ID NO:60、SEQ ID NO:61、SEQ ID NO:62、SEQ ID NO:63、SEQ ID NO:64、SEQ ID NO:65、SEQ ID NO:66和SEQ ID NO:67，SEQ ID NO:46、SEQ ID NO:55、SEQ ID NO:56、SEQ ID NO:57、SEQ ID NO:58、SEQ ID NO:59、SEQ ID NO:60、SEQ ID NO:61、SEQ ID NO:62、SEQ ID NO:63、SEQ ID NO:64、SEQ ID NO:65、SEQ ID NO:66或SEQ ID NO:67经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:46、SEQ ID NO:55、SEQ ID NO:56、SEQ ID NO:57、SEQ ID NO:58、SEQ ID NO:59、SEQ ID NO:60、SEQ ID NO:61、SEQ ID NO:62、SEQ ID NO:63、SEQ ID NO:64、SEQ ID NO:65、SEQ ID NO:66或SEQ ID NO:67功能相同的氨基酸序列，以及与SEQ ID NO:46、SEQ ID NO:55、SEQ ID NO:56、SEQ ID NO:57、SEQ ID NO:58、SEQ ID NO:59、SEQ ID NO:60、SEQ ID NO:61、SEQ ID NO:62、SEQ ID NO:63、SEQ ID NO:64、SEQ ID NO:65、SEQ ID NO:66或SEQ ID NO:67具有至少85％序列同一性的氨基酸序列；或者The amino acid sequence of the heavy chain variable region is selected from SEQ ID NO:45, SEQ ID NO:106, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53 and SEQ ID NO:54, SEQ ID NO:45, SEQ ID NO:106, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53 or SEQ ID NO:54 substituted, missing or obtained by adding one or more amino acids and is compatible with SEQ ID NO:45, SEQ ID NO:106, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53 or SEQ ID NO: 54 functionally identical amino acid sequences, and SEQ ID NO:45, SEQ ID NO:106, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53 or SEQ ID NO:54 An amino acid sequence having at least 85% sequence identity, and the amino acid sequence of the light chain variable region is selected from the group consisting of SEQ ID NO:46, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO: 66 and SEQ ID NO:67, SEQ ID NO:46, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66 or SEQ ID NO: 67 are substituted, deleted or added with one or more amino acid obtained and with SEQ ID NO:46, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID The functionally identical amino acid sequence of NO:66 or SEQ ID NO:67, and SEQ ID NO:46, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO :59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, or SEQ ID NO:67 Amino acid sequences having at least 85% sequence identity; or
    所述重链可变区的氨基酸序列选自SEQ ID NO:68、SEQ ID NO:72、SEQ ID NO:73、SEQ ID NO:74和SEQ ID NO:75，SEQ ID NO:68、SEQ ID NO:72、SEQ ID NO:73、SEQ ID NO:74或SEQ ID NO:75经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:68、SEQ ID NO:72、SEQ ID NO:73、SEQ ID NO:74或SEQ ID NO:75功能相同的氨基酸序列，以及与SEQ ID NO:68、SEQ ID NO:72、SEQ ID NO:73、SEQ ID NO:74或SEQ ID NO:75具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列选自SEQ ID NO:69、SEQ ID NO:76、SEQ ID NO:77、SEQ ID NO:78和SEQ ID NO:79，SEQ ID NO:69、SEQ ID NO:76、SEQ ID NO:77、SEQ ID NO:78或SEQ ID NO:79经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:69、SEQ ID NO:76、SEQ ID NO:77、SEQ ID NO:78或SEQ ID NO:79功能相同的氨基酸序列，以及与SEQ ID NO:69、SEQ ID NO:76、SEQ ID NO:77、SEQ ID NO:78或SEQ ID NO: 79具有至少85％序列同一性的氨基酸序列；或者The amino acid sequence of the heavy chain variable region is selected from SEQ ID NO:68, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74 and SEQ ID NO:75, SEQ ID NO:68, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74 or SEQ ID NO:75 obtained by substitution, deletion or addition of one or more amino acids and with SEQ ID NO:68, SEQ ID NO:72, SEQ ID An amino acid sequence functionally identical to NO:73, SEQ ID NO:74 or SEQ ID NO:75, and an amino acid sequence identical to SEQ ID NO:68, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74 or SEQ ID NO : 75 amino acid sequences having at least 85% sequence identity, and the amino acid sequence of the light chain variable region is selected from the group consisting of SEQ ID NO: 69, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78 and SEQ ID NO:79, SEQ ID NO:69, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78 or SEQ ID NO:79 obtained by substitution, deletion or addition of one or more amino acids and with The functionally identical amino acid sequence of SEQ ID NO:69, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78 or SEQ ID NO:79, and SEQ ID NO:69, SEQ ID NO:76, SEQ ID NO:76, An amino acid sequence having at least 85% sequence identity to ID NO:77, SEQ ID NO:78 or SEQ ID NO:79; or
    所述重链可变区的氨基酸序列选自SEQ ID NO:93、SEQ ID NO:97、SEQ ID NO:98、SEQ ID NO:99、SEQ ID NO:100和SEQ ID NO:101，SEQ ID NO:93、SEQ ID NO:97、SEQ ID NO:98、SEQ ID NO:99、SEQ ID NO:100或SEQ ID NO:101经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:93、SEQ ID NO:97、SEQ ID NO:98、SEQ ID NO:99、SEQ ID NO:100或SEQ ID NO:101功能相同的氨基酸序列，以及与SEQ ID NO:93、SEQ ID NO:97、SEQ ID NO:98、SEQ ID NO:99、SEQ ID NO:100或SEQ ID NO:101具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列选自SEQ ID NO:94、SEQ ID NO:102、SEQ ID NO:103、SEQ ID NO:104和SEQ ID NO:105，SEQ ID NO:94、SEQ ID NO:102、SEQ ID NO:103、SEQ ID NO:104或SEQ ID NO:105经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:94、SEQ ID NO:102、SEQ ID NO:103、SEQ ID NO:104或SEQ ID NO:105功能相同的氨基酸序列，以及与SEQ ID NO:94、SEQ ID NO:102、SEQ ID NO:103、SEQ ID NO:104或SEQ ID NO:105具有至少85％序列同一性的氨基酸序列。The amino acid sequence of the heavy chain variable region is selected from SEQ ID NO:93, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100 and SEQ ID NO:101, SEQ ID NO: 93, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100 or SEQ ID NO: 101 obtained by substitution, deletion or addition of one or more amino acids and identical to SEQ ID The functionally identical amino acid sequence of NO:93, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100 or SEQ ID NO:101, and SEQ ID NO:93, SEQ ID NO :97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100 or SEQ ID NO:101 has an amino acid sequence of at least 85% sequence identity, and the amino acid sequence of the light chain variable region is selected from SEQ ID NO:94, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104 and SEQ ID NO:105, SEQ ID NO:94, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104 or SEQ ID NO:105 obtained by substitution, deletion or addition of one or more amino acids and with SEQ ID NO:94, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104 or SEQ ID A functionally identical amino acid sequence of NO:105, and an amino acid sequence having at least 85% sequence identity to SEQ ID NO:94, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104 or SEQ ID NO:105 .
  4. 如权利要求1-3之任一项所述的抗CRTAM抗体或其抗原结合片段，其中所述重链可变区的氨基酸序列为SEQ ID NO:80，SEQ ID NO:80经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:80功能相同的氨基酸序列或与SEQ ID NO:80具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:81，SEQ ID NO:81经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:81功能相同的氨基酸序列或与SEQ ID NO:81具有至少85％序列同一性的氨基酸序列；The anti-CRTAM antibody or antigen-binding fragment thereof according to any one of claims 1-3, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 80, and SEQ ID NO: 80 is substituted, deleted or Adding one or more amino acids to obtain an amino acid sequence with the same function as SEQ ID NO: 80 or an amino acid sequence with at least 85% sequence identity to SEQ ID NO: 80, and the amino acid sequence of the light chain variable region is SEQ ID NO:81, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids of SEQ ID NO:81 and having the same function as SEQ ID NO:81 or having at least 85% sequence identity with SEQ ID NO:81 amino acid sequence;
    所述重链可变区的氨基酸序列为SEQ ID NO:87，SEQ ID NO:87经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:87功能相同的氨基酸序列或与SEQ ID NO:87具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的氨基酸序列为SEQ ID NO:90，SEQ ID NO:90经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:90功能相同的氨基酸序列或与SEQ ID NO:90具有至少85％序列同一性的氨基酸序列；或者The amino acid sequence of the heavy chain variable region is SEQ ID NO: 87, the amino acid sequence obtained by replacing, deleting or adding one or more amino acids of SEQ ID NO: 87 and having the same function as SEQ ID NO: 87 or the amino acid sequence with SEQ ID NO: 87 ID NO:87 has an amino acid sequence of at least 85% sequence identity, and the amino acid sequence of the light chain variable region is SEQ ID NO:90, and SEQ ID NO:90 is obtained by substitution, deletion or addition of one or more amino acids an amino acid sequence that is functionally identical to SEQ ID NO:90 or has at least 85% sequence identity to SEQ ID NO:90; or
    所述重链可变区的氨基酸序列为SEQ ID NO:87，SEQ ID NO:87经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:87功能相同的氨基酸序列或与SEQ ID NO:87具有至少85％序列同一性的氨基酸序列，且所述轻链可变区的 氨基酸序列为SEQ ID NO:92，SEQ ID NO:92经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO:92功能相同的氨基酸序列或与SEQ ID NO:92具有至少85％序列同一性的氨基酸序列。The amino acid sequence of the heavy chain variable region is SEQ ID NO: 87, the amino acid sequence obtained by replacing, deleting or adding one or more amino acids of SEQ ID NO: 87 and having the same function as SEQ ID NO: 87 or the amino acid sequence with SEQ ID NO: 87 ID NO:87 has an amino acid sequence of at least 85% sequence identity, and the amino acid sequence of the light chain variable region is SEQ ID NO:92, and SEQ ID NO:92 is obtained by substitution, deletion or addition of one or more amino acids An amino acid sequence that is functionally identical to SEQ ID NO:92 or an amino acid sequence that has at least 85% sequence identity to SEQ ID NO:92.
  5. 如权利要求1-4之任一项所述的抗CRTAM抗体或其抗原结合片段，其中所述抗体是鼠源单克隆抗体、嵌合抗体、人源化抗体、双特异性抗体或完全人抗体。The anti-CRTAM antibody or antigen-binding fragment thereof according to any one of claims 1-4, wherein the antibody is a murine monoclonal antibody, a chimeric antibody, a humanized antibody, a bispecific antibody or a fully human antibody .
  6. 一种分离的核酸，其编码如权利要求1-5之任一项所述的抗CRTAM抗体或其抗原结合片段。An isolated nucleic acid encoding the anti-CRTAM antibody or antigen-binding fragment thereof of any one of claims 1-5.
  7. 如权利要求6所述的核酸，其包含：The nucleic acid of claim 6, comprising:
    编码重链可变区如SEQ ID NO:80、SEQ ID NO:84、SEQ ID NO:85、SEQ ID NO:86或SEQ ID NO:87的核苷酸序列；和编码轻链可变区如SEQ ID NO:81、SEQ ID NO:88、SEQ ID NO:89、SEQ ID NO:90、SEQ ID NO:91或SEQ ID NO:92的核苷酸序列；或者A nucleotide sequence encoding a heavy chain variable region such as SEQ ID NO:80, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86 or SEQ ID NO:87; and a light chain variable region such as the nucleotide sequence of SEQ ID NO:81, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91 or SEQ ID NO:92; or
    编码重链可变区如SEQ ID NO:45、SEQ ID NO:106、SEQ ID NO:50、SEQ ID NO:51、SEQ ID NO:52，SEQ ID NO:53或SEQ ID NO:54的核苷酸序列；和编码轻链可变区如SEQ ID NO:46、SEQ ID NO:55、SEQ ID NO:56、SEQ ID NO:57、SEQ ID NO:58、SEQ ID NO:59、SEQ ID NO:60、SEQ ID NO:61、SEQ ID NO:62、SEQ ID NO:63、SEQ ID NO:64、SEQ ID NO:65、SEQ ID NO:66或SEQ ID NO:67的核苷酸序列；或者A nucleus encoding a heavy chain variable region such as SEQ ID NO:45, SEQ ID NO:106, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53 or SEQ ID NO:54 Nucleotide sequence; and coding light chain variable region such as SEQ ID NO:46, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID The nucleotide sequence of NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66 or SEQ ID NO:67 ;or
    编码重链可变区如SEQ ID NO:68、SEQ ID NO:72、SEQ ID NO:73、SEQ ID NO:74或SEQ ID NO:75的核苷酸序列；和编码轻链可变区如SEQ ID NO:69、SEQ ID NO:76、SEQ ID NO:77、SEQ ID NO:78或SEQ ID NO:79的核苷酸序列；或者A nucleotide sequence encoding a heavy chain variable region such as SEQ ID NO:68, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74 or SEQ ID NO:75; and a light chain variable region such as the nucleotide sequence of SEQ ID NO:69, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78 or SEQ ID NO:79; or
    编码重链可变区如SEQ ID NO:93、SEQ ID NO:97、SEQ ID NO:98、SEQ ID NO:99、SEQ ID NO:100或SEQ ID NO:101的核苷酸序列；和编码轻链可变区如SEQ ID NO:94、SEQ ID NO:102、SEQ ID NO:103、SEQ ID NO:104或SEQ ID NO:105的核苷酸序列。A nucleotide sequence encoding a heavy chain variable region such as SEQ ID NO:93, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100 or SEQ ID NO:101; and encoding A light chain variable region such as the nucleotide sequence of SEQ ID NO:94, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104 or SEQ ID NO:105.
  8. 一种表达载体，其包含如权利要求6或7所述的核酸。An expression vector comprising the nucleic acid according to claim 6 or 7.
  9. 一种宿主细胞，其转化如权利要求8所述的表达载体，所述宿主细胞选 自原核细胞和真核细胞，优先为哺乳动物细胞。A host cell, which transforms the expression vector according to claim 8, said host cell is selected from prokaryotic cells and eukaryotic cells, preferably mammalian cells.
  10. 制备权利要求1-5之任一项所述的抗CRTAM抗体或其抗原结合片段的方法，包括在如权利要求9所述的宿主细胞中表达抗体或其抗原结合片段，以及从宿主细胞中分离所述抗体或其抗原结合片段的步骤。The method for preparing the anti-CRTAM antibody or its antigen-binding fragment according to any one of claims 1-5, comprising expressing the antibody or its antigen-binding fragment in the host cell as claimed in claim 9, and isolating from the host cell The step of said antibody or antigen-binding fragment thereof.
  11. 一种药物组合物，其包含权利要求1-5之任一项所述的抗CRTAM抗体或其抗原结合片段和药学可接受的载体。A pharmaceutical composition comprising the anti-CRTAM antibody or antigen-binding fragment thereof according to any one of claims 1-5 and a pharmaceutically acceptable carrier.
  12. 如权利要求1-5之任一项所述的抗CRTAM抗体或其抗原结合片段或如权利要求11的药物组合物在制备预防和/或***的药物中的应用。Use of the anti-CRTAM antibody or antigen-binding fragment thereof according to any one of claims 1-5 or the pharmaceutical composition according to claim 11 in the preparation of drugs for preventing and/or treating tumors.
PCT/CN2022/102253 2021-06-30 2022-06-29 Anti-crtam antibody and application thereof WO2023274286A1 (en)

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Citations (5)

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US5686257A (en) * 1995-04-26 1997-11-11 Schering Corporation Binding compositions for mammalian T cell antigens and related reagents
US20050058642A1 (en) * 2003-07-25 2005-03-17 Galibert Laurent J. Antagonists and agonists of LDCAM and methods of use
CN101854949A (en) * 2007-08-30 2010-10-06 健泰科生物技术公司 Methods and compositions for modulating t cells
WO2016154341A1 (en) * 2015-03-23 2016-09-29 The Board Of Trustees Of The Leland Stanford Junior University Medical uses of crtam agonists
WO2019086878A1 (en) * 2017-11-02 2019-05-09 Oxford Biotherapeutics Ltd Antibodies and methods of use

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5686257A (en) * 1995-04-26 1997-11-11 Schering Corporation Binding compositions for mammalian T cell antigens and related reagents
US20050058642A1 (en) * 2003-07-25 2005-03-17 Galibert Laurent J. Antagonists and agonists of LDCAM and methods of use
CN101854949A (en) * 2007-08-30 2010-10-06 健泰科生物技术公司 Methods and compositions for modulating t cells
WO2016154341A1 (en) * 2015-03-23 2016-09-29 The Board Of Trustees Of The Leland Stanford Junior University Medical uses of crtam agonists
WO2019086878A1 (en) * 2017-11-02 2019-05-09 Oxford Biotherapeutics Ltd Antibodies and methods of use

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