WO2021147954A1 - Semg2抗体及其用途 - Google Patents

Semg2抗体及其用途 Download PDF

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WO2021147954A1
WO2021147954A1 PCT/CN2021/073100 CN2021073100W WO2021147954A1 WO 2021147954 A1 WO2021147954 A1 WO 2021147954A1 CN 2021073100 W CN2021073100 W CN 2021073100W WO 2021147954 A1 WO2021147954 A1 WO 2021147954A1
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amino acid
acid sequence
seq
sequence shown
semg2
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PCT/CN2021/073100
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English (en)
French (fr)
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李兆利
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上海柏全生物科技有限公司
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Priority to JP2022544425A priority Critical patent/JP2023511189A/ja
Priority to CN202180003362.4A priority patent/CN113939530A/zh
Priority to KR1020227023643A priority patent/KR20220131233A/ko
Priority to US17/794,598 priority patent/US20230080534A1/en
Priority to EP21744026.2A priority patent/EP4079758A4/en
Priority to AU2021209740A priority patent/AU2021209740A1/en
Priority to CA3161701A priority patent/CA3161701A1/en
Publication of WO2021147954A1 publication Critical patent/WO2021147954A1/zh

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    • 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
    • C07K16/2875—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF/TNF superfamily, e.g. CD70, CD95L, CD153, CD154
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    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
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    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
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    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids

Definitions

  • the invention relates to the field of biomedicine, in particular to a SEMG2 epitope peptide and its use.
  • the CD27 molecule belongs to the tumor necrosis factor receptor (TNFR) superfamily. It is a type I membrane protein with a molecular weight of about 55kDa, and exists as a dimer of two monomers connected by disulfide bonds. CD27 is mainly expressed on lymphocytes. Recent studies based on CD27 knockout mice have shown that activation of the CD27 signaling pathway can increase the infiltration of suppressor T cells (Treg) in solid tumors and reduce anti-tumor immunity (Claus C, Riether C, Schürch C, Matter MS, Hilmenyuk T, Ochsenbein AF. Cancer Res. 2012 Jul 15; 72(14): 3664-76).
  • Treg suppressor T cells
  • Treg cells in the skin tissues lose the expression of CD27 and cannot perform normal immune regulation functions (Remedios KA, Zirak B, Sandoval PM, Lowe MM, Boda D, Henley E, etc., Sci Immunol. 2018 Dec 21; 3(30).pii:eaau2042).
  • the activation of CD27 can increase the number of Tregs and reduce atherosclerosis in hyperlipidemia mice (Winkels H, Meiler S, Lievens D, Engel D, Spitz C,sum C, etc., Eur Heart J. 2017; 38(48):3590-3599).
  • CD70 is a 193 amino acid polypeptide with a hydrophilic N-terminal domain of 20 amino acids and a C-terminal domain containing two potential N-linked glycosylation sites. It belongs to the TNF family (Goodwin, RG et al. (1993) ) Cell 73: 447-56; Bowman et al. (1994) Immunol 152: 1756-61). These characteristics indicate that CD70 is a type II transmembrane protein with an extracellular C-terminal part. CD70 is transiently present on activated T and B lymphocytes and dendritic cells (Hintzen et al.
  • CD70 has been reported in different types of cancers, including renal cell carcinoma, metastatic breast cancer, brain tumors, leukemia, lymphoma, and nasopharyngeal carcinoma (Junker et al., J Urol. 2005; 173: 2150-3; Sloan et al., Am J Pathol. 2004; 164: 315-23; Held-Feindt and Mentlein, etc., Int J Cancer 2002; 98: 352-6).
  • CD70 and CD27 is a tumor immunotherapy strategy being studied.
  • the present invention aims to develop new anti-tumor treatments and drugs.
  • the present invention discloses a compound that agonizes or antagonizes the interaction between SEMG2 and CD27.
  • the amino acid positions at which the SEMG2 and CD27 interact are located at positions 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, and 508 of SEMG2, and the amino acid sequence of the SEMG2 protein is as shown in SEQ ID NO:1 is shown.
  • the compound is a small molecule inhibitor, polypeptide, antibody or antigen-binding fragment.
  • the present invention discloses a polypeptide which has or includes SEQ ID NO: 2 (QIEKLVEGKS), SEQ ID NO: 86 (QIEKLVEGKS(x)I(x)), SEQ ID NO: 87 (QIEKLVEGKS(x)I), or the amino acid sequence shown in the amino acid sequence shown in SEQ ID NO: 88 (QIEKLVEGKS(x)), preferably the polypeptide comprises SEQ ID NO: 3 (QIEKLVEGKSQIQ), SEQ ID NO: 4 (QIEKLVEGKSQ), or SEQ ID NO: 5 (QIEKLVEGKSQI), or an amino acid sequence that is at least 90% identical to any one of SEQ ID NO: 2-5.
  • the polypeptide is a polypeptide that stimulates the interaction between SEMG2 and CD27.
  • the amino acid positions of the interaction between SEMG2 and CD27 are located at positions 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, and 508 of SEMG2, and the amino acid sequence of the SEMG2 protein is as shown in SEQ ID NO:1 shows.
  • the present invention discloses an antibody capable of specifically binding to natural or mutant SEMG2 protein, said antibody binding to an epitope peptide derived from SEMG2 protein, said epitope peptide comprising SEQ ID NO: 2 (QIEKLVEGKS), SEQ ID NO: 3 (QIEKLVEGKSQIQ), SEQ ID NO: 4 (QIEKLVEGKSQ), or SEQ ID NO: 5 (QIEKLVEGKSQI).
  • the antibody is an antibody that antagonizes the interaction between SEMG2 and CD27.
  • the present invention discloses an antibody that can specifically bind to natural or mutant SEMG2 protein, and the antibody can recognize the 497, 498, 499, 500, 501, 502, 503, 504, At least one amino acid residue in positions 505, 506, and 508 or an amino acid residue identifying the corresponding position of the mutant SEMG2 protein, the amino acid sequence of the natural SEMG2 protein is shown in SEQ ID NO:1.
  • the antibody is an antibody that antagonizes the interaction between SEMG2 and CD27.
  • the present invention discloses an antibody capable of specifically binding to natural or mutant SEMG2 protein, wherein the antibody comprises a heavy chain variable region of HCDR1, HCDR2 and HCDR3 defined by IMGT; and comprising a heavy chain variable region according to IMGT
  • IMGT heavy chain variable region
  • the amino acid sequence of the HCDR1 has or includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 6-11, SEQ ID NOs: 60-61, and SEQ ID NO: 76;
  • the amino acid sequence of the HCDR2 has or includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 12-16 and SEQ ID NOs: 62-64;
  • the amino acid sequence of the HCDR3 includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 17-20, SEQ ID NOs: 65-67, and SEQ ID NOs: 77-81;
  • the amino acid sequence of the LCDR1 includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 21-25, SEQ ID NOs: 68-70, and SEQ ID NO: 82;
  • the amino acid sequence of the LCDR2 includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 26-29, SEQ ID NOs: 71-72, SEQ ID NOs: 83-84, and SEQ ID NO: 28;
  • the amino acid sequence of the LCDR3 includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 30-34, SEQ ID NOs: 73-75, SEQ ID NO: 85 and SEQ ID NO: 99.
  • the CDR sequence in the antibody is selected from any one of the combinations (a)-(k):
  • the amino acid sequence of HCDR1 includes the amino acid sequence shown in SEQ ID NO: 6; the amino acid sequence of HCDR2 includes the amino acid sequence shown in SEQ ID NO: 12; the amino acid sequence of HCDR3 includes the amino acid sequence shown in SEQ ID NO: The amino acid sequence shown in: 17; the amino acid sequence of LCDR1 includes the amino acid sequence shown in SEQ ID NO: 21; the amino acid sequence of LCDR2 includes the amino acid sequence shown in SEQ ID NO: 26; the amino acid sequence of LCDR3 includes The amino acid sequence shown in SEQ ID NO: 30;
  • the amino acid sequence of the HCDR1 includes the amino acid sequence shown in SEQ ID NO: 7; the amino acid sequence of the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 13; the amino acid sequence of the HCDR3 includes the amino acid sequence shown in SEQ ID NO: The amino acid sequence shown in: 18; the amino acid sequence of LCDR1 includes the amino acid sequence shown in SEQ ID NO: 22; the amino acid sequence of LCDR2 includes the amino acid sequence shown in SEQ ID NO: 27; the amino acid sequence of LCDR3 includes The amino acid sequence shown in SEQ ID NO: 31 or SEQ ID NO: 99;
  • the amino acid sequence of the HCDR1 includes the amino acid sequence shown in SEQ ID NO: 6; the amino acid sequence of the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 16; the amino acid sequence of the HCDR3 includes the amino acid sequence shown in SEQ ID NO The amino acid sequence shown in: 17; the amino acid sequence of LCDR1 includes the amino acid sequence shown in SEQ ID NO: 21; the amino acid sequence of LCDR2 includes the amino acid sequence shown in SEQ ID NO: 26; the amino acid sequence of LCDR3 includes The amino acid sequence shown in SEQ ID NO: 30;
  • the amino acid sequence of HCDR1 includes the amino acid sequence shown in SEQ ID NO: 8; the amino acid sequence of HCDR2 includes the amino acid sequence shown in SEQ ID NO: 13; the amino acid sequence of HCDR3 includes the amino acid sequence shown in SEQ ID NO: The amino acid sequence shown in: 18; the amino acid sequence of LCDR1 includes the amino acid sequence shown in SEQ ID NO: 23; the amino acid sequence of LCDR2 includes the amino acid sequence shown in SEQ ID NO: 27; the amino acid sequence of LCDR3 includes The amino acid sequence shown in SEQ ID NO: 32;
  • the amino acid sequence of the HCDR1 includes the amino acid sequence shown in SEQ ID NO: 9; the amino acid sequence of the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 14; the amino acid sequence of the HCDR3 includes the amino acid sequence shown in SEQ ID NO: The amino acid sequence shown in: 19; the amino acid sequence of LCDR1 includes the amino acid sequence shown in SEQ ID NO: 24; the amino acid sequence of LCDR2 includes the amino acid sequence shown in SEQ ID NO: 28; the amino acid sequence of LCDR3 includes The amino acid sequence shown in SEQ ID NO: 33;
  • the amino acid sequence of the HCDR1 includes the amino acid sequence shown in SEQ ID NO: 10; the amino acid sequence of the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 15; the amino acid sequence of the HCDR3 includes the amino acid sequence shown in SEQ ID NO: The amino acid sequence shown in: 20; the amino acid sequence of LCDR1 includes the amino acid sequence shown in SEQ ID NO: 25; the amino acid sequence of LCDR2 includes the amino acid sequence shown in SEQ ID NO: 29; the amino acid sequence of LCDR3 includes The amino acid sequence shown in SEQ ID NO: 34;
  • the amino acid sequence of the HCDR1 includes the amino acid sequence shown in SEQ ID NO: 11; the amino acid sequence of the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 15; the amino acid sequence of the HCDR3 includes the amino acid sequence shown in SEQ ID NO: The amino acid sequence shown in: 20; the amino acid sequence of LCDR1 includes the amino acid sequence shown in SEQ ID NO: 25; the amino acid sequence of LCDR2 includes the amino acid sequence shown in SEQ ID NO: 29; the amino acid sequence of LCDR3 includes The amino acid sequence shown in SEQ ID NO: 34;
  • the amino acid sequence of the HCDR1 includes the amino acid sequence shown in SEQ ID NO: 60; the amino acid sequence of the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 62; the amino acid sequence of the HCDR3 includes the amino acid sequence shown in SEQ ID NO: The amino acid sequence shown in: 65; the amino acid sequence of LCDR1 includes the amino acid sequence shown in SEQ ID NO: 68; the amino acid sequence of LCDR2 includes the amino acid sequence shown in SEQ ID NO: 71; the amino acid sequence of LCDR3 includes The amino acid sequence shown in SEQ ID NO: 73;
  • the amino acid sequence of the HCDR1 includes the amino acid sequence shown in SEQ ID NO: 61; the amino acid sequence of the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 63; the amino acid sequence of the HCDR3 includes the amino acid sequence shown in SEQ ID NO: The amino acid sequence shown in: 66; the amino acid sequence of LCDR1 includes the amino acid sequence shown in SEQ ID NO: 69; the amino acid sequence of LCDR2 includes the amino acid sequence shown in SEQ ID NO: 72; the amino acid sequence of LCDR3 includes The amino acid sequence shown in SEQ ID NO: 74;
  • the amino acid sequence of the HCDR1 includes the amino acid sequence shown in SEQ ID NO: 60; the amino acid sequence of the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 64; the amino acid sequence of the HCDR3 includes the amino acid sequence shown in SEQ ID NO: The amino acid sequence shown in: 67; the amino acid sequence of LCDR1 includes the amino acid sequence shown in SEQ ID NO: 70; the amino acid sequence of LCDR2 includes the amino acid sequence shown in SEQ ID NO: 28; the amino acid sequence of LCDR3 includes The amino acid sequence shown in SEQ ID NO: 75;
  • the amino acid sequence of the HCDR1 includes the amino acid sequence shown in SEQ ID NO: 60 or 76; the amino acid sequence of the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 64 or 62; the amino acid sequence of the HCDR3 includes The amino acid sequence shown in SEQ ID NO: 77, 78 or 79; and/or
  • the amino acid sequence of the LCDR1 includes the amino acid sequence shown in SEQ ID NO: 70 or 82; the amino acid sequence of the LCDR2 includes the amino acid sequence shown in SEQ ID NO: 28, 83 or 84; the amino acid sequence of the LCDR3 includes The amino acid sequence shown in SEQ ID NO: 75 or 85.
  • the present invention discloses an antibody capable of specifically binding to natural or mutant SEMG2 protein, wherein the antibody comprises a heavy chain variable region and a light chain variable region,
  • the amino acid sequence of the variable region of the heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 35-41, 48-51, 54-56 and 96-100 or has at least 70% of the sequence in the group. , 80%, 90%, 95% or 99% sequence identity;
  • the amino acid sequence of the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 42-47, 52-53, 57-69 and 101-103 or has at least 70% of the sequence in the group. , 80%, 90%, 95%, or 99% sequence identity.
  • variable region of the heavy chain and the variable region of the light chain are selected from any one of (a)-(o) combinations:
  • the amino acid sequence of the variable region of the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 35 or has at least 70%, 80%, 90%, 95% or 99% sequence identity with the sequence;
  • the amino acid sequence of the chain variable region includes the amino acid sequence shown in SEQ ID NO: 42 or has at least 70%, 80%, 90%, 95% or 99% sequence identity with the sequence;
  • the amino acid sequence of the variable region of the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 36 or has at least 70%, 80%, 90%, 95% or 99% sequence identity with the sequence;
  • the amino acid sequence of the chain variable region includes the amino acid sequence shown in SEQ ID NO: 43 or has at least 70%, 80%, 90%, 95% or 99% sequence identity with the sequence;
  • the amino acid sequence of the variable region of the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 37 or has at least 70%, 80%, 90%, 95% or 99% sequence identity with the sequence;
  • the amino acid sequence of the chain variable region includes the amino acid sequence shown in SEQ ID NO: 44 or has at least 70%, 80%, 90%, 95% or 99% sequence identity with the sequence;
  • the amino acid sequence of the variable region of the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 38 or has at least 70%, 80%, 90%, 95% or 99% sequence identity with the sequence;
  • the amino acid sequence of the chain variable region includes the amino acid sequence shown in SEQ ID NO: 45 or has at least 70%, 80%, 90%, 95%, or 99% sequence identity with the sequence;
  • the amino acid sequence of the variable region of the heavy chain includes the amino acid sequence shown in SEQ ID NO: 39 or has at least 70%, 80%, 90%, 95%, or 99% sequence identity with the sequence;
  • the amino acid sequence of the chain variable region includes the amino acid sequence shown in SEQ ID NO: 46 or has at least 70%, 80%, 90%, 95% or 99% sequence identity with the sequence;
  • the amino acid sequence of the variable region of the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 40 or has at least 70%, 80%, 90%, 95% or 99% sequence identity with the sequence;
  • the amino acid sequence of the chain variable region includes the amino acid sequence shown in SEQ ID NO: 47 or has at least 70%, 80%, 90%, 95% or 99% sequence identity with the sequence;
  • the amino acid sequence of the variable region of the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 41 or has at least 70%, 80%, 90%, 95% or 99% sequence identity with the sequence;
  • the amino acid sequence of the chain variable region includes the amino acid sequence shown in SEQ ID NO: 47 or has at least 70%, 80%, 90%, 95% or 99% sequence identity with the sequence;
  • the amino acid sequence of the variable region of the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 48, 49, 50, or 51 or has at least 70%, 80%, 90%, 95% or 99% of the sequence thereof Sequence identity;
  • the amino acid sequence of the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 52 or 53, or has at least 70%, 80%, 90%, 95% or 99% sequence identity with the sequence ;
  • the amino acid sequence of the variable region of the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 54 or has at least 70%, 80%, 90%, 95% or 99% sequence identity with the sequence;
  • the amino acid sequence of the chain variable region includes the amino acid sequence shown in SEQ ID NO: 57 or has at least 70%, 80%, 90%, 95%, or 99% sequence identity with the sequence;
  • the amino acid sequence of the variable region of the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 55 or has at least 70%, 80%, 90%, 95% or 99% sequence identity with the sequence;
  • the amino acid sequence of the chain variable region includes the amino acid sequence shown in SEQ ID NO: 58 or has at least 70%, 80%, 90%, 95%, or 99% sequence identity with the sequence;
  • the amino acid sequence of the variable region of the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 56 or has at least 70%, 80%, 90%, 95% or 99% sequence identity with the sequence;
  • the amino acid sequence of the chain variable region includes the amino acid sequence shown in SEQ ID NO: 59 or has at least 70%, 80%, 90%, 95% or 99% sequence identity with the sequence;
  • the amino acid sequence of the variable region of the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 96 or has at least 70%, 80%, 90%, 95% or 99% sequence identity with the sequence;
  • the amino acid sequence of the chain variable region includes the amino acid sequence shown in SEQ ID NO: 59, 101, 102 or 103 or has at least 70%, 80%, 90%, 95% or 99% sequence identity with the sequence;
  • the amino acid sequence of the variable region of the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 97 or has at least 70%, 80%, 90%, 95% or 99% sequence identity with the sequence;
  • the amino acid sequence of the chain variable region includes the amino acid sequence shown in SEQ ID NO: 59 or has at least 70%, 80%, 90%, 95%, or 99% sequence identity with the sequence;
  • the amino acid sequence of the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 98 or has at least 70%, 80%, 90%, 95% or 99% sequence identity with the sequence;
  • the amino acid sequence of the chain variable region includes the amino acid sequence shown in SEQ ID NO: 103 or has at least 70%, 80%, 90%, 95%, or 99% sequence identity with the sequence;
  • the amino acid sequence of the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 99 or 100 or has at least 70%, 80%, 90%, 95% or 99% sequence identity with the sequence; so
  • the amino acid sequence of the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 57 or has at least 70%, 80%, 90%, 95%, or 99% sequence identity with the sequence.
  • the antibody of the present invention may further comprise a coupling portion connected to a polypeptide, the coupling portion selected from radionuclides, drugs, toxins, cytokines, enzymes, luciferin, carrier proteins, lipids, and biotin.
  • a coupling portion connected to a polypeptide, the coupling portion selected from radionuclides, drugs, toxins, cytokines, enzymes, luciferin, carrier proteins, lipids, and biotin.
  • the linker is a peptide or a polypeptide.
  • the antibody is selected from monoclonal antibody, polyclonal antibody, antiserum, chimeric antibody, humanized antibody and human antibody.
  • the antibody is selected from the group consisting of multispecific antibodies, single chain Fv (scFv), single chain antibodies, anti-idiotypic (anti-Id) antibodies, diabodies, minibodies, nanobodies, single domain antibodies, Fab fragments, F (ab') Fragment, disulfide-linked bispecific Fv (sdFv) and intracellular antibody.
  • the present invention discloses an epitope peptide, wherein the epitope peptide is derived from the SEMG2 protein, and the amino acid of the epitope peptide comprises or has an amino acid selected from SEQ ID NO: 2 (QIEKLVEGKS), The amino acid sequence in the group consisting of SEQ ID NO: 3 (QIEKLVEGKSQIQ), SEQ ID NO: 4 (QIEKLVEGKSQ), and SEQ ID NO: 5 (QIEKLVEGKSQI).
  • the present invention discloses a protein whose amino acid sequence comprises SEQ ID NO: 2 (QIEKLVEGKS), SEQ ID NO: 86 (QIEKLVEGKS(x)I(x)), SEQ ID NO: 87 (QIEKLVEGKS(x) ) 1), or the amino acid sequence shown in the amino acid sequence shown in SEQ ID NO: 88 (QIEKLVEGKS(x)), preferably the polypeptide comprises SEQ ID NO: 3 (QIEKLVEGKSQIQ), SEQ ID NO: 4 (QIEKLVEGKSQ), Or SEQ ID NO: 5 (QIEKLVEGKSQI) or an amino acid sequence that is at least 90% identical to any one of SEQ ID NO: 2-5, more preferably SEQ ID NO: 89-94 and SEQ ID NO: 3( Corresponding to P1-P6, and P7) respectively; and N-terminal or C-terminal can choose to connect the tag sequence.
  • SEQ ID NO: 2 QIEKLVEGKS
  • tag protein includes but is not limited to C-Myc, His, GST (glutathione sulfhydryl transferase), HA , MBP (maltose binding protein), Flag, SUMO, eGFP/eCFP/eYFP/mCherry, etc.
  • polypeptide has the amino acid sequence shown in SEQ ID NO: 3 (P7: QIEKLVEGKSQIQ) or SEQ ID NO: 93 (P5).
  • the present invention also discloses a method for preparing antibodies or antigen-binding fragments thereof, wherein a protein is used as an immunogen to inject a subject such as a mouse or to screen a natural library to prepare an antibody, and the amino acid sequence of the protein comprises SEQ ID NO: 2(QIEKLVEGKS), SEQ ID NO: 86 (QIEKLVEGKS(x)I(x)), SEQ ID NO: 87(QIEKLVEGKS(x)I), or SEQ ID NO: 88(QIEKLVEGKS(x)) shown in the amino acid sequence
  • the amino acid sequence shown, preferably the polypeptide comprises SEQ ID NO: 3 (QIEKLVEGKSQIQ), SEQ ID NO: 4 (QIEKLVEGKSQ), or SEQ ID NO: 5 (QIEKLVEGKSQI) or any of SEQ ID NO: 2-5
  • the amino acid sequence with at least 90% or more sequence identity of the item sequence is more preferably as shown in SEQ ID NO: 93 (P5) or
  • a method of using SEMG2 and CD27 binding key epitope polypeptides as immunogens to obtain isolated antibodies through screening methods of murine hybridomas, phage display human origin and camel origin natural libraries is disclosed.
  • the present invention also discloses an isolated polynucleotide, which encodes the compound, antigenic peptide, or protein as described above.
  • the present invention discloses a recombinant vector, which comprises the polynucleotide and optional regulatory sequences; preferably, the recombinant vector is a cloning vector or an expression vector.
  • control sequence is selected from a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, a signal sequence, a transcription terminator, or any combination thereof.
  • the present invention discloses a host cell, which contains the aforementioned recombinant vector.
  • the host cell is a prokaryotic cell or a eukaryotic cell.
  • the present invention discloses a pharmaceutical composition, which comprises the aforementioned compound, the aforementioned antigen peptide, the aforementioned protein, the aforementioned polynucleotide, and the aforementioned recombinant vector , And one or more of the aforementioned host cells.
  • composition further includes a pharmaceutically acceptable carrier or excipient.
  • the present invention also discloses the aforementioned compound, the aforementioned antigen peptide, the aforementioned protein, the aforementioned polynucleotide, the aforementioned recombinant vector, or the aforementioned aforementioned
  • the present invention also discloses the aforementioned compound, the aforementioned antigen peptide, the aforementioned protein, the aforementioned polynucleotide, the aforementioned recombinant vector, or the aforementioned aforementioned
  • the host cell is used in the preparation of drugs for preventing or treating tumors or drugs for regulating the immune response caused by tumors.
  • the tumor is selected from colorectal cancer, lung cancer, melanoma, lymphoma, liver cancer, head and neck cancer, stomach cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, endometrial cancer, breast cancer
  • the tumor is selected from colorectal cancer, lung cancer, melanoma, lymphoma, liver cancer, head and neck cancer, stomach cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, endometrial cancer, breast cancer
  • the group consisting of cancer and ovarian cancer is selected from colorectal cancer, lung cancer, melanoma, lymphoma, liver cancer, head and neck cancer, stomach cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, endometrial cancer, breast cancer
  • the present invention also discloses a method for screening drugs or reagents for preventing or treating tumors, by screening inhibitors or antibodies that inhibit the interaction of SEMG2 and CD27, candidate drugs or reagents are obtained.
  • the invention also discloses a method for preventing or treating tumors, which includes:
  • the subject’s immune cells such as lymphocytes (T lymphocytes) or tumor cells are brought into contact with an effective dose of the compound as described in any one of the preceding items; wherein an effective amount of the compound is used with the subject’s immune cells and/or tumor Before cell contact, the expression of SEMG2 in tumor cells can be selectively detected.
  • T lymphocytes lymphocytes
  • SEMG2 SEMG2
  • the subject has received or is receiving or will receive additional anti-cancer therapy.
  • the additional anti-cancer therapy includes surgery, radiotherapy, chemotherapy, immunotherapy or hormone therapy.
  • the present invention also discloses a kit comprising the aforementioned compound, the aforementioned antigen peptide, the aforementioned protein, the aforementioned polynucleotide, the aforementioned recombinant vector, And one or more of the aforementioned host cells and contained in a suitable container.
  • the present invention also discloses a method for detecting the presence or absence of SEMG2 in a biological sample in vitro, which includes contacting the biological sample with the aforementioned compound.
  • a method of inhibiting the growth of tumor cells comprising the following steps: A) analysis of the expression of tumor cells in SEMG2; B) can be identified by using tumor cells in contact SEMG2 antibody, said antibody binding with SEMG2 KD ⁇ 2 ⁇ 10 - 8 ; C) Bring the T lymphocytes, the antibody and tumor cells into contact.
  • FIG 1 shows the results of the immunoprecipitation experiment, which is divided into the upper figure and the lower figure.
  • the above figure proves that human CD27 protein and SEMG2 (Flag labeled) are physically bound.
  • the figure below proves that the mouse CD27 protein and SEMG2 (Flag labeled) are physically bound.
  • Figure 2 shows the results of immunofluorescence staining and ELISA detection.
  • Figure 2(A) proves that CD27 protein and SEMG2 protein have significant co-localization after overexpression in tumor cells.
  • Figure 2(B) shows the results of the ELISA test, which proves that the concentration-dependent effect of the binding of CD27 protein and SEMG2 protein on the micro-reaction plate, CD27 protein does not bind to the negative control protein and there is no concentration effect.
  • Figure 3 shows the results of the immunoprecipitation experiment, which is used to detect whether the fragments of SEMG2 (ie P1 to P6) can bind to CD27. Among them, the P5 fragment detected obvious binding to CD27.
  • Figure 4 shows the results of co-immunoprecipitation experiments to detect whether fragments of SEMG2 (ie P4, P5, P6, P7) can bind to CD27.
  • the sequence of P7 comes from a part of P5, which is "QIEKLVEGKSQIQ".
  • the results show that the left and right images are included.
  • the left picture shows the binding of the above-mentioned SEMG2 fragment to human CD27
  • the right picture shows the binding of the SEMG2 fragment to murine CD27.
  • the results showed that both human and murine CD27 can bind to P5 and P7 fragments.
  • Figure 5 shows that the contribution of each amino acid of P7 to the binding of CD27 protein is accurately proved by the method of Alanine Scan. Including A picture and B picture.
  • Figure A shows the sequence generated after replacing each amino acid of P7 with glycine one by one, that is, the amino acid sequence of the mutant numbered 1-13.
  • Figure B is the result of the co-immunoprecipitation experiment, indicating the extent to which the fusion protein of mutant 1-13 polypeptide and GFP binds to CD27; Mutants 5 and 9 completely lose their binding to CD27; Mutants 11 and 13 have no effect on SEMG2 (497 -509) binding to CD27; mutants at other sites (1, 2, 3, 4, 6, 7, 8, 10, 12) weaken the binding of SEMG2 (497-509) to CD27 to a certain extent. It can be seen that the amino acids at positions 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, and 508 of SEMG2 have a significant effect on the binding of CD27.
  • Figure 6 shows the binding of SEMG2 epitope polypeptide to CD27 and its competitive inhibition of full-length SEMG2 binding to CD27.
  • the human SEMG2 (497-509) polypeptide coupled with BSA and the monkey SEMG2 polypeptide can bind to CD27 protein on the micro-reaction plate, and it is significantly higher than the negative BSA control. It can be seen that CD27 can be combined with the negative BSA control. Human and monkey SEMG2 (497-509) fragments were combined.
  • B is the inhibitory effect of peptides derived from SEMG2 and their derivatives QIEKLVEGKSQIQ, QIEKLVEGKSQI, QIEKLVEGKSQ, and QIAKLVEGKSQ on the binding of full-length SEMG2 to CD27.
  • the peptides of different concentrations are incubated and combined with CD27-Fc, and then added to the micro-reaction plate pre-coated with SEMG2 protein. After incubation, the unconjugated molecules are washed away, and the anti-Fc secondary antibody-HRP is used for detection and color development. .
  • the results show that the polypeptide molecule can inhibit the binding of full-length SEMG2 to CD27.
  • Figure 7 shows an apoptosis experiment of HCT116 cells stably transfected with SEMG2 or a control empty vector and activated human peripheral blood mononuclear cells co-cultured.
  • A is a representative photo of apoptosis detection, the green field shows apoptotic cells.
  • B is based on statistics from three independent biological experiments (error bars represent standard deviations).
  • Figure 8 shows an immunoblotting experiment to show the expression of SEMG2 protein in different tumor cells.
  • the name of the tumor cell is marked at the top (the font is tilted by 45 corners). It can be seen that about half of the tested cell lines have detectable expression of SEMG2 protein.
  • FIG. 9 shows the results of immunohistochemistry (IHC) experiments showing the expression of SEMG2 protein in different tumor tissues.
  • A shows the expression of SEMG2 in different colorectal cancer tumor tissues, with normal colorectal tissue as a control;
  • B shows the expression of SEMG2 in different lung cancer tissues with normal lung tissue as a control;
  • C shows a representative picture of the positive expression of SEMG2 in prostate cancer, melanoma, and gastric cancer. Due to space constraints, the test results of all tumor types are not listed here in detail;
  • D is the difference shown in SEMG2 The positive rate of expression in the tumor type. Positive expression is defined as a moderate or strong positive expression in frontal immunohistochemical staining.
  • the statistical results based on tissue chips are plotted as a percentage to show the positive expression ratio of SEMG2.
  • Figure 10 shows the statistical results of Kaplan-Meier factor survival analysis, suggesting that the high expression of SEMG2 (defined as medium and strong positive staining of SEMG2 immunohistochemical staining) is significantly associated with the shortening of the overall survival of patients with colorectal cancer. A P value below 0.001 suggests a highly significant association.
  • Figure 11 shows the results of the immunohistochemical experiment.
  • the above figure shows the statistical results of the correlation between T lymphocytes, namely Treg and SEMG2 staining in lung cancer.
  • the intensity of the SEMG2 immunohistochemical staining is divided into different levels, and each field of view is counted separately.
  • the following figures are representative pictures of Treg markers in the case of positive and negative expression of SEMG2, respectively.
  • Figure 12 shows the results of the ELISA experiment.
  • the ordinate is the normalized A405 absorption value as the reading of the ELISA experiment, showing the degree of binding of SEMG2 and CD27; the abscissa is the concentration of antibody added.
  • the solid line represents the blocking effect of the polyclonal antibody produced by SEMG2 (497-509) as an antigen; the dashed line represents the blocking effect of the polyclonal antibody produced by the full-length SEMG2 protein as an immunogen.
  • the polyclonal antibody produced by SEMG2 (497-509) requires a lower concentration to exert the blocking effect, that is, the blocking titer of the antibody produced by SEMG2 (497-509) is higher than the titer of the antibody produced by the full-length SEMG2 protein. This indicates that the recognition of the key epitope of SEMG2 (497-509) makes the development of blocking antibodies easier.
  • Figure 13 shows the number of blocking monoclonal antibodies and the total number of antibodies obtained after injection of the SEMG2 (497-509) epitope peptide and the full-length SEMG2 protein as immunogens into mice.
  • the murine monoclonal antibodies obtained through hybridoma fusion the antibodies that were confirmed to inhibit the binding of SEMG2 and CD27 through ELISA experiments were counted and displayed as black bar graphs. It can be seen that most of the antibodies prepared from SEMG2 (497-509) epitope fragments as immunogens can block the binding of SEMG2 to CD27, and the positive rate is significantly higher than that of antibodies prepared using full-length SEMG2 as immunogens.
  • Figure 14 shows the binding ability of the murine monoclonal antibody and the humanized murine monoclonal antibody to the SEMG2 protein.
  • the reading OD 450 absorbance value of the ELISA test is used as the ordinate, and the abscissa indicates the addition of different concentrations of antibodies.
  • the OD value gradually increased, indicating that the binding of SEMG2 to the murine monoclonal antibody ( Figure 14A) or the humanized monoclonal antibody ( Figure 14B) gradually increased.
  • the fitted curve is a representative result based on the statistics of three independent biological experiments.
  • Figure 15 shows the binding of murine monoclonal antibodies to BSA-SEMG2 (497-509) and blocking the binding function of SEMG2 to receptor proteins.
  • A The OD 450 absorbance value of the ELISA test is taken as the ordinate, and the abscissa indicates the addition of different concentrations of antibodies, indicating that the binding of SEMG2 (497-509) to the mouse monoclonal antibody is gradually increasing.
  • Figure B shows that the murine monoclonal antibody can block the binding of SEMG2 and CD27, and its blocking effect increases as the concentration increases.
  • the control mouse IgG antibody did not show blocking function.
  • the ordinate is the normalized blocking ratio as the blocking ratio; the abscissa indicates the different concentrations of antibody added. As the antibody concentration in the ELISA system increases, the binding of SEMG2 and CD27 gradually decreases.
  • the fitted curve is based on the statistics of three independent biological experiments (error bars represent standard deviations).
  • Figure 16 shows the results of the ELISA experiment.
  • the ordinate is the normalized A450 absorption value as the reading of the ELISA experiment, showing the degree of binding between SEMG2 (fixed on the surface of the ELISA plate) and the added CD27-Fc;
  • the abscissa shows the difference Experimental conditions, that is, different antibodies incubated together (both at a concentration of 10 ⁇ g/ml):
  • HPA042767 and HPA042835 are rabbit polyclonal antibodies against the epitopes of SEMG2 (354-403) and SEMG2 (563-574) respectively;
  • MM02, MM05 , MM07, MM08, MM13, MM14 are murine monoclonal antibodies against the epitope of SEMG2 (497-509).
  • Figure 17 shows the effects of different types of antibodies on the killing of tumor cells by T cells.
  • Activated PBMC human peripheral blood mononuclear cells were co-cultured with A375 human melanoma cells or LOVO colorectal cancer cells that highly express SEMG2, and different antibodies were added at the same time: unrelated mouse IgG, HPA042767, HPA042835, MM02, MM05, MM07, MM08, MM13, or MM14.
  • the ordinate is the percentage of apoptotic tumor cells; the abscissa is different experimental treatment conditions, that is, different antibodies added.
  • the murine monoclonal antibodies (MM02, MM05, MM07, MM08, MM13, or MM14) against the epitope of SEMG2 (497-509) significantly promoted the killing of tumors by T cells, while the control irrelevant IgG or against SEMG2 (354 -403) and SEMG2 (563-574) epitope HPA042767 and HPA042835 antibodies did not show this function.
  • SEMG2 (497-509) epitope is the key site of the immune escape function of SEMG2 expressed by tumor cells, and the antibodies against this epitope belong to the same class in terms of anti-tumor immune regulation function.
  • Figure 18 shows the experimental results of T cell killing of different tumor cells in the presence of SEMG2 blocking antibodies.
  • A375 and LOVO are tumor cells that highly express SEMG2 protein, while DLD1, NCM460 and NCI-H1975 are SEMG2 negative cells.
  • different antibodies were added, namely: irrelevant mouse IgG antibody, MM02 or MM05 mouse monoclonal antibody.
  • the abscissa represents different tumor cell lines, and the ordinate represents the percentage of tumor cell apoptosis. It can be seen that tumor cells (A375 and LOVO) with higher expression of SEMG2 can be more effectively killed by T cells after treatment with antibodies.
  • SEMG2 blocking antibodies MM02 and MM05 tumor cells that do not express SEMG2 (DLD1, NCM460 and NCI-H1975) did not significantly increase their apoptosis levels after administration of SEMG2 blocking antibodies MM02 and MM05. This indicates that the positive expression of SEMG2 can be used as a selective marker for the administration of SEMG2 blocking antibodies.
  • SEMG2 blocking antibody is used as an anti-tumor immune drug, the expression of SEMG2 has guiding significance for the selection of suitable patients.
  • Figure 19 shows the A450 absorbance value as a reading in an ELISA experiment to detect the degree of binding of SEMG2 to different antibodies.
  • Different antigens from SEMG2 (shown on the left) are coated on ELISA plates and combined with HPA04276, HPA042835, MM02, MM05, MM07, MM08, MM13, MM14, and then anti-mouse secondary antibodies (for HPA04276 , HPA042835, MM02, MM05, MM07, MM08) or anti-rabbit secondary antibodies (for HPA04276, HPA042835) to detect bound antibodies.
  • anti-mouse secondary antibodies for HPA04276 , HPA042835, MM02, MM05, MM07, MM08
  • anti-rabbit secondary antibodies for HPA04276, HPA042835
  • MM02, MM05, MM07, MM08, MM13, MM14 all bind to the SEMG2 (497-509) epitope, belonging to the same class; HPA04276 binds to the SEMG2 (354-403) epitope, and HPA042835 binds to the SEMG2 (563-574) epitope. Bit.
  • Figure 20 shows the value detected by the ELISA experiment, that is, the OD450 absorption value.
  • SEMG2 (497-509) epitope peptide and its glycine scanning mutant (that is, amino acid replaced by glycine one by one) polypeptide were immobilized on an ELISA plate, and further combined with different antibodies as shown in the figure.
  • This experiment is used to determine the precise amino acid epitopes that different monoclonal antibodies bind to, and the relative importance of each amino acid to the binding antibody.
  • control antibodies HPA04276 and HPA042835 bind to the above-mentioned epitopes and mutants in steps, and the amino acids at different positions contribute to the antibody binding differently, which can block the binding of SEMG2 and CD27 antibodies (MM02, MM05, MM07, MM08, MM13, MM14). )
  • the combined important amino acids are similar. This indicates that antibodies with blocking function belong to the same class in terms of binding epitopes.
  • Figure 21 shows the results of the ELISA test, indicating the concentration-dependent effect of fully human antibodies H88-67, H88-93, H88-96 and affinity matured fully human antibodies binding to SEMG2 and BSA-SEMG2 (497-509) .
  • the reading OD 450 absorbance value of the ELISA test is used as the ordinate, and the abscissa indicates the addition of different concentrations of antibodies.
  • Figure 22 shows the ELISA results, indicating the concentration-dependent effect of the competitive binding of the fully human antibody and the murine monoclonal antibody to SEMG2.
  • the ordinate is the ratio of fully human antibody blocking the binding of SEMG2 to the murine antibody. As the concentration of the fully human antibody increases, the signal detecting the murine antibody bound to SEMG2 gradually weakens.
  • Figure 23 shows the results of an ELISA test, indicating the blocking effects of different human antibodies H88-93, H88-96, and H88-67 on the binding of SEMG2 and CD27. All antibody concentrations are 10 ⁇ g/ml. Antibody clones H88-93, H88-96, and H88-67 are all fully human antibodies screened in the natural phage library using SEMG2 (497-509) epitope.
  • Figure 24 shows the killing degree of T cells on co-cultured A375 and LOVO tumor cells, and the effect of human antibodies H88-93, H88-96, and H88-67 on the killing effect.
  • the results show that these three antibodies against the epitope of SEMG2 (497-509) can significantly promote the killing of tumor cells expressing SEMG2 by T cells.
  • Figure 25 shows the biological membrane interference technique to measure the binding of SEMG2 to fully human antibody molecules, indicating the changes in the binding and dissociation of the fully human antibody in the solution and the SEMG2 protein molecules immobilized on the biosensor, and the whole human is calculated based on this.
  • Figure 26 shows that the SEMG2 antibody significantly inhibited tumor growth in the A375 melanoma mouse in vivo model.
  • Figure 27 shows the phenotypic analysis results of homozygous knockout of mouse gene Svs3a corresponding to human SEMG2 and wild-type mice, including gross morphology, tissue and organ biopsy, T lymphocyte pressure ratio analysis of different subtypes, blood Specific results of biochemical, liver function, and blood routine tests.
  • the term “subject” includes any human or non-human animal.
  • non-human animals includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, rats, mice, amphibians, reptiles Wait.
  • the terms “patient” or “subject” are used interchangeably.
  • the preferred subject is a human.
  • SEMG2 is human seminal vesicle gland coagulation protein 2, one of the main components of human semen, which is secreted by the seminal vesicle gland to form a gelatinous substance that coats sperm cells and restricts their movement.
  • the proteolytic enzyme and plasmin secreted by the prostate in the semen can break down the coagulation protein of the seminal vesicle gland and promote the liquefaction of the semen, so that the sperm can move more freely.
  • the mechanism by which SEMG2 inhibits sperm motility may also include its binding and influence on sperm cell membranes.
  • the "SgII A" polypeptide isolated from the SEMG2 protein has antibacterial properties. Activity, the sequence is H-KQEGRDHDKSKGHFHMIVIHHKGGQAHHG-OH. It should be noted that the antimicrobial peptide sequence is different from the key amino acid sequence of the binding of SEMG2 and CD27 of the present invention, and is located in a completely different region of SEMG2. See AM, Malm J, Frohm B, Martellini JA, Giwercman A, M, et al., J Immunol.
  • SEMG2 has also been reported to bind zinc ions and affect the activity of the proteolytic enzyme PSA. See Jonsson M, Linse S, Frohm B, Lundwall A, Malm J. Biochem J. 2005; 387(Pt 2):447-53.
  • the term “antibody” includes intact antibodies and any antigen-binding fragments (ie, “antigen-binding portions") or single chains thereof.
  • Antibody refers to a protein comprising at least two heavy chains (H) and two light chains (L) connected to each other by disulfide bonds, or an antigen-binding portion thereof.
  • Each heavy chain is composed of a heavy chain variable region (abbreviated as VH herein) and a heavy chain constant region.
  • the heavy chain constant region is composed of three domains, CH1, CH2 and CH3.
  • Each light chain consists of a light chain variable region (abbreviated as VL herein) and a light chain constant region.
  • the light chain constant region consists of a domain CL.
  • VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDR), interspersed with more conservative regions called framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminal to the carboxy terminal: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain binding domains that interact with antigens.
  • antibody refers to an immunoglobulin or its fragments or derivatives thereof, and includes any polypeptide of the antigen binding site it contains, regardless of whether it is produced in vitro or in vivo.
  • the term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, non-specific, humanized, single-stranded, chimeric, synthetic, recombinant, hybrid, Mutant, grafted antibodies.
  • antibody also includes antibody fragments such as Fab, F(ab')2, FV, scFv, Fd, dAb and other antibody fragments that retain the antigen-binding function, that is, can specifically bind to PD-1. Normally, such fragments will include antigen-binding fragments.
  • antigen-binding fragment refers to an antibody molecule that contains amino acids responsible for the binding between a specific antibody and an antigen.
  • the antigen-binding fragment only binds a part of the antigen. That is, the part of the antigen molecule responsible for the specific interaction with the antigen-binding fragment is called "epitope” or "antigenic determinant”.
  • An antigen-binding fragment usually includes an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), however, it does not necessarily include both.
  • VL antibody light chain variable region
  • VH antibody heavy chain variable region
  • a so-called Fd antibody fragment consists of only the VH domain, but still retains some of the antigen-binding functions of a complete antibody.
  • epitope is defined as an antigenic determinant, which specifically binds/recognizes the binding fragment.
  • the binding fragment can specifically bind/react with a conformational or continuous epitope that is unique to the target structure.
  • the conformational or discontinuous epitope is characterized in that the polypeptide antigen is separated by two or more discrete amino acid residues in the primary sequence However, when the polypeptides are folded into natural proteins/antigens, they are gathered together on the surface of the molecule. Two or more discrete amino acid residues of an epitope are present in independent parts of one or more polypeptide chains.
  • treatment and “method of treatment” refer to both therapeutic treatment and preventive/preventive measures.
  • Those in need of treatment include individuals who already have a specific medical condition, and those who may eventually get the condition.
  • vector refers to a molecular tool that transports, transduces, and expresses contained exogenous genes of interest (such as the polynucleotides of the present invention) in target cells, and the tools provide suitable molecular tools in target cells.
  • exogenous genes of interest such as the polynucleotides of the present invention
  • the nucleotide sequence that initiates transcription that is, the promoter.
  • tag protein and “protein tag” are interchangeable, and refer to a polypeptide or protein expressed by fusion with the target protein by using DNA in vitro recombination technology to facilitate protein expression, detection, tracing, and purification.
  • Tag proteins include but are not limited to His6, Flag, GST, MBP, HA, GFP, Myc.
  • human HEK239 cells were co-transfected, including a complex of pcDNA3-Flag-SEMG2 plasmid and pcDNA3-HA-CD27 plasmid. After 48 hours, the cell lysate was collected, and The standard immunoprecipitation procedure was used to enrich the CD27 in the lysate.
  • the antibody used for precipitation is Flag antibody, and the control group uses IgG non-specific antibody. After that, Western Blot was performed. HA antibody was used to detect the amount of co-precipitation of CD27, and Flag antibody was used to detect the amount of precipitation of SEMG2.
  • the cells were dissolved in 1% Triton X-100 (TBS pH 7.6) with Roche complete protease inhibitor on ice for 30 minutes, and then the insoluble matter was granulated by centrifugation. The lysate was heated to 100°C in 50mM DTT SDS sample buffer for 10 minutes, separated by SDS-PAGE, and transferred to PVDF membrane (micropore). In 5% bovine serum albumin (BSA), the cell membrane was blocked in TBS, the cell membrane was probed with the indicated antibody, and the reaction zone was observed with West Pico (Thermo Fisher Scientific).
  • BSA bovine serum albumin
  • SEMG2 protein amino acid sequence SEQ ID NO:1
  • SEMG2-P1, SEMG2-P2, SEMG2-P3, SEMG2-P4, SEMG2-P5, SEMG2-P6 see Table 1 for specific sequences, and the corresponding ones are abbreviated as P1-P6, respectively. Plasmids expressing these amino acid sequences were co-transfected with CD27 into HEK293 cells, and co-immunoprecipitation experiments were performed to identify the main fragments where SEMG2 binds to CD27.
  • SEQ ID NO:1 human SEMG2
  • SEMG2-P7 the specific sequence is QIEKLVEGKSQIQ, abbreviated as P7 or SP7.
  • SEMG2-P7 497-509, SEMG2-P5 (positive control), SEMG2-P4 (negative control), SEMG2-P6 (negative control) were co-transfected with CD27 into HEK293 cells, including human CD27 and murine CD27.
  • the subsequent immunoprecipitation experiment results showed that both SEMG2-P7 and SEMG2-P5 combined with CD27, and the results of human-derived CD27 and mouse-derived CD27 were the same.
  • the experimental results are shown in Figure 4. This co-immunoprecipitation experiment confirmed that SEMG2 (497-509) is the main structure that binds to human and murine CD27.
  • Example 3 Using glycine scanning method to accurately characterize the key amino acids that SEMG2 binds to CD27
  • each amino acid of SEMG2 (497-509) is replaced one by one with Glycine, the resulting sequence is the amino acid sequence of the mutant numbered 1-13 (see Figure 5).
  • These mutant plasmids and CD27 expression vector were co-transfected into HEK293 cells, and the degree of binding of the fusion protein of variant 1-13 polypeptide and GFP to CD27 was detected by immunoprecipitation experiment. The experimental results are shown in Figure 5.
  • Example 4 SEMG2 expressed by tumor cells inhibits the killing effect of immune cells on tumors
  • HCT116 human colorectal cancer cells were stably transfected with SEMG2 expression vector or a control empty vector, and the proportion of apoptotic cells after co-cultivation of activated PBMC was determined by the caspase3/7 lysis method (green fluorescence method). Specifically, HCT116 cells stably expressing SEMG2 were seeded in 96-well plates.
  • Tumor SW1116 colorectal cancer, DLD1 colorectal cancer, HEK293 human renal epithelial cell line, HepG2 hepatocellular carcinoma, NCM460 human normal colonic epithelial cells, NCI-H1975 human non-small cell lung adenocarcinoma, CaCo2 colon adenocarcinoma, HT29 nodule Rectal adenocarcinoma, SW1990 human pancreatic cancer, AGS human gastric adenocarcinoma, SW480 colorectal cancer, SaOS2 osteosarcoma, GES-1 human gastric mucosal cells, etc.
  • the cells were dissolved in 1% Triton X-100 (TBS pH 7.6) with Roche complete protease inhibitor on ice for 30 minutes, and then the insoluble matter was granulated by centrifugation. The lysate was heated to 100°C in 50mM DTT SDS sample buffer for 10 minutes, separated by SDS-PAGE, and transferred to PVDF membrane (micropore). In 5% bovine serum albumin (BSA), the cell membrane was blocked in TBS, the cell membrane was probed with the specific primary antibodies of the shown SEMG2 and GAPDH internal controls, and the primary antibody was labeled with the HRP-conjugated secondary antibody, West Pico( Thermo Fisher Scientific) observe the reaction zone.
  • BSA bovine serum albumin
  • Example 6 Using immunohistochemistry (IHC) to detect the expression of SEMG2 in different tumor tissues.
  • tissue specimens were cultured with anti-SEMG2 (HPA042767, purchased from Sigma Aldrich, diluted 1:100) with a biotin-conjugated secondary antibody, and then cultured with anti-biotin-biotin-peroxidase complex to cultivate. Observe with aminoethylcarbazole developer.
  • staining intensity is divided into four groups: high (3), medium (2), low (1) and negative (0).
  • Example 8 Demonstration of the correlation between high expression of SEMG2 and Treg regulatory T lymphocyte infiltration with immunosuppressive function
  • Antigen preparation synthesis of peptides with SEMG2 (497-509) or "QIEKLVEGKSQIQ" sequence, and coupling to VLP carrier for immunization; the other group uses full-length SEMG2 protein (purchased from Cusabio, Product number CSB-YP021002HU) was used as the immunogen.
  • Second immunization Use scissors to cut off part of the rabbit hair on the two hind feet of the rabbit, disinfect the skin with alcohol and iodine, and use a 2 mL syringe to suck 1 mL of the antigen solution emulsified with Freund's complete adjuvant (FCA), each side Inject 0.5 mL into the sole of the foot.
  • FCA Freund's complete adjuvant
  • Second immunization 10-14 days later, inject antigen solution into the swollen lymph nodes in both fossa and groin, 0.1 mL for each lymph node, and 1 mL subcutaneously near the lymph nodes. If the lymph nodes are not enlarged or the enlargement is not obvious, inject directly into the fossa on both sides and subcutaneously in the groin.
  • Second immunization 10-14 days later, inject antigen solution into the swollen lymph nodes in both fossa and groin, 0.1 mL for each lymph node, and 1 mL subcutaneously near the lymph nodes. If the lymph nodes are not enlarged or the enlargement is not obvious, inject directly into the fossa on both sides and subcutaneously in the groin.
  • collect 0.5-1.0 mL blood from the ear vein separate the serum, determine the serum titer, use the indirect ELISA method, coat with 10 ⁇ g/mL antigen, and collect blood with a t
  • titer does not meet the requirements, use the antigen solution without adjuvant to inject intravenously into the ear for immunization. That is, 3 injections within 1 week, respectively, 0.1, 0.3, 0.5mL. Try the blood again at 1 week intervals. If the titer reaches the requirement, blood should be taken immediately, and all antiserum should be collected.
  • the specific experimental steps of polyclonal antibody purification include: (1) Prepare protein A sepharose CL-4B affinity column. Prepare 10mL protein A sepharose CL-4B filler, mix an equal volume of filler and TBS buffer solution in a vacuum flask, and stir. Vacuum for 15 minutes to remove air bubbles in the packing. Slowly add Protein A Sepharose CL-4B filler to the glass column, use a pump to control the filling speed of 1mL/min-2mL/min to avoid column dryness, and equilibrate the column with a pre-cooled TBS buffer solution that is 10 times the bed volume. (2) Preparation of antiserum.
  • the antiserum into ice water or a refrigerator at 4°C to thaw slowly to avoid protein aggregation.
  • the aggregation that occurs during the thawing of the protein can be dissolved by preheating at 37°C.
  • the antibody was diluted with TBS buffer solution at a ratio of 1:5, and then filtered with a filter. Load the antiserum onto the column at a rate of 0.5 mL per minute.
  • Example 10 Comparing the blocking effects of SEMG2 (497-509) and full-length SEMG2 as immunogens for preparing antibodies
  • SEMG2 (497-509) sequence fragment is the key epitope for SEMG2 to bind to CD27 and has a relatively short sequence
  • SEMG2 (497-509) as an immunogen to prepare antibodies is theoretically better than using full-length SEMG2 to prepare antibodies. It is easy to obtain functional antibody molecules with the function of blocking the binding of SEMG2 and CD27.
  • the ELISA experiment described in the foregoing implementation examples was used to verify the difference in the effective concentration of antibodies produced by the two methods.
  • the antibody prepared with SEMG2 (497-509) as the immunogen and the antibody prepared with the full-length SEMG2 were added to the enzyme-linked immunosorbent assay (ELISA) reaction system (10 ⁇ -2,10 ⁇ -1,10) at different concentrations.
  • ELISA enzyme-linked immunosorbent assay
  • the specific steps of the enzyme-linked immunosorbent assay are as follows: (1) Dissolve the SEMG2 protein antigen with 50 mM carbonate coating buffer (pH 9.6) to make the antigen concentration 10 ⁇ g/mL, and add 100 ⁇ L/well to the 96-well ELISA plate ( (Purchased from Corning Company), placed overnight at 4°C. (2) After discarding the coating solution the next day, wash with PBST 3 times, add 150 ⁇ L of 1% BSA to each well and block at 37°C for 2 hours.
  • SEMG2 (497-509) as an antibody produced by the antigen can reduce the binding of SEMG2 and CD27 detected by ELISA by 50% at a lower concentration, while the full-length SEMG2 protein as an immunogen produces more Antibiotics can only exert this effect at higher concentrations (the required dose is more than 300 times that of the former). That is, the blocking titer of the antibody produced by SEMG2 (497-509) is more than 300 times higher than the titer of the antibody produced by the full-length SEMG2 protein. This indicates that the recognition of the key epitope of SEMG2 (497-509) makes the development of blocking antibodies easier, and makes it easier for those skilled in the art to obtain antibodies that can block the binding of SEMG2 and CD27.
  • Example 11 Preparation of mouse monoclonal antibody using SEMG2 (497-509) epitope peptide and SEMG2 full-length protein
  • mice with higher titers were selected for hybridoma fusion screening. After subcloning, the binding of monoclonal antibodies to the target antigen was detected by ELISA, and the function of different monoclonal antibodies to block the binding of SEMG2 to CD27 was tested by ELISA.
  • the monoclonal antibodies produced by hybridomas were screened by ELISA experiments.
  • the monoclonal antibodies prepared with SEMG2 (497-509) as the immunogen the first 27 strains of antibodies were verified to have blocking function (inhibit the binding of SEMG2 and CD27)
  • the monoclonal antibodies prepared with the SEMG2 full-length protein immunogen only one antibody with blocking function was obtained after a total of 108 antibodies were verified in batches, as shown in Figure 13.
  • the ELISA plate was coated with the SEMG2 protein, and the mouse monoclonal antibody of gradient dilution was used as the primary antibody, and the binding ability of the mouse monoclonal antibody to SEMG2 was tested with the anti-mouse secondary antibody.
  • Figure 14A Show that the mouse monoclonal antibody has a good affinity for the SEMG2 protein.
  • the mouse anti-SEMG2 monoclonal antibody MM05 was humanized to reduce immunogenicity when used in human patients.
  • the sequences of the heavy and light chain variable regions (VH and VL) are compared with the human antibody sequences in the protein database (PDB), and a homology model is established.
  • the CDRs in the heavy and light chains of the mouse mAb are grafted into the human framework region, which is most likely to maintain the proper structure required for antigen binding.
  • reverse mutations or other mutations from human residues to mouse residues are designed. For example, the amino acid at position 95 of the humanized light chain VL-V2 is mutated from K to Q, and the corresponding light chain VL-V2 is changed from K to Q.
  • the CDR3 sequence of the chain is converted to QQSYSLPWT (SEQ ID NO: 95) according to IMGT analysis.
  • the humanized VH and VL regions are fused to the constant regions of human IgG1 heavy chain and kappa light chain, respectively.
  • the construct vector corresponding to the mAb sequence was used for transient transfection in 293E cells, and the binding ability of the purified mAb with the SEMG2 protein was analyzed by ELISA. The results are shown in absorbance, where a higher absorbance indicates a higher level of interaction between the humanized antibody and SEMG2.
  • Fig. 14B shows the fitting curve of the binding of the humanized monoclonal antibody to the SEMG2 protein in gradient dilutions. The results show that the humanized antibody maintains the binding ability of the murine monoclonal antibody to the SEMG2 protein.
  • Example 13 Comparison of the functions of SEMG2 (497-509) epitope-specific antibodies and other epitope-specific antibodies in blocking the binding of SEMG2 and CD27
  • SEMG2 (497-509) epitope-specific antibodies such as MM02, MM05
  • other epitope-specific antibodies such as HPA042767
  • the binding of the above antibodies to the SEMG2 (497-509) epitope was confirmed by ELISA experiments: MM02 and MM05 can bind to SEMG2 (497-509), but HPA042767 cannot bind to this epitope in a large concentration range, as shown in Figure 15A Show.
  • the ELISA experiment described in Example 11 analyzed the blocking function of different antibodies (unrelated murine IgG, MM02, MM05, HPA042767) on the binding of SEMG2 and CD27.
  • Example 14 Comparison of the effects of SEMG2 (497-509) epitope-specific antibodies and other epitope-specific antibodies on the killing of tumor cells by activated PBMC.
  • SEMG2 showed the function of inhibiting activated PBMC to kill tumor cells. Since SEMG2 may exert the above effects by binding to CD27, and the SEMG2 (497-509) epitope is the key site for binding CD27, the SEMG2 (497-509) epitope specific antibody may neutralize the effect of SEMG2 on PBMC killing tumor cells. Influence.
  • Example 15 Verification of the correlation between the expression of SEMG2 and the function of blocking antibodies in promoting PBMC to kill tumor cells
  • SEMG2 Since the expression of SEMG2 is a prerequisite for suppressing tumor-specific immunity, the expression of SEMG2 is also a potential condition suitable for the administration of SEMG2 blocking antibodies.
  • tumor cells with high expression of SEMG2 will relatively increase the killing of tumor cells by PBMC after neutralizing the activity of SEMG2; tumor cells that do not express SEMG2 may not rely on SEMG2 to exert immune escape function, so it neutralizes the activity of SEMG2 The killing of tumor cells by PBMC may not change significantly.
  • Example 16 Accurate definition of related epitopes of SEMG2 and CD27 binding blocking antibodies
  • MM02, MM05, MM07, MM08, MM13, MM14 all bind to the SEMG2 (497-509) epitope
  • HPA042767 binds to the SEMG2 (354-403) epitope
  • HPA042835 binds to the SEMG2 (497-509) epitope.
  • Epitope, and none of the above antibodies bind to the SEMG2 (442-453) control fragment.
  • the substitution of amino acids at positions 507 and 509 did not significantly affect the binding of MM02 and similar antibodies; the substitution of amino acids at positions 501 and 506 significantly affected the binding of MM02 and similar antibodies (a decrease of more than 70%) ); After the substitution of amino acids at other positions, it affects the binding of MM02 and similar antibodies to a certain extent.
  • the above results accurately define the epitope amino acids related to MM02 and similar antibodies (that is, antibodies that block the binding of SEMG2 and CD27), and the contribution of each amino acid to the binding.
  • the above-mentioned reference SEMG2 has high consistency with the key amino acids that bind to the blocking antibody and the key amino acids involved in the binding of CD27, which indicates that MM02 and its similar antibodies compete with CD27 to bind to SEMG2, verifying the molecular mechanism of the antibody's action.
  • Example 17 Preparation and screening of fully human antibodies using SEMG2 (497-509) epitope to block the binding of SEMG2 and CD27 and promote the killing of tumor cells by PBMC
  • the SEMG2 (497-509) epitope plays an important role in the preparation of blocking antibodies, and this epitope is applied to the screening of fully human antibodies.
  • the preparation of the polypeptide antigen and the screening of the human natural antibody library are first carried out.
  • the SEMG2 (497-509) polypeptide was synthesized and coupled to BSA and KLH, respectively, and screened in the fully human phage display antibody library.
  • Use ELISA experiments to select clones that can bind to the antigen epitope for preliminary screening. After sequencing individual clones, different unique sequences are obtained, and affinity ranking is performed.
  • Full-length antibodies are constructed for antigen-binding fragments (Fab) with higher affinity, and then expressed and purified.
  • the binding ability test and the blocking function test are to determine the effect of the antibody on the binding of SEMG2 and CD27 through the aforementioned ELISA experiment.
  • the binding ability of fully human antibodies and mouse antibodies to SEMG2 was tested. That is, in a 96-well microplate coated with SEMG2, the mouse antibodies MM02 and MM05 were mixed with the fully human antibody H88-93 with gradient dilutions. The primary antibody, the murine monoclonal antibody bound to SEMG2 was assayed with the anti-mouse secondary antibody HRP. Calculate the blocking percentage according to the following formula:
  • Blocking percentage [1-(experimental antibody A450-blank control)/(positive control antibody A450-blank control A450)] ⁇ 100%
  • H88-93 can compete with MM02 and MM05 for binding to SEMG2, as shown in Figure 22. It shows that fully human antibodies and murine monoclonal antibodies are the same type of antibodies that can bind to SEMG2, and since MM02, MM05 and H88-93 can all bind to the short peptide SEMG2 (497-509), this type of antibody can be defined as A type of antibody that binds to SEMG2 (497-509).
  • Example 17 Determination of the binding kinetics of the monoclonal antibody of the present invention to the antigen by the bio-optical interferometry method
  • the equilibrium dissociation constant (KD) of the antibody of the present invention bound to human SEMG2 is determined by the biological membrane interferometry method (ForteBio Bltz or Gator instrument). For example, ForteBio affinity determination is carried out according to the existing method, that is, half an hour before the start, according to the number of samples, take the appropriate amount of AMQ (Pall, 1506091) (for sample detection) or AHQ (Pall, 1502051) (for positive control detection) ) The sensor is immersed in SD buffer (PBS 1 ⁇ , BSA 0.1%, Tween-200.05%). Take 100 ⁇ l of SD buffer, antibody, and SEMG2 respectively and add them to 96-well black polystyrene half-volume microtiter plates.
  • the plasmids constructed with the VH and VL coding sequences of the fully human antibodies H88-96 and H88-67 as templates, the plasmids were obtained through gene synthesis, and then single-point and double-point saturation mutations were performed, and then the antibody genes were recombined by in vitro connection Finally, the recombinant antibody Fab gene sequence was inserted into the vector and transformed to obtain 4 mutant phage affinity mature antibody libraries with a storage capacity higher than 10 8 CFU. The antibody mutation library was screened by the immune tube gradient screening method, and the mutants with better affinity than the wild-type were obtained.
  • anti-human SEMG2 monoclonal antibodies with increased affinity such as the affinity maturation heavy chain numbered 67-3 and the affinity maturation number 67-3, 67-4, 67-5, 67-6
  • the light chain sequence combination constitutes 67-3-67-3, 67-3-67-4, 67-3-67-5, 67-3-67-6, the heavy chain numbered 67-9 and the heavy chain numbered 67-
  • the light chain combination of 3 constitutes antibody 67-9-67-3
  • the combination of light chain and heavy chain numbered 67-6 constitutes antibody 67-6-67-6
  • the heavy chain numbered 96-10R and 96-10V The antibodies 96-10R-10 and 96-10V-10 reconstituted with the light chain of H88-96L.
  • Example 19 Verification of the anti-tumor effect of the SEMG2 antibody in the PBMC immune system humanized mouse model xenograft model of human malignant melanoma A375 cells
  • mice Thirty 6-8 week old male NPSG mouse models were weighed. A375 cells (endogenous expression of SEMG2 has been confirmed) were cultured in vitro to obtain 1.8 ⁇ 10 8 cells. After 30 mice were inoculated with PBMC, they were inoculated with A375 tumor cells on the third day. After that, the proportion of hCD45+ cells in the blood of the mice and their body weight were measured once a week. After inoculation, the tumor volume was measured once a week. When the average tumor volume reached about 40-80mm 3 , the proportion of hCD45+ cells in the blood of the mice was measured. According to the tumor volume and the proportion of hCD45+ cells in the blood of the mice, the mice were randomly divided into groups and the administration was started immediately.
  • mice The start date of dosing is regarded as day 0.
  • Dosing regimen SEMG2 antibody (MM05 clone) was injected intraperitoneally at 5 mg/kg three times a week. After the administration started, the mice were observed the tumor growth status every week. After the tumor grew, the body weight and tumor volume were measured 3 times a week, and the relative counts of hCD45+ cells in the blood of the mice were monitored by flow cytometry 3 times a week. When the tumor volume reached the endpoint standard, blood was taken to test the same indicators as above, and the experiment ended. Observations on mice include: daily observation, after inoculation, observation of the disease and death of animals every working day.
  • Example 20 Knockout of the corresponding gene Svs3a in mice proves that the side effects are not significant after the function of SEMG2 is blocked
  • mice take anticoagulated whole blood for flow cytometry experiment to analyze the proportion of CD8.CD4.CD3.CD27 in the blood and the proportion of positive cells. After the mice were resting for 2 days, the blood samples of the anticoagulant whole blood were taken from the inner canthus to test the blood routine. After the mice rested for 3 days, the mice were weighed and anesthetized, and the mice were photographed in general; the mice were taken from the eyeballs and blood was collected and the serum was separated. The mouse was euthanized after the eyeball was removed and blood was taken.
  • Brain remove the intact brain, separate it in the mid-sagittal shape, fix on the left side and quick-frozen on the right side; liver: remove the intact liver, fix the left lobe, and quick-frozen the rest; spleen: remove the intact spleen , One is divided into two, half fixed, half quick-frozen; kidney: remove the left kidney to fix, remove the right kidney to quick-freeze; stomach: remove the intact stomach, separate sagittal, the greater curvature part is fixed, the small curvature part is quick-frozen; large intestine: take away Lower the intact large intestine, all fixed with Swiss roll; small intestine: remove the intact small intestine and fix it in three sections (duodenum, ileum, jejunum) with Swiss roll; lung: remove the left lung for fixation, remove the right lung for quick freezing; heart : Remove the entire heart and fix it after diastole.

Abstract

提供了一种激动或拮抗SEMG2和CD27相互作用的化合物，包括小分子抑制剂、多肽、抗体或抗原结合片段。该多肽作为免疫原制备用于阻断SEMG2与CD27结合的抗体，进而促进抗肿瘤免疫。还公开了通过可阻断SEMG2与CD27结合筛选治疗性药物的筛选方法。

Description

SEMG2抗体及其用途 技术领域
本发明涉及生物医药领域，具体涉及一种SEMG2抗原表位肽及其用途。
背景技术
CD27分子属于肿瘤坏死因子受体(TNFR)超家族的成员，是分子量约为55kDa的I型膜蛋白，并作为两个单体通过二硫键连接的二聚体形式存在。CD27主要表达在淋巴细胞上，近期基于CD27敲除小鼠的研究表明，CD27信号途径的激活可增加抑制性T细胞(Treg)在实体瘤的浸润，降低抗肿瘤免疫(Claus C,Riether C,Schürch C,Matter MS,Hilmenyuk T,Ochsenbein AF.Cancer Res.2012 Jul 15；72(14):3664-76)。与此相一致，研究还发现皮肤组织内的Treg细胞失去CD27的表达后无法发挥正常的免疫调控功能(Remedios KA,Zirak B,Sandoval PM,Lowe MM,Boda D,Henley E等，Sci Immunol.2018 Dec 21；3(30).pii:eaau2042)。此外，CD27的激活可增加Treg的数量并减少高脂血症小鼠的动脉粥样硬化(Winkels H,Meiler S,Lievens D,Engel D,Spitz C,Bürger C,等，Eur Heart J.2017；38(48):3590-3599)。上述的最新研究一致表明，CD27在特定Treg(包括肿瘤浸润的Treg)细胞的功能激活中发挥重要作用，因此避免肿瘤浸润Treg细胞表达的CD27激活是潜在的癌症治疗策略。
与配体的结合可激活CD27下游信号转导，而目前已知的CD27配体分子是CD70。CD70为193个氨基酸的多肽，其具有20个氨基酸的亲水性N端结构域和包含2个潜在的N连接糖基化位点的C端结构域，属于TNF家族(Goodwin，R.G.等(1993)Cell 73：447-56；Bowman等(1994)Immunol 152：1756-61)。这些特性表明CD70是具有细胞外C端部分的II型跨膜蛋白。CD70在活化的T和B淋巴细胞以及树突状细胞上瞬时地存在(Hintzen等(1994)J.Immunol.152：1762-1773；Oshima等 (1998)Int.Immunol.10：517-26；Tesselaar等(2003)J.Immunol.170：33-40)。除了在正常细胞上表达外，已经在不同类型的癌症中报道了CD70的表达，所述癌症包括肾细胞癌、转移性乳腺癌、脑肿瘤、白血病、淋巴瘤和鼻咽癌(Junker等，J Urol.2005；173：2150-3；Sloan等，Am J Pathol.2004；164：315-23；Held-Feindt和Mentlein等，Int J Cancer 2002；98：352-6)。目前，阻断CD70与CD27的结合是一种正在被研究的肿瘤免疫治疗策略。
既往的研究并未提示CD27还存在CD70以外的其它配体。但免疫检查点通路受体的新配体(尤其是肿瘤细胞表达特异性比较高的新配体)，对于开发更有效的抗肿瘤治疗方法具有重要的意义。本发明旨在开发新的抗肿瘤治疗和药物。
发明内容
一方面，本发明公开了一种激动或拮抗SEMG2和CD27相互作用的化合物。其中，其中所述SEMG2和CD27相互作用的氨基酸位点位于SEMG2的第497、498、499、500、501、502、503、504、505、506、508位，所述SEMG2蛋白的氨基酸序列如SEQ ID NO:1所示。其中，所述化合物为小分子抑制剂、多肽、抗体或抗原结合片段。
在一个实施例中，本发明公开了一种多肽，所述多肽具有或包含如SEQ ID NO:2(QIEKLVEGKS)、SEQ ID NO:86(QIEKLVEGKS(x)I(x))、SEQ ID NO:87(QIEKLVEGKS(x)I)、或SEQ ID NO:88(QIEKLVEGKS(x))所示氨基酸序列所示的氨基酸序列，优选所述多肽包含如SEQ ID NO:3(QIEKLVEGKSQIQ)、SEQ ID NO:4(QIEKLVEGKSQ)、或SEQ ID NO:5(QIEKLVEGKSQI)或与SEQ ID NO:2-5任一项序列至少90％以上序列一致性的氨基酸序列。其中，所述多肽是一种激动SEMG2和CD27相互作用的多肽。其中，所述SEMG2和CD27相互作用的氨基酸位点位于SEMG2的第497、498、499、500、501、502、503、504、505、506、508位，所述SEMG2蛋白的氨基酸序列如SEQ ID NO:1所示。
在一个实施例中，本发明公开了一种能够特异性结合天然或突变SEMG2蛋白的抗体，所述抗体结合源自SEMG2蛋白的抗原表位肽，所述抗原表位肽包含SEQ ID NO:2(QIEKLVEGKS)、SEQ ID NO:3 (QIEKLVEGKSQIQ)、SEQ ID NO:4(QIEKLVEGKSQ)、或SEQ ID NO:5(QIEKLVEGKSQI)所示的氨基酸序列。其中，所述抗体是一种拮抗SEMG2和CD27相互作用的抗体。
在一个实施例中，本发明公开了一种能够特异性结合天然或突变SEMG2蛋白的抗体，所述抗体能够识别天然SEMG2蛋白的第497、498、499、500、501、502、503、504、505、506、508位点中的至少一个氨基酸残基或识别突变SEMG2蛋白相应位置的氨基酸残基，所述天然SEMG2蛋白的氨基酸序列如SEQ ID NO:1所示。其中，所述抗体是一种拮抗SEMG2和CD27相互作用的抗体。
在一个实施例中，本发明公开了一种能够特异性结合天然或突变SEMG2蛋白的抗体，其中所述抗体包含根据IMGT定义的HCDR1、HCDR2和HCDR3序列的重链可变区；以及包含根据IMGT定义的LCDR1、LCDR2和LCDR3序列的轻链可变区，
所述HCDR1的氨基酸序列具有或包含选自由SEQ ID NOs:6-11、SEQ ID NOs:60-61、和SEQ ID NO:76所组成的组中的氨基酸序列；
所述HCDR2的氨基酸序列具有或包含选自由SEQ ID NOs:12-16和SEQ ID NOs:62-64所组成的组中的氨基酸序列；
所述HCDR3的氨基酸序列包含选自由SEQ ID NOs:17-20、SEQ ID NOs:65-67、和SEQ ID NOs:77-81所组成的组中的氨基酸序列；
所述LCDR1的氨基酸序列包含选自由SEQ ID NOs:21-25、SEQ ID NOs:68-70、和SEQ ID NO:82所组成的组中的氨基酸序列；
所述LCDR2的氨基酸序列包含选自由SEQ ID NOs:26-29、SEQ ID NOs:71-72、SEQ ID NOs:83-84、和SEQ ID NO:28所组成的组中的氨基酸序列；
所述LCDR3的氨基酸序列包含选自由SEQ ID NOs:30-34、SEQ ID NOs:73-75、SEQ ID NO:85和SEQ ID NO:99所组成的组中的氨基酸序列。
在一个具体实施例中，所述抗体中的CDR序列选自(a)-(k)组合中的任一种：
(a)所述HCDR1的氨基酸序列包含如SEQ ID NO:6所示氨基酸序列；所述HCDR2的氨基酸序列包含如SEQ ID NO:12所示氨基酸序列；所述 HCDR3的氨基酸序列包含如SEQ ID NO:17所示氨基酸序列；所述LCDR1的氨基酸序列包含如SEQ ID NO:21所示氨基酸序列；所述LCDR2的氨基酸序列包含如SEQ ID NO:26所示氨基酸序列；所述LCDR3的氨基酸序列包含如SEQ ID NO:30所示氨基酸序列；
(b)所述HCDR1的氨基酸序列包含如SEQ ID NO:7所示氨基酸序列；所述HCDR2的氨基酸序列包含如SEQ ID NO:13所示氨基酸序列；所述HCDR3的氨基酸序列包含如SEQ ID NO:18所示氨基酸序列；所述LCDR1的氨基酸序列包含如SEQ ID NO:22所示氨基酸序列；所述LCDR2的氨基酸序列包含如SEQ ID NO:27所示氨基酸序列；所述LCDR3的氨基酸序列包含如SEQ ID NO:31或SEQ ID NO:99所示氨基酸序列；
(c)所述HCDR1的氨基酸序列包含如SEQ ID NO:6所示氨基酸序列；所述HCDR2的氨基酸序列包含如SEQ ID NO:16所示氨基酸序列；所述HCDR3的氨基酸序列包含如SEQ ID NO:17所示氨基酸序列；所述LCDR1的氨基酸序列包含如SEQ ID NO:21所示氨基酸序列；所述LCDR2的氨基酸序列包含如SEQ ID NO:26所示氨基酸序列；所述LCDR3的氨基酸序列包含如SEQ ID NO:30所示氨基酸序列；
(d)所述HCDR1的氨基酸序列包含如SEQ ID NO:8所示氨基酸序列；所述HCDR2的氨基酸序列包含如SEQ ID NO:13所示氨基酸序列；所述HCDR3的氨基酸序列包含如SEQ ID NO:18所示氨基酸序列；所述LCDR1的氨基酸序列包含如SEQ ID NO:23所示氨基酸序列；所述LCDR2的氨基酸序列包含如SEQ ID NO:27所示氨基酸序列；所述LCDR3的氨基酸序列包含如SEQ ID NO:32所示氨基酸序列；
(e)所述HCDR1的氨基酸序列包含如SEQ ID NO:9所示氨基酸序列；所述HCDR2的氨基酸序列包含如SEQ ID NO:14所示氨基酸序列；所述HCDR3的氨基酸序列包含如SEQ ID NO:19所示氨基酸序列；所述LCDR1的氨基酸序列包含如SEQ ID NO:24所示氨基酸序列；所述LCDR2的氨基酸序列包含如SEQ ID NO:28所示氨基酸序列；所述LCDR3的氨基酸序列包含如SEQ ID NO:33所示氨基酸序列；
(f)所述HCDR1的氨基酸序列包含如SEQ ID NO:10所示氨基酸序列；所述HCDR2的氨基酸序列包含如SEQ ID NO:15所示氨基酸序列；所述HCDR3的氨基酸序列包含如SEQ ID NO:20所示氨基酸序列；所述LCDR1的氨基酸序列包含如SEQ ID NO:25所示氨基酸序列；所述LCDR2的氨基酸序列包含如SEQ ID NO:29所示氨基酸序列；所述LCDR3的氨基酸序列包含如SEQ ID NO:34所示氨基酸序列；
(g)所述HCDR1的氨基酸序列包含如SEQ ID NO:11所示氨基酸序列；所述HCDR2的氨基酸序列包含如SEQ ID NO:15所示氨基酸序列；所述HCDR3的氨基酸序列包含如SEQ ID NO:20所示氨基酸序列；所述LCDR1的氨基酸序列包含如SEQ ID NO:25所示氨基酸序列；所述LCDR2的氨基酸序列包含如SEQ ID NO:29所示氨基酸序列；所述LCDR3的氨基酸序列包含如SEQ ID NO:34所示氨基酸序列；
(h)所述HCDR1的氨基酸序列包含如SEQ ID NO:60所示氨基酸序列；所述HCDR2的氨基酸序列包含如SEQ ID NO:62所示氨基酸序列；所述HCDR3的氨基酸序列包含如SEQ ID NO:65所示氨基酸序列；所述LCDR1的氨基酸序列包含如SEQ ID NO:68所示氨基酸序列；所述LCDR2的氨基酸序列包含如SEQ ID NO:71所示氨基酸序列；所述LCDR3的氨基酸序列包含如SEQ ID NO:73所示氨基酸序列；
(i)所述HCDR1的氨基酸序列包含如SEQ ID NO:61所示氨基酸序列；所述HCDR2的氨基酸序列包含如SEQ ID NO:63所示氨基酸序列；所述HCDR3的氨基酸序列包含如SEQ ID NO:66所示氨基酸序列；所述LCDR1的氨基酸序列包含如SEQ ID NO:69所示氨基酸序列；所述LCDR2的氨基酸序列包含如SEQ ID NO:72所示氨基酸序列；所述LCDR3的氨基酸序列包含如SEQ ID NO:74所示氨基酸序列；
(j)所述HCDR1的氨基酸序列包含如SEQ ID NO:60所示氨基酸序列；所述HCDR2的氨基酸序列包含如SEQ ID NO:64所示氨基酸序列；所述HCDR3的氨基酸序列包含如SEQ ID NO:67所示氨基酸序列；所述LCDR1的氨基酸序列包含如SEQ ID NO:70所示氨基酸序列；所述LCDR2的氨基 酸序列包含如SEQ ID NO:28所示氨基酸序列；所述LCDR3的氨基酸序列包含如SEQ ID NO:75所示氨基酸序列；
(k)所述HCDR1的氨基酸序列包含如SEQ ID NO:60或76所示氨基酸序列；所述HCDR2的氨基酸序列包含如SEQ ID NO:64或62所示氨基酸序列；所述HCDR3的氨基酸序列包含如SEQ ID NO:77、78或79所示氨基酸序列；和/或
所述LCDR1的氨基酸序列包含如SEQ ID NO:70或82所示氨基酸序列；所述LCDR2的氨基酸序列包含如SEQ ID NO:28、83或84所示氨基酸序列；所述LCDR3的氨基酸序列包含如SEQ ID NO:75或85所示氨基酸序列。
在一个实施例中，本发明公开了一种能够特异性结合天然或突变SEMG2蛋白的抗体，其中所述抗体包含重链可变区和轻链可变区，
所述重链可变区的氨基酸序列包含选自由SEQ ID NOs﹕35-41、48-51、54-56和96-100所组成的组中的氨基酸序列或与组中的序列具有至少70％、80％、90％、95％或99％序列同一性；
所述轻链可变区的氨基酸序列包含选自由SEQ ID NOs﹕42-47、52-53、57-69和101-103所组成的组中的氨基酸序列或与组中的序列具有至少70％、80％、90％、95％或99％序列同一性。
在一个具体实施例中，所述重链可变区和轻链可变区选自(a)-(o)组合中的任一种：
(a)所述重链可变区的氨基酸序列包含如SEQ ID NO:35所示氨基酸序列或与其序列具有至少70％、80％、90％、95％或99％序列同一性；所述轻链可变区的氨基酸序列包含如SEQ ID NO:42所示的氨基酸序列或与其序列具有至少70％、80％、90％、95％或99％序列同一性；
(b)所述重链可变区的氨基酸序列包含如SEQ ID NO:36所示氨基酸序列或与其序列具有至少70％、80％、90％、95％或99％序列同一性；所述轻链可变区的氨基酸序列包含如SEQ ID NO:43所示的氨基酸序列或与其序列具有至少70％、80％、90％、95％或99％序列同一性；
(c)所述重链可变区的氨基酸序列包含如SEQ ID NO:37所示氨基酸序列 或与其序列具有至少70％、80％、90％、95％或99％序列同一性；所述轻链可变区的氨基酸序列包含如SEQ ID NO:44所示的氨基酸序列或与其序列具有至少70％、80％、90％、95％或99％序列同一性；
(d)所述重链可变区的氨基酸序列包含如SEQ ID NO:38所示氨基酸序列或与其序列具有至少70％、80％、90％、95％或99％序列同一性；所述轻链可变区的氨基酸序列包含如SEQ ID NO:45所示的氨基酸序列或与其序列具有至少70％、80％、90％、95％或99％序列同一性；
(e)所述重链可变区的氨基酸序列包含如SEQ ID NO:39所示氨基酸序列或与其序列具有至少70％、80％、90％、95％或99％序列同一性；所述轻链可变区的氨基酸序列包含如SEQ ID NO:46所示的氨基酸序列或与其序列具有至少70％、80％、90％、95％或99％序列同一性；
(f)所述重链可变区的氨基酸序列包含如SEQ ID NO:40所示氨基酸序列或与其序列具有至少70％、80％、90％、95％或99％序列同一性；所述轻链可变区的氨基酸序列包含如SEQ ID NO:47所示的氨基酸序列或与其序列具有至少70％、80％、90％、95％或99％序列同一性；
(g)所述重链可变区的氨基酸序列包含如SEQ ID NO:41所示氨基酸序列或与其序列具有至少70％、80％、90％、95％或99％序列同一性；所述轻链可变区的氨基酸序列包含如SEQ ID NO:47所示的氨基酸序列或与其序列具有至少70％、80％、90％、95％或99％序列同一性；
(h)所述重链可变区的氨基酸序列包含如SEQ ID NO:48、49、50、或51所示氨基酸序列或与其序列具有至少70％、80％、90％、95％或99％序列同一性；所述轻链可变区的氨基酸序列包含如SEQ ID NO:52或53所示的氨基酸序列或与其序列具有至少70％、80％、90％、95％或99％序列同一性；
(i)所述重链可变区的氨基酸序列包含如SEQ ID NO:54所示氨基酸序列或与其序列具有至少70％、80％、90％、95％或99％序列同一性；所述轻链可变区的氨基酸序列包含如SEQ ID NO:57所示的氨基酸序列或与其序列具有至少70％、80％、90％、95％或99％序列同一性；
(j)所述重链可变区的氨基酸序列包含如SEQ ID NO:55所示氨基酸序列或与其序列具有至少70％、80％、90％、95％或99％序列同一性；所述轻 链可变区的氨基酸序列包含如SEQ ID NO:58所示的氨基酸序列或与其序列具有至少70％、80％、90％、95％或99％序列同一性；
(k)所述重链可变区的氨基酸序列包含如SEQ ID NO:56所示氨基酸序列或与其序列具有至少70％、80％、90％、95％或99％序列同一性；所述轻链可变区的氨基酸序列包含如SEQ ID NO:59所示的氨基酸序列或与其序列具有至少70％、80％、90％、95％或99％序列同一性；
(l)所述重链可变区的氨基酸序列包含如SEQ ID NO:96所示氨基酸序列或与其序列具有至少70％、80％、90％、95％或99％序列同一性；所述轻链可变区的氨基酸序列包含如SEQ ID NO:59、101、102或103所示的氨基酸序列或与其序列具有至少70％、80％、90％、95％或99％序列同一性；
(m)所述重链可变区的氨基酸序列包含如SEQ ID NO:97所示氨基酸序列或与其序列具有至少70％、80％、90％、95％或99％序列同一性；所述轻链可变区的氨基酸序列包含如SEQ ID NO:59所示的氨基酸序列或与其序列具有至少70％、80％、90％、95％或99％序列同一性；
(n)所述重链可变区的氨基酸序列包含如SEQ ID NO:98所示氨基酸序列或与其序列具有至少70％、80％、90％、95％或99％序列同一性；所述轻链可变区的氨基酸序列包含如SEQ ID NO:103所示的氨基酸序列或与其序列具有至少70％、80％、90％、95％或99％序列同一性；
(o)所述重链可变区的氨基酸序列包含如SEQ ID NO:99或100所示氨基酸序列或与其序列具有至少70％、80％、90％、95％或99％序列同一性；所述轻链可变区的氨基酸序列包含如SEQ ID NO:57所示的氨基酸序列或与其序列具有至少70％、80％、90％、95％或99％序列同一性。
本发明中的抗体可进一步包含连接至多肽的偶联部分，所述偶联部分选自放射性核素、药物、毒素、细胞因子、酶、荧光素、载体蛋白、脂类、和生物素中的一种或多种，其中所述多肽或抗体与所述偶联部分可选择性通过连接子相连，优选所述连接子为肽或多肽。
其中所述抗体选自单克隆抗体、多克隆抗体、抗血清、嵌合抗体、人源化抗体和人抗体。
其中所述抗体选自多特异性抗体、单链Fv(scFv)、单链抗体、抗独特型(抗-Id)抗体、双抗体、微型抗体、纳米抗体、单结构域抗体、Fab片段、F(ab’)片段、二硫化物连接的双特异性Fv(sdFv)和胞内抗体。
又一方面，本发明公开了一种抗原表位肽，其中，所述抗原表位肽源自SEMG2蛋白，所述抗原表位肽的氨基酸包含或具有选自由SEQ ID NO:2(QIEKLVEGKS)、SEQ ID NO:3(QIEKLVEGKSQIQ)、SEQ ID NO:4(QIEKLVEGKSQ)、SEQ ID NO:5(QIEKLVEGKSQI)组成的组中的氨基酸序列。
本发明公开了一种蛋白，所述蛋白的氨基酸序列包含如SEQ ID NO:2(QIEKLVEGKS)、SEQ ID NO:86(QIEKLVEGKS(x)I(x))、SEQ ID NO:87(QIEKLVEGKS(x)I)、或SEQ ID NO:88(QIEKLVEGKS(x))所示氨基酸序列所示的氨基酸序列，优选所述多肽包含如SEQ ID NO:3(QIEKLVEGKSQIQ)、SEQ ID NO:4(QIEKLVEGKSQ)、或SEQ ID NO:5(QIEKLVEGKSQI)或与SEQ ID NO:2-5任一项序列至少90％以上序列一致性的氨基酸序列，更优选如SEQ ID NO:89-94和SEQ ID NO:3(分别对应P1-P6，以及P7)所示；以及N端或C端可选择连接的标签序列。其中本领域技术人员应理解标签蛋白的添加并不会影响所制备的抗体参与SEMG2与CD27结合，该标签蛋白包括但不限于C-Myc、His、GST(谷胱甘肽巯基转移酶)、HA、MBP(麦芽糖结合蛋白)、Flag、SUMO、eGFP/eCFP/eYFP/mCherry等。
在一个具体实施例中，所述多肽具有SEQ ID NO:3(P7：QIEKLVEGKSQIQ)或SEQ ID NO:93(P5)所示氨基酸序列。
本发明还公开了一种制备抗体或其抗原结合片段的方法，其中使用蛋白作为免疫原注射受试者例如小鼠或筛选天然库以制备抗体，所述蛋白的氨基酸序列包含如SEQ ID NO:2(QIEKLVEGKS)、SEQ ID NO:86(QIEKLVEGKS(x)I(x))、SEQ ID NO:87(QIEKLVEGKS(x)I)、或SEQ ID NO:88(QIEKLVEGKS(x))所示氨基酸序列所示的氨基酸序列，优选所述多肽包含如SEQ ID NO:3(QIEKLVEGKSQIQ)、SEQ ID NO:4(QIEKLVEGKSQ)、或SEQ ID NO:5(QIEKLVEGKSQI)或与SEQ ID NO:2-5任一项序列至少90％以上序列一致性的氨基酸序列，更优选如SEQ  ID NO:93(P5)或SEQ ID NO:3(P7)所示。
一个优选的实施方案中，公开了利用SEMG2和CD27结合关键表位多肽作为免疫原，通过鼠杂交瘤、噬菌体展示人源和骆驼源天然库筛选的方法获得分离的抗体的方法。
再一方面，本发明还公开了一种分离的多核苷酸，其编码如前所述的化合物、抗原肽、或蛋白。
本发明公开了一种重组载体，其包含所述的多核苷酸，以及任选的调控序列；优选所述重组载体为克隆载体或表达载体。
其中，所述调控序列选自前导序列、多聚腺苷酸化序列、前肽序列、启动子、信号序列、转录终止子，或其任何组合。
本发明公开了一种宿主细胞，其包含如前所述的重组载体。
其中，所述宿主细胞为原核细胞或真核细胞。
本发明公开了一种药物组合物，其包含如前所述的化合物、如前所述的抗原肽、如前所述的蛋白、如前所述的多核苷酸，如前所述的重组载体、和如前所述的宿主细胞中的一种或更多种。
其中，所述组合物还包含药学上可接受的载体或辅料。
本发明还公开了如前所述的化合物、如前所述的抗原肽、如前所述的蛋白、如前所述的多核苷酸，如前所述的重组载体、或如前所述的宿主细胞在制备用于激动或拮抗SEMG2和CD27相互作用的产品中的用途，优选SEMG2表达于肿瘤细胞，CD27表达于免疫细胞。
本发明还公开了如前所述的化合物、如前所述的抗原肽、如前所述的蛋白、如前所述的多核苷酸，如前所述的重组载体、或如前所述的宿主细胞在在制备用于预防或治疗肿瘤的药物或用于调节针对肿瘤所引起的免疫反应的药物中的用途。
在一个具体实施例中，其中所述肿瘤选自由结直肠癌、肺癌、黑色素瘤、淋巴瘤、肝癌、头颈癌、胃癌、肾癌、膀胱癌、前列腺癌、睾丸癌、子宫内膜癌、乳腺癌、和卵巢癌组成的组中的一种或多种。
本发明还公开了一种筛选用于预防或治疗肿瘤的药物或试剂的方法，通过筛选抑制SEMG2和CD27相互作用的抑制剂或抗体，获得候选药物或试剂。
本发明还公开了一种预防或治疗肿瘤的方法，其包括：
使受试者的免疫细胞诸如淋巴细胞(T淋巴细胞)或肿瘤细胞与有效剂量的如前任一项所述的化合物接触；其中在使用有效量的化合物和受试者的免疫细胞和/或肿瘤细胞接触前，可选择性的检测SEMG2在肿瘤细胞的表达。
其中所述受试者已经接受或正在接受或将要接受额外的抗癌疗法。
其中所述额外的抗癌疗法包括手术、放疗、化疗、免疫疗法或激素疗法。
本发明还公开了一种试剂盒，其包含前所述的化合物、如前所述的抗原肽、如前所述的蛋白、如前所述的多核苷酸，如前所述的重组载体、和如前所述的宿主细胞中的一种或更多种，并容纳于合适的容器中。
本发明还公开了一种体外检测生物样本中SEMG2存在与否的方法，包括：使生物样本与如前所述的化合物接触。
一种抑制肿瘤细胞生长的方法，包括以下步骤：A)分析SEMG2在肿瘤细胞的表达；B)利用可以识别SEMG2的抗体与肿瘤细胞接触，所述的抗体与SEMG2的结合KD<2×10 -8；C)使T淋巴细胞、所述抗体和肿瘤细胞相接触。其中所述KD<2×10 -8、<1×10 -8、<9×10 -9、<8×10 -9、<7×10 -9、<6×10 - 9、<5×10 -9、<4×10 -9、<3×10 -9、<2×10 -9、<1×10 -9、<1×10 -10。
附图说明
图1示出免疫共沉淀实验结果，分为上图和下图。上图证明人源CD27蛋白和SEMG2(Flag标记的)存在物理结合。下图证明鼠源CD27蛋白和SEMG2(Flag标记的)存在物理结合。
图2示出免疫荧光染色与ELISA检测结果，图2(A)证明CD27蛋白和SEMG2蛋白在肿瘤细胞内过表达后存在显著的共定位。图2(B)示出ELISA试验的结果，证明CD27蛋白和SEMG2蛋白在微量反应板上结合的浓度依赖效应，CD27蛋白不与阴性对照蛋白结合且不存在浓度效应。
图3示出免疫共沉淀实验结果，用于检测SEMG2的片段(即P1至P6)是否能够与CD27发生结合。其中P5片段检测到了与CD27的明显结合。
图4示出免疫共沉淀实验结果，用于检测SEMG2的片段(即P4、P5、P6、P7)是否能够与CD27发生结合。其中，P7的序列来自P5的一部分，为“QIEKLVEGKSQIQ”。结果表明包括左右两图。左图显示的是上述SEMG2的片段与人源CD27的结合，右图显示的是SEMG2的片段与鼠源CD27的结合。结果表明，人源和鼠源的CD27均能够和P5、P7片段进行结合。
图5示出通过甘氨酸扫查(Alanine Scan)的方法准确地证明P7的每一个氨基酸对结合CD27蛋白质的贡献。包括A图和B图。A图显示的是将P7的每个氨基酸逐个替换为甘氨酸之后产生的序列，即编号为1-13的突变体氨基酸序列。B图是免疫共沉淀实验结果，表明突变体1-13多肽与GFP的融合蛋白结合CD27的程度；其中突变体5、9完全丧失了与CD27的结合；突变体11、13没有影响SEMG2(497-509)与CD27的结合；其他位点的突变体(1、2、3、4、6、7、8、10、12)在一定程度上减弱了SEMG2(497-509)与CD27的结合。由此可见，SEMG2位于497、498、499、500、501、502、503、504、505、506、508位点的氨基酸对于与CD27的结合具有明显的影响。
图6示出SEMG2表位多肽与CD27的结合，及其对全长SEMG2结合CD27的竞争抑制。(A)是偶联BSA的人源SEMG2(497-509)多肽以及猴源SEMG2多肽可以分别与CD27蛋白在微量反应板上结合，且显著高于与阴性BSA对照，由此可见CD27均能够与人源和猴源的SEMG2(497-509)片段进行结合。(B)是SEMG2来源的多肽及衍生物QIEKLVEGKSQIQ、QIEKLVEGKSQI、QIEKLVEGKSQ、QIAKLVEGKSQ对全长SEMG2与CD27结合的抑制作用。如图所示的不同浓度多肽先与CD27-Fc共同孵育结合，再加入预先包被SEMG2蛋白的微量反应板，孵育后洗去未接合的分子，并以抗Fc二抗-HRP检测并显色。结果表明所述的多肽分子能够抑制全长SEMG2与CD27的结合。
图7示出稳定转染SEMG2或对照空载体的HCT116细胞与激活的人外周血单核细胞共培养凋亡实验。(A)是代表性的细胞凋亡检测照片，绿色视野显示凋亡的细胞。(B)是基于三次独立生物学实验的统计(误差线代表标准差)。
图8示出免疫印迹实验，用于显示SEMG2蛋白质在不同的肿瘤细胞中的表达。肿瘤细胞的名称标记在上方(字体倾斜了45独角)。可见约半数被检测的细胞系有可检测的SEMG2蛋白质表达。
图9示出显示SEMG2蛋白质在不同的肿瘤组织中表达的免疫组织化学(IHC)实验结果。(A)显示了不同的结直肠癌肿瘤组织中SEMG2的表达情况，以正常结直肠组织作为对照；(B)显示了不同的肺癌组织中SEMG2的表达情况，以正常的肺组织作为对照；(C)显示了前列腺癌、黑恶色素瘤、胃癌中SEMG2阳性表达的代表性图片，由于空间所限，此处不详尽列出所有肿瘤类型的检测结果；(D)是SEMG2在所示的不同肿瘤类型中表达的阳性率。阳性表达被定义为额免疫组织化学染色结果为中等或强阳性表达。基于组织芯片(每个芯片包括超过50个组织样本)的统计结果以百分比的形式做图，以显示SEMG2的阳性表达比率。
图10示出Kaplan-Meier因素生存分析的统计结果，提示SEMG2的高表达(定义为SEMG2免疫组织化学染色的中等、强阳性染色)与结直肠癌患者总体生存期的缩短显著关联。P值低于0.001提示高度显著的关联。
图11示出免疫组化实验的结果，上图为调节T淋巴细胞即Treg与SEMG2在肺癌中染色相关性的统计结果，将SEMG2免疫组化染色的强度分为不同级别，分别统计每个视野中Treg(以Foxp3抗体标记)的个数，并进行对比。下图分别为SEMG2阳性和阴性表达情况下Treg标记的代表性图片。
图12示出ELISA实验的结果，纵坐标是标准化后的A405吸收值作为ELISA实验的读数，显示SEMG2和CD27的结合程度；横坐标是抗体加入的浓度。实线表示SEMG2(497-509)作为抗原产生的多抗产生的阻断效应；虚线表示SEMG2全长蛋白作为免疫原产生多抗的阻断效应。可见SEMG2(497-509)产生的多抗发挥阻断效应所需要的浓度更低，即SEMG2(497-509)产生抗体的阻断效价比SEMG2全长蛋白产生抗体的效价更高。这表明SEMG2(497-509)这一关键作用表位的识别使阻断抗体的研发变得更加容易。
图13示出由SEMG2(497-509)表位肽和全长SEMG2蛋白作为免疫原注射 小鼠后获得的阻断性单克隆抗体的数量和抗体总数量。经过杂交瘤融合获得的鼠单克隆抗体中，经过ELISA实验确认能够抑制SEMG2和CD27结合的抗体经统计并显示为黑色柱状图。可见由SEMG2(497-509)表位片段作为免疫原制备的抗体大多数可阻断SEMG2与CD27的结合，阳性率显著高于使用全长SEMG2做免疫原制备的抗体。
图14示出鼠单克隆抗体以及人源化后的鼠单克隆抗体与SEMG2蛋白的结合能力。ELISA试验的读数OD 450吸收值作为纵坐标，横坐标表明了加入不同浓度的抗体。随着抗体在ELISA系统中浓度的增加，OD值逐渐增加，表明SEMG2和鼠源单克隆抗体(图14A)或人源化单克隆抗体(图14B)的结合逐渐增加。拟合的曲线是基于三次独立生物学实验的统计中的一次代表性结果。
图15示出鼠单克隆抗体与BSA-SEMG2(497-509)的结合以及阻断SEMG2与受体蛋白的结合功能。(A)将ELISA试验的读数OD 450吸收值作为纵坐标，横坐标表明了加入不同浓度的抗体，表明SEMG2(497-509)和鼠单克隆抗体的结合逐渐增加。图B则显示鼠单克隆抗体可以阻断SEMG2和CD27的结合，且其阻断作用随着浓度升高而增强。对照的小鼠IgG抗体并未显示阻断功能。纵坐标是标准化后阻断比值作为阻断比率；横坐标表明了加入的不同浓度的抗体。随着抗体在ELISA系统中浓度的增加，SEMG2和CD27的结合逐渐减少。拟合的曲线是基于三次独立生物学实验的统计(误差线代表标准差)。
图16示出ELISA实验的结果，纵坐标是标准化后的A450吸收值作为ELISA实验的读数，显示SEMG2(被固定在ELISA板表面)和加入的CD27-Fc的结合程度；横坐标显示了不同的实验条件，即共同孵育的不同抗体(浓度均为10微克/毫升)：HPA042767和HPA042835为分别针对SEMG2(354-403)和SEMG2(563-574)抗原表位的兔多克隆抗体；MM02、MM05、MM07、MM08、MM13、MM14为针对SEMG2(497-509)表位的鼠单克隆抗体。结果表明，上述针对SEMG2(497-509)表位的鼠单克隆抗体能够阻断SEMG2和CD27的结合；而针对其它表位的抗体不具备此功能。该实验表明，针对SEMG2(497-509)表位的鼠单克隆抗体在功能上属于同 一类抗体。
图17示出不同类型的抗体对T细胞杀伤肿瘤细胞作用的影响。经过激活的PBMC人外周血单核细胞分别和高表达SEMG2的A375人黑色素瘤细胞或LOVO结直肠癌细胞共培养，同时加入不同的抗体：不相关的鼠IgG、HPA042767、HPA042835、MM02、MM05、MM07、MM08、MM13、或MM14。纵坐标是凋亡肿瘤细胞的百分比；横坐标是不同的实验处理条件，即加入的不同抗体。可见针对SEMG2(497-509)表位的鼠单克隆抗体(MM02、MM05、MM07、MM08、MM13、或MM14)显著促进了T细胞对肿瘤的杀伤，而对照的不相关IgG或针对SEMG2(354-403)和SEMG2(563-574)抗原表位的HPA042767和HPA042835抗体未显示此功能。这表明SEMG2(497-509)表位是肿瘤细胞表达的SEMG2免疫逃逸功能的关键位点，而针对该表位的抗体在抗肿瘤免疫调节功能方面属于同一类。
图18示出不同肿瘤细胞在SEMG2阻断性抗体存在的情况下T细胞杀伤的实验结果。其中A375和LOVO是高表达SEMG2蛋白的肿瘤细胞，而DLD1，NCM460和NCI-H1975是SEMG2阴性的细胞。针对上述肿瘤细胞进行T细胞杀伤实验的过程中，加入不同的抗体，即：不相关的鼠IgG抗体、MM02或MM05鼠单抗。横坐标表示不同的肿瘤细胞系，而纵坐标表示肿瘤细胞凋亡的百分比。可见表达SEMG2较高的肿瘤细胞(A375和LOVO)经过抗体处理后可更有效地被T细胞杀伤。而不表达SEMG2的肿瘤细胞(DLD1，NCM460和NCI-H1975)在施用了SEMG2阻断性抗体MM02和MM05后凋亡水平没有明显增加。这表明，SEMG2的阳性表达可做为施用SEMG2阻断性抗体的选择性标志物。在SEMG2阻断性抗体作为抗肿瘤免疫药物使用时，SEMG2的表达对于适用的患者的选择具有指导意义。
图19示出A450吸收值作为ELISA实验的读数，用于检测SEMG2与不同抗体结合的程度。不同的来自于SEMG2的抗原(如左侧所示)包被在ELISA板上，并以HPA04276、HPA042835、MM02、MM05、MM07、MM08、MM13、MM14进行结合，再采用抗鼠二抗(针对HPA04276、HPA042835、MM02、MM05、MM07、MM08)或抗兔二抗(针对HPA04276、 HPA042835)检测结合的抗体。可见MM02、MM05、MM07、MM08、MM13、MM14都结合在SEMG2(497-509)表位，属于同一类；HPA04276结合在SEMG2(354-403)表位，HPA042835结合在SEMG2(563-574)表位。
图20示出ELISA实验检测的数值，即OD450吸收值。该实验将SEMG2(497-509)表位肽及其甘氨酸扫查突变体(即逐个氨基酸替换为甘氨酸)多肽固定在ELISA板上，进一步结合如图所示的不同抗体。该实验用以确定不同单克隆抗体结合的精确氨基酸表位，以及每个氨基酸对结合抗体的相对重要性。可见对照抗体HPA04276、HPA042835步结合上述表位和突变体，而不同位点的氨基酸对抗体结合贡献度不同，可阻断SEMG2和CD27结合的各抗体(MM02、MM05、MM07、MM08、MM13、MM14)所结合的重要氨基酸类似。这表明具备阻断功能的抗体在结合表位方面属于同一类。
图21示出ELISA试验的结果，表明全人源抗体H88-67,H88-93,H88-96以及亲和力成熟的全人源抗体与SEMG2以及BSA-SEMG2(497-509)多肽结合的浓度依赖效应。ELISA试验的读数OD 450吸收值作为纵坐标，横坐标表明了加入不同浓度的抗体。
图22示出的ELISA结果，表明全人源抗体与鼠单克隆抗体对SEMG2的竞争结合的浓度依赖效应。纵坐标是全人源抗体阻断SEMG2与鼠抗结合的比率。随着全人源抗体的浓度的增加，检测到结合到SEMG2的鼠抗的信号逐渐减弱。
图23示出ELISA试验的结果，表明不同的人源抗体H88-93,H88-96,H88-67对于SEMG2和CD27结合的阻断效果。所有的抗体浓度均为10微克/毫升。抗体克隆H88-93、H88-96、H88-67均为使用SEMG2(497-509)表位在噬菌体天然库中所筛选到的全人源抗体。
图24示出T细胞对共培养的A375和LOVO肿瘤细胞杀伤程度，以及人源抗体H88-93、H88-96、H88-67对于该杀伤作用的影响。结果表明这三种针对SEMG2(497-509)表位的抗体均能够显著促进T细胞对表达SEMG2的肿瘤细胞的杀伤。
图25示出生物膜干涉技术测定SEMG2与全人源抗体分子的结合，表明溶液中的全人源抗体与固定到生物传感器的SEMG2蛋白分子的结合与解离的变化，据此计算出全人源抗体与SEMG2的亲和常数。
图26示出SEMG2抗体在A375黑色素瘤小鼠体内模型中显著抑制肿瘤生长。
图27示出人类SEMG2对应的小鼠基因Svs3a纯合敲除和野生型小鼠对比的表性分析结果，包括大体形态、各组织器官切片检查、T淋巴细胞压不同亚型的比例分析、血液生化、肝功能、血常规检查的具体结果。
具体实施方式
下面将通过具体描述，对本发明作进一步的说明。
除非另有限定，本文中所使用的所有技术和科学术语具有与本发明所属技术领域的普通技术人员通常理解相同的含义。
本申请中，单数形式“一个”、“该”包括复数对象，除非上下文另外清楚规定。
如本文所用，术语“受试者”包括任何人或非人动物。术语“非人类动物”包括所有脊椎动物，例如哺乳动物和非哺乳动物，例如非人灵长类动物、绵羊、狗、猫、马、牛、鸡、大鼠、小鼠、两栖动物、爬行动物等。除非另有说明，否则术语“患者”或“受试者”可互换使用。在本发明中，优选的受试者是人类。
如本文所用，术语“SEMG2”是人精囊腺凝固蛋白2，人类精液的主要成分之一，由精囊腺分泌，形成胶状物质包被精子细胞并限制其运动。精液中由前列腺分泌的蛋白水解酶和纤维蛋白溶酶可以分解精囊腺凝固蛋白并促使精液液化，使精子更自由地运动。SEMG2抑制精子运动的机制还可能包括对精子细胞膜的结合与影响。参见Yoshida K,Karzai ZT,Krishna Z,Yoshika M,Kawano N,Yoshida M,等，Cell Motil Cytoskeleton.2009；66(2):99-108.从SEMG2蛋白中分离出的“SgII A”多肽具有抗菌活性，序列为H-KQEGRDHDKSKGHFHMIVIHHKGGQAHHG-OH。需要说明的 是，该抗菌肽序列与本发明所述的SEMG2和CD27结合的关键氨基酸序列不同，位于SEMG2的完全不同区域。参见
Figure PCTCN2021073100-appb-000001
AM,Malm J,Frohm B,Martellini JA,Giwercman A,
Figure PCTCN2021073100-appb-000002
M,等，J Immunol.2008；181(5):3413-21.此外，SEMG2还被报道结合锌离子并影响前列腺蛋白水解酶PSA的活性。参见Jonsson M,Linse S,Frohm B,Lundwall A,Malm J.Biochem J.2005；387(Pt 2):447-53。
如本文所用，术语如本文中所使用，术语“抗体”包括完整抗体和任何抗原结合片段(即“抗原结合部分”)或其单链。“抗体”是指包含至少两条重链(H)和两条轻链(L)并通过二硫键相互连接的，或其抗原结合部分的蛋白质。每条重链由重链可变区(本文缩写为VH)和重链恒定区组成。重链恒定区由三个结构域，CH1，CH2和CH3组成。每条轻链由轻链可变区(本文缩写为VL)和轻链恒定区的。轻链恒定区由一个结构域CL组成。VH和VL区可以进一步细分成高变区，称为互补决定区(CDR)，与更保守的称为构架区(FR)的区域散布。每个VH和VL由三个CDR和四个FR组成，从氨基末端到羧基末端以下面的顺序排列：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。重链和轻链的可变区包含与抗原相互作用的结合结构域。
术语“抗体”，在本申请中所用的是指免疫球蛋白或其片段或它们的衍生物，并且包括其包含的抗原结合位点的任何多肽，而不管其是否是在体外或体内产生。该术语包括，但不限于，多克隆、单克隆、单特异性的、多特异性的、非特异性的、人源化、单链的、嵌合的、合成的、重组的、杂合的、突变的、嫁接的抗体。术语“抗体”还包括抗体片段例如Fab、F(ab')2、FV、scFv、Fd、dAb和其它保留抗原结合功能的抗体片段，即，能够与PD-1的特异性结合。通常情况下，这样的片段将包括抗原结合片段。
术语“抗原结合片段”、“抗原结合结构域”和“结合片段”是指一种抗体分子，其包含负责具体的抗体和抗原之间的结合的氨基酸。例如，其中的抗原是大的，抗原结合片段只结合抗原的一部分。即抗原分子中负责与抗原结合片段特异性相互作用的部分被称为“表位”或“抗原决定簇”。
抗原结合片段通常包括抗体轻链可变区(VL)和抗体重链可变区(VH)，然而，它不一定必须包括两者。例如，一个所谓的Fd抗体片段仅由VH结构域组成，但仍保留了完整抗体的一些抗原结合功能。
上述术语“表位”定义为抗原决定簇，其特异性结合/识别结合片段。结合片段可以特异性与针对靶结构独特的构象或连续表位进行结合/反应，构象或不连续表位的特征在于多肽抗原在一级序列中是分离的两个或多个离散的氨基酸残基，但多肽折叠成天然蛋白/抗原时是一起聚集在在分子的表面上的。表位的两个或多个离散的氨基酸残基存在于一个或多个多肽链的独立部分。当多肽链折叠成三维结构，这些残基聚集在分子表面以构成表位。与此相反，由两个或多个离散的氨基酸残基组成的连续或线性表位，其存在于多肽链的单个线性区段。
术语“治疗”和“治疗方法”是指治疗性治疗和预防性/预防措施。那些需要治疗的包括已具有特定医学病症，以及那些可能最终获得该病症的个体。
本文所用的术语“载体”是指运输、转导和在靶细胞表达被包含的外源目的基因(例如本发明所述的多核苷酸)的分子工具，所述工具提供合适的在靶细胞中起始转录的核苷酸序列，即启动子。
本发明中术语“标签蛋白”和“蛋白标签”可互换，是指利用DNA体外重组技术,与目的蛋白一起融合表达的一种多肽或者蛋白,以便于蛋白表达、检测、示踪、纯化。标签蛋白包括但不限于His6、Flag、GST、MBP、HA、GFP、Myc。
实施例
下述实施例中的实验方法，如无特殊说明，均为常规方法。将参照下述非限制性实验实施例进一步理解本发明。
实施例1：SEMG2和CD27蛋白质结合的检测
在直径为10厘米的培养平皿中，对人HEK239细胞进行共转染，包括pcDNA3-Flag-SEMG2质粒和pcDNA3-HA-CD27质粒的复合物共转染细胞后48小时，收集细胞裂解液，并采用标准的免疫沉淀流程对裂解液中的CD27进行富集。用于沉淀的抗体是Flag抗体，对照组采用IgG非特异 性抗体。其后进行免疫印迹实验(Western Blot)，采用HA抗体检测CD27的共沉淀量，并采用Flag抗体检测SEMG2的沉淀量。在免疫印迹法中，细胞在1％Triton X-100(TBS pH7.6)中用罗氏完全蛋白酶抑制剂在冰上溶解30分钟，然后通过离心使不溶性物质颗粒化。在50mM DTT的SDS样品缓冲液中将溶解物加热到100℃，10分钟，用SDS-PAGE分离，并转移到PVDF膜(微孔)。在5％的牛血清白蛋白(BSA)中，在TBS中阻断细胞膜，用所示抗体探测细胞膜，并用West Pico(Thermo Fisher Scientific)观察反应带。
在共免疫沉淀实验中，细胞在IP缓冲液(Thermo Scientific)和罗氏完全蛋白酶抑制剂中溶解10分钟，然后在室温下添加苯并酶(sigma)25分钟。然后在4℃下以15000转/分的速度离心溶解液以去除沉淀。然后将上清液用一级抗体在4℃下缓慢游动培养过夜，然后添加蛋白质A或蛋白质G dynabeads，在4℃下培养2小时。在PBST中洗涤4次(PBS含0.01％吐温20)，在SDS样品缓冲液中用50mM DTT在100℃下洗脱10分钟，用SDS分离，如前所述进行免疫印迹。
结果显示，利用Flag抗体进行沉淀，可以使沉淀复合物同时含有SEMG2和CD27，而对照组中不含有SEMG2和CD27。实验结果见图1。该实验表明SEMG2和人源CD27的物理结合。此外，将SEMG2和鼠源CD27的结合进行检测，实验方法同上。该实验中用鼠源CD27载体替代人源CD27转染HEK293细胞，其它实验条件不变。实验结果见图1。该实验表明SEMG2和鼠源CD27的物理结合。
在上述共转染实验条件下，取10厘米平皿中预先放置的细胞爬片，进行固定、透膜、封闭处理，进一步用含有HA标签(鼠抗)和Flag标签(兔抗)的抗体同时对CD27和SEMG2进行免疫标记，再用二抗分别标记显色为红色和绿色。利用荧光共聚焦显微镜观察SEMG2和CD27在细胞中的共定位情况。结果见图2。共表达的SEMG2和CD27蛋白质在细胞中显示了非常明显的共定位，甚至定位模式接近完全相同。这符合SEMG2和CD27蛋白质相结合的发现。
实施例2：SEMG2(497-509)片段与CD27蛋白质的结合
为了进一步确定SEMG2的哪一部分和CD27发生结合，进一步设计SEMG2蛋白质的片段。将全长的SEMG2蛋白质氨基酸序列(SEQ ID NO:1)进行分割后产生6段序列，分别与GFP融合，命名为SEMG2-P1、SEMG2-P2、SEMG2-P3、SEMG2-P4、SEMG2-P5、SEMG2-P6(具体序列见表1，对应的分别简称为P1-P6)。将表达这些氨基酸序列的质粒与CD27共转染HEK293细胞，并进行免疫共沉淀实验，识别SEMG2与CD27发生结合的主要片段。免疫共沉淀的结果如图3。只有SEMG2-P5与CD27存在明显结合，而SEMG2-P1、SEMG2-P2、SEMG2-P3、SEMG2-P4、SEMG2-P6均未与CD27发生结合。上述结果表明，SEMG2-P5片段是结合CD27的主要部分。
SEQ ID NO:1(人SEMG2)：
Figure PCTCN2021073100-appb-000003
表1：构建SEMG2表达片段对应的氨基酸序列
Figure PCTCN2021073100-appb-000004
Figure PCTCN2021073100-appb-000005
为了进一步明确SEMG2-P5序列中与CD27结合的关键氨基酸，选取了SEMG2(497-509)片段并命名为SEMG2-P7(具体序列为QIEKLVEGKSQIQ，简称为P7或者SP7)。将SEMG2-P7(497-509)、SEMG2-P5(阳性对照)、SEMG2-P4(阴性对照)、SEMG2-P6(阴性对照)分别与CD27共转染HEK293细胞，包括人源的CD27和鼠源的CD27。其后进行的免疫共沉淀实验结果表明SEMG2-P7、SEMG2-P5均和CD27发生结合，并且人源的CD27和鼠源的CD27结果相同。实验结果见图4。这个免疫共沉淀实验确认了SEMG2(497-509)是与人源和鼠源CD27结合的主要结构。
实施例3：利用甘氨酸扫查的方法精确表征SEMG2结合CD27的关键氨基酸
为了以更高的分辨率表征SEMG2结合CD27的表位，更准确地说明SEMG2(497-509)的每一个氨基酸对结合CD27蛋白质的贡献，将SEMG2(497-509)的每个氨基酸逐个替换为甘氨酸，产生的序列即编号为1-13的突变体氨基酸序列(见图5)。将这些突变体质粒和CD27表达载体共转染HEK293细胞，并利用免疫共沉淀实验检测变体1-13多肽与GFP的融合蛋白结合CD27的程度。实验结果见图5，其中突变体5、9完全丧失了与CD27的结合；突变体11、13没有影响SEMG2(497-509)与CD27的结合；其他位点的突变体(1、2、3、4、6、7、8、10、12)在一定程度上减弱了SEMG2(497-509)与CD27的结合。由此可见，SEMG2位于497、498、499、500、501、502、503、504、505、506、508位点的氨基酸对于与CD27的结合具有明显的影响。将多肽序列497-509偶联到BSA，包被96孔微量板，以不同浓度的CD27-hFc为一抗，检测CD27结合到多肽序列的能力，实验结果见图6。结果表明CD27可以结合到SEMG2具有浓度依赖效应。
实施例4：肿瘤细胞表达的SEMG2抑制免疫细胞对肿瘤的杀伤作用
T细胞介导的肿瘤细胞杀伤试验。用SEMG2表达载体或对照空载体稳定转染HCT116人结直肠癌细胞，用caspase3/7裂解法(绿色荧光法)测定活化PBMC共培养后凋亡细胞的比例。具体地，将稳定表达SEMG2的HCT116细胞接种在96孔板中。用100ng/mL的CD3抗体、100ng/mL的CD28抗体和10ng/mL的IL2(#317303；#302913；#589102，BioLegend)分别激活人外周血单核细胞(PBMC；#70025，Stem Cell)，并在荧光caspase-3/7底物存在下以10:1的比例与上述大肠癌细胞共培养(#4440，Essen Bioscience)。10小时后，在荧光显微镜下观察细胞。结果如图7所示，相对于对照细胞，SEMG2过表达的肿瘤细胞在与活化PBMC共培养后凋亡的发生显著减少。本实验的结果支持SEMG2具有抑制免疫细胞功能的作用。
实施例5：检测SEMG2在不同肿瘤细胞中的表达
在含有5％二氧化碳的37℃细胞培养箱中，利用含有10％小牛血清的DMEM培养基孵育人的不同类型肿瘤细胞，包括LOVO结直肠癌、RKO结直肠癌、PC3前列腺癌、A375恶性黑色素瘤、SW1116结直肠癌、DLD1结直肠癌、HEK293人肾上皮细胞系、HepG2肝细胞癌、NCM460人正常结肠上皮细胞、NCI-H1975人非小细胞肺腺癌细胞、CaCo2结肠腺癌、HT29结直肠腺癌、SW1990人胰腺癌、AGS人胃腺癌、SW480结直肠癌、SaOS2骨肉瘤、GES-1人胃粘膜细胞等。
在免疫印迹法中，细胞在1％Triton X-100(TBS pH7.6)中用罗氏完全蛋白酶抑制剂在冰上溶解30分钟，然后通过离心使不溶性物质颗粒化。在50mM DTT的SDS样品缓冲液中将溶解物加热到100℃，10分钟，用SDS-PAGE分离，并转移到PVDF膜(微孔)。在5％的牛血清白蛋白(BSA)中，在TBS中阻断细胞膜，用所示SEMG2和GAPDH内参的特异性一抗分别探测细胞膜，并用HRP偶联的二抗标记一抗，West Pico(Thermo Fisher Scientific)观察反应带。结果见图8，表明在GES-1人胃黏膜细胞、NCM460人正常结肠上皮细胞中未见SEMG2的表达，但是在多种恶性肿瘤细胞中 存在可见的SEMG2表达，包括LOVO结直肠癌、RKO结直肠癌、PC3前列腺癌、A375恶性黑色素瘤、SW1116结直肠癌、HEK293人肾上皮细胞系、HepG2肝细胞癌、CaCo2结肠腺癌、HT29结直肠腺癌、AGS人胃腺癌、SW480结直肠癌、SaOS2骨肉瘤。该结果表明，SEMG2是一种肿瘤中普遍表达的蛋白质。
实施例6：利用免疫组织化学(IHC)检测SEMG2在不同肿瘤组织中的表达。
免疫组织化学染色中，我们从上海芯超生物技术公司获得了多种肿瘤的组织芯片。简单地描述，组织标本用抗SEMG2(HPA042767，购自Sigma Aldrich，通过1:100稀释)的抗体和生物素结合的二级抗体培养，然后用抗生物素-生物素-过氧化物酶复合物培养。用氨基乙基咔唑显色剂进行观察。如组织学评分，染色强度分为高(3)、中(2)、低(1)和阴性(0)四组。
首先，染色了结直肠癌肿瘤组织芯片中SEMG2的表达情况，以正常结直肠组织作为对照；发现在肺癌组织中存在比较广泛的SEMG2高表达情况。结果见图9。
其次，染色了不同的肺癌组织中SEMG2的表达情况，以正常的肺组织作为对照；发现在肺癌组织中存在比较广泛的SEMG2高表达情况。结果见图9。
再次，染色了前列腺癌、黑恶色素瘤、胃癌中SEMG2阳性表达的情况，结果见图9。
最后，基于上述组织芯片的染色，计算了SEMG2在所示的不同肿瘤类型中表达的阳性率。阳性表达被定义为额免疫组织化学染色结果为中等或强阳性表达。基于组织芯片(每个芯片包括超过50个组织样本)的统计结果以百分比的形式显示在图9中。
实施例7：证明SEMG2高表达和肿瘤预后不良之间的关联
免疫组织化学染色中，我们从上海芯超生物技术公司获得了多种肿瘤 的组织芯片，均带有生存期信息的随访资料。通过实施例6所述的方法进行免疫组化检测。以结直肠癌为例，依据SEMG2的表达量对患者进行分类，分为SEMG2高(免疫组化评分为2、3)和SEMG2低(免疫组化评分为0、1)两组，通过Kaplan-Meier方法比较两组患者的总体生存期，结果如图10所示，发现SEMG2高表达的患者生存期显著短于SEMG2低表达的肿瘤患者。其他肿瘤如肺癌(P<0.05)、胃癌(P<0.05)等均存在此显著关联。上述结果提示SEMG2是肿瘤免疫逃避关键分子，并可能作为新的抗肿瘤靶标。
实施例8：证明SEMG2高表达和具有免疫抑制功能的Treg调节性T淋巴细胞浸润的相关性
为了分析肿瘤组织中SEMG2表达和具有免疫抑制功能的Treg调节性T淋巴细胞浸润的相关性，我们用免疫组织化学方法检测了购自上海芯超生物技术公司的多种肿瘤组织组织芯片，以肺癌为例，依据SEMG2的表达量分别对Treg在肿瘤组织中的浸润(Foxp3抗体标记)进行对比，发现SEMG2表达量越高，Treg的浸润越多(各组织间差异存在统计学显著性，P<0.05)，见图11。该结果表明，SEMG2的表达和肿瘤局部免疫微环境显著相关，SEMG2的表达可作为免疫抑制状态的生物标志物，以及肿瘤免疫调节剂使用的伴随诊断标志物。
实施例9：以SEMG2(497-509)片段做免疫原制备抗体
具体包括以下步骤：(1)抗原制备，以SEMG2(497-509)即“QIEKLVEGKSQIQ”序列合成多肽，并偶联至VLP载体用于免疫；另一组使用全长的SEMG2蛋白质(购自Cusabio,货号CSB-YP021002HU)作为免疫原。(2)第一次免疫:用剪刀剪去家兔两后脚掌的部分兔毛，以酒精及碘酒消毒皮肤，用2mL注射器吸取弗氏完全佐剂(FCA)乳化的抗原液1mL，每侧脚掌皮下各注入0.5mL。(3).第二次免疫：间隔10-14天后，于两侧窝及鼠蹊部肿大的淋巴结内注入抗原液，每个淋巴结注0.1mL，其余注入淋巴结附近皮下共1mL。如淋巴结未肿大或肿大不明显时，直接注入两侧窝及鼠蹊部皮下。(4)间隔7-10天后，从耳静脉采血0.5～1.0 mL，分离血清，测定血清效价，使用间接ELISA方法，用10μg/mL抗原包被，效价1:64000以上进行取血。(5)若效价未达到要求，用不加佐剂的抗原液耳静脉内注射免疫。即于1周内注射3次，分别为0.1、0.3、0.5mL。间隔1周再试血。如效价达到要求应立即取血，收集全部抗血清。
多克隆抗体纯化的具体实验步骤包括：(1)准备蛋白A sepharose CL-4B亲和柱。准备10mL蛋白A sepharose CL-4B填料，在真空瓶中将等体积的填料和TBS缓冲溶液混合，搅拌。抽真空15分钟以除去填料中的气泡。将蛋白A sepharose CL-4B填料缓慢加入玻璃柱中，利用泵控制填充速度为1mL/分-2mL/分，避免柱干，利用10倍于床体积并经过预冷的TBS缓冲溶液平衡柱子。(2)制备抗血清。将抗血清放入冰水或4℃冰箱中缓慢解冻以避免蛋白质的聚集。在蛋白质解冻过程中出现的聚集可通过37℃预热而溶解。加入固体叠氮化钠至浓度为0.05％，4℃，15,000×g离心5分钟，移出澄清的抗血清再经过滤器过滤除去多余的脂。(3)亲和层析。将抗体用TBS缓冲溶液以1：5的比例进行稀释，再用过滤器进行过滤。以每分钟0.5mL的速度将抗血清上到柱上,为保证抗血清与填料的结合，需连续上柱2次并保留上样流出液。用TBS缓冲溶液清洗柱子至Aλ280nm<0.008后加Ph 2.7洗脱缓冲溶液，以0.5mL/min的速度洗脱至所有蛋白均流下来。用已经加入100μL中和缓冲溶液的1.5mL EP管分管收集洗脱液，混匀后用pH试纸检查洗脱液的pH，如果pH低于7可利用中和缓冲液调至约pH7.4以防止抗体的变性。在柱中加入10mL，pH1.9洗脱缓冲溶液，按上述方法收集洗脱液至Aλ280nm<0.008。利用分光光度计测定各管中蛋白质的含量。
实施例10：对比SEMG2(497-509)和全长SEMG2作为免疫原制备抗体所具备的阻断作用
因为SEMG2(497-509)序列片段是SEMG2结合CD27的关键表位，而且具有比较短的序列，因此用SEMG2(497-509)作为免疫原制备抗体，在理论上比用全长SEMG2制备抗体更容易获得具备阻断SEMG2和CD27结合功能的功能抗体分子。为了进行直接比较，采用前述实施实例中描述的ELISA实验，验证两种方法产生抗体的有效浓度的差别。将SEMG2(497- 509)作为免疫原制备的抗体、用全长SEMG2制备的抗体都在不同的浓度下加入酶联免疫吸附试验(ELISA)反应体系(10^-2,10^-1,10^0,10^1,10^2,10^3,10^4μg/mL)，并测量ELISA结合数值。酶联免疫吸附试验具体步骤如下：(1)用50mM的碳酸盐包被缓冲液(pH 9.6)溶解SEMG2蛋白质抗原，使抗原浓度为10μg/mL，加100μL/孔到96孔酶标板(购自康宁公司)，4℃放置过夜。(2)第二天弃去包被液后，用PBST洗涤3次，每孔加入150μL的1％BSA 37℃封闭2小时。(3)PBST洗涤3次后，每孔加入所示的抗体(以SEMG2(497-509)作为免疫原产生的多克隆抗体、以全长SEMG2作为免疫原生产的多克隆抗体)至如图12所示的不同终浓度，并加入10μg/ml的CD27-Fc融合蛋白(即人源CD27蛋白质的胞外区融合人抗体Fc段)，在37℃孵育2小时。(4)PBST洗涤5次后，加入100μl稀释后的HRP标记的抗人Fc二抗，37℃孵育1小时。(5)PBST洗涤5次后，显色剂显色20min后，酶标仪上读取A450吸收值。
实验结果如图10所示，SEMG2(497-509)作为抗原产生的抗体，在更低的浓度即可使ELISA检测的SEMG2和CD27结合降低50％，而SEMG2全长蛋白作为免疫原产生的多抗在更高的浓度才能发挥此作用(所需剂量超过前者的300倍)。即SEMG2(497-509)产生抗体的阻断效价比SEMG2全长蛋白产生抗体的效价高300倍以上。这表明SEMG2(497-509)这一关键作用表位的识别使阻断抗体的研发变得更加容易，使本领域的技术人员能够更容易地获得可阻断SEMG2和CD27结合的抗体。
实施例11：利用SEMG2(497-509)表位肽和SEMG2全长蛋白制备小鼠单克隆抗体
将SEMG2(497-509)序列合成多肽，并偶联至VLP载体用于免疫；利用HEK293细胞表达SEMG2全长蛋白质，经过检测达到92％纯度，并利用ELISA实验验证了SEMG2蛋白质具备结合CD27的活性。利用蛋白和多肽抗原分别免疫10只小鼠，进行多次免疫以增强效果：(1)初次免疫，抗原50μg/只，加福氏完全佐剂皮下多点注射，间隔3周；(2)第二次免疫，剂量途径同上，加福氏不完全佐剂，间隔3周；(3)第三次免疫，剂量同上，不加佐剂，腹腔注射间隔3周；(4)加强免疫，剂量50μg，腹 腔注射。最后一次注射3天后采血测其效价，检测免疫效果，分别选择效价较高的小鼠进行杂交瘤融合筛选。亚克隆后通过ELISA检测单克隆抗体与目标抗原的结合，并通过ELISA实验检测不同的单克隆抗体阻断SEMG2与CD27结合的功能。
对杂交瘤产生的单克隆进行ELISA实验的筛选，在以SEMG2(497-509)为免疫原制备的单克隆抗体中，首批验证的27株抗体中具备阻断功能(抑制SEMG2和CD27结合)的抗体为19株，如图13所示。而以SEMG2全长蛋白免疫原制备的单克隆抗体中，分批验证了共108株抗体后仅得到1株具备阻断功能的抗体，如图13所示。
因此，以SEMG2(497-509)表位肽为免疫原制备单克隆抗体，显著地提高了发现阻断性抗体的效率，鼠源单抗的亚型(表2)与序列(表3)如下表所示。
表2.鼠源单抗的亚型
抗体克隆号 亚型 轻链
MM02 mIgG2b kappa
MM05 mIgG1 kappa
MM07 mIgG2b kappa
MM08 mIgG1 kappa
MM13 mIgG1 kappa
MM14 mIgG2b kappa
MM15 mIgG2a kappa
表3.鼠源单抗的重链和轻链可变区序列
Figure PCTCN2021073100-appb-000006
Figure PCTCN2021073100-appb-000007
表4.鼠源抗体的CDR氨基酸序列
Figure PCTCN2021073100-appb-000008
Figure PCTCN2021073100-appb-000009
以SEMG2蛋白包被ELISA板，将梯度稀释的鼠单克隆抗体作为一抗，用抗鼠二抗检测了上述鼠单克隆抗体对SEMG2的结合能力，结果如图14A所示。显示鼠单克隆抗体对SEMG2蛋白具有很好的亲和能力。
实施例12：抗SEMG2mAb的人源化
小鼠抗SEMG2单克隆抗体MM05被人源化，以降低用于人类患者时的免疫原性。将重链和轻链可变区(VH和VL)的序列与蛋白质数据库(PDB)中的人抗体序列进行比较，并建立同源性模型。将小鼠mAb的重链和轻链中的CDR移植到人框架区中，该框架区最有可能维持抗原结合所需的适当结构。在必要时，设计了从人残基到小鼠残基的反向突变或其他突变，例如：人源化后的轻链VL-V2的第95位氨基酸从K突变为Q，相应的该轻链的CDR3序列根据IMGT分析转变为QQSYSLPWT(SEQ ID NO:95)。人源化的VH和VL区分别与人IgG1重链和κ轻链的恒定区融合。使用对应于mAb序列的构建载体在293E细胞中进行瞬时转染，并使用ELISA分析纯化的mAb 与SEMG2蛋白的结合能力。结果以吸光度显示，其中较高的吸光度表示较高的人源化抗体与SEMG2相互作用水平。本发明获得的8个人源化抗体的CDR、轻链可变区和重链可变区，轻链和重链的氨基酸序列参见如上文所述的表4以及如下表5。图14B显示了梯度稀释的人源化单克隆抗体对SEMG2蛋白结合的拟合曲线，结果显示人源化抗体保持了鼠源单克隆抗体对SEMG2蛋白的结合能力。
表5.MM05人源化抗体的VH与VL氨基酸序列
Figure PCTCN2021073100-appb-000010
实施例13：对比SEMG2(497-509)表位特异性抗体和其它表位的特异性抗体在阻断SEMG2和CD27结合方面的功能
为了证明SEMG2(497-509)表位对于制备阻断性抗体的重要性，进一步对比了针对不同SEMG2表位的抗体在阻断SEMG2和CD27结合方面的功能。已知现有的HPA042767和HPA042835商业化抗体(均购于Sigma Aldrich公司)，是分别针对SEMG2(354-403)和SEMG2(563-574)抗原表位的兔多克隆抗体。
首先，在不同浓度范围内对比SEMG2(497-509)表位特异性抗体(例如 MM02、MM05)与其它表位的特异性抗体(例如HPA042767)在阻断SEMG2和CD27结合方面的功能。上述抗体对SEMG2(497-509)表位的结合通过ELISA实验进行了确认：MM02和MM05能够结合SEMG2(497-509)，而HPA042767在大浓度范围内均不能结合该表位，如图15A所示。如实施例11所述的ELISA实验分析不同的抗体(不相关的鼠IgG，MM02，MM05，HPA042767)对SEMG2和CD27结合的阻断功能。如图15B所示，随着MM02和MM05浓度的升高，SEMG2和CD27的结合逐渐降低，而该现象在不相关的鼠IgG和HPA042767抗体均为观察到，表明后者在大浓度范围内不具备阻断SEMG2和CD27的结合的功能。这些结果支持SEMG2(497-509)表位对于制备阻断性抗体的重要性。
进一步地，在相同抗体浓度的条件下，对比不同抗体对SEMG2和CD27结合的影响。在上述的ELISA实验中，采用相同浓度(10微克/毫升)抗体，并检测SEMG2和CD27结合的强度。结果如图16所示，针对SEMG2(497-509)表位的MM02、MM05、MM07、MM08、MM13、MM14抗体均显著地降低了SEMG2和CD27的结合；而针对SEMG2其它表位的HPA042767和HPA042835抗体均不能降低SEMG2和CD27的结合。
实施例14：对比SEMG2(497-509)表位特异性抗体和其它表位的特异性抗体对活化的PBMC杀伤肿瘤细胞的影响。
在前述的实施例中，SEMG2显示出抑制活化的PBMC杀伤肿瘤细胞的功能。由于SEMG2可能通过结合CD27来发挥上述作用，且SEMG2(497-509)表位是结合CD27的关键位点，因此SEMG2(497-509)表位特异性抗体可能中和SEMG2对PBMC杀伤肿瘤细胞的影响。
为了验证上述假设，对比了不同表位特异性的抗体对于活化的PBMC杀伤肿瘤细胞的影响。具体地，将高表达SEMG2的A375人黑色素瘤和LOVO人结直肠癌细胞接种在96孔板中。用100ng/mL的CD3抗体、100ng/mL的CD28抗体和10ng/mL的IL2(#317303；#302913；#589102，BioLegend)分别激活人外周血单核细胞(PBMC；#70025，Stem Cell)，并在荧光caspase-3/7底物存在下以10:1的比例与上述肿瘤细胞共培养(#4440， Essen Bioscience)。10小时后，在荧光显微镜下观察细胞。结果如图17所示，HPA042767和HPA042835抗体均不能影响活化的PBMC杀伤肿瘤细胞；而针对SEMG2(497-509)表位的MM02、MM05、MM07、MM08、MM13、MM14抗体显著地提高了凋亡的肿瘤细胞比率。上述结果表明，SEMG2(497-509)表位特异性抗体能够中和SEMG2的活性(即消除SEMG2对PBMC杀伤肿瘤细胞的抑制作用)。
实施例15：验证SEMG2的表达量与阻断性抗体促进PBMC杀伤肿瘤细胞功能的相关性
由于SEMG2的表达是其发挥抑制肿瘤特异性免疫的前提，因此SEMG2的表达也是适合施用SEMG2阻断性抗体的潜在条件。在理论上，表达SEMG2高的肿瘤细胞，中和SEMG2活性以后PBMC对肿瘤细胞的杀伤会有相对的升高；而不表达SEMG2的肿瘤细胞可能不依赖SEMG2发挥免疫逃逸功能，因此中和SEMG2活性后PBMC对肿瘤细胞的杀伤可能不会产生显著变化。
为了验证上述假设，选取了高表达SEMG2的肿瘤细胞(A375，LOVO)，以及SEMG2阴性的肿瘤细胞(DLD1，NCM460和NCI-H1975)。针对上述肿瘤细胞进行PBMC杀伤实验的过程中，加入不同的抗体(不相关的鼠IgG抗体，MM02或MM05抗体)。结果如图18所示，MM02和MM05抗体显著升高了活化的PBMC对SEMG2阳性肿瘤细胞(A375，LOVO)的杀伤，而对SEMG2阴性的肿瘤细胞(DLD1，NCM460和NCI-H1975)的杀伤无明显影响。因此，上述实验结果表明SEMG2的阳性表达是施用SEMG2和CD27阻断性抗体的筛选条件，即相应的生物标志物。
实施例16：SEMG2和CD27结合阻断性抗体相关表位的准确定义
为了明确地区分阻断性抗体(即可以抑制SEMG2和CD27结合的抗体)与非阻断性抗体在结合表位上的区别，建立了相应的ELISA分析方法。具体地，分别将SEMG2全长蛋白(1-582)、SEMG2(354-403)片段、SEMG2(442-453)片段、SEMG2(497-509)片段、SEMG2(563-574)片段固定在ELISA板上，并加入相同浓度的抗体(MM02、MM05、MM07、MM08、 MM13、MM14、HPA042767和HPA042835)，再用抗鼠或抗兔的二抗检测结合的相应抗体。结果如图19所示，MM02、MM05、MM07、MM08、MM13、MM14均结合在SEMG2(497-509)表位，HPA042767结合在SEMG2(354-403)表位，HPA042835结合在SEMG2(497-509)表位，而上述抗体均未结合SEMG2(442-453)对照片段。这些结果支持上述抗体的标为特异性。
为了进一步准确地定义阻断性抗体MM02、MM05、MM07、MM08、MM13、MM14结合的准确表位(具体到单个氨基酸的水平)，建立了相应的ELISA分析方法。如图20所示，SEMG2(497-509)多肽以及逐个氨基酸替换为甘氨酸的一组多肽序列(甘氨酸突变扫查序列组)被固定在ELISA板上，并分别加入相同浓度的抗体(MM02、MM05、MM07、MM08、MM13、MM14、HPA042767和HPA042835)，再用抗鼠或抗兔的二抗检测结合的相应抗体。结果如图20所示，HPA042767和HPA042835抗体未结合上述序列，表明该实验的特异性以及这两种抗体不同的表位类别。同时，SEMG2(497-509)序列内不同的氨基酸被替换为甘氨酸后，对同类阻断性抗体(MM02、MM05、MM07、MM08、MM13、MM14)的结合产生了不同程度的影响。例如：位于507和509位点的氨基酸被替换后没有明显影响MM02及同类抗体的结合；位于501和506位点的氨基酸被替换后比较显著地影响了MM02及同类抗体的结合(降幅超过70％)；其它位点的氨基酸被替换后，在一定程度上影响了MM02及同类抗体的结合。上述结果精确地定义了MM02及同类抗体(即可阻断SEMG2和CD27结合的抗体)相关的表位氨基酸，以及各个氨基酸对结合的贡献度。此外，上述参SEMG2与结合阻断性抗体的关键氨基酸和参与结合CD27的关键氨基酸存在较高的一致性，这表明MM02及其同类抗体是与CD27竞争结合SEMG2，验证了抗体作用的分子机制。
实施例17：利用SEMG2(497-509)表位制备和筛选全人源抗体，用于阻断SEMG2和CD27的结合，并促进PBMC对肿瘤细胞的杀伤
由前述实施例的结果得知SEMG2(497-509)表位在制备阻断性抗体中的重要作用，将这一表位应用于全人源抗体的筛选。具体地，首先进行多 肽抗原制备及人天然抗体库筛选。将SEMG2(497-509)多肽合成后分别偶联到BSA和KLH上，在全人源噬菌体展示抗体库中进行筛选。利用ELISA实验挑选可结合抗原表位的克隆进行初筛，对单个克隆后测序得到不同的独特序列，进行亲和力排序，对亲和力较高的抗原结合片段(Fab)构建全长抗体，表达纯化后进行结合能力的测试以及阻断功能的检测，即通过前述的ELISA实验确定抗体对SEMG2和CD27结合的影响。
在同一批次的筛选中，得到能够结合SEMG2(497-509)表位并抑制SEMG2和CD27的结合的抗体独特序列共3种，三株克隆分别命名为：H88-93、H88-96、H88-67。相应的全长抗体结合SEMG2的浓度效应见图22A。三株全人源抗体对应的VH与VL的氨基酸序列以及相应的CDR序列如表6与表7所示。
表6.全人源抗体的可变区序列
Figure PCTCN2021073100-appb-000011
表7.人源抗体的CDR氨基酸序列
Figure PCTCN2021073100-appb-000012
Figure PCTCN2021073100-appb-000013
全人源抗体与鼠抗对SEMG2的结合能力进行了测试，即在SEMG2包被的96孔微量板中，将鼠源抗体MM02与MM05与浓度梯度稀释的全人源抗体H88-93混合加入作为一抗，用抗鼠二抗HRP测定结合到SEMG2的鼠源单克隆抗体。根据以下公式算阻断百分率：
阻断百分率＝[1-(实验组抗体A450-空白对照)/(阳性对照抗体A450-空白对照A450)]×100％
结果显示H88-93可以与MM02以及MM05竞争结合到SEMG2，如图22所示。表明全人源抗体与鼠源单克隆抗体是同一类可以结合到SEMG2的抗体，又由于MM02与MM05以及H88-93均可以结合到短肽SEMG2(497-509)，这一类抗体可以定义为结合到SEMG2(497-509)的一类抗体。
而进一步地，通过ELISA实验检测了人源抗体H88-93,H88-96,H88-67对于SEMG2和CD27结合的阻断效果。所有的抗体浓度均为10微克/毫升是，不同程度地抑制SEMG2和CD27的结合，如图23所示。
为了验证上述人源抗体对活化的PBMC杀伤肿瘤细胞功能的影响，将A375和LOVO细胞与活化的PBMC共培养，同时分别加入H88-93,H88-96,或H88-67抗体，并检测肿瘤细胞凋亡比例。结果如图24所示，表明这三种针对SEMG2(497-509)表位的全人源抗体均能够显著促进PBMC细胞 对表达SEMG2的肿瘤细胞的杀伤。
实施例17：生物光干涉测定法测定本发明的单克隆抗体与抗原的结合动力学
采用生物膜干涉测定法(ForteBio Bltz或Gator仪器)测定本发明抗体结合人SEMG2的平衡解离常数(KD)。如ForteBio亲和力测定按照现有的方法实验，即开始前半个小时，根据样品数量，取合适数量的AMQ(Pall，1506091)(用于样品检测)或AHQ(Pall，1502051)(用于阳性对照检测)传感器浸泡于SD buffer(PBS 1×，BSA0.1％，Tween-200.05％)中。取100μl的SD缓冲液、抗体、SEMG2分别加入到96孔黑色聚苯乙烯半量微孔板中。根据样品位置布板，选择传感器位置。使用分子相互作用分析软件分析KD值。在以上测定法所述的实验中，鼠源单克隆抗体与人源抗体H88-67、H88-93与H88-96的亲和力常数如表8所示，SEMG2与相应蛋白的亲和解离曲线如图25所示。
表8.生物膜层光学干涉检测抗原抗体结合的亲和力常数(平衡解离常数)
抗体 KD(M)
MM02 1.33×10 -9
MM05 5.28×10 -9
MM07 1.82×10 -9
MM08 2.34×10 -9
MM13 6.93×10 -10
MM14 1.44×10 -9
H88-67 2.84×10 -8
H88-93 4.60×10 -9
H88-96 1.40×10 -8
实施例18：全人源单克隆抗体的亲和力成熟
以全人源抗体H88-96与H88-67的VH与VL编码序列构建的质粒为模板，通过基因合成获得质粒，然后进行单点及双点饱和突变，再进行体外连接的方法重组抗体基因，最后将重组后的抗体Fab基因序列插入载体，转化进而获得突变的4个库容高于10 8CFU的噬菌体亲和力成熟抗体库。 通过免疫管梯度筛选法对抗体突变文库进行筛选，得到亲和力比野生型有较好提升的突变体。然后根据测得的Fab序列或者Fab序列中VH、VL序列的重新组合构建了全长亲和力成熟人源抗体。来源于H88-67的VH与VL序列以及H88-96的VH序列的CDR区亲和力成熟后见表9，抗体的轻、重链CDR区见表9。
表9.亲和力成熟全人源抗体对应的CDR序列
Figure PCTCN2021073100-appb-000014
表10.亲和力成熟全人源抗体对应的VH和VL序列
Figure PCTCN2021073100-appb-000015
Figure PCTCN2021073100-appb-000016
通过以上亲和力成熟过程，我们获得了亲和力提高的抗人SEMG2单克隆抗体，如编号为67-3亲和力成熟重链与编号为67-3、67-4、67-5、67-6的亲和力成熟轻链序列组合构成67-3-67-3、67-3-67-4、67-3-67-5、67-3- 67-6，编号为67-9的重链与编号为67-3的轻链组合构成抗体67-9-67-3，编号为67-6的轻链与重链组合构成抗体67-6-67-6，以及编号为96-10R和96-10V的重链与H88-96L的轻链重新构成的抗体96-10R-10与96-10V-10，这些轻重链组合的重组单克隆抗体对SEMG2以及BSA-S2(497-509)(即BSA-SP7)的亲和力较母本抗体提高10倍以上(见图21B-D)。
实施例19:验证SEMG2抗体在人恶性黑色素瘤A375细胞的PBMC免疫系统人源化小鼠模型异体移植模型中的抗肿瘤作用
将30只6-8周龄雄性NPSG小鼠模型称重。将A375细胞(已确认内源表达SEMG2)进行体外培养，获得1.8×10 8细胞。30只小鼠接种PBMC后,第3天接种A375肿瘤细胞，其后每周一次测定小鼠血液中hCD45+细胞比例及体重。接种后，每周1次测量肿瘤体积，当平均肿瘤体积达到约40-80mm 3时测量小鼠血液中hCD45+细胞比例。依据肿瘤体积和小鼠血液中hCD45+细胞比例，小鼠随机分组，随即开始给药。给药开始日期视为第0天。给药方案：SEMG2抗体(MM05克隆)腹腔注射5mg/kg每周三次。给药开始后，小鼠每周观测瘤体生长状况，瘤体生长后，每周3次测量体重和瘤体积，每周3次流式监控小鼠血液中hCD45+细胞相对计数。当肿瘤体积达到终点标准后，取血检测同上指标，结束实验。对小鼠的观察包括：日常观察，接种后，每工作日观察动物发病及死亡情况。肿瘤体积测量：接种后至分组前，当肿瘤可见时，每周一次测量实验动物肿瘤体积，接种分组后，实验中的动物肿瘤体积每周测量2次。肿瘤体积测量采用双向测量法，首先利用游标卡尺测量肿瘤长短径，再使用公式TV＝0.5*a*b2计算肿瘤体积。其中a是肿瘤的长径，b是肿瘤的短径。实验结果如图26所示，SEMG2抗体显著地抑制了肿瘤在小鼠体内的生长。该结果表明SEMG2是抗肿瘤的有效靶点。
实施例20:敲除小鼠的对应基因Svs3a证明SEMG2功能阻断后副作用不显著
为了证明通将SEMG2作为药物靶点进行功能阻断之后可能产生的毒副作用，将小鼠的相应基因Svs3a进行全身敲除，具体方案如下：本项目采用CRISPR/Cas9技术，利用非同源重组修复引入突变的方式，造成Svs3a 基因蛋白读码框移码，功能缺失。简要过程如下：通过体外转录的方式，获得Cas9 mRNA和gRNA；将Cas9 mRNA和gRNA显微注射到C57BL/6J小鼠的受精卵中，获得F0代小鼠。PCR扩增及测序鉴定阳性的F0代小鼠与C57BL/6J小鼠交配获得阳性F1代小鼠。
gRNAs序列(5’-3’)：
gRNA1，CAGCCGCAGAGAGGCACTCA GGG；
gRNA2，ATGCACCACCAAGAAACACTGGG。
敲除前后序列比对：
野生型:
Figure PCTCN2021073100-appb-000017
突变型:TGAGTTCAGGGAGCAGCCGCAAGAGAGG…(-1006bp)…GAGGTGCATGGTGGGCTCCCTGTGCCCGCAGTGC。
后续繁殖：将获得的基因敲除杂合子小鼠(gene+/-)分成两部分：一部分杂合子小鼠与野生型小鼠交配，扩群繁育较多的杂合子小鼠；一部分杂合子小鼠自交，获得基因敲除纯合子小鼠(gene-/-)，进行基因敲除效果验证和后续的表型分析。
表型分析：小鼠取抗凝全血进行流式实验，分析血液中CD8.CD4.CD3.CD27，阳性细胞所占比例。小鼠休息2天后，内眦取抗凝全血交分子部检测血常规。小鼠休息3天后，小鼠称重、麻醉，小鼠大体拍照；小鼠摘眼球取血并分离血清，血清交分子部检测血生化指标。摘眼球取血后小鼠安乐死取材：大脑：取下完整大脑,矢状正中分开，左侧固定、右侧速冻；肝脏：取下完整肝脏，左叶固定，其余速冻；脾脏：取下完整脾脏，一分为二，一半固定，一半速冻；肾脏：取下左肾固定，取下右肾速冻；胃：取下完整胃，矢状分开，大弯部分固定，小弯部分速冻；大肠：取下完整大肠，全部做瑞士卷固定；小肠：取下完整小肠，分三段(十二指肠、回肠、空肠)做瑞士卷固定；肺：取下左肺固定，取下右肺速冻；心脏：取下整个心脏舒张后固定。所有固定样品送病理进行石蜡包埋，其中一只KO鼠 (#98)的11个脏器(脑、心、肺、肾、脾、肝、胃、十二指肠、空肠、回肠、结肠)进行切片、HE染色、读片分析。
野生型(WT)和基因敲除纯合子小鼠(KO)的表型分析结果见图27结果表明，Svs3a纯合敲除的小鼠交配后无下一代出生，而杂合敲除情况下未见影响生育功能。其它方面的各项分析均未见到异常。因此，Svs3a功能的完全缺失或阻断可能影响生育，但对其他系统不产生明显的毒副作用。这提示SEMG2靶点的阻断可能产生的毒性或副作用是有限的，具备较高的安全性。
以上，发明人对基于本发明的实施方式进行了说明，但本发明不限定于此，本领域的技术人员应该明白，在本发明的主旨范围内能够以进行变形和变更的方式实施，这样的变形和变更的方式，理应属于本发明的保护范围。

Claims (36)

  1. 一种激动或拮抗SEMG2和CD27相互作用的化合物。
  2. 如权利要求1所述的化合物，其中所述SEMG2和CD27相互作用的氨基酸位点位于SEMG2的第497、498、499、500、501、502、503、504、505、506、508位，所述SEMG2蛋白的氨基酸序列如SEQ ID NO:1所示。
  3. 如权利要求1所述的化合物，其中所述化合物为小分子抑制剂、多肽、抗体或抗原结合片段。
  4. 如权利要求3所述的化合物，其中所述多肽包含如SEQ ID NO:2(QIEKLVEGKS)、SEQ ID NO:86(QIEKLVEGKS(x)I(x))、SEQ ID NO:87(QIEKLVEGKS(x)I)、或SEQ ID NO:88(QIEKLVEGKS(x))所示氨基酸序列所示的氨基酸序列，优选所述多肽包含如SEQ ID NO:2、SEQ ID NO:3(QIEKLVEGKSQIQ)、SEQ ID NO:4(QIEKLVEGKSQ)、或SEQ ID NO:5(QIEKLVEGKSQI)或与SEQ ID NO:2-5任一项序列至少90％以上序列一致性的氨基酸序列，其中x选自任意氨基酸。
  5. 如权利要求3所述的化合物，其中所述抗体为一种能够特异性结合天然或突变SEMG2蛋白的抗体，所述抗体结合源自SEMG2蛋白的抗原表位肽，所述抗原表位肽包含SEQ ID NO:2(QIEKLVEGKS)、SEQ ID NO:3(QIEKLVEGKSQIQ)、SEQ ID NO:4(QIEKLVEGKSQ)、或SEQ ID NO:5(QIEKLVEGKSQI)所示的氨基酸序列。
  6. 如权利要求3所述的化合物，其中所述抗体为一种能够特异性结合天然或突变SEMG2蛋白的抗体，所述抗体能够识别天然SEMG2蛋白的第497、498、499、500、501、502、503、504、505、506、508位点中的至少一个氨基酸残基或识别突变SEMG2蛋白相应位置的氨基酸残基，所述天然SEMG2蛋白的氨基酸序列如SEQ ID NO:1所示。
  7. 如权利要求3所述的化合物，其中所述抗体包含根据IMGT定义的HCDR1、HCDR2和HCDR3序列的重链可变区；以及包含根据IMGT定义的LCDR1、LCDR2和LCDR3序列的轻链可变区，
    所述HCDR1的氨基酸序列具有或包含选自由SEQ ID NOs:6-11、SEQ  ID NOs:60-61、和SEQ ID NO:76所组成的组中的氨基酸序列；
    所述HCDR2的氨基酸序列具有或包含选自由SEQ ID NOs:12-16和SEQ ID NOs:62-64所组成的组中的氨基酸序列；
    所述HCDR3的氨基酸序列包含选自由SEQ ID NOs:17-20、SEQ ID NOs:65-67、和SEQ ID NOs:77-81所组成的组中的氨基酸序列；
    所述LCDR1的氨基酸序列包含选自由SEQ ID NOs:21-25、SEQ ID NOs:68-70、和SEQ ID NO:82所组成的组中的氨基酸序列；
    所述LCDR2的氨基酸序列包含选自由SEQ ID NOs:26-29、SEQ ID NOs:71-72、SEQ ID NOs:83-84、和SEQ ID NO:28所组成的组中的氨基酸序列；
    所述LCDR3的氨基酸序列包含选自由SEQ ID NOs:30-34、SEQ ID NOs:73-75、SEQ ID NO:85和和SEQ ID NO:95所组成的组中的氨基酸序列。
  8. 如权利要求7所述的化合物，其中所述抗体包含根据IMGT定义的HCDR1、HCDR2和HCDR3序列的重链可变区；以及包含根据IMGT定义的LCDR1、LCDR2和LCDR3序列的轻链可变区，所述抗体中的CDR序列选自(a)-(k)组合中的任一种：
    (a)所述HCDR1的氨基酸序列包含如SEQ ID NO:6所示氨基酸序列；所述HCDR2的氨基酸序列包含如SEQ ID NO:12所示氨基酸序列；所述HCDR3的氨基酸序列包含如SEQ ID NO:17所示氨基酸序列；所述LCDR1的氨基酸序列包含如SEQ ID NO:21所示氨基酸序列；所述LCDR2的氨基酸序列包含如SEQ ID NO:26所示氨基酸序列；所述LCDR3的氨基酸序列包含如SEQ ID NO:30所示氨基酸序列；
    (b)所述HCDR1的氨基酸序列包含如SEQ ID NO:7所示氨基酸序列；所述HCDR2的氨基酸序列包含如SEQ ID NO:13所示氨基酸序列；所述HCDR3的氨基酸序列包含如SEQ ID NO:18所示氨基酸序列；所述LCDR1的氨基酸序列包含如SEQ ID NO:22所示氨基酸序列；所述LCDR2的氨基酸序列包含如SEQ ID NO:27所示氨基酸序列；所述LCDR3的氨基酸序列包含如SEQ ID NO:31或SEQ ID NO:95所示氨基酸序列；
    (c)所述HCDR1的氨基酸序列包含如SEQ ID NO:6所示氨基酸序列；所述HCDR2的氨基酸序列包含如SEQ ID NO:16所示氨基酸序列；所述HCDR3的氨基酸序列包含如SEQ ID NO:17所示氨基酸序列；所述LCDR1的氨基酸序列包含如SEQ ID NO:21所示氨基酸序列；所述LCDR2的氨基酸序列包含如SEQ ID NO:26所示氨基酸序列；所述LCDR3的氨基酸序列包含如SEQ ID NO:30所示氨基酸序列；
    (d)所述HCDR1的氨基酸序列包含如SEQ ID NO:8所示氨基酸序列；所述HCDR2的氨基酸序列包含如SEQ ID NO:13所示氨基酸序列；所述HCDR3的氨基酸序列包含如SEQ ID NO:18所示氨基酸序列；所述LCDR1的氨基酸序列包含如SEQ ID NO:23所示氨基酸序列；所述LCDR2的氨基酸序列包含如SEQ ID NO:27所示氨基酸序列；所述LCDR3的氨基酸序列包含如SEQ ID NO:32所示氨基酸序列；
    (e)所述HCDR1的氨基酸序列包含如SEQ ID NO:9所示氨基酸序列；所述HCDR2的氨基酸序列包含如SEQ ID NO:14所示氨基酸序列；所述HCDR3的氨基酸序列包含如SEQ ID NO:19所示氨基酸序列；所述LCDR1的氨基酸序列包含如SEQ ID NO:24所示氨基酸序列；所述LCDR2的氨基酸序列包含如SEQ ID NO:28所示氨基酸序列；所述LCDR3的氨基酸序列包含如SEQ ID NO:33所示氨基酸序列；
    (f)所述HCDR1的氨基酸序列包含如SEQ ID NO:10所示氨基酸序列；所述HCDR2的氨基酸序列包含如SEQ ID NO:15所示氨基酸序列；所述HCDR3的氨基酸序列包含如SEQ ID NO:20所示氨基酸序列；所述LCDR1的氨基酸序列包含如SEQ ID NO:25所示氨基酸序列；所述LCDR2的氨基酸序列包含如SEQ ID NO:29所示氨基酸序列；所述LCDR3的氨基酸序列包含如SEQ ID NO:34所示氨基酸序列；
    (g)所述HCDR1的氨基酸序列包含如SEQ ID NO:11所示氨基酸序列；所述HCDR2的氨基酸序列包含如SEQ ID NO:15所示氨基酸序列；所述HCDR3的氨基酸序列包含如SEQ ID NO:20所示氨基酸序列；所述LCDR1的氨基酸序列包含如SEQ ID NO:25所示氨基酸序列；所述LCDR2的氨基 酸序列包含如SEQ ID NO:29所示氨基酸序列；所述LCDR3的氨基酸序列包含如SEQ ID NO:34所示氨基酸序列；
    (h)所述HCDR1的氨基酸序列包含如SEQ ID NO:60所示氨基酸序列；所述HCDR2的氨基酸序列包含如SEQ ID NO:62所示氨基酸序列；所述HCDR3的氨基酸序列包含如SEQ ID NO:65所示氨基酸序列；所述LCDR1的氨基酸序列包含如SEQ ID NO:68所示氨基酸序列；所述LCDR2的氨基酸序列包含如SEQ ID NO:71所示氨基酸序列；所述LCDR3的氨基酸序列包含如SEQ ID NO:73所示氨基酸序列；
    (i)所述HCDR1的氨基酸序列包含如SEQ ID NO:61所示氨基酸序列；所述HCDR2的氨基酸序列包含如SEQ ID NO:63所示氨基酸序列；所述HCDR3的氨基酸序列包含如SEQ ID NO:66所示氨基酸序列；所述LCDR1的氨基酸序列包含如SEQ ID NO:69所示氨基酸序列；所述LCDR2的氨基酸序列包含如SEQ ID NO:72所示氨基酸序列；所述LCDR3的氨基酸序列包含如SEQ ID NO:74所示氨基酸序列；
    (j)所述HCDR1的氨基酸序列包含如SEQ ID NO:60所示氨基酸序列；所述HCDR2的氨基酸序列包含如SEQ ID NO:64所示氨基酸序列；所述HCDR3的氨基酸序列包含如SEQ ID NO:67所示氨基酸序列；所述LCDR1的氨基酸序列包含如SEQ ID NO:70所示氨基酸序列；所述LCDR2的氨基酸序列包含如SEQ ID NO:28所示氨基酸序列；所述LCDR3的氨基酸序列包含如SEQ ID NO:75所示氨基酸序列；
    (k)所述HCDR1的氨基酸序列包含如SEQ ID NO:60或76所示氨基酸序列；所述HCDR2的氨基酸序列包含如SEQ ID NO:64或62所示氨基酸序列；所述HCDR3的氨基酸序列包含如SEQ ID NO:77、78或79所示氨基酸序列；和/或
    所述LCDR1的氨基酸序列包含如SEQ ID NO:70或82所示氨基酸序列；所述LCDR2的氨基酸序列包含如SEQ ID NO:28、83或84所示氨基酸序列；所述LCDR3的氨基酸序列包含如SEQ ID NO:75或85所示氨基酸序列。
  9. 如权利要求3所述的化合物，其中所述抗体包含重链可变区和轻链可变区，
    所述重链可变区的氨基酸序列包含选自由SEQ ID NOs﹕35-41、48-51、54-56和96-100所组成的组中的氨基酸序列或与组中的序列具有至少70％、80％、90％、95％或99％序列同一性；
    所述轻链可变区的氨基酸序列包含选自由SEQ ID NOs﹕42-47、52-53、57-69和101-103所组成的组中的氨基酸序列或与组中的序列具有至少70％、80％、90％、95％或99％序列同一性。
  10. 如权利要求9所述的化合物，其中所述抗体包含重链可变区和轻链可变区，所述重链可变区和轻链可变区选自(a)-(o)组合中的任一种：
    (a)所述重链可变区的氨基酸序列包含如SEQ ID NO:35所示氨基酸序列或与其序列具有至少70％、80％、90％、95％或99％序列同一性；所述轻链可变区的氨基酸序列包含如SEQ ID NO:42所示的氨基酸序列或与其序列具有至少70％、80％、90％、95％或99％序列同一性；
    (b)所述重链可变区的氨基酸序列包含如SEQ ID NO:36所示氨基酸序列或与其序列具有至少70％、80％、90％、95％或99％序列同一性；所述轻链可变区的氨基酸序列包含如SEQ ID NO:43所示的氨基酸序列或与其序列具有至少70％、80％、90％、95％或99％序列同一性；
    (c)所述重链可变区的氨基酸序列包含如SEQ ID NO:37所示氨基酸序列或与其序列具有至少70％、80％、90％、95％或99％序列同一性；所述轻链可变区的氨基酸序列包含如SEQ ID NO:44所示的氨基酸序列或与其序列具有至少70％、80％、90％、95％或99％序列同一性；
    (d)所述重链可变区的氨基酸序列包含如SEQ ID NO:38所示氨基酸序列或与其序列具有至少70％、80％、90％、95％或99％序列同一性；所述轻链可变区的氨基酸序列包含如SEQ ID NO:45所示的氨基酸序列或与其序列具有至少70％、80％、90％、95％或99％序列同一性；
    (e)所述重链可变区的氨基酸序列包含如SEQ ID NO:39所示氨基酸序列或与其序列具有至少70％、80％、90％、95％或99％序列同一性；所述轻链可变区的氨基酸序列包含如SEQ ID NO:46所示的氨基酸序列或与其序列具有至少70％、80％、90％、95％或99％序列同一性；
    (f)所述重链可变区的氨基酸序列包含如SEQ ID NO:40所示氨基酸序列或与其序列具有至少70％、80％、90％、95％或99％序列同一性；所述轻链可变区的氨基酸序列包含如SEQ ID NO:47所示的氨基酸序列或与其序列具有至少70％、80％、90％、95％或99％序列同一性；
    (g)所述重链可变区的氨基酸序列包含如SEQ ID NO:41所示氨基酸序列或与其序列具有至少70％、80％、90％、95％或99％序列同一性；所述轻链可变区的氨基酸序列包含如SEQ ID NO:47所示的氨基酸序列或与其序列具有至少70％、80％、90％、95％或99％序列同一性；
    (h)所述重链可变区的氨基酸序列包含如SEQ ID NO:48、49、50、或51所示氨基酸序列或与其序列具有至少70％、80％、90％、95％或99％序列同一性；所述轻链可变区的氨基酸序列包含如SEQ ID NO:52或53所示的氨基酸序列或与其序列具有至少70％、80％、90％、95％或99％序列同一性；
    (i)所述重链可变区的氨基酸序列包含如SEQ ID NO:54所示氨基酸序列或与其序列具有至少70％、80％、90％、95％或99％序列同一性；所述轻链可变区的氨基酸序列包含如SEQ ID NO:57所示的氨基酸序列或与其序列具有至少70％、80％、90％、95％或99％序列同一性；
    (j)所述重链可变区的氨基酸序列包含如SEQ ID NO:55所示氨基酸序列或与其序列具有至少70％、80％、90％、95％或99％序列同一性；所述轻链可变区的氨基酸序列包含如SEQ ID NO:58所示的氨基酸序列或与其序列具有至少70％、80％、90％、95％或99％序列同一性；
    (k)所述重链可变区的氨基酸序列包含如SEQ ID NO:56所示氨基酸序列或与其序列具有至少70％、80％、90％、95％或99％序列同一性；所述轻链可变区的氨基酸序列包含如SEQ ID NO:59所示的氨基酸序列或与其序列具有至少70％、80％、90％、95％或99％序列同一性；
    (l)所述重链可变区的氨基酸序列包含如SEQ ID NO:96所示氨基酸序列或与其序列具有至少70％、80％、90％、95％或99％序列同一性；所述轻链可变区的氨基酸序列包含如SEQ ID NO:59、101、102或103所示的氨基酸序列或与其序列具有至少70％、80％、90％、95％或99％序列同一性；
    (m)所述重链可变区的氨基酸序列包含如SEQ ID NO:97所示氨基酸序列或与其序列具有至少70％、80％、90％、95％或99％序列同一性；所述轻链可变区的氨基酸序列包含如SEQ ID NO:59所示的氨基酸序列或与其序列具有至少70％、80％、90％、95％或99％序列同一性；
    (n)所述重链可变区的氨基酸序列包含如SEQ ID NO:98所示氨基酸序列或与其序列具有至少70％、80％、90％、95％或99％序列同一性；所述轻链可变区的氨基酸序列包含如SEQ ID NO:103所示的氨基酸序列或与其序列具有至少70％、80％、90％、95％或99％序列同一性；
    (o)所述重链可变区的氨基酸序列包含如SEQ ID NO:99或100所示氨基酸序列或与其序列具有至少70％、80％、90％、95％或99％序列同一性；所述轻链可变区的氨基酸序列包含如SEQ ID NO:57所示的氨基酸序列或与其序列具有至少70％、80％、90％、95％或99％序列同一性。
  11. 如权利要求5至10任一项所述的化合物，其中，所述抗体可进一步包含连接至多肽的偶联部分，所述偶联部分选自放射性核素、药物、毒素、细胞因子、酶、荧光素、载体蛋白、脂类、和生物素中的一种或多种，其中所述多肽或抗体与所述偶联部分可选择性通过连接子相连，优选所述连接子为肽或多肽。
  12. 如权利要求5至10任一项所述的化合物，其中所述抗体选自单克隆抗体、多克隆抗体、抗血清、嵌合抗体、人源化抗体和人抗体。
  13. 如权利要求5至10任一项所述的化合物，其中所述抗体选自多特异性抗体、单链Fv(scFv)、单链抗体、抗独特型(抗-Id)抗体、双抗体、微型抗体、纳米抗体、单结构域抗体、Fab片段、F(ab’)片段、二硫化物连接的双特异性Fv(sdFv)和胞内抗体。
  14. 一种抗原表位肽，其中，所述抗原表位肽源自SEMG2蛋白，所述抗原表位肽的氨基酸包含选自由SEQ ID NO:2(QIEKLVEGKS)、SEQ ID NO:3(QIEKLVEGKSQIQ)、SEQ ID NO:4(QIEKLVEGKSQ)和SEQ ID NO:5(QIEKLVEGKSQI)组成的组中的氨基酸序列。
  15. 一种蛋白，其中，所述蛋白含有如权利要求14所述的抗原表位肽以及 N端或C端可选择连接的标签序列。
  16. 如权利要求15所述的蛋白，所述蛋白的氨基酸序列包含如SEQ ID NO:2(QIEKLVEGKS)、SEQ ID NO:86(QIEKLVEGKS(x)I(x))、SEQ ID NO:87(QIEKLVEGKS(x)I)、或SEQ ID NO:88(QIEKLVEGKS(x))所示氨基酸序列所示的氨基酸序列，优选所述多肽包含如SEQ ID NO:3(QIEKLVEGKSQIQ)、SEQ ID NO:4(QIEKLVEGKSQ)、或SEQ ID NO:5(QIEKLVEGKSQI)或与SEQ ID NO:2-5任一项序列至少90％以上序列一致性的氨基酸序列，更优选如89-94和SEQ ID NO:3所示。
  17. 一种制备抗体的方法，包括使用如权利要求14所述的抗原表位肽或如权利要求15或16所述的蛋白作为免疫原免疫哺乳动物或者在天然抗体库筛选获得。
  18. 一种分离的多核苷酸，其编码3至13中任一项所述的化合物、如权利要求14所述的抗原肽、或如权利要求15或16所述的蛋白。
  19. 一种重组载体，其包含权利要求18所述的多核苷酸，以及任选的调控序列；优选所述重组载体为克隆载体或表达载体。
  20. 如权利要求19所述的重组载体，其中，所述调控序列选自前导序列、多聚腺苷酸化序列、前肽序列、启动子、信号序列、转录终止子，或其任何组合。
  21. 一种宿主细胞，其包含权利要求19或20所述的重组载体。
  22. 如权利要求21所述的宿主细胞，其中，所述宿主细胞为原核细胞或真核细胞。
  23. 一种药物组合物，其包含如1至13中任一项所述的化合物、如权利要求14所述的抗原肽、如权利要求15或16所述的蛋白、如权利要求18所述的多核苷酸，如权利要求19或20所述的重组载体、和如权利要求21或22所述的宿主细胞中的一种或更多种。
  24. 如权利要求23所述的药物组合物，其中，所述组合物还包含药学上可接受的载体或辅料。
  25. 如1至13中任一项所述的化合物、如权利要求14所述的抗原肽、如权利要求15或16所述的蛋白、如权利要求18所述的多核苷酸，如权利要求19或20所述的重组载体、或如权利要求21或22所述的宿主细胞在制备用于激动或拮抗SEMG2和CD27相互作用的产品中的用途，优选SEMG2表达于肿瘤细胞，CD27表达于免疫细胞。
  26. 如1至13中任一项所述的化合物、如权利要求14所述的抗原肽、如权利要求15或16所述的蛋白、如权利要求18所述的多核苷酸，如权利要求19或20所述的重组载体、或如权利要求21或22所述的宿主细胞在制备用于预防或治疗肿瘤的药物的用途。
  27. 如1至13中任一项所述的化合物、如权利要求14所述的抗原肽、如权利要求15或16所述的蛋白、如权利要求18所述的多核苷酸，如权利要求19或20所述的重组载体、或如权利要求21或22所述的宿主细胞在制备用于调节针对肿瘤的免疫反应中的药物的用途。
  28. 如权利要求26或27所述的用途，其中所述肿瘤选自结直肠癌、肺癌、黑色素瘤、淋巴瘤、肝癌、头颈癌、胃癌、肾癌、膀胱癌、前列腺癌、睾丸癌、子宫内膜癌、乳腺癌、和卵巢癌中的一种或多种。
  29. 一种筛选用于预防或治疗肿瘤的药物或试剂的方法，通过筛选抑制SEMG2和CD27相互作用的抑制剂或抗体，获得候选药物或试剂。
  30. 一种预防或治疗肿瘤的方法，其包括：
    使受试者的免疫细胞诸如淋巴细胞和/或肿瘤细胞与有效剂量的如权利要求1至13中任一项所述的化合物接触。
  31. 如权利要求30所述的方法，其中在使用有效量的化合物和受试者的免疫细胞和/或肿瘤细胞接触前，检测SEMG2在肿瘤细胞的表达。
  32. 如权利要求30所述的方法，其中所述受试者已经接受或正在接受或将要接受额外的抗癌疗法。
  33. 如权利要求30所述的方法，其中所述额外的抗癌疗法包括手术、放疗、化疗、免疫疗法或激素疗法。
  34. 一种试剂盒，其包含如1至13中任一项所述的化合物、如权利要求14所述的抗原肽、如权利要求15或16所述的蛋白、如权利要求18所述的多核苷酸，如权利要求19或20所述的重组载体、和如权利要求21或22所述的宿主细胞中的一种或更多种，并容纳于合适的容器中。
  35. 一种体外检测生物样本中SEMG2存在与否的方法，包括：使生物样本与如权利要求1-13任一项所述的化合物接触。
  36. 一种抑制肿瘤细胞生长的方法，包括以下步骤：A)分析SEMG2在肿瘤细胞的表达；B)利用可以识别SEMG2的抗体与肿瘤细胞接触，所述的抗体与SEMG2的结合KD<2×10 -8；C)使T淋巴细胞、所述抗体和肿瘤细胞相接触。
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KR1020227023643A KR20220131233A (ko) 2020-01-21 2021-01-21 Semg2 항체 및 이의 용도
US17/794,598 US20230080534A1 (en) 2020-01-21 2021-01-21 Semg2 antibody and use thereof
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