WO2022042673A1 - 用于降低异源多肽末端异质性的信号肽 - Google Patents

用于降低异源多肽末端异质性的信号肽 Download PDF

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WO2022042673A1
WO2022042673A1 PCT/CN2021/114911 CN2021114911W WO2022042673A1 WO 2022042673 A1 WO2022042673 A1 WO 2022042673A1 CN 2021114911 W CN2021114911 W CN 2021114911W WO 2022042673 A1 WO2022042673 A1 WO 2022042673A1
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antibody
seq
amino acid
acid sequence
heavy chain
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PCT/CN2021/114911
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French (fr)
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周松涛
刘洵
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江苏恒瑞医药股份有限公司
上海恒瑞医药有限公司
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Priority to US18/023,301 priority Critical patent/US20230312725A1/en
Priority to CN202180050556.XA priority patent/CN116234814A/zh
Priority to CA3191184A priority patent/CA3191184A1/en
Priority to JP2023512724A priority patent/JP2023539581A/ja
Priority to EP21860507.9A priority patent/EP4206222A4/en
Publication of WO2022042673A1 publication Critical patent/WO2022042673A1/zh

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    • C—CHEMISTRY; METALLURGY
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
    • C07K16/065—Purification, fragmentation
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    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—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 immunoglobulin superfamily
    • C07K16/283—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 immunoglobulin superfamily against Fc-receptors, e.g. CD16, CD32, CD64
    • A—HUMAN NECESSITIES
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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
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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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
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    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

Definitions

  • the present disclosure relates to the fields of molecular biology and protein engineering, and in particular to methods for reducing heterologous polypeptide terminal heterogeneity.
  • Signal peptides are short peptide chains that direct the transfer of newly synthesized proteins to the secretory pathway.
  • the signal peptide is located at the N-terminus of the secreted protein. Generally composed of 15 to 30 amino acids. It usually consists of three regions: a positively charged N-terminus, called the basic amino-terminus; an intermediate hydrophobic sequence, dominated by neutral amino acids, which can form an ⁇ -helix structure, which is the main functional region of the signal peptide; A longer negatively charged C-terminus, containing small amino acids, is the signal sequence cleavage site, also known as the processing region.
  • the signal peptide sequence When the signal peptide sequence is synthesized, it is recognized by the signal recognition granule (SRP), protein synthesis is suspended or slowed down, the signal recognition granule carries the ribosome to the endoplasmic reticulum, and the protein synthesis restarts. Under the guidance of the signal peptide, the newly synthesized protein enters the endoplasmic reticulum cavity, and the signal peptide sequence is cleaved under the action of the signal peptidase.
  • SRP signal recognition granule
  • the efficient cleavage of the signal peptide can ensure that both the light and heavy chain polypeptides of the antibody can be accurately cleaved, which is very important for mammalian cell-mediated antibody expression.
  • the cleavage site may change, resulting in the extension or truncation of the light and heavy amino acid chains of the recombinant antibody.
  • changes in their structure will lead to changes in their affinity, thereby affecting antibody activity; therefore, it is necessary to avoid the occurrence of amino acid chain extension or truncation during antibody production.
  • the present disclosure relates to signal peptides and uses thereof for reducing terminal heterogeneity of heterologous polypeptides.
  • the present disclosure provides a method of reducing the N-terminal heterogeneity of the heavy chain of an antibody and/or the light chain of an antibody, comprising
  • the host cell comprises: (1) a first polynucleotide encoding a heavy chain of an antibody and a first signal peptide operatively linked to the N-terminus of the heavy chain; wherein the first signal The peptide comprises the amino acid sequence of SEQ ID NO:55 or SEQ ID NO:56; and/or
  • SEQ ID NO: 55 is shown as MEWSWVFLFFLSLTGX 1 HX 2 , wherein X 1 is V, A, S, T, G, C, L or I, and X 2 is A, G, S, C, T or Q ;
  • SEQ ID NO: 56 is set forth as MSVPTQVLGLLLLWLTDX 3 RX 4 , wherein X 3 is V, A, S, T, G, C, L or I; X 4 is A, G, S, C, T or Q, and When X3 is selected from A, X4 is not C.
  • the method for reducing the N-terminal heterogeneity of an antibody heavy chain and/or an antibody light chain as previously described wherein the expressed heavy chain of the antibody has less than 3%, 2.5 %, 2%, 1.5% or 1% end extension ratio and/or the light chain of the antibody has a terminal extension ratio of less than 3%, 2.5%, 2%, 1.5% or 1%; in some embodiments, the expression The resulting antibody heavy chain has a terminal extension ratio of less than 1% and/or the antibody light chain has a terminal extension ratio of less than 1%.
  • the end extension ratio is calculated based on a peptide mapping detection method.
  • the expressed antibody heavy chain is substantially free of terminal residues and/or the antibody light chain is substantially free of terminal residues; in some embodiments, the expressed antibody The heavy chain has a terminal extension ratio of 0% and/or the light chain of the antibody has a terminal extension ratio of 0%.
  • the method for reducing the N-terminal heterogeneity of the heavy chain of an antibody and/or the light chain of an antibody as previously described wherein the first signal peptide or the second signal peptide each independently comprises a selected From the following peptides:
  • SEQ ID NO 57 MEWSWVFLFFLSLTGVHA
  • SEQ ID NO 58 MEWSWVFLFFLSLTGVHG
  • SEQ ID NO 59 MEWSWVFLFFLSLTGVHS
  • SEQ ID NO 60 MEWSWVFLFFLSLTGVHC
  • SEQ ID NO 61 MEWSWVFLFFLSLTGVHT
  • SEQ ID NO 62 MEWSWVFLFFLSLTGVHQ;
  • SEQ ID NO 64 MEWSWVFLFFLSLTGAHG
  • SEQ ID NO 65 MEWSWVFLFFLSLTGAHS
  • SEQ ID NO 66 MEWSWVFLFFLSLTGAHC
  • SEQ ID NO 68 MEWSWVFLFFLSLTGAHQ;
  • SEQ ID NO 69 MEWSWVFLFFLSLTGSHA;
  • SEQ ID NO 70 MEWSWVFLFFLSLTGSHG
  • SEQ ID NO 72 MEWSWVFLFFLSLTGSHC
  • SEQ ID NO 73 MEWSWVFLFFLSLTGSHT
  • SEQ ID NO 74 MEWSWVFLFFLSLTGSHQ;
  • SEQ ID NO 75 MEWSWVFLFFLSLTGTHA
  • SEQ ID NO 76 MEWSWVFLFFLSLTGTHG;
  • SEQ ID NO 77 MEWSWVFLFFLSLTGTHS
  • SEQ ID NO 78 MEWSWVFLFFLSLTGTHC
  • SEQ ID NO 80 MEWSWVFLFFLSLTGTHQ
  • SEQ ID NO 81 MEWSWVFLFFLSLTGGHA
  • SEQ ID NO 82 MEWSWVFLFFLSLTGGHG
  • SEQ ID NO 84 MEWSWVFLFFLSLTGGHC
  • SEQ ID NO 86 MEWSWVFLFFLSLTGGHQ;
  • SEQ ID NO 88 MEWSWVFLFFLSLTGCHG
  • SEQ ID NO 90 MEWSWVFLFFLSLTGCHC
  • SEQ ID NO 92 MEWSWVFLFFLSLTGCHQ;
  • SEQ ID NO 96 MEWSWVFLFFLSLTGLHC
  • SEQ ID NO 98 MEWSWVFLFFLSLTGLHQ;
  • SEQ ID NO 99 MEWSWVFLFFLSLTGIHA
  • SEQ ID NO 100 MEWSWVFLFFLSLTGIHG
  • SEQ ID NO 101 MEWSWVFLFFLSLTGIHS
  • SEQ ID NO 102 MEWSWVFLFFLSLTGIHC;
  • SEQ ID NO 103 MEWSWVFLFFLSLTGIHT
  • SEQ ID NO 104 MEWSWVFLFFLSLTGIHQ;
  • SEQ ID NO 105 MSVPTQVLGLLLLWLTDVRA;
  • SEQ ID NO 106 MSVPTQVLGLLLLWLTDVRG;
  • SEQ ID NO 107 MSVPTQVLGLLLLWLTDVRS
  • SEQ ID NO 108 MSVPTQVLGLLLLWLTDVRC;
  • SEQ ID NO 109 MSVPTQVLGLLLLWLTDVRT;
  • SEQ ID NO 110 MSVPTQVLGLLLLWLTDVRQ
  • SEQ ID NO 111 MSVPTQVLGLLLLWLTDARA;
  • SEQ ID NO 112 MSVPTQVLGLLLLWLTDARG;
  • SEQ ID NO 113 MSVPTQVLGLLLLWLTDARS;
  • SEQ ID NO 114 MSVPTQVLGLLLLWLTDART
  • SEQ ID NO 116 MSVPTQVLGLLLLWLTDSRA;
  • SEQ ID NO 118 MSVPTQVLGLLLLWLTDSRS;
  • SEQ ID NO 119 MSVPTQVLGLLLLWLTDSRC;
  • SEQ ID NO 120 MSVPTQVLGLLLLWLTDSRT;
  • SEQ ID NO 121 MSVPTQVLGLLLLWLTDSRQ;
  • SEQ ID NO 122 MSVPTQVLGLLLLWLTDTRA
  • SEQ ID NO 124 MSVPTQVLGLLLLWLTDTRS
  • SEQ ID NO 126 MSVPTQVLGLLLLWLTDTRT;
  • SEQ ID NO 127 MSVPTQVLGLLLLWLTDTRQ;
  • SEQ ID NO 128 MSVPTQVLGLLLLWLTDGRA;
  • SEQ ID NO 129 MSVPTQVLGLLLLWLTDGRG;
  • SEQ ID NO 130 MSVPTQVLGLLLLWLTDGRS
  • SEQ ID NO 131 MSVPTQVLGLLLLWLTDGRC;
  • SEQ ID NO 132 MSVPTQVLGLLLLWLTDGRT;
  • SEQ ID NO 133 MSVPTQVLGLLLLWLTDGRQ;
  • SEQ ID NO 134 MSVPTQVLGLLLLWLTDCRA;
  • SEQ ID NO 135 MSVPTQVLGLLLLWLTDCRG;
  • SEQ ID NO 136 MSVPTQVLGLLLLWLTDCRS
  • SEQ ID NO 137 MSVPTQVLGLLLLWLTDCRC;
  • SEQ ID NO 138 MSVPTQVLGLLLLWLTDCRT
  • SEQ ID NO 139 MSVPTQVLGLLLLWLTDCRQ;
  • SEQ ID NO 140 MSVPTQVLGLLLLWLTDLRA;
  • SEQ ID NO 141 MSVPTQVLGLLLLWLTDLRG
  • SEQ ID NO 142 MSVPTQVLGLLLLWLTDLRS
  • SEQ ID NO 143 MSVPTQVLGLLLLWLTDLRC;
  • SEQ ID NO 144 MSVPTQVLGLLLLWLTDLRT;
  • SEQ ID NO 145 MSVPTQVLGLLLLWLTDLRQ;
  • SEQ ID NO 146 MSVPTQVLGLLLLWLTDIRA
  • SEQ ID NO 147 MSVPTQVLGLLLLWLTDIRG;
  • SEQ ID NO 148 MSVPTQVLGLLLLWLTDIRS
  • SEQ ID NO 149 MSVPTQVLGLLLLWLTDIRC;
  • SEQ ID NO 150 MSVPTQVLGLLLLWLTDIRT.
  • SEQ ID NO 151 MSVPTQVLGLLLLWLTDIRQ.
  • the method for reducing the N-terminal heterogeneity of an antibody heavy chain and/or an antibody light chain as previously described wherein the first signal peptide or the second signal peptide each independently comprises SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO :101, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:146, SEQ ID NO:147 or the amino acid sequence of SEQ ID NO: 148.
  • the method for reducing the N-terminal heterogeneity of the heavy chain of an antibody and/or the light chain of an antibody as previously described wherein the first signal peptide comprises SEQ ID NO:57 or SEQ ID NO:105
  • the amino acid sequence, and/or the second signal peptide comprises the amino acid sequence of SEQ ID NO:57 or SEQ ID NO:105.
  • the method for reducing the N-terminal heterogeneity of the heavy chain of an antibody and/or the light chain of an antibody as previously described wherein the first signal peptide comprises the amino acid sequence of SEQ ID NO:57, and/or Or the second signal peptide comprises the amino acid sequence of SEQ ID NO:57.
  • the method for reducing the N-terminal heterogeneity of the heavy chain of an antibody and/or the light chain of an antibody as previously described wherein the first signal peptide comprises the amino acid sequence of SEQ ID NO:57, and/or Or the second signal peptide comprises the amino acid sequence of SEQ ID NO:105.
  • the method for reducing the N-terminal heterogeneity of the heavy chain of an antibody and/or the light chain of an antibody as previously described wherein the first signal peptide comprises the amino acid sequence of SEQ ID NO: 105, and/or Or the second signal peptide comprises the amino acid sequence of SEQ ID NO:57.
  • the method for reducing the N-terminal heterogeneity of the heavy chain of an antibody and/or the light chain of an antibody as previously described wherein the first signal peptide comprises the amino acid sequence of SEQ ID NO: 105, and/or Or the second signal peptide comprises the amino acid sequence of SEQ ID NO:105.
  • each of the first polynucleotide or the second polynucleotide An independent encoded polypeptide comprising a polypeptide selected from the group consisting of SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: Amino acid sequences of SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 171 and SEQ ID NO: 172.
  • the method for reducing the N-terminal heterogeneity of an antibody heavy chain and/or an antibody light chain as previously described wherein the host cell is a eukaryotic host cell; in some embodiments, The eukaryotic host cells are CHO cells or yeast.
  • the aforementioned method for reducing the N-terminal heterogeneity of the heavy chain of an antibody and/or the light chain of an antibody wherein the antibody is a murine antibody, a chimeric antibody, a humanized antibody Antibodies, human antibodies, affinity matured antibodies or multispecific antibodies.
  • the aforementioned method for reducing the heterogeneity of the N-terminus of the heavy chain of an antibody and/or the light chain of an antibody wherein the antibody is selected from anti-TLR7 antibody, anti-HER2 (ErbB2) antibody , Anti-Claudin18.2 Antibody, Anti-EGFR Antibody, Anti-B7H3 Antibody, Anti-c-Met Antibody, Anti-HER3(ErbB3) Antibody, Anti-HER4(ErbB4) Antibody, Anti-CD3 Antibody, Anti-CD20 Antibody, Anti-CD22 Antibody, Anti-CD30 Antibody , Anti-CD33 Antibody, Anti-CD38 Antibody, Anti-CD44 Antibody, Anti-CD47 Antibody, Anti-CD56 Antibody, Anti-CD70 Antibody, Anti-CD73 Antibody, Anti-CD105 Antibody, Anti-CEA Antibody, Anti-A33 Antibody, Anti-Cripto Antibody, Anti-SOST Antibody, Antibody EphA2 Antibody, Anti-G250 Antibody
  • the aforementioned method for reducing the N-terminal heterogeneity of the heavy chain of an antibody and/or the light chain of an antibody wherein the antibody is selected from Trastuzumab, Pertuzumab, Nimotuzumab, Enoblituzumab, Emibetuzumab, Inotuzumab, Pinatuzumab, Brentuximab, Gemtuzumab, Bivatuzumab, Lorvotuzumab, cBR96, Glematumamab, anti-Claudin18.2 antibody and anti-FcRn antibody, wherein the heavy chain of the anti-Claudin18.2 antibody comprises the amino acid sequence of SEQ ID NO: 49 and the light chain comprises the amino acid sequence of SEQ ID NO: 49 The amino acid sequence of SEQ ID NO:47; the heavy chain of the anti-FcRn antibody comprises the amino acid sequence of SEQ ID NO:167, and the light chain comprises the amino acid sequence of SEQ ID NO
  • a method for reducing the N-terminal heterogeneity of an antibody heavy chain and/or an antibody light chain as previously described comprising cloning a light chain plasmid into a heavy chain plasmid to construct a full-length antibody plasmid.
  • the method introduces the plasmid into the host cell by electrotransformation.
  • the present disclosure also provides a signal peptide comprising or consisting of the amino acid sequence of SEQ ID NO:55 or SEQ ID NO:56.
  • the polypeptide is a signal peptide, the amino acid sequence of which is set forth in any of SEQ ID NO:55 to SEQ ID NO:151.
  • the polypeptide as previously described, wherein the signal peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO57 to SEQ ID NO:151.
  • the polypeptide as previously described wherein the signal peptide comprises SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:140, SEQ ID The amino acid sequence of NO: 141, SEQ ID NO: 142, SEQ ID NO: 146, SEQ ID NO: 147 or SEQ ID NO: 148.
  • the polypeptide as previously described, wherein the signal peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:57 and SEQ ID NO:105.
  • the polypeptide as previously described wherein the polypeptide comprises the group consisting of SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 163, SEQ ID NO: 163, Amino acid sequences of the group consisting of ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171 and SEQ ID NO: 172.
  • the present disclosure also provides a nucleic acid molecule encoding the polypeptide of any preceding item.
  • the present disclosure also provides a host cell comprising the nucleic acid molecule as previously described.
  • the host cell as previously described, wherein the host cell is a eukaryotic host cell; in some embodiments, the eukaryotic host cell is a CHO cell or yeast.
  • the present disclosure also provides a composition comprising a heterologous polypeptide having a terminal extension ratio of less than 3%, 2.5%, 2%, 1.5%, or 1%; in some embodiments, the expression yields
  • the heterologous polypeptide has a terminal extension ratio of less than 1%; in some embodiments, the expressed heterologous polypeptide has a terminal extension ratio of less than 1%, and the terminal extension ratio is based on peptide mapping detection
  • the expressed heterologous polypeptide is substantially free of residues of terminal residues; in some embodiments, the expressed heterologous polypeptide has a terminal extension ratio of 0%.
  • the aforementioned composition wherein the heterologous polypeptide is an antibody heavy chain and/or an antibody light chain; in some embodiments, the antibody is selected from Trastuzumab, Pertuzumab, An anti-Claudin18.2 antibody and an anti-FcRn antibody, wherein the heavy chain of the anti-Claudin18.2 antibody comprises the amino acid sequence of SEQ ID NO:49, and the light chain comprises the amino acid sequence of SEQ ID NO:47.
  • the antibody is selected from an anti-FcRn antibody, wherein the heavy chain of the anti-FcRn antibody comprises the amino acid sequence of SEQ ID NO: 167 and the light chain comprises the amino acid sequence of SEQ ID NO: 168.
  • composition as hereinbefore described which is prepared by a method as described in any preceding item.
  • the signal peptide provided by the present disclosure has the effect of stably reducing the heterologous terminal heterogeneity of the heterologous polypeptide, and is suitable for the large-scale expression of the heterologous polypeptide.
  • Figure 1 FACS detection of humanized antibody binding to human Claudin18.2 at the cellular level.
  • FIG. 1 NUGC4 endocytosis experiments with humanized antibodies.
  • Figure 3A to Figure 3C Detection of ADCC effect of antibody in NUGC4 cells with different Claudin18.2 expression levels;
  • Figure 3A is the detection of ADCC effect of antibody in wild-type NUGC4 cells (low expression of Claudin18.2);
  • Figure 3B is antibody Detection of ADCC effect in Claudin18.2 medium expressing NUGC4 cells;
  • Figure 3C is the ADCC effect detection of antibody in Claudin18.2 highly expressing NUGC4 cells.
  • Figure 4A to Figure 4D Figure 4A is the mass spectrum of the deglycosylated molecular weight of the light chain of Pertuzumab-1 antibody; Figure 4B is the mass spectrum of the deglycosylated molecular weight of the light chain of Pertuzumab-3 antibody; Figure 4C is the deglycosylated molecular weight of the heavy chain of Pertuzumab-1 antibody Reduced molecular weight mass spectrum; Figure 4D is the reduced molecular weight mass spectrum of the heavy chain of Pertuzumab-3 antibody.
  • Figure 5A to Figure 5D Figure 5A is the mass spectrum of the deglycosylated molecular weight of the light chain of Pertuzumab-2 antibody; Figure 5B is the mass spectrum of the deglycosylated molecular weight of the light chain of Pertuzumab-4 antibody; Figure 5C is the deglycosylation of the heavy chain of the Pertuzumab-2 antibody Reduced molecular weight mass spectrum; Figure 5D is the reduced molecular weight mass spectrum of the heavy chain of Pertuzumab-4 antibody.
  • Figure 6A to Figure 6D Figure 6A is the mass spectrum of the deglycosylation reduction molecular weight of the light chain of the h1902-5-1 antibody; Figure 6B is the mass spectrum of the deglycosylation reduction molecular weight of the h1902-5-2 antibody light chain; Figure 6C is the h1902-5-1 Figure 6D shows the mass spectrogram of the desugar-reduced molecular weight of the heavy chain of the antibody;
  • Figure 7A to Figure 7D Figure 7A is the mass spectrum of FcRn-1 antibody light chain desugar-reduced molecular weight; Figure 7B is the mass spectrum of FcRn-2 antibody light chain desugar-reduced molecular weight; Figure 7C is FcRn-1 antibody heavy chain deglycosylation Mass spectrum of reduced molecular weight; FIG. 7D is the mass spectrum of reduced molecular weight of FcRn-2 antibody heavy chain deglycosylation.
  • Antibody as used in this disclosure is used herein in the broadest sense and includes various antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies, murine antibodies, chimeric antibodies, humanized antibodies , multispecific antibodies (eg, bispecific antibodies) and antibody fragments so long as they exhibit the desired antigen-binding activity and specificity.
  • antibody fragment refers to a molecule other than an intact antibody that comprises a portion of the intact antibody that specifically binds the antigen to which the intact antibody specifically binds.
  • antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') 2 , diabodies, linear antibodies, single chain antibody molecules (eg, scFv or scFab), single domain antibodies (dAbs) , and multispecific antibodies formed from antibody fragments.
  • the "chimeric antibody” described in the present disclosure is an antibody obtained by fusing the variable region of a murine antibody with the constant region of a human antibody, which can reduce the immune response induced by the murine antibody.
  • the "humanized antibody” described in this disclosure also known as CDR-grafted antibody, refers to the transplantation of murine CDR sequences into the framework of human antibody variable regions, that is, different types of antibodies.
  • Antibodies raised in human germline antibody framework sequences The heterologous reaction induced by chimeric antibodies can be overcome because they carry a large amount of murine protein components. In order to avoid the decrease in activity while reducing immunogenicity, minimal reverse mutation or back mutation can be performed on the framework sequence of the variable region of the human antibody to maintain or enhance the activity.
  • the humanized antibodies of the present disclosure also include humanized antibodies that have been further subjected to affinity maturation mutation of the CDRs by yeast display.
  • human antibody (HuMAb)
  • human antibody “human antibody”, “fully human antibody”, “fully human antibody” are used interchangeably, the amino acid sequence of which corresponds to the amino acid sequence of an antibody produced by a human or human cell, or derived from amino acid sequences of non-human origin using human antibody repertoires or other human antibody coding sequences.
  • This definition of human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues.
  • an “antibody fragment” as used in the present disclosure refers to a molecule other than an intact antibody that comprises a portion of the intact antibody that specifically binds to the antigen to which the intact antibody specifically binds.
  • antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') 2 , diabodies, linear antibodies, single chain antibody molecules (eg, scFv or scFab), single domain antibodies (dAbs) , and multispecific antibodies formed from antibody fragments.
  • the "antibody” of the present disclosure includes “whole antibodies” and "antibody fragments" thereof.
  • the heavy chain of the antibody or the light chain of the antibody described in the present disclosure includes a complete heavy chain or a complete light chain, and also includes a heavy chain fragment or a light chain fragment in an antibody fragment.
  • FR complementarity determining region
  • the FRs of the variable domains generally consist of four FR domains: FR1, FR2, FR3 and FR4.
  • CDR and FR sequences generally appear in VH (or VL) in the following order:
  • CDR complementarity determining region
  • HCDR1, HCDR2, HCDR3 three CDRs in each heavy chain variable region and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable region.
  • LCDR1, LCDR2, LCDR3 three CDRs in each light chain variable region.
  • the amino acid sequence boundaries of CDRs can be determined using any of a variety of well-known schemes, including the "Kabat” numbering convention (see Kabat et al.
  • VH variable domain
  • VL variable domain
  • CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50 -56 (LCDR2) and 89-97 (LCDR3).
  • CDR amino acids in VH are numbered 26-32 (HCDR1), 52-56 (HCDR2) and 95-102 (HCDR3); and amino acids in VL Residue numbers are 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3).
  • the CDRs are defined by amino acid residues 26-35 in human VH (HCDR1 ), 50-65 (HCDR2) and 95-102 (HCDR3) and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3) in human VL.
  • VH The CDR amino acid residue numbers in VL are approximately 26-35 (CDR1), 51-57 (CDR2) and 93-102 (CDR3), and the CDR amino acid residue numbers in VL are approximately 27-32 (CDR1), 50-52 (CDR2) and 89-97 (CDR3).Following the IMGT rules, the CDR region of the antibody can be determined using the program IMGT/DomainGap Align.Unless otherwise stated, the antibody variable regions and CDR sequences involved in the embodiments of the present disclosure are all applicable to "Kabat" "Numbering rules.
  • antibodies bind with an affinity (KD) of less than about 10-8 M, eg, about less than 10-9 M, 10-10 M, 10-11 M, 10-12 M, or less.
  • KD affinity
  • KD refers to the dissociation equilibrium constant for a particular antibody-antigen interaction.
  • an antibody of the present disclosure binds an antigen with a dissociation equilibrium constant (KD) of less than about 10-7 M, eg, less than about 10-8 M or 10-9 M, eg, the affinity of the antibody for a cell surface antigen in the present disclosure KD value was determined by FACS method.
  • Polynucleotide or “nucleic acid molecule,” as used interchangeably in this disclosure, refer to polymers of nucleotides of any length, and include DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any that can be incorporated into polymers by DNA or RNA polymerases or by synthetic reactions. substrate. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and analogs thereof. If present, modifications to the nucleotide structure can be performed before or after assembly of the polymer. The sequence of nucleotides can be interrupted by non-nucleotide components.
  • operably linked refers to the juxtaposition of two or more components, wherein the components are in a relationship that allows them to function in their intended manner.
  • a promoter is operably linked to a coding sequence if it acts in cis to control or regulate the transcription of the linked sequence.
  • "operably linked" DNA sequences are contiguous, and where necessary to join two protein coding regions or in the case of a secretory leader sequence, contiguous and in open reading frame.
  • an operably linked promoter is usually located upstream of the coding sequence, it is not necessarily adjacent to it.
  • An operably linked enhancer can be located upstream, within, or downstream of the coding sequence and at a comparable distance from the promoter.
  • Ligation is accomplished by recombinant methods known in the art, eg, using PCR methods, by annealing, or by ligation at convenient restriction sites. If convenient restriction sites do not exist, synthetic oligonucleotide adaptors or linkers are routinely practiced.
  • one way of linking the signal peptide to the heterologous polypeptide is direct linking.
  • a “host cell” as used in this disclosure refers to a cell that has been genetically altered or capable of being genetically altered by the introduction of an exogenous polynucleotide, such as a recombinant plasmid or vector. It should be understood that such terms are intended to refer not only to a particular individual cell, but also to the progeny of such cells. Such progeny may actually differ from the parental cell due to certain modifications resulting from mutations or environmental influences that can occur in successive generations, and still be included within the scope of the term "host cell” as used in this disclosure.
  • Host cells can be microorganisms (such as eukaryotic microorganisms) or animal cells, suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris; suitable animal host cell lines include CHO (Chinese hamster ovary cell line), 293 cells and NS0 cells.
  • suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris
  • suitable animal host cell lines include CHO (Chinese hamster ovary cell line), 293 cells and NS0 cells.
  • Polypeptide as used in this disclosure generally refers to peptides and proteins from any cellular source having more than about 10 amino acids.
  • Heterologous polypeptides are those that are foreign to the host cell used, such as a human protein produced by the host cell. Heterologous polypeptides can be prokaryotic or eukaryotic, such as mammalian or human. A heterologous polypeptide can be a recombinantly produced polypeptide or a recombinant polypeptide.
  • heterologous polypeptides include transmembrane molecules (eg, receptors, such as receptor tyrosine kinases) or ligands, such as growth factors.
  • exemplary heterologous polypeptides include molecules such as: renin; growth hormone, including human growth hormone and bovine growth hormone; growth hormone releasing factor; parathyroid hormone; thyroid stimulating hormone; lipoprotein; alpha 1-antitrypsin; insulin A chain; insulin B chain; proinsulin; follicle-stimulating hormone; calcitonin; luteinizing hormone; glucagon; coagulation factors such as factor VIIIC, factor IX, tissue factor (TF), and von Willebrands factor; anticoagulant factors , such as protein C; atrial natriuretic peptide; pulmonary surfactant; plasminogen activator, such as urokinase or human urine or tissue plasminogen activator (t-PA); bombesin; thrombin; hematopoietic growth factor; tumor
  • Heterologous polypeptides can also be antibodies, exemplary of which targets include, but are not limited to, A33, BMPI, BMP2, BMP3B (GDFIO), BMP4, BMP6, BMP8, CSFI (M-CSF), CSF2 (GM-CSF) ), CSF3(G-CSF), EPO, FGFI(aFGF), FGF2(bFGF), FGF3(int-2), FGF4(HST), FGF5, FGF6(HST-2), FGF7(KGF), FGF9, FGF10 , FGF11, FGF12, FGF12B, FGF14, FGF16, FGF17, FGF19, FGF20, FGF21, FGF23, IGF1, IGF2, IFNAI, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNBI, IFNG, IFNWI, FELI, FELI (EPSELON) , FELI(ZETA), IL1A, IL
  • the antibody is selected from Trastuzumab, Pertuzumab, Nimotuzumab, Enoblituzumab, Emibetuzumab, Inotuzumab, Pinatuzumab, Brentuximab, Gemtuzumab, Bivatuzumab, Lorvotuzumab, cBR96, Glematumamab, and an anti-Claudin18.2 antibody, wherein the anti-Claudin18.2 antibody is heavy
  • the chain is shown in SEQ ID NO:49 and the light chain is shown in SEQ ID NO:47.
  • Constant modification or “conservative substitution or substitution” refers to the replacement of amino acids in a protein by other amino acids with similar characteristics (eg, charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.) such that frequent Changes are made without altering the biological activity of the protein.
  • Those skilled in the art are aware that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., 224, (4th ed.).
  • substitution of structurally or functionally similar amino acids is unlikely to disrupt biological activity. Exemplary conservative substitutions are set forth below.
  • terminal extension ratio refers to the ratio of expression products having terminal extension sequences in the expression product population.
  • An expression product with a terminal extension sequence is a variant of the target expression product with additional amino acid residues attached in addition to the desired target sequence.
  • 1% of the light chain sequence variants have other amino acid residues connected to the N-terminus, and the expression product has a terminal extension ratio of 1%.
  • terminal extension sequences in the present disclosure are derived from residues of signal peptide amino acids.
  • compositions of the present disclosure comprise the desired expression product of interest and the expression product with terminal extension sequences.
  • the presence of amino-terminal extensions in the composition can be detected by a variety of analytical techniques, including but not limited to N-terminal sequence analysis, assays of charge heterogeneity (eg, cation exchange chromatography or capillary zone electrophoresis), mass spectrometry, peptide Image detection, etc.
  • the amount of antibody variant in the composition generally ranges from an amount that constitutes the lower detection limit of any assay used to detect the variant (eg, a cation exchange assay) to less than the amount of the main species of antibody.
  • 3% or less eg. about 3%, 2.5%, 2%, 1.5%, 1%, 0.5%, 0%
  • Such percentage amounts can be determined by mass spectrometry, peptide mapping detection.
  • the proportion of heterologous polypeptides containing terminal extensions in the sample exceeds 1% of the total, it can be effectively identified based on the peptide map detection method, and the corresponding peaks in the peak map of the reduced molecular weight mass spectrometry can be quantitatively identified at the same time.
  • Example 1-1 Construction of a cell line highly expressing Claudin18.2
  • the pCDH-hClaudin18.2 lentiviral expression vector plasmid and the pVSV-G, pCMV-dR8.91 lentiviral system packaging vector were transfected into virus packaging cells 293T; the medium supernatant containing the virus was collected. , filter and perform ultracentrifugation; use the concentrated virus to infect human gastric signet ring cell cancer cell line NUGC4, screen for two to three weeks with puromycin, and then perform FACS single-cell sorting.
  • the degree of Claudin18.2 expression was differentiated according to tumor IHC score.
  • the cells with the same level of Claudin18.2 expression as the tumor with a tumor IHC score of 3 are high-expressing cells, and the cells with a comparable expression level of Claudin18.2 with the tumor with a tumor IHC score of 2 are medium-expressing cells.
  • the NUGC4/hClaudin18.2 monoclonal cell line with high Claudin18.2 expression was selected.
  • the expression of Claudin18.2 on the surface of wild-type NUGC4 cells was detected by FACS, and the NUGC4 clone cell line with moderate expression of Claudin18.2 was selected.
  • the selected monoclonal cell lines are expanded and cultured, and the library is frozen for subsequent experiments.
  • Example 1-2 Anti-human claudin18.2 monoclonal antibody production
  • Anti-human Claudin18.2 monoclonal antibody was produced by immunizing mice.
  • mice SJL white mice were used in the experiment, female, 6-8 weeks old (Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., animal production license number: SCXK (Beijing) 2012-0001). Breeding environment: SPF grade. After the mice were purchased, they were reared in a laboratory environment for 1 week, regulated by a 12/12 hour light/dark cycle, the temperature was 20-25°C, and the humidity was 40-60%. The acclimated mice were immunized according to the following protocol. The immunization antigen was huClaudin18.2-HEK293 cells (HEK-293 stable cell line transfected with human Claudin18.2 plasmid).
  • mice with high antibody titers in serum and titers approaching a plateau were selected for splenocyte fusion.
  • Boosters were injected intraperitoneally (IP) with 1 x 10< 7 > cells 3 days prior to splenocyte fusion.
  • a PEG-mediated fusion procedure was used to combine spleen lymphocytes with myeloma cells Sp2/0 cells ( CRL-8287 TM ) was fused to obtain hybridoma cells.
  • Hybridoma cells were resuspended in complete medium (IMDM medium containing 20% FBS, 1 ⁇ HAT, 1 ⁇ OPI) at a density of 0.5-1 ⁇ 10 6 /ml, and 100 ⁇ l/well were seeded in 96-well plates, 37 After incubating for 3-4 days at °C and 5% CO 2 , supplement 100 ⁇ l/well of HAT complete medium, and continue to culture for 3-4 days until colonies are formed. Remove the supernatant, add 200 ⁇ l/well of HT complete medium (IMDM medium containing 20% FBS, 1 ⁇ HT and 1 ⁇ OPI), culture at 37° C. and 5% CO 2 for 3 days before ELISA detection.
  • IMDM medium containing 20% FBS, 1 ⁇ HT and 1 ⁇ OPI
  • the culture supernatant was detected by binding ELISA method.
  • the monoclonal hybridoma cell lines mAb1901 and mAb1902 with high in vitro activity were selected; the monoclonal antibody sequences were cloned, and then humanization, recombinant expression and activity evaluation were carried out.
  • the procedure for cloning sequences from hybridomas is as follows. Hybridoma cells in logarithmic growth phase were collected, RNA was extracted with Trizol (Invitrogen, 15596-018) (according to the kit instructions) and reverse transcribed (PrimeScript TM Reverse Transcriptase, Takara, cat#2680A). The cDNA obtained by reverse transcription was amplified by PCR using mouse Ig-Primer Set (Novagen, TB326Rev.B 0503), and sent to a sequencing company for sequencing. The amino acid sequences corresponding to the obtained DNA sequences are shown in SEQ ID NOs: 3-6:
  • the above-mentioned murine heavy chain variable regions and light chain variable regions were linked to the heavy chain constant regions and human kappa light chain constant regions of the following human IgG1 antibodies, respectively, to form chimeric antibodies ch1901 and ch1902.
  • the constant region is selected from the following sequences:
  • Murine monoclonal antibodies were humanized as described in many literatures in the art. Briefly, CDR grafting was performed using human constant domains in place of the parental (murine antibody) constant domains, and human germline antibody sequences selected based on the homology of the murine and human antibodies. The present disclosure selects candidate molecules with good activity for humanization, and the results are as follows.
  • amino acid residues of the VH/VL CDRs in Table 2 were identified and annotated by the Kabat numbering system.
  • the sequences of the variable regions of the heavy and light chains were compared with the antibody Germline database to obtain a human germline template with high homology.
  • the human germline light chain framework region is derived from the human kappa light chain gene.
  • the chain variable region sequence is SEQ ID NO: 24 and the light chain variable region sequence is SEQ ID NO: 21; and then recombined with the IgG constant region to form a complete antibody.
  • back-mutation is performed on the FR region in the V region of the humanized antibody. Exemplary back-mutation methods and combinations are as follows:
  • the corresponding heavy chain variable region in the above table can be connected with the human IgG1 heavy chain constant region shown in SEQ ID NO:7 to form the heavy chain of the full-length antibody, and the light chain variable region and the human kappa shown in SEQ ID NO:8.
  • the light chain constant regions are linked to form the light chain of a full-length antibody.
  • the heavy and light chain variable regions can also be linked to other heavy and light chain constant regions, respectively, to form full-length antibodies.
  • the chain variable region sequence is SEQ ID NO: 31 and the light chain variable region sequence is SEQ ID NO: 28; recombined with the IgG constant region to form a complete antibody.
  • back-mutation is performed on the FR region in the V region of the humanized antibody. Exemplary back-mutation methods and combinations are as follows:
  • the corresponding heavy chain variable region is connected with the human IgG1 heavy chain constant region shown in SEQ ID NO:7 to form the heavy chain of the full-length antibody, and the light chain variable region is connected with the human kappa light shown in SEQ ID NO:8.
  • the chain constant regions are linked to form the light chain of a full-length antibody.
  • antibody full-length sequence is as follows:
  • ch1901 heavy chain (SEQ ID NO:35)
  • the positive control antibody of the present disclosure is IMAB-362 (from WO2016166122):
  • the above antibodies were cloned, expressed and purified by conventional gene cloning and recombinant expression methods, respectively.
  • Test Example 1 Biological evaluation of in vitro activity of anti-claudin18.2 antibody
  • a cell-based ELISA assay was used to examine the binding properties of the Claudin18.2 antibody.
  • the NUGC4 cells stably expressing Claudin18.2 were cultured in a 96-well cell plate (Corning, 3599), and when the cells were grown to 90% density, 4% paraformaldehyde was added to fix the cells for 1 hour, and PBST buffer (pH 7.4 PBS containing After washing the plate 3 times with 0.05% Tween-20), add 200 ⁇ l/well of blocking solution of 5% skim milk (bright skim milk powder) diluted with PBS, incubate at 37°C for 2.5 hours or place at 4°C overnight (16-18 hours) to be closed.
  • PBST buffer pH 7.4 PBS containing After washing the plate 3 times with 0.05% Tween-20
  • sample diluent pH 7.4 PBS containing 1% skim milk
  • sample diluent pH 7.4 PBS containing 1% skim milk
  • HRP-labeled goat anti-human secondary antibody Jackson Immuno Research, 109-035-003
  • NUGC4 cells stably expressing Claudin18.2 were prepared into 1 ⁇ 10 6 /ml cell suspension with FACS buffer (2% fetal bovine serum (Gibco, 10099141) pH7.4 PBS (Sigma, P4417-100TAB)), 100 ⁇ l /well was added to a 96-well round bottom plate (Corning, 3795). After removing the supernatant by centrifugation, 50 ⁇ l/well of Claudin18.2 antibody to be tested at different concentrations diluted with FACS buffer was added, and incubated in a refrigerator at 4°C for 1 hour in the dark.
  • FACS buffer 2% fetal bovine serum (Gibco, 10099141) pH7.4 PBS (Sigma, P4417-100TAB)
  • the Claudin18.2 antibody to be tested pre-labeled with DyLight 488 NHS Ester was added to 1 ⁇ 10 6 /ml NUGC4 cells stably expressing Claudin18.2 at a final concentration of 5 ⁇ g/ml, and placed on ice to avoid Incubate in light for 1 hour, centrifuge and wash 3 times with pre-cooled FACS buffer (pH 7.4PBS, 2% fetal bovine serum), remove the supernatant, add pre-warmed complete medium, and put it into 37°C 5% CO 2 for cell culture box. Cells were removed after 0, 0.5, 1, 2, and 4 hours, respectively, and stored on ice to protect from light.
  • Test Example 1-4 Determination of Antibody Affinity Based on Flow Cytometry
  • HEK293/hClaudin18.2 cells were collected in a U-bottom 96-well plate, with 1 ⁇ 10 5 to 2 ⁇ 10 5 cells per well.
  • Claudin18.2 antibody with an initial concentration of 5 ⁇ g/ml, 2 ⁇ gradient dilution (12 concentration points), and incubate at 4°C for 1 hour.
  • the positive control is IMAB362, and a negative control without antibody is set at the same time.
  • the antibody was removed by centrifugation, then 100 ⁇ l/well of FITC anti-human IgG Fc antibody (200 ⁇ ) was added, incubated at 4°C for 30 minutes in the dark, washed twice with PBS+2% FBS, and ready for flow cytometry detection.
  • Test Example 1-5 Evaluation of ADCC Effect of Antibody
  • the antibody was diluted in the above-mentioned phenol red-free medium to prepare a 3 ⁇ antibody dilution, and 25 ⁇ l/well of the antibody was added to the cell plate. Incubate for 0.5 h in a 37 °C, 5% CO2 incubator.
  • the effector cells (FcrR3A-V158-NFAT-RE-Jurkat cells) were collected, centrifuged at 1000 rpm, resuspended and counted. Cells were resuspended in phenol red-free RPMI 1640 supplemented with 10% FBS (New Zealand ultra-low IgG fetal bovine serum) at a density of 3 x 10 6 cells/ml, and 25 ⁇ l of cells (7.5 x 10 4 ) were added to each well of the assay plate. cells/well). Incubate for 6 h in a 37 °C, 5% CO2 incubator.
  • FBS New Zealand ultra-low IgG fetal bovine serum
  • Example 2-1 Design and expression of Pertuzumab fused with different signal peptides
  • MEWSWVFLFFLSVTTGVHS SEQ ID NO: 152
  • MSVPTQVLGLLLLWLTDARC SEQ ID NO: 153
  • MEWSWVFLFFLSVTTGVHS SEQ ID NO: 152
  • MEWSWVFLFFLSVTTGVHS SEQ ID NO: 152
  • MEWSWVFLFFLSLTGVHA SEQ ID NO: 57
  • MEWSWVFLFFLSLTGVHA SEQ ID NO: 57
  • MSVPTQVLGLLLLWLTDVRA SEQ ID NO: 105
  • MEWSWVFLFFLSLTGVHA SEQ ID NO: 57
  • the above signal peptide combinations were placed at the N-terminals of the heavy and light chain amino acids of the Pertuzumab antibody respectively, and corresponding new heavy and light chain sequences were designed.
  • the designed sequence is as follows:
  • Pertuzumab-1 antibody heavy chain amino acid sequence with signal peptide SEQ ID NO: 1544
  • Pertuzumab-1 antibody light chain amino acid sequence with signal peptide SEQ ID NO: 155)
  • Pertuzumab-2 antibody heavy chain amino acid sequence with signal peptide SEQ ID NO: 1544
  • Pertuzumab-2 antibody light chain amino acid sequence with signal peptide SEQ ID NO: 1536
  • Pertuzumab-3 antibody heavy chain amino acid sequence with signal peptide SEQ ID NO: 1557
  • Pertuzumab-3 antibody light chain amino acid sequence with signal peptide SEQ ID NO: 1578
  • Pertuzumab-4 antibody heavy chain amino acid sequence with signal peptide SEQ ID NO: 157)
  • Pertuzumab-4 antibody light chain amino acid sequence with signal peptide SEQ ID NO: 159
  • Pertuzumab-5 antibody heavy chain amino acid sequence with signal peptide SEQ ID NO: 160
  • Pertuzumab-5 antibody light chain amino acid sequence with signal peptide SEQ ID NO: 159
  • the gene fragments of each heavy chain and light chain were synthesized, the heavy chain gene was cloned into PXC18.4 to form a heavy chain plasmid, and the light chain gene was cloned into PXC17.4 to form a light chain plasmid.
  • the light chain plasmid was cloned into the heavy chain plasmid through the same restriction site in the plasmid, and a full-length antibody plasmid was constructed.
  • the full-length antibody plasmid was directly electroporated into CHO cells, and the stable transfected cells containing the plasmid were obtained after screening.
  • the stably transfected cells were cultured, and the cultured supernatant was purified with a Protein A affinity column to obtain the expression product.
  • Example 2-2 Design and expression of h1902-5 fused with different signal peptides
  • MEWSWVFLFFLSVTTGVHS SEQ ID NO: 152
  • MEWSWVFLFFLSVTTGVHS SEQ ID NO: 152
  • MEWSWVFLFFLSLTGVHA SEQ ID NO: 57
  • MSVPTQVLGLLLLWLTDVRA SEQ ID NO: 105
  • MSVPTQVLGLLLLWLTDVRA SEQ ID NO: 105
  • MEWSWVFLFFLSLTGVHA SEQ ID NO: 57
  • the above signal peptides were combined and placed at the N-terminus of the heavy chain and light chain of the h1902-5 antibody, respectively, and corresponding new heavy and light chain sequences were designed.
  • the designed sequence is as follows:
  • the gene fragments of each heavy chain and light chain were synthesized based on the above-mentioned sequence of h1902-5-1 or h1902-5-2, and constructed into an expression vector to obtain a full-length antibody plasmid.
  • the full-length antibody plasmid was directly electroporated into CHO cells, and the stable transfected cells containing the plasmid were obtained after screening.
  • the stably transfected cells were cultured, and the cultured supernatant was purified with a Protein A affinity column to obtain the expression product.
  • Anti-FcRn antibody heavy and light chain amino acid sequences are as follows:
  • MEWSWVFLFFLSVTTGVHS SEQ ID NO: 152
  • MEWSWVFLFFLSVTTGVHS SEQ ID NO: 152
  • MEWSWVFLFFLSLTGVHA SEQ ID NO: 57
  • MEWSWVFLFFLSLTGVHA SEQ ID NO: 57
  • FcRn-1 Antibody Heavy Chain Amino Acid Sequence with Signal Peptide SEQ ID NO: 169
  • FcRn-1 Antibody Light Chain Amino Acid Sequence with Signal Peptide SEQ ID NO: 170
  • FcRn-2 signal peptide-containing antibody heavy chain amino acid sequence (SEQ ID NO: 171)
  • FcRn-2 signal peptide-containing antibody light chain amino acid sequence (SEQ ID NO: 172)
  • the gene fragments of each heavy chain and light chain were synthesized based on the sequences of FcRn-1 and FcRn-2, and constructed into expression vectors to obtain full-length antibody plasmids.
  • the full-length antibody plasmid was directly electroporated into CHO cells, and a stable cell pool containing the plasmid was obtained after screening.
  • the stably transfected cell pool was cultured, and the cultured supernatant was purified with a Protein A affinity column to obtain the expression product.
  • Test Example 2 Terminal heterogeneity of expression products using different signal peptides
  • Test Example 2-1 Deglycosylated Reduction Molecular Weight and Amino Acid Sequence Detection of Pertuzumab Fused with Different Signal Peptides
  • Pertuzumab-1 and Pertuzumab-3 are shown in Figure 4A to Figure 4D:
  • Pertuzumab-1 samples detected 4.7% signal peptide amino acid (RC) residues in the light chain; while Pertuzumab-3 samples had no signal in the light chain Peptide amino acid residues.
  • No signal peptide amino acid residues were detected in the heavy chains of Pertuzumab-1 and Pertuzumab-3, but only a small amount of glycosylation modification.
  • the Glu C enzyme is used for enzyme digestion to cut the amino acid R in the sequence RC, and the residue amino acid C indicates the RC amino acid residue.
  • Pertuzumab-2 and Pertuzumab-4 are shown in Figure 5A to Figure 5D:
  • Pertuzumab-2 samples detected 5.8% signal peptide amino acid (VHS) residues in the light chain; while Pertuzumab-4 samples had no light chain signal Peptide amino acid residues.
  • VHS signal peptide amino acid
  • pertuzumab-2 was denatured with urea, it was reduced to light and heavy chains by adding DTT, and then Glu C enzyme was added for enzymatic digestion.
  • the molecular weight data of the obtained sample was collected by LC-MS, and the obtained data was analyzed by UNIFI software. Amino acid sequence information.
  • Test Example 2-2 Desaccharide reduction molecular weight and amino acid sequence detection of h1902-5 fused with different signal peptides
  • N-glycosyl carried by h1902-5-1 and h1902-5-2 samples was excised by PNGase F glycosidase (NEB, P0708), and then reduced to light and heavy chains by DTT (Sigma, 43815), and finally converted to light and heavy chains by LC- MS (Waters, ACQUITY UPLC H-Class/XeVo G2-XS QTOF) was used to detect the desugar-reduced molecular weight of the sample, and the data was processed and analyzed by UNIFI software.
  • PNGase F glycosidase N-glycosyl
  • DTT Sigma, 43815
  • LC- MS Waters, ACQUITY UPLC H-Class/XeVo G2-XS QTOF
  • h1902-5-1 was denatured with urea
  • DTT was added to reduce it into light and heavy chains
  • Glu C enzyme was added for enzymatic digestion.
  • the molecular weight data of the obtained sample was collected by LC-MS, and the obtained data was analyzed by UNIFI software. Amino acid sequence information of the sample.
  • Test Example 2-3 Deglycosylated Reduction Molecular Weight and Amino Acid Sequence Detection of Anti-FcRn Antibody Fused with Different Signal Peptides
  • N-glycosyl group carried by FcRn-1 and FcRn-2 samples was excised by PNGase F glycosidase (NEB, P0708), and then reduced to light and heavy chains by DTT (Sigma, 43815), and finally by LC-MS (Waters, ACQUITY UPLC H-Class/XeVo G2-XS QTOF) to detect the deglycosylated reduction molecular weight of the sample, and data processing and analysis are carried out by UNIFI software.

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Abstract

涉及用于降低异源多肽末端异质性的信号肽。具体地，涉及用于蛋白表达的信号肽，还涉及重组多肽、其制备方法及含有重组多肽的组合物。

Description

用于降低异源多肽末端异质性的信号肽 技术领域
本披露涉及分子生物学和蛋白质工程领域，具体涉及降低异源多肽末端异质性的方法。
背景技术
这里的陈述仅提供与本披露有关的背景信息，而不必然地构成现有技术。
信号肽是引导新合成的蛋白质向分泌通路转移的短肽链。信号肽位于分泌蛋白质的N末端。一般由15～30个氨基酸组成。通常包括了三个区：一个带正电的N末端，称为碱性氨基末端；一个中间疏水序列，以中性氨基酸为主，能够形成一段α螺旋结构，它是信号肽的主要功能区；一个较长的带负电荷的C末端，含小分子氨基酸，是信号序列切割位点，也称加工区。当信号肽序列合成后，被信号识别颗粒(SRP)所识别，蛋白质合成暂停或减缓，信号识别颗粒将核糖体携带至内质网上，蛋白质合成重新开始。在信号肽的引导下，新合成的蛋白质进入内质网腔，而信号肽序列则在信号肽酶的作用下被切除。
通常信号肽的高效切割，可以确保抗体轻、重链多肽均可被准确切割，这对于哺乳动物细胞介导的抗体表达非常重要。但是，在信号肽切割过程中，切割位点可能会发生变化，导致了重组抗体轻、重氨基酸链的延长或截短。截短或延长的抗体，其结构的变化会导致其亲和性发生变化，从而影响抗体活性；因此在抗体生产过程中需要避免氨基酸链延长或截短的发生。此外，部分的产物截短或延长，使得生产过程中不同批次产品产生差异，需要进行额外的质检，且从工艺上很难解决此类问题。因此，生产出具有N末端同质性的轻、重链抗体是目前工业界的普遍共识。
发明内容
本披露涉及信号肽及其用途，其中所述信号肽用于降低异源多肽的末端异质性。
本披露提供一种降低抗体的重链和/或抗体的轻链的N末端异质性的方法，其包括
培养宿主细胞，其中所述宿主细胞包含：(1)第一多核苷酸，其编码抗体的重链及与该重链的N端有效连接的第一信号肽；其中，所述第一信号肽包含SEQ ID NO:55或SEQ ID NO:56的氨基酸序列；和/或
(2)第二多核苷酸，其编码抗体的轻链及与该轻链的N端有效连接的第二信号肽；其中，所述第二信号肽包含SEQ ID NO:55或SEQ ID NO:56的氨基酸序列；
表达所述抗体的重链和/或抗体的轻链；
其中，SEQ ID NO:55如MEWSWVFLFFLSLTGX 1HX 2所示，其中，X 1是V、A、S、T、G、C、L或I，X 2是A、G、S、C、T或Q；
SEQ ID NO:56如MSVPTQVLGLLLLWLTDX 3RX 4所示，其中，X 3是V、A、S、T、G、C、L或I；X 4是A、G、S、C、T或Q，并且当X 3选自A时，X 4不是C。
在一些实施方案中，如前所述的降低抗体的重链和/或抗体的轻链的N末端异质性的方法，其中，表达得到的所述抗体的重链具有低于3％、2.5％、2％、1.5％或1％的末端延伸比率和/或抗体的轻链具有低于3％、2.5％、2％、1.5％或1％的末端延伸比率；在一些实施方案中，表达得到的所述抗体重链具有低于1％的末端延伸比率和/或抗体的轻链具有低于1％的末端延伸比率。在一些实施方案中，该末端延伸比率是基于肽图检测方法测算的。在一些实施方案中，表达得到的所述抗体重链基本上没有末端残基的残留和/或抗体的轻链基本上没有末端残基的残留；在一些实施方案中，表达得到的所述抗体重链具有0％的末端延伸比率和/或抗体的轻链具有0％的末端延伸比率。
在一些实施方案中，如前所述的降低抗体的重链和/或抗体的轻链的N末端异质性的方法，其中，所述第一信号肽或第二信号肽各自独立的包含选自如下的肽：
SEQ ID NO 57:MEWSWVFLFFLSLTGVHA；
SEQ ID NO 58:MEWSWVFLFFLSLTGVHG；
SEQ ID NO 59:MEWSWVFLFFLSLTGVHS；
SEQ ID NO 60:MEWSWVFLFFLSLTGVHC；
SEQ ID NO 61:MEWSWVFLFFLSLTGVHT；
SEQ ID NO 62:MEWSWVFLFFLSLTGVHQ；
SEQ ID NO 63:MEWSWVFLFFLSLTGAHA；
SEQ ID NO 64:MEWSWVFLFFLSLTGAHG；
SEQ ID NO 65:MEWSWVFLFFLSLTGAHS；
SEQ ID NO 66:MEWSWVFLFFLSLTGAHC；
SEQ ID NO 67:MEWSWVFLFFLSLTGAHT；
SEQ ID NO 68:MEWSWVFLFFLSLTGAHQ；
SEQ ID NO 69:MEWSWVFLFFLSLTGSHA；
SEQ ID NO 70:MEWSWVFLFFLSLTGSHG；
SEQ ID NO 71:MEWSWVFLFFLSLTGSHS；
SEQ ID NO 72:MEWSWVFLFFLSLTGSHC；
SEQ ID NO 73:MEWSWVFLFFLSLTGSHT；
SEQ ID NO 74:MEWSWVFLFFLSLTGSHQ；
SEQ ID NO 75:MEWSWVFLFFLSLTGTHA；
SEQ ID NO 76:MEWSWVFLFFLSLTGTHG；
SEQ ID NO 77:MEWSWVFLFFLSLTGTHS；
SEQ ID NO 78:MEWSWVFLFFLSLTGTHC；
SEQ ID NO 79: MEWSWVFLFFLSLTGTHT;
SEQ ID NO 80: MEWSWVFLFFLSLTGTHQ;
SEQ ID NO 81: MEWSWVFLFFLSLTGGHA;
SEQ ID NO 82: MEWSWVFLFFLSLTGGHG;
SEQ ID NO 83: MEWSWVFLFFLSLTGGHS;
SEQ ID NO 84: MEWSWVFLFFLSLTGGHC;
SEQ ID NO 85: MEWSWVFLFFLSLTGGHT;
SEQ ID NO 86: MEWSWVFLFFLSLTGGHQ;
SEQ ID NO 87: MEWSWVFLFFLSLTGCHA;
SEQ ID NO 88: MEWSWVFLFFLSLTGCHG;
SEQ ID NO 89: MEWSWVFLFFLSLTGCHS;
SEQ ID NO 90: MEWSWVFLFFLSLTGCHC;
SEQ ID NO 91: MEWSWVFLFFLSLTGCHT;
SEQ ID NO 92: MEWSWVFLFFLSLTGCHQ;
SEQ ID NO 93: MEWSWVFLFFLSLTGLHA;
SEQ ID NO 94: MEWSWVFLFFLSLTGLHG;
SEQ ID NO 95: MEWSWVFLFFLSLTGLHS;
SEQ ID NO 96: MEWSWVFLFFLSLTGLHC;
SEQ ID NO 97: MEWSWVFLFFLSLTGLHT;
SEQ ID NO 98: MEWSWVFLFFLSLTGLHQ;
SEQ ID NO 99: MEWSWVFLFFLSLTGIHA;
SEQ ID NO 100: MEWSWVFLFFLSLTGIHG;
SEQ ID NO 101: MEWSWVFLFFLSLTGIHS;
SEQ ID NO 102: MEWSWVFLFFLSLTGIHC;
SEQ ID NO 103: MEWSWVFLFFLSLTGIHT;
SEQ ID NO 104: MEWSWVFLFFLSLTGIHQ;
SEQ ID NO 105: MSVPTQVLGLLLLWLTDVRA;
SEQ ID NO 106: MSVPTQVLGLLLLWLTDVRG;
SEQ ID NO 107: MSVPTQVLGLLLLWLTDVRS;
SEQ ID NO 108: MSVPTQVLGLLLLWLTDVRC;
SEQ ID NO 109: MSVPTQVLGLLLLWLTDVRT;
SEQ ID NO 110: MSVPTQVLGLLLLWLTDVRQ;
SEQ ID NO 111: MSVPTQVLGLLLLWLTDARA;
SEQ ID NO 112: MSVPTQVLGLLLLWLTDARG;
SEQ ID NO 113: MSVPTQVLGLLLLWLTDARS;
SEQ ID NO 114: MSVPTQVLGLLLLWLTDART;
SEQ ID NO 115: MSVPTQVLGLLLLWLTDARQ;
SEQ ID NO 116: MSVPTQVLGLLLLWLTDSRA;
SEQ ID NO 117: MSVPTQVLGLLLLWLTDSRG;
SEQ ID NO 118: MSVPTQVLGLLLLWLTDSRS;
SEQ ID NO 119: MSVPTQVLGLLLLWLTDSRC; 
SEQ ID NO 120:MSVPTQVLGLLLLWLTDSRT；
SEQ ID NO 121:MSVPTQVLGLLLLWLTDSRQ；
SEQ ID NO 122:MSVPTQVLGLLLLWLTDTRA；
SEQ ID NO 123:MSVPTQVLGLLLLWLTDTRG；
SEQ ID NO 124:MSVPTQVLGLLLLWLTDTRS；
SEQ ID NO 125:MSVPTQVLGLLLLWLTDTRC；
SEQ ID NO 126:MSVPTQVLGLLLLWLTDTRT；
SEQ ID NO 127:MSVPTQVLGLLLLWLTDTRQ；
SEQ ID NO 128:MSVPTQVLGLLLLWLTDGRA；
SEQ ID NO 129:MSVPTQVLGLLLLWLTDGRG；
SEQ ID NO 130:MSVPTQVLGLLLLWLTDGRS；
SEQ ID NO 131:MSVPTQVLGLLLLWLTDGRC；
SEQ ID NO 132:MSVPTQVLGLLLLWLTDGRT；
SEQ ID NO 133:MSVPTQVLGLLLLWLTDGRQ；
SEQ ID NO 134:MSVPTQVLGLLLLWLTDCRA；
SEQ ID NO 135:MSVPTQVLGLLLLWLTDCRG；
SEQ ID NO 136:MSVPTQVLGLLLLWLTDCRS；
SEQ ID NO 137:MSVPTQVLGLLLLWLTDCRC；
SEQ ID NO 138:MSVPTQVLGLLLLWLTDCRT；
SEQ ID NO 139:MSVPTQVLGLLLLWLTDCRQ；
SEQ ID NO 140:MSVPTQVLGLLLLWLTDLRA；
SEQ ID NO 141:MSVPTQVLGLLLLWLTDLRG；
SEQ ID NO 142:MSVPTQVLGLLLLWLTDLRS；
SEQ ID NO 143:MSVPTQVLGLLLLWLTDLRC；
SEQ ID NO 144:MSVPTQVLGLLLLWLTDLRT；
SEQ ID NO 145:MSVPTQVLGLLLLWLTDLRQ；
SEQ ID NO 146:MSVPTQVLGLLLLWLTDIRA；
SEQ ID NO 147:MSVPTQVLGLLLLWLTDIRG；
SEQ ID NO 148:MSVPTQVLGLLLLWLTDIRS；
SEQ ID NO 149:MSVPTQVLGLLLLWLTDIRC；
SEQ ID NO 150:MSVPTQVLGLLLLWLTDIRT；和
SEQ ID NO 151:MSVPTQVLGLLLLWLTDIRQ。
在一些实施方案中，如前所述的降低抗体的重链和/或抗体的轻链的N末端异质性的方法，其中，所述第一信号肽或第二信号肽各自独立的包含SEQ ID NO:57、SEQ ID NO:58、SEQ ID NO:59、SEQ ID NO:93、SEQ ID NO:94、SEQ ID NO:95、SEQ ID NO:99、SEQ ID NO:100、SEQ ID NO:101、SEQ ID NO:105、SEQ ID NO:106、SEQ ID NO:107、SEQ ID NO:140、SEQ ID NO:141、SEQ ID NO:142、SEQ ID NO:146、SEQ ID NO:147或SEQ ID NO:148的氨基酸序列。
在一些实施方案中，如前所述的降低抗体的重链和/或抗体的轻链的N末端异 质性的方法，其中第一信号肽包含SEQ ID NO:57或SEQ ID NO:105的氨基酸序列，和/或第二信号肽包含SEQ ID NO:57或SEQ ID NO:105的氨基酸序列。
在一些实施方案中，如前所述的降低抗体的重链和/或抗体的轻链的N末端异质性的方法，其中，第一信号肽包含SEQ ID NO:57的氨基酸序列，和/或第二信号肽包含SEQ ID NO:57的氨基酸序列。
在一些实施方案中，如前所述的降低抗体的重链和/或抗体的轻链的N末端异质性的方法，其中，第一信号肽包含SEQ ID NO:57的氨基酸序列，和/或第二信号肽包含SEQ ID NO:105的氨基酸序列。
在一些实施方案中，如前所述的降低抗体的重链和/或抗体的轻链的N末端异质性的方法，其中，第一信号肽包含SEQ ID NO:105的氨基酸序列，和/或第二信号肽包含SEQ ID NO:57的氨基酸序列。
在一些实施方案中，如前所述的降低抗体的重链和/或抗体的轻链的N末端异质性的方法，其中，第一信号肽包含SEQ ID NO:105的氨基酸序列，和/或第二信号肽包含SEQ ID NO:105的氨基酸序列。
在一些实施方案中，如前所述的降低抗体的重链和/或抗体的轻链的N末端异质性的方法，其中，所述第一多核苷酸或第二多核苷酸各自独立的编码多肽，所述多肽包含选自SEQ ID NO:157、SEQ ID NO:158、SEQ ID NO:159、SEQ ID NO:160、SEQ ID NO:163、SEQ ID NO:164、SEQ ID NO:165、SEQ ID NO:166、SEQ ID NO:171和SEQ ID NO:172的氨基酸序列。
在一些实施方案中，如前所述的降低抗体的重链和/或抗体的轻链的N末端异质性的方法，其中，所述宿主细胞是真核宿主细胞；在一些实施方案中，所述真核宿主细胞是CHO细胞或酵母。
在一些实施方案中，如前所述的降低抗体的重链和/或抗体的轻链的N末端异质性的方法，其中，所述的抗体是鼠源抗体、嵌合抗体、人源化抗体、人抗体、亲和力成熟抗体或多特异性抗体。
在一些实施方案中，如前所述的降低抗体的重链和/或抗体的轻链的N末端异质性的方法，其中，所述的抗体选自抗TLR7抗体、抗HER2(ErbB2)抗体、抗Claudin18.2抗体、抗EGFR抗体、抗B7H3抗体、抗c-Met抗体、抗HER3(ErbB3)抗体、抗HER4(ErbB4)抗体、抗CD3抗体、抗CD20抗体、抗CD22抗体、抗CD30抗体、抗CD33抗体、抗CD38抗体、抗CD44抗体、抗CD47抗体、抗CD56抗体、抗CD70抗体、抗CD73抗体、抗CD105抗体、抗CEA抗体、抗A33抗体、抗Cripto抗体、抗SOST抗体、抗EphA2抗体、抗G250抗体、抗MUCl抗体、抗Lewis Y抗体、抗VEGFR抗体、抗GPNMB抗体、抗凝血酶抗体、抗Aβ抗体、抗Integrin抗体、抗ANGPTL3抗体、抗PSMA抗体、抗PD-1抗体、抗PD-L1抗体、抗LAG3抗体、抗IL-5抗体、抗IL-15抗体、抗IL-4R抗体、抗IL-6R抗体、抗TIGHT抗体、抗Tenascin-C抗体、抗SLC44A4抗体、抗PCSK9抗体、抗EpCAM 抗体、抗CTGF抗体、抗TSLP抗体、抗CEA抗体、抗Mesothelin抗体和抗FcRn抗体。
在一些实施方案中，如前所述的降低抗体的重链和/或抗体的轻链的N末端异质性的方法，其中，所述的抗体选自Trastuzumab、Pertuzumab、Nimotuzumab、Enoblituzumab、Emibetuzumab、Inotuzumab、Pinatuzumab、Brentuximab、Gemtuzumab、Bivatuzumab、Lorvotuzumab、cBR96、Glematumamab、抗Claudin18.2抗体和抗FcRn抗体，其中所述抗Claudin18.2抗体的重链包含SEQ ID NO:49的氨基酸序列、轻链包含SEQ ID NO:47的氨基酸序列；所述抗FcRn抗体的重链包含SEQ ID NO:167的氨基酸序列，轻链包含SEQ ID NO:168的氨基酸序列。
在一些实施方案中，如前所述的降低抗体的重链和/或抗体的轻链的N末端异质性的方法，所述方法包括将轻链质粒克隆至重链质粒中构建全长抗体质粒。在一些实施方案中，所述方法通过电转化将质粒导入宿主细胞。
本披露还提供一种信号肽，其包含SEQ ID NO:55或SEQ ID NO:56的氨基酸序列，或由其组成。在一些实施方案中，所述的多肽是信号肽，其氨基酸序列如SEQ ID NO:55至SEQ ID NO:151任一所示。
一种多肽，其包含信号肽，所述信号肽包含SEQ ID NO:55或SEQ ID NO:56的氨基酸序列。
在一些实施方案中，如前所述的多肽，其中，所述多肽还包含与所述信号肽有效连接的异源多肽；在一些实施方案中，所述异源多肽是抗体的重链或抗体的轻链；在一些实施方案中，所述信号肽与抗体的重链或抗体的轻链的N端有效连接。
在一些实施方案中，如前所述的多肽，其中，所述信号肽包含选自SEQ ID NO57至SEQ ID NO:151组成的组的氨基酸序列。
在一些实施方案中，如前所述的多肽，其中，所述信号肽包含SEQ ID NO:57、SEQ ID NO:58、SEQ ID NO:59、SEQ ID NO:93、SEQ ID NO:94、SEQ ID NO:95、SEQ ID NO:99、SEQ ID NO:100、SEQ ID NO:101、SEQ ID NO:105、SEQ ID NO:106、SEQ ID NO:107、SEQ ID NO:140、SEQ ID NO:141、SEQ ID NO:142、SEQ ID NO:146、SEQ ID NO:147或SEQ ID NO:148的氨基酸序列。
在一些实施方案中，如前所述的多肽，其中，所述信号肽包含选自如SEQ ID NO:57和SEQ ID NO:105所示的氨基酸序列。
在一些实施方案中，如前所述的多肽，其中，多肽包含选自SEQ ID NO:157、SEQ ID NO:158、SEQ ID NO:159、SEQ ID NO:160、SEQ ID NO:163、SEQ ID NO:164、SEQ ID NO:165、SEQ ID NO:166、SEQ ID NO:169、SEQ ID NO:170、SEQ ID NO:171和SEQ ID NO:172组成的组的氨基酸序列。
本披露还提供一种核酸分子，其编码如前任一项所述的多肽。
本披露还提供一种宿主细胞，其包含如前所述的核酸分子。
在一些实施方案中，如前所述的宿主细胞，其中，所述宿主细胞是真核宿主细胞；在一些实施方案中，所述真核宿主细胞是CHO细胞或酵母。
本披露还提供一种组合物，其含有异源多肽，所述异源多肽具有低于3％、2.5％、2％、1.5％或1％的末端延伸比率；在一些实施方案中，表达得到的所述异源多肽具有低于1％的末端延伸比率；在一些实施方案中，表达得到的所述异源多肽具有低于1％的末端延伸比率，并且该末端延伸比率是基于肽图检测方法测算的；在一些实施方案中，表达得到的所述异源多肽基本上没有末端残基的残留；在一些实施方案中，表达得到的所述异源多肽具有0％的末端延伸比率。
在一些实施方案中，如前所述的组合物，其中，所述的异源多肽是抗体的重链和/或抗体的轻链；在一些实施方案中，所述抗体选自Trastuzumab、Pertuzumab、抗Claudin18.2抗体和抗FcRn抗体，其中所述抗Claudin18.2抗体的重链包含SEQ ID NO:49的氨基酸序列，轻链包含SEQ ID NO:47的氨基酸序列。
在一些实施方案中，所述抗体选自抗FcRn抗体，其中所述抗FcRn抗体的重链包含SEQ ID NO:167的氨基酸序列，轻链包含SEQ ID NO:168的氨基酸序列。
在一些实施方案中，如前所述的降低抗体的重链和/或抗体的轻链的N末端异质性的方法，其中，表达得到的所述抗体的重链和/或抗体的轻链具有低于3％、2.5％、2％、1.5％或1％的末端延伸比率；在一些实施方案中，表达得到的所述抗体重链和/或抗体的轻链具有低于1％的末端延伸比率；在一些实施方案中，表达得到的所述抗体重链和/或抗体的轻链具有低于1％的末端延伸比率，并且该末端延伸比率是基于肽图检测方法测算的；在一些实施方案中，表达得到的所述抗体重链和/或抗体的轻链基本上没有末端残基的残留；在一些实施方案中，表达得到的所述抗体重链和/或抗体的轻链具有0％的末端延伸比率。
在一些实施方案中，如前所述的组合物，其是由如前任一项所述的方法制备得到的。
本披露提供的信号肽具有稳定的降低异源多肽末端异质性的效果，适用于大规模表达异源多肽。
附图说明
图1：在细胞水平上，人源化抗体与人Claudin18.2结合的FACS检测结果。
图2：人源化抗体的NUGC4细胞内吞实验。
图3A至图3C：在不同Claudin18.2表达程度的NUGC4细胞中，抗体的ADCC效应检测；图3A为抗体在野生型NUGC4细胞(Claudin18.2低表达)中的ADCC效应检测；图3B为抗体在Claudin18.2中等表达NUGC4细胞中的ADCC效应检测；图3C为抗体在Claudin18.2高表达NUGC4细胞中的ADCC效应检测。
图4A至图4D：图4A为Pertuzumab-1抗体轻链去糖还原分子量质谱图；图4B为Pertuzumab-3抗体轻链去糖还原分子量质谱图；图4C为Pertuzumab-1抗体 重链链去糖还原分子量质谱图；图4D为Pertuzumab-3抗体重链去糖还原分子量质谱图。
图5A至图5D：图5A为Pertuzumab-2抗体轻链去糖还原分子量质谱图；图5B为Pertuzumab-4抗体轻链去糖还原分子量质谱图；图5C为Pertuzumab-2抗体重链链去糖还原分子量质谱图；图5D为Pertuzumab-4抗体重链去糖还原分子量质谱图。
图6A至图6D：图6A为h1902-5-1抗体轻链去糖还原分子量质谱图；图6B为h1902-5-2抗体轻链去糖还原分子量质谱图；图6C为h1902-5-1抗体重链链去糖还原分子量质谱图；图6D为h1902-5-2抗体重链去糖还原分子量质谱图。
图7A至图7D：图7A为FcRn-1抗体轻链去糖还原分子量质谱图；图7B为FcRn-2抗体轻链去糖还原分子量质谱图；图7C为FcRn-1抗体重链链去糖还原分子量质谱图；图7D为FcRn-2抗体重链去糖还原分子量质谱图。
具体实施方式
发明详述
术语
为了更容易理解本披露，以下具体定义了某些技术和科学术语。除非在本文中另有明确定义，本文使用的所有其它技术和科学术语都具有本披露所属领域的一般技术人员通常理解的含义。
本披露所用氨基酸三字母代码和单字母代码如J.biol.chem，243，p3558(1968)中所述。
本披露所述的“抗体”在本文中以最广意义使用，并且包括不同的抗体结构，包括，但不限于，单克隆抗体，多克隆抗体，鼠源抗体、嵌合抗体、人源化抗体、多特异性抗体(例如，双特异性抗体)和抗体片段，只要它们显示所需的抗原结合活性和特异性即可。
术语“抗体片段”是指不同于完整抗体的分子，其包含完整抗体的部分，所述部分与完整抗体特异性结合的抗原特异性结合。抗体片段的实例包括但不限于Fv，Fab，Fab'，Fab’-SH，F(ab') 2，双抗体，线性抗体，单链抗体分子(例如scFv或scFab)，单域抗体(dAbs)，和由抗体片段形成的多特异性抗体。
本披露所述的“嵌合抗体(chimeric antibody)”，是将鼠源性抗体的可变区与人抗体的恒定区融合而成的抗体，可以减轻鼠源性抗体诱发的免疫应答反应。
本披露所述的“人源化抗体(humanized antibody)”，也称为CDR移植抗体(CDR-grafted antibody)，是指将鼠的CDR序列移植到人的抗体可变区框架，即不同类型的人种系抗体框架序列中产生的抗体。可以克服嵌合抗体由于携带大量鼠蛋白成分，从而诱导的异源性反应。为避免免疫原性下降的同时，引起的活性下降，可对所述的人抗体可变区框架序列进行最少反向突变或回复突变，以保持 或增强活性。本披露的人源化抗体也包括进一步由酵母菌展示对CDR进行亲和力成熟突变后的人源化抗体。
本披露中“人抗体(HuMAb)”、“人源抗体”、“全人抗体”、“完全人抗体”可以互换使用，其氨基酸序列对应于由人或人细胞产生的抗体的氨基酸序列、或衍生自利用人抗体组库或其它人抗体编码序列的非人来源的氨基酸序列。该人抗体的定义明确排除了包含非人抗原结合残基的人源化抗体。
本披露所述的“抗体片段”是指不同于完整抗体的分子，其包含完整抗体的部分，所述部分与完整抗体特异性结合的抗原特异性结合。抗体片段的实例包括但不限于Fv，Fab，Fab'，Fab’-SH，F(ab') 2，双抗体，线性抗体，单链抗体分子(例如scFv或scFab)，单域抗体(dAbs)，和由抗体片段形成的多特异性抗体。本披露的“抗体”包含“完整抗体”及其“抗体片段”。相应的，本披露所述的抗体的重链或抗体的轻链包含完整的重链或完整的轻链，也包含抗体片段中的重链片段或轻链片段。
术语“框架”或“FR”指除了互补决定区(CDR)残基以外的可变域残基。可变域的FR一般由四个FR域组成：FR1、FR2、FR3和FR4。因而，CDR和FR序列一般以下面的次序在VH(或VL)中出现：
FR1-HCDR1(LCDR1)-FR2-HCDR2(LCDR2)-FR3-HCDR3(LCDR3)-FR4。
术语“互补决定区”、“CDR”或“高变区”是指抗体的可变结构域内主要促成抗原结合的6个高变区之一。通常，每个重链可变区中存在三个CDR(HCDR1、HCDR2、HCDR3)，每个轻链可变区中存在三个CDR(LCDR1、LCDR2、LCDR3)。可以使用各种公知方案中的任何一种来确定CDR的氨基酸序列边界，包括“Kabat”编号规则(参见Kabat等(1991)，“Sequences of Proteins of Immunological Interest”，第5版，Public Health Service，National Institutes of Health，Bethesda，MD)、“Chothia”编号规则(参见Al-Lazikani等人，(1997)JMB 273：927-948)和ImMunoGenTics(IMGT)编号规则(Lefranc M.P.，Immunologist，7，132-136(1999)；Lefranc，M.P.等，Dev.Comp.Immunol.，27，55-77(2003)等。例如，对于经典格式，遵循Kabat规则，所述重链可变域(VH)中的CDR氨基酸残基编号为31-35(HCDR1)、50-65(HCDR2)和95-102(HCDR3)；轻链可变域(VL)中的CDR氨基酸残基编号为24-34(LCDR1)、50-56(LCDR2)和89-97(LCDR3)。遵循Chothia规则，VH中的CDR氨基酸编号为26-32(HCDR1)、52-56(HCDR2)和95-102(HCDR3)；并且VL中的氨基酸残基编号为26-32(LCDR1)、50-52(LCDR2)和91-96(LCDR3)。通过组合Kabat和Chothia两者的CDR定义，CDR由人VH中的氨基酸残基26-35(HCDR1)、50-65(HCDR2)和95-102(HCDR3)和人VL中的氨基酸残基24-34(LCDR1)、50-56(LCDR2)和89-97(LCDR3)构成。遵循IMGT规则，VH中的CDR氨基酸残基编号大致为26-35(CDR1)、51-57(CDR2)和93-102(CDR3)，VL中的CDR氨基酸残基编号大致为27-32(CDR1)、 50-52(CDR2)和89-97(CDR3)。遵循IMGT规则，抗体的CDR区可以使用程序IMGT/DomainGap Align确定。除非另有说明，本披露实施例涉及的抗体可变区和CDR序列均适用“Kabat”编号规则。
术语“特异性结合”、“选择性结合”、“选择性地结合”和“特异性地结合”是指抗体对预先确定的抗原上的表位的结合。通常，抗体以大约小于10 -8M，例如大约小于10 -9M、10 -10M、10 -11M、10 -12M或更小的亲和力(KD)结合。
术语“KD”是指特定抗体-抗原相互作用的解离平衡常数。通常，本披露的抗体以小于大约10 -7M，例如小于大约10 -8M或10 -9M的解离平衡常数(KD)结合抗原，例如，在本披露中抗体与细胞表面抗原的亲和力采用FACS法测定KD值。
本披露中可互换使用的“多核苷酸”或“核酸分子”指任意长度的核苷酸聚合物，且包括DNA和RNA。核苷酸可以是脱氧核糖核苷酸、核糖核苷酸、修饰的核苷酸或碱基、和/或其类似物，或可以通过DNA或RNA聚合酶或通过合成反应掺入聚合物的任意底物。多核苷酸可以包含修饰的核苷酸，如甲基化的核苷酸及其类似物。如果存在，则对核苷酸结构的修饰可以在组装聚合物之前或之后进行。核苷酸的序列可以由非核苷酸成分中断。
本披露中所述的“有效连接的”指两种或多种成分的并列，其中所述成分处于允许它们以其预期方式发挥作用的关系中。例如，如果启动子顺式作用来控制或调节所连接的序列的转录，则它与编码序列有效连接。通常，但并非必然，“有效连接的”DNA序列是相邻的，且在有必要连接两个蛋白质编码区时或在分泌前导序列的情况下是相邻和符合可读框的。但是，虽然有效连接的启动子通常位于编码序列的上游，但它并非必然与它相邻。有效连接的增强子可以位于编码序列上游、编码序列内或编码序列下游，且距启动子相当的距离。通过本领域已知重组方法来实现连接，例如，使用PCR方法，通过退火，或通过在方便的限制位点处连接。如果不存在方便的限制位点，则按照常规实施使用合成的寡核苷酸衔接子或接头。本披露中，信号肽与异源多肽的一种连接方式是直接连接。
本披露中所述的“宿主细胞”指已进行遗传改变或能够通过引入外源多核苷酸(如重组质粒或载体)进行遗传改变的细胞。应理解，这类术语旨在不仅指具体个体细胞，还指这种细胞的后代。由于连续世代中可发生由突变或环境影响引起的某些修饰，这种后代实际上可以与亲本细胞不同，但仍包括在本披露所用的术语“宿主细胞”的范围内。宿主细胞可以微生物(如真核微生物)或动物细胞，适当的微生物包括酿酒酵母(Saccharomyces cerevisiae)和毕赤酵母(Pichia pastoris)；适当的动物宿主细胞系包括CHO(中国仓鼠卵巢细胞系)、293细胞和NS0细胞。
本披露中所述的“多肽”泛指来自任意细胞来源的具有超过约10个氨基酸的肽和蛋白质。“异源”多肽是对所利用的宿主细胞而言为外来的那些多肽，如通过宿主细胞产生的人蛋白质。异源多肽可以是原核的或真核的，如哺乳动物的或人的。异源多肽可以是重组产生的多肽或重组多肽。
示例性异源多肽包括跨膜分子(例如受体，如受体酪氨酸激酶)或配体，如生长因子。示例性异源多肽包括诸如以下的分子：肾素；生长激素，包括人生长激素和牛生长激素；生长激素释放因子；甲状旁腺激素；促甲状腺激素；脂蛋白；α1-抗胰蛋白酶；胰岛素A链；胰岛素B链；胰岛素原；促卵泡激素；降钙素；黄体生成素；胰高血糖素；凝血因子，如因子VIIIC、因子IX、组织因子(TF)和von Willebrands因子；抗凝血因子，如蛋白C；心房钠尿肽；肺表面活性剂；纤溶酶原激活物，如尿激酶或人尿或组织型纤溶酶原激活物(t-PA)；铃蟾肽；凝血酶；造血生长因子；肿瘤坏死因子-α和-β；脑啡肽酶；RANTES(regulated onactivation normally T-cell expressed and secreted)；人巨噬细胞炎症蛋白(MIP-1-α)；血清白蛋白，如人血清白蛋白；Muellerian抑制物质；松弛素A链；松弛素B链；松弛素原；小鼠促性腺激素相关肽；微生物蛋白质，如β-内酰胺酶；DNA酶；IgE；细胞毒性T淋巴细胞相关抗原(CTLA)，如CTLA-4；抑制素；激活蛋白；血管内皮生长因子(VEGF)；激素或生长因子的受体；蛋白A或D；类风湿因子；神经营养因子，如骨衍生神经营养因子(BDNF)，神经营养蛋白-3、-4、-5或-6(NT-3、NT-4、NT-5或NT-6)，或神经生长因子如NGFβ；血小板衍生生长因子(PDGF)；成纤维细胞生长因子，如aFGF和bFGF；表皮生长因子(EGF)；转化生长因子(TGF)，如TGF-α和TGF-β，包括TGF-β1、TGF-β2、TGF-β3、TGF-β4或TGF-β5；胰岛素样生长因子-I和-II(IGF-I和IGF-II)；des(1-3)-IGF-I(脑IGF-I)，胰岛素样生长因子结合蛋白；CD蛋白，如CD3、CD4、CD8、CD19、CD20和CD40；促红细胞生成素；骨诱导因子；免疫毒素；骨形态发生蛋白(BMP)；干扰素，如干扰素-α、-β和-γ；集落刺激因子(CSF)，例如M-CSF、GM-CSF和G-CSF；白介素(IL)，例如IL-1至IL-10；超氧化物歧化酶；T-细胞受体；表面膜蛋白；衰变加速因子；病毒抗原，例如AIDS被膜的部分；转运蛋白；归巢受体；地址素；调节蛋白；整联蛋白，如CD11a、CD11b、CD11c、CD18、ICAM、VLA-4和VCAM；肿瘤相关抗原，如HER2、HER3或HER4受体；及任意上列多肽的片段。
异源多肽也可以是抗体，示例性的，所述抗体的靶标包括但不限于A33、BMPI、BMP2、BMP3B(GDFIO)、BMP4、BMP6、BMP8、CSFI(M-CSF)、CSF2(GM-CSF)、CSF3(G-CSF)、EPO、FGFI(aFGF)、FGF2(bFGF)、FGF3(int-2)、FGF4(HST)、FGF5、FGF6(HST-2)、FGF7(KGF)、FGF9、FGF10、FGF1 1、FGF12、FGF12B、FGF14、FGF16、FGF17、FGF19、FGF20、FGF21、FGF23、IGF1、IGF2、IFNAI、IFNA2、IFNA4、IFNA5、IFNA6、IFNA7、IFNBI、IFNG、IFNWI、FELI、FELI(EPSELON)、FELI(ZETA)、IL1A、IL1B、IL2、IL3、IL4、IL5、IL6、IL7、IL8、IL9、IL10、IL11、IL12A、I L12B、IL13、IL14、IL15、I L16、IL17、IL17B、IL18、IL19、IL20、IL22、IL23、IL24、IL25、IL26、IL27、IL28A、IL28B、I L29、IL30、PDGFA、PDGFB、TGFA、TGFB1、TGFB2、TGFB3、LTA(TNF-b)、LTB、TNF(TNF-a)、TNFSF4(OX40配体)、TNFSF5(CD40配体)、TNFSF6(FasL)、TNFSF7(CD27配体)、 TNFSF8(CD30配体)、TNFSF9(4-1BB配体)、TNFSFIO(TRAIL)、TNFSF1I(TRANCE)、TNFSF12(AP03L)、TNFSF13(April)、TNFSF13B、TNFSF14(HVEM-L)、TNFSF15(VEGI)、TNFSF18、HGF(VEGFD)、VEGF、VEGFB、VEGFC、ILIR1、IL1R2、IL1RL1、IL1RL2、IL2RA、IL2RB、IL2RG、IL3RA、IL4R、IL5RA、IL6R、IL7R、IL8RA、IL8RB、IL9R、ILIORA、ILIORB、IL1 1RA、IL12RB1、IL12RB2、IL13RA1、IL13RA2、IL15RA、IL17R、IL18R1、IL20RA、IL21R、IL22R、IL1HY1、IL1RAP、IL1RAPL1、IL1RAPL2、IL1RN、IL6ST、IL18BP、IL18RAP、IL22RA2、AIFI、HGF、LEP(瘦蛋白)、PTN、THPO、CCLI(I-309)、CCL2(MCP-1/MCAF)、CCL3(MIP-la)、CCL4(MIP-lb)、CCL5(RANTES)、CCL7(MCP-3)、CCL8(mcp-2)、CCLH(eotaxin)、CCL13(MCP-4)、CCL15(MIP-ld)、CCL16(HCC-4)、CCL17(TARC)、CCL18(PARC)、CCL19(MDP-3b)、CCL20(MIP-3a)、CCL21(SLC/exodus-2)、CCL22(MDC/STC-I)、CCL23(MPIF-I)、CCL24(MPIF-2/eotaxin-2)、CCL25(TECK)、CCL26(eotaxin-3)、CCL27(CTACK/ILC)、CCL28、CXCLI(GROI)、CXCL2(GR02)、CXCL3(GR03)、CXCL5(ENA-78)、CXCL6(GCP-2)、CXCL9(MIG)、CXCL10(IP 10)、CXCL1 1(I-TAC)、CXCL12(SDFI)、CXCL13、CXCL14、CXCL16、PF4(CXCL4)、PPBP(CXCL7)、CX3CL1(SCYDI)、SCYEI、XCLI(lymphotactin)、XCL2(SCM-lb)、BLRI(MDR15)、CCBP2(D6/JAB61)、CCR1(CKRI/HM145)、CCR2(mcp-IRB/RA)、CCR3(CKR3/CMKBR3)、CCR4、CCR5(CMKBR5/ChemR13)、CCR6(CMKBR6/CKR-L3/STRL22/DRY6)、CCR7(CKR7/EBII)、CCR8(CMKBR8/TERI/CKR-LI)、CCR9(GPR-9-6)、CCRLI(VSHKI)、CCRL2(L-CCR)、XCRI(GPR5/CCXCRI)、CMKLRI、CMKORI(RDCI)、CX3CR1(V28)、CXCR4、GPR2(CCRIO)、GPR31、GPR81(FKSG80)、CXCR3(GPR9/CKR-L2)、CXCR6(TYMSTR/STRL33/Bonzo)、HM74、IL8RA(IL8Ra)、IL8RB(IL8Rb)、LTB4R(GPR16)、TCPIO、CKLFSF2、CKLFSF3、CKLFSF4、CKLFSF5、CKLFSF6、CKLFSF7、CKLFSF8、BDNF、C5R1、CSF3、GRCCIO(CIO)、EPO、FY(DARC)、GDF5、HDFIA、DL8、PRL、RGS3、RGS13、SDF2、SLIT2、TLR2、TLR4、TREMI、TREM2、VHL、ABCFI、ACVRI、ACVRIB、ACVR2、ACVR2B、ACVRLI、AD0RA2A、Aggrecan、AGR2、AICDA、AIFI、AIGI、AKAPI、AKAP2、AMH、AMHR2、ANGPTI、ANGPT2、ANGPTL3、ANGPTL4、ANPEP、APC、APOCI、AR、AZGPI(锌-a-糖蛋白)、Aβ、B7.1、B7.2、B7H3、BAD、BAFF(BLys)、BAGI、BAH、BCL2、BCL6、BDNF、BLNK、BLRI(MDR15)、BMPI、BMP2、BMP3B(GDFIO)、BMP4、BMP6、BMP8、BMPRIA、BMPRIB、BMPR2、BPAGI(网蛋白)、BRCAI、C19orflO(IL27w)、C3、C4A、C5、C5R1、CANTI、CASP1、CASP4、CAVI、CCBP2(D6/JAB61)、CCLI(1-309)、CCLII(eotaxin)、CCL13(MCP-4)、CCL15(MlP-ld)、CCL16(HCC-4)、CCL17(TARC)、CCL18(PARC)、CCL19(MIP-3b)、CCL2(MCP-1)、MCAF、CCL20(MIP-3a)、CCL21(MTP-2)、SLC、exodus-2、CCL22(MDC/STC-I)、 CCL23(MPIF-1)、CCL24(MPIF-2/eotaxin-2)、CCL25(TECK)、CCL26(eotaxin-3)、CCL27(CTACK/ILC)、CCL28、CCL3(MTP-la)、CCL4(MDP-lb)、CCL5(RANTES)、CCL7(MCP-3)、CCL8(mcp-2)、CCNAI、CCNA2、CCNDI、CCNEI、CCNE2、CCRI(CKRI/HM145)、CCR2(mcp-IRB/RA)；CCR3(CKR3/CMKBR3)、CCR4、CCR5(CMKBR5/ChemR13)、CCR6(CMKBR6/CKR-L3/STRL22/DRY6)、CCR7(CKR7/EBII)、CCR8(CMKBR8/TERI/CKR-LI)、CCR9(GPR-9-6)、CCRLI(VSHKI)、CCRL2(L-CCR)、CD164、CD19、CD105、CDIC、CD20、CD200、CD22、CD24、CD28、CD3、CD33、CD37、CD38、CD3E、CD3G、CD3Z、CD4、CD40、CD40L、CD44、CD47、CD45RB、CD52、CD56、CD69、CD70、CD72、CD73、CD74、CD79A、CD79B、CD8、CD80、CD81、CD83、CD86、CDHI(E-钙黏着蛋白)、CDH10、CDH12、CDH13、CDH18、CDH19、CDH20、CDH5、CDH7、CDH8、CDH9、CDK2、CDK3、CDK4、CDK5、CDK6、CDK7、CDK9、CDKNIA(p21Wapl/Cipl)、CDKNIB(p27Kipl)、CDKNIC、CDKN2A(P16INK4a)、CDKN2B、CDKN2C、CDKN3、CEA、CEBPB、CERI、CHGA、CHGB、几丁质酶、CHST10、CKLFSF2、CKLFSF3、CKLFSF4、CKLFSF5、CKLFSF6、CKLFSF7、CKLFSF8、CLDN3、CLDN7、Claudin18.2、CLN3、CLU(簇蛋白)、c-Met、CMKLRI、CMKORI(RDCI)、CNRI、COL18A1、COLIAI、COL4A3、COL6A1、CR2、Cripto、CRP、CSFI(M-CSF)、CSF2(GM-CSF)、CSF3(GCSF)、CTGF、CTLA4、CTNNBI(b-联蛋白)、CTSB(组织蛋白酶B)、CX3CL1(SCYDI)、CX3CR1(V28)、CXCLI(GROI)、CXCL10(IP-10)、CXCLII(l-TAC/IP-9)、CXCL12(SDFI)、CXCL13、CXCL14、CXCL16、CXCL2、(GR02)、CXCL3(GR03)、CXCL5(ENA-78/LIX)、CXCL6(GCP-2)、CXCL9(MIG)、CXCR3(GPR9/CKR-L2)、CXCR4、CXCR6(TYMSTR/STRL33/Bonzo)、CYB5、CYCI、CYSLTRI、DAB2IP、DES、DKFZp451J01 18、DNCLI、DPP4、E2F1、ECGFI、EDGI、EFNAI、EFNA3、EFNB2、EGF、EGFR、ELAC2、ENG、EN01、EN02、EN03、EpCAM、EPHB4、EPO、ERBB2(Her-2)、ERBB3(Her-3)、ERBB4(Her-4)、EREG、ERK8、ESRI、ESR2、F3(TF)、FADD、FasL、FASN、FCERIA、FCER2、FCGR3A、FGF、FGFI(aFGF)、FGF10、FGF1 1、FGF12、FGF12B、FGF13、FGF14、FGF16、FGF17、FGF18、FGF19、FGF2(bFGF)、FGF20、FGF21、FGF22、FGF23、FGF3(int-2)、FGF4(HST)、FGF5、FGF6(HST-2)、FGF7(KGF)、FGF8、FGF9、FGFR3、FIGF(VEGFD)、FELI(EPSILON)、FILI(ZETA)、FLJ12584、FLJ25530、FLRTI(纤连蛋白)、FLTI、FOS、FOSLI(FRA-I)、FY(DARC)、G250、GABRP(GABAa)、GAGEBI、GAGECI、GALNAC4S-6ST、GAT A3、GDF5、GFI1、GGT1、GM-CSF、GNASI、GNRHI、GPR2(CCRIO)、GPR31、GPR44、GPR81(FKSG80)、GRCCIO(CIO)、GRP、GPNMB、GSN(绒毛蛋白)、GSTPI、HAVCR2、HDAC4、HDAC5、HDAC7A、HDAC9、HGF、HIFIA、HDPI、组胺和组胺受体、HLA-A、HLA-DRA、HM74、HMOXI、HUMCYT2A、ICEBERG、ICOSL、ID2、 IFN-a、IFNAI、IFNA2、IFNA4、IFNA5、IFNA6、IFNA7、IFNB1、IFNγ、DFNWI、IGBPI、IGFI、IGFIR、IGF2、IGFBP2、IGFBP3、IGFBP6、IL-I、IL10、MORA、IL10RB、IL1 1、IL1 1RA、IL-12、IL12A、IL12B、IL12RB1、IL12RB2、IL13、IL13RA1、IL13RA2、IL14、IL15、IL15RA、IL16、IL17、IL17B、IL17C、IL17R、IL18、IL18BP、IL18R1、IL18RAP、IL19、IL1A、IL1B、ILIF10、IL1F5、IL1F6、IL1F7、IL1F8、IL1F9、IL1HYI、IL1Rl、IL1R2、IL1RAP、IL1RAPL1、IL1RAPL2、IL1RL1、IL1RL2、ILIRN、IL2、IL20、IL20RA、IL21R、IL22、IL22R、IL22RA2、IL23、IL24、IL25、IL26、IL27、I L28A、IL28B、IL29、IL2RA、IL2RB、IL2RG、IL3、IL30、IL3RA、IL4、IL4R、IL5、IL5RA、IL6、IL6R、IL6ST(糖蛋白130)、EL7、EL7R、EL8、EphA2、IL8RA、DL8RB、IL8RB、DL9、DL9R、DLK、INHA、INHBA；INSL3、INSL4、IRAKI、Integrin、ERAK2、ITGAI、ITGA2、ITGA3、ITGA6(a6整联蛋白)、ITGAV、ITGB3、ITGB4(b4整联蛋白)、JAGI、JAKI、JAK3、JUN、K6HF、KAN、KDR、KITLG、KLF5(GC Box BP)、KLF6、KLKIO、KLK12、KLK13、KLK14、KLK15、KLK3、KLK4、KLK5、KLK6、KLK9、KRT1、KRT19(角蛋白19)、KRT2A、KHTHB6(毛发特异性H型角蛋白)、LAG3、LAMAS、LEP(瘦蛋白)、Lewis Y、Lingo-p75、Lingo-Troy、LPS、LTA(TNF-b)、LTB、LTB4R(GPR16)、LTB4R2、LTBR、MACMARCKS、MAG或Omgp、MAP2K7(c-Jun)、MDK、Mesothelin、MIBI、MUCl、中期因子、MEF、MIP-2、MKI67、(Ki-67)、MMP2、MMP9、MS4A1、MSMB、MT3(metallothionectin-lll)、MTSSI、MUCI(黏蛋白)、MYC、MYD88、NCK2、neurocan、NFKBI、NFKB2、NGFB(NGF)、NGFR、NgR-Lingo、NgR-Nogo66(Nogo)、NgR-p75、NgR-Troy、NMEI(NM23A)、N0X5、NPPB、NROBI、NR0B2、NRIDI、NR1D2、NR1H2、NR1H3、NR1H4、NR1I2、NR1I3、NR2C1、NR2C2、NR2E1、NR2E3、NR2F1、NR2F2、NR2F6、NR3C1、NR3C2、NR4A1、NR4A2、NR4A3、NR5A1、NR5A2、NR6A1、NRPI、NRP2、NT5E、NTN4、ODZI、OPRDI、P2RX7、PAP、PARTI、PATE、PAWR、PCA3、PCNA、PCSK9、PD-1、PD-L1、PDGFA、PDGFB、PECAMI、PF4(CXCL4)、PGF、PGR、phosphacan、PIAS2、PIK3CG、PLAU(uPA)、PLG、PLXDCI、PPBP(CXCL7)、PPID、PRI、PRKCQ、PRKDI、PRL、PROC、PROK2、PSAP、PSCA、PSMA、PTAFR、PTEN、PTGS2(COX-2)、PTN、RAC2(p21Rac2)、RARB、RGSI、RGS13、RGS3、RNFIIO(ZNF144)、ROB02、S100A2、SCGB1D2(亲脂素B)、SCGB2A1(mammaglobin2)、SCGB2A2(mamnnaglobin 1)、SCYEI(内皮单核细胞激活细胞因子)、SDF2、SERPINAI、SERPINA3、SERP1NB5(乳腺丝抑蛋白)、SERPINEI(PAI-I)、SERPDMF1、SHBG、SLA2、SLC2A2、SLC33A1、SLC43A1、SLC44A4、SLIT2、SOST、SPPI、SPRRIB(Sprl)、ST6GAL1、STABI、STAT6、STEAP、STEAP2、TB4R2、TBX21、TCPIO、TDGFI、TEK、TGFA、TGFBI、TGFBIII、TGFB2、TGFB3、TGFBI、TGFBRI、TGFBR2、TGFBR3、THIL、THBSI(血小板反应蛋白-1)、THBS2、THBS4、THPO、TIE(Tie-1)、TIGHT、Tenascin-C、 TMP3、组织因子、TLRIO、TLR2、TLR3、TLR4、TLR5、TLR6、TLR7、TLR8、TLR9、TNF、TNF-a、TNFAEP2(B94)、TNFAIP3、TNFRSFIIA、TNFRSFIA、TNFRSFIB、TNFRSF21、TNFRSF5、TNFRSF6(Fas)、TNFRSF7、TNFRSF8、TNFRSF9、TNFSFIO(TRAIL)、TNFSFI 1(TRANCE)、TNFSF12(AP03L)、TNFSF13(April)、TNFSF13B、TNFSF14(HVEM-L)、TNFSF15(VEGI)、TNFSF18、TNFSF4(OX40配体)、TNFSF5(CD40配体)、TNFSF6(FasL)、TNFSF7(CD27配体)、TNFSF8(CD30配体)、TNFSF9(4-1BB配体)、TOLLIP、Toll样受体、TOP2A(拓扑异构酶Ea)、TP53、TPMI、TPM2、TRADD、TRAFI、TRAF2、TRAF3、TRAF4、TRAF5、TRAF6、TREMI、TREM2、TRPC6、TSLP、TWEAK、VEGF、VEGFB、VEGFC、VEGFR、多能聚糖、VHL C5、VLA-4、XCLI(lymphotactin)、XCL2(SCM-lb)、XCRI(GPR5/CCXCRI)、YYI、ZFPM2和凝血酶。
示例性的是，抗体选自Trastuzumab、Pertuzumab、Nimotuzumab、Enoblituzumab、Emibetuzumab、Inotuzumab、Pinatuzumab、Brentuximab、Gemtuzumab、Bivatuzumab、Lorvotuzumab、cBR96、Glematumamab和抗Claudin18.2抗体，其中所述抗Claudin18.2抗体的重链如SEQ ID NO:49所示，轻链如SEQ ID NO:47所示。
“保守修饰”或“保守置换或取代”是指具有类似特征(例如电荷、侧链大小、疏水性/亲水性、主链构象和刚性等)的其它氨基酸置换蛋白中的氨基酸，使得可频繁进行改变而不改变蛋白的生物学活性。本领域技术人员知晓，一般而言，多肽的非必需区域中的单个氨基酸置换基本上不改变生物学活性(参见例如Watson等(1987)Molecular Biology of the Gene，The Benjamin/Cummings Pub.Co.，第224页，(第4版))。另外，结构或功能类似的氨基酸的置换不大可能破环生物学活性。示例性保守取代于下方陈述。
表1.氨基酸保守取代
原始残基 保守取代
Ala(A) Gly；Ser
Arg(R) Lys；His
Asn(N) Gln；His；Asp
Asp(D) Glu；Asn
Cys(C) Ser；Ala；Val
Gln(Q) Asn；Glu
Glu(E) Asp；Gln
Gly(G) Ala
His(H) Asn；Gln
Ile(I) Leu；Val
Leu(L) Ile；Val
Lys(K) Arg；His
Met(M) Leu；Ile；Tyr
Phe(F) Tyr；Met；Leu
Pro(P) Ala
Ser(S) Thr
Thr(T) Ser
Trp(W) Tyr；Phe
Tyr(Y) Trp；Phe
Val(V) Ile；Leu
本披露中所述的“末端延伸比率”是指表达产物群体中，具有末端延伸序列的表达产物所占的比率。具有末端延伸序列的表达产物是目标表达产物的变体，其除具有所期望的目标序列外，还连接有其他氨基酸残基。示例性的，抗体轻链表达产物中，1％的轻链序列变体的N末端还连接有其他氨基酸残基，则该表达产物的末端延伸比率为1％。当表达产物群体中含有具有末端延伸序列的表达产物时，即表示该表达产物群体具有末端异质性。示例性地，本披露中的末端延伸序列来源于信号肽氨基酸的残留。
本披露中的组合物包含所期望的目标表达产物及具有末端延伸序列的表达产物。组合物中氨基末端延伸的存在可通过多种分析技术来检测，包括但不限于N-末端序列分析、电荷异质性的测定法(例如阳离子交换层析或毛细管区带电泳)、质谱、肽图检测等。组合物中抗体变体的量通常在如下范围内，从构成用于检测变体的任何测定法(例如阳离子交换分析)的检测下限的量至少于主要种类抗体量的量。组合物中约3％或更少(例如约3％、2.5％、2％、1.5％、1％、0.5％、0％)的抗体轻链或抗体重链包含氨基末端延伸。这样的百分比量可以通过质谱、肽图检测法来测定。当样品内含有末端延伸的异源多肽比例超过总量1％时，可以基于肽图检测法有效的将其鉴定出，并同时对还原分子量质谱检测峰图中对应的峰进行定量标识。
在以上说明书中提出了本披露一种或多种实施方式的细节。虽然可使用与本文所述类似或相同的任何方法和材料来实施或测试本披露，但是以下描述的方法和材料。通过说明书和权利要求书，本披露的其他特点、目的和优点将是显而易见的。在说明书和权利要求书中，除非上下文中有清楚的另外指明，单数形式包括复数指代物的情况。除非另有定义，本文使用的所有技术和科学术语都具有本披露所属领域普通技术人员所理解的一般含义。说明书中引用的所有专利和出版物都通过引用纳入。提出以下实施例是为了更全面地说明本披露的可选实施方式。这些实施例不应以任何方式理解为限制本披露的范围，本披露的范围由权利要求书限定。
实施例
以下结合实施例进一步描述本披露，但这些实施例并非限制着本披露的范围。 本披露实施例中未注明具体条件的实验方法，通常按照常规条件，如冷泉港的抗体技术实验手册，分子克隆手册；或按照原料或商品制造厂商所建议的条件。未注明具体来源的试剂，为市场购买的常规试剂。
实施例1：抗claudin18.2抗体的制备
实施例1-1：构建高表达Claudin18.2的细胞株
用Lipofectamine 3000转染试剂，将pCDH-hClaudin18.2慢病毒表达载体质粒与pVSV-G,pCMV-dR8.91慢病毒系统包装载体转染至病毒包装细胞293T中；收集含有病毒的培养基上清，过滤并进行超高速离心；使用浓缩后的病毒感染人胃印戒细胞癌细胞株NUGC4，经嘌呤霉素筛选两至三周，再进行FACS单细胞分选。
根据肿瘤IHC评分来区分Claudin18.2表达程度。与肿瘤IHC评分为3分的肿瘤Claudin18.2表达水平相当的细胞为高表达细胞，与肿瘤IHC评分为2分的肿瘤Claudin18.2表达水平相当的细胞为中等表达细胞。根据通过FACS检测慢病毒感染的NUGC4细胞表面的Claudin18.2表达，挑选出Claudin18.2表达量高的NUGC4/hClaudin18.2单克隆细胞株。同时通过FACS检测野生型NUGC4细胞表面的Claudin18.2表达，挑选出Claudin18.2表达量中等的NUGC4克隆细胞株，野生型NUGC4为Claudin18.2低表达量细胞。
将挑选出的单克隆细胞株扩大培养，冻存备库以便后续实验。
Claudin18.2序列Genbank:NP_001002026：(SEQ ID NO:1)
Figure PCTCN2021114911-appb-000001
Claudin18.2 DNA序列：(SEQ ID NO:2)
Figure PCTCN2021114911-appb-000002
Figure PCTCN2021114911-appb-000003
实施例1-2：抗人claudin18.2单克隆抗体产生
1免疫
通过免疫小鼠产生抗人Claudin18.2单克隆抗体。
实验用SJL白小鼠，雌性，6-8周龄(北京维通利华实验动物技术有限公司，动物生产许可证号：SCXK(京)2012-0001)。饲养环境：SPF级。小鼠购进后，实验室环境饲养1周，12/12小时光/暗周期调节，温度20-25℃；湿度40-60％。将已适应环境的小鼠按以下方案免疫。免疫抗原为huClaudin18.2-HEK293细胞(转染人Claudin18.2质粒的HEK-293稳转细胞株)。
免疫方案：首次免疫细胞前，用
Figure PCTCN2021114911-appb-000004
Gold Adjuvant(Sigma Cat No.T2684)0.1ml/只注射小鼠腹膜内(IP)；半小时后每只小鼠腹膜内(IP)注射0.1ml生理盐水稀释至1×10 8/ml浓度的细胞液。细胞吹散均匀后进行接种，时间为第0、14、28、42、56天。于第21，35，49，63天取血，用ELISA方法确定小鼠血清中的抗体滴度。在第4-5次免疫以后，选择血清中抗体滴度高并且滴度趋于平台的小鼠进行脾细胞融合。在进行脾细胞融合前3天加强免疫，腹膜内(IP)注射1×10 7细胞。
2脾细胞融合
采用PEG介导的融合步骤将脾淋巴细胞与骨髓瘤细胞Sp2/0细胞(
Figure PCTCN2021114911-appb-000005
CRL-8287 TM)进行融合得到杂交瘤细胞。杂交瘤细胞以0.5-1×10 6/ml的密度用完全培养基(含20％FBS、1×HAT、1×OPI的IMDM培养基)重悬，100μl/孔种于96孔板中，37℃，5％CO 2孵育3-4天后，补充HAT完全培养基100μl/孔，继续培养3-4天至形成克隆。去除上清，加入200μl/孔的HT完全培养基(含20％FBS、1×HT和1×OPI的IMDM培养基)，37℃，5％CO 2培养3天后进行ELISA检测。
3杂交瘤细胞筛选
根据杂交瘤细胞生长密度，用结合ELISA方法检测培养上清。选择与huClaudin18.2-HEK293细胞结合能力强，同时与HEK293细胞没有结合的细胞，及时进行扩增冻存；经过二到三次亚克隆直至获得单细胞克隆。
每次亚克隆细胞均需进行细胞结合实验。通过以上实验筛选得到杂交瘤克隆，用无血清细胞培养法进一步制备抗体，纯化抗体，供在检测例中使用。
实施例1-3：鼠源抗体的人源化
挑选出体外活性高的单克隆杂交瘤细胞株mAb1901，mAb1902；克隆其中的单克隆抗体序列，再进行人源化、重组表达和活性评价。
从杂交瘤中克隆序列的过程如下。收集对数生长期杂交瘤细胞，用Trizol(Invitrogen,15596-018)提取RNA(按照试剂盒说明书步骤)并进行反转录(PrimeScript TM Reverse Transcriptase，Takara,cat#2680A)。将反转录得到的cDNA采用mouse Ig-Primer Set(Novagen,TB326Rev.B 0503)进行PCR扩增，送测序公司测序。得到的DNA序列对应的氨基酸序列SEQ ID NO:3-6所示：
mAb1901鼠源重链可变区(SEQ ID NO:3)
Figure PCTCN2021114911-appb-000006
mAb1901鼠源轻链可变区(SEQ ID NO:4)
Figure PCTCN2021114911-appb-000007
mAb1902鼠源重链可变区(SEQ ID NO:5)
Figure PCTCN2021114911-appb-000008
mAb1902鼠源轻链可变区(SEQ ID NO:6)
Figure PCTCN2021114911-appb-000009
上述鼠源重链可变区和轻链可变区，分别与下述人IgG1抗体的重链恒定区和人源κ轻链恒定区连接，形成嵌合抗体ch1901和ch1902。
恒定区选自以下序列：
人IgG1抗体的重链恒定区：(SEQ ID NO:7)
Figure PCTCN2021114911-appb-000010
人源κ轻链恒定区：(SEQ ID NO:8)
Figure PCTCN2021114911-appb-000011
如本领域许多文献公示的方法，对鼠源单克隆抗体进行人源化。简言之，使用人恒定结构域替代亲本(鼠源抗体)恒定结构域，根据鼠源抗体和人抗体的同源性选择人种系抗体序列，进行CDR移植。本披露选择活性好的候选分子进行人源化，结果如下。
1.鼠源抗体的CDR区
表2中VH/VL CDR的氨基酸残基由Kabat编号系统确定并注释。
鼠源抗体的CDR序列如表2所述：
表2.鼠源抗体的CDR序列
抗体 mAb1901
HCDR1 DYGIH(SEQ ID NO:9)
HCDR2 YISRGSSTIYYADTVKG(SEQ ID NO:10)
HCDR3 GGYDTRNAMDY(SEQ ID NO:11)
LCDR1 KSSQSLLNSGNQKNYLA(SEQ ID NO:12)
LCDR2 GASTRAS(SEQ ID NO:13)
LCDR3 QNDLYYPLT(SEQ ID NO:14)
抗体 mAb1902
HCDR1 SYWMH(SEQ ID NO:15)
HCDR2 MIHPNSGSTNYNEKFKGR(SEQ ID NO:16)
HCDR3 LKTGNSFDY(SEQ ID NO:17)
LCDR1 KSSQSLLNSGNQKNYLT(SEQ ID NO:18)
LCDR2 WASTRES(SEQ ID NO:19)
LCDR3 QNAYTYPFT(SEQ ID NO:20)
2.选择人种系FR区序列
在所获得的鼠源抗体VH/VLCDR典型结构的基础上，将重、轻链可变区序列与抗体Germline数据库比较，获得同源性高的人种系模板。其中人类种系轻链框 架区来自人κ轻链基因。
2.1 mAb1901的人源化改造和回复突变设计
选择适当的人抗体种系，对mAb1901鼠源抗体进行人源化改造，将鼠源抗体mAb1901的CDR区移植到选择的人源化模板上，得到人源化可变区，其人源化重链可变区序列为SEQ ID NO:24和轻链可变区序列为SEQ ID NO:21；再与IgG恒定区重组，形成完整抗体。同时，对人源化抗体的V区中FR区进行回复突变，示例性回复突变方式及组合如下：
表3.mAb1901人源化抗体及回复突变 *
Figure PCTCN2021114911-appb-000012
*表格中所有氨基酸位置编号为Kabat编号规则的编号，重链可变区的N82T中，82为Kabat规则的第82A位。
表4.mAb1901人源化抗体轻链可变区和重链可变区序列
Figure PCTCN2021114911-appb-000013
上表中对应重链可变区可与SEQ ID NO:7所示的人IgG1重链恒定区连接形成全长抗体的重链，轻链可变区与SEQ ID NO:8所示的人κ轻链恒定区连接形成全长抗体的轻链。在其他实施方案中，重链可变区和轻链可变区也可与其他重链恒定区和轻链恒定区分别连接形成全长抗体。
2.2 mAb1902的人源化改造和回复突变设计
选择适当的人抗体种系，对mAb1902鼠源抗体进行人源化改造，将鼠源抗体 mAb1902的CDR区移植到选择的人源化模板上，得到人源化可变区，其人源化重链可变区序列为SEQ ID NO:31和轻链可变区序列为SEQ ID NO:28；再与IgG恒定区重组，形成完整抗体。同时，对人源化抗体的V区中FR区进行回复突变，示例性回复突变方式及组合如下：
表5.mAb1902人源化抗体及其回复突变设计 *
Figure PCTCN2021114911-appb-000014
*表格中所有氨基酸位置编号为Kabat编号规则的编号。
表6.mAb1902人源化抗体轻链可变区和重链可变区序列
Figure PCTCN2021114911-appb-000015
上表中对应重链可变区与SEQ ID NO:7所示的人IgG1重链恒定区连接形成全长抗体的重链，轻链可变区与SEQ ID NO:8所示的人κ轻链恒定区连接形成全长抗体的轻链。
示例性的，抗体全长序列如下：
嵌合抗体ch1901：
ch1901重链：(SEQ ID NO:35)
Figure PCTCN2021114911-appb-000016
Figure PCTCN2021114911-appb-000017
ch1901轻链(SEQ ID NO:36)
Figure PCTCN2021114911-appb-000018
嵌合抗体ch1902：
ch1902重链(SEQ ID NO:37)
Figure PCTCN2021114911-appb-000019
ch1902轻链(SEQ ID NO:38)
Figure PCTCN2021114911-appb-000020
表7.mAb1901人源化抗体
Figure PCTCN2021114911-appb-000021
全长抗体轻重链序列如下所示：
表8.mAb1901人源化抗体轻链和重链序列
Figure PCTCN2021114911-appb-000022
Figure PCTCN2021114911-appb-000023
表9.mAb1902人源化抗体
Figure PCTCN2021114911-appb-000024
全长抗体轻重链序列如下所示：
表10.mAb1902人源化抗体轻链和重链序列
Figure PCTCN2021114911-appb-000025
Figure PCTCN2021114911-appb-000026
本披露阳性对照抗体为IMAB-362(来自WO2016166122)：
IMAB-362重链(SEQ ID NO:53)
Figure PCTCN2021114911-appb-000027
IMAB-362轻链(SEQ ID NO:54)
Figure PCTCN2021114911-appb-000028
用常规的基因克隆、重组表达的方法分别克隆、表达、纯化上述抗体。
测试例1：抗claudin18.2抗体的体外活性生物学评价
测试例1-1：Cell水平ELISA结合实验
基于细胞的ELISA实验被用来检测Claudin18.2抗体的结合特性。将稳转表达Claudin18.2的NUGC4细胞培养于96孔细胞板(Corning，3599)中，待生长至90％密度时加入4％多聚甲醛固定细胞1小时，用PBST缓冲液(pH 7.4PBS含0.05％ Tween-20)洗板3次后，加入用PBS稀释的5％脱脂牛奶(光明脱脂奶粉)封闭液200μl/孔，37℃孵育箱孵育2.5小时或4℃放置过夜(16-18小时)进行封闭。封闭结束后，弃去封闭液，并用PBST缓冲液洗板3次后，加入50μl/孔用样品稀释液(pH7.4PBS含1％脱脂乳)稀释的不同浓度待测抗体，放于37℃孵育箱孵育2小时。孵育结束后用PBST洗板5次，加入100μl/孔用样品稀释液稀释的HRP标记的羊抗人二抗(Jackson Immuno Research,109-035-003)，37℃孵育1小时。用PBST洗板6次后，加入50μl/孔TMB显色底物(KPL,52-00-03)，于室温孵育10-15min，加入50μl/孔1M H 2SO 4终止反应，用MD Versa Max TM酶标仪在450nm处读取吸收值，计算Claudin18.2抗体对Claudin18.2的结合EC50值(结果见下表)。
表11抗体的结合活性
抗体 IMAB362 ch1901 ch1902
Emax 1.175 1.399 1.272
EC50(nM) 0.108 0.098 0.074
表12-1.mAb1901人源化抗体的结合活性
抗体 Emax EC50(nM)
IMAB362 1.115 0.086
h1901-2 1.039 0.076
h1901-3 1.1055 0.22
h1901-4 0.986 0.201
h1901-6 0.937 0.091
h1901-7 0.921 0.166
h1901-8 1.047 0.091
h1901-11 1.44 0.076
h1901-12 1.22 0.116
表12-2.mAb1902人源化抗体的结合活性
抗体 Emax EC50(nM)
IMAB362 0.88 0.187
h1902-1 0.87 0.113
h1902-2 0.88 0.107
h1902-3 0.84 0.175
h1902-4 0.82 0.087
h1902-5 0.9 0.098
h1902-6 0.78 0.141
h1902-7 0.75 0.121
h1902-8 0.89 0.132
h1902-9 0.75 0.137
h1902-10 0.89 0.133
测试例1-2：抗体细胞水平结合实验
将稳转表达Claudin18.2的NUGC4细胞用FACS缓冲液(2％胎牛血清(Gibco，10099141)pH7.4PBS(Sigma,P4417-100TAB))制备成1×10 6/ml的细胞悬液，100μl/孔加入96孔圆底板(Corning，3795)中。离心去除上清后加入50μl/孔用 FACS缓冲液稀释的不同浓度待测Claudin18.2抗体，放于4℃冰箱中避光孵育1小时。以FACS缓冲液300g离心洗涤3次后，加入工作浓度的Alexa Fluor 488包被的抗人IgG(H+L)(invitrogen,A-11013)，放于4℃冰箱中避光孵育40分钟。以FACS缓冲液300g离心洗涤3次后，在BD FACS CantoII流式细胞仪上检测几何平均数荧光强度，计算Claudin18.2抗体对稳转表达Claudin18.2的NUGC4细胞的结合EC50值，结果见图1。
测试例1-3：抗体内吞实验
将预标记了DyLight 488 NHS Ester(thermofisher，46403)的待测Claudin18.2抗体，以5μg/ml终浓度加入1×10 6/ml稳转表达Claudin18.2的NUGC4细胞中，放于冰上避光孵育1小时，以预冷的FACS缓冲液(pH7.4PBS，2％胎牛血清)离心洗涤3次，去上清后加入预热的完全培养基，放入37℃5％CO 2细胞培养箱。分别在0、0.5、1、2、4小时后取出细胞，放置于冰上避光保存。待样品全部收集后，300g低温离心去除上清，加入洗脱缓冲液(pH1.7 0.05M甘氨酸，0.1M氯化钠)后，室温孵育7分钟，以FACS缓冲液300g离心洗涤1次，在BD FACS CantoII流式细胞仪上检测几何平均数荧光强度，计算Claudin18.2抗体对稳转表达Claudin18.2的NUGC4细胞的内吞效率。结果显示(见图2)，人源化抗体具有良好的细胞内吞效率。
测试例1-4：基于流式细胞技术测定抗体亲和力
实验当天收集HEK293/hClaudin18.2细胞于U底96孔板中，每孔1×10 5至2×10 5个细胞。加入起始浓度5μg/ml，2×梯度稀释(12个浓度点)的Claudin18.2抗体，4℃孵育1小时，阳性对照为IMAB362，同时设置不加抗体的阴性对照。离心去除抗体，再加入100μl/孔FITC抗人IgG Fc抗体(200×)，4℃避光孵育30分钟，用PBS+2％FBS清洗两遍后准备进行流式细胞检测。启动BD FACS CantoII，预热完成后打开BD FACSDiva软件，建立一个新的实验，检测HEK293/hClaudin18.2阴性对照样品，调节FSC及SSC电压至适当的数值并保存。根据Quantum TM FITC-5MESF Kit说明书，分别检测空白样品B及标准曲线1，调节FITC电压至适当的数值并保存。在保存的电压下检测U底96孔板中的样品，记录数据。使用Flowjo软件分析实验数据得到Geo Mean数值，根据Quantum TM FITC-5MESF Kit说明书拟合MESF-Geo Mean标准曲线，根据FITC抗人IgG Fc抗体的浓度荧光值计算出与HEK293/hClaudin18.2细胞结合的Claudin18.2抗体的摩尔浓度及游离抗体浓度，利用Scatchard作图法计算抗体的Bmax和解离常数KD。结果见表13。
表13.人源化抗体细胞水平亲和力
抗体 IMAB362 h1901-11 h1902-5
KD(nM) 10.2 6.8 1.64
测试例1-5：抗体的ADCC效应评价
消化各种NUGC4细胞(高中低表达Claudin18.2)，1000rpm离心后，重悬计数。将细胞以3×10 5细胞/ml的密度重悬在添加10％FBS(新西兰超低IgG胎牛血清，Gibco,1921005PJ)的无酚红RPMI 1640中(Gibco,11835-030)。在96孔板(Corning,3903)中，每孔加入25μl细胞(7500个/孔)。将抗体稀释在上述无酚红培养基中，配制成3×的抗体稀释液，向细胞板中加入25μl/孔的抗体。在37℃、5％CO 2培养箱中孵育0.5小时。
收集效应细胞(FcrR3A-V158-NFAT-RE-Jurkat细胞)，1000rpm离心后，重悬计数。将细胞以3×10 6细胞/ml的密度重悬在添加10％FBS(新西兰超低IgG胎牛血清)的无酚红RPMI 1640中，在实验板中每孔加入25μl细胞(7.5×10 4个细胞/孔)。在37℃、5％CO 2培养箱中孵育6小时。
向实验板的每个孔中加入75μl/孔的Bright-Glo(Promega,E2610)，用酶标仪(PerkinElmer,VITOR3)检测化学发光(luminescence)。
结果显示(见表14和图3A-图3C)，在低中高不同程度Claudin18.2表达的NUGC4细胞中，抗体h1901-11和h1902-5均显示出很强的ADCC活性。
表14.抗体在Claudin18.2不同表达程度的NUGC4细胞中的ADCC效应
Figure PCTCN2021114911-appb-000029
实施例2：信号肽的设计与应用
实施例2-1：融合不同信号肽的Pertuzumab的设计与表达
设计融合如下信号肽的抗体，其信号肽序列组合如下：
1.MEWSWVFLFFLSVTTGVHS(SEQ ID NO:152)与MSVPTQVLGLLLLWLTDARC(SEQ ID NO:153)
2.MEWSWVFLFFLSVTTGVHS(SEQ ID NO:152)与MEWSWVFLFFLSVTTGVHS(SEQ ID NO:152)
3.MEWSWVFLFFLSLTGVHA(SEQ ID NO:57)与MSVPTQVLGLLLLWLTDVRA(SEQ ID NO:105)
4.MEWSWVFLFFLSLTGVHA(SEQ ID NO:57)与MEWSWVFLFFLSLTGVHA(SEQ ID NO:57)
5.MSVPTQVLGLLLLWLTDVRA(SEQ ID NO:105)与MEWSWVFLFFLSLTGVHA(SEQ ID NO:57)
将上述信号肽组合，分别置于Pertuzumab抗体重、轻链氨基酸N端，并设计相应的新的重、轻链序列。所设计的序列如下：
Pertuzumab-1含信号肽的抗体重链氨基酸序列(SEQ ID NO:154)
Figure PCTCN2021114911-appb-000030
Pertuzumab-1含信号肽的抗体轻链氨基酸序列(SEQ ID NO:155)
Figure PCTCN2021114911-appb-000031
Pertuzumab-2含信号肽的抗体重链氨基酸序列(SEQ ID NO:154)
Pertuzumab-2含信号肽的抗体轻链氨基酸序列(SEQ ID NO:156)
Figure PCTCN2021114911-appb-000032
Pertuzumab-3含信号肽的抗体重链氨基酸序列(SEQ ID NO:157)
Figure PCTCN2021114911-appb-000033
Pertuzumab-3含信号肽的抗体轻链氨基酸序列(SEQ ID NO:158)
Figure PCTCN2021114911-appb-000034
Pertuzumab-4含信号肽的抗体重链氨基酸序列(SEQ ID NO:157)
Pertuzumab-4含信号肽的抗体轻链氨基酸序列(SEQ ID NO:159)
Figure PCTCN2021114911-appb-000035
Pertuzumab-5含信号肽的抗体重链氨基酸序列(SEQ ID NO:160)
Figure PCTCN2021114911-appb-000036
Pertuzumab-5含信号肽的抗体轻链氨基酸序列(SEQ ID NO:159)
(备注：下划线为信号肽。)
基于上述Pertuzumab-1至Pertuzumab-4的序列合成各重链和轻链的基因片段，将重链基因克隆到PXC18.4形成重链质粒，轻链基因克隆到PXC17.4形成轻链质粒。通过质粒中的相同酶切位点，将轻链质粒克隆至重链质粒中，并构建出全长抗体质粒。将全长抗体质粒直接电转CHO细胞，经筛选后获得了包含该质粒的稳转细胞。培养稳转细胞，将培养后的上清用Protein A亲和柱进行纯化，获得表达产物。
实施例2-2：融合不同信号肽的h1902-5的设计与表达
设计融合如下信号肽的抗体，其信号肽序列组合如下：
1.MEWSWVFLFFLSVTTGVHS(SEQ ID NO:152)与MEWSWVFLFFLSVTTGVHS(SEQ ID NO:152)
2.MEWSWVFLFFLSLTGVHA(SEQ ID NO:57)与MSVPTQVLGLLLLWLTDVRA(SEQ ID NO:105)
3.MSVPTQVLGLLLLWLTDVRA(SEQ ID NO:105)与MEWSWVFLFFLSLTGVHA(SEQ ID NO:57)与
将上述信号肽组合，分别置于h1902-5抗体重链、轻链N端，并设计相应的新的重、轻链序列。所设计的序列如下：
h1902-5-1含信号肽的抗体重链氨基酸序列(SEQ ID NO:161)
Figure PCTCN2021114911-appb-000037
Figure PCTCN2021114911-appb-000038
h1902-5-1含信号肽的抗体轻链氨基酸序列(SEQ ID NO:162)
Figure PCTCN2021114911-appb-000039
h1902-5-2含信号肽的抗体重链氨基酸序列(SEQ ID NO:163)
Figure PCTCN2021114911-appb-000040
h1902-5-2含信号肽的抗体轻链氨基酸序列(SEQ ID NO:164)
Figure PCTCN2021114911-appb-000041
h1902-5-3含信号肽的抗体重链氨基酸序列(SEQ ID NO:165)
Figure PCTCN2021114911-appb-000042
h1902-5-3含信号肽的抗体轻链氨基酸序列(SEQ ID NO:166)
Figure PCTCN2021114911-appb-000043
(备注：下划线为信号肽。)
基于上述的h1902-5-1或h1902-5-2的序列合成各重链和轻链的基因片段，将其构建到表达载体上获得全长抗体质粒。将全长抗体质粒直接电转CHO细胞，经筛选后获得了包含该质粒的稳转细胞。培养稳转细胞，将培养后的上清用Protein A亲和柱进行纯化，获得表达产物。
实施例2-3：融合不同信号肽的FcRn的设计与表达
抗FcRn抗体重、轻链氨基酸序列如下：
重链(SEQ ID NO:167)
Figure PCTCN2021114911-appb-000044
轻链(SEQ ID NO:168)
Figure PCTCN2021114911-appb-000045
设计融合如下信号肽的抗体，其信号肽序列组合如下：
1.MEWSWVFLFFLSVTTGVHS(SEQ ID NO:152)与MEWSWVFLFFLSVTTGVHS(SEQ ID NO:152)
2.MEWSWVFLFFLSLTGVHA(SEQ ID NO:57)与MEWSWVFLFFLSLTGVHA(SEQ ID NO:57)
所设计的序列如下：
FcRn-1含信号肽的抗体重链氨基酸序列(SEQ ID NO:169)
Figure PCTCN2021114911-appb-000046
Figure PCTCN2021114911-appb-000047
FcRn-1含信号肽的抗体轻链氨基酸序列(SEQ ID NO:170)
Figure PCTCN2021114911-appb-000048
FcRn-2含信号肽的抗体重链氨基酸序列(SEQ ID NO:171)
Figure PCTCN2021114911-appb-000049
FcRn-2含信号肽的抗体轻链氨基酸序列(SEQ ID NO:172)
Figure PCTCN2021114911-appb-000050
基于FcRn-1和FcRn-2的序列合成各重链和轻链的基因片段，将其构建到表达载体上获得全长抗体质粒。将全长抗体质粒直接电转CHO细胞，经筛选后获得了包含该质粒的稳转细胞池。培养稳转细胞池，将培养后的上清用Protein A亲和柱进行纯化，获得表达产物。
测试例2：采用不同信号肽的表达产物的末端异质性
测试例2-1：融合不同信号肽的Pertuzumab去糖还原分子量及氨基酸序列检测
通过PNGase F糖苷酶(NEB，P0708)切除Pertuzumab-1、Pertuzumab-2、Pertuzumab-3，Pertuzumab-4样品所携带的N-糖基，再经DTT(Sigma,43815)还原成轻、重链，最后由LC-MS(Waters，ACQUITY UPLC H-Class/XeVo G2-XS QTOF)对样品的去糖还原分子量进行检测，并通过UNIFI软件进行数据处理及 分析。
Pertuzumab-1及Pertuzumab-3的检测结果如图4A至图4D所示：Pertuzumab-1样品中检测到轻链中含有4.7％信号肽氨基酸(RC)残留；而Pertuzumab-3样品中轻链无信号肽氨基酸残留。Pertuzumab-1及Pertuzumab-3的重链均未检测到信号肽氨基酸残留，仅有少量糖化修饰。
Pertuzumab-1经尿素变性处理后，加入DTT还原成轻、重链，之后加入Glu C酶(Promega,V1651)进行酶切，所得样品经LC-MS采集分子量数据，所获得的数据通过UNIFI软件进行分析，得到样品的氨基酸序列信息。
结果显示Pertuzumab-1中在去糖还原分子量检测中所出现的异常峰，经肽图分析验证，确认为RC氨基酸残留。具体结果如表15所示。
表15.Pertuzumab-1轻链N端信号肽肽图检测数据
Figure PCTCN2021114911-appb-000051
注：本测试例采用Glu C酶进行酶切使序列RC中的氨基酸R被切割，该残留氨基酸C指征RC氨基酸残留。
Pertuzumab-2及Pertuzumab-4的检测结果如图5A至图5D所示：Pertuzumab-2样品中检测到轻链中含有5.8％信号肽氨基酸(VHS)残留；而Pertuzumab-4样品中轻链无信号肽氨基酸残留。两种抗体重链均未检测到信号肽氨基酸残留，仅有少量糖化修饰。
Pertuzumab-2经尿素变性处理后，加入DTT还原成轻、重链，之后加入Glu C酶进行酶切，所得样品经LC-MS采集分子量数据，所获得的数据通过UNIFI软件进行分析，得到样品的氨基酸序列信息。
结果显示，Pertuzumab-2中在去糖还原分子量检测中所出现的异常峰，经肽图分析验证，确认为VHS氨基酸残留。具体结果如表16显示。
表16.Pertuzumab-1轻链N端信号肽肽图检测数据
Figure PCTCN2021114911-appb-000052
测试例2-2：融合不同信号肽的h1902-5去糖还原分子量及氨基酸序列检测
通过PNGase F糖苷酶(NEB,P0708)切除h1902-5-1、h1902-5-2样品所携带的N-糖基，再经DTT(Sigma,43815)还原成轻、重链，最后由LC-MS(Waters，ACQUITY UPLC H-Class/XeVo G2-XS QTOF)对样品的去糖还原分子量进行检测，并通过UNIFI软件进行数据处理及分析。
结果如图6A至图6D所示，h1902-5-1样品中检测到重链中含有3.1％信号肽氨基酸(VHS)残留；而h1902-5-2样品中重链无信号肽氨基酸残留。两种抗体轻链均未检测到信号肽氨基酸残留，仅有少量糖化修饰。
h1902-5-1经尿素变性处理后，加入DTT还原成轻、重链，之后加入Glu C酶进行酶切，所得样品经LC-MS采集分子量数据，所获得的数据通过UNIFI软件进行分析，得到样品的氨基酸序列信息。
结果显示，h1902-5-1中于去糖还原分子量检测中所出现的异常峰，经肽图分析验证，确认为VHS氨基酸残留。具体结果如下表17显示。
表17.h1902-5-1重链N端信号肽肽图检测数据
Figure PCTCN2021114911-appb-000053
测试例2-3：融合不同信号肽的抗FcRn抗体去糖还原分子量及氨基酸序列检测
通过PNGase F糖苷酶(NEB,P0708)切除FcRn-1、FcRn-2样品所携带的N-糖基，再经DTT(Sigma,43815)还原成轻、重链，最后由LC-MS(Waters，ACQUITY UPLC H-Class/XeVo G2-XS QTOF)对样品的去糖还原分子量进行检测，并通过UNIFI软件进行数据处理及分析。
结果如图7A至图7D所示，FcRn-1样品中检测到重链中含有3.4％信号肽氨基酸(VHS)残留；而FcRn-2样品中重链无信号肽氨基酸残留。两种抗体轻链均未检测到信号肽氨基酸残留，仅有少量糖化修饰。
FcRn-1经尿素变性处理后，加入DTT还原成轻、重链，之后加入Glu C酶进行酶切，所得样品经LC-MS采集分子量数据，所获得的数据通过UNIFI软件进行分析，得到样品的氨基酸序列信息。
结果显示，FcRn-1中于去糖还原分子量检测中所出现的异常峰，经肽图分析验证，确认为VHS氨基酸残留。具体结果如下表显示。
表18.FcRn重链N端信号肽肽图检测数据
Figure PCTCN2021114911-appb-000054
虽然为了清楚的理解，已经借助于附图和实例详细描述了上述发明，但是描述和实例不应当解释为限制本披露的范围。本文中引用的所有专利和科学文献的公开内容通过引用完整地清楚结合。

Claims (15)

  1. 降低抗体的重链和/或抗体的轻链的N末端异质性的方法，其包括
    培养宿主细胞，其中所述宿主细胞包含
    (1)第一多核苷酸，其编码抗体的重链及与该重链的N端有效连接的第一信号肽；其中，所述第一信号肽包含SEQ ID NO:55或SEQ ID NO:56的氨基酸序列；和/或
    (2)第二多核苷酸，其编码抗体的轻链及与该轻链的N端有效连接的第二信号肽；其中，所述第二信号肽包含SEQ ID NO:55或SEQ ID NO:56的氨基酸序列；
    表达所述抗体的重链和/或抗体的轻链。
  2. 根据权利要求1所述的方法，其中，表达得到的所述抗体的重链和/或抗体的轻链具有低于3％的末端延伸比率；优选地，表达得到的所述抗体的重链和/或抗体的轻链具有低于1％的末端延伸比率。
  3. 根据权利要求1或2所述的方法，其中所述第一信号肽或所述第二信号肽各自独立的包含SEQ ID NO:57、SEQ ID NO:58、SEQ ID NO:59、SEQ ID NO:93、SEQ ID NO:94、SEQ ID NO:95、SEQ ID NO:99、SEQ ID NO:100、SEQ ID NO:101、SEQ ID NO:105、SEQ ID NO:106、SEQ ID NO:107、SEQ ID NO:140、SEQ ID NO:141、SEQ ID NO:142、SEQ ID NO:146、SEQ ID NO:147或SEQ ID NO:148的氨基酸序列；
    优选地，所述第一信号肽包含SEQ ID NO:57或SEQ ID NO:105的氨基酸序列，和/或所述第二信号肽包含SEQ ID NO:57或SEQ ID NO:105的氨基酸序列。
  4. 根据权利要求1至3任一项所述的方法，其中所述的抗体是鼠源抗体、嵌合抗体、人源化抗体、人抗体、亲和力成熟抗体或多特异性抗体。
  5. 根据权利要求1至4任一项所述的方法，其中所述的抗体选自抗TLR7抗体、抗HER2(ErbB2)抗体、抗Claudin18.2抗体、抗EGFR抗体、抗B7H3抗体、抗c-Met抗体、抗HER3(ErbB3)抗体、抗HER4(ErbB4)抗体、抗CD3抗体、抗CD20抗体、抗CD22抗体、抗CD30抗体、抗CD33抗体、抗CD38抗体、抗CD44抗体、抗CD47抗体、抗CD56抗体、抗CD70抗体、抗CD73抗体、抗CD105抗体、抗CEA抗体、抗A33抗体、抗Cripto抗体、抗SOST抗体、抗EphA2抗体、抗G250抗体、抗MUCl抗体、抗Lewis Y抗体、抗VEGFR抗体、抗GPNMB抗体、抗凝血酶抗体、抗Aβ抗体、抗Integrin抗体、抗ANGPTL3抗体、抗PSMA抗体、抗PD-1抗体、抗PD-L1抗体、抗LAG3抗体、抗IL-5抗体、抗IL-15抗体、抗IL-4R抗体、抗IL-6R抗体、抗TIGHT抗体、抗Tenascin-C抗体、抗SLC44A4 抗体、抗PCSK9抗体、抗EpCAM抗体、抗CTGF抗体、抗TSLP抗体、抗CEA抗体、抗Mesothelin抗体和抗FcRn抗体；
    优选地，所述的抗体选自Trastuzumab、Pertuzumab、Nimotuzumab、Enoblituzumab、Emibetuzumab、Inotuzumab、Pinatuzumab、Brentuximab、Gemtuzumab、Bivatuzumab、Lorvotuzumab、cBR96、Glematumamab、抗Claudin18.2抗体和抗FcRn抗体，其中所述抗Claudin18.2抗体的重链包含SEQ ID NO:49的氨基酸序列、轻链包含SEQ ID NO:47的氨基酸序列；所述抗FcRn抗体的重链包含SEQ ID NO:167的氨基酸序列，轻链包含SEQ ID NO:168的氨基酸序列。
  6. 一种多肽，其包含信号肽，所述信号肽包含SEQ ID NO:55或SEQ ID NO:56的氨基酸序列。
  7. 根据权利要求6所述的多肽，其中，所述多肽还包含与所述信号肽有效连接的异源多肽；
    优选地，所述异源多肽是抗体的重链或抗体的轻链；
    更优选地，所述信号肽与抗体的重链或抗体的轻链的N端有效连接。
  8. 根据权利要求6或7所述的多肽，其中所述信号肽包含SEQ ID NO:57、SEQ ID NO:58、SEQ ID NO:59、SEQ ID NO:93、SEQ ID NO:94、SEQ ID NO:95、SEQ ID NO:99、SEQ ID NO:100、SEQ ID NO:101、SEQ ID NO:105、SEQ ID NO:106、SEQ ID NO:107、SEQ ID NO:140、SEQ ID NO:141、SEQ ID NO:142、SEQ ID NO:146、SEQ ID NO:147或SEQ ID NO:148的氨基酸序列；
    优选地，所述信号肽包含SEQ ID NO:57或SEQ ID NO:105的氨基酸序列。
  9. 根据权利要求6至8任一项所述的多肽，其中所述多肽包含选自由SEQ ID NO:157、SEQ ID NO:158、SEQ ID NO:159、SEQ ID NO:160、SEQ ID NO:163、SEQ ID NO:164、SEQ ID NO:165、SEQ ID NO:166、SEQ ID NO:171和SEQ ID NO:172组成的组的氨基酸序列。
  10. 一种核酸分子，其编码权利要求6至9任一项所述的多肽。
  11. 一种宿主细胞，其包含如权利要求10所述的核酸分子。
  12. 根据权利要求11所述的宿主细胞，所述宿主细胞是真核宿主细胞；优选地，所述真核宿主细胞是CHO细胞或酵母。
  13. 一种组合物，其含有抗Claudin18.2抗体，所述抗Claudin18.2抗体的重链 包含SEQ ID NO:49的氨基酸序列，轻链包含SEQ ID NO:47的氨基酸序列；该组合物中的抗Claudin18.2抗体存在末端异质性，并且所述抗Claudin18.2抗体的重链和/或轻链具有低于3％的末端延伸比率；
    优选地，所述抗Claudin18.2抗体的重链具有低于1％的末端延伸比率。
  14. 一种组合物，其含有抗FcRn抗体，所述抗FcRn抗体的重链包含SEQ ID NO:167的氨基酸序列，轻链包含SEQ ID NO:168的氨基酸序列；该组合物中的抗FcRn抗体存在末端异质性，并且所述抗FcRn抗体的重链和/或轻链具有低于3％的末端延伸比率；
    优选地，所述抗FcRn抗体的重链具有低于1％的末端延伸比率。
  15. 根据权利要求13或14所述的组合物，其是由权利要求1至3任一项所述的方法制备得到的。
PCT/CN2021/114911 2020-08-28 2021-08-27 用于降低异源多肽末端异质性的信号肽 WO2022042673A1 (zh)

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