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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French (fr)
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周松涛
刘洵
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Jiangsu Hengrui Medicine Co Ltd
Shanghai Hengrui Pharmaceutical Co Ltd
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Jiangsu Hengrui Medicine Co Ltd
Shanghai Hengrui Pharmaceutical Co Ltd
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Priority to JP2023512724A priority Critical patent/JP7817241B2/ja
Priority to US18/023,301 priority patent/US20230312725A1/en
Priority to CA3191184A priority patent/CA3191184A1/en
Priority to EP21860507.9A priority patent/EP4206222A4/en
Priority to CN202180050556.XA priority patent/CN116234814A/zh
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    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/06—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies from serum
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    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], 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 [IG], 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 [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IG], 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 [IG], 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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    • 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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    • 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]
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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
    • C07K2317/567—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
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    • C07K2317/77—Internalization into the cell
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    • 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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