Signal peptides for reducing terminal heterogeneity of heteropolypeptides - Patent Application 20070122999

Abstract

The present disclosure relates to signal peptides for reducing terminal heterogeneity of heterologous polypeptides, particularly signal peptides for protein expression, and further to recombinant polypeptides, methods for their preparation, and compositions comprising the recombinant polypeptides.

Description

[Technical Field]

[0001] The present disclosure relates to the fields of molecular biology and protein engineering, and in particular to methods for reducing terminal heterogeneity of heterologous polypeptides. [Background technology]

[0002] Nothing herein necessarily constitutes prior art, but rather merely provides background information relevant to the present disclosure.

[0003] Signal peptides are short peptides that direct newly synthesized proteins into the secretory pathway. They are located at the N-terminus of secretory proteins and typically consist of 15 to 30 amino acids. Signal peptides typically contain three regions: a positively charged N-terminus (called the basic amino terminus); a hydrophobic middle region (mainly composed of neutral amino acids) that can form a single alpha helix (the main functional region of the signal peptide); and a long, negatively charged C-terminus (also called the processing region) that contains small amino acids and serves as the signal sequence cleavage site. After synthesis, the signal peptide sequence is recognized by the signal recognition particle (SRP), which halts or slows down protein synthesis. The SRP then imports ribosomes into the endoplasmic reticulum, where protein synthesis begins anew. Under the guidance of the signal peptide, newly synthesized proteins enter the endoplasmic reticulum lumen, where the signal peptide sequence is removed by signal peptidases.

[0004] Typically, highly efficient cleavage of signal peptides ensures accurate cleavage of both antibody light and heavy chain polypeptides, which is crucial for mammalian cell-mediated antibody expression. However, during signal peptide cleavage, the cleavage site can change, potentially resulting in elongation or truncation of the light and heavy amino acid chains of recombinant antibodies. Because truncated or elongated antibodies alter their affinity due to structural changes, affecting antibody activity, elongation or truncation of the amino acid chains must be avoided during antibody production. Furthermore, truncation or elongation of some products can lead to differences between product lots during production, requiring additional quality testing and making the process difficult to resolve. Therefore, the production of light and heavy chain antibodies with N-terminal homogeneity is now a universal consensus in the industry. Summary of the Invention

[0005] The present disclosure relates to signal peptides and their uses for reducing terminal heterogeneity of heterologous polypeptides.

[0006] The present disclosure provides a method for reducing N-terminal heterogeneity of an antibody heavy chain and / or an antibody light chain, comprising: (1) a first polynucleotide encoding a heavy chain of an antibody and a first signal peptide comprising the amino acid sequence of SEQ ID NO: 55 or SEQ ID NO: 56, which is operatively linked to the N-terminus of the heavy chain; and / or (2) culturing a host cell containing a second polynucleotide encoding a light chain of the antibody and a second signal peptide comprising the amino acid sequence of SEQ ID NO: 55 or SEQ ID NO: 56, operatively linked to the N-terminus of the light chain; Expression of the antibody heavy chain and / or the antibody light chain; Including, wherein SEQ ID NO: 55 is represented by MEWSWVFLFFLSLTGX1HX2, wherein X1 is V, A, S, T, G, C, L or I, and X2 is A, G, S, C, T or Q; Sequence number 56 is represented by MSVPTQVLGLLLLWLTDX3RX4, wherein X3 is V, A, S, T, G, C, L or I, and X4 is A, G, S, C, T or Q, and when X3 is selected from A, X4 is not C.

[0007] In some embodiments, in the method for reducing N-terminal heterogeneity of an antibody heavy chain and / or an antibody light chain as described above, the expressed antibody heavy chain has a terminal elongation of less than 3%, 2.5%, 2%, 1.5%, or 1%, and / or the antibody light chain has a terminal elongation of less than 3%, 2.5%, 2%, 1.5%, or 1%. In some embodiments, the expressed antibody heavy chain has a terminal elongation of less than 1% and / or the antibody light chain has a terminal elongation of less than 1%. In some embodiments, the terminal elongation is measured and calculated by peptide mapping detection. In some embodiments, the expressed antibody heavy chain has essentially no terminal residues remaining and / or the antibody light chain has essentially no terminal residues remaining. In some embodiments, the expressed antibody heavy chain has a terminal elongation of 0% and / or the antibody light chain has a terminal elongation of 0%.

[0008] In some embodiments, in the method of reducing N-terminal heterogeneity of an antibody heavy chain and / or an antibody light chain as described above, the first signal peptide or the second signal peptide each independently comprise: 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, and SEQ ID NO: 151: MSVPTQVLGLLLLWLTDIRQ The peptides include those selected from the group consisting of:

[0009] In some embodiments, in the methods of reducing N-terminal heterogeneity of an antibody heavy chain and / or an antibody light chain as described above, the first signal peptide or the second signal peptide each independently comprise the amino acid sequence of 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 SEQ ID NO:148.

[0010] In some embodiments, in the methods of reducing N-terminal heterogeneity of an antibody heavy chain and / or an antibody light chain as described above, the first signal peptide comprises the amino acid sequence of SEQ ID NO:57 or SEQ ID NO:105, and / or the second signal peptide comprises the amino acid sequence of SEQ ID NO:57 or SEQ ID NO:105.

[0011] In some embodiments, in the methods of reducing N-terminal heterogeneity of an antibody heavy chain and / or an antibody light chain as described above, the first signal peptide comprises the amino acid sequence of SEQ ID NO: 57 and / or the second signal peptide comprises the amino acid sequence of SEQ ID NO: 57.

[0012] In some embodiments, in the methods of reducing N-terminal heterogeneity of an antibody heavy chain and / or an antibody light chain as described above, the first signal peptide comprises the amino acid sequence of SEQ ID NO: 57 and / or the second signal peptide comprises the amino acid sequence of SEQ ID NO: 105.

[0013] In some embodiments, in the methods of reducing N-terminal heterogeneity of an antibody heavy chain and / or an antibody light chain as described above, the first signal peptide comprises the amino acid sequence of SEQ ID NO: 105 and / or the second signal peptide comprises the amino acid sequence of SEQ ID NO: 57.

[0014] In some embodiments, in the methods of reducing N-terminal heterogeneity of an antibody heavy chain and / or an antibody light chain as described above, the first signal peptide comprises the amino acid sequence of SEQ ID NO: 105 and / or the second signal peptide comprises the amino acid sequence of SEQ ID NO: 105.

[0015] In some embodiments, in the methods of reducing N-terminal heterogeneity of an antibody heavy chain and / or an antibody light chain as described above, the first polynucleotide or the second polynucleotide each independently encodes a polypeptide, wherein the polypeptide comprises an amino acid sequence selected from 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, and SEQ ID NO:172.

[0016] In some embodiments, in the methods of reducing N-terminal heterogeneity of an antibody heavy chain and / or an antibody light chain as described above, the host cell is a eukaryotic host cell, and in some embodiments, the eukaryotic host cell is a CHO cell or yeast.

[0017] In some embodiments, in the methods of reducing the N-terminal heterogeneity of an antibody heavy chain and / or an antibody light chain as described above, the antibody is a murine antibody, a chimeric antibody, a humanized antibody, a human antibody, an affinity matured antibody, or a multispecific antibody.

[0018] In some embodiments, in the methods of reducing N-terminal heterogeneity of an antibody heavy chain and / or an antibody light chain as described above, the antibody is selected from the group consisting of an anti-TLR7 antibody, an anti-HER2 (ErbB2) antibody, an anti-Claudin18.2 antibody, an anti-EGFR antibody, an anti-B7H3 antibody, an anti-c-Met antibody, an anti-HER3 (ErbB3) antibody, an anti-HER4 (ErbB4) antibody, an anti-CD3 antibody, an anti-CD20 antibody, an anti-CD22 antibody, an anti-CD30 antibody, an anti-CD33 antibody, an anti-CD38 antibody, an anti-CD44 antibody, an anti-CD47 antibody, an anti-CD56 antibody, an anti-CD70 antibody, an anti-CD73 antibody, an anti-CD105 antibody, an anti-CEA antibody, an anti-A33 antibody, an anti-Cripto antibody, an anti-SOST antibody, an anti-EphA2 antibody, an anti-G250 antibody, an anti-MUCl antibody, an anti-Lewis antibody, an anti-TNF-α ... Y antibody, anti-VEGFR antibody, anti-GPNMB antibody, anti-thrombin antibody, anti-Aβ antibody, anti-Integrin antibody, anti-ANGPTL3 antibody, anti-PSMA antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-LAG3 antibody, anti-IL-5 antibody, anti-IL-15 antibody, anti-IL-4 R antibody, anti-IL-6R antibody, anti-TIGHT antibody, anti-Tenascin-C antibody, anti-SLC44A4 antibody, anti-PCSK9 antibody, anti-EpCAM antibody, anti-CTGF antibody, anti-TSLP antibody, anti-CEA antibody, anti-Mesothelin antibody, and anti-FcRn antibody.

[0019] In some embodiments, in the method for reducing N-terminal heterogeneity of an antibody heavy chain and / or an antibody light chain as described above, the antibody is selected from Trastuzumab, Pertuzumab, Nimotuzumab, Enoblituzumab, Emibetuzumab, Inotuzumab, Pinatuzumab, Brentuximab, Gemtuzumab, Bivatuzumab, Lorvotuzumab, cBR96, Glematumamab, an anti-Claudin18.2 antibody, and an anti-FcRn antibody, wherein the anti-Claudin18.2 antibody has a heavy chain comprising the amino acid sequence of SEQ ID NO: 49 and a light chain comprising the amino acid sequence of SEQ ID NO: 47, and the anti-FcRn antibody has a heavy chain comprising the amino acid sequence of SEQ ID NO: 167 and a light chain comprising the amino acid sequence of SEQ ID NO: 168.

[0020] In some embodiments, there is provided a method for reducing N-terminal heterogeneity of an antibody heavy chain and / or an antibody light chain as described above, the method comprising cloning a light chain plasmid into a heavy chain plasmid to construct a full-length antibody plasmid, and in some embodiments, the method comprises introducing the plasmid into a host cell by electroporation.

[0021] The disclosure further provides a signal peptide comprising or consisting of the amino acid sequence of SEQ ID NO: 55 or SEQ ID NO: 56. In some embodiments, the polypeptide is a signal peptide, the amino acid sequence of which is set forth in any one of SEQ ID NO: 55 through SEQ ID NO: 151.

[0022] It is a polypeptide comprising a signal peptide comprising the amino acid sequence of SEQ ID NO:55 or SEQ ID NO:56.

[0023] In some embodiments, in the polypeptide described above, the polypeptide further comprises a heteropolypeptide that is operatively linked to the signal peptide, and in some embodiments, the heteropolypeptide is an antibody heavy chain or an antibody light chain, and in some embodiments, the signal peptide is operatively linked to the N-terminus of the antibody heavy chain or the antibody light chain.

[0024] In some embodiments, in such a polypeptide, the signal peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:57 to SEQ ID NO:151.

[0025] In some embodiments, in such polypeptides, the signal peptide comprises the amino acid sequence of 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 SEQ ID NO:148.

[0026] In some embodiments, in such a polypeptide, the signal peptide comprises an amino acid sequence selected from the amino acid sequences set forth in SEQ ID NO:57 and SEQ ID NO:105.

[0027] In some embodiments, in such a polypeptide, the polypeptide comprises an amino acid sequence 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:165, SEQ ID NO:166, SEQ ID NO:169, SEQ ID NO:170, SEQ ID NO:171 and SEQ ID NO:172.

[0028] The present disclosure further provides a nucleic acid molecule encoding any one of the above polypeptides.

[0029] The present disclosure further provides a host cell comprising such a nucleic acid molecule.

[0030] In some embodiments, in a host cell as described above, the host cell is a eukaryotic host cell, and in some embodiments, the eukaryotic host cell is a CHO cell or a yeast cell.

[0031] The present disclosure further provides compositions comprising heteropolypeptides having a terminal elongation of less than 3%, 2.5%, 2%, 1.5%, or 1%, wherein in some embodiments, the expressed heteropolypeptides have a terminal elongation of less than 1%, and in some embodiments, the expressed heteropolypeptides have a terminal elongation of less than 1%, and the terminal elongation is measured and calculated by peptide mapping detection, and in some embodiments, the expressed heteropolypeptides have essentially no terminal residues remaining, and in some embodiments, the expressed heteropolypeptides have a terminal elongation of 0%.

[0032] In some embodiments, in the composition described above, the heteropolypeptide is an antibody heavy chain and / or an antibody light chain, and in some embodiments, the antibody is selected from Trastuzumab, Pertuzumab, an anti-Claudin18.2 antibody, and an anti-FcRn antibody, wherein the anti-Claudin18.2 antibody has a heavy chain comprising the amino acid sequence of SEQ ID NO: 49 and a light chain comprising the amino acid sequence of SEQ ID NO: 47.

[0033] In some embodiments, the antibody is selected from anti-FcRn antibodies, 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.

[0034] In some embodiments, in the methods for reducing N-terminal heterogeneity of antibody heavy chains and / or antibody light chains described above, the expressed antibody heavy chains and / or antibody light chains have a terminal elongation of less than 3%, 2.5%, 2%, 1.5%, or 1%; in some embodiments, the expressed antibody heavy chains and / or antibody light chains have a terminal elongation of less than 1%; in some embodiments, the expressed antibody heavy chains and / or antibody light chains have a terminal elongation of less than 1%, and the terminal elongation is measured and calculated by peptide mapping detection; in some embodiments, the expressed antibody heavy chains and / or antibody light chains have essentially no terminal residues remaining; in some embodiments, the expressed antibody heavy chains and / or antibody light chains have a terminal elongation of 0%.

[0035] In some embodiments, such compositions are prepared by any one of the methods described above.

[0036] The signal peptide provided by the present disclosure has the effect of stably reducing the terminal heterogeneity of heteropolypeptides and is applicable to the large-scale expression of heteropolypeptides. [Brief explanation of the drawings]

[0037] [Figure 1] This shows the results of FACS detection of the binding of humanized antibodies to human Claudin 18.2 at the cellular level. [Figure 2] NUGC4 cell endocytosis experiment of humanized antibody. [Figure 3A] Figure 3A shows the ADCC effect of the antibody in wild-type NUGC4 cells (low Claudin18.2 expression), Figure 3B shows the ADCC effect of the antibody in NUGC4 cells with moderate Claudin18.2 expression, and Figure 3C shows the ADCC effect of the antibody in NUGC4 cells with high Claudin18.2 expression. [Figure 3B]Figure 3A shows the ADCC effect of the antibody in wild-type NUGC4 cells (low Claudin18.2 expression), Figure 3B shows the ADCC effect of the antibody in NUGC4 cells with moderate Claudin18.2 expression, and Figure 3C shows the ADCC effect of the antibody in NUGC4 cells with high Claudin18.2 expression. [Figure 3C] Figure 3A shows the ADCC effect of the antibody in wild-type NUGC4 cells (low Claudin18.2 expression), Figure 3B shows the ADCC effect of the antibody in NUGC4 cells with moderate Claudin18.2 expression, and Figure 3C shows the ADCC effect of the antibody in NUGC4 cells with high Claudin18.2 expression. [Figure 4A] Figure 4A is a deglycosylated, reduced molecular weight mass spectrum diagram of the light chain of Pertuzumab-1 antibody, Figure 4B is a deglycosylated, reduced molecular weight mass spectrum diagram of the light chain of Pertuzumab-3 antibody, Figure 4C is a deglycosylated, reduced molecular weight mass spectrum diagram of the heavy chain of Pertuzumab-1 antibody, and Figure 4D is a deglycosylated, reduced molecular weight mass spectrum diagram of the heavy chain of Pertuzumab-3 antibody. [Figure 4B] Figure 4A is a deglycosylated, reduced molecular weight mass spectrum diagram of the light chain of Pertuzumab-1 antibody, Figure 4B is a deglycosylated, reduced molecular weight mass spectrum diagram of the light chain of Pertuzumab-3 antibody, Figure 4C is a deglycosylated, reduced molecular weight mass spectrum diagram of the heavy chain of Pertuzumab-1 antibody, and Figure 4D is a deglycosylated, reduced molecular weight mass spectrum diagram of the heavy chain of Pertuzumab-3 antibody. [Figure 4C]Figure 4A is a deglycosylated, reduced molecular weight mass spectrum diagram of the light chain of Pertuzumab-1 antibody, Figure 4B is a deglycosylated, reduced molecular weight mass spectrum diagram of the light chain of Pertuzumab-3 antibody, Figure 4C is a deglycosylated, reduced molecular weight mass spectrum diagram of the heavy chain of Pertuzumab-1 antibody, and Figure 4D is a deglycosylated, reduced molecular weight mass spectrum diagram of the heavy chain of Pertuzumab-3 antibody. [Figure 4D] Figure 4A is a deglycosylated, reduced molecular weight mass spectrum diagram of the light chain of Pertuzumab-1 antibody, Figure 4B is a deglycosylated, reduced molecular weight mass spectrum diagram of the light chain of Pertuzumab-3 antibody, Figure 4C is a deglycosylated, reduced molecular weight mass spectrum diagram of the heavy chain of Pertuzumab-1 antibody, and Figure 4D is a deglycosylated, reduced molecular weight mass spectrum diagram of the heavy chain of Pertuzumab-3 antibody. [Figure 5A] Figure 5A is a deglycosylated, reduced molecular weight mass spectrum diagram of the light chain of Pertuzumab-2 antibody, Figure 5B is a deglycosylated, reduced molecular weight mass spectrum diagram of the light chain of Pertuzumab-4 antibody, Figure 5C is a deglycosylated, reduced molecular weight mass spectrum diagram of the heavy chain of Pertuzumab-2 antibody, and Figure 5D is a deglycosylated, reduced molecular weight mass spectrum diagram of the heavy chain of Pertuzumab-4 antibody. [Figure 5B] Figure 5A is a deglycosylated, reduced molecular weight mass spectrum diagram of the light chain of Pertuzumab-2 antibody, Figure 5B is a deglycosylated, reduced molecular weight mass spectrum diagram of the light chain of Pertuzumab-4 antibody, Figure 5C is a deglycosylated, reduced molecular weight mass spectrum diagram of the heavy chain of Pertuzumab-2 antibody, and Figure 5D is a deglycosylated, reduced molecular weight mass spectrum diagram of the heavy chain of Pertuzumab-4 antibody. [Figure 5C]Figure 5A is a deglycosylated, reduced molecular weight mass spectrum diagram of the light chain of Pertuzumab-2 antibody, Figure 5B is a deglycosylated, reduced molecular weight mass spectrum diagram of the light chain of Pertuzumab-4 antibody, Figure 5C is a deglycosylated, reduced molecular weight mass spectrum diagram of the heavy chain of Pertuzumab-2 antibody, and Figure 5D is a deglycosylated, reduced molecular weight mass spectrum diagram of the heavy chain of Pertuzumab-4 antibody. [Figure 5D] Figure 5A is a deglycosylated, reduced molecular weight mass spectrum diagram of the light chain of Pertuzumab-2 antibody, Figure 5B is a deglycosylated, reduced molecular weight mass spectrum diagram of the light chain of Pertuzumab-4 antibody, Figure 5C is a deglycosylated, reduced molecular weight mass spectrum diagram of the heavy chain of Pertuzumab-2 antibody, and Figure 5D is a deglycosylated, reduced molecular weight mass spectrum diagram of the heavy chain of Pertuzumab-4 antibody. [Figure 6A] Figure 6A is a deglycosylated, reduced molecular weight mass spectrum of the h1902-5-1 antibody light chain, Figure 6B is a deglycosylated, reduced molecular weight mass spectrum of the h1902-5-2 antibody light chain, Figure 6C is a deglycosylated, reduced molecular weight mass spectrum of the h1902-5-1 antibody heavy chain, and Figure 6D is a deglycosylated, reduced molecular weight mass spectrum of the h1902-5-2 antibody heavy chain. [Figure 6B] Figure 6A is a deglycosylated, reduced molecular weight mass spectrum of the h1902-5-1 antibody light chain, Figure 6B is a deglycosylated, reduced molecular weight mass spectrum of the h1902-5-2 antibody light chain, Figure 6C is a deglycosylated, reduced molecular weight mass spectrum of the h1902-5-1 antibody heavy chain, and Figure 6D is a deglycosylated, reduced molecular weight mass spectrum of the h1902-5-2 antibody heavy chain. [Figure 6C] Figure 6A is a deglycosylated, reduced molecular weight mass spectrum of the h1902-5-1 antibody light chain, Figure 6B is a deglycosylated, reduced molecular weight mass spectrum of the h1902-5-2 antibody light chain, Figure 6C is a deglycosylated, reduced molecular weight mass spectrum of the h1902-5-1 antibody heavy chain, and Figure 6D is a deglycosylated, reduced molecular weight mass spectrum of the h1902-5-2 antibody heavy chain. [Figure 6D]Figure 6A is a deglycosylated, reduced molecular weight mass spectrum of the h1902-5-1 antibody light chain, Figure 6B is a deglycosylated, reduced molecular weight mass spectrum of the h1902-5-2 antibody light chain, Figure 6C is a deglycosylated, reduced molecular weight mass spectrum of the h1902-5-1 antibody heavy chain, and Figure 6D is a deglycosylated, reduced molecular weight mass spectrum of the h1902-5-2 antibody heavy chain. [Figure 7A] Figure 7A is a mass spectrogram of the deglycosylated and reduced molecular weight of the light chain of FcRn-1 antibody, Figure 7B is a mass spectrogram of the deglycosylated and reduced molecular weight of the light chain of FcRn-2 antibody, Figure 7C is a mass spectrogram of the deglycosylated and reduced molecular weight of the heavy chain of FcRn-1 antibody, and Figure 7D is a mass spectrogram of the deglycosylated and reduced molecular weight of the heavy chain of FcRn-2 antibody. [Figure 7B] Figure 7A is a mass spectrogram of the deglycosylated and reduced molecular weight of the light chain of FcRn-1 antibody, Figure 7B is a mass spectrogram of the deglycosylated and reduced molecular weight of the light chain of FcRn-2 antibody, Figure 7C is a mass spectrogram of the deglycosylated and reduced molecular weight of the heavy chain of FcRn-1 antibody, and Figure 7D is a mass spectrogram of the deglycosylated and reduced molecular weight of the heavy chain of FcRn-2 antibody. [Figure 7C] Figure 7A is a mass spectrogram of the deglycosylated and reduced molecular weight of the light chain of FcRn-1 antibody, Figure 7B is a mass spectrogram of the deglycosylated and reduced molecular weight of the light chain of FcRn-2 antibody, Figure 7C is a mass spectrogram of the deglycosylated and reduced molecular weight of the heavy chain of FcRn-1 antibody, and Figure 7D is a mass spectrogram of the deglycosylated and reduced molecular weight of the heavy chain of FcRn-2 antibody. [Figure 7D] Figure 7A is a mass spectrogram of the deglycosylated and reduced molecular weight of the light chain of FcRn-1 antibody, Figure 7B is a mass spectrogram of the deglycosylated and reduced molecular weight of the light chain of FcRn-2 antibody, Figure 7C is a mass spectrogram of the deglycosylated and reduced molecular weight of the heavy chain of FcRn-1 antibody, and Figure 7D is a mass spectrogram of the deglycosylated and reduced molecular weight of the heavy chain of FcRn-2 antibody. DETAILED DESCRIPTION OF THE INVENTION

[0038] Details of the invention term In order that the present disclosure may be more readily understood, certain technical and scientific terms are specifically defined below. Unless expressly defined otherwise herein, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art.

[0039] The three-letter and one-letter codes for amino acids used in this disclosure are as described in J. Biol. Chem, 243, p. 3558 (1968).

[0040] "Antibody" as used in this disclosure is used herein in the broadest sense and encompasses different antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, murine antibodies, chimeric antibodies, humanized antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity and specificity.

[0041] The term "antibody fragment" refers to a molecule distinct from an intact antibody and includes a portion of the intact antibody that specifically binds to an antigen specifically bound by the intact antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv and scFab), single-domain antibodies (dAbs), and multispecific antibodies formed from antibody fragments.

[0042] A "chimeric antibody" as described in the present disclosure is an antibody in which the variable region of a mouse antibody is fused with the constant region of a human antibody, and can reduce the immune response elicited by a mouse antibody.

[0043] The "humanized antibody" described in the present disclosure, also known as a CDR-grafted antibody, refers to an antibody produced by grafting mouse CDR sequences onto a human antibody variable region framework, i.e., a framework sequence of a different human germline antibody. This overcomes the heterologous reactivity induced by chimeric antibodies containing a large amount of mouse protein components. To avoid reduced immunogenicity and reduced activity, minimal back mutations or reverse mutations can be made to the human antibody variable region framework sequence to maintain or improve activity. The humanized antibody of the present disclosure also includes humanized antibodies that have undergone affinity maturation mutations on the CDRs and are displayed in yeast.

[0044] In this disclosure, the terms "human antibody (HuMAb)," "human antibody," "fully human antibody," and "fully human antibody" may be used interchangeably to refer to antibodies whose amino acid sequences correspond to those of antibodies produced by humans or human cells, or that are derived from human antibody libraries or other non-human sources whose sequences are encoded by human antibodies. The definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

[0045] As used herein, an "antibody fragment" refers to a molecule distinct from a complete antibody and includes a portion of the complete antibody that specifically binds to an antigen that specifically binds to the complete antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv and scFab), single-domain antibodies (dAbs), and multispecific antibodies formed from antibody fragments. "Antibodies" according to the present disclosure include "complete antibodies" and "binding fragments" thereof. Accordingly, the heavy chain or light chain of an antibody described herein includes the complete heavy chain or the complete light chain, and also includes the heavy chain fragment or light chain fragment in an antibody fragment.

[0046] The term "framework" or "FR" refers to variable region residues excluding complementarity-determining region (CDR) residues. The FR of a variable region generally consists of four FR regions, FR1, FR2, FR3, and FR4. Thus, the CDR and FR sequences generally appear in VH (or VL) in the following order: FR1-HCDR1(LCDR1)-FR2-HCDR2(LCDR2)-FR3-HCDR3(LCDR3)-FR4.

[0047] The term "complementarity-determining region," "CDR," or "hypervariable region" refers to one of the six major hypervariable regions in an antibody variable domain that mediate antigen binding. Typically, each heavy chain variable region has three CDRs (HCDR1, HCDR2, and HCDR3), and each light chain variable region has three CDRs (LCDR1, LCDR2, and LCDR3). The amino acid sequence boundaries of the CDRs can be determined by any one of a variety of known methods, including the "Kabat" numbering convention (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD), the "Chothia" numbering convention (see Al-Lazikani et al., (1997) JMB 273:927-948), and the ImMunoGenTics (IMGT) numbering convention (see Lefranc MP, Immunologist, 7, 132-136 (1999); Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003)). For example, in a typical format, according to the Kabat rules, the CDR amino acid residues in the heavy chain variable region (VH) are numbered 31 to 35 (HCDR1), 50 to 65 (HCDR2), and 95 to 102 (HCDR3), and the CDR amino acid residues in the light chain variable region (VL) are numbered 24 to 34 (LCDR1), 50 to 56 (LCDR2), and 89 to 97 (LCDR3). According to the Chothia rules, the CDR amino acid residues in the VH are numbered 26 to 32 (HCDR1), 52 to 56 (HCDR2), and 95 to 102 (HCDR3), and the amino acid residues in the VL are numbered 26 to 32 (LCDR1), 50 to 52 (LCDR2), and 91 to 96 (LCDR3).According to the combined Kabat and Chothia CDR definition, a CDR consists of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in human VH, and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in human VL. According to the IMGT rules, the CDR amino acid residues in VH are numbered approximately 26-35 (CDR1), 51-57 (CDR2), and 93-102 (CDR3), and those in VL are numbered approximately 27-32 (CDR1), 50-52 (CDR2), and 89-97 (CDR3). According to the IMGT rules, the CDR regions of an antibody can be determined using the program IMGT / DomainGapAlign. Unless otherwise stated, all antibody variable region and CDR sequences according to the examples of this disclosure follow the "Kabat" numbering convention.

[0048] The terms "specific binding," "selective binding," "selectively binds," and "specifically binds" refer to the binding of an antibody to a predetermined epitope on an antigen. Typically, an antibody binds to an epitope of about 10 -8 Less than m, e.g., about 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 It binds with an affinity (KD) of less than M or less.

[0049] The term "KD" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction. Typically, antibodies of the present disclosure have a dissociation equilibrium constant of about 10 -7 Less than m, e.g., about 10 -8 M or 10 -9 It binds to an antigen with a dissociation equilibrium constant (KD) of less than M. For example, in the present disclosure, the affinity of an antibody to a cell surface antigen is measured as a KD value by the FACS method.

[0050] The terms "polynucleotide" and "nucleic acid molecule," as used interchangeably in this disclosure, refer to a polymer of nucleotides of any length, and include DNA and RNA. The nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modifications to the nucleotide structure can be made before or after incorporation into the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. As used herein, "operatively linked" refers to the juxtaposition of two or more components, wherein the components are in a relationship that allows them to function in their desired manner. For example, a promoter is operatively linked to a coding sequence if it controls or regulates the transcription of the linked sequence through cis-acting. Usually, but not necessarily, "operatively linked" DNA sequences are contiguous, and when necessary to link two protein-coding regions, or in the case of a secretory leader sequence, this applies to both contiguous and open reading frames. However, an operatively linked promoter is usually upstream of the coding sequence, but not necessarily adjacent to it. An operatively linked enhancer may be upstream of, within, or downstream of the coding sequence, and may be spaced a considerable distance from the promoter. Linking can be achieved by recombinant methods known in the art, such as PCR, by annealing, or by ligation at convenient restriction sites. If convenient restriction sites are not available, synthetic oligonucleotide adapters or linkers are commonly used. In the present disclosure, one method of linking a signal peptide to a heteropolypeptide is direct ligation.

[0051] As used herein, a "host cell" refers to a cell that has been genetically altered or can be genetically altered by introducing an exogenous polynucleotide (e.g., a recombinant plasmid or vector). It should be understood that such terms are intended to refer not only to a specific individual cell but also to the progeny of that cell. Because some modifications due to mutations or environmental influences may occur in successive generations, such progeny may differ substantially from the parent cell, yet still be included within the scope of the term "host cell" as used herein. Host cells may be microbial (e.g., eukaryotic) 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.

[0052] As used herein, "polypeptide" refers collectively to peptides and proteins of about 10 or more amino acids derived from any cell. "Heterologous" polypeptides are polypeptides foreign to the host cell in which they are utilized, such as human proteins produced by the host cell. Heterologous polypeptides may be prokaryotic or eukaryotic, e.g., mammalian or human. Heterologous polypeptides may be recombinantly produced or recombinant polypeptides.

[0053] Exemplary heteropolypeptides include ligands such as transmembrane molecules (e.g., receptors such as receptor tyrosine kinases) or growth factors. Illustrative heteropolypeptides include, for example, the following molecules: renin, growth hormones, including human growth hormone and bovine growth hormone, growth hormone-releasing factor, parathyroid hormone, thyroid-stimulating hormone, lipoproteins, α1-antitrypsin, insulin A chain, insulin B chain, proinsulin, follicle-stimulating hormone, calcitonin, luteinizing hormone, glucagon-like peptides, blood clotting factors such as factor VIIIC, factor IX, tissue factor (TF) and von Willebrands factor, anticoagulants such as protein C, atrial natriuretic peptide, pulmonary surfactant, plasminogen activators such as urokinase or human urinary or tissue plasminogen activator (t-PA), bombesin, thrombin, hematopoietic growth factors, tumor necrosis factor-α and -β, neprilysin, RANTES (regulated onactivation of normally T-cell expressed andsecreted), human macrophage inflammatory protein (MIP-1-α), serum albumins such as human serum albumin, Muellerian inhibitory substance, relaxin A chain, relaxin B chain, prorelaxin, mouse gonadotropin-related peptide, microbial proteins such as β-lactamase, deoxyribonuclease, IgE, cytotoxic T lymphocyte-associated antigen (CTLA) such as CTLA-4, inhibin, activating proteins, vascular endothelial growth factor (VEGF), hormones or Neurotrophic factors such as growth factor receptors, protein A or D, rheumatoid factor, bone-derived neurotrophic factor (BDNF), neurotrophic protein-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6), or nerve growth factors such as NGFβ; platelet-derived growth factor (PDGF), fibroblast growth factors such as aFGF and bFGF; epidermal growth factor (EGF), TGF-α and TGF-β, including TGF-β1, TGF-β2, TGF-β3, TGF-β4, and TGF-β5; transforming growth factors (TGFs), insulin-like growth factors-I and -II (IGF-I and IGF-II), des(1-3)-IGF-I (brain IGF-I), insulin-like growth factor binding proteins, CD proteins such as CD3, CD4, CD8, CD19, CD20, and CD40, erythropoietin, osteoinductive factors, immunotoxins, bone morphogenetic proteins (BMPs), interferons such as interferon-α, -β, and -γ, colony-stimulating factors such as M-CSF, GM-CSF, and G-CSF (CSF), interleukins (IL) such as IL-1 through IL-10, superoxide dismutase, T-cell receptors, surface membrane proteins, lysis-promoting factors, viral antigens such as portions of the AIDS envelope, transport proteins, homing receptors, addressins, regulatory proteins, integrins such as CD11a, CD11b, CD11c, CD18, ICAM, VLA-4 and VCAM, tumor-associated antigens such as HER2, HER3 and HER4 receptors, and fragments of any of the above polypeptides.

[0054] The heteropolypeptide may be an antibody, and illustratively the target of said antibody is A33, BMPI, BMP2, BMP3B (GDF1O), BMP4, BMP6, BMP8, CSFI (M-CSF), CSF2 (GM-CSF), CSF3 (G-CSF), EPO, FGF1 (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, FGF22, FGF23, FGF24, FGF25, FGF26, FGF27, FGF28, FGF29, FGF30, FGF31, FGF32, FGF33, FGF34, FGF35, FGF36, FGF37, FGF38, FGF39, FGF40, FGF41, FGF42, FGF43, FGF44, FGF45, FGF46, FGF47, FGF48, FGF49, FGF50, FGF51, FGF52, FGF53, FGF54, FGF55, FGF56, FGF57, FGF58, FGF5 ...1, FGF52, FGF53, FGF54, F GF21, 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, IL12B, IL13, IL14, IL15, IL16, IL17, IL17B, IL18, IL19, IL20, IL22, IL23, IL24, IL25, IL26, IL27, IL28A, IL28B, IL29, IL30, PDGFA, PDGFB, TGFA, TGFB1, TGFB2, TGFB3, LTA (TNF-b), LTB, TNF (TNF-a), TNFSF4 (OX40 ligand), TNFSF5 (CD40 ligand), TNFSF6 (FasL), TNFSF7 (CD27 ligand), TNFSF8 (CD30 ligand), TNFSF9 (4-1BB ligand), TNFSFIO (TRAIL), TNFSF1I (TRANCE), TNFSF12 (AP03L), TNFSF13 (April), TNFSF13B, TNF SF14(HVEM-L), TNFSF15(VEGI), TNFSF18, HGF(VEGFD), VEGF, VEGFB, VEGFC, ILIR1, IL1R2, IL1RL1, IL1RL2, IL2RA, IL2RB, IL2RG, IL3RA, IL4R, IL5RA, I L6R, IL7R, IL8RA, IL8RB, IL9R, ILIORA, ILIORB, IL11RA, IL12RB1, IL12RB2, IL13RA1, IL13RA2, IL15RA, IL17R, IL18R1, IL20RA, IL21R, IL22R, IL1HY1,IL1RAP、IL1RAPL1、IL1RAPL2、IL1RN、IL6ST、IL18BP、IL18RAP、IL22RA2、AIFI、HGF、LEP(レプチン)、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)、CXCL11(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、CKLFSF 5、CKLFSF6、CKLFSF7、CKLFSF8、BDNF、C5R1、CSF3、GRCCIO(CIO)、EPO、FY(DA RC)、GDF5、HDFIA、DL8、PRL、RGS3、RGS13、SDF2、SLIT2、TLR2、TLR4、TREMI、T REM2, VHL, ABCFI, ACVRI, ACVRIB, ACVR2, ACVR2B, ACVRLI, AD0RA2A, Aggreca n、AGR2、AICDA、AIFI、AIGI、AKAPI、AKAP2、AMH、AMHR2、ANGPTI、ANGPT2、ANG PTL3, ANGPTL4, ANPEP, APC, APOCI, AR, AZGPI(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, BPAG I(プレクチン), BRCAI, C19orflO(IL27w), C3, C4A, C5, C5R1, CANTI, CASP1, CASP 4、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、CC L22(MDC / STC-I)、CCL23(MPIF-1)、CCL24(MPIF-2 / eotaxin-2)、CCL25(TECK)、CCL26(eotaxin-3)、CCL27(CTACK / ILC)、CCL28、MTPCL3(MTPCla-4)(MDCCL) P-lb)、CCL5(RANTES)、CCL7(MCP-3)、CCL8(mcp-2)、CCNAI、CCNA2、CCNDI、C CNEI、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, CD 45RB, CD52, CD56, CD69, CD70, CD72, CD73, CD74, CD79A, CD79B, CD8, CD80, CD81, CD83, CD86, CDHI(E-カドヘリン), CDH10, CDH12, CDH13, CDH18, CDH19, CDH2 0. 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, キチナーゼ, CHST10, CKLFSF2, CKLFSF3, CKLFSF4, CKLFSF5, CKLFSF6, CKLFSF7, CKLFSF8, CLDN3, CLDN7, Claud in18.2, CLN3, CLU(クラステリン), c-Met, CMKLRI, CMKORI(RDCI), CNRI, COL18A1, COLIAI, COL4A3, COL6A1, CR2, Cripto, CRP, CSFI(M-CSF), CSF2(GM-CSF), C SF3 (GCSF), CTGF, CTLA4, CTNNBI (b-Catenin), CTSB (Organization Proteazer 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, EN G, EN01, EN02, EN03, EpCAM, EPHB4, EPO, ERBB2(Her-2), ERBB3(Her-3), ERBB4(H). er-4) EREG、ERK8、ESRI、ESR2、F3(TF)、FADD、FasL、FASN、FCERIA、FCER2、FCGR 3A, FGF, FGFI(aFGF), FGF10, FGF11, FGF12, FGF12B, FGF13, FGF14, FGF16, and 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(E PSILON), FILI(ZETA), FLJ12584, FLJ25530, FLRTI (Fox), FLTI, FOS, FOSL I(FRA-I), FY(DARC), G250, GABRP(MARKET), GAGEBI, GAGECI, GALNAC4S-6ST, GAT A3、GDF5、GFI 1, GGT1, GM-CSF, GNASI, GNRHI, GPR2(CCRIO), GPR31, GPR44, GPR81(FKSG80), GRCCIO(CIO), GRP, GPNMB, GSN(ビリン), GS TPI、HAVCR2、HDAC4、HDAC5、HDAC7A、HDAC9、HGF、HIFIA、HDP I. HLA-A, HLA-DRA, HM74, HMOXI, HUMCYT2 A、ICEBERG、ICOSL、ID2、IFN-a、IFNAI、IFNA2、IFNA4、IFNA5、IFNA6、IFNA7、IFNB1、IFNγ、DFNWI、IGBPI、IGFI、IGFIR、I GF2, IGFBP2, IGFBP3, IGFBP6, IL-I, IL10, MORA, IL10RB, IL11, IL11RA, IL-12, IL12A, IL12B, IL12RB1, IL12RB2, IL13IL13RA1、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, IL28A, IL28B, IL29, IL2RA, IL2RB, IL2RG, IL3, IL30, IL3RA, IL4, IL4R, IL5, IL5RA, IL6, IL6R, IL6ST (Glycoprotein 130), EL7, EL7R, EL8, EphA2, IL8RA, DL8RB, IL8RB, DL9, DL9R, DLK, INHA, INHBA, INSL3, INSL4, IRAKI, Integrin, ERAK2, ITGAI, ITGA2, ITGA3, ITGA6 (a6 integrin), ITGAV, ITGB3, ITGB4 (b4 integrin), 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 (Keratin 19), KRT2A, KHTHB6 (Hair-specific type H keratin), LAG3, LAMAS, LEP (Leptin), Lewis Y, Lingo-p75, Lingo-Troy, LPS, LTA (TNF-b), LTB, LTB4R (GPR16), LTB4R2, LTBR, MACMARCKS, MAG or Omgp, MAP2K7(c-Jun), MDK, Mesothelin, MIBI, MUCl MEF, MIP-2, MKI67, (Ki-67), MMP2, MMP9, MS4A1, MSMB, MT 3(metallothionectin-lll), MTSSI, MUCI(MYC, MYD88, NCK2, neurocan, NFKBI, NFKB2, NGFB(NGF), NGFR, Ng R-Lingo, NgR-Nogo66(Nogo), NgR-p75, NgR-Troy, NMEI(NM23A), N0X5, NPPB, NROBI, NR0B2, NRIDI, NR1D2, NR1H2, NR1H 3. 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, PD GFA, 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 (French B), SCGB2A1 (mammaglobin2), SCGB2A2 (mamnnaglobin).1), SCYEI (endothelial monocyte-activating cytokine), SDF2, SERPINAI, SERPINA3, SERP1NB5 (maspin), 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, TGFBR 2, TGFBR3, THIL, THBSI (thrombospondin-1), THBS2, THBS4, THPO, TIE (Tie-1), TIGHT, Tenascin-C, TMP3, tissue factor, 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 ligand), TNFSF5 (CD40 ligand), TNFSF6 (FasL), TNFSF7 (CD27 ligand), TNFSF8 (CD30 ligand), TNFSF9 (4-1BB ligand), TOLLIP, Toll-like receptor, TOP2A (topoisomerase Ea), TP53, TPMI, TPM2, TRADD, TRAFI, TRAF2, TRAF3, TRAF4, TRAF5, TRAF6, TREMI, TREM2, TRPC6, TSLP, TWEAK, VEGF, VEGFB, VEGFC, VEGFR, versican, VHL These include, but are not limited to, C5, VLA-4, XCLI (lymphotactin), XCL2 (SCM-1b), XCRI (GPR5 / CCXCRI), YYI, ZFPM2 and thrombin.

[0055] Illustratively, the antibody is selected from Trastuzumab, Pertuzumab, Nimotuzumab, Enoblituzumab, Emibetuzumab, Inotuzumab, Pinatuzumab, Brentuximab, Gemtuzumab, Bivatuzumab, Lorvotuzumab, cBR96, Glematumamab and anti-Claudin18.2 antibody, wherein the heavy chain of the anti-Claudin18.2 antibody is represented by sequence number 49 and the light chain is represented by sequence number 47.

[0056] "Conservative modification" or "conservative substitution or substitution" refers to the substitution of an amino acid in a protein with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation and rigidity, etc.), which frequently allows for variation without altering the biological activity of the protein. As is known to those skilled in the art, generally, a single amino acid substitution in a non-essential region of a polypeptide does not fundamentally alter the biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224 (4th edition)). Furthermore, substitution of amino acids with similar structures or functions is unlikely to destroy biological activity. Exemplary conservative substitutions are described as follows:

[0057] [Table 1]

[0058] The "terminal extension rate" described in this disclosure refers to the proportion of expression products having a terminal extension sequence in a population of expression products. Expression products having a terminal extension sequence are variants of the target expression product, and in addition to having the desired target sequence, are further linked to other amino acid residues. For example, if 1% of antibody light chain expression products have the N-terminus of the light chain sequence variants further linked to other amino acid residues, the terminal extension rate of the expression product is 1%. The presence of expression products having terminal extension sequences in a population of expression products indicates that the population of expression products has terminal heterogeneity. For example, the terminal extension sequence in this disclosure is derived from a residual signal peptide amino acid.

[0059] The compositions of the present disclosure contain a desired target expression product and an expression product having a terminal extension sequence. The presence of an amino-terminal extension in a composition can be detected by various analytical techniques, including, but not limited to, N-terminal sequence analysis, charge heterogeneity measurements (e.g., cation exchange chromatography or capillary zone electrophoresis), mass spectrometry, and peptide mapping. The amount of antibody variant in a composition generally ranges from an amount that constitutes the lower limit of detection of any measurement method for detecting the variant (e.g., cation exchange analysis) to an amount less than the amount of the predominant type of antibody. Approximately 3% or less (e.g., about 3%, 2.5%, 2%, 1.5%, 1%, 0.5%, or 0%) of the antibody light chains or antibody heavy chains in the composition contain an amino-terminal extension. Such percentages can be measured by mass spectrometry and peptide mapping. When the proportion of heteropolypeptides containing a terminal extension in a sample exceeds 1% of the total amount, peptide mapping can effectively identify it and quantitatively identify the corresponding peak in a reduced molecular weight mass spectrum.

[0060] The above specification provides details of one or more embodiments of the present disclosure. Although the present disclosure can be practiced or tested using any methods and materials similar or equivalent to those described herein, such methods and materials are described below. Other features, objects, and advantages of the present disclosure will become apparent from the specification and claims. Throughout the specification and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Unless otherwise defined, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art to which the present disclosure belongs. All patents and publications cited in the specification are incorporated by reference. The following examples are presented to more fully illustrate alternative embodiments of the present disclosure. These examples should not be construed in any way as limiting the scope of the present disclosure, which is limited only by the claims. [Example]

[0061] The present disclosure will be further explained below in conjunction with examples, but these examples do not limit the scope of the present disclosure. Experimental methods for which specific conditions are not specified in the examples of the present disclosure generally follow standard conditions, such as those in the Reisenko Antibody Technology Experimental Manual and Molecular Cloning Manual, or conditions recommended by the manufacturers of raw materials or products. Reagents for which specific sources are not specified are standard commercially available reagents.

[0062] Example 1: Preparation of anti-claudin18.2 antibody Example 1-1: Construction of a cell line that highly expresses Claudin18.2 The pCDH-hClaudin18.2 lentiviral expression vector plasmid and the pVSV-G and pCMV-dR8.91 lentiviral packaging vectors were transfected into 293T virus packaging cells using Lipofectamine 3000 transfection reagent. The virus-containing culture supernatant was collected, filtered, and centrifuged at ultra-high speed. The concentrated virus was then used to infect the human gastric signet ring cell carcinoma cell line NUGC4. After selection with puromycin for 2 to 3 weeks, FACS single-cell sorting was performed.

[0063] Claudin18.2 expression levels were determined based on tumor IHC scores. Cells with a tumor IHC score of 3 were considered high expressing cells, while cells with a tumor IHC score of 2 were considered moderate expressing cells. NUGC4 / hClaudin18.2 monoclonal cell lines with high Claudin18.2 expression were selected by detecting Claudin18.2 expression on the surface of lentivirus-infected NUGC4 cells using FACS. At the same time, NUGC4 clonal cell lines with moderate Claudin18.2 expression were selected by detecting Claudin18.2 expression on the surface of wild-type NUGC4 cells using FACS, while wild-type NUGC4 cells were considered low Claudin18.2 expressing cells.

[0064] The selected monoclonal cell lines were expanded and stored frozen in a refrigerator for subsequent experiments.

[0065] Claudin18.2 sequence Genbank: NP_001002026: (SEQ ID NO: 1) MAVTACQGLGFVVSLIGIAGIIAATCMDQWSTQDLYNNPVTAVFNYQGLWRSCVRESSGFTECRGYFTLLGLPAMLQAVRALMIVGIVLGAIGLLVSIFALKCIRIGSMEDSAKANMTLTSGIMFIVSGLCAIAGVSVFANMLVTNFWMSTANMYTGMGGMVQTVQTRYTFGAALFVGWVAGGLTLIGGVMMCIACRGLAPEETNYKAVSYHASGHSVAYKPGGFKASTGFGSNTKNKKIYDGGARTEDEVQSYPSKHDYV。

[0066] Claudin18.2 DNA sequence: (SEQ ID NO: 2) 1 AGAATTGCGC TGTCCACTTG TCGTGTGGCT CTGTGTCGAC ACTGTGCGCC ACCATGGCCG 61 TGACTGCCTG TCAGGGCTTG GGGTTCGTGG TTTCACTGAT TGGGATTGCG GGCATCATTG 121 CTGCCACCTG CATGGACCAG TGGAGCACCC AAGACTTGTA CAACAACCCC GTAACAGCTG 181 TTTTCAACTA CCAGGGGCTG TGGCGCTCCT GTGTCCGAGA GAGCTCTGGC TTCACCGAGT 241 GCCGGGGCTA CTTCACCCTG CTGGGGCTGC CAGCCATGCT GCAGGCAGTG CGAGCCCTGA 301 TGATCGTAGG CATCGTCCTG GGTGCCATTG GCCTCCTGGT ATCCATCTTT GCCCTGAAAT 361 GCATCCGCAT TGGCAGCATG GAGGACTCTG CCAAAGCCAA CATGACACTG ACCTCCGGGA 421 TCATGTTCAT TGTCTCAGGT CTTTGTGCAA TTGCTGGAGT GTCTGTGTTT GCCAACATGC 481 TGGTGACTAA CTTCTGGATG TCCACAGCTA ACATGTACAC CGGCATGGGT GGGATGGTGC 541 AGACTGTTCA GACCAGGTAC ACATTTGGTG CGGCTCTGTT CGTGGGCTGG GTCGCTGGAG 601 GCCTCACACT AATTGGGGGT GTGATGATGT GCATCGCCTG CCGGGGCCTG GCACCAGAAG 661 AAACCAACTA CAAAGCCGTT TCTTATCATG CCTCAGGCCA CAGTGTTGCC TACAAGCCTG 721 GAGGCTTCAA GGCCAGCACT GGCTTTGGGT CCAACACCAA AAACAAGAAG ATATACGATG 781 GAGGTGCCCG CACAGAGGAC GAGGTACAAT CTTATCCTTC CAAGCACGAC TATGTGTAAT 841 GCTCTAAGAC CTCTCAGCAC GGGCGGAAGA AACTCCCGGA GAGCTCACCC AAAAAACAAG 901 GAGATCCCAT CTAGATTTCT TCTTGCTTTT GACTCACAGC TGGAAGTTAG AAAAGCCTCG 961 ATTTCATCTT TGGAGAGGCC AAATGGTCTT AGCCTCAGTC TCTGTCTCTA AATATTCCAC 1021 CATAAAACAG CTGAGTTATT TATGAATTAG AGGCTATAGC TCACATTTTC AATCCTCTAT 1081 TTCTTTTTTT AAATATAACT TTCTACTCTG ATGAGAGAAT GTGGTTTTAA TCTCTCTCTC 1141 ACATTTTGAT GATTTAGACA GACTCCCCCT CTTCCTCCTA GTCAATAAAC CCATTGATGA 1201 TCTATTTCCC AGCTTATCCC CAAGAAAACT TTTGAAAGGA AAGAGTAGAC CCAAAGATGT 1261 TATTTTCTGC TGTTTGAATT TTGTCTCCCC ACCCCCAACT TGGCTAGTAA TAAACACTTA 1321 CTGAAGAAGA AGCAATAAGA GAAAGATATT TGTAATCTCT CCAGCCCATG ATTCCGGTTT 1381 TCTTACACTG TGATCTTAAA AGTTACCAAA CCAAAGTCAT TTTCAGTTTG AGGCAACCAA 1441 ACCTTTCTAC TGCTGTTGAC ATCTTCTTAT TACAGCAACA CCATTCTAGG AGTTTCCTGA 1501 GCTCTCCACT GGAGTCCTCT TTCTGTCGCG GGTCAGAAAT TGTCCCTAGA TGAATGAGAA 1561 AATTATTTTTT TTTAATTTAA GTCCTAAATA TAGTTAAAAT AAATAATGTT TTAGTAAAAT 1621 GATACACTAT CTCTGTGAAA TAGCCTCACC CCTACATGTG GATAGAAGGA AATGAAAAAA 1681 TAATTGCTTT GACATTGTCT ATATGGTACT TTGTAAAGTC ATGCTTAAGT ACAAATTCCA 1741 TGAAAAGCTC ACTGATCCTA ATTCTTTCCC TTTGAGGTCT CTATGGCTCT GATTGTACAT 1801 GATAGTAAGT GTAAGCCATG TAAAAGTAA ATAATGTCTG GGCACAGTGG CTCACGCCTG 1861 TAATCCTAGC ACTTTGGGAG GCTGAGGAGG AAGGATCACT TGAGCCCAGA AGTTCGAGAC 1921 TAGCCTGGGC AACATGGAGA AGCCCTGTCT CTACAAAATA CAGAGAGAAA AAATCAGCCA 1981 GTCATGGTGG CCTACACCTG TAGTCCCAGC ATTCCGGGAG GCTGAGGTGG GAGGATCACT 2041 TGAGCCCAGG GAGGTTGGGG CTGCAGTGAG CCATGATCAC ACCACTGCAC TCCAGCCAGG 2101 TGACATAGCG AGATCCTGTC TAAAAAAATA AAAAATAAAT AATGGAACAC AGCAAGTCCT 2161 AGGAAGTAGG TTAAAACTAA TTCTTTAAAA AAAAAAAAAA GTTGAGCCTG AATTAAATGT 2221 AATGTTTCCA AGTGACAGGT ATCCACATTT GCATGGTTAC AAGCCACTGC CAGTTAGCAG 2281 TAGCACTTTC CTGGCACTGT GGTCGGTTTT GTTTTGTTTT GCTTTGTTTA GAGACGGGGT 2341 CTCACTTTCC AGGCTGGCCT CAAACTCCTG CACTCAAGCA ATTCTTCTAC CCTGGCCTCC 2401 CAAGTAGCTG GAATTACAGG TGTGCGCCAT CACAACTAGC TGGTGGTCAG TTTTGTTACT 2461 CTGAGAGCTG TTCACTTCTC TGAATTCACC TAGAGTGGTT GGACCATCAG ATGTTTGGGC 2521 AAAACTGAAA GCTCTTTGCA ACCACACACC TTCCCTGAGC TTACATCACT GCCCTTTTGA 2581 GCAGAAAGTC TAAATTCCTT CCAAGACAGT AGAATTCCAT CCCAGTACCA AAGCCAGATA 2641 GGCCCCCTAG GAAACTGAGG TAAGAGCAGT CTCTAAAAAC TACCCACAGC AGCATTGGTG 2701 CAGGGGAACT TGGCCATTAG GTTATTATTT GAGAGGAAG TCCTCACATC AATAGTACAT 2761 ATGAAAGTGA CCTCCAAGGG GATTGGTGAA TACTCATAAG GATCTTCAGG CTGAACAGAC 2821 TATGTCTGGG GAAAGAACGG ATTATGCCCC ATTAAATAAC AAGTTGTGTT CAAGAGTCAG 2881 AGCAGTGAGC TCAGAGGCCC TTCTCACTGA GACAGCAACA TTTAAACCAA ACCAGAGGAA 2941 GTATTTGTGG AACTCACTGC CTCAGTTTGG GTAAAGGATG AGCAGACAAG TCAACTAAAG 3001 AAAAAAGAAA AGCAAGGAGG AGGGTTGAGC AATCTAGAGC ATGGAGTTTG TTAAGTGCTC 3061 TCTGGATTTG AGTTGAAGAG CATCCATTTG AGTTGAAGGC CACAGGGCAC AATGAGCTCT 3121 CCCTTCTACC ACCAGAAAGT CCCTGGTCAG GTCTCAGGTA GTGCGGTGTG GCTCAGCTGG 3181 GTTTTTAATT AGCGCATTCT CTATCCCAACA TTTAATTGTT TGAAAGCCTC CATATAGTTA 3241 GATTGTGCTT TGTAATTTTG TTGTTGTTGC TCTATCTTAT TGTATATGCA TTGAGTATTA 3301 ACCTGAATGT TTTGTTACTT AAAATTAAA AACACTGTTA TCCTACAGTT

[0067] Example 1-2: Production of anti-human claudin18.2 monoclonal antibody 1 immunity Anti-human Claudin18.2 monoclonal antibodies were produced by immunizing mice.

[0068] The experimental SJL white mice were female and 6-8 weeks old (Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., Animal Production License Number: SCXK(King)2012-0001). The breeding environment was SPF grade. After purchase, the mice were housed in a laboratory environment for one week, with a 12 / 12 hour light / dark cycle, a temperature of 20-25°C, and a humidity of 40-60%. Once acclimatized, the mice were immunized using the following protocol: huClaudin18.2-HEK293 cells (a stable HEK-293 cell line transfected with the human Claudin18.2 plasmid) were used as the immunizing antigen.

[0069] Immunization protocol: Before priming with cells, mice were injected intraperitoneally (IP) with 0.1 mL of TiterMax® Gold Adjuvant (Sigma Cat No. T2684) per mouse. Thirty minutes later, each mouse was injected IP with 1 × 10 TiterMax® Gold Adjuvant in 0.1 mL of saline. 8 The cells were injected into the mice after diluting them to a concentration of 1 × 10 / mL. After uniformly scattering the cells, they were inoculated on days 0, 14, 28, 42, and 56. Blood was collected on days 21, 35, 49, and 63, and the antibody titers in the mouse serum were determined by ELISA. After the fourth and fifth immunizations, mice with high and stable serum antibody titers were selected and subjected to splenocyte fusion. Three days before splenocyte fusion, a booster was administered, and 1 × 10 cells were injected intraperitoneally (IP). 7 of cells were injected.

[0070] 2. Splenocyte Fusion Hybridoma cells were obtained by fusing splenic lymphocytes with myeloma cells Sp2 / 0 cells (ATCC® CRL-8287™) using a PEG-mediated fusion process. Hybridoma cells were obtained at a concentration of 0.5 × 10 6 / mL ~ 1 × 10 6 The cells were resuspended in complete medium (IMDM medium containing 20% ​​FBS, 1x HAT, and 1x OPI) at a density of 1 / mL and seeded at 100 μL / well into a 96-well plate. After incubation at 37°C and 5% CO2 for 3-4 days, 100 μL / well of HAT complete medium was added and cultured for 3-4 days until clones were formed. The supernatant was removed, and 200 μL / well of HT complete medium (IMDM medium containing 20% ​​FBS, 1x HT, and 1x OPI) was added. After incubation at 37°C and 5% CO2 for 3 days, ELISA detection was performed.

[0071] 3. Hybridoma cell selection Depending on the growth density of the hybridoma cells, the culture supernatant was detected using a binding ELISA method. Cells that strongly bound to huClaudin18.2-HEK293 cells but did not bind to HEK293 cells were selected, amplified, cryopreserved, and subcloned two to three times until single-cell clones were obtained.

[0072] Each time the cells were subcloned, a cell binding experiment had to be performed. Hybridoma clones were selected through the above experiments, and antibodies were further prepared using serum-free cell culture methods, purified, and prepared for use in the detection example.

[0073] Example 1-3: Humanization of mouse antibodies Monoclonal hybridoma cell lines mAb1901 and mAb1902 with high in vitro activity were selected, and the monoclonal antibody sequences were cloned, humanized, recombinantly expressed, and evaluated for activity.

[0074] The process for cloning sequences from hybridomas is as follows: Hybridoma cells in the logarithmic growth phase were harvested, and RNA was extracted with Trizol (Invitrogen, 15596-018) (following the steps in the reagent kit's instruction manual) and reverse transcribed (PrimeScript™ Reverse Transcriptase, Takara, cat # 2680A). The cDNA obtained by reverse transcription was PCR amplified using a mouse Ig-Primer Set (Novagen, TB326 Rev.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 to 6.

[0075] mAb1901 mouse heavy chain variable region (SEQ ID NO: 3) EVQLMESGGGLVKPGGSLKLSCAASGFTFSDYGIHWVRQAPEMGLEWIAYISRGSSTIYYADTVKGRFTMSRDNAKNTLFLQMTSLRSEDTAMYYCARGGYDTRNAMDYWGQGTSVTVSS.

[0076] mAb1901 mouse light chain variable region (SEQ ID NO: 4) DIVMTQSPSSLSVSAGEKVTMSCKSSQSLLNSGNQKNYLAWYQQKPGQPPKLLIYGASTRASGVPDRFTGSGSGTDFTLTISSVQAEDLAIYHCQNDLYYPLTFGAGTKLELK.

[0077] mAb1902 mouse heavy chain variable region (SEQ ID NO: 5) EVQLQESGAELVKPGASVKLSCKASGYIFTSYWMHWVKQRPGQGLEWIGMIHPNSGSTNYNEKFKGKATLTLDKSSSTAYMQLSSLPSEDSAVYYCARLKTGNSFDYWGQGTTLTVSS.

[0078] mAb1902 mouse light chain variable region (SEQ ID NO: 6) DIVLTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAIYYCQNAYTYPFTFGSGTKLEIK.

[0079] The above mouse heavy chain variable region and light chain variable region were connected to the heavy chain constant region of a human IgG1 antibody and the human κ light chain constant region, respectively, to form chimeric antibodies ch1901 and ch1902, which will be described later.

[0080] The constant region is selected from the following sequences:

[0081] Heavy chain constant region of human IgG1 antibody: (SEQ ID NO: 7) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0082] Human kappa light chain constant region: (SEQ ID NO: 8) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0083] Following the methodology disclosed in many publications in this field, a mouse monoclonal antibody was humanized. That is, a human constant domain was used instead of the parent (mouse antibody) constant domain, and based on the homology between mouse and human antibodies, a human germline sequence was selected and CDR-grafted. In this disclosure, a candidate molecule with good activity was selected and humanized, and the results are as follows:

[0084] 1. Mouse antibody CDR region In Table 2, the amino acid residues of the VH / VL CDRs are determined and annotated according to the Kabat numbering system.

[0085] The CDR sequences of the mouse antibodies are shown in Table 2.

[0086] [Table 2]

[0087] 2. Selection of human germline FR region sequences Based on the obtained mouse antibody VH / VL CDR typical structures, the heavy and light chain variable region sequences were compared with the antibody Germline database to obtain highly homologous human germline templates, among which the human germline light chain framework regions were derived from the human kappa light chain gene.

[0088] 2.1 Design of humanized modifications and back mutations of mAb1901 An appropriate human antibody germline was selected, and the mAb1901 mouse antibody was humanized. The CDR regions of the mouse antibody mAb1901 were grafted onto the selected humanized template to obtain a humanized variable region with a humanized heavy chain variable region sequence of SEQ ID NO: 24 and a humanized light chain variable region sequence of SEQ ID NO: 21. These humanized variable regions were then recombined with an IgG constant region to form a complete antibody. At the same time, the FR regions in the V regions of the humanized antibody were backmutated. Exemplary backmutation patterns and combinations are as follows:

[0089] [Table 3-1] [Table 3-2] *In the table, all amino acid position numbers are based on the Kabat numbering convention; in N82T of the heavy chain variable region, 82 is position 82A according to the Kabat convention.

[0090] [Table 4]

[0091] In the above table, the heavy chain variable region can be joined to the human IgG1 heavy chain constant region shown in SEQ ID NO: 7 to form the heavy chain of a full-length antibody, while the light chain variable region can be joined to the human κ light chain constant region shown in SEQ ID NO: 8 to form the light chain of a full-length antibody. In other embodiments, the heavy chain variable region and the light chain variable region can be joined to other heavy chain constant regions and light chain constant regions, respectively, to form a full-length antibody.

[0092] 2.2 Humanization and back-mutation design of mAb1902 An appropriate human antibody germline was selected, and the mAb1902 mouse antibody was humanized. The CDR regions of the mouse antibody mAb1902 were grafted onto the selected humanized template to obtain a humanized variable region with a humanized heavy chain variable region sequence of SEQ ID NO: 31 and a humanized light chain variable region sequence of SEQ ID NO: 28. These were then recombined with an IgG constant region to form a complete antibody. At the same time, the FR region in the V region of the humanized antibody was backmutated. Exemplary backmutation patterns and combinations are as follows:

[0093] [Table 5] *All amino acid position numbers in the table are according to the Kabat numbering convention.

[0094] [Table 6] In the above table, the heavy chain variable region is connected to the human IgG1 heavy chain constant region shown in SEQ ID NO: 7 to form the heavy chain of the full-length antibody, while the light chain variable region is connected to the human κ light chain constant region shown in SEQ ID NO: 8 to form the light chain of the full-length antibody.

[0095] Exemplary antibody full length sequences are as follows:

[0096] Chimeric antibody ch1901: ch1901 heavy chain: (SEQ ID NO: 35) EVQLMESGGGLVKPGGSLKLSCAASGFTFSDYGIHWVRQAPEMGLEWIAYISRGSSTIYYADTVKGRFTMSRDNAKNTLFLQMTSLRSEDTAMYYCARGGYDTRNAMDYWGQ GTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0097] ch1901 light chain (SEQ ID NO: 36) DIVMTQSPSSLSVSAGEKVTMSCKSSQSLLNSGNQKNYLAWYQQKPGQPPKLLIYGASTRASGVPDRFTGSGSGTDFTLTISSVQAEDLAIYHCQNDLYYPLTFGAGTKL ELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0098] Chimeric antibody ch1902: ch1902 heavy chain (SEQ ID NO: 37) EVQLQESGAELVKPGASVKLSCKASGYIFTSYWMHWVKQRPGQGLEWIGMIHPNSGSTNYNEKFKGKATLTLDKSSSTAYMQLSSLPSEDSAVYYCARLKTGNSFDYWGQGT TLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0099] ch1902 light chain (SEQ ID NO: 38) DIVLTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAIYYCQNAYTYPFTFGSGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0100] [Table 7]

[0101] The light and heavy chain sequences of the full-length antibody are as follows:

[0102] [Table 8-1] [Table 8-2] [Table 8-3]

[0103] [Table 9]

[0104] The light and heavy chain sequences of the full-length antibody are as follows:

[0105] [Table 10-1] [Table 10-2] [Table 10-3]

[0106] The positive control antibody of the present disclosure is IMAB-362 (according to WO2016166122).

[0107] IMAB-362 heavy chain (SEQ ID NO: 53) 1 QVQLQQPGAE LVRPGASVKL SCKASGYTFT SYWINWVKQR PGQGLEWIGN 51 IYPSDSYTNY NQKFKDKATL TVDKSSSTAY MQLSSPTSED SAVYYCTRSW 101 RGNSFDYWGQ GTTLTVSSAS TKGPSVFPLA PSSKSTSGGT AALGCLVKDY 151 FPEPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSVVTVP SSSLGTQTYI 201 CNVNHKPSNT KVDKRVEPKS CDKTHTCPPC PAPELLGGPS VFLFPPKPKD 251 TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST 301 YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY 351 TLPPSREEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD 401 SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK.

[0108] IMAB-362 light chain (SEQ ID NO: 54) 1 DIVMTQSPSS LTVTAGEKVT MSCKSSQSLL NSGNQKNYLT WYQQKPGQPP 51 KLLIYWASTR ESGVPDRFTG SGSGTDFTLT ISSVQAEDLA VYYCQNDYSY 101 PFTFGSGTKL EIKRTVAAPS VFIFPPSDEQ LKSGTASVVC LLNNFYPREA 151 KVQWKVDNAL QSGNSQESVT EQDSKDSTYS LSSTLTLSKA DYEKHKVYAC 201 EVTHQGLSSP VTKSFNRGEC.

[0109] The above antibodies were cloned, expressed, and purified using standard gene cloning and recombinant expression methods.

[0110] Test Example 1: Biological evaluation of in vitro activity of anti-claudin 18.2 antibody Test Example 1-1: Cell-level ELISA binding experiment A cell-based ELISA was used to detect the binding properties of the Claudin18.2 antibody. NUGC4 cells stably transfected with Claudin18.2 and expressing it were cultured in 96-well cell plates (Corning, 3599). When the cells reached 90% confluency, they were fixed with 4% paraformaldehyde for 1 hour. After washing the plates three times with PBST buffer (PBS, pH 7.4, containing 0.05% Tween-20), 200 μL of 5% nonfat milk (Kwongming nonfat dry milk) diluted in PBS was added to each well. The plates were then incubated at 37°C for 2.5 hours or left overnight (16–18 hours) at 4°C for blocking. After blocking, the blocking solution was discarded and the plate was washed three times with PBST buffer. 50 μL / well of the antibody to be detected, diluted in sample diluent (1% nonfat milk in PBS, pH 7.4), was added at different concentrations and incubated in an incubator at 37°C for 2 hours. After incubation, the plate was washed five times with PBST and 100 μL / well of HRP-conjugated goat anti-human secondary antibody (Jackson Immuno Research, 109-035-003) diluted in sample diluent was added and incubated at 37°C for 1 hour. After washing the plate six times with PBST, 50 μL / well of TMB chromogenic substrate (KPL, 52-00-03) was added and incubated at room temperature for 10 to 15 minutes. The reaction was stopped by adding 50 μL / well of 1 M H2SO4. The absorbance value at 450 nm was read using an MD Versa Max™ microplate reader, and the binding EC50 value of the Claudin18.2 antibody to Claudin18.2 was calculated (see the table below for the results).

[0111] [Table 11]

[0112] [Table 12]

[0113] [Table 13]

[0114] Test Example 1-2: Antibody cell-level binding experiment NUGC4 cells stably transfected to express Claudin18.2 were cultured at 1 × 10 in FACS buffer (PBS (Sigma, P4417-100TAB) with 2% fetal bovine serum (Gibco, 10099141) pH 7.4). 6 A cell suspension was prepared at 1 / mL and added at 100 μL per well to a 96-well round-bottom plate (Corning, 3795). After centrifugation and removal of the supernatant, 50 μL per well of the target Claudin18.2 antibody diluted in FACS buffer was added at different concentrations and incubated in a refrigerator at 4°C in the dark for 1 hour. After washing by centrifugation three times at 300 g with FACS buffer, a working concentration of Alexa Fluor 488-coated anti-human IgG (H+L) (Invitrogen, A-11013) was added and incubated in a refrigerator at 4°C in the dark for 40 minutes. After washing by centrifugation three times at 300 g with FACS buffer, the geometric mean fluorescence intensity was detected using a BD FACS Canto II flow cytometer, and the binding EC50 value of the Claudin18.2 antibody to NUGC4 cells stably transfected with Claudin18.2 was calculated. The results are shown in Figure 1.

[0115] Test Example 1-3: Antibody endocytosis experiment The Claudin 18.2 antibody ready for detection, pre-labeled with DyLight 488 NHS Ester (thermofisher, 46403), was added to a final concentration of 5 μg / mL at 1 × 10 6NUGC4 cells stably transfected with Claudin18.2 were added with 1 mL of Claudin18.2 and incubated on ice in the dark for 1 hour. The cells were then washed three times by centrifugation with pre-chilled FACS buffer (PBS, pH 7.4, 2% fetal bovine serum). After removing the supernatant, pre-warmed complete medium was added and the cells were placed in a 37°C, 5% CO2 cell incubator. After 0, 0.5, 1, 2, and 4 hours, the cells were removed and stored on ice in the dark. After collecting all the samples, they were centrifuged at 300 g at low temperature to remove the supernatant, and then elution buffer (0.05 M glycine, 0.1 M sodium chloride, pH 1.7) was added. The cells were incubated at room temperature for 7 minutes, and then centrifuged once with FACS buffer at 300 g to wash. The geometric mean fluorescence intensity was measured using a BD FACS Canto II flow cytometer to calculate the endocytosis efficiency of the Claudin18.2 antibody in NUGC4 cells stably transfected to express Claudin18.2. The results (see Figure 2) indicate that the humanized antibody has good cellular endocytosis efficiency.

[0116] Test Example 1-4: Measurement of antibody affinity by flow cytometry On the day of the experiment, HEK293 / hClaudin18.2 cells were collected into a U-bottom 96-well plate, and 1 × 10 cells were added to each well. 5 pieces~2×10 5100 cells were added. Claudin18.2 antibody was added at an initial concentration of 5 μg / mL, diluted 2x (12 concentration points), and incubated at 4°C for 1 hour. IMAB362 was used as a positive control, and a no-antibody negative control was used. After centrifugation to remove the antibody, 100 μL / well of FITC anti-human IgG Fc antibody (200x) was added and incubated at 4°C in the dark for 30 minutes. After washing twice with PBS + 2% FBS, the cells were prepared for flow cytometry analysis. After preheating, a BD FACS Canto II was started, and a new experiment was established using BD FACSDiva software. The HEK293 / hClaudin18.2 negative control sample was detected, and the FSC and SSC voltages were adjusted to the appropriate values. Blank sample B and standard curve 1 were detected according to the Quantum™ FITC-5 MESF Kit instructions, and the FITC voltage was adjusted to the appropriate values ​​and saved. Samples in a U-bottom 96-well plate were detected at the stored voltage, and data were recorded. The experimental data was analyzed using Flowjo software to obtain GeoMean values. The MESF-GeoMean standard curve was fitted according to the Quantum™ FITC-5 MESF Kit instruction manual. The molar concentration and free antibody concentration of Claudin18.2 antibody bound to HEK293 / hClaudin18.2 cells were calculated based on the fluorescence concentration of the FITC anti-human IgG Fc antibody. The antibody Bmax and dissociation constant KD were calculated using the Scatchard plot method. The results are shown in Table 13.

[0117] [Table 14]

[0118] Test Example 1-5: Evaluation of ADCC effect of antibody Various NUGC4 cells (high / moderate / low Claudin18.2 expression) were digested, centrifuged at 1000 rpm, resuspended, and counted. 3 × 10 cells were used. 5Cells were resuspended at a density of 1000 cells / mL in phenol red-free RPMI 1640 (Gibco, 11835-030) supplemented with 10% FBS (New Zealand Ultra-Low IgG Fetal Bovine Serum, Gibco, 1921005PJ). 25 μL of cells (7500 cells / well) were added to each well of a 96-well plate (Corning, 3903). Antibodies were diluted in the above phenol red-free medium to prepare a 3x antibody dilution solution, and 25 μL of antibody was added to the cell plate at each well. The plate was incubated at 37°C in a 5% CO2 incubator for 0.5 hours.

[0119] Effector cells (FcrR3A-V158-NFAT-RE-Jurkat cells) were collected and centrifuged at 1000 rpm, then resuspended and counted. 3 × 10 cells were collected. 6 Resuspend the cells in phenol red-free RPMI 1640 supplemented with 10% FBS (New Zealand ultra-low IgG fetal bovine serum) at a density of 7.5 × 10 cells / mL and place 25 μL of cells (7.5 × 10 cells) into each well of an experimental plate. 4 Cells (cells / well) were added and incubated at 37°C in a 5% CO2 incubator for 6 hours.

[0120] Bright-Glo (Promega, E2610) was added at 75 μL / well to each well of the experimental plate, and chemiluminescence was detected using a microplate reader (PerkinElmer, VITOR3).

[0121] The results (see Table 14 and Figures 3A to 3C) show that both antibodies h1901-11 and h1902-5 exhibit very strong ADCC activity in NUGC4 cells expressing Claudin18.2 at different levels: low, moderate, and high.

[0122] [Table 15]

[0123] Example 2: Design and application of signal peptides Example 2-1: Design and expression of Pertuzumab fused with various signal peptides Antibodies were designed to which the following signal peptides were fused, and the combinations of the signal peptide sequences were as follows:

[0124] MEWSWVFLFFLSVTTGVHS (SEQ ID NO: 152) and MSVPTQVLGLLLLWLTDARC (SEQ ID NO: 153) MEWSWVFLFFLSVTTGVHS (SEQ ID NO: 152) and MEWSWVFLFFLSVTTGVHS (SEQ ID NO: 152) MEWSWVFLFFLSLTGVHA (SEQ ID NO: 57) and MSVPTQVLGLLLLWLTDVRA (SEQ ID NO: 105) MEWSWVFLFFLSLTGVHA (SEQ ID NO: 57) and MEWSWVFLFFLSLTGVHA (SEQ ID NO: 57) MSVPTQVLGLLLLWLTDVRA (SEQ ID NO: 105) and MEWSWVFLFFLSLTGVHA (SEQ ID NO: 57) The combination of the above signal peptides was placed at the N-terminus of the heavy and light chains of the Pertuzumab antibody, and corresponding new heavy and light chain sequences were designed. The designed sequences are as follows:

[0125] Antibody heavy chain amino acid sequence containing signal peptide of Pertuzumab-1 (SEQ ID NO: 154) MEWSWVFLFFLSVTTGVHSEVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Antibody light chain amino acid sequence containing signal peptide of Pertuzumab-1 (SEQ ID NO: 155) MSVPTQVLGLLLLWLTDARC DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Antibody heavy chain amino acid sequence containing signal peptide of Pertuzumab-2 (SEQ ID NO: 154) Antibody light chain amino acid sequence containing signal peptide of Pertuzumab-2 (SEQ ID NO: 156) MEWSWVFLFFLSVTTGVHSDIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Antibody heavy chain amino acid sequence containing signal peptide of Pertuzumab-3 (SEQ ID NO: 157) MEWSWVFLFFLSLTGVHA EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Antibody light chain amino acid sequence containing signal peptide of Pertuzumab-3 (SEQ ID NO: 158) MSVPTQVLGLLLLWLTDVRADIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Antibody heavy chain amino acid sequence containing signal peptide of Pertuzumab-4 (SEQ ID NO: 157) Antibody light chain amino acid sequence containing signal peptide of Pertuzumab-4 (SEQ ID NO: 159) MEWSWVFLFFLSLTGVHA DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Antibody heavy chain amino acid sequence containing signal peptide of Pertuzumab-5 (SEQ ID NO: 160) MSVPTQVLGLLLLWLTDVRAEVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Antibody light chain amino acid sequence containing signal peptide of Pertuzumab-5 (SEQ ID NO: 159) (Note: The underlined part is the signal peptide.) Based on the sequences of Pertuzumab-1 to Pertuzumab-4, heavy and light chain gene fragments 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 using the same enzyme cleavage site in the plasmid to construct a full-length antibody plasmid. The full-length antibody plasmid was directly electroporated into CHO cells, and after selection, stable transformants containing the plasmid were obtained. The stable transformants were cultured, and the culture supernatant was purified using a Protein A affinity column to obtain the expression product.

[0126] Example 2-2: Design and expression of h1902-5 fused with various signal peptides Antibodies were designed to which the following signal peptides were fused, and the combinations of the signal peptide sequences were as follows:

[0127] MEWSWVFLFFLSVTTGVHS (SEQ ID NO: 152) and MEWSWVFLFFLSVTTGVHS (SEQ ID NO: 152) MEWSWVFLFFLSLTGVHA (SEQ ID NO: 57) and MSVPTQVLGLLLLWLTDVRA (SEQ ID NO: 105) MSVPTQVLGLLLLWLTDVRA (SEQ ID NO: 105) and MEWSWVFLFFLSLTGVHA (SEQ ID NO: 57) and The above signal peptide combinations were placed at the N-terminus of the heavy and light chains of the h1902-5 antibody, and corresponding new heavy and light chain sequences were designed. The designed sequences are as follows:

[0128] Antibody heavy chain amino acid sequence containing signal peptide of h1902-5-1 (SEQ ID NO: 161) MEWSWVFLFFLSVTTGVHS EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQRLEWMGMIHPNSGSTNYNEKFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCARLKTGNSFDYWGQGT TVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Antibody light chain amino acid sequence containing signal peptide of h1902-5-1 (SEQ ID NO: 162) MEWSWVFLFFLSVTTGVHSDIVLTQSPDSLAVSLGERATINCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNAYTYPFTFGQGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Antibody heavy chain amino acid sequence containing signal peptide of h1902-5-2 (SEQ ID NO: 163) MEWSWVFLFFLSLTGVHA EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQRLEWMGMIHPNSGSTNYNEKFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCARLKTGNSFDYWGQGT TVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Antibody light chain amino acid sequence containing signal peptide of h1902-5-2 (SEQ ID NO: 164) MSVPTQVLGLLLLWLTDVRADIVLTQSPDSLAVSLGERATINCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNAYTYPFTFGQGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Antibody heavy chain amino acid sequence containing signal peptide of h1902-5-3 (SEQ ID NO: 165) MSVPTQVLGLLLLWLTDVRA EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQRLEWMGMIHPNSGSTNYNEKFKGRVTITRDTSASTAYMELSSLRSEDTAVYYCARLKTGNSFDYWGQGT TVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Antibody light chain amino acid sequence containing signal peptide of h1902-5-3 (SEQ ID NO: 166) MEWSWVFLFFLSLTGVHADIVLTQSPDSLAVSLGERATINCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNAYTYPFTFGQGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Note: The underlined part is the signal peptide.) Based on the above-mentioned h1902-5-1 or h1902-5-2 sequences, gene fragments for each heavy and light chain were synthesized and constructed into expression vectors to obtain full-length antibody plasmids. The full-length antibody plasmids were directly electroporated into CHO cells, and after selection, stable transformants containing the plasmids were obtained. The stably transformed cells were cultured, and the culture supernatant was purified using a Protein A affinity column to obtain the expression product.

[0129] Example 2-3: Design and expression of FcRn fused with various signal peptides The heavy and light chain amino acid sequences of the anti-FcRn antibody are as follows:

[0130] Heavy chain (SEQ ID NO: 167) EVQLVQSGAEVKKPGESLKISCKGSGYNFNKHYIAWVRQMPGKGLEWMGIIYPDNSNTIYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARFGGPTFAQWYFDYW GQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKY GPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK Light chain (SEQ ID NO: 168) NFMLTQPHSVSESPGKTVTISCTGSSGSIASNYVQWYQQRPGSSPTTVIYEDNQRASGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDSSSHNWVFGGGTKL TVLGQPKANTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS Antibodies were designed to which the following signal peptides were fused, and the combinations of the signal peptide sequences were as follows:

[0131] MEWSWVFLFFLSVTTGVHS (SEQ ID NO: 152) and MEWSWVFLFFLSVTTGVHS (SEQ ID NO: 152) MEWSWVFLFFLSLTGVHA (SEQ ID NO: 57) and MEWSWVFLFFLSLTGVHA (SEQ ID NO: 57) The designed sequences are as follows:

[0132] Antibody heavy chain amino acid sequence containing the signal peptide of FcRn-1 (SEQ ID NO: 169) MEWSWVFLFFLSVTTGVHS EVQLVQSGAEVKKPGESLKISCKGSGYNFNKHYIAWVRQMPGKGLEWMGIIYPDNSNTIYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARFGGPTFAQWYFDYW GQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKY GPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK Antibody light chain amino acid sequence containing the signal peptide of FcRn-1 (SEQ ID NO: 170) MEWSWVFLFFLSVTTGVHS NFMLTQPHSVSESPGKTVTISCTGSSGSIASNYVQWYQQRPGSSPTTVIYEDNQRASGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDSSSHNWVFGGGTKL TVLGQPKANTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS Antibody heavy chain amino acid sequence containing the signal peptide of FcRn-2 (SEQ ID NO: 171) MEWSWVFLFFLSLTGVHAEVQLVQSGAEVKKPGESLKISCKGSGYNFNKHYIAWVRQMPGKGLEWMGIIYPDNSNTIYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARFGGPTFAQWYFDYW GQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKY GPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK Antibody light chain amino acid sequence containing the signal peptide of FcRn-2 (SEQ ID NO: 172) MEWSWVFLFFLSLTGVHA NFMLTQPHSVSESPGKTVTISCTGSSGSIASNYVQWYQQRPGSSPTTVIYEDNQRASGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDSSSHNWVFGGGTKL TVLGQPKANTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS Based on the above FcRn-1 and FcRn-2 sequences, gene fragments for each heavy and light chain were synthesized and constructed into expression vectors to obtain full-length antibody plasmids. The full-length antibody plasmids were directly electroporated into CHO cells, and after selection, a pool of stable transformants containing the plasmids was obtained. The stable transformant pool was cultured, and the culture supernatant was purified using a Protein A affinity column to obtain the expression product.

[0133] Test Example 2: Terminal heterogeneity of expression products using various signal peptides Test Example 2-1: Detection of deglycosylated reduced molecular weight and amino acid sequence of Pertuzumab fused with various signal peptides The N-glycosyl groups on samples Pertuzumab-1, Pertuzumab-2, Pertuzumab-3, and Pertuzumab-4 were removed using PNGase F glycosidase (NEB, P0708) and then reduced to light and heavy chains with DTT (Sigma, 43815). Finally, the deglycosylated and reduced molecular weights of the samples were detected by LC-MS (Waters, ACQUITY UPLC H-Class / XeVo G2-XS QTOF), and data processing and analysis were performed using UNIFI software.

[0134] As shown in Figures 4A to 4D, the detection results for Pertuzumab-1 and Pertuzumab-3 showed that sample Pertuzumab-1 contained 4.7% residual signal peptide amino acids (RC) in the light chain, while sample Pertuzumab-3 contained no residual signal peptide amino acids in the light chain. The heavy chains of both Pertuzumab-1 and Pertuzumab-3 contained no residual signal peptide amino acids and only a small amount of glycosylation modification.

[0135] Pertuzumab-1 was denatured with urea, then reduced to light and heavy chains by adding DTT, and then enzymatically cleaved with GluC enzyme (Promega, V1651). The resulting sample was analyzed by LC-MS to obtain molecular weight data. The data was analyzed using UNIFI software to obtain the amino acid sequence information of the sample.

[0136] As a result, the abnormal peaks observed in the deglycosylated reduced molecular weight detection of Pertuzumab-1 were confirmed to be residual RC amino acids by peptide mapping analysis. The specific results are shown in Table 15.

[0137] [Table 16]

[0138] Note: In this test example, the amino acid R in the sequence RC is cleaved by enzymatic cleavage with the GluC enzyme, and the remaining amino acid C represents the residue of the RC amino acid.

[0139] As shown in Figures 5A to 5D, the detection results for Pertuzumab-2 and Pertuzumab-4 showed that sample Pertuzumab-2 contained 5.8% residual signal peptide amino acids (VHS) in the light chain, while sample Pertuzumab-4 contained no residual signal peptide amino acids in the light chain. The heavy chains of both antibodies contained no residual signal peptide amino acids and only a small amount of glycosylation.

[0140] Pertuzumab-2 was denatured with urea, then reduced to light and heavy chains by adding DTT, and then enzymatically cleaved by adding GluC enzyme. The resulting sample was analyzed by LC-MS to obtain molecular weight data, which was then analyzed using UNIFI software to obtain the amino acid sequence information of the sample.

[0141] As a result, the abnormal peaks observed in the deglycosylated reduced molecular weight detection of Pertuzumab-2 were confirmed to be residual VHS amino acids by peptide mapping analysis. The specific results are shown in Table 16.

[0142] [Table 17]

[0143] Test Example 2-2: Detection of deglycosylated reduced molecular weight and amino acid sequence of h1902-5 fused with various signal peptides The N-glycosyl groups in samples h1902-5-1 and h1902-5-2 were removed using PNGase F glycosidase (NEB, P0708) and then reduced to light and heavy chains with DTT (Sigma, 43815). Finally, the deglycosylated and reduced molecular weights of the samples were detected by LC-MS (Waters, ACQUITY UPLC H-Class / XeVo G2-XS QTOF), and data processing and analysis were performed using UNIFI software.

[0144] As shown in Figures 6A to 6D, sample h1902-5-1 was found to contain 3.1% residual signal peptide amino acids (VHS) in the heavy chain, while sample h1902-5-2 had no residual signal peptide amino acids in the heavy chain. Neither of the light chains of the two antibodies had any detected residual signal peptide amino acids and only a small amount of glycosylation.

[0145] h1902-5-1 was denatured with urea, then reduced to light and heavy chains by adding DTT, and then cleaved with GluC enzyme. The resulting sample was analyzed by LC-MS to obtain molecular weight data. The data was analyzed using UNIFI software to obtain the amino acid sequence information of the sample.

[0146] As a result, the abnormal peaks that appeared in the deglycosylated reduced molecular weight detection of h1902-5-1 were confirmed to be residual VHS amino acids by peptide mapping analysis. The specific results are shown in Table 17 below.

[0147] [Table 18]

[0148] Test Example 2-3: Detection of deglycosylated reduced molecular weight and amino acid sequence of anti-FcRn antibodies fused with various signal peptides The N-glycosyl groups on the FcRn-1 and FcRn-2 samples were removed using PNGase F glycosidase (NEB, P0708) and then reduced to light and heavy chains using DTT (Sigma, 43815). Finally, the deglycosylated and reduced molecular weights of the samples were detected using LC-MS (Waters, ACQUITY UPLC H-Class / XeVo G2-XS QTOF), and data processing and analysis were performed using UNIFI software.

[0149] As shown in Figures 7A to 7D, sample FcRn-1 was found to contain 3.4% residual signal peptide amino acids (VHS) in the heavy chain, whereas sample FcRn-2 contained no residual signal peptide amino acids in the heavy chain. Neither of the light chains of the two antibodies contained any detected residual signal peptide amino acids, and only a small amount of glycosylation was observed.

[0150] FcRn-1 was denatured with urea, then reduced to light and heavy chains by adding DTT, and then enzymatically cleaved by adding GluC enzyme. The resulting sample was analyzed by LC-MS to obtain molecular weight data, which was then analyzed using UNIFI software to obtain the amino acid sequence information of the sample.

[0151] As a result, the abnormal peaks that appeared in the deglycosylated reduced molecular weight detection of FcRn-1 were confirmed to be residual VHS amino acids by peptide mapping analysis. The specific results are shown in the table below.

[0152] [Table 19]

[0153] It is to be clearly understood that while the foregoing invention has been described in detail by way of illustration and example, such descriptions and examples should not be construed as limiting the scope of the present disclosure. The entire disclosures of the patent and scientific literature cited herein are hereby incorporated in their entirety and expressly by reference.

Claims

1. 1. A method for reducing N-terminal heterogeneity of an antibody heavy chain and / or an antibody light chain, comprising: (1) a first polynucleotide encoding a heavy chain of an antibody and a first signal peptide operably linked to the N-terminus of the heavy chain, the first signal peptide consisting of the amino acid sequence of SEQ ID NO: 57 or SEQ ID NO: 105; and (2) culturing a host cell containing a second polynucleotide encoding a light chain of the antibody and a second signal peptide consisting of the amino acid sequence of SEQ ID NO: 57 or SEQ ID NO: 105, operably linked to the N-terminus of the light chain; Expression of the antibody heavy chain and / or the antibody light chain; wherein the host cell is a CHO cell.

2. 2. The method of claim 1, wherein the antibody is a murine antibody, a chimeric antibody, a humanized antibody, a human antibody, an affinity matured antibody, or a multispecific antibody.

3. The antibodies include 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, and 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, anti-EphA2 antibody, anti-G250 antibody, anti-MUCl antibody, anti-Lewis Y antibody, anti-VEGFR antibody, anti-GPNMB antibody, anti-thrombin antibody, anti-Aβ antibody, anti-Integrin antibody, anti-ANGPTL3 antibody, anti-PSMA antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-LAG3 antibody, anti-IL-5 antibody, anti-IL-15 antibody, anti-IL-4R antibody, anti-I The method according to claim 1, which is selected from L-6R antibody, anti-TIGHT antibody, anti-Tenascin-C antibody, anti-SLC44A4 antibody, anti-PCSK9 antibody, anti-EpCAM antibody, anti-CTGF antibody, anti-TSLP antibody, anti-CEA antibody, anti-Mesothelin antibody and anti-FcRn antibody.

4. 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 anti-Claudin18.2 antibody has a heavy chain comprising the amino acid sequence of SEQ ID NO: 49 and a light chain comprising the amino acid sequence of SEQ ID NO: 47, and the anti-FcRn antibody has a heavy chain comprising the amino acid sequence of SEQ ID NO: 167 and a light chain comprising the amino acid sequence of SEQ ID NO:

168. The method of claim 1.

5. A polypeptide comprising a signal peptide consisting of the amino acid sequence of SEQ ID NO: 57 or SEQ ID NO:

105.

6. the polypeptide further comprises a heteropolypeptide operably linked to the signal peptide; the heteropolypeptide is an antibody heavy chain or an antibody light chain, the signal peptide is operably linked to the N-terminus of the antibody heavy chain or the antibody light chain; The polypeptide of claim 5.

7. 6. The polypeptide of claim 5, wherein the polypeptide comprises an amino acid sequence 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:165, SEQ ID NO:166, SEQ ID NO:171 and SEQ ID NO:

172.

8. A nucleic acid molecule encoding the polypeptide according to any one of claims 5 to 7.

9. A host cell which is a CHO cell and which comprises the nucleic acid molecule of claim 8.

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