WO2020038404A1 - Anticorps monoclonal anti-claudine 18,2 humaine et son utilisation - Google Patents

Anticorps monoclonal anti-claudine 18,2 humaine et son utilisation Download PDF

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WO2020038404A1
WO2020038404A1 PCT/CN2019/101785 CN2019101785W WO2020038404A1 WO 2020038404 A1 WO2020038404 A1 WO 2020038404A1 CN 2019101785 W CN2019101785 W CN 2019101785W WO 2020038404 A1 WO2020038404 A1 WO 2020038404A1
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Prior art keywords
seq
light chain
heavy chain
cdr2
cdr3
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PCT/CN2019/101785
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English (en)
Chinese (zh)
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吴燕燕
崔文俊
胡琛霏
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瑞阳(苏州)生物科技有限公司
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Priority claimed from CN201910767966.0A external-priority patent/CN110857322A/zh
Application filed by 瑞阳(苏州)生物科技有限公司 filed Critical 瑞阳(苏州)生物科技有限公司
Priority to US17/267,637 priority Critical patent/US20210261658A1/en
Priority to JP2021508276A priority patent/JP7192092B2/ja
Publication of WO2020038404A1 publication Critical patent/WO2020038404A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells

Definitions

  • the invention relates to the field of biomedicine, in particular to an anti-human claudin 18.2 monoclonal antibody and application thereof.
  • the Claudins family is a tightly-linked backbone protein between epithelial cells and participates in the formation of tightly-connected cells. Tightly-connected cells maintain the polar arrangement of cells and barrier functions. The abnormal expression of these cells leads to the destruction of tightly-connected structures and functions, which is the basis of many diseases. Studies have shown that the Claudins family is abnormally expressed in a variety of epithelial-derived tumors, and is closely related to the occurrence and development of these tumors. hot spot.
  • the human Claudin18 gene has two different first exons, so two subtypes, Claudin 18.1 and Claudin 18.2, can be produced.
  • the N-terminal 69 amino acids of these two molecules have different structures and are located in the first extracellular region of the cyclic structure.
  • the two subtypes of Claudin18 are transcribed and amplified in different tissues respectively. Among them, Claudin 18.1 is mainly expressed in lung tissue, while Claudin 18.2 is specifically expressed in gastric tissue.
  • Claudin 18.2 expression was present in 77% of primary gastric adenocarcinoma tissues, and it was extremely important that 56% of the tissues expressed more than 60%. Consistent with the above findings, the expression of Claudin 18.2 protein is down-regulated in intestinal gastric cancer, while the expression of Claudin 18.2 protein is higher in diffuse gastric cancer. However, Claudin 18.2 protein is not expressed in normal tissues such as pancreas, esophagus, and ovary, while Claudin 18.2 protein can be expressed in large amounts in corresponding tumor tissues. Therefore, Claudin 18.2 protein can be used as a target for clinical tumor diagnosis and treatment. However, no antibody has been developed that targets this protein.
  • the invention provides an anti-human claudin 18.2 monoclonal antibody with good specificity, high affinity and stability.
  • the first object of the present invention is to provide the amino acid sequence of an anti-human claudin 18.2 monoclonal antibody, which is as follows:
  • the heavy chain CDR1 is selected from: SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 13, SEQ ID NO: 19, SEQ ID NO: 25, SEQ ID NO: 31, SEQ ID NO: 37, SEQ ID NO: 43, SEQ ID: NO: 49, SEQ ID: NO: 55, SEQ ID: NO: 61, SEQ ID: NO: 67, SEQ ID: NO: 73 or a homology greater than 50% sequence;
  • the heavy chain CDR2 is selected from: SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 14, SEQ ID NO: 20, SEQ ID NO: 26, SEQ ID NO: 32, SEQ ID NO: 38, SEQ ID NO: 44, SEQ ID: NO: 50, SEQ ID: NO: 56, SEQ ID: NO: 62, SEQ ID: NO: 68, SEQ ID: NO: 74 or a homology with one of which is greater than 50% sequence;
  • the heavy chain CDR3 is selected from: SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 21, SEQ ID NO: 27, SEQ ID NO: 33, SEQ ID NO: 39, SEQ ID NO: 45, SEQ ID: NO: 51, SEQ ID: NO: 57, SEQ ID: NO: 63, SEQ ID: NO: 69, SEQ ID: NO: 75 or a homology with one of which is greater than 50% sequence;
  • the light chain CDR1 is selected from: SEQ ID NO: 4, SEQ ID NO: 10, SEQ ID NO: 16, SEQ ID NO: 22, SEQ ID NO: 28, SEQ ID NO: 34, SEQ ID NO: 40, SEQ ID NO: 46, SEQ ID: NO: 52, SEQ ID: NO: 58, SEQ ID: NO: 64, SEQ ID: NO: 70, SEQ ID: NO: 76 or a homology with one that is greater than 50% sequence;
  • the light chain CDR2 is selected from: SEQ ID NO: 5, SEQ ID NO: 11, SEQ ID NO: 17, SEQ ID NO: 23, SEQ ID NO: 29, SEQ ID NO: 35, SEQ ID NO: 41, SEQ ID NO: 47, SEQ ID: NO: 53, SEQ ID: NO: 59, SEQ ID: NO: 65, SEQ ID: NO: 71, SEQ ID: NO: 77 or a homology with one of which is greater than 50% sequence;
  • the light chain CDR3 is selected from: SEQ ID NO: 6, SEQ ID NO: 12, SEQ ID NO: 18, SEQ ID NO: 24, SEQ ID NO: 30, SEQ ID NO: 36, SEQ ID NO: 42, SEQ ID NO: 48, SEQ ID: NO: 54, SEQ ID: NO: 60, SEQ ID: NO: 66, SEQ ID: NO: 72, SEQ ID: NO: 78 or a homology with one of which is greater than 50% sequence.
  • the heavy chain CDR1 is SEQ ID NO: 1
  • the heavy chain CDR2 is SEQ ID NO: 2
  • the heavy chain CDR3 is SEQ ID NO: 3
  • the light chain CDR1 is SEQ ID NO: 4
  • the light chain CDR2 is SEQ ID NO: 5
  • light chain CDR3 is SEQ ID NO: 6
  • heavy chain CDR1 is SEQ ID NO: 7
  • heavy chain CDR2 is SEQ ID NO: 8
  • heavy chain CDR3 9, and light chain CDR1 is SEQ ID NO: 10
  • light chain CDR2 is SEQ ID NO: 11
  • light chain CDR3 is SEQ ID NO: 12
  • heavy chain CDR1 is SEQ ID NO: 13
  • heavy chain CDR2 is SEQ ID NO: 14
  • heavy chain CDR3 Is SEQ ID NO: 15, and light chain CDR1 is SEQ ID NO: 16, light chain CDR2 is SEQ ID NO: 17, light chain CDR3 is SEQ ID NO: 18; or heavy chain CDR1 is SEQ
  • the light chain CDR3 is SEQ ID NO: 30; or the heavy chain CDR1 is SEQ ID NO: 31, and the heavy chain CDR2 is SEQ I DNO: 32, heavy chain CDR3 is SEQ ID NO: 33, and light chain CDR1 is SEQ ID NO: 34, light chain CDR2 is SEQ ID NO: 35, light chain CDR3 is SEQ ID NO: 36; or heavy chain CDR1 Is SEQ ID NO: 37, heavy chain CDR2 is SEQ ID NO: 38, heavy chain CDR3 is SEQ ID NO: 39, and light chain CDR1 is SEQ ID NO: 40, light chain CDR2 is SEQ ID NO: 41, light chain CDR3 is SEQ ID NO: 42; or heavy chain CDR1 is SEQ ID NO: 43, heavy chain CDR2 is SEQ ID NO: 44, heavy chain CDR3 is SEQ ID NO: 45, and light chain CDR1 is SEQ ID NO: 46, Light chain CDR2 is SEQ ID NO: 47, light chain CDR3 is SEQ ID
  • the heavy chain variable region comprises the aforementioned heavy chain CDR1, CDR2 and CDR3; the light chain variable region comprises the aforementioned light chain CDR1, CDR2 and CDR3.
  • the amino acid sequence of the heavy chain variable region is selected from the group consisting of: SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85; SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID: NO: 91, SEQ ID: NO: 93, SEQ ID: NO: 95, SEQ ID: NO: 97, SEQ ID: NO: 99, SEQ ID: NO: 101 or SEQ ID: NO: 103;
  • the amino acid sequence of the light chain variable region is selected from the group consisting of: SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86; SEQ ID NO: 88, SEQ ID NO: 90, SEQ One of ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, or SEQ ID NO: 104.
  • the heavy chain variable region is SEQ ID NO: 79 and the light chain variable region is SEQ ID NO: 80; or
  • the heavy chain variable region is SEQ ID NO: 81 and the light chain variable region is SEQ ID NO: 82; or
  • the heavy chain variable region is SEQ ID NO: 83 and the light chain variable region is SEQ ID NO: 84; or
  • the heavy chain variable region is SEQ ID NO: 85 and the light chain variable region is SEQ ID NO: 86; or
  • the heavy chain variable region is SEQ ID NO: 87 and the light chain variable region is SEQ ID NO: 88; or
  • the heavy chain variable region is SEQ ID NO: 89 and the light chain variable region is SEQ ID NO: 90; or
  • the heavy chain variable region is SEQ ID NO: 91 and the light chain variable region is SEQ ID NO: 92; or
  • the heavy chain variable region is SEQ ID NO: 93 and the light chain variable region is SEQ ID NO: 94; or
  • the heavy chain variable region is SEQ ID NO: 95 and the light chain variable region is SEQ ID NO: 96; or
  • the heavy chain variable region is SEQ ID NO: 97 and the light chain variable region is SEQ ID NO: 98; or
  • the heavy chain variable region is SEQ ID NO: 99 and the light chain variable region is SEQ ID NO: 100; or
  • the heavy chain variable region is SEQ ID NO: 101 and the light chain variable region is SEQ ID NO: 102; or
  • the heavy chain variable region is SEQ ID NO: 103 and the light chain variable region is SEQ ID NO: 104.
  • the sequence of the amino acid in the variable region of the heavy chain is a similar sequence having at least 50% homology with the sequence a, wherein the sequence a is SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85; SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID One of NO: 101 or SEQ ID NO: 103.
  • the amino acid sequence of the light chain variable region is a similar sequence having at least 50% homology with sequence b, which is SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86; SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID Either NO: 102 or SEQ ID: NO: 104.
  • sequence b is SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86; SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID Either NO: 102 or SEQ ID: NO: 104.
  • the amino acid sequence of the heavy chain includes heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, and light chain CDR1, light chain CDR2, and light chain CDR3; heavy chain CDR1 is SEQ ID NO: 1, and heavy chain CDR2 is SEQ ID NO: 2, The heavy chain CDR3 is SEQ ID NO: 3, and the light chain CDR1 is SEQ ID NO: 4, the light chain CDR2 is SEQ ID NO: 5, and the light chain CDR3 is SEQ ID NO: 6; or the heavy chain CDR1 is SEQ ID NO: 7.
  • the heavy chain CDR2 is SEQ ID NO: 8
  • the heavy chain CDR3 is SEQ ID NO: 9
  • the light chain CDR1 is SEQ ID NO: 10
  • the light chain CDR2 is SEQ ID NO: 11
  • the light chain CDR3 is SEQ ID NO.
  • heavy chain CDR1 is SEQ ID NO: 13
  • heavy chain CDR2 is SEQ ID NO: 14
  • heavy chain CDR3 is SEQ ID NO: 15
  • light chain CDR1 is SEQ ID NO: 16
  • light chain CDR2 is SEQ ID NO: 17
  • light chain CDR3 is SEQ ID NO: 18
  • heavy chain CDR1 is SEQ ID NO: 19
  • heavy chain CDR2 is SEQ ID NO: 20
  • heavy chain CDR3 is SEQ ID NO: 21
  • light chain CDR2 is SEQ ID NO: 23
  • light chain CDR3 is SEQ ID NO: 24
  • heavy chain CDR1 is SEQ ID NO: 25
  • heavy chain CDR2 is SEQ ID NO: 26
  • the light chain CDR3 is SEQ ID NO: 30; or the heavy chain CDR1 is SEQ ID NO: 31; the heavy chain CDR2 is SEQ ID NO: 32, the heavy chain CDR3 is SEQ ID NO: 33, and the light chain CDR1 is SEQ ID NO: 34, light chain CDR2 is SEQ ID NO: 35, light chain CDR3 is SEQ ID NO: 36; or heavy chain CDR1 is SEQ ID NO: 37, heavy chain CDR2 is SEQ ID NO: 38, heavy chain CDR3 Is SEQ ID NO: 39, and light chain CDR1 is SEQ ID NO: 40, light chain CDR2 is SEQ ID NO: 41, light chain CDR3 is SEQ ID NO: 42, or heavy chain CDR1 is SEQ ID NO: 43, heavy Chain CDR2 is SEQ ID NO: 44, heavy chain CDR3 is SEQ ID NO: 45, and light chain CDR1 is SEQ ID NO: 46, light chain CDR2 is SEQ ID NO: 47, and light chain CDR3 is
  • the light chain CDR3 is SEQ ID NO: 54; or the heavy chain CDR1 is SEQ ID NO: 55, the heavy chain CDR2 is SEQ ID NO: 56, the heavy chain CDR3 is SEQ ID NO: 57, and the light chain CDR1 is SEQ ID NO: 58, light chain CDR2 is SEQ ID NO: 59, light chain CDR3 is SEQ ID NO: 60; or heavy chain CDR1 Is SEQ ID NO: 61, heavy chain CDR2 is SEQ ID NO: 62, heavy chain CDR3 is SEQ ID NO: 63, and light chain CDR1 is SEQ ID NO: 64, light chain CDR2 is SEQ ID NO: 65, light chain CDR3 is SEQ ID NO: 66; or heavy chain CDR1 is SEQ ID NO: 67, heavy chain CDR2 is SEQ ID NO: 68, heavy chain CDR3 is SEQ ID NO: 69, and light chain CDR1 is SEQ ID NO: 70, Light chain CDR2 is SEQ ID NO:
  • amino acid sequence of the antibody includes the heavy chain variable region and the light chain variable region described above.
  • a second object of the present invention is to provide a nucleic acid sequence of an anti-human claudin 18.2 monoclonal antibody, specifically as follows:
  • a nucleic acid molecule comprising a nucleic acid sequence capable of encoding a heavy chain CDR and / or a light chain CDR.
  • the heavy chain CDR1 is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 13, and SEQ ID NO: 19.SEQ ID NO: 25, SEQ ID NO: 31, SEQ ID NO: 37, SEQ ID NO: 43, SEQ ID NO: 49, SEQ ID NO: 55, SEQ ID NO: 61, SEQ ID NO: 67, One of SEQ ID NO: 73 or a similar sequence having a homology of greater than 50% with one of them;
  • the heavy chain CDR2 is selected from: SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 14, SEQ ID NO: 20, SEQ ID NO: 26, SEQ ID NO: 32, SEQ ID NO: 38, SEQ ID NO: 44, SEQ ID: NO: 50, SEQ ID: NO: 56, SEQ ID: NO: 62, SEQ ID: NO: 68, SEQ ID: NO: 74 or a homology with one of which is greater than 50% sequence;
  • the heavy chain CDR3 is selected from: SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 21, SEQ ID NO: 27, SEQ ID NO: 33, SEQ ID NO: 39, SEQ ID NO: 45, SEQ ID: NO: 51, SEQ ID: NO: 57, SEQ ID: NO: 63, SEQ ID: NO: 69, SEQ ID: NO: 75 or a homology with one of which is greater than 50% sequence;
  • the light chain CDR1 is selected from: SEQ ID NO: 4, SEQ ID NO: 10, SEQ ID NO: 16, SEQ ID NO: 22, SEQ ID NO: 28, SEQ ID NO: 34, SEQ ID NO: 40, SEQ ID NO: 46, SEQ ID: NO: 52, SEQ ID: NO: 58, SEQ ID: NO: 64, SEQ ID: NO: 70, SEQ ID: NO: 76 or a homology with one that is greater than 50% sequence;
  • the light chain CDR2 is selected from: SEQ ID NO: 5, SEQ ID NO: 11, SEQ ID NO: 17, SEQ ID NO: 23, SEQ ID NO: 29, SEQ ID NO: 35, SEQ ID NO: 41, SEQ ID NO: 47, SEQ ID: NO: 53, SEQ ID: NO: 59, SEQ ID: NO: 65, SEQ ID: NO: 71, SEQ ID: NO: 77 or a homology with one of which is greater than 50% sequence;
  • the light chain CDR3 is selected from: SEQ ID NO: 6, SEQ ID NO: 12, SEQ ID NO: 18, SEQ ID NO: 24, SEQ ID NO: 30, SEQ ID NO: 36, SEQ ID NO: 42, SEQ ID NO: 48, SEQ ID: NO: 54, SEQ ID: NO: 60, SEQ ID: NO: 66, SEQ ID: NO: 72, SEQ ID: NO: 78 or a homology with one of which is greater than 50% sequence.
  • a nucleic acid molecule characterized in that it contains a nucleic acid sequence capable of encoding a heavy chain variable region and / or a light chain variable region, and the amino acid sequence of the heavy chain variable region is selected from the group consisting of SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85; SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, or SEQ ID NO: 103;
  • the amino acid sequence of the light chain variable region is selected from the group consisting of: SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86; SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92.
  • SEQ ID NO: 80 SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86; SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92.
  • the present invention also provides a vector containing any of the nucleic acid molecules described above.
  • the present invention also provides a host cell containing the amino acid sequence, nucleic acid molecule, or vector described above.
  • the present invention also provides a conjugate containing the antibody according to any one of the above.
  • the present invention also provides a composition containing a main component and an auxiliary component, the main component containing the antibody or the nucleic acid molecule or the vector, the host cell, or the couple
  • auxiliary ingredients are selected from pharmaceutically acceptable carriers or excipients, and optionally other biologically active substances.
  • the other biologically active substances include, but are not limited to, other antibodies, fusion proteins, or drugs (for example, antitumor drugs such as radiotherapy and chemotherapy drugs).
  • the invention also provides the application of the above-mentioned antibodies, nucleic acid molecules, vectors, host cells, conjugates, and compositions in the preparation of drugs or detection reagents for treating diseases.
  • the disease is a malignant tumor, a cardiovascular disease or an inflammatory disease.
  • the present invention also provides a diagnostic reagent or kit containing the antibody according to any one of the above.
  • the diagnostic reagent or kit is used to diagnose a disease related to claudin 18.2 in vitro (e.g., a cell or tissue) or in vivo (e.g., a human or animal model).
  • antibody as referred to herein includes whole antibodies and any antigen-binding fragment (ie, "antigen-binding portion") or single chain thereof.
  • an “antibody” refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains linked to each other by a disulfide bond, or an antigen-binding portion thereof.
  • Each heavy chain consists of a heavy chain variable region and a heavy chain constant region.
  • homology refers to the sequence similarity between two polynucleotide sequences or between two polypeptide sequences at the most preferred alignment.
  • the invention discloses a monoclonal antibody and its application, and those skilled in the art can learn from the content of this article and appropriately improve the process parameters for implementation.
  • all similar replacements and modifications will be apparent to those skilled in the art, and they are all considered to be included in the present invention.
  • the method and application of the present invention have been described through the preferred embodiments. Obviously, relevant persons can obviously modify or appropriately modify and combine the methods and applications described herein without departing from the content and scope of the present invention to implement and apply the present invention. Invention technology.
  • the monoclonal antibody provided by the present invention and the raw materials and reagents used in the application can be purchased from the market.
  • the partial sequence of the first extracellular domain of human claudin 18.2 was constructed on the pGEX-KG vector (antigen sequence 1), and the constructed expression vector was transferred into E. coli strain TG1, and cultured with LB medium and induced with IPTG Method to express antigenic protein.
  • the collected E.coli TG1 bacterial cells were broken by a homogenizer, Glutathione Sepharose 4Fast Flow (GE, Cat # 17-5132-01) was used for affinity purification, and HiTrap Capto Q (GE, Cat # 11001302 )
  • the column was subjected to secondary purification and the protein was dialyzed into PBS, pH 7.4 to obtain an antigen protein with a purity of> 85%.
  • the partial or full-length sequence of the first extracellular domain of claudin 18.2 was constructed on the pcDNA3.1 vector as an immunogen (antigen sequence 2, antigen sequence 3).
  • the full-length sequence of human claudin 18.2 was cloned into an adenoviral vector, and the constructed expression vector was transferred into CHO-K1 cells using Lipofectamine TM 3000 Transfection Reagent (Thermo, Cat # L3000150) reagent, and 5 ⁇ g / mL puromycin (Gibco, Cat # 10131-027). After Q-PCR and FACS screening and verification, stable expression of CHO-K1_human claudin 18.2 cell line was obtained.
  • mice In order to obtain high-affinity and high-specificity antibodies against human claudin 18.2, a series of antigen combinations (DNA immunity, protein immunity, and cell line whole cell immunity) were designed in this experiment to immunize several Balb / c mice and C57BL / 6 cells. Mice are immunized using the classic immunization schedule, which triggers an immune response in mice. Mouse serum was collected, and GST-ECD protein cell line was used as an antigen. ELISA method or CHO-K1_human claudin 18.2 cell line was used for FACS to determine the serum titer. The corresponding mice with high titer were selected for fusion.
  • Antigen combinations DNA immunity, protein immunity, and cell line whole cell immunity
  • Electrofusion instrument fusion to ensure that the ratio of the number of splenocytes or lymph node cells to SP2 / 0 cells is between 10: 1 and 5: 1, and the number of splenocytes spread per well does not exceed 1x105, and the supernatant is harvested 7 days after fusion The medium was changed, and the collected supernatant was tested for cell binding activity by ELISA binding method and flow cytometry to obtain hybridoma cell lines that were positive in all three screening experiments.
  • the HEK293F_human claudin 18.1, HEK293F_human claudin 18.2 cell line and CHO-K1_human claudin 18.2 cell line were subjected to flow cytometry experiments with the hybridoma supernatant.
  • the human clone was specifically cloned as the target clone.
  • the primary antibody was the hybridoma supernatant, positive antibody, isotype control and PBS. After incubation with the primary antibody, the cells were washed, and Goat anti-Mouse IgG iFlour 647 (GenScript, 3 ⁇ g / ml) was used. Incubate as a secondary antibody and read the fluorescence signal by flow cytometry after washing.
  • RNA of hybridoma cells was extracted by Trizol (Ambion 15596-026) kit, and then the total RNA was reverse transcribed using Takara PrimeScript 1 st Strand cDNA synthesis kit to prepare cDNA, which were amplified using degenerate primers, respectively.
  • the heavy and light chain variable region sequences of an antibody was extracted by Trizol (Ambion 15596-026) kit, and then the total RNA was reverse transcribed using Takara PrimeScript 1 st Strand cDNA synthesis kit to prepare cDNA, which were amplified using degenerate primers, respectively.
  • the heavy and light chain variable region sequences of an antibody was extracted by Trizol (Ambion 15596-026) kit, and then the total RNA was reverse transcribed using Takara PrimeScript 1 st Strand cDNA synthesis kit to prepare cDNA, which were amplified using degenerate primers, respectively.
  • the heavy and light chain variable region sequences of an antibody was extracted by Trizol (Ambion 15596-0
  • the murine anti-light chain variable region sequence was spliced with human IgG kappa CL region sequence to obtain the full-length light chain sequence, which was constructed on the pcDNA3.1 vector containing the nitrogen-terminal signal peptide by whole gene synthesis to obtain a light chain expression vector.
  • the murine anti-heavy chain variable region sequence was spliced with the CH1, CH2 and CH3 region sequences of human IgG4S228P to obtain the full-length heavy chain sequence.
  • the heavy chain was constructed on the pcDNA3.1 vector containing the nitrogen terminal signal peptide. Expression vector.
  • HEK293F cells were transiently transfected by co-transfection of light and heavy chain expression vectors.
  • the 293F cells were cultured for 7 days after transfection, and the culture supernatant was collected. Then the chimeric antibody in the supernatant was purified by protein A affinity chromatography column and dialyzed into PBS pH 7.4. The concentration was determined by A280 method and analyzed by SDS-PAGE.
  • the primary antibody was purified human-mouse chimeric antibody, positive antibody (IMAB362 and Anti-Claudin18antibody [34H14L15] (abcam, Cat # ab203563)), isotype control and 1 % BSA in PBS, after primary antibody incubation, wash cells, and use Goat anti-human IgG Fc (FITC) (abcam, Cat # ab97224) and goat Anti-Rabbit IgG H & L (Alexa 488) (abcam, Cat # ab150077) was incubated as a secondary antibody, and the fluorescence signal was read by a flow cytometer after washing.
  • FITC Goat anti-human IgG Fc
  • FITC goat Anti-Rabbit IgG H & L
  • Alexa 488 abcam, Cat # ab150077
  • the primary antibody was human-mouse chimeric antibody, positive antibody (IMAB362), isotype control and 1% BSA in PBS. After the primary antibody was incubated, the cells were washed, and Goat anti-human IgG Fc (FITC) (abcam, Cat # ab97224) was incubated as a secondary antibody, and the fluorescence signal was read by flow cytometry after washing.
  • IMAB362 human-mouse chimeric antibody
  • FITC Goat anti-human IgG Fc
  • Specific amino acids in the region were restored for mutation, and then the CDR region sequence of the mouse antibody was grafted with the framework region sequence of the selected human germline gene, and the light and heavy chain variable region sequences of the humanized antibody were synthesized by gene synthesis after splicing
  • the expression vectors were constructed on the basis of human IgG4 heavy chain Fc and human IgG kappa CL.
  • the constructed humanized antibody light and heavy chain vectors were used for transient expression of HEK293F in one-to-one pair. After affinity purification and dialysis of antibodies in the culture supernatant of transient cells, the concentrations were determined by A280 method and analyzed by SDS-PAGE. After purity and purity, the affinity with HEK293F_human claudin 18.2 cell line was determined by FACS method.
  • the HEK293F_human claudin 18.2 cell line was used to determine the humanized antibody of the invention after FPLC purification to mediate complement-dependent cytotoxicity.
  • Effector cells PBMC are derived from fresh blood samples provided by healthy blood donors. Fresh blood is diluted 4 times with DPBS (Gibco, 14190-144), and Ficoll-Paque Plus (GE, Healthcare) density gradient centrifugation method to extract effector cell PBMC.
  • DPBS Gibco, 14190-144
  • Ficoll-Paque Plus GE, Healthcare
  • the medium (RPMI1640medium (Gibco, 11835-030) + 1% FBS (Gibco, 10099-141)) was used to dilute the concentration of the antibody to be detected and the control antibody to a final concentration of 10ug / ml, and then diluted 5 times in 8 gradients.
  • RPMI1640medium Gibco, 11835-030
  • FBS Gibco, 10099-141
  • Into a 96-well cell U-shaped culture plate (Corning, 3799).
  • HEK293F_human claudin 18.2 cell line stably expressing CLDN18A2 was used as a target cell.
  • the medium (RPMI1640medium (Gibco, 11835-030) + 1% FBS (Gibco, 10099-141)) was used to dilute the concentration of the antibody to be detected and the control antibody to a final concentration of 50ug / mL, and then diluted 5 times in 8 gradients.
  • Complement Quidel, A113 was diluted to a final concentration of 1:50 (V / V). Add 40 ⁇ l / well of pre-diluted antibody to a 96-well cell culture plate (Corning, 3917).
  • the epitope mapping method of the antigen recognized by the antibody in this invention is performed according to the "epitope mapping method" in “Methods of Molecular Biology” by Johan Rockberg (ISBN978-1-4939-7839-7).
  • the peptides of the epitope-mapped overlapping peptide library were designed according to the antigen sequence and synthesized in GenScript.
  • the peptides were coated with a high affinity ELISA plate, and first buffered with Na2CO 3 / NaHCO 3 , pH 9.59.
  • the peptide was diluted with solution and coated at 37 ° C for about 2 hours.
  • the coating solution in the well was discarded, and then the peptide was diluted with coating buffer with PBS, pH 7.4 into the well, and then coated twice at 37 ° C for about 2 hours.
  • the coating solution in the well was discarded, and finally the polypeptide diluted with ddH 2 O was added to the well and coated for a third time at 4 ° C. overnight.
  • the species-binding properties of the humanized antibody were detected by flow cytometry according to the method in Example 4.
  • the pcDNA3.1 (+) vector was used to construct CLDN18A2 expression vectors for mice, rats, rabbits and orangutans.
  • the expression vectors were transiently transfected into HEK293F cells using ExpiFectamine TM 293 Transfection Kit (gibico, Cat # A14524). Samples were taken for measurement 48 hours after dyeing. Blank HEK293F cells were used as negative controls, and HEK293F cell lines stably expressing human CLDN18A1 and human CLDN18A2 were used as positive controls for flow cytometry analysis.
  • the primary antibody was the humanized antibody of the present invention purified by FPLC, and the positive antibody (IMAB362 and Anti-Claudin18 antibody [34H14L15] (abcam, Cat # ab203563)), Isotype control and 1% BSA in PBS, after primary antibody incubation, cells were washed, and Goat anti-human IgG Fc (FITC) (abcam, Cat # ab97224) and goat Anti-Rabbit IgG H & L (Alexa 488) (abcam, Cat # ab150077) was incubated as a secondary antibody, and the fluorescence signal was read by a flow cytometer after washing.
  • FITC Goat anti-human IgG Fc
  • FITC abcam, Cat # ab97224
  • goat Anti-Rabbit IgG H & L Alexa 488)
  • a HEK293F_human claudin 18.2 cell line stably expressing CLDN18A2 was used as a detection cell.
  • the medium FreeStyle TM 293 Expression Medium (Gibco, 12338-018)) was used to dilute the concentration of the antibody to be detected and the isotype control antibody to a final concentration of 15 ug / ml, and then diluted 8.16 times by 8 gradients.
  • a HEK293F_human claudin 18.2 cell line stably expressing CLDN18A2 was used as a detection cell. Dilute the concentration of the antibody to be detected and the isotype control antibody to 40 nM using a medium (FreeStyle TM 293 Expression Medium (Gibco, 12338-018)), 120ul Fab-ZAP (Advanced Targeting Systems, IT-51) + 120uL antibody or Saporin (Advanced Targeting Systems, PR-1), mix, incubate at 37 ° C, 5% CO2 for 1 hour, and then dilute 8 gradients by 2.5 times. Add 50 ⁇ l / well of pre-diluted antibody to a 96-well cell culture plate (Corning, 3610).
  • mice Fifty female BALB / c nude mice were inoculated with human gastric cancer cell NUGC-4 subcutaneously in the right armpit.
  • 36 tumor-bearing mice with a tumor volume of 37.54 to 124.64 mm3 were selected and randomly divided into 6 groups: 1- negative control group, 2- positive control group (0.2mg / head), 3-RB0011-FK-13 group (0.2mg / head), 4-RB0011-FK-1 group (0.2mg / head), 5- In the RB0011-FK-2 group (0.2 mg / head) and 6-186CG4-mut group (0.2 mg / head), 6 animals in each group were administered twice a week after grouping for a total of 4 weeks by intravenous injection.
  • Tumor proliferation rate T / C%.
  • the two humanized antibodies and the HEK293F_human claudin 18.2 cell line showed ADCC effects, with EC50s of 0.019 ⁇ g / ml and 0.003 ⁇ g / ml, respectively. See attached picture 1.
  • the two humanized antibodies and the HEK293F_human claudin 18.2 cell line did not show a significant CDC effect, see FIG. 2.
  • the two humanized antibodies did not show a significant proliferation inhibitory effect on the HEK293F_human claudin 18.2 cell line. See Figure 3.
  • This experiment successfully established a subcutaneous transplanted tumor model of human gastric cancer NUGC-4 in BALB / c nude mice.
  • positive control substances test substances RB0011-FK-13, RB0011-FK-1, RB0011-FK-2, and 186CG4-mut were administered at a dose of 2 mg / head twice a week for a total intravenous administration.
  • the drug had inhibitory effects on the growth of human gastric cancer NUGC-4 BALB / c subcutaneously transplanted tumors in nude mice for 4 weeks. See Figure 5 and Figure 6.

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Abstract

L'invention concerne un anticorps monoclonal anti-claudine 18,2 humaine et son utilisation. L'anticorps possède trois régions déterminant la complémentarité de chaîne lourde (chaîne lourde CDR1, chaîne lourde CDR2 et chaîne lourde CDR3) et trois régions déterminant la complémentarité de chaîne légère (chaîne légère CDR1, chaîne légère CDR2 et chaîne légère CDR3).
PCT/CN2019/101785 2018-08-22 2019-08-21 Anticorps monoclonal anti-claudine 18,2 humaine et son utilisation WO2020038404A1 (fr)

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WO2022122709A1 (fr) 2020-12-07 2022-06-16 Sotio Biotech A.S. Conjugués anticorps-médicament à base d'anticorps cldn18.2 humanisés
WO2022136642A1 (fr) 2020-12-23 2022-06-30 Sotio Biotech A.S. Conjugués anticorps-médicament anti-claudine 18.2 spécifiques d'une tumeur
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WO2022122709A1 (fr) 2020-12-07 2022-06-16 Sotio Biotech A.S. Conjugués anticorps-médicament à base d'anticorps cldn18.2 humanisés
WO2022136642A1 (fr) 2020-12-23 2022-06-30 Sotio Biotech A.S. Conjugués anticorps-médicament anti-claudine 18.2 spécifiques d'une tumeur
WO2022253156A1 (fr) * 2021-05-31 2022-12-08 Shijiazhuang Yiling Pharmaceutical Co., Ltd. Anticorps monoclonaux contre cldn18.2 et versions à région fc modifiée de ceux-ci

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