WO2023045997A1 - Anticorps anti-claudine 18.2 et son utilisation - Google Patents

Anticorps anti-claudine 18.2 et son utilisation Download PDF

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WO2023045997A1
WO2023045997A1 PCT/CN2022/120428 CN2022120428W WO2023045997A1 WO 2023045997 A1 WO2023045997 A1 WO 2023045997A1 CN 2022120428 W CN2022120428 W CN 2022120428W WO 2023045997 A1 WO2023045997 A1 WO 2023045997A1
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seq
amino acid
antibody
acid sequence
variable region
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Chinese (zh)
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周金花
秦春铃
吴崇兵
朱彩琳
姜晓玲
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盛禾(中国)生物制药有限公司
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Priority to CN202280052157.1A priority Critical patent/CN117897403A/zh
Publication of WO2023045997A1 publication Critical patent/WO2023045997A1/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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells

Definitions

  • the invention belongs to the field of biomedicine, and in particular relates to an anti-Claudin18.2 antibody and its application.
  • Intercellular tight junction is a kind of transmembrane protein complex.
  • the stability of tight junction needs the coordinated activities of several different proteins to maintain, and Claudin protein is the main protein that ensures the specificity of tight junction permeability. So far, 24 Claudin family members have been found in mammals, and each protein is specifically expressed in a certain tissue.
  • the extracellular loop structure of the claudin protein connects to this structure of adjacent cells, bridging the cell layer and regulating the parallel traffic between the compartment and the basal layer.
  • Claudin18 protein includes four transmembrane regions and two extracellular loops. Its N-terminal and C-terminal are in the cytoplasm. The molecular weight of the protein is about 26KD.
  • the Claudin protein can be transformed into Claudin with different characteristics by selective cleavage. Isotypes: Claudin18.1 and Claudin18.2. Although there is only eight amino acid differences between the first extracellular domain of Claudin18.1 and Claudin18.2, the expression distribution is different.
  • Claudin18.1 is selectively expressed in normal lung and gastric epithelium, while Claudin18.2 is only expressed in Expressed on transiently differentiated gastric epithelial cells, completely undetectable in any other normal human organ, but Claudin18.2 is significantly upregulated in a variety of malignant tumors, including 80% of gastrointestinal adenomas, 60% of pancreatic tumors , 30% esophageal cancer and 25% non-small cell lung cancer. In tumors, the tight junctions between cells are disrupted, and Claudin18.2 cannot perform its normal function. Therefore, Claudin18.2 is a suitable target for tumor therapy.
  • IMAB362 also known as IMAB362
  • IgG1 chimeric antibody against Claudin18.2 recognizes the first extracellular domain (ECD1) of Claudin18.2 with high affinity and specificity.
  • ECD1 extracellular domain
  • IMAB362 mediates cell killing via ADCC, CDC, induction of apoptosis via target cross-linking on the surface of tumor cells, and proliferation.
  • ADCC extracellular domain
  • CDC induction of apoptosis via target cross-linking on the surface of tumor cells
  • IMAB362 efficiently lyses Claudin18.2-positive cells, including human gastric cancer cell lines in vitro and in vivo. Mice with Claudin18.2-positive cancer cell lines had a survival benefit, and when treated with IMAB362, up to 40% of mice showed regression of their tumors.
  • Gastric cancer and gastroesophageal junction (GEJ) adenocarcinoma is one of the most unmet medical needs of malignant tumors, and gastric cancer is also the third leading cause of cancer death worldwide. Therefore, the development of anti-Claudin18.2 antibodies and other drugs such as multifunctional and bifunctional antibodies has great significance for the treatment of some solid tumors, especially stomach-related tumors. However, there is currently no corresponding antibody developed and marketed.
  • the inventors developed an anti-Claudin18.2 antibody with good performance, which does not bind to Claudin18.1, but can bind to Claudin18.2 with high affinity and specificity, and mediate Killing of Claudin 18.2 expressing target cells (eg tumor cells) by effector cells.
  • target cells eg tumor cells
  • the present invention provides an anti-Claudin18.2 antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region comprises heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3,
  • the light chain variable region comprises light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein,
  • HCDR1 of the heavy chain variable region is selected from any amino acid sequence of SEQ ID NO: 1, 9, 15, 19, or has at least one amino acid sequence with any amino acid sequence of SEQ ID NO: 1, 9, 15, 19 A sequence that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical, or to SEQ ID NO: 1, Amino acid sequences with one or more (preferably 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to any amino acid sequence of 9, 15 and 19;
  • HCDR2 of the heavy chain variable region selected from any amino acid sequence of SEQ ID NO: 2, 10, 16, 20, 21, 22, 23, 24, 25, or with SEQ ID NO: 2, 10, Any amino acid sequence of 16, 20, 21, 22, 23, 24, 25 has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, A sequence of 98%, 99% or more identity, or one or more (preferably 2 or 3) amino acid sequences of conservative amino acid mutations (preferably substitutions, insertions or deletions);
  • HCDR3 of the heavy chain variable region selected from any amino acid sequence of SEQ ID NO: 3, 11, 17, 26, 27, 63, or with SEQ ID NO: 3, 11, 17, 26, 27, Any amino acid sequence of 63 is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical, Or an amino acid having one or more (preferably 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to any amino acid sequence of SEQ ID NO: 3, 11, 17, 26, 27, 63 sequence;
  • LCDR1 of the light chain variable region is selected from any amino acid sequence of SEQ ID NO: 4, 28, or has at least 80%, 85%, 90% of any amino acid sequence of SEQ ID NO: 4, 28 , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical sequences, or compared with any amino acid sequence of SEQ ID NO: 4, 28 Amino acid sequence with one or more (preferably 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions);
  • LCDR2 of the light chain variable region is selected from any amino acid sequence of SEQ ID NO: 5, or has at least 80%, 85%, 90%, 91%, any amino acid sequence with any amino acid sequence of SEQ ID NO: 5 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical sequences, or have one or more ( Amino acid sequence of preferably 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions); and/or
  • LCDR3 of the light chain variable region is selected from any amino acid sequence of SEQ ID NO: 6, 12, or has at least 80%, 85%, 90% of any amino acid sequence of SEQ ID NO: 6, 12 , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical sequences, or compared with any amino acid sequence of SEQ ID NO: 6, 12
  • An amino acid sequence with one or more (preferably 2 or 3) conservative amino acid mutations preferably substitutions, insertions or deletions).
  • the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region, and the LCDR1, LCDR2, and LCDR3 of the light chain variable region are selected from any amino acid sequence in the following (1)-(11):
  • HCDR1 shown in SEQ ID NO: 1 HCDR2 shown in SEQ ID NO: 2
  • HCDR3 shown in SEQ ID NO: 3 LCDR1 shown in SEQ ID NO: 4, shown in SEQ ID NO: 5
  • LCDR2 shown in SEQ ID NO: 6 LCDR3 shown in SEQ ID NO: 6;
  • HCDR1 shown in SEQ ID NO: 19 HCDR2 shown in SEQ ID NO: 20, HCDR3 shown in SEQ ID NO: 63, LCDR1 shown in SEQ ID NO: 28, shown in SEQ ID NO: 5 LCDR2, LCDR3 shown in SEQ ID NO: 6;
  • HCDR1 shown in SEQ ID NO: 19 HCDR2 shown in SEQ ID NO: 21, HCDR3 shown in SEQ ID NO: 63, LCDR1 shown in SEQ ID NO: 28, shown in SEQ ID NO: 5 LCDR2, LCDR3 shown in SEQ ID NO: 6;
  • HCDR1 shown in SEQ ID NO: 19 HCDR2 shown in SEQ ID NO: 22, HCDR3 shown in SEQ ID NO: 26, LCDR1 shown in SEQ ID NO: 28, shown in SEQ ID NO: 5 LCDR2, LCDR3 shown in SEQ ID NO: 6;
  • HCDR1 shown in SEQ ID NO: 19 HCDR2 shown in SEQ ID NO: 23, HCDR3 shown in SEQ ID NO: 63, LCDR1 shown in SEQ ID NO: 28, shown in SEQ ID NO: 5 LCDR2, LCDR3 shown in SEQ ID NO: 6;
  • HCDR1 shown in SEQ ID NO: 19 HCDR2 shown in SEQ ID NO: 24, HCDR3 shown in SEQ ID NO: 63, LCDR1 shown in SEQ ID NO: 28, shown in SEQ ID NO: 5 LCDR2, LCDR3 shown in SEQ ID NO: 6;
  • HCDR1 shown in SEQ ID NO: 19 HCDR2 shown in SEQ ID NO: 25, HCDR3 shown in SEQ ID NO: 63, LCDR1 shown in SEQ ID NO: 28, shown in SEQ ID NO: 5 LCDR2, LCDR3 shown in SEQ ID NO: 6;
  • HCDR1 shown in SEQ ID NO: 19 HCDR2 shown in SEQ ID NO: 23, HCDR3 shown in SEQ ID NO: 27, LCDR1 shown in SEQ ID NO: 28, shown in SEQ ID NO: 5 LCDR2, LCDR3 shown in SEQ ID NO: 6;
  • HCDR1 shown in SEQ ID NO: 19 HCDR2 shown in SEQ ID NO: 24, HCDR3 shown in SEQ ID NO: 27, LCDR1 shown in SEQ ID NO: 28, shown in SEQ ID NO: 5 LCDR2, LCDR3 shown in SEQ ID NO:6.
  • the present invention also provides an anti-Claudin18.2 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein:
  • said heavy chain variable region has any amino acid sequence given by SEQ ID NO: 7, 13, 18, 29, 30, 31, 33, 34, 35, 36, 37,
  • amino acid sequences of conservative amino acid mutations preferably substitutions, insertions or deletions
  • said light chain variable region has any amino acid sequence given by SEQ ID NO: 8, 14, 32, 38;
  • the heavy chain variable region and the light chain variable region are selected from any amino acid sequence in the following (1)-(11):
  • the antibody is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.
  • the antibody is a monoclonal antibody.
  • the anti-Claudin18.2 antibody or antigen-binding fragment thereof further comprises an Fc region selected from mouse IgG1, IgG2a, IgG2b and/or IgG3, or selected from rat IgG1, IgG2a , IgG2b and/or IgG2c.
  • the anti-Claudin18.2 antibody or antigen-binding fragment thereof further comprises an Fc region selected from human IgG1, IgG2, IgG3 and/or IgG4.
  • the present invention also provides a nucleic acid molecule encoding the anti-Claudin18.2 antibody or antigen-binding fragment thereof described in any one of the above.
  • the present invention also provides a multifunctional fusion protein comprising the anti-Claudin18.2 antibody or antigen-binding fragment thereof described in any one of the above.
  • the anti-Claudin18.2 antibody or antigen-binding fragment thereof further comprises one or more second antibodies or antigen-binding portions thereof that specifically bind to other antigens.
  • the antigen that binds the second antibody or antigen-binding portion thereof is selected from tumor-associated antigens (TAAs) or immune checkpoints.
  • TAAs tumor-associated antigens
  • immune checkpoints immune checkpoints
  • the immune checkpoint is PD-L1, CTLA4, PD-L2, PD-1, 4-1BB, CD47, TIGIT, GITR, TIM3, ILT4, TNFR2, TREM2, LAG3, CD27, B7H3, or B7H4.
  • cytokines are also included.
  • the cytokine is selected from IL-1, IL-2, IL-2R ⁇ , IL-2R ⁇ , IL-3, IL-3R ⁇ , IL-4, IL-4R ⁇ , IL-5, IL- 5R ⁇ , IL-6, IL-6R ⁇ , IL-7, IL-7R ⁇ , IL-8, IL-9, IL-9R ⁇ , IL-10, IL-10R1, IL-10R2, IL-11, IL-11R ⁇ , IL-12, IL-12R ⁇ , IL-12R ⁇ 2, IL-12R ⁇ 1, IL-13, IL-13R ⁇ , IL-13R ⁇ 2, IL-14, IL-15, IL-15R ⁇ sushi, IL-16, IL-17, IL- 18.
  • IL-19 IL-20, IL-20R1, IL-20R2, IL-21, IL-21R ⁇ , IL-22, IL-23, IL-23R, IL-27R, IL-31R, TGF, VEGF, IFN ⁇ , IFN ⁇ or GM-CSF.
  • the present invention also provides the use of any one of the anti-Claudin18.2 antibodies or antigen-binding fragments thereof or any one of the multifunctional fusion proteins described above in the preparation of drugs for treating cancer.
  • the cancer is lung cancer, liver cancer, melanoma, glioblastoma, head and neck cancer, esophageal cancer, gastric cancer, prostate cancer, ovarian cancer, bladder cancer, pancreatic cancer, gastric cancer, colon cancer, cervical cancer, or associated tumors.
  • the use is achieved by one or more of tumor immunotherapy, cell therapy, and gene therapy.
  • the present invention also provides a pharmaceutical composition, which comprises the anti-Claudin18.2 antibody or antigen-binding fragment thereof described in any one of the above and a pharmaceutically acceptable carrier, diluent or excipient.
  • the present invention also provides a pharmaceutical composition, which comprises any one of the above-mentioned multifunctional fusion proteins and a pharmaceutically acceptable carrier, diluent or excipient.
  • mAb monoclonal antibody
  • VH Antibody heavy chain variable region
  • VL antibody light chain variable region
  • FR Antibody framework region, that is, the amino acid residues in the antibody variable region except CDR residues
  • IgG Immunoglobulin G
  • antibody refers to natural immunoglobulins or immunoglobulins prepared by partial or complete synthesis. Antibodies can be reconstituted and isolated from natural resources such as plasma or serum where the antibodies naturally exist, or culture supernatants of hybridoma cells producing antibodies, animal immune serum, or phage library screening. Alternatively, it can be partially or completely synthesized by using a technique of gene recombination or the like. Preferred antibodies include, for example, antibodies to immunoglobulin isotypes or subclasses of these isotypes. Known human immunoglobulins include nine classes (isotypes) of IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, and IgM. Among these isotypes, antibodies of the invention may comprise IgG1, IgG2, IgG3 and/or IgG4.
  • the term “monoclonal antibody” refers to a homogeneous antibody directed only against a specific antigenic epitope. In contrast to common polyclonal antibody preparations, which typically include different antibodies directed against different antigenic determinants (epitopes), each monoclonal antibody is directed against a single antigenic determinant on the antigen.
  • the modifier "monoclonal” indicates the homogeneous character of the antibody and is not to be construed as requiring that the antibody be produced by any particular method.
  • the monoclonal antibodies of the invention are preferably produced by recombinant DNA methods, or obtained by screening methods as described elsewhere herein.
  • murine antibody in the present invention refers to a monoclonal antibody prepared according to the knowledge and skills in the art. In preparation, test subjects are injected with the antigen, and hybridomas expressing antibodies having the desired sequence or functional properties are isolated.
  • chimeric antibody is an antibody that fuses the variable region of a murine antibody with the constant region of a human antibody, which can reduce the immune response induced by the murine antibody.
  • To establish a chimeric antibody it is necessary to first establish a hybridoma that secretes a mouse-derived specific monoclonal antibody, then clone the variable region gene from the mouse hybridoma cell, and then clone the constant region gene of the human antibody as required, and then clone the variable region gene of the mouse
  • the gene is connected with the human constant region gene to form a chimeric gene and then inserted into a human vector, and finally the chimeric antibody molecule is expressed in a eukaryotic industrial system or a prokaryotic industrial system.
  • humanized antibody also known as CDR-grafted antibody, refers to an antibody produced by grafting mouse CDR sequences into human antibody variable region frameworks, that is, different types of human germline antibody framework sequences. It can overcome the strong heterologous reaction induced by chimeric antibodies due to carrying a large number of mouse protein components.
  • Partial antibodies as used herein are immunoglobulin molecules composed of two pairs of polypeptide chains, each pair having one light chain (LC) and one heavy chain (HC).
  • Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • the heavy chain constant region consists of 3 domains (CH1, CH2 and CH3).
  • Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL), or only a light chain constant region (CL).
  • the light chain constant region consists of one domain, CL.
  • the constant domains are not directly involved in antibody-antigen binding, but exhibit various effector functions, such as mediating immunoglobulin interactions with host tissues or factors, including various cells of the immune system (e.g., effector cells) and classical complement Binding of the first component (C1q) of the system.
  • the VH and VL regions can also be subdivided into regions of high variability called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and VL is composed of 3 CDRs and 4 FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxy terminus.
  • the variable regions (VH and VL) of each heavy chain/light chain pair form the antigen binding site, respectively.
  • antigen-binding fragment of an antibody refers to a polypeptide fragment of an antibody, such as a polypeptide fragment of a full-length antibody, that retains the ability to specifically bind to the same antigen to which the full-length antibody binds, and/or competes with the full-length antibody for binding of the antigen. specifically binds, which is also referred to as an "antigen-binding moiety".
  • Antigen-binding fragments of antibodies can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies.
  • Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb and complementarity determining region (CDR) fragments, single chain antibodies (e.g., scFv), chimeric antibodies, diabodies (diabody), linear antibody (linear antibody), nanobody (such as technology from Ablynx), domain antibody (such as technology from Domantis), and such polypeptides, which comprise at least a portion of an antibody sufficient to confer polypeptide-specific antigen-binding capacity .
  • CDR complementarity determining region
  • polypeptide refers to a chain of amino acids of any length, regardless of modifications such as phosphorylation or glycosylation.
  • polypeptide includes proteins and fragments thereof.
  • Polypeptides may be "exogenous” in the sense that they are “heterologous”, ie, foreign to the host cell utilized, eg, human polypeptides produced by bacterial cells.
  • a polypeptide is disclosed herein as a sequence of amino acid residues. Those sequences are written left to right in amino-terminal to carboxy-terminal orientation.
  • amino acid residue sequences are designated by three-letter or one-letter codes, as follows: alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), asparagine Aspartic acid (Asp, D), cysteine (Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), Isoleucine (Ile, I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F) , proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y) and valine (Val, V).
  • host cell refers to a cell that has been or can be transformed with a nucleic acid sequence and thereby express a selected gene of interest.
  • the term includes progeny of a parental cell, whether or not the progeny is morphologically or genetically identical to the original parental cell, as long as the progeny has the selected gene of interest.
  • Commonly used host cells include bacteria, yeast, mammalian cells, and the like.
  • vector refers to a nucleic acid molecule capable of multiplying another nucleic acid to which it has been linked.
  • the term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked, and are referred to herein as "expression vectors.”
  • pharmaceutically acceptable carrier includes any standard pharmaceutical carrier, such as phosphate buffered saline, water, and emulsions, as well as various types of wetting agents.
  • percent (%) amino acid sequence identity is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in a reference polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to obtain the maximum percent sequence identity . Alignment for purposes of determining percent amino acid sequence identity can be performed in various ways that are within the skill in the art, for example, using publicly available computer software such as the BLAST software or the FASTA program package.
  • the CDR of the anti-Claudin18.2 antibody of the present invention refers to the hypervariable region of the heavy chain and light chain of the immunoglobulin, wherein, SEQ ID NO: 1, 2, 3, 4, 5, 6, 9, 10, 11, 12
  • SEQ ID NO: 1, 2, 3, 4, 5, 6, 9, 10, 11, 12 The CDR amino acid positions of , 15, 16, and 17 are defined according to Kabat, and the CDR amino acid positions of SEQ ID NO: 19, 20, 21, 22, 23, 24, 25, 26, 27, and 63 are defined according to AbM.
  • the term "specific" means that one of the molecules involved in specific binding does not show any significant binding to a molecule or molecules other than the binding partner molecules.
  • the term is used when a domain comprising an antibody variable region is specific for a particular epitope among epitopes in an antigen.
  • an antigen-binding molecule comprising an antibody variable region-containing domain can bind various antigens having the epitope.
  • epitope refers to an antigenic determinant in an antigen, and refers to an antigenic site to which a domain of an antigen-binding molecule comprising an antibody variable region disclosed in this specification binds. Therefore, an epitope can be defined according to its structure. In addition, the epitope can also be defined based on the antigen-binding activity of the antigen-binding molecule that recognizes the epitope. When the antigen is a peptide or polypeptide, the epitope can be specified by the amino acid residues forming the epitope; when the epitope is a sugar chain, the epitope can be determined by its specific sugar chain structure.
  • positive control antibody refers to natural or engineered cells capable of binding to or expressing a target protein.
  • isotype control refers to the use of the same species source, the same subtype, the same dose, the same immunoglobulin and subtype of immunoglobulin, the same marker, etc. as the experimental sample in the same experiment, used in the elimination experiment.
  • the effect of the experimental background of the non-specific binding sample on the experimental value is used as a negative control to illustrate the experimental effect.
  • the murine, chimeric or humanized monoclonal antibody combined with Claudin18.2 provided by the present invention has higher Claudin18.2 binding activity, ADCC activity and CDC activity than the prior art antibody.
  • the anti-Claudin18.2 antibody of the present invention is humanized, and the modified antibody has Claudin18.2 binding activity equivalent to that of the original antibody, and its affinity is relatively stable, which can ensure the specificity of the antibody. Its CDC activity is comparable to that of the original antibody. Also basically the same.
  • Figure 1 shows the binding activity of murine antibodies 1, 2, and 3 to human Claudin18.2-PANC1.
  • Figure 2 shows the binding activity of chimeric antibodies 4 and 5 to human Claudin18.2-CHOK1.
  • Figure 3 shows the binding activity of chimeric antibody 6 to human Claudin18.2-CHOK1.
  • Figure 4 shows the binding activity of chimeric antibodies 4 and 5 to human Claudin18.2-PANC1.
  • Figure 5 shows the binding activity of humanized antibodies 7, 8, and 9 to human Claudin18.2-CHOK1.
  • Fig. 6 shows the binding activity of humanized antibody 10 to human Claudin18.2-CHOK1.
  • Fig. 7 shows the binding activity of humanized antibodies 11 and 12 to human Claudin18.2-CHOK1.
  • Figure 8 shows the CDC activity of murine antibodies 1, 2, and 3 against human Claudin18.2-CHOK1.
  • Figure 9 shows the CDC activity of chimeric antibodies 4 and 5 against human Claudin18.2-CHOK1.
  • Figure 10 shows the CDC activity of chimeric antibody 6 against human Claudin18.2-CHOK1.
  • Figure 11 shows the CDC activity of humanized antibody 10 against human Claudin18.2-CHOK1.
  • Figure 12 shows the CDC activity of humanized antibodies 11 and 12 against human Claudin18.2-CHOK1.
  • Figure 13 shows the ADCC activity of chimeric antibodies 4 and 5 against human Claudin18.2-PANC-1.
  • Figure 14 shows the ADCC activity of chimeric antibody 6 against human Claudin18.2-PANC-1.
  • Figure 15 shows the binding activity of chimeric antibodies A, B, C, D, E, F, G to human Claudin18.2-CHOK1.
  • Figure 16 shows the binding activity of humanized antibodies H, I, J, K, L, M, N, O, P, Q, R to human Claudin18.2-CHOK1, with Z1 as the control antibody.
  • Figure 17 shows the binding activity of humanized antibodies H, I, J, K, L, M, N, O, P, Q, R to human Claudin18.2-CHOK1, using IMAB362 as a control antibody.
  • Figure 18 is the CDC activity of humanized antibodies H, J, L on human Claudin18.2-CHOK1.
  • 293 cells overexpressing human Claudin18.2 were used as an immunogen to immunize Balb/c mice for 3-4 times, one week after each immunization with CHOK1 and cells coated with CHOK1 overexpressing human Claudin18.2 -ELISA to detect serum titer. After the titer reached the requirement for fusion, the cells were used for booster immunization 2 weeks after the last immunization.
  • Antibody 12 SEQ ID NO: 50 SEQ ID NO: 49
  • the control antibody of antibody 1-6 is the FUT8 gene silenced IMAB362 obtained by the sugar-knocking process described in patent application 202110752316.6, and the control antibody of humanized antibody 7-12 is IMAB362
  • the isotype control antibody start at 10ug/ml, dilute 3 times, perform 7 serial dilutions, set 2 parallel wells, 50ul/well.
  • NK cell count of the target cells Take out the NK cell count of the target cells, and set the number of cells to 5 ⁇ 10 4 /50ul/well. Centrifuge at 1000rpm for 4min. Incubate at 37°C for 3.25h. Set ST well (only target cells), SE well (only effector cells), BV well (200ul dilution), BM well (200ul dilution) and M well (50ul effector cells + 150ul dilution). Add 20ul of lysate to the BM and BV wells, and incubate at 37°C for 45min.
  • the results of ADCC activity showed that chimeric antibodies 4, 5, and 6 had similar inhibitory effects on ADCC activity in vitro as the control antibody.
  • the control antibody is FUT8 gene-silenced IMAB362 obtained according to the sugar knockout process described in patent application 202110752316.6. Compared with IMAB362 without FUT8 gene knockout, the ADCC effect of FUT8 gene-silenced IMAB362 is increased by 5 times.
  • the in vitro ADCC activity inhibitory effects of chimeric antibodies 4, 5, and 6 are equivalent to those of the control antibody. It can be seen that the in vitro ADCC activity inhibitory effects of chimeric antibodies 4, 5, and 6 significantly.
  • the binding activity of chimeric antibodies A, B, C, D, E to human Claudin18.2-CHOK1 was basically the same as that of the control antibody to human Claudin18.2-CHOK1, and the binding activity of chimeric antibody F and chimeric antibody G to human The binding activity of Claudin18.2-CHOK1 was lower than that of the control antibody to human Claudin18.2-CHOK1.
  • the binding activity of humanized anti-H, I, J, K, L, M, N, O, P, Q, R to human Claudin18.2-CHOK1 and the binding activity of control antibody Z1 to human Claudin18.2-CHOK1 Basically the same;
  • the binding activity of humanized antibodies H, I, J, K, L, M, N, O, P, Q, R to human Claudin18.2-CHOK1 is the same as that of the control antibody IMAB362 to human Claudin18.2-CHOK1
  • the combined activity effect is basically the same.

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Abstract

La présente invention concerne un anticorps anti-claudine 18.2 ou un fragment de liaison à l'antigène de celui-ci et son utilisation. La présente invention concerne également un anticorps bispécifique, un anticorps multispécifique, une protéine de fusion multifonctionnelle et une composition associée contenant l'anticorps, ainsi qu'une utilisation dans la préparation d'un médicament pour le traitement du cancer.
PCT/CN2022/120428 2021-09-24 2022-09-22 Anticorps anti-claudine 18.2 et son utilisation WO2023045997A1 (fr)

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104321345A (zh) * 2012-05-09 2015-01-28 加尼梅德药物公司 用于癌症诊断的针对密蛋白18.2的抗体
CN109762067A (zh) * 2019-01-17 2019-05-17 北京天广实生物技术股份有限公司 结合人Claudin 18.2的抗体及其用途
CN110857322A (zh) * 2018-08-22 2020-03-03 瑞阳(苏州)生物科技有限公司 抗人claudin 18.2单克隆抗体及其应用
CN112574307A (zh) * 2019-09-29 2021-03-30 迈威(上海)生物科技股份有限公司 抗人Claudin18.2抗体及其应用
WO2021130291A1 (fr) * 2019-12-23 2021-07-01 SOTIO a.s. Anticorps anti-claudin 18.2 spécifiques d'une tumeur
WO2021160155A1 (fr) * 2020-02-10 2021-08-19 上海诗健生物科技有限公司 Anticorps anti-claudine 18.2 et utilisation associée
EP3872093A1 (fr) * 2018-10-22 2021-09-01 Shanghai Genbase Biotechnology Co., Ltd. Anticorps anti-cldn28.2 et ses applications

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104321345A (zh) * 2012-05-09 2015-01-28 加尼梅德药物公司 用于癌症诊断的针对密蛋白18.2的抗体
CN110857322A (zh) * 2018-08-22 2020-03-03 瑞阳(苏州)生物科技有限公司 抗人claudin 18.2单克隆抗体及其应用
EP3872093A1 (fr) * 2018-10-22 2021-09-01 Shanghai Genbase Biotechnology Co., Ltd. Anticorps anti-cldn28.2 et ses applications
CN109762067A (zh) * 2019-01-17 2019-05-17 北京天广实生物技术股份有限公司 结合人Claudin 18.2的抗体及其用途
CN112574307A (zh) * 2019-09-29 2021-03-30 迈威(上海)生物科技股份有限公司 抗人Claudin18.2抗体及其应用
WO2021130291A1 (fr) * 2019-12-23 2021-07-01 SOTIO a.s. Anticorps anti-claudin 18.2 spécifiques d'une tumeur
WO2021160155A1 (fr) * 2020-02-10 2021-08-19 上海诗健生物科技有限公司 Anticorps anti-claudine 18.2 et utilisation associée

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