WO2020043044A1 - 一种抗Claudin18.2抗体及其应用 - Google Patents
一种抗Claudin18.2抗体及其应用 Download PDFInfo
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- WO2020043044A1 WO2020043044A1 PCT/CN2019/102502 CN2019102502W WO2020043044A1 WO 2020043044 A1 WO2020043044 A1 WO 2020043044A1 CN 2019102502 W CN2019102502 W CN 2019102502W WO 2020043044 A1 WO2020043044 A1 WO 2020043044A1
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- 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
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- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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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
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- C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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- C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
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- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
Definitions
- the invention relates to the technical field of antibody drugs, in particular to an anti-Claudin 18.2 antibody or an antigen-binding fragment thereof, a pharmaceutical composition containing the anti-Claudin 18.2 antibody or an antigen-binding fragment thereof, and applications thereof.
- Gastric cancer is the fifth highest cancer type in the world, and its fatality rate is the third highest.
- Chemotherapy is currently the standard first-line treatment for advanced or recurrent gastric cancer. More than 90% of cancer patients will receive ordinary chemotherapy and targeted chemotherapy, but chemotherapy generally does not cure cancer. It can only prolong patient survival and improve quality of life, and is prone to drug resistance.
- Bio-targeted therapy usually targets tumor-specific antigens or tumor-associated antigens. Depending on the binding or blocking of antibodies to the targets, it triggers the body's immune response and selectively kills tumors without affecting surrounding normal cells and tissues. Targeted therapy has strong specificity, few side effects, and accurate curative effect.
- Claudins are the most important backbone proteins that determine the structure of tight junctions between cells. They are involved in adhesion junctions and play an important role in the metastasis and invasion of tumor cells. Claudin protein is widely present in mammalian epithelium and endothelial cells, and its distribution is mainly on the side of epithelial cells and on the plasma membrane of basal cells.
- the Claudin18 (CLDN18) gene is located at 3q22.3, molecular weight is 24 kDa, contains 261 amino acid residues, and belongs to the Claudins superfamily. Its protein structure includes 2 extracellular Ring and 4 transmembrane regions.
- the two subtypes of human CLDN18 protein are CLDN18.1 (NM_016369.3) and CLDN18.2 (NM_001002026.2).
- CLDN18.1 NM_016369.3
- CLDN18.2 NM_001002026.2
- CLDN18 proteins are expressed in different tissues and exert physiological functions.
- CLDN18.1 is constitutively expressed in normal lung tissue cells; CLDN18.2 protein is expressed in normal gastric epithelial cell membranes, and is not expressed or extremely low expressed in various tissues except the stomach.
- CLDN was 18.2 positive, and the overall positive rate was 62.7%.
- Other studies have shown that the CLDN18.2 protein expressed by normal gastric parietal cells has a uniform glycosylation modification and a dense intercellular structure. Gastric cancer cells have a loose structure and are susceptible to invasion and metastasis. The CLDN18.2 protein expressed on them is not completely glycosylated, which may lead to target exposure (WO2004 / 047863). Therefore, CLDN 18.2 may be an ideal tumor-associated antigen target.
- Astellas Pharma Inc. has developed a monoclonal antibody drug Zolbetuximab (also known as IMAB362) that targets human Claudin 18.2 in clinical efficacy.
- Phase II clinical trials show that the combination of monoclonal antibodies and chemical drugs in the treatment of advanced or recurrent gastric cancer has a median survival of 13.2 patients Months, significantly longer than 8.4 months with chemotherapy alone. It has been initially confirmed that specific antibodies targeting CLDN18.2 can significantly improve the progression-free survival and overall survival of patients with advanced gastric and gastroesophageal junction adenocarcinoma.
- WO2004 / 047863 describes that the preparation of a human-mouse chimeric monoclonal antibody is the world's first therapeutic antibody developed against the human Claudin 18.2 target (hereinafter referred to as anti-CLDN18.2 antibody).
- the antibody described in this patent application mainly kills CLDN18.2-positive tumor cells through an antibody-dependent cell-mediated cytotoxicity (ADCC) pathway.
- ADCC antibody-dependent cell-mediated cytotoxicity
- the specific recognition and killing between the antibody and tumor cells described in this patent application mainly depends on the abundance of the CLDN18.2 target on tumor cells. When the surface of the cells is high, CLDN18.2 is more obvious. Binding and killing activity. This shows that it is necessary to prepare a high-affinity monoclonal antibody to improve its ability to selectively kill tumor cells.
- the present invention now provides a highly efficient CLDN18.2 antibody or antigen-binding fragment thereof, which has excellent antitumor activity.
- One aspect of the present invention provides an anti-CLDN18.2 antibody or an antigen-binding fragment thereof comprising a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region comprises a complement of a heavy chain variable region Determinant region 1 (HCDR1), complementarity determining region 2 (HCDR2) of a heavy chain variable region, and / or complementarity determining region 3 (HCDR3) of a heavy chain variable region, said light chain variable region comprising a light chain variable region Complementarity determining region 1 (LCDR1), complementarity determining region 2 (LCDR2) of the light chain variable region, and / or complementarity determining region 3 (LCDR3) of the light chain variable region.
- HCDR1 Heavy chain variable region Determinant region 1
- HCDR2 complementarity determining region 2
- HCDR3 complementarity determining region 3
- the invention provides an anti-CLDN18.2 antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein:
- the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3:
- the amino acid sequence of the HCDR1 is selected from the group consisting of SEQ ID NO 101, SEQ ID NO 102, SEQ ID NO 103, and an amino acid sequence having at least 85% sequence identity with them;
- amino acid sequence of the HCDR2 is selected from the group consisting of SEQ ID NO 104, SEQ ID NO 105, and an amino acid sequence having at least 85% sequence identity with them;
- the amino acid sequence of the HCDR3 is selected from the group consisting of SEQ ID NO 106, SEQ ID NO 107, and an amino acid sequence having at least 85% sequence identity with them;
- the light chain variable region includes LCDR1, LCDR2 and LCDR3, wherein:
- the amino acid sequence of the LCDR2 is selected from the group consisting of SEQ ID NO 109, SEQ ID NO 110, SEQ ID NO 111, and an amino acid sequence having at least 85% sequence identity with them;
- the amino acid sequence of the LCDR3 is selected from the group consisting of SEQ ID NO 112, SEQ ID NO 113, SEQ ID 114, SEQ ID NO 115, SEQ ID NO 116, and an amino acid sequence having at least 85% sequence identity with them.
- the present invention provides an anti-CLDN18.2 antibody or an antigen-binding fragment thereof, which has the HCDR1, HCDR2, and HCDR3 as SEQ ID NO 101, 105, and 106 or SEQ ID NO 101,
- the light chain variable region of a CDR with an amino acid sequence shown at 115 having at least 85% sequence identity.
- the anti-CLDN18.2 antibody or antigen-binding fragment thereof according to the present invention is a monoclonal antibody or an antigen-binding fragment thereof.
- the anti-CLDN18.2 antibody or antigen-binding fragment thereof according to the present invention is a murine antibody or an antigen-binding fragment thereof, a chimeric antibody or an antigen-binding fragment thereof, or a humanized antibody or an antigen-binding fragment thereof. .
- the present invention provides an anti-CLDN18.2 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein
- amino acid sequence of the variable region of the heavy chain is selected from:
- (b2) an amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequence shown in (b1) and having the same or similar function to the amino acid sequence shown in (b1);
- amino acid sequence of the light chain variable region is selected from:
- the present invention provides an anti-CLDN18.2 antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the variable region of the heavy chain is SEQ ID NO: 50, SEQ ID NO: 50 is substituted, The amino acid sequence obtained by deleting or adding one or more amino acids and having the same function as SEQ ID NO: 50 or having at least 85% sequence identity with SEQ ID NO: 50, and the amino acid of the light chain variable region
- the sequence is SEQ ID NO: 51
- SEQ ID NO: 51 is obtained by substitution, deletion or addition of one or more amino acids and has the same function as SEQ ID NO: 51 or has at least 85% of the sequence of SEQ ID NO: 51 Identity amino acid sequence.
- the present invention provides an anti-CLDN18.2 antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the variable region of the heavy chain is SEQ ID NO: 50, SEQ ID NO: 50 is substituted, The amino acid sequence obtained by deleting or adding one or more amino acids and having the same function as SEQ ID NO: 50 or having at least 85% sequence identity with SEQ ID NO: 50, and the amino acid of the light chain variable region
- the sequence is SEQ ID NO: 70
- SEQ ID NO: 70 is obtained by substitution, deletion or addition of one or more amino acids and has the same function as SEQ ID NO: 70 or has at least 85% of the sequence of SEQ ID NO: 70 Identity amino acid sequence.
- the present invention provides an anti-CLDN18.2 antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the variable region of the heavy chain is SEQ ID NO: 50, SEQ ID NO: 50 is substituted, The amino acid sequence obtained by deleting or adding one or more amino acids and having the same function as SEQ ID NO: 50 or having at least 85% sequence identity with SEQ ID NO: 50, and the amino acid of the light chain variable region
- the sequence is SEQ ID NO: 71, SEQ ID NO: 71 is obtained by substitution, deletion or addition of one or more amino acids and has the same function as SEQ ID NO: 71 or has at least 85% of the sequence of SEQ ID NO: 71 Identity amino acid sequence.
- the present invention provides an anti-CLDN18.2 antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 72, SEQ ID NO: 72 is substituted, An amino acid sequence obtained by deleting or adding one or more amino acids and having the same function as SEQ ID NO: 72 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 72, and the amino acid of the light chain variable region
- the sequence is SEQ ID NO: 73
- SEQ ID NO: 73 is obtained by substitution, deletion or addition of one or more amino acids and has the same function as SEQ ID NO: 73 or has at least 85% of the sequence of SEQ ID NO: 73 Identity amino acid sequence.
- the present invention provides an anti-CLDN18.2 antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the variable region of the heavy chain is SEQ ID NO: 74, SEQ ID NO: 74 is substituted, An amino acid sequence obtained by deleting or adding one or more amino acids and having the same function as SEQ ID NO: 74 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 74, and the amino acid of the light chain variable region
- the sequence is SEQ ID NO: 75, SEQ ID NO: 75 is obtained by substitution, deletion or addition of one or more amino acids and has the same function as SEQ ID NO: 75 or has at least 85% of the sequence of SEQ ID NO: 75 Identity amino acid sequence.
- the present invention provides an anti-CLDN18.2 antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the variable region of the heavy chain is SEQ ID NO: 50, SEQ ID NO: 50 is substituted, The amino acid sequence obtained by deleting or adding one or more amino acids and having the same function as SEQ ID NO: 50 or having at least 85% sequence identity with SEQ ID NO: 50, and the amino acid of the light chain variable region
- the sequence is SEQ ID NO: 76, SEQ ID NO: 76 is obtained by substitution, deletion or addition of one or more amino acids and has the same function as SEQ ID NO: 76 or has at least 85% of the sequence of SEQ ID NO: 76 Identity amino acid sequence.
- the present invention provides an anti-CLDN18.2 antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the variable region of the heavy chain is SEQ ID NO: 77, SEQ ID NO: 77 is substituted, An amino acid sequence obtained by deleting or adding one or more amino acids and having the same function as SEQ ID NO: 77 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 77, and an amino acid of the light chain variable region
- the sequence is SEQ ID NO: 78, SEQ ID NO: 78 is obtained by substitution, deletion or addition of one or more amino acids and has the same function as SEQ ID NO: 78 or has at least 85% of the sequence of SEQ ID NO: 78 Identity amino acid sequence.
- the present invention provides an anti-CLDN18.2 antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the variable region of the heavy chain is SEQ ID NO: 74, SEQ ID NO: 74 is substituted, An amino acid sequence obtained by deleting or adding one or more amino acids and having the same function as SEQ ID NO: 74 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 74, and the amino acid of the light chain variable region
- the sequence is SEQ ID NO: 70, SEQ ID NO: 70 is obtained by substitution, deletion or addition of one or more amino acids and has the same function as SEQ ID NO: 70 or has at least 85% of the sequence of SEQ ID NO: 70 Identity amino acid sequence.
- the present invention provides an anti-CLDN18.2 antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the variable region of the heavy chain is SEQ ID NO: 74, SEQ ID NO: 74 is substituted, An amino acid sequence obtained by deleting or adding one or more amino acids and having the same function as SEQ ID NO: 74 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 74, and the amino acid of the light chain variable region
- the sequence is SEQ ID NO: 71, SEQ ID NO: 71 is obtained by substitution, deletion or addition of one or more amino acids and has the same function as SEQ ID NO: 71 or has at least 85% of the sequence of SEQ ID NO: 71 Identity amino acid sequence.
- the present invention provides an anti-CLDN18.2 antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the variable region of the heavy chain is SEQ ID NO: 74, SEQ ID NO: 74 is substituted, An amino acid sequence obtained by deleting or adding one or more amino acids and having the same function as SEQ ID NO: 74 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 74, and the amino acid of the light chain variable region
- the sequence is SEQ ID NO: 76, SEQ ID NO: 76 is obtained by substitution, deletion or addition of one or more amino acids and has the same function as SEQ ID NO: 76 or has at least 85% sequence with SEQ ID NO: 76 Identity amino acid sequence.
- the present invention provides an anti-CLDN18.2 antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is SEQ ID NO: 72, SEQ ID NO: 72 is substituted, An amino acid sequence obtained by deleting or adding one or more amino acids and having the same function as SEQ ID NO: 72 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 72, and the amino acid of the light chain variable region
- the sequence is SEQ ID NO: 76
- SEQ ID NO: 76 is obtained by substitution, deletion or addition of one or more amino acids and has the same function as SEQ ID NO: 76 or has at least 85% sequence with SEQ ID NO: 76 Identity amino acid sequence.
- the present invention provides an anti-CLDN18.2 antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the variable region of the heavy chain is SEQ ID NO: 77, SEQ ID NO: 77 is substituted, An amino acid sequence obtained by deleting or adding one or more amino acids and having the same function as SEQ ID NO: 77 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 77, and an amino acid of the light chain variable region
- the sequence is SEQ ID NO: 70, SEQ ID NO: 70 is obtained by substitution, deletion or addition of one or more amino acids and has the same function as SEQ ID NO: 70 or has at least 85% of the sequence of SEQ ID NO: 70 Identity amino acid sequence.
- the present invention provides an anti-CLDN18.2 antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the variable region of the heavy chain is SEQ ID NO: 77, SEQ ID NO: 77 is substituted, An amino acid sequence obtained by deleting or adding one or more amino acids and having the same function as SEQ ID NO: 77 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 77, and an amino acid of the light chain variable region
- the sequence is SEQ ID NO: 76, SEQ ID NO: 76 is obtained by substitution, deletion or addition of one or more amino acids and has the same function as SEQ ID NO: 76 or has at least 85% sequence with SEQ ID NO: 76 Identity amino acid sequence.
- the present invention provides an anti-CLDN18.2 antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the variable region of the heavy chain is SEQ ID NO: 79, SEQ ID NO: 79 is substituted,
- the amino acid sequence obtained by deleting or adding one or more amino acids and having the same function as SEQ ID NO: 79 or having at least 85% sequence identity with SEQ ID NO: 79, and the amino acid of the light chain variable region
- the sequence is SEQ ID NO: 70
- SEQ ID NO: 70 is obtained by substitution, deletion or addition of one or more amino acids and has the same function as SEQ ID NO: 70 or has at least 85% of the sequence of SEQ ID NO: 70 Identity amino acid sequence.
- the present invention provides an anti-CLDN18.2 antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the variable region of the heavy chain is SEQ ID NO: 79, SEQ ID NO: 79 is substituted,
- the amino acid sequence obtained by deleting or adding one or more amino acids and having the same function as SEQ ID NO: 79 or having at least 85% sequence identity with SEQ ID NO: 79, and the amino acid of the light chain variable region
- the sequence is SEQ ID NO: 76, SEQ ID NO: 76 is obtained by substitution, deletion or addition of one or more amino acids and has the same function as SEQ ID NO: 76 or has at least 85% sequence with SEQ ID NO: 76 Identity amino acid sequence.
- the present invention provides an anti-CLDN18.2 antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the variable region of the heavy chain is SEQ ID NO: 50, SEQ ID NO: 50 is substituted, The amino acid sequence obtained by deleting or adding one or more amino acids and having the same function as SEQ ID NO: 50 or having at least 85% sequence identity with SEQ ID NO: 50, and the amino acid of the light chain variable region
- the sequence is SEQ ID NO: 80
- SEQ ID NO: 80 is obtained by substitution, deletion, or addition of one or more amino acids and has the same function as SEQ ID NO: 80 or has at least 85% of the sequence of SEQ ID NO: 80 Identity amino acid sequence.
- the present invention provides an anti-CLDN18.2 antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the variable region of the heavy chain is SEQ ID NO: 50, SEQ ID NO: 50 is substituted, The amino acid sequence obtained by deleting or adding one or more amino acids and having the same function as SEQ ID NO: 50 or having at least 85% sequence identity with SEQ ID NO: 50, and the amino acid of the light chain variable region
- the sequence is SEQ ID NO: 81, SEQ ID NO: 81 is obtained by substitution, deletion or addition of one or more amino acids and has the same function as SEQ ID NO: 81 or has at least 85% of the sequence of SEQ ID NO: 81 Identity amino acid sequence.
- the present invention provides an anti-CLDN18.2 antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the variable region of the heavy chain is SEQ ID NO: 79, SEQ ID NO: 79 is substituted,
- the amino acid sequence obtained by deleting or adding one or more amino acids and having the same function as SEQ ID NO: 79 or having at least 85% sequence identity with SEQ ID NO: 79, and the amino acid of the light chain variable region
- the sequence is SEQ ID NO: 80, SEQ ID NO: 80 is obtained by substitution, deletion, or addition of one or more amino acids and has the same function as SEQ ID NO: 80 or has at least 85% of the sequence of SEQ ID NO: 80 Identity amino acid sequence.
- the present invention provides an anti-CLDN18.2 antibody or an antigen-binding fragment thereof, wherein the amino acid sequence of the variable region of the heavy chain is SEQ ID NO: 79, SEQ ID NO: 79 is substituted,
- the amino acid sequence obtained by deleting or adding one or more amino acids and having the same function as SEQ ID NO: 79 or having at least 85% sequence identity with SEQ ID NO: 79, and the amino acid of the light chain variable region
- the sequence is SEQ ID NO: 81, SEQ ID NO: 81 is obtained by substitution, deletion or addition of one or more amino acids and has the same function as SEQ ID NO: 81 or has at least 85% of the sequence of SEQ ID NO: 81 Identity amino acid sequence.
- the present invention provides an anti-CLDN18.2 antibody or an antigen-binding fragment thereof, wherein the antibody further contains a heavy chain constant region of human IgG1 or a variant thereof, a kappa chain of human origin or A variant of the light chain constant region.
- the present invention provides an anti-CLDN18.2 antibody or an antigen-binding fragment thereof, and the following amino acid substitutions are made to the heavy chain variable region shown in SEQ ID ID NO 79:
- amino acid sequence of the light chain variable region shown in SEQ ID NO 81 is made as follows:
- the present invention provides an anti-CLDN18.2 antibody or an antigen-binding fragment thereof, wherein:
- amino acid sequence of the variable region of the heavy chain is selected from:
- (c2) an amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequence shown in (c1) and having the same or similar function as the amino acid sequence shown in (c1);
- the present invention provides an anti-CLDN18.2 antibody or an antigen-binding fragment thereof.
- the amino acid sequence of the heavy chain variable region is SEQ ID NO 82, SEQ ID NO 82 is substituted, deleted, or added An amino acid sequence obtained from one or more amino acids and having the same function as SEQ ID NO 82 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO 82, and the amino acid sequence of the light chain variable region is SEQ ID NO 84, SEQ ID NO 84 is an amino acid sequence obtained by substitution, deletion, or addition of one or more amino acids and having the same function as SEQ ID NO 84 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO 84.
- the present invention provides an anti-CLDN18.2 antibody or an antigen-binding fragment thereof.
- the amino acid sequence of the heavy chain variable region is SEQ ID NO 82, SEQ ID NO 82 is substituted, deleted, or added
- SEQ ID NO 86 is an amino acid sequence obtained by substitution, deletion, or addition of one or more amino acids and having the same function as SEQ ID NO 86 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO 86.
- the present invention provides an anti-CLDN18.2 antibody or an antigen-binding fragment thereof.
- the amino acid sequence of the variable region of the heavy chain is SEQ ID NO 88, and SEQ ID NO 88 is substituted, deleted, or added
- An amino acid sequence obtained from one or more amino acids and having the same function as SEQ ID NO 88 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO 88, and the amino acid sequence of the light chain variable region is SEQ ID NO 84
- SEQ ID NO 84 is an amino acid sequence obtained by substitution, deletion, or addition of one or more amino acids and having the same function as SEQ ID NO 84 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO 84.
- the present invention provides an anti-CLDN18.2 antibody or an antigen-binding fragment thereof.
- the amino acid sequence of the variable region of the heavy chain is SEQ ID NO 88, and SEQ ID NO 88 is substituted, deleted, or added
- An amino acid sequence obtained from one or more amino acids and having the same function as SEQ ID NO 88 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO 88, and the amino acid sequence of the light chain variable region is SEQ ID NO 86.
- SEQ ID NO 86 is an amino acid sequence obtained by substitution, deletion, or addition of one or more amino acids and having the same function as SEQ ID NO 86 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO 86.
- the present invention provides an anti-CLDN18.2 antibody or an antigen-binding fragment thereof.
- the amino acid sequence of the heavy chain variable region is SEQ ID NO 82, SEQ ID NO 82 is substituted, deleted, or added An amino acid sequence obtained from one or more amino acids and having the same function as SEQ ID NO 82 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO 82, and the amino acid sequence of the light chain variable region is SEQ ID NO 90, SEQ ID NO 90 is an amino acid sequence obtained by substitution, deletion, or addition of one or more amino acids and having the same function as SEQ ID NO 90 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO 90.
- the present invention provides an anti-CLDN18.2 antibody or an antigen-binding fragment thereof, and the amino acid sequence of the variable region of the heavy chain is SEQ ID NO 88 SEQ ID NO 88 is substituted, deleted or added one or An amino acid sequence obtained by multiple amino acids and having the same function as SEQ ID NO 88 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO 88, and the amino acid sequence of the light chain variable region is SEQ ID NO 90, SEQ ID NO 90 is an amino acid sequence obtained by substitution, deletion, or addition of one or more amino acids and having the same function as SEQ ID NO 90 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO 90.
- the present invention provides an anti-CLDN18.2 antibody or an antigen-binding fragment thereof, which comprises a heavy chain amino acid sequence of SEQ ID NO 92 and SEQ ID NO 92 substituted, deleted, or added one or more
- the present invention provides an anti-CLDN18.2 antibody or an antigen-binding fragment thereof, which comprises a heavy chain amino acid sequence of SEQ ID NO 96, and SEQ ID NO 96 is substituted, deleted, or added with one or more An amino acid sequence obtained by amino acid and having the same function as SEQ ID NO 96 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO 96, and the light chain amino acid sequence is SEQ ID NO 98, SEQ ID NO 98 has been replaced or deleted Or an amino acid sequence obtained by adding one or more amino acids and having the same function as SEQ ID NO 98 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO 98.
- Another aspect of the invention provides an isolated nucleotide encoding an anti-CLDN 18.2 antibody or an antigen-binding fragment thereof of the invention.
- the present invention provides a nucleotide sequence encoding an anti-CLDN18.2 antibody or an antigen-binding fragment thereof of the present invention, wherein,
- nucleotide sequence encoding the heavy chain amino acid sequence is shown in SEQ ID NO 93 or SEQ ID NO 97;
- nucleotide sequence encoding the light chain amino acid sequence is shown in SEQ ID NO 95 or SEQ ID NO 99.
- the present invention provides a nucleotide sequence encoding an anti-CLDN18.2 antibody or an antigen-binding fragment thereof of the present invention, wherein,
- nucleotide sequence encoding the heavy chain amino acid sequence SEQ ID NO 92 is shown in SEQ ID NO 93;
- the present invention provides a nucleotide sequence encoding an anti-CLDN18.2 antibody or an antigen-binding fragment thereof of the present invention, wherein,
- nucleotide sequence encoding the heavy chain amino acid sequence SEQ ID NO 96 is shown in SEQ ID NO 97;
- Another aspect of the present invention provides an expression vector expressing the anti-CLDN18.2 antibody or antigen-binding fragment thereof of the present invention.
- An expression vector according to the present invention comprises an isolated nucleotide molecule of the present invention.
- Another aspect of the present invention provides a host cell transformed with an expression vector as described above.
- the host cell according to the invention is selected from a prokaryotic cell and a eukaryotic cell.
- the host cell is a bacterium, preferably E. coli.
- the host cell is a mammalian cell.
- Another aspect of the present invention provides a method for preparing an anti-CLDN18.2 antibody or an antigen-binding fragment thereof of the present invention, comprising the steps of expressing the antibody in the host cell and isolating the antibody from the host cell.
- Another aspect of the present invention provides a pharmaceutical composition comprising the anti-CLDN 18.2 humanized antibody or antigen-binding fragment thereof of the present invention and a pharmaceutically acceptable carrier.
- the present invention provides a pharmaceutical composition comprising the anti-CLDN 18.2 humanized antibody or antigen-binding fragment thereof of the present invention, and further comprises other active components, such as other antibodies, targeted drugs, and the like.
- the pharmaceutically acceptable carrier is selected from the group consisting of antioxidants, polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, sugar alcohols, ions, and surfactants.
- the pharmaceutically acceptable carrier is a buffered aqueous solution.
- the pharmaceutically acceptable carrier is in the form of a liposome.
- the anti-CLDN18.2 humanized antibody or antigen-binding fragment thereof of the present invention can be mixed with a pharmaceutically acceptable carrier, diluent or excipient to prepare a pharmaceutical preparation, which is suitable for oral or parenteral administration.
- Methods of administration include, but are not limited to, the oral, intradermal, intramuscular, intraperitoneal, intravenous, brain, intraocular, intratracheal, subcutaneous, intranasal routes.
- the formulations can be administered by any route, such as by infusion or bolus, by absorption through the epithelium or skin mucosa (eg, oral mucosa or rectum, etc.). Administration can be systemic or local.
- the formulations can be prepared by methods known in the art and include carriers, diluents or excipients conventionally used in the field of pharmaceutical formulations.
- Another aspect of the invention provides a method of targeting CLDN18.2 therapy, which method comprises administering to an individual in need thereof an anti-CLDN18.2 antibody or antigen-binding fragment thereof or a pharmaceutical composition of the invention.
- Another aspect of the present invention provides the use of the anti-CLDN18.2 antibody of the present invention or an antigen-binding fragment thereof or the pharmaceutical composition of the present invention in the preparation of a medicament targeting CLDN18.2.
- the drug that targets CLDN 18.2 is used to treat gastric cancer, esophageal cancer, pancreatic cancer, lung cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer, and gallbladder cancer.
- the present invention provides the use of the above-mentioned anti-CLDN18.2 antibody or antigen-binding fragment thereof or the pharmaceutical composition of the present invention in the preparation of an antitumor medicament.
- the tumor is selected from the group consisting of gastric cancer, esophageal cancer, Pancreatic, lung, ovarian, colon, liver, head and neck, and gallbladder cancer.
- the anti-CLDN18.2 antibody or antigen-binding fragment thereof provided by the present invention has significant anti-tumor effect, high affinity, strong ability of ADCC to kill tumor cells in vitro, and significant technical advantages.
- the term "at least 80% sequence identity” means at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity.
- the term “at least 85% sequence identity” means at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity.
- sequence identity of the present invention may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100 %. Sequence comparison and percent identity determination between two sequences can be performed by the BLASTN / BLASTP algorithm on the National Center for Biotechnology Institute website.
- the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged at positions relative to each other in a three-dimensional space to form an antigen-binding surface.
- the antigen-binding surface is complementary to the three-dimensional surface of the bound antigen, and the three hypervariable regions of each heavy and light chain are called "complementarity determining regions" or "CDRs.”
- CDRs complementarity determining regions
- the "antigen-binding fragment” in the present invention refers to a Fab fragment, a Fab 'fragment, an F (ab') 2 fragment, an Fv fragment, and an scFv fragment that bind to human Claudin 18.2, which have antigen-binding activity.
- the Fv fragment contains the variable region of the heavy chain and light chain of the antibody, but has no constant region and has the smallest antibody fragment with all antigen-binding sites.
- Fv antibodies also contain a polypeptide linker between the VH and VL domains and are capable of forming the structure required for antigen binding.
- the variable regions of two antibodies can also be linked into a single polypeptide chain with different linkers, called single-chain antibodies or single-chain Fv (scFv).
- the antibody in the present invention refers to an immunoglobulin molecule or an immunologically active portion thereof, that is, a molecule comprising an antigen binding site that specifically binds an antigen (immunoreactively reacts with it).
- "Specific binding” refers to an antibody that reacts with one or more epitopes of an antigen without reacting with other polypeptides or binding other polypeptides with very low affinity (Kd> 10-6).
- Antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, dAb (domain antibody), single chain, Fab, Fab 'and F (ab') 2 fragments, Fv, scFv, and Fab expression libraries.
- Monoclonal antibodies are antibodies obtained from a single cloned cell line, which is not limited to eukaryotic, prokaryotic, or phage cloned cell lines. Monoclonal antibodies or antigen-binding fragments can be recombined using, for example, hybridoma technology, recombinant technology, phage display technology, and synthetic technology such as CDRgrafting or other existing technologies.
- the "murine antibody” according to the present invention is a monoclonal antibody to human CLDN18.2 prepared according to the knowledge and skills in the art. Test subjects are injected with CLDN 18.2 antigen during preparation, and then hybridomas expressing antibodies with the desired sequence or functional characteristics are isolated.
- the "chimeric antibody” is an antibody obtained by fusing the variable region of a murine antibody with the constant region of a human antibody, and can reduce the immune response response induced by the murine antibody.
- To establish a chimeric antibody first establish a hybridoma that secretes a mouse-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 according to the needs, and the mouse variable region The gene is linked to the human constant region gene into a chimeric gene and inserted into an expression vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic industrial system or a prokaryotic industrial system.
- the "humanized antibody” according to the present invention is also called a CDR-grafted antibody, and is an antibody produced by transplanting a mouse CDR sequence into a human antibody variable region framework (FR).
- FR human antibody variable region framework
- Such variable region framework sequences can be obtained from public DNA databases or published references, such as from the ImMunoGeneTics (IMGT) website http://imgt.cines.fr or from the Journal of Immunoglobulins, 2001 ISBN012441351.
- Figure 1 shows the binding of chimeric antibodies to CLDN18.2 cells, where the abscissa is the antibody concentration in ng / ml; the ordinate is the OD value at 450nm.
- Figure 2 shows the binding of chimeric antibodies to CLDN18.1 cells, where the abscissa is the antibody concentration in ng / ml; the ordinate is the OD value at 450nm.
- Figure 3 is the binding of phage Fab to CLDN18.2 protein, where the abscissa is the phage titer; the ordinate is the OD value at 450nm.
- Figure 4 shows the binding of humanized antibodies to CHO-K1-CLDN18.2 cells, where the abscissa is the antibody concentration in mg / ml; the ordinate is the average fluorescence intensity MFI bound to the cells.
- Figure 5 shows the binding of humanized antibodies to CHO-K1-CLDN18.1 cells, where the abscissa is the antibody concentration in mg / ml; the ordinate is the average fluorescence intensity MFI bound to the cells.
- Figure 6 is the ADCC activity of a humanized antibody.
- Nanjing Kingsray Biotechnology Co., Ltd. was commissioned for codon optimization and synthesized in pcDNA3.
- the constructed plasmids include pcDNA3.1-hG1 and pcDNA3.1-hK.
- the plasmid was co-transfected with expiCHO cells at a ratio of 1: 1 for transient expression. The cells were cultured for 4 days at a speed of 110 rpm, 37 ° C, and 8% CO 2 , and then transferred to a 32 ° C shaker for 8-10 days.
- the antibody sequence of the positive antibody variable region sequence combined with the mouse IgG2a constant region is SEQ ID NO 13 and SEQ ID NO 14. After codon optimization, it was synthesized in pcDNA3.1 (+) vector.
- the constructed plasmids include pcDNA3.1-mG2a and pcDNA3.1-mK. The plasmid was co-transfected into expiCHO cells at a 1: 1 ratio to transiently express positive antibody proteins.
- the harvested supernatant protein was purified by ProteinA affinity chromatography: 0.1M NaOH solution was used to clean the AKTA chromatography system and the column for 30 minutes, and the ultrapure water was washed away; 20 mM sodium phosphate buffer at pH 7.4 was equilibrated to 10 column volumes; Load the sample at a constant flow rate of 2 ml / min; wash the chromatography column with 10 volumes of the equilibrium solution; elute 3-5 column volumes of 50 mM acetic acid buffer at pH 3.4 to collect the eluted protein; ultrafiltration and concentration to replace the antibody at pH 7 .0 in 20 mM phosphate buffer.
- the fusion protein sequence of the human CLDN18.2 antigen designed above was optimized for eukaryotic codons, and a restriction site such as NotI and a tPA signal peptide sequence were added to the 5 'end of the gene, and an XhoI site was added to the 3' end of the gene. And Myc-His fusion protein tags.
- the above SEQ ID No. 1, 3, 5, and 6 sequences were synthesized and constructed into the pcDNA3.1 (+) vector to obtain a DNA plasmid expressing the human CLDN18.2 antigen. Plasmids include: pcDNA3.1-loop1, pcDNA3.1-TML, pcDNA3.1-CpG-loop1, and pcDNA3.1-CLDN18.2-FL.
- the above SEQ ID NO 7 and 9 sequences were optimized and synthesized by Bao Biological Engineering (Dalian) Co., Ltd. (hereinafter referred to as: Bao Biological Company), and constructed into the lentiviral vector pLVX-NS-ZsGreen (Bao Biological Company).
- the constructed lentiviral plasmids pLVX-CLDN18.2-ZsGreen and pLVX-CLDN18.1-ZsGreen are used to further construct a HEK293-CLDN18.2 fusion green fluorescent protein stable expression cell line, and to construct a HEK293-CLDN18.1 fusion green fluorescent protein to stabilize Expressing cell lines.
- the amplified cultured plasmid was used for enzyme digestion identification.
- the identification results showed that the digested target band was consistent with the expected size, indicating that the construction was correct.
- Example 2 The plasmid amplified and cultured in Example 2 was transiently transfected into HEK293T cells (Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, hereinafter referred to as Shanghai Cell Institute) for transient identification in vitro and identified at the protein level.
- the specific experimental steps are as follows: one day before transfection, HEK293T cells were plated in a six-well plate at a seeding density of 3 ⁇ 10 5 cells / cm 2 , and cultured (medium composition: 10% fetal bovine serum DMEM medium + 1%) Penicillin-streptomycin) overnight, so that the cells are completely adherent on the day of transfection, and the cell abundance is 70% -90% (the size of each well of a six-well plate is 9cm 2 , and the cells are about 2-2.5 ⁇ 10 6 Per well); use PEI cationic transfection reagent (sigma) or liposome (Lipo3000, Thermo) to mix with the DNA plasmid to prepare the DNA plasmid transfection complex, add the cells and leave to stand at room temperature for 5-20 minutes, 37 ° C, 5 % CO 2 was incubated for 4-5 hours; supplemented with a complete medium containing serum to a six-well plate, and cultured for 48-
- Collect transiently transfected HEK293T cells hybridize the target protein with rabbit anti-human Claudin18 antibody (Thermo, Cat: 700178) at room temperature for 2 hours; wash 3 times with PBST, and alkaline phosphatase-labeled goat anti-rabbit IgG (Thermo, Cat : 31346) Hybridization at room temperature for 1 hour; Western Blot immunoblotting to identify the expression of the target protein: using Lipo liposome transfection system, the plasmid can successfully express the target proteins CLDN18.2-loop1 and CLDN18.2-TM-loop1, molecular weights are respectively For 11KD and 14KD, the pcDNA3.1-CLDN18.2-FL plasmid successfully expressed the target protein CLDN18.2 with a molecular weight of 29KD.
- rabbit anti-human Claudin18 antibody Thermo, Cat: 700178
- PBST wash 3 times with PBST, and alkaline phosphatase-lab
- Loop1 pcDNA3.1-loop1
- TML pcDNA3.1-TM-Loop1
- (Loop1-C) pcDNA3.1-CpG-loop1
- DNA plasmids pass the living gene
- the introduction instrument (Shanghai Tarisa Biotechnology Co., Ltd.) was injected into the muscles of the left and right hind limbs of the mouse at two points, and an equal amount of DNA plasmid was injected at each point, and the injection volume was 50 ⁇ l.
- the electrodes of the instrument were used to input electric pulse stimulation to the injection site of the mouse, and the voltage was set to 36V for a total of 6 pulses.
- Electric pulse stimulation can promote the absorption of DNA plasmids in mouse muscle cells and increase the level of mouse immune response, especially humoral immune response.
- DNA immunization was injected every 2 weeks for a total of three immunizations. Seven days after the second and third immunizations, blood was collected from the fundus venous plexus of mice for flow analysis. When a clear CLDN18-positive signal was detected in serum antibodies, HEK293-CLDN18.2 cells were used as the antigen for the mice. A 5 ⁇ 10 7 cell / ml cell suspension was prepared, and mice were injected intraperitoneally with 100 ⁇ l, that is, 5 ⁇ 10 6 cells to enhance immunity.
- mice After the second DNA immunization, a significant positive signal shift was detected in the # 0103 mouse, indicating that the mouse produced an anti-CLDN18-specific antibody response.
- all mice After the third DNA immunization, all mice detected positive signals, among which # 0101, # 0102, # 0103, # 0203, # 0301, and # 0303 mice had a positive rate of CLDN18 greater than 50%, showing the third DNA After immunization, these mice produced a significant antibody immune response, and higher levels of anti-CLDN18 antibodies were present in the serum.
- Mouse myeloma cells SP2 / 0 were resuscitated two weeks in advance, cultured in 1640 medium containing 10% fetal bovine serum at 37 ° C, 5% CO 2 , and passaged. On the day of fusion, observe the state of SP2 / 0 cells. The confluence of SP2 / 0 cells was examined by microscopy at 70% -80%, and the cells were judged to be in the logarithmic growth phase.
- mice Preparation of mouse feeder layer cell suspensions: One day before fusion, 20 healthy Balb / c mice aged 10-12 weeks were sacrificed by pulling the neck. Sterile surgical scissors were used to open the abdominal skin of the mice, and a small cut was made in the abdomen. , And then cut longitudinally to the chest to expose the abdomen of the mouse. Collect peritoneal macrophages: first disinfect the mouse abdomen with an alcohol cotton ball, and then inject 5 ml of 1640 basal medium into the abdominal cavity of the mouse with a syringe, gently massage the abdominal cavity of the mouse for 5 minutes, and then suck the fluid in the abdominal cavity with the syringe.
- mice Preparation of mouse spleen lymphocyte suspension: On day 3-4 after cell booster immunization, spleen cells from immunized mice were fused with SP2 / 0. Mice were sacrificed by pulling the neck, the peritoneum was aseptically cut, the spleen was removed and the surrounding connective tissue was stripped.
- a 70 ⁇ m sieve was filtered into a 50 ml centrifuge tube containing 15 ml of 1640 medium in advance to prepare a single cell suspension; 1500 rpm, 5 min, 1640 medium was washed, and the supernatant was discarded; 4 ml of red blood cell lysate (Sigma ) Lyse red blood cells for about 2 min; add an equal volume of 1640 medium to 1500 rpm, centrifuge for 5 min, discard the upper red blood cell layer; resuspend in 20 ml PBS, wash twice, and centrifuge at 1500 rpm for 5 min. After the second wash, resuspend the cells with 15ml PBS.
- CHO-K1 cells (Shanghai Institute of Cells) were revived one week in advance, and cultured in DME / F12 complete medium containing 10% fetal bovine serum, 37 ° C, 5% CO 2 .
- the plasmid for transfected cells was pcDNA3.1-CLDN18.2-FL (see SEQ ID NO 6 for the synthetic sequence).
- the growth density of CHO-K1 cells reached 80% -90%.
- the plasmid was transferred into CHO-K1 cells, and all the cells after transfection were transferred to the pre-warming before complete culture.
- PCR program was set at 98 ° C for 30 seconds and 60 ° C for 30 seconds. Extend at 72 ° C for 1 minute and perform 30 cycles of amplification to obtain the CLDN18.1-FL target gene fragment (see SEQ ID NO 17). BamHI and EcoRI double-digested the target gene and pcDNA3.1 (+) vector, and T4 ligase (Takara) was ligated overnight at 16 ° C. The ligated product was transformed into E.
- the plasmid was transfected into CHO-K1 cells according to the above steps.
- the cells were cultured and passaged in G418-resistant medium.
- WesternBlot results showed that the cells could be positively stained with anti-CLDN18 antibody (Thermo) after transfection.
- the CLDN18.2 positive antibody IMAB362-similar-hG1 showed a negative fluorescence signal after incubation at a concentration of 10 ⁇ g / ml. This indicates that the transfected CLDN18.1 cells were successfully constructed.
- the first round of primary screening of hybridoma cells obtained after fusion is performed.
- the screening methods include cell ELISA and FACS to detect the culture supernatant of hybridoma cells, and to select CLDN18-positive hybridoma mother clones. After the primary screening showed that the CLDN18-positive clones were expanded, a second round of re-screening was performed to select the hybridoma mother clones whose FACS showed CLDN18.2-positive and CLDN18.1-negative.
- a single clone was prepared from the hybridoma cells selected and obtained in Example 6. Resuspend the hybridoma cells in a 24-well plate to prepare a cell suspension. Count each cloned cell, adjust the final cell concentration to 1 ⁇ 10 4 cells / ml in 1640 complete medium; take 60 ⁇ l of cell suspension and add 140 ⁇ l of 1640 complete medium, a total of 200 ⁇ l, mix well; remove -20 ° C one day in advance The stored semi-solid medium D (StemCell) was thawed at 4 ° C, and it was left at 37 ° C for 1 hour to warm up before use.
- SteCell stored semi-solid medium D
- the specificity of the CLDN18.2 antibody of the subcloned cell culture supernatant was identified.
- the screening method used was cell ELISA to detect the positive binding of the supernatant antibody to CLDN18.2 and CLDN18.1 stable transfected cells, respectively.
- the specific implementation method is as follows:
- HEK293-CLDN18.1 and HEK293-CLDN18.2 cells were cultured in DMEM complete medium containing 10% fetal calf serum at 37 ° C and 5% CO 2 , respectively.
- DMEM complete medium containing 10% fetal calf serum at 37 ° C and 5% CO 2 , respectively.
- 0.5 mM EDTA-PBS trypsin-free digestion solution was used to digest the cells at room temperature, centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded.
- DMEM complete medium was resuspended, and the cell concentration was adjusted to 6 ⁇ 10 5 cells / ml.
- the cells were plated in a 96-well cell plate (Corning) pre-coated with polylysine, and 100 ⁇ l per well was cultured at 37 ° C. and 5% CO 2 for 24 hours, so that the cells were evenly spread on the bottom of the wells.
- the culture supernatant was aspirated, the cells were fixed with 4% paraformaldehyde at room temperature for 20 minutes, and washed with 200 ⁇ l of PBS 3 times for 2 minutes each time. 200 ⁇ l 3% BSA-PBS blocked well plate at 37 ° C. for 1 hour.
- 100 ⁇ l of the subclone culture supernatant was added to a well plate, incubated at 37 ° C. for 1 hour, and washed 5 times with PBS.
- the HRP-anti mouse IgG + IgM (Jackson) diluted 1: 0000 in blocking solution was incubated at 37 ° C for 1 hour. Wash 5 times in PBS. Add 100 ⁇ l TMB single-component coloring solution, protect from light for 5-10 minutes at room temperature, stop with 2M sulfuric acid, and read by OD450 on a microplate reader. The results are shown in Table 3.
- the hybridoma culture supernatant combined with CLDN18.2 strongly positive subclones including: S3F10C5, S5H3A6, S5H3B6, S5H3D6, S6F10E7, S6H9B8, S6H9C8, C40B10D9, C42B12D10, and C58B11E11.
- S5H3A6, S5H3B6, S5H3D6, C40B10D9, C42B12D10, and C58B11E11 showed negative binding to CLDN18.1 cells.
- Subclones B4B10G3, S5H3B6, S6H9B8, C40B10D9, C42B12D10, and C58B11E11 were cloned for expansion. Resuspend cells in a 96-well plate with a 200 ⁇ L pipette. Transfer all the cell suspension to a 24-well cell plate (Corning), add 1 ml of 1640 medium containing 2% CloneEasy, 10% fetal bovine serum, and place at 37 ° C. Incubate in a 5% CO 2 incubator for 3-4 days.
- Mouse Antibody Subtype Identification Kit SBA Clonotyping System (Southern Biotech) add the subclone cell culture supernatant to be tested to identify the mouse antibody subtype.
- the identification results are as follows:
- Subclone B4B10G3 the antibody heavy chain constant region subtype is mouse IgG2b; the antibody light chain constant region subtype is mouse Kappa chain;
- Subclone S5H3B6 the antibody heavy chain constant region subtype is mouse IgM, and the antibody light chain constant region subtype is mouse Kappa chain;
- Subclone S6H9B8 antibody heavy chain constant region subtype is murine IgG3, antibody light chain constant region subtype is murine Kappa chain;
- the antibody heavy chain constant region subtype is mouse IgG2c, and the antibody light chain constant region subtype is mouse Kappa chain;
- the antibody heavy chain constant region subtype is mouse IgM
- the antibody light chain constant region subtype is mouse Kappa chain
- Subclone C42B12D10 the antibody heavy chain constant region subtype is mouse IgM, and the antibody light chain constant region subtype is mouse Kappa chain.
- Monoclonal hybridoma cells in a 24-well plate were collected, adjusted to 1 ⁇ 10 6 cells / ml, 1 ml in total, centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded.
- QIAGEN's RNA extraction kit (Cat: # 74134)
- small amounts of total RNA were extracted from the cells, and the RNA concentration was measured using a NanoDrop nucleic acid quantitative analyzer.
- 2 ⁇ g of total RNA was reverse transcribed into cDNA, and stored at -80 ° C until use.
- the PCR system for sequencing light and heavy chains of antibodies was prepared according to the PremixTaq enzyme operating instructions of Takara Company, and the program of the PCR instrument was set.
- the primers used in the PCR system were synthesized from Suzhou Jinweizhi Company.
- the specific primer sequences are as follows:
- the heavy chain sequencing primer VH-F includes:
- VH-F1 (including primer sequences SEQ ID NO 18 and SEQ ID NO 19), VH-F2 (including primer sequences SEQ ID NO 20, SEQ ID NO 21, SEQ ID NO 22, and primer sequences SEQ ID ID 23, SEQ ID NO 24, SEQ ID NO 25), VH-F3 (primer sequence SEQ ID NO 26)
- the heavy chain sequencing primer VH-R includes:
- VH-R1 (primer sequence SEQ ID NO 27), VH-R2 (primer sequence SEQ ID NO 28)
- Light chain sequencing primers VL-F include:
- VL-F1 (primer sequence SEQ ID NO 29), VL-F2 (including primer sequences SEQ ID NO 30 and SEQ ID 31), VL-F3 (including primer sequence SEQ ID NO 32, SEQ ID NO 33, SEQ ID NO 34 )
- Light chain sequencing primers VL-R include:
- VL-R (primer sequence SEQ ID NO 35)
- the human IgG1 constant region was combined with the murine antibody variable region, and a human-mouse chimeric antibody was constructed by gene synthesis to further study antibody affinity and antibody properties.
- the chimeric antibodies were named 1CHI, 3CHI, 7CHI, 17CHI, 18CHI, 19CHI.
- the sequence of the variable region of the antibody corresponding to each chimeric antibody is shown in Table 5 below:
- the plasmid containing the light and heavy chain genes of the aforementioned chimeric antibody was transiently transfected into HEK293E cells (Shanghai Institute of Cellular) at a ratio of 1: 1.
- the rotation speed was 110 rpm, 37 ° C, and 8% CO 2 with shaking culture for 7 days. Centrifuge at 4500 rpm for 20 minutes to harvest the supernatant protein.
- Purified by ProteinA affinity chromatography Wash the AKTA chromatography system and column for 30min with 0.1M NaOH solution, rinse with ultrapure water; equilibrate 10 column volumes with 20mM sodium phosphate buffer at pH7.4; set the flow rate to 2ml / min Load the sample; equilibrate the column with 10 column volumes; wash 3-5 column volumes with 50 mM acetic acid buffer at pH 3.4 to collect the eluted protein; ultrafiltration and concentration to replace the antibody with 50 mM vinegar at pH 5.55 Store in salt buffer.
- HEK293-CLDN18.2 and HEK293-CLDN18.1 cells were resuscitated two weeks in advance, and cultured in DMEM complete medium containing 10% fetal bovine serum at 37 ° C and 5% CO 2 .
- DMEM complete medium containing 10% fetal bovine serum at 37 ° C and 5% CO 2 .
- 0.5 mM EDTA-PBS Sigma
- digested the cells at room temperature centrifuged at 1000 rpm, 5 minutes, and discarded the supernatant.
- DMEM complete medium was resuspended, and the cell concentration was adjusted to 6 ⁇ 10 5 cells / ml.
- Cells were plated on 96-well cell plates pre-coated with 2.5 ⁇ g / well of polylysine (Corning), 100 ⁇ l per well, cultured at 37 ° C. and 5% CO 2 for 24 hours, so that the cells evenly covered the bottom of the wells. .
- the culture supernatant was aspirated, the cells were fixed with 4% paraformaldehyde at room temperature for 20 minutes, and washed with 200 ⁇ l of PBS 3 times for 2 minutes each time. 200 ⁇ l 3% BSA-PBS blocked well plate at 37 ° C. for 1 hour.
- Each chimeric antibody was diluted to a concentration of 6 ⁇ g / ml with 3% BSA-PBS, and diluted in a 6-fold gradient, and a total of 7 concentrations were diluted.
- IMAB362-similar-hG1 diluted with the same concentration gradient was used as a positive control
- human IgG1, kappa isotype control antibodies hereinafter referred to as Isotype, provided by Sino-American Crown Scientific Biotechnology (Taicang) Co., Ltd.
- Isotype human IgG1, kappa isotype control antibodies
- -CLDN18.2 and HEK293-CLDN18.1 cell plates Incubate at 37 ° C for 1 hour and wash 5 times with PBS.
- Example 11 the chimeric antibody 3CHI with the closest affinity to the positive control antibody was selected for affinity maturation and screening in vitro.
- the phage Fab antibody library is mainly used to screen for antibodies that can maintain the specificity of CLDN18.2 and improve affinity.
- the affinity of the above-mentioned anti-CLDN18.2 antibodies at the protein level was verified by a Fortebio molecular interaction instrument: First, an antibody solution and an antigen dilution solution were prepared, and a chimeric solution was prepared using SD buffer (PBS solution containing 0.02% Tween20 and 0.1% BSA) The antibody was diluted to a final concentration of 5 ⁇ g / ml; the antigen protein Claudin 18.2-His was diluted with SD buffer in a 4-fold concentration gradient to a concentration of 10 ⁇ g / ml, 2.5 ⁇ g / ml, 0.625 ⁇ g / ml, and 0 ⁇ g / ml, respectively. . After that, the AHC sensor was used to cure the antibody, and then combined with different concentrations of antigen, and the affinity was determined according to the operating instructions of Octet RED96.
- CHO-K1-CLDN 18.2 prepared in Example 5 cells were digested, the cells were resuspended in complete medium, the cells were counted, and the density was adjusted to 1 ⁇ 10 5 cells / ml, 100 ⁇ l / The wells were inoculated into a 96-well plate and cultured overnight at 37 ° C in a 5% CO 2 incubator. On the day of the test, the phenol red-free 1640 medium (Gibco) was incubated to 37 ° C, and the antibody was diluted by a 5-fold gradient.
- CHO-K1-CLDN 18.2 96-well plate, discard the culture medium, add 25 ⁇ l of phenol red-free 1640 to each well, and add the diluted antibody to the sample wells so that the final antibody concentrations are 10 ⁇ g / ml and 2 ⁇ g / ml, respectively. , 0.4 ⁇ g / ml, 0.08 ⁇ g / ml, 0.016 ⁇ g / ml, and 0.0032 ⁇ g / ml; equal amounts of IMAB362-similar-hG1 and human IgG1 isotype control antibody (CrownBio) were added to the control wells at 37 ° C, 5% CO 2 Incubate in the incubator for 1 h;
- Jurkat cells (Jurkat-CD16A / NF) stably expressing human CD16A protein and NFAT reporter gene protein as effector cells, transfer them to a centrifuge tube, centrifuge at 1000 rpm for 5 min at room temperature, discard the supernatant; 5ml phenol red-free 1640 culture Basal resuspension was washed once, centrifuged at 1000 rpm for 5 min, and the supernatant was discarded; the cells were resuspended without phenol red 1640, and the cell density was adjusted to 8 ⁇ 10 6 cells / ml;
- effector to target ratio i.e. the ratio of the number of effector cells to target cells was 20: 1
- Jurkat-CD16A / NF was added to the cell plate in 2 ⁇ 10 5 th, 200 g of, centrifuged for 2 minutes to effect target cell Full contact; incubate for 5-6 hours in a 37 ° C, 5% CO 2 incubator; 30 minutes before detection, return the cell plate to room temperature and follow the Bio-Turbo Firefly Luciferase Assay Kit (Jiangsu Ruian Biotechnology Co., Ltd.)
- Add detection reagents to the instruction manual transfer the supernatant to be tested to a white flat bottom 96-well plate, and use a Thermo Floskan Ascent FL fluorescence microplate reader to detect the fluorescence signal of the well plate.
- the original data was substituted into Graphpad 5.0 software for calculation and analysis. The results are shown in Table 11.
- the fluorescence signal of each chimeric antibody was dose-dependent with concentration gradient.
- the EC50 of the chimeric antibody 330CHI was 0.22 nM and the EC50 of the chimeric antibody 305CHI was 0.28 nM.
- the ADCC EC50 activity of the antibody was more than three times higher than that of the positive control antibody IMAB362-similar-hG1 (0.72 nM). It can be seen that at the same time that the cell binding activity and affinity of the chimeric antibody against CLDN18.2 are improved, it can also effectively improve the ADCC activity of the antibody in vitro.
- the in vitro affinity matured antibody 330CHI in Example 12 was optimized for humanized sequences. Humanized sequence optimization and antibody physicochemical properties identification were commissioned by Hangzhou Haoyang Biotechnology Co., Ltd. Humanization sequence optimization
- the immunogenic gene database (IMGT) was used to confirm the mouse-derived sequence source of the 3CHI antibody and 330CHI antibody variable region. After homology alignment, the source of the FR region of the heavy chain variable region sequence of 3CHI antibody and 330CHI antibody The mouse antibody IGHV5-15 * 01; the FR sequence of the antibody light chain variable region was derived from the mouse antibody IGKV8-19 * 01. The CDR region of the mouse antibody and the FR region of the human antibody are recombined to form a humanized antibody.
- the light and heavy chain template of the human antibody used has a sequence similarity of 65% or more with the corresponding murine anti-variable region. For the source of the human antibody, see Table 12. The names and sequences of the final humanized antibodies are shown in Table 13.
- a plasmid vector containing humanized antibody light and heavy chain genes was co-transfected into HEK293E cells at a ratio of 1: 1, and cultured at 110 rpm, 37 ° C, and 8% CO 2 for 7 days with shaking. Centrifuge at 4500 rpm for 20 minutes to harvest the supernatant protein.
- Purified by Protein A affinity chromatography Wash the AKTA chromatography system and column for 30 min with 0.1M NaOH solution, rinse with ultrapure water; equilibrate 10 column volumes with 20 mM sodium phosphate buffer at pH 7.4; set flow rate 2ml / min, load; wash the chromatography column with 10 volumes of equilibration solution; elute 3-5 column volumes with 50mM acetic acid buffer at pH 3.4 to collect the eluted protein; ultrafiltration and concentration to replace the antibody with 20mM at pH 6.5 Store in phosphate buffered saline. The purity of the purified antibodies was determined by size exclusion high performance liquid chromatography (SEC-HPLC).
- Hu330-1, Hu330-2, and Hu330-4 all had a purity of more than 95%.
- Hu330-1 As an example, the heavy chain molecular weight of an antibody is 48.72KD and the light chain molecular weight is 24.21KD.
- the digestion fluid of gastric cancer cells was digested and treated with CHO-K1-CLDN18.2 cells and CHO-K1-CLDN18.1 cells (prepared in Example 5), and digested at 37 ° C for 15 minutes; the complete medium was terminated, and centrifuged at 1000 rpm for 5 minutes.
- the Kd value of the humanized antibody Hu330-1 was 1.7 nM, which was significantly lower than the positive control antibody (28.7 nM), indicating that humanized The binding activity of the antibody to cells expressing CLDN18.2 was about 15 times higher than that of the positive control antibody IMAB362-similar-hG1.
- Example 16 Fortebio detects the specificity and affinity of humanized antibodies for binding to human CLDN 18.2 at the protein level
- Another method is to verify the affinity of the aforementioned humanized antibodies at the protein level, select AHC sensor-cured antibodies, and bind to different concentrations of antigens, and perform affinity determination according to the instructions of Octet RED96.
- the results are shown in Table 15.
- the equilibrium dissociation constant (KD) of the humanized antibody Hu330-1 bound to the CLDN18.2 protein at 37 ° C was 5.26 nM. Compared with the positive control, its antibody affinity was significantly increased by about 18 Times.
- Example 17 Determination of ADCC activity of humanized antibodies in vitro by luciferase labeling
- CHO-K1-CLDN18.2 cells (prepared in Example 5) were used as target cells, the cell density was adjusted to 1 ⁇ 10 5 cells / ml, and 100 ⁇ l / well was seeded into a 96-well plate, and cultured at 37 ° C.
- the humanized antibodies Hu330-1 and Hu330-2 Compared with the IMAB362-similar-hG1 positive antibody, the humanized antibodies Hu330-1 and Hu330-2 reduced EC50 by more than 6 times, and were almost the same as the chimeric antibody ADCC activity. This shows that while humanized antibodies have high affinity, they also show higher ADCC biological activity than positive antibodies.
- Example 18 Determination of ADCC killing activity of humanized antibodies on tumor cells in vitro by RTCA
- Collect human peripheral blood to isolate PBMC cells add an equal volume of PBS diluted blood sample containing 2% fetal bovine serum; add 15 ml of Lymphoprep density gradient centrifuge (StemCell) to a 50 ml centrifuge tube, add the diluted blood sample, and turn off the centrifuge brake. Centrifuge at 1200g for 10 minutes at room temperature. After centrifugation, the white membrane layer is found, which contains the enriched PBMC cells. Aspirate the white membrane layer and transfer it to a new centrifuge tube.
- the red blood cell lysate (Sigma) to lyse the red blood cells at room temperature.
- Fig. 6 The results are shown in Fig. 6.
- the tumor cell killing rate of each group increased with time.
- the maximum killing rate of humanized antibody Hu330-1 to NUGC4 cells was 45%, which was 10% higher than ADCC killing activity of tumor cells by IMAB362-similar-hG1 positive antibody (P ⁇ 0.05).
- the humanized antibody Hu330-1 can specifically target CLDN18.2, and its binding activity and affinity are 15-18 times higher than the positive antibody IMAB362-similar-hG1 at the molecular and cellular levels,
- the antibody-mediated ADCC killing effect on target cells in vitro exhibits antibody concentration-dependent properties. At lower concentrations, Hu330-1 can exert a certain killing effect on tumor cells.
- the Hu330-1 humanized antibody variable region sequence was combined with an IgG1 constant region sequence containing 5 amino acid site mutations to construct an Fc mutant humanized antibody Hu330-1-5M (see Table 14 for sequence); another construct A positive antibody 5M (see Table 17 for the sequence) containing this Fc mutation served as a control for the Fc segment.
- the cells were resuspended with 2% fetal bovine serum-PBS dilution to adjust the cells to 1 ⁇ 10 6 cells / ml, 100 ⁇ l of cell suspension was added to each well of a 96-well U-bottom plate; the antibody was diluted with 2% fetal bovine serum-PBS to a final concentration of 5 ⁇ g / ml, 1 ⁇ g / ml, 0.2 ⁇ g / ml, 0.04 ⁇ g / ml and 0.008 ⁇ g / ml, mix the cells with equal volume, and incubate at 4 ° C for 1 hour; centrifuge at 1000rpm for 5 minutes, discard the supernatant; add 200 ⁇ l Cell Stain Buffer (Biolegend) to each well and wash twice at 1000rpm for 5 minutes Centrifuge and discard the supernatant; add 100 ⁇ l of PE-anti human Fc antibody (Biolegend), incubate at 4 ° C for 1 hour; wash twice with
- CHO-K1-CLDN18.2 cells (prepared in Example 5) were used as target cells, the cell density was adjusted to 1 ⁇ 10 5 cells / ml, and 100 ⁇ l / well was seeded into a 96-well plate, and cultured at 37 ° C.
- the Fc mutation-positive antibody (5M) and humanized Hu330-1 antibody increased ADCC activity by 3 times compared to IMAB362-similar-hG1, while the Fc-mutated Hu330-1-5M antibody had an EC50 of 0.17 nM, which was higher than that of the positive control antibody.
- the ADCC activity of IMAB362-similar-hG1 was increased by more than 8 times, which obviously optimized the ADCC enhancement effect in vitro.
- Collect human peripheral blood to isolate PBMC cells as effector cells add an equal volume of 2% fetal bovine serum in PBS to dilute blood samples; add 15 ml Lymphoprep density gradient centrifuge (StemCell) to a 50 ml centrifuge tube, add the diluted blood sample, and close the centrifuge The machine brakes and centrifuges at 1200g for 10 minutes at room temperature. After centrifugation, the white membrane layer is found, which contains the enriched PBMC cells. Aspirate the white membrane layer and transfer it to a new centrifuge tube.
- the red blood cell lysate (Sigma) to lyse the red blood cells at room temperature.
- heat-inactivated medium 1640 (hereinafter referred to as heat-inactivated medium) containing 2% fetal bovine serum, and the cells were resuspended.
- the Fc-mutated humanized antibody Hu330-1-5M can kill tumor cells NUGC4 in vitro, and its ADCC killing effect is dose-dependent with antibody concentration.
- the calculated EC50 (0.3nM) is higher than that of IMAB362-similar-hG1 positive antibody 8 times; at the same time, the maximum killing rate of Hu330-1-5M to tumor cells is 130%, compared with 80% of positive antibodies, showing significantly enhanced ADCC killing effect in vitro.
- the Fc-mutated Hu330-1-5M humanized antibody on the one hand, has a 15-fold higher affinity for the targeted binding ability to CLDN18.2 than the positive antibody IMAB362-similar-hG1; on the other hand After Fc optimization, ADCC's ability to kill tumor cells in vitro has been further enhanced. Its EC50 activity is 8 times higher than that of positive antibodies, and its maximum killing effect is increased by more than 60%.
- the CLDN18.2 antibody provided by the present invention has significant binding activity and affinity to CLDN18.2, and at the same time has a highly effective ADCC killing activity against tumor cells in vitro, suggesting that the antibody has a highly effective antitumor effect and can be used in the preparation
- the application in antitumor drugs has broad market prospects.
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Abstract
Description
序号 | 抗体名称 | 重链可变区 | 轻链可变区 |
1 | 301CHI | SEQ ID NO 50 | SEQ ID NO 70 |
2 | 302CHI | SEQ ID NO 50 | SEQ ID NO 71 |
3 | 303CHI | SEQ ID NO 72 | SEQ ID NO 73 |
4 | 304CHI | SEQ ID NO 74 | SEQ ID NO 75 |
5 | 305CHI | SEQ ID NO 50 | SEQ ID NO 76 |
6 | 306CHI | SEQ ID NO 77 | SEQ ID NO 78 |
7 | 307CHI | SEQ ID NO 74 | SEQ ID NO 70 |
8 | 308CHI | SEQ ID NO 74 | SEQ ID NO 71 |
9 | 309CHI | SEQ ID NO 74 | SEQ ID NO 76 |
10 | 312CHI | SEQ ID NO 72 | SEQ ID NO 76 |
11 | 313CHI | SEQ ID NO 77 | SEQ ID NO 70 |
12 | 315CHI | SEQ ID NO 77 | SEQ ID NO 76 |
13 | 319CHI | SEQ ID NO 79 | SEQ ID NO 70 |
14 | 321CHI | SEQ ID NO 79 | SEQ ID NO 76 |
15 | 322CHI | SEQ ID NO 50 | SEQ ID NO 80 |
16 | 324CHI | SEQ ID NO 50 | SEQ ID NO 81 |
17 | 328CHI | SEQ ID NO 79 | SEQ ID NO 80 |
18 | 330CHI | SEQ ID NO 79 | SEQ ID NO 81 |
Claims (16)
- 一种抗Claudin18.2抗体或其抗原结合片段,其包含重链可变区和轻链可变区,其中:(1)所述重链可变区包含HCDR1、HCDR2和HCDR3,其中:(a1)所述HCDR1的氨基酸序列选自SEQ ID NO 101、SEQ ID NO 102、SEQ ID NO 103和与它们具有至少85%序列同一性的氨基酸序列;(a2)所述HCDR2的氨基酸序列选自SEQ ID NO 104、SEQ ID NO 105和与它们具有至少85%序列同一性的氨基酸序列;(a3)所述HCDR3的氨基酸序列选自SEQ ID NO 106、SEQ ID NO 107和与它们具有至少85%序列同一性的氨基酸序列;和(2)所述轻链可变区包含LCDR1、LCDR2和LCDR3,其中:(a4)所述LCDR1的氨基酸序列为SEQ ID NO 108;(a5)所述LCDR2的氨基酸序列选自SEQ ID NO 109、SEQ ID NO 110、SEQ ID NO 111和与它们具有至少85%序列同一性的氨基酸序列;(a6)所述LCDR3的氨基酸序列选自SEQ ID NO 112、SEQ ID NO 113、SEQ ID NO 114、SEQ ID NO 115、SEQ ID NO 116和与它们具有至少85%序列同一性的氨基酸序列。
- 如权利要求1所述的抗Claudin18.2抗体或其抗原结合片段,其中:(1)所述重链可变区包含氨基酸序列如SEQ ID NO 101所示的HCDR1、氨基酸序列如SEQ ID NO 105所示的HCDR2和氨基酸序列如SEQ ID NO 106所示的HCDR3;和(2)所述轻链可变区包含氨基酸序列如SEQ ID NO 108所示的LCDR1、氨基酸序列如SEQ ID NO 111所示的LCDR2和氨基酸序列如SEQ ID NO 115所示的LCDR3。
- 如权利要求1或2所述的抗Claudin18.2抗体或其抗原结合片段,其中所述抗体是鼠源抗体、嵌合抗体或人源化抗体。
- 如权利要求1-3之任一项所述的抗Claudin18.2抗体或其抗原结合片段,其中:(1)所述重链可变区的氨基酸序列选自:(b1)如SEQ ID NO 50、SEQ ID NO 72、SEQ ID NO 74、SEQ ID NO 77、SEQ ID NO 79、SEQ ID NO 82、SEQ ID NO 88所示的氨基酸序列;(b2)(b1)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(b1)所示的氨基酸序列功能相同或相似的氨基酸序列;和(b3)与(b1)所示的氨基酸序列具有至少80%序列同一性的氨基酸序列;和(2)所述轻链可变区的氨基酸序列选自:(b4)如SEQ ID NO 51、SEQ ID NO 70、SEQ ID NO 71、SEQ ID NO 73、SEQ ID NO 75、SEQ ID NO 76、SEQ ID NO 78、SEQ ID NO 80、SEQ ID NO 81、SEQ ID NO 84、SEQ ID NO 86、SEQ ID NO 90所示的氨基酸序列;(b5)(b4)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(b4)所示的氨基酸序列功能相同或相似的氨基酸序列;和(b6)与(b4)所示的氨基酸序列具有至少80%序列同一性的氨基酸序列。
- 如权利要求4所述的抗Claudin18.2抗体或其抗原结合片段,其中所述重链可变区的氨基酸序列为SEQ ID NO 79,SEQ ID NO 79经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO 79功能相同的氨基酸序列或与SEQ ID NO 79具有至少85%序列同一性的氨基酸序列,且所述轻链可变区的氨基酸序列为SEQ ID NO 81,SEQ ID NO 81经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO 81功能相同的氨基酸序列或与SEQ ID NO 81具有至少85%序列同一性的氨基酸序列。
- 如权利要求4所述的抗Claudin18.2抗体或其抗原结合片段,其中:(1)所述重链可变区的氨基酸序列选自:(c1)如SEQ ID NO 82、SEQ ID NO 88所示的氨基酸序列;(c2)(c1)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(c1)所示的氨基酸序列功能相同或相似的氨基酸序列;和(c3)与(c1)所示的氨基酸序列具有至少80%序列同一性的氨基酸序列;和(2)所述轻链可变区的氨基酸序列选自:(c4)SEQ ID NO 84、SEQ ID NO 86、SEQ ID NO 90所示的氨基酸序列;(c5)(c4)所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的、且与(c4)所示的氨基酸序列功能相同或相似的氨基酸序列;和(c6)与(c4)所示的氨基酸序列具有至少80%序列同一性的氨基酸序列。
- 如权利要求6所述的抗Claudin18.2抗体或其抗原结合片段,其中:所述重链可变区的氨基酸序列为SEQ ID NO 82,SEQ ID NO 82经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO 82功能相同的氨基酸序列或与SEQ ID NO 82具有至少85%序列同一性的氨基酸序列,且所述轻链可变区的氨基酸序列为SEQ ID NO 84,SEQ ID NO 84经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO 84功能相同的氨基酸序列或与SEQ ID NO 84具有至少85%序列同一性的氨基酸序列;所述重链可变区的氨基酸序列为SEQ ID NO 82,SEQ ID NO 82经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO 82功能相同的氨基酸序列或与SEQ ID NO 82具有至少85%序列同一性的氨基酸序列,且所述轻链可变区的氨基酸序列为SEQ ID NO 86,SEQ ID NO 86经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO 86功能相同的氨基酸序列或与SEQ ID NO 86具有至少85%序列同一性的氨基酸序列;所述重链可变区的氨基酸序列为SEQ ID NO 88,SEQ ID NO 88经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO 88功能相同的氨基酸序列或与SEQ ID NO 88具有至少85%序列同一性的氨基酸序列,且所述轻链可变区的氨基酸序列为SEQ ID NO 84,SEQ ID NO 84经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO 84功能相同的氨基酸序列或与SEQ ID NO 84具有至少85%序列同一性的氨基酸序列;所述重链可变区的氨基酸序列为SEQ ID NO 88,SEQ ID NO 88经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO 88功能相同的氨基酸序列或与SEQ ID NO 88具有至少85%序列同一性的氨基酸序列,且所述轻链可变区的氨基酸序列为SEQ ID NO 86,SEQ ID NO 86经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO 86功能相同的氨基酸序列或与SEQ ID NO 86具有至少85%序列同一性的氨基酸序列;所述重链可变区的氨基酸序列为SEQ ID NO 82,SEQ ID NO 82经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO 82功能相同的氨基酸序列或与SEQ ID NO 82具有至少85%序列同一性的氨基酸序列,且所述轻链可变区的 氨基酸序列为SEQ ID NO 90,SEQ ID NO 90经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO 90功能相同的氨基酸序列或与SEQ ID NO 90具有至少85%序列同一性的氨基酸序列;或所述重链可变区的氨基酸序列为SEQ ID NO 88,SEQ ID NO 88经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO 88功能相同的氨基酸序列或与SEQ ID NO 88具有至少85%序列同一性的氨基酸序列,且所述轻链可变区的氨基酸序列为SEQ ID NO 90,SEQ ID NO 90经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO 90功能相同的氨基酸序列或与SEQ ID NO 90具有至少85%序列同一性的氨基酸序列。
- 如权利要求7所述的抗Claudin18.2抗体或其抗原结合片段,其中:所述重链氨基酸序列为SEQ ID NO 92,SEQ ID NO 92经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO 92功能相同的氨基酸序列或与SEQ ID NO 92具有至少85%序列同一性的氨基酸序列,且所述轻链氨基酸序列为SEQ ID NO 94,SEQ ID NO 94经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO 94功能相同的氨基酸序列或与SEQ ID NO 94具有至少85%序列同一性的氨基酸序列;或所述重链氨基酸序列为SEQ ID NO 96,SEQ ID NO 96经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO 96功能相同的氨基酸序列或与SEQ ID NO 96具有至少85%序列同一性的氨基酸序列,且所述轻链氨基酸序列为SEQ ID NO 98,SEQ ID NO 98经取代、缺失或添加一个或多个氨基酸获得的且与SEQ ID NO 98功能相同的氨基酸序列或与SEQ ID NO 98具有至少85%序列同一性的氨基酸序列。
- 一种分离的核苷酸,其编码权利要求1-8之任一项所述的抗Claudin18.2抗体或其抗原结合片段。
- 如权利要求9所述的核苷酸,其中:(1)编码所述重链氨基酸序列的核苷酸序列如SEQ ID NO 93或SEQ ID NO 97所示;和(2)编码所述轻链氨基酸序列的核苷酸序列如SEQ ID NO 95或SEQ ID NO 99所示。
- 一种表达载体,其包含如权利要求9或10所述的核苷酸。
- 一种宿主细胞,其转化如权利要求11所述的表达载体,所述宿主细胞选自原核细胞和真核细胞,优先为哺乳动物细胞。
- 制备权利要求1-8任一项所述的抗Claudin18.2抗体或其抗原结合片段的方法,包括在如权利要求12所述的宿主细胞中表达抗体,以及从宿主细胞中分离所述抗体的步骤。
- 一种药物组合物,其包含权利要求1-8之任一项所述的抗Claudin18.2抗体或其抗原结合片段和药学可接受的载体。
- 如权利要求1-8之任一项所述的抗Claudin18.2抗体或其抗原结合片段在制备靶向Claudin18.2的药物中的应用。
- 如权利要求15所述的应用,所述靶向Claudin18.2的药物用于治疗胃癌、食管癌、胰腺癌、肺癌、卵巢癌、结肠癌、肝癌、头颈癌和胆囊癌。
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Cited By (7)
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CN111704669A (zh) * | 2020-07-13 | 2020-09-25 | 北京凯因科技股份有限公司 | 一种用于治疗晚期胃癌的抗cldn18全人源化抗体 |
CN111777681A (zh) * | 2020-07-06 | 2020-10-16 | 上海岺樾生物医药科技有限公司 | 一种结合紧密连接蛋白-18.2的抗体及其用途 |
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CN111704669A (zh) * | 2020-07-13 | 2020-09-25 | 北京凯因科技股份有限公司 | 一种用于治疗晚期胃癌的抗cldn18全人源化抗体 |
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WO2022068914A1 (zh) * | 2020-09-30 | 2022-04-07 | 江苏恒瑞医药股份有限公司 | 一种含抗体药物偶联物的药物组合物及其用途 |
WO2022122709A1 (en) | 2020-12-07 | 2022-06-16 | Sotio Biotech A.S. | Antibody-drug conjugates based on humanized cldn18.2 antibodies |
WO2022136642A1 (en) | 2020-12-23 | 2022-06-30 | Sotio Biotech A.S. | Tumor-specific claudin 18.2 antibody-drug conjugates |
WO2022253284A1 (zh) | 2021-06-02 | 2022-12-08 | 百奥泰生物制药股份有限公司 | 药物偶联物及其用途 |
CN114836388A (zh) * | 2022-06-07 | 2022-08-02 | 江苏亲科生物研究中心有限公司 | Claudin18.2单克隆抗体及其制备方法和用途 |
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CN110862454A (zh) | 2020-03-06 |
CA3110593A1 (en) | 2020-03-05 |
US20220119517A1 (en) | 2022-04-21 |
KR20210050547A (ko) | 2021-05-07 |
CN112654638A (zh) | 2021-04-13 |
CN112654638B (zh) | 2022-12-30 |
EP3878863A4 (en) | 2022-06-22 |
EP3878863A1 (en) | 2021-09-15 |
JP2021535744A (ja) | 2021-12-23 |
TW202023613A (zh) | 2020-07-01 |
CN110862454B (zh) | 2022-12-30 |
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