WO2021115303A1 - Anticorps monoclonal anti-claudin18.2, son procédé de préparation et son utilisation - Google Patents

Anticorps monoclonal anti-claudin18.2, son procédé de préparation et son utilisation Download PDF

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WO2021115303A1
WO2021115303A1 PCT/CN2020/134775 CN2020134775W WO2021115303A1 WO 2021115303 A1 WO2021115303 A1 WO 2021115303A1 CN 2020134775 W CN2020134775 W CN 2020134775W WO 2021115303 A1 WO2021115303 A1 WO 2021115303A1
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seq
claudin
antibody
monoclonal antibody
amino acid
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PCT/CN2020/134775
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Chinese (zh)
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姜伟东
陈奕颖
曾琪铃
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上海复宏汉霖生物技术股份有限公司
上海复宏汉霖生物制药有限公司
上海复宏汉霖生物医药有限公司
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Priority to CN202080085731.4A priority Critical patent/CN114761433A/zh
Publication of WO2021115303A1 publication Critical patent/WO2021115303A1/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

Definitions

  • the present invention relates to the field of biotechnology, in particular to an anti-Claudin 18.2 monoclonal antibody, its preparation method and application.
  • Humanized antibodies are derived from non-human species, and their sequence similarity with natural antibodies in humans is increased by modifying the protein sequence.
  • the "humanization” process usually involves the development of monoclonal antibodies for use in humans, such as the development of antibodies as anticancer drugs.
  • the humanization process is necessary for the production of specific antibodies in the immune system of non-humans (e.g., mice).
  • the protein sequence of the antibody produced in this way is partially different from the homologous antibody naturally occurring in humans, so it is potentially immunogenic when administered to a human patient.
  • Targeted therapy is one of the main ways to treat cancer with drugs. Others include hormone therapy and cytotoxic chemotherapy. As a form of molecular medicine, targeted therapy prevents the growth of cancer cells by interfering with specific targeted molecules required for canceration and tumor growth, rather than simply interfering with all rapidly dividing cells (such as traditional chemotherapy). Because most drugs used for targeted therapy are biological drugs, when used for cancer treatment, the term "biological therapy” is sometimes synonymous with targeted therapy, which is different from chemotherapy (ie, cytotoxic therapy). However, these methods can be used in combination. Antibody-drug conjugates combine biological and cytotoxic mechanisms into a targeted therapy.
  • Bispecific monoclonal antibody also known as bifunctional antibody
  • BsMAb Bispecific monoclonal antibody
  • BsMabs can be manufactured in a variety of structural forms, and applications for cancer immunotherapy and drug delivery have been explored.
  • GC Gastric cancer
  • AGC advanced gastric cancer
  • trastuzumab a monoclonal antibody targeting HER2
  • trastuzumab Developed by Genentech Trastuzumab is a humanized monoclonal antibody targeting HER2.
  • trastuzumab combined with paclitaxel was approved by the US FDA as a first-line treatment for HER2/neu overexpressing metastatic breast cancer, or as a treatment for HER2/neu overexpressing metastatic breast cancer after at least one chemotherapy cycle Single drug.
  • HER2-directed therapies for HER2-positive breast cancer and non-small cell lung cancer have been approved, including trastuzumab, pertuzumab, T-DM1, lapatinib, and afatin Nylon (tyrosine kinase inhibitor).
  • Claudin is a family of proteins first discovered by Shorichiro Tsukita and others. It is an important part of forming tight junctions of cells. It establishes a paracellular barrier and controls the flow of molecules between cells.
  • the transmembrane domain of Claudin includes N-terminal and C-terminal in the cytoplasm. Different Claudin proteins are expressed on different tissues, and their altered functions are related to the formation of cancer in their respective tissues. It has been shown that Claudin-1 is expressed in colon cancer, Claudin-18 is in gastric cancer, and Claudin-10 has prognostic value in hepatocellular carcinoma. UgurSahin et al.
  • Claudin-18 isoform 2 (CLDN18.2) is a highly selective cell lineage marker, and its expression in normal tissues is strictly limited to epithelial cells differentiated from the gastric mucosa, but in the gastric stem cell area. does not exist.
  • Claudin 18.2 remains in malignant transformation and is expressed in most primary gastric cancers and their metastatic cancer types.
  • ectopic activation of Claudin 18.2 is often observed in pancreatic cancer, esophageal cancer, ovarian cancer and lung cancer.
  • the correlation between Claudin protein and isotype 2, especially gastric cancer and its metastatic cancer, has led to the development of specific antibodies against Claudin 18.2 as a targeted therapy for gastric cancer and other human solid malignancies.
  • Claudiximab is a new type of chimeric IgG1 antibody with high specificity to Claudin 18.2.
  • the clinical phase IIa (MONO) study aims to determine the safety and effectiveness of multiple doses of Claudiximab as a monotherapy in patients with metastatic, refractory, and recurrent gastric or lower esophageal adenocarcinoma.
  • the median PFS was 102 days (95% CI, 70-146 days). All adverse reactions observed were grade 1-3. The most common grade 3 adverse reaction was vomiting, with 31 cases. No grade 4 adverse reactions occurred.
  • the subsequent clinical phase IIb (FAST) study evaluated Claudiximab as a first-line drug in patients with advanced/recurrent gastroesophageal cancer.
  • the patients included in the study were: ⁇ 40% of tumor cells expressing ⁇ 2+ CLDN18.2 (verified by CLAUDETECT TM 18.2 kit), Eastern Cooperative Oncology Group (ECOG) score of 0-1 and not suitable for trastuzumab Treated patients.
  • CLAUDETECT TM 18.2 kit The subsequent clinical phase IIb (FAST) study evaluated Claudiximab as a first-line drug in patients with advanced/recurrent gastroesophageal cancer.
  • the patients included in the study were: ⁇ 40% of tumor cells expressing ⁇ 2+ CLDN18.2 (verified by CLAUDETECT TM 18.2 kit), Eastern Cooperative Oncology Group (ECOG) score of 0-1 and not suitable for trastuzumab Treated patients.
  • ECOG Eastern Cooperative Oncology Group
  • PFS progression-free survival
  • Interleukin 15 is a 12-14kD cytokine discovered by Grabstein et al. in 1994. It can play a role in the body's normal immune response, such as promoting T cells, B cells, and natural killer (NK) cell proliferation.
  • IL-15 belongs to a member of the four small alpha-helical bundle cytokine families (small four-helix bundle cytokine family). IL-15 requires receptor binding to exert its biological activity.
  • the IL-15 receptor is composed of three receptor subunits: IL-15 receptor ⁇ (IL-15R ⁇ ), IL-2 receptor ⁇ (IL-2R ⁇ , also known as IL-15R ⁇ or CD122) and ⁇ c (also known as CD132).
  • IL-15R ⁇ contains a Sushi domain, which can bind to IL-15 and is necessary for the combined IL-15 to perform biological functions.
  • IL-15 forms a complex with the receptor IL-15R ⁇ , it can significantly enhance the biological activity of IL-15.
  • the ability of IL-15/IL-15R ⁇ complex to stimulate memory CD8+ T cell proliferation and maintain its survival is more than 10 times stronger than that of IL-15 alone, and its mechanism may be related to delivery.
  • the first objective of the present invention is to provide a new anti-Claudin 18.2 monoclonal antibody.
  • the second objective of the present invention is to provide a nucleic acid molecule encoding the anti-Claudin 18.2 monoclonal antibody.
  • the third object of the present invention is to provide an expression vector containing the nucleic acid molecule.
  • the fourth object of the present invention is to provide a host cell containing the expression vector.
  • the fifth object of the present invention is to provide a composition containing the anti-Claudin 18.2 monoclonal antibody.
  • the sixth objective of the present invention is to provide the application of the anti-Claudin 18.2 monoclonal antibody.
  • an anti-Claudin 18.2 monoclonal antibody which includes a heavy chain variable region and a light chain variable region, and the heavy chain variable region includes SEQ ID NO. 15, 16,
  • the heavy chain variable region shown in 17, 18 or 19 has HCDR1, HCDR2 and HCDR3 regions with the same sequence, and the light chain variable region comprises the same sequence as the light chain variable shown in SEQ ID NO. 20, 21, 22 or 23.
  • the zones have the same sequence of LCDR1, LCDR2 and LCDR3 zones.
  • the anti-Claudin 18.2 monoclonal antibody includes:
  • LCDR1, LCDR2, LCDR3, the LCDR1 has an amino acid sequence as shown in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10, LCDR2 has the amino acid sequence shown in SEQ ID NO: 11 or SEQ ID NO: 12, and the LCDR3 has the amino acid sequence shown in SEQ ID NO: 13 or SEQ ID NO: 14.
  • the anti-Claudin 18.2 monoclonal antibody includes a heavy chain variable region and a light chain variable region, and the heavy chain variable region has a variable region such as SEQ ID NO: 15, SEQ ID NO: 16, SEQ The amino acid sequence shown in ID NO: 17, SEQ ID NO: 18 or SEQ ID NO: 19, or a sequence with at least 85% homology with the above sequence, such as 85%, 90%, 92%, 94%, 95 %, 96%, 97%, 98%, or 99% homology; the light chain variable region has a sequence such as SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 or SEQ ID NO : The amino acid sequence shown in 23, or a sequence with at least 85% homology with the above sequence, such as 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% homology Source sequence.
  • the anti-Claudin 18.2 monoclonal antibody includes a light chain and a heavy chain, and the heavy chain has such features as SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32 or SEQ ID NO: 34, or a sequence with at least 85% homology to the above sequence, such as 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98 % Or 99% homology;
  • the light chain has an amino acid sequence as shown in SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31 or SEQ ID NO: 33, or has an amino acid sequence with the above sequence A sequence with at least 85% homology, such as a sequence with 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% homology.
  • the anti-Claudin 18.2 monoclonal antibody may be a murine, human, chimeric or humanized antibody, preferably a humanized antibody.
  • the anti-Claudin 18.2 monoclonal antibody is preferably a defucosylated antibody.
  • the anti-Claudin 18.2 monoclonal antibody further includes a Fab fragment comprising the aforementioned heavy chain variable region and light chain variable region, scFv, and the Fab or the scFv binding to Claudin 18.2 Part of the antigen-binding fragment, bispecific antibody or multispecific antibody.
  • nucleic acid molecule encoding any of the aforementioned anti-Claudin 18.2 monoclonal antibodies.
  • the preparation method of the nucleotide molecule of the present invention is a conventional preparation method in the field, and preferably includes the following preparation method: obtain the nucleotide molecule encoding the above-mentioned monoclonal antibody by gene cloning technology such as PCR method, or by The method of artificial full-sequence synthesis obtains the nucleotide molecule encoding the above-mentioned monoclonal antibody.
  • nucleotide sequence encoding the amino acid sequence of the monoclonal antibody can be replaced, deleted, changed, inserted or added as appropriate to provide a polynucleotide homolog.
  • the polynucleotide homologues of the present invention can be prepared by replacing, deleting or adding one or more bases of the monoclonal antibody gene encoding the monoclonal antibody within the scope of maintaining the activity of the antibody.
  • an expression vector containing the aforementioned nucleic acid molecule there is provided an expression vector containing the aforementioned nucleic acid molecule.
  • the expression vector may be a conventional expression vector in the art, which means that it contains appropriate regulatory sequences, such as promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and/or sequences, and other appropriate Expression vector of the sequence.
  • the host cell containing the above-mentioned expression vector.
  • the host cell is a CHO-S cell.
  • a pharmaceutical composition comprising the aforementioned anti-Claudin 18.2 monoclonal antibody of the present invention and a pharmaceutically acceptable carrier.
  • the above composition may further comprise other agents, such as anti-Her2 monoclonal antibody, IL-15, or IL-15/IL-15R ⁇ complex.
  • agents such as anti-Her2 monoclonal antibody, IL-15, or IL-15/IL-15R ⁇ complex.
  • the use of the above-mentioned anti-Claudin 18.2 monoclonal antibody or the above-mentioned pharmaceutical composition in the preparation of a medicine for the treatment of cancer is provided.
  • the cancer is breast cancer, gastric cancer, and pancreatic cancer.
  • the present invention also provides a method for treating cancer using the above-mentioned anti-Claudin 18.2 monoclonal antibody or the above-mentioned pharmaceutical composition containing the antibody.
  • the anti-Claudin 18.2 monoclonal antibody can be used in combination with other cancer treatment methods, including but not limited to: administration of targeted therapeutic agents, radiotherapy, surgery, or hormone removal.
  • the anti-Claudin 18.2 antibody is used in combination with other targeted therapeutic agents, and the preferred targeted therapeutic agent is anti-Her2 monoclonal antibody, IL-15, IL-15/IL-15R ⁇ complex .
  • the order of administration of different treatment methods at different time points may be the same or different; when multiple agents are administered, the time and order of administration of different agents may be the same or different, or in any way Combined administration depends on the clinical treatment plan.
  • the anti-Claudin 18.2 monoclonal antibody of the present invention can specifically bind to human Claudin 18.2, but does not bind to human Claudin 18.1; the present invention finds that the anti-Claudin 18.2 monoclonal antibody can be used in combination with the anti-Her-2 monoclonal antibody. .2 positive and Her-2 positive gastric tumor cells produced more significant killing activity; and it was also found that the addition of IL-15 to the anti-Claudin 18.2 monoclonal antibody can further enhance its ADCC-mediated cytotoxicity on cancer cells.
  • FIG 3 ADCC of human peripheral blood mononuclear cells (PBMC) of the humanized anti-Claudin 18.2 antibody of the present invention ( Figure 3A: h20D5, Figure 3B: h20D5-3) and anti-Her2 antibody (trastuzumab)
  • NC means anti-VEGF antibody (does not bind Claudin 18.2 and Her2).
  • Figure 5A shows the combined application of parent antibody h20D5-3 and mutant h20D5-3mu with trastuzumab and IL15;
  • Figure 5B shows the killing effect of h20D5-3mu, trastuzumab and IL15 in combination
  • Figure 7 The results of the six-week tumor suppression test in a gastric cancer model.
  • variable region of an antibody refers to the variable region (VL) of the antibody light chain or the variable region (VH) of the antibody heavy chain, alone or in combination.
  • VL variable region
  • VH variable region
  • the variable regions of the heavy chain and the light chain each consist of 4 framework regions (FR) connected by 3 complementarity determining regions (CDR) (also called hypervariable regions).
  • FR framework regions
  • CDR complementarity determining regions
  • the CDRs in each chain are held together tightly by FRs and together with the CDRs from the other chain contribute to the formation of the antigen binding site of the antibody.
  • There are at least two techniques for determining CDRs (1) Methods based on cross-species sequence variability (ie, Kabat et al.
  • CDR may refer to a CDR determined by either method or a combination of the two methods.
  • antibody framework or "FR region” refers to a part of a variable domain VL or VH, which serves as a scaffold for the antigen binding loop (CDR) of the variable domain. Essentially, it is a variable domain without CDRs.
  • CDR complementarity determining region
  • HCDR1, HCDR2, HCDR3 three CDRs in each heavy chain variable region and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable region.
  • Any one of various well-known schemes can be used to determine the amino acid sequence boundaries of CDRs, including the "Kabat” numbering rule (see Kabat et al.
  • the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3);
  • the CDR amino acid residues in the chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3).
  • the CDR amino acid numbers in VH are 26-32 (HCDR1), 52-56 (HCDR2) and 95-102 (HCDR3); and the amino acid residue numbers in VL are 26-32 (LCDR1), 50- 52 (LCDR2) and 91-96 (LCDR3).
  • CDR is defined by amino acid residues 26-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3) in human VH and amino acid residues 24-35 in human VL.
  • 34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3) constitute.
  • the CDR amino acid residue numbers in VH are roughly 26-35 (CDR1), 51-57 (CDR2) and 93-102 (CDR3)
  • the CDR amino acid residue numbers in VL are roughly 27-32 (CDR1) ), 50-52 (CDR2) and 89-97 (CDR3).
  • the CDR regions of antibodies can be determined using the program IMGT/DomainGap Align.
  • Claudin 18.2 refers to Claudin 18.
  • the term includes variants, homologs, orthologs and paralogs.
  • IL-15 is a human cytokine with NK cell proliferation and activating activity, and refers to human interleukin 15 and the functionality of human IL-15 extracellular domain or IL-15 extracellular domain. Variants and IL-15/IL-15R ⁇ complexes that retain IL-15 and enhance the immune response.
  • IL-15 functional variants include human IL-15 truncation, amino acid substitutions, deletions and additions and still retain all or part of IL-15 to enhance the immune response variants
  • exemplary IL-15 functional variants include But it is not limited to patent publication numbers WO2008143794A1, WO2012040323A2, US8940288B2, WO2012175222A1, WO2016095642A1, WO2015103928A1, WO2019204592, US201902907 34A1, CA3034912A1, US20190209653A1, US20180312560A1, and US20180200366A1 that retain variants of human IL-15 that have enhanced immune responses.
  • IL-15R ⁇ refers to the ⁇ receptor and its functional variants that can interact with IL-15 to form a complex. After IL-15R ⁇ and IL-15 form a complex, it can enhance the stability of IL-15. To further enhance the immune response effect of IL-15.
  • IL-15R ⁇ functional variant refers to a fragment containing the sushi domain in the extracellular region of IL-15R ⁇ , which retains the interaction with IL-15 and enhances the stability of IL-15.
  • Exemplary IL-15R ⁇ functional variants include, but are not limited to, patent publication numbers WO2008143794A1, WO2012040323A2, US8940288B2, WO2012175222A1, WO2016095642A1, WO2015103928A1, WO2019204592A1, CA3034912A1, US20190290734A1, US20190209653A1, US20180312560A1, and RUS20180200366A1, and other disclosed functional variants of human IL-15 .
  • the chimeric human Claudin 18.2 antibody was transiently expressed in CHO-S cells.
  • polyethyleneimine (PEI) was used to transfect CHO-S cells with the resulting vector, and the ratio of DNA:PEI was 1:3.
  • the total DNA used for transfection was 1.5 ⁇ g/ml.
  • the transfected CHO-S cells were cultured in a 37°C, 5% CO 2 incubator at 120 RPM. After 10-12 days, the cell culture supernatant was collected, centrifuged at 3500 rpm for 5 minutes, and filtered through a 0.22 ⁇ m capsule to remove cell debris to purify the antibody.
  • the antibody was purified using pre-equilibrated Protein-A (GE; USA; Cat#: 17040501; Lot#: 10252250) and eluted with an elution buffer (20mM citric acid, pH3.0-pH3.5). In addition to the buffer exchange, the antibody is stored in PBS buffer (pH 7.0), and its concentration is determined by a NanoDrop instrument. The purified monoclonal antibody was further characterized.
  • Antibodies are screened for affinity matured modified antibodies by phage display
  • clone 20D5 chose to undergo affinity maturation modification through phage display technology. Simply put, 3D structural modeling simulations were performed to identify residues in the heavy and light chain CDRs of clone 20D5 that may be important for binding affinity. The identified CDR residues are mutated by PCR, using primers specially designed for point mutations and standard method steps. A phage display library was constructed, and as described above, CHO-S cells stably overexpressing human Claudin 18.2 or Claudin 18.1 were used for biological screening. After 3 rounds of biological screening, high-binding clones were selected, collected and infected with bacterial cells.
  • Balb/c mice female, 8 weeks old, weighing about 20g, purchased from Shanghai Slack Laboratory Animal Co., Ltd.;
  • Bovine hyaluronidase Sigma H3506; CHO-S cell line: invitrogen;
  • Anti-Her2 antibody Trastuzumab, prepared by our company through cloning and synthesis according to the amino acid sequence.
  • the heavy chain has the amino acid sequence shown in SEQ ID NO: 35
  • the light chain has the amino acid sequence shown in SEQ ID NO: 36 Amino acid sequence
  • Human IL-15 (hIL-15): purchased from Peprotech article number 200-15, the sequence is shown in SEQ ID NO: 37;
  • mIL15Ra-Fc purchased from biolegend, catalog number 761606;
  • Human IL-15R ⁇ The sequence of hIL15R ⁇ (human IL15R ⁇ sushi domain) is shown in SEQ ID NO: 38;
  • Positive control antibody IMAB362 (disclosed from WO2014/146672A1): It was prepared by our company through cloning and synthesis according to the amino acid sequence.
  • the heavy chain amino acid sequence is shown in SEQ ID NO: 39, and the light chain amino acid sequence is shown in SEQ ID NO: 40.
  • mice Eighteen Balb/c mice were immunized with the plasmid expression vector encoding the fully human Claudin 18.2 and electroporated. 100 ⁇ g plasmid and 20U bovine hyaluronidase were injected intramuscularly on day 1, 75 ⁇ g plasmid and 20U bovine hyaluronidase were injected on day 14, and 50 ⁇ g plasmid and 20U bovine were injected on day 28, 42 and 56 respectively. Hyaluronidase, and finally 5 ⁇ 10 6 Claudin18.2 transfected CHO-S cells were injected on the 65th day to strengthen immunity, and splenectomy was performed two days later to produce monoclonal antibodies. The anti-Claudin 18.2 antibody produced in the mouse serum was monitored by flow cytometry (FACS) on 28, 42, 56 and 65 days, respectively.
  • FACS flow cytometry
  • mice numbered No. 2, 8, and 12 were used for the production of CRO hybridomas. Based on the high titer of the anti-Claudin 18.2 antibody and the low titer of the anti-Claudin 18.1 antibody, the resulting hybridomas were screened to produce Claudin 18.2 specific IgG.
  • Antibody 20D5 includes: heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO: 1 or SEQ ID NO: 2, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 3, and the amino acid sequence of HCDR3 is shown in SEQ ID NO: 3.
  • the amino acid sequence is shown in SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6; the light chain complementarity determining region LCDR1, LCDR2 or LCDR3, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO: 7, SEQ ID NO: 8.
  • the amino acid sequence of LCDR2 is shown in SEQ ID NO: 9 or SEQ ID NO: 10
  • the amino acid sequence of LCDR2 is shown in SEQ ID NO: 11 or SEQ ID NO: 12
  • the amino acid sequence of LCDR3 is shown in SEQ ID NO: 13 or SEQ ID NO: 14 shown.
  • the humanized anti-Claudin 18.2 antibody 20D5 (h20D5) was produced using the human germ cell line variable light chain (IGKV4-1*01) and the human germ cell line heavy chain variable region (IGHV4-4*08). In short, humanization is accomplished by grafting the CDR residues from the light and heavy chains of the chimeric antibody 20D5 to the similar light and heavy chain frameworks of human immunoglobulin.
  • Humanized antibody libraries grafted with CDRs can be generated for further affinity maturation based on in vitro phage display to enhance the affinity for their antigens.
  • the heavy chain variable region of each antibody and the antibody heavy chain constant region (SEQ ID NO: 24) are connected to form an antibody full-length heavy chain, and the light chain variable region of each antibody and the antibody light chain constant region (SEQ ID NO: 25) Linked to form the full-length light chain of the antibody.
  • amino acid sequence of part of the formed full-length antibody is shown in Table 1 below:
  • Antibody Heavy chain variable region Light chain variable region Heavy chain Light chain h20D5 SEQ ID NO: 15 SEQ ID NO: 20 SEQ ID NO: 26 SEQ ID NO: 27 h20D5-1 SEQ ID NO: 16 SEQ ID NO: 21 SEQ ID NO: 28 SEQ ID NO: 29 h20D5-2 SEQ ID NO: 17 SEQ ID NO: 22 SEQ ID NO: 30 SEQ ID NO: 31
  • Protein A affinity chromatography to extract fusion protein or antibody with Fc tag Protein A affinity chromatography to extract fusion protein or antibody with Fc tag
  • the supernatant of the cell culture expressing the Fc fusion protein or antibody is centrifuged at a high speed to collect the supernatant.
  • the ProteinA affinity column was washed 3-5 times the column volume with 0.1M NaOH, and then washed 3-5 times the column volume with 1 ⁇ PBS.
  • the cell supernatant is loaded and bound at a low flow rate, and the flow rate is controlled so that the retention time is about 1 min or longer.
  • the column is washed with 1 ⁇ PBS (pH 7.4) by 3-5 times the column volume until the UV absorption falls back to the baseline .
  • the sample was eluted with 0.1M glycine sodium chloride (pH3.0-3.5) buffer, and the elution peaks were collected according to UV detection.
  • the eluted product was quickly adjusted to pH 5-6 with 1M Tris-HCl (pH8.0). Save.
  • the eluted product can be replaced by a method well known to those skilled in the art, such as using an ultrafiltration tube for ultrafiltration and concentration and replacing the solution to the required buffer system, or using molecular exclusion such as G-25 desalting to replace it with the required Buffer system, or use a high-resolution molecular exclusion column such as Superdex 200 to remove the aggregate components in the eluted product to improve the purity of the sample.
  • a method well known to those skilled in the art such as using an ultrafiltration tube for ultrafiltration and concentration and replacing the solution to the required buffer system, or using molecular exclusion such as G-25 desalting to replace it with the required Buffer system, or use a high-resolution molecular exclusion column such as Superdex 200 to remove the aggregate components in the eluted product to improve the purity of the sample.
  • Flow cytometry was used to detect the presence of anti-CLD18 antibodies in the serum of immunized mice or the binding of monoclonal antibodies to living cells expressing CLD18.
  • Table 2 where the binding force is expressed as ++++, +++, ++, +,-from strong to weak.
  • the present invention constructs a Fab phage library for affinity maturation, selects 3 clones by affinity selection, and incubates the supernatant of Fab clone 20D5 hybridoma cells or purified antibodies (h20D5, h20D5-3 and IMAB362, concentration 20 ⁇ g/ ml).
  • the CHO-S cells transfected with Claudin 18.1 or 18.2 were incubated at 4°C for 30 minutes, washed with 2% FBS+PBS buffer, and stained with a secondary antibody FITC-labeled anti-human or mouse Fc antibody.
  • This example analyzes and detects the ability of antibodies to induce antibody-dependent cellular cytotoxicity (ADCC) of NUGC4 (JCRB0834) gastric cancer cells (Claudin 18.2-NUGC4) stably expressing human Claudin 18.2.
  • ADCC antibody-dependent cellular cytotoxicity
  • the target cells (1.5 ⁇ 10 5 /well) were pre-plated on a 96-well plate in RPMI-1640+2% FBS in a 37°C incubator overnight.
  • serially diluted antibodies were added to the 96-well plate containing the target cells and incubated at 37°C for 5 hours.
  • the lysis buffer LH cytotoxicity detection kit DOJINDO MOLECULAR TECHNOLOGIES
  • the maximum release amount is determined by adding lysis buffer to the target cells; the spontaneous release amount is measured in the absence of antibodies and effector cells and only target cells.
  • the combined benefits of the antibodies h20D5 and h20D5-3 and trastuzumab were tested.
  • Trastuzumab was serially diluted (200 ⁇ g/ml, 5-fold dilution) and added to 6 rows of wells.
  • the h20D5 and h20D5-3 antibodies were fixed at different concentrations in each row of wells.
  • the ADCC test results are shown in Figures 3A and 3B.
  • the results show that the combination of anti-Claudin 18.2 antibody and trastuzumab can enhance the ADCC lethality mediated by trastuzumab antibody, and even reach the maximum lethality rate.
  • Example 8 ADCC activity detection of anti-Claudin 18.2 antibody in combination with IL15
  • h20D5 was combined with IL15 (Peprotech Catalog No. 200-15) to test its combined benefits.
  • the h20D5 antibody was serially diluted (200 ⁇ g/ml, 5-fold dilution). The antibody was added to 6 rows of wells.
  • IL15 was Fixed in different concentrations in each row. The results are shown in Table 3 and Figure 4A.
  • +IL15 represents the combination of h20D5 antibody and IL15.
  • Example 9 ADCC activity detection of anti-Claudin 18.2 antibody in combination with anti-Her2 antibody and IL15
  • h20D5-3 mutant has similar combinatorial advantages compared with the parent antibody h20D5-3.
  • Trastuzumab antibody is serially diluted (200 ⁇ g/ml, 5-fold dilution), h20D5-3 or its mutant h20D5-3mu antibody is fixed at 0.016 ⁇ g/ml, and IL15 is fixed at 5ng/ml.
  • Figure 5A and Figure 5B show that the mutant antibody h20D5-3mu of the present invention has a combination advantage comparable to the parent antibody h20D5-3, and the combination of these two antibodies with the anti-Her2 antibody and IL15 can significantly enhance the ADCC lethality.
  • Figure 5B shows that the combination of h20D5-3mu, trastuzumab and IL15 shows a stronger cell killing rate than the combination of trastuzumab and IL15.
  • This example evaluates the drug trastuzumab, h20D5-3, hIL15-mIL15R ⁇ in The anti-tumor effect of gastric cancer GA0006 model Balb/c nude female mouse subcutaneous xenograft tumor model.
  • BALB/c nude mice were subcutaneously inoculated with GA0006 model tumor masses to establish Gastric cancer GA0006 tumor model.
  • the experiment is divided into 6 groups, and the drug dosage and administration method are shown in Table 4 below. Except for the 6 rats in the fourth group, 8 rats in each group were administered by intraperitoneal injection, twice a week, for a total of 8 times for 4 weeks. After the dosing cycle is completed (see Figure 6 for the results), select 4 mice in each of group 1, group 3, group 4, and group 6, and extend the administration for 2 weeks, where hIL15-mIL15R ⁇ is changed to BIW administration. Medicine (see Figure 7 for the results). Efficacy was evaluated based on the relative tumor inhibition rate (TGI%), and the safety was evaluated based on changes in animal weight and death.
  • TGI% tumor inhibition rate
  • the h20D5-3, hlL15-mIL15R ⁇ , and trastuzumab single-agent groups did not show tumor-inhibiting effects compared with the control group.
  • the trastuzumab+h20D5-3 combination group also failed to show tumor suppressive effect.
  • the combination of trastuzumab+h20D5-3+hlL15-mIL15R ⁇ showed a slight anti-tumor effect, but did not reach statistical difference.
  • Test drug trastuzumab, h20D5-3, hlL15-mIL15R ⁇ , in the gastric cancer GA0006 model because the model is a cachexia model, individual mice in each group have lost weight, but no animal died in each treatment group, and there was no obvious drug toxicity, and it was well tolerated during the treatment.
  • test drug h20D5-3 (20mg/kg), hIL15-mIL15R ⁇ (2 ⁇ g+9 ⁇ g/mouse) and trastuzumab (2mg/kg) combined treatment Gastric cancer GA0006 model has a significant inhibitory effect on tumor growth (P ⁇ 0.01). Tumor-bearing mice tolerated well to h20D5-3, hIL15-mIL15R ⁇ and trastuzumab.

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Abstract

L'invention concerne un anticorps monoclonal anti-Claudin18.2 et son utilisation. L'invention concerne également une molécule d'acide nucléique codant pour l'anticorps, un vecteur d'expression et une cellule hôte pour exprimer l'anticorps, et une composition pharmaceutique contenant l'anticorps. L'invention concerne en outre l'utilisation de l'anticorps seul ou en combinaison avec d'autres agents, tels qu'un anticorps anti-Her2 et/ou IL15, dans la préparation d'un médicament anticancéreux.
PCT/CN2020/134775 2019-12-11 2020-12-09 Anticorps monoclonal anti-claudin18.2, son procédé de préparation et son utilisation WO2021115303A1 (fr)

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