WO2020228447A1 - 抗cldn抗体及其药物组合物和检测方法 - Google Patents

抗cldn抗体及其药物组合物和检测方法 Download PDF

Info

Publication number
WO2020228447A1
WO2020228447A1 PCT/CN2020/083570 CN2020083570W WO2020228447A1 WO 2020228447 A1 WO2020228447 A1 WO 2020228447A1 CN 2020083570 W CN2020083570 W CN 2020083570W WO 2020228447 A1 WO2020228447 A1 WO 2020228447A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
antibody
cells
screened
tumor
Prior art date
Application number
PCT/CN2020/083570
Other languages
English (en)
French (fr)
Inventor
屈向东
潘琴
金后聪
都业杰
郑翰
Original Assignee
启愈生物技术上海有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 启愈生物技术上海有限公司 filed Critical 启愈生物技术上海有限公司
Priority to BR112021022757A priority Critical patent/BR112021022757A2/pt
Priority to JP2021568696A priority patent/JP7249696B2/ja
Priority to US17/611,370 priority patent/US20220235128A1/en
Priority to EP20804959.3A priority patent/EP3971207A4/en
Priority to KR1020217041245A priority patent/KR20220010002A/ko
Publication of WO2020228447A1 publication Critical patent/WO2020228447A1/zh

Links

Images

Classifications

    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/515—Complete light chain, i.e. VL + CL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants

Definitions

  • the present invention relates to an anti-CLDN antibody.
  • the present invention also relates to a pharmaceutical composition containing the anti-CLDN antibody.
  • the present invention also relates to a method for detecting the presence of CLDN in a biological sample.
  • Tight junction plays a key role in the flow of material between cells. It also maintains cell polarity by blocking the radial diffusion of membrane proteins and membrane lipids. It also participates in the recruitment of signal molecules that regulate cell proliferation, differentiation and movement. . Tight junctions are formed by Claudin (CLDN), and the Claudin family consists of more than 20 protein molecules, all of which contain a four-time transmembrane domain and similar amino acid sequences, but the tissue distribution has a certain specificity Sex. Human CLDN genes are distributed in pairs on different chromosomes, which implies that some CLDN genes are derived from gene duplication.
  • the molecular weight of CLDN protein is mostly in the range of 20-34kDa. The biggest difference is the sequence and size of the intracellular C-terminus. This sequence contains a PDZ domain binding motif, which allows CLDN protein to directly interact with the cytoplasm. Link related proteins such as ZO-1, ZO-2, ZO-3 and MUPP1 interact. In addition, this sequence contains post-transcriptional modification sites such as phosphorylation sites, which can affect the positioning and function of protein molecules.
  • MAPK mitogen-activated protein kinase
  • PKC protein kinase C
  • CLDN5 phosphorylation can promote the barrier function of CLDN protein
  • PKA-mediated CLDN16 phosphorylation can enhance magnesium Ion transport.
  • CLDN plays a key role in regulating the selective penetration of the cell bypass.
  • CLDN2 and CLDN15 participate in the formation of cation channels and cation pores, and CLDN4/7/10 participates in the formation of anion channels and pores.
  • Claudin protein is highly expressed in some cell lines, which affects transmembrane resistance and permeability. In cultured epidermal-derived cells, CLDN1/4/5/7 can increase transmembrane resistance, but CLDN2 and CLDN10 do the opposite.
  • CLDN gene mutations are thought to be related to many diseases. CLDN1 mutations may cause sclerosing cholangitis and ichthyosis, CLDN16 and CLDN19 mutations are thought to be related to hypomagnesemia and hypercalcinuria.
  • CLDN1 and CLDN7 are down-regulated in invasive breast cancer, prostate cancer and esophageal cancer, while CLDN3/4 are found to be up-regulated to varying degrees in cervical cancer, colon cancer, esophageal cancer, gastric cancer and other cancers.
  • Sahin et al. found that in normal tissues, the isoform 2 subtype of CLDN18 (CLDN18.2) is only expressed in differentiated epidermal cells of the gastric mucosa, but not expressed in the area of gastric stem cells, but in primary gastric cancer and its metastases Abnormally high expression was found in both.
  • the purpose of the present invention is to provide an anti-CLDN antibody and its pharmaceutical composition and detection method.
  • An anti-CLDN antibody characterized in that it comprises a heavy chain and a light chain:
  • the heavy chain of the antibody contains one or more CDRs, and the CDRs of the heavy chain correspond to the CDRs in any one of SEQ ID No. 1 to SEQ ID No. 7 or SEQ ID No. 15 to SEQ ID No. 30 Compared with the sequence, it is almost three amino acids,
  • the light chain of an antibody contains one or more than one CDR, and the CDR of the light chain corresponds to the CDR sequence of any one of SEQ ID No. 8 to SEQ ID No. 14 or SEQ ID No. 31 to SEQ ID No. 46 In comparison, it is almost three amino acids.
  • the anti-CLDN antibody of the present invention also has the feature that the heavy chain of the antibody is selected from any one of SEQ ID No. 1 to No. 7.
  • the anti-CLDN antibody of the present invention also has the feature that the light chain of the antibody is selected from any one of SEQ ID No. 8 to SEQ ID No. 14 or SEQ ID No. 31 to SEQ ID No. 46.
  • the anti-CLDN antibody of the present invention also has the feature that the heavy chain of the antibody is selected from any one of SEQ ID No. 15 to SEQ ID No. 30.
  • the anti-CLDN antibody of the present invention also has the characteristic that the light chain of the antibody is selected from any one of SEQ ID No. 31 to SEQ ID No. 46.
  • the anti-CLDN antibody of the present invention also has the characteristic that the combination of the heavy chain and light chain of the antibody is:
  • the present invention also provides a polynucleotide encoding the antibody as described above.
  • the present invention also provides a pharmaceutical composition comprising the antibody described in any one of the above.
  • the present invention also provides the application of any one of the above antibodies in the preparation of anti-tumor drugs.
  • the present invention also provides a method for detecting the presence of CLDN in a biological sample, which is characterized in that it comprises the step of administering the antibody of any one of the above to the biological sample, wherein the antibody has a detectable marker, and detecting whether There is the detectable marker, or the step of detecting the content of the detectable marker.
  • the anti-CLDN antibody of the present invention has a stronger ability to bind to cells than IMAB362.
  • the antibody of the present invention has a better effect of inhibiting tumor growth than IMAB362 in animal efficacy in vivo.
  • Figure 1a shows the binding ability of control antibody QP024025 to CHOS cells
  • Figure 1b shows the binding ability of antibody QP188189 screened by hybridoma to CHOS cells
  • Figure 1c shows the ability of the antibody QP190191 screened by hybridomas to bind to CHOS cells
  • Figure 1d shows the ability of the antibody QP192193 screened by hybridomas to bind to CHOS cells
  • Figure 1e shows the ability of the antibody QP196198 screened by hybridomas to bind to CHOS cells
  • Figure 1f shows the ability of the antibody QP199200 screened by hybridomas to bind to CHOS cells
  • Figure 1g shows the binding ability of the antibody QP201202 screened by hybridomas to CHOS cells
  • Figure 1h shows the ability of the antibody QP207208 screened by hybridomas to bind to CHOS cells
  • Figure 2a shows the ability of the antibody QP10731074 screened by phage to bind to CHOS cells.
  • Figure 2b shows the ability of the antibody QP10791080 screened by phage to bind to CHOS cells.
  • Figure 2c shows the ability of the antibody QP10851086 screened by phage to bind to CHOS cells.
  • Figure 2d shows the binding ability of antibody QP10911092 screened by phage to CHOS cells.
  • Figure 2e shows the ability of the antibody QP10971098 screened by phage to bind to CHOS cells.
  • Figure 2f shows the binding ability of the antibody QP10991100 screened by phage to CHOS cells.
  • Figure 3a shows the ability of the antibody QP11051106 screened by phage to bind to CHOS cells.
  • Figure 3b shows the ability of the antibody QP11071108 screened by phage to bind to CHOS cells.
  • Figure 3c shows the binding ability of the antibody QP11091110 screened by phage to CHOS cells.
  • Figure 3d shows the binding ability of the antibody QP11111112 screened by phage to CHOS cells.
  • Figure 3e shows the ability of the antibody QP11131114 screened by phage to bind to CHOS cells.
  • Figure 3f shows the binding ability of the antibody QP11151116 screened by phage to CHOS cells.
  • Figure 4a shows the ability of the antibody QP11171118 screened by phage to bind to CHOS cells.
  • Figure 4b shows the ability of the antibody QP11031104 screened by phage to bind to CHOS cells.
  • Figure 4c shows the binding ability of the antibody QP10451046 screened by phage to CHOS cells.
  • Figure 4d shows the ability of the antibody QP10471048 screened by phage to bind to CHOS cells.
  • Figure 5a shows the binding ability of the control antibody QP024025 to different 293T transient cell lines.
  • Figure 5b shows the binding ability of antibody QP188189 screened by hybridomas to different 293T transient cell lines
  • Figure 5c shows the binding ability of antibody QP190191 screened by hybridoma to different 293T transient cell lines
  • Figure 5d shows the binding ability of antibody QP192193 screened by hybridomas to different 293T transient cell lines.
  • Figure 5e shows the binding ability of the antibody QP196198 screened by hybridomas to different 293T transient cell lines.
  • Figure 5f shows the binding ability of antibody QP199200 screened by hybridomas to different 293T transient cell lines.
  • Figure 5g shows the binding ability of antibody QP201202 screened by hybridomas to different 293T transient cell lines.
  • Figure 5h shows the binding ability of the antibody QP207208 screened by hybridomas to different 293T transient cell lines.
  • Figure 6a shows the binding ability of antibody QP10451046 screened by phage to different 293T transient cell lines.
  • Figure 6b shows the binding ability of antibody QP10711072 screened by phage to different 293T transient cell lines.
  • Figure 6c shows the binding ability of antibody QP10731074 screened by phage to different 293T transient cell lines.
  • Figure 6d shows the binding ability of antibody QP10851086 screened by phage to different 293T transient cell lines.
  • Figure 6e shows the binding ability of antibody QP10911092 screened by phage to different 293T transient cell lines.
  • Figure 6f shows the binding ability of antibody QP10991100 screened by phage to different 293T transient cell lines.
  • Figure 6g shows the binding ability of antibody QP11031104 screened by phage to different 293T transient cell lines.
  • Figure 6h shows the binding ability of antibody QP11051106 screened by phage to different 293T transient cell lines.
  • Figure 7a shows the binding ability of antibody QP11071108 screened by phage to different 293T transient cell lines.
  • Figure 7b shows the binding ability of antibody QP11091110 screened by phage to different 293T transient cell lines.
  • Figure 7c shows the binding ability of antibody QP11111112 screened by phage to different 293T transient cell lines.
  • Figure 7d shows the binding ability of antibody QP11131114 screened by phage to different 293T transient cell lines.
  • Figure 7e shows the binding ability of antibody QP11151116 screened by phage to different 293T transient cell lines.
  • Figure 7f shows the binding ability of antibody QP11171118 screened by phage to different 293T transient cell lines.
  • Figure 8a is the curve of IMAB362 of the control group in the experiment of binding ability of control antibody to gastric cancer PDX model GA0006 tumor cells.
  • Fig. 8b is the curve of QP190191 in the experiment of binding ability of antibody to gastric cancer PDX model GA0006 tumor cells.
  • Fig. 8c is the curve of QP192193 in the binding ability experiment of the antibody and gastric cancer PDX model GA0006 tumor cells.
  • Figure 8d is the curve of QP201202 in the experiment of the binding ability of antibodies to gastric cancer PDX model GA0006 tumor cells.
  • Figure 8e is the curve of QP207208 in the experiment of binding ability of antibody to gastric cancer PDX model GA0006 tumor cells.
  • Figure 8f is the curve of QP11091110 in the binding ability experiment of the antibody and gastric cancer PDX model GA0006 tumor cells.
  • Figure 8g is the curve of QP11131114 in the binding ability experiment of the antibody and gastric cancer PDX model GA0006 tumor cells.
  • Fig. 8h is the curve of QP11151116 in the binding ability experiment of the antibody and gastric cancer PDX model GA0006 tumor cells.
  • Fig. 9 is a pharmacodynamic test of antibodies against gastric cancer PDX model GA0006.
  • Fig. 10A is the tumor growth curve after treatment in the PBS group (negative control).
  • Figure 10B is the tumor growth curve after treatment in the QP192193 group.
  • Fig. 10C is the tumor growth curve after treatment in the QP207208 group.
  • Figure 10D is the tumor growth curve after treatment in the QP11151116 group.
  • Figure 10E is the tumor growth curve after treatment in the control antibody IMAB362 group.
  • Figure 11 shows the weight of tumor D31 in each group of mice added with antibodies of the present invention and control antibodies.
  • Figure 12 is a real shot diagram of tumor volume in each experimental group.
  • Figure 13 is the body weight curve of each group of mice.
  • Figure 14 is a curve of the weight change rate of each group of mice.
  • the antibodies of the present invention include but are not limited to human antibodies.
  • the more convenient hybridoma screening antibody is usually preferred.
  • the antigen of this target claudin 18.2
  • the phage library is also used to screen antibodies.
  • the sequence of the anti-claudin18.2 antibody screened in this embodiment is as follows:
  • VH variable region of the heavy chain
  • VL light chain variable region
  • VH variable region of the heavy chain
  • VL light chain variable region
  • a) Prepare a gentleMACS TM C Tube and 3ml digestion solution for each tumor.
  • the digestion solution is prepared according to the instructions of the Tumor Dissociation Kit (miltenyibiotech, 130-096-730), and it is ready for use;
  • mice were killed, the tumors were removed with clean tools, and the blood vessels, fat, fascia and other tissues attached to the tumor surface were removed after washing with 1 ⁇ PBS.
  • Each digestive tube can digest tumor tissue no larger than 0.8g to ensure that the tumor tissue is completely digested by the digestive juice;
  • hybridoma clone in CHOS system and phage clone in CHOS system in the experimental drawings respectively refer to the ability of antibodies screened by hybridomas and phage to bind to CHOS cells.
  • CHO18.2 CHOS-CLDN18.2 stably transfected cells
  • CHO18.1 CHOS-CLDN18.1 stably transfected cells
  • CHOS CHOS transfected with an empty vector.
  • CHOS is a cell line obtained by immortalizing hamster ovary cells. FACS (Flow Cytometry) is used to detect the ability of antibodies to bind to the cell line.
  • Figures 1a to 1h show the binding ability of antibodies screened by hybridomas to CHOS cells.
  • Figures 2a to 2h, Figures 3a to 3h, and Figures 4a to 4d show the binding ability of antibodies screened by phage to CHOS cells.
  • each panel is composed of a combination of the FACS test results of three CHOS cell lines with a test antibody, in which:
  • the red curve 3 indicates the binding ability of the test antibody to the CHOS cell line (CHOS) transfected with the empty vector, that is, the negative control;
  • the blue curve 2 indicates the binding ability of the antibody to be tested on the CHOS cell line transfected with claudin 18.1 (CHO18.1), that is, non-specific binding is detected;
  • Yellow curve 1 indicating the binding ability of the antibody to be tested on the CHOS cell line transfected with claudin 18.2 (CHO18.2), that is, the binding ability of the target protein to be detected.
  • Figures 5a to 5h show the binding ability of antibodies screened by hybridomas to 293T cells.
  • Figures 6a to 6h and Figures 7a to 7f show the binding ability of antibodies screened by phage to CHOS cells.
  • 293T-QD210 293T-CLDN18.2-18.1ECD1 transiently transformed cells; the first extracellular domain of CLDN18.2 was replaced with the first extracellular domain of 18.1
  • Hybridom clone in 293T and phage clone in 293T similarly refer to the ability of antibodies screened by hybridoma and phage to bind different 293T transient cell lines.
  • the red curve 5 indicates the binding ability of the antibody to be tested to the 293T cell line with transient empty vector, that is, the negative control;
  • the blue curve 4 indicating the binding of the antibody to be tested to the 293T cell line (293T-QD010) transiently transfected with claudin 18.2, to detect the binding ability of the target protein;
  • the orange curve 3 indicates the binding of the antibody to be tested to the transiently transfected claudin18.1 293T cell line (293T-QD012) to detect non-specific binding;
  • Figures 8a to 8h show the binding ability experiment of the antibody with gastric cancer PDX model GA0006 tumor cells.
  • Table 1 The binding ratio of antibodies to gastric cancer PDX model GA0006 tumor cells
  • the present invention also provides a polynucleotide encoding the antibody as described above.
  • the polynucleotide encoding the antibody provided by the present invention is provided in the form of DNA, it may be included in The non-coding sequence that will be removed in the subsequent transcription and editing process may also only include the sequence encoding the antibody provided in the embodiment of the present invention and the sequence necessary for protein expression.
  • the present invention also provides a pharmaceutical composition containing the antibody described in any one of the above.
  • the pharmaceutical composition provided by the present invention may only contain any one or at least two of the antibodies provided in the embodiments. .
  • the present invention also provides a method for detecting the presence of CLDN in a biological sample, which is characterized in that it comprises:
  • mice were randomly divided into groups of 6 mice. Weigh the body weight of all animals and measure the tumor volume with vernier calipers. According to the tumor volume, a random grouping method is used to group to ensure that the tumor volume is similar between different groups.
  • Dose (mg/kg) Mode of administration Dosing cycle 1 6 Vehicle control - i.p. BIW ⁇ 4 weeks 2 6 QP192193 10 i.p. BIW ⁇ 4 weeks 3 6 QP207208 10 i.p. BIW ⁇ 4 weeks 4 6 QP11151116 10 i.p. BIW ⁇ 4 weeks 5 6 IMAB362 10 i.p. BIW ⁇ 4 weeks
  • the administration volume is 10 ⁇ L/g.
  • the meaning of the antibody numbers in Table 1 and Table 2, for example, QP190191, means the combination of a heavy chain and a light chain.
  • tumor volume (mm 3 ) 1/2 ⁇ (a ⁇ b 2 ) (where a represents the long diameter and b represents the short diameter).
  • the experiment was terminated one week after the last administration, the mice were sacrificed, the tumors were taken out, weighed, and photographed.
  • T/C Relative tumor proliferation rate
  • T/C (%) is the percentage value of the treatment group and the control group relative to the tumor volume or tumor weight at a certain point in time.
  • the average tumor volume of mice in the PBS control group was 911.16 ⁇ 177.81 mm 3 on the 31st day after administration.
  • the average tumor volume of antibody molecules QP192193 (10mg/kg), QP207208 (10mg/kg), and QP11151116 (10mg/kg) groups on the 31st day after administration were 580.97 ⁇ 67.97mm 3 , 680.28 ⁇ 193.50mm 3 , and 722.38, respectively ⁇ 118.07mm 3 , TGI were 36.34%, 25.34%, and 20.72%
  • the control molecule IMAB362 (10mg/kg) on the 31st day after administration the average tumor volume was 661.28 ⁇ 104.49mm 3
  • TGI was 27.42% (see below table).
  • the three selected molecules inhibited tumor growth to a certain extent. Although there was no statistically significant difference, QP192193 performed better than the control molecule IMAB362. QP207208 and QP11151116 also showed the same level of inhibition of tumor growth as IMAB362. ability. In addition, the weight of the mice did not decrease significantly during the administration process, indicating that the antibody molecules have no obvious toxic side effects on the mice.
  • the analysis result of tumor weight is similar to the result of tumor volume.
  • the average tumor weight of the mice in the PBS control group was 722.57 ⁇ 176.32 mg on the 31st day after administration, and the average tumor weight of the antibody molecules QP192193, QP207208, and QP11151116 treatment groups were 455.5 ⁇ 46.42 mg and 391.93 on the 31st day after the end of administration, respectively ⁇ 111.15mg and 432.03 ⁇ 66.25mg, TGI were 36.96%, 45.76% and 40.21%, respectively; the control molecule IMAB362 treatment group had an average tumor weight of 435.78 ⁇ 91 mg and TGI of 39.69% on the 31st day after administration. (See table below)
  • Figs. 9-14 The experimental results are shown in Figs. 9-14, among which the results of the efficacy test of the antibody on the gastric cancer PDX model GA0006 are shown in Fig. 9.
  • Fig. 10A is the tumor growth curve after treatment in the PBS group (negative control).
  • Figure 10B is the tumor growth curve after treatment in the QP192193 group.
  • Fig. 10C is the tumor growth curve after treatment in the QP207208 group.
  • Figure 10D is the tumor growth curve after treatment in the QP11151116 group.
  • Figure 10E is the tumor growth curve after treatment in the control antibody IMAB362 group.
  • Figure 11 shows the weight of tumor D31 in each group of mice added with antibodies of the present invention and control antibodies.
  • Figure 12 is a real shot diagram of tumor volume in each experimental group.
  • Figure 13 is the body weight curve of each group of mice.
  • Figure 14 is a curve of the weight change rate of each group of mice. It can be seen from the experimental results that the invented antibody has a better effect on inhibiting tumor growth than IMAB362 in animal efficacy in vivo.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Cell Biology (AREA)
  • Urology & Nephrology (AREA)
  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Physics & Mathematics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biotechnology (AREA)
  • Veterinary Medicine (AREA)
  • Microbiology (AREA)
  • Oncology (AREA)
  • Food Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Biophysics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Hospice & Palliative Care (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

提供一种抗CLDN18.2的抗体及其药物组合物和检测方法，其中，抗体的重链选自SEQ ID NO：1-7或SEQ ID NO：15-30中的任意一条，轻链选自SEQ ID NO：8-14或SEQ ID NO：31-46中的任意一条。抗体与细胞系及肿瘤组织细胞结合的能力较现有抗体IMAB362强，并且抗肿瘤效果也较现有抗体IMAB362强。

Description

抗CLDN抗体及其药物组合物和检测方法 技术领域
本发明涉及一种抗CLDN抗体，本发明还涉及一种包含抗CLDN抗体的药物组合物，本发明还涉及检测生物样品中是否存在CLDN的方法。
背景技术
紧密连接(tight junction,TJ)在细胞间物质流转中起到关键作用，还通过阻断膜蛋白和膜脂径向扩散维持细胞极性，此外还参与招募调控细胞增殖、分化和运动的信号分子。紧密连接由紧密连接蛋白(Claudin,CLDN)形成，而紧密连接蛋白家族由超过20种蛋白分子组成，其成员均含有一个四次跨膜的结构域和相似的氨基酸序列，但组织分布具有一定特异性。人类CLDN基因以成对形式分布在不同染色体上，这暗示了某些CLDN基因来源于基因复制。
CLDN蛋白分子量大多处于20-34kDa范围内，最大区别是胞内C-端的序列和大小，该段序列包含1个PDZ结构域结合基序，该基序使得CLDN蛋白能够直接和胞质内的紧密连接相关蛋白，如ZO-1、ZO-2、ZO-3以及MUPP1相互作用。此外，这段序列种含有磷酸化位点等转录后修饰位点，能够影响蛋白分子的定位和功能。MAPK(mitogen-activated protein kinase)或者PKC(protein kinase C)能够磷酸化CLDN1，cAMP(cyclicAMP)诱导CLDN5磷酸化，都能促进CLDN蛋白的屏障功能，而PKA介导的CLDN16磷酸化则能增强镁离子运输。
CLDN在调控细胞旁路的选择性渗透中起到关键作用，CLDN2和CLDN15参与形成阳离子通道和阳离子孔隙，CLDN4/7/10则参与构成阴离子通道和孔隙。在某些细胞系中claudin蛋白高表达，从而影响跨膜电阻和渗透性。在培养的表皮来源细胞中，CLDN1/4/5/7能够提高跨膜电阻，而CLDN2和CLDN10反之。
CLDN基因突变被认为与多种疾病有关。CLDN1突变可能会导致硬化性胆管炎(sclerosing cholangitis)和鱼鳞癣(ichthyosis)，CLDN16和CLDN19突变则被认为与低镁症(hypomagnesemia)和高钙尿症(hypercalcinuria)有关。
CLDN蛋白的差异性表达被认为与多种癌症相关联。CLDN1和CLDN7在侵入性乳腺癌、前列腺癌和食管癌中发生下调，而在宫颈癌、结肠癌、食管癌、胃癌等多种癌症中发现CLDN3/4发生不同程度上调。Sahin等发现在正常组织中，CLDN18的isoform 2亚型(CLDN18.2)只在胃黏膜的分化后表皮细胞中表达，在胃干细胞区域则未见表达，但在原发性胃癌及其转移灶中均发现异常高表达。在胰腺癌、食管癌和肺癌中也有CLDN18.2高表达的报道。由于CLDN18.2定位于细胞膜表面，其生物学功能和特征决定了它是一种理想的治疗靶点，而近年来针对该靶点也有单抗面世，其中发展最快的是Ganymed公司的IMAB362(Claudiximab)。IMAB362在肿瘤细胞表面结合CLDN18.2，诱导抗体依赖的细胞毒性作用(antibody-dependent cytotoxicity，ADCC)和补体依赖的细胞毒性作用(complement dependent cytotoxicity，CDC)从而杀伤肿瘤细胞。而与化疗结合使用时，IMAB362还能增强T细胞浸润及上调促炎症因子。
发明内容
本发明的目的在于提供一种抗CLDN抗体及其药物组合物和检测方法。
本发明采用了如下技术方案：
一种抗CLDN抗体，其特征在于，包括重链和轻链：
其中，抗体的重链包含一种或一种以上的CDR，重链的CDR与SEQ ID No.1至SEQ ID No.7中或者SEQ IDNo.15至SEQ ID No.30中的任意一条的CDR序列相比，相差不多于三个氨基酸，
抗体的轻链包含一种或一种以上的CDR，轻链的CDR与SEQ ID No.8至SEQ ID No.14中或者SEQ ID No.31至SEQ ID No.46中的任意一条的CDR序列相比，相差不多于三个氨基酸。
进一步，本发明的抗CLDN抗体，还具有这样的特征：抗体的重链选自SEQ ID No.1至No.7中的任意一条。
进一步，本发明的抗CLDN抗体，还具有这样的特征：抗体的轻链选自如SEQ ID No.8至 SEQ ID No.14中或者SEQ ID No.31至SEQ ID No.46中的任意一条。
进一步，本发明的抗CLDN抗体，还具有这样的特征：抗体的重链选自SEQ ID No.15至SEQ ID No.30中的任意一条。
进一步，本发明的抗CLDN抗体，还具有这样的特征：抗体的轻链选自SEQ ID No.31至SEQ ID No.46中的任意一条。
进一步，本发明的抗CLDN抗体，还具有这样的特征：其中，抗体的重链和轻链的组合有：
SEQ ID No.1和SEQ ID N0.8，SEQ ID No.2和SEQ ID N0.9，SEQ ID No.3和SEQ ID No.10，
SEQ ID No.4和SEQ ID No.11，SEQ ID No.5和SEQ ID No.12，SEQ ID No.6和SEQ ID No.13，
SEQ ID No.7和SEQ ID No.14，SEQ ID No.15和SEQ ID No.31，SEQ ID No.16和SEQ ID No.32，
SEQ ID No.17和SEQ ID No.33，SEQ ID No.18和SEQ ID No.34，SEQ ID No.19和SEQ ID No.35，
SEQ ID No.20和SEQ ID No.36，SEQ ID No.21和SEQ ID No.37，SEQ ID No.22和SEQ ID No.38，
SEQ ID No.23和SEQ ID No.39，SEQ ID No.24和SEQ ID No.40，SEQ ID No.25和SEQ ID No.41，
SEQ ID No.26和SEQ ID No.42，SEQ ID No.27和SEQ ID No.43，SEQ ID No.28和SEQ ID No.44，
SEQ ID No.29和SEQ ID No.45，SEQ ID No.30和SEQ ID No.46。
本发明还提供一种编码如上所述的抗体的多核苷酸。
本发明还提供一种包含上述任意一项所述的抗体的药物组合物。
本发明还提供上述任意一项所述的抗体，在制备抗肿瘤药物中的应用。
本发明还提供一种检测生物样品中是否存在CLDN的方法，其特征在于，包括：给予生物样品如上述任意一项所述的抗体的步骤，其中所述抗体具有可检测标志物，以及检测是否存在所述可检测标志物，或者检测所述可检测标志物含量的步骤。
发明的有益效果
本发明的抗CLDN抗体，与细胞结合的能力较IMAB362强。并且本发明的抗体比IMAB362在体内动物药效中表现出更好的抑制肿瘤生长的效果。
附图说明
图1a是对照抗体QP024025对CHOS细胞的结合能力
图1b是杂交瘤筛选出的抗体QP188189对CHOS细胞的结合能力
图1c是杂交瘤筛选出的抗体QP190191对CHOS细胞的结合能力
图1d是杂交瘤筛选出的抗体QP192193对CHOS细胞的结合能力
图1e是杂交瘤筛选出的抗体QP196198对CHOS细胞的结合能力
图1f是杂交瘤筛选出的抗体QP199200对CHOS细胞的结合能力
图1g是杂交瘤筛选出的抗体QP201202对CHOS细胞的结合能力
图1h是杂交瘤筛选出的抗体QP207208对CHOS细胞的结合能力
图2a是噬菌体筛选出的抗体QP10731074对CHOS细胞的结合能力。
图2b是噬菌体筛选出的抗体QP10791080对CHOS细胞的结合能力。
图2c是噬菌体筛选出的抗体QP10851086对CHOS细胞的结合能力。
图2d是噬菌体筛选出的抗体QP10911092对CHOS细胞的结合能力。
图2e是噬菌体筛选出的抗体QP10971098对CHOS细胞的结合能力。
图2f是噬菌体筛选出的抗体QP10991100对CHOS细胞的结合能力。
图3a是噬菌体筛选出的抗体QP11051106对CHOS细胞的结合能力。
图3b是噬菌体筛选出的抗体QP11071108对CHOS细胞的结合能力。
图3c是噬菌体筛选出的抗体QP11091110对CHOS细胞的结合能力。
图3d是噬菌体筛选出的抗体QP11111112对CHOS细胞的结合能力。
图3e是噬菌体筛选出的抗体QP11131114对CHOS细胞的结合能力。
图3f是噬菌体筛选出的抗体QP11151116对CHOS细胞的结合能力。
图4a是噬菌体筛选出的抗体QP11171118对CHOS细胞的结合能力。
图4b是噬菌体筛选出的抗体QP11031104对CHOS细胞的结合能力。
图4c是噬菌体筛选出的抗体QP10451046对CHOS细胞的结合能力。
图4d是噬菌体筛选出的抗体QP10471048对CHOS细胞的结合能力。
图5a是对照抗体QP024025对不同293T瞬转细胞株的结合能力。
图5b是杂交瘤筛选出的抗体QP188189对不同293T瞬转细胞株的结合能力
图5c是杂交瘤筛选出的抗体QP190191对不同293T瞬转细胞株的结合能力
图5d是杂交瘤筛选出的抗体QP192193对不同293T瞬转细胞株的结合能力。
图5e是杂交瘤筛选出的抗体QP196198对不同293T瞬转细胞株的结合能力。
图5f是杂交瘤筛选出的抗体QP199200对不同293T瞬转细胞株的结合能力。
图5g是杂交瘤筛选出的抗体QP201202对不同293T瞬转细胞株的结合能力。
图5h是杂交瘤筛选出的抗体QP207208对不同293T瞬转细胞株的结合能力。
图6a是噬菌体筛选出的抗体QP10451046对不同293T瞬转细胞株的结合能力。
图6b是噬菌体筛选出的抗体QP10711072对不同293T瞬转细胞株的结合能力。
图6c是噬菌体筛选出的抗体QP10731074对不同293T瞬转细胞株的结合能力。
图6d是噬菌体筛选出的抗体QP10851086对不同293T瞬转细胞株的结合能力。
图6e是噬菌体筛选出的抗体QP10911092对不同293T瞬转细胞株的结合能力。
图6f是噬菌体筛选出的抗体QP10991100对不同293T瞬转细胞株的结合能力。
图6g是噬菌体筛选出的抗体QP11031104对不同293T瞬转细胞株的结合能力。
图6h是噬菌体筛选出的抗体QP11051106对不同293T瞬转细胞株的结合能力。
图7a是噬菌体筛选出的抗体QP11071108对不同293T瞬转细胞株的结合能力。
图7b是噬菌体筛选出的抗体QP11091110对不同293T瞬转细胞株的结合能力。
图7c是噬菌体筛选出的抗体QP11111112对不同293T瞬转细胞株的结合能力。
图7d是噬菌体筛选出的抗体QP11131114对不同293T瞬转细胞株的结合能力。
图7e是噬菌体筛选出的抗体QP11151116对不同293T瞬转细胞株的结合能力。
图7f是噬菌体筛选出的抗体QP11171118对不同293T瞬转细胞株的结合能力。
图8a是对照抗体与胃癌PDX模型GA0006肿瘤细胞结合能力实验中,对照组IMAB362的曲线。
图8b是抗体与胃癌PDX模型GA0006肿瘤细胞结合能力实验中QP190191的曲线。
图8c是抗体与胃癌PDX模型GA0006肿瘤细胞结合能力实验中QP192193的曲线。
图8d是抗体与胃癌PDX模型GA0006肿瘤细胞结合能力实验中QP201202的曲线。
图8e是抗体与胃癌PDX模型GA0006肿瘤细胞结合能力实验中QP207208的曲线。
图8f是抗体与胃癌PDX模型GA0006肿瘤细胞结合能力实验中QP11091110的曲线。
图8g是抗体与胃癌PDX模型GA0006肿瘤细胞结合能力实验中QP11131114的曲线。
图8h是抗体与胃癌PDX模型GA0006肿瘤细胞结合能力实验中QP11151116的曲线。
图9是抗体对胃癌PDX模型GA0006的药效试验。
图10A是PBS组(阴性对照)处理在分组后的肿瘤生长曲线。
图10B是QP192193组处理在分组后的肿瘤生长曲线。
图10C是QP207208组处理在分组后的肿瘤生长曲线。
图10D是QP11151116组处理在分组后的肿瘤生长曲线。
图10E是对照抗体IMAB362组处理在分组后的肿瘤生长曲线。
图11是加入本发明各抗体及对照抗体各组小鼠肿瘤D31重量。
图12是各实验组的肿瘤体积实拍图。
图13是各组小鼠的体重曲线。
图14是各组小鼠的体重变化率曲线。
具体实施方式
以下结合附图来说明本发明的具体实施方式。
本发明的抗体，包括但不限于人源抗体。本发明中所提供的各种抗体的筛选方法，通常优先采用较为便利的杂交瘤筛选抗体，然而这个靶点的抗原(claudin18.2)较难获得，因此也同时采用了噬菌体库筛选抗体。本实施方式中筛选得到的抗claudin18.2的抗体序列如下所示：
杂交瘤筛选的抗体，重链可变区(VH)CDR如序列中带下划线的部分所示
Figure PCTCN2020083570-appb-000001
轻链序列：
其中，轻链可变区(VL)CDR如序列中带下划线的部分所示。
Figure PCTCN2020083570-appb-000002
Figure PCTCN2020083570-appb-000003
噬菌体库筛选的抗体，重链可变区(VH)CDR如序列中带下划线的部分所示
Figure PCTCN2020083570-appb-000004
Figure PCTCN2020083570-appb-000005
轻链序列：
其中，轻链可变区(VL)CDR如序列中带下划线的部分所示。
Figure PCTCN2020083570-appb-000006
Figure PCTCN2020083570-appb-000007
抗体结合能力实验：
实验一：FACS检测抗体对肿瘤细胞系结合能力
a)1.96孔板铺细胞，每孔2×10 5个细胞，1000×rpm离心5分钟，1×PBS细胞清洗一次，吸去上清多余液体；
b)加入3％BSA-PBS溶液封闭细胞，4℃封闭60分钟；
c)封闭液稀释待测抗体至5ug/ml，加入到孔中，4℃孵育60分钟；
d)吸去抗体，每孔220ul 1×PBS细胞清洗三次；
e)每孔加入50ul PE-anti-human FC(1：200稀释)二抗，4℃避光孵育40分钟；
f)吸去抗体，每孔1×PBS细胞清洗四次；
g)上机检测。
实验二：FACS检测抗体对PDX组织肿瘤细胞结合能力
a)每个瘤子准备一个gentleMACS TMC Tube和3ml消化液，消化液按照Tumor Dissociation Kit(miltenyibiotech，130-096-730)说明书配制，现配现用；
b)处死小鼠，用洁净工具取瘤，1×PBS清洗后去除附着在肿瘤表面的血管、脂肪、筋膜等组织。每根消化管可消化不大于0.8g的肿瘤组织，以保证肿瘤组织被消化液完全消化；
c)将肿瘤组织置于6孔板的孔中，加入消化液，用洁净镊子和解剖刀将肿瘤组织切割为约1mm 3小块；
d)将组织块放入gentleMACS TMC Tube，使用残留消化液清洗孔板并一起转移至消化管中，放置于冰面上；
e)将消化管倒置放入gentleMACS全自动组织处理器，选择程序37_c_m_TDK_1进行消化。程序结束后，取下消化管，300×g瞬时离心收集细胞和剩余组织；
f)弃上清，用1×PBS重悬细胞和组织，将细胞悬液加至细胞过滤网上，置于50ml离心管 中，用10ml PBS使悬液过筛网成为单细胞悬液；
g)300×g离心5分钟，去上清，5ml PBS重悬，细胞计数并调整细胞浓度至每份样品1×10 6个细胞；
h)每份样品加入100ul浓度为1μg/ml Mouse BD Fc Block(CAT#553141)PBS溶液重悬细胞，加入20ul human FcR Blocking Reagent，混匀，室温避光孵育10分钟；
i)加入5ug/ml一抗，4℃避光孵育60分钟；
j)加入2ml FACS wash buffer，轻柔重悬细胞，300×g离心5分钟，去上清，重复2次；
k)加入100ul含PE标记的human IgG Fc二抗和染料的FACS wash buffer避光孵育细胞60分钟；
l)加入2ml FACS wash buffer，轻柔重悬细胞，300×g离心5分钟，去上清，重复2次；
m)200ul FACS wash buffer重悬细胞，上机检测。
实验附图中hybridoma clone in CHOS system和phage clone in CHOS system即分别指杂交瘤和噬菌体筛选出的抗体对CHOS细胞的结合能力。图中的CHO18.2：CHOS-CLDN18.2稳转细胞，CHO18.1：CHOS-CLDN18.1稳转细胞，CHOS：CHOS转染空载体。
CHOS是仓鼠卵巢细胞永生化得到的细胞系，使用FACS(流式细胞荧光分选技术)来检测抗体对细胞系结合能力。
图1a到图1h显示了杂交瘤筛选出的抗体对CHOS细胞的结合能力。
图2a到图2h，图3a到图3h以及图4a到图4d显示了噬菌体筛选出的抗体对CHOS细胞的结合能力。
实验目的：在CHOS细胞系中验证抗体与claudin18.1(非特异性结合)和claudin18.2的结合能力。
在CHOS细胞系的附图中，每一个panel由一种待测抗体对3种CHOS细胞系的FACS检测结果组合而成，其中：
1.红色曲线③，表明待测抗体对转染空载体的CHOS细胞系(CHOS)的结合能力，即阴性对照；
2.蓝色曲线②，表明待测抗体对转染claudin18.1的CHOS细胞系(CHO18.1)的结合能力，即检测非特异性结合；
3.黄色曲线①，表明待测抗体对转染claudin18.2的CHOS细胞系(CHO18.2)的结合能力，即检测目的蛋白结合能力。
图5a到图5h，显示了杂交瘤筛选出的抗体对293T细胞的结合能力。
图6a到图6h以及图7a到图7f显示了噬菌体筛选出的抗体对CHOS细胞的结合能力。
图中，293T-QD012：293T-CLDN18.1瞬转细胞
293T-QD010：293T-CLDN18.2瞬转细胞
实验目的：在293-T细胞系中验证抗体与claudin18.1(非特异性结合)和claudin18.2的结合能力。
293T-QD210:293T-CLDN18.2-18.1ECD1瞬转细胞；CLDN18.2的第1个胞外结构域更换为18.1的第1个胞外结构域
293T-QD211:293T-CLDN18.1-18.2ECD1瞬转细胞；CLDN18.1的第1个胞外结构域更换为18.2的第1个胞外结构域
实验目的：在293-T细胞系中验证抗体结合区域是claudin18.2的exon-1编码的结构域。
hybridom clone in 293T及phage clone in 293T同理分别指杂交瘤和噬菌体筛选出的抗体对不同293T瞬转细胞株的结合能力。
在293T中实验目的与在CHO细胞系中相同，各个曲线及其代表的涵义解释如下。
1.红色曲线⑤，表明待测抗体对瞬转空载体的293T细胞系的结合能力，即阴性对照；
2.蓝色曲线④，表明待测抗体对瞬转claudin18.2的293T细胞系(293T-QD010)的结合，检测目的蛋白结合能力；
3.橙色曲线③，表明待测抗体对瞬转claudin18.1的293T细胞系(293T-QD012)的结合，检测非特异性结合；
4.深绿曲线②，表明待测抗体对瞬转claudin18.1-18.2ECD1(CLDN18.1的第1个胞外结构域更换为18.2的第1个胞外结构域，后者是设计抗体结合位点所在区域)的293T细胞系(293T-QD211)的结合，验证抗体结合位点所在结构域；
5.浅绿曲线①，表明待测抗体对瞬转claudin18.2-18.1ECD1(CLDN18.2的第1个胞外结构域更换为18.1的第1个胞外结构域，前者是设计抗体结合位点所在区域)的293T细胞系(293T-QD210)的结合，验证该结构域对抗体结合的必要性。
图8a到图8h显示了抗体与胃癌PDX模型GA0006肿瘤细胞结合能力实验。
表1：抗体与胃癌PDX模型GA0006肿瘤细胞结合比例
Figure PCTCN2020083570-appb-000008
以上实验结果表明，本发明所提供的抗体，与蛋白的结合能力更好，并且非特异性结合的情况较低。
在其它的实施方式中，本发明还提供一种编码如上所述的抗体的多核苷酸，这些编码本发明所提供的抗体的多核苷酸，当是以DNA的形式提供时，其可以包含在后续的转录和编辑过程中会被去除的非编码序列，也可以只包含编码与本发明实施例中所提供的抗体相对应的序列以及在进行蛋白表达时所必须的序列。
本发明还提供一种包含上述任意一项所述的抗体的药物组合物，本发明所提供的药物组合物，可以仅包含实施方式中所提供抗体中的任意一种或者至少两种的组合物。
本领域的技术人员应当清楚的是，对于药物组合物，还包含药物上可接受的赋形剂，利用制成粉剂或者片剂等其它剂型所需要的常规赋形剂均应当作为制药过程中所应当添加的成分。
本发明还提供一种检测生物样品中是否存在CLDN的方法，其特征在于，包括：
给予生物样品如上述任意一项所述的抗体的步骤，其中所述抗体具有可检测标志物，以及检测是否存在所述可检测标志物，或者检测所述可检测标志物含量的步骤。
动物药效试验
a)从胃癌异种移植模型GA0006荷瘤小鼠收取肿瘤组织，切成直径为2-3mm的瘤块接种 于Balb/c裸小鼠右前肩胛处皮下；
b)当Balb/c裸小鼠平均肿瘤体积到达约100mm 3时，将小鼠随机分组，每组6只。称量所有动物的体重，并用游标卡尺测量肿瘤体积。根据肿瘤体积，采用随机分组方法进行分组，保证不同组别间的肿瘤体积相似。各组内的肿瘤体积的变异系数(CV)用公式CV＝SD/MTV×100％计算，应小于40％。随机分组使用StudyDirectorTM完成。分组当天为D0，并于当天开始给药。详细的给药方法、给药剂量和给药途径见下表。
表2：动物药效试验的给药参数
组别 动物数 给药组 剂量(mg/kg) 给药方式 给药周期
1 6 Vehicle control -- i.p. BIW×4周
2 6 QP192193 10 i.p. BIW×4周
3 6 QP207208 10 i.p. BIW×4周
4 6 QP11151116 10 i.p. BIW×4周
5 6 IMAB362 10 i.p. BIW×4周
给药体积为10μL/g.
表1和表2中的抗体编号的含义，例如QP190191，表示一条重链和一条轻链的组合。
c)开始给药后，每周测量两次小鼠体重和肿瘤体积。肿瘤体积计算公式：肿瘤体积(mm 3)＝1/2×(a×b 2)(其中a表示长径，b表示短径)。当最后一次给药后一星期终止实验，处死小鼠，取瘤称重、拍照。选用以下分析方法进行数据分析：
相对肿瘤增殖率，T/C(％)，即在某一时间点，治疗组和对照组相对肿瘤体积或瘤重的百分比值。计算公式为：T/C％＝TRTV/CRTV×100％(TRTV：治疗组平均RTV；CRTV：对照组平均RTV；RTV＝Vt/V0，V0为分组时该动物的瘤体积，Vt为治疗后该动物的瘤体积)；
相对肿瘤抑制率，TGI(％)，计算公式为：TGI％＝(1-T/C)×100％(T和C分别为治疗组和对照组在某一特定时间点的相对肿瘤体积(RTV))。
PBS对照组小鼠在给药后第31天平均肿瘤体积为911.16±177.81mm 3。抗体分子QP192193(10mg/kg)、QP207208(10mg/kg)、和QP11151116(10mg/kg)组在给药后第31天平均肿瘤体积分别为580.97±67.97mm 3、680.28±193.50mm 3、和722.38±118.07mm 3，TGI分别为36.34％、25.34％、和20.72％，对照分子IMAB362(10mg/kg)在给药后第31天平均肿瘤体积为661.28±104.49mm 3，TGI为27.42％(见下表)。三 个筛选出的分子均在一定程度上抑制了肿瘤的生长，虽然没有统计学显著性差异，但QP192193表现优于对照分子IMAB362的趋势，QP207208和QP11151116也表现出与IMAB362相同水平的抑制肿瘤生长能力。而且小鼠体重在给药过程中没有发生明显下降，说明抗体分子对小鼠没有明显毒副作用。
Figure PCTCN2020083570-appb-000009
肿瘤重量的分析结果与肿瘤体积的结果相似。PBS对照组小鼠在给药后第31天平均肿瘤重量为722.57±176.32mg，抗体分子QP192193、QP207208、和QP11151116治疗组在结束给药后第31天平均肿瘤重量分别为455.5±46.42mg、391.93±111.15mg和432.03±66.25mg，TGI分别为36.96％、45.76％和40.21％；对照分子IMAB362治疗组在给药后第31天平均肿瘤重量为435.78±91mg，TGI为39.69％。(见下表)
Figure PCTCN2020083570-appb-000010
因此通过PDX动物药效试验，我们发现筛选出的抗体分子抗体分子具有比对照分子IMAB362 更优或相同水平的体内抑制肿瘤能力。
实验结果如图9至图14所示，其中，抗体对胃癌PDX模型GA0006的药效试验的结果如图9所示。图10A是PBS组(阴性对照)处理在分组后的肿瘤生长曲线。图10B是QP192193组处理在分组后的肿瘤生长曲线。图10C是QP207208组处理在分组后的肿瘤生长曲线。图10D是QP11151116组处理在分组后的肿瘤生长曲线。图10E是对照抗体IMAB362组处理在分组后的肿瘤生长曲线。图11是加入本发明各抗体及对照抗体各组小鼠肿瘤D31重量。图12是各实验组的肿瘤体积实拍图。图13是各组小鼠的体重曲线。图14是各组小鼠的体重变化率曲线。从实验结果中可见，发明的抗体比IMAB362在体内动物药效中表现出更好的抑制肿瘤生长的效果。
Figure PCTCN2020083570-appb-000011
Figure PCTCN2020083570-appb-000012
Figure PCTCN2020083570-appb-000013
Figure PCTCN2020083570-appb-000014
Figure PCTCN2020083570-appb-000015
Figure PCTCN2020083570-appb-000016
Figure PCTCN2020083570-appb-000017
Figure PCTCN2020083570-appb-000018
Figure PCTCN2020083570-appb-000019
Figure PCTCN2020083570-appb-000020
Figure PCTCN2020083570-appb-000021
Figure PCTCN2020083570-appb-000022
Figure PCTCN2020083570-appb-000023
Figure PCTCN2020083570-appb-000024
Figure PCTCN2020083570-appb-000025
Figure PCTCN2020083570-appb-000026
Figure PCTCN2020083570-appb-000027
Figure PCTCN2020083570-appb-000028
Figure PCTCN2020083570-appb-000029
Figure PCTCN2020083570-appb-000030
Figure PCTCN2020083570-appb-000031
Figure PCTCN2020083570-appb-000032

Claims (10)

  1. 一种抗CLDN抗体，其特征在于，包括重链和轻链：
    其中，所述抗体的重链包含一种或一种以上的CDR，重链的CDR与SEQ ID No.1至SEQ ID No.7中或者SEQ ID No.15至SEQ ID No.30中的任意一条的CDR序列相比，相差不多于三个氨基酸，
    所述抗体的轻链包含一种或一种以上的CDR，轻链的CDR与SEQ ID No.8至SEQ ID No.14中或者SEQ ID No.31至SEQ ID No.46中的任意一条的CDR序列相比，相差不多于三个氨基酸。
  2. 如权利要求1所述的抗CLDN抗体，其特征在于：
    抗体的重链选自SEQ ID No.1至No.7中的任意一条。
  3. 如权利要求2所述的抗CLDN抗体，其特征在于：
    抗体的轻链选自如SEQ ID No.8至SEQ ID No.14中或者SEQ ID No.31至SEQ ID No.46中的任意一条。
  4. 如权利要求1所述的抗CLDN抗体，其特征在于：
    抗体的重链选自SEQ ID No.15至SEQ ID No.30中的任意一条。
  5. 如权利要求4所述的抗CLDN抗体，其特征在于：
    抗体的轻链选自SEQ ID No.31至SEQ ID No.46中的任意一条。
  6. 如权利要求1所述的抗CLDN抗体，其特征在于：
    其中，抗体的重链和轻链的组合有：
    SEQ ID No.1和SEQ ID N0.8，
    SEQ ID No.2和SEQ ID N0.9，
    SEQ ID No.3和SEQ ID No.10，
    SEQ ID No.4和SEQ ID No.11，
    SEQ ID No.5和SEQ ID No.12，
    SEQ ID No.6和SEQ ID No.13，
    SEQ ID No.7和SEQ ID No.14，
    SEQ ID No.15和SEQ ID No.31，
    SEQ ID No.16和SEQ ID No.32，
    SEQ ID No.17和SEQ ID No.33，
    SEQ ID No.18和SEQ ID No.34，
    SEQ ID No.19和SEQ ID No.35，
    SEQ ID No.20和SEQ ID No.36，
    SEQ ID No.21和SEQ ID No.37，
    SEQ ID No.22和SEQ ID No.38，
    SEQ ID No.23和SEQ ID No.39，
    SEQ ID No.24和SEQ ID No.40，
    SEQ ID No.25和SEQ ID No.41，
    SEQ ID No.26和SEQ ID No.42，
    SEQ ID No.27和SEQ ID No.43，
    SEQ ID No.28和SEQ ID No.44，
    SEQ ID No.29和SEQ ID No.45，
    SEQ ID No.30和SEQ ID No.46。
  7. 一种编码如权利要求1-6中任意一项所述的抗体的多核苷酸。
  8. 一种包含如权利要求1-6中任意一项所述的抗体的药物组合物。
  9. 如权利要求权利要求1-6中任意一项所述的抗体，在制备抗肿瘤药物中的应用。
  10. 一种检测生物样品中是否存在CLDN的方法，其特征在于，包括：
    给予生物样品如权利要求1-6中任意一项所述的抗体的步骤，其中所述抗体具有可检测标志物，
    以及检测是否存在所述可检测标志物，或者检测所述可检测标志物含量的步骤。
PCT/CN2020/083570 2019-05-16 2020-04-07 抗cldn抗体及其药物组合物和检测方法 WO2020228447A1 (zh)

Priority Applications (5)

Application Number Priority Date Filing Date Title
BR112021022757A BR112021022757A2 (pt) 2019-05-16 2020-04-07 Anticorpo anti-cldn e composição farmacêutica do mesmo e método de detecção para o mesmo
JP2021568696A JP7249696B2 (ja) 2019-05-16 2020-04-07 抗ｃｌｄｎ抗体並びにその医薬組成物および検出方法
US17/611,370 US20220235128A1 (en) 2019-05-16 2020-04-07 Anti-cldn antibody and pharmaceutical composition thereof and detection method therefor
EP20804959.3A EP3971207A4 (en) 2019-05-16 2020-04-07 ANTI-CLDN ANTIBODY, ASSOCIATED PHARMACEUTICAL COMPOSITION AND CORRESPONDING DETECTION METHOD
KR1020217041245A KR20220010002A (ko) 2019-05-16 2020-04-07 항-cldn 항체 및 이의 약학적 조성물과 검출 방법

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201910410255.8A CN111944048B (zh) 2019-05-16 2019-05-16 抗cldn抗体及其药物组合物和检测方法
CN201910410255.8 2019-05-16

Publications (1)

Publication Number Publication Date
WO2020228447A1 true WO2020228447A1 (zh) 2020-11-19

Family

ID=73290063

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/083570 WO2020228447A1 (zh) 2019-05-16 2020-04-07 抗cldn抗体及其药物组合物和检测方法

Country Status (7)

Country Link
US (1) US20220235128A1 (zh)
EP (1) EP3971207A4 (zh)
JP (1) JP7249696B2 (zh)
KR (1) KR20220010002A (zh)
CN (3) CN111944048B (zh)
BR (1) BR112021022757A2 (zh)
WO (1) WO2020228447A1 (zh)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11407828B2 (en) 2019-02-01 2022-08-09 Novarock Biotherapeutics, Ltd. Anti-CLauDiN 18 antibodies and methods of use thereof
EP4144759A4 (en) * 2020-04-27 2024-05-29 Qure Biotechnology Shanghai Co Ltd BISPECIFIC ANTIBODY TARGETING HUMAN CLAUDIN AND HUMAN PDL1 PROTEINS, AND APPLICATION THEREOF

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IL303273A (en) * 2020-11-30 2023-07-01 Cspc Megalith Biopharmaceutical Co Ltd ANTI-CLDN18.2 antibody, drug conjugation and its preparation and use
CN114634566B (zh) * 2020-12-16 2023-09-19 广州百暨基因科技有限公司 一种抗Claudin18_2的抗原结合片段、抗体及其应用
WO2022143794A1 (zh) * 2020-12-30 2022-07-07 百奥泰生物制药股份有限公司 抗cldn18.2抗体及其制备方法和应用
CN112940124B (zh) * 2021-04-14 2022-04-05 南京凯地医疗技术有限公司 靶向Claudin18.2的人源化单克隆抗体及其制备方法和应用
CN114181311B (zh) * 2021-12-20 2023-06-20 华东师范大学 一种全人源抗DLL3 scFv及其在CART细胞治疗中的应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105073777A (zh) * 2013-02-20 2015-11-18 咖尼米德制药股份公司 用于治疗癌症的涉及抗密蛋白18.2抗体的组合疗法
CN105189554A (zh) * 2013-03-18 2015-12-23 咖尼米德制药股份公司 用于治疗癌症的涉及抗密蛋白18.2的抗体的疗法
CN107667118A (zh) * 2015-04-15 2018-02-06 加尼梅德药物有限公司 包含针对密封蛋白18.2之抗体的药物缀合物
WO2018054973A1 (en) * 2016-09-23 2018-03-29 Biontech Ag Bispecific trivalent antibodies binding to claudin6 or claudin18.2 and cd3 for treatment of claudin expressing cancer diseases

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1790664A1 (en) * 2005-11-24 2007-05-30 Ganymed Pharmaceuticals AG Monoclonal antibodies against claudin-18 for treatment of cancer
WO2013167153A1 (en) * 2012-05-09 2013-11-14 Ganymed Pharmaceuticals Ag Antibodies useful in cancer diagnosis
WO2013174404A1 (en) * 2012-05-23 2013-11-28 Ganymed Pharmaceuticals Ag Combination therapy involving antibodies against claudin 18.2 for treatment of cancer
JP6515111B2 (ja) * 2013-11-06 2019-05-22 アッヴィ・ステムセントルクス・エル・エル・シー 新規の抗クローディン抗体および使用方法
CN110698560B (zh) * 2015-12-24 2021-11-26 凯惠科技发展(上海)有限公司 一种tpbg抗体及其制备方法、其偶联物和应用
WO2018026742A1 (en) * 2016-08-01 2018-02-08 Askgene Pharma Inc. Novel antibody-albumin-drug conjugates (aadc) and methods for using them
CN113321738A (zh) * 2020-02-27 2021-08-31 启愈生物技术(上海)有限公司 肿瘤靶向、抗cd3和t细胞激活三功能融合蛋白及其应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105073777A (zh) * 2013-02-20 2015-11-18 咖尼米德制药股份公司 用于治疗癌症的涉及抗密蛋白18.2抗体的组合疗法
CN105189554A (zh) * 2013-03-18 2015-12-23 咖尼米德制药股份公司 用于治疗癌症的涉及抗密蛋白18.2的抗体的疗法
CN107667118A (zh) * 2015-04-15 2018-02-06 加尼梅德药物有限公司 包含针对密封蛋白18.2之抗体的药物缀合物
WO2018054973A1 (en) * 2016-09-23 2018-03-29 Biontech Ag Bispecific trivalent antibodies binding to claudin6 or claudin18.2 and cd3 for treatment of claudin expressing cancer diseases

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP3971207A4 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11407828B2 (en) 2019-02-01 2022-08-09 Novarock Biotherapeutics, Ltd. Anti-CLauDiN 18 antibodies and methods of use thereof
EP4144759A4 (en) * 2020-04-27 2024-05-29 Qure Biotechnology Shanghai Co Ltd BISPECIFIC ANTIBODY TARGETING HUMAN CLAUDIN AND HUMAN PDL1 PROTEINS, AND APPLICATION THEREOF

Also Published As

Publication number Publication date
JP7249696B2 (ja) 2023-03-31
EP3971207A4 (en) 2023-09-06
CN117264059A (zh) 2023-12-22
CN111944048A (zh) 2020-11-17
KR20220010002A (ko) 2022-01-25
EP3971207A1 (en) 2022-03-23
CN111944048B (zh) 2023-10-03
BR112021022757A2 (pt) 2022-02-15
US20220235128A1 (en) 2022-07-28
JP2022532784A (ja) 2022-07-19
CN117659190A (zh) 2024-03-08

Similar Documents

Publication Publication Date Title
WO2020228447A1 (zh) 抗cldn抗体及其药物组合物和检测方法
JP6563472B2 (ja) 抗ＥｒｂＢ３抗体およびその使用
KR101652125B1 (ko) 헤파린-결합 표피 성장 인자-유사 성장 인자 항원 결합 단백질
EP2835380B2 (en) Method of blocking vascular leakage using an anti-Ang2 antibody
WO2020025013A1 (zh) 结合人her2的抗体、其制备方法和用途
CN103201290A (zh) S100a4抗体及其治疗用途
KR20130105782A (ko) ErbB/ErbB 리간드 관련된 질병을 치료하기 위한 분자 및 이들의 사용 방법
US9884910B2 (en) Anti-PDGF-C antibodies
JP7094329B2 (ja) 低悪性度漿液性癌におけるｈｅｒ３阻害
WO2011007853A1 (ja) 癌特異的アイソフォームに対するモノクローナル抗体
WO2022188856A1 (en) Method of treating diseases using gremlin1 antagonists
CN103417965B (zh) 一种含有抗vegf抗体的药物组合物
US11753477B2 (en) Preparation and use of anti-MET-and-RON bispecific antibody and antibody-drug conjugate thereof
RU2801315C2 (ru) Анти-cldn антитело, его фармацевтическая композиция и способ его обнаружения
TW201716439A (zh) Ｈｅｒ３抗體
US20130330353A1 (en) Regimens for treatments using anti-igf antibodies
CN113226369A (zh) 给药
CN107683292A (zh) 特异性结合erbb3的抗体及其用途

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20804959

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2021568696

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

REG Reference to national code

Ref country code: BR

Ref legal event code: B01A

Ref document number: 112021022757

Country of ref document: BR

ENP Entry into the national phase

Ref document number: 20217041245

Country of ref document: KR

Kind code of ref document: A

REG Reference to national code

Ref country code: BR

Ref legal event code: B01E

Ref document number: 112021022757

Country of ref document: BR

Free format text: COM BASE NA PORTARIA 56 DE 27/12/2021, SOLICITA-SE QUE SEJA APRESENTADO, EM ATE 60 (SESSENTA) DIAS, NOVO CONTEUDO DE LISTAGEM DE SEQUENCIA POIS O CONTEUDO APRESENTADO NA PETICAO NO 870210115507 DE 15/12/2021 NAO POSSUI TODOS OS CAMPOS OBRIGATORIOS INFORMADOS, NAO CONSTANDO O CAMPO 140 / 141 .

ENP Entry into the national phase

Ref document number: 2020804959

Country of ref document: EP

Effective date: 20211216

ENP Entry into the national phase

Ref document number: 112021022757

Country of ref document: BR

Kind code of ref document: A2

Effective date: 20211111