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WO2024232668A1 - Anticorps monoclonal anti-ptgfrn et son utilisation - Google Patents

Anticorps monoclonal anti-ptgfrn et son utilisation Download PDF

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Publication number
WO2024232668A1
WO2024232668A1 PCT/KR2024/006210 KR2024006210W WO2024232668A1 WO 2024232668 A1 WO2024232668 A1 WO 2024232668A1 KR 2024006210 W KR2024006210 W KR 2024006210W WO 2024232668 A1 WO2024232668 A1 WO 2024232668A1
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cancer
ptgfrn
cells
cancer cells
blood
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Korean (ko)
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류춘제
이정호
이현민
서효선
천유진
최문주
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Industry Academy Cooperation Foundation of Sejong University
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Industry Academy Cooperation Foundation of Sejong University
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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • 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
    • 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
    • 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
    • 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

Definitions

  • the present invention relates to an anti-PTGFRN monoclonal antibody for determining the level of blood cancer cells, treating or preventing cancer, predicting the prognosis of cancer metastasis, and screening for anticancer drugs.
  • Hepatocellular carcinoma accounts for more than 90% of liver cancers and is the third leading cause of cancer-related mortality.
  • the main treatments for HCC include surgical resection, local resection for early-stage HCC, or liver transplantation.
  • most patients with early-stage HCC are already diagnosed with advanced disease, and survival is limited to palliative treatment such as transarterial chemoembolization, systemic treatment with tyrosine kinase inhibitors, and selective internal radiotherapy.
  • HCC has an exceptionally high recurrence rate, and the treatment effect is not satisfactory.
  • the diagnosis and monitoring of HCC mainly depend on serum biomarker detection, pathological examination, and image analysis.
  • AFP a common serum marker
  • DCP des-gamma-carboxy-prothrombin
  • Circulating tumor cells are cancer cells found in the peripheral blood of cancer patients, and they are cancer cells that move through the blood. Metastasis can be the direct cause of most cancer-related deaths, and circulating tumor cells are the seeds of this metastasis phenomenon and are cells that spread cancer. They form homo- and hetero-clusters to evade immune cells and internal and external inhibitory signals to metastasize, and their expression level is related to the survival rate of cancer. Since an increase in the number of circulating tumor cells in the peripheral blood indicates that metastasis is progressing in cancer patients, a method has been developed to use circulating tumor cells as a cancer metastasis diagnostic marker. In addition, circulating tumor cells are associated with a poor prognosis for cancer patients.
  • blood cancer cells have been separated and studied using a cell search system using EpCAM, a microfluidics system based on cell size and physical characteristics, and a flow cytometer.
  • EpCAM EpCAM
  • a microfluidics system based on cell size and physical characteristics
  • a flow cytometer a flow cytometer
  • EpCAM Epithelial Cell Adhesion Molecule
  • ASGPR asialoglycoprotein receptor
  • the proportion of 85 hepatocellular cancer patients with more than 1 circulating cancer cell per 5 ml of blood was approximately 81%, or 69 cases.
  • interest in EpCAM-positive blood cancer cells exhibiting stem cell characteristics has emerged in the field of hepatocellular carcinoma, based on research results showing that EpCAM is a marker of hepatocellular carcinoma stem cells that induce metastasis of hepatocellular carcinoma or induce recurrence of treatment.
  • the rate of detecting more than 1 circulating cancer cell per 7.5 ml of blood in 50 hepatocellular carcinoma patients using an EpCAM-based circulating cancer cell detection method was only 28%, or 14 cases.
  • the EpCAM-based circulating cancer cell detection method did not have a detection rate exceeding 50%. This is because EpCAM expression decreases due to epithelial-mesenchymal transition that occurs during the metastatic process of cancer, and is a fundamental limitation of the EpCAM-based circulating cancer cell detection method.
  • Epithelial-mesenchymal transition refers to the entire process by which adherent epithelial cancer cells transition into mesenchymal cancer cells with motility and invasiveness.
  • epithelial cell markers may be down-regulated. Therefore, if circulating cancer cells are detected solely based on epithelial cell markers, most circulating cancer cells may not be detected.
  • EpCAM is used as a marker to detect circulating cancer cells, only 50-70% of all circulating cancer cells can be detected in lung cancer, breast cancer, cervical cancer, and nasopharyngeal cancer, and in the case of liver cancer, only 25% of the circulating cancer cells are detected.
  • the results of analyzing the circulating cancer cells with mixed and mesenchymal markers in hepatocellular carcinoma showed that patients with more circulating cancer cells had stronger invasive ability, and these patients showed higher clinical stages.
  • the results of analyzing the circulating cancer cells and circulating cancer cell clusters collected from the peripheral blood of 214 hepatocellular carcinoma patients showed that the circulating cancer cell clusters were closely related to the overall survival (OS) or progression-free survival (PFS) and showed a poor prognosis.
  • OS overall survival
  • PFS progression-free survival
  • EMT electrospray
  • mesenchymal cells are known to have a hybrid epithelial/mesenchymal state or a partial phenotype that has both epithelial and mesenchymal characteristics, and they are known to have a tendency to form clusters and have immune-evading abilities to evade the host's immune system. Therefore, in order to detect all circulating cancer cells in a patient, it is necessary to discover markers that can detect not only epithelial circulating cancer cells, but also circulating cancer cells showing an EMT phenotype, circulating cancer cells showing partial EMT, and entirely new types of circulating cancer cells.
  • PTGFRN Prostaglandin F2 Receptor Negative Regulator, EWI-F, CD9P-1, CD315) is a type I transmembrane protein of the immunoglobulin superfamily that negatively affects the Prostaglandin F2 Receptor.
  • PTGFRN is a component of the Tetraspanin Enriched Microdomain (TEM), which consists of tetraspanins, integrins, signaling enzymes, and proteoglycans, and interacts with tetraspanin proteins such as CD9 and CD81.
  • TEM Tetraspanin Enriched Microdomain
  • PTGFRN is expressed in large quantities as a structural protein in exosomes, and its separation function through exosomes is being studied.
  • PTGFRN is involved in the regeneration of muscle cells, the growth of glioma, and metastasis.
  • PTGFRN is a molecule that is up-regulated in many cancer cells, including gliomas, and is known to be overexpressed in glioblastoma multiforme (GBM), a malignant brain tumor, where it promotes cell growth and radioresistance through PI3K-AKT signaling.
  • GBM glioblastoma multiforme
  • the present inventors have prepared 70 kinds of monoclonal antibodies that bind to the surface of human embryonic stem cells, and one of them, 63-D7, binds well to the surface of various human cancer cells including human embryonic stem cells, embryonic cancer cells, and liver cancer cells, and does not bind to normal cells such as human peripheral blood mononuclear cells (PBMC), fetal lung fibroblasts (MRC5), and normal hepatocytes.
  • PBMC peripheral blood mononuclear cells
  • MRC5 fetal lung fibroblasts
  • PTGFRN recognized by 63-D7 was highly co-expressed with B7-H3 (57.6%), MVP (31.9%), TGF ⁇ R1 (51.8%), PD-L1 (37.8%), PD-L2 (17.5%), and CD47 (38.1%), which are associated with drug resistance and immune evasion in circulating cancer cells, showing that the expression of PTGFRN is closely related to metastasis or recurrence, and suggesting that these molecules can induce immune evasion of cancer cells.
  • 63-D7 is an antibody that induces internalization in liver cancer cells and pancreatic cancer cells, and in fact, through these properties, we suggest that 63-D7 can be developed as a cancer treatment agent in the future by inducing liver cancer cell death using an Antibody-Drug Conjugate (ADC).
  • ADC Antibody-Drug Conjugate
  • the present invention aims to provide a use of PTGFRN as a surface molecular marker of cancer cells.
  • the present invention aims to provide a monoclonal antibody 63-D7 that specifically binds to PTGFRN.
  • the present invention aims to provide the use of monoclonal antibody 63-D7 as an agonistic antibody against PTGFRN that increases mobility and invasiveness in cancer cells.
  • the present invention aims to provide a use of PTGFRN as a surface molecular marker of blood cancer cells.
  • Another object of the present invention is to provide a use of the monoclonal antibody 63-D7 as a substance that specifically binds to PTGFRN to isolate, purify or detect PTGFRN protein.
  • An anti-PTGFRN monoclonal antibody comprising a heavy chain variable region comprising HCDR1 consisting of the amino acid sequence of SEQ ID NO: 3, HCDR2 consisting of the amino acid sequence of SEQ ID NO: 4, and HCDR3 consisting of the amino acid sequence of SEQ ID NO: 5; and a light chain variable region comprising LCDR1 consisting of the amino acid sequence of SEQ ID NO: 6, LCDR2 consisting of the amino acid sequence of SEQ ID NO: 7, and LCDR3 consisting of the amino acid sequence of SEQ ID NO: 8.
  • the heavy chain variable region is an anti-PTGFRN monoclonal antibody comprising the amino acid sequence of sequence number 1.
  • An antibody-drug conjugate comprising a drug conjugated to any one of the above 1 to 3 anti-PTGFRN monoclonal antibodies.
  • a pharmaceutical composition for treating or preventing cancer comprising any one of the above 1 to 3 anti-PTGFRN monoclonal antibodies.
  • the cancer is selected from the group consisting of brain and spinal tumors, head and neck cancer, lung cancer, breast cancer, thymoma, mesothelioma, esophageal cancer, stomach cancer, colon cancer, liver cancer, thyroid cancer, pancreatic cancer, biliary tract cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, germ cell tumor, ovarian cancer, cervical cancer, endometrial cancer, lymphoma, acute leukemia, chronic leukemia, multiple myeloma, sarcoma, malignant melanoma, neuroblastoma, glioblastoma, and skin cancer, a pharmaceutical composition for treating or preventing cancer.
  • composition for detecting PTGFRN comprising any one of the above 1 to 3 anti-PTGFRN monoclonal antibodies.
  • a method for providing information for diagnosing cancer comprising the step of treating a sample isolated from an individual with any one of the above 1 to 3 anti-PTGFRN monoclonal antibodies.
  • a method for providing information necessary for determining the level of cancer cells in blood comprising the step of treating a sample isolated from an individual with any one of the above 1 to 3 anti-PTGFRN monoclonal antibodies.
  • a method for providing information necessary for determining the level of blood cancer cells wherein the sample is selected from the group consisting of blood, plasma, bone marrow fluid, lymph, saliva, tears, urine, mucous membrane fluid, and amniotic fluid in the above 10.
  • a method for providing information necessary for determining the level of cancer cells in blood further comprising the step of treating a sample with a substance that specifically binds to EpCAM in the above 10.
  • the blood cancer cells are blood cancer cells that have undergone epithelial-mesenchymal transition or are undergoing epithelial-mesenchymal transition.
  • a method for providing information necessary for determining the level of blood cancer cells wherein the blood cancer cells are not epithelial cells or mesenchymal cells.
  • the blood cancer cells are blood cancer cells in the blood of a patient with liver cancer, lung cancer, colon cancer, thyroid cancer, pancreatic cancer, malignant melanoma, breast cancer, neuroblastoma or glioblastoma, a method for providing information necessary for determining the level of blood cancer cells.
  • a method for providing information necessary for determining the level of blood cancer cells wherein blood cancer cells in the above 10 more express B7-H3 (CD276) protein.
  • a method for providing information necessary for predicting the prognosis of cancer metastasis comprising the step of detecting PTGFRN by treating a sample isolated from an individual with any one of the above 1 to 3 anti-PTGFRN monoclonal antibodies.
  • a method for providing information necessary for predicting the prognosis of cancer metastasis further comprising a step of providing information that if the level of detected PTGFRN is higher than that of the control group, the prognosis of cancer metastasis is worse than that of the control group.
  • a method for screening the efficacy of an anticancer drug comprising the step of detecting PTGFRN by treating a sample isolated from a patient administered an anticancer drug with any one of the anti-PTGFRN monoclonal antibodies 1 to 3 above.
  • a method for screening the efficacy of an anticancer drug further comprising a step of predicting that the anticancer drug has a better efficacy in inhibiting cancer metastasis than the control group if the level of PTGFRN detected in the above 20 is lower than that of the control group.
  • composition for detecting cancer cells in blood comprising a substance that binds to PTGFRN.
  • the anti-PTGFRN monoclonal antibody 63-D7 of the present invention can be used for determining the level of blood cancer cells, treating or preventing cancer, predicting the prognosis of cancer metastasis, and screening for anticancer drugs.
  • the anti-PTGFRN monoclonal antibody of the present invention can specifically bind to PTGFRN on the surface of various cancer cells, including liver cancer.
  • anti-PTGFRN monoclonal antibody of the present invention various antibody-drug conjugates can be developed and used as anticancer agents.
  • the anti-PTGFRN monoclonal antibody of the present invention stimulates PTGFRN to increase the mobility and invasiveness of cancer cells
  • the anti-PTGFRN monoclonal antibody of the present invention can be used to prevent and inhibit the cancer growth of primary cancer by promoting the migration of primary cancer cells.
  • the anti-PTGFRN monoclonal antibody of the present invention can detect blood cancer cells that are neither epithelial nor mesenchymal.
  • the anti-PTGFRN monoclonal antibody of the present invention can partially detect circulating cancer cells that have undergone epithelial-mesenchymal transition.
  • the anti-PTGFRN monoclonal antibody of the present invention can detect blood cancer cells with high efficiency in liver cancer patients.
  • Blood cancer cells detected by the anti-PTGFRN monoclonal antibody of the present invention are measured at a higher level in patients with secondary metastatic cancer than in patients with primary cancer, so that the stage of cancer can be determined through the anti-PTGFRN monoclonal antibody of the present invention.
  • the anti-PTGFRN monoclonal antibody of the present invention can be internalized into cancer cells, thereby effectively delivering the drug into cancer cells.
  • the present invention also provides PTGFRN as a surface marker of blood cancer cells.
  • Anti-PTGFRN monoclonal antibodies can be used for the isolation, purification, and detection of PTGFRN protein through highly specific binding to PTGFRN protein.
  • the anti-PTGFRN monoclonal antibody binds to epithelial-mesenchymal transition circulating cancer cells and partially epithelial-mesenchymal circulating cancer cells that express PTGFRN on their surface, thereby enabling the detection of previously undetectable circulating cancer cells.
  • Anti-PTGFRN monoclonal antibodies may be useful in the diagnosis of metastatic cancer by detecting cancer cells in the blood.
  • Fig. 1a is a graph showing the results of flow cytometry, showing the degree of binding of antibodies 246-D7, 247-B9 and monoclonal antibody 63-D7 of the present invention to human embryonic stem cell H9, two types of liver cancer cells (Huh7, HepG2), two types of lung cancer cells (A549, NCI-H358), and peripheral blood mononuclear cells (PBMC), respectively.
  • Fig. 1b is a table showing the graph of Fig. 1a.
  • Fig. 1b is a table showing the graph of Fig. 1a.
  • FIG. 1c is a result of immunocytochemical staining, in order to confirm the detection of cancer cells in the blood, double staining was performed using monoclonal antibodies 63-D7, 246-D7, 247-B9 and CD45 antibody labeled with biotin in the blood of eight liver cancer patients and one normal person, respectively, and the nuclei were stained with DAPI, and a Merge of these was illustrated.
  • Figure 1d shows the results of Figure 1c in a table.
  • Figure 2 shows the results of flow cytometry, which shows that the monoclonal antibody 63-D7 of the present invention strongly binds to various cancer cells (SK-Hep1, A549, NCI-H358, NCI-H460, NCI-H1703, NCI-H23, Colo205, HCT116, SNU790, 8505C, BxPC3, A375, MCF-7, MDA-MB-435, SH-SY5Y, U87-MG) including human pluripotent stem cells (H9), human embryonic carcinoma cells (NT2/D1), and liver cancer cells (Huh7, HepG2, SNU387, SNU449) and human embryonic kidney cells (HEK293FT), but weakly binds to bone marrow-derived mesenchymal stem cells (BM-MSC) and human dermal fibroblasts (HDF), and human peripheral blood fibroblasts (PBS).
  • BM-MSC bone marrow-derived mesenchymal stem cells
  • HDF human dermal
  • PBMCs blood mononuclear cells
  • MRC5 human fetal lung fibroblasts
  • DPCs dental pulp cells
  • normal hepatocytes normal hepatocytes.
  • the solid line is monoclonal antibody 63-D7 and the shaded background is a negative control containing only the secondary antibody.
  • Figure 3a shows the results of separating proteins immunoprecipitated using monoclonal antibody 63-D7 from cell lysates of NT-2/D1 human embryonic cancer stem cells (NT-2/D1-biotin) whose cell surface was labeled with biotin, by 10% SDS-PAGE, Western blotting, transferring to a nitrocellulose membrane, and analyzing by reaction with streptavidin-HRP (SA-HRP).
  • SA-HRP streptavidin-HRP
  • Figure 3b shows the results of separating proteins immunoprecipitated in the same manner as in Figure 3a by 10% SDS-PAGE, and staining the polyacrylamide gel with PageBlue.
  • 1X means that 3 mg of cell lysate was used, and 3x means that three times that amount was used.
  • the protein within the dotted box in Fig. 3b was extracted and subjected to LC-MS/MS.
  • Fig. 3c shows the result of analyzing the protein recovered after immunoprecipitation with monoclonal antibody 63-D7 in Fig. 3b by LC-MS/MS, and the part of the amino acid sequence of the 63-D7 antigen that matches the amino acid sequence of the PTGFRN protein is underlined.
  • Figure 4a shows the results of Western blotting using mouse ⁇ -PTGFRN antibody after immunoprecipitation of proteins and a negative control protein without antibody (No Ab) using mouse anti-PTGFRN monoclonal antibody ( ⁇ -PTGFRN) and mouse 63-D7 monoclonal antibody from biotin-labeled human non-small cell lung cancer A549 cell lysates, respectively, separated by 10% SDS-PAGE.
  • Figure 4b shows the results of analysis using streptavidin-HRP (SA-HRP) after removing the nitrocellulose membrane of Figure 4a with a signal removing solution, confirming that PTRFRN is a cell surface molecule recognized by 63-D7.
  • SA-HRP streptavidin-HRP
  • Figure 4c shows the results of immunoprecipitation and Western blotting of the cell lysate using a known mouse anti-FLAG monoclonal antibody ( ⁇ -FLAG), a known mouse anti-PTGFRN monoclonal antibody ( ⁇ -PTGFRN), and monoclonal antibody 63-D7 to reconfirm that the antigen of monoclonal antibody 63-D7 is PTGFRN.
  • WB stands for Western blot.
  • Figure 5a shows the results of a PTGFRN knockdown experiment performed in hepatoma cell lines Huh7 and SNU449.
  • the expression of PTGFRN mRNA was analyzed by qPCR in hepatoma cells treated with a negative control siRNA (siCon) and two types of siRNA against PTGFRN (siPTGFRN#1 and siPTGFRN#2), respectively, to perform knockdown.
  • Figure 5b shows the results of flow cytometry to determine the extent of binding of mouse PTGFRN monoclonal antibody ( ⁇ -PTGFRN) to PTGFRN on the surface of knocked-down Huh7 and SNU449 cells.
  • siCon negative control siRNA
  • siPTGFRN#1 and siPTGFRN#2 two types of siRNA against PTGFRN
  • FIG. 5c is a graph statistically analyzing Figure 5b.
  • Figure 5d shows the results of measuring cancer cell clonogenic survival in PTGFRN knockdown Huh7 and SNU449, and the surviving cell clones 8 days after cell inoculation were stained with crystal violet.
  • Figure 5e is a graph statistically analyzing Figure 5d, and * indicates a p value of p ⁇ 0.05.
  • Figure 6a shows the results of a PTGFRN knockdown experiment performed in hepatoma cell lines Huh7 and SNU449.
  • the human embryonic carcinoma cell line HEK293FT was transfected with a negative control scrambled shRNA (shScramble) or two shRNAs for PTGFRN (shPTGFRN#1, shPTGFRN#2), and the lentivirus was recovered and treated to Huh7 and SNU449 hepatoma cells to perform knockdown.
  • PTGFRN protein expression was confirmed by Western blotting.
  • Figure 6b shows the results of analyzing the decrease in PTGFRN expression in Huh7 and SNU449 hepatoma cells using a flow cytometer.
  • Figure 6c is a graph analyzing the statistical data of Figure 6b.
  • Figure 6d shows the results of measuring cancer cell clonogenic survival in SNU449 knocked down with shPTGFRN#1. The cell clones that survived 8 days after cell inoculation were stained with crystal violet.
  • shScramble is a control.
  • Figure 6e is the result of statistical analysis of Figure 6d, where *** indicates p ⁇ 0.005 versus the control group (shScramble).
  • Figure 6f is the result of measuring cancer cell clonogenic survival in SNU449 knocked down with shPTGFRN#2.
  • Figure 6g is the result of statistical analysis of Figure 6f, where *** indicates p ⁇ 0.005.
  • Figure 7a is a photograph of the results of measuring the degree of migration of liver cancer cells using a negative control shRNA (shScramble) or two shRNAs against PTGFRN (shPTGFRN#1, shPTGFRN#2).
  • PTGFRN knockdown liver cancer cells Huh7 and SNU449 were dispensed into a 24-well transwell chamber and cultured for two days. The cells that had migrated to the lower chamber were stained with crystal violet and taken.
  • Figure 7b is a graph that statistically processed the experiment in Figure 7a after repeating it three times. *** indicates p ⁇ 0.001, ** indicates p ⁇ 0.01, and * indicates p ⁇ 0.05.
  • Figure 7c is a photograph of cells that had invaded the lower layer after being stained with crystal violet after being seeded on a Matrigel-coated transwell chamber and cultured for two days, measuring the invasiveness of cancer cells using a negative control shRNA (shscramble) or shRNA against two types of PTGFRN (shPTGFRN#1, shPTGFRN#2).
  • Figure 7d is a graph that is the result of statistical processing after repeating the experiment in Figure 7c three times. *** indicates p ⁇ 0.001, ** indicates p ⁇ 0.01, and * indicates p ⁇ 0.05.
  • Figure 8a is a photograph of attached liver cancer cells observed under a microscope after knockdown was performed by treating liver cancer cells with a negative control scrambled shRNA (shScramble) or two types of shRNA against PTGFRN (shPTGFRN#1, shPTGFRN#2) in the liver cancer cell line Huh7, and the degree of cell attachment was analyzed by reacting the PTGFRN knockdown liver cancer cells to a 12-well cell culture plate coated with Matrigel, CollagenI, CollagenIV, and gelatin.
  • Figure 8b is a graph showing the degree of cell binding statistically processed by analyzing the microscope photographs using Image J after repeating the experiment in Figure 8a.
  • Figure 8c is a photograph of attached liver cancer cells observed under a microscope after knockdown was performed by treating liver cancer cells with a negative control scrambled shRNA (shScramble) or two shRNAs against PTGFRN (shPTGFRN#1, shPTGFRN#2) in the liver cancer cell line SNU449, and the degree of cell attachment was analyzed by reacting the PTGFRN knockdown liver cancer cells to a 12-well cell culture plate coated with Matrigel, CollagenI, CollagenIV, and gelatin.
  • shScramble negative control scrambled shRNA
  • shPTGFRN#1, shPTGFRN#2 shRNAs against PTGFRN
  • Figure 8d is a graph showing the degree of cell binding statistically processed by analyzing the microscope photographs using Image J after repeating the experiment in Figure 8c. *** indicates p ⁇ 0.001, ** indicates p ⁇ 0.01, * indicates p ⁇ 0.05, and ns indicates no statistical significance.
  • Figure 9a shows the results of measuring the immune evasion ability of PTGFRN knockdown liver cancer cells against NK cells.
  • SNU449 liver cancer cells were treated with negative control scrambled shRNA (shscramble) or shPTGFRN#2 to perform knockdown, and the PTGFRN knockdown liver cancer cells were co-cultured with NK92, and the remaining SNU449 cells were stained with crystal violet. 1:1, 2.5:1, and 5:1 represent the ratios (E:T ratios) of NK92, which is an effector cell, and SNU449, which is a target cell.
  • Figure 9b is a graph showing the results of quantifying the absorbance at OD540 after melting the cells in Figure 9a.
  • Figure 9c is a graph showing the degree of cell death by expressing the OD540 results measured in Figure 9b as an E:T ratio.
  • Figure 10 shows the results of Western blot analysis of the changes in the expression of B7-H3, FAK, Src proteins, and phosphorylated p-FAK (Y397), p-Src (Y416) proteins in the protein lysates of PTGFRN knockdown hepatoma cells Huh7 and SNU449, which were treated with a negative control shRNA (shScramble) or two types of shRNA against PTGFRN (shPTGFRN#1, shPTGFRN#2). GAPDH is the control, and the numbers are quantified using Image J.
  • Figure 11a shows the results of immunoprecipitation of the lysate obtained after co-transfection of HEK293FT cells with pcDNA3.1(+)PTGFRN-FLAG vector and pcDNA3.1(+)PTGFRN-myc vector to confirm the cis interaction of the same PTGFRN in one cell with mouse anti-FLAG, rabbit anti-myc, and 63-D7, and Western blotting with anti-FLAG and anti-myc antibodies. No Ab is the control, and the arrow indicates the immunoprecipitated PTGFRN.
  • Figure 11b shows the results of mixing the lysates of HEK293FT cells transfected with pcDNA3.1(+)PTGFRN-FLAG vector and pcDNA3.1(+)PTGFRN-myc vector, respectively, and immunoprecipitating them with mouse anti-FLAG, rabbit anti-myc, and 63-D7 to confirm trans-interactions between different cells, and performing Western blotting with anti-FLAG and anti-myc antibodies. No Ab is the control, and the arrow indicates immunoprecipitated PTGFRN.
  • Figure 11c shows the results of Western blotting with ⁇ -PTGFRN after immunoprecipitation with mouse anti-PTGFRN monoclonal antibody ( ⁇ -PTGFRN), mouse anti-63-D7 monoclonal antibody, mouse anti-SLC3A2, and mouse anti-B7-H3, respectively, in hepatoma cell SNU449, showing that PTGFRN is immunoprecipitated by SLC3A2 (CD98hc) and B7-H3.
  • ⁇ -PTGFRN mouse anti-PTGFRN monoclonal antibody
  • mouse anti-63-D7 monoclonal antibody mouse anti-SLC3A2
  • mouse anti-B7-H3 mouse anti-B7-H3
  • Figure 11d shows the results of co-transfecting HEK293FT cells with pcDNA3.1(+)PTGFRN-myc vector and pcDNA3.1(+)B7-H3-FLAG vector, followed by immunoprecipitation with anti-FLAG, anti-B7-H3, anti-myc, and 63-D7, and detection with anti-myc and anti-FLAG antibodies, confirming that they interact with each other.
  • HC stands for immunoglobulin heavy chain.
  • WB stands for western blot.
  • Figures 12a and 12b show the results of measuring cell adhesion on a Materigel-coated plate after sorting 63-D7 positive and negative cells with 63-D7 antibody and magnetic beads in liver cancer cell lines Huh7 and HepG2, respectively.
  • Figure 12c shows the results of flow cytometry analysis to compare the cancer cell binding ability of 63-D7 with that of CD133, CD44, and EpCAM, which are cancer stem cell positive markers, when they are adherent cells, after culturing Huh7 as tumor cells to increase cancer stem cell potential.
  • Figure 12d is a graph statistically analyzing Figure 12c. *** indicates p ⁇ 0.005.
  • FIG. 12e is a graph showing the results of measuring the clonogenic survival of 63-D7 positive and negative cancer cells in the liver cancer cell line Huh7 sorted with 63-D7, showing the cancer stemness of the cell clones that survived 8 days after inoculation, stained with crystal violet.
  • Fig. 12f is a graph showing the statistical results of Fig. 12e. *** indicates p ⁇ 0.005 compared to the control group.
  • Fig. 12g is a graph showing the results of measuring the clonogenic survival of 63-D7 positive and negative cancer cells in the liver cancer cell line HepG2 sorted with 63-D7.
  • Fig. 12h is a graph showing the statistical results of Fig. 12g. *** indicates p ⁇ 0.005 compared to the control group.
  • Figures 13a and 13b show the results of analyzing the internalization of monoclonal antibody 63-D7 into human liver cancer cells Huh7, HepG2, SNU449 and human pancreatic cancer cells SNU213 and BxPC3 by flow cytometry.
  • the solid line is the monoclonal antibody and 63-D7 reacted at 4°C
  • the dotted line is the 63-D7 antibody reacted at 37°C after the reaction at 4°C
  • the shaded background includes only the secondary antibody.
  • Figure 13c is a graph comparing the degree of attachment of 63-D7 to the cell surface of each cancer cell by statistically processing it as the relative average fluorescence intensity by repeating the same experiment as Figure 13a three times.
  • Figure 13d is a graph comparing the degree of attachment of 63-D7 to the cell surface of each cancer cell by statistically processing the relative average fluorescence intensity by repeating the same experiment as Figure 13b three times. **** indicates p ⁇ 0.001, *** indicates p ⁇ 0.005, ** indicates p ⁇ 0.01, and * indicates p ⁇ 0.05.
  • Figures 14a and 14b show the results of measuring the cell viability of Huh7 cells treated with 63-D7 or mouse IgG isotype antibody. After Huh7 cells were treated with 0 nM to 100 nM of 63-D7 or mouse isotype antibody, respectively, Figure 14a shows that they were treated with ⁇ -mFc-CL-DMDM and Figure 14b shows that they were treated with ⁇ -mFc-CL-MMAF for 48 hours, and the cell viability was measured using CCK-8.
  • * indicates p ⁇ 0.05
  • ** indicates p ⁇ 0.01
  • **** indicates p ⁇ 0.001.
  • Fig. 15a is a drawing showing a method for detecting blood cancer cells recognized by the PTGFRN-specific monoclonal antibody 63-D7 of the present invention in the blood of a liver cancer patient, and shows a method for removing CD45 positive cells and staining the remaining cells with Dylight488-conjugated 63-D7 and another marker (antibody).
  • Fig. 15b is a graph showing the recovery rate measured by counting the Huh7 cells recovered after removing CD45 positive cells as shown in 15a by adding 0, 10, 30, 50, and 100 Huh7 cells to PBMC.
  • Fig. 15c shows the blood cancer cells recovered as shown in Fig. 15a, stained with Dylight649-conjugated anti-mouse IgG and Dylight 488-conjugated 63-D7, and DAPI that stains the nucleus, and Merge that merges them.
  • FIGS. 16A to 16C are confocal microscopy images showing blood cancer cells double-stained with the PTGFRN-specific monoclonal antibody 63-D7 of the present invention and anti-MVP, anti-HSA, anti-PanCK, anti-E-cadherin, anti-EpCAM, 63-D7, anti-Vimentin, anti-Twist, and anti-ZEB1 antibodies in the blood of a liver cancer patient, and DAPI that stains the nucleus and Merge that merges them.
  • Figure 17 is a graph comparing the number of all blood cancer cells (63-D7+ cells/ml) recognized by the monoclonal antibody 63-D7 of the present invention in the blood of liver cancer patients (53 primary HCC and 42 metastatic HCC) and normal people and hepatitis patients (26 non-neoplastic) through a comparative test (Kruskal-Wallis Test) among the three groups (p value ⁇ 0.0001). It shows that the p-values for each group are all ⁇ 0.0001, indicating that there is significance in the differences among all three groups.
  • FIG. 18a is a confocal microscope image showing circulating cancer cells triple-stained with the PTGFRN-specific monoclonal antibody 63-D7 of the present invention, anti-EpCAM, and anti-Vimentin antibodies in the blood of a liver cancer patient, together with DAPI, which stains the nucleus, and a Merge, which merges them.
  • BF stands for bright field.
  • 18b is a confocal microscope image showing circulating cancer cells triple-stained with the PTGFRN-specific monoclonal antibody 63-D7 of the present invention, anti-MVP, and anti-B7-H3 antibodies in the blood of a liver cancer patient, together with DAPI, which stains the nucleus, and a Merge, which merges them.
  • BF stands for bright field.
  • BF stands for bright field.
  • Figure 19 is a confocal microscope image showing blood cancer cells triple-stained with the PTGFRN-specific monoclonal antibody 63-D7 of the present invention, anti-TGF ⁇ R1, and anti-B7-H3 antibodies in the blood of a liver cancer patient, along with DAPI that stains the nucleus and a Merge that combines them.
  • BF stands for bright field.
  • FIG. 20a is a confocal microscope image of circulating cancer cells triple-stained with the PTGFRN-specific monoclonal antibody 63-D7 of the present invention, anti-ULBP1, and anti-B7-H3 antibodies in the blood of a liver cancer patient, together with DAPI, which stains the nucleus, and a Merge, which merges them.
  • BF stands for bright field.
  • 20b is a confocal microscope image of circulating cancer cells triple-stained with the PTGFRN-specific monoclonal antibody 63-D7 of the present invention, anti-MICA/B, and anti-B7-H3 antibodies in the blood of a liver cancer patient, together with DAPI, which stains the nucleus, and a Merge, which merges them.
  • BF stands for bright field.
  • FIG. 21a is a confocal microscope image showing circulating cancer cells triple-stained with the PTGFRN-specific monoclonal antibody 63-D7 of the present invention, anti-PD-L1, and anti-B7-H3 antibodies in the blood of a liver cancer patient, together with DAPI that stains the nucleus and a Merge that merges them.
  • BF stands for bright field.
  • FIG. 21b is a confocal microscope image showing circulating cancer cells triple-stained with the PTGFRN-specific monoclonal antibody 63-D7 of the present invention, anti-PD-L2, and anti-B7-H3 antibodies in the blood of a liver cancer patient, together with DAPI that stains the nucleus and a Merge that merges them.
  • BF stands for bright field.
  • Figure 22 is a confocal microscope image showing blood cancer cells triple-stained with the PTGFRN-specific monoclonal antibody 63-D7 of the present invention, anti-CD47, and anti-B7-H3 antibodies in the blood of a liver cancer patient, along with DAPI that stains the nucleus and a Merge that combines them.
  • BF stands for bright field.
  • Figure 23 shows the base sequence and amino acid sequence of the heavy chain gene variable region of the monoclonal antibody 63-D7, with the CDR (Complementarity Determining Region) that binds to the antigen indicated in bold.
  • Figure 24 shows the base sequence and amino acid sequence of the light chain gene variable region of the monoclonal antibody 63-D7, with the CDR (Complementarity Determining Region) that binds to the antigen indicated in bold.
  • the present invention provides an anti-PTGFRN monoclonal antibody for determining the level of blood cancer cells, treating or preventing cancer, predicting the prognosis of cancer metastasis, and screening for anticancer drugs.
  • the present invention provides an anti-PTGFRN monoclonal antibody comprising a heavy chain variable region comprising HCDR1 consisting of the amino acid sequence of SEQ ID NO: 3, HCDR2 consisting of the amino acid sequence of SEQ ID NO: 4, and HCDR3 consisting of the amino acid sequence of SEQ ID NO: 5; and a light chain variable region comprising LCDR1 consisting of the amino acid sequence of SEQ ID NO: 6, LCDR2 consisting of the amino acid sequence of SEQ ID NO: 7, and LCDR3 consisting of the amino acid sequence of SEQ ID NO: 8.
  • antibody as used herein includes a complete antibody and any antigen-binding fragment (i.e., "antigen-binding portion") or single chain thereof.
  • An antibody refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains joined to each other by disulfide bonds, or an antigen-binding portion thereof.
  • Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • the heavy chain constant region is comprised of three domains, CH1, CH2, and CH3.
  • Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region is comprised of one domain, CL.
  • the VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs).
  • CDRs complementarity determining regions
  • Each VH and VL is composed of three CDRs and four FRs, namely FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, arranged in the following order from amino terminus to carboxy terminus.
  • the variable regions of the heavy and light chains comprise a binding domain that interacts with an antigen.
  • the constant regions of the antibody can mediate binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.
  • the CDRs included in the VH region are called heavy chain complementary determining region 1 (HCDR1), HCDR2, and HCDR3, and the CDRs included in the VL region are called light chain complementary determining region 1 (LCDR1), LCDR2, and LCDR3.
  • antibody portion refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., PTGFRN). It has been determined that the antigen binding function of an antibody can also be exerted by fragments of full-length antibodies.
  • an antigen e.g., PTGFRN
  • binding fragments encompassed by the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of one arm of an antibody; (v) a dAb fragment consisting of a VH domain [Ward et al., (1989) Nature 341:544-546]; and (vi) an isolated complementarity determining region (CDR) or (vii) a combination of two or more isolated CDRs, which may, in some cases, be linked by a synthetic linker.
  • CDR complementarity determining region
  • the two domains of the Fv fragment namely the VL and VH
  • they can be joined by a synthetic linker, which can be prepared by recombinant methods, into a single protein chain in which the VL and VH regions pair to form a monovalent molecule (called single-chain Fv (scFv)).
  • single-chain Fv single-chain Fv
  • Such single-chain antibodies are also included in the term "antigen-binding portion" of an antibody.
  • antibody fragments can be obtained by conventional techniques known to those skilled in the art, and these fragments are screened for utility in the same manner as complete antibodies.
  • the antigen-binding portion can be prepared by recombinant DNA techniques, or by enzymatic or chemical cleavage of complete immunoglobulins.
  • the antibody herein may be a monoclonal antibody or a polyclonal antibody.
  • the antibody may be produced using various techniques known in the art for producing humanized antibodies.
  • monoclonal antibody refers to an antibody that exhibits a single binding specificity and exhibits affinity for a particular epitope.
  • PTGFRN-specific monoclonal antibodies can be efficiently produced in virtually unlimited quantities in a highly purified form. PTGFRN-specific monoclonal antibodies can specifically bind to a specific epitope of PTGFRN.
  • the antibodies herein that bind to PTGFRN can be produced by viral or oncogene transformation of B cells fused to immortalized cells obtained from a nonhumanized animal having a genome comprising a heavy chain transgene and a light chain transgene, phage display techniques using a library of human antibody genes, somatic cell hybridization techniques, etc.
  • Methods for producing hybridomas in animal systems, including immunization protocols and isolation and fusion techniques of immunized spleen cells, for producing monoclonal antibodies are well known in the art.
  • Antibodies or antigen-binding fragments thereof can be labeled with radionuclides, fluorescors, enzymes, etc.
  • epitopes refers to a portion of an antigen to which an immunoglobulin or antibody specifically binds.
  • Epitopes may be formed from adjacent amino acids or from non-adjacent amino acids that are juxtaposed by tertiary folding of the protein. Epitopes formed from adjacent amino acids are generally retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are generally lost upon treatment with denaturing solvents.
  • Epitopes typically comprise at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in a unique spatial conformation.
  • the epitope (or antigenic determinant site) functions as a conformational epitope in that no additional increase in affinity for the epitope/antigenic determinant site is observed even when it is within a larger amino acid segment (e.g., a protein having a three-dimensional structure).
  • isotype refers to the class of antibodies (e.g., IgM or IgG1) encoded by the heavy chain constant region genes.
  • the monoclonal antibodies of the invention are of the IgG1 isotype.
  • the present invention produces a monoclonal antibody that binds to the surface of human embryonic stem cells, and searches for an antigen of monoclonal antibody 63-D7, which binds to the surface of human embryonic stem cells, embryonic cancer cells, and various cancer cells, but does not bind to normal cells such as human peripheral mononuclear cells or human hepatocytes.
  • the 63-D7 monoclonal antibody recognizes the cell surface protein PTGFRN (Prostaglandin F2 Receptor Negative Regulator, CD315).
  • PTGFRN Prostaglandin F2 Receptor Negative Regulator, CD315
  • PTGFRN recognized by the 63-D7 monoclonal antibody is expressed in cell lines such as liver cancer, lung cancer, colon cancer, pancreatic cancer, and thyroid cancer.
  • the results of an experiment on 95 patients with hepatocellular carcinoma confirmed that the 63-D7 monoclonal antibody can detect circulating cancer cells with an efficiency of 97%. Since the 63-D7 antibody is co-expressed with Vimentin, an EMT marker, at a ratio of about 53% in primary HCC and about 28% in secondary HCC, it was confirmed that the 63-D7 antibody can be used to detect circulating cancer cells showing an EMT phenotype. In addition, circulating cancer cells recognized by 63-D7 can also detect intermediate circulating cancer cells that do not express either EpCAM or Vimentin, and specifically, it was confirmed that expression was found at a ratio of about 46% in primary HCC and about 72% in secondary HCC.
  • the 63-D7 antibody is a new marker that can detect both intermediate phenotype and EMT phenotype circulating cancer cells that could not be identified by existing EpCAM marker-based circulating cancer cell diagnostic technology.
  • PTGFRN Prostaglandin F2 Receptor Negative Regulator, EWI-F, CD9P-1, CD315) is a type I transmembrane protein of the immunoglobulin superfamily, which negatively affects the Prostaglandin F2 Receptor.
  • PTGFRN includes a full-length sequence, a fragment that performs an equivalent function, or a polypeptide having a continuous amino acid sequence of PTGFRN (e.g., the PTGFRN amino acid sequence disclosed in FIG. 3c).
  • PTGFRN may be, but is not limited to, a PTGFRN derived from a vertebrate, more specifically, a human or mouse.
  • the present invention provides an anti-PTGFRN monoclonal antibody, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 1.
  • the present invention may be an antibody comprising a single chain variable region (scFv) comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or more identical to the heavy chain variable region amino acid sequence (SEQ ID NO: 1) produced by the anti-PTGFRN monoclonal antibody clone 63-D7 of the present invention.
  • scFv single chain variable region
  • the present invention provides an anti-PTGFRN monoclonal antibody, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 2.
  • the anti-PTGFRN monoclonal antibody of the present invention may be an antibody comprising a single chain variable region (scFv) comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or more identical to the light chain variable region amino acid sequence (SEQ ID NO: 2) produced by clone 63-D7.
  • scFv single chain variable region
  • the present invention provides an antibody-drug conjugate (ADC) in which a drug is conjugated to the PTGFRN monoclonal antibody of the present invention.
  • ADC antibody-drug conjugate
  • the monoclonal antibody of the present invention can promote cellular internalization of PTGFRN in various cancer cells, and is therefore suitable as an antibody used in antibody-drug conjugates.
  • the drug can be selected by those skilled in the art according to the purpose without limitation in type.
  • it can be a known anticancer drug, but is not limited thereto.
  • the method of conjugation can be selected without limitation by a method known in the art as long as it can bring the antibody and the drug into contact.
  • the present invention provides a pharmaceutical composition for treating or preventing cancer comprising the anti-PTGFRN monoclonal antibody of the present invention.
  • the present invention provides a method for treating, preventing or diagnosing cancer comprising the anti-PTGFRN monoclonal antibody of the present invention.
  • cancer may be any one selected from the group consisting of brain and spinal tumors, head and neck cancer, lung cancer, breast cancer, thymoma, mesothelioma, esophageal cancer, stomach cancer, colon cancer, liver cancer, thyroid cancer, pancreatic cancer, biliary tract cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, germ cell tumor, ovarian cancer, cervical cancer, endometrial cancer, lymphoma, acute leukemia, chronic leukemia, multiple myeloma, sarcoma, malignant melanoma, neuroblastoma, glioblastoma, and skin cancer, but is not limited thereto.
  • Metastatic cancer is cancer that has spread from one organ to another, including the lymph nodes.
  • the cancer present in the organ where the cancer has spread is called the primary cancer.
  • metastases can be brain metastases, bone metastases, liver metastases, or lung metastases.
  • Metastatic cancer can be a malignant tumor.
  • a malignant tumor can be a brain or spinal tumor, head and neck cancer, lung cancer, breast cancer, thymoma, mesothelioma, esophageal cancer, stomach cancer, colon cancer, liver cancer, pancreatic cancer, biliary tract cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, germ cell tumor, ovarian cancer, cervical cancer, endometrial cancer, lymphoma, acute leukemia, chronic leukemia, multiple myeloma, sarcoma, malignant melanoma, skin cancer, or a combination of these.
  • the monoclonal antibody of the present invention is an agonistic antibody for the cancer, and can stimulate PTGFRN on the surface of cancer cells to promote the movement and invasiveness of cancer cells.
  • the monoclonal antibody can promote the movement of cancer cells in primary cancer, thereby preventing the occurrence of early primary cancer or inhibiting its growth.
  • the present invention provides a hybridoma producing the above anti-PTGFRN monoclonal antibody.
  • a hybridoma may include a hybridoma cell or a hybridoma cell line.
  • the hybridoma is a cell created by artificially fusing two different types of cells, and refers to a cell or cell line in which two or more homologous or heterologous cells are fused using a substance that causes cell fusion, such as polyethylene glycol (PEG) or a certain type of virus, thereby integrating different functions of each cell into a single cell.
  • PEG polyethylene glycol
  • a hybridoma that secretes a monoclonal antibody can be cultured in large quantities in vitro or in vivo.
  • the present invention provides a composition for detecting PTGFRN comprising the anti-PTGFRN monoclonal antibody of the present invention.
  • the above antigen-antibody complex can be detected using a detection label.
  • the detection label can be selected from enzymes, fluorescent substances, ligands, luminescent substances, microparticles, redox molecules, or radioisotopes, but is not particularly limited thereto.
  • the present invention provides a method for providing information for diagnosing cancer, comprising the step of treating a separated sample with an anti-PTGFRN monoclonal antibody of the present invention.
  • an entity may be a mammal, including a human.
  • a biological sample refers to a sample obtained from a living organism.
  • the biological sample may be, for example, blood, plasma, bone marrow fluid, lymph, saliva, tears, urine, mucous membrane fluid, amniotic fluid, or a combination thereof.
  • the substance that specifically binds to PTGFRN may be an anti-PTGFRN antibody or an antigen-binding fragment thereof or an aptamer.
  • An aptamer refers to an oligonucleic acid or peptide that binds to a target molecule.
  • the present invention provides a method for providing information necessary for determining the level of blood cancer cells, comprising the step of treating a sample isolated from an individual with an anti-PTGFRN monoclonal antibody of the present invention.
  • it can provide information necessary for diagnosing metastatic cancer, including the steps of contacting a biological sample isolated from an individual with the anti-PTGFRN monoclonal antibody of the present invention to bind blood cancer cells in the sample to the anti-PTGFRN monoclonal antibody of the present invention; detecting the blood cancer cells from the reaction mixture; and determining, if the blood cancer cells are detected, that the individual has or is likely to have metastatic cancer.
  • the anti-PTGFRN monoclonal antibody of the present invention can specifically bind to PTGFRN, PTGFRN can be detected, and furthermore, PTGFRN can be separated or purified using the composition.
  • PTGFRN By contacting the anti-PTGFRN monoclonal antibody of the present invention with a sample and detecting the formation of an antigen-antibody complex, the PTGFRN protein can be separated, identified, or detected.
  • the antigen-antibody complex refers to a combination of PTGFRN and a monoclonal antibody that recognizes it for the purpose of confirming the presence or absence of PTGFRN in a sample.
  • the antigen-antibody complex can be detected using a detection label.
  • the label can be selected from an enzyme, a fluorescent substance, a ligand, a luminescent substance, a microparticle, a redox molecule, or a radioisotope, but is not particularly limited thereto.
  • antigen-antibody complexes can be detected using a colorimetric method, an electrochemical method, a fluorescence method, a luminometry method, a particle counting method, a visual assessment, or a scintillation counting method.
  • a colorimetric method an electrochemical method, a fluorescence method, a luminometry method, a particle counting method, a visual assessment, or a scintillation counting method.
  • it can be detected by flow cytometry, immunocytochemistry, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), Western blotting, immunoprecipitation assay, immunodiffusion assay, complement fixation assay, protein chip, etc.
  • immune precipitation and immunoblotting which are useful for recovering a small amount of target protein.
  • an antigen-antibody complex can be detected using an enzyme-linked immunosorbent assay (ELISA).
  • the ELISA includes various ELISA methods, such as a direct ELISA using a labeled antibody that recognizes an antigen attached to a solid support, an indirect ELISA using a labeled antibody that recognizes a capture antibody in a complex of antibodies that recognize the antigen attached to the solid support, a direct sandwich ELISA using another labeled antibody that recognizes an antigen in a complex of an antibody and an antigen attached to the solid support, and an indirect sandwich ELISA using a labeled secondary antibody that recognizes the antibody after reacting with another antibody that recognizes the antigen in a complex of an antibody and an antigen attached to the solid support.
  • Circulating tumor cells are rare tumor cells that exist in the blood and circulate in the body after undergoing a tumor invasion process. Circulating tumor cells are known to be a factor involved in cancer metastasis and recurrence. Circulating tumor cells may be circulating tumor cells that have undergone epithelial-mesenchymal transition.
  • Epithelial-mesenchymal transition is the process by which epithelial cells lose cell polarity and cell-to-cell adhesion and transform into mesenchymal cells that are mobile and invasive. Epithelial-mesenchymal transition is essential for many developmental processes, including mesoderm formation and neural tube formation, and is known to occur in wound healing, organ fibrosis, and in the initiation of metastasis during cancer progression.
  • the circulating cancer cells may be circulating cancer cells that have undergone epithelial-mesenchymal transition, circulating cancer cells that have not undergone epithelial-mesenchymal transition, or circulating cancer cells that are undergoing epithelial-mesenchymal transition, and are preferably, but not limited to, circulating cancer cells that have undergone epithelial-mesenchymal transition or are undergoing epithelial-mesenchymal transition.
  • Cancer cells in the blood may be neither epithelial nor mesenchymal cells.
  • detection of blood cancer cells comprises separating a complex in which blood cancer cells and a substance that specifically binds to PTGFRN are combined from a biological sample.
  • the detection method comprises electron microscopy, microfiltration, centrifugation, microfluidics, immunostaining, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), flow cytometry, fluorescense activated cell sorting (FACS), or a combination thereof.
  • the detection method may be polymerase chain reaction (PCR), electrophoresis, northern blotting, western blotting, or a combination thereof.
  • PTGFRN was confirmed to be expressed in liver cancer, lung cancer, colon cancer, pancreatic cancer, and thyroid cancer cell lines, and 97% of hepatocellular carcinoma patients were detected with circulating cancer cells.
  • an EMT marker in about 53% of primary HCCs and about 28% of secondary HCCs, it was confirmed to be useful for detecting circulating cancer cells showing an EMT phenotype and can be used for diagnosing metastatic cancer.
  • circulating cancer cells recognized by 63-D7 were simultaneously expressed in about 46% of primary HCCs and about 72% of secondary HCCs, even in intermediate circulating cancer cells that did not express either EpCAM or Vimentin, so it can be usefully used for detecting circulating cancer cells by complementing the shortcomings of existing EpCAM marker-based circulating cancer cell diagnosis technology.
  • the blood cancer cells may be derived from a primary cancer selected from the group consisting of brain and spinal cancer, head and neck cancer, lung cancer, breast cancer, thymoma, mesothelioma, esophageal cancer, stomach cancer, colon cancer, liver cancer, pancreatic cancer, biliary tract cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, germ cell tumor, ovarian cancer, cervical cancer, endometrial cancer, lymphoma, acute leukemia, chronic leukemia, multiple myeloma, sarcoma, malignant melanoma, and skin cancer.
  • a primary cancer selected from the group consisting of brain and spinal cancer, head and neck cancer, lung cancer, breast cancer, thymoma, mesothelioma, esophageal cancer, stomach cancer, colon cancer, liver cancer, pancreatic cancer, biliary tract cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, germ cell tumor, ovarian cancer, cervical
  • the present invention provides a method for providing information necessary for determining the level of cancer cells in blood, which further includes a step of treating a sample isolated from an individual with a substance that specifically binds to EpCAM.
  • the present invention provides a method for providing information necessary for determining the level of cancer cells in blood, wherein the substance that specifically binds to EpCAM is an anti-EpCAM antibody or an antigen-binding fragment thereof.
  • EpCAM is a transmembrane glycoprotein that mediates Ca 2+ -dependent homotypic cell-cell adhesion in epithelial cells. EpCAM is known to be involved in cell signaling, migration, proliferation, and differentiation. EpCAM can be human or mouse EpCAM. For example, EpCAM can be a polypeptide having an amino acid sequence of GenBank Accession No. NP_002345. For example, EpCAM can be a polypeptide encoded by a nucleotide sequence of GenBank Accession No. NM_002354. The substance that specifically binds to EpCAM can be an anti-EpCAM antibody or an antigen-binding fragment thereof. The antibody or antigen-binding fragment thereof is as described above.
  • the blood cancer cells may be blood cancer cells undergoing epithelial-mesenchymal transition or undergoing epithelial-mesenchymal transition.
  • the blood cancer cells may be in a hybrid E/M state.
  • the blood cancer cells may be neither epithelial cells nor mesenchymal cells.
  • the blood cancer cells may be blood cancer cells in the blood of a patient with liver cancer, lung cancer, colon cancer, thyroid cancer, pancreatic cancer, malignant melanoma, breast cancer, neuroblastoma, or glioblastoma.
  • the blood cancer cells can further express B7-H3 (CD276) protein.
  • the present invention provides a method for providing information necessary for predicting the prognosis of cancer metastasis, comprising the step of detecting PTGFRN by treating a sample isolated from an individual with the anti-PTGFRN monoclonal antibody of the present invention.
  • the present invention provides a method for providing information necessary for predicting the prognosis of cancer metastasis, further comprising a step of providing information that if the level of detected PTGFRN is higher than that of the control group, the prognosis of cancer metastasis is worse than that of the control group.
  • the present invention provides a method for screening the efficacy of an anticancer drug, comprising the step of detecting PTGFRN by treating a sample isolated from a patient administered an anticancer drug with an anti-PTGFRN monoclonal antibody of the present invention.
  • the present invention provides a method for screening the efficacy of an anticancer drug, further comprising a step of predicting that the anticancer drug will have a better efficacy in inhibiting cancer metastasis compared to a control group if the level of detected PTGFRN is lower than that of a control group.
  • the present invention provides a composition for detecting cancer cells in blood, comprising a substance that binds to PTGFRN.
  • the present invention provides a composition for isolating, purifying, detecting and detecting the concentration of PTGFRN protein, which comprises a substance that binds to PTGFRN.
  • composition for detecting blood cancer cells of the present invention may further include a substance that specifically binds to epithelial cell adhesion molecule (EpCAM).
  • EpCAM epithelial cell adhesion molecule
  • the substance binding to PTGFRN may be an anti-PTGFRN antibody or an aptamer.
  • the anti-PTGFRN antibody may be a monoclonal antibody or a polyclonal antibody.
  • the circulating cancer cells may be, but are not limited to, circulating cancer cells in the blood of a patient with liver cancer, lung cancer, colon cancer, thyroid cancer, pancreatic cancer, melanoma, breast cancer, neuroblastoma, or glioblastoma.
  • the circulating cancer cells may be circulating cancer cells that have undergone epithelial-mesenchymal transition or are undergoing epithelial-mesenchymal transition.
  • the circulating cancer cells may be in a hybrid E/M state.
  • the substance binding to PTGFRN may be an anti-PTGFRN antibody of the present invention.
  • the composition for detecting blood cancer cells in blood of the present invention may be included in the form of a kit.
  • the kit of the present invention when the kit of the present invention is applied to an immunoassay, the kit of the present invention may optionally include a secondary antibody and a substrate of a label.
  • the kit according to the present invention may be manufactured into a plurality of separate packages or compartments containing the above-mentioned reagent components.
  • the composition for detecting blood cancer cells of the present invention may be included in the form of a microarray.
  • the monoclonal antibody is used as a hybridizable array element and is immobilized on a substrate.
  • a preferred substrate is a suitable rigid or semi-rigid support, such as a membrane, a filter, a chip, a slide, a wafer, a fiber, a magnetic bead or a non-magnetic bead, a gel, a tubing, a plate, a polymer, a microparticle, and a capillary.
  • the hybridizable array element is arranged and immobilized on the substrate, and such immobilization may be performed by a chemical bonding method or a covalent bonding method such as UV.
  • the hybridizable array element may be bonded to a glass surface modified to include an epoxy compound or an aldehyde group, and may also be bonded to a polylysine-coated surface by UV.
  • the hybridizable array element may be bonded to the substrate through a linker (e.g., ethylene glycol oligomer and diamine).
  • the present invention provides a composition for monitoring cancer metastasis comprising a substance that binds to PTGFRN.
  • the substance is not limited as long as it can measure the level of PTGFRN.
  • Human embryonic stem cells H9 were purchased from the Wicell Research Institute and cultured according to the provided protocol.
  • the culture medium contained DMEM/F12 (Invitrogen, Seoul, Korea), 20% Knockout SR (Invitrogen), 0.1 mM ⁇ -mercaptoethanol (Sigma, St Luis, MO, USA), 2 mM glutamine (Invitrogen), 0.1 mM nonessential amino acids (Invitrogen), 100 U/ml penicillin G (Sigma), 100 ⁇ g/ml streptomycin (Sigma), and 4 ng/ml bFGF (PeproTech, Rocky Hill, NJ).
  • Human embryonic carcinoma NT-2/D1 cells were purchased from ATCC (Manassas, VA, USA) and cultured according to the provided protocol.
  • Human hepatocarcinoma Human hepatocarcinoma (Huh7, HepG2, SNU387, SNU449), non-small cell lung cancer (A549, NCI-H358, NCI-H460, NCI-H1703, NCI-H23), and thyroid cancer (SNU-790) cells were purchased from Korea Cell Line Bank (KCLB, Seoul, Korea).
  • Human normal lung fetal fibroblast (MRC5), colon cancer (Colo205), and pancreatic cancer (BxPC3) cells were purchased from ATCC, and human hepatocytes were purchased from Termo Fisher Scientific (Waltham, USA) and cultured according to the provided protocol.
  • PBMCs Human peripheral blood mononuclear cells
  • Human embryonic stem cells (H9) and human peripheral mononuclear cells (PBMC) were also used in the analysis.
  • PBA 1% bovine serum albumin, 0.02% NaN 3 in PBS, pH 7.4
  • the antibodies were reacted for 30 minutes at 4 °C, respectively.
  • the primary antibody and the corresponding anti-mouse IgG-FITC were reacted for another 30 minutes at 4 °C.
  • the antibody reaction was analyzed for propidium iodide (PI)-negative cells using FACS Calibur and Cell Quest software (BD sciences).
  • 63-D7, 246-D7, and 247-B9 antibodies that bound to human embryonic stem cells (H9), liver cancer cell lines (Huh7, HepG2), and lung cancer cell lines (A549) but not to PBMCs were selected (Fig. 1a, Fig. 1b).
  • 246-D7 and 247-B9 bound to the epithelial lung cancer cell line NCI-H358, but 63-D7 did not bind.
  • 63-D7, 246-D7, and 247-B9 antibodies were purified by protein G-agarose column chromatography, and the purified antibodies were biotinylated according to the protocol provided using the DSB-XTM Biotin Protein Labeling Kit (Molecular Probes, Seoul, Korea).
  • PBMCs were isolated from the blood of one healthy adult and eight patients with hepatocellular carcinoma (HCC) using the same method as in Example 1-2.
  • the recovered PBMC cells were divided into 2 to 4 cells depending on the number of cells and attached to poly-L-lysine-coated slides.
  • the prepared slides were treated with 4% paraformaldehyde (PFA) for 10 minutes at room temperature to fix the cells.
  • PFA paraformaldehyde
  • PBS pH 7.4 containing 1 mM Ca 2+ and 0.5 mM Mg 2+ was always used in all PBS used for cell staining.
  • the cells were blocked with blocking solution (10% horse serum, 0.1% BSA, PBS, pH 7.4) for 1 hour. Then, the plate was reacted with mouse antibody CD45 antibody (BD Biosciences, Seoul Korea) at room temperature, blocked from light, for 1 hour, followed by anti-mouse IgG-Alexa488 (Invitrogen, 1:3000) at room temperature, blocked from light, for another 1 hour. After washing with PBS (pH 7.4), 5 ⁇ g of 63-D7-biotin, 246-D7-biotin, and 247-B9-biotin antibodies were added respectively to stain the antigens recognized by each antibody and reacted for 12 hours at room temperature, blocked from light, 4°C.
  • blocking solution 10% horse serum, 0.1% BSA, PBS, pH 7.4
  • Example 3 Analysis of 63-D7 binding to various cells using a flow cytometer
  • various cancer cells including embryonic cancer (NT2/D1), metastatic liver cancer (SNU387, SNU449), lung cancer (NCI-H460, NCI-H1703, NCI-H23), colon cancer (Colo205, HCT116), thyroid cancer (SNU790), and pancreatic cancer (BxPC3), as well as human embryonic kidney cells (HEK293FT), bone marrow-derived mesenchymal stem cells (BM-MSC), human dermal fibroblasts (HDF), and human peripheral blood (PB) cells.
  • HEK293FT human embryonic kidney cells
  • BM-MSC bone marrow-derived mesenchymal stem cells
  • HDF human dermal fibroblasts
  • PB peripheral blood
  • PBMCs primary mononuclear cells
  • MRC5 normal lung fetal fibroblasts
  • DPCs dental pulp cells
  • hepatocytes normal hepatocytes. Specifically, cells were detached by treating with 0.05% trypsin, washed with phosphate-buffered saline (PBS), and filtered using a 40 ⁇ m strainer (BD Biosciences) to separate single cells. 5 ⁇ 10 5 cells were mixed with PBA (1% bovine serum albumin, 0.02% NaN 3 in PBS, Ph 7.4), and then reacted with 63-D7 antibody at 4 °C for 30 min.
  • PBA 1% bovine serum albumin, 0.02% NaN 3 in PBS, Ph 7.4
  • the primary antibody and corresponding anti-mouse IgG-FITC (BD Biosciences) were reacted for another 30 min at 4 °C.
  • the antibody reaction to propidium iodide (PI)-negative cells was analyzed using FACS Calibur and Cell Quest software (BD sciences).
  • PI propidium iodide
  • the 63-D7 antibody binds to all cancer cells including hepatoma cells, but does not bind to epithelial lung cancer cells H358.
  • it does not bind to normal cells such as PBMC, dental pulp cells (DPC), and MRC5, and does not bind to normal hepatocytes, demonstrating cancer cell-specific binding (Fig. 2).
  • the experimental results are shown in Fig. 2 and Table 1 below.
  • NT-2/D1 cancer cells that bind well were washed twice with PBS (pH 7.4), and a solution of NHS-Sulfo-LC-biotin (Pierce) dissolved in PBS (pH 8.0) was added and reacted at 4°C for 30 minutes, and then washed twice with PBS (pH 8.0).
  • lysis buffer 25 mM Tris-HCl, pH 7.5, 250 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 2 ⁇ g/ml aprotinin, 100 ⁇ g/ml PMSF, 5 ⁇ g/ml leupeptin, 1 mM NaF, 1 mM Na 3 VO 4 ), centrifuged at 12,000 rpm for 40 minutes to remove nuclei, and stored at -70°C until use to prepare a protein solution.
  • lysis buffer 25 mM Tris-HCl, pH 7.5, 250 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 2 ⁇ g/ml aprotinin, 100 ⁇ g/ml PMSF, 5 ⁇ g/ml leupeptin, 1 mM NaF, 1 mM Na 3 VO 4 .
  • protein-G-plus agarose (Merck millipore, Darmstadt, Germany) was added to the prepared protein solution.
  • 20 ⁇ l of protein-G-agarose was added to the cell extract of about 1 ⁇ 10 7 cells to remove molecules that bind nonspecifically, and the mixture was reacted at 4 °C for 2 hours.
  • the molecules that bind to protein-G-plus agarose were removed through centrifugation, and the remaining supernatant was collected and used to immunoprecipitate molecules that bind to 63-D7.
  • the membrane was blocked with 5% skim milk powder for 2 hours at room temperature, washed three times with PBST [phosphate buffer solution (PBS), pH 7.4 containing 0.05% Tween 20], and the Western membrane was reacted with streptavidin-HRP (Streptavidin-HRP, GE healthcare) at room temperature for 1 hour to bind to the surface antigen immunoprecipitated by 63-D7 that was biotin-labeled. After washing with PBST, the bound protein was confirmed using an ECL detection kit (GE healthcare). As shown in Fig. 3a, in NT-2/D1 cells, the 63-D7 antibody recognizes and immunoprecipitates a surface antigen labeled with biotin with a size of approximately 130 kDa (Fig. 3a).
  • the polyacrylamide gel containing the protein immunoprecipitated by 63-D7 was stained with PageBlue Protein Staining Solution (Thermo Fischer Scientific) according to the supplier's protocol (Fig. 3b).
  • the protein band stained at the 130 kDa position which was assumed to be the protein immunoprecipitated by 63-D7, was excised from the gel and subjected to LC-MS/MS (Liquid Chromatography with Tandem Mass Spectrometry) analysis (ProteomeTech, Seoul, Korea).
  • the ProFound search engine (http:/129.85.19.192/profound_bin/WebProFound.exe) developed by Rockefeller University was used to identify proteins from the analyzed mass spectrum.
  • the antigen protein recognized by 63-D7 was confirmed to be Homo sapiens Prostaglandin F2 receptor negative regulator (PTGFRN) (Fig. 3c).
  • the underlined amino acids in Figure 3c actually represent the peptides identified by mass spectrometry, showing that 71 amino acids are identical to PTGFRN.
  • antigen protein of monoclonal antibody 63-D7 is PTGFRN
  • immunoprecipitation, Western blotting, gene knockdown by RNA interference, and flow cytometry were performed using commercially available antibodies, gene expression vectors, siRNA, and shRNA against PTGFRN.
  • a mouse anti-PTGFRN monoclonal antibody ( ⁇ -PTGFRN, R&D system) was purchased and used for immunoprecipitation and Western blotting.
  • a cell lysate (150 ⁇ g) was prepared from A549 cells labeled with biotin as described in Example 4, and the lysate was immunoprecipitated with the monoclonal antibody 63-D7 (5 ⁇ g) and ⁇ -PTGFRN (2.5 ⁇ g) as described above.
  • the membrane was reacted with gentle shaking in Striping solution (100 mM 2ME, 2% SDS, 62.5 mM Tris-HCl, pH6.7) at 50 °C for 30 min, washed twice with the above PBST for 10 min, and blocked with 5% skim milk powder for 1 h at room temperature. After washing three times with 0.1% PBST, streptavidin-HRP (SA-HRP, 1:5000) was added and reacted at room temperature for 1 h. Then, it was washed three times with PBST, and the biotin-labeled protein was confirmed with an ECL detection kit.
  • Striping solution 100 mM 2ME, 2% SDS, 62.5 mM Tris-HCl, pH6.7
  • PTGFRN with a size of approximately 130 kDa identical to cell surface PTGFRN, was detected in the immunoprecipitate of 63-D7 and ⁇ -PTGFRN (Fig. 4b). These results show that 63-D7 binds to cell surface PTGFRN.
  • PTGFRN gene expression vector pcDNA3.1(+)-PTGFRN-DYK (GeneScript, Piscataway, NJ, USA) was purchased, and recombinant PTGFRN tagged with a FLAG tag was overexpressed in human embryonic kidney cells HEK293FT.
  • the cell lysate was immunoprecipitated and analyzed by Western blotting using mouse anti-FLAG monoclonal antibody ( ⁇ -FLAG) (Invitrogen), mouse anti-PTGFRN monoclonal antibody ( ⁇ -PTGFRN) (AbCAM), and 63-D7.
  • HEK293FT human embryonic kidney cells HEK293FT
  • PEI polyetherimide
  • the cell lysate was immunoprecipitated with 63-D7, anti-FLAG antibody, and ⁇ -PTGFRN as described above, and the eluted protein and the negative control protein without antibody (No Ab) were separated using 10% SDS-PAGE and transferred to a nitrocellulose membrane.
  • the membrane was blocked with 5% skim milk powder for 1 hour at room temperature. After washing three times with 0.1% TBST, the known mouse anti-FLAG antibody, or ⁇ -PTGFRN, 63-D7 was reacted at 4 °C for 16 hours. After washing three times with 0.1% TBST, the membrane was further reacted with anti-mouse IgG-HRP (1:10,000; Millipore) for 1 hour at room temperature.
  • PTGFRN the antigen of antibody 63-D7
  • two kinds of siRNA (Bioneer, Daejeon, Korea) targeting PTGFRN were purchased and transiently knocked down in hepatocellular carcinoma cell lines Huh7 and SNU449.
  • siRNA-lipofectamine RNAiMAX (Invitrogen) was each diluted in TOM (Welgene, Gyeongsan, Korea) and incubated for 5 min at room temperature. Then, each siRNA dilution and RNAiMAX dilution were mixed and reacted for 20 min at room temperature. The siRNA-lipofectamine mixture was added to each cell medium and reacted for 24 hours.
  • qPCR was performed using primers targeting PTGFRN and GAPDH and Power SYBR Green PCR Master Mix (applied biosystem), and GAPDH was used as a reference gene.
  • the specific sequences of the primers used are shown in Table 2 below.
  • the knocked-down cancer cells showed a reduction efficiency of PTGFRN mRNA of 85-88% and 71-72% in Huh7 and SNU449, respectively, compared to the negative control group (Fig. 5a).
  • Fig. 5a when flow cytometry analysis was performed using cells with PTGFRN knocked-down as in Example 3, it was observed that the binding of 63-D7 antibody was reduced by 51-75% and 71-78% in Huh7 and SNU449, respectively, indicating that PTGFRN expression was successfully knocked down (Fig. 5b, Fig. 5c).
  • a clonogenic survival assay was performed using the PTGFRN knockdown Huh7 cells and SNU449 cells described above.
  • the PTGFRN knockdown Huh7 cells prepared through the above experiment were detached with 0.05% Trypsin-EDTA (Welgene, Gyeongsan, Korea) and neutralized with cell culture medium containing 10% fetal bovine serum (Corning).
  • the cells were passed through a 40 ⁇ m strainer (SPL, Pocheon, Korea) to prepare single cells. 2.0 ⁇ 103 cells per well were plated in a 6-well plate, and the cells were allowed to disperse and attach as single cells.
  • Cells inserted with the target gene were selected for one week with RPMI-1640 medium containing 10% fetal bovine serum (Corning) and 2 ⁇ g/ml puromycin (Gibco). After selection, the cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (Corning) and 100 ng/ml doxycycline (Sigma) to induce knockdown, and then cultured for 72 h. The cultured cells were detached with 0.05% Trypsin-EDTA (Welgene), neutralized with cell culture medium containing 10% fetal bovine serum (Corning), and then harvested.
  • RPMI-1640 medium containing 10% fetal bovine serum (Corning) and 2 ⁇ g/ml puromycin (Gibco). After selection, the cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (Corning) and 100 ng/ml doxycycline (Sigma) to induce knockdown, and then cultured for 72 h
  • PTGFRN protein expression in the harvested cells was analyzed by Western blot, and PTGFRN expression was observed to be reduced by 57-84% and 88-89% in Huh7 and SNU449, respectively (Fig. 6a).
  • PTGFRN#1, shPTGFRN#2 two types of shPTGFRN
  • PTGFRN#1 reduced by 90% (Fig. 6d, Fig. 6e) and shPTGFRN#2 reduced by 47% (Fig. 6f, Fig. 6g).
  • transwell experiments were performed using Huh7 and SNU449 cells in which PTGFRN was knocked down with the above shPTGFRN#1 and shPTGFRN#2.
  • the PTGFRN knocked down Huh7 cells prepared through the above experiment were detached with 0.05% Trypsin-EDTA (Welgene), neutralized with cell culture medium containing 10% fetal bovine serum (Corning), and then passed through a 40 ⁇ m strainer (SPL) to prepare single cells.
  • Trypsin-EDTA Trypsin-EDTA
  • SPL 40 ⁇ m strainer
  • RPMI-1640 solution containing 250 ⁇ g/ml Matrigel was coated on transwells for 2 hours at 37°C, and the same experiment as above was performed. Invasiveness was reduced by 82-93% in PTGFRN knockdown Huh7 cells and by 57-91% in SNU449 cells (Fig. 7c, 7d).
  • PTGFRN deficiency reduced motility and invasiveness in both Huh7 and SNU449 cells, and these results indicate that PTGFRN is an important cell surface molecule that promotes migration and invasiveness, which are important for metastasis in hepatocellular carcinoma cells.
  • PTGFRN knockdown liver cancer cells Huh7 and SNU449 bind well to various extracellular matrices (ECM).
  • ECM extracellular matrices
  • 200 ⁇ g/ml Matrigel, 20 ⁇ g/ml CollagenI, 20 ⁇ g/ml CollagenIV, and 0.1% Gelatin were coated and cultured in an incubator at 37°C with 5% CO2 and 95% air for 2 hours.
  • PTGFRN knockdown liver cancer cells Huh7 and SNU449 were prepared, diluted to a concentration of 5.0 X 104 cells/ml, and then dispensed onto plates that were washed twice with PBS (pH 7.4) and cultured for 20-60 minutes.
  • PTGFRN knockdown SNU449 cells manufactured through the above experiment were detached with 0.05% Trypsin-EDTA (Welgene, Gyeongsan, Korea) and neutralized with cell culture medium containing 10% fetal bovine serum (Corning) and 200 ng/ml doxycycline (Sigma). Then, 2.0 X 104 cells were dispensed per well in a 12-well plate and cultured in an incubator at 37°C and 5% CO2 for one day.
  • NK92 effector cells
  • target cells shPTGFRN-SNU449
  • a medium containing 400 U/ml IL2 (Peprotech), 10% fetal bovine serum (Corning), and 200 ng/ml doxycycline (Sigma) dispensed, and cultured for 2 days at 37°C, 5% CO2 .
  • floating NK92 cells were washed and removed, and the SNU449 target cells that remained attached to the bottom and survived were stained with 0.5% (w/v) crystal violet solution.
  • NK cytotoxicity (%) ⁇ 1 - (target OD value/No NK OD value) ⁇ *100
  • PTGFRN acts as an immune checkpoint molecule in cancer cells to suppress apoptosis by NK cells, thereby providing cancer cells with the ability to evade the host's immunity.
  • the knocked-down cells were harvested, washed twice with phosphate buffer (Ph 7.4), and lysed using RIPA lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% Deoxycholate). The cells were centrifuged at 4°C, 12,000 rpm for 30 minutes, and the supernatant was mixed with 5X sample buffer to prepare a protein sample. Afterwards, they were separated on an 8-10% SDS-PAGE gel and transferred to a nitrocellulose membrane for Western blotting.
  • RIPA lysis buffer 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% Deoxycholate.
  • the cells were centrifuged at 4°C, 12,000 rpm for 30 minutes, and the supernatant was mixed with 5X sample buffer to prepare a protein sample. Afterwards, they were separated on an 8-10% SDS
  • Blocking was performed using 5% skim milk powder, and each target antibody, rabbit anti-PTGFRN polyclonal antibody (abcam, 97567), rabbit anti-B7-H3 polyclonal antibody (sinobiological, 11188-RP02), rabbit anti-FAK polyclonal antibody (Cell Signaling Technology, 3285), rabbit anti-p-FAK polyclonal antibody (CST, 3283), rabbit anti-Src monoclonal antibody (CST, 2109), rabbit anti-p-Src monoclonal antibody (CST, 6943), rabbit anti-GAPDH polyclonal antibody (CSB, PA00025A0Rb), was mixed in 5% BSA and reacted at 4°C for 16 hours.
  • rabbit anti-PTGFRN polyclonal antibody (abcam, 97567)
  • rabbit anti-B7-H3 polyclonal antibody sinobiological, 11188-RP02
  • rabbit anti-FAK polyclonal antibody Cell Signaling Technology, 3285
  • PTGFRN regulates the migration and invasiveness of cancer cells through the FAK signaling pathway.
  • PTGFRN expressed in the cell membrane contributes to forming clusters between cells
  • PTGFRN expressed in circulating cancer cells will help in the growth or survival and immune evasion of cancer cells.
  • 2.5 X 10 5 cells were seeded per well in a 6-well plate, and the next day, 12 ⁇ g of PEI, pcDNA3.1(+)PTGFRN-FLAG vector, and pcDNA3.1(+)-PTGFRN-myc vector were diluted together in TOM (Welgene) to 4 ⁇ g each, and reacted at room temperature for 5 minutes.
  • the diluted PEI solution and the diluted vector solution were mixed and reacted at room temperature for 20 minutes, and then treated together in one HEK293FT cell and cultured for 24 hours in an incubator with 37°C and 5% CO 2 .
  • pcDNA3.1(+)PTGFRN-FLAG vector and pcDNA3.1(+)-PTGFRN-myc vector were prepared respectively and reacted separately in two HEK293FT cell wells, and after 24 hours, the culture medium was replaced with new one, cultured for another 48 hours, and cell lysates were prepared by treating cell lysis buffer in the same manner as in Example 4.
  • each cell lysate was prepared using BCA (Thermo), and each lysate was immunoprecipitated using mouse anti-FLAG monoclonal antibody, rabbit anti-myc tag monoclonal antibody, and mouse 63-D7 antibody, and then analyzed by Western blotting with anti-FLAG monoclonal antibody and rabbit anti-myc tag monoclonal antibody.
  • SNU449 cell lysates were prepared as in Example 4, and then immunoprecipitated using mouse anti-PTGFRN monoclonal antibody, mouse 63-D7 monoclonal antibody, mouse anti-SLC3A2 monoclonal antibody, and mouse anti-B7-H3 monoclonal antibody.
  • Western blotting was performed using mouse anti-PTGFRN monoclonal antibody ( ⁇ -PTGFRN). As a result, it was confirmed that PTGFRN binds to SLC3A2 (CD98hc) and B7-H3 (CD276) (Fig. 11c).
  • B7-H3 is known to be an immune checkpoint regulator in cancer cells
  • HEK293FT cells were seeded at 2.5 X 10 5 cells per well in a 6-well plate.
  • 12 ⁇ g of PEI, PTGFRN vector with myc tag (pcDNA3.1(+)PTGFRN-myc) and PTGFRN vector with FLAG tag (pcDNA3.1(+)-B7-H3-FLAG) were diluted together with 4 ⁇ g each in TOM (Welgene, Gyeongsan, Korea) and reacted at room temperature for 5 minutes.
  • the diluted PEI solution and the diluted vector solution were mixed and reacted at room temperature for 20 minutes, treated to transfect HEK293FT cells, and cultured in an incubator with 5% CO 2 at 37°C for 24 hours. After 24 hours, the culture medium was replaced with new medium, and after culturing for another 48 hours, the cell lysate was prepared as in Example 4. Using BCA (Thermo Fischer Scientific), 50 ⁇ g of each cell lysate was prepared, and then immunoprecipitated with anti-FLAG, anti-B7-H3, anti-myc, and 63-D7. The immunoprecipitates were detected with anti-myc and anti-FLAG antibodies.
  • BCA Thermo Fischer Scientific
  • PTGFRN was detected in the immunoprecipitate by anti-FLAG antibody, and B7-H3 was detected in the immunoprecipitate by anti-myc antibody. Therefore, it can be seen that PTGFRN and B7-H3 physically bind and interact with each other (Fig. 11d). These results suggest that PTGFRN is involved in immune regulation through B7-H3, which is known as an immune checkpoint regulatory molecule.
  • Example 7 Analysis of adhesion, cancer stemness, and clonogenicity of 63-D7 positive liver cancer cells
  • Huh7 and HepG2 cells were sorted into 63-D7 positive and negative using magnetic beads with 63-D7 antibody.
  • 63-D7-biotin antibody was separated using Neutravidin-conjugated magnetic beads (Thermo Fishcer Scientific) according to the manufacturer's protocol. Cells attached to the antibody were separated as 63-D7(+), and non-attached cells were prepared as 63-D(-). Next, 12-well plates were coated with Matrigel and blocked in serum-free DMEM/F12 medium containing 5% BSA at 37°C for at least 1 hour. 63-D7(+), 63-D7(-) hepatoma cells were seeded at 105 cells per well and reacted at 37°C for 6 hours. To remove non-attached cells, the plates were washed with PBS (pH 7.4).
  • Adherent cells were stained with 0.5% crystal violet (Sigma-Aldrich) in 2% ethanol for 30 min at room temperature, and the stained cells were dissolved in 0.1% SDS and measured for absorbance at 570 nm.
  • 63-D7(+) Huh7 cells showed a 43% increase in adhesion compared to 63-D7(-) Huh7 cells
  • 63-D7(+) HepG2 cells showed a 245% increase in adhesion compared to 63-D7(-) HepG2 cells, suggesting that 63-D7-positive hepatoma cells have significantly better adhesion to the extracellular matrix than do negative hepatoma cells (Fig. 12a, Fig. 12b).
  • tumorspheres that induce cancer stemness were cultured.
  • attached and growing Huh7 liver cancer cells were detached with trypsin-EDTA (Welgene, Daegu, Korea), filtered through a 40 ⁇ m strainer, and centrifuged at 1500 rpm for 3 minutes at room temperature.
  • the cells were seeded at 2.1 x 10 3 cells/cm 2 on very low adherent plates (Corning, SEOUL, KOREA) and cultured in DMEM/F12 medium (Corning) supplemented with 20 ng/ml fibroblast growth factor 2 (FGF2, R&D systems, Seoul, Korea), 20 ng/ml epidermal growth factor (EGF, PeproTech, Seoul, Korea), and 1 x B27 supplement (Life Technologies). The medium was changed every 3 days, and the culture was performed for at least 9 days.
  • the expression of CD133, CD44, and EpCAM known as representative liver cancer stem cell markers, and PTGFRN, a 63-D7 antigen, was analyzed by flow cytometry in the same manner as in Example 3.
  • the cancer stem cell-positive markers CD133, CD44, and EpCAM increased by about 33%, 43%, and 59%, respectively, compared to the adherent cells, and at this time, the binding of 63-D7 also increased by 61% (Fig. 12c, Fig. 12d).
  • Example 6-2 To further analyze whether 63-D7-positive liver cancer cells show liver cancer stem cell properties, a clonogenic survival experiment showing cancer stem cell properties was performed as in Example 6-2 using liver cancer cells sorted with 63-D7 in the same manner as above. As a result, it was observed that clonogenicity increased by 492% in Huh7 (Fig. 12e, Fig. 12f) and by 56% in HepG2 (Fig. 12g, Fig. 12h). These results suggest that 63-D7-positive liver cancer cells show remarkable cancer stem cell properties compared to negative liver cancer cells and have cancer stem cell properties with excellent clonogenic survival ability.
  • Example 8 Internalization analysis after 63-D7 treatment in liver and pancreatic cancer cells
  • Cells were detached with 0.05% Trypsin-EDTA (Welgene) and neutralized with cell culture medium containing 10% fetal bovine serum (FBS; VWR, PA, USA), and then passed through a 40 ⁇ m strainer to prepare single cells. Approximately 1 ⁇ 10 5 cells per ml of each single cell were mixed with PBA (1% bovine serum albumin, 0.02% NaN 3 in PBS) and reacted with antibody 63-D7 at 4 °C for 30 min. To induce antibody internalization, cells were washed once with PBA, and then cells were suspended in 100 ⁇ l of culture medium and reacted for 30 min at 37 °C to allow cell membrane internalization.
  • PBA 1% bovine serum albumin, 0.02% NaN 3 in PBS
  • 63-D7 antibody bound to the surface of cancer cells at 4°C is internalized into the cells by cell activity at 37°C, thereby reducing binding on the surface, suggesting that 63-D7 is an antibody that can be applied to the development of antibody therapeutics using ADC.
  • Example 8 it was confirmed that the 63-D7 antibody having a mouse IgG1 constant region bound to the surface of liver cancer cells and was internalized into the cells at a temperature of 37°C. Based on the above characteristics, it was assumed that when an anticancer drug is conjugated to the 63-D7 antibody, the drug delivered via the 63-D7 antibody would exhibit cytotoxicity in cancer cells, and an experiment was performed to confirm this.
  • ⁇ -mFc-CL-DMDM AM-102DD, Moradec, USA
  • ⁇ -mFc-CL-MMAF AM-102AF, Moradec
  • Huh7 cells were dispensed into each well of a 96-well plate (SPL) using RPMI-1640 (Biowest, France) medium containing 10% FBS, and then cultured for 24 hours. The next day, the existing culture medium was removed, and 63-D7 antibody or mouse IgG isotype control (Invitrogen, 31903) diluted in the culture medium at various concentrations was dispensed into each well at 100 nM, 10 nM, 1 nM, 0.1 nM, and 0.01 nM, and the antibody was allowed to bind to the cell surface for 10 minutes at 37°C.
  • the drug-conjugated secondary antibody at a concentration of 12.7 nM was diluted in the culture medium, the primary antibody was removed, and the antibody was dispensed into each well. After culturing the cells for 48 hours under 5% CO2 conditions at 37°C, Cell Counting Kit-8 (CCK-8, K1018, APExBIO, USA) was used to determine the cell viability. 10 ⁇ l of CCK-8 was added to each well, reacted for 3 hours, and the absorbance at OD450nm was measured using a plate reader.
  • 63-D7 antibody is internalized into liver cancer cells, and based on this characteristic, it can exhibit anticancer effects by effectively delivering drugs into liver cancer cells expressing PTGFRN.
  • Example 10 Analysis of 63-D7 antigen in blood cancer cells from liver cancer patients
  • PTGFRN promotes survival, migration, invasion, cell adhesion, and immune evasion of hepatocellular carcinoma (HCC) cells, which are biological characteristics of circulating cancer cells that are important for metastasis (Figs. 5 to 9). Therefore, we hypothesized that PTGFRN could serve as a surface marker to detect circulating cancer cells in HCC patients, and indeed, we confirmed this possibility in preliminary experiments to detect circulating cancer cells (Figs. 1b and 1c).
  • peripheral blood mononuclear cells PBMCs
  • PBMCs peripheral blood mononuclear cells
  • Enrichment of circulating cancer cells by depletion of CD45-positive cells from these leukocyte lineages was performed using a Human CD45 depletion kit (EasySep®, Stem Cells Technologies, Vancouver, BC, Canada) according to the provided protocol.
  • EasySep® Stem Cells Technologies, Vancouver, BC, Canada
  • anti-CD45 antibody was added to the cell suspension and reacted at room temperature for 15 minutes, then dextran-coated magnetic nanoparticles were reacted with the cells at room temperature for 10 minutes, and the cell suspension was placed in an EasySep® big easy magnet (StemCell Technologies, Vancouver, Canada) at room temperature for 10 minutes.
  • the unbound cell fraction was then transferred to a clean tube and collected, and the collected cells were centrifuged at 3,560 ⁇ g for 5 minutes, resuspended in 400 ⁇ l of RPMI1640 medium, divided into eight fractions, and finally seeded onto glass slides coated with 0.1 ⁇ g/mL of poly-L-lysine.
  • the cells were reacted at room temperature for 2-4 hours to induce spontaneous binding of the cells to the glass slides. Unbound cells were washed with PBS (pH 7.4) before fixation.
  • the bound cells were fixed in 3.7% paraformaldehyde (PFA) and stored in the refrigerator before analysis by confocal microscopy.
  • PFA paraformaldehyde
  • CD45-depleted cells were fixed with 3.7% PFA, blocked with 10% normal horse serum, and then reacted with Dylight 649-conjugated anti-mouse IgG (Vector laboratories) and Dylight 488-conjugated 63-D7. After washing with PBS, nuclei were stained with DAPI (4,6-diamidino-2-phenylindole). Fluorescent signals were detected with a Leica TCS SP5 confocal microscope (Leica Microsystems, Seoul, Korea). As a result, all 63-D7-positive cells were confirmed to be CD45-negative (lower panel of Fig. 15c, Table 3). The expression profiles of EMT markers in 63-D7+ nucleated CTCs of HCC patients by double immunofluorescence staining are specifically shown in Table 3 below.
  • HCC liver cancer patients
  • HCC liver cancer patients
  • Normal normal people
  • hepatitis patients are shown in Table 4 below.
  • HCC M 69 3.75 11 21 14 1.90 2 HCC F 63 1.13 10 15 11 1.50 3 HCC F 62 1.05 9.4 28 22 2.98 4 HCC M 65 3.2 10 61 46 6.10 5 HCC M 64 2.35 10.6 44 31 4.15 6 HCC M 54 2.43 10 3 2 0.30 7 HCC M 68 3.54 10 38 29 3.80 8 HCC M 64 2 10.2 14 10 1.37 9 HCC M 73 1.68 10 33 25 3.30 10 HCC M 54 2.08 8 15 14 1.88 11 HCC F 60 1.48 10.1 22 16 2.18 12 HCC F 62 2.23 10.4 26 19 2.50 13 HCC F 45 3.63 9.4 24 19 2.55 14 HCC M 60 1.87 10.1 6 5 0.59 15 HCC M 72 1.5 10.2 25 18 2.45 16 HCC M 45 1.45 8 20 19 2.50 17 HCC M 68 1.375 9 28
  • 63-D7-positive circulating cancer cells were heterogeneous, ranging from 18 ⁇ m to 40 ⁇ m, and they had hyperchromatic nuclei and a high nuclear-to-cytoplasmic ratio (Figs. 16a to 16c).
  • 63-D7-positive circulating cancer cells were detected in all HCC patients, and 92 of 95 patients (approximately 97%) showed a higher cell number than that of normal adults (Fig. 17, Table 4) (p ⁇ 0.0001).
  • the number of cells isolated from HCC patients ranged from 0.1 to 50.72 per ml, and the average was 6.13 per ml (Table 4). Since 63-D7-positive circulating cancer cells were not found in patients with chronic hepatitis and cirrhosis (Table 4), it shows that they are circulating cancer cells caused by cancer development, not inflammatory cells caused by hepatitis, etc.
  • CD45-depleted cells were fixed with 3.7% PFA, blocked with 10% normal horse serum, and then reacted with anti-MVP (Aviva systems, San Diego, CA, USA), anti-CD44 (BD bioscience, Seoul, Korea), anti-CD90 (BD bioscience), Alexa 555-conjugated anti-EpCAM (BD Biosciences), Alexa 555-conjugated anti-E-cadherin (Cell Signaling Technology, Beverly, MA, USA), and anti-HSA (Novus, Litteleton, CO, USA), followed by additional reaction with Dylight 650-conjugated anti-rabbit IgG (Thermo Fischer Scientific), and finally Dylight 488-conjugated 63-D7, and stained as in Example 10-1.
  • anti-MVP Aviva systems, San Diego, CA, USA
  • anti-CD44 BD bioscience, Seoul, Korea
  • anti-CD90 BD bioscience
  • Alexa 555-conjugated anti-EpCAM BD Biosciences
  • CD45-depleted cells were fixed and permeabilized with 0.1% Triton X-100 before blocking and reacted with anti-vimentin (Santa Cruz Biotechnology), anti-Twist1 (AbCAM, Cambridge, UK), anti-ZEB1 (AbCAM), or phycoerythrin-conjugated anti-panCK antibodies (BD biosciences, San Jose, CA, USA).
  • the cells were incubated with Dylight 650-conjugated anti-rabbit IgG (Thermo Fischer Scientific) depending on the conjugation status of the primary antibody and finally reacted with Dylight 488-conjugated 63-D7, and the fluorescence signals were detected and analyzed by a Leica TCS SP5 confocal microscope (Leica Microsystems).
  • MVP cell surface major vault protein
  • 63-D7/HSA staining approximately 25% (50/203) of the 63-D7-positive circulating cancer cells were HSA-positive in both primary and secondary HCC patients (Fig. 16a, Table 3).
  • 63-D7/E-cadherin staining all circulating cancer cells were E-cadherin-negative (0/18) in all primary HCC patients (Fig. 16b, Table 3).
  • 63-D7/panCK (pan-cytokeratin) staining all circulating cancer cells were panCK-negative in both primary (0/104) and secondary HCC patients (0/24) (Fig. 16a, Table 3).
  • 63-D7/EpCAM staining approximately 15% (67/442) of 63-D7-positive cells were EpCAM-positive in both primary and secondary HCC patients (Fig. 16b, Table 3).
  • epithelial markers such as PanCK or E-cadherin are not expressed in PTGFRN-positive circulating cancer cells recognized by 63-D7, regardless of whether they are primary or secondary metastatic/recurrent cancers. This suggests that 63-D7-positive circulating cancer cells are at least not epithelial circulating cancer cells.
  • 63-D7-positive circulating cancer cells exhibit a mesenchymal phenotype
  • representative mesenchymal markers Vimentin, Twist, and ZEB1 were observed in primary HCC patients.
  • Approximately 24% (43/180) of 63-D7-positive circulating cancer cells were vimentin-positive (Fig. 16c, Table 3).
  • Twist (3.2%, 6/189) and ZEB1 (5%, 3/60) positivity were also observed in small fractions in 63-D7-positive circulating cancer cells (Fig. 16c, Table 3).
  • 63-D7-positive circulating cancer cells exhibit a significantly predominant mesenchymal phenotype when comparing the proportions of epithelial and mesenchymal phenotypes, it can be seen that in most cases, they are a partial intermediate-stage cell population in which the distinction between epithelial and mesenchymal phenotypes is unclear.
  • triple fluorescence staining for 63-D7/EpCAM/vimentin was performed using CD45-depleted circulating cancer cells, using the epithelial representative marker EpCAM and the mesenchymal representative marker Vimentin (Fig. 18a, Table 6). The results of the triple fluorescence staining are specifically shown in Table 6 below.
  • CD45-depleted cells were fixed and permeabilized with 0.1% Triton X-100 before blocking and reacted first with anti-Vimentin antibody (Santa Cruz Biotechnology). Cells were then reacted with Dylight 650-conjugated anti-rabbit IgG (Thermo Scientific), Alexa 555-conjugated anti-EpCAM (Cell Signaling Technology), and Dylight 488-conjugated 63-D7, washing cells four times with PBS containing Ca 2+ and Mg 2+ between each step. Nuclei were stained with DAPI, and fluorescence signals were detected by confocal microscopy (Leica Microsystems).
  • the 63-D7 + EpCAM + Vimentin + triple-positive circulating cancer cells were 9.1% (47/518) in primary HCC patients, whereas 0.3% (3/933) in secondary HCC patients.
  • MVP-positive circulating cancer cells 51.3% were MVP-single positive in MVP/EpCAM/Vimentin triple staining (Lee, Joh et al. 2017), so we performed MVP/EpCAM/Vimentin triple staining using the same method.
  • MVP single-positive circulating cancer cells were classified into primary HCC and secondary HCC, MVP single-positive circulating cancer cells were 53.5% in primary HCC patients and 50.3% in secondary HCC patients (Table 6).
  • MVP single-positive circulating cancer cells were found in all secondary HCC patients (9/9 patients, 100%) (Table 6).
  • MVP 50.3%) or 63-D7 (72.3%) single-positive circulating cancer cells were EpCAM-negative and Vimentin-negative and were predominantly present in all secondary HCC patients. Therefore, PTGFRN recognized by 63-D7 is a novel marker representing most circulating cancer cells in patients with HCC that are neither epithelial nor mesenchymal, and it can be seen as an excellent diagnostic marker observed in all patients, especially in patients with secondary metastatic/recurrent HCC.
  • the cells were then reacted with Dylight 650-conjugated anti-rabbit B7-H3 (Sinobiological, Beijing, China) and Dylight 488-conjugated 63-D7 antibody. Between each step, cells were washed four times with PBS containing Ca 2+ and Mg 2+ . Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI) and fluorescence signals were detected with a Leica TCS SP5 confocal microscope. The expression profiles of EMT and immune checkpoint markers in nucleated CTCs from secondary HCC patients by triple immunofluorescence staining are shown in Table 7 below.
  • EMT provides cancer cells with metastatic potential, immunosuppressive potential, and cancer stemness.
  • EMT can induce the expression of immunosuppressive molecules such as PD-L1 and CD47 in cancer stem cells and circulating cancer cells.
  • immunosuppressive molecules such as PD-L1 and CD47 in cancer stem cells and circulating cancer cells.
  • Approximately 53% of 63-D7-positive circulating cancer cells were EMT phenotype circulating cancer cells in primary HCC patients, but the proportion of EMT phenotype circulating cancer cells decreased to 27.6% in secondary HCC patients (Table 6). Instead, most 63-D7-positive circulating cancer cells (72.3%) were partial/intermediate phenotype circulating cancer cells in secondary HCC patients (Table 6).
  • TGF ⁇ /TGF ⁇ R1 signaling exerts multiple immunosuppressive effects in CSCs.
  • TGF ⁇ is also required for the suppression of NK cell function.
  • TGF ⁇ /TGF ⁇ R1 signaling provides the high metastasis-initiating ability of breast CSCs.
  • TGF ⁇ 1 signaling also promotes T cell-mediated tumor evasion in colorectal cancer through increased B7-H3 expression. Therefore, although the number of EMT phenotype CSCs was reduced in PTGFRN-positive CSCs from secondary HCC patients, we investigated to what extent TGF ⁇ R1 expression was positive in 63-D7-positive CSCs.
  • circulating cancer cells were simultaneously triple-stained with anti-ULBP1/63-D7/anti-B7-H3 antibodies or anti-MICA/B/63-D7/anti-B7-H3 antibodies (Fig. 20a, Fig. 20b, Table 7).
  • ULBP1 and MICA/B are known as ligands for the activating receptor NKG2D of NK and CD8 T cells, and are molecules that promote immunity.
  • ULBP1/63-D7/B7-H3 staining 4.1% (9/222) of total PTGFRN-positive circulating tumor cells were ULBP1-positive in secondary HCC patients, whereas 50.5% (112/222) of total PTGFRN-positive circulating tumor cells were B7-H3-positive.
  • MICA/B/63-D7/B7-H3 staining all patients (18/18) had MICA/B-positive circulating tumor cells.
  • PD-L1 and PD-L2 are well-known ligands for the immunosuppressive receptor PD-1 on NK and CD8 T cells. Therefore, circulating cancer cells were simultaneously triple-stained with anti-PD-L1/63-D7/anti-B7-H3 antibodies or anti-PD-L2/63-D7/anti-B7-H3 antibodies, respectively (Fig. 21a, Fig. 21b, Table 7).
  • PD-L1/63-D7/B7-H3 staining 37.8% (197/521) of the total PTGFRN-positive circulating cancer cells in patients with secondary hepatocellular carcinoma were PD-L1 positive, and 55.7% (290/521) of the total PTGFRN were B7-H3 positive.
  • PD-L2/63-D7/B7-H3 staining 17.5% (69/395) of the total PTGFRN-positive circulating cancer cells in patients with secondary hepatocellular carcinoma were PD-L2 positive, and 56.2% (222/395) of the total PTGFRN were B7-H3 positive (Fig. 21a, Fig. 21b, Table 7). Meanwhile, all patients (22/22) had PD-L1 or PD-L2 positive circulating cancer cells.
  • CD47 is a “don’t eat me” signal for macrophages and inhibits phagocytic innate immune surveillance.
  • CD47/63-D7/B7-H3 staining 38.1% (115/302) of the total PTGFRN-positive CBCTs in patients with secondary HCC were CD47-positive, and 51.7% (156/302) were B7-H3-positive (Fig. 22 , Table 7 ). And all patients (14/14) had CD47-positive CBCTs.
  • immunostimulatory receptors such as ULBP1 (4.1%) and MICA/B (34.7%) were detected in PTGFRN-positive circulating cancer cells
  • immune checkpoint regulatory molecules such as TFG ⁇ R1 (51.8%), PD-L1 (37.8%), PD-L2 (17.5%), CD47 (38.1%), and B7-H3 (50.5–65.3%) were more widely detected in PTGFRN-positive circulating cancer cells from secondary HCC patients.
  • B7-H3 was predominantly expressed in all PTGFRN-positive circulating cancer cells (average 57.6%).
  • PTGFRN is responsible for immune evasion of circulating cancer cells in secondary HCC patients, and its expression is closely associated with immunosuppressive receptors and plays a role in immune evasion.
  • PTGFRN recognized by 63-D7 antibody in circulating cancer cells may serve as an excellent diagnostic marker for indicating the stage or progression of liver cancer.
  • Vigorously growing hybridoma 63-D7 cells (5 ⁇ 10 6 cells) were harvested by centrifugation, washed twice with cold PBS, and total RNA was extracted using RNAiso plus reagent (TaKaRa, Otsu, Japan) according to the manufacturer's protocol. The A260 of total RNA was measured using Nanodrop to quantify the amount of RNA.
  • 1 unit of DNase I (TaKaRa) per 1 ⁇ g of total RNA was added and reacted at 37 °C for 30 minutes to remove residual DNA.
  • 1 ⁇ l of 50 mM EDTA was added and reacted at 65 °C for 10 minutes to inactivate DNase I and denature total RNA.
  • a reverse transcription polymerase chain reaction mixture was prepared using total RNA and Prime Script RT reagent Kit (TaKaRa), and cDNA was synthesized according to the manufacturer's protocol.
  • a 5'-ggt gtc gac GGA TAC AGT TGG TGC AGC ATC-3' (SEQ ID NO: 23) oligonucleotide primer corresponding to the kappa chain constant region and a 5'MK 5'-cgg aag ctt GAY ATT GTG MTS ACM CAR WCT MCA-3' (SEQ ID NO: 24) oligonucleotide primer corresponding to the N-terminus of the kappa chain variable region were used.
  • an EcoRI restriction site was provided at the end of the 5'-primer for the heavy chain and a SalI restriction site was provided at the end of the 3'-primer.
  • a HindIII restriction site was provided at the end of the 5'-primer and a SalI restriction site was provided at the end of the 3'-primer.
  • amplified DNA was obtained at a position corresponding to approximately 400 bp, which is estimated to be a DNA fragment corresponding to the heavy chain variable region, and approximately 390 bp, which is estimated to be a DNA fragment corresponding to the light chain variable region.
  • the heavy chain of the polymerase chain reaction product was treated with EcoRI and SalI, and the light chain was treated with HindIII and SalI, and then developed on a 1.0% (w/v) agarose gel.
  • DNA corresponding to approximately 400 bp and 390 bp was isolated using FavorPrep GEL PCR Purification Kit (Favorgen, Pingtung, Taiwan).
  • the vector pBluescript KS+ for cloning the heavy chain gene was treated with EcoRI and SalI
  • the vector pBluescript KS+ for cloning the light chain gene was treated with HindIII and SalI, and then isolated using FavorPrep GEL PCR Purification Kit.
  • the heavy chain corresponded to subgroup IIB (Fig. 23) and the light chain corresponded to subgroup V (Fig. 24).
  • the antigen binding sites, CDR 1, 2, and 3 are indicated in bold for each sequence.
  • the results of comparing the amino acid sequences of the antibodies using BLAST confirmed that 63-D7 is a new antibody that was not previously known.

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Abstract

La présente invention concerne un anticorps monoclonal anti-PTGFRN pour déterminer le taux sanguin de cellules cancéreuses, traiter ou prévenir le cancer, prédire le pronostic de métastases cancéreuses, et cribler un agent anticancéreux et, plus particulièrement, un anticorps monoclonal anti-PTGFRN comprenant : une région variable de chaîne lourde comprenant HCDR1 consistant en une séquence d'acides aminés de SEQ ID NO : 3, HCDR2 constituée d'une séquence d'acides aminés de SEQ ID NO : 4, et HCDR3 constituée d'une séquence d'acides aminés de SEQ ID NO : 5 ; et une région variable de chaîne légère comprenant LCDR1 constituée d'une séquence d'acides aminés de SEQ ID NO : 6, LCDR2 constituée d'une séquence d'acides aminés de SEQ ID NO : 7, et LCDR3 constituée d'une séquence d'acides aminés de SEQ ID NO : 8.
PCT/KR2024/006210 2023-05-09 2024-05-09 Anticorps monoclonal anti-ptgfrn et son utilisation Pending WO2024232668A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20190365914A1 (en) * 2013-07-11 2019-12-05 Universite Francois Rabelais Novel antibody-drug conjugates and the use of same in therapy
KR20200071740A (ko) * 2017-09-28 2020-06-19 임팩트-바이오 리미티드. 저해성 키메라 항원 수용체 (icar)를 제조하기 위한 보편적 플랫폼
US20210188972A1 (en) * 2017-10-13 2021-06-24 A&G Pharmaceutical, Inc. Monoclonal antibodies and conjugates against prostaglandin f2 receptor inhibitor and uses thereof

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20190365914A1 (en) * 2013-07-11 2019-12-05 Universite Francois Rabelais Novel antibody-drug conjugates and the use of same in therapy
KR20200071740A (ko) * 2017-09-28 2020-06-19 임팩트-바이오 리미티드. 저해성 키메라 항원 수용체 (icar)를 제조하기 위한 보편적 플랫폼
US20210188972A1 (en) * 2017-10-13 2021-06-24 A&G Pharmaceutical, Inc. Monoclonal antibodies and conjugates against prostaglandin f2 receptor inhibitor and uses thereof

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LIN DANFENG, SHEN LESANG, LUO MENG, ZHANG KUN, LI JINFAN, YANG QI, ZHU FANGFANG, ZHOU DAN, ZHENG SHU, CHEN YIDING, ZHOU JIAOJIAO: "Circulating tumor cells: biology and clinical significance", SIGNAL TRANSDUCTION AND TARGETED THERAPY, vol. 6, no. 1, 1 December 2021 (2021-12-01), XP055954502, DOI: 10.1038/s41392-021-00817-8 *
MARQUEZ JORGE, DONG JIANPING, DONG CHUN, TIAN CHANGSHENG, SERRERO GINETTE: "Identification of Prostaglandin F2 Receptor Negative Regulator (PTGFRN) as an internalizable target in cancer cells for antibody-drug conjugate development", PLOS ONE, PUBLIC LIBRARY OF SCIENCE, US, vol. 16, no. 1, 27 January 2021 (2021-01-27), US , pages e0246197, XP093235777, ISSN: 1932-6203, DOI: 10.1371/journal.pone.0246197 *

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