WO2024232668A1 - Anti-ptgfrn monoclonal antibody and use thereof - Google Patents

Abstract

The present invention relates to an anti-PTGFRN monoclonal antibody for determining the blood level of cancer cells, treating or preventing cancer, predicting the prognosis of cancer metastasis, and screening for an anticancer agent and, more specifically, to an anti-PTGFRN monoclonal antibody comprising: a heavy chain variable region comprising HCDR1 consisting of an amino acid sequence of SEQ ID NO: 3, HCDR2 consisting of an amino acid sequence of SEQ ID NO: 4, and HCDR3 consisting of an amino acid sequence of SEQ ID NO: 5; and a light chain variable region comprising LCDR1 consisting of an amino acid sequence of SEQ ID NO: 6, LCDR2 consisting of an amino acid sequence of SEQ ID NO: 7, and LCDR3 consisting of an amino acid sequence of SEQ ID NO: 8.

Description

Anti-PTGFRN monoclonal antibodies and uses thereof

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 (HCC) 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. However, 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. However, HCC has an exceptionally high recurrence rate, and the treatment effect is not satisfactory. Currently, the diagnosis and monitoring of HCC mainly depend on serum biomarker detection, pathological examination, and image analysis. However, the diagnostic performance of AFP, a common serum marker, is poor at around 58-66%, and the sensitivity of AFP in combination with another marker, des-gamma-carboxy-prothrombin (DCP), is only around 82% when diagnosed. Moreover, imaging and pathology tests have limitations in diagnostic accuracy and sensitivity, so better approaches are needed for the diagnosis and monitoring of HCC.

Circulating tumor cells (CTCs) 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. Unlike tissue examinations, detection of circulating tumor cells is a simple method for diagnosing cancer through liquid biopsy, and is therefore being presented as a new alternative for cancer diagnosis. And through animal models, it has been known that blood cancer cells are transmitted from the primary cancer through blood from the early stage of cancer, and blood cancer cells are detected from the early stage when metastasis is not observed in hepatocellular carcinoma. Therefore, early detection and management methods of hepatocellular carcinoma through blood cancer cell diagnosis have been proposed. However, blood cancer cells are very small in number, so it is not easy to effectively separate them from the blood. In addition, it is estimated that different blood cancer cells will come out and move from various types of cancer, so it is not easy to accurately detect and separate blood cancer cells that are of various sizes and very small in number. To date, 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. However, due to the diversity of blood cancer cells, there are blood cancer cells similar in size to leukocytes, and there are cells that cannot be separated due to physical characteristics, so it is important to separate blood cancer cells using surface biomarkers that reflect the characteristics of hepatocellular carcinoma.

Among markers that can isolate circulating cancer cells of various cancers, the one currently approved by the US FDA is the Epithelial Cell Adhesion Molecule (EpCAM), which is of epithelial origin. However, although the liver is an epithelial-derived organ, the majority of hepatocytes and hepatocellular cancer are EpCAM-negative, and in fact, only about 15-30% of hepatocellular cancer tissues are EpCAM-positive when stained with an immunohistochemical method. Therefore, a method of detecting circulating cancer cells positive for the asialoglycoprotein receptor (ASGPR) that binds to asialofetuin, a marker other than EpCAM, was used in hepatocellular cancer. 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. However, there was no clear correlation between the number of circulating cancer cells detected by ASGPR and hepatocellular cancer metastasis related to the stage. Meanwhile, 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.

Fudan University in China reported that among 123 patients with hepatocellular carcinoma before surgery, only 51 patients (41%) had more than 2 EpCAM-positive circulating cancer cells per 7.5 ml of blood, but the rate of recurrence after surgery was significantly higher (87.5% vs. 15.5%) compared to patients with 2 or fewer circulating cancer cells. In another study, only 18 patients (31%) had more than 1 EpCAM-positive circulating cancer cells per 7.5 ml of blood among 59 patients with hepatocellular carcinoma, but EpCAM-positive circulating cancer cells were observed in intermediate or higher stages and were closely related to the survival rate. In another study, 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. Overall, 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 (EMT) refers to the entire process by which adherent epithelial cancer cells transition into mesenchymal cancer cells with motility and invasiveness. During the EMT process, 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. In fact, when 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. In addition, 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.

Meanwhile, recent studies have shown that EMT can be reversible and temporary, and that partial EMT occurs rather than cells transforming into mesenchymal cells through complete EMT. In fact, most circulating cancer 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. PTGFRN is expressed in large quantities as a structural protein in exosomes, and its separation function through exosomes is being studied. In addition, the expression of 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.

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. In this invention, after finding that the 63-D7 antibody recognizes blood cancer cells in liver cancer patients, it was revealed that its target molecule is PTGFRN. 63-D7 recognized PTGFRN in liver cancer, lung cancer, colon cancer, thyroid cancer, and pancreatic cancer cells. In experiments on viability, invasion, and migration using liver cancer cells, it was confirmed that inhibition of PTGFRN gene expression inhibits the clonal survival, migration, and invasion of cancer cells, and consequently inhibits cancer metastasis. In addition, in an analysis of 95 patients with liver cancer, the 63-D7 antibody could detect circulating cancer cells in 92 patients (97%, 92/95), and the detectable rate further increased in patients with metastasis or recurrence. In addition, PTGFRN recognized by 63-D7 was highly co-expressed with B7-H3 (57.6%), MVP (31.9%), TGFβR1 (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. In addition, we revealed that 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). In addition, we were able to confirm that the invasiveness of liver cancer cells treated with 63-D7 increased, so the 63-D7 antibody itself is an agonistic antibody that promotes the migration and invasiveness of liver cancer cells. By utilizing these properties, it is expected that the 63-D7 antibody can be applied to promote the migration of cancer cells in primary cancers to prevent the occurrence of early primary cancers or to interfere with the growth of primary cancers.

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.

1. 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.

2. In the above 1, the heavy chain variable region is an anti-PTGFRN monoclonal antibody comprising the amino acid sequence of sequence number 1.

3. An anti-PTGFRN monoclonal antibody according to claim 1, wherein the light chain variable region comprises the amino acid sequence of sequence number 2.

4. An antibody-drug conjugate comprising a drug conjugated to any one of the above 1 to 3 anti-PTGFRN monoclonal antibodies.

5. A pharmaceutical composition for treating or preventing cancer comprising any one of the above 1 to 3 anti-PTGFRN monoclonal antibodies.

6. In the above 5, 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.

7. A hybridoma producing any one of the above 1 to 3 anti-PTGFRN monoclonal antibodies.

8. A composition for detecting PTGFRN comprising any one of the above 1 to 3 anti-PTGFRN monoclonal antibodies.

9. 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.

10. 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.

11. 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.

12. 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.

13. A method for providing information necessary for determining the level of cancer cells in blood, wherein in the above 12, the substance that specifically binds to EpCAM is an anti-EpCAM antibody or an antigen-binding fragment thereof.

14. In the above 10, a method for providing information necessary for determining the level of blood cancer cells, wherein the blood cancer cells are blood cancer cells that have undergone epithelial-mesenchymal transition or are undergoing epithelial-mesenchymal transition.

15. In the above 10, 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.

16. In the above 10, 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.

17. 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.

18. 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.

19. 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.

20. 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.

21. 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.

22. A 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.

Using the anti-PTGFRN monoclonal antibody of the present invention, various antibody-drug conjugates can be developed and used as anticancer agents.

By utilizing the fact that 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. 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). It shows no binding to blood mononuclear cells (PBMCs), human fetal lung fibroblasts (MRC5), dental pulp cells (DPCs), and normal hepatocytes. Here, 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). The same experiment performed without adding antibody (No Ab) was used as a negative control (Con). The arrow indicates the protein immunoprecipitated by 63-D7. 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. Afterwards, 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 α-PTGFRN antibody after immunoprecipitation of proteins and a negative control protein without antibody (No Ab) using mouse anti-PTGFRN monoclonal antibody (α-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. Figure 4c shows the results of immunoprecipitation and Western blotting of the cell lysate using a known mouse anti-FLAG monoclonal antibody (α-FLAG), a known mouse anti-PTGFRN monoclonal antibody (α-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 (α-PTGFRN) to PTGFRN on the surface of knocked-down Huh7 and SNU449 cells. The solid line is the monoclonal antibody, and the shaded background is the negative control (isotype) data including only the secondary antibody. Figure 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. *** indicates p<0.001, ** indicates p<0.01, * indicates p<0.05, and ns indicates no statistical significance. 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. 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 α-PTGFRN after immunoprecipitation with mouse anti-PTGFRN monoclonal antibody (α-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. 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. In Figures 11a to 11d, 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. In Figures 13a and 13b, 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, and 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. **** indicates p<0.001, *** indicates p<0.005, ** indicates p<0.01, and * indicates p<0.05. 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 α-mFc-CL-DMDM and Figure 14b shows that they were treated with α-mFc-CL-MMAF for 48 hours, and the cell viability was measured using CCK-8. In Figures 14a and 14b, * indicates p<0.05, ** indicates p<0.01, and **** 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. FIG. 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-TGFβR1, 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. FIG. 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.

The term "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).

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.

The term "antigen binding portion" of an antibody (or simply "antibody portion") as used herein 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.

Examples of 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. Also, although the two domains of the Fv fragment, namely the VL and VH, are encoded by separate genes, 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)). Such single-chain antibodies are also included in the term "antigen-binding portion" of an antibody. Such 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.

The term "monoclonal antibody" as used herein refers to an antibody that exhibits a single binding specificity and exhibits affinity for a particular epitope.

In the present disclosure, 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.

The term "epitope" or "antigenic determining portion" as used herein 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. Methods for determining the spatial conformation of an epitope include techniques known in the art and techniques described herein, such as x-ray crystallography and two-dimensional nuclear magnetic resonance. In this regard, reference may be made, for example, to the literature [Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996)]. In a preferred embodiment, 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).

The term "isotype" as used herein refers to the class of antibodies (e.g., IgM or IgG1) encoded by the heavy chain constant region genes. In one embodiment, 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.

In the present invention, it was revealed that the 63-D7 monoclonal antibody recognizes the cell surface protein PTGFRN (Prostaglandin F2 Receptor Negative Regulator, CD315). In addition, it was confirmed that 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.

In the present invention, 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.

Therefore, it can be seen that 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. In the present specification, 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.

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.

The present invention provides an antibody-drug conjugate (ADC) in which a drug is conjugated to the PTGFRN monoclonal antibody of the present invention.

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.

In the antibody-drug conjugate, the drug can be selected by those skilled in the art according to the purpose without limitation in type. For example, it can be a known anticancer drug, but is not limited thereto. In addition, 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.

In the present specification, 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. Depending on the tissue to which it has spread, 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. In addition, 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.

In this specification, 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. 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. For example, 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.

In this specification, an entity may be a mammal, including a human.

In this specification, 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.

In the present specification, 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.

For example, 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.

Since 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. 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. For example, 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.

The formation of 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. For example, 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. More specifically, it can be detected by immune precipitation and immunoblotting, which are useful for recovering a small amount of target protein.

Specifically, 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 (CTCs) 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 (EMT) 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.

In the present specification, 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. In addition, the detection method may be polymerase chain reaction (PCR), electrophoresis, northern blotting, western blotting, or a combination thereof.

In addition, according to one specific example of the present invention, 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. At the same time, since it was confirmed to be simultaneously expressed with Vimentin, 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. In addition, 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.

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.

In the method for providing information necessary for determining the level of blood cancer cells of the present invention, the blood cancer cells may be blood cancer cells undergoing epithelial-mesenchymal transition or undergoing epithelial-mesenchymal transition.

In the method for providing information necessary for determining the level of blood cancer cells of the present invention, the blood cancer cells may be in a hybrid E/M state.

In the method for providing information necessary for determining the level of blood cancer cells of the present invention, the blood cancer cells may be neither epithelial cells nor mesenchymal cells.

In the method for providing information necessary for determining the level of blood cancer cells of the present invention, 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.

In the method for providing information necessary for determining the level of blood cancer cells of the present invention, 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.

The composition for detecting blood cancer cells of the present invention may further include a substance that specifically binds to epithelial cell adhesion molecule (EpCAM).

In the composition for detecting blood cancer cells of the present invention, 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.

In the composition for detecting blood cancer cells of the present invention, 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. For example, 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. Furthermore, 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. In the 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. For example, 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. In addition, 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.

Example 1. Cancer cell line culture and research ethics

1-1. Cancer cell line culture

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 β-mercaptoethanol (Sigma, St Luis, MO, USA), 2 mM glutamine (Invitrogen), 0.1 mM nonessential amino acids (Invitrogen), 100 U/㎖ penicillin G (Sigma), 100 ㎍/㎖ streptomycin (Sigma), and 4 ng/㎖ 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 (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.

1-2. Research ethics and patient consent

Human peripheral blood mononuclear cells (PBMCs) were isolated from donors by Ficoll-Paque gradient centrifugation using the method provided by the Ficoll-Paque Plus method (GE Healthcare, Seoul, Korea). Blood samples from liver cancer patients undergoing liver resection in the Department of Surgery, Samsung Hospital, Seoul were collected into heparin-coated tubes after obtaining informed consent. All procedures were approved by the IRB of Seoul Samsung Hospital and were transferred to Sejong University in good faith for analysis there. Cancer staging was analyzed using the criteria specified by the American Joint Committee on Cancer (AJCC, 2010).

Example 2. Selection of 63-D7 antibody

In order to observe the degree of binding to two liver cancer cells (Huh7, HepG2) and two lung cancer cells (A549, NCI-H358) among the 70 antibodies specific for human embryonic stem cells produced in a previous study (Choi HS, et al., Cell Tissue Res. vol. 333, pp. 197-206, 2008), flow cytometry was performed. Specifically, Huh7, HepG2, A549, and NCI-H358 cancer cells were treated with 0.05% trypsin to detach the cells, washed with PBS (pH 7.4), and then filtered using a 40 μm strainer (BD Biosciences) to separate them into single cells. Human embryonic stem cells (H9) and human peripheral mononuclear cells (PBMC) were also used in the analysis. After mixing 5 × 10 ⁵ cells with 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. After washing twice with PBA, the primary antibody and the corresponding anti-mouse IgG-FITC (BD Biosciences) were reacted for another 30 minutes at 4 °C. After washing twice with PBA, the antibody reaction was analyzed for propidium iodide (PI)-negative cells using FACS Calibur and Cell Quest software (BD sciences). As a result, 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). Among these, 246-D7 and 247-B9 bound to the epithelial lung cancer cell line NCI-H358, but 63-D7 did not bind. To analyze the characteristics of the antibodies, 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).

To investigate whether three antibodies can detect circulating cancer cells in the blood of patients with liver cancer, 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. To prevent cell loss, 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. After washing with PBS, 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 μg 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. After washing with PBS, the plate was reacted with Cy3-conjugated streptavidin (Vector Laboratories, BURLINGAME, CA, USA, 1:3000) at room temperature, blocked from light, for 1 hour. After washing with PBS, nuclei were stained with DAPI (4,6-diamidino-2-phenylindole). When counting circulating cancer cells positive for antibodies in cells recovered from blood, only DAPI-positive cells were counted to eliminate nonspecific binding caused by blood. As a result, antibody-positive cells were detected in the blood of most liver cancer patients, but not in the blood of normal people. In particular, the 63-D7 antibody was selected as an antibody requiring further analysis because it could detect a total of 51 circulating cancer cells in 6 out of 8 patients (Fig. 1c, Fig. 1d).

Example 3. Analysis of 63-D7 binding to various cells using a flow cytometer

To determine whether monoclonal antibody 63-D7 binds to various cancer cells and normal cells in addition to embryonic stem cells (H9), liver cancer (Huh7, HepG2), and lung cancer (A549, NCI-H358), various cancer cells (SK-Hep1, 8505C, A375, MCF-7, MDA-MB-435, SH-SY5Y, U87-MG) 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. Flow cytometry was performed using primary mononuclear cells (PBMCs), normal lung fetal fibroblasts (MRC5), dental pulp cells (DPCs), and normal hepatocytes. Specifically, cells were detached by treating with 0.05% trypsin, washed with phosphate-buffered saline (PBS), and filtered using a 40 μm 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. After washing twice with PBA, the primary antibody and corresponding anti-mouse IgG-FITC (BD Biosciences) were reacted for another 30 min at 4 °C. After washing twice with PBA, the antibody reaction to propidium iodide (PI)-negative cells was analyzed using FACS Calibur and Cell Quest software (BD sciences). As shown in Fig. 2, the 63-D7 antibody binds to all cancer cells including hepatoma cells, but does not bind to epithelial lung cancer cells H358. In addition, 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.

Cell lines 63-D7 H9 Human embryonic stem cell ++ NT2/D1 Human embryonal carcinoma + HepG2 Human hepatocellular carcinoma ++ Huh7 Human hepatocellular carcinoma +++ SNU-387 Human hepatocellular carcinoma ++ SNU-449 Human hepatocellular carcinoma ++ SK-Hep-1 Human hepatic adenocarcinoma + A549 Human non-small cell lung adenocarcinoma ++ NCI-H358 Human non-small cell lung adenocarcinoma - NCI-H460 Human non-small cell lung adenocarcinoma ++ NCI-H1703 Human non-small cell lung adenocarcinoma + NCI-H23 Human non-small cell lung adenocarcinoma + Colo205 Human colorectal adenocarcinoma +++ HCT116 Human colorectal carcinoma +++ SNU-790 Human thyroid carcinoma ++ 8805C Human thyroid carcinoma ++ BxPC-3 Human pancreatic carcinoma +++ A375 Human melanoma +++ MCF-7 Human breast adenocarcinoma ++ MDA-MB-435 Human breast metastatic carcinoma +++ SH-SY-5Y Human neuroblastoma, Bone + U87-MG Human glioblastoma +++ HEK293FT Human embryonic kidney cell +++ BM-MSC Human bone marrow-mesenchymal stem cell + HDF Human dermal fibroblast cell + PBMC Human peripheral blood mononuclear cells - MRC5 Human normal lung fibroblast - DPC Human dental pulp cells - Hepatocyte Human normal hepatocyte -

Example 4. Isolation of antigen bound by antibody 63-D7

To analyze the cell surface antigen recognized by the 63-D7 antibody, an immunoprecipitation method was performed in which only the cell surface proteins were labeled with biotin and then precipitated with 63-D7 to isolate them. 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). Afterwards, the solution was incubated at 4°C for 30 minutes using lysis buffer (25 mM Tris-HCl, pH 7.5, 250 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 2 μg/ml aprotinin, 100 μg/ml PMSF, 5 μg/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. After adding protein-G-plus agarose (Merck millipore, Darmstadt, Germany) to the prepared protein solution, 20 μl 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. To immunoprecipitate the surface antigen recognized by 63-D7, 5 μg of antibody was added to the collected supernatant and reacted at 4 °C for 12 hours, and then 20 μl of protein-G-plus agarose was added and reacted at 4 °C for 3 hours. The immunoprecipitated immune mixture was washed five times using a dissolution solution. After that, 5× sample buffer was added to elute the antigen bound to the antibody, boiled at 100 °C for 10 minutes, developed using 10% SDS-PAGE, and Western blotting was performed with a nitrocellulose membrane. 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).

Example 5. Identification and validation of antigen immunoprecipitated by antibody 63-D7

5-1. Identification of antigen immunoprecipitated by antibody 63-D7

To identify the antigen recognized by 63-D7, 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. As a result, 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.

5-2. Verification of antigen immunoprecipitated by antibody 63-D7

To confirm that the 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.

To confirm whether the protein immunoprecipitated by the monoclonal antibody 63-D7 was PTGFRN, a mouse anti-PTGFRN monoclonal antibody (α-PTGFRN, R&D system) was purchased and used for immunoprecipitation and Western blotting. A cell lysate (150 μg) 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 μg) and α-PTGFRN (2.5 μg) as described above. Thereafter, the eluted protein and the negative control protein without antibody (No Ab) were separated through 10% SDS-PAGE, transferred to a nitrocellulose membrane, and Western blotting was performed with a known mouse α-PTGFRN antibody. As a result, approximately 130 kDa PTGFRN immunoprecipitated from 63-D7 and α-PTGFRN was detected simultaneously, confirming that they could recognize and immunoprecipitate PTGFRN (Fig. 4a). The membrane was reacted with gentle shaking in Striping solution (100 mM 2ME, 2% SDS, 62.5 mM Tris-HCl, pH6.7) at 50 ℃ 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. As a result, PTGFRN with a size of approximately 130 kDa, identical to cell surface PTGFRN, was detected in the immunoprecipitate of 63-D7 and α-PTGFRN (Fig. 4b). These results show that 63-D7 binds to cell surface PTGFRN.

An experiment was performed to distinguish whether antibody 63-D7 directly recognizes and immunoprecipitates PTGFRN or indirectly recognizes and precipitates it. 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 (α-FLAG) (Invitrogen), mouse anti-PTGFRN monoclonal antibody (α-PTGFRN) (AbCAM), and 63-D7. To overexpress PTGFRN in human embryonic kidney cells HEK293FT, first, 2.5 X 10 ⁵ HEK293FT cells per well in a 6-well cell culture plate were cultured for 24 hours, 2 μg of the pcDNA3.1(+)-PTGFRN-DYK vector DNA was mixed with 6 μg of polyetherimide (PEI) (DNA:PEI = 1:3), and the mixture was reacted at room temperature for 20 minutes, added to the cell culture medium, and transfection was performed. After additional culture for 72 hours, a cell lysate was prepared from the cells using a cell lysis buffer as described in Example 4. The cell lysate was immunoprecipitated with 63-D7, anti-FLAG antibody, and α-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 α-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. After washing three times with 0.1% TBST, the membrane was confirmed using an ECL detection kit (Fig. 4c). As a result, it was observed that each protein immunoprecipitated by anti-FLAG, α-PTGFRN, and 63-D7 was recognized by anti-FLAG (Fig. 4c top), α-PTGFRN (Fig. 4c middle), and 63-D7 (Fig. 4c bottom). From these results, it was verified that the monoclonal antibody 63-D7 of the present invention is an antibody that directly recognizes the PTGFRN protein present on the surface of the human embryonic carcinoma cell line NT-2/D1.

Example 6. Functional analysis of PTGFRN in liver cancer cells

6-1. Knockdown using siRNA in liver cancer cell lines

To investigate how PTGFRN, the antigen of antibody 63-D7, affects the growth of hepatocellular carcinoma cells, two kinds of siRNA (Bioneer, Daejeon, Korea) targeting PTGFRN were purchased and transiently knocked down in hepatocellular carcinoma cell lines Huh7 and SNU449. After seeding the cells at 8.0 × 10 ⁵ cells/well in a 6-well plate, 100 nM of negative control siRNA (siCon) (Genolution, Seoul, Korea) and two kinds of siRNA against PTGFRN (siPTGFRN#1, siPTGFRN #2) (Table 2) and Lipofectamine RNAiMAX (Invitrogen) were 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. After 24 hours, the medium was replaced with fresh culture medium and cultured for an additional 48 hours. Seventy-two hours after transfection, the cells were detached with 0.05% trypsin-EDTA (Welgene, Gyeongsan, Korea), neutralized with culture medium containing 10% fetal bovine serum (Corning), and washed twice with PBS (pH 7.4). After that, RNA was isolated from 3.0 x 10 ⁵ cells using RNAiso (Takara) according to the manufacturer's manual, and cDNA was synthesized using PrimeScript (Takara) according to the manufacturer's manual. After that, 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.

Name Sequence Reference
/Supplier Primers for human PTGFRN Forward: 5'-ACAACAGCTGGGTGAAAGC-3'
(sequence number 9)
Reverse: 5'-TTTCATTGGGACTGGAGAGG-3'
(sequence number 10) Primers for human GAPDH Forward: 5'-ACAGCGACACCCACTCCTCC-3'
(Sequence number 11)
Reverse: 5'-GAGGTCCACCACCCTGTTGC-3'
(sequence number 12) siControl 5'-UUCUCCGAACGUGUCACGUtt-3'
(Sequence number 13) siPTGFRN#1 5'-CUCCUACAUUUACUGGUUtt-3'
(Sequence number 14) Bioneer #5738 siPTGFRN#2 5'-GUCCCAAUGAAACGAAGUAtt-3'
(Sequence number 15) Bionics #2079 shScramble Tet-pLKO-puro-scrambled Addgene #47541 shPTGFRN#1 5'-ctagcGCCTTTGATGTGTCCTGGTTTTtactagtAAACCAGGACACATCAAAGGCTTTTTg-3'
(Sequence number 16) TRCN0000057448 5'-aattcAAAAAAGCCTTTGATGTGTCCTGGTTTTactagtaAAACCAGGACACATCAAAGGCg-3'
(Sequence number 17) shPTGFRN#2 5'-ctagcCCTATTGAGATAGACTTCCAAtactagtTTGGAAGTCTATCTCAATAGGTTTTTTg-3'
(Sequence number 18) TRCN0000057452 5'-aattcAAAAAACCTATTGAGATAGACTTCCAAactagtaTTGGAAGTCTATCTCAATAGGg-3'
(Sequence number 19)

As a result, 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). In addition, 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).

6-2. Clonogenic survival analysis of cancer cells in PTGFRN knockdown liver cancer cells using siRNA and shRNA

Next, to investigate the role of PTGFRN in the survival and proliferation of liver cancer cells, 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 μm 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. After culturing for 14 days at 37°C under 5% _CO2 conditions, the formed colonies were stained with 0.5% (w/v) crystal violet solution, and the total number of colonies was analyzed. Clonogenic survival assays in PTGFRN knockdown Huh7 cells and SNU449 cells showed 41-43% and 29-35% decreases, respectively (Fig. 5d, Fig. 5e). These results demonstrate that PTGFRN is an important cell surface molecule that enhances hepatoma cell survival and promotes proliferation under low-density stress.

To analyze how PTGFRN, the antigen of antibody 63-D7, affects the clonogenic survival of liver cancer cells in the long term, continuous gene knockdown was performed in liver cancer cell lines using shRNA targeting the PTGFRN gene. The negative target scramble and shRNA sequences #1 and #2 (TRCN0000057448, TRCN00000057452) targeting PTGFRN were inserted into the EZ-tet-PLKO vector (Addgene, 85996) and transfected with psPAX2 and pMD2G into human embryonic kidney cells HEK293FT cells (Table 2). Approximately 48 and 72 hours after transfection, the cell culture medium was collected and infected into liver cancer cell lines Huh7 and SNU449. Cells inserted with the target gene were selected for one week with RPMI-1640 medium containing 10% fetal bovine serum (Corning) and 2 μg/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. 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). When the binding of 63-D7 was analyzed using the flow cytometry method of Example 3 on the same cells, it was confirmed that the cell surface PTGFRN expression was well knocked down by 44-49% and 52-61% in both Huh7 and SNU449 liver cancer cells by two types of shPTGFRN (shPTGFRN#1, shPTGFRN#2), respectively (Fig. 6b, Fig. 6c). Among these, a clonogenic survival assay was performed in the same manner as above using SNU449 cells, and as a result, it was observed that shPTGFRN#1 reduced by 90% (Fig. 6d, Fig. 6e) and shPTGFRN#2 reduced by 47% (Fig. 6f, Fig. 6g). These results show that PTGFRN is an important cell surface molecule that continuously provides cancer stemness in liver cancer cells, enhances liver cancer cell survival, and promotes proliferation.

6-3. Analysis of cancer cell migration and invasion in PTGFRN knockdown liver cancer cell lines

Next, to investigate the role of PTGFRN in the migration and metastasis of liver cancer cells, 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 μm strainer (SPL) to prepare single cells.

To evaluate cell motility, 3.0 x 10 ⁴ cells were diluted in RPMI1640 medium and placed into a transwell chamber, and RPMI-1640 medium containing 10% Fetal Bovine Serum (Corning) was used as a chemoattractant to induce migration. After culturing for 37 h in a 37 ℃ incubator supplied with 5% CO ₂ and 95% air, the cells were washed twice with PBS (pH 7.4), and non-migrated cells were removed with a cotton swab and fixed with 4% PFA. The fixed cells were stained with 0.5% crystal violet solution, which was repeated three times and presented statistically. As a result, migration was reduced by 72% in PTGFRN knockdown Huh7 cells and 74-85% in SNU449 cells (Fig. 7a, Fig. 7b).

To investigate the invasiveness for metastasis of liver cancer cells, 0.1 ml of RPMI-1640 solution containing 250 μg/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).

Thus, 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.

6-4. Cell proliferation analysis of PTGFRN knockdown liver cancer cells

To analyze whether PTGFRN knockdown liver cancer cells Huh7 and SNU449 bind well to various extracellular matrices (ECM), a cell spreading assay experiment was performed. For the cell spreading experiment, 200 μg/ml Matrigel, 20 μg/ml CollagenⅠ, 20 μg/ml CollagenⅣ, and 0.1% Gelatin were coated and cultured in an incubator at 37℃ with 5% _CO2 and 95% air for 2 hours. After that, 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. After fixing with 4% paraformaldehyde for 10 minutes at room temperature, the cells were washed with PBS (pH 7.4) and observed under a microscope. The microscopic cell photographs were analyzed using the Image J program. In the Huh7 cell analysis, in shScramble, there were many spread cells that were attached to the bottom, but in the two types of shPTGFRN, there were many unspread cells that were round and floating because the cells did not attach well to the bottom (Fig. 8a, Fig. 8b). When the same experiment was performed on PTGFRN knockdown SNU449 cells, it was found that the binding to the extracellular matrix was reduced (Fig. 8c, Fig. 8d). When the results of repeating the same experiment three times were statistically analyzed, in Huh7, regardless of the type of shPTGFRN, the cell proliferation ability of liver cancer cells was observed to decrease in Collagen I, Collagen IV, and Gelatin extracellular matrices, except for Matrigel, in the case of knockdown (Fig. 8b). In SNU449, there was no difference in cell proliferation ability on Matrigel when shPTGFRN#2 was knocked down, but a decrease in cell proliferation ability on the remaining three types of extracellular matrices was observed (Fig. 8d). These results show that PTGFRN is important for the attachment and proliferation of hepatoma cells to most types of extracellular matrices.

6-5. Analysis of the immune evasion ability of PTGFRN knockdown liver cancer cells against NK cells

Since cancer cells can grow well when they evade the cytotoxicity of immune cells, most advanced cancer cells have the ability to avoid attacks by immune cells attacking them by expressing immune checkpoint molecules. To confirm whether the PTGFRN molecule expressed in liver cancer cells serves as an immune checkpoint molecule that plays an important role in immune evasion, the degree of cytotoxicity was analyzed when PTGFRN knockdown SNU449 cells generated using the above shScramble and shPTGFRN#2 were co-cultured with NK92, an NK cell line.

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. One day later, the effector cells (NK92):target cells (shPTGFRN-SNU449) were mixed at an E:T ratio of 1:1, 2.5:1, and 5:1 in 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 . After culture, 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. After drying the plate, the crystal violet was dissolved in 50% ethanol, 0.1 M sodium citrate solution, and the absorbance (OD540) was measured at 540 nm. When comparing the relative OD540, shPTGFRN#2 showed about 50% lower OD540 than shScramble due to low clonogenic viability, and showed an additional about 50% lower OD540 when co-cultured with NK92 (Fig. 9a, Fig. 9b). These results show that NK cell-mediated hepatoma cell killing is further increased when PTGFRN is deficient. Next, when the number of NK cells was increased by changing the E:T ratio to 1:1, 2.5:1, and 5:1, the degree of hepatoma cell killing was analyzed using the following formula.

[formula]

NK cytotoxicity (%) = {1 - (target OD value/No NK OD value)}*100

As a result, it can be seen that as the number of NK92 cells increases, the degree of apoptosis of liver cancer cells increases (Fig. 9c). These results suggest that 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.

6-6. Analysis of changes in signaling substances due to PTGFRN deficiency in liver cancer

As in Example 6-3 above, the expression of FAK and Src, which are key signaling molecules important for cancer metastasis, and B7-H3, known as an immune checkpoint molecule, was confirmed using the knocked-down cells.

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. 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. After washing three times with 0.1% TBST (Tris-Buffered Saline, 0.1% tween) for 10 minutes each, the sample was reacted with anti-rabbit IgG-HRP (1:12,000) at room temperature for one hour, and the target protein was confirmed using an ECL detection kit (Advansta) (Fig. 10).

As a result, the decrease in PTGFRN decreased the expression of B7-H3, an immune checkpoint regulatory molecule, and decreased the expression of p-FAK and p-Src, FAK signaling pathways. These results suggest that PTGFRN regulates the migration and invasiveness of cancer cells through the FAK signaling pathway.

6-7. Measurement of cis and trans binding between PTGFRN in the cell membrane

If 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. To observe this, 2.5 X 10 ⁵ cells were seeded per well in a 6-well plate, and the next day, 12 μg of PEI, pcDNA3.1(+)PTGFRN-FLAG vector, and pcDNA3.1(+)-PTGFRN-myc vector were diluted together in TOM (Welgene) to 4 μg each, and reacted at room temperature for 5 minutes. After that, 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 . Also, 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. 50 μg of 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.

As a result, both FLAG and myc tags were detected in the cell lysate co-expressing PTGFRN (Fig. 11a), but when they were transfected individually and then mixed to perform immunoprecipitation, FLAG-tagged PTGFRN was not detected in PTGFRN immunoprecipitated with anti-myc antibody, and myc-tagged PTGFRN was not detected in PTGFRN immunoprecipitated with anti-FLAG antibody (Fig. 11b). Therefore, this confirms that PTGFRN interacts with each other in a cis- rather than a trans- form on the cell membrane.

6-8. Analysis of PTGFRN binding substances in the cell membrane

To find other proteins that bind to PTGFRN in the cell membrane, 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 (α-PTGFRN). As a result, it was confirmed that PTGFRN binds to SLC3A2 (CD98hc) and B7-H3 (CD276) (Fig. 11c).

Since B7-H3 is known to be an immune checkpoint regulator in cancer cells, in order to verify the interaction between B7-H3 and PTGFRN once again, HEK293FT cells were seeded at 2.5 X 10 ⁵ cells per well in a 6-well plate. The next day, 12 μg 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 μg each in TOM (Welgene, Gyeongsan, Korea) and reacted at room temperature for 5 minutes. After that, 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 μg 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. 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

To analyze how the antigen PTGFRN of 63-D7 affects the adhesion of hepatoma cells, Huh7 and HepG2 cells were sorted into 63-D7 positive and negative using magnetic beads with 63-D7 antibody.

For sorting, 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. As a result, 63-D7(+) Huh7 cells showed a 43% increase in adhesion compared to 63-D7(-) Huh7 cells, and 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).

Meanwhile, to observe whether 63-D7 antigen-positive cancer cells have cancer stemness and are related to cancer recurrence and drug resistance, tumorspheres that induce cancer stemness were cultured. To generate tumorspheres, attached and growing Huh7 liver cancer cells were detached with trypsin-EDTA (Welgene, Daegu, Korea), filtered through a 40 μm 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. As a result, in the tumor cell culture with increased cancer stem cell properties, 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). These results suggest that PTGFRN, a 63-D7 antigen, acts as a new liver cancer stem cell marker.

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

When treated with antibodies, internalization of antibodies into cancer cells is a key prerequisite for the development of anticancer antibody therapeutics using antibody-drug conjugates (ADCs). To confirm the internalization of monoclonal antibody 63-D7 into various cancer cells, flow cytometry analysis was performed.

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 μm 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 μl of culture medium and reacted for 30 min at 37 °C to allow cell membrane internalization. After washing once with PBA, anti-mouse IgG-Alexa flour 488 (Invitrogen) corresponding to the primary antibody was further reacted at 4 °C for 20 min. After washing twice with PBA, antibody reactivity was analyzed for PI (propidium iodide)-negative cells using FACSCalibur and Cell Quest software (BD sciences). When the fluorescence intensity of monoclonal antibody 63-D7 bound to the cell surface at 4 °C was compared by flow cytometry to the degree of fluorescence intensity of 63-D7 when reacted at 37 °C, it could be seen that the binding of monoclonal antibody 63-D7 was markedly reduced in human hepatoma cells Huh7, HepG2, SNU387, SNU449 and human pancreatic cancer cells BxPC3, SNU213 (Fig. 13a, Fig. 13b). This means that the 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 9. Cytotoxicity of drug-conjugated 63-D7 antibody in hepatoma cells

Through the above 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.

In the experiment, drug-conjugated mouse IgG Fc region-specific secondary antibodies [anti-mouse IgG (Fc Specific) antibody-drug conjugates (ADCs)] were used. Specifically, α-mFc-CL-DMDM (AM-102DD, Moradec, USA) antibody conjugated with duocarmycin, a DNA alkylation agent, and α-mFc-CL-MMAF (AM-102AF, Moradec) antibody conjugated with monomethyl auristatin, a tubulin polymerization inhibitor, were used. First, 5,000 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℃. After that, 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℃, Cell Counting Kit-8 (CCK-8, K1018, APExBIO, USA) was used to determine the cell viability. 10 μl of CCK-8 was added to each well, reacted for 3 hours, and the absorbance at OD450nm was measured using a plate reader.

As a result, it was confirmed that the cell viability decreased by 40% when 100 nM 63-D7 antibody was treated with α-mFc-CL-DMDM ADC, and by 20% when 10 nM 63-D7 antibody was treated (Fig. 14a). When the same experiment was performed using α-mFc-CL-MMAF, the viability of liver cancer cells decreased by 40% at 100 nM and by 20% at 10 nM (Fig. 14b). However, the viability of cells treated with mouse-derived isotype antibody did not show a significant change. This proves that the drug-conjugated secondary antibody used in the experiment specifically binds to the mouse Fc region and enters the cell together with the mouse antibody 63-D7 to induce cytotoxicity.

Therefore, it can be seen that 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. These results suggest that the ADC strategy using 63-D7 antibody can be used to induce apoptosis of liver cancer cells and pancreatic cancer cells.

Example 10. Analysis of 63-D7 antigen in blood cancer cells from liver cancer patients

10-1. Detection of 63-D7 positive blood cancer cells in the blood of liver cancer patients by immunocytochemical method

We confirmed that 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).

To efficiently detect circulating cancer cells in HCC patients, we chose a strategy to remove red blood cells, then further remove CD45-positive cells by a CD45 depletion protocol, and analyze the remaining cells (Fig. 15c). Specifically, peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood of healthy donors, hepatitis patients, or HCC patients by Ficoll-Paque Plus (GE Healthcare, Seoul, Korea) gradient centrifugation within 6 h after collecting the blood of HCC patients. PBMCs were washed with PBS containing 0.5% bovine serum albumin (BSA) and 2 mM EDTA and resuspended in this buffer at a concentration of 1 × 10 ⁸ cells/mL. 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. First, 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 ㎕ of RPMI1640 medium, divided into eight fractions, and finally seeded onto glass slides coated with 0.1 ㎍/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.

To test the sensitivity and specificity of the CD45-positive leukocyte depletion method, increasing numbers of Huh7 cells were added to PBMCs, and the CD45 depletion protocol described above was applied to remove CD45-positive cells. The number of remaining 63-D7-positive Huh7 cells was measured after blocking with 10% normal horse serum. At this time, Dylight488-conjugated 63-D7 labeled with 63-D7 antibody was stained for Huh7 by immunocytochemical method using Dylight488-conjugation Kit (Vector laboratories, Burlingame, CA, USA). Linear regression of the number of Huh7 cells detected versus the number of spiked Huh7 cells yielded an ^R2 of 0.9801. The average recovery of Huh7 cells was approximately 72%, showing that more than 70% of cells can be detected by this method (Fig. 15b).

For double staining of 63-D7 and CD45 to confirm whether CD45 was well removed, 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.

Markers Primary HCC Secondary HCC Patients with marker-positive CTCs Marker-positive CTCs Patients with marker-positive CTCs Marker-positive CTCs DAPI+ (63-D7-) ND ND 22/22 (100%) 453/3307(13.7%) 63-D7+ CD45+ 0/20 (0.0%) 0/123 (0.0%) 0/2 (0.0%) 0/24(0.0%) 63-D7+ MVP+ 16/20 (80.0%) 100/363 (27.5%) 5/5(100%) 35/147(23.8%) 63-D7+ HSA+ 3/4 (75.0%) 29/118 (24.6%) 2/2(100%) 21/85(24.7%) 63-D7+ PanCK+ 0/4 (0.0%) 0/104 (0.0%) 0/1(0%) 0/24(0%) 63-D7+ E-cadherin+ 0/1 (0.0%) 0/18 (0.0%) ND ND 63-D7+ EpCAM+ 20/20 (100%) 46/331 (13.9%) 4/4(100%) 19/111(17.1%) 63-D7+ Vimentin+ 7/8 (100%) 43/180 (23.9%) ND ND 63-D7+ Twist+ 6/10 (60.0%) 6/189 (3.2%) ND ND 63-D7+ ZEB1+ 3/5 (60.0%) 3/60 (5.0%) ND ND

Next, blood samples from 21 healthy volunteers and 95 HCC patients (53 primary HCC patients and 42 secondary HCC patients) were collected by the same method as described above to remove CD45-positive cells, and the remaining cells were attached to slides and analyzed by confocal microscopy. The numbers of all 63-D7-positive circulating cancer cells in the blood of liver cancer patients (HCC, HCC (meta)), normal people (Normal), and hepatitis patients are shown in Table 4 below.

Number Cancer Sex Age PBMC(X107) blood(ml) total 63-D7 63-D7/7.5ml 63-D7/ml 1 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 23 3.11 18 HCC M 45 1.45 9.8 39 30 3.98 19 HCC M 48 0.5 9.8 6 5 0.61 20 HCC M 59 0.825 9.8 18 14 1.84 21 HCC M 56 1 9 13 11 1.44 22 HCC M 58 1.075 10 30 23 3.00 23 HCC F 58 2.5 10.4 40 29 3.85 24 HCC F 62 0.75 9 18 15 2.00 25 HCC M 59 0.875 8 28 26 3.50 26 HCC M 55 1.125 9 43 36 4.78 27 HCC M 75 1.51 10.5 14 10 1.33 28 HCC M 65 1.375 10 1 1 0.10 28 HCC M 50 1.575 9.5 23 18 2.42 30 HCC M 41 1.8 10.3 41 30 3.98 31 HCC M 45 1.5 10.1 18 13 1.78 32 HCC M 72 1.55 10 33 25 3.30 33 HCC M 75 1.49 10 13 10 1.30 34 HCC M 57 1.5 10.5 65 46 6.19 35 HCC M 64 1.15 8.6 36 31 4.19 36 HCC M 60 2.75 10.5 32 23 3.05 37 HCC M 59 2.55 10 30 23 3.00 38 HCC M 54 1.5 9 24 20 2.67 39 HCC M 38 10 10.5 26 19 2.48 40 HCC M 56 0.64 8.5 27 24 3.18 41 HCC M 68 6.43 9.2 22 18 2.39 42 HCC F 44 1.23 10 34 26 3.40 43 HCC M 66 0.96 10.5 24 17 2.29 44 HCC M 49 1.06 8.3 50 45 6.02 45 HCC M 78 2.1 10.4 14 10 1.35 46 HCC M 39 2.5 10.5 20 14 1.90 47 HCC F 60 2.05 11 29 20 2.64 48 HCC M 55 0.79 11 5 3 0.45 49 HCC M 58 1 10.7 25 18 2.34 50 HCC M 59 1.31 10.4 11 8 1.06 51 HCC M 57 4.25 10.6 13 9 1.23 52 HCC M 56 1.35 8.8 17 14 1.93 53 HCC M 39 1.25 9.2 3 2 0.33 54 HCC(meta) F 68 1.2 9.5 63 50 6.63 55 HCC(meta) M 57 5.25 10 56 42 5.60 56 HCC(meta) F 68 3 9.8 36 28 3.67 57 HCC(meta) M 57 2.38 10.5 32 23 3.05 58 HCC(meta) M 62 3.1 9.5 28 22 2.95 59 HCC(meta) M 59 0.825 9.8 16 12 1.63 60 HCC(meta) F 44 1.375 9 24 20 2.67 61 HCC(recu) M 67 1.13 8.5 44 39 5.18 62 HCC(meta) F 61 0.75 8 15 14 1.88 63 HCC(meta) F 56 2.15 9.5 24 19 2.53 64 HCC(meta) M 58 8.4 10.4 15 11 1.44 65 HCC(meta) F 67 2.75 8 65 61 8.13 66 HCC(meta) M 50 0.61 8 170 159 21.25 67 HCC(meta) F 38 1.25 9.5 70 55 7.37 68 HCC(meta) M 66 0.813 11 40 27 3.63 69 HCC(meta) M 76 1.13 10.5 50 36 4.76 70 HCC(meta) M 66 3.2 10 70 52.5 7.00 71 HCC(meta) F 70 1.3 11 558 380.5 50.72 72 HCC M 57 1.53 10.5 100 71.43 9.52 73 AGML M 33 5 9 39 33 4.33 74 HCC(meta) M 0.85 10.5 55 39.3 5.24 75 HCC(meta) F 0.55 8.5 125 110.3 14.71 76 HCC(meta) M 0.9 10.5 141 100.7 13.43 77 HCC(meta) F 1.38 9 90 75.0 10.00 78 HCC(meta) M 1.55 10 164 123.0 16.40 79 HCC(meta) M 1.13 8.3 91 82.2 10.96 80 HCC(meta) M 2.1 10.5 135 96.4 12.86 81 HCC(meta) M 2.3 10.3 162 118.0 15.73 82 HCC(meta) M 1.75 9.5 123 97.1 12.95 83 HCC(meta) M 1.85 9.3 159 128.2 17.10 84 HCC(meta) M 1.7 10 118 88.5 11.80 85 HCC(meta) F 1.95 10 158 118.5 15.80 86 HCC(meta) M 1.47 10.5 143 102.1 13.62 87 HCC(meta) F 2.15 10.5 139 99.3 13.24 88 HCC(meta) F 2 10.7 122 85.5 11.40 89 HCC(meta) M 1.9 10 188 141.0 18.80 90 HCC(meta) M 2 10.5 72 51.4 6.86 91 HCC(meta) M 1.75 10 132 99.0 13.20 92 HCC(meta) M 1.35 8.5 160 141.2 18.82 93 HCC(meta) M 2.25 8.5 106 93.5 12.47 94 HCC(meta) M 1.9 9.7 140 108.2 14.43 95 HCC(meta) M 0.8 9.5 130 102.6 13.68 Number Normal Sex Age PBMC(X107) blood(ml) total 63-B6 63-B6/7.5ml 63-B6/ml 1 Normal M 52 2.5 10.8 0 0 0.00 2 Normal M 63 2.375 10.3 4 3 0.39 3 Normal M 54 1.875 10.1 2 1 0.20 4 Normal M 31 3.575 10 0 0 0.00 5 Normal M 28 2.9 10.8 0 0 0.00 6 Normal M 32 1.75 10.8 0 0 0.00 7 Normal F 25 1 10.2 3 2 0.29 8 Normal F 36 1.33 8.2 0 0 0.00 9 Normal F 26 0.83 8 0 0 0.00 10 Normal M 30 2.25 8.3 0 0 0.00 11 Normal F 23 2.5 8.3 1 1 0.12 12 Normal M 35 3.75 8.2 2 2 0.24 13 Normal M 33 2 8.3 0 0 0.00 14 Normal M 28 2.25 8.4 0 0 0.00 15 Normal M 33 1.5 8.4 0 0 0.00 16 Normal M 29 2.5 8 0 0 0.00 17 Normal F 23 3 8.5 0 0 0.00 18 Normal M 54 1.4 7.5 0 0 0.00 19 Normal M 27 1 8 0 0 0.00 20 Normal F 25 1.85 8.3 0 0 0.00 21 Normal M 47 1.2 7.3 0 0 0.00 22 Chronic hepatitis M 45 1.03 8.7 0 0 0.00 23 Chronic hepatitis M 54 0.625 8.5 0 0 0.00 24 Cirrhosis M 61 1.625 8.2 0 0 0.00 25 Cirrhosis F 71 0.8 8 0 0 0.00 26 Acute hepatitis F 61 7.5 8.3 0 0 0.00

Although a few 63-D7-positive cells were detected in 5 of 21 normal adults, the measured cell numbers were very rare (≤ 0.39 cells/ml) (Table 4). When blood cells from HCC patients were stained with 63-D7 and other antibodies, 13.7% of the cells were 63-D7-negative and DAPI-positive, indicating that about 14% of the circulating cancer cells were not detected by 63-D7 (Fig. 15c upper panel, Table 3). When cells isolated from HCC patients were stained with 63-D7 and CD45 antibodies, all of the 63-D7-positive cells were CD45-negative (Fig. 15c lower panel, Table 3), suggesting that 63-D7 can detect about 86% of CD45-negative and DAPI-positive circulating cancer cells. And the size of 63-D7-positive circulating cancer cells was heterogeneous, ranging from 18 μm to 40 μm, 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.

The associations between 63-D7 and clinical pathological variables are shown in Table 5.

Variable Category Total
(n=53) 63-D7
<2.45
(n=26) 63-D7 ≥2.45
(n=27) p-value Age 1:≤60yrs 33 (62.26) 16 (61.54) 17 (62.96) 0.9148 2:>60yrs 20 (37.74) 10 (38.46) 10 (37.04) sex 1:M 44 (83.02) 23 (88.46) 21 (77.78) 0.4672 2:F 9 (16.98) 3 (11.54) 6 (22.22) Edmondson grade 1:1+2 48 (90.57) 23 (88.46) 25 (92.59) 0.6687 2:3+4 5 (9.43) 3 (11.54) 2 (7.41) Tumor_size 1:<3 28 (52.83) 14 (53.85) 14 (51.85) 0.8644 2:3~5 18 (33.96) 8 (30.77) 10 (37.04) 3:≥5 7 (13.21) 4 (15.38) 3 (11.11) Microvessel invasion 0 17 (32.08) 14 (53.85) 3 (11.11) 0.0009 1 36 (67.92) 12 (46.15) 24 (88.89) Portal vein invasion 0 51 (96.23) 25 (96.15) 26 (96.3) 1.0000 1 2 (3.77) 1 (3.85) 1 (3.7) Bile duct invasion 0 52 (98.11) 25 (96.15) 27 (100) 0.4906 1 1 (1.89) 1 (3.85) 　 Intrahepatic metastasis 0 45 (84.91) 24 (92.31) 21 (77.78) 0.2501 1 8 (15.09) 2 (7.69) 6 (22.22) Multicentric occurrence 0 47 (88.68) 23 (88.46) 24 (88.89) 1.0000 1 6 (11.32) 3 (11.54) 3 (11.11) Serosa invasion 0 51 (96.23) 25 (96.15) 26 (96.3) 1.0000 1 2 (3.77) 1 (3.85) 1 (3.7) Pathologic T stage (modified UICC, 2000) 1:pT1+pT2 19 (35.85) 14 (53.85) 5 (18.52) 0.0073 2:pT3+pT4 34 (64.15) 12 (46.15) 22 (81.48) Pathologic T stage (AJCC, 2010) 1:pT1+pT2 51 (96.23) 25 (96.15) 26 (96.3) 1.0000 2:pT3a 2 (3.77) 1 (3.85) 1 (3.7) Etiology 1:HBV 48 (90.57) 22 (84.62) 26 (96.3) 0.1917 2:HCV+other 5 (9.43) 4 (15.38) 1 (3.7) Child-Pugh class 1:A 50 (94.34) 24 (92.31) 26 (96.3) 0.6104 2:B+C 3 (5.66) 2 (7.69) 1 (3.7) Clinical Stage (AJCC) 1:1+2 51 (96.23) 25 (96.15) 26 (96.3) 1.0000 2:3a 2 (3.77) 1 (3.85) 1 (3.7) Clinical stage (UICC) 1:1+2 20 (37.74) 15 (57.69) 5 (18.52) 0.0033 2:3+4 33 (62.26) 11 (42.31) 22 (81.48) Milan_Criteria 0 40 (75.47) 20 (76.92) 20 (74.07) 0.8096 1 13 (24.53) 6 (23.08) 7 (25.93) AFP(ng/ml) 1:≤8.1 28 (52.83) 15 (57.69) 13 (48.15) 0.4865 2:>8.1 25 (47.17) 11 (42.31) 14 (51.85) PIVKA II(mAU/ml) 1:<40 31 (58.49) 13 (50) 18 (66.67) 0.2183 2:≥40 22 (41.51) 13 (50) 9 (33.33)

In a clinicopathological analysis of 53 patients with primary hepatocellular carcinoma, there was a statistically significant correlation between the number of 63-D7-positive circulating cancer cells and the advanced tumor stage group, with microvascular invasion, pathological stage, and clinical stage, indicating that patients with 63-D7-positive cancer cells had microvascular invasion and advanced to worse pathological and clinical stages (Table 5).

This means that the higher the number of 63-D7-positive circulating cancer cells, the worse the pathological and clinical condition. When the patient group was divided into primary HCC and secondary metastatic and recurrent HCC, the number of 63-D7-positive circulating cancer cells was more increased in the secondary metastatic and recurrent HCC patient group than in the primary HCC (Fig. 17, Table 4) (p<0.0001). Therefore, the number of 63-D7-positive circulating cancer cells (normal vs. primary HCC = ≤0.39 vs. 2.54 cells/ml) is an excellent diagnostic marker for diagnosing primary HCC patients (Table 4), and it is an excellent diagnostic marker closely related to disease progression, with the number further increasing in metastasis or recurrence (average 10.7 cells/ml) (Table 4).

10-2. Characteristics of 63-D7 positive blood cancer cells by double staining

To further characterize the 63-D7-positive blood cancer cells, cells were simultaneously stained with 63-D7 and various antibodies.

To double stain circulating cancer cells with 63-D7 and various surface markers, 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. Also, for double staining of Dylight 488-conjugated 63-D7 and intracellular markers, 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). Then, 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).

In our previous study, we found that cell surface major vault protein (MVP) was detected in approximately 90% of circulating cancer cells from HCC patients and further increased in metastatic cancer cells (Lee, et al., Sci Rep. vol. 7, 13201, 2017). Therefore, when circulating cancer cells were simultaneously stained with 63-D7 and MVP antibodies, approximately 28% (100/363) of 63-D7-positive circulating cancer cells were MVP-positive in primary HCC patients, and approximately 24% (35/147) of 63-D7-positive circulating cancer cells were MVP-positive in secondary HCC patients (Fig. 16a, Table 3). In 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). In 63-D7/E-cadherin staining, all circulating cancer cells were E-cadherin-negative (0/18) in all primary HCC patients (Fig. 16b, Table 3). In 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). In 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). Thus, 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.

Therefore, to determine whether 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). In addition, 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). Therefore, although 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.

10-3. Characteristics of 63-D7 positive blood cancer cells by triple staining

To further analyze the expression patterns of epithelial and mesenchymal phenotype markers in 63-D7-positive circulating cancer cells from primary and secondary HCC patients, 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.

Markers Primary HCC Secondary HCC Patients with marker-positive CTCs Marker-positive CTCs Patients with marker-positive CTCs Marker-positive CTCs 63-D7 ⁺ EpCAM ⁺ Vimentin ⁺ 7/21 (33.3%) 47/518 (9.1%) 3/30 (0.1%) 3/933 (0.3%) 63-D7 ⁺ EpCAM ⁺ Vimentin ^- 2/21 (9.5%) 3/518 (0.6%) 0/30 (0.0%) 0/933 (0.0%) 63-D7 ⁺ EpCAM ^- Vimentin ⁺ 18/21 (85.7%) 229/518 (44.2%) 27/30 (90.0%) 255/933 (27.3%) 63-D7 ⁺ EpCAM ^- Vimentin ^- 19/21 (90.5%) 239/518 (46.1%) 30/30 (100%) 675/933 (72.3%) MVP ⁺ EpCAM ⁺ Vimentin ⁺ 0/5 (0.0%) 0/144 (0.0%) 0/9 (0.0%) 0/304 (0.0%) MVP ⁺ EpCAM ⁺ Vimentin ^- 0/5 (0.0%) 0/144 (0.0%) 0/9 (0.0%) 0/304 (0.0%) MVP ⁺ EpCAM ^- Vimentin ⁺ 3/5 (60.0%) 67/144 (46.5%) 9/9 (100.0%) 151/304 (49.7%) MVP ⁺ EpCAM ^- Vimentin ^- 4/5 (80.0%) 77/144 (53.5%) 9/9 (100.0%) 153/304 (50.3%)

For triple staining of 63-D7, Vimentin, and EpCAM, 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).

As a result, 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. The 63-D7 ⁺ EpCAM ⁺ Vimentin ^- circulating cancer cells, representing epithelial cells, were 0.6% (3/518) in primary HCC patients, whereas there were none (0/933) in secondary HCC patients. The 63-D7 ⁺ EpCAM ^- Vimentin ⁺ circulating cancer cells, representing mesenchymal cells, were 44.2% (229/518) in primary HCC patients, whereas 27.3% (255/933) in secondary HCC patients (Fig. 18a, Table 6).

Thus, 0.6% of 63-D7-positive circulating cancer cells in primary HCC were of the epithelial phenotype, whereas 53.3% (9.1% + 44.2%) were of the semimesenchymal (both EpCAM and vimentin positive) and mesenchymal (vimentin positive) phenotypes. Interestingly, 63-D7 single-positive circulating cancer cells with a partial or intermediate phenotype without EpCAM or Vimentin were 46.1% in primary HCC patients and a whopping 72.3% (675/933) in secondary HCC patients. In addition, this intermediate phenotype was observed in all investigated secondary HCC patients (30/30 patients), suggesting that the intermediate phenotype expressing only 63-D7 is a key circulating cancer cell marker in patients with recurrent metastatic HCC (Table 6).

In a previous study, 51.3% of MVP-positive circulating cancer cells 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. When 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). In addition, MVP single-positive circulating cancer cells were found in all secondary HCC patients (9/9 patients, 100%) (Table 6).

Therefore, 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.

10-4. Analysis of immune checkpoint molecules in 63-D7 positive blood cancer cells

Numerous studies have established a strong correlation between B7-H3 expression and poor clinical outcomes in various cancers, including HCC. Since we experimentally confirmed that PTGFRN interacts with B7-H3 in HCC cells (Fig. 11c, Fig. 11d), we analyzed the expression of PTGFRN, B7-H3, and other immune-regulatory molecules in circulating cancer cells by triple staining.

For triple staining of 63-D7, B7-H3, and immune-regulatory molecules, cells were adhered to poly-L-lysine-coated glasses, then CD45-depleted cells were fixed, blocked, and reacted with anti-poly rabbit MVP antibody (Aviva bioscience), ULBP1 (Abcam), MICA/B (Bioss), and TGFβR1 (Atlas). They were then reacted with Alexa 568-conjugated anti-rabbit IgG (Thermo Fischer Scientific) or Alexa 594-conjugated mouse monoclonal PD-L1 (Biolegend), PD-L2 (Biolegend), or phycoerythrin-conjugated CD47 (Biolegend). 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.

Markers Patients with marker-positive CTCs (%) Marker-positive CTCs
(% marker/CTCs) MVP ⁺ 63-D7 ⁺ B7-H3 ⁺ 22/22 (100.0%) 57/458 (12.4%) MVP ⁺ 63-D7 ⁺ B7-H3 ^- 20/22 (90.9%) 54/458 (11.8%) MVP ⁺ 63-D7 ^- B7-H3 ⁺ 6/22 (27.3%) 15/458 (3.3%) MVP ⁺ 63-D7 ^- B7-H3 ^- 0/22 (0%) 0/458 (0%) MVP ^- 63-D7 ⁺ B7-H3 ⁺ 22/22 (100.0%) 160/458 (34.9%) MVP ^- 63-D7 ⁺ B7-H3 ^- 22/22 (100.0%) 77/458 (16.8%) MVP ^- 63-D7 ^- B7-H3 ⁺ 20/22 (90.9%) 60/458 (13.1%) MVP- 63-D7- B7-H3- (DAPI only) 18/22 (81.8%) 35/458 (7.6%) MVP ⁺ /total PTGFRN ⁺
or B7-H3 ⁺ /total PTGFRN ⁺ 111/348 (31.9%) or 217/348 (62.4%) TGFβR1+ 63-D7 ⁺ B7-H3 ⁺ 18/18 (100.0%) 140/787 (17.8%) TGFβR1+ 63-D7 ⁺ B7-H3 ^- 18/18 (100.0%) 109/787 (13.9%) TGFβR1+ 63-D7 ^- B7-H3 ⁺ 18/18 (100.0%) 79/787 (10.0%) TGFβR1+ 63-D7 ^- B7-H3 ^- 18/18 (100.0%) 54/787 (6.9%) TGFβR1- 63-D7 ⁺ B7-H3 ⁺ 18/18 (100.0%) 139/787 (17.7%) TGFβR1- 63-D7 ⁺ B7-H3 ^- 18/18 (100.0%) 93/787 (11.8%) TGFβR1- 63-D7 ^- B7-H3 ⁺ 18/18 (100.0%) 108/787 (13.7%) TGFβR1 ^- 63-D7 ^- B7-H3 ^- (DAPI only) 16/18 (88.9%) 65/787 (8.3%) TGFβR1 ⁺ /total PTGFRN ⁺
or B7-H3 ⁺ /total PTGFRN ⁺ 249/481 (51.8%) or 279/481 (58.0%), ULBP1 ⁺ 63-D7 ⁺ B7-H3 ⁺ 1/14 (7.1%) 1/404 (0.24%) ULBP1 ⁺ 63-D7 ⁺ B7-H3 ^- 8/14 (57.1%) 8/404 (2.0%) ULBP1 ⁺ 63-D7 ^- B7-H3 ⁺ 11/14 (78.6%) 17/404 (4.2%) ULBP1 ⁺ 63-D7 ^- B7-H3 ^- 14/14 (100%) 26/404 (6.4%) ULBP1 ^- 63-D7 ⁺ B7-H3 ⁺ 14/14 (100.0%) 111/404 (27.5%) ULBP1 ^- 63-D7 ⁺ B7-H3 ^- 14/14 (100.0%) 102/404 (25.2%) ULBP1 ^- 63-D7 ^- B7-H3 ⁺ 14/14 (100.0%) 99/404 (24.5%) ULBP1- 63-D7- B7-H3- (DAPI only) 14/14 (100.0%) 40/404 (9.9%) ULBP1 ⁺ /total PTGFRN ⁺
or B7-H3 ⁺ /total PTGFRN ⁺ 9/222 (4.1%) or 112/222 (50.5%) MICA/B ⁺ 63-D7 ⁺ B7-H3 ⁺ 18/18 (100.0%) 116/675 (17.2%) MICA/B ⁺ 63-D7 ⁺ B7-H3 ^- 16/18 (88.9%) 26/675 (3.9%) MICA/B ⁺ 63-D7 ^- B7-H3 ⁺ 17/18 (94.4%) 29/675 (4.3%) MICA/B ⁺ 63-D7 ^- B7-H3 ^- 18/18 (100%) 33/675 (4.9%) MICA/B ^- 63-D7 ⁺ B7-H3 ⁺ 18/18 (100.0%) 151/675 (22.4%) MICA/B ^- 63-D7 ⁺ B7-H3 ^- 18/18 (100.0%) 116/675 (17.2%) MICA/B ^- 63-D7 ^- B7-H3 ⁺ 18/18 (100%) 142/675 (21.0%) MICA/B ^- 63-D7 ^- B7-H3 ^- (DAPI only) 16/18 (88.9%) 65/675 (9.6%) MICA/B ⁺ /total PTGFRN ⁺
or B7-H3 ⁺ /total PTGFRN ⁺ 142/409 (34.7%) or 267/409 (65.3%) PD-L1 ⁺ 63-D7 ⁺ B7-H3 ⁺ 22/22 (100.0%) 114/950 (12.0%) PD-L1 ⁺ 63-D7 ⁺ B7-H3 ^- 19/22 (86.4%) 83/950 (8.7%) PD-L1 ⁺ 63-D7 ^- B7-H3 ⁺ 20/22 (90.9%) 80/950 (8.4%) PD-L1 ⁺ 63-D7 ^- B7-H3 ^- 22/22 (100.0%) 64/950 (6.7%) PD-L1 ^- 63-D7 ⁺ B7-H3 ⁺ 22/22 (100.0%) 176/950 (18.5%) PD-L1 ^- 63-D7 ⁺ B7-H3 ^- 22/22 (100.0%) 148/950 (15.6%) PD-L1 ^- 63-D7 ^- B7-H3 ⁺ 21/22 (95.5%) 208/950 (21.9%) PD-L1 ^- 63-D7 ^- B7-H3 ^- (DAPI only) 20/22 (90.9%) 77/950 (8.1%) PD-L1 ⁺ /total PTGFRN ⁺
or B7-H3 ⁺ /total PTGFRN ⁺ 197/521 (37.8%) or 290/521 (55.7%) PD-L2 ⁺ 63-D7 ⁺ B7-H3 ⁺ 21/22 (95.5%) 39/719 (5.4%) PD-L2 ⁺ 63-D7 ⁺ B7-H3 ^- 19/22 (86.4%) 30/719 (4.2%) PD-L2 ⁺ 63-D7 ^- B7-H3 ⁺ 19/22 (86.4%) 28/719 (3.9%) PD-L2 ⁺ 63-D7 ^- B7-H3 ^- 22/22 (100.0%) 39/719 (5.4%) PD-L2 ^- 63-D7 ⁺ B7-H3 ⁺ 22/22 (100.0%) 183/719 (25.5%) PD-L2 ^- 63-D7 ⁺ B7-H3 ^- 22/22 (100.0%) 143/719 (19.9%) PD-L2 ^- 63-D7 ^- B7-H3 ⁺ 22/22 (100%) 180/719 (25.0%) PD-L2 ^- 63-D7 ^- B7-H3 ^- (DAPI only) 20/22 (90.9%) 77/719 (10.7%) PD-L2 ⁺ /total PTGFRN ⁺
or B7-H3 ⁺ /total PTGFRN ⁺ 69/395 (17.5%) or 222/395 (56.2%) CD47 ⁺ 63-D7 ⁺ B7-H3 ⁺ 14/14 (100.0%) 78/497 (15.7%) CD47 ⁺ 63-D7 ⁺ B7-H3 ^- 14/14 (100.0%) 37/497 (7.4%) CD47 ⁺ 63-D7 ^- B7-H3 ⁺ 14/14 (100.0%) 41/497 (8.2%) CD47 ⁺ 63-D7 ^- B7-H3 ^- 14/14 (100.0%) 35/497 (7.0%) CD47 ^- 63-D7 ⁺ B7-H3 ⁺ 14/14 (100.0%) 78/497 (15.7%) CD47 ^- 63-D7 ⁺ B7-H3 ^- 14/14 (100.0%) 109/497 (21.9%) CD47 ^- 63-D7 ^- B7-H3 ⁺ 14/14 (100.0%) 62/497 (12.5%) CD47 ^- 63-D7 ^- B7-H3 ^- (DAPI only) 14/14 (100.0%) 57/497 (11.5%) CD47 ⁺ /total PTGFRN ⁺
or B7-H3 ⁺ /total PTGFRN ⁺ 115/302 (38.1%) or 156/302 (51.7%)

In our previous study, we reported that MVP is responsible for the survival, proliferation, migration, and invasion of HCC cells in a stressful environment. In addition, B7-H3 has been known to interact with MVP and promote cancer stem cell enrichment in breast cancer cells. Therefore, to determine how many PTGFRN-positive circulating cancer cells were MVP-positive in patients with secondary HCC, we simultaneously stained circulating cancer cells with 63-D7, anti-B7-H3, and anti-MVP antibodies (Fig. 18b, Table 7). About 32% (111/348) of the total PTGFRN-positive circulating cancer cells were MVP-positive, and about 62% (217/348) of the total PTGFRN-positive circulating cancer cells were B7-H3-positive. Therefore, a subset of PTGFRN-positive circulating cancer cells may likely be associated with the roles of B7-H3 and MVP in cell survival, proliferation, migration, and invasion in the peripheral blood of secondary HCC patients.

Recent studies have shown that EMT provides cancer cells with metastatic potential, immunosuppressive potential, and cancer stemness. In addition, EMT can induce the expression of 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). Recent studies suggest that partial/intermediate phenotype cancer cells mediate tumor immune escape by avoiding immune cell cytotoxicity and polarizing immune cells toward an immunosuppressive phenotype. Therefore, 63-D7-positive circulating cancer cells with partial/intermediate phenotype are expected to play an important role in patients with secondary liver cancer.

TGFβ/TGFβR1 signaling exerts multiple immunosuppressive effects in CSCs. TGFβ is also required for the suppression of NK cell function. In addition, TGFβ/TGFβR1 signaling provides the high metastasis-initiating ability of breast CSCs. TGFβ1 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 TGFβR1 expression was positive in 63-D7-positive CSCs. When CSCs were stained with anti-TGFβR1, 63-D7, and anti-B7-H3 antibodies, all secondary HCC patients had TGFβR1-positive CSCs (Fig. 19, Table 7). Detailed analysis revealed that 51.8% (249/481) of all PTGFRN-positive circulating cancer cells were TGFβR1-positive, and 58% (279/481) of all PTGFRN-positive circulating cancer cells were B7-H3-positive, suggesting that PTGFRN-positive circulating cancer cells likely have a TGFβR1- and B7-H3-mediated immunosuppressive phenotype.

To further investigate how PTGFRN-positive circulating cancer cells are associated with the host immune response in patients with secondary HCC, 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. In 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. In MICA/B/63-D7/B7-H3 staining, all patients (18/18) had MICA/B-positive circulating tumor cells. In patients with secondary HCC, 34.7% (142/409) of total PTGFRN-positive circulating cancer cells were MICA/B-positive, and 65.3% (267/409) of total PTGFRN-positive circulating cancer cells were B7-H3-positive. These results suggest that secondary circulating cancer cells express some immunoactivating molecules, such as ULBP1 (4.1%) or MICA/B (34.7%), but at the same time, most of them preferentially express B7-H3 (65.3%), which is known as an immune checkpoint regulatory molecule.

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).

In 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. In 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.

Finally, we investigated whether PTGFRN-positive CBCTs were associated with CD47, because CD47 is a “don’t eat me” signal for macrophages and inhibits phagocytic innate immune surveillance (Matlung, Szilagyi et al. 2017). In 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.

Although 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 TFGβR1 (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. In particular, B7-H3 was predominantly expressed in all PTGFRN-positive circulating cancer cells (average 57.6%). Therefore, these results suggest that 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. These results suggest that 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.

Example 11. Analysis of monoclonal antibody 63-D7 gene and amino acid

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 μg of total RNA was added and reacted at 37 °C for 30 minutes to remove residual DNA. 1 μl 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. In order to perform heavy chain cloning using the synthesized cDNA, 25 pmole of an oligonucleotide having the polymerase chain reaction primer corresponding to the IgG1 constant region, 5'-gga gtc gac ATA GAC AGA TGG GGG TGT CGT TTT GGC-3' (SEQ ID NO: 20), and 25 pmole of an oligonucleotide having the base sequence 5'MH1 5'-ctt ccg gaa ttc SAR GTN MAG CTG SAG SAG TC-3' (SEQ ID NO: 21) and 25 pmole of an oligonucleotide 5'MH2 5'-ctt ccg gaa ttc SAR GTN MAG CTG SAG SAG TCW GG-3' (SEQ ID NO: 22) corresponding to the N-terminus of the heavy chain antibody variable region were added to prepare a chain polymerization reaction mixture. For light chain cloning, 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. For efficient cloning of polymerase chain reaction products, 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. For the light chain, 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. After mixing the heavy chain or light chain polymerase reaction primers and 1 unit of i-pfu DNA Polymerase (iNtRON Biotechnology, Seoul, Korea) respectively, the reaction was performed once at 95 °C for 5 minutes, and then 30 times at 95 °C for 1 minute, 35 °C for 1 minute, and 72 °C for 1 minute. As a result, 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.

To clone the amplified 63-D7 gene, first, 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, and 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. These two DNAs were ligated with T4 DNA ligase (New England Biolab, Massachusetts, USA) and transformed into E. coli DH5α using the CaCl ₂ method. For DNA sequence analysis of antibody genes, the above-mentioned multiple clones were cultured overnight in 3 ml of LB medium containing 100 ㎍/㎖ of ampicillin. Plasmid DNA was isolated using a DNA-Spin plasmid mini prep kit (Intron, Korea) according to the manufacturer's protocol. For the heavy chain, E. coli clones with DNA inserts of approximately 400 bp in size were selected, and for the light chain, E. coli clones with DNA inserts of approximately 390 bp in size were selected. The base sequence of each DNA insert was confirmed by nucleotide sequencing (Bionics, Seoul, Korea).

After translating the base sequences of the heavy and light chain DNA into amino acids and organizing the antigen recognition determining sites according to the antibody structure using Kabat numbering, 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.

Claims (22)

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. An anti-PTGFRN monoclonal antibody according to claim 1, wherein the heavy chain variable region comprises an amino acid sequence of sequence number 1. An anti-PTGFRN monoclonal antibody according to claim 1, wherein the light chain variable region comprises an amino acid sequence of sequence number 2. An antibody-drug conjugate comprising a drug conjugated to the anti-PTGFRN monoclonal antibody of any one of claims 1 to 3. A pharmaceutical composition for treating or preventing cancer comprising an anti-PTGFRN monoclonal antibody according to any one of claims 1 to 3. A pharmaceutical composition for treating or preventing cancer according to claim 5, wherein 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 hybridoma producing a PTGFRN monoclonal antibody of any one of claims 1 to 3. A composition for detecting PTGFRN comprising a PTGFRN monoclonal antibody according to any one of claims 1 to 3. A method for providing information for diagnosing cancer, comprising the step of treating a sample isolated from an individual with an anti-PTGFRN monoclonal antibody according to any one of claims 1 to 3. 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 an anti-PTGFRN monoclonal antibody according to any one of claims 1 to 3. A method for providing information necessary for determining the level of cancer cells in blood, wherein the sample is selected from the group consisting of blood, plasma, bone marrow fluid, lymph, saliva, tears, urine, mucosal fluid, and amniotic fluid, according to claim 10. A method for providing information necessary for determining the level of cancer cells in blood, further comprising the step of treating the sample with a substance that specifically binds to EpCAM according to claim 10. A method for providing information necessary for determining the level of cancer cells in blood, wherein the substance that specifically binds to EpCAM according to claim 12 is an anti-EpCAM antibody or an antigen-binding fragment thereof. A method for providing information necessary for determining the level of blood cancer cells according to claim 10, wherein 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 according to claim 10, wherein the blood cancer cells are not epithelial cells or mesenchymal cells. A method for providing information necessary for determining the level of blood cancer cells according to claim 10, wherein 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, wherein the blood cancer cells further express B7-H3 (CD276) protein according to claim 10. 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 an anti-PTGFRN monoclonal antibody according to any one of claims 1 to 3. 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 the 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 an anti-PTGFRN monoclonal antibody according to any one of claims 1 to 3. 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 compared to the control group if the level of the detected PTGFRN is lower than that of the control group in claim 20. A composition for detecting cancer cells in blood comprising a substance that binds to PTGFRN.

Images