The Anti-tumour Effects Of LunX And EpCAM Antibody

Malignant tumor is one of the most serious diseases in the world, which can lead to high mortality in the human. Therefore, improvement in malignant tumor treatment is urgently required. In recent years, with further understanding of pathogenesis of cancer from the cell level to molecular level and the techniques of molecular biology, molecular targeted therapy achieved remarkable curative effect in clinic. Monoclonal antibodies for malignant tumor therapy have achieved great achievements in recent years. Antibody conjugates are effective new treatment options for solid tumours and lymphomas and immunomodulatory.Antibody therapy for tumor has been established over the past15years, and is an important and sucessuful stratey in treating tumor patient. Using monoclonal antibodies to kill tumor cells can result from blockade or activation of receptor, payload delivery, immunity cell-edited killing mechanisms, blockade of tumour angiogenesis, and targeted-stroma cells.The efficacy and safety of therapeutic antibody vary depending on cell surface antigens. Ideally, the targeted-antigen should be accessible, abundant, special, consistent and homogeneous on tumor cell membrane.Lung cancer has become one of the highest incidence and mortality rates of malignant solid tumors. So far, haven’t found a specific target in the treatment of lung cancer, it is urgent to find a typical target for lung cancer, which is conducive to the early diagnosis and targeted therapy of lung cancer. Lung specific X protein (LunX) belongs to the PLUNC family. The previous research found that LunX mRNA can be used as the specific biomarker of lung cancer. So, whether LunX protein is widely expressed in lung cancer tissues; promotes the occurrence and development of lung cancer; and can be as biological target for the therapy of lung cancer or not?Epithelial cell adhesion molecule (EpCAM) as malignant tumor associated antigen, is expressed in lung cancer, liver cancer, gastric cancer and other malignant solid tumor, also in most normal cells; the previous study found that EpCAM can be as stem cell markers of multiple solid tumors, has become the target of tumor immunotherapy. Until recetly, EpCAM is reported only in solid tumors, and the correlation between EpCAM and leukemia has not been verified. If the expression of EpCAM in leukemia cells, EpCAM may be a potential therapeutic drug for the treatment of leukemia.This paper mainly launches the research of the therapeutic targets for lung cancer and blood tumor. The restults as follows:Ⅰ、Targeting LunX inhibits lung cancer growth and metastasisThe study was designed to analyze LunX protein expression in150randomly collected primary NSCLC patient samples and to evaluate the correlations between LunX overexpression and clinical factors in NSCLCs. Through IP and immunoblotting, I wanted to find LunX binding protein and downstream signaling pathways. To explore whether LunX is an effective therapeutic target in lung cancer, we tested the relationship between LunX levels and tumor progression by conducting multiple lung cancer xenograft experiments using LunX-silenced or LunX-overexpressing lung cancer cells. At last, we screened and generated a therapeutic LunX antibody and tested its antitumor effect in vitro and in vivo.1. LunX is overexpressed in non-small cell lung cancer cells, especially in lymph node metastaseizing tumor cell.150cases from NSCLC patients were analyized by immunohistochemical analysis,90%of which showed that LunX is overexpressed in lung cancer cells, especially in Lymph node metastasis of tumor cells. Through Kaplan-Meier curves assessment in108cases, we detected higher LunX immunoreactivity in NSCLC patients, accompanied with significantly lower rate of postsurgery survival.2. LunX protein promotes NSCLC growth, migration and invasion by binding to14-3-3protein and activating14-3-3pathways.LunX interacted with14-3-3θ and14-3-3ζ and maintained the proteins in an active state to promote lung cancer cell migration and proliferation in vitro. LunX-targeted small interfering RNA reduced tumor growth and metastasis by disrupting primary tumor growth, local invasion, micrometastasis formation and metastatic colonization.3. LunX antibody reduces lung cancer growth, metastasis and invasion LunX expression was detected in tumor cell membranes; therefore, a LunX-targeted antibody was screened and generated, which can reduce proliferation and migration of lung cancer cells by inducing LunX protein down-regulation and blocking its downstream pathways. The antibody slowed the growth of subcutaneous lung cancer xenograft, and moreover, blocked tumor metastasis and improved mouse survival in lung cancer xenograft model induced by tail vein injection. Thus, LunX is a potential therapeutic target in lung cancer.Ⅱ、Targeting EpCAM reverses chemoresistance and eradicates malignance in leukemiaThe study was designed to analyze EpCAM expression in bone marrow and PBMC of leukemia patients, and to analyze the changes of the percentage of EpCAM positive cells before and after chemotherapy. Using clone formation in vitro and xenograft formation in vivo, I want to verify whether EpCAM protmots blood tumor formation or not. At last, we screened and generated a therapeutic EpCAM antibody and tested its antitumor effect in vitro and in vivo.The results as follows:1. EpCAM is overexpressed in AML and MM cells LunX is overexpressed in bone marrow and PBMC samples from AML and MM patients, but not in normal person. To confirm EpCAM expression in leukemia cells, EpCAM protein was also detected in myeloid leukemia cells, K562and HL60.2. EpCAM+leukemia cells resistant to chemotherapy In the same patient samples, EpCAM+cells accounted for the proportion of abnormal cells were significantly increased after chemotherapy, and EpCAM-cells have more sensitivity of chemotherapeutic drug. EpCAM promote leukemia cells resistant to chemotherapy.3. EpCAM antibody inhibits the formation and growth of blood tumor In subcutaneous tumor model of NOD-SCID mice, EpCAM antibody inhibits tumor growth. With antibody up to80mg/kg (body weight), the tumor can be completely removed. In the xenograft mouse model of intravenous inoculation, EpCAM antibody inhibits tumor formation in cervical lymph nodes and bone marrow, and reduced the number of tumor cells in peripheral blood. In this model, the antibody could significantly prolong the survival of mice.4. Macrophage mediated EpCAM antibody anti-tumor effect. In subcutaneous xenograft model, EpCAM antibody treatment promoted the migration of F/80+Gr-1-macrophage from spleen to tumor tissues, and tumour-infiltrating macrophages have anti-tumor effect.In summary, based on EpCAM overexpression and carcinogenesis in leukemia, a EpCAM-targeted antibody was screened and generated, which can inhibit blood tumor formation and growth. Thus, EpCAM is a potential therapeutic target in leukemia. The study of humanized EpCAM antibodies may have important significance for clinical treatment of leukemia.