Development Adn Applicationt Of Human Anti-Trop-2 Fab In Targeted Therapy Of Breast Cancer

Breast cancer is one of the neoplasms that have greatest negative psychological impact on the sufferers. Although China is among the low morbidity countries of breast cancer, its yearly increasing rate in China is 1-2% higher than the average rate of the world, especially among urban women, the rate jumped sharply over the past decade. Due to its largest population in the world. China tops the world in its breast cancer cases. Despite the marked improvements in diagnosis and treatment with the development of endoscope. the survival rate of breast cancer were on the rise, the five-year survival rate for metastasis breast cancer was only 20%. In order to explore new effective therapeutic strategy for this disease, it is of important significance to profoundly understand the molecular mechanism of breast cancer metastasis and to find new drugs.The human trophoblast cell surface antigen 2 (Trop-2) is one of the first tumor associated antigens (TAA) identified by monoclonal antibodies. Trop-2 is a cell surface glycoprotein that was originally identified on human trophoblast and choriocarcinoma cell lines. Trop-2 has been cloned and shown to encode a type-1 transmembrane protein with a single transmembrane domain. Furthermore, research had shown Trop-2 to be involved in the transduction of an intracellular calcium signal, although the exact physiological mechanism has not yet to be elucidated. Its relatively low expression levels in normal epithelial cells and overexpression in various types of human carcinomas as well as its association with decreased overall patient survival and increased tumor grade and metastasis make it an important target for cancer immunotherapy. Currently. Trop-2 had been applied in clinic or pre-clinic as tumor marker for diagnosis and prognosis. The role of Trop-2 in breast cancer and in particular, its molecular mechanisms remained unclear. To explore whether or not Trop-2 expression is related to malignant phenotype of breast cancer in China, we investigated the expression and the role of Trop-2 in human breast cancer of China. On the base of the technology of our key laboratory of antibody techniques, human anti-Trop-2 Fab had been prepared and its characteristics analyzed, then it had been used as a drug alone to examine the therapeutic effects on the breast cancer cells. Some possible mechanisms of anti-Trop-2 Fab involved are also our interests.[Objectives]1 To investigate the expression of Trop-2 in breast cancer cells and the relationship of expression intensity with the clinicopathologic characteristics of breast cancer. especially the relationship with metastasis.2 To select and prepare from human phage display library, to express and purify fully human anti-Trop-2 Fab. and to analyze its characteristics.3 To examine the effects of anti-Trop-2 Fab on the malignant phenotypes, especially invasion and metastasis of breast cancer cells by expressional and functional studies in vitro and in vivo.[Methods]1 The subcellular location and expression of Trop-2 in breast cancer, including metastatic and non-metastatic cases were determined by one-step RT-qPCR and immunohistochemistry assay. Univariate and multivariate Cox regression analyze whether Trop-2 is a prognostic factor in breast cancer.2 Fully human anti-Trop-2 Fab was selected and prepared with phage display technology. Fab characters was analyzed by ELISA、FACS、IF and IP-MS.3 The breast cancer cells incubated with Fab and the control cells were depicted by MTT assay, wound healing assay. Flow cytometery (FCM) with AV/PI staining, invasion assay, immnofluorescence staining and western blot.5 Mice xenograft tumor model were used to observe the inhibition of Fab by tail vein in vivo and the changes of clinicopathological features by immnohistochemistry.[Results]1 Trop-2 was over-expressed in breast cancer tissues, especially in metastatic breast cancer. Fifteen fresh frozen breast cancer speciments processed Real-time quality PCR showed Trop-mRNA expression was 2.74 fold higher in breast cancer than in normal breast tissue:Immunohistochemistry carried out on 82 paraffin-embedded breast cancer sections showed that Trop-2 was predominantly expressed in the membrane of epithelial cells and occasionally diffused into cytoplasm. In 56.10% breast cancer tissues. Trop-2 was overexpressed. significantly higher than that in nonneoplastic tissues. The relationship between Trop-2 expression and clinicopathological features of the patients analysed by Chi-square test revealed Trop-2 overexpression was significantly correlated with Cyclin D1. P53. Univariate and multivariate Cox regression analysis of prognostic factors indicated that seven-year survival was associated with Trop-2 expression. Cyclin Dl expression. Trop-2 and Cyclin D1 co-expression and lymph-node metastasis. These results demonstrated that Trop-2 can be used as a molecular prognostic marker in breast cancer.2 Fully human antibody fragment Fab that specifically binding to Trop-2 was selected from phage display antibody library. Positive phage-display antibody clones were selected on live cells and immobilized protein. The purified Fab was verified by SDS-PAGE and Western blot, which showed two bands at the expected sizes. FACS and immnofluorescence staining showed the Fab could bind native Trop-2 specifically on the breast cancer cell SK-BR-3 surface. IP-MS showed that the protein which the Fab bound is Trop-2 protein.3 Anti-Trop-2 Fab inhibited proliferation, migratory and invasion abilities of breast cancer cells in vitro. MTS assay showed that growth of five breast cancer cells incubated with Fab01 all decreased. Under 2-D culture, the wound healing assay revealed that migration rate of tested MDA-MB-231 was lower than that of control cells. Direct immnotluorescence staining showed that the F-actin expression was down regulated, it indicated that anti-Trop-2 Fab may decrease the migration through affecting cytoskeloton rebuild. FCM analysis showed cell cycle arrest by Fab. that is. tested MDA-MB-231 in G1 phrase and S phrase reduced. Indirect immnofluorescence staining showed that Bax up-expressed and Bcl-2 down-expressed. The influence of anti-Trop-2 Fab on the invasive ability of MDA-MB-231 by invasion assay. It was found that anti-Trop-2 Fab produced a marked inhibition of invasive ability of MDA-MB-231 through matrigel on Corning chamber assay. Under 3-D culture, the volumes of MTS incubated with Fab01 were smaller than that of nomal cells.4 Compared with effects of PBS and another unrelated Fab (anti-RV Fab) inoculated throuth tail vein. anti-Trop-2 Fab induced an inhibition the tumor growth in nude mice with exograft breast cancer.ConclusionsWe provide evidence that Trop-2 is highly expressed in breast cancer tissues. Incubation with anti-Trop-2 Fab prepared can reverse the malignant potential of breast cancer cells to some degree including the abilities of proliferation, migration and invasion in vitro and in vivo. These findings demonstrated that Trop-2 is a novel, important determinant of breast tumor growth, as well as of metastatic cancer, and candidate Trop-2 for novel diagnostic and therapeutic procedures.