Application Of Human Anti-Trop-2Fab In Targeted Therapy Of Pancreatic Cancer

The pancreatic cancer is a malignant tumor in digestive system characterized by high malignant degree, rapid progress and poor prognosis, its fatality rate is more than90%, and the median survival of pancreatic cancer patients without surgical intervention is less than six months. About20million people died of the disease within the scope of world each year, and it showed a yearly increasing tendency recently. At present, surgery was the only means to cure pancreatic cancer. However, due to the early symptoms being non-obvious, most of the patients in the time of diagnosis had been ill in the late, the5year survival rate only%1～3%. And that about80%of patients had local recurrence after tumor resection. Only by taking a variety of treatment including surgery, chemotherapy, radiation therapy, and targeted therapy, supportive care to prolong the survival time of patients, to reduce the clinical symptoms and improve the quality of life. Although the traditional chemotherapy drugs significantly improved the treatment of pancreatic cancer, extended survival time of patients, but resistance and a variety of acute and chronic toxic ide effects severely limit its clinical application. Molecular targeted therapy is to conjugate tumor cells surface specific molecular ligand with chemotherapy drugs, radioactive nuclide or biological toxins, and killing tumor cells specifically. Due to the clear role sites, drugs usually has the very high selectivity, can effectively kill or inhibit the target cells.But to the normal tissue cells, those drugs do not produce or only produce little side effects. Therefore, the developments of molecular targeted drugs become the hot spots of cancer clinical research.Human trophoblast cell surface antigen2(human trophoblast cell the surface antigen, Trop-2) is a cell surface glycoprotein, belonging to the TACSTD gene family, which is highly expressed in a wide variety of epithelial cancers, such as breast cancer, pancreatic cancer, prostate cancer, ovarian cancer, and so on, but there is little or no expression in adult somatic tissue. Clinical data shows that Trop-2expression level has a positive correlation with the invasion and metastasis of tumors, while a negative correlation with the prognosis of patients. Trop-2is not only related with the intracellular calcium ion concentration and cyclin D1(cyclin D1) and kinase C (PKC) regulation, but also may activate the ERK pathway, thereby regulating tumor cell growth, invasion and metastasis. Latest research shows that Trop-2also has cancer stem cells characteristics. Therefore, Trop-2may be a candidate molecule for the early diagnosis and targeted therapy of many malignant tumors. The whole molecule antibody ability of tissue penetration is poor and slower clearance in the blood and non-tumor tissue, therefore leading to relatively low T/NT ratio. Fab, a molecular weight of50Kd, is1/3of full IgG, and has no Fc section, has good penetration and pharmacokinetic characteristics, and it can maintain the activity of the bound antigen. Therefore, it becomes a kind of genetic engineering antibodies and be used more often. Fab can not be combined with cell Fc receptors, theoretically reducing the occurrence of non-specific binding and the opportunity of rejection. Since there was no Fc, it showed short half-life and turnover quickly. So it is helpful for radiation immune imaging to examination tumors. Compared with complete antibody, Fab has no antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), it can be used as a carrier molecule for disease video diagnostic and targeted therapy.In this study, the expression vector of anti-Trop-2Fab which screened from the antibody phage display library was transferred into Escherichia coli Top10. The expression and purification conditions were optimized gradually and high purity anti-Trop-2of Fab antibody were prepared. Based on the analysis of Trop-2Fab antibody immunological characteristics, its impact on the proliferation, migration and invasion of pancreatic cancer cell were evaluated on the cellular level. After conjugated with doxorubicin (DOX), the impaction of Fab-DOX was evaluated on pancreatic cancer growth in vitro and in vivo.[Objectives]1. To select the best condition for the anti-Trop-2antibody fragment Fab expression in Escherichia coli and to optimize the conditions of protein expression and purification.2. To analysis anti-Trop-2antibody fragment Fab immunological characteristics and to prepare a large number of anti-Trop-2antibodies fragment Fab.3. To evaluate its impact on the proliferation, migration and invasion of pancreatic cancer cell on the cellular level.4. To conjugate anti-Trop-2antibody fragment Fab with doxorubicin (DOX) and analyze its affinity and specificity.5. To evaluate its impact on pancreatic cancer proliferation in vitro and in vivo respectively[Methods]1. The expression plasmid of anti-Trop-2Fab antibody was transformed into E.coli TOP10F’. It was tested to replace the culture medium before induction of anti-Trop-2Fab expression.The effects were compared in different induction temperatures, induction times and additional glucose before induction on protein expression. The expressed anti-Trop-2Fab were purified by affinity chromatography and evalued its immunological characteristics were evalued by immunofluorescence and flow cytometry.2. The influence of anti-Trop-2Fab antibodies on the proliferation of two strains of pancreatic cancer cell lines was detected by MTT assay. According to the results, a cell line was chosed in the following experimens. The influence of Trop-2Fab antibodies on pancreatic cancer cells migrating ability were evaluated with wound healing experiment, and the change of invasion of pancreatic cancer cells Were tested with Transwell method.3. To conjugate anti-Trop-2antibody fragment Fab with doxorubicin (DOX) by the chemical synthesis, its affinity and specificity were analyzed with ELISA and direct immune fluorescence, and the transport proceeding of DOX-anti-Trop-2Fab into pancreatic cancer cells BXPC3and NIH3T3cells were detected by direct immune fluorescence.[Results] 1. In the culture medium, adding5g/L of glucose, the expression of anti-Trop-2Fab was significantly increased. Decreasing the temperature as16℃could increase the expression of Fab than other temperatures. The expression of anti-Trop-2Fab reached peak after adding inducer for12hours. FACS results showed that the combination signal of anti-Trop2Fab is strong in pancreatic cancer cells BxPC3, but the signal of NIH-3T3cells which did not express Trop-2, is weak; immunofluorescence results showed that anti-Trop-2antibody Fab could combined with BxPc3cells, positive signals were mainly displayed on the cell membrane, while the negative control cells NIH3T3almost without signals. All the results above suggested that anti-Trop-2Fab antibodies can specifically bind to the natural conformation Trop-2protein on the cell membrane surface of pancreatic cancer cells BxPc3.2. MTT assay results showed that anti-Trop-2antibody Fab fragment can inhibit the proliferation of human pancreatic cancer cells; wound healing experiments showed anti-Trop-2antibody Fab fragment can inhibit pancreatic cancer cell migration; Transewell results showed that anti-Trop-2antibody Fab fragment can inhibit the invasion of pancreatic cancer cell. However, all experimental groups require a relatively high concentration of antibodies.3. The results of cell ELISA showed that the affinity of anti-Trop-2antibody Fab had no significant decline after conjugated with DOX; cell Immunofluorescence results showed that anti-Trop-2antibody Fab could combined with BxPc3cells, positive signals were mainly displayed on the cell membrane, while the negative control cells NIH3T3almost had no signal can be seen. These results showed that even after conjugated with DOX, anti-Trop-2Fab antibodies could specifically bind to the natural conformation Trop-2protein on the cell membrane surface of pancreatic cancer cells BxPc3. Cell Immune fluorescence determine the transport process of DOX-anti-Trop-2Fab within pancreatic cancer cells, the results suggested that compared with doxorubicin, DOX-anti-Trop-2Fab showed a certain lag, but after four hours, positive signal had no obvious difference in two different cells.4. The results of experiments in vitro showed that DOX-anti-Trop-2Fab could inhibit the proliferation of pancreatic cancer significantly, and is superior to free doxorubicin. MTT assay showed that DOX-anti-Trop-2Fab showed a certain lag compared with doxorubicin, but72hours after treatment, the inhibition rate of DOX-anti-Trop-2Fab on pancreatic cancer cells has been close to90%, while in the same experimental conditions, the same concentration of doxorubicin was only75%, and anti-Trop-2Fab was55%. It showed that conjugate anti-Trop-2antibody fragment Fab with doxorubicin (DOX) could effectively improve the inhibition rate of doxorubicin and anti-Trop-2Fab on pancreatic cancer cells.[Conclusions]1. Different induction temperature, induction time and additional glucose had a significant effect on the expression of anti-Trop-2antibody Fab.2. Anti-Trop-2antibody Fab can inhibit pancreatic cancer cell proliferation, invasion and migration, but requires a relatively high concentration.3. The affinity and specificity of anti-Trop-2antibody Fab has no significant changes even after conjugated with DOX.4. DOX-anti-Trop-2Fab could significantly inhibit the proliferation of pancreatic cancer in vitro, and more effective than that of free doxorubicin. After72hours, the inhibition rate of DOX-anti-Trop-2Fab already nearly90%, while the same concentration of doxorubicin only75%, anti-Trop-2Fab was only55%.