| Nasopharyngeal cancer (NPC) is the most common cancer in the Department of Otolaryngology and remains a serious threat to the health and lives of the people in China. It is the primacy incidence in head and neck cancer and a high incidence in southern China. The pharyngeal recess is a good site of NPC. The location of pharyngeal recess is deep in neck and around of the important tissue and nerve. It is a plurality of lymphatic figured, so the most of NPC were found adjacent areas and lymph nodes involved in the early stage the disease. The clinical symptoms ofpatient are not typical and often prone to misdiagnosis and missed diagnosis. The invasion and spread of cancer cells to other parts of the body, known as metastasis, is a principal cause of death in patients diagnosed with. Most patients of NPC are difficult to operation because of the hidden tumor site and its infiltration growth. Radiotherapyor chemotherapy are more sensitive for NPC, but radiotherapy, chemotherapy or radiotherapy and chemotherapy combined treatment is not satisfactory for NPC. With the development of molecular biology and immunology techniques, the molecular targeted therapy of NPC has become a hot research in the field.The molecular targeted therapy is a emerging model of Cancer Biotherapy.It is showing a better therapeutic effect and lower toxicity as compared with the conventional treatment methods and the first condition is to find an important target for the development and tumorigenesis.c-Met is the product of proto-met and belongs to the growth factor receptor family with tyrosine kinase activity. The expression of c-Met was found higher in various cancer than in normal tissue. Hepatocyte growth factor (HGF) and its receptor c-Met play an important role in a series of complex intracellular pathways by specifically binding with each other to regulate tumor invasion, metastasis, and angiogenesis and has been a novel target of tumor diagnosis and therapy. Currently, monoclonal antibodies as therapeutics have been used in patients and obtain good results, but it is restricted such as thehuman anti-mouse antibody (HAMA) and so promoted the study on humanized monoclonal antibody. For example the humanized monoclonal antibody can not only recognize the whole molecule of tumor cell surface antigens, and with the specific binding in vivo to prevent their binding to the ligand, And they are all in the Fc portion of the molecule can induce antibody-dependent cell-mediated cytotoxicity of (antibody-dependent cell-mediated cytotoxicity, ADCC)and apoptosis.We built a fully human Fab antibody library in early. In this present study, we prepare a whole humanied molecule anti-c-Met antibody with high neutralizing activity useing genetic-engineering technique and identify its mmunological activity and observe the biological characteristics of NPC, our study may provide a target therapy for NPC.Objective:1. Expression and purification of humanized whole molecule anti-c-Met antibodies and finish its identification.So we detect its’affinity.2. Deternin effect of humanized whole molecule anti-c-Met antibody on the proliferation, migration and invasion of NPC.Methods:1. Constructeukaryotic expression vector of recombinant human whole molecule anti-c-Met antibody:Amplificate the heavy and light chain variable region sequence humanized antibody anti-c-Met Fab antibody; Cloning the PCR products of the antibody variable region gene into the eukaryotic expression plasmid. After transformation of E. coli DH5a, positive clones were screened and identified by restriction enzyme digestion and identify the correct clone and sequenced to confirm the sequence.2. Obtained the humanized whole molecule anti-c-Met antibody:Transfected eukaryotic recombinant vector into 293 Freestyle (293F) cells and collected the cell culture supernatant6 days later. Obtained the purified antibody by the AKTA protein purification system and pre Hitrap Protein A column.3. Identified the Immunological Characteristics of the humanized whole molecule c-Met antibody:the immunological Characteristics of the humanized whole molecule c-Met antibody was confirmed by ELISA, Western blot and immunofluorescence.4. According to molecular interaction principle of BLItz system to analysis the results of affinity with BLItz Pro1.0 software.5. Cell viability of whole molecule anti-c-Met antibody on NPC were determined in triplicate via using a CCK-8 kit.6. Wound healing assay and transwell were used to determined the effect of the humanized whole molecule anti-c-Met antibody on the migration and invasion of NPC.Results:1. Amplified the 348bp length sequence of VH gene and the 324bp length sequence of VL gene. By sequence alignment, design of primers and restriction digestion of vectorlnfusion PCR obtained by c-Met light and heavy chain recombinant vector and transformed into E. coli DH5a after sequencing correct.2. Obtained the whole humanized anti-c-Met antibody molecule using the 293 Free style Expression System and the yield was about 2mg/100ml, SDS-PAGE results identified the high antibody purity. The heavy and light chains wereabout 55kD and 25kD respectively.3. ELISA showed that the full humanized anti-c-Met antibodies canspecifically binded tothe recombinant c-Met protein with good dose-dependent effect; Western blotresults showed that the humanized whole molecule anti-c-Met antibody can bind with the positive PCN cell; Immunofluorescence showed that the whole humanized anti-c-Met antibody can recognized the c-Met protein in NPC cell surface.4. BLItz Pro1.0 Software Analysis:KD is 5.56×10-10M5. CCK-8 results showed that the proliferation of high c-Met expression NPC cell was significantly higher than the negative cell with significantly statistically difference (P<0.05).6. Wound healing assay showed that NPC positive cells with higher healing rate compared to the NPC negtive cells(P<0.05); The migration and invasion assays showed thataverage penetratedcell number was morein NPC positive cells compared with the control group (P<0.05), but there was not statistically significant in the NPC negative cell (P>0.05).Conclusion:1. This study successfully constructed humanized whole molecule anti-c-Met antibodies of the recombinant expression vector, have succeeded in obtaining purified source whole molecule anti-c-Met antibody that specifically recognizes a cell surface nasopharyngeal c-Met protein. The antibody has a higher affinity.2. Humanied whole molecule anti-c-Met antibodies nasopharyngeal carcinoma cell proliferation, migration and invasion of a certain extent. |
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