| γδT lymphocyte belong to a small subpopulation of T lymphocytes which mainly locate in the skin, intestine, lung and other mucosal tissues. γδT cells are involved in immune regulation by recognizing and killing infected cells and abnormal cells, therefore leading to the enhancement of non-specific immune defense. Numerous studies demonstrate that γδT cell receptor(TCRγδ) from tumor-infiltrating y8T lymphocytes are able to specifically recognize the tumor antigens, suggesting that γδT lymphocytes play an important role in tumor immunity. In our previous studies, we focus on the binding function of epitope in CDR3δ, and constructed a humanized CDR3δ-grafted antibody, so as to provide γδTCR-contained targeted antibodies for development and application of tumor therapeutic antibodies. We identified a predominant complementary determining region3(CDR3) of δ1chain in γδT lymphocytes from three patients with gastric cancer and constructed a humanized CDR3δ1-grafted antibody named TCRγ4δ1-Fc (GTM-Fc) in which the CDR3of TCRδ1was replaced by GTM using gene recombination technology. In addition, we also obtained a humanized CDR3δ2-grafted antibody which named TCRy9δ2-Fc (OT3-Fc) according to a predominant TCR82CDR3chain of γδT lymphocytes from patients with epithelial ovarian cancers.Our previous studies showed that OT3-Fc could promote the cytotoxicity of peripheral blood mononuclear cells (PBMCs) to ovarian cancer cells AKOV3and ES-2which was also confirmed in tumor-bearing nude mice experiments. Furthermore, OT3-Fc could bind to ovarian carcinoma cells and a variety of other cancer cell lines, suggesting OT3-Fc possesses a broad spectrum in recognizing tumors. Dr. Yan Jiang also proved that synthetic peptides GTM-Fc could bind to different cancer cell lines, such as GC-823, G401, GLC-82, HT-29and SKOV3cancer cells, and some cancer tissues such as gastric cancer, kidney cancer, lung cancer, colorectal cancer and ovarian cancer. Therefore, we proposed that GTM-Fc might be a potential candidate for cancer therapy like OT3-Fc.Liver cancer is one of the most common malignancy and high mortality diseases. Current treatments for liver cancer include surgery, chemotherapy, radiotherapy and biological therapy and etc. The biologic therapy has been playing more and more important roles in facilitating the treatment of liver cancer as a new therapeutic approach.Antibody therapy is divided into polyclonal antibody therapy and monoclonal antibody therapy. Because of the poor specificity and severe side effects, polyclonal antibody therapy has rarely been used in clinic. The monoclonal antibody plays an important role in the clinical cancer therapy because of their characteristic with high purity, high titer, specificity and less cross-reactivity, etc. In the past twenty years, therapeutic monoclonal antibody drugs approved for use in clinic have been up to dozens, but monoclonal antibody drugs for the treatment of liver cancer are still very little, which include anti-alpha fetal protein (AFP) antibodies, bevacizumab antibodies and anti-ferritin antibodies. Exploring more antibody drugs for the treatments of liver cancer is very important for clinical therapy.This study includes two parts. The first part is designed to study the possibility of GTM-Fc and OT3-Fc to treat liver cancer as monoclonal antibody drugs. First, we detected the binding capacities of GTM-Fc and OT3-Fc antibodies to three kinds of hepatoma cells HepG2, Hep3b and HepG2.2.15by flow cytometry (FCM). The results showed that GTM-Fc combined to three types of hepatoma cells with high levels, whereas OT3-Fc displayed low level binding activity to two hepatoma cells except for HepG2. Thus, We selected GTM-Fc as a target antibody for the following studies.After confirming the binding ability of GTM-Fc with hepatoma cells, we investigated the roles of GTM-Fc infacilitating PBMC to kill liver cancer cells through antibody-dependent cell-mediated cytotoxicity (ADCC) in vitro and the anti-tumor effect in bearing HepG2.2.15-tumor nude mice in vivo. The results showed that GTM-Fc could promote PBMC to kill three hepatoma cells in vitro and enhance the anti-tumor effects of PBMC in bearing HepG2.2.15-tumor nude mice in vivo. GTM-Fc could thus participate in the treatments of liver cancer as candidate therapeutic monoclonal antibody applied to the biotherapy in liver cancer.Autoimmune disease (AID) is one type of diseases that have damage to body caused by the disorder of autoimmune, which include systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), hyperthyroidism and chronic lymphocytic thyroiditis (CLT) and etc.In our early studies, we identified a ligand binding protein CCT5from the HK-2cells in human kidney which could be recognized and bound by TCR82CDR3sequences which was predominately expressed in SLE patients. CCT5(Chaperonin containing T-complex polypeptide-1, zeta) belongs to chaperon family and was confirmed as one of ligands of γδTCR. Some researches demonstrate that the abnormal number and the expression of γδT lymphocytes clone could present in some AIDs, indicating that γδT lymphocyte may be involved in the occurrence and development of AIDs. The expression of CCT5, as a candidate ligand molecule for γδTCR, may be altered in AIDs. To investigate the change of CCT5in the AIDs would help us to understand the role of γδT lymphocytes in AIDs and may provide a new target for the diagnosis or treatment of AIDs in clinic.Therefore, in the second part of this research, we detected the expression levels of CCT5in SLE patients and RA patients as well as healty controls. Enzyme-linked immune sorbent assay (ELISA) was performed to detect the levels of CCT5in healthy people, RA patients and SLE patients. The results showed that there was significant difference in the positive rates and levels of CCT5between healthy controls, SLE patients and RA patients. The CCT5’s positive rate in healthy people was43.75%significant higher than those of SLE patients and RA patients. The positive rates of CCT in SLE patients and RA patients were less than20%. It indicates that CCT5may be useful as a molecular biomarker for the diagnosis of SLE and RA disease.In summary, we have got the main conclusions for this research:1. The humanized antibody GTM-Fc could bind to three hepatoma cell lines HepG2, Hep3b and HepG2.2.15in vitro, and displayed stronger binding ability than OT3-Fc. GTM-Fc could promote the cytotoxicity of PBMC to three hepatoma cells in vitro. In tumor-bearing Balb/c nude mice, GTM-Fc enhanced the PBMC-mediated inhibition of HepG2.2.15liver cancer cells in vivo, suggesting that GTM-Fc monoclonal antibody may be a therapeutic candidate for the treatment of liver cancer.2. The positive rate and the level of CCT5in healthy people was significant higher than those of SLE patients or RA patients, while there was no significant difference between SLE patients and RA patients. It indicates that CCT5may be useful as a molecular biomarker for the diagnosis of SLE and RA disease. |
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