Studies On The Functional Monoclonal Antibody Against Lung Cancer And Its Target Antigen

Lung cancer is the most common malignant tumor, its incidence rate and mortality rate account for the first of all malignant cases in the worldwide. Every year nearly60million people died of lung cancer in China. Unfortunately, the present three main types of cancer treatment have only limited effect on reducing lung cancer mortality. Molecular target therapy has been considered as a promising method for treating cancer, because of low-toxic side effects. The prerequisite and key factor for targeting therapy is to identify the tumor specific molecules or genes. Therefore, based on successfully obtaining the22monoclonal antibodies against lung cancer, we further screen and identify functional monoclonal antibodies, which can inhibit the malignant biological behavior in vitro and effectively suppress tumor growth in vivo. Then, we applied mass spectrometry to identify functional target antigen gene, which might provide valuable functional monoclonal antibody and their targets.Firstly, we selected10monoclonal antibodies from22lung cancer tissue specific monoclonal antibodies to study their biological properties. The expression and localization of targeted antigen of10mAb in3lung cancer cell lines were examined by fixed and viable immunofluorescence assay. The results showed that the antigens recognized by9monoclonal antibodies were expressed on the membrane, which might be preferred mAb for targeting therapy. The immunohistochemical analyses showed that4of9monoclonal antibodies could specifically react with human lung cancer tissues. Then.4monoclonal antibodies were further studied in a large number of lung cancer tissues. The result revealed that those4monoclonal antibodies could specifically react with lung cancer tissues with82.1-89.8%specificity and88.9-91.9%positive rate and display excellent targeting effect.Functional experiments indicated that40E11mAb is a functional monoclonal antibody, which could significantly inhibit proliferation of tumor cells in vitro, with inhibition rate being27.4%.24D4mAb could obviously inhibit invasion of tumor cells in vitro, with inhibition rate being40.0%. Moreover, we applied the co-inoculation model to test the inhibitory effect of40E11and24D4mAbs on tumor growth in nude mice. The results showed that40E11mAb could significantly suppress the xenograft tumor growth, and the tumor weight inhibition rate was about64.4%. However,24D4mAb had little effect on tumor growth. To further prove the efficacy of40E11targeted therapy for lung cancer, we purified40E11mAb and conducted antibody treatment experiment. The results indicated that40E11mAb has dose-related inhibitory effects on xenograft tumor growth, with inhibition rate being68.2%. Above results indicated that40E11mAb could be used as a good candidate monoclonal antibody for lung cancer target treatment.Then we applied the methods to identify the functional target antigen recognized by40E11. Firstly, we validated the molecular weight of target antigen were130KD,95KD and70KD by western blot. Peptides of this antigen were generated by in-gel tryptic digestion, and the mixture of peptides was analyzed by ion-trap electrospray mass spectrometry. The deduced sequences of these peptides confirmed the identity of this antigen as an H gene. Bioinformatic analysis of the functional role of gene H in lung cancer progression has not been reported in the worldwide. To confirm the result of mass spectrometry, we applied two color immunofluorescence and confocal microscopy to study the expression and localization of H gene. The results showed that the antigen recognized by40E11mAb and commercial antibody H were co-located on the cell membrane. Co-immunoprecipitation experiment also verified that the antigen gene recognized by the40E11mAb and commercial antibody H was the same. Then, we synthesized two small interfering RNA (siRNA) based on the H sequences to down-regulate the expression of gene H. Then the expression of H gene was tested by immunofluorescence by using40E11mAb and commercial antibody H. The result demonstrated that the expression of H gene was consistently decreased in siRNA-H-cells.In order to study the function of the H gene, we applied transient RNAi to knock down the gene H in lung cancer cells and detect the effect of down-regulation of H on lung cancer cell characteristic including proliferation, adhesion, invasion, migration, and other functions. The result revealed that downregulation of gene H in lung cancer cells leaded to growing at a rate57.4%slower than control cells. Knockdown of H also decreased adhesion with matrix matrigel, with inhibitory rate being20.1%. However, downregulation of H did not affect cell invasion and migration. To further study the mechanism of H gene on lung cancer cell proliferation, we assayed the cell cycle after RNAi and revealed that H gene knockdown led to G2arrest.Together, these results not only provide valuable candidate antibody drug, but also provide potentially useful molecular target for lung cancer targeted therapy.