The Screening And Identification Of Tumor Associated Antigens In Lung Cancer

Background and ObjectiveAlthough there has been a long time to search for tumor antigens, major progress has only recently been made in the molecular definition of human tumor antigens. In 1995, Sahin developed an approach that can be used to identify tumor antigens in human cancers. This approach is designed as SEREX (Serological analysis of recombinant cDNA expression libraries), which defines antigens by the reaction with autologous or xenogenic patient sera. Many antigens were defined by this method. SEREX makes a great progress in defining human tumor associated antigens.Lung cancer is one of the most common malignancies, both the incidence rate and fatality rate of lung cancer are the first among various kinds of malignancies. The majority of lung cancers remain clinically undetectable until patients have developed to the advanced - stage disease, and the five-year survival rate of lung cancer only accounts for less than 15%. The major reason for such a high mortality rate is that lung cancer is difficult to be early diagnosed and to further to be treated at its earlier period, and the prognosis of lung cancer is very poor. Therefore, it is crucial to search for the tumor associated antigens for early diagnosis and immunotherapy of lung cancer. It is rarely reported about the screening and identification of lung cancer antigen using SEREX in the world. SEREX was applied to screen the phage cDNA expression library of lung cancer with sera of lung cancer patients in our study. Moreover, some clones encoding lung cancer associated antigens identified in this study were evaluated for the frequency of IgG antibody responses in xenogenic sera. We hope that the identified antigens can not only provide new markers for the early diagnosis and prognosis of lung cancer, but also make foundation for the advanced development of lung cancer vaccine and the monitoring of immune effects. And this will contribute to clarify the molecular mechanism of lung cancer.MethodsPart one: the screening of tumor associated antigens in lung cancer with patients' sera1. E.coli transfected with recombinant cDNA phages were plated onto LB-agar plates. Plaques were transferred onto the nitrocellulose membranes, and expression of recombinant proteins was induced by IPTG. The membranes were blocked with 3% non-fat milk and incubated with 1:100 diluted patients' sera, which had been preabsorbed with E.coli transfected with wild phage. And then they were incubated with a 1:5000 dilution of the horseradish peroxidase (HRP)-labeled antibody specific for human IgG. Reactive clones were visualized by staining with DAB; these clones were re-screened three times until to obtain 100% purity.2. Positive clone plaques were picked out, stored in SM buffer at 4℃. All of these positive clones were rescured into pBluescript phagemids forms by in vivo excision.3. The plasmids were extracted and the size of the inserted cDNA was determined primarily by double restriction enzyme digestion with EcoR I and Xho I .4. The positive clones were sequenced with T3, T7 primers, and the analysis of the cDNA sequences was performed by using Genbank database and BLAST program to search for homologs.Part two: initial sero-reaction analysis of lung cancer associated antigens having been screened1.E.coli transfected with positive recombinant phages which were obtained from the three-round screening of cDNA expression library were plated onto LB-agar plates. Plaques were transferred onto the nitrocellulose membranes, and expression of recombinant proteins were induced by IPTG. The membranes were blocked with 3% non-fat milk and cut into 100 pieces, the size of which is 1cm×2cm, then incubated with 50 sera examples from patients with lung cancer and 50 normal human sera samples, which had been pre-absorbed with the lysate of E.coli. And then they were incubated with a 1:5000 dilution of the horseradish peroxidase (HRP)-labeled antibody specific for human IgG. The nitrocellulose membrane of positive reaction was visualized by staining with DAB.2. Statistical analysis: To compare the positive rate of every clone between tumor patients' and normal human sera samples. The positive rate of clone which is higher in tumor patients' sera samples than in normal human sera samples is considered as positive reaction clone. The sensitivity and specificity of the positive reaction clone were evaluated. SPSS11.0 was used to analyze all data. In the statistics analysis, x~2 test and exact propability of fourfold table were used.Results1. Three rounds of serological screening of 4×10~5 pfu from primary library with five lung cancer patients' sera yielded 70 positive cDNA clones, the size of cDNA inserts were in the range of about 300bp～2800bp and the average length of the cDNA inserts was about 1.1kb. These positive clones present 63 different genes, including 26 genes whose biological function have not been reported yet; 2 insert fragments which have no obvious homolog in GeneBank indicate that they are probably novel genes; 35 genes whose biological function have been reported, one of which was reported to be closely related to the occurrence and development of lung cancer, 34 of which were reported relating to differentiation, metabolism, signal transduction of cells and also relating to occurrence and development of other tumors.2. Five clones were elected from the 35 genes whose biological function have been reported, and IgG immune reactions which were mediated by the 5 clones were detected and analyzed with 50 lung cancer sera and 50 normal human sera. The positive rate of IgG antibody of 3 clones in lung cancer patients was much higher than that in normal donors.3. When combinational analysis of 3 antigen-antibody systems was performed, the sensitivity increased obviously with invariable specificity on the whole, suggesting there have a higher clinical diagnostic value.ConclusionsThis study demonstrated that the identification of lung cancer associated antigens, and subsequent serological analysis of antibodies against these antigens might contribute to the understanding of pathogenesis in lung cancer, and could provide candidate markers in early diagnosis,combinational detection and immunotherapy of lung cancer, and also highlight a new direction of early diagnosis of lung cancer.