| Lung cancer is a common kind of malignancy and non-small cell lung cancer accounts for 80% of lung cancers. Currently, the incidence of lung cancer has showed an increasing trend year by year, and most of lung cancer patients have reached advanced stage before diagnosis, so it is a very serious threat to human health. Because diagnosis of early lung cancer is not easy and the effective rates of main treatments such as surgery, chemotherapy and external radiotherapy for lung cancer are not very ideal with many adverse reactions, there is an urgent need to research new and effective ways for the diagnosis and treatment of lung cancer. At present, the radioimmunoimaging and radioimmunotherapy are the hot spots of many scholars at home and abroad. The specific antibodies are labeled with radionuclide, which can be oriented and introduced into the body by antigen-antibody immune response mechanism. On one hand, the external radiation detection instrument can be used to diagnose the disease. On the other hand, radionuclide can release radiation to destroy the surrounding lesions and play the role of therapy. The radioimmunoimaging and radioimmunotherapy have been a very important direction in the field of biological therapy, and have become more and more important in the early diagnosis, correct staging and effective treatment of tumor.For the radioimmunoimaging and radioimmunotherapy, it is a key to choose a good target and prepare specific antibodies. Hypoxia inducible factor 1 is a important transcription factor in tumor hypoxia, and HIF-1αis the primary functional subunit. It is confirmed by many studies that HIF-lαhas high expression in non-small cell lung cancer, and is closely related to the growth, invasion and metastasis of tumor. It may be new and effective target for the diagnosis and treatment of lung cancer. In the choice of antibodies, after abandoning the human anti-mouse antibody response of monoclonal antibody, phage single chain antibody is a completed human antibody. Meanwhile, compared with monoclonal antibody, it has greater advantages, such as relatively small molecular weight, good penetration, low immunogenicity and easy removing, and is expected to be more widespread in clinical application.In order to find new and effective diagnosis and treatment of lung cancer, we attempted to screen out lung adenocarcinoma human single-chain antibody of specific anti-HIF-1αfrom mass phage antibody library, which is labeled by radionuclides before the radioimmunoimaging in the lung adenocarcinoma of nude mice. Objective: To prepare anti-HIF-1αlung adenocarcinoma human single chain antibody by phage antibody library and study radioimmunoimaging in the lung adenocarcinoma of nude mice, to provide a new and effective method for the immune imaging and treatment of lung cancerMethods and Results:Part I Preparation of anti-HIF-1αlung adenocarcinoma human single-chain antibody1. Screening of lung adenocarcinoma A549 cells and HIF-1αon phage antibody library: After normal bronchial epithelial cells HBE16 adsorption, lung cancer A549 cells and HIF-1αwere taken as the antigens on the phage antibody library to carry out four rounds of "adsorption - elution - amplification" enrichment screening. And then, recovery rate of phage was increased gradually, the final round of the output about 5.9×10~5 cfu/ml enriched the phage antibody.2. Identification of phage antibody by ELISA and immunocytochemistry: Single colony was picked out to prepare phage antibody. In ELISA detection, 5 of 10 had the positive HIF-1αantigen reaction with the positive rate of 50%. Immunocytochemical identification showed that the phage antibody screened obviously stained the lung adenocarcinoma A549 cells, which confirmed that this antibody would specifically combine with lung adenocarcinoma A549 cells, but not with breast cancer MDA-MB-435s cells. 3. Expression and purification of soluble anti-HIF-1αsingle chain antibody: After ELISA identification, the strong positive recombinant phage infected E.coli HB2151, and expressed soluble scFv by IPTG induction. The bacteria were identified out soluble scFv expression by SDS-PAGE and were purified by HiTrapTM Anti-E Tag. Purified soluble scFv was identified by Western-blot, and was further confirmed the success of soluble scFv expression and purification. After the determination, relative molecular weight of soluble scFv was about 30kDa.4. Identification of immune activity of soluble scFv by ELISA: ELISA showed that D(450) of lung adenocarcinoma A549 cells was 0.61±0.08, D(450) of breast cancer MDA-MB-435s cells was 0.30±0.06, and D(450) of normal bronchial epithelial HBE16 cells was 0.29±0.07. It was confirmed that soluble scFv had a higher immune activity and could combine with the lung cancer A549 cells, but not with the breast cancer MDA-MB-435s cells and normal bronchial epithelial HBE16 cells. Competitive inhibition ELISA showed that soluble scFv brought free HIF-1αwith inhibitory effect, which was related to the dose-dependent of free HIF-1α, and thus confirmed that soluble scFv had higher combination activity with HIF-1α.Part II Radioimmunoimaging and image fusion of 131I-soluble scFv in human lung adenocarcinoma in nude mice1. Radionuclides labeling purification and identification of soluble scFv anti-HIF-1α: The chloramine T method was used to carry out 131I labeling of prepared anti-HIF-1αsoluble scFv, and then the labled fluid was purified through Sephadex G200 column. The labeling yield of 131I-labeled soluble scFv by trichloroacetic acid precipitation method was 79.6±4.9%. The results of paper chromatography analysis showed that radiochemical purity was 94.4±4.4%, and specific activity was 3.2±0.2MBq/μg. In each time point during setting aside at room temperature for 24h and incubating in fresh human serum for 48h,the determined radiochemical purities were all above 90%.2. Radioimmunoimaging and image fusion: After the establishment of model of human lung adenocarcinoma in nude mice, injection of 131I-soluble scFv, SPECT planar static imaging and ROI measurement of T/NT (radioactive count ratio of tumor and non-tumor area), the results showed that there was selective uptake of radioactivity in nude mice tumor area, and radioactivity reached the peak in 72h with T/NT of up to 3.19. 72h SPECT/CT fusion image showed directly the clear image of tumor area.Conclusion: Anti-HIF-1αlung adenocarcinoma human single chain antibody has been successfully prepared by phage antibody library, 131I labeled the scFv could make the tumor area clearly visualized in RII and fusion image of nude mice xenografted with A549 cells tumor to lay the foundation for the early immune imaging and treatment of lung cancer.
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