Evaluation Of MICA As A Potential Target For Ovarian Cancer Imaging

MICA is a member of non-classical HLA-I gene family located in the MHC-I region on No. 6 chromosome, which was found by Tom Spies et al. in 1994. The gene is polymorphic. MICA protein is a cellular stress molecule, which is expressed little on the surface of normal tissue, while some amount on the cellular surface of the gastrointestinal tract. Its expression was also documented on the surface of many kinds of tumor cells, especially the epithelial tumor cells, such as in many carcinomas of the lung, breast, kidney, ovary, prostate and colon. By interacting with the NKG2D receptor on the surface of NK cell,γδT cell and CD8⁺ T cell, MICA can be recognized by those effect cells, resulting activation of the killing function of those cells.In the previous work, we obtained recombinant MICAα1-2, and the corresponding monoclonal antibody (mAb) 7B6, 10C6 and 12B7. Those mAbs could combine with native MICA on the cells and tissues specifically. Based on those results, we studied whether MICA could be recarded as a kind of TAA, targeted by its MAbs in vivo. Radionuclides ^99mTc and ¹³¹I were selected as labels. Our work provided the possibility of MICA as a target for tumor imaging and therapy on the early stage of disease. Furthermore, the corresponding anti-MICA mAbs 10C6 and 7B12 had the potential application in clinic. This paper included three parts:1. The study of ^99mTc-mAb 10C6 for the diagnosis in nude mice bearing MICA-positive tumorThe affinities of 7B6, 10C6 and 12B7 were determined by ELISA. 10C6, which was with the highest affinity, was labeled with ^99mTc by the direct labeling method. The biodistribution and the uptake of tumor tissue were detected. Gamma camera was used for the whole body image. At the same time, the clearance of the radiolabeled mAb in the whole body and the blood was detected at the appointed time. Furthermore, mAb 10C6 was labeled with ¹³¹I by NBS, in order to compare the imaging effect of different radionuclides. The results showed that MICA could be regarded as a TAA, which might be used as a target for tumor imaging and therapy at early stage. And for its lower energy and relatively pureγradiation, ^99mTc was considered as a more suitable imaging reagent for tumor in short term.2. The selection of anti-MICA mAb with high affinityrMICAα1-2 was expressed and purified. Then a series specific anti-MICA mAbs were obtained as follows: After immunized for 4 times with purified rMICAal-2, the mice were sacrificed and spleen cells were isolated in sterile condition. The spleen cells were hybridized with mouse myeloma SP2/0 cells through routine cell hybridization method. Positive clones were screened by conditional cultivation with HAT. Finally, hybridized myeloma cell clone 7B12 excreting monoclonal antibody with high affinity was selected by indirect ELISA, Western blot and flow cytometry. And its K value was obatianed by noncompetitive ELISA method.3. The study of ^99mTc-mAb 7B12 Fab' fragment for the diagnosis in nude mice bearing MICA-positive tumorThe F(ab')₂ of IgG 7B12, which was obtained by enzyme-digestion with pepsin, was labeled with ^99mTc directly. The biodistribution and the uptake of tumor tissue were detected. Gamma camera was used for the whole body image. At the same time, the clearance of the radiolabeled mAb fragment in the whole body and the blood was detected at the appointed time. The results showed that ^99mTc-Fab' had lower tumor targeting than the whole IgG, but had a better blood clearance, which might be related with its lower avidity, biochemical character and the labeling procedure with ^99mTc.In this paper, innovative ideas included the following points: primarily, as a TAA, MICA was firstly proposed as a target for tumor imaging and therapy at early stage of diseases. Anti-MICA mAbs 10C6 and 7B12 have the potential application in clinic. And we provided a procedure of selecting mAb with high affinity in a relative shortterm.