Development of anti c-kit monoclonal antibody probe for SPECT imaging of gastrointestinal stromal tumor

Aim: Gastrointestinal stromal tumor (GIST) is a most common mesenchymal tumor arising from the human gastrointestinal tract. Overexpression of c-kit, encoding type III receptor tyrosine kinase, and its functional mutation is present in most GIST. 18F-fluoro-deoxyglucose (FDG) has been reported to highly accumulate in GISTs, and positron emission tomography using FDG (FDG-PET) is very useful for tumor detection and also for the assessment of early response to treatments using Imatinib (Gleevec). However, FDG-PET, in some cases, fails to detect relapse of GIST at early time point. If we could develop a specific method to detect c-kit expression, it can be used for the evaluation of the early response and relapse of GISTs. In this study, we labeled anti-c-kit antibodies with 125I and 111In, and assessed its in vitro and in vivo characteristics.Materials & methods: We constructed an expressing vector of mutated c-kit and transfected it to HEK293 human embryonic kidney cells and isolated several stable clones. To select highly expressing cell clones, we determined c-kit expression using western blot analysis and immunofluorescent staining. The cell suspension (1x107) was mixed with Matrigel, and injected subcutaneously in BALB/c-nu/nu male mice. We radiolabeled two anti-c-kit murine monoclonal antibodies (Mab1 and Mab2) with 125I using the chloramine-T or 3\u27-lodohippuryl-Ne-maleoyl-L-lysine (HML), and 111In using the 2-(p-isothiocyanatobenzyl) cyclohexyl-diethyl-enetriaminepentaacetic acid (CHX-A"-DTPA). We performed cell binding, competitive inhibition and internalization assays in vitro, and biodistribution and SPECT imaging in tumor-bearing mice.Results: We isolated 18 c-kit stably expressing cell clones, and determined 6 of them highly express c-kit using western blotting analysis. Immunofluorescent staining of c-kit highly expressing cells showed that c-kit is primarily localized on the cell membrane like the original GIST cell line, and developed tumors in mice. 125I-MAb1, 125I-MAb1(HML), 125I-Mab2, 111In-MAb1 and 111In-MAb2 specifically bound to c-kit expressing cells with affinity constants of 4.3x108M-1, 5.2x108M-1, 3.5x108M-1, 3.4x108M-1 and 3.9x108M-1, respectively. These antibodies were internalized, and 111In-labeled Mab1 highly accumulated in xenografted tumors, which were visualized by SPECT.Conclusion: These results showed that the radiolabeled c-kit antibody specifically accumulated in xenografted tumors and would be useful for the PET/SPECT imaging of GIST.Annual Congress of the European Association of Nuclear Medicine 200