Chongjiao Li1, Yongxue Zhang2, Lifei Wang3, Hongyan Feng2, Xiaotian Xia2, Juan Ma3, Hui Yuan2, Bin Gao4, Xiaoli Lan5. 1. Department of Nuclear Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Hubei Province Key Laboratory of Molecular Imaging, Wuhan, PR China; Department of Nuclear Medicine, Zhongnan Hospital of Wuhan University, Wuhan, PR China. 2. Department of Nuclear Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Hubei Province Key Laboratory of Molecular Imaging, Wuhan, PR China. 3. CAS Key Laboratory of Pathogenic Microbiology and Immunology (CASPMI), Centre for Molecular Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, PR China; College of Life Sciences, University of Science and Technology of China, Hefei 230026, PR China. 4. CAS Key Laboratory of Pathogenic Microbiology and Immunology (CASPMI), Centre for Molecular Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, PR China; College of Life Sciences, University of Science and Technology of China, Hefei 230026, PR China; China-Japan Joint Laboratory of Molecular Immunology and Microbiology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, PR China. Electronic address: gaob@im.ac.cn. 5. Department of Nuclear Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Hubei Province Key Laboratory of Molecular Imaging, Wuhan, PR China. Electronic address: LXL730724@hotmail.com.
Abstract
INTRODUCTION: The C4b binding protein (C4bp) α/β-chain C-terminal effectively induces polymerization during protein synthesis. Using this fragment and the single-domain antibody EG2, which targets the epidermal growth factor receptor (EGFR), we generated the novel multimeric antibody EG2-C4bpα. We radiolabeled EG2-C4bpα with (99m)Tc and evaluated its targeting efficiency and pharmacokinetics in tumor xenografts. METHODS: EGFR expression and EGFR-EG2-C4bpα binding was evaluated in A431 and OCM-1 cells by Western blotting and flow cytometry, respectively. EG2-C4bpα was radiolabeled with [(99m)Tc(CO)3(OH2)3](+) using a tricarbonyl vial followed by purification on a PD-10 column. In vitro studies with (99m)Tc-EG2-C4bpα were performed in A431 and/or OCM-1 cells. Single photon emission computed tomography (SPECT) imaging and biodistribution studies were carried out in (99m)Tc-EG2-C4bpα-injected mice bearing A431- and OCM-1-derived tumors. EGFR immunofluorescent staining in A431 and OCM-1 tumors was performed. RESULTS: A431 cells showed higher EGFR expression levels than OCM-1 cells, and flow cytometry confirmed EG2-C4bpα bound more A431 cells than OCM-1 cells. (99m)Tc-EG2-C4bpα was successfully prepared with radiochemical yields of 30.3-50.4%. The binding affinity of (99m)Tc-EG2-C4bpα to A431 cells was approximately 20 nM. (99m)Tc-EG2-C4bpα specifically bound A431 cells and this binding was blocked by 41% in the presence of 50 nM excess unlabeled EG2-C4bpα. In vivo radioactivity uptake in A431 tumors was detected 2h after (99m)Tc-EG2-C4bpα administration and sustained up to 18h. The highest ratio of A431 tumor-to-muscle and tumor-to-blood was 3.69 ± 0.48 at 10h and 0.77 ± 0.14 at 20 h, respectively. Excess unlabeled EG2-C4bpα blocked radioactivity uptake in A431 tumors by 55% at 10h. (99m)Tc-EG2-C4bpα was barely detectable in OCM-1 tumors, and biodistribution analysis confirmed that radioactivity uptake was significantly lower than in A431 tumors. CONCLUSIONS: (99m)Tc-EG2-C4bpα specifically and efficiently targets EGFR over-expressing tumors suggesting that EG2-C4bpα may be a promising antibody alternative for future diagnostic application and potential radioimmunotherapy. However, the high activity in the blood and liver, and the relative low ratio of tumor-to-blood should be noticed and improved.
INTRODUCTION: The C4b binding protein (C4bp) α/β-chain C-terminal effectively induces polymerization during protein synthesis. Using this fragment and the single-domain antibody EG2, which targets the epidermal growth factor receptor (EGFR), we generated the novel multimeric antibody EG2-C4bpα. We radiolabeled EG2-C4bpα with (99m)Tc and evaluated its targeting efficiency and pharmacokinetics in tumor xenografts. METHODS: EGFR expression and EGFR-EG2-C4bpα binding was evaluated in A431 and OCM-1 cells by Western blotting and flow cytometry, respectively. EG2-C4bpα was radiolabeled with [(99m)Tc(CO)3(OH2)3](+) using a tricarbonyl vial followed by purification on a PD-10 column. In vitro studies with (99m)Tc-EG2-C4bpα were performed in A431 and/or OCM-1 cells. Single photon emission computed tomography (SPECT) imaging and biodistribution studies were carried out in (99m)Tc-EG2-C4bpα-injected mice bearing A431- and OCM-1-derived tumors. EGFR immunofluorescent staining in A431 and OCM-1 tumors was performed. RESULTS: A431 cells showed higher EGFR expression levels than OCM-1 cells, and flow cytometry confirmed EG2-C4bpα bound more A431 cells than OCM-1 cells. (99m)Tc-EG2-C4bpα was successfully prepared with radiochemical yields of 30.3-50.4%. The binding affinity of (99m)Tc-EG2-C4bpα to A431 cells was approximately 20 nM. (99m)Tc-EG2-C4bpα specifically bound A431 cells and this binding was blocked by 41% in the presence of 50 nM excess unlabeled EG2-C4bpα. In vivo radioactivity uptake in A431 tumors was detected 2h after (99m)Tc-EG2-C4bpα administration and sustained up to 18h. The highest ratio of A431 tumor-to-muscle and tumor-to-blood was 3.69 ± 0.48 at 10h and 0.77 ± 0.14 at 20 h, respectively. Excess unlabeled EG2-C4bpα blocked radioactivity uptake in A431 tumors by 55% at 10h. (99m)Tc-EG2-C4bpα was barely detectable in OCM-1 tumors, and biodistribution analysis confirmed that radioactivity uptake was significantly lower than in A431 tumors. CONCLUSIONS: (99m)Tc-EG2-C4bpα specifically and efficiently targets EGFR over-expressing tumors suggesting that EG2-C4bpα may be a promising antibody alternative for future diagnostic application and potential radioimmunotherapy. However, the high activity in the blood and liver, and the relative low ratio of tumor-to-blood should be noticed and improved.
Authors: Nairo Brilhante-da-Silva; Rosa Maria de Oliveira Sousa; Andrelisse Arruda; Eliza Lima Dos Santos; Anna Carolina Machado Marinho; Rodrigo Guerino Stabeli; Carla Freire Celedonio Fernandes; Soraya Dos Santos Pereira Journal: Mol Diagn Ther Date: 2021-06-19 Impact factor: 4.074
Authors: Paolo Rainone; Benedetta Riva; Sara Belloli; Francesco Sudati; Marilena Ripamonti; Paolo Verderio; Miriam Colombo; Barbara Colzani; Maria Carla Gilardi; Rosa Maria Moresco; Davide Prosperi Journal: Int J Nanomedicine Date: 2017-05-02