title: A novel multivalent (99m)Tc-labeled EG2-C4bpα antibody for targeting the epidermal growth factor receptor in tumor xenografts.
abstract: BACKGROUND
  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.affiliation: 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.
authors.forename: Chongjiao
authors.initials: C
authors.lastname: Li
authors.affiliation: 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.
authors.forename: Yongxue
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: 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.
authors.forename: Lifei
authors.initials: L
authors.lastname: Wang
authors.affiliation: 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.
authors.forename: Hongyan
authors.initials: H
authors.lastname: Feng
authors.affiliation: 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.
authors.forename: Xiaotian
authors.initials: X
authors.lastname: Xia
authors.affiliation: 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.
authors.forename: Juan
authors.initials: J
authors.lastname: Ma
authors.affiliation: 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.
authors.forename: Hui
authors.initials: H
authors.lastname: Yuan
authors.affiliation: 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.
authors.forename: Bin
authors.initials: B
authors.lastname: Gao
authors.affiliation: 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.
authors.forename: Xiaoli
authors.initials: X
authors.lastname: Lan
mesh_terms: D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000494:pharmacology*; D015153:Blotting, Western; D002294:Carcinoma, Squamous Cell / Q000188:drug therapy* / Q000276:immunology / Q000378:metabolism; D066246:ErbB Receptors / Q000037:antagonists & inhibitors* / Q000276:immunology*; D006801:Humans; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D015609:Organotechnetium Compounds / Q000737:chemistry / Q000493:pharmacokinetics / Q000494:pharmacology*; D011854:Radiochemistry; D019275:Radiopharmaceuticals / Q000737:chemistry / Q000493:pharmacokinetics / Q000494:pharmacology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000494:pharmacology*; D014018:Tissue Distribution; D015899:Tomography, Emission-Computed, Single-Photon / Q000379:methods; D014407:Tumor Cells, Cultured; D023041:Xenograft Model Antitumor Assays
chemical_list: D000906:Antibodies; D015609:Organotechnetium Compounds; D019275:Radiopharmaceuticals; D011993:Recombinant Fusion Proteins; C512478:EGFR protein, human; D066246:ErbB Receptors
keywords: Biodistribution; C4b binding protein; Epidermal growth factor receptor; Multimerization; Single photon emission computed tomography; Single-domain antibody