Fazekas-Singer Judit, Berroterán-Infante Neydher, Rami-Mark Christina, Dumanic Monika, Matz Miroslawa, Willmann Michael, Andreae Fritz, Singer Josef, Wadsak Wolfgang, Mitterhauser Markus, Jensen-Jarolim Erika
Comparative Medicine, The Interuniversity Messerli Research Institute of the University of Veterinary Medicine Vienna, Medical University Vienna, and University of Vienna, Vienna, Austria.
Institute of Pathophysiology and Allergy Research, Center of Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.
Oncotarget. 2017 Sep 15;8(47):83128-83141. doi: 10.18632/oncotarget.20914. eCollection 2017 Oct 10.
Due to large homology of human and canine EGFR, dogs suffering from spontaneous EGFR+ cancer can be considered as ideal translational models. Thereby, novel immunotherapeutic compounds can be developed for both human and veterinary patients. This study describes the radiolabeling of a canine anti-EGFR IgG antibody (can225IgG) with potential diagnostic and therapeutic value in comparative clinical settings. Can225IgG was functionalized with DTPA for subsequent chelation with the radionuclide Tc. Successful coupling of 10 DTPA molecules per antibody on average was proven by significant mass increase in MALDI-TOF spectroscopy, gel electrophoresis and immunoblots. Following functionalization and radiolabeling, Tc-DTPA-can225IgG fully retained its binding capacity towards human and canine EGFR in flow cytometry, immuno- and radioblots, and autoradiography. The affinity of radiolabeled can225IgG was determined to K 0.8 ±0.0031 nM in a real-time kinetics assay on canine carcinoma cells by a competition binding technique. Stability tests of the radiolabeled compound identified TRIS buffered saline as the ideal formulation for short-term storage with 87.11 ±6.04% intact compound being still detected 60 minutes post radiolabeling. High stability, specificity and EGFR binding affinity pinpoint towards Tc-radiolabeled can225IgG antibody as an ideal lead compound for the first proof-of-concept diagnostic and therapeutic applications in canine cancer patients.
由于人类和犬类表皮生长因子受体（EGFR）具有高度同源性，患有自发性EGFR阳性癌症的犬类可被视为理想的转化模型。因此，可以为人类和兽医患者开发新型免疫治疗化合物。本研究描述了一种具有潜在诊断和治疗价值的犬抗EGFR IgG抗体（can225IgG）在比较临床环境中的放射性标记。Can225IgG用二乙三胺五乙酸（DTPA）进行功能化，以便随后与放射性核素锝（Tc）螯合。通过基质辅助激光解吸电离飞行时间质谱（MALDI-TOF）光谱、凝胶电泳和免疫印迹中显著的质量增加，证明平均每个抗体成功偶联了10个DTPA分子。在功能化和放射性标记后，Tc-DTPA-can225IgG在流式细胞术、免疫印迹和放射自显影中完全保留了其对人类和犬类EGFR的结合能力。通过竞争结合技术在犬癌细胞的实时动力学分析中，确定放射性标记的can225IgG的亲和力为K 0.8±0.0031 nM。放射性标记化合物的稳定性测试确定，三羟甲基氨基甲烷（TRIS）缓冲盐水是短期储存的理想制剂，放射性标记后60分钟仍能检测到87.11±6.04%的完整化合物。高稳定性、特异性和EGFR结合亲和力表明，Tc放射性标记的can225IgG抗体是犬类癌症患者首个概念验证诊断和治疗应用的理想先导化合物。
