Development of 90 Y-DOTA-nimotuzumab Fab fragment for radioimmunotherapy Luis M. Alonso Martı ´nez • Abmel Xiques Castillo • Victoria N. Calzada Falco ´n • Marylaine Pe ´rez-Malo Cruz • Rene ´ Leyva Montan ˜a • Minely Zamora Barrabı ´ • Ignacio Herna ´ndez Gonza ´lez • Mariela Leo ´n Pe ´rez • Alejandro Arbesu ´ Valdivia Received: 18 November 2013 / Published online: 24 August 2014 Ó Akade ´miai Kiado ´, Budapest, Hungary 2014 Abstract Yttrium-90-( 90 Y) labeled monoclonal antibod- ies prepared with a chelating agent, 1,4,7,10-tetra- azacyclododecane-1,4,7,10-tetraacetic acid (DOTA), have been used for radioimmunotherapy of cancer. In the present work, the Fab fragment of anti-EGFR monoclonal antibody nimotuzumab was prepared with high purity, integrity and biological activity. The Fab fragment with high specific recognition of EGFR in NCI-H125 human lung adenocar- cinoma cells was derivatized with DOTA-NHS applying a simple procedure. DOTA-nimotuzumab Fab fragment was successfully radiolabeled with 90 Y with high radiochemical yield. The in vitro stability of labeled product was optimal over 24 h in buffered solution at 37 °C. Biodistribution and pharmacokinetic studies correctly evaluated the in vivo non-tumor uptake, dosage regimen and excretion pathway in normal Wistar rats. Keywords 90 Y  Radioimmunotherapy  Nimotuzumab Fab fragment  DOTA  Radiopharmaceutical development Introduction Epidermal growth factor receptors (HER1/erbB1/EGFR) with tyrosine kinase activity are involved in transmission of signals controlling normal cell development, growth, dif- ferentiation, and survival. These receptors are found in various combinations in different tissues and have been thus identified as a promising target for several therapeutic drugs, such as EGFR directed tyrosine kinase inhibitors (TKIs) and anti-EGFR monoclonal antibodies (mAbs) [1]. Nimotuzumab is a genetically engineered humanized mAb (IgG 1 ) that recognizes an epitope located in the extracellular domain of human EGFR. Nimotuzumab blocks the binding of EGF, leads to the inhibition of cell proliferation and pro- apoptotic signals, and decreases VEGF production [2, 3]. Intact nimotuzumab labelled with 99m Tc has been exten- sively studied for immunoscintigraphy of nasopharyngeal-, head and neck-, esophageal-, breast-, prostate- and uterine cervical cancer [3]. Its 188 Re-labeled version has shown promising results for therapy in a Phase I trial for the radiation treatment of gliomas via an in-dwelling catheter [4]. Addi- tionally, there are a number of ongoing pre-clinical studies with its 90 Y- and 177 Lu-DOTA- radioimmunoconjugates [5–10]. IgG molecules are large proteins of approximate 150 kDa mass. Accordingly, intact IgG antibodies would be expected to have significantly slower kinetics of distri- bution and severely limited tissue penetration properties as L. M. Alonso Martı ´nez (&)  A. Xiques Castillo  M. Pe ´rez-Malo Cruz  R. Leyva Montan ˜a Deparment of Radiopharmacy, Center of Isotopes, Ave Monumental y Carr. La Rada, Km 3, 22 Guanabacoa, Havana, Cuba e-mail: lmichel@centis.edu.cu A. Xiques Castillo Jubilant DraxImage, 16751 Trans-Canada Highway, Kirkland, QC H9H 4J4, Canada V. N. Calzada Falco ´n Centro de Investigaciones Nucleares, Universidad de la Repu ´blica, Mataojo 2055, 11400 Montevideo, Montevideo, Uruguay M. Zamora Barrabı ´  A. Arbesu ´ Valdivia Deparment of Quality Control, Center of Isotopes, Ave Monumental y Carr. La Rada, Km 3, 22 Guanabacoa, Havana, Cuba I. Herna ´ndez Gonza ´lez  M. Leo ´n Pe ´rez Deparment of Development, Center of Isotopes, Ave Monumental y Carr. La Rada, Km 3, 22 Guanabacoa, Havana, Cuba 123 J Radioanal Nucl Chem (2014) 302:49–56 DOI 10.1007/s10967-014-3402-9