Radioimmunotherapy of MCF7 breast cancer cell line with ¹³¹I-PR81 monoclonal antibody against MUC1: Comparison of direct and indirect radioiodination methods

Article type: Research Article

Authors: Mohammadnejad, Javad^a | Rasaee, Mohammad Javad^{a; *} | Babaei, Mohammad Hosein^b | Paknejad, Malihe^c | Hasan, Zahir Mohammad^d | Salouti, Mojtaba^e | Gandomkar, Mostafa^b | Sadri, Keyvan^f

Affiliations: [a] Department of Clinical Biochemistry, School of Medical Sciences, Tarbiat Modarres University, Tehran, Iran | [b] Department of Radioisotope, Nuclear Research Center, Atomic Energy Organization of Iran, Tehran, Iran | [c] Department of Biochemistry, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran | [d] Department of Immunology, School of Medical Sciences, Tarbiat Modarres University, Tehran, Iran | [e] Department of Biology, Faculty of Sciences, Islamic Azad University of Zanjan Branch, Zanjan, Iran | [f] Department of Radiopharmacy, School of Pharmacy, Medical Sciences/University of Tehran, Tehran, Iran

Correspondence: [*] Corresponding author. Fax: +98 21 8013030; E-mail: Rasaee_m@modares.ac.ir.

Abstract: PR81 is a monoclonal antibody that binds with high affinity to MUC1, which is over expressed on breast and other tumors. The objective of this study was to compare the two labeling methods (direct and indirect radioiodination) for application of this antibody against MUC1 as a radioimmunotherapeutical agent.Monoclonal antibody (PR81) against the tandem repeat of the core protein (MUC1) was prepared, characterized, purified, and labeled with 131I using the direct (chloramin-T) and indirect (Fmoc-D-Tyr (tBu)-D-Tyr (tBu)-D-Lys (Boc)-OH (YYK) attached to N-hydroxysuccinimide as a linker between PR81 and 131I) methods. The immunoreactivity of 131I-PR81 and 131I-TP-PR81 complexes with MUC1 (the native protein), BSA-P20 (a 20 amino acid corresponding the tandem repeat of MUC1) and MCF7 cell line were performed by RIA. In vitro stability of 131I-PR81 and 131I-YYK-peptide-PR81 complexes in human serum was determined by thin layer chromatography (TLC). Cell toxicity and in vitro internalization studies were performed with the MCF7 cell line, and the tissue biodistribution of the 131I-PR81 and 131I- YYK-peptide -PR81 complexes was evaluated in normal BALB/c mice at 4, 24 and 48 hrs. The labeling efficiency was determined by measuring the percentage recovery of radioactivity in the final product relative to the initial activity in the shipment vial, was found to be 59.9% ± 7.9% for direct and 50% ± 3.2% for indirect methods. 131I-PR81 and 131I- YYK- peptide -PR81 complexes showed high immunoreactivity towards MUC1 protein, BSA-P20 and MCF7 cell line. In vitro stability of the labeled products in human serum which was measured by thin layer chromatography (TLC) was found to be more than 50% over 24 hr for 131I-PR81 and 70% for 131I- YYK-peptide -PR81 complexes. Cell toxicity and in vitro nternalization studies showed that the 131I-PR81 and 131I- YYK-peptide -PR81 complexes inhibited 80% growth of the MCF7 cultured cell lines in vitro in a high concentration and up to 40% of the 131I-PR81 and 60% of the 131I- YYK-peptide -PR81 complexes internalized after 24 h. Biodistribution studies were performed in normal BALB/c mice at 4, 24 and 48 hrs post-injection. Thyroid and stomach levels from PR81 labeled with 131I- YYK-peptide were two- to three- fold less than those with directly labeled 131I-PR81, suggesting low recognition of its D-iodotyrosine residue by endogenous deiodinase. These results show that the indirect labeling was better than the indirect labeling and 131I- YYK-peptide -PR81 may be considered as a promising candidate for therapy of breast cancer.

Keywords: Radioiodination, PR81 mAb, MUC1, radioimmunotherapy

DOI: 10.3233/HAB-2010-0216

Journal: Human Antibodies, vol. 19, no. 1, pp. 15-25, 2010