Synthesis and biodistribution studies of iodine-131 D-amino acid YYK peptide as a potential therapeutic agent for labeling an anti-CD20 antibody K. Sadri, a M. Gandomkar, b M. H. Babaei, b R. Najafi, b S. R. Zakavi, c and S. E. Sadat Ebrahimi aà A major drawback of conventionally radioiodinated monoclonal antibodies for radioimmunotherapy is in vivo dehalogenation of iodine as a result of deiodinase recognition. To solve this problem we have synthesized a YYK tri-peptide consisting of non-metabolizable D-amino acids modified with the N-succinimidyl (N-Succ) function. The chemical purity of the synthesized peptide as assessed by analytical high performance liquid chromatography was 95%. Labeling of the Fmoc-D-Tyr( t Bu)-D-Tyr( t Bu)-D-Lys(Boc)-N-Succ was performed using the chloramine-T method and the conventional extraction, resulting in a radiochemical yield of 50–71% and a radiochemical purity of 495%. Radioiodination of the peptide was followed by conjugation to anti-CD20 antibody with 65–75% labeling efficiency and 90% radiochemical purity. The effect of radioiodinated peptide on the biological behavior of the conjugate was evaluated through biodistribution studies in normal Lewis rats. Thyroid and stomach levels from Rituximab labeled with [ 131 I]-YYK-peptide were two- to four- fold less than those with directly labeled [ 131 I]-Rituximab, suggesting low recognition of its D-iodotyrosine residue by endogenous deiodinases. The favorable in vitro/in vivo stability and biodistribution profiles suggest that this radioiodine- labeled YYK peptide is a good candidate for further exploration of its potential clinical application. Keywords: radioiodination; radioimmunotherapy; dehalogenation; D-amino acid Introduction Non-Hodgkin lymphoma (NHL) involves a heterogeneous group of malignancies derived from lymphoid tissues, which have the ability to spread to other organs, with differences in appearance behavior and response to therapy. 1 Studies indicated that NHL can be treated effectively by radioimmunotherapy (RIT) for several reasons, including the inherent radiosensitivity of lymphocyte, the vascular accessibility of these malignancies and the large number of target antigens on the surface of lymphocytes. 2 The integral membrane protein CD20 has been identified as an important therapeutic target in the treatment of NHL. 3 CD20 is a 35kD hydrophobic transmembrane protein expressed on pre-B- cells and mature B-lymphocyte. It is expressed on more than 90% of B-cell NHL. 4 The absence of antigen expression on stem cells allows for the recovery of normal B-cell following RIT, which leads to the destruction of both malignant and normal B-cells. 5 The first antibody to be approved in 1997 for treating NHL was Rituximab, a chimeric anti-CD20 immunoglobulin. 6 Rituximab acts via various mechanisms to kill tumor cells, including complement-dependent cytotoxicity, antibody- dependent cell cytotoxicity and induction of apoptosis. The next agent to be approved was 90 Y-ibritumomab tiuxetan (Zevalin). 7 This was followed approximately 1.5 years later by 131 I-tositumomab (Bexxar). 8 Iodine-131 is a relatively inexpensive and readily available nuclide for labeling of radiopharmaceuticals. It has a long half- life of 8.1 days with beta emission of 0.69 MeV for therapy, and a gamma emission for imaging, which is useful for making dosimetry estimates. 5 The widespread use of iodine-131 for the labeling of tumor-associated antibodies has shown that this nuclide suffers substantially from undesirable physical and biological properties, principally the rapid and persistent in vivo dehalogenation of the radiolabeled antibody. 9 Such in vivo removal of radioiodine from target cells within the first 24–120 h postinjection of the labeled antibodies reduces tumor to non- tumor ratio, which is important for radiodiagnosis. In addition, it reduces the residence time of radioiodine in target cells, which significantly affects radiotherapy effectiveness. 10 Research Article Received 26 July 2008, Revised 19 January 2009, Accepted 2 March 2009 Published online 19 May 2009 in Wiley Interscience (www.interscience.wiley.com) DOI: 10.1002/jlcr.1600 J. Label Compd. Radiopharm 2009, 52 289–294 Copyright r 2009 John Wiley & Sons, Ltd. 289 a Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran b Nuclear Science Research School, Nuclear Science and Technology Research Institute (NSTRI), Atomic Energy Organization of Iran, Tehran, Iran c Nuclear Medicine Department of Imamreza Hospital, Mashad University of Medical Sciences, Mashad, Iran *Correspondence to: S. E. Sadat Ebrahimi, Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran. E-mail: sesebrahimi@sina.tums.ac.ir, sesebrahimi@yahoo.com