Abstract
1488
Objectives The ease of radiolabeling of peptides or antibodies via direct iodination has made isotopes of iodine the nuclides of choice for several medical applications. However, radioiodinated antibodies are not ideal for in vivo applications owing to the rapid deiodination, proteolysis and catabolism which result in loss of radioactive label from the target tissue. In order to improve in vivo stability of radioiodinated antibodies, we have developed a novel prosthetic group 2-(3-iodophenyl)-N-(4-isothiocyanatobenzyl)acetamide.
Methods To a tube containing tin-precursor, [131I]-NaI was added. ChloramineT added to the vial and after 5 min later, cold NaI was added. Sodium metabisulfite was added after additional 5 min incubation. The purity and yield were determined using HPLC. For antibody conjugation, Cetuximab in borate was added to the mixture and incubated at 37 °C over night. The labeled protein was purified using a PD-10 column with PBS as the eluant. Lindmo and internalization assays were performed on EGFR positive PC9 cells with direct- and indirect-radioiodinated antibody to compare and confirm retention of biological activity of the antibody.
Results The radioiodination of the bifunctional conjugate was highly efficient and addition of cold NaI results in complete consumption of residual precursors. Immunoreactivity of direct- and indirect-radioiodinated antibody was 45% and 60% respectively. Time dependent internalization of antibody was observed with a maximum of 90 % internalization 4 h post incubation.
Conclusions We have developed a novel bifunctional conjugate and a HPLC free method for radioiodination of antibodies. Our in vitro studies on cetuximab on EGFR expressing PC9 cells, indicates that the immunoreactivity of the antibody is retained.