Characterization of a Radiolabeled Small Molecule Targeting Leukocyte Function-associated Antigen-1 Expression in Lymphoma and Leukemia

Objective: Leukocyte function-associated antigen-1 (LFA-1) is constitutively expressed on leukocytes, including overexpression on lymphomas and leukemias. We have developed a derivative of BIRT 377, an allosteric inhibitor of LFA-1, which may be chemically tagged without affecting binding. In this study, we modified this derivative, (R)-1-(4-aminobutyl)-5-(4-bromobenzyl)-3-(3,5-dichlorophenyl)-5-methylimidazolidine- 2,4-dione (butylamino-NorBIRT), and demonstrated its potential as a noninvasive imaging agent.

Methods: Specific binding of fluorescein-labeled butylamino-NorBIRT to both human and murine cells was demonstrated using equilibrium binding and dissociation techniques. A radiometal, lutetium-177 (Lu-177), was incorporated into the butylamino-NorBIRT through 1,4,7,10-tetraazacyclododecane-N,N',N",N'''- tetraacetic acid (DOTA) as a chelator.

Results: Equilibrium-binding experiments demonstrated that fluorescein- labeled butylamino-NorBIRT specifically binds human and murine LFA-1 with affinity constants of 135 and 186 nM, respectively. Dissociation kinetic experiments demonstrated an off-rate of 0.168/second(1) on murine cells, consistent with the observed affinity constant. Lutetium-177 was used for labeling, with > or =99.99% radiochemical purity and incorporation yield. This radiolabeled derivative exhibited high stability in fetal bovine serum (FBS) at 37 degrees C over 72 hours. (177)Lu-DOTA-butylamino-NorBIRT showed a binding affinity of 235 nM to human LFA-1 for equilibrium binding and competitive binding experiments.

Conclusions: The radiolabeled DOTA-butylamino-NorBIRT may have potential as a noninvasive imaging or therapeutic agent in both human and mouse models.