Phosphorylatable (33P)IL-2 & Anti-Tac MAb in ATL Therapy

DESCRIPTION (provided by applicant): lnterleukin-2 (IL-2) and Anti-Tac monoclonal antibodies (MAb; targeting IL-2 receptor alpha subunit CD25) bind with high affinity to malignant immune cells because these cells markedly over express the alpha subunit of the high affinity IL-2 receptor. We propose to develop multiple novel therapeutic proteins, 33P-IL-2 and 33P-Anti-Tac MAb, for targeting and selective destruction of malignant cells of acute T cell leukemia (ATL). Our strategy addresses significant limitations of other isotope-coupling chemistries by utilizing enzymatic labeling of biotherapeutics with 33P. In contrast to competitive technologies used to radiolabel protein therapeutics, this gentle, enzymatic, site-specific phosphorylation procedure 1) allows the protein to retain nearly quantitative bioactivity and specificity for the receptor, 2) permits improved product reproducibility in manufacture (moving toward a well-characterized radiolabeled biomolecule), and 3) may lead to decreased side effects. The specific aims of this proposal are: 1) Use molecular modeling to select phosphorylation sites and to optimize the predicted stability of the radiophosphate linkages on IL-2 and Anti-Tac constructs; 2) Introduce phosphorylation sites into Anti-Tac monoclonal antibody and IL-2 through genetic engineering; produce stable cells expressing candidate MAb constructs; express and purify at least two constructs each of phosphorylatable IL-2 and phosphorylatable Anti-Tac; 3) Examine and compare the bioactivity of native, phosphorylatable, and phosphorylated IL-2; examine binding capacity of parental and phosphorylated Anti-Tacs to purified antigen and/or to cells in tissue culture; 4) Determine the stability of the phosphate in vitro on IL-2 and Anti-Tac protein constructs. Should this approach deliver viable preclinical molecules for evaluation in vivo and subsequent treatment of malignancies abnormally expressing IL-2R alpha, the human benefit and commercialization potential are significant. These results will be used to leverage extension of this labeling strategy to a variety of other types of malignancies using either 33P- or 32P-labeled proteins.