Effective therapy of human lymphoma xenografts with a novel recombinant ribonuclease/anti-CD74 humanized IgG4 antibody immunotoxin

Ranpirnase (Rap) is a cytotoxic ribonuclease (RNase) isolated from frog oocytes. Here we describe high antitumor activity of a novel immunotoxin, 2L-Rap-hLL1-gamma 4P, composed of 2 Rap molecules, each fused to the N terminus of the light chain of hLL1, an internalizing anti-CD74 humanized antibody. To reduce unwanted side effects, the constant region of hLL1 was changed from gamma 1 to gamma 4 and further to gamma 4P by replacing serine(228) to proline to prevent the formation of a half immunoglobulin G (IgG) common for IgG(4). In vitro, 2L-Rap-hLL1-gamma 4P retained RNase activity, specific binding to CD74, and was significantly more potent against CD74(+) cell lines (Daudi, Raji, and MC/CAR) than naked hLL1. In vivo, the pharmacokinetic profile of 2L-Rap-hLL1-gamma 4P was similar to that of naked hLL1. The maximum tolerated dose of 2L-Rap-hLL1-gamma 4P in severe combined immunodeficient mice (SCID) or BALB/c mice was 50 mu g per mouse. In Raj! and Daudi Burkitt lymphoma xenograft models, treatment with a single 5 to 50 mu g dose of 2L-Rap-hLL1-gamma 4P, given as early or delayed treatment, resulted in cures of most animals. Treatment with 2L-Rap-hLL1-gamma 4P was significantly better than all controls, including saline, naked hLL1, and nonspecific immunotoxin. In conclusion, 2L-RaphLL1-gamma 4P demonstrated excellent in vitro and in vivo efficacy and thus merits further consideration as a therapeutic for CD74(+) tumors.