The Novel De-Immunized Anti-CD20-IL-2 Immunocytokine DI-Leu16-IL-2 Is Effective in SCID Mice Bearing Rituximab-Resistant Human Lymphoma Xenografts.

Abstract

Objectives:Tumor-targeted MAb-cytokine fusion proteins, such as IL-2 based immunocytokines, have demonstrated potent anti-tumor activity in several pre-clinical models as well as early stage clinical trials. For the engineering of anti-CD20-IL-2, the parental murine anti-CD20 MAb Leu16 was deimmunized by removal of potential helper T cell epitopes and then humanized by combination of recombinant V regions with human L chain and H chain constant regions. Fusion of the H chain to human IL-2 resulted in the de-immunized immunocytokine DI-Leu16-IL-2 which retained full CD20 binding activity and showed enhanced ADCC effector function relative to a control MAb (rituximab). This novel molecule has been demonstrated to be effective in vivo in a Daudi Burkitt lymphoma model. We investigated the therapeutic potential in a rituximab resistant CD20+ human lymphoma xenograft mouse model.Materials & Methods:The mature plasmocytic B-cell line ARH77 was stably transfected with the firefly luciferase-gene using electroporation. 2x106 luciferase-transfected ARH77 cells (ARH77:luc) were inoculated s.c. into the right flank of 6–8 week old SCID mice. Groups of 8 ARH77:luc tumor bearing mice were injected i.v. with 3 doses of DI-Leu16-IL2 (25μg), Rituximab (20μg) or PBS on day 0, 2, and 4. Tumor burden was monitored by both physical measurements and bioluminescence imaging. Mice were to be sacrificed at a tumor volume > 2000mm3.Results:In this regimen DI-Leu16-IL2 treated mice demonstrated significant reduction in tumor growth whereas rituximab or the PBS control had no effect on ARH77:luc tumor progression. An increase of survival from 28 to 44 days, relative to rituximab treated mice and PBS-control, was seen. Re-growth of tumors in the DI-Leu16-IL2 group was observed after an average delay of 16 days. There were no signs of toxicity. Multiple regression analyses between tumor volume and light emission were performed which revealed a correlation between tumor mass and bioluminescence for the DI-Leu16-IL-2 group (r2=0.64), rituximab group (r2=0.61), PBS-group (r2=0.82), and the combined data sets (r2=0.7).Conclusions:DI-Leu16-IL2 demonstrated significant anti-tumor activity against rituxan-resistant human lymphoma xenografts using both physical measures and bioluminescence imaging. Despite a shorter half-life it was more effective than rituximab. Previous studies have shown the anti-tumor effects of DI-Leu16-IL2 were not entirely dependent on FcR binding suggesting that both ADCC and IL-2 targeting contribute to the superior anti-tumor activity of the immunocytokine. Ongoing studies include the use of repeat dosing cycles of this novel immunocytokine for best response using the SCID mouse/ARH77:luc tumor model. DI-Leu16-IL2 may offer promising therapeutic potential with presumed low immunogenicity for patients with CD20+ lymphoma.