Lenalidomide, an immunomodulatory drug (IMiD), is a Thalidomide analogue with anti-tumor activity against various hematological malignancies. In murine models, we demonstrated that IMiDs enhance rituximab-associated biological activity against B-cell lymphoma. In our present work, we attempt to further define the mechanisms by which lenalidomide increases peripheral pool of natural killer (NK) cells. Dendritic cells (DC) were isolated from hematopoietic cells obtained from femurs of SCID mice by co-culturing them in vitro with GM-CSF (20nm/ml) and IL-2 (10u/ml) for 72 hours. Subsequently, DC were co-cultured with splenocytes derived from healthy 6-8 week old SCID mice with or without lenalidomide (10µg/ml) for up to five days. Stimulated splenocytes were used in immunological assays to study rituximab-associated antibody dependent cellular cytotoxicity (ADCC). 51Cr-labeled Raji cells were exposed rituximab (10μg/ml) and lenalidomide-stimulated or unstimulated control splenocytes (Effector: Target ratio 40:1). 51Cr-release was measured and the percentage of lysis was calculated. Statistical analysis of results was performed using the Chi-square test. Cytokine release was measured by luminex assay in the DC culture supernatant. For in vivo studies, SCID mice were inoculated with 1x106 Raji cells subcutaneously (sq). Once tumors reached 1cm3 in size, animals were treated with lenalidomide or DMSO vehicle-control administered intraperitoneally (ip) for four doses. Twenty four hours after the last dose of lenalidomide, animals were sacrificed and tumors were evaluated for degree of NK-cell infiltration (CD49) and vascular density (CD31) by immunohistochemistry (IHC). In vitro co-stimulation of splenocytes by DC and lenalidomide significantly enhanced rituximab-associated ADCC (% specific lyses: 95% CI for placebo control was 7.7+1.6 and Lenalidomide was 15.3+4.3) (P=0.007). In vitro exposure of DC to lenalidomide resulted in an increase in m-IFN-γ (P=0.04), m-TNF-α (P=0.004) and m-MCP-1(P=0.002) when compared to unstimulated control DCs. In vivo therapy of lymphoma-bearing SCID mice with lenalidomide resulted in specific changes within the tumor-bed: 1) infiltration by NK-cells 2) decrease in vascular density as demonstrated by CD31 staining (P=0.03). Our data suggests that lenalidomide enhances rituximab- associated ADCC by in vivo expansion, activation and tumor infiltration of NK cells. Furthermore, the tumor microenvironment is altered by the secretion of IFN-γ, TNF-α, and MCP-1 by DC. Finally, lenalidomide is associated with significant anti-angiogenic activity. Understanding the MOA of IMiDs is a fundamental step for the rational design of future clinical studies combining lenalidomide with other biological agents (supported by PO1 grant-CA103985-1)

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA