Targeting CD20 and CD22 with Rituximab in Combination with CMC-544 Results in Improved Anti-Tumor Activity Against Non-Hodgkin's Lymphoma (NHL) Pre-Clinical Models.

Abstract

CMC-544 is an IgG4 CD22-humanized targeted immunoconjugate of calicheamicin. Upon CD22 biding, CMC-544 internalizes inducing cell apoptosis in vitro/in vivo against NHL with minimal modulation of the immune system. In addition to direct cell-death, rituximab, anti-CD20 monoclonal antibody (mAb), can induce complement-dependent cytotoxicity (CDC) and antibody dependent cellular cytotoxicity (ADCC). Combination of mAbs directed against unique tumor-associated targets can potentially enhance anti-tumor activity. Objectives: To study the effects of targeting CD20 and CD22 using rituximab in combination with CMC-544. Material and Methods: For in vitro studies, a panel of NHL cell lines [Raji, SUDHL-4, SU-DHL-10, Ramos, and two rituximab resistant cell lines (RRCL), [2R and 4RH] were exposed to CMC-544 (0.4pg/ml to 0.4μg/ml) +/− Rituximab (10μg/ml) or appropriate controls for 24 or 48 hrs. DNA synthesis was quantified by [H3]Thymidine incorporation and apoptosis was detected by multiparameter flow cytometric analysis and Poly(ADP-ribose) polymerase (PARP) cleavage. For ADCC/CMC studies, 51Cr-labeled NHL cells were exposed to CMC-544 (0.4μg/ml) prior to treatment with rituximab (10mg/ml) and peripheral blood mononuclear cells (Effector: Target ratio 40:1) or human serum, respectively. 51Cr-release was measured and the percentage of lysis was calculated. Statistical analysis of results was performed using the Chi-square test. For in vivo studies, 6–8-week old SCID mice were inoculated via tail vein injection (iv) with Raji cells (1 × 106 on day 0). Animals were assigned to receive 8 doses of either saline, CMC-544 (40μg/kg/dose), rituximab (10mg/kg/dose iv), or rituximab alternating with CMC-544. Antibodies were administered iv every other day starting on day +5. Survival analysis was performed by Kaplan-Meier curves and p values calculated using log rank test. Results: Exposure of various NHL cells to CMC-544 resulted in a dose-dependent significant decrease in cell proliferation and apoptosis. Rituximab-associated CMC or ADCC was not further enhanced by CMC-544 in vitro. In vivo studies demonstrated that the mean survival for animals treated with CMC-544 increased when compared to control mice [64 days (95% C.I. 48–80; 22 days (95% C.I. 21–23), respectively, P=0.001]. To a lesser degree rituximab monotherapy resulted in significant anti-tumor effects and the median survival reached 44 days (95% C.I. 31–56). Notably, animals treated with rituximab in combination with CMC-544 had the longest median survival (not reached at +90 days, log rank P=0.007). Conclusions: Our data suggest targeting CD20 and CD22 by rituximab and CMC-544 resulted in a longer survival in lymphoma-bearing SCID mice. Clinical trials with this combination will be needed to confirm these results for the treatment of lymphoma patients