Establishing mechanisms of resistance to rituximab, is necessary to further improve its therapeutic efficacy. In an attempt to define mechanisms by which B-cell NHL may escape rituximab immunotherapy, we developed several rituximab resistant cell lines (RRCL, Raji-2R, Raji-4RH and RL-4RH) generated from rituximab sensitive cell lines (RSCL) Raji and RL cells. Previously we demonstrated an up-regulation of CD52 upon development of rituximab-resistance. CD52 function is largely unknown although it is postulated to regulate normal T-cell activation. In our current work, we further examined the significance of CD52 in rituximab-associated CMC in and the biological effects of alemtuzumab against RRCL. To this end, the surface expression of CD20, CD52 and the complement inhibitory proteins (CIP) CD55, CD46 and CD59 was studied in RSCL and RRCL by flow cytometric analysis. CD52 F(ab’)2 fractions obtained by pepsin digestion of alemtuzumab (CAMPATH-1H), were used to block surface CD52 in RRCL. Following complete CD52 blocking (confirmed by flow cytometry) standard functional CMC and ADCC assays were performed using rituximab or isotype (10μg/ml) in 51Cr-labeled RRCL, CD52 unblocked RRCL served as controls. To asses the biological effects of alemtuzumab in RRCL functional assays for ADCC and CMC were performed. Rituximab resistance was associated with a CD20 down-regulation and up-regulation of CD52 and the CIP CD55 and CD59. In vitro blocking of CD52 with CD52 F(ab’)2 fractions in RRCL improved rituximab-associated CMC when compared to unblocked RRCL. In CD52 blocked RL-4RH or Raji-2H cells the mean % of rituximab-associated CMC was higher (19.7, +/- 0.66% and 19.45 +/- 3.84%) when compared to unblocked cells (3.59 +/- 1% and 5.4 +/- 1/7%) (P = 0.009 and P = 0.022). In vitro exposure of RRCL to alemtuzumab resulted in significant CMC and ADCC. The mean % of CMC lysis for alemtuzumab treated 2R and 4RH was 38% [95% C.I. 32.6-45%] and 84.7% [95% C.I. 71.1-98.4%] respectively. In addition, ADCC was demonstrated in 48% [95% C.I. 19.9-77.3%] of Raji-2R cells exposed to alemtuzumab. In contrast no significant alemtuzumab associated CMC or ADCC was demonstrated in RSCL. In summary, we demonstrated that CD52 up-regulation may play a significant role in rituximab-associated CMC, possibly serving as a CIP. In addition, alemtuzumab is biologically active and induces effective CMC and ADCC in RRCL. Our data provides a basis for the future development and study of alemtuzumab-based clinical trials for rituximab-resistant NHL-patients.
98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA