THE CHIMERIC MONOCLONAL ANTIBODY C2B8 INHIBITS GROWTH OF EBV INFECTED B CELLS FROM PATIENTS WITH PTLD

Abstract

543 Post transplant lymphoproliferative disorder (PTLD) is a major complication of organ transplantation, and is associated with significant morbidity and mortality. Current therapeutic strategies for the treatment of PTLD are limited. The "humanized" mAb C2B8, which recognizes the CD20 molecule on human B cells has been used successfully to treat non-Hodgkin's B cell lymphomas and shows promise in the treatment of PTLD. The mechanism of C2B8 however remains largely unknown. The purpose of this study is to determine the effect of C2B8 on Epstein-Barr virus (EBV) infected B cells derived from PTLD patients. CD20 expression was determined on four EBV infected spontaneous lymphoblastoid cell lines (SLCL) from PTLD patients, as well as the Burkitt's lymphoma cell lines Daudi and Raji by indirect immunofluoresence and flow cytometry using the C2B8 parent murine mAb 2B8. All cell lines express CD20, although to varying degrees. To determine the effect of C2B8 on cell growth, C2B8 (10 ug/ml) or control mAb was added to SLCL cultures and proliferation assessed at 3-5 days (Figure 1). C2B8 inhibited proliferation in all SLCL tested (range 23-61%) as well as the Raji cell line (42%). In contrast C2B8 had a modest effect on Daudi (16%). Interestingly, JB7 had the highest degree of CD20 expression and inhibition by C2B8. To test the possibility that C2B8 induces apoptosis, SLCL were cultured with C2B8 or anti-Fas mAb (CH11) and stained with propidium iodide. Cell cycle analysis after 18 hrs. of anti-Fas exposure revealed apoptosis in 13% of MF4 cells and 17% of VB5 cells. Daudi was resistant to Fas mediated apoptosis, while JB7 and JC62 were minimally affected (2%). All SLCL express Fas on the cell surface as determined by staining with anti-Fas mAb. Importantly, all SLCL undergo apoptosis when treated with staurosporine, which acts independent of the Fas pathway. In addition, the JB7 cell line undergoes apoptosis when treated with C2B8 (1 ug/ml). Thus, EBV infected B cells have differential sensitivity to apoptotic signals and further studies are underway to dissect the pathways involved. Furthermore, C2B8 inhibits proliferation and may induce apoptosis in EBV infected B cells from PTLD patients.Figure 1