Apoptosis and complement-mediated lysis of myeloma cells by polyclonal rabbit antithymocyte globulin

Abstract

Current monoclonal antibody therapies for multiple myeloma have had limited success, perhaps due to narrow target specificity. We have previously described the ability of polyclonal rabbit antithymo-cyte globulin (rATG) to induce caspase-and cathepsin-mediated apoptosis in human B and plasma cells. We now extend this observation to myeloma cells. Complement independent cell death was measured after addition of rATG (1-1000 g/mL) to cultures of myeloma cell lines or primary CD138 isolates from patient bone marrow aspirates. rATG induced significant levels of apoptosis in my-eloma cells as assayed by caspase induction , annexin V binding, subdiploid DNA fragmentation, plasma-membrane per-meability, and loss of mitochondrial-membrane potential. Addition of complement greatly augmented myeloma-cell death. Binding of rATG to individual my-eloma cell-surface proteins, primarily CD38, CD52, CD126, and CD138, was demonstrated by competitive inhibition experiments with targeted monoclonal antibod-ies. Three pathways of cell death were identified involving caspase activation, cathepsin D, and the genistein sensitive tyrosine kinase pathway. F(ab) 2 fragments of rATG had reduced proapoptotic activity, which was restored by coincuba-tion with Fc fragments, and anti-CD32 or anti-CD64 antibodies. We conclude that rATG is an effective agent for in vitro induction of apoptosis in multiple myeloma, and that exploratory clinical trials may be warranted. (Blood. 2006;107:2895-2903)

Thymoglobulin targets multiple plasma cell antigens and has in vitro and in vivo activity in multiple myeloma

“…The cytotoxic effects of the Thymoglobulin on myeloma cells are also clearly observed in the absence of complement as in this study. [4][5][6] Importance of the caspases has been highlighted in a previous study where the cytotoxicity was significantly reduced in the presence of caspase inhibitors 5 and a second study that demonstrated activation of caspases 3, 8 and 9 in several myeloma cell lines. 6 The current study found that Thymoglobulin was able to induce apoptosis of myeloma cells resistant to conventional anti-myeloma agents, demonstrating non-overlapping mechanisms of action.…”

“…A role for complement has been suggested by previous studies that showed enhanced cytotoxicity of Thymoglobulin when cells are incubated in the presence of complement. 4,6 In addition, Zand et al 4 demonstrated decreased activity when cells were treated with the F(ab) 2 fragments instead of the whole antibody, an effect that was reversed by Fc ligation using other antibodies. The cytotoxic effects of the Thymoglobulin on myeloma cells are also clearly observed in the absence of complement as in this study.…”

“…These results confirm previous observations that suggested induction of apoptosis when treated with 0.1-1 mg/ml of Thymoglobulin. 5,6 Previous studies have implicated various mechanisms for the anti-myeloma activity of Thymoglobulin. A role for complement has been suggested by previous studies that showed enhanced cytotoxicity of Thymoglobulin when cells are incubated in the presence of complement.…”

Mini–Midi–Maxi? How to harness the graft-versus-myeloma effect and target molecular remission after allogeneic stem cell transplantation

“…Thymoglobulin has broader activity on T-cell depletion, whereas ATG-Fresenius mainly Table 2 Allogeneic reduced-intensity conditioning after prior cytoreductive autograft (auto-allo approach) 39,40 More recently, ATG preparations as polyclonal antibody have shown in vitro complement-dependent and apoptosis-mediated cytotoxicity on myeloma cells. 41,42 Therefore, ATG might be able to improve remission rate after transplantation. However, the role of ATG in allogeneic stem cell transplantation of myeloma patients needs to be determined.…”

Filgrastim enhances T-cell clearance by antithymocyte globulin exposure after unrelated cord blood transplantation

“…After binding to its targets, ATG mediates its cytotoxicity either through direct apoptosis via the Fas/FasL pathway, complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC) by natural killer (NK) cells or neutrophils, and antibody-dependent cellular phagocytosis (ADCP) by monocytes and macrophages. [16][17][18][19][20] ATG affects early T-cell reconstitution by depleting graft-derived T cells that are important for T-cell recovery through homeostatic peripheral expansion. The CBgraft cells, most of which are naive, might contain more epitopes for ATG, which may make them more susceptible to ATG-mediated cytotoxicity than BM-graft cells.…”