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Induction of myeloma cell apoptosis by polyclonal rabbit anti-thymocyte globulin (rATG).

2552 Background: Myelomatous plasma cells have a variable surface phenotype which depends on the stage of differentiation arrest. Polyclonal anti-thymocyte globululin (rATG) is generally thought of as an anti-T cell lineage reagent, although it has potent activity against many cell surface antigens expressed on B cells. We describe the use of rATG to induce in vitro apoptosis of CD19+ plasmablasts, CD138+ cell lines and freshly isolated myeloma cells.


METHODS
We tested the ability of rATG (Thymoglobulin; Sangstat), anti-CD20 (rituximab), and anti-CD52 (Campath-1H) to induce apoptosis in human plasmablasts, myeloma cell lines (RPMI 8226, NCI-H929, U266) and freshly isolated myeloma cells from bone marrow aspirates of 5 untreated patients isolated by CD138 magnetic bead affinity columns. Surface marker phenotyping was performed for all cells. Cells were incubated with rATG (0.01 - 1000 mcg/ml), anti-CD20 (0.01-10 mcg/ml) anti-CD52 (0.01-100 mcg/ml), and pooled rabbit immunoglobulin in media containing heat inactivated sera. Apoptosis was assessed at 18 hours post-exposure by annexin-V plus TOPRO-3 staining, nuclear fragmentation, caspase 3/8/9 staining, mitochondiral membrane potential, and sub-diploid DNA quantitation.


RESULTS
All myeloma cells and cell lines were phenotyped by FACS and found to express surface at least 3 antigens known to have reactivity with rATG (MHC class I, CD80, CD38, CD40, CD45). No myeloma cells, cell lines, or pre-plasmablasts expressed significant expression of the target antigens for rituximab (CD20) or campath-1H (CD52). Compared with controls, rATG at 100 mcg/ml induced significant apoptosis of naive B cells (94±11%, p<0.001), CD40L stimulated plasmablasts (99±12%, p<0.001), all myeloma cell lines (93±7%, p<0.001), and myeloma cell isolates (91±24%, p<0.001). Neither CD40L stimulated pre-plasmablasts, myeloma cells or cell lines had apoptotic levels statistically different from controls.


CONCLUSIONS
These results indicate that rATG, but not campath-1H or rituximab, is an active agent against multiple myeloma tumor cells. Further testing in a rigorous clinical trial to determine in vivo efficacy is warranted. [Table: see text].