A promising strategy to control Graft versus Host disease (GvHD) in allogeneic stem cell transplantation is the use of donor T cells, genetically modified to express the Herpes Simplex Thymidine Kinase (HSVtk) gene. The HSVtk+ donor T cells can be selectively eliminated by ganciclovir (GCV) in vivo. Despite the encouraging clinical results limitations have been observed with this system. The viral HSVtk gene is immunogenic, GCV-activity is limited to dividing cells, and aberrant splicing of the HSVtk mRNA occurs. The human CD20 molecule, in combination with the humanized CD20 Ab Rituximab (RTX), was recently proposed as a novel ‘suicide’ system (Introna et al., Hum Gene Ther 2000). CD20 is non-immunogenic and both resting and dividing CD20+ cells can be eliminated in vivo by RTX, which triggers the complement system and recruits effector cells. However, for a safe and effective clinical application a high, stable and homogenous CD20 expression by human T cells is required. We constructed different CD20-encoding retroviral vectors. To enhance CD20 expression we included the WPRE element, which is widely used to increase transgene expression. Chromatin insulators separate differentially regulated loci, shield promoters from neighboring regulatory elements and protect against transcriptional silencing. Thus, insulators may protect against insertional mutagenesis and provide a more homogenous and stable expression of the transgene. We therefore included insulators into the vectors.
For this, CEM T cells were transduced with the different CD20-vectors at an MOI of 1 and a mean transduction efficiency of 22.9±5.1% was obtained. Results showed a significantly more homogenous CD20 expression for cells containing the insulators. Also after CD20 magnetic bead selection the CD20+ insulator containing cells had a more homogenous CD20 expression pattern. Surprisingly, the CD20 expression pattern became 2-fold more homogenous after continuous culturing for 3 months. In contrast, 60% of the cells without insulators lost CD20 expression. Inclusion of the WPRE element increased the CD20 expression level. CD20-vectors containing both the insulator and the WPRE element gave the highest, most homogenous and stable CD20 expression in CEM T cells.
In addition, we generated individual CEM-CD20 clones with different CD20 expression levels, to analyze the efficacy of killing by RTX in a complement dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC) and a combination of both assays. The CDC assay showed that RTX-mediated kill depends on the level of CD20 expression. We observed a clear, significant correlation between number of CD20 molecules per cells and the extent of CDC (p<0.001). In a newly developed ADCC assay the CEM-CD20 clones were equally sensitive to cytokine-activated PBMCs in the presence of RTX (49.0±10.6% survival), independent of the CD20 expression level. Combination of both effector mechanisms resulted in an even better cell kill than that with either of the assays alone, suggesting that CDC and ADCC exhibit an additive effect.
In summary, this is the first time that the combination of insulators and the WPRE element results in a higher, more homogeneous and stable retroviral transgene expression in T cells, which makes the T cells more sensitive to RTX. We also show for the first time that the level of CD20 expression is important for efficient CDC and not for ADCC and that CDC and ADCC synergize in Rituximab-mediated kill of CD20+ cells.