The CD20/αCD20 ‘suicide’ system: novel vectors with improved safety and expression profiles and efficient elimination of CD20-transgenic T cells

Abstract

Adoptive transfer of T lymphocytes is an attractive strategy for many experimental treatment strategies for cancer. Unfortunately, manipulated T cells could be responsible for serious adverse events. Retroviral CD20-transduced T cells may be able to control these unwanted effects. CD20-positive cells are sensitive to rituximab (RTX), a monoclonal antibody specific for CD20. This permits their selective elimination in vivo in case of adverse events. To this end, a system is required that permits efficient and safe transduction of donor T cells and effective elimination of CD20-positive T cells. We constructed different CD20-encoding retroviral vectors and investigated the impact of inclusion of the woodchuck posttranscriptional regulatory element (WPRE) and the chicken hypersensitivity site 4 insulator elements on the levels, homogeneity and stability of CD20 expression. Importantly, inclusion of either WPRE or insulator elements in the retroviral vector resulted in a dramatic improvement in the stability of CD20 expression. The insulator element also led to a much more homogeneous level of CD20 expression. We also show the efficient elimination of the CD20-transgenic T cells via RTX by different effector mechanisms. In conclusion, we have constructed CD20-encoding retroviral vectors with improved efficiency and safety profiles, which can be used as a suicide strategy. Gene Therapy (2006) 13, 789-797.

Forced overexpression of either of the two common human Foxp3 isoforms can induce regulatory T cells from CD4+CD25– cells

“…No Foxp3 expression was detected in these cells by quantitative RT-PCR (data not shown). After 48 h, cells were harvested and retrovirally transduced using retronectin-coated tissue culture plates as described elsewhere [37]. Retroviral transduction was repeated the next day.…”

HuMab-7D8, a monoclonal antibody directed against the membrane-proximal small loop epitope of CD20 can effectively eliminate CD20low expressing tumor cells that resist rituximab-mediated lysis

“…Cell kill was analyzed by propidium iodide staining as previously described. 29,38 HuMab-7D8 and rituximab alone did not induce lysis of CEM-CD20 cells after prolonged incubation in the absence of complement (up to 72 h), indicating that none of the antibodies induced apoptosis under the conditions used.…”

“…29,38 Based on optimization assays, we used 10 mg/mL of anti-CD20 mAb, 20% normal human serum as source of complement, and incubated for 30 min at 37°C.…”

“…For in vivo imaging, CEM cells were transduced with a CD20 expression vector (CD20-WPRE-INS) and a retroviral vector expressing luciferase and eGFP (LucR-IRESeGFP) 38 ( Figure 2A). The CD20-WPRE-INS retroviral vector was selected because of its highly stable CD20 expression in vitro.…”

Adoptive cellular immunotherapy for childhood malignancies

“…120 Gene transfer can also be undertaken to render T cells resistant to the anti-inflammatory tumor micro-environment, such as by expressing a dominant-negative TGF-b receptor 121,122 or decreasing the sensitivity of the adoptively-transferred T cells to Fas-induced apoptosis. 123 Finally, given the potential for gene transfer to cause unwanted genotoxicity, investigators have included suicide genes [124][125][126][127][128][129][130][131][132] which conditionally render the cells sensitive to ablation in the event of an adverse event such as GVHD. [133][134][135][136] Adoptive cellular immunotherapy in the future There are a number of challenges to broadening the application of adoptive cellular immunotherapy for childhood cancers.…”