Retroviral transfer of human CD20 as a suicide gene for adoptive T-cell therapy

Abstract

The aim of adoptive T-cell therapy of cancer is to selectively confer immunity against tumor cells. Autoimmune side effects, however, remain a risk, emphasizing the relevance of a suicide mechanism allowing in vivo elimination of infused T cells. We investigated the use of human CD20 as suicide gene in T-lymphocytes. Potential effects of forced CD20 expression on T-cell function were investigated by comparing CD20- and mock-transduced cytomegalovirus (CMV) specific T cells for cytolysis, cytokine release and proliferation. The use of CD20 as suicide gene was investigated in CMV specific T cells and in T cells genetically modified with an antigen specific T-cell receptor. No effect of CD20 on T-cell function was observed. CD20-transduced T cells with and without co-transferred T-cell receptor were efficiently eliminated by complement dependent cytotoxicity induced by therapeutic anti-CD20 antibody rituximab. The data support the broad value of CD20 as safety switch in adoptive T-cell therapy.

Figure 1.

Delivery of human CD20 by retroviral vectors. Jurkat-T cells were transduced with MP71-CD20-IRES-NGFR and LZRS-CD20-IRES-NGFR retroviral vectors. Indicated are the mean fluorescence intensities after staining with PE-labeled anti-CD20 and APC-labeled anti-NGFR antibodies.

Figure 2.

Human CD20 as suicide gene in cytomegalovirus specific T cells. CD20- and mock-transduced T-cell clones specific for pp50-VTE, IE1-ELR and IE1-QIK were isolated and compared for functional activity upon stimulation with autologous EBV-LCL pulsed with titrated CMV peptides. T cells specific for pp50-VTE are shown as representative examples. (A) Flow cytometric analysis after staining with CMV tetramers and antibodies against CD20 and NGFR. (B) T cells were tested for cytolytic activity in ⁵¹Cr-release assays at E:T ratios of 10:1 (upper), specific release of IFN-γ in ELISA (middle) and proliferative capacity by flow cytometry (lower). (C) CD20- and mock-transduced CMV specific T-cell clones were compared for RTX-induced CDC. T cells were treated with RTX and BRC or heat inactivated HS for 1 h at 37°C and analyzed for percentages of viable (PI−) cells relative to cultures incubated in medium with heat inactivated HS without RTX (100%) (upper). After CDC, T cells were washed and tested for cytolytic activity against peptide-pulsed autologous EBV-LCL in ⁵¹Cr-release assays at E:T ratios of 10:1 as counted prior to CDC (lower).

Figure 3.

Human CD20 as suicide gene in TCR gene transfer. (A) CD20-transduced peptide-stimulated CD8⁺ cells from a healthy donor were analyzed by flow cytometry before (upper) as well as one week after (lower) isolation by magnetic beads and subsequent in vitro expansion. (B) CD20-isolated T cells were restimulated and transduced with the HA-2 TCR. CD20⁺ HA-2-tetramer⁺ T cells were sorted by flow cytometry. Indicated is the MFI of anti-CD20 and HA-2 tetramer staining one week after sorting (upper). As a control, MFI values are shown for a parental HA-2 specific CTL (lower). (C) T cells co-transduced with CD20 and HA-2 TCR (left) and the parental HA-2 specific CTL (right) were compared for RTX-induced CDC. T cells were treated with RTX and BRC or heat inactivated HS for 1 h at 37°C. After CDC, T cells were washed and analyzed for cytolytic activity against HA-2 peptide-pulsed EBV-Z (HLA-A*0201⁺ HA-2⁻) and EBV-RZ (HLA-A*0201⁺ HA-2⁺) in 51Cr-release assay at E:T ratios of 10:1 as determined prior to CDC.