Characterization of CD20-Transduced T Lymphocytes as an Alternative Suicide Gene Therapy Approach for the Treatment of Graft-Versus-Host Disease

Abstract

We have previously proposed the CD20 molecule as a novel suicide gene for T lymphocytes in the context of allogeneic bone marrow transplantation, because CD20 can be used both as a selection marker and as a killer gene after exposure to the anti-CD20 therapeutic antibody rituximab. We now report on preclinical studies using this novel system, in which the best transduction protocol, reproducibility, yield, feasibility, and functionality of the transduced T lymphocytes have been investigated with a large donor series. Wild-type human CD20 cDNA was transduced into human T lymphocytes, using a Moloney-derived retroviral vector. Alternative protocols were tested by employing either one or four spinoculations (in which cells are centrifuged in the presence of retroviral vector supernatant) and stimulating T cells with phytohemagglutinin (PHA) or anti-CD3/CD28. One spinoculation alone was sufficient to obtain approximately 30% CD20-positive cells within four experimental days. Four spinoculations significantly increased transduction to 60%. A small difference in transduction efficiency was observed between the two stimulation methods, with PHA being superior to anti-CD3/CD28. Transduced cells could be purified on immunoaffinity columns, with purity reaching 98% and yield being on average 50%. Finally, 86-97% of immunoselected T lymphocytes could be killed in vitro with rituximab and complement. More importantly, the CD20 transgene did not alter the functionality of T lymphocytes with respect to allogeneic recognition and cytotoxic response, anti-Epstein-Barr virus cytotoxic response, antigenic response to tetanus toxoid antigen, interleukin 2 (IL-2), IL-4, and interferon gamma production; chemotaxis in the presence of stromal cell-derived factor 1, phenotype for several activation markers including HLA-DR, CD25, CD69, and CD95, and T cell repertoire.

A transgene-encoded cell surface polypeptide for selection, in vivo tracking, and ablation of engineered cells

“…[29][30][31][32] We demonstrated that Erbitux bound to huEGFRt expressed on T cells directs the cytotoxicity of ADCC effector cells and that in vivo engraftment of huEGFRt ϩ T cells can be inhibited by systemic administration of Erbitux. Unlike the suicide strategy based on CD20 expression and the use of rituximab, [33][34][35][36] our suicide system would not ablate B cells and would be compatible for T-cell transfer in patients who are receiving rituximab as part of the lymphoma/leukemia therapy. It is also important to note that CD20 is a tetraspan transmembrane protein, and there have not been CD20 truncations described that retain rituximab binding.…”

CD3+CD20+ cells may be an artifact of flow cytometry: Comment on the article by Wilk et al

“…Several flow cytometry-based studies have shown the existence of a small compartment of CD3ϩCD20ϩ cells in normal donors (2,7,8). We became interested in using CD20 as a transgene to combine the selection and suicide gene functions (9)(10)(11), and thus we wished to determine the extent to which this small T cell subset could interfere with the suicide gene transfer approach. Accordingly, we sought to phenotypically and functionally characterize this T cell subset.…”

An Inducible Caspase 9 Safety Switch Can Halt Cell Therapy-Induced Autoimmune Disease

“…More importantly, unwanted elimination of the transferred T cells as a consequence of immune responses toward the HSV-TK gene product has been observed in a substantial fraction of patients, likely limiting the use of this safety switch to patients who are immune suppressed at the time of T cell infusion (11,12). As a first possible nonimmunogenic alternative safety switch, a human CD20 molecule has been validated in preclinical studies (13,14). Exposure of patients to anti-CD20 mAb could then be used to induce killing of T cells that express this safety switch, but would in clinical practice also lead to an unwanted and prolonged loss of B cells.…”