Anti-CD19 Chimeric Antigen Receptor Controlled By The Suicide Gene HSVsr39TK In Hematopoietic Stem Cells For Immunotherapy Of B-Lineage Malignancies

• Published in: Blood 2013-11, Vol.122 (21), p.1659-1659
• Main Authors: Larson, Sarah M., Tu, Andy, Senadheera, Shanta, Ho, Michelle, Kohn, Donald B., deOliveira, Satiro
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V122.21.1659.1659

Although significant improvements have been made, patients with relapsed or refractory B-cell malignancies continue to have unfavorable clinical outcomes. We hypothesize that transduction of hematopoietic stem cells (HSCs) with an anti-CD19 Chimeric Antigen Receptor (CAR) will produce a multi-lineage, persistent immunotherapy that can be controlled by the HSVsr39TK suicide gene. First generation anti-CD19 CAR lentiviral constructs containing the HSVsr39TK suicide gene were developed to compare vectors containing the human elongation factor alpha short (EFS) or myeloproliferative sarcoma virus U3 (MNDU3) promoters for transduction efficiency, antigen-specific cytotoxicity and ganciclovir (GCV)-induced cell death in primary human T-cells. The CD28 costimulatory domain was added to the selected construct, and high titer lentiviral vectors were generated to evaluate transduction of human umbilical cord blood (UCB) HSCs for in vitro and in vivo assays. In vitro assays were performed after culture under myeloid differentiation conditions, followed by assessment of phenotype, transduction efficiency, cytotoxic function and GCV-induced cell death. In vivo assays were conducted through transplantation of gene-modified human HSCs into irradiated NSG pups, compared to humanized NSG injected with non-modified human HSCs. Once engraftment was identified, mice from each cohort were further separated into GCV treated and untreated groups. Following GCV administration, mice were harvested to evaluate the presence of human and CAR-modified cells in the bone marrow, spleen and peripheral blood. In human primary T cells, the MNDU3 promoter resulted in higher percentage of CAR expressing cells and mean fluorescence intensity compared to the EFS promoter. Cytotoxicity by the transduced T cells against the huCD19+Raji cell line showed similar target cell specific lysis among the constructs. Treatment with GCV effectively decreased the in vitro survival of the cells containing the HSVsr39TK gene compared to the non-transduced and control vector. The construct with MNDU3 promoter was then used with a CD28-containing second-generation anti-CD19 CAR (CCL-MND-αCD19/z/28-sr39). Once transduction efficiency and CAR function were validated in primary human T cells, this vector was used to transduce human UCB CD34+ cells. Following transduction, these cells were evaluated in vitro and in vivo. The cells used for the in vitro studies were cultured under myeloid differentiation conditions