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. The average number of CAR expressing cells was 45% at the clinically relevant vector copy number of 0.5-1 copies/cell. The myeloid cells transduced with the CCL-MND-αCD19/z/28-sr39 vector demonstrated CD19-specific killing and were eliminated by GCV. In vivo studies demonstrated successful engraftment of transduced HSC with CAR-expressing cells in the different hematopoietic lineages (T, NK, myeloid) detected among human cells in the bone marrow (1.2-15.4%, mean 7.6%), spleen (0.3-15.4%, mean 5.6%), and peripheral blood (0.5-30%, mean 9.2%). Mice engrafted with anti-CD19 CAR-modified HSCs exhibited decreased huCD19+ populations, compared to the mice engrafted with non-modified HSCs. Treatment with GCV resulted in significant decrease in CAR-expressing cells only in the mice transplanted with CD34+ cells transduced with the HSVsr39TK-containing vector.
Here we demonstrate that HSCs can be effectively transduced with an anti-CD19 CAR linked to the HSVsr39TK suicide gene. The CAR was detected in human cells in the bone marrow, spleen and peripheral blood and resulted in decreased B-lineage populations as an index of antigen-specific cytotoxicity; the HSVsr39TK gene conferred sensitivity to ganciclovir which eliminated transduced cells. These results provide pre-clinical support for the use of a CD19 targeted CAR in HSCs for the treatment of B-cell malignancies.
Larson:Millenium: Speakers Bureau.
Asterisk with author names denotes non-ASH members.