Oncoretroviral and Lentiviral Transduction of Donor T Cells to Facilitate Engraftment of Dog Leukocyte Antigen (DLA)-Haploidentical T-Cell-Depleted Marrow.

Abstract

Several clinical studies of adoptive immunotherapy with genetically modified (GM) donor T cells infused after allogeneic hematopoietic cell transplantation (HCT) showed limited in vivo function of the transduced T cells. Factors that have hindered successful translation to clinical trials include insufficient preclinical data in large animal models and the need for prolonged cell culture - up to 2 to 3 weeks for optimal oncoretroviral (OR) vector transduction and selection of T cells. In preparation for in vivo studies of GM T cells to facilitate engraftment in the preclinical dog model of allogeneic HCT, we compared transduction protocols with OR and lentiviral (LV) vectors that aimed to decrease the duration of ex vivo T cell culture necessary for stable transduction while maintaining T cell alloreactivity. Vectors expressed enhanced yellow fluorescent protein (YFP) under a constitutive promoter. We compared vectors pseudotyped with viral glycoproteins (GP) including vesicular stomatitis virus (VSV)-G (LV only), feline endogenous virus (RD114), and chimeric RD114 envelope GP fused with murine leukemia virus-A cytoplasmic tail (RD114/TR). Although T cells transduced with LV vectors without prior mitogenic or allogeneic stimulation had 14% – 30% transduction efficiency of predominantly CD4+ cells, transgene expression was not sustained in CD8+ cells after allogeneic stimulation (n=3). In order to transduce T cells that could generate GM alloreactive cytotoxic T lymphocytes (CTL), freshly isolated T cells were stimulated with allogeneic dendritic cells (DC) for 4 days prior to transduction. VSV-G, RD114 or RD114/TR pseudotyped LV had primary transduction efficiency of 1.2 to 9% (n=5). Only cells that were transduced with RD114 or RD114/TR pseudotyped vectors maintained stable YFP expression after 2° allogeneic stimulation. Next, OR YFP vector pseudotyped with RD114 transduced 15 to 36% of DC allo-stimulated T cells (n=3). Both CD4+ and CD8+ cell populations were transduced (CD4+: CD8+ ratio 1.4:1) and the mean YFP fluorescence intensity was increased 0.6-log compared to LV vectors (p=0.01). We then evaluated T cells transduced with OR RD114 pseudotyped vector in vivo. To determine if short-term culture and transduction of T cells facilitated engraftment of CD3-depleted marrow in the DLA-haploidentical HCT model, donor T cells were collected on day −7 prior to HCT, cultured with recipient CD34+ derived DC, and on day −4 cells were transduced with RD114 pseudotyped YFP OR vector. To date, one dog was transplanted after 920cGy total body irradiation with 2x108 CD3+ donor cells/kg (1:1 CD4:CD8 ratio) 25% YFP expression and 2-log CD3-depleted marrow (4x108 TNC/kg). No post-grafting immunosuppression was given. Donor YFP transduced T cells were detected in the peripheral blood daily after HCT, and peaked on day +7. After engraftment on day +8, GVHD developed and the dog was euthanized on day +21 with all-donor chimerism. YFP+ T cells were detected in GVHD affected target organs. Previously, transduced donor CTL cultured for 4 weeks and transplanted with CD3-depleted marrow in this HCT model failed to engraft in all 5 dogs studied. These preliminary results support the hypothesis that short-term culture and transduction of donor T cells with RD114 pseudotyped OR vector maintain in vivo alloreactivity and facilitate engraftment of CD3-depleted marrow in MHC-mismatched recipients.