Abstract
Abstract 3580
Poster Board III-517
Immune cell expression of programmed death ligand-1 (PD-L1) represents a particularly important molecular mechanism responsible for control of auto- and allo-immunity mediated by effector memory T cells expressing PD1 receptor. As such, we have reasoned that an immuno-gene therapy approach that enables T cell expression of PD-L1 will represent a novel method of immune regulation. Advantageous features of this proposed therapy include a capacity to: (1) enforce long-term, stable expression of PD-L1; (2) build-in an independent surface marker to allow specific transduced cell enrichment; (3) utilize cellular delivery vehicles comprised of highly functional T cells that persist in vivo after adoptive transfer; and (4) incorporate an enhanced cell fate control or ‘suicide’ gene to permit in vivo control of the immuno-gene therapy. Given these considerations, we developed a recombinant lentiviral vector (LV) incorporating an EF1-α promoter that first encodes the cDNA for a fusion protein consisting of human CD19 (truncated, non-signaling) combined with mutated human TMPK that efficiently activates AZT as a pro-drug (Sato et al; Mol Therapy, 2007); then, after an IRES element, the vector encodes full-length human PD-L1. LV was made after transfection of 293T cells and then concentrated and titered. Initial experiments used Jurkat cells to optimize virus infection and to confirm co-expression of CD19 and PD-L1 by flow cytometry. In previous work, we have demonstrated that ex vivo T cell expansion in rapamycin induces an anti-apoptotic phenotype that permits enhanced in vivo T cell persistence in murine models and human-into-mouse xenogeneic transplant models. As such, we established the goal of infecting primary human CD4+ T cells manufactured using ex vivo co-stimulation (anti-CD3, anti-CD28), Th1-type polarization (inclusion of IFN-α), and exposure to high-dose rapamycin (1 μM); using a 6-day culture system and subsequent anti-CD19 column purification, >90% of resultant transduced T cells expressed PD-L1. Next, we utilized a xenogeneic transplantation model (Rag2−/−γc−/− hosts) to assess in vivo persistence of the gene-modified T cells and transgene expression (10,000 T cells transferred i.v. into each host). In vivo experiment #1 demonstrated that recipients of gene-modified T cells had increased numbers of human T cells in the spleen that co-expressed CD19 and PD-L1 relative to recipients of non-transduced but identically expanded human T cells (harvested at day 5 after adoptive transfer; 38,000 cells/spleen vs. 1000 cells/spleen, p=0.02). Such in vivo harvested T cells were secondarily co-stimulated ex vivo and propagated for an additional 5 days: co-expression of CD19 and PD-L1 persisted in ∼ 50% of T cells harvested from the gene-modified T cell cohort, and T cell numbers were maintained ex vivo (yield of CD19+PD-L1+ cells, 28,600 vs. 1500; p=0.0001). In vivo experiment #2 confirmed and extended these results. At day 21 after adoptive transfer, recipients of gene-modified T cells had increased numbers of human T cells that co-expressed CD19 and PD-L1 relative to recipients of non-transduced but identically expanded human T cells in both the spleen (2800 cells/spleen vs. 390 cells/spleen, p=0.01; n=10 per cohort) and bone marrow (71,600 cells/marrow vs. 6500 cells/marrow, p=0.0001; n=10 per cohort). Such in vivo harvested T cells at day 21 after adoptive transfer were secondarily co-stimulated ex vivo and propagated for an additional 6 days: co-expression of CD19 and PD-L1 persisted in ∼ 50% of T cells harvested from the gene-modified T cell cohort, and T cell numbers were maintained ex vivo (yield of CD19+PD-L1+ cells harvested from spleen, 71,200 vs. 1800, p=0.0008; yield of CD19+PD-L1+ cells harvested from marrow, 226,000 vs. 1400, p=0.0001). Because the rapamycin-resistant T cell vehicle utilized in these experiments manifests an anti-apoptotic phenotype that confers long-term engraftment potential, it is likely that the demonstrated durability in transgene expression relates both to the efficiency of the LV method utilized and to a T cell pro-survival function. In conclusion, the LV-mediated transfer of this novel combination of CD19/TMPK fusion protein and PD-L1 results in stable transgene expression in primary human T cells in vitro and in vivo, thereby opening an avenue to assess PD-L1 mediated immuno-gene therapy under cell fate control.
Disclosures:
No relevant conflicts of interest to declare.
Ex vivo activated and transduced human T cells generate lethal GVHD in a mouse model, and are efficiently eliminated in vivo with suicide gene therapy