Manufacture of Clinical-Grade CD19-Specific T Cells Stably Expressing Chimeric Antigen Receptor Using Sleeping Beauty System and Artificial Antigen Presenting Cells

Abstract

Adoptive transfer of T cells expressing a CD19-specific chimeric antigen receptor (CAR) is being evaluated in multiple clinical trials. Our current approach to adoptive immunotherapy is based on a second generation CAR (designated CD19RCD28) that signals through a CD28 and CD3-ζ endodomain. T cells are electroporated with DNA plasmids from the Sleeping Beauty (SB) transposon/transposase system to express this CAR. Stable integrants of genetically modified T cells can then be retrieved when co-cultured with designer artificial antigen presenting cells (aAPC) in the presence of interleukin (IL)-2 and 21. Here, we reveal how the platform technologies of SB-mediated transposition and CAR-dependent propagation on aAPC were adapted for human application. Indeed, we have initiated clinical trials in patients with high-risk B-lineage malignancies undergoing autologous and allogeneic hematopoietic stem-cell transplantation (HSCT). We describe the process to manufacture clinical grade CD19-specific T cells derived from healthy donors. Three validation runs were completed in compliance with current good manufacturing practice for Phase I/II trials demonstrating that by 28 days of co-culture on γ-irradiated aAPC ∼1010 T cells were produced of which >95% expressed CAR. These genetically modified and propagated T cells met all quality control testing and release criteria in support of infusion.

Engineering of PEDF-Expressing Primary Pigment Epithelial Cells by the SB Transposon System Delivered by pFAR4 Plasmids

“…The SB100X transposase meditates the stable genomic integration of the expression cassette into the host cell’s genome, which allows for long-term transgene expression. Effective SB -mediated gene transfer has been reported for various in vitro and in vivo disease models,41, 42, 43 and it has been approved for clinical application of genetically modified T cells to treat B-lymphoid malignancies 44, 45. We have reported long-term and continuous PEDF secretion by RPE cells that were transfected with the PEDF transgene using the SB100X transposon system 25 and demonstrated that the subretinal transplantation of PEDF-transfected cells in a rat model of CNV reduces neovascularization and inhibits new vessel formation 46 …”

Dihydrofolate Reductase and Thymidylate Synthase Transgenes Resistant to Methotrexate Interact to Permit Novel Transgene Regulation

“…A construct to select for inducible caspase 9 (iC9) FLAG-DHFR and 3Ј to the IL-12p40 stop site ligated the three components into the TS SS G backbone in a four-part ligation. All constructs described integrate into the cellular genome with the aid of Sleeping Beauty transposase (20).…”

Bioengineering T cells to target carbohydrate to treat opportunistic fungal infection

“…We genetically modified primary circulating T cells using our SB transposon/transposase system that we adapted for human gene transfer. Indeed, we have achieved institutional and federal regulatory approvals for four clinical trials (NCT00968760, NCT01497184, NCT01362452, NCT01653717) infusing autologous and allogeneic T cells genetically modified with an SB encoding a CD19-specific CAR and propagation on aAPC (clone #4) (21,22,37). To generate clinically relevant numbers of D-CAR + T cells for adoptive transfer, T cells were expanded numerically on "designer" aAPC.…”

“…35050-061; Life Technologies) and 10% heat-inactivated FBS (HyClone). D-CAR + T cells were generated as previously described for CD19-specific CAR + T cells (37). In brief, T cells were electroporated with DNA plasmids from the SB system coding for D-CAR and SB11 and were propagated on aAPCs (clone #4) preloaded with Dectin-1-specific mAb in the presence of IL-2 and IL-21.…”