Clinical Application of <em>Sleeping Beauty</em> and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

Abstract

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR [1][2][3] . This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10 th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application [4][5][6][7][8] . Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2 nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats [9][10][11] . To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-γ receptor 1) for the loading of monoclonal antibodies (mAb) 12 . In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR + T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of γ-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-21 13. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.

Engineering cord blood to improve engraftment after cord blood transplant

“…The same group has also developed a TCR-like antibody that recognizes PR-1/HLA2 complex on acute myeloid leukemia cells and generated a second generation CAR using CBMC (111). In order to avoid using viral supernatant for transduction, the group at MD Anderson used electro-transfer of non-viral plasmids of the gene of interest (112). They adapted a Sleeping Beauty transposon and transposase along with artificial APCS to scale up the manufacture of CD19-directed CAR T cells from CBMCs.…”

Phase I trials using Sleeping Beauty to generate CD19-specific CAR T cells

“…To produce CAR T cells for infusion, peripheral blood (PB) mononuclear cells (PBMCs) simply obtained by venipuncture (Supplemental Table 1), were electroporated with SB transposon and transposase plasmids encoding CD19RCD28 and SB11. Electroporated cells were expanded on γ-irradiated clone 4 AaPCs for a median of 28 days (autologous: average 29 days, SD 1.4 days; allogeneic: average 28.5 days, SD 3.9 days) with IL-2 and IL-21, enabling selective growth of T cells stably encoding the CD19-specific CAR (Figure 2A), following methods reported previously (34)(35)(36)(37)(38). This process resulted in the selective outgrowth of T cells with integrated CAR (average cell expansion: ~2,200-fold autologous and ~2,500-fold allogeneic).…”

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.…”

“…After sequence validation, the FLAG 3(CoOp) -D-CAR-HA 3 fragment was subcloned into the SB transposon DNA plasmid CoOp CD19RCD28/pSBSO (used to express CD19RCD28 for human application), by restriction digestion at SpeI-NruI and NheI-SmaI sites, thus replacing the CD19RCD28 CAR fragment with the FLAG 3(CoOp) -D-CAR-HA 3 fragment to create the final plasmid CoOpD D-CAR/pSBSO (designated "pSBD-CAR"). The DNA plasmid pCMV-SB11 expresses the SB11 transposase (21).…”

“…Thus, using a gene transfer and propagation approach adapted for human application (21,22), we have demonstrated that an innate immune response to fungi can be harnessed by bioengineering cytotoxic T cells. This demonstration provides the foundation for testing whether D-CAR + T cells can be infused to improve the survival of immunocompromised patients who develop opportunistic IA infection.…”