Adoptive Therapy Using Sleeping Beauty Gene Transfer System and Artificial Antigen Presenting Cells to Manufacture T Cells Expressing CD19-Specific Chimeric Antigen Receptor

Abstract

Objectives: T cells can be genetically modified ex vivo to redirect specificity upon expression of a chimeric antigen receptor (CAR) that recognizes tumor-associated antigen (TAA) independent of human leukocyte antigen. We employ non-viral gene transfer using the Sleeping Beauty (SB) transposon/transposase system to stably express a 2nd generation CD19-specific CAR- (designated CD19RCD28 that activates via CD3z/CD28) in patient (pt)- or donor-derived T cells for patients with advanced B-cell malignancies.Methods: T cells were electroporated using a Nucleofector device to synchronously introduce two DNA plasmids coding for SB transposon (CD19RCD28) and hyperactive SB transposase (SB11). T cells stably expressing the CAR were retrieved over 28 days of co-culture by recursive additions of designer g-irradiated activating and propagating cells (AaPC) in presence of soluble recombinant interleukin (IL)-2 and IL-21. The aAPC were derived from K562 cells and genetically modified to co-express the TAA CD19 as well as the co-stimulatory molecules CD86, CD137L, and a membrane-bound protein of IL-15. The dual platforms of the SB system and aAPC are illustrated in figure below.Results: To date we have successfully manufactured product for 42 pts with multiply-relapsed ALL (n=19), NHL (n=17), or CLL (n=5) on 4 investigator-initiated trials at MD Anderson Cancer Center to administer thawed pt- and donor-derived CD19-specific T cells as planned infusions in the adjuvant setting after autologous (n=5), allogeneic (n=21) or umbilical cord (n=4) hematopoietic cell transplantation (HCT), or for the treatment of active disease (n=12). Each clinical-grade T-cell product was subjected to a battery of in-process and final release testing. Adjuvant trials: Twelve pts have been infused with donor-derived CAR+ T cells following allogeneic HCT, including 2 pts with cord blood-derived T cells (ALL, n=10; NHL, n=2), beginning at a dose of 106 and escalating to 5x107 modified T cells/m2. Three pts, all with ALL, remain alive and in remission at median 5 months following T cell infusion. Five pts with NHL have been treated with pt-derived modified T cells following autologous HCT at a dose of 5x108 T cells/m2, and 4 pts remain in remission at median 12 months following T-cell infusions. Relapse trials: Thirteen pts have been treated for active disease (ALL, n=8; NHL, n=3; CLL, n=2) with pt or donor-derived (if prior allo-HCT) modified T cells at doses 106-5x107/m2, and 3 remain alive and in remission at median 3 months following T-cell infusions. No acute or late toxicities, including excess GVHD, have been noted.Conclusion: We report the first human application of the SB and AaPC systems to genetically modify clinical-grade cells. Furthermore, infusing CD19-specific CAR+ T cells in the adjuvant HCT setting and thus targeting minimal residual disease may provide an effective and safe approach for maintaining remission in pts at high risk for relapse.Next steps: The SB system serves as a nimble and cost-effective platform for genetic engineering of T cells. We are implementing next-generation clinical T-cell trials targeting ROR1, releasing T cells for infusion within days after electro-transfer of SB DNA plasmid coding for CAR and mRNA coding for transposase, and infusing T cells modified with CAR designs with improved therapeutic potential. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.