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.
Manufacture of CD19-specific T cells from peripheral and umbilical cord blood mononuclear cells by electro-transfer of SB plasmids and selective propagation of CAR+ T cells on AaPC/IL-2/IL-21.
Manufacture of CD19-specific T cells from peripheral and umbilical cord blood mononuclear cells by electro-transfer of SB plasmids and selective propagation of CAR+ T cells on AaPC/IL-2/IL-21.
No relevant conflicts of interest to declare.
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