Adoptive Immunotherapy Following Umbilical Cord Blood Transplantation Using The Sleeping Beauty System and Artificial Antigen Presenting Cells To Generate Donor-Derived T Cells Expressing a CD19-Specific Chimeric Antigen Receptor (2013)

Abstract Background The ability to transplant across HLA disparities makes allogeneic umbilical cord blood (UCB) an attractive graft source for hematopoietic stem-cell transplantation (HSCT). Disease relapse remains a limitation, and adoptive transfer of tumor-specific T cells post UCB HSCT has not been feasible due to the functionally naïve CB T cells, and the small size as well as anonymity of the donor. We report a new approach to non-viral gene transfer using the Sleeping Beauty (SB) transposon/transposase system to stably express a 2nd generation CD19-specific chimeric antigen receptor (CAR, designated CD19RCD28) on UCB-derived T cells manufactured in compliance with current good manufacturing practice (cGMP). Methods After thawed UCB units are washed for clinical infusion 5% to 10% of cells are used to generate CAR+ T cells. The mononuclear cells are 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 are retrieved over 28 days of co-culture by recursive additions of g-irradiated artificial antigen presenting cells (aAPC) in presence of soluble recombinant interleukin (IL)-2 and IL-21. The aAPC (designated clone #4) were derived from K562 cells and genetically modified to co-express the CD19 as well as the co-stimulatory molecules CD86, CD137L, and a membrane-bound protein of IL-15. Enrolled patients on our phase I trial receive two UCB units, thus two genetically modified T-cell products are made for each patient. We infuse thawed donor-derived CD19-specific CAR+ T cells from the dominant CB unit based on peripheral blood chimerism on days 40-100 post transplant in the adjuvant setting after double UCB HSCT Results To date we have successfully manufactured 8 products for 4 patients (ALL n=3, NHL=1) enrolled on trial. The median number of T cells in the starting CB aliquot was 8.6x106 (range, 2.5x106 to 54.8x106) with final modified T cell count at median 3x109 (range,1.7x108 to 4.1x1010) at time of cryopreservation days 28-32. In the final product, the median CD19-CAR+ cell purity by flow was 88% (range, 81.9% to 95.8%). The modified T cell product consisted of median 97.3% CD3+, 2.7 CD3-/CD56+ cells. All of the products exhibited CD19-specific killing by chromium assay as illustrated (Figure). Each clinical-grade T-cell product was subjected to a battery of in-process testing to complement release testing. One patient with ALL has been infused to date with a T cell dose of 106T cells/m2 and no toxicity has been observed. The patient remains alive and in continued molecular remission at 111 days post HSCT. Conclusion We combined the SB system and aAPC-mediated propagation of T cells to successfully manufacture disease-specific T cells from small aliquots of UCB used to restore hematopoiesis. Importantly, this approach allows us to employ adoptive therapy to enhance the graft-versus-tumor effect in UCB HSCT as an approach to improve overall survival for these recipients. Accrual to the trial continues and updated results will be presented at the meeting. Disclosures: No relevant conflicts of interest to declare.