Reprogramming CD19-Specific T Cells with IL-21 Signaling Can Improve Adoptive Immunotherapy of B-Lineage Malignancies

Abstract

Improving the therapeutic efficacy of T cells expressing a chimeric antigen receptor (CAR) represents an important goal in efforts to control B-cell malignancies. Recently an intrinsic strategy has been developed to modify the CAR itself to improve T-cell signaling. Here we report a second extrinsic approach based on altering the culture milieu to numerically expand CAR+ T cells with a desired phenotype. For, the addition of IL-21 to tissue culture improves CAR-dependent T-cell effector functions. We used electrotransfer of Sleeping Beauty (SB) system to introduce a CAR transposon and selectively propagate CAR+ T cells on CD19+ artificial antigen-presenting cells (aAPC). When IL-21 was present, there was preferential numeric expansion of CD19-specific T cells which lysed and produced IFN-γ in response to CD19. Populations of these numerically expanded CAR+ T cells displayed an early memory surface phenotype characterized as CD62L+CD28+ and a transcriptional profile of naïve T cells. In contrast, T cells propagated with only exogenous IL-2 tended to result in an overgrowth of CD19-specific CD4+ T cells. Furthermore, adoptive transfer of CAR+ T cells cultured with IL-21 exhibited improved control of CD19+ B-cell malignancy in mice. To provide coordinated signaling to propagate CAR+ T cells, we developed a novel mutein of IL-21 bound to the cell surface of aAPC that replaced the need for soluble IL-21. Our findings demonstrate that IL-21 can provide an extrinsic reprogramming signal to generate desired CAR+ T cells for effective immunotherapy.

T-cell and natural killer cell therapies for hematologic malignancies after hematopoietic stem cell transplantation: enhancing the graft-versus-leukemia effect

“…62 Additionally, to further improve the efficacy of CAR-modified T cells some groups are exploring the incorporation of two or more co-stimulatory domains (socalled third-generation CAR) 62,63 or combining them with other antibody recognition domains (so-called tandem CAR). 64 Addition of cytokine signals such as IL-21 65 and IL-15 66 has also been explored. Other attempts to improve in vivo persistence involve utilizing the endogenous signaling provided by latent viruses 67,68 which have been explored clinically in neuroblastoma and B-cell leukemias/lymphomas.…”

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

“…Here we show that CD19-specific T cells could be generated from mononuclear cells (MNC) derived from PB or UCB using SB transposition to introduce the CAR followed by addition of aAPC to numerically expand the T cells in a CAR-dependent manner (Figures 1, 4) 13,18 . Ten cuvettes (2x10 7 MNC/cuvette) are electroporated for each recipient using 15 μg of DNA plasmid (CD19RCD28/pSBSO) coding for transposon (CAR) and 5 μg of DNA plasmid (pCMV-SB11) coding for transposase (SB11).…”

“…These genetically modified T cells express an introduced CAR that recognizes the TAA CD19, independent of major histocompatibility complex. The SB-derived DNA plasmids to express the (i) transposon, a 2 nd generation CAR (CD19RCD28) that signals through CD28 and CD3-ε 14 , and (ii) transposase, SB11 15 , have been previously described 13 , 16,17 . The plasmids used in the current study were produced commercially by Waisman Clinical Biomanufacturing Facility (Madison, WI).…”

“…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.…”

Sleeping Beauty transposon vectors for therapeutic applications: advances and challenges

“…Co-expression of CD19 serves to specifically propagate the genetically modified T cells, leaving those cells that did not integrate the transposon to die from neglect. This method of expansion strategy can efficiently overcome the toxicity of nucleofection and yield sufficient numbers of CD19-specific CAR + T cells for clinical applications [82,83].…”