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To generate membrane-bound IL-21 (mIL-21), the GM-CSF (granulocyte macrophage-colony-stimulating factor) signal peptide sequence was directly fused to the coding sequence of mature human IL-21 which was attached via a modified 12 aa IgG4 hinge region (aa 99–110), to the 5′ end of a human immunoglobulin γ-4 chain C H2 and C H3 regions (aa 111–327, UniProtKB P01861), that was fused in frame to human CD4 transmembrane domain (aa 397–418, UniProtKB P01730). The SB transposon CoOpCD19RCD28/pSBSO expresses the human codon optimized (CoOp) second generation CoOpCD19RCD28 CAR under human elongation factor 1-α (hEF-1α) promoter, flanked by the SB inverted repeats ( 6). To translate this CAR to clinical trials, we established a platform for nonviral gene transfer using the Sleeping Beauty (SB) system and subsequent selective expansion of CAR + T cells recursively cocultured upon CD19 + artificial antigen-presenting cells (aAPC) modified from K562 to express CD19 and desired costimulatory molecules ( 5–7). In response, we developed a second-generation CD19-specific CAR to activate T cells through both CD3-ζ and CD28 endodomains (signals 1 and 2, respectively) to improve T-cell activation ( 4). These clinical data demonstrated that infused T cells were short lived due, in part, to the use of a first-generation chimeric antigen receptor (CAR) that recognized CD19 independent of MHC via chimeric CD3-ζ (signal 1).
#LINEAGE W PUBLISHER TRIAL#
An initial clinical trial showed the safety and feasibility of redirecting T cell specificity to CD19, a TAA expressed on B-cell malignancies ( 1–3).
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To overcome immune tolerance to human tumor-associated antigens (TAA), investigators have redirected specificity through the introduction of immunoreceptors. ©2011 AACR.Īdoptive transfer of antigen-specific T cells has been used to treat and prevent malignancies and opportunistic infections. Our findings show that IL-21 can provide an extrinsic reprogramming signal to generate desired CAR + T cells for effective immunotherapy. 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. Furthermore, adoptive transfer of CAR + T cells cultured with IL-21 exhibited improved control of CD19 + B-cell malignancy in mice. In contrast, T cells propagated with only exogenous IL-2 tended to result in an overgrowth of CD19-specific CD4 + T cells. 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. 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. We used electrotransfer of Sleeping Beauty system to introduce a CAR transposon and selectively propagate CAR + T cells on CD19 + artificial antigen-presenting cells (aAPC). 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 interleukin (IL)-21 to tissue culture improves CAR-dependent T-cell effector functions. Recently an intrinsic strategy has been developed to modify the CAR itself to improve T-cell signaling. Improving the therapeutic efficacy of T cells expressing a chimeric antigen receptor (CAR) represents an important goal in efforts to control B-cell malignancies.