BACKGROUND.T cells expressing antigen-specific chimeric antigen receptors (CARs) improve outcomes for CD19-expressing B cell malignancies. We evaluated a human application of T cells that were genetically modified using the Sleeping Beauty (SB) transposon/transposase system to express a CD19-specific CAR. METHODS.T cells were genetically modified using DNA plasmids from the SB platform to stably express a second-generation CD19-specific CAR and selectively propagated ex vivo with activating and propagating cells (AaPCs) and cytokines. Twenty-six patients with advanced non-Hodgkin lymphoma and acute lymphoblastic leukemia safely underwent hematopoietic stem cell transplantation (HSCT) and infusion of CAR T cells as adjuvant therapy in the autologous (n = 7) or allogeneic settings (n = 19).RESULTS. SB-mediated genetic transposition and stimulation resulted in 2,200-to 2,500-fold ex vivo expansion of genetically modified T cells, with 84% CAR expression, and without integration hotspots. Following autologous HSCT, the 30-month progression-free and overall survivals were 83% and 100%, respectively. After allogeneic HSCT, the respective 12-month rates were 53% and 63%. No acute or late toxicities and no exacerbation of graft-versus-host disease were observed. Despite a low antigen burden and unsupportive recipient cytokine environment, CAR T cells persisted for an average of 201 days for autologous recipients and 51 days for allogeneic recipients.CONCLUSIONS. CD19-specific CAR T cells generated with SB and AaPC platforms were safe, and may provide additional cancer control as planned infusions after HSCT. These results support further clinical development of this nonviral gene therapy approach.
Chimeric antigen receptors (CAR) combine an antigenbinding domain with a CD3-Z signaling motif to redirect Tcell specificity to clinically important targets. First-generation CAR, such as the CD19-specific CAR (designated CD19R), may fail to fully engage genetically modified T cells because activation is initiated by antigen-dependent signaling through chimeric CD3-Z, independent of costimulation through accessory molecules. We show that enforced expression of the fulllength costimulatory molecule CD28 in CD8 + CD19R + CD28À T cells can restore fully competent antigen-dependent T-cell activation upon binding CD19 + targets expressing CD80/CD86. Thus, to provide costimulation to T cells through a CD19-specific CAR, independent of binding to CD80/CD86, we developed a second-generation CAR (designated CD19RCD28), which includes a modified chimeric CD28 signaling domain fused to chimeric CD3-Z. CD19R + and CD19RCD28 + CD8 + T cells specifically lyse CD19 + tumor cells. However, the CD19RCD28 + CD8 + T cells proliferate in absence of exogenous recombinant human interleukin-2, produce interleukin-2, propagate, and up-regulate antiapoptotic Bcl-X L after stimulation by CD19 + tumor cells. For the first time, we show in vivo that adoptively transferred CD19RCD28 + T cells show an improved persistence and antitumor effect compared with CD19R + T cells. These data imply that modifications to the CAR can result in improved therapeutic potential of CD19-specific T cells expressing this second-generation CAR. (Cancer Res 2006; 66(22): 10995-1004)
Many tumors over express tumor-associated antigens relative to normal tissue, such as epidermal growth factor receptor (EGFR). This limits targeting by human T cells modified to express chimeric antigen receptors (CARs) due to potential for deleterious recognition of normal cells. We sought to generate CAR+ T cells capable of distinguishing malignant from normal cells based on the disparate density of EGFR expression by generating two CARs from monoclonal antibodies which differ in affinity. T cells with low affinity Nimo-CAR selectively targeted cells over-expressing EGFR, but exhibited diminished effector function as the density of EGFR decreased. In contrast, the activation of T cells bearing high affinity Cetux-CAR was not impacted by the density of EGFR. In summary, we describe the generation of CARs able to tune T-cell activity to the level of EGFR expression in which a CAR with reduced affinity enabled T cells to distinguish malignant from non-malignant cells.
Genetic modification of clinical-grade T cells is undertaken to augment function, including redirecting specificity for desired antigen. We and others have introduced a chimeric antigen receptor (CAR) to enable T cells to recognize lineage-specific tumor antigen, such as CD19, and early-phase human trials are currently assessing safety and feasibility. However, a significant barrier to next-generation clinical studies is developing a suitable CAR expression vector capable of genetically modifying a broad population of T cells. Transduction of T cells is relatively efficient but it requires specialized manufacture of expensive clinical grade recombinant virus. Electrotransfer of naked DNA plasmid offers a cost-effective alternative approach, but the inefficiency of transgene integration mandates ex vivo selection under cytocidal concentrations of drug to enforce expression of selection genes to achieve clinically meaningful numbers of CAR +
Adoptive immunotherapy retargeting T cells to CD19 via a chimeric antigen receptor (CAR) is an investigational treatment capable of inducing complete tumor regression of B-cell malignancies when there is sustained survival of infused cells. T-memory stem cells (TSCM) retain superior potential for long-lived persistence, but challenges exist in manufacturing this T-cell subset because they are rare among circulating lymphocytes. We report a clinically relevant approach to generating CAR+T cells with preserved TSCMpotential using theSleeping Beautyplatform. Because IL-15 is fundamental to T-cell memory, we incorporated its costimulatory properties by coexpressing CAR with a membrane-bound chimeric IL-15 (mbIL15). The mbIL15-CAR T cells signaled through signal transducer and activator of transcription 5 to yield improved T-cell persistence independent of CAR signaling, without apparent autonomous growth or transformation, and achieved potent rejection of CD19+leukemia. Long-lived T cells were CD45ROnegCCR7+CD95+, phenotypically most similar to TSCM, and possessed a memory-like transcriptional profile. Overall, these results demonstrate that CAR+T cells can develop long-term persistence with a memory stem-cell phenotype sustained by signaling through mbIL15. This observation warrants evaluation in clinical trials.
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.
Adoptive transfer of T cells expressing a CD19-specific chimeric antigen receptor (CAR) is being evaluated in multiple clinical trials. Our current approach to adoptive immunotherapy is based on a second generation CAR (designated CD19RCD28) that signals through a CD28 and CD3-ζ endodomain. T cells are electroporated with DNA plasmids from the Sleeping Beauty (SB) transposon/transposase system to express this CAR. Stable integrants of genetically modified T cells can then be retrieved when co-cultured with designer artificial antigen presenting cells (aAPC) in the presence of interleukin (IL)-2 and 21. Here, we reveal how the platform technologies of SB-mediated transposition and CAR-dependent propagation on aAPC were adapted for human application. Indeed, we have initiated clinical trials in patients with high-risk B-lineage malignancies undergoing autologous and allogeneic hematopoietic stem-cell transplantation (HSCT). We describe the process to manufacture clinical grade CD19-specific T cells derived from healthy donors. Three validation runs were completed in compliance with current good manufacturing practice for Phase I/II trials demonstrating that by 28 days of co-culture on γ-irradiated aAPC ∼1010 T cells were produced of which >95% expressed CAR. These genetically modified and propagated T cells met all quality control testing and release criteria in support of infusion.
Significance Patients with compromised T-cell function are at risk for opportunistic fungal infections. We have developed a novel approach to restore immunity by using a fungal pattern-recognition receptor Dectin-1 to redirect T-cell specificity to carbohydrate antigen in the fungal cell wall. We did so by genetically modifying T cells using the nonviral Sleeping Beauty gene-transfer system to enforce expression of a chimeric antigen receptor (CAR) that recapitulates the specificity of Dectin-1 (D-CAR). The D-CAR + T cells can be electroporated and propagated on artificial activating and propagating cells in a manner suitable for human application, enabling this immunology to be translated into immunotherapy. This approach has implications for genetically modifying T cells to express CARs with specificity for carbohydrate and thus broadening their application in the investigational treatment of pathogens and malignancies.
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