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Many tumors overexpress tumor-associated antigens relative to normal tissue, such as EGFR. This limits targeting by human T cells modified to express chimeric antigen receptors (CAR) 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 that differ in affinity. T cells with low-affinity nimotuzumab-CAR selectively targeted cells overexpressing EGFR, but exhibited diminished effector function as the density of EGFR decreased. In contrast, the activation of T cells bearing high-affinity cetuximab-CAR was not affected 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 nonmalignant cells., (©2015 American Association for Cancer Research.)

The treatment of osteosarcoma pulmonary metastases remains a challenge. T cells genetically modified to express a chimeric antigen receptor (CAR), which recognizes a tumor-associated antigen, have shown activity against hematopoietic malignancies in clinical trials, but this requires the identification of a specific receptor on the tumor cell. In the current study, we found that interleukin (IL)-11Rα was selectively expressed on 14 of 16 osteosarcoma patients' lung metastases and four different human osteosarcoma cell lines, indicating that IL-11Rα may be a novel target for CAR-specific T-cell therapy. IL-11Rα expression was absent or low in normal organ tissues, with the exception of the gastrointestinal tract. IL-11Rα-CAR-specific T cells were obtained by non-viral gene transfer of Sleeping Beauty DNA plasmids and selectively expanded ex vivo using artificial antigen-presenting cells derived from IL-11Rα + K562 cells genetically modified to coexpress T-cell costimulatory molecules. IL-11Rα-CAR(+) T cells killed all four osteosarcoma cell lines in vitro; cytotoxicity correlated with the level of IL-11Rα expression on the tumor cells. Intravenous injection of IL-11Rα-CAR(+) T cells into mice resulted in the regression of osteosarcoma pulmonary metastases with no organ toxicity. Together, the data suggest that IL-11Rα-CAR T cells may represent a new therapy for patients with osteosarcoma pulmonary metastases., (©2011 AACR.)

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 interleukin (IL)-21 to tissue culture improves CAR-dependent T-cell effector functions. 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). 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 show that IL-21 can provide an extrinsic reprogramming signal to generate desired CAR(+) T cells for effective immunotherapy., (©2011 AACR)

Allogeneic hematopoietic stem-cell transplantation can cure some patients with high-risk B-cell malignancies, but disease relapse following transplantation remains a significant problem. One approach that could be used to augment the donor T-cell-mediated antitumor effect is the infusion of allogeneic donor-derived T cells expressing a chimeric antibody receptor (CAR) specific to the B-cell antigen CD19. However, the use of such cells might result in toxicity in the form of graft-versus-host disease mediated by CD19-specific (CD19-CAR) T cells possessing alloreactive endogenous T-cell receptors. We therefore investigated whether nonalloreactive tumor-specific human T cells could be generated from peripheral blood mononuclear cells of healthy donors by the combination of CD19 redirection via CAR expression and subsequent alloanergization by allostimulation and concomitant blockade of CD28-mediated costimulation. Alloanergization of CD19-CAR T cells resulted in efficient and selective reduction of alloresponses in both CD4(+) and CD8(+) T cells, including allospecific proliferation and cytokine secretion. Importantly, T-cell effector functions including CAR-dependent proliferation and specific target cytolysis and cytokine production were retained after alloanergization. Our data support the application of CD19 redirection and subsequent alloanergization to generate allogeneic donor T cells for clinical use possessing increased antitumor activity but limited capacity to mediate graft-versus-host disease. Immunotherapy with such cells could potentially reduce disease relapse after allogeneic transplantation without increasing toxicity, thereby improving the outcome of patients undergoing allogeneic transplantation for high-risk B-cell malignancies., ((c)2010 AACR.)

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(+) T cells. We report a new approach to efficiently generating T cells with redirected specificity, introducing DNA plasmids from the Sleeping Beauty transposon/transposase system to directly express a CD19-specific CAR in memory and effector T cells without drug selection. When coupled with numerical expansion on CD19(+) artificial antigen-presenting cells, this gene transfer method results in rapid outgrowth of CD4(+) and CD8(+) T cells expressing CAR to redirect specificity for CD19(+) tumor cells.