Background There is an increasing demand for chimeric antigen receptor (CAR) T cell products from patients and care givers. Here, we established an automated manufacturing process for CAR T cells on the CliniMACS Prodigy platform that is scaled to provide therapeutic doses and achieves gene-transfer with virus-free Sleeping Beauty (SB) transposition. Methods We used an advanced CliniMACS Prodigy that is connected to an electroporator unit and performed a series of small-scale development and large-scale confirmation runs with primary human T cells. Transposition was accomplished with minicircle (MC) DNA-encoded SB100X transposase and pT2 transposon encoding a CD19 CAR. Results We defined a bi-pulse electroporation shock with bi-directional and unidirectional electric field, respectively, that permitted efficient MC insertion and maintained a high frequency of viable T cells. In three large scale runs, 2E8 T cells were enriched from leukapheresis product, activated, gene-engineered and expanded to yield up to 3.5E9 total T cells/1.4E9 CAR-modified T cells within 12 days (CAR-modified T cells: 28.8%±12.3%). The resulting cell product contained highly pure T cells (97.3±1.6%) with balanced CD4/CD8 ratio and a high frequency of T cells with central memory phenotype (87.5%±10.4%). The transposon copy number was 7.0, 9.4 and 6.8 in runs #1–3, respectively, and gene analyses showed a balanced expression of activation/exhaustion markers. The CD19 CAR T cell product conferred potent anti-lymphoma reactivity in pre-clinical models. Notably, the operator hands-on-time was substantially reduced compared with conventional non-automated CAR T cell manufacturing campaigns. Conclusions We report on the first automated transposon-based manufacturing process for CAR T cells that is ready for formal validation and use in clinical manufacturing campaigns. This process and platform have the potential to facilitate access of patients to CAR T cell therapy and to accelerate scaled, multiplexed manufacturing both in the academic and industry setting.

T cell-based immunotherapy has demonstrated great therapeutic potential in recent decades, on the one hand, by using tumor-infiltrating lymphocytes (TILs) and, on the other hand, by engineering T cells to obtain anti-tumor specificities through the introduction of either engineered T cell receptors (TCRs) or chimeric antigen receptors (CARs). Given the distinct design of both receptors and the type of antigen that is encountered, the requirements for proper antigen engagement and downstream signal transduction by TCRs and CARs differ. Synapse formation and signal transduction of CAR T cells, despite further refinement of CAR T cell designs, still do not fully recapitulate that of TCR T cells and might limit CAR T cell persistence and functionality. Thus, deep knowledge about the molecular differences in CAR and TCR T cell signaling would greatly advance the further optimization of CAR designs and elucidate under which circumstances a combination of both receptors would improve the functionality of T cells for cancer treatment. Herein, we provide a comprehensive review about similarities and differences by directly comparing the architecture, synapse formation and signaling of TCRs and CARs, highlighting the knowns and unknowns. In the second part of the review, we discuss the current status of combining CAR and TCR technologies, encouraging a change in perspective from “TCR versus CAR” to “TCR and CAR”.

Acute myeloid leukemia (AML), a fast-progressing hematological malignancy affecting myeloid cells, is typically treated with chemotherapy or hematopoietic stem cell transplantation. However, approximately half of the patients face relapses and 5-year survival rates are poor. With the goal to facilitate dual-specificity, boosting anti-tumor activity, and minimizing the risk for antigen escape, this study focused on combining chimeric antigen receptor (CAR) and T cell receptor (TCR) technologies. CAR’TCR-T cells, co-expressing a CD33-CAR and a transgenic dNPM1-TCR, revealed increased and prolonged anti-tumor activity in vitro, particularly in case of low target antigen expression. The distinct transcriptomic profile suggested enhanced formation of immunological synapses, activation, and signaling. Complete elimination of AML xenografts in vivo was only achieved with a cell product containing CAR’TCR-T, CAR-T, and TCR-T cells, representing the outcome of co-transduction with two lentiviral vectors encoding either CAR or TCR. A mixture of CAR-T and TCR-T cells, without CAR’TCR-T cells, did not prevent progressive tumor outgrowth and was comparable to treatment with CAR-T and TCR-T cells individually. Overall, our data underscore the efficacy of co-expressing CAR and transgenic TCR in one T cell, and might open a novel therapeutic avenue not only for AML but also other malignancies.

Solid tumors consist of malignant and nonmalignant cells that together create the local tumor microenvironment (TME). Additionally, the TME is characterized by the expression of numerous soluble factors such as TGF-β. TGF-β plays an important role in the TME by suppressing T cell effector function and promoting tumor invasiveness. Up to now CAR T cells exclusively target tumor-associated antigens (TAA) located on the cell membrane. Thus, strategies to exploit soluble antigens as CAR targets within the TME are needed. This study demonstrates a novel approach using Adapter CAR (AdCAR) T cells for the detection of soluble latent TGF-β within the TME of a pancreatic tumor model. We show that AdCARs in combination with the respective adapter can be used to sense soluble tumor-derived latent TGF-β, both in vitro and in vivo. Sensing of the soluble antigen induced cellular activation and effector cytokine production in AdCAR T cells. Moreover, we evaluated AdCAR T cells for the combined targeting of soluble latent TGF-β and tumor cell killing by targeting CD66c as TAA in vivo. In sum, our study broadens the spectrum of targetable moieties for AdCAR T cells by soluble latent TGF-β.

Chimeric antigen receptor (CAR) T cell therapy has emerged as an attractive strategy for cancer immunotherapy. Despite remarkable success for hematological malignancies, excessive activity and poor control of CAR T cells can result in severe adverse events requiring control strategies to improve safety. This work illustrates the feasibility of a zinc finger-based inducible switch system for transcriptional regulation of an anti-CD20 CAR in primary T cells providing small molecule-inducible control over therapeutic functions. We demonstrate time- and dose-dependent induction of anti-CD20 CAR expression and function with metabolites of the clinically-approved drug tamoxifen, and the absence of background CAR activity in the non-induced state. Inducible CAR T cells executed fine-tuned cytolytic activity against target cells both in vitro and in vivo, whereas CAR-related functions were lost upon drug discontinuation. This zinc finger-based transcriptional control system can be extended to other therapeutically important CARs, thus paving the way for safer cellular therapies.

Acute myeloid leukemia (AML) is a heterogeneous malignancy that requires further therapeutic improvement, especially for the elderly and for subgroups with poor prognosis. A recently discovered T cell receptor (TCR) targeting mutant nucleophosmin 1 (ΔNPM1) presents an attractive option for the development of a cancer antigen-targeted cellular therapy. Manufacturing of TCR-modified T cells, however, is still limited by a complex, time-consuming, and laborious procedure. Therefore, this study specifically addressed the requirements for a scaled manufacture of ΔNPM1-specific T cells in an automated, closed, and good manufacturing practice-compliant process. Starting from cryopreserved leukapheresis, 2E8 CD8-positive T cells were enriched, activated, lentivirally transduced, expanded, and finally formulated. By adjusting and optimizing culture conditions, we additionally reduced the manufacturing time from 12 to 8 days while still achieving a clinically relevant yield of up to 5.5E9 ΔNPM1 TCR-engineered T cells. The cellular product mainly consisted of highly viable CD8-positive T cells with an early memory phenotype. ΔNPM1 TCR CD8 T cells manufactured with the optimized process showed specific killing of AML in vitro and in vivo. The process has been implemented in an upcoming phase 1/2 clinical trial for the treatment of NPM1-mutated AML.