Clinical development of chimeric antigen receptor (CAR)-T-cell therapy has been enabled by advances in synthetic biology, genetic engineering, clinical-grade manufacturing, and complex logistics to distribute the drug product to treatment sites. A key ambition of the CARAMBA project is to provide clinical proof-of-concept for virus-free CAR gene transfer using advanced Sleeping Beauty (SB) transposon technology. SB transposition in CAR-T engineering is attractive due to the high rate of stable CAR gene transfer enabled by optimized hyperactive SB100X transposase and transposon combinations, encoded by mRNA and minicircle DNA, respectively, as preferred vector embodiments. This approach bears the potential to facilitate and expedite vector procurement, CAR-T manufacturing and distribution, and the promise to provide a safe, effective, and economically sustainable treatment. As an exemplary and novel target for SB-based CAR-T cells, the CARAMBA consortium has selected the SLAMF7 antigen in multiple myeloma. SLAMF7 CAR-T cells confer potent and consistent anti-myeloma activity in preclinical assays in vitro and in vivo. The CARAMBA clinical trial (Phase-I/IIA; EudraCT: 2019-001264-30) investigates the feasibility, safety, and anti-myeloma efficacy of autologous SLAMF7 CAR-T cells. CARAMBA is the first clinical trial with virus-free CAR-T cells in Europe, and the first clinical trial that uses advanced SB technology worldwide.

Aspergillus fumigatus is a ubiquitous mold that can cause severe infections in immunocompromised patients, typically manifesting as invasive pulmonary aspergillosis (IPA). Adaptive and innate immune cells that respond to A. fumigatus are present in the endogenous repertoire of patients with IPA but are infrequent and cannot be consistently isolated and expanded for adoptive immunotherapy. Therefore, we gene-engineered A. fumigatus –specific chimeric antigen receptor (Af-CAR) T cells and demonstrate their ability to confer antifungal reactivity in preclinical models in vitro and in vivo. We generated a CAR targeting domain AB90-E8 that recognizes a conserved protein antigen in the cell wall of A. fumigatus hyphae. T cells expressing the Af-CAR recognized A. fumigatus strains and clinical isolates and exerted a direct antifungal effect against A. fumigatus hyphae. In particular, CD8 ⁺ Af-CAR T cells released perforin and granzyme B and damaged A. fumigatus hyphae. CD8 ⁺ and CD4 ⁺ Af-CAR T cells produced cytokines that activated macrophages to potentiate the antifungal effect. In an in vivo model of IPA in immunodeficient mice, CD8 ⁺ Af-CAR T cells localized to the site of infection, engaged innate immune cells, and reduced fungal burden in the lung. Adoptive transfer of CD8 ⁺ Af-CAR T cells conferred greater antifungal efficacy compared to CD4 ⁺ Af-CAR T cells and an improvement in overall survival. Together, our study illustrates the potential of gene-engineered T cells to treat aggressive infectious diseases that are difficult to control with conventional antimicrobial therapy and support the clinical development of Af-CAR T cell therapy to treat IPA.

B-cell maturation antigen (BCMA) is the lead antigen for chimeric antigen receptor (CAR) T-cell therapy in multiple myeloma (MM). A challenge is inter- and intra-patient heterogeneity in BCMA expression on MM cells and BCMA downmodulation under therapeutic pressure. Accordingly, there is a desire to augment and sustain BCMA expression on MM cells in patients that receive BCMA-CAR T-cell therapy. We used all-trans retinoic acid (ATRA) to augment BCMA expression on MM cells and to increase the efficacy of BCMA-CAR T cells in pre-clinical models. We show that ATRA treatment leads to an increase in BCMA transcripts by quantitative reverse transcription polymerase chain reaction and an increase in BCMA protein expression by flow cytometry in MM cell lines and primary MM cells. Analyses with super-resolution microscopy confirmed increased BCMA protein expression and revealed an even distribution of non-clustered BCMA molecules on the MM cell membrane after ATRA treatment. The enhanced BCMA expression on MM cells after ATRA treatment led to enhanced cytolysis, cytokine secretion and proliferation of BCMA-CAR T cells in vitro, and increased efficacy of BCMA-CAR T-cell therapy in a murine xenograft model of MM in vivo (NSG/MM.1S). Combination treatment of MM cells with ATRA and the γ- secretase inhibitor crenigacestat further enhanced BCMA expression and the efficacy of BCMA-CAR T-cell therapy in vitro and in vivo. Taken together, the data show that ATRA treatment leads to enhanced BCMA expression on MM cells and consecutively, enhanced reactivity of BCMA-CAR T cells. The data support the clinical evaluation of ATRA in combination with BCMA-CAR T-cell therapy and potentially, other BCMA-directed immunotherapies.

The successful application of CAR-T cells in the treatment of hematologic malignancies has fundamentally changed cancer therapy. With increasing numbers of registered CAR-T cell clinical trials, efforts are being made to streamline and reduce the costs of CAR-T cell manufacturing while improving their safety. To date, all approved CAR-T cell products have relied on viral-based gene delivery and genomic integration methods. While viral vectors offer high transfection efficiencies, concerns regarding potential malignant transformation coupled with costly and time-consuming vector manufacturing are constant drivers in the search for cheaper, easier-to-use, safer, and more efficient alternatives. In this review, we examine different non-viral gene transfer methods as alternatives for CAR-T cell production, their advantages and disadvantages, and examples of their applications. Transposon-based gene transfer methods lead to stable but non-targeted gene integration, are easy to handle, and achieve high gene transfer rates. Programmable endonucleases allow targeted integration, reducing the potential risk of integration-mediated malignant transformation of CAR-T cells. Non-integrating CAR-encoding vectors avoid this risk completely and achieve only transient CAR expression. With these promising alternative techniques for gene transfer, all avenues are open to fully exploiting the potential of next-generation CAR-T cell therapy and applying it in a wide range of applications.

Although the approval of new drugs has improved the clinical outcome of multiple myeloma (MM), it was widely regarded as incurable over the past decades. However, recent advancements in groundbreaking immunotherapies, such as chimeric antigen receptor T cells (CAR-T), have yielded remarkable results in heavily pretreated relapse/refractory patients, instilling hope for a potential cure. CAR-T are genetically modified cells armed with a novel receptor to specifically recognize and kill tumor cells. Among the potential targets for MM, the B-cell maturation antigen (BCMA) stands out since it is highly and almost exclusively expressed on plasma cells. Here, we review the currently approved BCMA-directed CAR-T products and ongoing clinical trials in MM. Furthermore, we explore innovative approaches to enhance BCMA-directed CAR-T and overcome potential reasons for treatment failure. Additionally, we explore the side effects associated with these novel therapies and shed light on accessibility of CAR-T therapy around the world.