Abstract
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.
Highlights
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
CAR-T-cell therapy targeting the CD19 antigen is an approved treatment for acute lymphoblastic leukemia (ALL), non-Hodgkin lymphoma (NHL), and mantle cell lymphoma, and CAR-T-cell products targeting several alternative antigens are under investigation in hematology and oncology
The current ex vivo, autologous CAR-T-cell therapy paradigm comprises a labor, time, and cost-intensive supply chain of harvesting the patient’s T cells at a leukapheresis center, shipping to a CARAMBA: a first-in-human clinical trial with SLAMF7 CAR-T cells prepared by virus-free Sleepingy centralized manufacturing facility to perform CAR gene transfer and T-cell expansion, and return shipment of the cryopreserved cell product to the hospital, where the therapy is administered (Fig. 1)
Summary
Key technology in CARAMBA: virus-free gene transfer using the Sleeping Beauty transposon system. The transposase enzyme mediates the excision of the element from the donor vector, followed by integration of the transposon into a chromosomal locus (Fig. 2) This feature uniquely positions transposons as non-viral gene delivery systems capable of efficient genomic integration that can be used as tools for versatile applications in genetic engineering, including gene therapy (reviewed in [3]). Electroporation of primary human cells, including hematopoietic stem and progenitor cells and T cells, with mRNA was shown to cause significantly reduced cellular toxicity as compared to nucleofection with plasmid DNA [39, 40, 46] This is likely explained by the inability of RNA to provoke a cellular IFN response in transfected T cells [38]. The CARAMBA consortium has settled to apply a unique combination of mRNA encoding the SB100X hyperactive transposase [37] and an MC vector carrying an SB transposon equipped with a SLAMF7 CAR
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