SB transposition-based nonviral gene delivery has an outstanding potential to provide innovative and potentially curative treatments for an array of monogenetic disorders and cancer (recently reviewed in Amberger and Ivics33). SB successfully entered the clinical stage in 2011 with two clinical trials as the first nonviral vector being used to generate CD19-specific CAR-T cells for adjuvant immunotherapy targeting minimal residual disease of non-Hodgkin's lymphoma (NHL) and ALL following hematopoietic stem cell transplantation (HSCT).37 Here, SB was successfully used to shuttle a second-generation, CD19-specific CAR cassette in a classic double-plasmid delivery setting.38

The trials resulted in 30-month progression-free rates of 83% for patients who received autologous HSCT and 12-month progression-free rates of 53% for patients who received allogenic HSCT. Overall survival rates were 100% for the autologous and 63% for the allogenic HSCT group. Neither transgene integration hotspots nor acute or late toxicities or exacerbation of graft-versus-host disease (GVHD) was observed. These pilot studies established safety and illustrated the potential to use SB in CAR-T cell engineering.

Furthermore, the CARAMBA clinical trial (Phase-I/IIA; EudraCT: 2019-001264-30) investigates the feasibility, safety, and antimyeloma efficacy of autologous SLAMF7 CAR-T cells. CARAMBA is the first clinical trial relying on SB technology for CAR-T cell manufacturing in Europe, and the first clinical trial that uses advanced SB technology (hyperactive SB100X transposase encoded as synthetic mRNA in conjunction with CAR transposon supplied as minicircle vectors) worldwide.39

In addition, SB has been used to generate allogeneic “off-the-shelf” CAR-Natural Killer (NK) cells that have shown encouraging results against solid tumors.40 Cytokine-induced killer (CIK) cells, effector lymphocytes displaying a mixed T and NK phenotype with nonhuman leukocyte antigen (HLA)-restricted cytotoxicity and minimal alloreactivity, have also been generated with SB to target CD19, CD123, and CD33.41,42 Importantly, it has been recently reported that allogeneic, donor-derived CD19 CAR-CIK cells engineered with the SB system demonstrated high expansion, low toxicity, and complete remission in patients with relapsed and refractory ALL in a Phase I/II trial.43 Finally, SB was shown to be successful for T cell receptor (TCR) engineering,44,45 a strategy that enables to broaden the spectrum of targets as the major histocompatibility complex-TCR interaction also permits recognition of epitopes from intracellular proteins. There are currently a total of 14 active clinical trials in gene therapy making use of SB gene transfer technology (recently reviewed in Amberger and Ivics33).

A first-in-human Phase-I study conducted in Australia (The CARTELL Study, NHMRC identifier: 1102172) introduced PB into the clinics in 2016, used to manufacture CD19-specific CAR-T cells infused to patients suffering from relapsed/refractory CD19+ malignancies, namely B cell ALL and NHL. Preliminary reports suggest similar results as expected with viral vector-generated CD19 CAR-T cells.46 Two additional clinical trials with centers in Japan (UMIN Clinical Trials registry ID: UMIN000030984) and China (clinicaltrials.gov ID: {"type":"clinical-trial","attrs":{"text":"NCT04289220","term_id":"NCT04289220"}}NCT04289220) are currently being conducted/planned, making use of the PB system as well, to manufacture CD19-specific CAR-T cells to treat CD19+ B cell malignancies.

Furthermore, expanding on CAR-T cell targets, Poseida Therapeutics, Inc. is sponsoring two U.S.-based clinical trials making use of PB technology to manufacture BCMA-specific CAR-T cells for patients with relapsed/refractory multiple myeloma (clinicaltrials.gov ID: {"type":"clinical-trial","attrs":{"text":"NCT03288493","term_id":"NCT03288493"}}NCT03288493) and prostate-specific, membrane antigen-specific CAR-T cells for patients with metastatic castration-resistant prostate cancer (clinicaltrials.gov ID: {"type":"clinical-trial","attrs":{"text":"NCT04249947","term_id":"NCT04249947"}}NCT04249947). Reports of the Phase-I BCMA-specific CAR-T cell trial show highly encouraging results, with significant efficacy, low rates of cytokine release syndrome (CRS), and neurotoxicity.47 Accordingly, a subsequent Phase-II study has begun with a planned sample number of 100 patients in an outpatient setting given the unique safety profile observed in Phase-I.48

Unfortunately, The CARTEL Study has recently produced unexpected serious adverse events. Following CAR-T cell infusion, two patients developed T cell lymphoma leading to the death of one of the patients.49,50 Analysis of the first lymphoma showed a high CAR copy number, but no insertion into typical oncogenes. A detailed analysis of the samples revealed transcriptional changes of many genes driven by the transgene promoter despite insulator sequences surrounding the transgene. However, these changes correlated with genomic copy number variations rather than with the PB vector insertion sites.49 Although the molecular events that led to these adverse events are currently unknown, the potential oncogenic activity of the PB system needs to be rigorously addressed.