CARAMBA: a first-in-human clinical trial with SLAMF7 CAR-T cells prepared by virus-free Sleeping Beauty gene transfer to treat multiple myeloma

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.

Fig. 1. CAR-T-cell manufacturing pipeline.

Patients undergo leukapheresis in the clinic to extract T cells, which are transported to a GMP facility, where manufacturing takes place. The CAR gene is introduced into the cells’ genome, leading to receptor expression and conversion into a CAR-T cell. CAR-T cells are then expanded in order to reach therapeutically relevant numbers. Finally, product formulation takes place, and the manufactured product is transported back to the clinic to be infused into the patient. CAR composition: antigen binding domain (1), transmembrane domain (2) spanning the cell membrane (3), costimulatory domain (4), and signaling domain (5).

Fig. 2. Schematic overview of gene delivery with Sleeping Beauty transposition.

The SB transposase is introduced into a cell in form of DNA (expression plasmid), mRNA, or recombinant protein along with donor DNA in which the transposon to be mobilized is located. After binding within the terminal inverted repeats of the transposon (TIRs, yellow rectangles) flanking a gene of interest (GOI, green rectangle), SB transposase (blue circles) performs the excision of the transposon from the donor DNA (black strand) and integrates it into a site in the genomic target DNA (purple strand).

Fig. 3. Schematic of the CARAMBA CAR construct and manufacturing process.

A Schematic of the transposon construct: an EF1α promoter drives expression (arrow) of the SLAMF7 CAR (modeled after the medicinal antibody elotuzumab) and, by means of a T2A element, the human truncated EGFR (huEGFRt). GMCSFRss: GM-CSF receptor-α chain signal sequences. B Flowchart of CARAMBA IMP manufacturing. Manufacturing steps are (1) apheresis, (2) immunomagnetic selection and CD3/CD28 activation, (3) nucleofection with SB100X transposase mRNA and SLAMF7 CAR-tEGFR transposon, (4) CD3/CD28-bead removal and transfer to G-Rex culture flasks, (5) half-medium change, and (6) harvest and formulation.

Fig. 4. Multiple myeloma cells expressing SLAMF7 and targeting them with CAR-T cells.

A dSTORM image showing SLAMF7 molecules expressed on MM.1S (ATCC ref. CRL-2974) cells. Cells were stained with anti-SLAMF7 Alexa Fluor 647 antibody. The inset displays a bright field image of the same cells. B Confocal microscope image showing the interaction between a SLAMF7 CAR-T cell and a MM.1S target cell. Cells were stained with phalloidin Atto643 for actin (green), with anti-CAR CF568 antibody (orange) and Hoechst 34580 for nuclei (blue).