Nanoporous Substrates to Advance CAR-T Cell Production for Advanced Cell Therapies
Authors: Zünd Tamara , Lickert Sebastian , Strebel Anja , Murner Marcia , Willms Tido , Simsek Hasan , Du Weijie , Wagner Dimitrios Laurin , Vogel Viola , Klotzsch Enrico 
Published: December 2023

Background: CAR-T cell therapy has transformed blood cancer treatment with promising clinical trial outcomes. However, access to this therapy has been limited due lengthy and complex CAR-T cell production process, resulting in a very costly therapy. To enhance patient welfare, establishment of innovative point-of-care solutions with a simplified and shortened manufacturing processes is crucial to ensure higher cell quality and a shorter vein to vein time. immUni technology, a novel nano-engineered substrate will allow to combine and enhance key steps of the CAR-T production - from T cell activation, gene transfer to expansion. immUni platform integrates a unique combination of biochemical and physical stimulation. The biophysical activation is achieved without the need for a TCR agonist, resulting in a striking balance between robust T cell activation and proliferation as well as T cell effector function and a less differentiated phenotype required for CAR-T cell therapy. immUni technology is an easy-to-use platform that minimizes benchwork.
Aims: To provide a novel technology to produce optimal CAR-T cell therapies.
Methods:
Human naïve pan T cells isolated from healthy donors were used for the following experiments.
After 24h Il-2 secretion was measured using an ELISA assay and CD69 expression via flowcytometry as a readout for activation.
The gene transfer of the CD19-specific 2 nd generation CAR construct was conducted 48 hours after activation, employing both lentiviral transduction and non-viral CRISPR/Cas9 gene editing into the TRAC locus.
For T cell culture and expansion, the medium was supplemented with IL-7 and IL-15. CAR expression was measured 6 days post activation. The T cell phenotype was identified and quantified after 10 days of expansion with markers against CD95, CD45RA, CCR7 as well as CD4, CD8 and CD3.
Results: For a head-to-head comparison between immUni substrates and established activation methods for CAR-T production, we benchmarked our solely aCD28-antibody coated immUni surface, against Dynabeads TM (Thermo Fisher Scientific) and against aCD3/aCD28 antibodies immobilized on flat surfaces
When measuring CD69 expression after activation on immUni substrates, T cells showed high percentages of CD69 expressing cells (&gt;80%) while maintaining lower MFI values compared to the two other activation methods. In contrast, Il-2 secretion was significantly enhanced on immUni substrates (120%, Fig. 1A). Furthermore, we conducted T cell expansion and differentiation experiments, which demonstrated a remarkable 3x higher fold expansion after 10 days when activated on immUni substrates compared to activation via Dynabeads TM. On top of their outstanding effector functions, 80% of the T cells maintain a less differentiated CCR7 positive phenotype (Fig. 1B). This outcome holds great promise for long-term tumor control.
To further validate this novel activation method for CAR-T cell production, we measured gene transfer efficiency using lentiviral transduction and non-viral CRISPR/Cas9 gene editing via electroporation. Lentiviral transduction resulted in at least 90% more CD19 CAR-positive CAR-T cells after 6 days post activation (n=2) compared to Dynabeads TM and flat substrates. CRISPR/Cas9 knock-in to the TRAC locus resulted in an improvement of 50% when compared to antibody-coated flat substrates (n=2), holding a great promise in further combining activation and gene transfer steps.
Conclusion: Our preliminary data strongly supports the potential of our innovative immUni technology to enhance CAR-T cell production. Comparative studies against Dynabeads TM demonstrate that our technology outperforms current methods with abbreviated T cell activation time, enhanced gene transfer, and robust cytokine production. The accelerated expansion of CAR-T cells, coupled with the retention of a memory-related phenotype, holds great promise for effective tumor eradication, currently under preclinical validation. immUni user-friendly, off-the-shelf product is ready for R&D use in this field. A seamless integration into existing production lines, enhancing accessibility and adoption is currently under development.