Synthetic Viscoelastic Activating Cells for T Cell Engineering and Cancer Therapy

Introduction: T cell-based therapies, particularly CAR-T cell therapy, hold great promise for cancer treatment. However, current methods for T cell activation and expansion, such as antibody-coated paramagnetic polystyrene beads (e.g., Dynabeads), often result in suboptimal cell subpopulations, low efficiency of CAR introduction into T cells, low stemness, and limited durability for long-lasting therapeutic effect. One reason is that Dynabeads are stiff and have a very different mechanical property in comparison to antigen-presenting cells (APCs) which are much softer and have the viscoelastic property that is critical to mediate ligand-receptor engagement and activation.

Materials and

Methods: To address this challenge, we propose to engineer synthetic viscoelastic activating cells (SynVACs), i.e., alginate microspheres with well-controlled mechanical properties (particularly viscoelasticity) and bioactivity that mimic the native APCs, and therefore enhance T cell activation, differentiation, expansion, and cancer cell-killing efficiency (Figure 1). The alginate microbeads are fabricated using a microfluidic device based on a pH-induced internal gelation method. The stiffness and viscoelasticity are measured using AFM and a mechanical tester. Anti-CD3 and anti-CD28 antibodies are conjugated to the surface of SynVACs using EDC/NHS chemistry and click-chemistry. The ligand density are measured using confocal imaging and calibration beads. SynVACs are then cocultured with human PBMCs for 14 days, during which they are transduced with CAR.

Results, Conclusions, and Discussions: Our preliminary data have shown that SynVACs increase CAR transduction rate to >90%, promote the stemness of expanded CAR-T cells, and boost the cancer-killing ability of CAR-T cells in vitro and in vivo. Based on these findings, we hypothesize that the stiffness and viscoelasticity of SynVACs regulate the T cell receptor (TCR) activation and thus T cell expansion and differentiation, which in turn boosts the cancer-killing activity of SynVAC-expanded CAR-T cells for long-lasting immunity against cancer.

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