A Simple and Robust Single-Step Method for CAR-Vδ1 γδT Cell Expansion and Transduction for Cancer Immunotherapy.

The γδT cell subset of peripheral lymphocytes exhibits potent cancer antigen recognition independent of classical peptide MHC complexes, making it an attractive candidate for allogeneic cancer adoptive immunotherapy. The Vδ1-T cell receptor (TCR)-expressing subset of peripheral γδT cells has remained enigmatic compared to its more prevalent Vγ9Vδ2-TCR and αß-TCR-expressing counterparts. It took until 2021 before a first patient was dosed with an allogeneic adoptive Vδ1 cell product despite pre-clinical promise for oncology indications stretching back to the 1980s. A contributing factor to the paucity of clinical progress with Vδ1 cells is the lack of robust, consistent and GMP-compatible expansion protocols. Herein we describe a reproducible one-step, clinically translatable protocol for Vδ1-γδT cell expansion from peripheral blood mononuclear cells (PBMCs), that is further compatible with high-efficiency gene engineering for immunotherapy purposes. Briefly, αßTCR- and CD56-depleted PBMC stimulation with known-in-the-art T cell stimulators, anti-CD3 mAb (clone: OKT-3) and IL-15, leads to robust Vδ1 cell expansion of high purity and innate-like anti-tumor efficacy. These Vδ1 cells can be virally transduced to express chimeric antigen receptors (CARs) using standard techniques, and the CAR-Vδ1 exhibit antigen-specific persistence, cytotoxicity and produce IFN-γ. Practicable, GMP-compatible engineered Vδ1 cell expansion methods will be crucial to the wide-spread clinical testing of these cells for oncology indications.

In-Plate Cryopreservation of 2D and 3D Cell Models: Innovative Tools for Biomedical Research and Preclinical Drug Discovery.

Cell-based assays performed in multiwell plates are utilized in basic and translational research in a variety of cell models. The assembly of these multiwell platforms and their use is often laboratory specific, preventing the standardization of methods and the comparison of outputs across different analytical sites. Moreover, when cell models are based on primary cells with specialized culture requirements, including three-dimensional (3D) cell culture, their complexity and the need for manipulation by experienced operators can add significant cost and introduce long lead times to analysis, both of which are undesirable in any preclinical situation. To address this issue, we explored adaptations of cryopreservation technology that allow cells to be cryopreserved in-plate, ready for use in analysis, and have developed a method applicable to cells from different origins and different culture formats. Here we describe the application of this technology to conventional two-dimensional (2D) monolayers of human mesenchymal stem cells (MSCs) and human macrophages derived from primary monocytes, and to 3D cultures of hepatic organoids, colon organoids, and colon tumor organoids, each presented for cryopreservation in their obligate extracellular matrix. We demonstrated that cell viability, cell physiology, and cytotoxic sensitivity were maintained after cryopreservation, such that the models offer the means to uncouple model assembly from analytical use and to standardize cell models in product form for distribution to end users.