Search

Introduction For chimeric antigen receptor T cell-based (CAR-T) and engineered T cell receptor (TCR) immunotherapies, T cell expansion methods and phenotype/s of transplanted T cells may heavily influence clinical outcomes. Much current focus is on the potential of defined CD4+/CD8+ T cell populations vs bulk, and on the potential superiority of CAR-T cells from naïve (TN) or central memory (TCM) versus effector memory (TEM) cells. Many commercial T cell activation and expansion methods utilize rigid magnetic beads bound to antibodies against CD3 and CD28 as substrates. These methods are often associated with high costs and licensing restrictions for clinical and commercial applications. Additionally, de-beading processes can be highly complex and inefficient, adding additional time, costs and risks. It has been shown that substrate rigidity influences T cell expansion and phenotype. We hypothesized that a novel phase-change substrate could modulate expanded T cell phenotype/s and address de-beading challenges. Methods An alginate-based phase-change hydrogel was synthesized and coated onto magnetic beads to form hydrogel-coated particles of approximately 10 µm diameter. This hydrogel, in the presence of chelating agents, rapidly dissolves, enabling removal magnetic bead removal. The coated particles were conjugated with streptavidin (SA) and bound to biotinylated antibodies against CD3 (OKT3) and CD28 (28.2) to form CD3/CD28 hydrogel particles (CD3/CD28-HP). Human CD3+ T cells from peripheral blood were seeded (Day 0) at 1x10E6 cells/mL in 24 well plates (n=3) in complete RPMI medium supplemented with IL-2. To each well, 25 µL of CD3/CD28-HP were added per 0.5x10E6 cells in a single stimulation. Media addition or change of culture vessel occurred each 2-3 days. Following expansion, chelating agent was added and magnetic beads removed. Flow cytometry was used to assess cell viability and expression of phenotypic markers including CD3, CD4, CD8, CD45RA and CCR7. ELISA was used to measure secretion of IL-2, IL-4, and IFNγ. Residual magnetic beads were counted via hemocytometer. Results CD3/CD28-HP promoted significant T cell expansion of 0.3, 1.4, 2.4, 4.8 and 6.6 population doublings (PD) by Days 2, 5, 6, 9, and 13 respectively (p Phenotypic markers were assessed on Days 6 and 13. Expansion using CD3/CD28-HP led to significantly more CD8+ cells and significantly fewer CD4+ cells versus the starting population on both days (p Following de-beading of expanded cells, cell recovery was 96% for the CD3/CD28-HP-expanded cells and 93% for cells expanded using commercial magnetic bead-based expansion product. Additionally, in de-beaded cells, fewer residual magnetic particles were present in the CD3/CD28-HP-expanded population than in cells expanded via the commercial magnetic bead-based expansion product. Conclusions These data demonstrate the utility of a novel phase-change hydrogel system to efficiently induce T cell proliferation, promote expansion of functional T cells expressing markers associated with CD8+, TN and TCM phenotypes, and to separate expanded cells efficiently from magnetic beads. In future studies, we will determine if T cells expanded using this method show increased stemness and persistence in in vivo models, and further explore the possibilities of this novel system for rapid expansion and recovery of specific T cell subtypes. Disclosures Jesuraj: Quad Technologies: Employment, Other: stock options. Cole:Quad Technologies: Employment, Other: Stock Options. Wells:Quad Technologies: Employment, Other: Stock Options. Qin:Quad Technologies: Employment, Other: Stock options. Kevlahan:Quad Technologies: Employment, Equity Ownership. Maus:Novartis: Patents & Royalties: related to CTL019, Research Funding. Ball:Quad Technologies: Employment, Other: Stock Options.