SignificanceEx vivo manipulation of primary cells is critical to the success of this emerging generation of cell-based therapies, such as chimeric antigen receptor T cells for the treatment of cancer and CRISPR for the correction of developmental diseases. However, the limitations of existing delivery approaches may dramatically restrict the impact of genetic engineering to study and treat disease. In this paper, we compared electroporation to a microfluidic membrane deformation technique termed “squeezing” and found that squeezed cells had dramatically fewer side effects than electroporation and gene expression profiles similar to those of unmanipulated cells. The significant differences in outcomes from the two techniques underscores the importance of understanding the impact of intracellular delivery methods on cell function for research and clinical applications.

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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<0.01-p<0.001 vs Day 0). Similarly, CD3/CD28-HP-induced expansion in a separate lab using a different T cell donor yielded 4.7 PD by Day 9 (p<0.001 vs Day 0). 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<0.05-p<0.001). When compared to a commercially available magnetic CD3/CD28 bead product, CD3/CD28-HP produced a significantly larger CD8+ population on Days 6 (p<0.05)and 13 (p<0.001), and a smaller population of CD4+ T cells on Day 13 (p<0.01). CD3/CD28-HP-based expansion significantly increased the percentage of CD3/CD45RA expressing T cells compared with the magnetic bead-based product on Day 6 (p<0.05). Also, on Day 6, T cells expanded using CD3/CD28-HP showed increased CD8/CD45RA/CCR7 expression when compared to T cells expanded with the commercial magnetic bead product (p<0.05). Cytokine secretion was assessed on Days 6 and 13. Cells expanded using both expansion methods secreted IL-2, IL-4, and IFNγ, with no significant differences in secretory function observed between expansion methods. 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.

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Background: Tumor-specific T cells possess unique potential for cancer therapy but are limited by T cell exhaustion and anergy induced in the tumor microenvironment. Ex vivo manipulation of these T cells to maintain their full function is critical to their success clinically. Yet, limitations of existing ex vivo delivery approaches dramatically restrict their function and thus limit their therapeutic use. Methods: Genome-wide profiling was used to identify the impact of optimized electroporation treatment and the SQZ cell therapy platform on gene expression in human T cells. The profiling was paired with a 42 key T cell cytokine-multiplex analysis comprised of to assess perturbation of cytokine secretion. We then compared the in vivo functionality of immune checkpoint deleted antigen-specific T cells, modified by either electroporation or SQZ delivery of CRISPR/Cas9, and adoptively transferred into tumor bearing mice. Finally, genomic editing of tumor infiltrating leukocyte (TIL) derived T cells was compared using either electroporation or SQZ and subsequent effector response upon re-exposure to tumor cells. Results: Impactful disruptions in transcript expression after treatment with electroporation (17% of genes mis-regulated, FDR q &lt;0.1) we identified, whereas cells treated with SQZ had similar expression profiles to untreated control cells (0% of genes mis-regulated, FDR q &lt;0.1). These genetic disruptions result in concomitant perturbation of cytokine secretion and effector response. Ultimately, the effects at the transcript and protein level resulted in functional deficiencies in vitro and in vivo with electroporated antigen-specific and TIL derived T cells failing to demonstrate sustained antigen-specific effector responses and tumor control with or without immune checkpoint editing. Conclusions: This work demonstrates that functional modifications to tumor-specific T cells ex vivo can restore and improve their function upon re-exposure to tumor cells but that the delivery mechanism used is critical to the desired phenotype. The significant differences in outcomes from the two techniques tested here underscores the importance of understanding the impact of intracellular delivery methods on cell function for research and clinical applications. For both research and therapeutic applications with primary T cells, the functional consequences of the selected intracellular delivery technique and its impact on cell phenotype should be carefully evaluated. Citation Format: Luke Cassereau, Julie M. Cole, Roslyn Yi, Jacquelyn L. Hanson, Josh Bugge, Tia DiTommaso, Howard Bernstein, Armon Sharei. Tumor-specific T cell engineering for enhanced effector function via microfluidic delivery of bioactive molecules [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1445.