Pooled screening for CAR function identifies novel IL13Rα2-targeted CARs for treatment of glioblastoma.

Chimeric antigen receptor therapies have demonstrated potent efficacy in treating B cell malignancies, but have yet to meaningfully translate to solid tumors. Here, we utilize our pooled screening platform, CARPOOL, to expedite the discovery of CARs with anti-tumor functions necessary for solid tumor efficacy. We performed selections in primary human T cells expressing a library of 1.3×10 6 3 rd generation CARs targeting IL13Rα2, a cancer testis antigen commonly expressed in glioblastoma. Selections were performed for cytotoxicity, proliferation, memory formation, and persistence upon repeated antigen challenge. Each enriched CAR robustly produced the phenotype for which it was selected, and one enriched CAR triggered potent cytotoxicity and long-term proliferation upon in vitro tumor rechallenge. It also showed significantly improved persistence and comparable antigen-specific tumor control in a microphysiological human in vitro model and a xenograft model of human glioblastoma. Taken together, this work demonstrates the utility of extending CARPOOL to diseases beyond hematological malignancies and represents the largest exploration of signaling combinations in human primary cells to date.

G9a/GLP inhibition during ex vivo lymphocyte expansion increases in vivo cytotoxicity of engineered T cells against hepatocellular carcinoma.

Engineered T cells transiently expressing tumor-targeting receptors are an attractive form of engineered T cell therapy as they carry no risk of insertional mutagenesis or long-term adverse side-effects. However, multiple rounds of treatment are often required, increasing patient discomfort and cost. To mitigate this, we sought to improve the antitumor activity of transient engineered T cells by screening a panel of small molecules targeting epigenetic regulators for their effect on T cell cytotoxicity. Using a model for engineered T cells targetting hepatocellular carcinoma, we find that short-term inhibition of G9a/GLP increases T cell antitumor activity in in vitro models and an orthotopic mouse model. G9a/GLP inhibition increases granzyme expression without terminal T cell differentiation or exhaustion and results in specific changes in expression of genes and proteins involved in pro-inflammatory pathways, T cell activation and cytotoxicity.

Isotropic myosin-generated tissue tension is required for the dynamic orientation of the mitotic spindle.

The ability of cells to divide along their longest axis has been proposed to play an important role in maintaining epithelial tissue homeostasis in many systems. Because the division plane is largely set by the position of the anaphase spindle, it is important to understand how spindles become oriented. While several molecules have been identified that play key roles in spindle orientation across systems, most notably Mud/NuMA and cortical dynein, the precise mechanism by which spindles detect and align with the long cell axis remain poorly understood. Here, in exploring the dynamics of spindle orientation in mechanically distinct regions of the fly notum, we find that the ability of cells to properly reorient their divisions depends on local tissue tension. Thus, spindles reorient to align with the long cell axis in regions where isotropic tension is elevated, but fail to do so in elongated cells within the crowded midline, where tension is low, or in regions that have been mechanically isolated from the rest of the tissue via laser ablation. Importantly, these differences in spindle behavior outside and inside the midline can be recapitulated by corresponding changes in tension induced by perturbations that alter nonmuscle myosin II activity. These data lead us to propose that isotropic tension within an epithelium provides cells with a mechanically stable substrate upon which localized cortical motor complexes can act on astral microtubules to orient the spindle.