Background The success of chimeric antigen receptor (CAR) T cell therapy for hematological malignancies has not yet translated into long-term elimination of solid tumors indicating the need for adequately tuning CAR T cell functionality. Methods We leveraged a translational pipeline including biophysical characterization and structural prediction of the CAR binding moiety, evaluation of cellular avidity, synapse formation, T cell motility, and functional capacities under repetitive target challenge and in sustained tumor control. Results As an example of clinical relevance, we derived a panel of anti-Her2 CARs covering a 4-log affinity range, all expected to target the same Her2 epitope. The same scFv mutations increased both antigen-specific affinity, cellular avidity, and antigen-independent “tonic” signaling; above a minimum threshold, raise in affinity translated into functional avidity in a non-linear fashion. In this case, replacement by amino acids of higher hydrophobicity within the scFv coincidentally augmented affinity, non-specific binding, spontaneous CAR clustering, and tonic signaling, all together relating to T cell functionality in an integrated fashion. Conclusions Data emphasize that tonic signaling is not always due to the positive charge but can be driven by hydrophobic interactions of the scFv. CAR binding affinity above the threshold and tonic signaling are required for sustained T cell functionality in antigen rechallenge and long-term tumor control.

Background/Objectives: Monoclonal antibody therapies for HER2-positive tumors frequently encounter resistance, requiring alternative treatment strategies. This study investigates the use of natural killer (NK) cells expressing HER2-specific chimeric antigen receptor (CAR) to address this issue. CAR NK cells have several benefits over CAR T cells: they are less likely to cause severe side effects such as cytokine release syndrome and neurotoxicity, can be sourced from various origins, and do not trigger Graft versus Host Disease, making them ideal for “off-the-shelf” applications. Methods: We have generated NK-92 cell lines expressing first, second and third-generation HER2-specific CARs with CD28 and/or 41BB costimulatory domains using a retroviral transduction system, followed by FACS sorting and expansion to obtain pure HER2-CAR NK-92 cell products for functional benchmarking. Results: In vitro tests showed that these CAR NK cells were effective against both trastuzumab-sensitive (CD44⁻) and -resistant (CD44⁺) tumors in monolayer cultures. However, in three-dimensional spheroid models and in vivo xenografts, they were less effective against CD44+ trastuzumab-resistant tumors. Conclusions: This reduced efficacy highlights the significant role of the tumor microenvironment, particularly the extracellular matrix, in hindering the therapeutic potential of CAR NK cells. Despite the promising in vitro performance of CAR NK cells, this study emphasizes the need for improved strategies to enhance their penetration and effectiveness in resistant tumors: optimizing CAR constructs and devising methods to overcome extracellular matrix barriers are crucial for advancing CAR NK cell therapies in oncology.

Although hyperpolarization-activated and cyclic nucleotide-gated 2 channels (HCN2) are expressed in multiple cell types in the gut, the role of HCN2 in intestinal motility is poorly understood. HCN2 is down-regulated in intestinal smooth muscle in a rodent model of ileus. Thus, the purpose of this study was to determine the effects of HCN inhibition on intestinal motility. HCN inhibition with ZD7288 or zatebradine significantly suppressed both spontaneous and agonist-induced contractile activity in the small intestine in a dose-dependent and tetrodotoxin-independent manner. HCN inhibition significantly suppressed intestinal tone but not contractile amplitude. The calcium sensitivity of contractile activity was significantly suppressed by HCN inhibition. Inflammatory mediators did not affect the suppression of intestinal contractile activity by HCN inhibition but increased stretch of the intestinal tissue partially attenuated the effects of HCN inhibition on agonist-induced intestinal contractile activity. HCN2 protein and mRNA levels in intestinal smooth muscle tissue were significantly down-regulated by increased mechanical stretch compared to unstretched tissue. Increased cyclical stretch down-regulated HCN2 protein and mRNA levels in primary human intestinal smooth muscle cells and macrophages. Overall, our results suggest that decreased HCN2 expression induced by mechanical signals, such as intestinal wall distension or edema development, may contribute to the development of ileus.