Biology subjects

Brezinger-Dayan, K.

Publications and source records attributed to Brezinger-Dayan, K..

3 recordsLinked to original sources

bioRxiv2026

A Lymphomimetic Synthetic Immune Niche, Consisting of CCL21 and ICAM1, Accelerates the Expansion of Potent CAR T-cells

Background: Chimeric Antigen Receptor (CAR) T-cell therapy has transformed the treatment of hematologic malignancies, yet, its broader clinical application often faces major challenges, including slow expansion rates, variable transduction efficiency, exhaustion, and functional heterogeneity. Recent studies have demonstrated that a Synthetic Immune Niche (SIN) composed of immobilized CCL21 and ICAM1 promotes the proliferation of murine and human T-cells while preserving their cytotoxic potency. In this study, we explored the capacity of immobilized CCL21 and ICAM1 to enhance the production of highly potent CAR T-cells by facilitating both their expansion and cytotoxic capacity. Methods: CD19-directed CAR T-cells were generated from PBMCs of healthy donors using a clinical-grade protocol. Following retroviral transduction, the cells were expanded on CCL21 and ICAM1 coated plates, or on uncoated control plates. The effects of the SIN treatment on CAR T-cell expansion, morphology, physical properties, phenotypic markers, and potency were systematically evaluated. Results: SIN exposure significantly enhanced CAR T-cell expansion, achieving a 9.3-fold higher total cell yield by day 13, compared with control cultures. This proliferative advantage persisted even after withdrawal of the cells from the synthetic niche on day 10. SIN stimulation preferentially expanded the CAR-transduced population, increasing CAR T-cell frequencies from 45.4% to 77.6%, resulting in a 14.6-fold increase in the absolute number of CAR T-cells compared with untreated cultures. Morphological and phenotypic analyses revealed distinct activated cell morphology, manifested by increased cell size, polarity and granularity, and elevated expression of the activation markers CD137 and CD69. Importantly, CAR T-cells transiently exposed to the SIN retained cytokine secretion and cytotoxic activity comparable to that of continuously SIN-treated cells, indicating that the SIN effect is persistent. Overall, SIN-conditioned CAR T-cells displayed robust antigen-dependent IFN-{gamma} secretion and cytotoxicity against CD19-expressing target cells. Conclusion: Stimulation of CAR T-cells with a synthetic immune niche consisting of immobilized CCL21 and ICAM1 enhances overall expansion while selectively enriching the CAR-transduced population, thereby substantially increasing both the number and prominence of therapeutically relevant CAR T-cells. This effect is accompanied by a persistent activation phenotype and high functional potency. We propose that incorporating SIN stimulation into CAR T-cell manufacturing represents a simple and scalable strategy for improving CAR T-cell yield while maintaining high cytotoxic efficacy.

immunology↗

bioRxiv2026

Context-dependent determinants of CRISPR-Cas9 editing efficiency revealed through cross-species endogenous editing analysis

Accurate prediction of CRISPR-Cas9 guide RNA (gRNA) editing efficiency remains limited, particularly outside human systems, where models trained on exogenous human datasets show poor generalization. We analyzed Cas9 efficiency and repair outcomes using novel endogenous editing data from four human cell types, two tomato cell types, and cells from giant river prawn and black soldier fly. While integrating publicly available predictors via ensemble frameworks improved performance, our analysis revealed hundreds of novel features affecting activity. Crucially, dominant features related to sites competition for gRNA, and local geometric properties varied across systems, highlighting the strong context dependence of Cas9 efficiency and arguing against a universal model. Interestingly, codon usage bias-based features also emerged as informative predictors, as they are proxies for chromatin accessibility. In contrast, trends in repair outcomes remained conserved. This work provides essential resources for more generalizable CRISPR guide design.

synthetic biology↗

bioRxiv2025

Differential effects of immobilized CCL21 and ICAM1 on TILs with distinct expansion properties

Adoptive T cell therapy (ACT), particularly tumor-infiltrating lymphocyte (TIL), holds great promise for cancer treatment, yet it still faces major challenges such as variability in expansion rates, cytotoxic potency and immune suppression. Recent studies suggest that a "synthetic immune niche" (SIN), composed of immobilized CCL21 and ICAM-1, enhances both the expansion and cytotoxicity of murine and patient-derived T cells. Here, we investigate the mechanism underlying the expansion variability by identifying morphological and molecular markers that distinguish low- and high-expanding TILs and predict their ex vivo expansion potential. We further developed novel SIN-based strategies that differentially reinforce the efficacy of both low- and high-expanding TILs. We demonstrate that a 14-day REP with feeder cells and SIN facilitates the proliferation of the low-expanding cells, while the high-expanding counterparts benefit from a sequential expansion protocol of 7 days with feeder cells only, followed by 7 days with SIN treatment. At the end of the REP both TIL populations display high levels of granzyme B and perforin and reduced levels of exhaustion markers. In conclusion, our findings demonstrate that the refined CCL21+ICAM1 SIN treatment improves expansion rates and activation profiles of both TIL populations, thereby enabling personalized SIN-enhanced protocols for TIL-based immunotherapy.

bioengineering↗