Biology subjects

Zoabi, R.

Publications and source records attributed to Zoabi, R..

4 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

Decoupling T Cell Cytotoxicity: A CCL21+ICAM1-Based Synthetic Immune Niche Enhances Tumor Elimination by Accelerating Lytic Hit Delivery

Preserving T cell cytotoxic function during ex vivo expansion remains a major challenge for adoptive cancer immunotherapy. A synthetic immune niche (SIN) composed of immobilized CCL21 and ICAM1 was shown to improve T cell expansion while preserving cytotoxicity; however, it remains unclear which specific step of the T cell killing process is enhanced by the SIN stimulation. Here, we combined advanced imaging based on time-lapse microscopy with a mean-field model to analyze the distinct stages in killing of B16 melanoma cells by CD8+ T cells. This framework enabled us to resolve the tumor cell killing process into discrete steps throughout target cell engagement with SIN-treated T cells and lytic hit delivery. We found that tumor cell death is best explained by a multi-hit process, requiring approximately four discrete hits to trigger cell death. Model-based analysis identified an increase in the lytic hit delivery as the parameter that best accounts for the enhanced cytotoxicity of SIN-treated T cells, a difference not explained by changes in target encounter frequency or conjugate stability. Global sensitivity analysis further showed that tumor control is more strongly improved by enhancing lytic hit delivery than by increasing target encounter rates. These findings fundamentally reorient our understanding of optimized T cell manufacturing, suggesting that the lytic execution step may be the primary rate-limiting bottleneck in this in-vitro system, and that engineering strategies targeting granule polarization or discharge warrant prioritization alongside affinity-enhancement approaches.

immunology↗

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↗

bioRxiv2024

Molecular mechanisms underlying the modulation of T-cell proliferation and cytotoxicity by immobilized CCL21 and ICAM1

BackgroundAdoptive cancer immunotherapy, using engineered T-cells, expressing chimeric antigen receptor (CARs) or autologous tumor infiltrating lymphocytes (TILs) became, in recent years, a major therapeutic approach for diverse types of cancer. However, despite the transformative potential of adoptive cancer immunotherapy, this field still faces a major challenge manifested by the complex interplay between the proliferation rate and cytotoxic capacity of effector CD8+ T cells. MethodsWe performed integrated analysis of specific differentiation markers via flow cytometry, together with gene expression profiling to explore the molecular mechanisms through which a "synthetic immune niche" (SIN), composed of immobilized CCL21 and ICAM1, modulates the interplay between the proliferation and cytotoxic potency of effector CD8+ T cells. ResultsOn day 3, the transcriptomic effect induced by the SIN was largely similar for both DC/OVA and anti CD3/CD28-activated cells. Cell proliferation increased and the cells exhibited high killing capacity. On day 4 and on, the proliferation/cytotoxicity phenotypes were radically "activation-specific"; The DC/OVA-activated cells lost their cytotoxic activity, which, in turn, was rescued by the SIN treatment. Upon longer incubation, the cytotoxic activity further declined, and on day7, could not be rescued by the SIN. SIN stimulation following activation with antiCD3/CD28 beads resulted in a highly proliferative phenotype with low cytotoxicity, yet the cells regained killing activity on day 7. Potential molecular regulations of the SIN effects were identified, based on transcriptomic and multispectral imaging profiling. ConclusionsThese data indicate that cell proliferation and cytotoxicity are negatively correlated, and the interplay between them is differentially regulated by the mode of initial activation. The SIN stimulation greatly enhanced the cell expansion, following both activation modes, while maintaining high cytotoxic potency, suggesting that it could reinforce adoptive cancer immunotherapy.

immunology↗