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

Sofi Yado¹Rawan Zoabi¹Karin Brezinger-Dayan²Shira Albeck¹Tamar Unger¹Moran Meiron³Alessio David Nahmad²Aya Tzur Gilat³Michal J Besser²Benjamin Geiger^1*

• ¹Weizmann Institute of Science, Rehovot, Israel
• ²Rabin Medical Center, Petah Tikva, Israel
• ³Orgenesis Biotech, Misgav, Israel

Adoptive T cell therapy (ACT), particularly tumor-infiltrating lymphocyte (TIL)-based therapy holds great promise for cancer treatment, yet it still faces major challenges such as patient-to-patient variability in expansion rates and, cytotoxic potency and immune suppression. Recent studies suggest that a "synthetic immune niche" (SIN), composed of immobilized CCL21 and ICAM-1, can enhance both the expansion and cytotoxicity of murine and patient-derived T cells. To explore the mechanisms underlying the variability of expansion and cytotoxic potency variability, we conducted morphological and molecular phenotyping of multiple TIL specimens from different donors immediately following the pre-Rapid Expansion Protocol (pre-REP) stage, enabling us to predict their expansion potential. We further developed novel SIN-based strategies that differentially reinforce the efficacy of both low-and high-expanding TILs. Our findings experiments revealed two distinct TIL groups subsets with either low-or high-proliferation properties, identified across cultures derived from different patients. We further demonstrate that a 14-day REP with feeder cells and SIN facilitates the proliferation of the low-expanding cells, while the expansion of high-expanding TILs benefits from a sequential expansion protocol, consisting 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. Importantly, functional cytotoxicity assays using autologous tumor targets demonstrated that SIN stimulation improved the tumor-killing capacity of low-expanding TILs, while preserving the potent cytotoxicity of the high-expanding TILs. These data indicate that the refined CCL21+ICAM1 SIN treatment improves expansion rates and activation profiles of both 4 TIL populations, thereby enabling a powerful, personalized SIN-enhanced protocol for TIL-based immunotherapy.