Abstract 3185: Optimizing Stim-RTM, a synthetic stimulatory agent, for feeder-free tumor-infiltrating lymphocyte manufacturing

Abstract The choice of stimulation agent (e.g. irradiated feeder cells) utilized to expand cells for adoptive cell therapy may limit both scalability and potency of the product. Synthetic cell stimulating agents are tunable, consistent, scalable, and can be adapted to a range of cell therapies. Previously, we reported the development of Stim-RTM for use in melanoma tumor infiltrating lymphocyte (TIL) manufacturing. To broaden the potential application of the technology, we describe optimization of Stim-R in the TIL manufacturing process in additional cancer types. To achieve robust TIL expansion from multiple types of cancer, screens for additional feeder activation cues were conducted by assessing expression of T-cell stimulating ligands on the feeder cell surface, assessing the secretome of feeder cells, and assessing expression of stimulable surface receptors on the initial TIL populations. These data were utilized to develop a new, optimized Stim-R process, which incorporated both new surface cues on the Stim-R platform and soluble signals in the TIL culture media. The optimized Stim-R process was utilized to replace feeder cells in the second expansion step of the TIL manufacturing process. TIL products manufactured with the optimized Stim-R process or feeder cells were evaluated for total cell yield, the expression of stemness markers using flow cytometry, and retention of putative tumor-reactive clones, assessed as the highest frequency clones from the starting tumor material using T-cell receptor sequencing. We developed a new formulation of Stim-R, Stim-RV2, which engages CD2, CD3, CD28, CD137 (41BB), and ICOS, and a set of feeder-secreted proteins (FSPs), which are supplemented in the TIL culture media. An optimized Stim-R process that incorporates both Stim-RV2 and the FSPs resulted in increased TIL expansion from all melanoma and NSCLC tumors evaluated compared to products generated using feeder cells. TIL products generated with the optimized Stim-R process demonstrated similar CD8 T-cell stemlike phenotypes to feeder-produced TIL and retained a similar proportion of the tumor reactive cells compared to a feeder-based production process. Stim-R can serve as a synthetic replacement for feeder cells in TIL manufacturing. We have optimized soluble and tethered signals incorporated into the Stim-R process to increase TIL expansion in multiple types of tumors. TIL produced with the optimized Stim-R process were phenotypically comparable to TIL produced with a feeder-based process and retained a similar proportion of the putative tumor reactive clones. The Stim-R technology has potential to overcome scalability limitations in TIL manufacturing and may make TIL therapy accessible for more patients. Furthermore, we demonstrate the versatility of the Stim-R platform as a stimulating agent that can be tuned with activation signals based on cell population and desired phenotype. Citation Format: Riley P. Allen, Jason Duecker, Mikayla Min, Samantha Chan, Christina Ta, Benjamin Harris, Emily Fu-Sum, Stefan Siebert, Purnima Sundar, Yogin Patel, Shobha Potluri, Suman Vodnala, Gary Lee, Alexander Cheung, Aileen Li. Optimizing Stim-R, a synthetic stimulatory agent, for feeder-free tumor-infiltrating lymphocyte manufacturing. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3185.