Abstract B001: Effective generation of potent tumor-infiltrating lymphocytes (TIL) expressing regulatable membrane-bound IL15 (mbIL15) and LIGHT (TNFSF14) from immune-excluded chondrosarcoma
Authors: Ao Zheng , Al-Marayaty Rusul , Aksoy Bulent Arman , Passaro Carmela , Obeidin Farres , Burga Rachel , Roy Nishita , Attar Samer , Peabody Terrance , Ng Juliana , Jing Weiqing , Shinglot Himaly , Zhang Ali , Ocando Alonso Villasmil , Alvarado Andres , Sun Dexue , Sethi Dhruv , Meulen Jan ter , Ols Michelle , Pollack Seth M 
Published: February 2025

Background: TIL cell therapy can induce durable disease regression in solid tumors. TIL engineered with regulatable mbIL15 (cytoTIL15TM cells) demonstrate interleukin 2-independent expansion and function (Burga SITC 2023) and can lead to durable clinical responses (Amaria ASCO 2024). Furthermore, TIL engineered with co-regulated expression of mbIL15 and LIGHT show enhanced efficacy in fibroblast-containing, immunologically cold tumors (Koscso AACR 2024). Chondrosarcomas are often of mesenchymal stem-cell origin and show similar transcriptional signature as fibrotic tumors, including high expression of LTBR and HVEM (TNFRSF14; LIGHT co-receptor). Herein, we sought to determine if tumor-reactive TIL engineered with mbIL15 and LIGHT could be produced from immune-excluded tumor types, such as chondrosarcoma. Methods: This retrospective study used tumors from patients with chondrosarcoma treated at Northwestern University to characterize the tumor microenvironment using immunohistochemistry, TCR sequencing, and spatial transcriptomics. Punches from intratumoral and peritumoral areas of selected tumor sections were compared. We also prospectively collected chondrosarcoma tumors to expand TIL using a conventional (non-engineered) process or the Obsidian process. TIL were characterized via flow cytometry, TCR sequencing, and single-cell sequencing. TIL function was evaluated with LIGHT functional assays as well as co-cultures with autologous tumor spheroids, with cytotoxicity marked by caspase 3/7 staining. Results: Chondrosarcoma tumors had significantly higher peritumoral levels of cells expressing nearly every immunologic marker tested, including CD45, CD68, CD4, CD8, CD20, CD56, CD14, CD206, and MECA-79. Gene expression analysis showed that while large sections of chondrosarcomas have a more inflammatory gene expression profile compared with other similar sarcomas, these genes are confined to the tumor's periphery. TCR sequences detected in the intratumoral area appeared to be from semi-invariant alpha-beta TCR-expressing, innate-like MAIT cells, whereas a more polyclonal T-cell fraction was present in the peritumoral area (2–959 unique complementarity-determining region 3 [uCDR3] in peritumoral region, vs 460–18989 uCDR3 in peritumoral region immune clusters, n=5). Additionally, chondrosarcoma showed 2.58-fold higher expression of HVEM compared with other sarcoma types, providing a rationale for LIGHT-engineered TIL. Co-culture experiments with TIL engineered with co-regulated mbIL15 and LIGHT manufactured from chondrosarcomas demonstrated enhanced cytotoxicity against autologous spheroids compared with non-engineered TIL (ANOVA, p&lt;0.005). Conclusions: While chondrosarcoma is usually classified as an immunologically cold tumor, we observe peritumoral immune cells in chondrosarcoma, and using the Obsidian TIL expansion process coupled with mbIL15 and LIGHT engineering generates potent tumor-specific lymphocytes. These findings support further efforts to develop TIL cell therapy for immunologically cold tumors such as chondrosarcoma.

Citation Format: Zheng Ao, Rusul Al-Marayaty, Bulent Arman Aksoy, Carmela Passaro, Farres Obeidin, Rachel Burga, Nishita Roy, Samer Attar, Terrance Peabody, Juliana Ng, Weiqing Jing, Himaly Shinglot, Ali Zhang, Alonso Villasmil Ocando, Andres Alvarado, Dexue Sun, Dhruv Sethi, Jan ter Meulen, Michelle Ols, Seth M Pollack. Effective generation of potent tumor-infiltrating lymphocytes (TIL) expressing regulatable membrane-bound IL15 (mbIL15) and LIGHT (TNFSF14) from immune-excluded chondrosarcoma [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr B001.