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Background: The autologous cell therapy field has investigated numerous novel strategies and processes to improve response rates and expand the use of adoptive cell therapies (ACT) to patient suffering from a broad range of cancers. Tumor infiltrating lymphocytes (TIL) can be harvested from tumors, expanded in vitro, and infused to patients leading to substantial clinical benefits in some patient populations. Tumor reactivity of TIL can vary greatly, representing a key limiting factor of bulk TIL therapies. The ability to generate an autologous cell product enriched in tumor reactive cells while limiting the presence of potentially detrimental and/or competitive non-reactive cells is highly desirable for next generation TIL products. Here we demonstrate that direct selection of antigen experienced TIL from tumor dissociates is feasible and allows to obtain functional cells enriched for tumor reactivity from various tumor indications. Methods: Patient-derived tumor material from various indications, including ovarian, kidney, colorectal cancer and lung tumors were first processed into single cell suspensions, maximizing TIL recovery and viability. Antigen experienced TIL were immediately selected based on the co-expression of PD-1 and CD39, using fluorescence-activated single cell sorting (FACS). The sorted cells or the unselected counterparts were expanded in a rapid expansion protocol (REP), then analyzed for phenotypic and functional characteristics and reactivity against autologous tumor cells. Results: The PD-1+ CD39+ selection strategy consistently allowed sorting of a population of viable TIL that was amenable to in vitro culture from tumors of various cancer indications. The selected TIL successfully expanded in a rapid expansion protocol. TCR sequencing analysis revealed that the PD-1+ CD39+ sorted populations were enriched in unique clones compared to the unselected populations. Expanded PD-1+ CD39+ selected cells demonstrated the ability to produce key effector cytokines upon restimulation in polyclonal assays. Importantly, the PD-1+ CD39+ expanded TIL were enriched for tumor reactive T cells and showed improved cytotoxic activity against autologous tumors. Conclusions: PD-1+ CD39+ selected TIL can be successfully isolated and expanded in vitro, generating a TIL product of superior reactivity in multiple cancer indications. PD-1+ CD39+ selected TIL showed increased cytokine secretion and cytotoxic activity against autologous material, indicating that this selection strategy enriches for functional tumor-reactive lymphocytes, which is likely to be a key feature of successful ACT. Citation Format: Christophe Pedros, Larissa Pikor, Anna Fritzsche, Zachary K. Jilesen, Bethany Macleod, Niloufar Khojandi, Bryant Thompson, Matthew Thayer, Nikolas Bryan, Emily Carron, Sowbarnika S Ratliff, April Fraley, Jake Nikota, Robert Fisher, Sebastien Delpeut, Anna Kluew, Madysson Scott, Christian Laing, Leo He, Antoine Bernard, Nathalie Brassard, Simon Turcotte, Timothy J Langer, David Stojdl, Stewart Abbot, Barbara Sennino. Direct selection of PD1+ CD39+ tumor infiltrating lymphocytes (TIL) from tumor dissociates enrich for functional tumor-reactive cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4052.

Background: Tumor infiltrating lymphocyte (TIL) therapy is capable of mediating durable complete responses in melanoma. While solid tumors such as colorectal cancer (CRC), non-small cell lung cancer (NSCLC), ovarian and breast have been shown to contain neoantigen reactive TIL, the success of bulk TIL therapy in these tumors has been limited. Enhancing tumor reactivity through the selective expansion of neoantigen-reactive subpopulations, has demonstrated success in cancers outside of melanoma underscoring the potential of a neoantigen selected TIL approach in indications with lower tumor mutational burdens. Here we demonstrate that the TIDAL-01 process, which utilizes tumor-specific mutation containing peptides to select neoantigen reactive TIL produces TIL products significantly enriched in neoantigen reactivity. Methods: Fresh tumors were cut into fragments or dissociated and cultured in a primary expansion (preREP). Antigen presenting cells (APCs) were isolated and expanded from patient matched blood. Whole exome and RNA sequencing was performed on tumor tissue and autologous PBMCs and used to predict and prioritize neoantigen mutations. Peptides encoding the mutations were synthesized, loaded onto APCs and co-cultured with autologous TIL. Neoantigen reactive TIL were selected by fluorescence activated cell sorting (FACS), based on the upregulation of the activation markers CD134 and CD137 and expanded with a rapid expansion protocol (REP). Bulk and unselected TIL were expanded alongside for comparison. Neoantigen reactivity was quantified and deconvoluted by cytokine secretion, degranulation, upregulation of CD134/CD137 by flow and when practical, killing of autologous tumor cell lines or organoids. Results: Successful TIL expansion was achieved in 31/34 (91%) tumors (14/17 CRC, 10/10 NSCLC, 3/3 ovarian and 3/3 melanoma) using both tumor fragments and dissociated tumors. CRC tumors accounted for half of the samples (17/34), and the tumor mutational burden within these samples varied substantially, ranging from 229 to 5436 mutations. Upregulation of CD134 and CD137 and increased IFN-γ production was observed in all samples upon co-culture with peptide loaded APCs. Peptide restimulation and deconvolution revealed that the TIDAL-01 process is capable of enriching for both CD4 and CD8 reactivities. Selected TIL products produced up to 50x more IFN-γ, TNF-α and Granzyme B than bulk TIL and at least 2x higher levels of degranulation, indicative of greater killing potential. Conclusions: TIL from metastatic CRC, melanoma, NSCLC and ovarian tumors were successfully expanded from the majority of patients. Co-culture of TIL and peptide loaded APCs followed by FACS significantly enriched for neoantigen reactivity compared to bulk TIL, demonstrating the potential of the TIDAL-01 process to produce selected TIL products for the treatment of non-melanoma tumors. Citation Format: Larissa A. Pikor, Antoine Bernard, Nathalie Brassard, Anna Fritzsche, Anna Kluew, Zachary K. Jilesen, Jake Nikota, Rohan Bareja, Christian Laing, David F. Stojdl, TJ Langer, Stewart Abbot, Barbara Sennino, Simon Turcotte. TIDAL-01: A selected TIL process that enriches for neoantigen reactive TIL in solid tumors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4049.