Abstract 3482: Enhanced targeting of HPV positive cancers using gamma delta 1 T cells armored with an HPV E7-specific TCR: a novel approach to overcome HLA downregulation

Abstract T-cell receptor (TCR) engineered T cells have emerged as a promising cancer therapeutic. While the clinical trial using ab T cells targeting the HPV E7 protein showed promise, it also highlighted a concerning issue: tumor escape due to the acquisition of defects in peptide-HLA presentation. As an alternative cellular platform, Gamma Delta One T cells (DOT) offer several advantages over alpha/beta T cells. These include their tissue-homing abilities, their use as allogeneic cells without GVHD risk, and their potential to target cells through both TCR and NK-like receptors (which can kill HLA Class-I deficient cells). The overall purpose of this study is to establish DOTs as an effective platform for TCR-T therapy targeting solid tumors, demonstrating enhanced therapeutic efficacy in overcoming tumor escape through mutations in the antigen-processing machinery (APM). We developed a novel protocol to isolate and expand DOTs from peripheral blood using a Delta 1 TCR-specific antibody and EasySep positive selection. By day 4, DOTs reached 37.3% (SD ± 10.3%) enrichment, increasing to 91.85% (SD ± 2.15%) by the end of culture. From transduction day, cells expanded over 100-fold by day 15, demonstrating scalability for therapeutic use. Efficiently transduced with an E7-specific TCR engineered to minimize mispairing (murine constant alpha and beta chains), we achieved transduction levels comparable to alpha/beta T cells. DOTs expressed high levels of NK-like receptors, including NKG2D (94.85% SD ± 0.55%), DNAM-1 (99.1% SD ± 0.3%), and NKp30 (76% SD ± 11%), crucial for targeting HLA-negative tumors. Using HPV+ E7+ SCC90 and CaSki cell lines with varying NKG2D ligand expression, we performed CRISPR/Cas9-mediated B2M knockout to generate HLA class I-deficient variants. Cytotoxicity was evaluated using in vitro chromium release (short-term) and IncuCyte (long-term) assays, where DOTs consistently outperformed alpha/beta T cells in killing HLA-heterogeneous tumors. DOT TCR-T cells exhibited comparable cytotoxicity to alpha/beta TCR-T cells against HLA-A*02:01+ E7+ targets while excelling against HLA-negative variants. These findings establish DOTs as a robust platform for TCR-engineered therapies, capable of overcoming tumor immune evasion across diverse HLA settings. We are currently validating these results in vivo using murine models. In summary, DOTs offer a promising platform for TCR-engineered therapies, excelling in targeting HLA-heterogeneous tumor populations through their dual recognition mechanisms. This work highlights their potential for developing off-the-shelf TCR-T therapies to overcome HLA downregulation and tumor heterogeneity in solid tumors. Citation Format: Andres Mosquera, Diego F. Chamorro, Paul Shafer, Lauren K. Somes, Emily Madaras, Valentina Hoyos. Enhanced targeting of HPV positive cancers using gamma delta 1 T cells armored with an HPV E7-specific TCR: a novel approach to overcome HLA downregulation [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 3482.