Invigorating pre-existing tumor-infiltrating expandable (TIE) T cell clonotypes in exceptional responders with dual PARP-PD-1 blockade in homologous recombination deficient (HRD) pancreatic cancer

Background: Most pancreatic cancers (PC) are inert to immunotherapy, yet a subset of metastatic PC derives durable benefit from PARP-PD-1 blockade (POLAR trial, NCT04666740). The 2-year survival rate was 56% for patients with HRD disease (N=33). Previously, we identified circulating TCR clonotypes that expanded on treatment in responders. These clonotypes constituted 0.05% of circulating TCRs at baseline and 10% were barcode-matched to intratumoral T cells by scTCR-seq, defining tumor-infiltrating expanding (TIE) clonotypes. TIE clonotypes were identified exclusively in 7 exceptional responders (minimum OS > 18m, median OS not reached). However, the T cell lineage, state and spatial organization of TIE clonotypes remain unknown. Methods: We performed Xenium spatial transcriptomics of 53 longitudinal primary and metastatic samples from 36 POLAR participants, including 5 exceptional responders using a customized 480-gene panel, including probes designed for specific TIE clonotypes (N=18) identification. High-quality neoepitopes from long-term responders were predicted from exome sequencing and subsequently generated for functional T-cell activation assays. Results: Over 3.2 million malignant and stromal cells were profiled spatially. TIE + clonotypes were detected at the time of diagnosis, prior to any systemic treatment, in both available samples (2/2) and persisted at least 3 years from initiation of PARP/PD-1 blockade. In the 5 exceptional responders, TIE + clonotypes accounted for 1.49% of the intratumoral CD8 T-cell compartment across all timepoints. Prior to disease progression, TIE + clonotypes were restricted to the CD8⁺ T cell lineage, and were spatially closer to tumor cells than TIE⁻ CD8⁺ T cells (median distance 62 vs 121 µm), while also displaying enhanced cytotoxicity (IFNG⁺), a type 1 effector profile (TBX21 + ), and chronic antigen-driven activation (ENTPD1 + , TOX + ). In contrast, at disease progression, the median distance between TIE + CD8⁺ T cells and tumor cells increased to 82 µm, along with TIE detection among FoxP3 + CTLA4 + Tregs (36% of TIE). Baseline resistance was associated with an MKI67^hi malignant cell state exhibiting enhanced KRAS signaling activity, coupled with myofibroblast-driven stromal remodeling that constrained T-cell infiltration. Predicted neopeptides in TIE + responders are undergoing functional T cell assays to elucidate specific immune responses. Conclusions: Exceptional benefit from PARP-PD-1 blockade in PC is associated with the expansion and sustained intratumoral positioning of rare, pre-existing tumor-reactive CD8⁺ T cell clonotypes. These data support a model in which select HRD tumors are immunogenic from disease onset and resistance emerges at least partly through stromal remodeling and spatial immune exclusion.