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TPS281 Background: Metastatic colorectal cancer (CRC) has poor prognosis in patients resistant or intolerant to oxaliplatin- and irinotecan-based chemotherapy, and durable responses to salvage regimens such as trifluridine/tipiracil plus bevacizumab remain limited. BTN1A1, a novel immune checkpoint implicated in tumor progression and immune evasion, is under clinical evaluation as a therapeutic target. Nelmastobart, a first-in-class anti-BTN1A1 monoclonal antibody, is being tested in the STCUBE-003 Phase Ib/II trial (NCT06873763) in combination with trifluridine/tipiracil and bevacizumab. This study aimed to validate BTN1A1 as a patient selection biomarker in CRC clinical trials and to explore its relationship with other immune and tumor biomarkers in the tumor microenvironment using ongoing Phase 1b patient samples. BTN1A1 expression was assessed by single-marker IHC for prevalence and multiplex IHC (Opal platform) for spatial profiling with immune markers. Methods: Archived tumor samples from Phase 1b CRC patients in the STCUBE-003 trial were analyzed. BTN1A1 expression was assessed by single-plex IHC using Tumor Proportion Score (TPS) and H-score (0–300). Multiplex IHC was performed to evaluate BTN1A1 with PD-L1, CD8, Ki-67, SLFN11, cytokeratin (CK), and YAP1, enabling assessment of co-expression, spatial distribution, and immune–tumor interactions. Results: The safety run-in portion of STCUBE-003 was conducted from June 17 to August 7, 2025. Six Phase 1b CRC tumor samples were evaluated by IHC. BTN1A1 expression was detected in all samples: one patient (16.7%) had low expression (TPS 20, H-score 20), while five (83.3%) had high expression (TPS ≥50). H-scores ranged from 20 to 300, strongly correlating with TPS (r = 0.84). These results were consistent with prior findings in CRC TMAs and previous BTN1A1 studies. At first response evaluation, two high-TPS patients achieved partial responses (52% and 31% reductions), while four experienced decreasing stable disease. No progressive disease was observed. Multiplex IHC revealed BTN1A1 spatial association with PD-L1, Ki-67, SLFN11, CK, and YAP1. Conclusions: BTN1A1 was consistently expressed across all tumors, with high TPS associated with favorable outcomes. These findings support BTN1A1 as a robust biomarker for patient selection and provide rationale for the ongoing BTN1A1-guided Phase 2 trial. Clinical trial information: NCT06873763 .

Abstract Butyrophilin 1A1 (BTN1A1) is a novel immune checkpoint protein belonging to the butyrophilinfamily. BTN1A1 is expressed in various human tumors, as well as on immune cells. Our findingsindicate a mutual interaction between BTN1A1 and Yes-associated protein 1 (YAP1) expressionin various human solid tumors. Furthermore, BTN1A1 was found to co-localize with nuclearYAP1 expression in cancer patient samples. Previous reports demonstrated that BTN1A1 playsan immunoregulatory role in T cell activation and proliferation. Here, we further show thatBTN1A1 is also involved in the activation and expression of YAP1.YAP1, a key effector of the Hippo signaling pathway, plays a critical role in regulating cellularproliferation, apoptosis, and tissue homeostasis. Dysregulation of YAP1 activity has beenimplicated in several malignancies, including lung and colon cancers. Functional assays revealedthat YAP1 overexpression enhances tumor cell proliferation, migration, and resistance tochemotherapeutic agents. Conversely, YAP1 knockdown or inhibition leads to reduced tumorgrowth and sensitization of cells to therapy.In our analysis, we observed a significant correlation between high levels of BTN1A1 and highlevels of nuclear YAP1 in several cancer patients. To further understand this interaction, weinvestigated the effects of the BTN1A1-YAP1 pathway on lung and colon cancer cells. A549lung cancer cell lines were labeled and cultured as 3D spheroids with human peripheral bloodmononuclear cells (hPBMC), hSTC810, and docetaxel. The A549 spheroids were examined forred fluorescent protein (RFP) disruption following treatment with hPBMC, hSTC810, anddocetaxel, by assessing spheroid size and formation rate. Similarly, LS1034 colon cancer celllines were labeled and cultured as 3D spheroids with hPBMC, hSTC810, and TAS-102. TheLS1034 spheroids were evaluated for RFP disruption after treatment with hPBMC, hSTC810,and TAS-102, by assessing spheroid size and formation rate. In all experiments, the synergisticeffects of hSTC810 in combination with other therapies were analyzed in the context of YAP1expression in the tumor.This study demonstrates that inhibition of BTN1A1 by hSTC810 in cancer cell lines leads tosignificant downregulation of genes associated with the YAP1 response and upregulation of Tcell activation. By simultaneously downregulating YAP1-driven oncogenic pathways andenhancing immune activation, BTN1A1 inhibition offers a novel dual mechanism for combatinglung and colon cancers. These findings pave the way for future clinical exploration of BTN1A1as a key target in cancer immunotherapy. Citation Format: Seung-Hoon Lee, Chunai Wu, Young-Seung Kim, Bong-Ki Hong, Andrew H. Park, Stephen S. Yoo. BTN1A1 and YAP1 crosstalk: A key mechanism in tumor immune evasion and therapeutic targeting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB035.

BackgroundWhile Programmed cell death protein 1 (PD-1)/programmed cell death-ligand 1 (PD-L1) blockade is a potent antitumor treatment strategy, it is effective in only limited subsets of patients with cancer, emphasizing the need for the identification of additional immune checkpoints. Butyrophilin 1A1 (BTN1A1) has been reported to exhibit potential immunoregulatory activity, but its ability to function as an immune checkpoint remains to be systematically assessed, and the mechanisms underlying such activity have yet to be characterized.MethodsBTN1A1 expression was evaluated in primary tumor tissue samples, and its ability to suppress T-cell activation and T cell-dependent tumor clearance was examined. The relationship between BTN1A1 and PD-L1 expression was further characterized, followed by the development of a BTN1A1-specific antibody that was administered to tumor-bearing mice to test the amenability of this target to immune checkpoint inhibition.ResultsBTN1A1 was confirmed to suppress T-cell activation in vitro and in vivo. Robust BTN1A1 expression was detected in a range of solid tumor tissue samples, and BTN1A1 expression was mutually exclusive with that of PD-L1 as a consequence of its inhibition of Janus-activated kinase/signal transducer and activator of transcription signaling-induced PD-L1 upregulation. Antibody-mediated BTN1A1 blockade suppressed tumor growth and enhanced immune cell infiltration in syngeneic tumor-bearing mice.ConclusionTogether, these results confirm that the potential of BTN1A1 is a bona fide immune checkpoint and a viable immunotherapeutic target for the treatment of individuals with anti-PD-1/PD-L1 refractory or resistant disease, opening new avenues to improving survival outcomes for patients with a range of cancers.

Abstract Background Immune checkpoints have emerged as key regulators of the immune responses against various tumor types. Butyrophilin 1A1 (BTN1A1) has been identified as a novel immune checkpoint protein that has the potential to be targeted for new immunotherapeutic treatment options. We have observed that BTN1A1 and PD-L1 expression are mutually exclusive in various human solid tumors. Recently, Nelmastobart (BTN1A1-targeting antibody, hSTC810) has successfully completed phase 1 clinical trials. To ensure the effectiveness of Nelmastobart, it is crucial to possess a profound comprehension of patients’ tumor microenvironment factors involved. Here, we report Opal multiplex immunofluorescence and Hyperion imaging mass cytometry approach that includes a deeper understanding of the spatial relationships in the tumor microenvironment. Methods In a first-of-its-kind Phase 1 clinical trial, we are to characterize the safety profile and determine the maximum tolerated dose (MTD) and/or recommended Phase 2 dose (RP2D). Nelmastobart is administered as monotherapy at increasing dose levels of 0.3, 1, 3, 6, 10, and 15 mg/kg on a Q2W schedule. To understand the heterogeneity of the interaction mechanisms between immune cells and cancer cells, Patients’ FFPE specimens were stained cancer and immune cells using Opal multiplex immunofluorescence and analyzed Hyperion imaging mass cytometer. Results We enrolled 45 pts with various cancers in a phase I trial of anti-BTN1A1, a novel immunotherapy. We tested doses from 0.3 to 15 mg/kg and found no DLTs. We report results for ovarian cancer. Opal multiplex immunofluorescence analysis showed that anti-BTN1A1 therapy increased the infiltration and activation of CD8+ T cells and NK cells and decreased the level of regulatory T cells in the tumor microenvironment. We also found the opposite expression of BTN1A1 in PD-L1-expressing cancer cells in KI67-negative tumors from ovarian cancer patients. Conclusions Nelmastobart treatment activates immune response pathways and enhances anti-tumor activity with anti-PD-L1 combination therapy in vitro, in vivo, and clinical settings. The results of the study suggest that BTN1A1 is a novel immune checkpoint that can be targeted to overcome resistance to conventional therapies in cancer patients. Citation Format: Stephen S Yoo, Youngseung Kim, Seung Hoon Lee, Soohyeon Lee, Sangjoon Shin, Patricia LoRusso, Hyunjin Jung, David Hong. Analysis of tumor microenvironments in ovarian cancer patients receiving anti-BTN1A1 immunotherapy combined with standard-of-care therapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A002.

206 Background: Human serum metabolome profiles have been analyzed to explore the molecular changes that occur with aging. We hypothesized that deep metabolic profiling of sera with different ages would allow the identification of distinct metabolic chronologic patterns as a normal biological baseline to study personal aging. We further hypothesized that metabolic assessment of this chronologic deviation, resulting from advanced precancerous lesion (APL) and stage I/II/III CRC, from the normal reference baseline, would be instrumental for prognosis of relapse revealing underlying pathophysiology. Methods: A cohort of normal (n=3,616, training; n=1,170, testing), 631 advanced adenoma, 1,019 stage I, 404 stage II and 417 stage III serum samples were assembled. Innovative global LCMS metabolomic production were applied to deep profile these subjects. Identification of the age-associated molecular patterns in normal subjects, modeled with an elastic net algorithm, established the reference baseline to mirror a metabolic clock. CRC associated deviation from the precise chronologically paced metabolic patterns was quantified to associate the clinical endpoints of relapse, OSF and PFS, and to identify the tightly associated metabolic pathways. Results: We observed that for those CRCs, the predicted metabolic age can differ from the chronological age with consistent variations, resulting “older” or “younger” metabolic age subgroup in reference to the chronological age. Significant disruptions from the normal baseline were observed in CRCs patients, and consistent stage specific patterns were observed. Outlier, “Older” or “younger” metabolic age subgroup, CRC patients were found with significant future relapse enrichment. Predictive models were derived to case find the patients at risk of future relapse at the CRC diagnosis timepoint. Conclusions: Deviations from the meticulously timed metabolic aging patterns may provide utility to allow prognosis of future clinical endpoints of relapse and overall survival. Close examination of the underlying metabolic pathways, associated with CRC stage specific metabolic patterns, disrupting the baseline ageotypes, not only may improve the sensitivity and specificity of prognostic tests of CRC relapse, but also shed new insights into CRC therapeutics.

Makes ArviZ an optional dependency and adds minimum Python version (3.6).

Shallow Water Equations on a square basin with stochastic deep learning parameterization of subgrid momentum forcing