Multiplexed immunohistochemistry (mIHC) analysis and clinical outcomes of nelmastobart in combination with trifluridine/tipiracil and bevacizumab in patients with refractory colorectal cancer (phase 1b/2).

3602 Background: Colorectal cancer (CRC) patients who are refractory or intolerant to oxaliplatin- and irinotecan-based regimens face limited treatment options and poor prognoses. BTN1A1, a novel immune checkpoint protein involved in tumor progression and immune evasion, presents a potential therapeutic target. Nelmastobart, a first-in-class anti-BTN1A1 monoclonal antibody, is being evaluated in combination with trifluridine/tipiracil and bevacizumab in the Phase 1b/2 STCUBE-003 trial (NCT06873763). This study employs multiplexed immunohistochemistry (mIHC) to characterize the tumor microenvironment (TME) and identify biomarkers predictive of therapeutic response in CRC patients. Methods: Tumor tissues from 120 CRC patients were prescreened for BTN1A1 expression via immunohistochemistry (IHC). A total of 61 patients with a tumor proportion score (TPS) ≥50 were enrolled in the Phase 2 study. Pathologists used the TPS to guide patient selection, with TPS ≥50 as the inclusion criterion. Selected samples were analyzed using mIHC (Opal and CellDIVE), utilizing a 20-protein panel relevant to cancer immunology. Protein co-expression and the spatial distribution of immune and malignant cells were evaluated to assess tumor-immune interactions. Results: Among 120 tumor samples analyzed by IHC, 78 (65%) exhibited TPS ≥50. Sixteen patients (13%) showed no expression (TPS 0), and 26 (21.6%) had low to medium expression (1 ≤ TPS ≤ 49). This expression profile aligns with previous CRC studies (STCUBE-001 and STCUBE-IIT-001). In the initial efficacy evaluation of 39 patients with TPS ≥50, 2 (4.8%) achieved a partial response (PR), 27 (69.2%) showed stable disease (SD), and 8 (20.5%) had progressive disease (PD). High BTN1A1 expression was significantly associated with a higher clinical response rate. mIHC analysis further revealed that BTN1A1 expression correlated with Ki-67, SLFN11, and YAP1, as well as distinct patterns of CD8⁺ T-cell infiltration. Conclusions: BTN1A1 is consistently and highly expressed in refractory CRC (median TPS 50). mIHC analysis confirms that BTN1A1 is closely integrated with key immune checkpoints and tumor markers within the TME. Initial clinical results, which show disease control in the majority of patients, particularly within the TPS ≥50 population, support the utility of BTN1A1 as a predictive biomarker for Nelmastobart combination therapy. These findings justify the ongoing biomarker-driven Phase 2 portion of the STCUBE-003 trial. Clinical trial information: NCT06873763 .