Abstract Introduction: Despite the clinical success of immune checkpoint inhibitors (ICIs), microsatellite-stable (MSS) tumors, which account for the majority of colorectal cancer (CRC), remain resistant to anti-PD-1 therapy. While VEGF/VEGFR-targeted combination therapies can provide partial remission, they fail to sufficiently reconstitute the immunosuppressive tumor microenvironment (TME). TGF-β signaling induces immune exclusion and stromal fibrosis in CRC, contributing to resistance to VEGF and PD-1 blockade. In this study, we investigated whether vactosertib (Vac), a TGF-β receptor type I inhibitor, could overcome this resistance when administered in combination with anti-PD-1 and VEGF inhibitors in the CT26 CRC model with PD-1 resistance. Methods and Results: To establish a clinically meaningful PD-1 resistance model, CT26 tumors that initially responded to anti-PD-1 therapy but later progressed according to RECIST criteria were serially passaged to establish the CT26-αPD-1/R2 cell line. Maintained PD-1 antibody binding during progression indicated that resistance was not due to reduced target engagement. BALB/c mice bearing tumors were treated with the FDA-approved pan-VEGFR inhibitor fruquintinib (Fru), an anti-PD-1 antibody, and/or Vac. Vac enhanced the efficacy of Fru (TGI 10% → 47%) and anti-PD-1 (TGI 17% → 42%) monotherapy. Triple combination therapy showed a complete response rate of 20-33%, demonstrating a significantly greater antitumor effect (TGI 86%) than dual blockade of anti-PD-1 and anti-VEGF (TGI 50%). Subsequent RNA-seq analysis revealed that the triple combination therapy was the only treatment that induced a robust intratumoral immune response. Specifically, the triplet activated T cells and upregulated genes involved in myeloid activation, phagocytosis, and antigen presentation, while also increasing B cell activation. TIL analysis showed that the triplet increased IFNγ+CD8+ T cells by 6.0-, 12.6-, and 3.9-fold compared to Vac + Fru, anti-PD-1 + Fru, and Vac + anti-PD-1 combination therapy, respectively. The ratios of activated CD4+ T cells and M1/M2 macrophages showed a similar pattern. Furthermore, the triplet preferentially enhanced effector memory T cells, suggesting persistent and memory-rich adaptive immunity. In a second trial using a VEGFR2-selective inhibitor, Vac similarly improved the efficacy of VEGFR2 inhibitor plus anti-PD-1 therapy (TGI 39% → 63.3%). Conclusions: Triple blockade using Vac, an anti-PD-1 inhibitor, and a VEGFR inhibitor achieved superior antitumor efficacy and broad immune activation compared to all other dual combination therapies in PD-1-resistant CRC. These results suggest that TGF-β inhibition is essential for overcoming treatment resistance in an immunosuppressive TME, supporting the clinical evaluation of this triple combination in refractory CRC. Citation Format: Seong-Jin Kim, Dong Woo Kang, Hye Jin Kim, Seonho Yoo, Jin Beom Bae, June Myoung Kim, Min Kyu Kim, Jinhwan Kim, Jin Su Kim, Ye Seon Kim, Jeong Su Lee, Kwiyeom Yoon, . Rewiring the immune-excluded tumor microenvironment: Vactosertib/anti-PD-1/VEGF inhibitor triplet therapy reinstates antitumor immunity in CRC abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1562.
Kim et al. (Fri,) studied this question.