Rewiring the immune-excluded tumor microenvironment: Vactosertib/anti-PD-1/VEGF inhibitor triplet therapy reinstates antitumor immunity in CRC

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-beta 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-beta 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-alphaPD-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 IFNgamma⁺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-beta inhibition is essential for overcoming treatment resistance in an immunosuppressive TME, supporting the clinical evaluation of this triple combination in refractory CRC.