Simultaneous PD-1 and VIP receptor blockade optimizes tumor immunotherapy in P815 Acute Myeloid Leukemia model

Abstract Anti-programmed death-1 (PD-1) therapy enhances anti-tumor immunity in many solid tumors, but is largely ineffective in acute myeloid leukemia (AML). CD47 blockade promotes phagocytic clearance of leukemic stem cells in AML, but its efficacy is limited by off-target binding to healthy cells. Vasoactive intestinal peptide (VIP), an immunosuppressive neuropeptide overexpressed in some AML, may serve as an alternative checkpoint. ANT308 is a potent VIP antagonist that promotes T-cell activation and downregulates immune checkpoint molecule expression, demonstrating single-agent anti-leukemia activity in murine leukemia models. We hypothesized that combining ANT308 with PD-1 or CD47 blockade would improve anti-leukemia immunity in mice. DBA2/J mice were subcutaneously inoculated with 5×10⁴ or 1×10⁵ P815 AML cells. One-week later, mice were treated with daily s.c. injections of ANT308 or scrambled peptide, or biweekly injections of long-acting ANT308 (ANT308 PEG, ANT308 IgG4 Fc fusion) for 2 weeks. For combination studies, ANT308 was co-administered with either anti-PD-1 (200 µg twice/week) or anti-CD47 (1.3 nmol every other day) for 2 weeks. Tumor size and body weight were measured twice/week; survival was monitored daily. On day 17, peripheral blood was analyzed by 26-color flow cytometry for T cell phenotypes. Before treatment, tumor volumes were comparable across groups (10.5 ± 1.1 mm³). In the control mice, tumors grew rapidly, leading to death or IUCAC-specific endpoint with a median survival time [MST] of 19 days. In contrast, ANT308 monotherapy achieved a 76.4% tumor volume relative reduction [TVRR] at one-week post-treatment, compared to tumor volumes at 21 days post-inoculation, and improved survival (MST 26 d), with 30% of mice tumor-free at 45 days. Twice-weekly injections of ANT308PEG (MST 29 d) and ANT308Fc (MST 51 d; 40% tumor-free survival) outperformed daily ANT308 treatment. Anti-CD47 monotherapy achieved 10% tumor free survival through 70 days (MST 27), but its combination with ANT308 reduced anti-leukemia activity (MST 27 d vs. 60 d for ANT308 alone), with only 10% leukemia-free survivors in the combination group. In contrast, single-agent ANT308 and anti-PD-1 both demonstrated significant anti-leukemia activity (50.4% and 61.9% one-week TVRR, respectively) and were more potent when combined, resulting in an 83% one-week TVRR and 40% tumor-free survival. Furthermore, flow cytometry revealed increased Ki67 and TCF1 expression on CD8⁺ T cells following anti-PD-1 and ANT308/anti-PD-1 therapy. CXCR3 was upregulated by both anti-PD-1 and the combination. CX3CR1⁺ cells were highest with ANT308 alone or in combination, whereas mice treated with anti-PD-1 exhibited lower levels than the control. Additionally, mice treated with the combination of ANT308 and anti-PD-1 had reduced levels of exhausted CD8⁺ T cells (PD-1⁺ Tim-3⁺) compared to controls. Notably, expression of VPAC2, a receptor for VIP, was induced on T cells in both the anti-PD-1 and ANT308/anti-PD-1 combination group. These findings suggest that ANT308 synergizes with PD-1 blockade to enhance AML immunity, likely by relieving VIP-receptor-mediated suppression during T-cell priming and activation, thereby enabling PD-1 blockade to maximize effector function. Long-acting ANT308 formulations further improved survival outcomes compared to daily dosing. The combination therapy also promoted the expansion of effector T cell phenotypes with chemokine receptors associated with homing to tumors (CX3CR1⁺, CXCR3⁺) and anti-tumor immunity. A strategy that targets both VIP and PD-1 pathways is a promising approach to immunotherapy in AML.