VIP-Receptor Antagonist Enhanced Antileukemic Activity In Mice
Authors: Li Jian-Ming , Petersen Christopher Thomas , Darlak Katarzyna Anna , Yun Hyun Don , Blazar Bruce R , Waller Edmund K. 
Published: October 2019

Background and Objective
We have previously published that antagonizing vasoactive intestinal peptide (VIP) receptors dramatically decreases PD-1 expression on activated CD8+ T-cells and increases antiviral immunity (Blood 2013, 121:2347-51; PLoS One 2013, 8: e63381). Herein we tested whether short-term pharmacological antagonism of VIP signaling could induce anti-tumor immune responses in mice.

Methods
B6 mice were inoculated with 0.5 - 2 x 106 luciferase+ murine acute myeloid leukemia cells (Luc+ C1498) and B10BR mice were injected with 3 x 106 luciferase+ murine acute T-cell lymphoma cells (Luc+ LBRM) through tail vein. Mice were treated with one or more daily s.c. injections of 10 μg VIPhyb. Survival of groups that received 1, 3 or 7 doses of VIPhyb were compared with PBS treated controls. Tumor growth was monitored weekly by bioluminescence imaging (BLI). Cytokine expression and expression of immune markers PD-1, PD-1H (VISTA), and PD-L1 on blood and spleen leukocytes were analyzed by flow cytometry.

Results
Long-term survival (day 80) of tumor-bearing B6 and B10BR mice that received a single-dose of VIPhyb one day before tumor inoculation was 80% for both mouse strains harboring both leukemia cells (p<0.01 vs. control, B6 n=10 and B10BR n=5, Figure 1). A single injection of VIPhyb was more effective than multiple doses in achieving long-term tumor-free survival, with 60% survival among C1498-tumor bearing mice (p<0.01 vs. controls, n=5) treated with 3 doses, and 46% survival in mice (p<0.01 vs. control, n=13) with C1498 and 40% survival in mice (p=0.06 vs. control, n=5,) with LBRM treated with 7 doses. None of the control mice inoculated with C1498 (n=21) or LBRM (n=10) that received PBS injections survived to day 55. To explore the therapeutic effect of VIPhyb on established tumors, B6 mice and B10BR were treated with 7 daily doses of VIPhyb starting 8 days or 15 days after inoculation with Luc+ C1498 or Luc+ LBRM, respectively. Survival of B6 mice bearing C1498 and B10BR mice bearing LBRM that received delayed administration of VIPhyb was 60% (p<0.001 vs. control, n=10) and 20% (p=0.039 vs. control, n=5), respectively, compared with 0 % survival (and faster tumor growth) among control mice (B6 n=10; B10BR n=5) that received PBS injections. Tumor burdens in VIPhyb treated mice measured by BLI showed slower tumor growth, and regression of established tumors compared with mice that received PBS (Figure 1). To elucidate the mechanisms whereby VIPhyb induced anti-tumor activities, expression of serum cytokines (IFN-γ, TNF-α, IL-10 and IL-13), expression of co-inhibitory molecules PD-1, PD-1H, PD-L1, and effector molecules Fas-L and granzyme B were measured in T-cells from VIPhyb- and PBS-treated tumor-bearing B10BR mice. Blood and splenic activated (CD62L-CD25+CD69+) and memory (CD62L+/-CD44+) CD8+ T-cells from VIPhyb-treated tumor–bearing mice expressed higher levels of IFN-γ, FAS-L and granzyme B, and lower levels of PD-1 (but not VISTA/PD-1H) in activated CD8+ T-cells compared with those from PBS-treated mice (Figure 2). Expression levels of TNF-α, IL-10, IL-13, and PD-L1 in blood and splenic dendritic cells were not different comparing with tumor-bearing VIPhyb-treated with PBS-treated control mice.

Conclusion
Treatment with a small molecule antagonist of VIP-receptor, VIPhyb, dramatically increased immune/T-cell specific anti-leukemic activity. The mechanism by which administration of a VIP receptor antagonist enhanced anti-tumor immunity includes increasing productions of IFN-γ, and expression of FAS-L and granzyme B in and decreasing expression of PD-1 in activated CD8+ T-cells, leading to enhance anti-tumor cytotoxicity.

Disclosures:
No relevant conflicts of interest to declare.