Purine Nucleoside Phosphorylase Inhibitors as Novel Immuno-Oncology Agent and Vaccine Adjuvant

Background: Purine Nucleoside Phosphorylase (PNP) deficiency in humans causes lymphopenia and this provided the rationale for developing PNP inhibitors as immunosuppressive agents. However, careful re-evaluation of clinical history of PNP deficient patients and clinical experience with PNP inhibitors together with new experimental data suggest inhibition of PNP may have immune activating effects through elevation of guanosine and activation of various tolllike receptors (TLRs). This paper proposes a mechanism of action for the immune activating effects of PNP inhibition. Methods: To evaluate in vitro TLR activation, by PNP inhibitor and the purine nucleosides that are elevated with PNP inhibition, HEK293 cells expressing various TLRs linked to the SEAP reporter group was used. To evaluate forodesine as vaccine adjuvant, well known mouse models of tetanus toxoid vaccine and Hepatitis B vaccine was used. To evaluate anti-tumor and anti-bacterial effects of forodesine, syngeneic B16F10 mouse melanoma model and L.monocytogenes bacterial infection mouse model were used. Results: Guanosine demonstrated significant and robust activation of TLR2 and TLR4, in HEK293 cells. Like other TLR activators, PNP inhibitor forodesine which elevates guanosine in vivo demonstrated statistically significant increases in antibody titers and, although not significant, a trend towards increase in interferon-γ (IFN-γ) levels compared to vaccine alone groups in tetanus toxoid and Hepatitis B vaccine mouse models. Similarly, forodesine treatment improved the mouse immune system as demonstrated by significant decrease in tumor growth in mouse melanoma model, inspite of lack of direct in vitro cytotoxic effects of forodesine on malignant cells. In addition, like other TLR agonists, forodesine demonstrated significant decrease in weight loss in L.monocytogenes bacterial infection model. Discussion: In PNP inhibitor clinical trials, immune activating effects have been noted which included increased response to tetanus toxoid vaccine, graft versus leukemia effects in post hematopoietic stem cell transplant relapse acute lymphoblastic leukemia patients, and in vivo effectiveness in non-leukemic cancers in spite of lack of direct in vitro cytotoxic effects on the malignant cells. Clinical findings in PNP deficient patients include autoimmune manifestations, elevation of IL-18 levels (an IFN-γ inducer), and neurological disorders which is consistent with immune activation. Lymphopenia noted in PNP deficient patients is primarily due to constitutive activation of immune system in these patients causing immune exhaustion and T-cell elimination. Genetic studies, clinical experience with PNP inhibitors together with the preclinical data presented here clearly support the role of PNP inhibitors as immune-activating agents. Contrary to prior literature, we have confirmed that PNP inhibition has immune activating effects. Conclusion: Clinical findings in PNP deficient patients along with clinical and preclinical experience with PNP inhibitor support the use of PNP inhibitors as an immuno-oncology agent and as a vaccine adjuvant.