Abstract LB-226: Depletion of kynurenine using an engineered therapeutic enzyme potently inhibits cancer immune checkpoints both as a monotherapy and in combination with anti-PD-1 (2015)

Abstract Introduction: The kynurenine pathway is of key importance for immune suppression in cancer, a function exerted primarily via kynurenine as an AhR ligand that potently impairs adaptive immune responses. Inhibition of kynurenine (Kyn) synthesis, mediated by IDO1, TDO (and possibly IDO2) is of great interest for cancer immune checkpoint inhibition, with 3-4 IDO1 inhibitors currently in clinical development. However: (1) IDO1 inhibition has marginal anti-cancer effects as a monotherapy; (2) IDO1 inhibitors only block one of the two major pathways for Kynureine synthesis (the other being via TDO) and (3) IDO1 inhibition does generally not impact the serum concentration of Kynurenine and thus no pharmacodynamic marker is available. Experimental: We postulated that administration of a therapeutic enzyme (Kynureninase) that can degrade Kyn into non-toxic and immunologically inactive metabolites (Alanine and anthranilic acid) may be able to: (a) potently relieve cancer immune suppression. An extensive protein engineering campaign was carried out to develop a Kynureninase suitable for therapeutic administration which was then PEGylated (Kynase-PEG) to enable long circulation persistence. Kynase-PEG was evaluated in the well-established B16-OVA melanoma model in wild type C57BL/6J mice as a monotherapy and in combination with anti-PD-1 administration. Quantitation of tumor size/regression, histology, as well as flow cytometric analyses assessing lymphocytes in the spleen, tumor (TILs) and tumor-draining lymph nodes (dLNs) was used to evaluate efficacy. Summary: Administration of KYNase-PEG in B16-OVA melanoma allografts in C57BL/6J mice reduced serum Kyn levels and resulted in significant tumor growth retardation and extended survival in a manner indistinguishable from that observed with immune checkpoint inhibitors anti-PD1 (clone RMP1-14) or anti-CTLA-4 (clone 9D9) antibodies. KYNase-PEG administration did not display anti-tumor activity in NOD-scid Il2Rγ-/- mice or in IDO1-/- mice revealing that the function of the enzyme is dependent on adaptive immune responses and also on the function of stromal IDO1. Consistent with the hypothesis that depletion of Kyn relieves immune inhibition, we observed a marked increase in CD8+ TILs expressing Gzm B + IFNγ, enhanced proliferation of CD4+ and CD8+ T cells in the tumor dLNs as well as changes in tumor neovascularization. Importantly, combination of KYNase-PEG and anti-PD-1 administration resulted in complete tumor eradication in 60% of the animals (n = 10 per group) for &gt;100 days, with all the surviving animals completely rejecting tumors following re-challenge. For comparison, anti-PD-1 treatment alone, only led to 20% long-term survival in this model. Conclusions: We demonstrated that unlike IDO1 inhibitors, KYNase-PEG displays a significant anti-tumor efficacy as a monotherapy as well as excellent synergism with anti-PD1. The observed increase in tumor infiltration and proliferation of cytotoxic CD8+ T cells argues that the mechanism of action of Kynase-PEG acts by relieving the immune suppression that normally occurs in the tumor microenvironment as a consequence of Kyn accumulation. Thus, KYNase-PEG represent a “first in class” immune checkpoint enzyme. Progress in developing a clinical candidate enzyme will be discussed. Citation Format: Everett Stone, Nicholas Marshall, Moses Donkor, Kendra Triplett, John Blazek, Todd Triplett, Lauren Ehrlich, George Georgiou. Depletion of kynurenine using an engineered therapeutic enzyme potently inhibits cancer immune checkpoints both as a monotherapy and in combination with anti-PD-1. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-226. doi:10.1158/1538-7445.AM2015-LB-226