Abstract 589: Discovery of NTX-001, a potent Cbl-b inhibitor with antitumor activity in syngeneic models

Abstract

Abstract Introduction: Casitas B-lineage lymphoma b (Cbl-b), a RING finger E3 ligase and a member of a highly conserved family of Cbl proteins, catalyzes the ubiquitination of substrate proteins to regulate multiple signaling events in a variety of cell types, including immune cells. In T cells, Cbl-b negatively regulates adaptive immune system signaling by establishing the threshold for the activation of antigen receptors. Additionally, Cbl-b regulates the function of other immune cell types, including NK cells, dendritic cells (DC) and monocytes. Cbl-b deficient T cells no longer require a costimulatory signal to be fully activated. Cbl-b KO mice spontaneously reject tumors via an enhanced immune response. Taken together, these findings point to Cbl-b inhibitors as having the potential to be highly efficacious immuno-oncology agents. Experimental Procedures: A structure-based drug design approach was used to identify potent inhibitors of Cbl-b. Biochemical and cellular binding assays, a cellular NFAT-luciferase reporter assay, as well as primary in vitro human and mouse T cell activation assays measuring IL-2 production were used to profile inhibitor compounds. In vivo activity of Cb-b inhibitors was evaluated using an anti-CD3 mouse model and a CT-26 syngeneic mouse model Results: Cbl-b inhibitor NTX-001 potently binds to Cbl-b, preventing Cbl-b phosphorylation and binding to E2. In cells, NTX-001 treatment results in enhanced phosphorylation of ZAP-70, a protein proximal to the TCR and reported to be a direct substrate of Cbl-b. Additionally, inhibition of Cbl-b potently enhances cytokine secretion from primary human and mouse T cells. In vivo, an increase in cytokines and T cell activation markers was observed after a single dose of NTX-001. Repeated dosing of NTX-001 showed dose-dependent anti-tumor activity in the colorectal CT-26 syngeneic model. Conclusions: NTX-001 is a potent Cbl-b inhibitor that demonstrated strong T cell activation and anti-tumor activity in a syngeneic tumor model. These data support Cbl-b inhibitors as a promising therapeutic opportunity for cancer treatment. Citation Format: Fred Csibi-Levin, Silvana Leit, David L. Laughton, Tom Baker, Suzanne L. Jacques, Beth Browning, Angela V. Toms, Samantha Garside, Simon D'Archivio, Katarzyna Kopycka, Xiaohua Zhu, Allan M. Jordan, Stuart Thomson, Christine Loh, Peter Tummino, David N. Ciccone. Discovery of NTX-001, a potent Cbl-b inhibitor with antitumor activity in syngeneic models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 589.