Abstract 661: An oral Cbl-b inhibitor with sustained T cell activation demonstrated robust anti-tumor efficacy along with enhanced infiltration and activation of functional T cells
Authors: Zhou Feng , Yang Guimei , Liu Yajing , Xue Liting , Chen Weijie , Li Zhengtao , Liu Xiaowu , Li Jian , Tang Renhong 
Published: March 2024

Abstract
The E3 ubiquitin ligase Casitas B-lineage lymphoma b (Cbl-b) is a member of the highly conserved family of Cbl (casitas b-lineage lymphoma) proteins, which associates with multi-immune response regulations including Teff and NK activation, Treg differentiation. Cbl-b KO mice showed enhanced immune responses and resistance to tumor transplantation. Previously, we reported a novel Cbl-b inhibitor, ZM-8026, which robustly activated T cells and efficiently inhibited tumor growth as monotherapy or in combination with anti-PD-1. Here, we further demonstrated that Cbl-b inhibition restores the Teff cells' function in immune suppression conditions, such as in the presence of PGE2, A2AR agonist, and TGF-β. CD8+T proliferation and cytokines induction were maintained in the presence of MDSC. Furthermore, CD8+T function was reversed from an exhausting status induced by a continuous anti-CD3/CD28 activation for a long period. To investigate whether continuous target occupancy was necessary for sustained TCR activation, the dynamic impacts of Cbl-b inhibition on CD8+T cell activation were explored using a washout assay. The results showed that the high-level activated T cells lasted for 48 h after more than 4hs preincubation with 1 µM Cbl-b inhibitor followed by a washout, however, short and weak activation states were found with less than 4hs preincubation or low inhibitor concentration treatment (&lt; 0.1 uM). This finding revealed sufficient inhibitor concentration and treatment time ensure high T-cell activation. Further, in vivo efficacy studies demonstrated less anti-tumor growth efficacy for intermittent dosing (Q2D, Q3D) compared with QD dosing. In vitro and in vivo pharmacokinetics studies exhibited favorable ADME profiles and bioavailability of our Cbl-b inhibitor for oral administration in mice, rats, and dogs. At least in two syngeneic models, our Cbl-b inhibitor exhibited efficient anti-tumor growth with more than 70% TGI (tumor growth inhibition). In a CT26 syngeneic model, complete tumor growth inhibition in 6 of 8 mice was found in the Cbl-b inhibitor combined with an anti-PD1 antibody. The complete regression mice were re-challenged with the same tumor cells, and no measurable tumors were found for up to 30 days, suggesting the treatment-induced immune memory. Tumor samples in mice were collected, RNAseq showed that activated functions of tumor-infiltrated T, NK, DC, and macrophage were dramatically increased in the Cbl-b inhibitor treatment group, and the T and NK activation signature genes such as Gzmb, Ifng were significantly upregulated by RT-PCR. In conclusion, our Cbl-b inhibitor exhibited a favorable PK profile in multi-preclinical animals. The sustained T cell activation in vitro was identified and robust anti-tumor growth in vivo was demonstrated with optimized dosing frequency.
Citation Format: Feng Zhou, Guimei Yang, Yajing Liu, Liting Xue, Weijie Chen, Zhengtao Li, Xiaowu Liu, Jian Li, Renhong Tang. An oral Cbl-b inhibitor with sustained T cell activation demonstrated robust anti-tumor efficacy along with enhanced infiltration and activation of functional T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 661.