Abstract 4042: Cbl inhibition increases the response of cytotoxic T cells to sub-optimal stimulation and drives enhanced anti-tumor responses alone or in combination with PD-1 blockade
Authors: Yamashiro Livia , Marubayashi Sachie , Rayaji Rameshwari , Battsooj Anudari , Mrouj Karim , Singh Hema , Lee Yue Tong , Chen Ada , Shen Lunda , Mailyan Artur , Yu Kai , Podunavac Masha , Yu Julie , Liu Dongdong , Hardman Clayton , Yin Johnny , Gangam Srikanth , Lawson Ken , Leleti Manmohan , Walters Matthew , DiRenzo Daniel 
Published: March 2024

Abstract
INTRODUCTION: Herein, we describe a series of experiments to define the unique biology of dual Cbl-b/c-Cbl (Cbl) inhibition in amplifying T cell activation and anti-tumor responses. Through their E3 ubiquitin ligase activity, Cbl-b and c-Cbl have been shown to negatively regulate multiple signaling proteins downstream of the T cell receptor (TCR), suggesting that inhibition may yield enhanced anti-tumor T cell activity. Indeed, using a dual Cbl-b/c-Cbl small molecule inhibitor, we demonstrate that Cbl blockade enhances T cell activity under standard and sub-optimal stimulation conditions. Moreover, Cbl inhibition increases mouse syngeneic tumor control alone or in combination with α-PD-1, as described below.
METHODS: Human CD8+ T cells were isolated from healthy human blood and activated using α-CD3 or α-CD3/CD28 stimulation. Mouse OT-I splenocytes were stimulated with SIINFEKL or lower affinity ovalbumin peptides and cytokine production was assessed. For tumor studies, mice were inoculated with syngeneic cancer cells and were treated with Cbl inhibitor (30 mg/kg, QD) +/- α-PD-1 (10 mg/kg, Q5D) starting at a tumor volume of 75 mm3.
RESULTS: Human CD8+ T cells, stimulated in the presence of a Cbl inhibitor, demonstrated a concentration-dependent increase in activation hallmarks including cytokine secretion (IL-2, IFN-γ and granzyme B), proliferation, and the presence of highly polyfunctional cells. Furthermore, using serially stimulated CD8+ T cells with reduced overall activity, Cbl inhibition showed a marked enhancement of IFN-γ production and cytotoxicity compared to controls upon re-stimulation. We then used an antigen-specific T cell assay where OT-I splenocytes were stimulated by peptides with optimal or reduced TCR affinities, Cbl inhibition produced significantly higher IFN-γ levels with all peptides relative to controls demonstrating that Cbl inhibition can increase the response of T cells to sub-optimal stimulation. Using mouse CT26 and MC38 tumor models, Cbl inhibition was able to significantly enhance tumor control in combination with α-PD-1 and as a monotherapy in CT26 tumors. Notably, a greater number of tumor antigen-specific cytotoxic T cells were found in Cbl inhibitor-treated tumors. Higher expression levels of the co-stimulatory molecule CD226 and the TCR in these anti-tumor cells were also observed, demonstrating the unique biology of Cbl inhibition in enhancing anti-tumor responses. Finally, Cbl inhibition had no effect on the growth or survival of cancer cells in vitro consistent with the notion that enhanced tumor control was driven by immune activity.
CONCLUSIONS: Altogether, these data demonstrate that pharmacologic Cbl inhibition robustly enhances T cell activity in vitro and in vivo and provide a mechanistic rationale for targeting Cbl-b/c-Cbl to amplify anti-tumor immune responses.
Citation Format: Livia Yamashiro, Sachie Marubayashi, Rameshwari Rayaji, Anudari Battsooj, Karim Mrouj, Hema Singh, Yue Tong Lee, Ada Chen, Lunda Shen, Artur Mailyan, Kai Yu, Masha Podunavac, Julie Yu, Dongdong Liu, Clayton Hardman, Johnny Yin, Srikanth Gangam, Ken Lawson, Manmohan Leleti, Matthew Walters, Daniel DiRenzo. Cbl inhibition increases the response of cytotoxic T cells to sub-optimal stimulation and drives enhanced anti-tumor responses alone or in combination with PD-1 blockade [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 4042.