Abstract 6733: Discovery and characterization of CBL-B intramolecular glue inhibitors that increase T cell activation and suppress tumor growth.

Abstract Background. CBL-B, a RING-type E3 ubiquitin ligase, is a master regulator of peripheral T-cell activation and a promising immuno-oncology (IO) target. Genetic ablation of CBL-B in mice leads to enhanced T-cell activation and spontaneous tumor rejection, suggesting that pharmacological inhibition of CBL-B could reverse immunosuppression in the tumor microenvironment and potentiate anti-tumor immunity. This work describes the rational discovery of intramolecular glue inhibitors targeting the auto-inhibited conformation of CBL-B. Methods. We designed and executed a high-throughput screening (HTS) campaign using a homogeneous time-resolved fluorescence (HTRF) assay to identify small-molecule inhibitors that stabilize the autoinhibited, closed conformation of CBL-B. Hits were validated through orthogonal HTRF and surface plasmon resonance (SPR) assays. The binding mode was elucidated by X-ray co-crystal structure. The series was optimized for affinity and properties through iterative cycles of medicinal chemistry. Lead compounds were evaluated for their ability to enhance T-cell activation and cytokine secretion in primary human T cells and for anti-tumor efficacy in a syngeneic mouse model. Results. HTS of 250,000 compounds yielded a singleton hit that inhibited CBL-B activation with KD = 24.12 ± 8.90 μM. Structure-guided optimization resulted in NRX-8766, with a KD of 0.033 ± 0.001 μM for CBL-B. Treatment of human peripheral blood mononuclear cells (hPBMCs), with NRX-8766 results in 3- to 4-fold increases in T-cell activation markers (CD25, CD69) and 2- to 25-fold increases in cytokine secretion (IL-2, IFN-γ, TNF-α) when stimulated with α-CD3 or α-CD33/α-CD28. In vivo, oral administration of NRX-8766 to mice bearing syngeneic CT26 tumors significantly increased T-cell activation and suppressed tumor growth. Conclusions. These studies demonstrate that pharmacological inhibition of CBL-B reproduces the reported mouse knockout phenotype. NX-1607, a first-in-class CBL-B inhibitor, is now in clinical development as a potential cancer immunotherapy. Citation Format: Stefan Gajewski, Asad Taherbhoy, Brandon Bravo, Ketki Dhamnaskar, Julie Sheung, Dhwani Haria, Austin Tenn-McClellan, Jennifa Gosling, Matthew C. Clifton, Mario Cardozo, Paul A. Barsanti, Jilliane R. Perkins, Kathleen Boyle, Thomas Cummins, Morgan Lawrenz, Marilena Gallotta, Jeffrey T. Mihalic, Nichole O'Connell, Sumit Prakash, Anjanabha Saha, Ryan Rountree, Chenbo Wang, Dahlia R. Weiss, Christoph W. Zapf, Frederick Cohen, . Discovery and characterization of CBL-B intramolecular glue inhibitors that increase T cell activation and suppress tumor growth abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6733.