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Background Casitas B-lineage Lymphoma proto-oncogene B (CBL-B) is an E3 ubiquitin ligase acting as a critical negative regulator of immune activation. 1 2 It dampens signaling in T cells and NK cells, limiting their cytotoxic potential and cytokine production. Tumors exploit this pathway as a mechanism of immune evasion, contributing to resistance against current immunotherapies. Inhibiting CBL-B presents a novel strategy to overcome this resistance by enhancing intrinsic immune cell function.3 4 Our project focuses on the discovery and preclinical development of a potent CBLB inhibitor designed to amplify anti-tumor immune responses. This approach aims to restore immune effector activity and improve clinical benefit for patients unresponsive to existing immunotherapies.Methods Several series of different scaffold molecules were designed via scaffold-hopping and evaluated using AIDD (artificial-intelligence drug design) via integrating machine learning, molecular docking, QSAR modeling, and FEP calculations to predict in vivo activity, pharmacokinetics, and ADME profiles. Over 100 promising compounds were selected based on these comprehensive assessments and advanced to enzymatic or cellular bio-assays for further validation. The ability of compounds to bind CBL-B protein was evaluated using TR-FRET assay. The immunomodulatory activity was assessed in vitro through human T cells or NK cells. As a proof of concept study in vivo, an experiment in CT26 syngeneic tumor mouse models was performed.Results Our lead candidate, HDM2021, demonstrates potent in vitro and in vivo activity as a CBL-B inhibitor (CBL-B displacement IC50 < 5 nM). Functionally, HDM2021 potently enhances IFNγ secretion in human immune effector cells, with a 38-fold increase in T cells and a ~3-fold increase in NK cells, significantly boosting their anti-tumor potential. Particularly, HDM2021 showed potent anti-tumor efficacy in vivo. In the CT-26 syngeneic mouse model. HDM2021 combined with anti-PD1 treatment induced statistically significant tumor growth inhibition (TGI > 80%), demonstrating substantial combinatorial activity.Conclusions HDM2021 has demonstrated a potent and effective CBL-B inhibitor, exhibiting significant immunomodulatory activity, enhancing T and NK cell function in vitro, and robust antitumor efficacy in in vivo models. These findings strongly support HDM2021 a novel immunomodulatory drug candidate for anti-tumor therapy. IND-enabling studies have been started for INDs application.References Venuprasad K. Cbl-b and itch: key regulators of peripheral T-cell tolerance. Cancer Res. 2010;70:3009-3012.Paolino M, Choidas A, Wallner S, et al. The E3 ligase Cbl-b and TAM receptors regulate cancer metastasis via natural killer cells. Nature. 2014;507:508-512.Augustin RC, Bao R, Luke JJ. Targeting Cbl-b in cancer immunotherapy. J Immunother Cancer. 2023;11:e006007.Fusco R, Saedi Z, Capriello I, Lubskyy A, Dömling A. CBL-B - an upcoming immune-oncology target. Expert Opin Ther Pat. 2025;35:47-64.