Background: Casitas B-lineage lymphoma b (Cbl-b), a RING finger E3 ligase, is a negative regulator of immune cell activation1. Genetic deletion or pharmacological inhibition of Cbl-b resulted in hyper-reactive and co-stimulation independent T cell activation and cytokine production1. In syngeneic tumor models, CD8 T-cell and NK-cell mediated rejection of tumours were observed1. These findings point to Cbl-b as a therapeutic target in cancer immunotherapy. Inhibition of Cbl-b also demonstrated the potential to enhance the efficacy of check-point blockers like anti-PD-1 antibody, an unmet need in the clinic. Methods: Using intuitive medicinal chemistry design supported by computational approaches, we identified a lead Cbl-b inhibitor. SAR was developed using a battery of biochemical assays, functional read-outs and primary human in vitro T-cell activation and exhaustion assays. In vivo efficacy was demonstrated in syngeneic mouse colon tumor model. Results: Our lead Cbl-b inhibitor demonstrated potent binding to Cbl-b, robust anti-tumor cytokine secretion in human and mouse T cells, whole blood and potent reversal of T cell exhaustion. A strong tumor growth inhibition was demonstrated by the lead compound in a mouse colon tumor model. Compared to single agent, a combination of the lead compound with anti-PD-1 antibody induced enhanced complete tumor rejections. Conclusions: We have identified a novel, potent and orally bioavailable Cbl-b inhibitor that demonstrated robust in vitro and in vivo anti-tumor profiles. Acknowledgements: We thank Sanjib Das, Ajit Patil, Gauri Gawas, Savita Pandita, Priya Yadav, Sneha Pusadkar, Mayura Behere, Subhadip Das, Shravankumar Kolli and Radheshyam Yadav for their contributions to the project References: 1. Clinical and Experimental Immunology, 204: 14-31, 2020 Citation Format: Murugan Chinnapattu, Sandeep Shelke, Prashant Ingale, Nayan Waghmare, Nanasaheb Kadlag, Manoj Pawar, Akshay Kangane, Sachin S. Chaudhari, Jagmohan Saini, Vidya Kattige, Arti Joshi, Colina Dutta, Debjyoti Boral, Sheetal Kadam, Varada Potdar, Jiju Mani, Pooja Sawant, Megha Marathe, Madhavi Mulay, Akshata Virdikar, Sravan Mandadi, Atul Akarte, Anuj Singh, Chandrasekhar Misra, Pandurang Lambade, Chaitanya Tirumalasetty, Raju Patole, Vikas Karande, Dayanidhi Behera, Pankaj Jain, Vishwanath Kurawattimath, Nagaraj Gowda, Pravin S. Iyer. A novel and potent Cblb inhibitor demonstrates robust immunological profile and anti-tumor efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 462.