Discovery and characterization of small molecule inhibitors of CBL-B that act as intramolecular glue to enhance T-cell anti-tumor activity

Gajewski, S.; Taherbhoy, A. M.; Boyle, K.; Gosling, J.; Perkins, J. R.; Dhamnaskar, K.; Sheung, J.; Baker, K.; O'Connell, N.; Bravo, B.; Mukerji, R.; Tenn-McClellan, A.; Kurylo, K.; Haria, D.; Gallotta, M.; Juan, J.; Tan, M.; Prakash, S.; Weiss, D. R.; Lawrenz, M.; Cardozo, M.; Wang, C.; Cummins, T.; Clifton, M. C.; Rountree, R.; Saha, A.; Zapf, C. W.; Hansen, G. M.; Sands, A. T.; Cohen, F.

doi:10.64898/2026.01.22.701125

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bioRxiv · 10.64898/2026.01.22.701125

Discovery and characterization of small molecule inhibitors of CBL-B that act as intramolecular glue to enhance T-cell anti-tumor activity

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Abstract

CBL-B is a RING-type E3 ubiquitin ligase that acts as a critical negative regulator of T-cell activation. It promotes T-cell anergy and suppresses immune responses through ubiquitin-mediated control of signaling proteins at the immunological synapse. T cells deficient in CBL-B activity lose their dependence on CD28 co-stimulation, exhibit heightened activation and increased cytokine production, and fail to re-establish anergy. In addition, mice deficient in CBL-B activity reject tumors. Together, this cellular mechanism and in vivo phenotype suggest inhibition of CBL-B may be a viable immuno-oncology therapeutic strategy. Here, we report the rational design and execution of a high-throughput screen (HTS) to identify small molecule inhibitors of CBL-B. This campaign led to the discovery of a scaffold that inhibits CBL-B E3 ligase activity with micromolar potency. Structural characterization revealed an intramolecular glue mechanism, in which the compound stabilizes the closed state of CBL-B, preventing phosphorylation of a tyrosine residue that is critical for activation and E2 binding. Iterative structure-activity optimization yielded compounds with nanomolar activity that enhanced T-cell activation and cytokine secretion in primary human T cells and suppressed tumor growth in a syngeneic colorectal mouse model. Together, these studies validate the biological rationale for pharmacological CBL-B inhibition and enabled the de novo discovery of intramolecular CBL-B glue inhibitors. This work culminated in the identification of NX-1607, a first-in-class oral CBL-B inhibitor now in clinical development for cancer immunotherapy.

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Gajewski, S., Taherbhoy, A. M., Boyle, K., Gosling, J., Perkins, J. R., Dhamnaskar, K., Sheung, J., Baker, K., O'Connell, N., Bravo, B., Mukerji, R., Tenn-McClellan, A., Kurylo, K., Haria, D., Gallotta, M., Juan, J., Tan, M., Prakash, S., Weiss, D. R., Lawrenz, M., Cardozo, M., Wang, C., Cummins, T., Clifton, M. C., Rountree, R., Saha, A., Zapf, C. W., Hansen, G. M., Sands, A. T., Cohen, F.. 2026-01-24. Discovery and characterization of small molecule inhibitors of CBL-B that act as intramolecular glue to enhance T-cell anti-tumor activity. https://doi.org/10.64898/2026.01.22.701125

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