Background Casitas B-lineage lymphoma-b (Cbl-b), a protein of the Cbl family of ring finger E3 ligases,¹ regulates innate and adaptive immune activation by mediating the ubiquitination and degradation of various signalling transducers.^{2 3} A master regulator downstream of TAM receptors (NK-cells)⁴ and CD28/CTLA4 (T-cells), Cbl-b promotes an immunosuppressive milieu in the TME (tumor microenvironment), limiting anti-tumor effector functions of T and NK cells.⁵ Lack of CD28 reportedly defines a severely exhausted T-cell phenotype contributing to resistance to anti-PD-1 therapy.^{6 7} Cbl-b inhibition activates T-cells even in absence of CD28 co-stimulation, thereby providing a potential advantage in immune-suppressive TME. Blockade of Cbl-b function by GRC 65327 translated to activation of immune functions resulting in efficacy in in-vivo mouse models of colon (CT26) cancer and enhanced efficacy in combination with ICI (Immune Checkpoint Inhibitors), thereby underlining the therapeutic potential of Cbl-b inhibition for cancer immunotherapy. With selectivity >25-fold over c-Cbl, GRC 65327 is expected to translate to better safety profile compared to non-selective inhibitors.

Methods GRC 65327, was evaluated in vitro for Cbl-b and c-Cbl activity inhibition in Homogeneous Time-Resolved Fluorescence assays, cytokine release from multiple cell-based/whole blood and reversal of immunosuppression. In-vivo efficacy was tested as monotherapy and in combination with ICI in mouse colon cancer CT26 tumor model. Its safety profile was further characterized through CEREP safety panel (Eurofins) and tolerability in CD1 mice.

Results GRC 65327 is a potent Cbl-b inhibitor with IC₅₀ of 4nM, >25-fold selectivity over c-Cbl, >100-fold selectivity in the CEREP safety panel and potentiates cytokine in PBMCs at EC₅₀ around 400nM It demonstrated reversal of T-cell exhaustion and PGE2/NECA-mediated immunosuppression. In CT26 colon tumor model, GRC 65327 displayed significant tumor growth inhibition as single agent and in combination with ICI. Further, our studies showed an immune-mediated mechanism involving cytokine release and lymphocyte infiltration. Animals showed complete response (CR) when re-challenged maintained rejection of tumors and exhibited memory immune response. GRC 65327 showed the absence of treatment-related toxicity in a repeat dose exploratory toxicity study in CD1 mice, with an achieved AUC of approximately 5-fold over ED_max.

Conclusions GRC 65327 is a novel, selective, orally active Cbl-b inhibitor that demonstrates good in vitro potency, in vivo efficacy with monotherapy, with potential to enhance checkpoint blocker-based immunotherapy regimens and acceptable safety profile in mice. IND enabling studies are underway.

Acknowledgements We thank Netra Deshpande, Debjyoti Boral, Priya Yadav, Rahulkumar Bhadane, Sanjay Gaikwad, Akshay Kangane, Sandip Patil for their contributions to the project.

References

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