Background Casitas B-lineage lymphoma proto-oncogene b (CBL-B) is an E3 ubiquitin-protein ligase that serves as a critical regulator of immunity. Substantial preclinical evidence supports CBL-B inhibition, as a potent driver of anti-tumor immunotherapy. HotSpot has previously disclosed our discovery of an orally bioavailable, selective, small molecule, allosteric CBL-B inhibitor (CBL-Bi), HST-1011, that has advanced into Phase 1/2 clinical trial (NCT05662397). As part of the translation of mechanism of action to clinical exploration, we sought to identify and characterize proximal biomarkers capable of monitoring CBL-Bi clinically.
Methods A phosphoproteomics study was conducted to investigate alterations in phosphorylation sites on proteins in Jurkat cells activated by anti-CD3 antibody cross-linking (anti-CD3), in the presence and absence of CBL-Bi. Selected phosphorylation changes were validated using a Meso Scale Discovery (MSD) assay, and flow cytometry in human T cells. A flow-based assay was employed to validate the biomarkers in mouse peripheral blood mononuclear cells (PBMCs) from animals treated with CBL-Bi. CBL-Bi’s impact on potential substrates of CBL-B’s E3 ligase activity was monitored for changes in treated human PBMCs using flow cytometry.
Results When comparing Jurkat cells treated with anti-CD3 to those treated with anti-CD3 and CBL-Bi, significant differences were observed in tyrosine, serine, and threonine phosphorylation. Among them were phosphorylation sites on ZAP70, CD3E, LAT, and PLCG1, key proteins downstream of T cell receptor (TCR) signaling. Examination of ZAP70 revealed dose-dependent increases in tyrosine phosphorylation in activated human T cells. This effect was also measured by flow cytometry in mouse PBMCs treated in vivo. Despite the desirability of ZAP70 phosphorylation as a proximal biomarker for CBL-Bi, quantifying phosphoproteins clinically poses significant challenges. Increased abundance of potential substrates of CBL-B’s E3 ligase activity was also observed upon CBL-Bi. Treatment of TCR-activated human PBMCs demonstrated dose-dependent increases in Notch1 and IGF1R on the surface of CD4+ and CD8+ T cells, measured by flow cytometry. CBL-Bi concentration-related changes in these markers following immune stimulation, measured by absolute levels and increases from baseline, suggested that one or both proteins could be used as proximal biomarkers for monitoring CBL-B pathway inhibition in patients treated with HST-1011.
Conclusions Evaluating the effect of CBL-Bi on TCR signaling enabled identification of a repertoire of proximal biomarkers with potential to monitor and optimize CBL-Bi clinically. We believe clinical validation and exploration are warranted to assess the utility of these proximal biomarkers and their association with downstream pharmacodynamics following treatment with HST-1011 in the clinic.
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