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55 Exploring proximal biomarkers of CBL-B inhibition in human peripheral blood mononuclear cells
  1. David Greco,
  2. Yilin Qi,
  3. Jun Kuai,
  4. Samira Jaeger,
  5. Yingzhi Bi,
  6. Huadong Sun,
  7. Wei Liu,
  8. Ken Carson,
  9. Timothy Reilly,
  10. Geraldine Harriman,
  11. Fang Wang and
  12. Hadi Danaee
  1. HoSpot Therapeutics, Boston, MA, USA
  • Journal for ImmunoTherapy of Cancer (JITC) preprint. The copyright holder for this preprint are the authors/funders, who have granted JITC permission to display the preprint. All rights reserved. No reuse allowed without permission.


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.

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See

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