Abstract 3036: Intracellular phospho-flow reveals novel insights to pathway modulation by tirabrutinib in combination with entospletinib and obinutuzumab
Authors: Yang Xiaoyun , Wehrman Tom , Laboriante Justin , Li Biao , Kutsch Nadine , Pallasch Christian , Eichhorst Barbara , Jürgensmeier Juliane M. 
Published: September 2020

Abstract
Tirabrutinib is a second-generation, potent, selective irreversible Bruton tyrosine kinase (BTK) inhibitor in clinical development for relapsed/refractory chronic lymphocytic leukemia (CLL) in combination with entospletinib and obinutuzumab. To study the modulation or inhibition of different signaling pathways in response to drug treatment, we developed a phospho-flow cytometric assay that can measure up to 110 intracellular signaling proteins. This method was validated in an in vitro setting to develop the pre-analytical sample handling, assay setup and analysis parameters. The optimized procedure was implemented in a phase II clinical study (NCT02983617). For the in vitro experiments, whole blood was collected from healthy volunteers and CLL patients. Following a stimulation with anti-IgD/anti-IgM, the blood was processed and stained with a panel of 110 phospho-specific antibodies and evaluated by flow cytometry. A panel of 43 phosphoproteins was selected based on their relevance to the inhibited pathways and/or their responsiveness to B cell receptor cross-linking. In the clinical study, whole blood was collected at baseline, week 2 and 25 post-treatment from 23 patients treated with tirabrutinib in combination with entospletinib and obinutuzumab. The samples were stimulated with anti-IgM/anti-IgD and stained with surface markers to identify the cancer cells and their signaling fingerprint was evaluated with the selected panel of phosphoprotein markers. The data from the in vitro experiments showed, at baseline, both CLL and healthy B cells had no or minimal constitutive activation of downstream BCR signaling proteins. In normal B cells, of the phospho-proteins analyzed, most showed great increase after activation of BCR. However, BCR-induced phosphorylation of most signaling proteins was significantly lower in the CLL cells from the patients. In addition, the fraction of cells responding to the in vitro stimulation was highly variable among the CLL patient samples compared to normal donors. Down-regulation of phosphoproteins, including pBTK (Y223)/pITK (Y180), pSyk (Y348), pSyk (Y525/Y526) was observed in most of the patients both in stimulated or un-stimulated conditions at week 2 and 25. The level of the phosphorylation of pBTK (Y223)/pITK (Y180), pSyk (Y348), pSyk (Y525/Y526) from stimulated blood was highly correlated with the blast counts in the periphery at week 1 and 2. These findings support the utility of this multi-parameter flow assay to detect and monitor immune signaling inside different immune cells. Evaluating the effects of targeted therapeutics following clinical administration can inform on disease and drug mechanisms, combination strategies, and identify potential biomarkers for pharmacodynamic or patient stratification that are otherwise inaccessible using less sensitive or non-functional analyses.
Citation Format: Xiaoyun Yang, Tom Wehrman, Justin Laboriante, Biao Li, Nadine Kutsch, Christian Pallasch, Barbara Eichhorst, Juliane M. Jürgensmeier. Intracellular phospho-flow reveals novel insights to pathway modulation by tirabrutinib in combination with entospletinib and obinutuzumab [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3036.