Abstract 4880: Anti-membrane IgM monoclonal antibody, WBMP-4, induces apoptosis in B-cell malignancies by modulation of kinase expression and phosphorylation (2023)

Abstract The concept that the B-cell Receptor Complex (BCRC) initiates the dominant driver pathway in leukemia-lymphoma is clinically validated. However, the BCRC has been overlooked as a therapeutic target due to homology between the BCRC’s membrane Ig (mIg) subunit and circulating serum Ig. Previously, we reported the generation of a first-in-class antibody, WBMP-4, to a neo-epitope specific to the mIgM-BCRC, which inhibits cell growth and induces apoptosis (Welt et al. 2016). Here we evaluate kinase activity in Burkitt lymphoma (CA46) cells treated with WBMP-4 (at 5 and 20 ug/mL, and at 6 and 24 hours) via the Pamgene phosphotyrosine kinase assay. This assay measures kinase activity in cell lysates using a microarray chip containing peptide sequences that serve as substrate for phophsotyrosine sites on well-characterized signaling proteins. We measure kinase activity at 189 phosphosites using this array, as well as protein expression and phosphorylation of key targets downstream of the BCRC using ELISA, flow cytometry, western blot, and immunohistochemistry, in WBMP-4-treated and untreated IgM-expressing B-cells. Our Pamgene analysis reveals widespread kinase modulation following WBMP-4 treatment, with &amp;gt;180 of 189 phosphosites showing a significant change in phosphorylation signal intensity (with &amp;gt;153 showing a significant decrease) upon treatment. Western blot analyses of downstream BCRC kinases similarly show changes in phosphorylation at analyzed phosphosites (ZAP70, mTOR, AKT, and MEK) with WBMP-4 treatment. However, we also find changes in protein expression levels of CD79b, ZAP70, BTK, MEK, Myc (all decreased), and LCK (increased) following WBMP-4 treatment. We show that the kinase activity of proteins in multiple signaling pathways is altered upon WBMP-4 binding to the BCRC’s mIgM, with the vast majority exhibiting a downregulation of phosphorylation. We also find that protein expression is impacted in several kinases directly under the control of the BCRC. We hypothesize that WBMP-4’s impact on protein expression contributes to BCRC signal disruption and the cytotoxic effect achieved by WBMP-4. For example, WBMP-4 induces a profound reduction in CD79b expression (undetectable by western blot following WBMP-4 treatment), which is consistent with the reduction in CD79a kinase activity found via Pamgene assay. These data support the concept that WBMP-4 binds an epitope on mIgM that is critical to its signal transduction to CD79a/b.Due to WBMP-4’s specific targeting of IgM-expressing B-cells, we expect that the toxicities associated with approved therapies upon their interaction with non-lymphatic tissue (e.g. bleeding, infection, cytopenia, arrhythmia, secondary malignancies) will be avoided. With potent anti-tumor activity and expected reduced toxicities, WBMP-4 is a compelling candidate for clinical development. Citation Format: Rachel S. Welt, Jonathan A. Welt, Virginia Raymond, David Kostyal, Sydney Welt. Anti-membrane IgM monoclonal antibody, WBMP-4, induces apoptosis in B-cell malignancies by modulation of kinase expression and phosphorylation. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4880.