Abstract PO020: Anti-membrane-IgM monoclonal antibody, mAb4, inhibits the BCRC, modulating downstream signaling pathways (2021)

Abstract The concept that the B-cell Receptor Complex (BCRC) initiates a driver pathway in leukemia-lymphoma has been validated by clinical data. Previously, we reported the generation of a first-in-class antibody, mAb4, to a neo-epitope specific to the BCRC’s membrane IgM (mIgM) subunit, which inhibits cell growth and induces apoptosis (Welt, et al. 2016; US Patent Nos. 9,926,381 and 10,227,419). mAb4 epitope mapping demonstrates that it does not bind to the complementarity-determining regions (CDRs), nor to the micro-clustering site of mIgM that is believed to play a role in BCRC signal amplification. To characterize the mechanism of action of this antibody, we are investigating the inhibition of tyrosine kinase activity of several BCRC downstream signaling pathways. We hypothesize that mAb4 inhibits signal transduction of multiple BCRC-dependent downstream pathways. We are currently evaluating kinase activity across three sites on BTK (Ser180, Tyr223, and Tyr551). Phosphorylation of other common clinical targets, including Syk, Lyn, BCL2, and PI3K, will also be studied. To do this, we are using antibody reagents against phosphorylation sites to assess activation or inhibition of these kinases by mAb4 compared to control mAb-treated IgM-expressing B-cells, and, where available, cells treated with drugs against these targets. In preliminary ELISA analyses we find that mAb4 and a BTK inhibitor drug, inhibit BTK phosphorylation at Tyr223 and Tyr551, but not Ser180, across all anti-phospho-tyrosine/serine antibody reagents tested (Ser180: clones 3i5 and 3D3; Tyr223: clones A16128B and 720101; and Tyr 551: clones A16064A, 797837, and M4G3LN). In vitro, mAb4-treated leukemia and lymphoma cells undergo growth inhibition and apoptosis while the BTK inhibitor drug mediates only cell growth inhibition, as reported by others. Here we show that mAb4 inhibits phosphorylation of BTK tyrosine upon binding to a determinant that spans the extracellular proximal domain and the constant domain 4 (μC4) regions of mIgM. While BTK inhibitor drugs induce a cell growth inhibitory effect, we find that mAb4 mediates BCRC internalization, and in low-density cultures, cell growth inhibition, anti-clonogenic activity, and apoptosis, and may therefore represent a next generation of BTK inhibitor. Though mAb4 does not interact with the mIgM’s CDRs nor the micro-clustering site on μC4, it modulates transmembrane signal transduction. We predict that continued analyses of additional downstream kinases (Syk, Lyn, BCL2, and PI3K) will similarly show an effect following BCRC inhibition by mAb4. These finding indicate that mAb4 may target mIgM at a critical structural site, which interrupts specific ligand binding signal transduction. Furthermore, mAb4 is highly specific to IgM-expressing B-cells, thus this novel approach to modulating B-cell signaling to induce a cytotoxic response may represent an exciting new direction in clinical development. Citation Format: Rachel S. Welt, Jonathan A. Welt, Virginia Raymond, David Kostyal, Sydney Welt. Anti-membrane-IgM monoclonal antibody, mAb4, inhibits the BCRC, modulating downstream signaling pathways [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO020.