Introduction: The concept that the B-cell receptor complex (BCRC) initiates a driver pathway in leukemia-lymphoma has been validated by clinical data. However, the BCRC has been overlooked as a therapeutic target due to homology between the BCRC's membrane IgM (mIgM) subunit and circulating serum IgM (sIgM). Previously, we reported the generation of a first-in-class antibody, mAb4, to a neoepitope specific to the BCRC, which inhibits cell growth (Welt et al. 2016; US Patent Nos. 9,926,381 and 10,227,419). Bruton's tyrosine kinase (BTK) inhibitors have emerged as valuable clinical therapies but can induce severe off-target effects. BTK is a downstream mediator of the BCRC's signaling pathway. We aim to characterize the impact of targeting the BCRC on BTK phosphorylation and its resulting biologic effects. We hypothesize that mAb4 binding to the BCRC initiates antitumor effects in part by inhibition of BTK phosphorylation.
Methods: Antibody reagents detecting phosphorylation of BTK at Tyr551 (clones A16064A, 797837, and M4G3LN), Tyr223 (clones A16128B and 720101), and Ser180 (clones 3i5 and 3D3), were used to compare inhibition of phosphorylation by a BTK inhibitor drug, and by mAb4, in Burkitt lymphoma cells (CA46) over 0-96 hours, by ELISA. Flow cytometry will be used to further confirm these biologic effects and examine cell cycle phase inhibition.
Results: We find that both mAb4 and a BTK inhibitor drug inhibit phosphorylation at Tyr551 and Tyr223, but not Ser180, across all Ab reagents used in ELISA. 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. Preliminary flow cytometry results suggest that mAb4 treatment induces a specific cell cycle arrest.
Conclusions: Here we show that phospho-tyrosine BTK inhibition is mediated upon mAb4 binding to the BCRC's mIgM. Though BTK exhibits identical patterns of phosphorylation inhibition following treatment with either mAb4 or a BTK inhibitor drug, each treatment results in different cell survival outcomes. While studies show that BTK inhibitors induce a cell growth inhibitory effect, we find that mAb4 mediates BCRC internalization, and in low-density cultures, cell growth inhibition, anticlonogenic activity, and apoptosis. Further studies will examine spleen tyrosine kinase (SYK) phosphorylation and regulation of CXCR4 (a chemokine receptor), as mobilization of tumor cells into the blood may additionally be an important clinical parameter in BTK inhibitor drug efficacy. Due to the specific targeting of mAb4 to IgM-expressing B-cells, we expect that the toxicities associated with small-molecule BTK inhibitors upon interaction with nonlymphatic tissue, including bleeding, infection, cytopenia, arrhythmia, and secondary malignancies, will be avoided with mAb4 treatment. With increased antitumor activity and expected reduced toxicities, mAb4 represents a compelling candidate for clinical development.
Citation Format: Rachel Welt, Jonathan Welt, Virginia Raymond, David Kostyal, Sydney Welt. Anti-membrane IgM monoclonal antibody, mAb4, is a novel, next-generation BTK inhibitor mediating B-cell leukemia and lymphoma cell death [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-59.