Background: The B-cell Receptor (BCR) initiates a driver pathway in B-cell lymphoma-leukemia. Inhibition of downstream pathway tyrosine kinases is therapeutic. Both vertical and horizontal membrane B-cell Receptor Complex (BCRC) interactions render this pathway complex. As a consequence of the BCR's sequence homology to serum IgM (sIgM), specific B-cell membrane IgM (mIgM) targeting in vivo was thought not to be feasible. A unique sequence identified in the membrane-bound BCR, designated proximal domain (PD), is not expressed in sIgM. Here we identify an additional conformational epitope discovered in the extracellular domains of mIgM, not detected in sIgM. Methods: We have generated high affinity anti-PD monoclonal antibodies (mAbs) by proprietary immunization techniques. These mAbs are shown by ELIZA, Westerns blots and Scanning Immuno-Electron Microscopy (SEM) to bind to mIgM protein and mIgM+ expressing cell lines (CA 46, SU-DHL-5, CRL-1596, CRL-1432). Using these high affinity anti-PD mAbs, mIgM was immune-affinity purified. Second generation antibodies detecting conformational epitopes on mBCR and not reacting with sIgM in ELIZA/Western/SEM assays were collected. Growth inhibition was assessed by MTT and clonogenic assays. Four mAbs were selected for further studies. Results: Cell surface binding assays demonstrated the specificity of these mAbs by testing against a panel of mIgM + vs mIgM- (mIgG+) cells. Normal or Waldenstrom's Macroglobulinemia sera failed to block or reduce mAb binding to mIgM+ cells and results were confirmed by SEM. At 37°C anti-PD mAbs (mAb1-1, mAb2-2b, mAb3-2b) internalize mIgM by 30 mins, but they do not modulate cell growth inhibition. Second generation mAb4-2b mediates BCR internalization and in low density cultures, cell growth inhibition, anti-clonogenic activity and apoptosis is observed. Apoptosis is seen in a variety of malignant B-cell lines including high and low mIgM/Igα/β expressers. Conclusions: These data support the contention that BCR internalization is insufficient to interrupt the BCRC signaling cascade despite the lack of detectable residual mIgM on the cell surface. Upon mAb4-2b binding to a non-ligand binding site on mIgM, it induces both BCR internalization and in another distinct event, apoptosis. By competitive mAb studies the apoptosis mediating conformational epitope appears to reside outside of the linear PD sequence. These mAbs are candidates for clinical development as drug/radioisotope targeting vehicles or as a mediator of inhibition of the BCR signaling pathway (mAb 4-2b). As these agents have a high level of specificity because they do not bind to non-mIgM B-cells, normal lymphocytes and non-lymphatic tissues may be spared toxicity.

Citation Format: Rachel S. Welt, Yamuna D. Gangadharan, Jonathan A. Welt, Virginia Raymond, David Kostyal, Sydney Welt. Antibody targeting the IgM B-cell receptor (mBCR) induces growth inhibition and apoptosis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3641. doi:10.1158/1538-7445.AM2014-3641