Abstract C165: Determining whether CD52 regulates homotypic cellular aggregation of human B-cells
Authors: Margulis Alexander , Radzwill Kristin A. , Criscione Stephen W. , Sancho Jose , Dwyer Markryan , Nguyen Cokey T. , Reczek David J. , Brondyk William , Campos-Rivera Juanita , Roberts Bruce L. , Shankara Srinivas , Morgenbesser Sharon D. 
Published: June 2012

Abstract
Human CD52 is a 12 amino acid, GPI-anchored cell surface glycoprotein that is expressed at very high levels on the surface of B-cells, T-cells, monocytes, and macrophages. It is a target for the Campath-1H (alemtuzumab) monoclonal antibody (mAb) that is approved for the treatment of B-cell chronic lymphocytic leukemia. Although some studies have suggested that CD52 plays a role in regulating B-cell signaling and survival, and in preventing cell aggregation, amongst other possibilities, the biological significance of CD52 remains poorly understood. The goal of this study is to further explore the potential role of CD52 in regulating cell aggregation, as it may expand our understanding of the use of anti-CD52 agents in the clinic. Towards this end, CD52 function was disrupted in several transformed CD52-positive human B-cell lines derived from a variety of hematologic malignancies, utilizing α-CD52 mAbs and F(ab)′2 fragments as blocking agents, or CD52 shRNAs to knockdown expression. A high-throughput quantitative aggregation assay was developed in-house that measures both the number and size of single cells as well as aggregates that can be classified as small (2–5 cells), medium (∼6–100 cells), and large cell clusters (&gt;100 cells). We have observed enhanced formation of tightly adherent clusters following the exposure of the BL31, DND39, Raji, and numerous other B-cell lines with Campath and several other -CD52 mAbs relative to isotype controls. Cell viability was also measured in these experiments and shown not to be substantially affected by exposure to these mAbs. To rule out Fc R (CD32) crosslinking as a potential mechanism for this effect, we have utilized two approaches. First, the Campath F(ab)′2 fragment was also used, and was shown to increase cellular aggregation. Second, we have determined which of these cell lines express CD32, established the appropriate concentration of an -CD32 mAb required for sufficient Fc R blocking, and have shown that aggregation was not disrupted by this -CD32 blocking mAb in -CD52 mAb-treated cells. Using shRNAs targeting two different sequences in the CD52 mRNA, we were able to significantly knockdown CD52 surface levels and generate dozens of low-expressing clones with either of these CD52 shRNAs, or high expressing clones with an shRNA negative control plasmid, from three of the aforementioned B-cell lines. Greater cell aggregation was observed in CD52 knockdown clones derived from BL31 and Raji relative to shRNA negative control clones when cultured in growth media. While we observed only moderate increases in the aggregation of some DND39 knockdown clones under these same conditions, when these clones were stimulated with an α-IgM antibody, CD52 knockdown significantly augmented their aggregation. Taken together, the results generated using these two complementary approaches provide strong evidence for a role of CD52 in negative regulation of B-cell aggregation. Additional studies are underway to better understand these observations.
Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C165.