Abstract 2523: The in vitro effects of immobilized epratuzumab, a non-blocking anti-CD22 humanized monoclonal antibody, on malignant B cells

Abstract

Abstract Epratuzumab (hLL2), a humanized anti-CD22 monoclonal antibody, is currently in advanced trials in non-Hodgkin lymphoma (NHL) and systemic lupus erythematosus (SLE) patients. As a single agent, hLL2 is well tolerated and depletes 30 to 50% of circulating B cells in patients with NHL and SLE. The mechanisms by which hLL2 depletes B cells in vivo is still largely unknown and continues to be a subject of current research interest. In vitro, hLL2 shows moderate antibody-dependent cell-mediated cytotoxicity, but no detectable complement-dependent toxicity, and displays cytotoxicity to CD22-expressing cells, for example, Ramos and Daudi human Burkitt lymphomas, only when immobilized onto plastic plates or in combination with a suboptimal amount (1 μg/mL) of anti-IgM plus a crosslinking secondary antibody. We have previously reported that hLL2, despite a lack of direct cytotoxicity, is effective in inducing the phosphorylation of CD22, translocation of CD22 to lipid rafts, and internalization of CD22. In this study, we evaluated the various effects of immobilized hLL2 on D(1-1), a subclone of Daudi selected for a higher expression of membrane IgM (mIgM). Our findings demonstrate for the first time that many of the intracellular events induced by binding CD22 to immobilized hLL2 were similar to those induced by binding mIgM with a sufficient high concentration (10 μg/mL) of anti-IgM; for example, both were found to induce phosphorylation of CD22, CD79a and CD79b, and the translocation of all three into the lipid rafts, which is critical for the observed cytotoxicity. Immunofluorescence analysis revealed CD22 colocalized with BCR and formed caps before being internalized. Like anti-IgM, immobilized hLL2 induced substantial apoptosis (25 to 40%) in D(1-1), which was caspase-dependent, accompanied by a pronounced increase in both pERKs and pJNKs with a concurrent decrease in the constitutively-activated p38, and preventable by inhibitors to JNKs or caspases. The involvement of mitochondria in the apoptotic pathway was evidenced by changes in mitochondrial membrane potential and generation of reactive oxygen species, as well as upregulation of Bax (pro-apoptotic) and downregulation of Bcl-xl, Mcl-1 and Bcl-2 (anti-apoptotic). Further studies are underway to elucidate how hLL2 interferes with B-cell survival in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2523. doi:1538-7445.AM2012-2523