Combination Therapy of a Defucosylated Anti-HM1.24 Monoclonal Antibody Plus Lenalidomide Induces Marked Antibody-Dependent Cellular Cytotoxicity and Inhibits the Clonogenic Potential of Myeloma Cancer Stem-Like Cells
Authors: Harada Takeshi , Ozaki Shuji , Oda Asuka , Iwasa Masami , Fujii Shiro , Nakamura Shingen , Miki Hirokazu , Kagawa Kumiko , Abe Masahiro , Shibata Hironobu , Ikegame Akishige , Tsuji Daisuke , Itoh Kohji , Ri Masaki , Iida Shinsuke , Shiotsu Yukimasa , Kawai Shigeto , Yamada-Okabe Hisafumi , Matsumoto Toshio 
Published: October 2019

Abstract
Abstract 1839
The implementation of hematopoietic stem cell transplantation and new agents such as bortezomib, thalidomide, and lenalidomide (Len) has dramatically improved survival in patients with multiple myeloma (MM). However, most MM patients eventually relapse after achieving of complete response. The existence of cancer stem cells is suggested to cause the relapse and considered as an important therapeutic target. We have demonstrated that HM1.24 (CD317) is selectively over-expressed on neoplastic plasma cells and that a defucosylated humanized monoclonal antibody (mAb) specific to HM1.24 (YB-AHM) is able to induce potent antibody-dependent cellular cytotoxicity (ADCC) against human MM cells in the presence of human effector cells. On the other hand, Len, an immunomodulatory drug, has been shown to activate NK cells to enhance their ADCC activity. Recently, we have reported that “side population (SP)” cells expelling a Hoechst 33342 dye represent a fraction with cancer stem cell-like property in MM cells. Moreover, we have found that MM cancer stem-like cells over-express pluripotency-associated transcription factors such as Sox2 and Nanog, and that these factors are useful for evaluating the potential of MM cancer stem-like cells. Here, we evaluated the efficacy of the combinatory therapy of YB-AHM and Len on MM cancer stem-like cells. We first examined the expression levels of the target molecule, HM1.24 on SP fraction of MM cells. Although SP cells expressed higher levels of ABC transporter ABCG2 compared with main population (MP) cells, HM1.24 was highly expressed in both SP and MP fractions in MM cells. We next examined the inhibitory effect of YB-AHM and Len on clonogenic activity of MM cell lines. RPMI 8226, U266, and OPM-2 cells were pre-incubated with YB-AHM (0.1 μg/ml) and Len (3 μM)-stimulated peripheral blood mononuclear cells (PBMCs) at an effector/target (E/T) ratio of 10 for 4 hours, and then were cultured into H4034 methylcellulose medium. Colony formation of MM cells was examined after 14 days. Treatment with YB-AHM and Len-stimulated PBMCs significantly suppressed the colony formation of MM cell lines compared with the no-treatment group (4±5 vs 62±2 colonies/well in RPMI 8226, p<0.01; 21±4 vs 43±8 colonies/well in U266, p<0.05; and 16±2 vs 84±4 colonies/well in OPM-2, p<0.01), suggesting that the combination therapy can target clonogenic MM cells. The mRNA expression levels of Sox2 and Nanog on MM cell lines and primary MM cells were also decreased when treated with YB-AHM (0.1 μg/ml) and Len-stimulated PBMCs (E/T=10) for 24 hours. Notably, this combination therapy decreased the mRNA expression of these transcription factors even in bortezomib-resistant MM cell line, OPM-2/BTZ. Finally, we examined the cytotoxic activity of YB-AHM plus Len using patients' bone marrow mononuclear cells (BMMCs) containing both target MM cells and autologous effector cells. BMMCs were stimulated with Len (3 μM) for 48 hours and further incubated with YB-AHM (0.1 μg/ml) for 24 hours. The cytotoxic activity was evaluated by counting CD38-positive MM cells in total BMMCs using flow cytometry. When we examined BMMCs from 10 MM patients, the combination of YB-AHM plus Len significantly induced MM cell death compared with controls (mean cytotoxicity, 46±23% vs 7±10%, p<0.01). Collectively, these results demonstrate that defucosylated HM1.24 mAb and Len in combination induce ADCC to eradicate MM cancer stem-like cells in bone marrow environment. Therefore, this combination might provide a novel therapeutic strategy targeting clonogenic drug-resistant clones in MM.

Disclosures:
Nakamura: Janssen Pharmaceutical K.K.: Honoraria. Abe:Janssen Pharmaceutical K.K.: Honoraria, Research Funding. Iida:Janssen Pharmaceutical K.K.: Honoraria.