Combination Therapy of a Defucosylated Anti-HM1.24 Monoclonal Antibody Plus Lenalidomide Significantly Enhances Myeloma Cell Cytotoxicity Via Antibody-Dependent Cellular Cytotoxicity
Authors: Harada Takeshi , Ozaki Shuji , Oda Asuka , Amou Hiroe , Fujii Shiro , Nakamura Shingen , Miki Hirokazu , Nakano Ayako , Kagawa Kumiko , Takeuchi Kyoko , Abe Masahiro , Shibata Hironobu , Kawai Shigeto , Yamada-Okabe Hisafumi , Matsumoto Toshio 
Published: October 2019

Abstract
Abstract 1833
Multiple myeloma (MM) is a B-cell malignancy characterized by the accumulation of neoplastic plasma cells in the bone marrow. During the last decade, treatment of MM has been improved by incorporating bortezomib, thalidomide, and lenalidomide (LEN) into conventional cytotoxic and transplantation regimens in newly diagnosed and relapsed/refractory MM patients. However, MM still remains incurable despite the implementation of these new treatment options, so more efficacious therapies are needed to further improve the prognosis of MM. Monoclonal antibody (mAb)-based immunotherapy has recently become an alternative strategy for treatment of cancers. Our previous studies have shown that HM1.24 (CD317) is selectively expressed on terminally differentiated normal and neoplastic plasma cells and, moreover, expressed on the side population of MM cells that represents MM cancer stem cells. We have previously generated a humanized mAb (AHM) specific to HM1.24 for the treatment of MM. AHM carries an Fc region derived from human IgG1-k and exhibits the ability to induce antibody-dependent cellular cytotoxicity (ADCC) against human MM cells in the presence of human effector cells. To improve the efficacy of AHM, we have developed a defucosylated mAb (YB-AHM) with a higher affinity to Fc gamma RIII. LEN is a structural analog of thalidomide with more potent immunomodulatory activities. Several studies have shown that LEN activates NK cell function and enhances NK cell-mediated lysis of both MM cell lines and patient MM cells in vitro. Here, we evaluated the efficacy of combination therapy of YB-AHM and LEN. First, we investigated whether LEN stimulates the expression of HM1.24 on MM cells. LEN alone did not affect HM1.24 expression, but in the presence of peripheral blood mononuclear cells (PBMCs) LEN augmented the expression of HM1.24 in MM cell lines and primary MM cells. In PBMCs, expression levels of CD56 increased after stimulation with LEN. These results suggest that LEN might augment the ADCC activity by enhancing HM1.24 antigen and NK activity. Next, we evaluated ADCC activity of YB-AHM against RPMI 8226 cells by using flow cytometric PKH-26 assay. When we used PBMCs from healthy donors (n=5) as effectors, ADCC activity of YB-AHM was increased in an E:T ratio-dependent manner. Importantly, YB-AHM induced significantly higher ADCC activity compared with AHM (24±6% vs 11±7%, p<0.05; mAb, 100 ng/mL; E:T ratio, 10). Treatment of PBMCs with LEN (3 micro M for 2 days) slightly enhanced ADCC activity of AHM (12±5%) and YB-AHM (30±6%). In PBMCs from MM patients (n=11), YB-AHM induced ADCC activity (36±15%) that was further enhanced by treatment with LEN (45±15%). To evaluate the efficacy of this combination therapy in a more physiological manner, we assessed the efficacy of YB-AHM using total bone marrow mononuclear cells (BMMCs) from MM patients that contained both MM cells and effector cells. BMMCs were stimulated with LEN (3 micro M) for 2 days and further incubated with YB-AHM for 24 hours. Cytotoxicity was evaluated by the number of CD38-positive MM cells in total BMMCs using flow cytometry. YB-AHM plus LEN significantly reduced the number of MM cells (10.3%) compared to YB-AHM alone (21.6%) in patient No.1. Finally, RPMI 8226 cells were co-cultured with YB-AHM and LEN-stimulated PBMCs from MM patients, and MM colony formation was examined using methylcellulose assay. Colony formation of RPMI 8226 was significantly suppressed by YB-AHM and LEN-stimulated PBMCs compared to control (14±8 vs 49±10 colonies, p<0.01), suggesting that this combination therapy can target MM cancer stem cells. Thus, these results indicate that combining defucosylated HM1.24 mAb with immunomodulatory drugs provides a novel therapeutic strategy in patients with MM.

Disclosures:
No relevant conflicts of interest to declare.