A Recombinant HLA Class I-Specific Single-Chain Fv Diabody Can Target Cancer Stem Cell-Like Side Population Cells in Multiple Myeloma.

Abstract 2799

Poster Board II-775

Multiple myeloma (MM) remains an incurable disease despite high overall response rates induced by combination therapy of chemotherapeutic drugs and new agents such as thalidomide, lenalidomide, and bortezomib. Recently, the existence of cancer stem cells is proposed for several tumors including MM, and such cells are considered as an important target for curative therapy. The side population (SP) cells are identified by their ability to efflux Hoechst 33342 dye, which represent a small fraction with stem cell properties. Our previous studies have demonstrated that MM cells expressed HLA class I at high levels than normal hematopoietic cells, and that antibodies against HLA class I specifically induced MM cell death by Rho-mediated actin aggregation. In this study, we characterized SP fraction in MM cell lines (RPMI 8226, U266, and MM.1S) and primary MM cells (n=3) by flow cytometry, and investigated the efficacy of chemotherapeutic drugs as well as a recombinant single-chain Fv diabody specific to HLA class I (C3B3-DB, Chugai Pharmaceutical Co., Ltd, Tokyo, Japan). MM cell lines and primary MM cells contained a distinct fraction of SP cells ranging from 0.01% to 0.6% of the gated cells, which was confirmed by disappearance after treatment with verapamil. Treatment with melphalan (10 μM, 48 hours) decreased the percentage of non-SP cells (16.7% to 3.9%) but not of SP cells (0.6% to 0.7%) in RPMI 8226. In contrast, treatment with C3B3-DB (1 μg/ml, 48 hours) caused a significant reduction in both non-SP cells (16.7% to 4.0%) and SP cells (0.6% to 0.3%) in RPMI 8226. Similar results were observed in primary MM cells enriched from patient bone marrow cells by negative selection with antibody cocktail. Next, we isolated SP cells and non-SP cells in RPMI 8226 using a cell sorter, and characterized in detail. SP cells exhibited elevated levels of ABCG2 and low levels of CD138 compared with non-SP cells, but HLA class I was expressed at high levels in both SP and non-SP cells by flow cytometry. Annexin/PI assay showed that SP cells were 1.5- and 2.0-fold more resistant to melphalan and bortezomib than non-SP cells. Whereas both SP cells and non-SP cells showed similar sensitivity to C3B3-DB. Methylcellulose colony-forming assay showed that SP cells have a higher potential for colony formation (numbers of colonies, 13.0±1.0) than non-SP cells (1.3±0.6), and the colony formation of SP cells was significantly inhibited by C3B3-DB (0.7±0.6, p<0.01). Notably, RPMI 8226 cells expressed the pluripotency-associated transcription factors including Oct3/4, Sox2, and Nanog as detected by RT-PCR, but only Sox2 mRNA expression was decreased at 6 hours after C3B3-DB treatment. Furthermore, when C3B3-DB-treated SP cells were inoculated subcutaneously in SCID mice (n=4), there was a significant decrease in a tumor volume as compared with untreated SP cells (679±148mm³ vs 3217±562 mm³, p<0.01). SP cell analysis of these tumors showed that the percentage of SP fraction of C3B3-DB-treated SP cell tumors was significantly low (0.01%) compared with that of untreated SP cell tumors (0.33%). These results indicate that C3B3-DB has a potential activity for eradicating MM cancer stem cell-like SP cells, and that the molecular targeting of such drug-resistant cells provides an important strategy for improving the efficacy of current therapies in MM.