Human-Like Mouse Models for Testing the Efficacy and Safety of Anti-β2-Microglobulin Monoclonal Antibodies to Treat Myeloma

Abstract

Multiple myeloma (MM) remains fatal in the majority of patients with the disease, even though the application of new therapeutic agents has greatly advanced the treatment of the disease. There is an urgent need to develop novel therapeutic agents. We recently demonstrated that anti-β2-microglobulin monoclonal antibodies (anti-β2M mAbs) have remarkably strong apoptotic effects on myeloma in vitro and in SCID mice. However, whether the mAbs will be therapeutic and safe in treating myeloma patients, in whom every tissue expresses low densities of MHC class I molecules and elevated levels of soluble β2M are present, remains to be determined. In this study, we examined the antimyeloma activity and potential toxicity of the mAbs in a human-like situation. We developed and used a myeloma-HLA-A2-transgenic NOD/SCID mouse model. After myeloma establishment in NOD/SCID mice transgenic for HLA-A2 α-chain, we found that all tissues express human HLA-A2 and β2M, and detected high levels (2 mg/mL) of circulating human β2M. After one intraperitoneal (i.p.) injection of the mAbs (1 mg per mouse), the mAbs could be detected on the surface of tumors and murine organs. Furthermore, murine splenocytes were incubated with immobilized mAb W6/32 to crosslink MHC class I. Western blotting analysis showed that cross-linking human MHC class I triggers phosphorylation of Lyn, Syk, and PLC-γ2 in the treated cells, indicating that indeed human MHC class I are functional and able to transduce signals in murine cells. Thus, these findings demonstrate that the mouse model is suitable for our studies because myeloma-derived human β2M was highly elevated in the serum, and bound with the α-chain to form mature and functional human MHC class I on all tissues. By using the model, we first examined the therapeutic efficacy of the mAbs on established myeloma. Myeloma-bearing mice were i.p. injected with the mAbs (1 mg per mouse) twice a week for a total of 4 injections. The results showed that the mAbs effectively suppress myeloma growth, and activated caspase-9 and -3 and induced myeloma cell apoptosis in vivo. We also examined whether the mAbs damage human MHC class I-expressing normal cells and tissues. After treatment with the mAbs, murine organs were removed for histological examination. We found that although the mAbs were detected on different organs, no tissue damage or cell apoptosis was observed in the mice. Furthermore, we elucidated the mechanisms underlying the selectivity of the mAbs in killing myeloma cells without damaging normal cells. By using the QuantiBRITE beads, we quantified the numbers of surface β2M and found that myeloma cells have approximately 265,918 surface β2M molecules whereas normal blood lymphocytes have approximately 79,521, indicating that myeloma cells express 3-fold more surface β2M than lymphocytes. Myeloma cells treated with specific siRNAs for human β2M expressed a similarly low level of β2M (91,723) and became resistant to mAb-induced apoptosis. Thus, our findings indicate that the mAbs may be safe and that the tissue-expressed and soluble β2M may not compromise their therapeutic effects in myeloma patients. This study provides further support for the future application of the mAbs as therapeutic agents for MM.