A Single-Chain Fv Diabody against Human Leukocyte Antigen-A Molecules Specifically Induces Myeloma Cell Death in the Bone Marrow Environment

Abstract

Cross-linked human leukocyte antigen (HLA) class I molecules have been shown to mediate cell death in neoplastic lymphoid cells. However, clinical application of an anti-HLA class I antibody is limited by possible side effects due to widespread expression of HLA class I molecules in normal tissues. To reduce the unwanted Fc-mediated functions of the therapeutic antibody, we have developed a recombinant single-chain Fv diabody (2D7-DB) specific to the A2 domain of HLA-A. Here, we show that 2D7-DB specifically induces multiple myeloma cell death in the bone marrow environment. Both multiple myeloma cell lines and primary multiple myeloma cells expressed HLA-A at higher levels than normal myeloid cells, lymphocytes, or hematopoietic stem cells. 2D7-DB rapidly induced Rho activation and robust actin aggregation that led to caspase-independent death in multiple myeloma cells. This cell death was completely blocked by Rho GTPase inhibitors, suggesting that Rho-induced actin aggregation is crucial for mediating multiple myeloma cell death. Conversely, 2D7-DB neither triggered Rho-mediated actin aggregation nor induced cell death in normal bone marrow cells despite the expression of HLA-A. Treatment with IFNs, melphalan, or bortezomib enhanced multiple myeloma cell death induced by 2D7-DB. Furthermore, administration of 2D7-DB resulted in significant tumor regression in a xenograft model of human multiple myeloma. These results indicate that 2D7-DB acts on multiple myeloma cells differently from other bone marrow cells and thus provide the basis for a novel HLA class I-targeting therapy against multiple myeloma. [Cancer Res 2007;67(3):1184-92]

Small molecule antibody targeting HLA class I inhibits myeloma cancer stem cells by repressing pluripotency-associated transcription factors

“…As reported previously, 27,28 this led to robust actin aggregation in the cytoplasm and eventually induces cell death (Figure 5b). To clarify the signaling pathway involved in this effect, we first examined the phosphorylation pattern of signal-associated factors such as p44/42 MAPK, STAT3 and Akt in the presence of C3B3 with or without stimulation of interleukin-6 or IGF-1.…”

“…26 Previously, we have demonstrated that HLA class I molecules are overexpressed in MM cells and that a recombinant single-chain Fv diabody against this molecule efficiently crosslinks HLA class I and induces rho-mediated actin aggregation, which leads to MM cell death without effector function. 27,28 In this study, we generated a new diabody specific to HLA class I molecule (C3B3) and investigated its potential on cancer stem cell-like fraction of MM cell lines and primary MM cells, focusing on the regulation of b-catenin and pluripotency-associated transcription factors.…”

Regulation of multiple myeloma survival and progression by CD1d

“…28,29 We found that ligation of CD1d in the surface of the KMS11-CD1d cell line also induces considerable cell aggregation compared with KMS11-vector control ( Figure 4A). …”

“…No correlation between extent of cell death and level of CD1d expression was found (r 2 ϭ 0.081). The reason for this is not clear, but lack of correlation between HLA level of expression and cell death has been also observed with the anti-HLA class I diabody remain 2D7 29,34 and may reflect the increased affinity of the corresponding mAb. Therefore, consistent with a role of CD1d in myeloma cell survival, its ligation on the surface of myeloma cell lines and primary myeloma cells induces cell death.…”

“…28,29 Although the in vivo significance of this phenomenon is not known, it has led to the development and validation of anti-HLA class I and II mAb with promising therapeutic potential. 30,31 In this study, we investigate the effect of CD1d ligation on the survival of myeloma cells and expression of CD1d during disease progression.…”