DKK1-Specific Cytotoxic T Lymphocytes Are Potent Effector Cells for the Treatment of Multiple Myeloma in SCID-hu Model.

Dickkopf-1 (DKK1), a secreted protein and Wnt signaling pathway inhibitor, is highly expressed by the tumor cells of most multiple myeloma (MM) patients and contributes to osteolytic bone disease by inhibiting the differentiation of osteoblasts. DKK1 has been targeted as a myeloma-associated antigen by immunotherapeutical approaches based on DKK1-directed monoclonal antibodies and DKK1-specific cytolytic T lymphocytes (CTLs). The present study was conducted to develop a treatment strategy through adoptive transfer of DKK1-specific CTLs in a SCID-hu system. Using DKK1 peptide-pulsed dendritic cells (DCs), we successfully generated DKK1-specific CTL lines and clones from HLA-A2⁺ MM patients in vitro. These CTLs not only had cytolytic activity against DKK1 peptide-pulsed T2 cells, but also significantly lysed autologous myeloma tumor cells, HLA-A2⁺ and DKK1⁺ MM cell lines U266 and IM-9, and primary tumor cells in vitro. No killing was observed against HLA-A2⁺ normal lymphocytes, including B cells and HLA-A2⁺ DKK1^- XG1 or HLA-A2^- myeloma cell lines or primary myeloma cells from patients. To determine the in vivo antitumor activity, SCID-hu mice were engrafted with primary MM cells expressing high levels of DKK1 from patients and treated with DKK1-specific CTLs after one month of tumor injection. Control mice were treated with naïve CD8⁺ T cells or PBS alone. All mice were examined by X-ray, tumor burden was measured according to levels of circulating human IgG, and survival rates were determined. All mice established human myeloma within one month after tumor inoculation, at which time the level of circulating human IgG was greater than 600 ng/ml in mouse serum. After another 3 weeks, the level of circulating human IgG was greater than 2,500 ng/ml in the serum of all control mice, while only 40% mice treated with DKK1-specific CTLs had high levels of circulating human IgG. X-ray examination showed that osteolytic bone disease and a large tumor burden were found in all control mice, while the established tumor was eradicated in 60% mice treated with DKK1-specific CTLs. Results of immunofluorescence studies showed that only tumor-reactive DKK1-specific CD8⁺ T cells were able to infiltrate into the tumor and mediate apoptosis in tumor cells. All control mice succumbed to myeloma within one month of establishment of the myeloma, while 9 out of 15 mice treated with DKK1-specific CTLs were macroscopically disease-free at the end of the experiment. These results suggest that DKK1-specific CTLs are able to eradicate established patient-derived primary myeloma in the host and that adoptive transfer of DKK1-specific CTLs may be used for myeloma therapy.