Active Vaccination with DKK1 Induces Protective and Therapeutic Antitumor Immunity In Multiple Myeloma

Abstract 3040

Dickkopf-1 (DKK1), a secreted protein and Wnt signaling pathway inhibitor, is highly expressed by the tumor cells of almost all patients with multiple myeloma (MM) and may be responsible for suppressed osteoblast formation. In our previous studies, we demonstrated that DKK-1 is a potent tumor-associated antigen in MM recognized by cytotoxic T lymphocytes (CTLs), which can effectively lyse autologous myeloma tumor cells in vitro (Qian et al., Blood 2007;110:1587-1594) and eradicate established patient-derived primary myeloma in SCID-hu mice upon adoptive transfer. To examine the potential of DKK1-based immunotherapy in MM, we investigated the efficacy of active vaccination with (murine) DKK1 DNA vaccine in a murine (MOPC-21) myeloma model. A plasmid DNA construct encoding defensin2-sFv was generated, and DKK1 full-length cDNA was cloned from DKK1-expressing mouse stromal cells by reverse transcription–polymerase chain reaction and genetically fused with defensin2 (DNA-vac). We first examined the ability of the vaccines to protect mice from developing myeloma. While 100% of mice vaccinated with vector control or PBS (10 mice for each group) developed tumors, 70% and 40% mice vaccinated with DNA-vac or DNA-vac with CpG (ODN 1826), respectively, developed tumors. These results clearly show that active vaccination with DKK1 DNA vaccines was able to protect mice from tumor challenge, and that combination with CpG was more effective than DNA vaccine alone. On day 90 after tumor challenge, all surviving tumor-free mice were rechallenged with the same myeloma cells and followed for tumor development. By day 180, All surviving mice that were vaccinated with DNA-vac or DNA-vac with CpG have no developed tumors, indicating that active vaccination with DNA vaccines induced strong tumor-specific memory immune responses to protect mice from tumor rechallenge. Next, we examined the therapeutic effects of DNA vaccines in our myeloma mouse model. DNA vaccine alone eradicated established myeloma in 1 out of 5 mice, while DNA vaccine plus CpG eradicated myeloma in 3 out of 5 mice bearing intermediate tumors (≥ 5 mm in diameter). These results indicate that DNA vaccine plus CpG was much more effective at eradicating established myeloma than DNA vaccine alone. Finally, the mechanisms of tumor protection induced by DNA vaccines were investigated. By depleting CD4⁺ or CD8⁺ T cells, we showed that CD8⁺ T cells are required for DNA-induced antimyeloma responses. In DNA-vaccinated mice, splenocytes contained increased numbers of DKK1-specific, IFN-g-secreting and proliferative T cells. The splenic CD8⁺ T cells exhibited enhanced cytotoxicity against myeloma cells. Furthermore, DKK1-specific CD8⁺ T cells were shown to be increased in DKK1-DNA vaccinated mice by using DKK1-peptide (P11, P15 and P210) tetramer staining. These results demonstrate the presence of myeloma-specific CTLs in vaccinated mice and show that DKK1 DNA vaccine can induce a potent CTL response capable of killing myeloma cells. Together, our study lays a basis for future clinical trials in MM by using DKK1 as a vaccine for all patients.