Identification of an immunogenic DKK1 long peptide for immunotherapy of human multiple myeloma

_Dnormal multiple ^ickkopf-1_tissues, myeloma ^(DKK1),_may^broadly (MM) _{be an} and _ideal^expressed other _target cancers _for^by_{immunotherapy.}^tumor but absent ^{cells from} from _Our^human most _previous studies have shown that DKK1 (peptide)-specific cytotoxic T lymphocytes can effectively lyse primary MM cells in vitro. To develop DKK1-based vaccines that can be easily and inexpensively made and used by all patients, we identified a DKK1 long peptide (LP), DKK1_3-76-LP, that contains 74 amino acids and epitopes that can potentially bind to all major MHC class I and II molecules. Using HLA-A*0201- and HLA-DR*4-transgenic mouse models, we found that DKK1-specific CD4⁺ and CD8⁺ T-cell responses, detected by DKK1 short peptide (P20 and P66v)-HLA-A*0201 tetramer staining and cytotoxic assay for CD8⁺ T cells or by carboxyfluorescein diacetate succinimidyl ester (CSFE) dilution and IFN-g secretion for CD4⁺ T cells, respectively, can be induced in vivo by immunizing mice with the DKK1_3-76-LP. In addition, DKK1_3-76-LP also induced anti-DKK1 humoral immunity in the transgenic mice and the DKK1 antibodies were functional. Finally, DKK1_3-76-LP stimulated human blood T cells ex vivo to generate DKK1-specific CD4⁺ and CD8⁺ T-cell responses from 8 out of 10 MM patients with different MHC backgrounds. The generated DKK1-specific CD8⁺ cells efficiently lysed autologous MM cells from these patients. Thus, these results confirm the immunogenicity of the DKK1_3-76-LP in eliciting DKK1-specific CD4⁺ and CD8⁺ T-cell responses in vitro and in vivo, and suggest that the DKK1_3-76-LP can be used for immunotherapy of MM and other cancers.