Novel DKK1-A2 CAR-T cell therpy for hematologic malignancies Free

Background DKK1 (Dickkopf-1) is a secreted protein that acts as an antagonist of the Wnt/β-catenin signaling pathway. DKK1 overexpression has been observed in various tumors, especially in multiple myeloma. Conversely, DKK1 has limited expression in normal human tissues. This reciprocal expression makes DKK1 an attractive target for cancer immunotherapy, particularly in developing CAR-T cell therapies. Previously, we have identified a DKK1 peptide, P20 (ALGGHPLLGV), which showed excellent binding affinity to HLA-A*0201 (HLA-A2), a widely distributed HLA serotype found in 30% to 50% of the population. We developed a specific C2 monoclonal antibody that recognizes the DKK1-P20 and-HLA-A2 (DKK1-A2) complex. In this study, we developed a novel CAR-T cell therapy targeting DKK1-A2 complex and the therapeutic efficacy and safety profile were evaluated.

Methods DKK1-A2 CAR consisted of the scFv derived from C2 antibody, 4-1BB and CD3ζ intracellular domains. Enhanced green fluorescent protein was co-expressed with CAR to track CAR-T cells. Human myeloma cell lines U266, KMS-26, ARP-1, KMS-11, human mantle cell lymphoma cell line JeKo-1 and human Burkitt's lymphoma Daudi were used. Cytolytic activity of DKK1-A2 CAR-T cells against tumor cells was determined by a luciferase-based cytotoxicity assay. Proliferation of CAR-T cells was corroborated by CFSE dilution assay. Cytokine secretion was determined by flow cytometry-based intracellular staining. Therapeutic efficacy of DKK1-A2 CAR-T cells was evaluated in human U266 and KMS-26 xenograft models by intravenously transferring CAR-T cells. Immunoglobulin lambda chain secreted by tumor cells was determined by ELISA as tumor burden and survival was monitored weekly. DKK1-A2 complex expression on normal tissues was assessed with commercially available tissue arrays. Potential hematological toxicity of DKK1-A2 CAR-T cells was evaluated by co-culture of CAR-T cells and peripheral blood mononuclear cell (PBMC) and bone marrow mononuclear cells (BMMC), and cell apoptosis was analyzed by flow cytometry. Similarly, potential efficacy and toxicity of DKK1-A2 CAR-T cells in immunocompetent mice was performed in DKK1 and HLA-A2 transgenic mice.

Results DKK1-A2 CAR-T cells were generated by retrovirus transduction of human T cells. The transduction efficiency of activated human T cells was generally greater than 90%. DKK1-A2 CAR-T cells significantly lysed HLA-A2 positive tumor cells JeKo-1, U266, and KMS-26 in a dose-dependent manner compared to CD19 or control CAR-T cells. DKK1-A2 CAR-T cells exhibited a strong proliferation in response to HLA-A2 positive tumor cells. The anti-tumor activity of DKK1-A2 CAR-T cells was further supported by IFN-γ and TNF-α production by both CD4⁺ and CD8⁺ DKK1-A2 CAR-T cells. U266 or KMS26 tumor cells were implanted intravenously into NSG mice. In the therapeutic experiments, DKK1-A2 CAR-T cells effectively controlled U266 or KMS-26 tumor cell growth and all mice remained alive at 7 weeks post CAR-T cell infusion, while CD19 CAR-T cells failed to do so. In the commercial normal tissue arrays, 35 different types of normal tissues from three HLA-A2 positive donors were stained with DKK1, HLA-A2 and DKK1-A2 antibodies and all human normal tissues were stained negative for DKK1-A2 complex except one tonsil tissue. In the hematological toxicity experiment, DKK1-A2 CAR-T cells did not cause significant cell death in cell populations of PBMC or BMMC. In the DKK1-A2 transgenic mice, murine DKK1-A2 CAR-T cells showed effective anti-tumor activity by inhibiting 5TGM1-DKK1-A2 tumor cell growth. Also, adoptively transfer of murine DKK1-A2 CAR-T cells did not significantly change body weight of tumor-bearing mice, or affect cell populations in blood, spleen or bone marrow.

Conclusions We developed CAR-T cells targeting DKK1-A2 complex for the treatment of human hematologic tumors. DKK1-A2 CAR-T cells exhibited significant therapeutic efficacy and favorable safety profiles without detectable on- or off-target toxicities against normal tissues. Our results suggest the further study of DKK1-A2 CAR-T cells in translational and clinical applications.