A Novel Bispecific Allogeneic CAR-DNT Cells Targeting BCMA and CD19 for Immunotherapy
Authors: Zhao Hui , Wang Liuyang , Liu Shuai , Wen Xiaojun , Xu Man , Yang Liming 
Published: December 2024

Introduction:
Although autologous CAR-T cells to hematological malignancy have achieved deep and durable responses with manageable safety, there is a pressing need for allogeneic chimeric antigen receptor (CAR)-immune cell therapies that are safe, effective, and affordable. We have reported before that the safety and potential efficacy of allogeneic DNT therapy for acute myeloid leukemia (AML) and B-NHL in phase I clinical trials with no signs of GvHD or other &gt;grade 2 treatment-associated adverse events. Here, we reported a successful development of a novel dual-target BCMA/CD19-mbIL15 CAR-DNT cells with superior antitumor activity in vitro and in vivo by comparison to dual-target BCMA/CD19-CAR-DNT cells, single-target BCMA-CAR-DNT cells and single-target CD19-CAR-DNT cell in short-term cytotoxicity, long-term recursive killing potential at high tumor cell loads as well as long-term proliferation and survival without exogenous IL-2 in vitro culture. The new designed CAR-DNT cells is expected to have superior safety and efficacy in clinic for multiple applications in treating hematological tumors and autoimmune diseases.
Methods:
The biological activity differences of dual-target BCMA/CD19-mbIL15 CAR-DNT cells, dual-target BCMA/CD19-CAR-DNT cells, single-target BCMA-CAR-DNT cells, and single-target CD19-CAR-DNT cell in short-term tumor kill, repeating tumor challenge killing and long-term survival without exogenous IL-2 in vitro were studied by firefly luciferase reporter gene assay and cell counting assay. Human cell line-derived xenograft animal model in NSG mice were utilized to study the anti-tumor efficacy and safety in vivo.
Results:
We demonstrated that BCMA/CD19-mbIL15 CAR-DNT cells and BCMA/CD19-CAR-DNT cells have more potent tumor killing effect than single target BCMA-CAR-DNT cells and single target CD19-CAR-DNT cells in a short-term cytotoxicity against multiple tumor cell lines in vitro. In the assay, 4 different designed CAR-DNT cells were co-cultured with H929-CD19-luciferase at an E/T ratio of 1:2 for 24 hours. The percentage of cytotoxicity of BCMA/CD19-mbIL15 CAR-DNT cells, BCMA/CD19-CAR-DNT cells, BCMA-CAR-DNT cells, and CD19-CAR-DNT cells were 52%, 56%, 10% ,and 33% respectively. To evaluate the long-term cytotoxic function and proliferative capacity of 4 different designed CAR-DNT cells in vitro, the cells were co-cultured over 12 days with additional tumor targets administered to the co-culture every 4 day. It is shown that the BCMA/CD19-mbIL15 CAR-DNT cells has lasting tumor-specific lysis than three other CAR-DNT cells in that the percentage of cytotoxicity is still more that 99% after 3 rounds tumor cell restimulation. In addition, BCMA/CD19-mbIL15 CAR-DNT cells can proliferate with &gt; 90% viability in the absence of exogenous IL-2 for at least 14 days and survive continuously for more than 50 days. In contrast, three other CAR-DNT cells undergo apoptosis rapidly after 7 days in vitro. In xenograft NSG mice model, BCMA/CD19-mbIL15 CAR-DNT cells showed the superior antitumor activities without observable adverse events.
Conclusions:
We have successfully developed a bispecific CAR-DNT cells targeting BCMA and CD19 with mbIL15 gene to support long-term proliferation and cytotoxicity both in vitro and in vivo without exogenous IL-2. The enhanced efficacy profile of BCMA/CD19-mbIL15 CAR-DNT cells warrant further clinical trials to evaluate its efficacy and safety in multiple applications for hematological and autoimmune diseases treatment.