Development of multi-targeting CAR-double-negative T cells for allogeneic immunotherapy in B-cell leukemia

Abstract Introduction Clinical breakthroughs achieved through Chimeric Antigen Receptor (CAR)-T cell therapy have revolutionized the treatment for B-cell acute lymphoblastic leukemia (B-ALL). Despite this, B-ALL still causes 7,000 deaths each year. Two major barriers limit the success of CAR-T. First, access remains limited due to a costly, time-consuming, and complex manufacturing process. Current CAR-T products are autologous and derived from heavily pre-treated patients, which delays production and results in a high price tag. Second, relapse remains common, affecting more than 50% of individuals. This can result from low CAR-T cell persistence or antigen escape. Since commercially available CAR-T cells target a single antigen, CD19, cancer cells can evade CAR-T cell-mediated immunity by downregulating the CAR-targeted antigen (Rampotas & Roddie, Blood 2025). Our group described donor-derived CD3+ CD4- CD8- Double-Negative T cells (DNTs), a rare type of mature peripheral T cell, as an off-the-shelf adoptive cellular therapy for acute myeloid leukemia (Tang et. al., Am J Hematol 2022). In clinical trials, allogeneic DNTs and CD19-CAR-DNT proved effective cancer-killing without inducing graft-versus-host disease (Xiao et. al., EClinicalMedicine 2024) (Tin, Lee, Zhang; Curr Opin Pharmacol 2025). These findings support DNTs as a safe and effective allogeneic platform for CAR-based therapies. Here, we evaluate the potential of arming DNTs with CAR constructs targeting multiple B-cell antigens to overcome relapse and broaden access to allogeneic CAR therapies for B-ALL. Methods DNTs were isolated from healthy donor peripheral blood and transduced with CAR constructs targeting CD19 or CD22. Following expansion, CAR-DNTs were cryopreserved for subsequent assays. Cytotoxicity was assessed by co-culturing individual or pooled CAR-DNTs with either primary B-ALL patient blasts or NALM-6 cells. A CD19-knockout NALM-6 line was generated via CRISPR-Cas9 to model antigen escape. Flow cytometry was used to evaluate killing efficiency, CAR-DNT phenotype, and antigen expression on target cells. Cytokine release profiles were analyzed using a Luminex multiplex assay. Results Allogeneic CAR-DNTs—single-targeting CD19 or pooled with CD22—exhibited superior cytotoxicity compared to untransduced DNTs in in vitro killing assays against NALM-6 cells and a primary B-ALL patient sample. Among them, CD19-CAR-DNTs showed the highest cytotoxic activity (p < 0.0004). Importantly, CAR-DNTs retained both cancer-killing function and >90% viability after cryopreservation, supporting their potential for “off-the-shelf” therapeutic use. When co-cultured with CD19-CAR-DNTs, leukemia cells showed marked downregulation of CD19 expression. In contrast, CD22 expression remained largely unchanged following exposure to CD22-CAR-DNTs. The functional relevance of antigen loss was confirmed using a CD19-knockout NALM-6 line: in this context, CD19-CAR-DNTs lost their cytotoxic advantage. To model long-term tumour control, a co-culture system using wild-type NALM-6 and CD19-CAR-DNTs at a low effector-to-target ratio was maintained and monitored for 79 days. CAR-DNTs effectively suppressed leukemia growth for over 60 days and remained viable for more than 78 days. Interestingly, in persisting viable NALM-6 cells, CD19 downregulation was observed early, but surface expression gradually recovered to baseline levels despite continuous CAR19-DNT exposure. Finally, cytokine profiling via Luminex revealed distinct effector signatures. While CAR-T conventional cells produced higher levels of IFN-γ and TNF-α, CAR-DNTs secreted more IL-8 and granzyme A (GZMA), suggesting a mechanism of action that favours localized cytotoxicity with potentially reduced systemic inflammation. Conclusion Our findings support the feasibility of using CAR-DNTs as an off-the-shelf immunotherapy for B-ALL, highlighting their capacity for cryopreservation, long-term storage, and sustained cytotoxic function. The potential for a “mix-and-match” approach, combining CAR-DNTs targeting different antigens, may be especially valuable in the context of relapse or antigen escape. Moving forward, we plan to expand our panel of CAR targets to broaden therapeutic coverage and to validate efficacy using additional B-chronic lymphocytic leukemia cell lines and B-ALL patient samples. Preclinical testing will continue in the established B-ALL xenograft model using NSG-GSM3 mice to further evaluate in vivo functionality and safety.

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