Jul 2026· Journal of Immunology· Vol 215· 0 citations
TL;DR
In vivo, CAR4-DNT achieved durable leukemia clearance and prolonged survival, whereas PBS-treated mice rapidly progressed and UT-DNT recipients showed transient disease control before relapse, establishing CAR4-DNTs as a promising immunotherapy candidate for AML, supporting their further preclinical and translational development.
Abstract
Acute myeloid leukemia (AML) is an aggressive hematologic malignancy with poor prognosis. Current Immunotherapies, including chimeric antigen receptor (CAR) T cell strategies, are limited by the scarcity of antigens that are present on AML cells but absent from hematopoietic stem and progenitor cells (HSPCs). To identify a potential therapeutic target, we analyzed two publicly available single-cell RNA sequencing datasets and found that CD4 is frequently expressed on AML blasts but absent from HSPCs, supporting its potential as a novel CAR target. We developed a CD4-targeted CAR using CD3+CD4⁻CD8⁻ double-negative T cells (CAR4-DNTs). DNTs offer a unique therapeutic platform: they naturally lack CD4, preventing fratricide, and elicit endogenous anti-leukemic activity. Their feasibility and safety as an allogeneic cell therapy platform was demonstrated in a Phase I trial. This work aims to establish CAR4-DNTs as a next-generation, off-the-shelf CAR-T cell therapy for AML.
Donor-derived DNTs were engineered to express a CD4-CAR. Cytotoxicity, persistence, and cytokine secretion were evaluated against AML cell lines, CD4+ and CD4-knockout AML variants, and primary AML samples. NSG-SGM3 mice engrafted with luciferase+ MV4-11 cells received CAR4-DNTs, untransduced (UT) DNTs, or PBS. Disease progression was monitored by bioluminescence imaging and survival analysis.
CAR4-DNTs were successfully manufactured without fratricide. They exhibited significantly enhanced and more durable cytotoxicity than UT-DNTs against AML cell lines and patient samples. Importantly, CAR4-DNTs retained endogenous cytotoxicity towards CD4-negative AML cells. In vivo, CAR4-DNT achieved durable leukemia clearance and prolonged survival, whereas PBS-treated mice rapidly progressed and UT-DNT recipients showed transient disease control before relapse.
These findings establish CAR4-DNTs as a promising immunotherapy candidate for AML, supporting their further preclinical and translational development.
Distinguished Doctoral Recruitment Scholarship, The C17 Council, Alberta Cancer Foundation, Alberta Children’s Hospital Research Institute
Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)
Chimeric antigen receptor (CAR)-T cell therapy has achieved remarkable success in B-cell malignancies, but its application in acute myeloid leukemia (AML) and renal cell carcinoma (RCC) remains challenging due to the lack of ideal target antigens. CD70 is highly expressed on AML and RCC tumor cells, while its expression in normal tissues is largely restricted to activated lymphocytes, making it a promising immunotherapeutic target. However, CD70 expression on activated T cells can trigger fratricide in CD70-targeted CAR-T cells, posing a major challenge for their preparation and efficacy. Using an analog of the clinical-stage anti-CD70 antibody ARGX-110 as a reference, we screened a phage display library from immunized mice and identified high-affinity anti-CD70 antibodies, which were then used to construct a panel of second-generation CARs. Functional characterization identified a lead candidate, A174-CAR-T, with high CAR expression, strong in vitro antitumor activity, and low fratricide. We further humanized its single-chain variable fragment to generate A174-hu1-CAR. Compared with the parental construct, A174-hu1-CAR-T cells exhibited enhanced expansion and markedly reduced surface expression of CD70, consistent with improved cis-masking (i.e., surface shielding on the same cell). Molecular docking and surface electrostatic potential analyses suggested that humanization optimized the scFv-CD70 binding interface, which together with increased CAR surface expression contributed to the enhanced masking and reduced fratricide. Functionally, A174-hu1-CAR-T cells maintained potent cytotoxicity and showed enrichment of proliferative and central memory T-cell subsets. In xenograft models, A174-hu1-CAR-T infusion demonstrated potent antitumor efficacy against both AML and RCC, with a favorable safety profile. Overall, we developed a humanized CD70-targeted CAR-T cell therapy, A174-hu1-CAR-T, with reduced fratricide and potent antitumor activity, providing a preclinical foundation to support further translational development.
Meng-Jia Zhang, Zhihao Wang, Cheng Gao et al.· International Immunopharmaco...· 0 citations
This first-in-human study demonstrated the feasibility of an "off-the-shelf" base-edited CAR T cell approach and informs future multiantigen strategies against AML.
C. Georgiadis, Robert Chiesa, Hebatalla Rashed et al.· Science Translational Medici...· 0 citations
Multiple myeloma (MM) is an aggressive blood cancer arising from plasma cells. B cell maturation antigen (BCMA)-targeted chimeric antigen receptor T cell (α-BCMA-CAR-T) immunotherapies currently provide life-saving treatment for MM patients. Unfortunately, severe toxicities along with the high cost and complexity of autologous CAR-T manufacturing remain important limitations. Novel research is underway to use CAR-expressing natural killer (NK) cells as an allogeneic CAR-T alternative, but studies have yet to evaluate long-term CAR-NK efficacy against MM.
NK cells were isolated, expanded via feeder-cell stimulation, and engineered to express α-BCMA-CAR and IL-15 co-expression. The functional characteristics of α-BCMA-CAR-IL15-expressing NK cells were initially assessed in vitro, followed by long-term testing in a luciferase-expressing MM-xenograft mouse model to examine the persistence and therapeutic effect of α-BCMA-CAR-IL15 NK.
α-BCMA-CAR NK cells have enhanced cytokine production and cytotoxicity against BCMA-high MM cells compared to untransduced NK cells, with IL-15 co-expression required for CAR-NK persistence. When injected into NSG mice, both α-BCMA-CAR and IL-15 expression were required for persistent restriction of MM growth. Despite near complete and sustained elimination of MM in hematopoietic tissues, long-term assessment of mice treated with α-BCMA-CAR-IL15 NK cells revealed the emergence of extramedullary disease (EMD) in the form of BCMA-positive MM plasmacytomas.
This study showcases α-BCMA-CAR-IL15 NK cell therapy as a potent anti-MM therapeutic, achieving sustained MM elimination from the bone marrow and greatly extending survival in a MM-xenograft model. However, α-BCMA-CAR-IL15 NK cells appeared ineffective at eliminating extramedullary disease. By demonstrating the strengths and weaknesses of α-BCMA-CAR-IL15 cells, our study provides a valuable pre-clinical MM model for studying and developing interventions for aggressive MM-EMD.
Canadian Institutes of Health Research
Translational and Interventional Immunology (TI)
Seung-Hwan Lee, Shelby Kaczmarek, Safa Ghaziasgar et al.· Journal of Immunology· 0 citations
Abstract Background Acute myeloid leukemia (AML) is an aggressive hematologic malignancy with dismal outcomes, especially in relapsed/refractory settings. Chimeric antigen receptor natural killer (CAR-NK) cell therapy holds promise but is constrained by the immunosuppressive tumor microenvironment (TME), where adenosine-mediated suppression is a key barrier. Objective To develop a novel CAR-NK construct cotargeting AML cells and the adenosine-rich TME to enhance antileukemia efficacy. Methods Ex vivo expanded primary NK cells were used to compare the effects of CD39 versus CD73 blockade on NK cell function via messenger RNA-electroporated antibodies. A CD33-CD73 dual-function CAR-NK construct (integrating CD33-specific lysis and anti-CD73scFv secretion for TME disruption) was designed and transduced into NK cells via retrovirus. Engineered NK cells were characterized for transduction efficiency, expansion, purity, viability, and CAR stability. In vitro cytotoxicity against AML cell lines and primary blasts was assessed, and in vivo efficacy was evaluated in a MOLM-13 xenograft mouse model. Results CD73 blockade more potently enhanced NK cell activity than CD39 blockade. Retroviral transduction achieved >50% efficiency, and expansion with K562-4-1BBL-mbIL-21/−15 feeder cells yielded NK cells with ≥6,000 fold expansion, >93% purity, >98% viability, and stable CAR expression. At an effector-to-target ratio of 0.5:1, CD33-CD73 CAR-NK cells mediated ~80% specific lysis, with superior cytotoxicity vs conventional CD33 CAR-NK cells. In xenografts, CD33-CD73 CAR-NK cells achieved robust tumor clearance, extended median survival by 24.5 days (59.5 vs 35 days) versus standard CD33 CAR-NK cells, and five out of six mice achieved long-term survival (>50 days). Conclusion The CD33-CD73 dual-targeting CAR-NK platform synergistically targets AML cells and the adenosine-rich TME, exhibiting superior anti-leukemia efficacy. This strategy advances AML immunotherapy and provides a translational blueprint for TME-targeted therapies in other cancers.
Lu Wang, S. Gong, Jun Wang et al.· Journal for ImmunoTherapy of...· 0 citations
Acute myeloid leukemia (AML) is the most common form of adult leukemia and has a poor prognosis with a 5-year overall survival of ∼30%. While CAR-T has revolutionized the treatment of other hematological malignancies, it has been difficult to replicate this same success in AML. Healthy myeloid cells often share expression of putative AML targets and have led to significant toxicity in clinical trials. These challenges necessitate examining unexplored target antigens with higher cancer specificity, such as lipids.
This project aims to target lipid antigens presented by CD1d, an MHC Class 1-like molecule that presents lipid antigens to unconventional T cells such as iNKT cells. We aim to develop a lipid-targeting cell therapy for AML by engineering conventional T cells with a CD1d-targeting TCR derived from iNKT cells. These engineered T cells (herein called CD1d-targeting T cells) were co-cultured with CD1d+ AML cell lines with a-GalCer, a canonical iNKT agonist, or DMSO as a vehicle control for up to 72 hours and at varying effector: target ratios. Cells were analyzed or cytotoxicity via flow cytometry and supernatant was collected for ELISA.
CD1d-targeting T cells demonstrated robust cytotoxicity against CD1d+ AML cell lines in the presence of a-GalCer. CD1d KO cells (THP-1) showed no killing in the presence of a-GalCer or DMSO. Cell lines with higher CD1d expression (OCI-AML3) displaying targeted killing without the presence of a-GalCer. IFN-y ELISA results corroborate results from our killing assays and show pronounced IFN-y production in culture conditions that have high CD1d expression and/or presence of a lipid antigen.
Our engineered T cells showed CD1d-specific killing that was dependent on CD1d expression, and in cases of high CD1d expression, may be dependent on endogenously self-lipids found in AML cells. Further work will be done to identify putative lipid antigen targets and establish in vivo efficacy and safety our CD1d-targeting T cells.
n/a
Vaccines and Immunotherapy (VAC)
Amandip Bangar, B. Linden, Andrew Kent et al.· Journal of Immunology· 0 citations
The potential of circRNA-based CD19-targeted CAR-NK therapy as an effective approach for enhancing the safety and efficacy of cancer immunotherapy is supported.
Qisheng Dong, Ying Liu, Na Chen et al.· International Immunopharmaco...· 0 citations
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