Jul 2026· International Immunopharmacology· Vol 187, pp.
117193
· 0 citations· 81 references
Medicine
TL;DR
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.
Abstract
Chimeric antigen receptor (CAR)-T cell therapy targeting CD19 has demonstrated notable clinical efficacy in the treatment of B-cell acute lymphoblastic leukemia (B-ALL), but its wider clinical applicability is constrained by long manufacturing processes, substantial costs, and severe adverse events. A potentially safer and more accessible alternative is provided by CAR-Natural killer (CAR-NK) cell therapy. Currently, most CAR-NK cells are generated using viral transduction, which is labor-intensive and associated with risks of genomic integration. Electroporation of CAR-encoding mRNA provides a non-integrating alternative but results in only transient CAR expression. Circular RNA (circRNA), owing to its enhanced stability and prolonged protein expression capacity, has recently emerged as a promising alternative to linear mRNA. To overcome the limitations of transient mRNA expression, we generated circRNA using a Group II intron-mediated cyclization system incorporating a newly selected Coccidioides immitis-derived Group II intron. The newly established Coccidioides immitis-derived Group II intron circularization system efficiently generated circRNA and supported more durable EGFP expression than linear mRNA in both HEK293T and NK92 cells. Using this system, we successfully developed a circRNA-based CD19-targeted CAR-NK platform. CircRNA-engineered CD19-targeted CAR-NK92 cells maintained more durable CAR expression and showed stronger antitumor activity at later time points. In mouse models of B-ALL, circRNA-engineered CAR-NK92 cells demonstrated better tumor control and extended survival compared with their linear mRNA-engineered counterparts. These results support the potential of circRNA-based CAR-NK therapy as an effective approach for enhancing the safety and efficacy of cancer immunotherapy.
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)
Shanshan Wang, Michele Nawata, Juan Arteaga et al.· Journal of Immunology· 0 citations
On the basis that T-cell acute lymphoblastic leukemia (T-ALL) cells overexpress IL7 receptor (IL7R), which promotes resistance to chemotherapy and disease relapse, here we develop IL7Rα (CD127)–targeted chimeric antigen receptor (CAR) T cells with low- and high-affinity single-chain variable fragments. We establish the antitumor efficacy of CD127 CAR against T-ALL cells in vitro, in female mouse models of T-ALL, and against blasts from patients with T-ALL using the patients’ own T cells transduced with CD127 CAR. Antitumor efficacy is higher with low-affinity CAR T cells than high-affinity CAR T cells, albeit with fratricide of CAR T cells following eradication of CD127-overexpressing blasts. CRISPR-Cas9 knockout of CD127 eliminates fratricide at the risk of prolonged lymphopenia in vivo. To overcome fratricide, we investigate short-term ( < 7 days) co-culture with or without dasatinib, a tyrosine kinase inhibitor, versus a natural selection method (10 days) and demonstrate that co-culturing with dasatinib facilitates higher CAR T-cell yield, improved fitness, and preserved functionality. In vivo, dasatinib can be used to temporarily and reversibly suppress CAR T-cell activity. With this supporting translational data, we are initiating a trial with low-affinity CD127 CAR T cells for adult and pediatric patients with relapsed or refractory T-ALL. Chimeric antigen receptor (CAR) T cell therapies for T-cell acute lymphoblastic leukemia are explored in the clinic. Here the authors propose IL7-Receptor (IL7R/CD127) as an alternative target for T-ALL, showing the antitumor efficacy of IL7Rα/CD127 CAR-T cells against T-ALL in preclinical models.
H. Hocine, Undrakh Ganbaatar, Alfredo Amador-Molina et al.· Nature Communications· 0 citations
Chimeric antigen receptor (CAR) T cell therapy has significantly advanced treatment of hematologic malignancies, including B-cell acute lymphoblastic leukemia (B-ALL), non-Hodgkin lymphoma (NHL), and multiple myeloma (MM). However, conventional autologous CAR-T therapies are hampered by logistical complexity, high costs, and lengthy manufacturing timelines, limiting accessibility–particularly for patients with aggressive disease. To address these challenges, we developed an in vivo CAR-T platform that directly engineers functional CAR T cells within the body, eliminating the need for ex vivo manipulation.
Here we report the development and validation of Ori-TTV, a rationally designed T cell—targeting lentiviral vector optimized for safe and efficient in vivo CAR-T generation. Key innovations of Ori-TTV include: (1) an Ori Multiple Targeting Domain (Ori-MTD) for selective T cell transduction; (2) a synthetic T cell—specific promoter for lineage-restricted CAR expression; (3) a phagocytosis-resistant modification to evade macrophage clearance; and (4) a de-targeted fusogen to enhance safety and reduce off-target effects. These features enable selective in vivo reprogramming of T cells.
Ori-TTV encodes a dual-target CAR recognizing both CD19 and BCMA, facilitating simultaneous elimination of malignant B and plasma cells. This expands therapeutic potential across relapsed/refractory B-ALL, NHL, and MM, and B/plasma cell—driven autoimmune disorders such as systemic lupus erythematosus (SLE), idiopathic inflammatory myopathy (IIM), and systemic sclerosis (SSc). In vitro, the T cell—specific promoter drives CAR expression at levels comparable to EF-1α, supporting robust and multi-round cytotoxicity against CD19+ and BCMA+ cells. In vivo, Ori-TTV administration induced significant and durable tumor regression in humanized PBMC and CD34+ mouse models.
In summary, Ori-TTV offers a safe, scalable, cost-effective platform to broaden access to CAR-T immunotherapy.
n/a
Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)
Xiaowen He, Xuhui Dong, Yongqin Li et al.· Journal of Immunology· 0 citations
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
Small cell lung cancer (SCLC) is an aggressive form of lung cancer, with most patients showing poor response rates to immunotherapies, resulting in limited treatment options. Profiling of the SCLC TIME shows immunologic heterogeneity, with the majority of SCLC being neuroendocrine (NE) subtype and exhibiting low MHC-I expression, and a minority being non-neuroendocrine (non-NE) subtype, with higher MHC-I expression. Recent incorporation of T cell engagers targeting DLL3 and CD3 have shown promising results, however, MHC-I downregulation can potentially limit endogenous T cell activity and therapeutic responses. Chimeric antigen receptor (CAR) based therapies enable the genetic engineering of T or NK cells to target tumor-specific antigens independently of MHC-I, representing a promising therapeutic strategy for SCLC. However, CAR-NK cells’ proliferation and lifespan is limited compared to CAR-T cells. CAR-T might benefit from a combination with CAR-NK providing a rapid antitumor response to promote infiltration and expansion. In this study we aimed to use 3D micro-physiological systems to test whether a combination of CAR-T and CAR-NK could overcome CAR therapy limitations in solid tumors.
DLL3 surface protein expression was characterized in SCLC cell lines by flow cytometry. Then, DLL3-targeting CAR-T and CAR-NK cells were generated through viral transduction and validated in both 2D and 3D tumor models. The combination of the CAR-T and CAR-NK was tested using a co-culture spheroids model in a 3D microfluidic device. Finally, infiltration of the CARs across a blood vessel barrier into the TME was investigated by creating a microphysiological system of SCLC TME.
We found that DLL3 expression was heterogenous across SCLC cell lines, with NE SCLC exhibiting higher DLL3 expression than non-NE SCLC. Both DLL3 CAR-T and CAR-NK cells demonstrated potent antitumor activity in 2D and 3D culture, with CAR-NK cells showing more rapid cytotoxic activity compared to CAR-T cells, killing via antigen recognition but also recognizing the absence of MHC-I expression. We then sought to combine both therapies to investigate cooperative activity between the two effector-cell platforms. At a 6-day timepoint, without exogenous IL-2 stimulation, we found that the DLL3 CAR-T and CAR-NK cell combination showed the greatest anti-tumor activity in the DLL3+ NE cell lines, compared to either therapy alone when tested in the 3D microfluidic model, suggesting synergistic and bystander effector function. These results correlated with higher IL-2 and IFN-g production detected in conditioned media only in the CAR-T and CAR-NK cell combination treatment.
These results suggest that DLL3-targeting CAR therapies represent a promising approach for treating SCLC, particularly given their ability to function independently of MHC-I. In addition, a combination of CAR-T and CAR-NKs could be an efficacious approach to overcoming current limitations of CAR therapies in solid tumors, including immune cell exclusion, persistence, and exhaustion.
Alan E. Bers, Ian Gillanders, Eden Bobilev, Carla Stornante, Mubin Tarannum, Navin R. Mahadevan, Rizwan Romee, Marco Campisi, David A. Barbie. DLL3 CAR-T and CAR-NK cell combination therapy promotes synergistic antitumor activity in small cell lung cancer [abstract]. In: Proceedings of AACR Drug Discovery and Development (AACR D3) Conference; 2026 Jul 21-24; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(14_Suppl):Abstract nr B056.
Alan E. Bers, I. Gillanders, Eden Bobilev et al.· Clinical Cancer Research· 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
