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.
Abstract
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)
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.
Shanshan Wang, Michele Nawata, Juan Arteaga et al.· Journal of Immunology· 0 citations
Multiple myeloma (MM) is a hematologic malignancy characterized by uncontrolled malignant plasma cell proliferation. B-cell maturation antigen (BCMA) is an attractive therapeutic target due to its high expression on malignant plasma cells. In this study, BCMA-directed CAR-NK-92 cells were generated via lentiviral transduction of a second-generation CAR construct. CAR expression was confirmed by flow cytometry (81% efficiency). Engineered cells were functionally evaluated against BCMA-positive (U266, RPMI-8226) myeloma cell lines, as well as BCMA-negative control cells (K562, Jurkat). CAR-NK-92 cells demonstrated significantly enhanced cytotoxic activity against BCMA-positive targets compared with parental NK-92 cells (e.g., 87% vs. 54% cytotoxicity at 1:1, p < 0.001). Enhanced antitumor activity was accompanied by increased CD107α surface expression and elevated secretion of perforin, granzyme B, TNF-α, and IFN-γ (p < 0.05). No significant differences in cytotoxicity or cytokine secretion were observed against BCMA-negative control cells (p > 0.05), supporting antigen-specific activity. Importantly, CAR-NK-92 cells also exhibited potent cytotoxicity and significantly elevated cytokine secretion against primary CD138+ myeloma cells isolated from patient bone marrow aspirates. These findings demonstrate that anti-BCMA CAR-NK-92 cells exhibit potent and selective antimyeloma activity in vitro, supporting CAR-NK-92 cells as an off-the-shelf immunotherapeutic platform for multiple myeloma.
Chimeric antigen receptor (CAR) T cell therapies have revolutionized treatment of hematologic malignancies such as lymphoma and multiple myeloma. However, their success is limited by high manufacturing costs, reliance on autologous T cells, variable product quality, and life-threatening toxicities like cytokine release syndrome. In contrast, natural killer (NK) cells offer a safer, more flexible alternative, but their clinical translation remains constrained by complex expansion protocols and high production costs. Here, we present a transformative approach using the human NK cell line YTS, which is amenable to large-scale culture, genetic manipulation, and cryopreservation. By introducing a CD19-specific CAR into YTS cells, we generate potent effector cells capable of selectively eliminating CD19-expressing targets. We demonstrate that CAR signaling in YTS cells requires intracellular activation and, in certain tumor settings, is enhanced by co-stimulation via the 2B4-CD48 pathway. Importantly, irradiation of YTS-CAR cells prevents proliferation without compromising their cytotoxic function even after freezing and thawing. In preclinical models, injections of irradiated YTS-CAR cells significantly reduced CD19+ tumor burden, underscoring their therapeutic promise. This work positions engineered YTS cells as a novel, scalable, and cost-effective "off-the-shelf" immunotherapy platform suitable for treating refractory leukemias and lymphomas. Future studies will be required to assess safety and to explore applicability to autoimmune diseases and solid tumors.
Hanan Jaber, Anas Abu Khalaf, L. Weiss et al.· Haematologica· 0 citations
Chimeric antigen receptor (CAR) therapies have shown great success in hematological malignancies but remain largely ineffective against solid tumors such as pancreatic ductal adenocarcinoma (PDAC). A key obstacle among various aspects, is the dense stromal barrier formed by cancer-associated fibroblasts (CAFs), providing a rationale for simultaneously targeting stroma and tumor cells. Using immunohistochemistry of primary PDAC tumors and liver metastases, we confirmed high mesothelin (MSLN) expression on tumor cells, and CD70 expression on tumor cells and predominantly CAFs. Based on these results and the favorable safety profile of CAR natural killer (NK) cells over CAR T cells, we generated MSLN- and CD70-targeting IL-15-armored CAR NK cells. Both constructs mediated cytotoxicity against different pancreatic cancer and CAF cell lines with varying antigen expression in vitro, demonstrating that both, the CAR-molecule and IL-15 were required to increase functionality against more treatment-resistant cell lines. Interestingly, pooled MSLN- and CD70-CAR NK cells did not significantly improve cytolysis compared to monotherapies in an advanced 3D in vitro model or in vivo. Together these findings highlight the limitations of dual-targeting approaches and underscore the need for advanced engineering strategies to improve CAR NK cells beyond antigen targeting and cytokine support in the PDAC microenvironment.
Laura Gehrcken, J. Ott, Astrid Van Den Eynde et al.· Molecular therapy. Oncology· 0 citations
Abstract Background Renal cell carcinoma (RCC) frequently exhibits upregulation of CD70, a tumor-associated antigen implicated in immune evasion and disease progression, making it an attractive therapeutic target. Cusatuzumab, an Fc-enhanced anti-CD70 monoclonal antibody, has demonstrated the ability to mediate antibody-dependent cellular cytotoxicity (ADCC); however, its clinical efficacy is limited by insufficient effector cell engagement, antigen heterogeneity, and adaptive resistance mechanisms within the tumor microenvironment. Methods To overcome these limitations, we developed a universal, off-the-shelf natural killer T (NKT) cell product derived from hematopoietic stem cells (HSCs) through genetic engineering of an invariant T cell receptor (TCR) and a high-affinity, non-cleavable CD16 receptor. These CD16-enhanced HSC-derived NKT (CD16HSC-NKT) cells were generated using a feeder-free differentiation platform, yielding high purity and scalability while preserving canonical NKT cell phenotypes and effector functions. Results Functionally, CD16HSC-NKT cells exhibited potent intrinsic cytotoxicity and multimodal tumor-targeting capabilities. In combination with cusatuzumab, these cells demonstrated robust ADCC against CD70-positive RCC cells both in vitro and in vivo, significantly enhancing tumor cell killing compared to antibody monotherapy. Moreover, CD16HSC-NKT cells displayed improved persistence and functional stability, supporting sustained antitumor activity. Importantly, CD16HSC-NKT cells contributed to remodeling of the tumor microenvironment by selectively depleting immunosuppressive populations, including tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs). Safety assessments indicated a favorable profile, with no evidence of uncontrolled activation or off-target toxicity. Conclusions Collectively, our findings establish CD16HSC-NKT cells as a versatile and scalable cellular platform that synergizes with cusatuzumab to enhance therapeutic efficacy against RCC. This combinatorial strategy represents a promising approach to overcoming current limitations of monoclonal antibody therapy and advancing next-generation immunotherapies for solid tumors.
Yan-Ruide Li, Yi-Chen Zhu, Yu-Ning Chen et al.· The Oncologist· 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
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