bims-nakice 2026-07-19 papers

Issue of 2026–07–19
six papers selected by
Santosh Phuyal, Oslo Universitetssykehus

bioRxiv. 2026 Jul 06. pii: 2026.07.03.736431. [Epub ahead of print]

The membrane distal domain of CD16a allosterically regulates NK cell ADCC.

Tania Cid, Monica Fernandez-Quintero, Hijab Fatima, Ema Robinson, Braden Christenson, Johannes Loeffler, Daniel Leaman, Ryan Lin, Kayla Xu, Jessica Matthias, Scott C Henderson, Kathryn Spencer, Joseph Jardine, Michael B Zwick, Andrew B Ward, Emily M Mace, Charles Daniel Murin.

Antibody-dependent cellular cytotoxicity (ADCC) by natural killer (NK) cells is mediated by the activating IgG receptor CD16a (FcγRIIIa), yet the molecular mechanisms governing receptor activation remain poorly understood. We demonstrate that the membrane-distal domain 1 (D1) of CD16a functions as an allosteric checkpoint that controls ADCC independently of IgG-Fc binding. A nanobody, C28, that binds an electronegative patch in D1 dose-dependently blocks NK cell ADCC against multiple therapeutic antibodies without affecting direct cytotoxicity. A second nanobody, C21, binding an adjacent D1 epitope has no such effect. Cryo-EM structures of the CD16a-IgG-nanobody complex reveal that C28 allosterically competes with core-fucosylated IgG and stabilizes a closed D1 conformation resembling unliganded receptor, even when Fc is bound. Molecular dynamics simulations show that occupation of the D1 epitope rigidifies the IgG-binding site, stabilizing CD16a overall in contrast with IgG binding alone. The nanobody C28 restricts CD3ζ phosphorylation in both resting and ADCC-activated NK cells, revealing tonic inhibitory control upstream of the signaling cascade. Using MINFLUX nanoscopy, we also show that CD16a forms dimers of ∼9 nm spacing on the NK cell surface, a geometry unaltered by the ADCC-enhancing L48H polymorphism. Drawing on structural parallels with the IgE receptor FcεRI, which is held inactive as a cholesterol-stabilized dimer, we propose that CD16a dimerization through D1 contacts represents a conserved autoinhibitory mechanism among Fc receptors. Consistent with this model, structure-guided disruption of the C28 epitope in NK-92 cells enhances ADCC potency and killing kinetics, providing a blueprint for engineering improved cellular immunotherapeutics.

DOI: https://doi.org/10.64898/2026.07.03.736431

Neuro Oncol. 2026 Jul 11. pii: noag154. [Epub ahead of print]

iPSC-derived NK cell therapy induces durable responses in glioblastoma and overcomes resistance via a B7-H3-targeted tri-specific killer engager.

BACKGROUND: Glioblastoma is the most aggressive primary brain tumor, with poor prognosis and limited treatment options. Natural killer (NK) cell therapy is a promising immunotherapeutic strategy, yet its efficacy remains limited. We evaluated FT538, a clinical-grade NK product derived from induced pluripotent stem cells (iPSCs), in glioblastoma models.
METHODS: FT538, engineered with a high-affinity non-cleavable CD16 Fc receptor, a membrane-bound IL-15/IL-15Rα fusion protein, and CD38 knockout, was tested against 13 patient-derived glioblastoma stem-like cells (GSCs) in vitro and in orthotopic xenograft models. Intracranial persistence and neurotoxicity were assessed in mice. Surface proteomics identified therapeutic targets, and a B7-H3-targeted tri-specific killer engager (TriKE) was evaluated with FT538 and NKG2C+ adaptive NK cells.
RESULTS: GSCs were classified as sensitive (38%), moderately sensitive (38%), or resistant (23%) to FT538. Intracranial administration in mice was well tolerated, persisted for at least 35 days, and caused no neurotoxicity. A single intratumoral dose induced complete regression in sensitive xenografts. Surface profiling identified B7-H3 as a target to overcome resistance. Combination therapy with FT538 and a B7-H3 TriKE enhanced antitumor efficacy in resistant models, an effect also observed with adaptive NK cells.
CONCLUSIONS: FT538 exhibits potent tumoricidal activity in 77% of GSC lines (NK-sensitive and moderately sensitive), with curative potential in sensitive models, and demonstrates favorable persistence and tolerability in vivo. B7-H3-targeted TriKE restores NK sensitivity in resistant tumors. These findings provide a strong preclinical rationale for further clinical evaluation of FT538, alone or combined with B7-H3-targeted TriKE, for glioblastoma and other solid tumors.

Keywords: B7-H3; FT538; Glioblastoma; NK cells; TriKE

DOI: https://doi.org/10.1093/neuonc/noag154

Front Immunol. 2026 ;17 1868673

Automated cytotoxicity assessment of natural killer cells by flow cytometry.

Aleksander Szarzynski, Valentin von Werz, Gregor Mattert, Werner Dammermann, Oliver Spadiut.

Natural killer (NK) cell-based therapies are emerging as highly promising candidates for cancer treatment, but their development and quality control depend on robust assessment of key critical quality attributes, particularly their cytotoxicity. Despite the availability of various approaches for assessing cytotoxicity, existing techniques often suffer from high data variability and show limited reproducibility. We compared commonly used approaches for NK-cell cytotoxicity assessment, including calcein release, lactate dehydrogenase release, and flow cytometry (FCM)-based analysis, using NK-92 effector and GFP-labelled K562 target cells. Our results provide insight into the shortcomings of these methods, as well as problems resulting from unharmonized evaluation criteria and limitations of endpoint measurements, which are commonly applied in literature. We selected FCM as the most suitable platform for standardized evaluation and developed an automated gating workflow for cytotoxicity analysis. The automated workflow was benchmarked against three independent manual evaluations to assess agreement, bias, and performance. The optimized workflow showed agreement with manual analysis while remaining essentially unbiased. In addition, automated analysis reduced evaluator dependence by producing deterministic outputs from identical input data. Further comparison revealed directional bias in manual gating, indicating that a relevant portion of measurement variability arose from manual evaluation, rather than biology alone. We present autogating as a fit-for-purpose, automated FCM-based strategy for NK-cell cytotoxicity evaluation that preserves agreement with manual analysis while improving standardization and reproducibility, thereby providing a practical route toward more harmonized cytotoxicity testing in cell therapy workflows.

Keywords: NK cells; NK-92 cell line; automatization; cytotoxicity; flow cytometry; gating strategy

DOI: https://doi.org/10.3389/fimmu.2026.1868673

Sci Adv. 2026 Jul 17. 12(29): eaed1645

Hyperwrinkling of the nuclear lamina is associated with attenuated mechanosensitivity in giant nucleated cancer cells.

Samere Abolghasemzade, Ryan Blanchard, Chance French, Christina R Dollahon, Harrison Pham, Aidan Atkins, Richard B Dickinson, Tanmay P Lele.

Polyploid giant cancer cells (PGCCs) are chemoresistant tumor cells associated with poor patient outcomes. PGCCs in tissue are identified by their extremely large sizes and the presence of massive or several nuclei. Although nuclear dysmorphia is also a common characteristic of tumor cells, little is known about the shape of nuclei in PGCCs. We show that the nuclear lamina in giant nucleated PGCCs is highly wrinkled across diverse cancer patient tissues. We investigated the cause of this hyperwrinkling in ovarian PGCCs in vitro. Laminar hyperwrinkling in PGCCs was independent of cell shape or cytoskeletal forces. Instead, measurements combined with computational modeling show that laminar hyperwrinkling is an intrinsic property of PGCCs, arising from a disproportionate amount of laminar excess area. PGCCs also displayed attenuated mechanosensitivity of cell spreading and YAP nuclear localization compared with control cells. Thus, laminar hyperwrinkling may disrupt PGCC mechanobiological pathways, slowing their proliferation.

DOI: https://doi.org/10.1126/sciadv.aed1645

Methods Cell Biol. 2026 ;pii: S0091-679X(26)00123-8. [Epub ahead of print]209 27-40

Sensitive detection of lysosomal membrane permeabilization using the galectin puncta assay.

Konstantinos Danias, Victoria Alexandra Bjerregaard, Marja Jäättelä.

The Lysosomal Galectin Puncta Assay is a microscopy-based technique able to detect even minor lysosomal leakage with high sensitivity. This protocol describes the detection of galectin puncta as markers of lysosomal membrane permeabilization, a process that relies on the high-affinity binding of the cytosolic galectins to the luminal glycans exposed on damaged lysosomes. Compared to traditional methods, the Galectin Puncta Assay offers high sensitivity, detects subtle lysosomal leakage, and enables analysis at single-lysosome level. Here, we provide a step-by-step protocol for this assay, covering sample preparation, immunostaining, imaging and image quantification.

Keywords: Galectin puncta; Lysosomal leakage; Lysosomal membrane permeabilization

DOI: https://doi.org/10.1016/bs.mcb.2026.04.002

Cytotherapy. 2026 Feb 23. pii: S1465-3249(26)00085-X. [Epub ahead of print]28(9): 102123

Anti-tumor effects of CD133-targeted CAR-NK92MI cells in colorectal cancer.

Yuyi Zhang, Zheng Huang, Cailing Tong, Zhongquan Qi, Shifeng Zhang.

BACKGROUND AIMS: Colorectal cancer (CRC), a highly aggressive malignancy, continues to threaten many lives. Chimeric antigen receptor-natural killer cell (CAR-NK) therapy has emerged as a promising therapeutic approach for CRC. CD133 is an important marker in many solid tumors, especially CRC.
METHODS: In this study, we generated CD133-targeted CAR-NK92MI cells using a lentiviral system and analyzed CAR expression by flow cytometry. The proliferation of NK92MI cells after CAR introduction was evaluated by CCK-8 assay. In vitro cytotoxicity against target cells was assessed by LDH release, Calcein-AM/PI staining, and TUNEL staining, and effector cell activation was evaluated by measuring IFN-γ secretion. The effects of CAR-NK92MI cells on target-cell invasion and migration were further assessed by Transwell invasion assays, wound-healing assays, and a 3D tumor-spheroid system. In vivo antitumor activity was also evaluated.
RESULTS: CAR was successfully expressed in NK92MI cells without significantly affecting cell proliferation. In vitro, CAR-NK92MI cells showed enhanced killing activity against target cells and increased IFN-γ secretion. CAR-NK92MI cells also suppressed target-cell invasion and spheroid growth. The in vivo findings were consistent with the in vitro results.
CONCLUSION: CD133-targeted CAR-NK92MI cells showed promising antitumor activity against colorectal cancer and may provide a potential strategy for developing an off-the-shelf cell therapy product for CRC.

Keywords: CAR-NK92MI; CD133; HCT116; colorectal cancer

DOI: https://doi.org/10.1016/j.jcyt.2026.102123