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References
Maude, S. L. et al. Tisagenlecleucel in children and young adults with B-cell lymphoblastic leukemia. N. Engl. J. Med. 378, 439–448 (2018).
Schuster, S. J. et al. Chimeric antigen receptor T cells in refractory B-cell lymphomas. N. Engl. J. Med. 377, 2545–2554 (2017).
Kingwell, K. CAR T therapies drive into new terrain. Nat. Rev. Drug Discov. 16, 301–304 (2017).
Chiossone, L., Dumas, P. Y., Vienne, M. & Vivier, E. Natural killer cells and other innate lymphoid cells in cancer. Nat. Rev. Immunol. 18, 671–688 (2018).
Davenport, A. J. et al. Chimeric antigen receptor T cells form nonclassical and potent immune synapses driving rapid cytotoxicity. Proc. Natl Acad. Sci. USA 115, E2068–E2076 (2018).
Watanabe, K., Kuramitsu, S., Posey, A. D. Jr. & June, C. H. Expanding the therapeutic window for CAR T cell therapy in solid tumors: the knowns and unknowns of CAR T cell biology. Front. Immunol. 9, 2486 (2018).
Lin, J. & Weiss, A. The tyrosine phosphatase CD148 is excluded from the immunologic synapse and down-regulates prolonged T cell signaling. J. Cell Biol. 162, 673–682 (2003).
Roda-Navarro, P. & Alvarez-Vallina, L. Understanding the spatial topology of artificial immunological synapses assembled in T cell-redirecting strategies: a major issue in cancer immunotherapy. Front. Cell Dev. Biol. 7, 370 (2019).
Nourry, C., Grant, S. G. & Borg, J. P. PDZ domain proteins: plug and play! Sci. STKE 2003, RE7 (2003).
Yeh, J. H., Sidhu, S. S. & Chan, A. C. Regulation of a late phase of T cell polarity and effector functions by Crtam. Cell 132, 846–859 (2008).
Humbert, P. O., Dow, L. E. & Russell, S. M. The Scribble and Par complexes in polarity and migration: friends or foes. Trends Cell Biol. 16, 622–630 (2006).
Arase, N. et al. Heterotypic interaction of CRTAM with Necl2 induces cell adhesion on activated NK cells and CD8+ T cells. Int. Immunol. 17, 1227–1237 (2005).
Chockley, P. J. et al. Epithelial–mesenchymal transition leads to NK cell-mediated metastasis-specific immunosurveillance in lung cancer. J. Clin. Invest. 128, 1384–1396 (2018).
Gwalani, L. A. & Orange, J. S. Single degranulations in NK cells can mediate target cell killing. J. Immunol. 200, 3231–3243 (2018).
Lafouresse, F. et al. Stochastic asymmetric repartition of lytic machinery in dividing CD8+ T cells generates heterogeneous killing behavior. eLife 10, e62691 (2021).
Vivier, E., Tomasello, E., Baratin, M., Walzer, T. & Ugolini, S. Functions of natural killer cells. Nat. Immunol. 9, 503–510 (2008).
Yi, Z., Prinzing, B. L., Cao, F., Gottschalk, S. & Krenciute, G. Optimizing EphA2-CAR T cells for the adoptive immunotherapy of glioma. Mol. Ther. Methods Clin. Dev. 9, 70–80 (2018).
Pasquale, E. B. Eph receptors and ephrins in cancer: bidirectional signalling and beyond. Nat. Rev. Cancer 10, 165–180 (2010).
Wykosky, J. & Debinski, W. The EphA2 receptor and ephrinA1 ligand in solid tumors: function and therapeutic targeting. Mol. Cancer Res. 6, 1795–1806 (2008).
Lanier, L. L., Chang, C., Spits, H. & Phillips, J. H. Expression of cytoplasmic CD3 epsilon proteins in activated human adult natural killer (NK) cells and CD3 gamma, delta, epsilon complexes in fetal NK cells. Implications for the relationship of NK and T lymphocytes. J. Immunol. 149, 1876–1880 (1992).
Zigmond, S. H. How WASP regulates actin polymerization. J. Cell Biol. 150, F117–F120 (2000).
Guillerey, C., Huntington, N. D. & Smyth, M. J. Targeting natural killer cells in cancer immunotherapy. Nat. Immunol. 17, 1025–1036 (2016).
Molon, B., Liboni, C. & Viola, A. CD28 and chemokine receptors: signalling amplifiers at the immunological synapse. Front. Immunol. 13, 938004 (2022).
Nguyen, P. et al. Route of 41BB/41BBL costimulation determines effector function of B7-H3-CAR.CD28ζ T cells. Mol. Ther. Oncolytics 18, 202–214 (2020).
Majzner, R. G. et al. CAR T cells targeting B7-H3, a pan-cancer antigen, demonstrate potent preclinical activity against pediatric solid tumors and brain tumors. Clin. Cancer Res. 25, 2560–2574 (2019).
Fauriat, C., Long, E. O., Ljunggren, H. G. & Bryceson, Y. T. Regulation of human NK-cell cytokine and chemokine production by target cell recognition. Blood 115, 2167–2176 (2010).
Barreda, D. et al. The Scribble complex PDZ proteins in immune cell polarities. J. Immunol. Res. 2020, 5649790 (2020).
Larson, R. C. et al. CAR T cell killing requires the IFNγR pathway in solid but not liquid tumours. Nature 604, 563–570 (2022).
Molgora, M., Cortez, V. S. & Colonna, M. Killing the invaders: NK cell impact in tumors and anti-tumor therapy. Cancers (Basel) 13, 595 (2021).
Rafei, H., Daher, M. & Rezvani, K. Chimeric antigen receptor (CAR) natural killer (NK)-cell therapy: leveraging the power of innate immunity. Br. J. Haematol. 193, 216–230 (2021).
Lee, D. A. Cellular therapy: adoptive immunotherapy with expanded natural killer cells. Immunol. Rev. 290, 85–99 (2019).
Liu, E. et al. Use of CAR-transduced natural killer cells in CD19-positive lymphoid tumors. N. Engl. J. Med. 382, 545–553 (2020).
Li, Y., Hermanson, D. L., Moriarity, B. S. & Kaufman, D. S. Human iPSC-derived natural killer cells engineered with chimeric antigen receptors enhance anti-tumor activity. Cell Stem Cell 23, 181–192 (2018).
Oelsner, S. et al. Continuously expanding CAR NK-92 cells display selective cytotoxicity against B-cell leukemia and lymphoma. Cytotherapy 19, 235–249 (2017).
Myers, J. A. & Miller, J. S. Exploring the NK cell platform for cancer immunotherapy. Nat. Rev. Clin. Oncol. 18, 85–100 (2021).
Gong, Y., Klein Wolterink, R. G. J., Wang, J., Bos, G. M. J. & Germeraad, W. T. V. Chimeric antigen receptor natural killer (CAR-NK) cell design and engineering for cancer therapy. J. Hematol. Oncol. 14, 73 (2021).
Majzner, R. G. & Mackall, C. L. Tumor antigen escape from CAR T-cell therapy. Cancer Discov. 8, 1219–1226 (2018).
Wagner, J., Wickman, E., DeRenzo, C. & Gottschalk, S. CAR T cell therapy for solid tumors: bright future or dark reality. Mol. Ther. 28, 2320–2339 (2020).
Yu, W. L. & Hua, Z. C. Chimeric antigen receptor T-cell (CAR T) therapy for hematologic and solid malignancies: efficacy and safety—a systematic review with meta-analysis. Cancers (Basel) 11, 47 (2019).
Tong, C. et al. Optimized tandem CD19/CD20 CAR-engineered T cells in refractory/relapsed B-cell lymphoma. Blood 136, 1632–1644 (2020).
Hegde, M. et al. Tandem CAR T cells targeting HER2 and IL13Rα2 mitigate tumor antigen escape. J. Clin. Invest. 126, 3036–3052 (2016).
Majzner, R. G. et al. Tuning the antigen density requirement for CAR T-cell activity. Cancer Discov. 10, 702–723 (2020).
Xiong, W. et al. Immunological synapse predicts effectiveness of chimeric antigen receptor cells. Mol. Ther. 26, 963–975 (2018).
Mata, M. et al. Inducible activation of MyD88 and CD40 in CAR T cells results in controllable and potent antitumor activity in preclinical solid tumor models. Cancer Discov. 7, 1306–1319 (2017).
Imai, C., Iwamoto, S. & Campana, D. Genetic modification of primary natural killer cells overcomes inhibitory signals and induces specific killing of leukemic cells. Blood 106, 376–383 (2005).
Gundry, M. C. et al. Highly efficient genome editing of murine and human hematopoietic progenitor cells by CRISPR/Cas9. Cell Rep. 17, 1453–1461 (2016).
Taylor, K. R. et al. Recurrent activating ACVR1 mutations in diffuse intrinsic pontine glioma. Nat. Genet. 46, 457–461 (2014).
He, C. et al. Patient-derived models recapitulate heterogeneity of molecular signatures and drug response in pediatric high-grade glioma. Nat. Commun. 12, 4089 (2021).
Chow, K. K. et al. T cells redirected to EphA2 for the immunotherapy of glioblastoma. Mol. Ther. 21, 629–637 (2013).
Liu, D., Paczkowski, P., Mackay, S., Ng, C. & Zhou, J. Single-cell multiplexed proteomics on the IsoLight resolves cellular functional heterogeneity to reveal clinical responses of cancer patients to immunotherapies. Methods Mol. Biol. 2055, 413–431 (2020).
Tonikian, R. et al. A specificity map for the PDZ domain family. PLoS Biol. 6, e239 (2008).
Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).
Witten, I. H., Frank, E., Hall, M. A. & Pal, C. J. Data Mining: Practical Machine Learning Tools and Techniques 4th edn (Morgan Kaufmann, 2016).
Ershov, D. et al. Bringing TrackMate in the era of machine-learning and deep-learning. Preprint at bioRxiv https://doi.org/10.1101/2021.09.03.458852 (2021).
Haydar, D. et al. Cell-surface antigen profiling of pediatric brain tumors: B7-H3 is consistently expressed and can be targeted via local or systemic CAR T-cell delivery. Neuro Oncol. 23, 999–1011 (2021).
Acknowledgements
We would like to acknowledge the technical support from H. Houke, C. O’Reilly and A. Chabot. This work was supported by the St. Jude Sumara Fellowship (P.C.), the American Lebanese Syrian Associated Charites (S.G.), the ChadTough Defeat DIPG Foundation (G.K.), National Institute of Neurological Disorders and Stroke grant R01NS121249 (G.K.), the Rally Foundation for Childhood Cancer Research (L.J.T.), the Garwood Postdoctoral Fellowship (J.I.V.) and National Institutes of Health (NIH)/National Cancer Institute (NCI) grant P30 CA021765. Animal imaging was performed by the Center for In Vivo Imaging and Therapeutics, which is supported, in part, by NIH grants P01CA096832 and R50CA211481. Cellular images were acquired at the St. Jude Childrenʼs Research Hospital Cell & Tissue Imaging Center, which is supported by St. Jude and NCI P30 CA021765. Gene editing of cell lines was performed by the Center for Advanced Genome Engineering (CAGE), which is supported, in part, by NCI P30 CA021765. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.
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Competing interests
S.G. and P.J.C. have patent applications in the fields of NK and T cell and/or gene therapy for cancer. S.G. has a research collaboration with TESSA Therapeutics, is a Data and Safety Monitoring Board member of Immatics and was on the scientific advisory board of Tidal. P.J.C. is a technical consultant for LUMICKS.
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Extended data
Extended Data Fig. 1 Primary NK cell transduction efficiency.
(a) Representative flow cytometry histogram plots detailing surface CAR expression. (b) Quantified flow cytometry data showing percent CAR positive NK cells of various donors. UN: n = 9, CAR Δ: n = 8, CAR: n = 9, CAR.PDZ: n = 6 donors, mean ± SEM shown. (c) Immunophenotype of NK cells via flow cytometry. n = 4 donors, mean ± SEM shown.
Extended Data Fig. 2 Scribble Polarization at 30 minutes.
(a) Confocal images as prepared in (Fig. 1) incubated for 30 minutes with NK cells in various groups quantified in (b). White bars indicate 10microns. Immunolabelling of Scribble in red, CD3ε in green, and filamentous actin (F-actin) in white. (b) Scribble polarization and accumulation at the immune synapse (IS). CAR Δ; n = 13, CAR; n = 7, CAR.PDZmut: n = 14, CAR.PDZ n = 9, One-Way ANOVA was used to determine statistical significance with Two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli to correct for FDR. mean ± SEM shown of one donor.
Extended Data Fig. 3 WASp Polarization at 15 and 30 minutes.
Confocal images as prepared in (Fig. 1) incubated for 15 and 30 minutes with NK cells in various groups. Quantified WASp polarization and accumulation at the immune synapse (IS). CAR Δ; n = 34 and 25, CAR; n = 42 and 25, CAR.PDZ n = 19 and 39, for 15 and 30 minutes, respectively. Two-Way ANOVA was used to determine statistical significance with Two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli to correct for FDR. Statistical difference delineated by q < 0.01 *, q < 0.0001 ****; mean ± SEM shown of one donor.
Extended Data Fig. 4 Live NK cell imaging reveals lysosomal condensing and enhanced synapse formation with increased calcium Flux.
Lysosomal coalescing from live cell imaging in Fig. 2g EphA2 targeting CARs UN; n = 27, CARΔ; n = 48, CAR; n = 44, CAR.PDZ; n = 48 cells. Peak lysosome signal was measured from the first calcium flux peak in each condition. One-Way ANOVA was used to determine statistical significance with Two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli to correct for FDR; mean ± SEM shown of one donor.
Extended Data Fig. 5 LM7 Avidity assessment with EphA2-targeting CARs.
Normalized fold change of CAR NK cell binding compared to untransduced NK cells. Arrowed lines indicate the point of statistical difference at CAR.PDZ vs. CAR Δ at 268 pN, CAR.PDZ vs CAR at 343 pN, CAR.PDZ vs CAR.PDZmut at 363 pN which continued to 1000 pN indicated by dashed arrow lines. The only exception to this significance was from 650 to 738 pN for CAR.PDZ vs CAR.PDZmut. Two-Way ANOVA was used to determine statistical significance with Two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli to correct for FDR; n = 1-3 donors, mean ± SEM shown.
Extended Data Fig. 6 B7-H3 CAR design with avidity, synapse, and calcium flux analyses.
(a) Chimeric antigen receptor design schemes. Antigen recognition domain (anti-B7-H3 scFv): goldenrod, hinge and transmembrane domains (CD8αH/TM): grey, CD28 co-stimulatory domain: purple, CD3ζ activation domain: blue, PDZbm scaffolding anchor domain: red. (b) Example flow cytometry plot detailing B7-H3 CAR expression. (c) Normalized fold change of CAR NK cell binding compared to untransduced NK cells. Bracketed line indicates the scale of statistical difference at CAR.PDZ vs. CAR Δ and CAR from 194 to 646 pN for both comparisons except for 205-215 pN. Two-Way ANOVA was used to determine statistical significance with Two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli to correct for FDR; q < 0.05 *, <0.001 ***, n = 3 donors, mean ± SEM shown. (d) Immune synapse area quantification of B7-H3 CAR and CAR.PDZ NK cells (n = 11 and 11 cells) Two-Way ANOVA was used to determine statistical significance with Uncorrected Fisher’s LSD test p < 0.05 * at minute 5 and 12 with area under the curve analysis. (e) Calcium flux quantification of B7-H3 CAR and CAR.PDZ NK cells (n = 15 and 14 cells) with Two-Way ANOVA was used to determine statistical significance with Uncorrected Fisher’s LSD test p < 0.05 * starting at minute 2; 1st peak AUC analysis with unpaired Student’s t-Test, mean ± SEM shown of one donor.
Extended Data Fig. 7 A549 and LM7 tumor rechallenge rejection.
(a) A549 tumor rechallenge timeline with identical initial cancer cell numbers. Indicated tumor volumes from palpable nodules overtime. (b) Intravital imaging of LM7 rechallenge with identical initial cancer cell numbers in complete responder mice. Color scale 1e6 to 1e7 of total photon flux(p/s). (c) Tumor flux values of weekly measurements.
Extended Data Fig. 8 CAR T cell phenotyping and cytokine production.
(a) Chimeric antigen receptor design schemes. Antigen recognition domain (anti-B7-H3 scFv): goldenrod, hinge and transmembrane domains (CD8αH/TM): grey, CD28 co-stimulatory domain: purple, CD3ζ activation domain: blue, PDZbm scaffolding anchor domain: red. (b) Transduction efficiencies of various CAR constructs in T cells CAR Δ; n = 7, CAR; n = 8, CAR.PDZ n = 5 donors mean ± SEM shown. (c) CD4/8 T cell analysis post transduction at Day 5-6, n = 2 donors mean ± SEM shown. (d) Immunophenotype of CD4 CAR T cells Day 5-6 and longitudinally Day 12-13, n = 2 donors mean ± SEM shown. (e) Immunophenotype of CD8 CAR T cells Day 5-6 and longitudinally Day 12-13, n = 2 donors mean ± SEM shown. (f) B7-H3 CAR T cells were co-cultured with A549, LM7, and 143b cancer cells lines at a 2:1 ratio for 24 hours for n = 3 experiments. Supernatant was collected and multiplex cytokine assessment was performed. One-Way ANOVA was used to determine statistical significance with Two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli to correct for FDR mean ± SEM.
Extended Data Fig. 9 B7-H3 CAR T cell synapse and calcium flux analyses.
(a) Immune synapse area quantification of B7-H3 CAR and CAR.PDZ T cells (n = 12 and 9 cells) co-cultured with LM7 cells with Area Under the Curve analysis. (b) Calcium flux quantification of B7-H3 CAR and CAR.PDZ T cell (n = 16 and 22 cells) co-cultured with LM7 cells with Two-Way ANOVA was used to determine statistical significance with Uncorrected Fisher’s LSD test p < 0.0001 **** at minute 4; 1st peak AUC analysis. mean ± SEM shown of one donor. (c) Calcium flux quantification of B7-H3 CAR and CAR.PDZ T cells (n = 42 and 15 cells) co-cultured with U87 cells. Two-Way ANOVA was used to determine statistical significance with Uncorrected Fisher’s LSD test p < 0.0001 **** starting at minute 1; 1st peak AUC analysis with unpaired Student’s t-Test. mean ± SEM shown of one donor. (d) Calcium flux quantification of B7-H3 CAR and CAR.PDZ T cells (n = 22 and 31 cells) co-cultured with DIPG007 cells. Two-Way ANOVA was used to determine statistical significance with Uncorrected Fisher’s LSD test p < 0.0001 **** starting at minute 1; 1st peak AUC analysis with unpaired Student’s t-Test. mean ± SEM shown of one donor.
Extended Data Fig. 10 B7-H3 expression on tumor cells.
143B, U87, LM7, DIPG7c, and DIPG007 tumor cells were analyzed for B7-H3 expression. Black histograms indicate Isotype controls and red histograms are immunolabeled cells. Each sample is 100% positive and the indicated gMFI values are delineated.
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Chockley, P.J., Ibanez-Vega, J., Krenciute, G. et al. Synapse-tuned CARs enhance immune cell anti-tumor activity.
Nat Biotechnol (2023). https://doi.org/10.1038/s41587-022-01650-2
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DOI: https://doi.org/10.1038/s41587-022-01650-2
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Peter J. Chockley