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Locoregionally administered B7-H3-targeted CAR T cells for treatment of atypical teratoid/rhabdoid tumors

Nature Medicine volume 26, pages 712–719 (2020)Cite this article

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Abstract

Atypical teratoid/rhabdoid tumors (ATRTs) typically arise in the central nervous system (CNS) of children under 3 years of age. Despite intensive multimodal therapy (surgery, chemotherapy and, if age permits, radiotherapy), median survival is 17 months1,2. We show that ATRTs robustly express B7-H3/CD276 that does not result from the inactivating mutations in SMARCB1 (refs. 3,4), which drive oncogenesis in ATRT, but requires residual SWItch/Sucrose Non-Fermentable (SWI/SNF) activity mediated by BRG1/SMARCA4. Consistent with the embryonic origin of ATRT5,6, B7-H3 is highly expressed on the prenatal, but not postnatal, brain. B7-H3.BB.z-chimeric antigen receptor (CAR) T cells administered intracerebroventricularly or intratumorally mediate potent antitumor effects against cerebral ATRT xenografts in mice, with faster kinetics, greater potency and reduced systemic levels of inflammatory cytokines compared to CAR T cells administered intravenously. CAR T cells administered ICV also traffic from the CNS into the periphery; following clearance of ATRT xenografts, B7-H3.BB.z-CAR T cells administered intracerebroventricularly or intravenously mediate antigen-specific protection from tumor rechallenge, both in the brain and periphery. These results identify B7-H3 as a compelling therapeutic target for this largely incurable pediatric tumor and demonstrate important advantages of locoregional compared to systemic delivery of CAR T cells for the treatment of CNS malignancies.

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Fig. 1: High levels of B7-H3 expression in ATRTs and during normal prenatal brain development.
Fig. 2: Locoregional administration of B7-H3 CAR T cells is more potent and results in lower systemic inflammatory cytokine levels than IV administration against aggressive orthotopic ATRT xenografts.
Fig. 3: Locoregionally administered B7-H3 CAR T cells home more rapidly to cerebral ATRT xenografts than those administered intravenously. B7-H3 CAR T cells administered intracerebroventricularly efficiently migrate out of the CNS.
Fig. 4: B7-H3 CAR T cells persist in the brain and lead to antigen-specific protection from tumor rechallenge.

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Data availability

All unprocessed western blot images and statistics are supplied as Source Data. All source data for Figs. 1–4 and Extended Data Figs. 1–8 are supplied in the Supplementary Dataset.

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Acknowledgements

J.Theruvath is supported by German Cancer Aid (Deutsche Krebshilfe) grant no. P-91650709. This work was supported by a St Baldrick’s/Stand Up To Cancer Pediatric Dream Team Translational Cancer Research Grant (C.L.M., A.D., P.H.S., M.M., R. G.M.). Stand Up To Cancer is a program of the Entertainment Industry Foundation administered by the American Association for Cancer Research. C.L.M. is a member of the Parker Institute for Cancer Immunotherapy, which supports the Stanford University Cancer Immunotherapy Program. The work was also supported by the Virginia and D.K. Ludwig Fund for Cancer Research (C.L.M. and M.M.) and by National Cancer Institute grant no. 5P30CA12443 (C.L.M.). R.G.M. is supported by the Be Brooks Brave Fund St. Baldrick’s Scholar Award. A.L. is supported by the Nuovo-Soldati Foundation and by ITMO Cancer AVIESAN (Alliance Nationale pour les Sciences de la Vie et de la Santé/National Alliance for the Life Sciences and Health) within the framework of the French Cancer Plan. M.A.F. and D.W. receive funding as part of the INSTINCT network program grant, cofunded by The Brain Tumour Charity, Great Ormond Street Hospital Children’s Charity and Children with Cancer UK (16/193). O.D., D.S. and S.Z. are supported by the Ligue Nationale Contre le Cancer (équipe labellisée) and by the following grants: ERA-NET TRANSCAN (JTC 2014, no. TRAN201501238), TRANSCAN JTC 2017 (no.TRANS201801292) and H2020-lMI2-JTl-201 5-07 (116064, ITCC-P4 project). F.B. is supported by the St Baldrick’s Foundation. M.H. is funded by Interdisziplinäre Zentrum für Klinische Forschung Münster (no. Ha3/017/20) and Deutsche Forschungsgemeinschaft (DFG, no. HA 3060/8-1). M.C.F. is funded by DFG no. 1516/4-1. D.W.Y. is supported by the Tashia and John Morgridge Endowed Postdoctoral Fellowship from the Child Health Research Institute at Lucile Packard Children’s Hospital as well as the National Institute of Neurological Disorders and Stroke of the National Institutes of Health under award no. R25NS065741. S.C. was supported by the Ludwig Cancer Center, Department of Neurosurgery at Stanford University, and the Huntsman Cancer Institute, Department of Neurosurgery, University of Utah. He was also supported as the Ty Louis Campbell St. Baldrick’s Foundation Scholar and Kathryn S.R. Lowry Endowed Chari in Neurosurgery. S.C. was also supported by kind gifts from the Victoria and Rider McDowell Family Foundation, Cancer-A-Gogo, J. and C. Fisher, and C. Comey and J. Huang. S.S.M. is supported by the Siebel Scholars Award, The Andrew McDonough B+ (Be Positive) Foundation, The Morgan Adams Brain Tumor Foundation, the American Cancer Society Institutional Research Grant and the Plachy-Rubin foundation. We thank Y.-J. Cho (Oregon Health & Science University) for providing the ATRT-CHB-1 cell line and E. Huellman (Amsterdam University) for providing the VU-397 cell line. We thank J. Huang for assistance with mouse colony maintenance. We thank the Stanford Human Immune Monitoring Center for their support with the Luminex assays. We thank the Stanford Neuropathology Department for their help acquiring tissues. The illustrations for Fig. 2b and Extended Data Fig. 3b were used and modified from Servier Medical ART, licensed under a Creative Commons Attribution 3.0 Generic License (http://smart.servier.com/).

Author information

Authors and Affiliations

  1. Department of Pediatrics, Stanford University School of Medicine, Stanford, CA, USA

    Johanna Theruvath, Elena Sotillo, Claus Moritz Graef, Sabine Heitzeneder, Louai Labanieh, Shaurya Dhingra, Amaury Leruste, Robbie G. Majzner, Peng Xu, Michelle Monje & Crystal L. Mackall

  2. Department of Neurology, Stanford University School of Medicine, Stanford, CA, USA

    Christopher W. Mount & Michelle Monje

  3. Department of Molecular Oncology, British Columbia Cancer Research Centre, Vancouver, British Columbia, Canada

    Alberto Delaidelli & Poul H. Sorensen

  4. Department of Bioengineering, Stanford University, Stanford, CA, USA

    Louai Labanieh

  5. Stanford Cancer Institute, Stanford University School of Medicine, Stanford, CA, USA

    Robbie G. Majzner, Michelle Monje & Crystal L. Mackall

  6. Department of Neurology, University of California San Francisco, San Francisco, CA, USA

    Sabine Mueller

  7. Department of Neurological Surgery, University of California San Francisco, San Francisco, CA, USA

    Sabine Mueller

  8. Department of Pediatrics, University of California San Francisco, San Francisco, CA, USA

    Sabine Mueller

  9. Department of Neurosurgery, Stanford University School of Medicine, Stanford, CA, USA

    Derek W. Yecies & Michelle Monje

  10. Wolfson Childhood Cancer Research Centre, Northern Institute for Cancer Research, Newcastle University, Newcastle upon Tyne, UK

    Martina A. Finetti & Daniel Williamson

  11. Hopp Children’s Cancer Center Heidelberg, Heidelberg, Germany

    Pascal D. Johann, Marcel Kool & Stefan Pfister

  12. Division of Pediatric Neurooncology, German Cancer Research Center and German Cancer Consortium, Heidelberg, Germany

    Pascal D. Johann, Marcel Kool & Stefan Pfister

  13. Department of Pediatric Hematology and Oncology, Heidelberg University Hospital, Heidelberg, Germany

    Pascal D. Johann & Stefan Pfister

  14. Institute of Neuropathology, Münster University Hospital, Münster, Germany

    Martin Hasselblatt

  15. University Children’s Hospital Augsburg, Swabian Children’s Cancer Center, Augsburg, Germany

    Michael C. Frühwald

  16. EU-RHAB Registry Center, Augsburg, Germany

    Michael C. Frühwald

  17. Paris Sciences Lettres Research University, INSERM U830, Paris, France

    Olivier Delattre, Didier Surdez, Franck Bourdeaut & Sakina Zaidi

  18. Paris Sciences Lettres Research University, SIREDO: Care, Innovation and Research for Children, Adolescents and Young Adults with Cancer, Institut Curie, Paris, France

    Olivier Delattre, Didier Surdez, Franck Bourdeaut & Sakina Zaidi

  19. Paris University, Necker-Enfants Malades Hospital, Department of Neurosurgery, Assistance Publique-Hôpitaux de Paris, Paris, France

    Stephanie Puget

  20. Pediatrics, University of Colorado Anschutz Medical Campus, Aurora, CO, USA

    Siddhartha S. Mitra

  21. Division of Pediatric Neurosurgery, Department of Neurosurgery, Primary Children’s Hospital and Huntsman Cancer Institute, University of Utah, Salt Lake City, UT, USA

    Samuel Cheshier

  22. Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA

    Michelle Monje

  23. Stanford Institute for Stem Cell and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, USA

    Michelle Monje

  24. Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA

    Crystal L. Mackall

Authors
  1. Johanna Theruvath
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  2. Elena Sotillo
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  3. Christopher W. Mount
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Contributions

J.T., E.S., C.L.M. and R.G.M. conceived and designed the study. J.T., E.S., C.W.M., C.M.G., S.H., L.L., P.X., S.M., D.W.Y., M.A.F., M.H., M.C.F., A.D., A.L., S.D., S.Z., O.D., D.S., F.B. and S.Puget collected and assembled the data. J.T., E.S., C.L.M., C.W.M., C.M.G., D.W., M.A.F., P.D.J., M.K., S.Pfister, S.S.M., S.C., A.D., P.H.S. and M.M. analyzed and interpreted the data. J.T., E.S. and C.L.M. wrote the manuscript.

Corresponding author

Correspondence to Crystal L. Mackall.

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Competing interests

R.G.M., L.L. and E.S. hold several patent applications in the area of CAR T cell immunotherapy. C.L.M. is an inventor on a patent application for B7-H3 CAR T cells and holds several patent applications in the area of CAR T cell immunotherapy. C.L.M. is a founder of, holds equity in and receives consulting fees from Lyell Immunopharma. C.L.M. has also received consulting fees from NeoImmune Tech, Nektar Therapeutics and Apricity Health and royalties from Juno Therapeutics for the CD22-CAR. R.G.M, E.S. and L.L. are consultants for Lyell Immunopharma. R.G.M. is a consultant for Xyphos, Illumina Radiopharmaceuticals, GammaDelta Therapeutics and PACT Pharma. J.T. is a consultant for Dorian Therapeutics.

Additional information

Peer review information Saheli Sadanand was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Regulation of B7-H3 expression in ATRT and during normal brain development.

a, Flow cytometric analysis of B7-H3 expression on ATRT cell lines. Representative of two independent experiments is shown b, Quantification of B7-H3 molecules per cell on ATRT cell lines using flow cytometry (TYR n = 3, SHH n = 3, MYC n = 3; TYR vs. SHH p = 0.700; TYR vs. MYC p = 0.400; SHH vs MYC p = 0.200) (Mann-Whitney test, two-tailed). c, B7-H3 mRNA upon SMARCB1 re-expression from datamining of Chauvin et al (GSE98277 Cell Rep1017) published inducible SMARCB1 system in a SMARCB1-deficient rhabdoid cell line (n = 3 for all time points) 0 days vs. 2 days **p = 0.007; 0 days vs. 4 days *p = 0.011; 0 days vs. 7 days *p = 0.012; 0 days vs. 14 days **p = 0.007) (unpaired t-test, two-tailed). d, ChipSeq H3K27Ac data of the promoter region of B7-H3 in primary ATRT tumors. e, ChipSeq SMARCA4 data at the promoter region of B7-H3 in primary ATRT tumors f, RT-qPCR analysis of B7-H3 and SMARCA4 mRNA expression levels in SMARCB1-deficient ATRT cell lines (BT12 and BT16) five days after shRNA SMARCA4 knock down (k.d.) with two different short hairpins. Graph represents ΔΔCt relative to cells transduced with empty vector. Triplicates were run in each experiment. Representative results of two independent experiments is shown g, Correlation of normalized SMARCA4 and normalized B7-H3 expression from primary ATRT tumors (GSE70678) (n = 49) (r = 0.32, p = 0.026) (Pearson correlation, two-sided) h, Correlation of mRNA expression of SMARCA4 and B7-H3 during normal brain development (prenatal n = 237; pediatric n = 178; adult n = 109) (r = 0.86; p = <1x10-15) (Pearson correlation, two-sided). (i) mRNA expression levels of miR29c during and after normal brain development (prenatal n = 237; pediatric n = 178; adult n = 109). j, miR29 nanostring counts in ATRT cells lines and mature and immature neurons. k, ChipSeq H3K27Ac data around miR29 locus in primary ATRT tumors. All data are means ± s.d.

Source data

Extended Data Fig. 2 B7-H3 CAR T cells are highly potent against ATRT cell lines in vitro.

a, Cytometric Bead Array of interferon γ release 24 hours after co-culture of B7-H3 CAR T cells or CD19 CAR T cells (control) with ATRT tumor cells (B7-H3 CAR T cells vs. CD19 CAR T cells: BT12 ****p = 5x10-7; BT16 p = 0.086; ATRT-CHB-1 ****p = 5x10-6; VU397 ****p = 5x10-8; CHLA-2 ****p = 9x10-6; CHLA-4 ****p = 5x10-7; CHLA-5 ***p = 9x10-4; ATRT13808 ****p = 7x10-5) (unpaired t-test, two-tailed). b, Killing assay of BT16 ATRT tumor cells when co-cultured with B7-H3 CAR T cells or CD19 CAR T cells (control) at different E:T ratios (B7-H3 CAR T cells vs. CD19 CAR T cells: 1:1 ****p = 5x10-5; 1:4 ***p = 3x10-4; 1:8 ****p = 2x10-6; 1:16: **p = 0.003) (Two-Way Anova). c, Representative images obtained 72 hours after co-culture of BT16 ATRT tumor cells (red) with B7-H3 or CD19 CAR T cells in a 1:4 ratio. d, Killing assay of BT12 ATRT tumor cells when co-cultured with B7-H3 CAR T cells or CD19 CAR T cells (control) at different E:T ratios (B7-H3 CAR T cells vs. CD19 CAR T cells: 5:1 ****p = 3x10-6; 1:1 ****p = 8x10-5; 1:5 ****p = 5x10-6) (Two-Way Anova). All data are means ± s.e.m. (a-d) n = 3 independent samples, experiments have been conducted three times.

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Extended Data Fig. 3 B7-H3 CAR T cells are highly potent against ATRT PDXs in vivo.

a, Representative IHC image of B7-H3 staining of IC-pPDX-69 and IC-pPDX-159 ATRT PDX after engraftment (IC-pPDX-69: H Score 210 and IC-pPDX-159: H score 210). Staining was performed one time. b, Experimental overview for the evaluation of in vivo efficacy of B7-H3 CAR T cells against IC-pPDX-69 c,d, and IC-pPDX-159 e,f, Summary (c,e) and individual (d,f) tumor measurements after 10x106 B7-H3 CAR T cells or CD19 CAR T cells (control) (c) p = 2x10-6; (e) p = 6x10-5 (Two-way Anova). All data are means ± s.e.m. Experiment has been performed one time (n = 10 mice per group).

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Extended Data Fig. 4 Locoregionally administered B7-H3 CAR T cells are highly potent against orthotopic ATRT xenografts in vivo.

a, Representative IHC image of B7-H3 staining of BT12 ATRT xenograft (H Score: 270). Staining was performed one time. b, IT Group (B7-H3 (1x106) and CD19 (1x106) CAR T cells), c, ICV Group (B7-H3 (1x106) and CD19 (1x106) CAR T cells), d, IV Group (1x106 or 10x106) B7-H3 and CD19 (10x106) CAR T cells). (c-e) Bioluminescence (BLI) was obtained serially and flux curve for individual mice are shown. Kaplan-Meier analysis (n = 1) of B7-H3 or CD19 (control) CAR T cell treated mice (IT p = 0.0023; ICV p = 0.0031); IV (1x106) p = 0.6015; IV (10x106) p = 0.0038). (Log-rank (Mantel-Cox) test, two-tailed). One mouse in the B7-H3 (10x106) IV group died while imaging and was censored from analysis. Mio=million.

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Extended Data Fig. 5 In vivo dose response testing demonstrates that ICV administered B7-H3 CAR T cells require lower dose for cure than those delivered via IV.

In vivo dose response testing of B7H3 CAR T cells administered ICV (a,b) or IV (c,d). Bioluminescence (BLI) was obtained serially and flux curve for individual mice are shown for ICV (a) or IV (c) treatment. Kaplan-Meier analysis of B7-H3 or CD19 (control) CAR T cell treated mice in ICV (b) or IV (d) treatment. Stated p values are compared to CD19 control. (Log-rank (Mantel-Cox) test, two-tailed) n = 5 biologically independent animals. Experiment has been performed one time. Mio=million.

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Extended Data Fig. 6 IV Administration of B7-H3 CAR T cells results in higher systemic inflammatory cytokines than ICV or IT administration.

a, INFγ, IL-4, IL-10 levels in serum and CSF from mice 7 days after CAR T cell treatment (For IT and ICV: 1x106 CAR T cells, IV: 10x106 CAR T cells). Significantly higher INFγ, IL-4 and IL-10 concentrations in the serum of the B7-H3 IV group compared to all other groups (Serum INFγ: B7-H3 IV vs. IT **p = 0.0033; B7-H3 IV vs. ICV **p = 0.0033; Serum IL-4: B7-H3 IV vs. IT ***p = 0.0008; B7-H3 IV vs. ICV ***p = 0.0008; Serum IL-10: B7-H3 IV vs. IT *p = 0.01; B7-H3 IV vs. ICV *p = 0.01). No significant differences in the CSF between B7-H3 CAR IV and locoregional treated mice (IT, ICV). (Ordinary one-way Anova). All data are means ± s.e.m. 3 mice per group. Serum from mice 7 days b, and 13 days c, after CAR T cell treatment (at indicated dose levels and CD19 IV 10x106 and ICV 1x106 CAR T cells) demonstrates significantly higher inflammatory cytokines on day7 after CAR T cell administration in B7-H3 IV group compared to B7H3 ICV. Strong reduction of cytokine levels on day 13 after CAR T cell administration. Z score values were calculated for each cytokine across the timepoints day 7 and day 13/all samples; for better visualization purposes z score was limited to 2 SD; 3 mice per group. Statistics were calculated using box plots. (Overall cytokine secretion in serum: Day7: Least curative CAR T cell dose: B7-H3 2.5x106 IV vs B7-H3 0.5 x106 ICV p = 3x10-13; B7-H3 2.5x106 IV vs B7-H3 0.25 x106 ICV p = 2x10-13; Day 7 vs. Day13: B7-H3 2.5 x106 p = 3x10-11; B7-H3 0.5 x106 ICV p = 0.9921, B7-H3 0.25 x106 ICV p = 0.9957) (Mann-Whitney Test, two-tailed).

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Extended Data Fig. 7 B7-H3 CARs demonstrate antigen-dependent proliferation and trafficking outside the CNS.

a, Flow cytometry demonstrating that fluorescent protein fused to CAR construct correlated with CAR surface expression. Experiment was performed two times. b, Representative immunofluorescence confocal microscopy of Ki67 staining of B7-H3 or CD19 CAR T cells in the brain. B7-H3 CAR T cells are positive for Ki 67, CD19 CAR T cells are negative. Scanning of 1:12 series from 3 independent mice/group. c, Quantification (n = 3 per group) and d, representative flow cytometry histogram of CAR T cells detected in single cell suspensions from mouse spleens 47 days after one time treatment (IT vs. ICV p = 0.200, IT vs. IV p = 0.800, ICV vs. IV p = 0.200) (Mann-Whitney test, two-tailed). e, Quantification of CAR T cell /T cell ratio in spleen after one time CAR T cell treatment (n = 3 per group) (ICV vs. IV *p = 0.041) (IT vs. IV p = 0.288) (unpaired t-test, two-tailed) All data are means ± s.e.m.

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Extended Data Fig. 8 CRISPR/Cas9 Knock out of B7-H3 slightly reduces the proliferation rate of ATRT cell lines in vitro and in vivo.

a, Proliferation assay of BT16 vs. B7-H3 KO BT16 tumor cells (p = 0.05) (Two-way Anova) n = 3 independent samples, experiment has been conducted three times. b, Representative images of BT16 and BT16 B7-H3 KO ATRT tumor cells. Experiment has been conducted two times c, Flux curve and survival d, of BT16 vs BT16 B7-H3 KO flank xenografts (p = 0.0031) (n = 5 independent animals) (Log-rank (Mantel-Cox) test, two-tailed). Experiment has been performed one time. All data are means ± s.e.m.

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Supplementary information

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Theruvath, J., Sotillo, E., Mount, C.W. et al. Locoregionally administered B7-H3-targeted CAR T cells for treatment of atypical teratoid/rhabdoid tumors. Nat Med 26, 712–719 (2020). https://doi.org/10.1038/s41591-020-0821-8

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