Locoregional delivery of chimeric antigen receptor (CAR) T cells has resulted in objective responses in adults with glioblastoma, but the feasibility and tolerability of this approach is yet to be evaluated for pediatric central nervous system (CNS) tumors. Here we show that engineering of a medium-length CAR spacer enhances the therapeutic efficacy of human erb-b2 receptor tyrosine kinase 2 (HER2)-specific CAR T cells in an orthotopic xenograft medulloblastoma model. We translated these findings into BrainChild-01 (NCT03500991), an ongoing phase 1 clinical trial at Seattle Children’s evaluating repetitive locoregional dosing of these HER2-specific CAR T cells to children and young adults with recurrent/refractory CNS tumors, including diffuse midline glioma. Primary objectives are assessing feasibility, safety and tolerability; secondary objectives include assessing CAR T cell distribution and disease response. In the outpatient setting, patients receive infusions via CNS catheter into either the tumor cavity or the ventricular system. The initial three patients experienced no dose-limiting toxicity and exhibited clinical, as well as correlative laboratory, evidence of local CNS immune activation, including high concentrations of CXCL10 and CCL2 in the cerebrospinal fluid. This interim report supports the feasibility of generating HER2-specific CAR T cells for repeated dosing regimens and suggests that their repeated intra-CNS delivery might be well tolerated and activate a localized immune response in pediatric and young adult patients.
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All requests for raw and analyzed data and materials will be promptly reviewed by the intellectual property office of Seattle Children’s Research Institute to verify if the request is subject to any intellectual property or confidentiality obligations. Raw preclinical and clinical data are stored at Seattle Children’s with indefinite appropriate backup. The full raw western blot is shown in Supplementary Fig. 3. Patient-related data not included in the paper were generated as part of clinical trials and might be subject to patient confidentiality. Any data and materials that can be shared will be released via a material transfer agreement.
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We thank the children and families who bravely shoulder the burden of their disease and place their trust in Seattle Children’s. We are indebted to our clinical research team, including H. Ullom, A. Thomsen, V. Weiss, K. Cilluffo, E. Stowe and G. Mun. We are grateful for the clinical expertise of our neuro-oncology team, including R. Geyer, J. Olson, S. Leary, N. Millard, A. Sato, E. Crotty, C. Hoeppner, S. Holtzclaw, S. Chaffee, A. Laurine, S. Stasi, B. Cole, F. Perez, M. Susun and W. Iwata. We thank J. Stevens, as well as the Seattle Children’s Hospital’s Department of Anatomic Pathology and Seattle Children’s Tumor Bank, for assistance in tissue collection and research coordination, and C. Schubert for editorial assistance. We thank the Therapeutic Cell Production Core for their tireless efforts to manufacture infusion products and the Correlative Studies Lab for their assistance in research coordination and correlative sample processing. Funding: We are grateful for generous funding from the Seattle Run of Hope (N.A.V.), the Pediatric Brain Tumor Research Fund Guild of Seattle Children’s Hospital (N.A.V.), the McKenna Claire Foundation (N.A.V.), Unravel Pediatric Cancer (N.A.V.), Team Cozzi Foundation (N.A.V.), Love for Lucy (N.A.V.), the Julianna Sayler Foundation (N.A.V.), the Avery Huffman DIPG Foundation (N.A.V.), Liv Like a Unicorn (N.A.V.), ImmunoMomentum! (N.A.V., N.P., M.C.J.), Amazon (N.A.V., M.C.J.), the DIPG All-In Initiative (N.A.V.) and St. Baldrick’s Stand Up to Cancer (SU2C) Dream Team Translational Cancer Research Grants (SU2C-AACR-DT-27-17 to N.A.V., R.J.O., R.G., M.C.J. and J.R.P.). Stand Up to Cancer is a division of the Entertainment Industry Foundation, and research grants are administered by the American Association for Cancer Research, the scientific partner of SU2C. Funding was also provided by Alex’s Lemonade Stand Foundation for Childhood Cancer (R.A.G.); by the German Research Foundation (DFG, Deutsche Forschungsgemeinschaft; KU-2906/1-1 to A.K.); and by the National Center for Advancing Translational Sciences of the National Institutes of Health (U01TR002487 to A.L.W., W.H., R.A.G. and J.R.P.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
M.C.J. has interests in Umoja Biopharma and Juno Therapeutics, a Bristol Myers Squibb company. He is a seed investor and holds ownership equity in Umoja, serves as a member of the Umoja Joint Steering Committee and is a Board Observer of the Umoja Board of Directors. M.C.J. also holds patents, some of which are licensed to Umoja Biopharma and Juno Therapeutics. R.A.G. serves on a study steering committee for and is an inventor on a patent licensed to Juno Therapeutics and has served on advisory boards for Novartis. A.J.J. is an inventor on issued and pending patents related to CAR T cell therapies, and R.J.O. receives research support from Lentigen Technology, a Miltenyi Biotec company, and is a consultant for Umoja Biopharma. All other authors declare no competing financial interests.
Peer review information Nature Medicine thanks Arzu Onar-Thomas and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. 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.
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a, Schematic of 2nd generation HER2CAR extracellular domain spacer variants: Short (S), IgG4-hinge; Medium (M), IgG4-hinge-CH3; Long (L), IgG4-hinge-CH2-CH3. b, Human CD8 + T cell surface expression of S, M and L spacer variants of CAR T (EGFRt+) cells detected by cetuximab and Protein-L. c, CAR expression detected by CD3ζ-specific western blot. This experiment was performed in duplicate and a representative image is shown.
a, Flow analysis of HER2+ (D283, Med411FH) and HER2- (D341) medulloblastoma cell lines. b, In vitro cytotoxicity of HER2-specific CAR T cells against target cell lines. c, Cytokine release assay of supernatants obtained from 24-hour co-cultures of CD8+ T cells expressing the HER2CAR extracellular spacer variants, with target cells at a 2:1 ratio. N = 3 technical replicates per condition and data presented as mean values + /− SD. d, Schematic of HER2t-CD19t components: In-frame fusion of GMCSFRss, HER2t (aa 563–653 of HER2) and CD19t (aa 20–323 of CD19). SS, signal sequence; TM, transmembrane domain. e, In vitro cytotoxicity of HER2-specific CAR T cells against a lymphoblastoid cell line (LCL) panel (parental, OKT3, HER2, and HER2t-CD19t). The x-axis shows the ratio of effector:target cells. f, Cytokine release assay of supernatants obtained from 24-hour co-cultures of CD8 + T cells expressing the HER2CAR extracellular spacer variants with LCL targets at a 2:1 ratio. N = 3 technical replicates per condition and data presented as mean values ± SD.
Extended Data Fig. 3 Medium-spacer HER2CAR T cells prolong survival and target metastases in an orthotopic xenograft model.
Groups of mice were inoculated with 0.2e6 D283 eGFP:ffluc medulloblastoma cells (Day 0) and 2e6 HER2CAR CD8 + T cells (Day 7 – dotted vertical line) (a-d) intracranially or (e) via intracerebroventricular injection (ICV). a,b, Serial bioluminescence imaging of tumor in groups of mice treated with Mock, Short, Medium, or Lmut spacer HER2CAR CD8+ T cells. Data presented as mean values ± SD. N = 5 animals per group. All Kaplan-Meier statistical significance determined by one-sided log-rank (Mantel-Cox) test (M-spacer versus mock (P = 0.0035), S-spacer (P = 0.0021) and Lmut-spacer (P = 0.025). c, Bioluminescence imaging measured in region of interest (head) for orthotopic intracranial model. d, Kaplan-Meier analysis of survival in treatment (Medium and Lmut) and control (Mock) groups from repeat experiments. e, Kaplan-Meier analysis of survival in treatment and control groups of ICV dosed tumor models: D283 eGFP:ffluc cells (no IL-2) and D283 eGFP:ffluc cells (IL-2). f, Serial bioluminescence imaging in treatment and control groups of ICV dosed tumor models: D283 eGFP:ffluc cells (no IL-2) and D283 eGFP:ffluc cells (IL-2). All in vivo data are representative of a minimum of two independent experiments.
a, NSG mice were injected with 2e6 mixed CD4+ :CD8+ HER2CAR T cells or un-transduced CD4+ :CD8+ Mock T cells 7 days post intracranial tumor inoculation with 0.2e6 D283 eGFP:ffluc medulloblastoma cells. Serial bioluminescence tumor imaging (left) and Kaplan-Meier analysis of survival (right) were performed. HER2CAR T cell treated mice survived significantly longer than the un-transduced Mock group (one-sided log-rank (Mantel-Cox) test, p = 0.002). Data presented as mean values ± SD. N = 5 animals per group. Bioluminescence images are from one representative experiment. b, Serial bioluminescence signal measured in region of interest (head) from mice treated with Mock or HER2CAR T cells. c, Individual serial bioluminescence imaging of tumor from un-transduced Mock (left) or HER2CAR T cell groups (right).
Flow diagram of the manufacturing process from apheresis to CAR T cell delivery.
Extended Data Fig. 6 Multiparameter co-expression profiles of activation/exhaustion markers for patient starting and final products.
Boolean gating was performed for activation markers of interest before visualization via SPICE analysis software. Final-02 and Final-03 refer to second and third manufacturing attempts for S003. Two-sided permutation tests performed with 10,000 iterations per the built-in SPICE analysis function found no significant differences between samples (p > 0.05). Representative flow gating strategy shown in Supplementary Fig. 1a,b.
CTCAE grading of all observed toxicities that constituted an increase from baseline. If the toxicity was not present pre-Course 1 Week 1 administration, then no baseline grade is shown.
Three adverse event (AE) types are plotted here: fever, headaches, and nervous system disorders (excluding headache). CAR T cell infusions indicated with arrows.
Extended Data Fig. 9 Pre versus post infusion detection of T cell populations in CSF via flow cytometry.
Representative pre and post infusion flow plots from S002 and S003 show no detectable CAR T (EGFRt+) cells, but detectable non-CAR T cells (both CD4+ and CD8+ T cells) in subject CSF post infusion. Representative flow gating strategy shown in Supplementary Fig. 1d.
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Vitanza, N.A., Johnson, A.J., Wilson, A.L. et al. Locoregional infusion of HER2-specific CAR T cells in children and young adults with recurrent or refractory CNS tumors: an interim analysis. Nat Med (2021). https://doi.org/10.1038/s41591-021-01404-8