{
  "abstract": "Background H3K27M-mutated diffuse midline gliomas (DMGs) are universally lethal cancers in children and young adults. Our team previously demonstrated efficacy of GD2-targeting chimeric antigen receptor (GD2-CAR) T-cells in preclinical models of DMG 1 and opened a Phase I clinical trial (NCT04196413) treating patients with intravenous (IV) followed by repeated infusions of intracerebroventricular (ICV) GD2-CAR T-cells.2 Encouragingly, 10/12 infused patients experienced neurologic benefit and/or volumetric tumor reductions. However, most patients had disease progression with varying durations of response, including limited duration <5 months (n=4), intermediate duration 5-12 months (n=4), and sustained response >12 months (n=4). To understand the mechanisms of resistance to CAR T-cells in patients, we employed a robust reverse translational platform of high-dimensional analyses of patient samples. Here, we present an in-depth evaluation of immune-mediated CAR T-cell rejection in patients treated on this GD2 CAR T-cell trial.Methods Single cell RNA-sequencing (scRNAseq) was conducted on 113 cerebrospinal fluid (CSF) samples from 12 patients, resulting in 614,133 single cells. We also developed and implemented a Humanized Anti-CAR Antibodies (HACA) assay on 332 patient plasma and CSF samples. Statistical analyses included Mann-Whitney and Spearman correlation analysis, as appropriate.Results In scRNAseq data, we identified that GD2-CAR T-cell infusion induces dynamic changes to the immune environment in CSF. Patients with limited response duration associated with increased regulatory T-cells early after IV GD2-CAR infusion (p-value=0.046), while loss of response associated with B-cell expansion early after ICV GD2-CAR infusion (p-value=0.062), statistics by Mann-Whitney U-test. HACA development was associated with increased number of GD2-CAR infusions (p-value=0.0541; Mann-Whitney). Additionally, HACA negatively correlated with peripheral blood CAR T-cell persistence (Spearman correlation coefficient = -0.4840). These findings were orthogonally validated using a cell-free ELISA of HACA. Several B-cell receptor clonotypes emerged after GD2-CAR T-cell therapy and were highly expanded and dominant. Studies are ongoing to identify T-cell-mediated immune rejection.Conclusions Together, these data identify immune rejection mechanisms in GD2-CAR T-cell-treated DMG patients, providing insights to optimize this therapy with hope to shift the paradigm of this fatal disease.References Mount CW, Majzner RG, Sundaresh S, Arnold EP, Kadapakkam M, Haile S, Labanieh L, Hulleman E, Woo PJ, Rietberg SP, Vogel H, Monje M, Mackall CL. Potent antitumor efficacy of anti-GD2 CAR T cells in H3-K27M + diffuse midline gliomas. Nat Med. 2018 May;24(5):572-579. doi: 10.1038/s41591-018-0006-x. Epub 2018 Apr 16. PMID: 29662203; PMCID: PMC6214371Monje M, Mahdi J, Majzner R, Yeom K, Schultz LM, Richards RM, Barsan V, Song KW, Kamens J, Baggott C, Kunicki M, Lim AS, Reschke A, Mavroukakis S, Egeler E, Moon J, Patel S, Chinnasamy H, Erickson C, Jacobs A, Duh AK, Rietberg SP, Tunuguntla R, Klysz DD, Fowler C, Green S, Beebe B, Carr C, Fujimoto M, Brown AK, Petersen AG, McIntyre C, Siddiqui A, Lepori-Bui N, Villar K, Pham K, Bove R, Musa E, Reynolds W, Kuo A, Prabhu S, Rasmussen L, Cornell TT, Partap S, Fisher PG, Campen CJ, Grant G, Prolo L, Ye X, Sahaf B, Davis KL, Feldman SA, Ramakrishna S, Mackall C. Sequential intravenous and intracerebroventricular GD2-CAR T-cell therapy for H3K27M-mutated diffuse midline gliomas. medRxiv [Preprint]. 2024 Jun 27:2024.06.25.24309146. doi: 10.1101/2024.06.25.24309146. PMID: 38978673; PMCID: PMC11230330",
  "authors": [
    {
      "affiliations": [
        "Stanford Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University, Stanford, CA, USA"
      ],
      "name": "Sneha Ramakrishna"
    },
    {
      "affiliations": [
        "Stanford Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University, Stanford, CA, USA"
      ],
      "name": "Yiyun Chen"
    },
    {
      "affiliations": [
        "Stanford Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University, Stanford, CA, USA"
      ],
      "name": "Kun-Wei Song"
    },
    {
      "affiliations": [
        "Stanford Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University, Stanford, CA, USA"
      ],
      "name": "Ying-Wen Huang"
    },
    {
      "affiliations": [
        "Stanford Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University, Stanford, CA, USA"
      ],
      "name": "Nadya Iswari"
    },
    {
      "affiliations": [
        "Stanford Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University, Stanford, CA, USA"
      ],
      "name": "Moksha Desai"
    },
    {
      "affiliations": [
        "Stanford Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University, Stanford, CA, USA"
      ],
      "name": "Zachary Ehlinger"
    },
    {
      "affiliations": [
        "Stanford Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University, Stanford, CA, USA"
      ],
      "name": "Hossein Daghagh"
    },
    {
      "affiliations": [
        "Stanford Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University, Stanford, CA, USA"
      ],
      "name": "Kevin Reynolds"
    },
    {
      "affiliations": [
        "Stanford Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University, Stanford, CA, USA"
      ],
      "name": "Maximilian Richard Andreas Koch"
    },
    {
      "affiliations": [
        "Baylor College of Medicine, Houston, TX, USA"
      ],
      "name": "Jasia Mahdi"
    },
    {
      "affiliations": [
        "Dana-Farber Cancer Institute, Boston, MA, USA"
      ],
      "name": "Robbie G Majzner"
    },
    {
      "affiliations": [
        "University of Wisconsin-Madison, Madison, WI, USA"
      ],
      "name": "Rebecca M Richards"
    },
    {
      "affiliations": [
        "Cincinnati Children’s Hospital, Cincinnati, OH, USA"
      ],
      "name": "Jennifer Kamens"
    },
    {
      "affiliations": [
        "Stanford Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University, Stanford, CA, USA"
      ],
      "name": "Valentin Barsan"
    },
    {
      "affiliations": [
        "Stanford Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University, Stanford, CA, USA"
      ],
      "name": "Cynthia Campen"
    },
    {
      "affiliations": [
        "Stanford Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University, Stanford, CA, USA"
      ],
      "name": "Sonia Partap"
    },
    {
      "affiliations": [
        "Stanford Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University, Stanford, CA, USA"
      ],
      "name": "Divya Madhav"
    },
    {
      "affiliations": [
        "Stanford Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University, Stanford, CA, USA"
      ],
      "name": "Jennifer Moon"
    },
    {
      "affiliations": [
        "Stanford Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University, Stanford, CA, USA"
      ],
      "name": "Christina Baggott"
    },
    {
      "affiliations": [
        "Stanford Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University, Stanford, CA, USA"
      ],
      "name": "Michael Kunicki"
    },
    {
      "affiliations": [
        "Stanford Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University, Stanford, CA, USA"
      ],
      "name": "Michelle Fujimoto"
    },
    {
      "affiliations": [
        "Stanford Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University, Stanford, CA, USA"
      ],
      "name": "Amy Li"
    },
    {
      "affiliations": [
        "Stanford Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University, Stanford, CA, USA"
      ],
      "name": "Sneha Jariwala"
    },
    {
      "affiliations": [
        "Stanford Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University, Stanford, CA, USA"
      ],
      "name": "Sharon Mavroukakis"
    },
    {
      "affiliations": [
        "Stanford Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University, Stanford, CA, USA"
      ],
      "name": "Emily Egeler"
    },
    {
      "affiliations": [
        "Stanford Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University, Stanford, CA, USA"
      ],
      "name": "Ashley Jacobs"
    },
    {
      "affiliations": [
        "Stanford Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University, Stanford, CA, USA"
      ],
      "name": "Courtney Erickson"
    },
    {
      "affiliations": [
        "Stanford Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University, Stanford, CA, USA"
      ],
      "name": "Aanchal Kaur"
    },
    {
      "affiliations": [
        "Stanford Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University, Stanford, CA, USA"
      ],
      "name": "Smriti Ramesh Soundaranayagi"
    },
    {
      "affiliations": [
        "Stanford Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University, Stanford, CA, USA"
      ],
      "name": "Kristin CY Tsui"
    },
    {
      "affiliations": [
        "Stanford Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University, Stanford, CA, USA"
      ],
      "name": "Kelvin C Mo"
    },
    {
      "affiliations": [
        "Stanford Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University, Stanford, CA, USA"
      ],
      "name": "Skyler Rietberg"
    },
    {
      "affiliations": [
        "Stanford Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University, Stanford, CA, USA"
      ],
      "name": "Liora Schultz"
    },
    {
      "affiliations": [
        "Stanford Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University, Stanford, CA, USA"
      ],
      "name": "Kara L Davis"
    },
    {
      "affiliations": [
        "Stanford Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University, Stanford, CA, USA"
      ],
      "name": "Steven A Feldman"
    },
    {
      "affiliations": [
        "Stanford Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University, Stanford, CA, USA"
      ],
      "name": "Sabine Heitzeneder"
    },
    {
      "affiliations": [
        "Stanford Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University, Stanford, CA, USA"
      ],
      "name": "Sean A Yamada-Hunter"
    },
    {
      "affiliations": [
        "Stanford Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University, Stanford, CA, USA"
      ],
      "name": "Elena Sotillo"
    },
    {
      "affiliations": [
        "Stanford Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University, Stanford, CA, USA"
      ],
      "name": "Zinaida Good"
    },
    {
      "affiliations": [
        "Stanford Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University, Stanford, CA, USA"
      ],
      "name": "Bita Sahaf"
    },
    {
      "affiliations": [
        "Stanford Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University, Stanford, CA, USA"
      ],
      "name": "Michelle Monje"
    },
    {
      "affiliations": [
        "Stanford Center for Cancer Cell Therapy, Stanford Cancer Institute, Stanford University, Stanford, CA, USA"
      ],
      "name": "Crystal L Mackall"
    }
  ],
  "title": "269 Humanized anti-CAR antibodies affect durable response to GD2-CAR T-cells in diffuse midline glioma",
  "uid": "f4830d6a-91f5-5aaa-ae1f-13b118f8b922"
}