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A single dose of peripherally infused EGFRvIII-directed CAR T cells mediates antigen loss and induces adaptive resistance in patients with recurrent glioblastoma

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Science Translational Medicine  19 Jul 2017:
Vol. 9, Issue 399, eaaa0984
DOI: 10.1126/scitranslmed.aaa0984
  • Fig. 1. Protocol design, consort diagram, and clinical outcomes in subjects infused with CART-EGFRvIII.

    (A) Protocol schema for EGFRvIII testing, leukapheresis, and manufacturing, treatment with CAR T cells directed to EGFRvIII, and follow-up. (B) Consort diagram indicating the number of subjects screened and enrolled on the study. (C) Swimmer’s plot describing time on study for each subject (black), duration of follow-up off study (that is, survival beyond progression or initiation of other therapy) (gray), and present status. Arrows indicate ongoing survival. Asterisks indicate surgical intervention. Dashed vertical line indicates 1-year time point. (D) OS plotted as Kaplan-Meier estimate for all subjects. X axis is shown in days. Tick marks indicate each censored subject (that is, subjects who are alive at the data cutoff point).

  • Fig. 2. Engraftment of CART-EGFRvIII and cytokine modulation in the peripheral blood.

    (A) CART-EGFRvIII engraftment and persistence in the peripheral blood by flow cytometry detecting the CAR on CD3+ T cells (left y axis, blue curve) and by qPCR detecting CAR sequences (right y axis, red curve) in PBMC genomic DNA. Pharmacokinetics over the first 30 days for each subject are shown. (B) Fold change in IL-6 levels in the peripheral blood of each subject over time (black squares, plotted on the left y axis). Baseline IL-6 levels for subjects 201, 202, 204, 205, 207, 209, 211, 213, 216, and 217 were 2.57, 7.5, 8.58, 4.90, 1.78, 4.40 , 0.35, 3.11, 8.89, and 33.85 pg/ml, respectively. Baseline level of IL-10 in subject 207 was 2.03 pg/ml. Data for all other cytokines are shown in table S6. C-reactive protein (CRP) levels are plotted in the subjects in whom it was measured over time (green triangles, right y axis). Significant clinical events such as seizures, surgery, or administration of siltuximab are noted on the x axis.

  • Fig. 3. Effects of CART-EGFRvIII on radiological and/or pathological assessments in two subjects.

    (A) MRI performed before and after administration of gadolinium (gad) in subject 205. T1 post-contrast and FLAIR images are shown for the indicated time points. (B) Histological analysis of surgical specimens obtained from subject 205, 2 months after CART-EGFRvIII infusion. Hematoxylin and eosin (H&E)–stained sections and immunohistochemistry for CD3 to demonstrate T cells are shown. Scale bar, 200 μm. (C) MRI (T1 post-contrast images) shown at the indicated time points for subject 209. This subject underwent surgical resection of one portion of the tumor after the 3-month scan.

  • Fig. 4. T cell trafficking and effect on EGFR mutations in brain tumors after intravenous infusion of CART-EGFRvIII.

    (A) Comparison of CART-EGFRvIII quantification in brain tumor specimens compared to peripheral blood in each of the seven patients who underwent surgical resection at various time points (subject # and day # indicated on the x axis) after CART-EGFRvIII infusion. Ratio is calculated on the basis of copies per microgram of genomic DNA in cells. (B) Levels of expression of EGFRvIII as determined by NGS of purified genomic DNA in brain tumor specimens obtained before or after CART-EGFRvIII infusion. Subject numbers are indicated along the right, and each subject is color-coded for clarity. Where more than one sample was obtained and tested separately, points show the mean. Bar indicates P value between pre- and post-CART levels in paired specimens by the Wilcoxon matched-pairs signed-rank test. (C) EGFR amplification in brain tumor specimens obtained pre– and post–CART-EGFRvIII infusion from all tissue samples tested. Bar indicates P value between pre- and post-CART levels in paired specimens by the Wilcoxon matched-pairs signed-rank test. Subject numbers are indicated along the right, and each subject is color-coded for clarity. (D) TCRβ CDR3 deep sequencing analysis of T cells infiltrating brain tumor specimens obtained before and after CART-EGFRvIII and (E) in the matching infusion product and post-CART brain tumor–infiltrating lymphocytes in three subjects (205, 209, and 211). Unique TCR sequences in the pre-infusion tumor biopsy are shown in the green box along the x axis in (D) and in the infusion product in the green box along the x axis in (E). In (D) and (E), these are compared with post-infusion tumor specimen clonotype repertoire shown in red along the y axis. Shared clonotypes are displayed in a blue box.

  • Fig. 5. Immunohistochemistry of the tumor microenvironment in GBM specimens before and after CART-EGFRvIII infusion.

    (A) T cell infiltration and phenotyping in pre– and post–CART-EGFRvIII infusion specimens from subject 216; pre-infusion specimens are from day −81, whereas post-infusion specimens are from day +13, relative to CART-EGFRvIII infusion at day 0. Top row shows low-power magnification of CD3 immunohistochemical stain, with high-power magnification as inset; ISH specifically for CAR sequences. T cell phenotyping is shown with ISH for IFN-γ and with immunohistochemistry for CD8, granzyme B (GRZMB), and the IL-2 receptor α chain (CD25). Scale bars, 4 mm (low-power graphs) and 200 μm (high-power graphs). (B) In situ assessment of immunosuppressive molecules in the tumor microenvironment is shown before and after CART-EGFRvIII infusion in patient 216, including IDO1, PD-L1, FoxP3, TDO, IL-10, and TGFβ. (C) Summary table with heat map of T cell infiltration, CART-EGFRvIII trafficking, and tumor microenvironment in seven subjects before and after treatment with CART-EGFRvIII. NP, not performed; 0, not detectable. Date indicates day of specimen relative to CART-EGFRvIII infusion, which was designated as day 0.

  • Fig. 6. Immunohistochemical colocalization of CD3/FoxP3 and CD8/Ki67.

    (A and B) Analysis of brain tumor samples performed pre– and post–CART-EGFRvIII infusion. Representative analysis of CD3/FoxP3 from patient 216 is shown in (A), with quantitative analysis of percent FoxP3+ CD3 cells shown for five subjects’ brain tumor samples in (B). Two-tailed paired t test analysis did not demonstrate statistical significance (P = 0.10). Hem, hematoxylin. (C) Representative analysis of CD8/Ki67 staining in patient 213, and (D) quantitative analysis of percent Ki67+ CD8 cells for five subjects’ brain tumor samples. Two-tailed paired t test analysis did not demonstrate statistical significance (P = 0.07).

  • Table 1. Patient and product characteristics.
    Median (n = 10)Range
    Age59.545–76
    Sex (male)50%
    Time from initial diagnosis to
    infusion (in days)
    358179–682
    Line of treatment at infusion
      220%
      340%
      440%
    Karnofsky performance status
      10020%
      9030%
      8030%
      7010%
      6010%
    Receiving steroids at infusion20%
    EGFRvIII expression71%(6–96%)
    % T cells transduced19.75%(4.8–25.6%)
    Dose of CART-EGFRvIII5 × 108(1.75 × 108–5 × 108)

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/9/399/eaaa0984/DC1

    Materials and Methods

    Fig. S1. Sample stain of peripheral blood T cells for CART-EGFRvIII.

    Fig. S2. Lack of correlation between engraftment and absolute lymphocyte count.

    Fig. S3. Histology and CD3 immunohistochemistry stain of pre- and post-CART infusion tumor specimens.

    Fig. S4. Validation of RNAscope ISH.

    Fig. S5. Validation of PD-L1 staining.

    Table S1. Individual patient characteristics.

    Table S2. Individual product characteristics.

    Table S3. Individual post-CART infusion events.

    Table S4. Adverse events at least possibly related to CART-EGFRvIII.

    Table S5. Immunohistochemical antibodies and ISH probes used.

    Table S6. Primary cytokine data (separate Excel file).

  • Supplementary Material for:

    A single dose of peripherally infused EGFRvIII-directed CAR T cells mediates antigen loss and induces adaptive resistance in patients with recurrent glioblastoma

    Donald M. O'Rourke, MacLean P. Nasrallah, Arati Desai, Jan J. Melenhorst, Keith Mansfield, Jennifer J. D. Morrissette, Maria Martinez-Lage, Steven Brem, Eileen Maloney, Angela Shen, Randi Isaacs, Suyash Mohan, Gabriela Plesa, Simon F. Lacey, Jean-Marc Navenot, Zhaohui Zheng, Bruce L. Levine, Hideho Okada, Carl H. June, Jennifer L. Brogdon, Marcela V. Maus*

    *Corresponding author. Email: mvmaus{at}mgh.harvard.edu

    Published 19 July 2017, Sci. Transl. Med. 9, eaaa0984 (2017)
    DOI: 10.1126/scitranslmed.aaa0984

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Sample stain of peripheral blood T cells for CART-EGFRvIII.
    • Fig. S2. Lack of correlation between engraftment and absolute lymphocyte count.
    • Fig. S3. Histology and CD3 immunohistochemistry stain of pre- and post-CART infusion tumor specimens.
    • Fig. S4. Validation of RNAscope ISH.
    • Fig. S5. Validation of PD-L1 staining.
    • Table S1. Individual patient characteristics.
    • Table S2. Individual product characteristics.
    • Table S3. Individual post-CART infusion events.
    • Table S4. Adverse events at least possibly related to CART-EGFRvIII.
    • Table S5. Immunohistochemical antibodies and ISH probes used.
    • Legend for table S6

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S6. Primary cytokine data (separate Excel file).

    [Download Table S6]

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