Research ArticleCancer Immunotherapy

CD19-Targeted T Cells Rapidly Induce Molecular Remissions in Adults with Chemotherapy-Refractory Acute Lymphoblastic Leukemia

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Science Translational Medicine  20 Mar 2013:
Vol. 5, Issue 177, pp. 177ra38
DOI: 10.1126/scitranslmed.3005930
  • Fig. 1

    Rapid antitumor effects mediated by 19-28z T cells. (A) BM aspirates before and after treatment with 19-28z T cells in two patients with morphologic chemotherapy-refractory B-ALL. Cyclophosphamide was given at day −1, and CD19 CAR-targeted T cells were infused on days 1 and 2. Left panels: BM before CAR-modified T cell therapy demonstrated predominant blast cells with an absence of normal BM precursors. For MSK-ALL04, the left panel includes an inset with ×100 magnification. Middle panels: BM aspirates done shortly after 19-28z T cell infusion and hypocellular with normal stromal elements, histiocytes, and no evidence of blasts. Right panels: By 1 to 2 months after CAR-modified T cell therapy, there is BM recovery with normal hematopoiesis and no evidence of abnormal blasts. (B) Flow cytometry for CD19 and CD10 expression in BM before and after treatment. Cells were gated on CD45+7AAD cells.

  • Fig. 2

    Cytokine release and T cell persistence were increased in patients with high tumor burdens. (A) Pre- and posttreatment serum from patients was evaluated for listed cytokines (pg/ml). The asterisk marks initiation of steroids for MSK-ALL04 and MSK-ALL05. MSK-ALL04 started dexamethasone (20 mg) every 8 hours on day 6, then held for 1 day, and restarted on day 8. Dexamethasone was then tapered off over 2 weeks. MSK-ALL05 was started on dexamethasone (20 mg) every 12 hours, and it was tapered off over 12 days. (B) 19-28z T cells in the blood were detected by quantitative PCR as described (8). From these results, absolute 19-28z T cell counts were calculated. MSK-ALL04 is missing a time point immediately before administration of steroids. (C) Correlation between the maximum IFN-γ (pg/ml) and tumor burden for each patient (left panel) and correlation between the maximum 19-28z T cell count and tumor burden for each patient were calculated as the Spearman rank correlation coefficient (r), which is listed on both panels. Tumor burden is the number of malignant IgH clonotypes identified in the pretreatment BM. Tumor burden, cytokines, and 19-28z T cell counts were rank-ordered to calculate the correlation coefficient. The Spearman rank correlation coefficients for tumor burden and maximum IP10 (r = 0.91), IL-2 (r = 0.88), and IL-6 (r = 0.72) were also calculated.

  • Fig. 3

    Persistent fevers in patients with high tumor burden after infusion with 19-28z CAR+ T cells. The maximum temperature (°C) in a 24-hour period is noted for all patients. Days listed range from day −1 (cyclophosphamide) to 14 days after CD19 CAR-targeted T cell infusion. The asterisk marks day 6 when MSK-ALL04 and MSK-ALL05 were both started on high-dose steroids. The green line marks the minimum temperature for a fever (38°C).

  • Fig. 4

    19-28z T cells could be detected in the blood of treated patients. Within 1 week of 19-28z T cell infusion, peripheral T cells were isolated from the blood and activated ex vivo as described in the Supplementary Materials and as reported elsewhere (8). Reactivated T cells were evaluated by flow cytometry for CD3 and 19-28z CAR expression. Cells displayed within the FACS plots have been gated as CD45+7AAD.

  • Fig. 5

    Relapsed B-ALL tumor cells from MSK-ALL04 retained CD19 expression and sensitivity to 19-28z T cell–mediated killing. (A) CD19 and CD10 expression of B-ALL tumor cells from the initial diagnostic whole-blood sample (left panel) and the post–19-28z relapsed sample. Displayed cells were gated on CD45+7AAD cells. (B) Untransduced (UNT) T cells from the leukapheresis product or 19-28z T cells from the end-of-product formulated cells were incubated with the post–19-28z relapsed B-ALL tumor cells. Effectors were incubated with tumor cells (radiolabeled with 51Cr) at a 36:1 effector/target ratio for 4 hours. 51Cr release was measured and calculated as a killing efficiency as described (8).

  • Table 1

    Patient characteristics and response summary. Cy, cyclophosphamide; Vinc, vincristine; Pred, prednisone; Etop, etoposide; Peg, pegylated asparaginase; Mito, mitoxantrone; CR1, first complete remission; MRD, minimal residual disease as assessed by deep sequencing (see Supplementary Materials); Allo-SCT, allogeneic stem cell transplant; FISH, fluorescence in situ hybridization. All patients were treated with cyclophosphamide before T cell infusion, either 1.5 g/m2 (MSK-ALL04, MSK-ALL05, and MSK-ALL06) or 3.0 g/m2 (MSK-ALL01 and MSK-ALL03).

    Patient
    ID*
    Age
    (years)
    FISH/cytogeneticsInitial
    therapy
    Duration
    of CR1
    Salvage
    therapy
    Disease response
    to salvage
    therapy
    Disease
    response to
    T cell therapy
    SteroidsOutcome
    MSK-ALL0166Normal
    karyotype
    Mito/Cy→
    Vinc/Pred→Cy→
    Etop/Cy
    27 weeksVinc/Pred/PegMRD+MRDNAllo-SCT
    MSK-ALL0356Normal
    karyotype
    Hyper-CVAD45 weeksInotuzumab
    ozogamicin→Vinc/
    Pred/Peg
    MRDMRDNAllo-SCT
    MSK-ALL0459t(9;11), 9p21
    deletion
    ECOG2993 (24)5 weeksVinc/PredRefractory
    disease,
    63% blasts
    in BM
    MRDYIneligible
    for Allo-SCT,
    relapse
    90 days
    MSK-ALL05589p21 deletionECOG299328 weeksHIDAC/MitoRefractory
    disease,
    70% blasts
    in BM
    MRDYAllo-SCT
    MSK-ALL0623Normal
    karyotype
    NYII (25)34 monthsModified NYII
    consolidation I (25)
    MRD+MRDNAllo-SCT

    *MSK-ALL02 patient was removed from the study before the planned T cell infusion because he deferred T cell infusion for an allo-SCT.

    †Disease status within 1 week of infusion with CD19-targeted T cells.

    ‡This patient’s T cells were harvested while in remission. All other patients listed had their T cells harvested while they had relapsed disease.

    • Table 2

      Deep sequencing for IgH rearrangements before and after CD19 CAR-targeted T cell therapy. Adaptive Biotechnologies performed multiplex PCR and deep sequencing on genomic DNA prepared from BM aspirated on the noted day (see Supplementary Materials for further detail). Malignant IgH rearrangement refers to IgH rearrangements associated with the B-ALL tumor cells. Total numbers of IgH rearrangements are derived from both malignant and nonmalignant B cells.

      Patient
      ID
      Day of
      treatment
      Total no.
      of IgH
      rearrangements
      Total no. of
      malignant IgH
      rearrangements
      MSK-
      ALL01*
      −157,48058
      5515,9250
      MSK-
      ALL03*
      −51,0840
      3000
      MSK-
      ALL04
      −42,430,0582,426,898
      112,4071,316
      241,144637
      59995,5630
      MSK-
      ALL05*
      −13,307,4943,300,732
      81,8800
      298,2700
      MSK-
      ALL06*
      −34255,301174,698
      185,4294
      391,866,8510

      *Patient has gone to allo-SCT and is off study.

      Additional Files

      • Supplementary Material for:

        CD19-Targeted T Cells Rapidly Induce Molecular Remissions in Adults with Chemotherapy-Refractory Acute Lymphoblastic Leukemia

        Renier J. Brentjens,* Marco L. Davila, Isabelle Riviere, Jae Park, Xiuyan Wang, Lindsay G. Cowell, Shirley Bartido, Jolanta Stefanski, Clare Taylor, Malgorzata Olszewska, Oriana Borquez-Ojeda, Jinrong Qu, Teresa Wasielewska, Qing He, Yvette Bernal, Ivelise V. Rijo, Cyrus Hedvat, Rachel Kobos, Kevin Curran, Peter Steinherz, Joseph Jurcic, Todd Rosenblat, Peter Maslak, Mark Frattini, Michel Sadelain*

        *Corresponding author. E-mail: brentjer@mskcc.org (R.B.); m-sadelain@ski.mskcc.org (M.S.)

        Published 20 March 2013, Sci. Transl. Med. 5, 177ra38 (2013)
        DOI: 10.1126/scitranslmed.3005930

        This PDF file includes:

        • Fig. S1. Trial schema for adult patients with relapsed B-ALL.
        • Fig. S2. Trial schema for adult patients with B-ALL in CR.
        • Fig. S3. Serum cytokine detection and correlation with tumor burden.
        • Fig. S4. Rapid hematopoietic recovery in MSK-ALL05 after infusion with 19-28z T cells.
        • Table S1. Characteristics of infused 19-28z CAR-transduced T cells; tumor burden in apheresis and BM.
        • Table S2. Adverse events.
        • Table S3. Percentage of 19-28z CAR+ T cells detected in the CD3+ T cells of the peripheral blood and of the BM (in parenthesis) of patients up to 57 days after CAR-modified T cell therapy.

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