Research ArticleLeukemia

T Cells with Chimeric Antigen Receptors Have Potent Antitumor Effects and Can Establish Memory in Patients with Advanced Leukemia

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Science Translational Medicine  10 Aug 2011:
Vol. 3, Issue 95, pp. 95ra73
DOI: 10.1126/scitranslmed.3002842
  • Fig. 1

    Schematic representation of the gene transfer vector and transgene, gene-modified T cell manufacturing, and clinical protocol design. (A) T cell manufacturing. Autologous cells were obtained via leukapheresis, and T cells were enriched by mononuclear cell elutriation, washed, and expanded by addition of anti-CD3/CD28–coated paramagnetic beads for positive selection and activation of T cells. Residual leukemic cells were depleted. The lentiviral vector was added at the time of cell activation and was washed out on day 3 after culture initiation. Cells were expanded on a rocking platform device (WAVE Bioreactor System) for 8 to 12 days. On the final day of culture, the beads were removed by passage over a magnetic field and the CART19 cells were harvested and cryopreserved in infusible medium. mAb, monoclonal antibody. (B) Clinical protocol design. Patients were given lymphodepleting chemotherapy as described, followed by CART19 infusion #1 by intravenous gravity flow drip over a period of 15 to 20 min. The infusion was given using a split-dose approach over 3 days (10, 30, and 60%) beginning 1 to 5 days after completion of chemotherapy. Endpoint assays were conducted on study week 4. At the conclusion of active monitoring, subjects were transferred to a destination protocol for long-term follow-up as per FDA guidance.

  • Fig. 2

    Sustained in vivo expansion and persistence in blood and marrow of CART19 cells. (A to D) qPCR analysis was performed on DNA isolated from whole blood (A to C) or bone marrow (BM) (D) samples obtained from UPN 01, UPN 02, and UPN 03 to detect and quantify CAR19 sequences. The frequency of CART19 cells is shown as average transgene copies (A), total calculated CART19 cells in circulation (B), or as a fraction of circulating white blood cells (WBCs) (C). (A) Copies CAR19/microgram DNA is calculated as described in Materials and Methods. (B) The total number of lymphocytes (total normal and CLL cells) versus total CART19+ cells in circulation is plotted for all three subjects using the absolute lymphocyte count from complete blood count values and assuming a 5.0-liter volume of peripheral blood. (C) % WBC is calculated as described in Materials and Methods. (D) Bulk qPCR analysis of marrow to quantify CART19 sequences. The data from patient UPN 03 in (A, C, and D) has been published in (9) and is reprinted here with permission. Each data point represents the average of triplicate measurements on 100 to 200 ng of genomic DNA, with maximal percent coefficient of variation (CV) less than 1.56%. Pass/fail parameters for the assay included preestablished ranges for slope and efficiency of amplification, and amplification of a reference sample. The lower limit of quantification for the assay established by the standard curve range was two copies of transgene per microgram of genomic DNA; sample values below that number are considered estimates and presented if at least two of three replicates generated a Ct value with percent CV for the values 15%. CART19 cells were infused at days 0, 1, and 2 for UPN 01 and 03 and at days 0, 1, 2, and 11 for UPN 02.

  • Fig. 3

    Serum and BM cytokines before and after CART19 cell infusion. (A to C) Longitudinal measurements of changes in serum cytokines, chemokines, and cytokine receptors in UPN 01 (A), UPN 02 (B), and UPN 03 (C) on the indicated day after CART19 cell infusion. (D) Serial assessments of the same analytes in the BM from UPN 03. Analytes with a greater than or equal to threefold change are indicated and plotted as relative change from baseline (A to C) or as absolute values (D). In (C) and (D), a subset of the cytokine data (IFN-γ, CXCL10, CXCL9, IL-2Rα, and IL-6) from UPN 03 have been published in (9) and are reprinted here with permission. Absolute values for each analyte at each time point were derived from a recombinant protein-based standard curve over a threefold eight-point dilution series, with upper and lower limits of quantification determined by the 80 to 120% observed/expected cutoff values for the standard curves. Each sample was evaluated in duplicate with average values calculated and percent CV in most cases less than 10%. To accommodate consolidated data presentation in the context of the wide range for the absolute values, data are presented as fold change over the baseline value for each analyte. In cases where baseline values were not detectable, half of the lowest standard curve value was used as the baseline value. Standard curve ranges for analytes and baseline (day 0) values (listed in parentheses sequentially for UPN 01, 02, and 03), all in pg/ml: IL-1Rα: 35.5 to 29,318 (689, 301, and 287); IL-6: 2.7 to 4572 (7, 10.1, and 8.7); IFN-γ: 11.2 to 23,972 (2.8, not detected, and 4.2); CXCL10: 2.1 to 5319 (481, 115, and 287); MIP-1β: 3.3 to 7233 (99.7, 371, and 174); MCP-1: 4.8 to 3600 (403, 560, and 828); CXCL9: 48.2 to 3700 (1412, 126, and 177); IL-2Rα: 13.4 to 34,210 (4319, 9477, and 610); IL-8: 2.4 to 5278 (15.3, 14.5, and 14.6); IL-10: 6.7 to 13,874 (8.5, 5.4, and 0.7); MIP-1α: 7.1 to 13,778 (57.6, 57.3, and 48.1).

  • Fig. 4

    Prolonged surface CAR19 expression and establishment of functional memory CART19 cells in vivo. (A and B) T cell immunophenotyping of CD4+ (A) and CD8+ (B) T cell subsets. Frozen peripheral blood (PB) samples from UPN 03 obtained at days 56 and 169 after T cell infusion were subjected to multiparametric immunophenotyping for expression of markers of T cell memory, activation, and exhaustion; data are displayed after biexponential transformation for objective visualization of events. (C) Functional competence of persisting CAR cells. Frozen PB samples from UPN 03 obtained at days 56 and 169 after T cell infusion were evaluated directly ex vivo for the ability to recognize CD19-expressing target cells using CD107 degranulation assays. Presented data are for the CD8+ gated population. The gating strategies for these figures are presented in fig. S2.

  • Fig. 5

    Evaluation of clinical responses after infusion of CART19 cells. (A) UPN 02 was treated with two cycles of rituximab and bendamustine with minimal response (R/B, arrows). CART19 cells were infused beginning 4 and 14 days after bendamustine only (B, arrow). The rituximab- and bendamustine-resistant leukemia was rapidly cleared from blood, as indicated by a decrease in the absolute lymphocyte count (ALC) from 60,600/μl to 200/μl within 18 days of the infusion. Corticosteroid treatment was started on day 18 after infusion because of malaise and noninfectious febrile syndrome. The reference line (dotted) indicates the upper normal limit for ALC. (B) Sequential BM biopsy or clot specimens from UPN 01 were stained for CD20. Leukemia infiltration was present before treatment was absent after treatment; normalization of cellularity and trilineage hematopoiesis were also observed. (C) Sequential CT imaging indicates rapid resolution of chemotherapy-resistant generalized lymphadenopathy. Bilateral axillary masses in UPN 01 resolved by 83 days after infusion, as indicated by arrows and circle.

  • Table 1

    Patient demographics and response. CR, complete response; PR, partial response; N/A, not available.

    Subject
    UPN
    Age/sex
    karyotype
    Previous therapiesCLL tumor burden at baselineTotal
    dose
    of CART19
    (cells/kg)
    Response day
    +30 (duration)
    BM (study day)Blood
    (study
    day)
    Nodes/spleen
    (study day)
    0165/M
    normal
    Fludarabine × four cycles
    (2002)
    Hypercellular 70% CLLN/A6.2 × 1011 to
    1.0 × 1012 CLL cells
    (day −37)
    1.1 × 109
    (1.6 × 107/kg)
    CR (11+ months)
    Rituximab/fludarabine ×
    four cycles (2005)
    2.4 × 1012 CLL cells
    (day −14)
    Alemtuzumab × 12 weeks
    (2006)
    1.7 × 1012 CLL cells
    (day −1)
    Rituximab (two courses,
    2008 to 2009)
    R-CVP × two cycles (2009)
    Lenalidomide (2009)
    PCR × two cycles (5/18/2010
    to 6/18/2010)
    Bendamustine × one cycle
    (7/31/10 to 8/1/10)
    pre-CART19
    0277/M del
    (17)(p13)*
    Alemtuzumab × 16 weeks
    (6/2007)
    Hypercellular
    >95% CLL
    2.75 × 1011
    CLL cells
    (day −1)
    1.2 × 1012 to
    2.0 × 1012 CLL cells
    (day −24)
    5.8 × 108
    (1.0 × 107/kg)
    PR (7 months)
    Alemtuzumab × 18 weeks
    (3/2009)
    3.2 × 1012 CLL cells
    (day −47)
    Bendamustine/rituximab:
    7/1/2010 (cycle 1)
    7/28/2010 (cycle 2)
    8/26/2010 (cycle 3) pre-CART19
    0364/M del
    (17)(p13)
    R-Fludarabine × two
    cycles (2002)
    Hypercellular
    40% CLL
    N/A3.3 × 1011 to
    5.5 × 1011 CLL cells
    (day −10)
    1.4 × 107
    (1.46 × 105/kg)
    CR (10+ months)
    R-Fludarabine × four cycles
    (10/06 to 1/07)
    8.8 × 1011 CLL cells
    (day −1)
    R-Bendamustine × one cycle
    (2/09)
    Bendamustine × three cycles
    (3/09 to 5/09)
    Alemtuzumab × 11 weeks
    (12/09 to 3/10)
    Pentostatin/cyclophosphamide
    (9/10/10) pre-CART19

    *UPN 02 karyotype [International System for Human Cytogenetic Nomenclature (ISCN)]: 45,XY,del(1)(q25),+del(1)(p13),t(2;20)(p13;q11.2),t(3;5)(p13;q35),add(9)(p22),?del(13)(q14q34),-14,del(17)(p13)[cp24].

    †UPN 03 karyotype (ISCN): 46,XY,del(17)(p12)[18]/44~46,idem,der(17)t(17;21)(p11.2;q11.2)[cp4]/40~45,XY,-17[cp3].

    ‡See the Supplementary Material for methods of tumor burden determination.

    • Table 2

      High-resolution characterization of residual tumor burden and normal B cells in UPN 01 and 03. Analyses were performed on DNA isolated from whole blood by high-throughput IgH immune profiling (Adaptive TcR Corp.). PB, peripheral blood; BM, bone marrow.

      Patient
      (UPN)
      TissueTime
      point
      Amount of DNA
      in PCR (ng)
      Cell equivalentsTotal productive
      sequence reads
      Total productive
      unique B cell
      clonotypes
      CLL IGH@
      CDR3
      clone reads
      Clone frequency
      (% productives)
      01PBPreinfusion1000158,730184,78624184,25699.713
      01PBDay −11000158,730408,57948407,59299.758
      01PBDay +281000158,7300000.000
      01PBDay +17650079,365285,3057,36200.000
      01BMDay +281000158,7300000.000
      01BMDay +1761000158,730202,5354,45100.000
      03PBPreinfusion2000317,46022,074,91258,23419,948,50890.367
      03PBDay −138661,2701,385,3404,5441,231,01888.860
      03PBDay +311000158,7300000.000
      03PBDay +1762000317,4600000.000
      03BMDay +311750277,7780000.000
      03BMDay +1761000158,7300000.000

    Additional Files

    • Supplementary Material for:

      T Cells with Chimeric Antigen Receptors Have Potent Antitumor Effects and Can Establish Memory in Patients with Advanced Leukemia

      Michael Kalos, Bruce L. Levine, David L. Porter, Sharyn Katz, Stephan A. Grupp, Adam Bagg, Carl H. June*

      *To whom correspondence should be addressed. E-mail: cjune{at}exchange.upenn.edu

      Published 10 August 2011, Sci. Transl. Med. 3, 95ra73 (2011)
      DOI: 10.1126/scitranslmed.3002842

      This PDF file includes:

      • Methods
      • Fig. S1. Prolonged surface CART19 expression and absent B cells in vivo in blood and marrow of UPN 03.
      • Fig. S2. Direct ex vivo detection of CART19-positive cells in UPN 01 PBMC 71 and 286 days after T cell infusion.
      • Fig. S3. Gating strategy to identify CART19 expression using polychromatic flow cytometry in UPN 03 blood specimens.
      • Table S1. Apheresis products and CART19 product release criteria.
      • Table S2. Longitudinal measurement of absolute levels for circulating cytokines/chemokines/growth factors in serum from patient UPN 01.
      • Table S3. Longitudinal measurement of absolute levels for circulating cytokines/chemokines/growth factors in serum from patient UPN 02.
      • Table S4. Longitudinalmeasurement of absolute levels for circulating cytokines/chemokines/growth factors in serumfrompatient UPN 03.
      • Table S5. Longitudinal measurement of absolute levels for marrow cytokines, chemokines, and growth factors in serum obtained from bone marrow samples from patients UPN 01, UPN 02, and UPN 03.
      • Table S6. Calculated CART19 effector/target ratios achieved in vivo.
      • Table S7. Percentage and mass of CLL in active bone marrow.
      • Table S8. Patient tumor volume in secondary lymphoid tissues.
      • Table S9. Bone marrow plasma cell percentages in patients UPN 01, UPN 02, and UPN 03.
      • Table S10. Serum immunoglobulin levels in UPN 01.
      • References

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