Research ArticleCancer

Immunotherapy of non-Hodgkin’s lymphoma with a defined ratio of CD8+ and CD4+ CD19-specific chimeric antigen receptor–modified T cells

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Science Translational Medicine  07 Sep 2016:
Vol. 8, Issue 355, pp. 355ra116
DOI: 10.1126/scitranslmed.aaf8621
  • Fig. 1. Heterogeneity in distribution of TN, TCM, and TEM/EMRA cells within CD4+ and CD8+ T cell subsets in normal donors and patients with NHL.

    (A) Representative fluorescence-activated cell sorting plots showing the proportion of TN (CD45RA+/CD62L+), TCM (CD45RA/CD62L+), and TEM/EMRA (CD62L) subsets in the CD3+/CD4+ and CD3+/CD8+ T cell populations in the blood of an NHL patient. (B) Absolute CD4+ and CD8+ T cell counts in blood from healthy individuals (n = 10) and NHL patients (n = 30). (C) Percentages of TN, TCM, and TEM/EMRA cells in the CD3+/CD4+ T cell population. (D) Percentages of TN, TCM and TEM/EMRA cells in the CD3+/CD8+ T cell population. Comparisons of continuous variables between two categories were made using the Wilcoxon rank-sum test.

  • Fig. 2. Increased CD19 CAR-T cell expansion and persistence after Cy/Flu lymphodepletion.

    (A and B) Peak CD4+/EGFRt+ (A) and CD8+/EGFRt+ (B) CAR-T cell numbers after the first CAR-T cell infusion in relation to the percentage of CD19+ cells (normal and malignant CD19+ B cells) in the bone marrow before lymphodepletion chemotherapy for each patient. (C) CAR-T cell persistence in the blood as integrated transgene copies per microgram of DNA in two patients who received a cycle of Cy or Cy/E lymphodepletion chemotherapy and CAR-T cell infusion followed by a second cycle of Cy lymphodepletion chemotherapy and CAR-T cell infusion. Integrated transgene copies were determined by quantitative real-time fluorescence polymerase chain reaction (QPCR) for distinct sequences located in the Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) or Flap/EF1α regions of the lentivirus. (D and E) Mean ± SEM of CD4+/EGFRt+ (D) and CD8+/EGFRt+ (E) CAR-T cell counts in the blood on the indicated days after CAR-T cell infusion for patients treated with 2 × 107 EGFRt+ cells/kg after either Cy or Cy/E lymphodepletion (No Flu) or Cy/Flu lymphodepletion (Cy/Flu). (F) CAR-T cell persistence in blood of patients who received Cy or Cy/E lymphodepletion (No Flu, black; n = 9) compared to Cy/Flu lymphodepletion (red; n = 18) is shown as FlapEF1α integrated transgene copies per microgram of DNA. CAR-T cell persistence data are truncated at the time of HCT for patients who underwent autologous or allogeneic HCT after CAR-T cell infusion.

  • Fig. 3. Improved clinical responses to CD19 CAR-T cell immunotherapy after Cy/Flu lymphodepletion.

    (A and B) Mean ± SEM of CD4+/EGFRt+ (A) and CD8+/EGFRt+ (B) CAR-T cell counts in the blood on the indicated days after CAR-T cell infusion for patients treated with Cy/Flu lymphodepletion and either 2 × 105 or 2 × 106 EGFRt+ cells/kg. (C) CAR-T cell persistence in the blood as integrated transgene copies per microgram of DNA in two patients who received a cycle of Cy/Flu lymphodepletion chemotherapy and CAR-T cell infusion followed by a second cycle of Cy/Flu lymphodepletion chemotherapy and CAR-T cell infusion. (D) Probability curves showing the likelihood of response (CR/PR) according to the peak CD4+/EGFRt+ and CD8+/EGFRt+ cell counts in blood, the AUC0–28, and Cmax. P values were reported from the Wilcoxon two-sample test. (E and F) OS and PFS for patients who received Cy/Flu compared to Cy or Cy/E (No Flu) lymphodepletion followed by infusion of CD19 CAR-T cells at ≤2 × 106 EGFRt+ cells/kg. The median OS follow-up times for No Flu and Cy/Flu are 25 and 6.3 months, respectively. The median PFS follow-up for Cy/Flu is 5.8 months. The median PFS for No Flu is 1.5 months. The numbers of patients at risk at each time point are indicated. Log-rank tests were used to compare between-group differences in survival curves. HR, hazard ratio; CI, confidence interval.

  • Fig. 4. Factors correlating with toxicity after CD19 CAR-T cell therapy.

    (A) Peak counts (median and interquartile range) of CD4+/EGFRt+ and CD8+/EGFRt+ cells in blood after CAR-T cell infusion and the AUC0–28 in patients with sCRS (requiring ICU) and without sCRS. (B) Peak concentrations (median and interquartile range) of serum IL-6, IFN-γ, ferritin, and CRP after CAR-T cell infusion in patients with sCRS, with mild CRS (signs and symptoms of CRS, but not requiring ICU admission), and without CRS. Units shown on the y axis are as follows: IL-6, pg/ml; IFN-γ, pg/ml; ferritin, ng/ml; CRP, mg/liter. **P ≤ 0.01; *P < 0.05, Wilcoxon two-sample test. P values are shown in table S6. (C) Peak concentrations of serum IL-6 and IFN-γ after CAR-T cell infusion in patients treated at dose level 1 (DL1), DL2, or DL3. (D) Peak counts (median and interquartile range) of CD4+/EGFRt+ and CD8+/EGFRt+ cells in the blood after CAR-T cell infusion and the AUC0–28 in patients with grade ≥3 neurotoxicity or with grade 0 to 2 neurotoxicity. (E) Peak concentrations (median and interquartile range) of serum IL-6, IFN-γ, IL-15, IL-2, IL-18, TIM-3, ferritin, CRP, and TGF-β after CAR-T cell infusion in patients with grade ≥3 neurotoxicity and with grade 0 to 2 neurotoxicity. Units shown on the y axis are as follows: cytokines (IL-6, IFN-γ, IL-15, IL-2, IL-18, TIM-3, and TGF-β), pg/ml; ferritin, ng/ml; CRP, mg/liter. **P ≤ 0.01; *P < 0.05, Wilcoxon two sample test. P values are shown in table S6.

  • Fig. 5. Prediction of subsequent toxicity using serum biomarkers collected within 24 hours of CAR-T cell infusion.

    (A) Concentrations (median and interquartile range) of serum IL-8, IFN-γ, IL-15, IL-10, and IL-6 on day 1 after CAR-T cell infusion in patients who did or did not subsequently develop sCRS. **P ≤ 0.01; *P < 0.05, Wilcoxon two-sample test. P values are shown in table S6. (B) Concentrations (median and interquartile range) of serum IL-15, TGF-β, IL-6, IL-10, IL-8, and IFN-γ on day 1 after CAR-T cell infusion in patients who developed grade ≥3 neurotoxicity or grade 0 to 2 neurotoxicity. **P ≤ 0.01; *P < 0.05, Wilcoxon two-sample test. P values are shown in table S6. (C to E) ROC curves demonstrating the interaction between serum IL-6, IL-15, and TGF-β concentrations on day 1 after CAR-T cell infusion and the occurrence of grade ≥3 neurotoxicity. The red point indicates the cut points for each assay at which the following sensitivities and specificities were obtained: IL-6, 15.2 pg/ml (sensitivity, 78%; specificity, 81%); IL-15, 76.7 pg/ml (sensitivity, 88%; specificity, 78%); TGF-β, 25,532 pg/ml (sensitivity, 88%; specificity, 78%). Data are summarized in table S5.

  • Table 1. Patient characteristics, toxicity, and response.
    Lymphodepletion
    CategoryCy or Cy/ECy/FluTotal
    PatientsEvaluablen = 12n = 20n = 32
    Age (years)Median (range)60 (52–68)53 (36–70)58 (36–70)
    SexMale11 (92%)16 (80%)27/32 (84%)
    Female1 (8%)4 (20%)5/32 (16%)
    Prior regimens≤24 (36%)3 (15%)7/32 (22%)
    3-42 (18%)5 (25%)7/32 (22%)
    ≥56 (50%)12 (60%)18/32 (56%)
    Prior autologous HCTYes6 (50%)8 (40%)14/32 (44%)
    No6 (50%)12 (60%)18/32 (56%)
    Prior allogeneic HCTYes1 (8%)3 (15%)4/32 (13%)
    No11 (92%)17 (85%)28/32 (88%)
    HistologyDe novo aggressive B cell lymphoma4 (33%)7 (35%)11/32 (34%)
    Transformed LBCL4 (33%)7 (35%)11/32 (34%)
    Follicular lymphoma2 (17%)4 (20%)6/32 (19%)
    Mantle cell lymphoma2 (17%)2 (10%)4/32 (13%)
    ChemorefractoryYes11 (92%)18 (90%)29/32 (91%)
    No/indeterminate1 (8%)2 (10%)3/32 (9%)
    ToxicityEvaluablen = 12n = 20n = 32
    sCRS2 × 105 EGFRt+ cells/kg0/2 (0%)0/3 (0%)0/5 (0%)
    2 × 106 EGFRt+ cells/kg0/7 (0%)1/11 (9%)1/18 (6%)
    2 × 107 EGFRt+ cells/kg0/3 (0%)3/6 (50%)3/9 (33%)
    Total0 (0%)4/20 (20%)4/32 (13%)
    Grade ≥3 neurotoxicity2 × 105 EGFRt+ cells/kg0/2 (0%)1/3 (33%)1/5 (20%)
    2 × 106 EGFRt+ cells/kg2/7 (29%)2/11 (18%)4/18 (22%)
    2 × 107 EGFRt+ cells/kg0/3 (0%)4/6 (67%)4/9 (44%)
    Total2/12 (17%)7/20 (35%)9/32 (28%)
    ResponseEvaluablen = 12n = 18n = 30
    CRDe novo aggressive B cell lymphoma0/4 (0%)2/7 (29%)2/11 (18%)
    Transformed LBCL1/4 (25%)5/6 (83%)6/10 (60%)
    Follicular lymphoma0/2 (0%)2/3 (67%)2/5 (40%)
    Mantle cell lymphoma0/2 (0%)0/2 (0%)0/4 (0%)
    2 × 105 EGFRt+ cells/kg0/2 (0%)1/3 (33%)1/5 (20%)
    2 × 106 EGFRt+ cells/kg1/7 (14%)7/11 (64%)8/18 (44%)
    2 × 107 EGFRt+ cells/kg0/3 (0%)1/4 (25%)1/7 (14%)
    Total1/12 (8%)9/18 (50%)10/30 (33%)
    ORRDe novo aggressive B cell lymphoma1/4 (25%)6/7 (86%)7/11 (64%)
    Transformed LBCL2/4 (50%)5/6 (83%)7/10 (70%)
    Follicular lymphoma2/2 (100%)2/3 (67%)4/5 (80%)
    Mantle cell lymphoma1/2 (50%)0/2 (0%)1/4 (25%)
    2 × 105 EGFRt+ cells/kg2/2 (100%)1/3 (33%)3/5 (60%)
    2 × 106 EGFRt+ cells/kg3/7 (43%)9/11 (82%)12/18 (67%)
    2 × 107 EGFRt+ cells/kg1/3 (33%)3/4 (75%)4/7 (57%)
    Total6/12 (50%)13/18 (72%)19/30 (63%)

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/8/355/355ra116/DC1

    Fig. S1. Plan of CD19 CAR-T cell manufacturing.

    Fig. S2. CAR-T cell product characterization.

    Fig. S3. Anti-CAR transgene product immune response.

    Fig. S4. Tumor regression after a second CAR-T cell infusion in patients who received Cy/Flu lymphodepletion.

    Fig. S5. Delayed normalization of imaging after CD19 CAR-T cell infusion.

    Table S1. Variable CD4+/CD8+ T cell ratio in blood of NHL patients.

    Table S2. Summary of anti-CAR transgene product immune responses.

    Table S3. CRS and neurotoxicity.

    Table S4. Multivariate analyses.

    Table S5. Simultaneous and sequential testing of cytokines to predict toxicity.

    Table S6. P values for Figs. 4 (B and E) and 5 (A and B).

    Reference (39)

  • Supplementary Material for:

    Immunotherapy of non-Hodgkin's lymphoma with a defined ratio of CD8+ and CD4+ CD19-specific chimeric antigen receptor–modified T cells

    Cameron J. Turtle,* Laïla-Aïcha Hanafi, Carolina Berger, Michael Hudecek, Barbara Pender, Emily Robinson, Reed Hawkins, Colette Chaney, Sindhu Cherian, Xueyan Chen, Lorinda Soma, Brent Wood, Daniel Li, Shelly Heimfeld, Stanley R. Riddell, David G. Maloney

    *Corresponding author. Email: cturtle{at}fhcrc.org

    Published 7 September 2016, Sci. Transl. Med. 8, 355ra116 (2016)
    DOI: 10.1126/scitranslmed.aaf8621

    This PDF file includes:

    • Fig. S1. Plan of CD19 CAR-T cell manufacturing.
    • Fig. S2. CAR-T cell product characterization.
    • Fig. S3. Anti-CAR transgene product immune response.
    • Fig. S4. Tumor regression after a second CAR-T cell infusion in patients who received Cy/Flu lymphodepletion.
    • Fig. S5. Delayed normalization of imaging after CD19 CAR-T cell infusion.
    • Table S1. Variable CD4+/CD8+ T cell ratio in blood of NHL patients.
    • Table S2. Summary of anti-CAR transgene product immune responses.
    • Table S3. CRS and neurotoxicity.
    • Table S4. Multivariate analyses.
    • Table S5. Simultaneous and sequential testing of cytokines to predict toxicity.
    • Table S6. P values for Figs. 4 (B and E) and 5 (A and B).
    • Reference (39)

    [Download PDF]

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