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

Abstract

CD19-specific chimeric antigen receptor (CAR)-modified T cells have antitumor activity in B cell malignancies, but factors that affect toxicity and efficacy have been difficult to define because of differences in lymphodepletion and heterogeneity of CAR-T cells administered to individual patients. We conducted a clinical trial in which CD19 CAR-T cells were manufactured from defined T cell subsets and administered in a 1:1 CD4(+)/CD8(+) ratio of CAR-T cells to 32 adults with relapsed and/or refractory B cell non-Hodgkin's lymphoma after cyclophosphamide (Cy)-based lymphodepletion chemotherapy with or without fludarabine (Flu). Patients who received Cy/Flu lymphodepletion had increased CAR-T cell expansion and persistence, and higher response rates [50% complete remission (CR), 72% overall response rate (ORR)] than patients who received Cy-based lymphodepletion without Flu (8% CR, 50% ORR). The CR rate in patients treated with Cy/Flu at the maximally tolerated dose was 64% (82% ORR; n = 11). Cy/Flu minimized the effects of an immune response to the murine single-chain variable fragment component of the CAR, which limited CAR-T cell expansion and clinical efficacy in patients who received Cy-based lymphodepletion without Flu. Severe cytokine release syndrome (sCRS) and grade ≥3 neurotoxicity were observed in 13 and 28% of all patients, respectively. Serum biomarkers, one day after CAR-T cell infusion, correlated with subsequent sCRS and neurotoxicity. Immunotherapy with CD19 CAR-T cells in a defined CD4(+)/CD8(+) ratio allowed identification of correlative factors for CAR-T cell expansion, persistence, and toxicity, and facilitated optimization of lymphodepletion that improved disease response and overall and progression-free survival.

Conflict of interest statement

CT and CB receive research funding from Juno Therapeutics and hold patents. DM receives research funding from Juno Therapeutics. SR receives research funding from and is a co-founder of Juno Therapeutics, and holds patents. MH holds a patent. DL is an employee of and has equity in Juno Therapeutics. FHCRC receives research funding from Juno Therapeutics.

Copyright © 2016, American Association for the Advancement of Science.

Figures

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 FACS 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 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) The percentages of TN, TCM, and TEM/EMRA cells in the CD3+/CD4+ T cell population. D) The 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. AB) 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 blood as integrated transgene copies/μg DNA in 2 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 QPCR for distinct sequences located in the WPRE or Flap/EF1α regions of the lentivirus. D–E) Mean+/−SEM CD4+/EGFRt+ (D) and CD8+/EGFRt+ (E) CAR-T cell counts in 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/μg 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–B) Mean +/− SEM CD4+/EGFRt+ (A) and CD8+/EGFRt+ (B) CAR-T cell counts in 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 blood as integrated transgene copies/μg DNA in 2 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–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 months 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/L. **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 (DL) 1, DL2, or DL3. D) 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 grade ≥ 3 neurotoxicity (NT) or with grade 0–2 NT. 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 0–2 neurotoxicity. Units shown on the y-axis are as follows: cytokines (IL-6, IFN-γ, IL-15, IL-2, IL-18, TIM3, TGF-β), pg/mL; ferritin, ng/mL; CRP, mg/L. **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