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.

Fig. 1

Heterogeneity in distribution of T_N, T_CM, and T_EM/EMRA cells within CD4⁺ and CD8⁺ T cell subsets in normal donors and patients with NHL. A) Representative FACS plots showing the proportion of T_N (CD45RA⁺/CD62L⁺), T_CM (CD45RA⁻/CD62L⁺), and T_EM/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 T_N, T_CM, and T_EM/EMRA cells in the CD3⁺/CD4⁺ T cell population. D) The percentages of T_N, T_CM and T_EM/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 AUC_0-28, and C_max. 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 AUC_0-28 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 AUC_0-28 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<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 0–2 neurotoxicity. **p≤0·01, *p<0·05, Wilcoxon two sample test. P values are shown in Table S6. C–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, 25532 pg/mL, sensitivity 88%, specificity 78%). Data are summarized in Table S5.