Preferential expansion of CD8+ CD19-CAR T cells postinfusion and the role of disease burden on outcome in pediatric B-ALL

Abstract

T cells expressing CD19-specific chimeric antigen receptors (CD19-CARs) have potent antileukemia activity in pediatric and adult patients with relapsed and/or refractory B-cell acute lymphoblastic leukemia (B-ALL). However, not all patients achieve a complete response (CR), and a significant percentage relapse after CD19-CAR T-cell therapy due to T-cell intrinsic and/or extrinsic mechanisms. Thus, there is a need to evaluate new CD19-CAR T-cell products in patients to improve efficacy. We developed a phase 1/2 clinical study to evaluate an institutional autologous CD19-CAR T-cell product in pediatric patients with relapsed/refractory B-ALL. Here we report the outcome of the phase 1 study participants (n = 12). Treatment was well tolerated, with a low incidence of both cytokine release syndrome (any grade, n = 6) and neurotoxicity (any grade, n = 3). Nine out of 12 patients (75%) achieved a minimal residual disease-negative CR in the bone marrow (BM). High disease burden (≥40% morphologic blasts) before CAR T-cell infusion correlated with increased side effects and lower response rate, but not with CD19-CAR T-cell expansion. After infusion, CD8+ CAR T cells had a proliferative advantage over CD4+ CAR T cells and at peak expansion, had an effector memory phenotype with evidence of antigen-driven differentiation. Patients that proceeded to allogeneic hematopoietic cell transplantation (AlloHCT) had sustained, durable responses. In summary, the initial evaluation of our institutional CD19-CAR T-cell product demonstrates safety and efficacy while highlighting the impact of pre-infusion disease burden on outcomes. This trial was registered at www.clinicaltrials.gov as #NCT03573700.

Graphical abstract

Figure 1.

Clinical outcomes after infusion of CD19-CAR T cells. (A) Swimmers plot depicting the clinical course for each patient, with each lane representing a single patient. Patient 6: the indication for a third CAR T-cell reinfusion was a loss of BCA; the patient achieved BCA again after infusion. (B) EFS and OS of the entire patient cohort (n = 12). EFS was defined as the time from CAR T-cell infusion to NR at 4 weeks after CAR T-cell infusion, relapse, or death, with censoring at the time of the last follow-up. OS was defined by the time from CAR T-cell infusion to death, censoring at the time of the last follow-up. EFS and OS rates were estimated by the Kaplan-Meier method and compared by the log-rank test. (C) Duration of BCA among patients achieving a CR after CAR T-cell therapy. (D) Graphical presentation of outcome and observed toxicities, including disease response in the BM at 4 weeks after CAR T-cell infusion, the highest grade of CRS/NTX, and presence of carHLH. Patients are grouped based on the level of leukemic disease in the BM before CAR T-cell infusion (high: ≥40% morphologic blasts; low: <40% morphologic blasts).

Figure 2.

Plasma chemokine, cytokine, and growth factor levels are influenced by disease burden, lymphodepleting chemotherapy, and CRS/ICANS. (A) Day −5 (before lymphodepleting chemotherapy), t test, ∗P < .05 and ∗∗P < .01. (B and C) Change in plasma chemokine, cytokine, and growth factor levels before and after lymphodepleting chemotherapy. (B) Individual chemokine, cytokine, and growth factor levels, t test, ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001. (C) Sum of all chemokine, cytokine, and growth factor levels (CytoSum), t test, ∗P < .05. (D) Principal component analysis. (E) Loess plots comparing plasma chemokine, cytokine, and growth factor levels in patients with or without CRS and/or carHLH.

Figure 3.

Expansion and persistence of infused CD19-CAR T cells does not correlate with disease burden or cell dose. CAR T-cell expansion was determined by qPCR for the CD19-CAR transgene. CAR expansion based on (A) high (≥40% morphologic blasts in the BM) and low disease burden or (B) cell dose (DL1, dose level 1 [1 × 10⁶ CAR⁺ T cells per kg]; DL2, dose level 2, 3 × 10⁶ CAR⁺ T cells per kg]). (C and D) Peak expansion is stratified for disease burden or cell dose. (E and F) The area under the curve analysis for the first 28 days after the infusion was stratified for disease burden or cell dose. (G and H) The ratio of qPCR for the CD19-CAR transgene and the TRAC locus at 4 weeks after infusion in PB, BM, and CSF stratified for disease burden or cell dose. For patient 12 (low disease burden), qPCR data were only available until day 7 after CAR T cell infusion due to coronavirus disease 2019 (COVID-19); qPCR data are shown in panels (A and B) but not included in the analysis; t test. ns, not significant. ∗∗P < .01 and ∗∗∗P < .001.

Figure 4.

CD8-positive CD19-CAR T cells preferentially expand in vivo and have an effector memory phenotype. The phenotype of CAR-positive and CAR-negative T cells was determined by flow cytometry in the infused cell product and at peak expansion (week [wk] 1 or 2 after infusion); n = 9 (patients 2 to 6, and 8 to 11). (A-D) Comparison of CAR-positive T-cell phenotype in cell product and at peak expansion (paired t test for CD4⁺/CAR⁺ GMP vs Peak and CD8⁺/CAR⁻ GMP vs Peak. ns: not significant. ∗P < .05, ∗∗P < .01, and ∗∗∗P < .01). (A) Percent CD4-positive and CD8-positive CAR T cells. (B) Percent naïve (TN), central memory (TCM), effector memory (TEM), and effector memory T cells reexpressing CD45RA (TEMRA) T cells. (C) Percent of transitional memory (Ttm) T cells. (D) Percent of PD1⁺ and TIM3⁺ CAR-positive T cells. (E-G) Comparison of CAR-positive and CAR-negative T-cell phenotype at peak expansion (paired t test for CD4⁺/CAR⁺ vs CD4⁺/CAR⁻ and CD8⁺/CAR⁺ vs CD8⁺/CAR⁻. ∗P < .05 and ∗∗P < .01): (E) TN, TCM, TEM, and effector memory T cells reexpressing CD45RA (TEMRA) T cells. (F) Percent of Ttm T cells. (G) Percent PD1- and/or TIM3-positive T cells.