Phenotypic Composition of Commercial Anti-CD19 CAR T Cells Affects In Vivo Expansion and Disease Response in Patients with Large B-cell Lymphoma

Abstract

Purpose: In clinical trials, the expansion and persistence of chimeric antigen receptor (CAR) T cells correlate with therapeutic efficacy. However, properties of CAR T cells that enable their in vivo proliferation have still to be consistently defined and the role of CAR T bag content has never been investigated in a real-life setting.

Experimental design: Residual cells obtained after washing 61 anti-CD19 CAR T product bags were analyzed to identify tisagenlecleucel/Tisa-cel and axicabtagene ciloleucel/Axi-cel phenotypic features associated with postinfusion CAR T-cell in vivo expansion and with response and survival.

Results: While Tisa-cel was characterized by a significant enrichment in CAR+CD4+ T cells with central memory (P < 0.005) and effector (P < 0.005) phenotypes and lower rates of CAR+CD8+ with effector memory (P < 0.005) and naïve-like (P < 0.05) phenotypes as compared with Axi-cel, the two products displayed similar expansion kinetics. In vivo CAR T-cell expansion was influenced by the presence of CAR T with a CD8+ T central memory signature (P < 0.005) in both Tisa-cel and Axi-cel infusion products and was positively associated with response and progression-free survival (P < 0.05).

Conclusions: Our data indicate that despite the great heterogeneity of Tisa-cel and Axi-cel products, the differentiation status of the infused cells mediates CAR T-cell in vivo proliferation that is necessary for antitumor response.

Figure 1.

Patients’ survival. Kaplan–Meier curve showing PFS (A) and OS (B) according to the product infused. Overall, median PFS was 10.1 months, median PFS for Tisa-cel was 6.4 and not reached for Axi-cel. Overall, median OS was not reached, median OS for Tisa-cel was 19.4 and not reached for Axi-cel. According to the Log-rank test data were not statistically different.

Figure 2.

Comparison of Tisa-cel and Axi-cel infusion products phenotypes by flow cytometry. Violin plots showing the comparison of CD3⁺ rates among CD45⁺ cells (A), CD19⁺CAR⁺ rates among CD3⁺ cells (B), and CD4⁺/CD8⁺ ratios (C) between Tisa-cel (n = 29) and Axi-cel (n = 32) infusion products. Violin plots showing the comparison of frequency distribution of T central memory (T_CM), T effector memory (T_EM), T effector (T_E), and T naïve-like (T_N-like) cells gated on CD4⁺ CAR⁺ cells (D–G) and on CD8⁺ CAR⁺ cells (H–K) between Tisa-cel (n = 19) and Axi-cel (n = 27) infusion products. Exact median values are reported. P values were calculated applying the Mann–Whitney test; *, P < 0.05; **, P < 0.01 or 0.005; ***, P < 0.001; ****, P < 0.0001.

Figure 3.

In vivo CAR T-cell expansion by flow cytometry and association with infusion products phenotypes. A, Line chart of overall CAR T-cell expansion kinetics (n = 53). Violin plots showing the comparison of frequency distribution of T central memory (T_CM), T effector memory (T_EM), T effector (T_E), and T naïve-like (T_N-like) cells gated on CD4⁺ CAR⁺ cells (B–E) and on CD8⁺ CAR⁺ cells (F–I) between infusion products from expanders (n = 21) or poor-expanders (n = 18). Exact median values are reported. P values were calculated applying the Mann–Whitney test; **, P < 0.01. J and K, CAR T-cell expansion kinetics in patients receiving Tisa-cel (blue curves, N = 26) and Axi-cel (orange curves, N = 27). L, Box-and-whiskers plots showing the comparison of expansion parameters, namely concentration of CAR T cells at day 10 after infusion (C₁₀), peak concentration of CAR T cells (C_max) and magnitude of expansion by day 30 after infusion expressed as area under the curve (AUC_0–30) between patients receiving Tisa-cel or Axi-cel. Exact median values are reported. Comparison was made by the Mann–Whitney test; ns, P > 0.05, not significantly different.

Figure 4.

In vivo CAR T-cell expansion by flow cytometry and association with response and survival. A and B, CAR T-cell expansion kinetics in responders to therapy by day 90 after infusion (RE, dark green curves, n = 31) and nonresponders (NR, dark red curves, n = 22). C, Box-and-whiskers plots showing the comparison of expansion parameters, namely concentration of CAR T cells at day 10 after infusion (C₁₀), peak concentration of CAR T cells (C_max), and magnitude of expansion by day 30 after infusion expressed as area under the curve (AUC_0–30) between RE and NR. Exact median values are reported. Comparison is made applying the Mann–Whitney test; **, P < 0.01 or 0.005; ***, P < 0.001. D, Alluvial plot representing how patients are distributed into the different groups analyzed. Tisa-cel (n = 26) and Axi-cel (n = 27) patients were equally divided into expanders [14 out of 26 Tisa-cel (53.8%) and 13 out of 27 Axi-cel (48.1%)] or poor-expanders [12 out of 26 Tisa-cel (46.2%) and 14 out of 27 Axi-cel (51.9%)], whereas the majority of expanders were RE by day 90 after therapy (21 RE out of 27 expanders, 77.8%) compared with poor-expanders (10 RE of 26 poor-expanders, 38.5%). E, Kaplan–Meier curve showing PFS according to CAR T-cell expansion. Median PFS for expanders was not reached, compared with 3.7 months for poor-expanders. Comparison is made applying the log-rank test; P < 0.05.