Background: The Phase Ib/II FELIX study (NCT04404660) evaluated obe-cel, a CD19-directed autologous CAR T-cell therapy with a fast off-rate binding domain designed to improve persistence and minimize severe immunotoxicity, in adults with relapsed/refractory B-cell acute lymphoblastic leukemia. Patients achieved a high overall remission rate (ORR) with low incidence of Grade ≥3 cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome. While CAR T-cell therapy has been used for over a decade, the association between specific drug product (DP)/leukapheresis product (LP) phenotypes and clinical outcomes remains poorly characterized. This analysis investigated how DP phenotypes correlate with treatment outcomes following infusion with obe-cel.

Methods: Phenotypic markers of memory, activation, and exhaustion were assessed via an 18-parameter flow cytometry panel in CD3+, CD4+, or CD8+ CAR+ cells from LP (47 phenotype features) and DP (48 phenotype features) samples in FELIX. Memory subsets were defined by CCR7/CD45RA expression; activation by CD25 and HLADR; and exhaustion by LAG3, TIM3, PD-1, or TIGIT positivity. Univariate Cox regression analysis (UVA) in the Phase Ib discovery cohort (n=16) was used to identify OS-associated features (p<0.1), which were validated in the Phase II validation cohort (n=107) and tested for association with event-free survival (EFS) and ORR, in the combined cohort (n=123). Multivariate analysis (MVA) models of the combined cohort were used to evaluate features associated with outcomes after adjustment for key clinical covariates that were previously described in Park et al. EHA 2025. The OS MVA model included: Philadelphia chromosome status, prior inotuzumab ozogamicin (InO), bone marrow blasts at lymphodepletion, and CAR T-cell persistence (time-varying). The EFS MVA model substituted prior InO with the number of prior lines of therapy and prior stem cell transplant. All continuous features were scaled and centered, therefore odds ratios (OR) and hazard ratios (HR) reflect one standard deviation from the mean.

Results: In the DP samples, 15 phenotypic features were associated with OS in the discovery cohort (p<0.1) and nine were confirmed in the validation cohort (p<0.1).The validated features associated with longer OS included: a higher percentage of central memory cells (Tcm; in CD3+, CD4+, and CD8+ cells), one T-cell activation phenotype (CD25+ HLADR+ in CD8+ cells), and four exhaustion phenotypes (measured by combinations of TIM3, PD-1 and LAG3). Having a higher percentage of terminal effector memory (TEMRA) T-cells in the DP was associated with shorter OS. None of the nine phenotypic features validated in the DP samples were also validated in the LP samples. In the LP samples (n=122), only the expression of CD25+ HLADR+ in CD4+ cells was associated with shorterOSin both the Phase Ib and II cohorts (UVA). In the UVA of the combined cohort (n=123), having a higher Tcm percentage in CD4+ cells in the DP was significantly associated with improving EFS (HR: 0.735, p=0.035) and ORR (OR: 2.061, p=0.026). A higher Tcm percentage in CD4+ cells in the DP was also an independent predictor of longer OS (HR: 0.673, p=0.008) when adjusted for previously identified key clinical covariates. In the LP, cell phenotypes only showed a weak correlation with those in the DP (median Spearman's rho: 0.173, range: –0.033–0.594). A higher percentage of CD25+ HLADR+ CD4+ cells in the LP independently predicted worse clinical outcomes (EFS HR: 1.510, p=1.54x10-6; OS HR: 1.248, p=0.008) after adjusting for key clinical covariates.

Conclusions: A higher Tcm percentage in the DP samples was an independent predictor of positive clinical outcomes, including OS, following obe-cel infusion. While the T-cell phenotype composition in the LP was weakly correlated with that in the DP, CD25+ HLADR+ CD4+ cells in the LP independently predicted less favorable clinical outcomes. However, other factors (e.g. tumor characteristics) are also likely to affect outcomes; therefore, further investigations are needed to better understand and predict favorable clinical outcomes, and to potentially guide studies of additional cell manipulations during CAR T-cell manufacturing.

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2025
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