CAR T-cell therapy is effective for CD19-dim B-lymphoblastic leukemia but is impacted by prior blinatumomab therapy

Abstract

Tisagenlecleucel, a chimeric antigen receptor (CAR) T-cell product targeting CD19 is approved for relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL). However, the impact of pretreatment variables, such as CD19 expression level, on leukemic blasts, the presence of CD19- subpopulations, and especially prior CD19-targeted therapy, on the response to CAR T-cell therapy has not been determined. We analyzed 166 patients treated with CAR T-cell therapy at our institution. Eleven patients did not achieve a minimal residual disease (MRD)- deep remission, whereas 67 patients had a recurrence after achieving a MRD- deep remission: 28 patients with CD19+ leukemia and 39 patients with CD19- leukemia. Return of CD19+ leukemia was associated with loss of CAR T-cell function, whereas CD19- leukemia was associated with continued CAR T-cell function. There were no significant differences in efficacy of CAR T cells in CD19-dim B-ALL, compared with CD19-normal or -bright B-ALL. Consistent with this, CAR T cells recognized and lysed cells with very low levels of CD19 expression in vitro. The presence of dim CD19 or rare CD19- events by flow cytometry did not predict nonresponse or recurrence after CAR T-cell therapy. However, prior therapy with the CD19-directed, bispecific T-cell engager blinatumomab was associated with a significantly higher rate of failure to achieve MRD- remission or subsequent loss of remission with antigen escape. Finally, immunophenotypic heterogeneity and lineage plasticity were independent of underlying clonotype and cytogenetic abnormalities.

Graphical abstract

Figure 1.

Clinicopathological characteristics of the CD19⁺MRD/relapse, CD19^–MRD/relapse, and no-MRD^–-remission categories after CAR T-cell therapy. Age, prior blinatumomab treatment, and CD19 expression levels on blasts, before CAR T-cell infusion are shown. HP-4 and H-53 had false-positive MRD at day 28 assessment and remained in remission without further therapy at data cutoff.

Figure 2.

Immunophenotypic and clonotypic comparison of representative CD19⁺MRD/relapse. (A) Early reappearance of normal hematogones in the bone marrow at 3 months after infusion in HP-181 who subsequently developed CD19⁺ MRD. Normal maturation pattern of B cells with progressive acquisition of CD45 and CD19 and decrease in CD10 as they mature from stage 1 (blue) to stage 2 (teal) to stage 3 (green) hematogones. Other markers such as CD34, CD24, CD22, and CD20 (not shown) were also consistent with normal maturation pattern. Events in green also include mature T cells (CD19^– events). Pre-CAR blasts (B) and 12-month post-CAR blasts (C) from HP-6 with identical CD45 dim variable; CD22+,CD10+,CD34 dim variable; CD20 variable; CD24 bright; and CD38 dim phenotype. Immune repertoire analysis showed identical CDR3 sequences (CARRGARIVVVPAAV_RAVAKYYYYGMDVW) and identical variable region (V) joining region (J) family usage (V4-31 J6) in pre-CAR blasts (D) and 12-month post-CAR blasts (E) from HP-51.

Figure 3.

CAR T cells are effective for CD19-dim B-ALL, but there is a higher rate of failure to achieve MRD^–remission and CD19^–MRD/relapse in blinatumomab-pretreated patients. (A) Distribution of CD19 expression before CAR T-cell therapy in all patients (n = 166). (B) Proportion of remission and NR/CD19-negMRD/R among the CD19-normal/bright and CD19-dim B-ALL cases. (C) Distribution of CD19 expression among the patients previously treated with blinatumomab (blina group; n = 16). (D) Proportion of remission and NR/CD19⁻ MRD/R among patients stratified by prior blinatumomab therapy. (E) Flow cytometric data from HP-12 at relapse (2 plots on the left) and postblinatumomab pre-CAR (2 plots on the right) time points show loss of CD19 expression after blinatumomab.

Figure 4.

CAR T cells recognize very low levels of CD19. (A) Expanded populations of CD19^– precursors (also CD22⁺CD24^–CD10 variable CD34 variable) that are seen after CAR T-cell therapy. (B) Flow cytometric estimation of CD19 expression (antigen-binding capacity) in CD19^– K562 cells transduced with increasing doses of CD19 mRNA (n = 3). (C) Significant CD69 expression in CAR T cells at 0.005 μg CD19 mRNA (n = 3). (D) Significant CD137 expression at 0.1 μg CD19 mRNA (n = 3). (E) Significant killing by CAR (19-BBz CAR construct transduced) at 0.02 μg CD19 mRNA at a 10:1 effector-to-target ratio (n = 3). Student t test; *P < .05, **P < .01, ***P < .001, and ****P < .0001.

Figure 5.

Immunophenotypic and clonotypic comparison of representative CD19^–recurrence. Immunophenotype of blasts pre-CAR (A) and 3 months after CAR MRD (B) from HP-15 showed complete loss of CD19. Immune repertoire analysis of IgH variable (V) region revealed identical CDR3 sequences (CARNWNYYFDYW) and identical VJ family usage (V6-1 J-4) in pre-CAR B-ALL (C) and post-CAR CD19^– leukemia (D) from HP-78.