CD19-targeted T cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia

Abstract

Adults with relapsed B cell acute lymphoblastic leukemia (B-ALL) have a dismal prognosis. Only those patients able to achieve a second remission with no minimal residual disease (MRD) have a hope for long-term survival in the context of a subsequent allogeneic hematopoietic stem cell transplantation (allo-HSCT). We have treated five relapsed B-ALL subjects with autologous T cells expressing a CD19-specific CD28/CD3ζ second-generation dual-signaling chimeric antigen receptor (CAR) termed 19-28z. All patients with persistent morphological disease or MRD(+) disease upon T cell infusion demonstrated rapid tumor eradication and achieved MRD(-) complete remissions as assessed by deep sequencing polymerase chain reaction. Therapy was well tolerated, although significant cytokine elevations, specifically observed in those patients with morphologic evidence of disease at the time of treatment, required lymphotoxic steroid therapy to ameliorate cytokine-mediated toxicities. Indeed, cytokine elevations directly correlated to tumor burden at the time of CAR-modified T cell infusions. Tumor cells from one patient with relapsed disease after CAR-modified T cell therapy, who was ineligible for additional allo-HSCT or T cell therapy, exhibited persistent expression of CD19 and sensitivity to autologous 19-28z T cell-mediated cytotoxicity, which suggests potential clinical benefit of additional CAR-modified T cell infusions. These results demonstrate the marked antitumor efficacy of 19-28z CAR-modified T cells in patients with relapsed/refractory B-ALL and the reliability of this therapy to induce profound molecular remissions, forming a highly effective bridge to potentially curative therapy with subsequent allo-HSCT.

Figures

Figure 1. Rapid anti-tumor effects mediated by 19-28z T cells

A. BM aspirates pre- and post-treatment with 19-28z T cells in 2 patients with morphologic chemotherapy-refractory B-ALL. Cyclophosphamide was given at Day −1 and CD19 CAR-targeted T cells were infused on Days 1 and 2. Left panels. BM prior to CAR modified T cell therapy demonstrated predominant blast cells with an absence of normal BM precursors. For MSK-ALL04 the left panel includes an inset with 100× magnification. Middle panels are BM aspirates done shortly after 19-28z T cell infusion and is hypocellular with normal stromal elements, histiocytes, and no evidence of blasts. Right panels. By 1 to 2 months after CAR modified T cell therapy there is BM recovery with normal hematopoiesis and no evidence of abnormal blasts. B. Flow cytometry for CD19 and CD10 expression in BM pre- and post-treatment. Cells were gated on CD45+7AAD− cells.

Figure 2. Cytokine release and T cell persistence are increased in patients with high tumor burdens

A) Pre- and post-treatment serum from patients were evaluated for listed cytokines (pg/mL). * marks initiation of steroids for MSK-ALL04 and MSK-ALL05. MSK-ALL04 started dexamethasone 20 mg every 8 hours on Day 6, then held for one day, and restarted on Day 8. It was tapered off over 2 weeks. MSK-ALL05 was started on dexamethasone 20 mg every 12 hours and it was tapered off over 12 days. B) 19-28z T cells in the blood were detected by qPCR as described (8). From these results absolute 19-28z T cell counts were calculated. MSK-ALL04 is missing a time-point immediately before administration of steroids. C) Correlation between maximum IFNγ (pg/mL) and tumor burden for each patient (left-panel) and between the maximum 19-28z T cell count and tumor burden for each patient were calculated as the Spearman rank correlation coefficient (r), which is listed on both panels. Tumor burden is the number of malignant IgH clonotypes identified in the pre-treatment BM. Tumor burden, cytokines, and 19-28z T cell counts were rank-ordered to calculate the correlation coefficient. The Spearman rank correlation coefficients for tumor burden and maximum IP10 (r=0.91), IL2 (r=0.88), and IL6 (r=0.72) were also calculated.

Figure 3. Persistent fevers in patients with high tumor burden after infusion with 19-28z CAR+ T cells

The maximum temperature (°C) in a 24-hour period is noted for all patients. Days listed range from Day −1 (cyclophosphamide) to 14 days after CD19 CAR-targeted T cell infusion. The * marks Day 6 when MSK-ALL04 and MSK-ALL05 were both started on high-dose steroids. The green line marks the minimum temperature for a fever (38°C).

Figure 4. 19-28z T cells can be detected in the blood of treated patients

Within 1 week of 19-28z T cell infusion, peripheral T cells are isolated from the blood and activated ex vivo as described in Supplementary Methods and as reported elsewhere (8). Re-activated T cells were evaluated by flow cytometry for CD3 and 19-28z CAR expression. Cells displayed within the FACs plots have been gated as CD45+7AAD-.

Figure 5. MSK-ALL04 relapsed B-ALL tumor cells retain CD19 expression and sensitivity to 19-28z T cell mediated killing

A. CD19 and CD10 expression of B-ALL tumor cells from the initial diagnostic whole blood sample (left panel) and the post 19-28z relapsed sample. Displayed cells have been gated on CD45+7AAD− cells. B. Untransduced (UNT) T cells from the leukapheresis product or 19-28z T cells from the End-of-Product formulated cells were incubated with the post 19-28z relapsed B-ALL tumor cells. Effectors were incubated with tumor cells (radiolabeled with 51Cr) at a 36:1 E:T ratio for 4 hours. 51Cr release was measured and calculated as a killing efficiency as described (8).