Patient characteristics
Overall, 22 B-ALL patients who relapsed after allo-HSCT, then received anti-CD19-CAR T-cell therapy were included in this study. The detailed characteristics of the patients are shown in Table 1. The median time from allo-HSCT to relapse was 13 months (IQR 7–15 months). The median proportion of leukemia cells was 44.7% (IQR 32.8–58.4%) in BM and 37.2% (IQR 25.8–45.5%) in the peripheral blood (PB) when patients were enrolled. The median percentage of donor BM chimerism at enrollment was 55.9%(IQR 48.2–71.3%). Patients had no GVHD or central nervous system disease at enrollment. Moreover, patients had not previously received blinatumomab or CD19-CAR T-cell therapy.
Clinical responses and donor chimerism analysis after anti-CD19-CAR T-cell therapy
In the first evaluation of therapy response on day 14 after anti-CD19-CAR T-cell infusion, 19 patients (19/22, 86.4%) achieved CR/CRi. Of these, 6(6/19, 31.6%) achieved CR, and 13 patients (13/19, 68.4%) achieved CRi. Seventeen patients (17/19, 89.5%) achieved minimal residual disease (MRD)-negative responses, whereas two patients were evaluated as CR/CRi with MRD-positive response (PtDSI 5,11).The other three patients were evaluated as NR owing to the progression of leukemia.
After 14 days of anti-CD19-CAR T-cell infusion, the donor chimerism in BM increased from 56.3 ± 22.9% to 99.9 ± 0.2% in the 19 patients who achieved CR/CRi. The donor chimerism in a patient evaluated as NR with extramedullary leukemia was 99.0%, whereas it was 10.7% and 29.2% in the other two patients who were evaluated as NR. Before or after anti-CD19-CAR T- cell therapy, there was no difference in donor chimerism in the DSI and DLI groups (Pbefore=0.056 and Pafter=0.828).
Maintenance therapy with DSI/DLI following the anti-CD19-CAR T-cell therapy
Of the patients achieving CR/CRi, 11 patients (Pt DSI 1-11) who had previously preserved frozen stem cells received DSI, whereas the other 8 patients (Pt DLI 1-8) received unfrozen DLI maintenance therapy. Only one patient (Pt DLI 7) received a second allo-HSCT after DLI. In the DSI group, six patients received one DSI treatment, whereas five received two DSI treatments. In the DLI group, three patients received one DLI treatment, whereas four patients received DLI twice and one patient received DLI thrice. As all patients received DSI/DLI for different times, we only compared the changes in various indicators after the first DSI/DLI therapy.
The median number of donor CD34+ cells infused in these patients was 1.3 (IQR 1.2–1.4)×105 cells/kg and that of CD3+ T cells was 2.2 (IQR 1.9–2.3)×107 cells/kg in DSI therapy. In the DLI group, the median number of donor CD3+ T cells infused was2.4(IQR 2.2–2.7)×107 cells/kg. There was no difference in the number of CD3+ T cell infusions between the two groups (P=0.351). The median percentage of CD19+ cells in the donor product in the DSI group was 0.02±0.03%, whereas it was 2.20±0.71% in the DLI group.
One patient who did not achieve CR/CRi with the anti-CD19-CAR T-cell therapy received DSI once (Pt 3). The number of donor CD34+ cells and CD3+ cells infused was1.4×105cells/kg and1.9×107 cells/kg, respectively.
aGVHD after anti-CD19-CAR T-cell therapy and after DSI/DLI therapy
After anti-CD19-CAR T-cell therapy, aGVHD was observed in 6 patients (6/11, 54.6%) from 19 to 48 days before DSI, and it was observed in 4 patients (4/8, 50.00%) before DLI. Only one patient (Pt DSI 5) developed grade III aGVHD, whereas no patient developed grade III-IV aGVHD in the DLI group. There was no difference in the grade of aGVHD between the two groups (P=0.732).
In the DSI group, aGVHD was observed in four patients (4/11, 36.4%) from 32 to 56 days after the first DSI. These four patients (PtDSI1, 2, 4, and 6) developed grade I-II aGVHD. Only one patient (PtDLI 3) developed grade II aGVHD after 52 days of the first DLI. There was no difference in aGVHD between the two groups after DSI/DLI therapy (P=0.636). None of the patients in the DSI and DLI groups developed grade III-IV aGVHD. Pt 3 did not develop any grade of aGVHD after DSI. None of the patients died because of aGVHD in our study.
Expansion of anti-CD19-CAR T-cell
The proportion of anti-CD19-CAR T-cell was detected by FCM at 0, 4, 7, 14, 21, 28, and 56 days after anti-CD19-CAR T-cell infusion and at 28, 42, and 56 days after the first DSI/DLI therapy (Figure 2 a). The median peak of the anti-CD19-CAR T-cell in CD3+ T cells in peripheral blood was 34.9±14.0% on day 8.3±2.7 of anti-CD19-CAR T-cell therapy in all 22 patients. In the process of anti-CD19-CAR T-cell therapy, there was no difference in the expansion peaks of anti-CD19-CAR T-cell in the DSI and DLI groups(P=0.124) (Figure2 b). From 28–56 days after DSI therapy, the expansion of anti-CD19-CAR T-cell increased again in 9 of 11 patients(Figure 2 a). The expansion peaks of anti-CD19-CAR T-cell in the DSI group were higher than those in the DLI group after DSI/DLI therapy (P<0.0001) (Figure 2 c).
In all patients with aGVHD after CAR-T cell therapy or DSI/DLI therapy, there was no difference in the expansion peaks of anti-CD19-CAR T-cell in the period of CAR-T cell therapy and in the period of DSI/DLI therapy between the DSI and DLI groups (PCAR-T=0.609 and PDSI/DLI=0.903)(Figure 2 d e).
In the period of CAR-T cell therapy, there was no difference in the expansion peaks of the anti-CD19-CAR T-cell between patients with and without aGVHD(P=0.474) (Figure 2 f). The mean peak of anti-CD19-CAR T-cell in patients with aGVHD in the period of aGVHD before DSI/DLI therapy was 5.4±3.9%, and was not higher than that the mean peak of anti-CD19-CAR T-cells in patients without aGVHD at 28 days after CAR-T cell infusion (3.2±1.4%) (P=0.128) (Figure 2 g).
Anti-CD19-CAR DNA changes in anti-CD19-CAR T-cell therapy and DSI/DLI therapy
In the two groups of patients, the copies of anti-CD19-CAR DNA were detected at 0, 4, 7, 14, 21, 28, and 56 days after CAR-T cell infusion and at 28, 42, and 56 days after the first DSI/DLI therapy (Figure 2h). In the process of anti-CD19-CAR T-cell therapy, there was no difference in the median peak of anti-CD19-CAR DNA in the DSI and DLI groups(P=0.070) (Figure 2i). From 28–56 days after the first DSI/DLI therapy, the median anti-CD19-CAR DNA peaks in the DSI group increased again after DSI therapy, but not in the DLI group (P<0.0001) (Figure 2j).
In all patients with aGVHD after CAR-T cell therapy or DSI/DLI therapy, there was no difference in the mean peak of anti-CD19-CAR DNA in the period of CAR-T cell therapy and in the period of DSI/DLI therapy between the two groups (PCAR-T=0.973 and PDSI/DLI=0.599) (Figure2 kl). In anti-CD19-CAR T-cell therapy and in DSI/DLI therapy, the mean peak of anti-CD19-CAR DNA showed the same trend as that of the mean peak of anti-CD19-CAR T-cells between patients with and without aGVHD(P=0.199 and P=0.059) (Figure 2 mn).
Cytokine levels in anti-CD19-CAR T-cell therapy and DSI/DLI
In the anti-CD19-CAR T-cell therapy, the cytokines reached their peaks at 7 to 10 days after anti-CD19-CAR T-cell infusion, and then declined from 14 to 18 days after infusion (Figure3 a).There was no difference in the peaks of IL-6 and TNF-α in the two groups in anti-CD19-CAR T-cell therapy and in the period of aGVHD before DSI/DLI therapy (Figure 3 b c). However, following DSI/DLI therapy, the IL-6 and TNF-α levels increased again in 9 of the 11 patients in the DSI group. All 4 patients who developed aGVHD after DSI therapy were included in these 9 patients. However, the levels of the two cytokines did not increase again in the DLI group after DLI (Figure 3 d).
Observation of AEs upon anti-CD19-CAR T-cell therapy and DSI/DLI therapy
In anti-CD19-CAR T-cell therapy, the clinical symptoms were similar to those reported in our previous studies[21]. These AEs resolved 14–18 days after anti-CD19-CAR T-cell infusion. There was no difference in the incidence of AEs between the two groups during this period (Table 2). In DSI/DLI therapy, at 28-56 days post DSI/DLI treatment, AEs reappeared in the two groups. There was no difference in the incidence of AEs between the two groups during this period as well (Table 2). The duration of AEs in the DSI/DLI treatment ranged from 7-15 days.
Eighteen patients who obtained CR/CRi (18/22, 81.8%) had grade 3-4 hematological toxicity after anti-CD19-CAR T-cell infusion. The other four patients only had grade 2 hematological toxicity. The hematological toxicity in all patients was recovered prior to their subsequent DSI/DLI therapy.
In anti-CD19-CAR T-cell therapy, 17 (17/22, 77.3%) patients were diagnosed with grade 0-2 of CRS, whereas 5 (5/22, 22.7%) patients were diagnosed with grade 3-4 of CRS. Only one patient in the DSI group and two patients in the DLI group developed grade 1 ICANS during this period. There were no differences in the grades of CRS and ICANS between the two groups in anti-CD19-CAR T-cell therapy (Figure 3 e f). No CRS or ICANS-related deaths were observed in our study.
Antipyretic drugs and methylprednisolone were administered to overcome AEs. Only four patients (Pt DSI 3,5 and Pt DLI 2,4) who developed grade 3-4 CRS received tocilizumab after anti-CD19-CAR T-cell therapy. None of the patients received tocilizumab after DSI/DLI therapy.
Survival after the anti-CD19-CAR T-cell therapy and DSI/DLI therapy
By November 30, 2021, 14 patients survived without leukemia. Anti-CD19-CAR T-cell therapy and DSI/DLI therapy, second allo-HSCT, response to anti-CD19-CAR T-cell therapy, occurrence of aGVHD, PFS and OS, and cause of death are shown in Figure 4a. The rates of PFS and OS at 180 days were 90.91% and 90.91%, respectively, and those at 365 days were 63.64% and 63.64%, respectively, in the DSI group. The PFS and OS rates at 180 days were 50.50% and 50.50% and those at 365 days were 12.50% and 12.50%, respectively, in the DLI group. Although the rates of PFS and OS in the DSI group were higher at 180 days, there was no difference in the rate of PFS or OS between the two groups at 180 days (PPFS=0.064 and POS=0.057). The PFS and OS rates in the DSI group were higher than those in the DLI group at 365 days(PPFS=0.010 and POS=0.009)(Figure 4 b c).Pt 1,2,and 3 did not respond to anti-CD19-CAR T-cell therapy and died of leukemia. Pt DSI 11 and PtDLI 6 died of disease recurrence with negative CD19 expression, whereas Pt DLI 5 and 8 died of disease recurrence with positive CD19 expression. Pt DSI 2 died of cytomegalovirus infection at 720 days post anti-CD19-CAR T-cell infusion and 620 days after the second DSI therapy. Pt DLI 4 died of sudden cardiac death at 83 days after anti-CD19-CAR T-cell infusion and 22 days after DLI therapy without aGVHD.