Evidence of long‐lasting anti‐CD19 activity of engrafted CD19 chimeric antigen receptor–modified T cells in a phase I study targeting pediatrics with acute lymphoblastic leukemia

Abstract Ninety percent of relapse/refractory B‐cell acute lymphatic leukemia (R/R B‐ALL) patients can achieve complete remission (CR) after CD19‐targeting chimeric antigen receptor T (CAR‐T) cell therapy. However, around 50% of them relapse in 1 year. Persistent CAR‐T cell engraftment is considered as the key to remain durable remission. Here, we initiated a phase I study to treat 10 pediatric B‐ALL patients using a CD19‐targeted second generation CAR with a 4‐1BB intracellular costimulatory domain. All patients received a standard fludarabine and cyclophosphamide (FC) preconditioning regiment, followed by a CAR‐T infusion with a median number of 0.5 (0.3‐1.58) × 106 CAR+ T cells/kg. The pretreatment tumor burdens were high with a median bone marrow (BM) blasts percentage of 59.2% (7.31%‐86.2%), excluding one patient only with brain infiltration of leukemia cells (0% BM blasts). The initial CR rate was 80% (n = 8/10). Four patients (40%) experienced serious (grade > 2) cytokine release syndrome (CRS) and three patients (30%) with obvious neurotoxicity. Monthly assessments of CD19+ minimal residual disease (MRD) and CAR‐T engraftment demonstrated the anti‐CD19 activity of long‐term engrafted CAR‐T cell clones in one patient for more than 2 years.


| INTRODUCTION
Since the first report of genetically engineered T cell with chimeric antigen receptor (CAR-T), CAR-T has become the hope for relapsed/refractory patients, especially for those with hematologic malignancy. 1 To date, more than 130 ongoing registered clinical trials are recruiting patients to investigate the safety and efficacy of variety design of CD19 CAR-T cells (clinicaltrial.gov). Several groups have reported extended regressions of B-cell malignancies in patients receiving infusions of anti-CD19 CAR-T cells at different ages. [2][3][4][5] Recent studies indicated high initial remission rates of B-cell acute lymphoblastic leukemias (B-ALL) in adults with response rate ranging Futian Ma and Jin-Yuan Ho contributed equally to the study. from 83% to 93%. [5][6][7] In contrast, children's response rate was lower than that of adults, expanding from 68% to 90%. 1,8,9 Although adults with B-ALL display 10% higher relapse rates compare with pediatric patients and experience long-term event-free survival of less than 50%, it is reported that approximately 25% of pediatric patients have a relapse with CD19 negative or CD19 low and resulted in a considerable number of all childhood cancer deaths. 10,11 Thus, developing efficient and molecular-targeted approaches to prolong the life-span of B-ALL children has become a vital priority in recent years. CAR-T cells targeting CD19 has successfully shown tremendous potential for Bcell lineage malignancies. 2,4,5,12 Here, we report the safety, efficacy, and correlative studies of CD19 CAR-T cells therapy in 10 pediatric patients in China.

| Design of CD19 CAR-T cells and patient enrollment
The sequence of CD19 chimeric antigen receptor is composed by CD19 scFv, CD28-hing, 4-1BB costimulatory region, and CD3ζ activation domains ( Figure 1A). T cells were activated by CD3/CD28 F I G U R E 1 The structure feature of CAR construct (A) including VH, heavy chain variable domain; VL, light chain variable domain, hinge, transmembrane domain, 4-1BB and CD3 ζ. (B) The screen, enrollment criteria, and the treatment microbead 2 days before CD19-CAR lentivirus infection. After transduction, the T cells were expanded for another 10 days before formulation and infusion. The detail of manufacturing of Senl-B19 was outlined in Figure S1.

| Demographics and baseline characteristics
Patients with relapsed or refractory B-ALL in Hebei Second Provincial People Hospital were recruited in the phase I/II clinical trial of CD19 CAR-T cell treatment (NCT02963038). The enrollment procedure is outlined in Figure 1B. Patients' characteristics are summarized in Table 1. The median age was 6.5 (range 3-13 y); none of them has prior hematopoietic stem cell transplant. All patients have experienced at least three courses of chemotherapy prior CAR-T infusion (median 4, range 3-10 times). Mean bone marrow (BM) blast was 40.8% (range 0% to 86.2%) at the first day (day −5) of standard preconditioning regiment (fludarabine 25 mg/m 2 ; cyclophosphamide 900 mg/m 2 ). All patients were carefully observed in the hospital for the first month to monitor the adverse effect and efficacy. Complete blood counts and cytokine concentration were examined at different time course, including day −1 to 0, day 1, day 4 or 5, day 7, day 10 or 11, day 14, day 21, and day 28. Afterwards, levels of CAR-T cells and residual leukemia cells in BM were analyzed monthly.

| Safety and efficacy
The median percentage of BM blasts (prior treatment) was 59.2% (7.31%-86.2%), excluding one who only with brain infiltration of leukemia cells (0% BM blasts). Patients were infused with various doses of CD19 CAR-T cells as shown in Table 2. The average dose of treatment was 0.71 × 10 6 cells/kg (range 0.3-1.58 × 10 6 cells/kg), and the average transfection rate was 32.0% ± 24.7%. Most patients received one infusion, except patient 21 infused twice. Cytokine release syndrome (CRS) is the major risk for patients receiving CAR-T cell therapy especially in hematologic malignancy. 13 In this report, six patients encountered grade 1/2 CRS, three patients with grade 3 CRS, and one with grade 4 ( Table 2). In addition, central nervous system (CNS) neurotoxicity also occurred in three patients (pts 4, 34, and 68). Toxicities were managed by supportive care ± tocilizumab and/or dexamethasone depending on physicians' instruction. Thirteen cytokines were examined, and six of them were with significantly elevated concentration along with CAR-T cell expansion. The dynamics of cytokine release (TNF-α, INF-γ, and IL-6) were summarized in Figure 2A. The peak of CAR-T cell expansion was around day 7 to day 10 and rapidly decreased within 5 to 10 days ( Figure 2B). Some patients revealed different adverse effects, which were summarized in Table S1.  The composition of infused CAR-T cells was further analyzed (Table S2). The average of CD3+, CD4+, and CD8+ T cells was 97.7%, 32.4%, and 66.8%, respectively ( Figure 2D). The expression of PD1 was around 0.24% to 2% (not available for pts 3, 9, and 57), which suggested the activity of infused T cells. Further analysis of the absolute number of CAR-T cells and CD4/CD8 ratio ( Figure 2E,F) revealed that the T cell population of most patients were CD8 dominant.

| Overall efficacy and CRS response
Three patients (pts) with more than 70% blast in BM receiving 0. CRS is suggested to be correlated with the blast amount. However, the dosages of infusion were sometimes adjusted to reduce the chance of severe CRS; thus, we did not observe a strong correlation between BM blast and CRS degree ( Figure S3). The peak concentration of IFN-γ and IL-6, but not IL-8 or IL-18, was slightly correlated with the degree of CRS ( Figure S4). In addition, we found that the

| Relapse and long-term survival
In this report, four patients were relapsed around 3 to 4 months after infusion. According to their last results of flow cytometry analysis and qPCR quantitation before relapse, the detection of CAR DNA suggested the persistence of CD19 CAR-T cells against CD19+ leukemia. The blast phenotypes of patients 4 and 44 were CD19-/CD22-; for patient 19, it was CD19-/CD22+; and for patient 26, it was a myeloid lineage relapse.
On the other hand, we analyze potential factors that may correlate to long-term survival from many aspects. We found two patients with long-term survival were happened to be high CD4 ratio ( Figure 2E were CD19 loss that suggests a strong correlation between the incidence of CD19-negative relapse and longer BCA duration. However, there were still 25% relapsed patients (2/8) who lost BCA in 2 months after CAR-T therapy were CD19 negative. Therefore, other tumor escape mechanisms besides sustained functional persistence of CAR-T cells cannot be excluded. The absence of subsequent allo-SCT may also increase the chances of antigen-loss relapse. In our study, no CR patients received further therapy including allo-HCT that might potentially lead to a high rate of CD19-negative relapse.
To be noted, pt 3 is still under CR with BCA for more than 2 years.
The persistence of CAR-T cells and the retainment of antileukemia functionality were detected as long as 2 years after treatment (Figure 4). Despite the long duration of BCA, we have not observed obvious serious adverse effects correlated with BCA with a closely following up during the past 2 years. During the time, and long-term persistence of functional CAR-T cells were detected as long as 2 years after treatment, which is consistent with previous speculate. She only encountered one pneumonia and stayed in the hospital for 10 days.
There were no other complications within all these days. On contrary, the other three sustained remission patients lost BCA at 2 to 4 months after CAR-T infusion ( To sum up, our study validates the safety and efficacy of CAR-T cell therapy targeting CD19 in 10 pediatric patients, which encourage us to explore more patients with relapse/refractory B-ALL in the future. Importantly, antileukemia activity lasting for more than 2 years demonstrates the maintenance of durable remission could be achieved by only single shot of CAR-T therapy.

| Generation of the lentiviral construct and package of lentivirus
The  And then the CAR-T cells were transferred to a culture bag on the 6th day and expanded in it for another 10 to 12 days before infusion.

| Cytokine measurement
The

| Statistics
We used descriptive statistics (means and standard deviations or median and ranges) to summarize the data. The relationships between overall survival time and the ratio of CD4+CAR-T/CD8+CAR-T as well as cytokines (IL-8, IL-18, and MCP-1) and the blast percentage in BM, respectively, were analyzed by linear regression models using the generalized estimating equations method. Pearson correlation was used to test the statistical relationship between two variables. Data were plotted using OriginPro7.5. The survival curve and progression-free survival (PFS) were determined by the Kaplan-Meier method. P value < .05 was considered to be statistically significant.

ACKNOWLEDGEMENT
Financial support for this work was provided by Hebei Senlang Bio.