Humanized <scp>CD19</scp> ‐targeted chimeric antigen receptor <scp>T</scp> ( <scp>CAR‐T</scp> ) cells for relapsed/refractory pediatric acute lymphoblastic leukemia
Authors: Shiyuan Wang, Xue Wang, Chunying Ye, Hai Cheng, Ming Shi, Wei Chen, Kunming Qi, Gang Wang, Qingyun Wu, Lingyu Zeng, Zhenyu Li, Guangjun Jing, Junnian Zheng, Kailin Xu, Jiang Cao
Journal: American Journal of Hematology (2021)

Acute lymphoblastic leukemia (ALL) is the most common malignancy occurring in childhood, accounting for approximately 30% of pediatric cancers and 10%–15% of children remain refractory or experience relapse.1 Clinical trials using CD19 chimeric antigen receptor T (CAR-T) cells have shown impressive results in treatment of B-cell malignancies such as relapsed/refractory (R/R) acute lymphoblastic leukemia.2 The single-chain variable fragments of CD19 CAR in published clinical trials are primarily derived from murine FMC63 or SJ25C1 antibodies. However, clinical studies on the humanized CD19 CAR-T treatment of pediatric R/R B-ALL are currently limited. Here, we performed a pilot phase I study to assess the efficacy and safety of the infusion of autologous humanized CD19-targeted CAR-T cells (hCART19s) for the treatment of pediatric R/R B-ALL (ClinicalTrials.gov # NCT02782351). This is different from our previous study with the inclusion of patients aged from 3 to 57 years old,3 in this report, we focused on the pediatric R/R B-ALL patients with an age less than 18 years old and analyzed the prognosis after receiving the infusion of hCART19s. Meanwhile, we also performed the short-term and long-term follow-up and analyzed the related factors which might affect the prognosis. This study was sponsored and designed by the Affiliated Hospital of Xuzhou Medical University. All clinical investigations were conducted according to the Declaration of Helsinki principle. Detailed information of trail design, treatment regimen, and clinical responses and toxicities assessment is provided in supplementary methods, Appendix S1. From March 11, 2016 to April 1, 2020, we admitted 30 children with R/R ALL. After screening, 27 were recruited, of whom 24 received hCRT19s infusion and were evaluated for efficacy and safety. Three were excluded due to rapid disease progression during the preparation of CAR-T cells. Among the subjects included, there were 14 males and 10 females, and the average age of these participants was 12 years. Twenty-four children had a median of three lines (range 2–6 lines) of therapy before enrollment, and two who experienced relapse post hematopoietic stem cell transplantation (HSCT) were also included. A total of four children had been exposed to the murine CD19 CAR-T cell product but then relapsed. The proportion of bone marrow (BM) blasts exceeded 20% in eight patients before hCART19s infusion, which were regarded as having high disease burden. Two subjects were diagnosed with Philadelphia (Ph)-positive ALL due to the presence of the Ph chromosome by routine karyotype analysis. The patients' baseline characteristics are listed in Table S1. One month after hCART19s treatment, 83.3% (20/24) of patients achieved complete remission (CR) or CR with incomplete count recovery (CRi), 18 of whom obtained a minimal residual disease (MRD) negative status assessed by flow cytometry (FCM) and two of whom obtained an MRD-positive status. 16.7% (4/24) of patients had no response (NR) to hCART19s treatment. Among these assessable patients who achieved CR or CRi, the rate of CR did not differ significantly in analyses performed according to gender, age, or previous transplantation state (p > .05) (Figure S1). However, our results showed that the CR rate of patients who had low disease burden tended to be higher than that of those with high disease burden [100% (16/16) vs. 50% (4/8) p = .001 < .05] (Figure 1). Of the four children with NR, three had previously received the first infusion of the murine CD19 CAR-T cell product and then hCART19s infusion, and another one had received a single infusion of hCART19s. The CR rate was 25% for patients receiving two times of CD19 CAR-T product (1/4) and 95% for patients receiving a single infusion of hCART19s (19/20). The CR rate was significantly different between patients who received a single infusion of hCART19s and those who received more than one time of CD19 CAR-T cell infusion (p < .05) (Figure 1). Over a median of 7 days after cell infusion (range 0–14 days), 18 of 20 (90%) evaluable patients who achieved CR or CRi developed cytokine release syndrome (CRS) that lasted for a median of 9 days (range 1 to 14 days). Among these patients, 15 (83.3%) developed grade 1–2 CRS, and only three (16.7%) developed grade 3 CRS (Table S2). Cytokine release syndrome was managed according to protocol-specific guidelines as previously published,4 and the CRS-associated symptoms were relieved in all patients after valid treatment. The plasma levels of C-reactive protein (CRP) and interleukin-6 (IL-6) were increased in all children who experienced CRS, and the median peak levels were 103.05 (98.1~108) mg/L and 93.94 (3.4~5000) pg/mL, respectively. Our results also demonstrated that the peak serum levels of IL-6 and CRP in patients who developed grade 3–5 CRS (n = 3) were higher than those with grade 1 or 2 CRS (n = 15) (Figure S1). Neurotoxicity symptoms occurred in 15% (3/20) of patients within 2 weeks after cell infusion. Among them, one (5%) child had grade 1 neurologic events, and two (10%) children had grade 3 neurotoxicity. No abnormalities were detected by computed tomography and magnetic resonance imaging, and all these symptoms disappeared after symptomatic treatment. During the median follow-up duration of 471 days (range 84 to 1353 days), of the 20 evaluable patients who achieved CR or CRi, eight underwent HSCT on the condition of MRD negativity from day 31 to day 177 after hCART19s infusion. Following HSCT, six of these patients remained alive and disease-free after experiencing a series of treatment-related and/or transplant-related adverse events; one died on day 29 after transplant following serious graft versus host disease (GVHD), and the other died of primary disease recurrence (CD19-positive relapse) on day 267 after HSCT. The remaining 12 patients did not receive HSCT while in remission due to economic factors, psychological factors and so on. Among these children, three remained in the CR state at the cutoff day, nine experienced primary disease relapse before receiving additional anticancer therapy, eight of whom experienced CD19-positive relapse, and one experienced CD19-negative relapse. After a series of chemotherapies and symptomatic treatments, two patients who experienced recurrence remained in the stable disease (SD) status, and seven patients died due to unmanageable progressive disease (PD). Of the four patients with NR, one was lost to follow-up on day 65, and three died due to rapid primary disease recurrence (Figure 1). The EFS and OS rates were 60% (95% confidence interval [CI], 36 to 77) and 63% (95% CI, 55 to 91), respectively, at one year and 37% (95% CI, 18 to 58) and 42% (95% CI, 26 to 71), respectively, at three years (Figure 1). Our results showed that patients who obtained MRD− CR tended to achieve longer EFS and OS than patients who obtained MRD+ CR (p < .05) (Figure 1). On the other hand, the EFS rate of patients undergoing HSCT was significantly higher than that of patients not undergoing HSCT (p < .05), but the OS rate of patients who received bridged transplantation was similar to that of patients who did not receive bridged transplantation (p > .05) (Figure 1). On the contrary, we found that age, gender, and disease burden were not associated with EFS or OS (p > .05) (Figure S2). In this study, we focused on the safety and efficacy of humanized CAR-T cells in pediatric R/R ALL. Our results showed 83.3% patients achieved CR or CRi one month after hCART19s treatment. The disease burden and times of CAR-T infusion had significant influence on CR rate. On the other hand, only 16.7% patients experienced the signs and symptoms of grade 3 CRS and 10% had grade 3 neurotoxicity. The short-term safety and efficacy data are encouraging. When it comes to long-term follow-up assessment, the rates of EFS and OS were 60% and 63% at one year and 37% and 42% at three years, which highlight the potential application of hCART19s in the treatment of R/R pediatric B-ALL. However, the long-term follow-up assessment showed that the time of durable remission in patients that did not receive HSCT after hCART19s infusion seems to be lower than that of a murine CAR-T product.5 Differences in the dosage of CAR-T product conducted on patients, the number of patients enrolled, and the duration of follow-up may account for this difference. On the cutoff day, our results showed 10 of the 20 patients who achieved CR experienced disease recurrence, and the majority presented with CD19+ relapse, which is consistent with previous studies. But there are some studies also showed that the proportion of patients with CD19− recurrence is higher, and the main relapse mechanism is tumor escape.2 In our study, quantitative real-time PCR (qPCR) analysis demonstrated that among the CR patients, the longest existence of CAR DNA copies is 2 years (data not shown) and the CAR copy numbers of these relapsed cases were lower than the detection limit before disease recurrence (Figure 1). We speculate that the recurrence of disease might be attributed to limited CAR-T cell persistence in vivo. Although the humanized modification may have reduced the immunogenicity of the murine CAR constructs, some mouse complementarity-determining regions (CDRs) sequences were retained and may have led to immune-mediated rejection. Taking the results together, our long-term follow-up study confirmed the potent efficacy and safety of hCART19s in pediatric patients with R/R ALL. Additional patient accrual and longer follow-up are required to gain an in-depth understanding of the factors that influence the efficacy of hCART19s therapy in patients with R/R B-ALL. This work was supported by grants from the National Natural Science Foundation of China (81930005), Jiangsu Province Social Development Key Projects (BE2017639, BE2018634), Key Research & Developement Plan of Xuzhou (KC18102). The authors declare no conflict of interest. Jiang Cao, Junnian Zheng and Kailin Xu designed the research; Shiyuan Wang, Xue Wang and Chunying Ye collected data; Hai Cheng, Ming Shi, Wei Chen and Lingyu Zeng analyzed and interrupted the results; Kunming Qi, Gang Wang, Qingyun Wu and Zhenyu Li designed the figures; Jiang Cao and Shiyuan Wang wrote the manuscript. All authors critically reviewed the final draft prior to submission. The data that support the findings of this study are available from the corresponding author upon reasonable request. Appendix S1. Supporting Information Figure S1. The peak serum levels of IL-6 and CRP in patients and comparison of the CR rate according to gender, age, or previous transplantation state. Figure S2. Comparison of the rates of EFS and OS according to gender, age and disease burden. Table S1. Basic patient information Table S2. Common symptoms of CRS Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.