Donor-derived CARCIK-CD19 cells engineered with Sleeping Beauty transposon in acute lymphoblastic leukemia relapsed after allogeneic transplantation

Abstract

Non-viral engineering can ease CAR-T cell production and reduce regulatory and cost requirements. We utilized Sleeping Beauty transposon to engineer donor-derived anti-CD19.CD28.OX40.CD3zeta T cells differentiated in cytokine-induced killer (CARCIK-CD19) for B-cell precursor acute lymphoblastic leukemia (BCP-ALL) patients relapsed after allogeneic hematopoietic stem cell transplantation (alloHSCT). We report the results of CARCIK-CD19 observed in 36 patients (4 children and 32 adults) treated according to the final recommended dose. Cytokine release syndrome of grade 2 or lower occurred in 15 patients, ICANS grade 2 in 1 patient, and late-onset peripheral neurotoxicity of grade 3 in 2 patients. GVHD never occurred after treatment with allogeneic CARCIK-CD19. Complete remission was achieved by 30 out of 36 patients (83.3%), with MRD negativity in 89% of responders. With a median follow-up of 2.2 years, the 1-year overall survival was 57.0%, and event-free survival was 32.0%. The median duration of response at 1 year was 38.6%. CAR-T cells expanded rapidly after infusion and remained detectable for over 2 years. Integration site analysis after infusion showed a high clonal diversity. These data demonstrated that SB-engineered CAR-T cells are safe and induce durable remission in heavily pretreated patients with BCP-ALL relapsed after alloHSCT. Trial registration: The phase 1/2 and phase II trials are registered at www.clinicaltrials.gov as NCT#03389035 and NCT#05252403.

Fig. 1. Outcomes after CARCIK-CD19 infusion.

A OS, and EFS, in the 36 patients who received CARCIK-CD19 at the recommended dose. Kaplan–Meier estimates of OS and EFS. EFS was estimated censoring at treatment shift and all estimates are reported with the corresponding SE. B DOR in the 30 patients who received CARCIK-CD19 and achieved CR. Kaplan–Meier estimates of DOR. DOR was estimated censoring at treatment shift and all estimates are reported with the corresponding SE. C OS among 34 patients who received CARCIK-CD19 according to number of prior alloHSCT and bone marrow burden post-lymphodepletion.

Fig. 2. CAR-T cell expansion and persistence.

A CAR-T cells in peripheral blood by flow cytometry. B–C AUC-d28 (B) and Cmax-d28 (C) according to the severity of CRS. D AUC-d28 in patients who experienced CR or NR after CAR-T cell treatment. E AUC-d28 in patients with BM blasts post lymphodepletion less than or higher than 5%. F AUC-d28 in patients treated with CAR-T cells manufactured from identical sibling donors (ISD), haploidentical donors (Haplo), and matched unrelated donors (MUD). G Normal B-cell and CAR-T cell engraftment in the peripheral lood of patient 5 at different time points by flow cytometry. H B-cell recovery in CR and NR patients.

Fig. 3. Integration site analysis in SB-engineered CAR T cells pre and post infusion.

A Clonal abundance as percentage of genomes with a specific IS over the total genomes represented over time in the cell product (time 0) and in the peripheral blood post infusion of patients 9 and 13; ribbons connect tracked clones between two consecutive time points. Below each plot, the ten most abundant clones annotated with the closest gene are reported. B Diversity index (Shannon entropy) computed for each time points in the peripheral blood. Each line represents a different patient. C Percentage of IS located in exonic, intronic, and intergenic genomic regions overtime in the cell product, in the BM, and in the peripheral blood post infusion.