Donor-Derived CD19 CAR Cytokine Induced Killer (CIK) Cells Engineered with Sleeping Beauty Transposon for Relapsed B-Cell Acute Lymphoblastic Leukemia (B-ALL)

Abstract

Background Immunotherapy using patient-derived CAR T cells has achieved complete remission and durable response in highly refractory populations. However, logistical complexity and high costs of manufacturing autologous viral products limit CAR T cell availability. Allogeneic Cytokine Induced Killer (CIK) cells, a T-cell population characterized by the enrichment of CD3+CD56+ cells, have demonstrated a high profile of safety in acute lymphoblastic leukemia (ALL) patients (Introna M et al. Biol Blood Marrow Transplant. 2017). CIK cells could be easily engineered by the non-viral Sleeping Beauty (SB) transposon for the clinical application (Magnani CF et al, Hum Gene Ther. 2018, Biondi A et al. J Autoimmun. 2017). Methods CIK cells were generated from 50 ml of donor-derived peripheral blood (PB) by electroporation with the GMP-grade CD19.CAR/pTMNDU3 and pCMV-SB11 plasmids according to the method enclosed in the filed patent EP20140192371. After lymphodepletion with Fludarabine (30 mg/m2/day) x 4 days and Cyclophosphamide (300 mg/m2/day) x 2 days, CARCIK-CD19 were infused in pediatric and adult B-cell ALL (B-ALL) patients relapsed after allogeneic hematopoietic stem cell transplantation (HSCT). The clinical trial follows a four-dose escalation scheme (1x106, 3x106, 7.5x106 and 15x106 transduced CAR+ T cells/kg) using the novel Bayesian Optimal Interval Design (BOIN). During the cell manufacturing period, bridging anti leukemic therapy from patient registration to the beginning of the lymphodepletion, was allowed. The primary endpoint was to define the Maximum Tolerated Dose (MTD) and a safety assessment. Key secondary endpoints included the assessment of complete hematologic response (CR), defined as < 5% bone marrow (BM) blasts, circulating blasts < 1%, no clinical evidence of extramedullary disease, as well as the characterization of CARCIK-CD19 persistence in PB and BM (NCT03389035). Results We manufactured eighteen batches by seeding a median of 126.8x106 allogeneicPBMCs. At the end of expansion, the mean harvesting was 6.46x109 nucleated cells (range 1.39 - 16.00x109). Manufactured cells were mostly CD3+ lymphocytes (mean 98.90% ±SE 0.30%). Of these, 43.57%±3.73% were CAR+, 47.07%±2.74% were CD56+, 80.44%±2.53% were CD8+. The quality requirements for batch release were met in 17 productions. As of the data cut-off date (July 19, 2019), 4 pediatric and 7 adult patients were infused with a single dose of CARCIK-CD19 (n=2 HLA identical sibling, n=4 MUD, n=5 haploidentical donor). The leukemic burden in the BM post lymphodepletion/pre-infusion ranged from 0% to 96%. CARCIK-CD19 were characterized by a high profile of safety in all treated patients. Toxicities reported were a grade I cytokine release syndrome and an infusion-related DMSO-associated seizure, with absence of dose-limiting toxicities, neurotoxicity and graft-versus-host disease (GvHD) in any of the treated patients. Four out of 5 patients, receiving the highest doses, achieved CR and CRi at day 28. The 3 patients in CR were also MRD- (by flow cytometry and RT-PCR) while the CRi was MRD+ and relapsed at day+49. Robust expansion was achieved in the majority of the patients as defined by detectable CAR T-cell detection (vector copy number VCN, range 4645-977992 transgene copies/ug) and flow (range 0.5-30%) in PB. The median time to peak engraftment was 14 days. The magnitude of expansion was correlated with the CD19+ burden in the BM at the time of the infusion (P value = 0.0006, R square 0.7469). CD8+ T cells represented the predominant CARCIK-CD19 T-cell subset (78.88%±5.33% d14 n=6) along with CD3+CD56+ CIK cells and CD4+ T cells to a lesser extent. The majority of CAR T cells had a central and effector memory phenotype. CAR T cells were measurable by VCN up to 6 months with a mean persistence of 70.5 ± 14.85 days (follow up ranging from 28 days to 1 year). No major difference was observed by integration analyses of the patients' PB and the cell products. The vector integration sites reflected the classical random distribution of SB without any tendency for gene dense regions. Conclusions Our ongoing phase I/II trial demonstrates that SB-engineered CARCIK-CD19 cells are able to expand and persist in pediatric and adult B-ALL patients relapsed after HSCT, with important implications for a non-viral technology. These encouraging results prompted us to expand our study. Disclosures Gritti: Autolus Ltd: Honoraria; Roche: Other: Not stated; Abbvie: Other: Not stated; Becton Dickinson: Other: Not stated. Rambaldi:Celgene: Membership on an entity's Board of Directors or advisory committees, Other: travel support, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Other: travel support, Research Funding, Speakers Bureau; Jazz: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau, travel support; Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead: Membership on an entity's Board of Directors or advisory committees, Other: travel support, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees, Other: travel support, Research Funding, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Other: travel support, Speakers Bureau; Italfarmaco: Membership on an entity's Board of Directors or advisory committees, Other: travel support, Research Funding, Speakers Bureau; Omeros: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Transposon-Based CAR T Cells in Acute Leukemias: Where Are We Going?

“…Expansion was associated with clear observations of engrafted CAR T-cell functionality and persistent B cell aplasia in the absence of life-threatening toxicities. No evidence of GvHD was found in any of the treated patients and integration site analysis of the patients' peripheral blood confirmed the unique safety of this engineering approach in terms of genotoxicity [71]. Given that our findings are based on a limited number of patients and short-term follow-up, these results should therefore be interpreted as suggestive rather than definitive.…”

“…All the GMP batches were released about 10 days after the end of production. These data demonstrate that our GMP platform is feasible and allows rapid and efficient expansion of highly potent CAR T-cells starting from easily available small amounts of PB [71].…”

Arming Immune Cells for Battle: A Brief Journey through the Advancements of T and NK Cell Immunotherapy

“…Compared with viral transduction, the SB method showed a significantly higher genomic integration profile using minicircle (MC) and SB mRNA compared to lentiviral integration in CD19-CAR-T cells [ 222 ]. Lower costs and reduced regulatory demands offer clear advantages for production of non-viral vector-based cell therapeutics under GMP, and successful clinical application has already been reported for CD19-CAR-CIK cells [ 223 , 224 ]. Furthermore, a SB-engineered signaling lymphocytic activation molecule (SLAM) family member 7 (SLAMF7)-specific T cell product for treatment of patients suffering from multiple myeloma is currently under investigation in a phase I/II clinical trial CARAMBA-1 (NCT04499339; EU Horizon 2020 project).…”

“…Furthermore, a SB-engineered signaling lymphocytic activation molecule (SLAM) family member 7 (SLAMF7)-specific T cell product for treatment of patients suffering from multiple myeloma is currently under investigation in a phase I/II clinical trial CARAMBA-1 (NCT04499339; EU Horizon 2020 project). Additionally, CIK cells can be CAR-redirected through lentiviral and non-viral transposon system [ 224 ], and can exert relevant ADCC upon incubation with clinical-grade mAbs due to CD16 expression [ 225 , 226 ].…”

Recent advances in chimeric antigen receptor natural killer cell therapy for overcoming intractable hematological malignancies

“…42 This clinical trial showed no significant side effects at doses of CD33‐CAR NK92 cells up to 5 × 10 9 per patient with a significant effect on minimal residual disease biomarkers. Magnani et al conducted a phase I/II trial of therapy using Sleeping Beauty‐engineered allogeneic donor‐derived CD19‐CAR CIK cells for relapsed B‐cell ALL patients 43 . Four pediatric and seven adult patients received a single dose of CD19‐CAR CIK cells that contained mostly CD3 + cells (47.1% CD56 + cells).…”