Donor-Derived CAR T Cells Engineered with Sleeping Beauty in Pediatric and Adult Patients with Acute Lymphoblastic Leukemia Relapsed Post-HSCT
Authors: Magnani Chiara F , Gaipa Giuseppe , Lussana Federico , Gritti Giuseppe , Belotti Daniela , Napolitano Sara , Buracchi Chiara , Borleri Gian Maria , Rambaldi Benedetta , Zaninelli Silvia , Rizzuto Giuliana , Cavallaro Gianluca , Tettamanti Sarah , Cazzaniga Giovanni , Galimberti Stefania , Ferrari Silvia , Introna Martino , Balduzzi Adriana , Valsecchi Maria Grazia , Dastoli Giuseppe , Rambaldi Alessandro , Biondi Andrea 
Published: November 2021

Abstract
Introduction
Allogeneic Chimeric Antigen Receptor (CAR) T cells engineered with non-viral methods offer a modality to reduce costs and logistical complexity of the viral process and allow lymphodepleted patients to access CAR T cell treatment. We recently proposed the use of Sleeping Beauty (SB) transposon to engineer donor-derived T cells differentiated according to the cytokine-induced killer (CIK) cell protocol (Magnani CF et al. J Clin Invest. 2021). We report here outcomes on B-cell acute lymphoblastic leukemia (B-ALL) patients, relapsing after transplantation, treated with donor-derived anti-CD19 CAR T cells (CARCIK-CD19).
Methods
We conducted an academic, multi-center, phase I/II dose-escalation trial in patients relapsed after allogeneic hematopoietic stem cell transplantation (HSCT). The infusion product was manufactured in-house starting from 50 mL of peripheral blood from the HSCT donor by electroporation with GMP-grade plasmids. All patients underwent lymphodepletion with Fludarabine (30 mg/m 2/day x 4 days) and Cyclophosphamide (500 mg/m 2/day x 2 days), before proceeding to CARCIK-CD19 infusion. We used the Bayesian Optimal Interval (BOIN) design to define a four-dose escalation scheme. Primary objectives were to define the Maximum Tolerated Dose (MTD), safety, and feasibility. Secondary objectives included the assessment of complete hematologic response (CR), duration of response (DOR), progression-free (PFS), event-free (EFS), and overall survival (OS). This study was registered at ClinicalTrials.gov, NCT03389035.
Results
From January 2018 to June 2021, a total of 32 patients were screened, 26 enrolled (6 children and 20 adults) and 21 infused (4 children and 17 adults). Reasons for not receiving infusion included consent withdrawal (N=1), disease progression not controlled by bridging therapy (N=3), acquisition of myeloid phenotype (N=1). The median number of prior therapies was 4 (range, 1-7) with a median time interval from HSCT to relapse of 9 months. The median BM blasts was 60% (range, 5-100%) at enrollment and 7% (range, 0-96%) post lymphodepletion. Of the 21 patients infused, CARCIK-CD19 were obtained by HLA-identical sibling (n=6, 29%), matched unrelated (n= 7, 33%), and haploidentical donors (n=8, 38%). Three patients (14%) received the first dose level of 1x10 6 CARCIK-CD19 cells/Kg, three (14%) the second of 3x10 6, and three (14%) the third of 7.5x10 6 whereas 12 patients (57%) received the fourth and last planned dose level of 15x10 6 cells/Kg, as no dose limiting toxicity (DLT) was observed. CRS was observed in six patients (three grade I and three grade II) and immune effector cell-associated neurotoxicity in two patients at the highest dose. Although 9 out of 21 had experienced acute or chronic graft-versus-host disease (GvHD) after the previous HSCT, secondary GvHD was never induced by CARCIK-CD19. Complete response was achieved by 13 out of 21 patients (61.9%, 95%CI=38-82%) and by 11 out of 15 patients treated with the 2 highest doses (73.3%, 95%CI=45-92%). Eleven of these responders were MRD-negative. Notably, the type of donor did not influence the achievement of CR 28 days post-infusion. At a median follow up of 21.6 months (range, 1.0-38.4 months), 10 patients (47.6%) are alive in CR (9 in the 2 highest dose levels). Overall, the median OS and EFS were 9.7 and 3.2 months, respectively, with a median DOR of 4.0 months (range, 1.0-23.5 months). Patients in CR at 28-days had a 6-months relapse-free survival of 48.4% (SE=14.9). EFS at 6 months was 26.5% (SE=9.9) and OS was 67.6% (SE=11.1). Among the 13 patients who achieved CR, two children underwent consolidation with a second allo-HSCT in complete remission. Adult patients did not receive any additional anti-leukemic therapies unless a relapse occurred, and four of them remained in remission and alive (+24, +9, +6, and +4 months). Robust CARCIK-CD19 cell expansion was achieved in most patients and CARCIK-CD19 cells were measurable for up to 22 months.
Conclusions
SB-engineered CAR T cells induce sustained responses in B-ALL patients relapsed after HSCT irrespective of the donor type and without severe toxicities.

Disclosures
Lussana: Incyte: Honoraria; Pfizer: Honoraria; Astellas Pharma: Honoraria; Amgen: Honoraria. Gritti: Takeda: Consultancy; Roche: Consultancy; Kite Gilead: Consultancy; IQvia: Consultancy; Italfarmaco: Consultancy; Clinigen: Consultancy. Biondi: Incyte: Consultancy, Other: Advisory Board; Bluebird: Other: Advisory Board; Novartis: Honoraria; Amgen: Honoraria; Colmmune: Honoraria.