Phase I/II Clinical Trial on CD19-Chimeric Antigen Receptor (CAR) T Cells Generated with an Automated Process, from Fresh Apheresis, and Reinfused As Fresh Drug Product for Pediatric/Young Adult Patients with Relapsed/Refractory (r/r) B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL)

CAR T cells have revolutionized the management of children and young adults with r/r BCP-ALL. However, the conventional, centralized manufacturing process of commercially available drug products (DP) is time-consuming and sometimes not suitable for the clinical need of patients (pts) with rapidly progressing disease. The point of care, automated manufacturing from a fresh apheresis and the infusion of a fresh DP can reduce the waiting window and increase the therapy accessibility.

We conducted a phase I/II trial on the use of T cells transduced with a 2^nd generation (4.1BB), lentiviral vector, CD19-CAR construct (CD19-CAR_Lenti), provided by Miltenyi Biotec®, for the treatment of BCP-ALL pts with one of the following: i) 1^st relapse with either very high risk (VHR) characteristics (unfavorable cytogenetic/molecular alterations, very early relapses) or refractoriness to the 2^nd line therapy; ii) 2^nd or subsequent relapse and rapidly progressing disease preventing access to the commercially available DP. CAR-T cells were manufactured with a 12-day process using CliniMACS Prodigy®, from a fresh apheresis, after the selection of CD4⁺/CD8⁺ cells, and infused as fresh DP. All pts received lymphodepletion based on fludarabine (30 mg/sqm, days -5, -4, -3) and cyclophosphamide (500 mg/sqm, days -4 and -3). Three dose levels were tested: 1.0 (DL1), 2.0 (DL2) and 3.0 (DL3) ×10⁶ CAR⁺ T cells/kg (NCT04787263).

Nineteen pts were enrolled between 03/2021 and 04/2024, 10 males, median age 7 yrs (3-25); 13/19 (68%) were in first relapse (VHR: 11/13; refractory: 2/13). Four patients had constitutional trisomy 21, 1 pt each t(1;19), t(11;19) and t(9;22). Five patients had combined relapse (2 CNS, 3 other sites) and 12/19 (63%) had high disease burden (Schultz LM, JCO 2022).

The designed dose was successfully produced for all pts, obtaining a median of 4.2 x 10⁹ (1.39– 5.48x10⁹) total cells, with 42.9% (range: 17.5%-56.2%) transduction efficiency. Vector copy number was 0.7 (0.3-1.3).

No dose-limiting toxicities were observed in the phase I portion of the study; therefore, the RP2D was 3.0x10⁶ CAR⁺ T cells/kg. The most common toxicity was hematological, with grade 4 neutropenia occurring in all pts. Cytokine release syndrome was observed in 13/19 (68%) pts, reaching a maximum of grade 3 (Hayden PJ, 2022) in 2 children; grade 1-2 immune effector cell-associated neurotoxicity syndrome (ICANS) occurred in 2 patients and resolved spontaneously within 48h. One patient with constitutional trisomy 21 developed prolonged grade 3 capillary leak syndrome (CLS) late after infusion, requiring management with steroids, tocilizumab and anakinra.

CD19-CAR_Lenti cells expanded significantly in all pts, reaching a mean peak of 90.17 (+86.38 CAR+ cells/mcl). Gene-modified cells were detectable in the peripheral blood by flow cytometry >3 months after infusion in 87.5% of pts who did not receive subsequent hematopoietic stem cell transplantation (HSCT), up to 12 months in one patient, with B-cell aplasia > 6 months in 6 of them, up to 24 and 36 months (last available follow-up) in 2 pts, respectively.

All pts achieved bone marrow complete remission (CR) with negative minimal residual disease (MRD); in the extramedullary compartment, only 1 pt, with t(1;19) ALL, achieved partial response of bone localization, which progressed early after HSCT. Eleven pts (58%) received consolidation with HSCT, 4/6 (67%) were children treated at the DL1 or DL2 and 7/13 (54%) at the DL3. Overall, with a median follow-up of 18 months (range: 3-39), CR was maintained in 14/19 patients (74%), 50% having received HSCT. Importantly, all patients treated with 3.0x10⁶ CAR⁺ T cells/kg maintain CR. The 2y-EFS and OS were both 100% for patients treated DL3, even after censoring for HSCT, whereas in the whole cohort, the 2y-OS is 75% and the 2y-EFS 63% (40% with censoring for HSCT).

Our data suggest that the point-of-care, automated manufacturing of autologous, anti-CD19 CAR T cells can successfully treat patients with BCP-ALL in 1^st relapse with VHR characteristics and/or refractory to 2^nd line and patients with subsequent relapses who cannot access commercial DPs. The toxicity profile compares favorably to the common toxicities observed with commercial CAR T cells, also in a more vulnerable population of pts as those with constitutional trisomy 21. Our results underline the importance of the dose of engineered T cells administered for the long-term control of the disease.