A B S T R A C T Cytotoxic chemotherapy and radiation can render lymphocyte repertoires qualitatively and quantitatively defective. Thus, heavily treated patients are often poor candidates for the manufacture of autologous chimeric antigen receptor (CAR)-T cell products. In the United States and Europe, children with high-risk neuroblastoma undergo apheresis early in the course of treatment to collect peripheral blood stem cells (PBSCs) for cryopreservation in preparation for high-dose chemotherapy followed by autologous stem cell rescue. Here, we investigate whether these cryopreserved chemotherapy and granulocyte colony-stimulating factor (G-CSF)-mobilized PBSCs can serve as starting material for CAR-T cell manufacturing. We evaluated T cell precursor subsets in cryopreserved PBSC units from 8 patients with neuroblastoma using fluorescent activated cell sorting-based analysis. Every cryopreserved unit collected early in treatment contained both CD4 and CD8 precursors with significant numbers of na€ ıve and central memory precursors. Significant numbers of Ki67 + /PD1 + T cells were detected, presumably the result of chemotherapy-induced lymphopenia and subsequent homeostatic proliferation. Cryopreserved PBSC units containing 56 to 112 £ 10 6 T cells were amenable to immunomagnetic selection, CD3 £ 28 bead activation, lentiviral transduction, and cytokine-driven expansion, provided that CD14 monocytes were depleted before the initiation of cultures. Second-and third-generation CD171 CAR + CD4 and CD8 effector cells derived from cryopreserved units displayed antineuroblastoma lytic potency and cytokine secretion comparable to those derived from a healthy donor and mediated in vivo antitumor regression in NSG mice. We conclude that cryopreserved PBSCs procured via standard methods during early treatment can serve as an alternative starting source for CAR-T cell manufacturing, extending the options for heavily treated patients.

T cells modified to express a chimeric-antigen receptor (CAR)targeting CD19 can induce potent and sustained responses in children with relapsed/refractory acute lymphoblastic leukemia (ALL). The durability of remission is related to the length of time the CAR T cells persist. Efforts to understand differences in persistence have focused on the CAR construct, in particular the co-stimulatory signaling module of the chimeric receptor. We previously reported a robust intent-to-treat product manufacturing success rate and remission induction rate in children and young adults with recurrent/refractory B-ALL using the SCRI-CAR19v1 product, a 2nd generation CD19-specific CAR with 4-1BB costimulation co-expressed with the EGFRt cell surface tag (NCT02028455). Following completion of the phase 1 study, two changes to CAR T-cell manufacturing were introduced: switching the T-cell activation reagent and omitting mid-culture EGFRt immunomagnetic selection. We tested the modified manufacturing process and resulting product, designated SCRI-CAR19v2, in a cohort of 21 subjects on the phase 2 arm of the trial. Here, we describe the unanticipated enhancement in product performance resulting in prolonged persistence and B-cell aplasia, and improved leukemia-free survival with SCRI-CAR19v2 as compared to SCRI-CAR19v1.

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Supplementary Data from Modified Manufacturing Process Modulates CD19CAR T-cell Engraftment Fitness and Leukemia-Free Survival in Pediatric and Young Adult Subjects

<div>Abstract<p>T cells modified to express a chimeric antigen receptor (CAR) targeting CD19 can induce potent and sustained responses in children with relapsed/refractory acute lymphoblastic leukemia (ALL). The durability of remission is related to the length of time the CAR T cells persist. Efforts to understand differences in persistence have focused on the CAR construct, in particular the costimulatory signaling module of the chimeric receptor. We previously reported a robust intent-to-treat product manufacturing success rate and remission induction rate in children and young adults with recurrent/refractory B-ALL using the SCRI-CAR19v1 product, a second-generation CD19-specific CAR with 4-1BB costimulation coexpressed with the EGFRt cell-surface tag (NCT02028455). Following completion of the phase I study, two changes to CAR T-cell manufacturing were introduced: switching the T-cell activation reagent and omitting midculture EGFRt immunomagnetic selection. We tested the modified manufacturing process and resulting product, designated SCRI-CAR19v2, in a cohort of 21 subjects on the phase II arm of the trial. Here, we describe the unanticipated enhancement in product performance resulting in prolonged persistence and B-cell aplasia and improved leukemia-free survival with SCRI-CAR19v2 as compared with SCRI-CAR19v1.</p></div>