In this global study of CAR T-cell therapy, a single infusion of tisagenlecleucel provided durable remission with long-term persistence in pediatric and young adult patients with relapsed or refractory B-cell ALL, with transient high-grade toxic effects. (Funded by Novartis Pharmaceuticals; ClinicalTrials.gov number, NCT02435849 .).

To establish a more appropriate animal recipient for xenotransplantation, NOD/ SCID/␥ c null mice double homozygous for the severe combined immunodeficiency (SCID) mutation and interleukin-2R␥ (IL-2R␥) allelic mutation (␥ c null ) were generated by 8 backcross matings of C57BL/6J-␥ c null mice and NOD/Shi-scid mice. When human CD34 ؉ cells from umbilical cord blood were transplanted into this strain, the engraftment rate in the peripheral circulation, spleen, and bone marrow were significantly higher than that in NOD/Shiscid mice treated with anti-asialo GM1 antibody or in the ␤2-microglobulindeficient NOD/LtSz-scid (NOD/SCID/ ␤2m null ) mice, which were as completely defective in NK cell activity as NOD/SCID/ ␥ c null mice. The same high engraftment rate of human mature cells was observed in ascites when peripheral blood mononuclear cells were intraperitoneally transferred. In addition to the high engraftment rate, multilineage cell differentiation was also observed.

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SummaryLifelong blood cell production is governed through the poorly understood integration of cell-intrinsic and -extrinsic control of hematopoietic stem cell (HSC) quiescence and activation. MicroRNAs (miRNAs) coordinately regulate multiple targets within signaling networks, making them attractive candidate HSC regulators. We report that miR-126, a miRNA expressed in HSC and early progenitors, plays a pivotal role in restraining cell-cycle progression of HSC in vitro and in vivo. miR-126 knockdown by using lentiviral sponges increased HSC proliferation without inducing exhaustion, resulting in expansion of mouse and human long-term repopulating HSC. Conversely, enforced miR-126 expression impaired cell-cycle entry, leading to progressively reduced hematopoietic contribution. In HSC/early progenitors, miR-126 regulates multiple targets within the PI3K/AKT/GSK3β pathway, attenuating signal transduction in response to extrinsic signals. These data establish that miR-126 sets a threshold for HSC activation and thus governs HSC pool size, demonstrating the importance of miRNA in the control of HSC function.

Globoid cell leukodystrophy (GLD; also known as Krabbe disease) is an invariably fatal lysosomal storage disorder caused by mutations in the galactocerebrosidase (GALC) gene. Hematopoietic stem cell (HSC)-based gene therapy is being explored for GLD; however, we found that forced GALC expression was toxic to HSCs and early progenitors, highlighting the need for improved regulation of vector expression. We used a genetic reporter strategy based on lentiviral vectors to detect microRNA activity in hematopoietic cells at single-cell resolution. We report that miR-126 and miR-130a were expressed in HSCs and early progenitors from both mice and humans, but not in differentiated progeny. Moreover, repopulating HSCs could be purified solely on the basis of miRNA expression, providing a new method relevant for human HSC isolation. By incorporating miR-126 target sequences into a GALC-expressing vector, we suppressed GALC expression in HSCs while maintaining robust expression in mature hematopoietic cells. This approach protected HSCs from GALC toxicity and allowed successful treatment of a mouse GLD model, providing a rationale to explore HSC-based gene therapy for GLD.

To investigate the events leading to the depletion of CD4(+) T lymphocytes during long-term infection of human immunodeficiency virus type 1 (HIV-1), we infected human CD34(+) cells-transplanted NOD/SCID/IL-2Rgamma(null) mice with CXCR4-tropic and CCR5-tropic HIV-1. CXCR4-tropic HIV-1-infected mice were quickly depleted of CD4(+) thymocytes and both CD45RA(+) naïve and CD45RA(-) memory CD4(+) T lymphocytes, while CCR5-tropic HIV-1-infected mice were preferentially depleted of CD45RA(-) memory CD4(+) T lymphocytes. Staining of HIV-1 p24 antigen revealed that CCR5-tropic HIV-1 preferentially infected effector memory T lymphocytes (T(EM)) rather than central memory T lymphocytes. In addition, the majority of p24(+) cells in CCR5-tropic HIV-1-infected mice were activated and in cycling phase. Taken together, our findings indicate that productive infection mainly takes place in the activated T(EM) in cycling phase and further suggest that the predominant infection in T(EM) would lead to the depletion of memory CD4(+) T lymphocytes in CCR5-tropic HIV-1-infected mice.

BACKGROUND Tisagenlecleucel is an FDA approved chimeric antigen receptor (CAR)-T cell therapy that reprograms T cells to eliminate CD19+ B cells. ELIANA (NCT02435849) is a phase 2 pivotal study of tisagenlecleucel in pediatric/young adult patients (pts) with CD19+ r/r B-cell acute lymphoblastic leukemia (ALL), the first global trial of a CAR-T cell therapy. The primary objective was met, with an overall remission rate (ORR) of 81% (complete remission [CR] + CR with incomplete blood count recovery [CRi]). Here we present an update of ELIANA, with additional pts and additional 11 mo follow-up from the previous report (Maude et al. N Engl J Med 2018). METHODS Eligible pts were aged ≥3 y at screening and ≤21 y at diagnosis and had ≥5% leukemic blasts in bone marrow. Tisagenlecleucel was centrally manufactured at 2 sites (Morris Plains, NJ, USA and Leipzig, Germany) by lentiviral transduction of autologous T cells with a vector encoding for a second generation 4-1BB anti-CD19 CAR and expanded ex vivo. Tisagenlecleucel was provided to pts at 25 study centers in 11 countries on 4 continents using cryopreserved apheresed mononuclear cells, central production facilities, and a global supply chain. The primary endpoint, ORR within 3 mo and maintained for ≥28 d among infused pts, was assessed by an independent review committee. Secondary endpoints included duration of remission (DOR), overall survival (OS), safety, and cellular kinetics. RESULTS As of April 13, 2018, 113 pts were screened and 97 enrolled. There were 8 manufacturing failures (8%) and 10 pts (10%) were not infused due to death or adverse events (AEs). Following lymphodepleting chemotherapy in most pts (76/79; fludarabine/cyclophosphamide [n=75]), 79 pts were infused with a single dose of tisagenlecleucel (median dose, 3.0×106 [range, 0.2-5.4×106] CAR-positive viable T cells/kg), and all had ≥3 mo of follow-up or discontinued earlier (median time from infusion to data cutoff, 24 mo [range, 4.5-35 mo]). Median age was 11 y (range, 3-24 y); 61% of pts had prior hematopoietic stem cell transplant (SCT). Among the 65 pts with CR/CRi, 64 (98%) were MRD- within 3 mo. Median DOR by K-M analysis was not reached (Figure): responses were ongoing in 29 pts (max DOR, 29 mo and ongoing); 19 pts relapsed before receiving additional anticancer therapy (13 died subsequently); 8 pts underwent SCT while in remission, 8 received additional anticancer therapy (non-SCT) and 1 discontinued while in remission. The probability of relapse-free survival at 18 mo was 66% (95% CI, 52%-77%). Median OS was not reached; OS probability at 18 mo was 70% (95% CI, 58%-79%). Cytokine release syndrome (CRS) occurred in 77% of pts (grade [G] 3/4; 48%; graded using the Penn scale); 39% of pts received tocilizumab for treatment of CRS with or without other anti-cytokine therapies; 48% of pts required ICU-level care for CRS, with a median ICU stay of 7 d. All cases of CRS were reversible. Most common G 3/4 nonhematologic AEs (>15%) other than CRS were neutropenia with a body temperature >38.3°C (62% within 8 wk of infusion), hypoxia (20%), and hypotension (20%). 13% of pts experienced G 3 neurological events, with no G 4 events or cerebral edema. Based on laboratory results, 43% and 54% of pts had G 3/4 thrombocytopenia and neutropenia not resolved by d 28; the majority of events resolved to G ≤2 by 3 mo. 25 post-infusion deaths were reported: 2 within 30 d (1 disease progression, 1 cerebral hemorrhage); 23 after 30 d of infusion (range, 53-859 d; 18 disease progression, 1 each due to encephalitis, systemic mycosis, VOD [hepatobiliary disorders related to allogeneic-SCT], bacterial lung infection, and an unknown reason after study withdrawal). Tisagenlecleucel expansion in vivo correlated with CRS severity, and persistence of tisagenlecleucel along with B-cell aplasia in peripheral blood was observed for ≥2.5 y in some responding pts. Analysis of B-cell recovery and correlation with relapse will be presented. CONCLUSIONS With longer follow-up, the ELIANA study continues to confirm the efficacy of a single infusion of tisagenlecleucel in pediatric and young adults with ALL without additional therapy. AEs were effectively and reproducibly managed globally by appropriately trained personnel at study sites. The achievement of high overall response rates and deep remissions, in combination with the median duration of response and overall survival not being reached, further corroborate previously reported results. Figure. Figure. Disclosures Grupp: Novartis Pharmaceuticals Corporation: Consultancy, Research Funding; Jazz Pharmaceuticals: Consultancy; Adaptimmune: Consultancy; University of Pennsylvania: Patents & Royalties. Maude:Novartis Pharmaceuticals Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees. Rives:Shire: Consultancy, Other: Symposia, advisory boards ; Jazz Pharma: Consultancy, Other: Symposia, advisory boards ; Novartis Pharmaceuticals Corporation: Consultancy, Other: Symposia, advisory boards ; Amgen: Consultancy, Other: advisory board . Baruchel:Celgene: Consultancy; Amgen: Consultancy; Roche: Consultancy; Jazz Pharmaceuticals: Consultancy, Honoraria, Other: Travel, accommodations or expenses; Novartis: Membership on an entity's Board of Directors or advisory committees; Shire: Research Funding; Servier: Consultancy. Bittencourt:Novartis Pharmaceuticals Corporation: Consultancy; Jazz Pharmaceuticals: Consultancy, Honoraria. Bader:Riemser: Research Funding; Cellgene: Consultancy; Medac: Patents & Royalties, Research Funding; Neovii: Research Funding; Novartis: Consultancy, Speakers Bureau. Laetsch:Bayer: Consultancy; Pfizer: Equity Ownership; Eli Lilly: Consultancy; Novartis Pharmaceuticals Corporation: Consultancy; Loxo Oncology: Consultancy. Stefanski:Novartis Pharmaceuticals Corporation: Consultancy, Honoraria, Speakers Bureau. Myers:Novartis Pharmaceuticals Corporation: Consultancy, Honoraria, Research Funding, Speakers Bureau. Qayed:Novartis: Consultancy. Pulsipher:CSL Behring: Consultancy; Amgen: Honoraria; Adaptive Biotech: Consultancy, Research Funding; Novartis: Consultancy, Honoraria, Speakers Bureau. Martin:Novartis Pharmaceuticals Corporation: Research Funding; Jazz Pharmaceuticals: Research Funding. Nemecek:Novartis Pharmaceuticals Corporation: Other: advisory boards. Boissel:Servier: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceuticals Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees. Leung:Novartis Pharmaceuticals Corporation: Employment. Eldjerou:Novartis Pharmaceuticals Corporation: Employment. Yi:Novartis Pharmaceuticals Corporation: Employment. Mueller:Novartis Institutes for Biomedical Research: Employment; Novartis Pharmaceuticals Corporation: Equity Ownership, Other: Patent pending. Bleickardt:Novartis Pharmaceuticals Corporation: Employment.