PiggyBac-Engineered T Cells Expressing CD19-Specific CARs that Lack IgG1 Fc Spacers Have Potent Activity against B-ALL Xenografts.

Clinical trials of CD19-specific chimeric antigen receptor (CAR19) T cells have demonstrated remarkable efficacy against relapsed and refractory B cell malignancies. The piggyBac transposon system offers a less complex and more economical means for generating CAR19 T cells compared to viral vectors. We have previously optimized a protocol for the generation of CAR19 T cells using the piggyBac system, but we found that CAR19 T cells had poor in vivo efficacy and persistence, probably due to deleterious FcγR interactions with the CAR's IgG1 Fc-containing spacer domain. We therefore designed three CD19-specifc CARs that lacked the IgG1 Fc region, and we incorporated combinations of CD28 or 4-1BB transmembrane and co-stimulatory domains. PiggyBac-generated CAR19 T cells expressing these re-designed constructs all demonstrated reactivity in vitro specifically against CD19+ cell lines. However, those combining CD28 transmembrane and co-stimulatory domains showed CD4 predominance and inferior cytotoxicity. At high doses, CAR19 T cells were effective against B-ALL in a xenograft mouse model, regardless of co-stimulatory domain. At diminishing doses, 4-1BB co-stimulation led to greater potency and persistence of CAR19 T cells, and it provided protection against B-ALL re-challenge. Production of potent CAR T cells using piggyBac is simple and cost-effective, and it may enable wider access to CAR T cell therapy.

Ultrafiltration versus Diuretics on Prognostic Cardiac and Renal Biomarkers in Acute Decompensated Heart Failure: A Systematic Review and Meta-Analysis.

Existing systematic reviews have insufficiently delineated the differing cardiac and renal profile of ultrafiltration compared to diuretics as a method of decongestion in acute decompensated heart failure. This meta-analysis will investigate the impact of ultrafiltration compared to diuretics on prognostic cardiac and renal biomarkers. We searched PubMed Central, Ovid MEDLINE®, Ovid Embase, all EBM reviews, and Web of Science Core Collection for randomised controlled trials published before 21 July 2022. Our main outcome measures were cardiac (brain natriuretic peptide and N-terminal pro-brain natriuretic peptide) and renal biomarkers (serum creatinine, serum sodium, and blood urea nitrogen). A total of 10 randomised trials were included in our analysis after screening. An inverse-variance random effects meta-analysis of the pooled results demonstrated no significant difference between ultrafiltration and diuretics for brain natriuretic peptide, N-terminal pro-brain natriuretic peptide, creatinine, sodium and long-term blood urea nitrogen. However, ultrafiltration produced statistically greater increases in blood urea nitrogen in the short-term (mean difference, 3.88; 95% confidence interval 0.59-7.17 mg/dL). Overall, ultrafiltration produces a similar impact on prognostic cardiac and renal biomarkers when compared to diuretic therapy. We highlight ultrafiltration's significant impact on short-term BUN and recommend further research to investigate more optimal protocols of ultrafiltration administration.

Priming Leukemia with 5-Azacytidine Enhances CAR T Cell Therapy.

PURPOSE: Despite the success of chimeric antigen receptor (CAR) T cells in clinical studies, a significant proportion of responding patients eventually relapsed, with the latter correlating with low CAR T cell expansion and persistence. METHODS AND RESULTS: Using patient-derived xenograft (PDX) mouse models of CD19+ B cell acute lymphoblastic leukemia (B-ALL), we show that priming leukemia-bearing mice with 5-azacytidine (AZA) enhances CAR T cell therapy. AZA given 1 day prior to CAR T cell infusion delayed leukemia growth and promoted CAR T cell expansion and effector function. Priming leukemia cells with AZA increased CAR T cell/target cell conjugation and target cell killing, promoted CAR T cell divisions and expanded IFNγ+ effector T cells in co-cultures with CD19+ leukemia Nalm-6 and Raji cells. Transcriptome analysis revealed activation of diverse immune pathways in leukemia cells isolated from mice treated with AZA. We propose that epigenetic priming with AZA induces transcriptional changes that sensitize tumor cells to subsequent CAR T cell treatment. Among the candidate genes up-regulated by AZA is TNFSF4 which encodes OX40L, one of the strongest T cell co-stimulatory ligands. OX40L binds OX40, the TNF receptor superfamily member highly specific for activated T cells. TNFSF4 is heterogeneously expressed in a panel of pediatric PDXs, and high TNFSF4 expression correlated with increased CAR T cell numbers identified in co-cultures with individual PDXs. High OX40L expression in Nalm-6 cells increased their susceptibility to CAR T cell killing while OX40L blockade reduced leukemia cell killing. CONCLUSION: We propose that treatment with AZA activates OX40L/OX40 co-stimulatory signaling in CAR T cells. Our data suggest that the clinical use of AZA before CAR T cells could be considered.