Enhancing chimeric antigen receptor T cell therapy by modulating the p53 signaling network with Δ133p53α.

Chimeric antigen receptor (CAR) T cell dysfunction is a major barrier to achieving lasting remission in hematologic cancers, especially in chronic lymphocytic leukemia (CLL). We have shown previously that Δ133p53α, an endogenous isoform of the human TP53 gene, decreases in expression with age in human T cells, and that reconstitution of Δ133p53α in poorly functional T cells can rescue proliferation [A. M. Mondal et al., J. Clin. Invest. 123, 5247-5257 (2013)]. Although Δ133p53α lacks a transactivation domain, it can form heterooligomers with full-length p53 and modulate the p53-mediated stress response [I. Horikawa et al., Cell Death Differ. 24, 1017-1028 (2017)]. Here, we show that constitutive expression of Δ133p53α potentiates the anti-tumor activity of CD19-directed CAR T cells and limits dysfunction under conditions of high tumor burden and metabolic stress. We demonstrate that Δ133p53α-expressing CAR T cells exhibit a robust metabolic phenotype, maintaining the ability to execute effector functions and continue proliferating under nutrient-limiting conditions, in part due to upregulation of critical biosynthetic processes and improved mitochondrial function. Importantly, we show that our strategy to constitutively express Δ133p53α improves the anti-tumor efficacy of CAR T cells generated from CLL patients that previously failed CAR T cell therapy. More broadly, our results point to the potential role of the p53-mediated stress response in limiting the prolonged antitumor functions required for complete tumor clearance in patients with high disease burden, suggesting that modulation of the p53 signaling network with Δ133p53α may represent a translationally viable strategy for improving CAR T cell therapy.

Engineered cellular immunotherapies in cancer and beyond.

This year marks the tenth anniversary of cell therapy with chimeric antigen receptor (CAR)-modified T cells for refractory leukemia. The widespread commercial approval of genetically engineered T cells for a variety of blood cancers offers hope for patients with other types of cancer, and the convergence of human genome engineering and cell therapy technology holds great potential for generation of a new class of cellular therapeutics. In this Review, we discuss the goals of cellular immunotherapy in cancer, key challenges facing the field and exciting strategies that are emerging to overcome these obstacles. Finally, we outline how developments in the cancer field are paving the way for cellular immunotherapeutics in other diseases.

Evaluation of a digital dispenser for direct curve dilutions in a vaccine potency assay.

Dilutions are a common source of analytical error, both in terms of accuracy and precision, and a common source of analyst mistakes. When serial dilutions are used, errors compound, even when employing laboratory automation. Direct point dilutions instead of serial dilutions can reduce error but is often impractical as they require either large diluent volumes or very small sample volumes when performed with traditional liquid handling equipment. We evaluated preparation of dilution curves using a picoliter digital dispenser, the HP, Inc. / TECAN D300 which is capable of accurately delivering picoliter volumes directly into sample wells filled with assay diluent. Dilution linearity and variability of the direct dilutions were similar to or less than those generated with a traditional liquid handler as measured using a fluorophore assay and an ELISA used to measure vaccine potency. Minimum concentrations for detergent in the dispensed sample were identified but no correlation with detergent characteristics was observed. The tolerance to protein in the sample was evaluated as well with up to 5% BSA having no impact on dispense linearity and precision. We found the digital dispenser to reduce automation complexity while maintaining or improving assay performance in addition to facilitating complex plate lay-outs.

Reduction of dilution error in ELISAs using an internal standard.

BACKGROUND: Dilution bias is a major cause of immunoassay variability due to the lack of an internal standard to determine the true versus the expected dilution value. METHODOLOGY: We used an internal control to measure dilution bias in an ELISA. Acridine-orange was added at the first dilution step and monitored throughout dilutions. Assay results were corrected using the fluorescent signal ratio between samples and reference. Acridine dilution correlated with analyte-specific assay measurements (R2 = 0.987). Correction of assay results with the measured dilution factor improved both accuracy and precision resulting in a reduction of >50% %CV reduction. CONCLUSION: Dilution correction can significantly improve accuracy and precision of immunoassays. Additional control strategies may further mitigate other sources of variability.

Mitigation of microtiter plate positioning effects using a block randomization scheme.

Microtiter plate-based assays are a common tool in biochemical and analytical labs. Despite widespread use, results generated in microtiter plate-based assays are often impacted by positional bias, in which variability in raw signal measurements are not uniform in all regions of the plate. Since small positional effects can disproportionately affect assay results and the reliability of the data, an effective mitigation strategy is critical. Commonly used mitigation strategies include avoiding the use of outer regions of the plate, replicating treatments within and between plates, and randomizing placement of treatments within and between plates. These strategies often introduce complexity while only partially mitigating positional effects and significantly reducing assay throughput. To reduce positional bias more effectively, we developed a novel block-randomized plate layout. Unlike a completely randomized layout, the block randomization scheme coordinates placement of specific curve regions into pre-defined blocks on the plate based on key experimental findings and assumptions about the distribution of assay bias and variability. Using the block-randomized plate layout, we demonstrated a mean bias reduction of relative potency estimates from 6.3 to 1.1 % in a sandwich enzyme-linked immunosorbent assay (ELISA) used for vaccine release. In addition, imprecision in relative potency estimates decreased from 10.2 to 4.5 % CV. Using simulations, we also demonstrated the impact of assay bias on measurement confidence and its relation to replication strategies. We outlined the underlying concepts of the block randomization scheme to potentially apply to other microtiter-based assays.