Engineering Improved CAR T Cell Products with A Multi-Cytokine Particle Platform for Hematologic and Solid Tumors.

Despite the remarkable clinical efficacy of chimeric antigen receptor (CAR) T cells in hematological malignancies, only a subset of patients achieves a durable complete response (dCR). DCR has been correlated with CAR T cell products enriched with T cells memory phenotypes. Therefore, reagents that consistently promote memory phenotypes during the manufacturing of CAR T cells have the potential to significantly improve clinical outcomes. A novel modular multi-cytokine particle (MCP) platform is developed that combines the signals necessary for activation, costimulation, and cytokine support into a single "all-in-one" stimulation reagent for CAR T cell manufacturing. This platform allows for the assembly and screening of compositionally diverse MCP libraries to identify formulations tailored to promote specific phenotypes with a high degree of flexibility. The approach is leveraged to identify unique MCP formulations that manufacture CAR T cell products from diffuse large B cell patients   with increased proportions of memory-like phenotypes MCP-manufactured CAR T cells demonstrate superior anti-tumor efficacy in mouse models of lymphoma and ovarian cancer through enhanced persistence. These findings serve as a proof-of-principle of the powerful utility of the MCP platform to identify "all-in-one" stimulation reagents that can improve the effectiveness of cell therapy products through optimal manufacturing.

Mass spectral enhanced detection of Ubls using SWATH acquisition: MEDUSA--simultaneous quantification of SUMO and ubiquitin-derived isopeptides.

Protein modification by ubiquitination and SUMOylation occur throughout the cell and are responsible for numerous cellular functions such as apoptosis, DNA replication and repair, and gene transcription. Current methods for the identification of such modifications using mass spectrometry predominantly rely upon tryptic isopeptide tag generation followed by database searching with in vitro genetic mutation of SUMO routinely required. We have recently described a novel approach to ubiquitin and SUMO modification detection based upon the diagnostic a' and b' ions released from the isopeptide tags upon collision-induced dissociation of reductively methylated Ubl isopeptides (RUbI) using formaldehyde. Here, we significantly extend those studies by combining data-independent acquisition (DIA) with alternative labeling reagents to improve diagnostic ion coverage and enable relative quantification of modified peptides from both MS and MS/MS signals. Model synthetic ubiquitin and SUMO-derived isopeptides were labeled with mTRAQ reagents (Δ0, Δ4, and Δ8) and subjected to LC-MS/MS with SWATH acquisition. Novel diagnostic ions were generated upon CID, which facilitated the selective detection of these modified peptides. Simultaneous MS-based and MS/MS-based relative quantification was demonstrated for both Ub and SUMO-derived isopeptides across three channels in a background of mTRAQ-labeled Escherichia coli digest.

Butyrolactone I Quantification from Lovastatin Producing Aspergillus terreus Using Tandem Mass Spectrometry-Evidence of Signalling Functions.

Aspergillus terreus is an industrially important filamentous fungus producing a wide spectrum of secondary metabolites, including lovastatin and itaconic acid. It also produces butyrolactone I which has shown potential as an antitumour agent. Additionally, butyrolactone I has been implicated to have a regulating role in the secondary metabolism and morphology of A. terreus. In this study, a quantitative time-course liquid chromatography-electrospray ionisation-tandem mass spectrometry (LC-ESI-MS-MS) analysis of butyrolactone I is reported for the first time in nine-day long submerged cultures of A. terreus. Butyrolactone I was fragmented in the mass analysis producing a reproducible fragmentation pattern of four main daughter ions (m/z 307, 331, 363 and 393) in all the samples tested. Supplementing the cultures with 100 nM butyrolactone I caused a statistically significant increase (up to two-fold) in its production, regardless of the growth stage but was constitutive when butyrolactone I was added at high cell density during the stationary phase. Furthermore, the extracellular butyrolactone I concentration peaked at 48 h post inoculation, showing a similar profile as has been reported for bacterial quorum sensing molecules. Taken together, the results support the idea of butyrolactone I as a quorum sensing molecule in A. terreus.

LC-ESI-Q-TOF-MS for faster and accurate determination of microcystins and nodularins in serum.

Microcystins (MC) and nodularins (Nod) are cyclic peptide hepatotoxins and tumour promoters produced by cyanobacteria. This study deals with liquid chromatography-mass spectrometry (LC-MS) analyses of 9 major cyanobacterial peptide toxins, starting with a comparison of six small particle size reversed-phase HPLC columns, from which one, Phenomenex Synergi Hydro-RP, was chosen for further chromatography with accurate mass MS studies in a complex biological fluid, serum. The instrumentation used for the serum sample analysis included a Bruker micrO-TOF-Q-MS coupled to an Agilent 1200RR LC system. Total analysis run time per sample was 8.5 min. The Q-TOF-MS instrument was operated on auto MS-MS mode to obtain fragment ions (such as the characteristic fragment m/z 135 from Adda amino acid residue) for toxin identification purposes. Detected mass errors in serum samples were in the range of from 0.3 mDa to 9.1 mDa. The narrow mass window (+/-20 mDa) for mass chromatograms used in quantitation gave benefits by background noise reduction. We conclude that a LC-ESI-Q-TOF-MS instrumentation is a powerful tool for identification and quantitation of cyanobacterial peptide toxins in a biological matrix.

Detection of free and covalently bound microcystins in animal tissues by liquid chromatography-tandem mass spectrometry.

Microcystins are cyanobacterial hepatotoxins capable of accumulation into animal tissues. The toxins act by inhibiting specific protein phosphatases and both non-covalent and covalent interactions occur. The 2-methyl-3-methoxy-4-phenylbutyric acid (MMPB) method determines the total, i.e. the sum of free and protein-bound microcystin in tissues. The aim of the method development in this paper was to tackle the problems with the MMPB methodology: the rather laborious workflow and the loss of material during different steps of the method. In the optimised workflow the oxidation recovery was of acceptable level (29-40%), the extraction efficiency good (62-97%), but the signal suppression effect from the matrix remained severe in our system (16-37% signal left). The extraction efficiency for the determination of the free, extractable microcystins, was found to be good, 52-100%, depending on the sample and the toxin variant and concentration.

Fast separation of microcystins and nodularins on narrow-bore reversed-phase columns coupled to a conventional HPLC system.

Six modern narrow-bore (50 mm length x 2-2.1 mm internal diameter) reversed-phase HPLC columns were tested in the rapid separation of ten microcystins and three nodularins, cyanobacterial peptide hepatotoxins. The columns had either a sub-3 microm particle size or were of monolithic silica technology for high efficiency and rapid run times. A standard low-pressure gradient HPLC equipment with diode-array UV detector was used for the chromatography. The gradient mobile phase consisted of water and acetonitrile, both acidified with trifluoroacetic acid, and the gradient rise times were 1-4 min. Chromatographic performance was assessed by visual judgement and by calculating parameters such as capacity factors, resolution, peak width, selectivity and peak form. Several chromatographic conditions produced excellent results. We recommend sub-3 microm particle-based or monolithic reversed-phase columns for routine use in microcystin and nodularin HPLC analyses.

Accumulation of free and covalently bound microcystins in tissues of Lymnaea stagnalis (Gastropoda) following toxic cyanobacteria or dissolved microcystin-LR exposure.

Accumulation of free microcystins (MCs) in freshwater gastropods has been demonstrated but accumulation of MCs covalently bound to tissues has never been considered so far. Here, we follow the accumulation of total (free and bound) MCs in Lymnaea stagnalis exposed to i) dissolved MC-LR (33 and 100 microg L(-1)) and ii) Planktothrix agardhii suspensions producing 5 and 33 microg MC-LR equivalents L(-1) over a 5-week period, and after a 3-week depuration period. Snails exposed to dissolved MC-LR accumulated up to 0.26 microg total MCs g(-1) dry weight (DW), with no detection of bound MCs. Snails exposed to MCs producing P. agardhii accumulated up to 69.9 microg total MCs g(-1) DW, of which from 17.7 to 66.7% were bound. After depuration, up to 15.3 microg g(-1) DW of bound MCs were detected in snails previously exposed to toxic cyanobacteria, representing a potential source of MCs transfer through the food web.

Separation of microcystins and nodularins by ultra performance liquid chromatography.

Four ultra performance liquid chromatography (UPLC) columns with different reversed-phase characteristics were tested in the chromatographic separation of 10 microcystins and three nodularins, cyanobacterial peptide toxins. The columns had been designed by the manufacturer to withstand the ultra-high pressure generated by sub-2microm stationary phase particles and the Waters ACQUITY UPLC system in ultra-fast separations. The gradient mobile phase consisted of water and acetonitrile, both acidified with trifluoroacetic acid, with three gradient rise times: 1, 1.5 and 2min. The UV detection of the toxins was performed by a photodiode array detector. The chromatographic performance was evaluated both visually and by calculating chromatographic parameters such as capacity factor, resolution, peak width at half height, selectivity and peak asymmetry. The best chromatographic performance as judged by visual inspection was given by the ACQUITY BEH Shield RP18 and ACQUITY BEH Phenyl columns. The BEH Shield RP18 column showed excellent selectivity and resolution of chosen peak pairs considered as critical. A further advantage of the UPLC system was the high sample throughput with a total analysis time of 3.12min (injection-to-injection) equalling to 461 separations per 24h.

Rapid LC-MS detection of cyanobacterial hepatotoxins microcystins and nodularins--comparison of columns.

Eight reversed-phase columns intended for rapid HPLC were assessed for the separation of thirteen microcystins and nodularins, cyclic peptidic hepatotoxins. The instrumentation consisted of an Agilent Technologies 1200 Rapid Resolution high performance liquid chromatography system coupled to a mass spectrometer, Bruker Daltonics Ultra Performance High Capacity Ion Trap MS (HCT Ultra) with electrospray ionisation (RRLC-ESI-IT-MS). The columns tested were 2-2.1 mm x 50 mm in diameter and length, and contained small particles (1.8-2.7 microm), or monolithic silica supports for fast performance. The shortest total run time achieved was 3 min 15 s including equilibration and injection. Critical microcystin pairs were still resolved. Several columns showed excellent performance.