The development of a 14-day non-viral engineered CAR T-cell process

Immunotherapy utilizing chimeric antigen receptor (CAR) T cells is a promising strategy for the treatment of several types of cancer. Many preclinical and clinical studies engineer CAR T cells through a viral vector, presenting the potential for genotoxicity or insertional mutagenesis. We propose a 14-day non-viral process where we introduce the gene of interest via electroporation; integration can be achieved with the Sleeping Beauty transposon system. Minicircle (MC) DNA constructs containing the CAR, a surface marker (EGFRt), and a double mutant of dihydrofolate reductase (DHFRdm) are electroporated into previously frozen, unstimulated CD4/CD8 T cells with an RNA construct coding for the Sleeping Beauty transposase. After electroporation, cells are bead-stimulated with CD3/CD28 without the use of feeder cells throughout the process. CAR+ cells expressing DHFRdm are rendered insensitive to an FDA-approved small molecule drug, methotrexate (MTX), which allows for chemical selection of the cells of interest while avoiding a magnetic bead sort. The entire process is completed in 2 weeks with a media formulation that contains a serum-free replacement. Please click Additional Files below to see the full abstract

Synthesis of Microgel Sensors for Spatial and Temporal Monitoring of Protease Activity

Proteases are involved in almost every important cellular activity, from embryonic morphogenesis to apoptosis. To study protease activity in situ, hydrogels provide a synthetic mimic of the extracellular matrix (ECM) and have utility as a platform to study activity, such as those related to cell migration, in three-dimensions. Although three-dimensional visualization of protease activity could prove quite useful to elucidate the proteolytic interaction at the interface between cells and their surrounding environment, there has been no versatile tool to visualize local proteolytic activity in real time. Here, micrometer-sized gels were synthesized by inverse suspension polymerization using thiol–ene photoclick chemistry. The size distribution was selected to avoid cellular uptake and to lower cytotoxicity, while simultaneously allowing the integration of peptide-based FRET sensors of local cell activity. Proteolytic activity of collagenase was detected within an hour via changes in fluorescence of embedded microgels; incubation of microgel sensors with A375 melanoma cells showed upregulated MMP activity in the presence of soluble fibronectins in media. The microgel sensors were readily incorporated into both gelatin and poly­(ethylene glycol) (PEG) hydrogels and used to successfully detect spatiotemporal proteolytic activity of A375 melanoma cells. Finally, a tumor model was constructed from a hydrogel microwell array that was used to aggregate A375 melanoma cells, and local variations in proteolytic activity were monitored as a function of distance from the cell aggregate center

Morphologies and cytoskeletal structure for HT-1080 fibrosarcoma cells (HT-1080s) and primary human dermal fibroblasts (hDFs) in synthetic extracellular matrix (ECM).

<div><p>(<b>A</b>) Schematic representation of synthetic extracellular matrix (synthetic ECM) formed through “thiol-ene” photopolymerization chemistry to couple norbornene C=C bonds on 4-arm poly(ethylene glycol) (PEG) molecules with thiol (-SH) bonds of cysteine-containing peptides. Crosslinks were formed using matrix metalloproteinase (MMP)-degradable peptides with cysteine groups on each end while adhesion was promoted using pendant RGD-containing peptides (C<b>RGDS</b>) with a single cysteine (2.5 or 3 wt% by mass PEG-NB + MMP-degradable crosslinking peptide, shear moduli = 140 Pa or 220 Pa respectively). Constant total pendant peptide was maintained using non-bioactive C<i><b>RDGS</b></i> (1500 μM active C<b>RGDS</b> + non-active C<i><b>RDGS</b></i>). </p> <p>(<b>B</b>-<b>I</b>) Projected z-stack immunofluorescence (IF) images illustrating hDFs and HT-1080s seeded in synthetic ECM (220 Pa, 1000 μM CRGDS). All overlay images are counterstained with TRITC-conjugated phalloidin (F-actin, red) and DAPI (nuclei, blue). (<b>B</b>,<b>C</b>) Overview to illustrate morphological differences (F-actin, red; Nuclei, blue) for (<b>B</b>) hDFs and (<b>C</b>) HT-1080s. (<b>D</b>-<b>F</b>) IF images illustrating myosin IIb expression for hDFs: (<b>D</b>) Overlay image (Myosin IIb, green; F-actin, red; Nuclei, blue); Single channel images (grayscale) illustrate (<b>E</b>) F-actin and (<b>F</b>) Myosin IIb. White arrows point to actomyosin filaments. (<b>G</b>-<b>I</b>) IF images illustrating myosin IIb expression for <b><i>HT-1080s</i></b>: (<b>G</b>) Overlay (Myosin IIb, green; F-actin, red; Nuclei, blue); Single channel images (grayscale) illustrate (<b>H</b>) F-actin and (<b>I</b>) Myosin IIb. </p> <p>(<b>J</b>-<b>L</b>) Comparison of quantified mean (<b>J</b>) circularity and (<b>K</b>) elongation for hDFs and HT-1080s calculated using Nikon NIS Elements software (n > 150 cells, ≥ 6 hydrogels, at least two separate experiments; *** = p<0.001). (<b>L</b>) Fraction of elongated (Elongation ≥ 3.0), middle (2.0 ≤ Elongation < 3.0), and rounded (Elongation < 2.0) cells. Differences in fraction of elongated and rounded hDFs compared to HT-1080s were each statistically significant (N ≥ 6 total gels, at least two separate experiments; *** = p<0.001). </p></div

Matrix influences on migration and morphologies for HT-1080s in synthetic ECM.

<p>(<b>A</b>) Cell speed and (<b>B</b>) directionality (DTO/TD) for HT-1080s as a function of matrix conditions (≥ 6 gels, ≥ 40 cells, at least two separate experiments; * = p<0.05; ** = p<0.01). X-axis: Modulus in Pa / RGD concentration in μM. <i>Box</i> and <i>whisker </i><i>plot </i><i>for </i><i>cell </i><i>speed</i>: White diamond = mean, white line = median, boxes = middle upper (top) and middle lower (bottom) quartile of the cell population, whiskers = highest (above) and lowest (below) migration speeds. Error bars for DTO/TD represent standard error of the mean for individual cells. There is also a statistical difference in cell speed for the 140 Pa (1500 μM RGD) and 220 Pa (250 μM RGD) conditions (p<0.05, not shown on graph for clarity). (<b>C</b>) A comparison of the fraction of rounded HT-1080s (Elongation Index < 2.0) as a function of synthetic ECM conditions (x-axis: Modulus in Pa / RGD concentration in μM; white bar = 140 Pa; black bars = 220 Pa). Error bars represent standard deviation for fraction of rounded cells per gel (≥ 6 gels, at least two separate experiments; * = p<0.05; ** = p<0.01; *** = p<0.001). </p

Qualitative and quantitative migration for HT-1080s and hDFs.

<div><p>Time-lapse images (10 min./frame) illustrating migration for (<b>A</b>) an hDF and (<b>B</b>) an HT-1080 in synthetic ECM (220 Pa, 1000 μM CRGDS). Image sequences in (<b>A</b>,<b>B</b>) are contrast and brightness enhanced for better display (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081689#pone.0081689.s013" target="_blank">Movies S2, S3</a> for unaltered images, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081689#pone.0081689.s015" target="_blank">Movie S4</a> for overview comparison of many cells). (<b>A</b>) Migration movement for hDF is characterized by: 1. Front end extension, 2. Attachment, 3. Cell body contraction, and 4. Rear-end release. (<b>B</b>) HT-1080 movement illustrates cell body contraction (white dashed lines and arrow) and simultaneous front end extension (red dashed lines and arrow). </p> <p>(<b>C</b>-<b>E</b>) Comparison of quantified 3D migration for HT-1080s and hDFs in synthetic ECM (220 Pa and 1000 μM CRGDS): (<b>C</b>) Cell speed (adjusted by a factor of √3/2, which was a 3D correction for analysis on 2D minimum intensity z-projections), (<b>D</b>) directionality (DTO/TD), and (<b>E</b>) fraction migrating cells. DTO/TD is a dimensionless parameter that provides a measure of directional motility analogous to persistence time (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081689#pone.0081689.s001" target="_blank">Fig. S1A</a>) that is calculated as the distance-to-origin (DTO) after 6 hours divided by the total path length (total distance, TD) (shown schematically, panel to right of C). Migration was calculated from images collected in 15 min. increments for 6 hours, with dividing or interacting cells excluded (≥200 cells, ≥ 3 separate experiments, ≥ 9 total hydrogels). <i>Box</i> and <i>whisker </i><i>plot </i><i>for </i><i>cell </i><i>speed</i>: White diamond = mean, white line = median, boxes = middle upper (top) and middle lower (bottom) quartile of the cell population, whiskers = highest (above) and lowest (below) migration speeds. Values for DTO/TD represent the mean for all cells while fraction migrating represents the mean for replicate experiments (N ≥ 3). Significance was calculated for hDF relative to HT-1080 migration for each parameter (*** = p<0.001). </p></div

Rounded proteolytic migration modes for tumor cells in synthetic ECM.

<p>Rounded tumor cells migrating in synthetic ECM (220 Pa, 1000 μM CRGDS): (<b>A</b>) Time-lapse images (15 min / frame; See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081689#pone.0081689.s022" target="_blank">Movie S11</a>) for a rounded HT-1080 after overnight swelling (day 1). (<b>B</b>) β1-integrin expression (immunofluorescence) for a rounded HT-1080. (<b>C</b>) Time-lapse images (10 min / frame; See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081689#pone.0081689.s023" target="_blank">Movie S12</a>) for a rounded HT-1080 shortly after encapsulation (Initial migration, Day 0). (<b>D</b>) Time-lapse images (30 min / frame) for a rounded WM239a melanoma cell after overnight swelling (day 1). Scale bars = 25 μm unless otherwise noted.</p

Comparison of adhesion properties for HT-1080s and hDFs.

<div><p>(<b>A</b>-<b>M</b>) Projected z-stack immunofluorescence (IF) images for hDFs and HT-1080s in synthetic ECM (220 Pa, 1000 μM CRGDS). All overlay images are counterstained with TRITC-conjugated phalloidin (F-actin, red) and DAPI (nuclei, blue). </p> <p>(<b>A</b>-<b>D</b>) IF images illustrating vinculin expression for <i><b>hDFs</b></i> (boxed region from A shown in B-D): (<b>A</b>) Overlay image (Vinculin, green; F-actin, red; Nuclei, blue). White arrows point to regions enriched with vinculin. (<b>B</b>) Overlay (Vinculin, green; F-actin, red); Single channel images (grayscale) illustrate (<b>C</b>) F-actin and (<b>D</b>) Vinculin. (<b>E</b>-<b>G</b>) IF images illustrating vinculin expression for <b><i>HT-1080s</i></b>: (<b>E</b>) Overlay image (Vinculin, green; F-actin, red; Nuclei, blue). Single channel images (grayscale) illustrate (<b>F</b>) F-actin and (<b>G</b>) Vinculin. </p> <p>(<b>H</b>-<b>J</b>) IF images illustrating β1-integrin expression for <i><b>hDFs</b></i>: (<b>H</b>) Overlay (β1-integrin, green; F-actin, red; Nuclei, blue); Single channel images (grayscale) illustrate (<b>I</b>) F-actin and (<b>J</b>) β1-integrin (White arrows point to punctate β1-integrin features; Also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081689#pone.0081689.s003" target="_blank">Fig. S3</a>). (<b>K</b>-<b>M</b>) IF images illustrating β1-integrin expression for <b><i>HT-1080s</i></b>: (<b>K</b>) Overlay (β1-integrin, green; F-actin, red; Nuclei, blue); Single channel images (grayscale) illustrate (<b>L</b>) F-actin and (<b>M</b>) β1-integrin. </p> <p>(<b>N</b>) Comparison of attachment for hDFs and HT-1080s on RGD-SAMs as a function of RGD density. Attachment was statistically significant (cell number > 0 RGD control spots) for HT-1080s on surfaces with ≥ 0.19% mol fraction RGD and for hDFs on surfaces ≥ 0.007% mol fraction RGD. Error bars represent standard error of the mean (SEM) for array spots at given RGD density. Significance for cell attachment on individual spots was calculated for hDFs relative to HT-1080s (* = p<0.05; ** = p<0.01; *** = p<0.001). </p></div

Contractile movement for HT-1080s in 3D culture.

<div><p>(<b>A</b>) Propagation of a constriction ring (arrow) for an HT-1080 migrating in synthetic ECM (220 Pa, 1000 μM CRGDS; 10 min / frame, see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081689#pone.0081689.s014" target="_blank">Movie S3</a>). (<b>B</b>) Propagation of a constriction ring (arrow) for an HT-1080 migrating in collagen (1.7 mg/mL; 15 min./frame, see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081689#pone.0081689.s020" target="_blank">Movie S9</a>). Scale bars = 25 μm.</p> <p>(<b>C</b>-<b>G</b>) Z-projected immunofluorescence images illustrating myosin IIb expression for an HT-1080 in synthetic ECM (220 Pa, 1000 μM CRGDS): (<b>C</b>) Overlay image illustrating myosin IIb (green), counterstained with TRITC-conjugated phalloidin (F-actin, red) and DAPI (nucleus, blue). Single channel images (grayscale) illustrate (<b>D</b>) F-actin and (<b>E</b>) Myosin IIb. Profile plots generated using ImageJ “Interactive 3D Surface Plot” function (“Spectrum” intensity scale, projection for middle 3 planes) illustrate (<b>F</b>) F-actin and (<b>G</b>) Myosin IIb. Two consecutive single plane images (grayscale) illustrate (<b>H</b>) F-actin and (<b>I</b>) Myosin IIb.</p></div