Chimeric Antigen Receptor T Cell Research For Clinical Applications

Genetically engineered T cells are a promising therapeutic tool for the treatment of cancer or infectious diseases. The generation of potent chimeric antigen receptor (CAR) T cells for clinical use, however, is associated with a plurality of challenges covering medical, economical as well as scientific aspects. Therefore, the present study focusses on specific medical and technical difficulties limiting the dissemination of adoptive cellular therapies (ACT). Furthermore, strategies aiming to improve the potential of CAR modified T cells, especially in a solid tumor setting, were investigated. To this effect, our work initially focused on the generation of a novel CD20-directed CAR which we evaluated in newly established in vitro and in vivo assays. After confirming the potency of the anti-CD20 CAR T cells, we focused on the manufacturing and consequently demonstrated that a current good manufacturing practice (cGMP)-compliant, automated T cell Transduction (TCT) Process is both reliable and applicable to manufacture CAR T cells in a clinically relevant scale and quality. Despite varying starting material, different operators or the use of other devices, the developed manufacturing process yielded a robust formulated product with regard to cellular composition, T cell phenotype or anti-tumor potency. Overall, the high reproducibility of the TCT Process proved the suitability to manufacture CD20-directed CAR T cells which are intended to be used in two clinical trials. In addition, this work focused on strategies to enhance the CAR-based immune response in a melanoma model by co-expressing a CAR and a chimeric co-stimulatory receptor (CCR) in the same T cell. This two-receptor approach includes a second generation CAR specific for chondroitin sulfate proteoglycan 4 (CSPG4) and a CCR that recognizes CD20. Here we show that, dependent on intracellular CCR design, only a simultaneous activation of both co-expressed chimeric receptors CAR as well as CCR resulted in a significantly enhanced immune response. Altogether, this data supports the idea of using an anti-CD20 CCR as a tool to increase the potential of CAR T cells

FAS-Based Cell Depletion Facilitates the Selective Isolation of Mouse Induced Pluripotent Stem Cells

Cellular reprogramming of somatic cells into induced pluripotent stem cells (iPSC) opens up new avenues for basic research and regenerative medicine. However, the low efficiency of the procedure remains a major limitation. To identify iPSC, many studies to date relied on the activation of pluripotency-associated transcription factors. Such strategies are either retrospective or depend on genetically modified reporter cells. We aimed at identifying naturally occurring surface proteins in a systematic approach, focusing on antibody-targeted markers to enable live-cell identification and selective isolation. We tested 170 antibodies for differential expression between mouse embryonic fibroblasts (MEF) and mouse pluripotent stem cells (PSC). Differentially expressed markers were evaluated for their ability to identify and isolate iPSC in reprogramming cultures. Epithelial cell adhesion molecule (EPCAM) and stage-specific embryonic antigen 1 (SSEA1) were upregulated early during reprogramming and enabled enrichment of OCT4 expressing cells by magnetic cell sorting. Downregulation of somatic marker FAS was equally suitable to enrich OCT4 expressing cells, which has not been described so far. Furthermore, FAS downregulation correlated with viral transgene silencing. Finally, using the marker SSEA-1 we exemplified that magnetic separation enables the establishment of bona fide iPSC and propose strategies to enrich iPSC from a variety of human source tissues

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Joining Forces for Cancer Treatment: From “TCR versus CAR” to “TCR and CAR”

T cell-based immunotherapy has demonstrated great therapeutic potential in recent decades, on the one hand, by using tumor-infiltrating lymphocytes (TILs) and, on the other hand, by engineering T cells to obtain anti-tumor specificities through the introduction of either engineered T cell receptors (TCRs) or chimeric antigen receptors (CARs). Given the distinct design of both receptors and the type of antigen that is encountered, the requirements for proper antigen engagement and downstream signal transduction by TCRs and CARs differ. Synapse formation and signal transduction of CAR T cells, despite further refinement of CAR T cell designs, still do not fully recapitulate that of TCR T cells and might limit CAR T cell persistence and functionality. Thus, deep knowledge about the molecular differences in CAR and TCR T cell signaling would greatly advance the further optimization of CAR designs and elucidate under which circumstances a combination of both receptors would improve the functionality of T cells for cancer treatment. Herein, we provide a comprehensive review about similarities and differences by directly comparing the architecture, synapse formation and signaling of TCRs and CARs, highlighting the knowns and unknowns. In the second part of the review, we discuss the current status of combining CAR and TCR technologies, encouraging a change in perspective from “TCR versus CAR” to “TCR and CAR”

Identification of differentially expressed surface markers.

<p>A) A single flow cytometric analysis is shown to exemplify SSEA1 and ITGAV expression properties (n.d.  =  not determined), both of which were previously shown to be differentially expressed between MEF and PSC. B) Expression frequencies of antibody-targeted surface markers were tested by flow cytometry comparing MEFs (CF1), ESC line HM1 and iPSC line LV1-7b (n = 4 for MEFs: mean +/− SD; n = 2 for ESC/iPSC each). Given are the percentages of positive cells for identified candidate markers (6 potential pluripotency associated markers on the left-hand side and 6 potential MEF associated markers on the right-hand side). Expression data of all antibodies tested in the screen can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102171#pone.0102171.s004" target="_blank">Table S1</a>, additional expression characteristics on OG2-MEFs are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102171#pone.0102171.s001" target="_blank">Figure S1</a>. C) Representative histograms are shown for selected markers.</p

Expression kinetics of some candidate markers correlate with reprogramming stages.

<p>A) Expression frequencies (mean +/− SD) of candidate markers on reprogramming subpopulations were investigated by flow cytometry over time (n = 3: mean +/− SD; for SSEA1 n = 1) (also see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102171#pone.0102171.s002" target="_blank">Figure S2</a>). Reprogramming subpopulations were defined as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102171#pone-0102171-g003" target="_blank">Fig. 3D</a>. B) Correlation of ITGAV, SSEA1, EPCAM and FAS with expression of OCT4 protein as analyzed by flow cytometry at day 12 p.t. C) Likewise, correlation of the selected candidate markers with the <i>Oct4</i>-GFP reporter system is shown at day 12 of reprogramming.</p

Oct4-GFP expression characteristics.

<p>A) Immunofluorescence of <i>Oct4</i>-GFP transgenic iPSC line LV1-7b cultured in non-differentiating conditions. Depicted are the <i>Oct4</i>-GFP marker, staining for OCT4 protein, a DAPI counterstain and phase contrast images. The overlay displays <i>Oct4</i>-GFP and OCT4 protein. B) The same iPSC line and analysis as in A cultured under differentiating conditions (2 day LIF withdrawal).</p

Opposing regulation of BIM and BCL2 controls glucocorticoid-induced apoptosis of pediatric acute lymphoblastic leukemia cells

© 2015 by The American Society of Hematology Glucocorticoids are critical components of combination chemotherapy regimens in pediatric acute lymphoblastic leukemia (ALL). The proapoptotic BIM protein is an important mediator of glucocorticoid-induced apoptosis in normal and malignant lymphocytes, whereas the antiapoptotic BCL2 confers resistance. The signaling pathways regulating BIM and BCL2 expression in glucocorticoid-treated lymphoid cells remain unclear. In this study, pediatric ALL patient-derived xenografts (PDXs) inherently sensitive or resistant to glucocorticoids were exposed to dexamethasone in vivo. Microarray analysis showed that KLF13 and MYB gene expression changes were significantly greater in dexamethasone-sensitive than -resistant PDXs. Chromatin immunoprecipitation (ChIP) analysis detected glucocorticoid receptor (GR) binding at the KLF13 promoter to trigger KLF13 expression only in sensitive PDXs. Next, KLF13 bound to the MYB promoter, deactivating MYB expression only in sensitive PDXs. Sustained MYB expression in resistant PDXs resulted in maintenance of BCL2 expression and inhibition of apoptosis. ChIP sequencing analysis revealed a novel GR binding site in a BIM intronic region (IGR) that was engaged only in dexamethasone-sensitive PDXs. The absence of GR binding at the BIM IGR was associated with BIM silencing and dexamethasone resistance. This study has identified novel mechanisms of opposing BCL2 and BIM gene regulation that control glucocorticoid-induced apoptosis in pediatric ALL cells in vivo.Link_to_subscribed_fulltex

Automated Manufacturing of Potent CD20-Directed Chimeric Antigen Receptor T Cells for Clinical Use

The clinical success of gene-engineered T cells expressing a chimeric antigen receptor (CAR), as manifested in several clinical trials for the treatment of B cell malignancies, warrants the development of a simple and robust manufacturing procedure capable of reducing to a minimum the challenges associated with its complexity. Conventional protocols comprise many open handling steps, are labor intensive, and are difficult to upscale for large numbers of patients. Furthermore, extensive training of personnel is required to avoid operator variations. An automated current Good Manufacturing Practice-compliant process has therefore been developed for the generation of gene-engineered T cells. Upon installation of the closed, single-use tubing set on the CliniMACS Prodigy, sterile welding of the starting cell product, and sterile connection of the required reagents, T cells are magnetically enriched, stimulated, transduced using lentiviral vectors, expanded, and formulated. Starting from healthy donor (HD) or lymphoma or melanoma patient material (PM), the robustness and reproducibility of the manufacturing of anti-CD20 specific CAR T cells were verified. Independent of the starting material, operator, or device, the process consistently yielded a therapeutic dose of highly viable CAR T cells. Interestingly, the formulated product obtained with PM was comparable to that of HD with respect to cell composition, phenotype, and function, even though the starting material differed significantly. Potent antitumor reactivity of the produced anti-CD20 CAR T cells was shown in vitro as well as in vivo. In summary, the automated T cell transduction process meets the requirements for clinical manufacturing that the authors intend to use in two separate clinical trials for the treatment of melanoma and B cell lymphoma