Biology subjects

Murren, N.

Publications and source records attributed to Murren, N..

3 recordsLinked to original sources

bioRxiv2025

Directing fratricide within T cell products using an anti-uPAR chimeric antigen receptor to drive the production of potent therapeutic cells

Cell therapy manufacturing of primary T cells often results in heterogeneous cell populations within a final product, with many cells lacking desired of receptor expression or those that have exhausted or other dysfunctional phenotypes. Here, we design a novel cell-intrinsic strategy to genetically reprogram primary human T cells to autonomously detect and eliminate dysfunctional cells. This integrated detection and elimination process, known as directed fratricide, is programmed via nonviral CRISPR genome-editing to eliminate the T cell receptor (TCR) alpha chain (TRAC gene knockout) and integrate a chimeric antigen receptor (CAR) against the urokinase-type plasminogen activator receptor (uPAR), also known as CD87. Within these cell products, strong T cell stimulation or activation during manufacturing causes a small subset of cells to express uPAR, which subsequently triggers CAR-mediated killing by a separate subset of cells within the product. This fratricide induces proliferation in the desired cells and destroys undesired cells, a process that could be modeled computationally and controlled robustly via supplements to the culture media. The strategy enabled enrichment of anti-uPAR and anti-GD2 CAR T cell products up to [≥]99% CAR+/TCR-, favoring a stem cell memory-like phenotype (CD45RAhigh/CD62Lhigh). Understanding growth dynamics among T cell subsets and reprogramming them via CRISPR could accelerate the biomanufacturing of potent cell products without extensive selection methods. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=90 SRC="FIGDIR/small/684419v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@199d85forg.highwire.dtl.DTLVardef@14c638aorg.highwire.dtl.DTLVardef@1b3322org.highwire.dtl.DTLVardef@1ba253d_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2025

Chimeric Antigen Receptors Transmit Co-stimulatory Domain Dependent Piconewton Forces to their Target

Chimeric antigen receptor (CAR) T cells promote tumor-specific cytotoxicity through engagement of a recombinant, synthetic receptor with target ligands expressed on cancer cells. Native T cells are mechanically active, both transmitting and sensing forces exceeding 19 piconewtons (pN) via transmembrane receptors, including the T cell receptor (TCR). Emerging evidence implicates mechanoactivity in CAR T cell biology, but CAR-transmitted T cell forces have not been directly measured. Here, we utilize DNA-based molecular tension probes (MTPs) conjugated to CAR target ligands, providing evidence of actin-polymerization dependent forces exceeding 4.7-19 pN borne by the CAR. We demonstrate force transmission by three clinically relevant CARs (CD123, CD33, and CD19), suggesting that these forces are generalizable across CAR targets and constructs. Additionally, we identify intracellular co-stimulatory domains as the main determinants of CAR-mediated forces, because first-generation CARs lacking co-stimulatory domains do not transmit measurable forces to their ligand. Finally, we demonstrate that CAR forces temporally precede Ca2+ signaling and are spatially correlated with phosphorylation of classical TCR-signaling machinery, indicating a link between CAR T cell forces and early biochemical signaling. Our study introduces CAR-mediated mechanobiology as a key correlate of early CAR T cell activation events. Significance StatementChimeric antigen receptor (CAR) T cell therapies have revolutionized treatment for several hematological malignancies. CARs are recombinant receptors containing domains derived from the T cell receptor complex machinery and other co-stimulatory proteins. Mechanical forces are believed to be important in T cell activation and antigen recognition. The role of mechanobiology in CAR T cell immunotherapy remains poorly understood. Here, using DNA-based molecular tension probes conjugated to CAR ligands, we provide direct evidence that CARs bear actin polymerization-dependent piconewton forces during antigen engagement that precede early signaling events. These forces depend on CAR co-stimulatory domains, and first-generation CARs lacking these domains fail to transmit detectable force. These findings suggest mechanobiology may be a key, tunable parameter for next-generation CAR T cells.

immunology↗

bioRxiv2024

Genome-wide CRISPRa screens nominate modulators of CAR T cell survival within distinct tumor cytokine milieus

Chimeric Antigen Receptor (CAR) T cell therapy has revolutionized the treatment of B cell malignancies and translating this success to other cancers remains an ongoing clinical objective. Next-generation T cell products in development aim to genetically modulate many facets of cell behavior, for which gene-nominating platforms provide a useful framework for prioritization. Among competing screening approaches, CRISPR activation (CRISPRa) technology permits gain-of-function (GoF) gene surveys at genome-wide scale, but routine implementation in primary T cells has been stymied by high cell requirements ([~]107 - 108) and abbreviated activity. Here, we describe a novel cell manufacturing schema using an all-in-one transposon-based gene delivery system coupled with CAR-restricted cell expansion to generate yields (109) of primary T cells bearing CAR and CRISPRa transgenes that are well above the threshold needed for genome-scale screening. CRISPRa activity is sustained via the inclusion of divergent, duplicate Elongation Factor 1 core/human T-cell leukemia virus (EF1-HTLV) hybrid promoters; while guide RNA representation is preserved through late lentiviral transduction, thus preventing bottlenecking and premature candidate pruning. CRISPRa-CAR T cells manufactured via this pipeline retain potent on-target gene-overexpression (>85% target+) across varied cell subsets (e.g. Tim-3+Lag3+ or serial-challenge) and timescales (>14 days). When deployed to survival-based genome-wide selection landscapes, CRISPRa-CAR pools nominate known and novel endogenous genes capable of enhancing CD8+ CAR T survival in cytokine-rich (e.g. MYC, FUT6, IRF4, GSE1) and cytokine-depleted (e.g. CSF2RB, STAT6, IRF4, GSE1) settings of tumor challenge. This system will have broad utility for therapy-enhancing gene discovery.

immunology↗