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bioRxiv · 10.1101/2025.10.24.684419

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

Sarko, L.·Givand, D.·Shepley, C.·Rattin, B.·Attar, A.·Taylor, R.·Kutler, B.·Traynor, R. M.·Upadhyaya, A.·Mnuk, M.·Gehrke, C.·Murren, N.·Ulland, T.·Kotanchek, T.·Saha, K.
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Abstract

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

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Sarko, L., Givand, D., Shepley, C., Rattin, B., Attar, A., Taylor, R., Kutler, B., Traynor, R. M., Upadhyaya, A., Mnuk, M., Gehrke, C., Murren, N., Ulland, T., Kotanchek, T., Saha, K.. 2025-10-25. Directing fratricide within T cell products using an anti-uPAR chimeric antigen receptor to drive the production of potent therapeutic cells. https://doi.org/10.1101/2025.10.24.684419

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