Engineered SNIPRs transform CAR T-cell precision for safer cancer therapy

by · News-Medical

Harnessing synthetic biology, researchers unveil SNIPRs—receptors that revolutionize CAR T-cell therapies by enhancing tumor precision and safety.

In a recent study published in the journal Nature, researchers from the United States of America developed the synthetic intramembrane proteolysis receptor (SNIPR) architecture to activate engineered therapeutic cells in response to soluble ligands. This novel receptor system operates via ligand-induced dimerization followed by endocytic proteolysis, offering high sensitivity and specificity for detecting soluble factors. They found that SNIPRs allow CAR T-cells to precisely localize to tumors and support synthetic intercellular signaling while avoiding off-target effects.

Background

The foundation of biochemical signaling lies in the ability of cells to sense and react to soluble molecules, enabling complex functions like immune responses and tissue development. Mimicking this in synthetic biology could potentially revolutionize therapeutic applications, such as creating engineered cells that respond to distant signals or communicate exclusively through artificial pathways. However, existing receptor systems for detecting soluble factors such as chimeric antigen receptor (CAR)-T cells face challenges like weak responses, limited ligand flexibility, and complex multi-component designs, which hinder clinical translation.

To address these limitations, researchers developed SNIPRs as compact, single-chain receptors. SNIPRs use a Notch-based architecture but uniquely bypass mechano-sensing filters, allowing them to detect soluble ligands with high sensitivity and low background activity. Unlike conventional synNotch receptors, SNIPRs can sense soluble factors via an alternate activation pathway involving ligand-triggered dimerization and subsequent endosomal signaling. Their modularity and efficiency make SNIPRs a promising tool for enabling precise therapeutic genetic programs in immune cells, overcoming the constraints of current receptor platforms. In the present study, researchers explored whether the SNIPR system could be engineered to detect soluble ligands, paving the way for diverse applications in therapeutic cell engineering and synthetic biology.

About the Study

Dual-functionality for therapeutic precision: SNIPRs can sense both natural and synthetic ligands, enabling dual applications in detecting disease states and creating bio-orthogonal cellular communication networks.

SNIPRs were designed to detect soluble tumor-associated cytokines like transforming growth factor β (TGF-β) and vascular endothelial growth factor α (VEGF) using single-chain variable fragments (scFvs) from antibodies against these cytokines. SNIPRs were integrated into human CD3+ T-cells and tested for activity upon ligand binding. Performance was tuned by adjusting scFv properties and hinge domains. For example, mutating a cysteine residue in the hinge domain to serine enhanced sensitivity and reduced background activation. SNIPR activation was studied using small molecule inhibitors like DAPT and chloroquine. “OrthoSNIPR” signaling was tuned with ligands of different valency and geometry, using synthetic heterodimers for bio-orthogonal communication.

To assess the therapeutic potential of soluble SNIPR-CAR circuits, SNIPRs were co-cultured with tumor cell lines, and ligand production and CAR expression were measured. This included testing SNIPRs against a variety of tumor-associated ligands in vitro and in vivo to confirm their selectivity and therapeutic relevance. In vitro, T-cell killing was assessed via live-cell imaging with different CARs. In vivo, SNIPR-CAR T-cell circuits were tested in mice with melanomas and lung adenocarcinomas, comparing effectiveness to standard CAR-T treatments. The therapeutic window was also tested using a cross-reactive CAR targeting human epidermal growth factor receptor 2 (Her2).

Results and Discussion

SNIPRs successfully activate a transcriptional response in human T-cells upon ligand exposure. Modifications to the scFv identity, orientation, and transactivation domains enhanced SNIPR potency while maintaining specificity. Additionally, SNIPRs detected other factors like fibroblast growth factor 2 and interferon-γ. The bio-orthogonal "orthoSNIPR" system using LHD heterodimers could enable precise control over engineered cellular signaling independent of natural proteins. This system enables "private" communication channels, where only engineered cells recognize and respond to specific synthetic signals.

Broad implications beyond cancer: SNIPRs could also be employed in tissue engineering, developmental biology, and autoimmunity research by detecting gradients of morphogens or cytokines in complex biological systems.

Activation of SNIPRs was shown to occur through an endocytic pathway, where ligand binding triggered receptor internalization, followed by proteolysis in the endosome. This was confirmed through confocal imaging, showing ligand-receptor colocalization in internal compartments. The use of designed synthetic ligands with varying valency and geometry further demonstrated the tunability of SNIPR responses, allowing for fine control of signaling strength and timing. The orthoSNIPR system showed tunability in its signaling response, influenced by ligand valency and geometry, and could support conditional signaling in which external factors modulate activation. Additionally, the system was found to enable autonomous signaling.

In vitro, SNIPR-T-cells responded to soluble factors like TGF-β1 and VEGFα in a dose-dependent manner, with clear correlations found between ligand production and SNIPR activation. SNIPR → CAR circuits demonstrated effective tumor cell killing, with Her2-specific CARs being particularly potent. These circuits showed slower killing kinetics due to delayed CAR expression but also excellent specificity. In vivo, SNIPR → CAR circuits improved safety and efficacy compared to constitutive CAR-T cells. For example, cross-reactive CAR circuits designed to reduce lung toxicity showed effective tumor control without the severe off-target effects observed with conventional CAR therapies.

Conclusion

In conclusion, the study suggests that soluble SNIPRs are promising and versatile tools for therapeutic bioengineering and biology, given their ability to sense gradients during development and report immune states in diseases such as cancer and autoimmunity. The compact and customizable design of SNIPRs supports complex biocomputational circuits and multi-receptor systems, enabling precise cellular control for therapy and research.

Future studies on SNIPRs could explore optimizing response profiles through modifications like hinge domain engineering, enabling finer control of receptor activity. Additionally, integrating SNIPRs with multi-receptor circuits or combining soluble and membrane-bound ligand detection could expand their applicability to dynamic and complex tissue environments. Investigating dual activation by soluble and membrane-bound ligands may enhance precision in targeting microenvironments. Additionally, integrating SNIPRs with multi-cell signaling networks could help unlock advanced therapeutic strategies for complex diseases. These applications extend beyond cancer therapy to developmental biology and tissue engineering, where controlled signaling gradients are critical.

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