Combination of Degron-based PROTACs and CAR-T
What are CAR-T and PROTAC?
Engineered cells can sense environmental signals, process them through genetic circuits, and respond therapeutically. Chimeric antigen receptors (CAR) consist of a custom extracellular domain (typically a single stranded variable fragment (scFv)) and a signaling domain from T cell receptors and associated co-stimulatory receptors. These synthetic receptors can redirect T cell signals to cancer cells, showing complete remission in more than 85 percent of patients with blood cancers that are resistant to other treatments such as chemotherapy. However, effective and consistent CAR signals can be a double-edged sword. High levels of chimeric antigen receptor T-cell (CAR-T) activity are associated with cytokine release syndrome (CRS), leading to severe inflammation, life-threatening shock, and organ failure.
Studies in preclinical models have shown that small-molecule-induced CAR protein degradation can inhibit signaling and therapeutic activity and alleviate CAR T cell failure. While these small molecule-based systems have reversible and efficient degradation capabilities, they systematically underregulate CAR expression in individual cell populations, potentially hindering therapeutic effectiveness. Regulating CAR expression through gene circuits enables intercellular decision-making to regulate T cell signals in response to local environmental signals, thereby improving the safety and effectiveness of therapy. A key example of a gene circuit in cell therapy is the AND logic gate, which utilizes antigen-sensing receptors such as synthetic Notch (synNotch) and synthetic intramembrane proteolytic receptors (SNIPR) to activate CAR expression. SynNotch and SNIPR are forced-sensitive chimeric receptors composed of extracellular antigen binding domains, transmembrane domains and near-membrane domains.
From mammalian Notch receptors and intracellular synthesis of transcription factors. Antigen binding triggers proteolytic cleavage and the release of transcription factors from the plasma membrane to activate the expression of custom genetic payloads. This topology with grid circuits improves the specificity and safety of CAR-T cells in mouse models and has even been shown to reduce depletion. Gene circuits have also been constructed with cell state promoters, or synnotches, to drive the production of cytokines in response to specific cellular environments to improve CAR-T cell tumor clearance. These examples demonstrate the potential of gene circuits to enhance engineered cell therapy.
The coupling of gene circuits with protein degradation could improve strategies for controlling signal transduction in CAR-T cells. However, existing small molecular-based protein degradation methods for controlling CAR are not suitable for incorporation into genetic circuits. Another way to induce degradation of the protein of interest (POI) is to use protein-based heterobifunctional molecules, known as bioPROTACs. BioPROTAC is a modular molecule consisting of one protein domain that binds POI and another that promotes POI ubiquitination. Endogenous protein-binding domains or mods and engineered proteins, such as DARPins, nanobodies and monomers, have been successfully used to target a variety of proteins, including HER2, PCNA, and KRAS, for degradation using bioPROTAC molecules. To facilitate POI ubiquitination, most bioPROTACs utilize truncated endogenous E3 ligases that, interestingly, degenerate, short degradation-inducing sequences, can replace these larger protein domains while maintaining bioPROTAC degradation activity. Degron is known to interact with E3 ligase and has been screened for use in developing bioPROTACs that target tau proteins. BioPROTACs are potentially powerful tools for cell engineering because they can be easily designed to target different POIs through regional switching and are genetically codible, enabling them to compose genetic circuits.
Recently, the journal ACS of the American Chemical Society published an article titled "Degron-Based bioPROTACs for Controlling Signaling in CAR T Cells" to introduce a novel bioPROTACs optimized for T cell engineering.
Schematic diagram of degron-based bioPROTACs. (Kim, M. S., 2024)
BioPROTACs degrade cytoplasmic proteins in T cells
In this work, the researchers developed a novel bioPROTACs designed to effectively degrade cytoplasmic proteins within human T cells. Conventional PROTACs rely on large E3 ubiquitin ligase fragments to guide the ubiquitin proteasome system (UPS) to degrade target proteins, however, this limits its application in cell engineering because E3 ubiquitin ligases and their domains can be quite large, which poses a potential challenge for delivering large genetic payloads into cells. To solve this problem, the researchers used smaller degron sequences, such as the RRRG short peptide sequence consisting of only four amino acids, which effectively promoted the ubiquitination and degradation of the target protein.
In the experiment, various degron sequences were fused to the C-terminal of green fluorescent protein (GFP), and Jurkat cells were transduced by lentiviral vector. Flow cytometry was used to monitor the change of fluorescence intensity of GFP, so as to evaluate the degradation efficiency of different degron. The results showed that RRRG degron significantly reduced the fluorescence signal of GFP, while other degrons did not show similar effects. Therefore, RRRG was chosen as the degron in subsequent experiments.
The team then designed a variety of bioPROTACs using vhhGFP4 nanobodies and synthetic leucine zips (SynZips) as binders between the target protein and degron. The adhesive is connected to the degron via 3x(GS) flexible connectors to form the bioPROTACs complex. In Jurkat T cells, after co-transducing lentiviruses encoding GFP and bioPROTACs, it was found that SynZip mediated bioPROTACs was effective in inducing GFP degradation, while the vhhGFP4 nanosome-based version was less effective, reducing GFP fluorescence by only 1.45 times.
To enhance the activity of bioPROTACs, the researchers introduced the K→R mutation, which replaces lysine residues with arginine, to reduce cis-ubiquitination, thereby avoiding its own degradation and promoting trans-ubiquitination of the target protein. This strategy significantly improved the performance of bioPROTACs, and for SynZip18, the activity increased to 12 times that of the wild type, and the GFP fluorescence decreased by 60 times. For vhhGFP4 nanoparticles, the activity increased by 100 times and the GFP fluorescence decreased by 204 times. These optimized bioPROTACs provide a powerful tool for the efficient degradation of target proteins in T cells and are expected to play an important role in the field of cell engineering.
BioPROTACs function through UPS and cullin ring ligases
To determine the broader applicability of these new bioPROTACs, the researchers tested them in multiple types of cells, including mESCs, human CD4+ T cells, 3T3 fibroblasts, HEK293T cells, and K562 leukemia cells. Co-transducing GFP reporter genes and bioPROTACs based on vhhGFP4 nanobodies or SynZip18 validate their potential for widespread application. SynZip18 bioPROTAC significantly reduced GFP fluorescence intensity by at least 33 times in all cell types, in contrast to the control group, while vhhGFP4 nanobody bioPROTAC showed similar activity levels. Further studies on the mechanism of bioPROTACs showed that the degradation of GFP was mainly driven by the cullin ring ligase mediated UPS pathway, while the lysosomal pathway contributed relatively little, which strengthened the key role of UPS in bioPROTACs function.
Plasma membrane localization of bioPROTACs enhances internalization of CAR
The research team explored the potential of bioPROTACs in promoting the internalization of CAR to regulate the precision of CAR-T cell therapies. By lentiviral transduction of Jurkat T cells to simultaneously express GFP, SynZip18 bioPROTAC, and SynZip17 labeled CAR, we evaluated the effect of bioPROTACs on the cytoplasmic endogenesis of anti-CD19 CAR and anti-HER2 CAR. Flow cytometry was used to detect the surface expression of myc epitope-labeled cars, and the results showed that SynZip18 bioPROTAC significantly reduced the surface expression of anti-CD19 by up to 80%, while it had little effect on HER2.
To further enhance the internalization effect of CAR and reduce potential signal interference, the researchers designed "membrane-based bioPROTAC" to increase its concentration on the plasma membrane by fusing SynZip18 bioPROTAC with membrane localization sequences, such as DAP10 signal sequences or lyn membrane targeting tags. This design optimizes the co-localization of bioPROTAC with CAR, thereby more effectively mediating CAR internalization. Compared to the original "cytoplasmic bioPROTAC," membrane-based bioPROTAC showed a significant 86% improvement in the internalization of anti-HER2 CAR, and achieved a more than 99% decrease in surface expression on anti-CD19 CAR.
Conclusion
CAR T cells have made a huge impact in the clinic, but strong signaling through CAR can be detrimental to the safety and efficacy of therapy. Using protein degradation to control CAR signaling can address these issues in preclinical models.
Existing strategies for regulating CAR stability rely on small molecule induced system degradation. Compared to small molecule regulation, genetic circuits provide a more precise way to control CAR signaling in an independent, cell-by-cell manner. Here, the authors describe a programmable protein degradation tool that utilizes a bioPROTACs framework for heterofunctional proteins consisting of target recognition domains fused into domains that recruit the endocystitic proteasome system. The researchers developed a novel bioPROTACs that utilize compact four-residue degraders and use nanosomes or synthetic leucine zippers as protein binders to degrade cytoplasmic and membrane protein targets. BioPROTACs showed effective degradation of CAR and inhibited CAR signaling in primary human T cells. Genetic circuits are then constructed to degrade tyrosine kinase ZAP70 in response to recognition of specific membrane binding antigens. ZAP70 is a cytoplasmic tyrosine kinase that is recruited to the plasma membrane and binds to phosphorylated immune receptor tyrosine activating motifs (ITAMs) found in TCR complexes and CARs. In addition, knocking out ZAP70 in CAR-T cells resulted in downstream signal propagation failure after CAR stimulation. Harnessing the important role of ZAP70, the bioPROTAC gene circuit degrades ZAP70 in response to cell-to-cell interactions, thereby weakening CAR-T cell signaling. These results suggest that bioPROTAC is a powerful tool that can expand the toolbox of cell engineering for CAR-T cells and help improve the safety and efficacy of CAR-T cell therapy.
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Reference:
- Kim, M. S., et al. Degron-based bioPROTACs for controlling signaling in CAR T cells. ACS Synthetic Biology. 2024.
