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Abstract The design of a fully synthetic, chemical “apoptosis‐inducing receptor” (AIR) molecule is reported that is anchored into the lipid bilayer of cells, is activated by the incoming biological input, and responds with the release of a secondary messenger—a highly potent toxin for cell killing. The AIR molecule has four elements, namely, an exofacial trigger group, a bilayer anchor, a toxin as a secondary messenger, and a self‐immolative scaffold as a mechanism for signal transduction. Receptor installation into cells is established via a robust protocol with minimal cell handling. The synthetic receptor remains dormant in the engineered cells, but is effectively triggered externally by the addition of an activating biomolecule (enzyme) or in a mixed cell population through interaction with the surrounding cells. In 3D cell culture (spheroids), receptor activation is accessible for at least 5 days, which compares favorably with other state of the art receptor designs.

Abstract The newest generation of cell‐based technologies relies heavily on methods to communicate to the engineered cells using artificial receptors, specifically to deactivate the cells administered to a patient in the event of adverse effects. Herein, artificial synthetic internalizing receptors are engineered that function in mammalian cells in 2D and in 3D and afford targeted, specific intracellular drug delivery with nanomolar potency in the most challenging cell type, namely primary, donor‐derived T cells. Receptor design comprises a lipid bilayer anchor for receptor integration into cell membrane and a small xenobiotic molecule as a recognition ligand. Artificial receptors are successfully targeted by the corresponding antibody–drug conjugate (ADC) and exhibit efficient cargo cell entry with ensuing intracellular effects. Receptor integration into cells is fast and robust and affords targeted cell entry in under 2 h. Through a combination of the receptor design and the use of ADC, combined benefits previously made available by chimeric artificial receptors (performance in T cells) and the chemical counterpart (robustness and simplicity) in a single functional platform is achieved. Artificial synthetic receptors are poised to facilitate the maturation of engineered cells as tools of biotechnology and biomedicine.

Epidermal growth factor receptor (EGFR) is a clinically validated target for a multitude of human cancers. The receptor is activated upon ligand binding through a critical dimerization step. Dimerization can be replicated in vitro by locally concentrating the receptor kinase domains on the surface of lipid-based vesicles. In this study we investigated the use of coiled coils to induce spontaneous receptor kinase domain dimerization in vitro to form non-membrane-bound artificial receptor mimics in solution. Two engineered forms of EGFR kinase domain fused to coiled coil complementary peptides were designed to self-associate upon mixing. Two fusion protein species (P3-EGFR and P4-EGFR) independently showed the same activity and polymerization profile known to exist with EGFR kinase domains. Upon mixing the two species, coiled coil heterodimers were formed that induced EGFR association to form dimers of the kinase domains. This was accompanied by 11.5-fold increase in the phosphorylation rate indicative of kinase domain activation equivalent to the levels achieved using vesicle localization and mimicking in vivo ligand-induced activation. This study presents a soluble tyrosine kinase receptor mimic capable of spontaneous in vitro activation that can facilitate functional and drug discovery studies for this clinically important receptor class., (© 2020 Wiley Periodicals LLC.)