Protein-based bandpass filters for controlling cellular signaling with chemical inputs.

Biological signal processing is vital for cellular function. Similar to electronic circuits, cells process signals via integrated mechanisms. In electronics, bandpass filters transmit frequencies with defined ranges, but protein-based counterparts for controlled responses are lacking in engineered biological systems. Here, we rationally design protein-based, chemically responsive bandpass filters (CBPs) showing OFF-ON-OFF patterns that respond to chemical concentrations within a specific range and reject concentrations outside that range. Employing structure-based strategies, we designed a heterodimeric construct that dimerizes in response to low concentrations of a small molecule (ON), and dissociates at high concentrations of the same molecule (OFF). The CBPs have a multidomain architecture in which we used known drug receptors, a computationally designed protein binder and small-molecule inhibitors. This modular system allows fine-tuning for optimal performance in terms of bandwidth, response, cutoff and fold changes. The CBPs were used to regulate cell surface receptor signaling pathways to control cellular activities in engineered cells.

Rational design of small-molecule responsive protein switches.

Small-molecule responsive protein switches are powerful tools for controlling cellular processes. These switches are designed to respond rapidly and specifically to their inducer. They have been used in numerous applications, including the regulation of gene expression, post-translational protein modification, and signal transduction. Typically, small-molecule responsive protein switches consist of two proteins that interact with each other in the presence or absence of a small molecule. Recent advances in computational protein design already contributed to the development of protein switches with an expanded range of small-molecule inducers and increasingly sophisticated switch mechanisms. Further progress in the engineering of small-molecule responsive switches is fueled by cutting-edge computational design approaches, which will enable more complex and precise control over cellular processes and advance synthetic biology applications in biotechnology and medicine. Here, we discuss recent milestones and how technological advances are impacting the development of chemical switches.

Rational Design of Chemically Controlled Antibodies and Protein Therapeutics.

Protein-based therapeutics, such as monoclonal antibodies and cytokines, are important therapies for various pathophysiological conditions such as oncology, autoimmune disorders, and viral infections. However, the wide application of such protein therapeutics is often hindered by dose-limiting toxicities and adverse effects, namely, cytokine storm syndrome, organ failure, and others. Therefore, spatiotemporal control of the activities of these proteins is crucial to further expand their application. Here, we report the design and application of small-molecule-controlled switchable protein therapeutics by taking advantage of a previously engineered OFF-switch system. We used the Rosetta modeling suite to computationally optimize the affinity between B-cell lymphoma 2 (Bcl-2) protein and a previously developed computationally designed protein partner (LD3) to obtain a fast and efficient heterodimer disruption upon the addition of a competing drug (Venetoclax). The incorporation of the engineered OFF-switch system into anti-CTLA4, anti-HER2 antibodies, or an Fc-fused IL-15 cytokine demonstrated an efficient disruption in vitro, as well as fast clearance in vivo upon the addition of the competing drug Venetoclax. These results provide a proof-of-concept for the rational design of controllable biologics by introducing a drug-induced OFF-switch into existing protein-based therapeutics.

Programmable DARPin-based receptors for the detection of thrombotic markers.

Cellular therapies remain constrained by the limited availability of sensors for disease markers. Here we present an integrated target-to-receptor pipeline for constructing a customizable advanced modular bispecific extracellular receptor (AMBER) that combines our generalized extracellular molecule sensor (GEMS) system with a high-throughput platform for generating designed ankyrin repeat proteins (DARPins). For proof of concept, we chose human fibrin degradation products (FDPs) as markers with high clinical relevance and screened a DARPin library for FDP binders. We built AMBERs equipped with 19 different DARPins selected from 160 hits, and found 4 of them to be functional as heterodimers with a known single-chain variable fragments binder. Tandem receptors consisting of combinations of the validated DARPins are also functional. We demonstrate applications of these AMBER receptors in vitro and in vivo by constructing designer cell lines that detect pathological concentrations of FDPs and respond with the production of a reporter and a therapeutic anti-thrombotic protein.

A rational blueprint for the design of chemically-controlled protein switches.

Small-molecule responsive protein switches are crucial components to control synthetic cellular activities. However, the repertoire of small-molecule protein switches is insufficient for many applications, including those in the translational spaces, where properties such as safety, immunogenicity, drug half-life, and drug side-effects are critical. Here, we present a computational protein design strategy to repurpose drug-inhibited protein-protein interactions as OFF- and ON-switches. The designed binders and drug-receptors form chemically-disruptable heterodimers (CDH) which dissociate in the presence of small molecules. To design ON-switches, we converted the CDHs into a multi-domain architecture which we refer to as activation by inhibitor release switches (AIR) that incorporate a rationally designed drug-insensitive receptor protein. CDHs and AIRs showed excellent performance as drug responsive switches to control combinations of synthetic circuits in mammalian cells. This approach effectively expands the chemical space and logic responses in living cells and provides a blueprint to develop new ON- and OFF-switches.

Synthetic Receptors for Sensing Soluble Molecules with Mammalian Cells.

Synthetic receptors control cell behavior in response to environmental stimuli for applications in basic research and cell therapy. However, the integration of synthetic receptors in unexplored contexts is cumbersome, especially for nonspecialist laboratories. Here, I provide a detailed protocol on how to use receptors of the generalized extracellular molecule sensor (GEMS) platform. GEMS is a modular receptor system that can be adapted to sense molecules of choice by using affinity domains that dimerize in response to the target. GEMS consist of an erythropoietin receptor scaffold that has been mutated to no longer bind to erythropoietin. N-terminal fusions with affinity domains, such as single chain variable fragments (scFvs), that bind to two epitopes on the same target activate the receptor. The intracellular receptor domain can be chosen from several signal transduction domains of single-pass transmembrane receptors to activate endogenous signaling pathways. As of now, GEMS have been used for sensing prostate specific antigen (PSA), the synthetic azo dye RR120, caffeine, nicotine, rapamycin, the SunTag peptide, and a de novo designed protein displaying two viral epitopes. The tested intracellular domains were derived from FGFR1, IL-6RB, and VEGFR2, and were used to drive transgene expression from reporter plasmids responsive to the endogenous transcription factors STAT3, NFAT, NF-κB, and a synthetic transcription factor activated by the MAPK pathway. In this protocol, I focus on transient transfections of HEK293T cells and include several general notes about cell handling. While the described methods are optimized for experiments with GEMS, most of the described techniques are general procedures to set up synthetic biology experiments in mammalian cell culture. I outline how to generate stable cell lines and share tips on how to adapt GEMS for new ligands. The main objective of this protocol is to make the GEMS technology accessible also to nonspecialist laboratories to facilitate the use of synthetic receptors in new research contexts.

Bottom-up de novo design of functional proteins with complex structural features.

De novo protein design has enabled the creation of new protein structures. However, the design of functional proteins has proved challenging, in part due to the difficulty of transplanting structurally complex functional sites to available protein structures. Here, we used a bottom-up approach to build de novo proteins tailored to accommodate structurally complex functional motifs. We applied the bottom-up strategy to successfully design five folds for four distinct binding motifs, including a bifunctionalized protein with two motifs. Crystal structures confirmed the atomic-level accuracy of the computational designs. These de novo proteins were functional as components of biosensors to monitor antibody responses and as orthogonal ligands to modulate synthetic signaling receptors in engineered mammalian cells. Our work demonstrates the potential of bottom-up approaches to accommodate complex structural motifs, which will be essential to endow de novo proteins with elaborate biochemical functions, such as molecular recognition or catalysis.

Calcium-Mediated Liposome Fusion to Engineer Giant Lipid Vesicles with Cytosolic Proteins and Reconstituted Mammalian Proteins.

Giant unilamellar lipid vesicles (GUVs) are widely used as model membrane systems and provide an excellent basis to construct artificial cells. To construct more sophisticated artificial cells, proteins-in particular membrane proteins-need to be incorporated in GUVs. However, current methods for protein reconstitution have limited throughput or are not generally applicable for all proteins because they depend on detergent solubilization. This limitation is addressed here by introducing calcium-mediated membrane fusion to transfer proteins between negatively charged GUVs and cell-derived plasma membrane vesicles (CDVs), derived from HEK293T cells overexpressing a membrane receptor protein. Fusion conditions are optimized using large unilamellar vesicles and GUVs containing phosphatidylserines and fusogenic lipids. The approach is then applied to induce lipid mixing and subsequent transfer of the overexpressed membrane receptor from CDVs into GUVs. The membrane receptor is detected by immunofluorescence on GUVs that underwent lipid mixing with CDVs. Those GUVs also exhibit esterase activity because cytosolic esterases entrapped in the CDVs are transferred during membrane fusion. Thus, content mixing is demonstrated. Using CDVs circumvents the need to purify or solubilize proteins. Moreover, calcium-mediated fusion allows transfer of lipids, water-soluble and membrane bound proteins in one step, resulting in a semi-synthetic cell.

Phosphoregulated orthogonal signal transduction in mammalian cells.

Orthogonal tools for controlling protein function by post-translational modifications open up new possibilities for protein circuit engineering in synthetic biology. Phosphoregulation is a key mechanism of signal processing in all kingdoms of life, but tools to control the involved processes are very limited. Here, we repurpose components of bacterial two-component systems (TCSs) for chemically induced phosphotransfer in mammalian cells. TCSs are the most abundant multi-component signal-processing units in bacteria, but are not found in the animal kingdom. The presented phosphoregulated orthogonal signal transduction (POST) system uses induced nanobody dimerization to regulate the trans-autophosphorylation activity of engineered histidine kinases. Engineered response regulators use the phosphohistidine residue as a substrate to autophosphorylate an aspartate residue, inducing their own homodimerization. We verify this approach by demonstrating control of gene expression with engineered, dimerization-dependent transcription factors and propose a phosphoregulated relay system of protein dimerization as a basic building block for next-generation protein circuits.

Genetically encoded betaxanthin-based small-molecular fluorescent reporter for mammalian cells.

We designed and engineered a dye production cassette encoding a heterologous pathway, including human tyrosine hydroxylase and Amanita muscaria 4,5-DOPA dioxygenase, for the biosynthesis of the betaxanthin family of plant and fungal pigments in mammalian cells. The system does not impair cell viability, and can be used as a non-protein reporter system to directly visualize the dynamics of gene expression by profiling absorbance or fluorescence in the supernatant of cell cultures, as well as for fluorescence labeling of individual cells. Pigment profiling can also be multiplexed with reporter proteins such as mCherry or the human model glycoprotein SEAP (secreted alkaline phosphatase). Furthermore, absorbance measurement with a smartphone camera using standard application software enables inexpensive, low-tech reporter quantification.

Rewiring of endogenous signaling pathways to genomic targets for therapeutic cell reprogramming.

Rewiring cellular sensors to trigger non-natural responses is fundamental for therapeutic cell engineering. Current designs rely on engineered receptors that are limited to single inputs, and often suffer from high leakiness and low fold induction. Here, we present Generalized Engineered Activation Regulators (GEARs) that overcome these limitations by being pathway-specific rather than input-specific. GEARs consist of the MS2 bacteriophage coat protein fused to regulatory or transactivation domains, and work by rerouting activation of the NFAT, NFκB, MAPK or SMAD pathways to dCas9-directed gene expression from genomic loci. This system enables membrane depolarization-induced activation of insulin expression in ß-mimetic cells and IL-12 expression in activated Jurkat cells, as well as IL-12 production in response to the immunomodulatory cytokines TGFß and TNFα in HEK293T cells. Engineered cells with the ability to reinterpret the extracellular milieu have potential for applications in immunotherapy and in the treatment of metabolic diseases.

A modular degron library for synthetic circuits in mammalian cells.

Tight control over protein degradation is a fundamental requirement for cells to respond rapidly to various stimuli and adapt to a fluctuating environment. Here we develop a versatile, easy-to-handle library of destabilizing tags (degrons) for the precise regulation of protein expression profiles in mammalian cells by modulating target protein half-lives in a predictable manner. Using the well-established tetracycline gene-regulation system as a model, we show that the dynamics of protein expression can be tuned by fusing appropriate degron tags to gene regulators. Next, we apply this degron library to tune a synthetic pulse-generating circuit in mammalian cells. With this toolbox we establish a set of pulse generators with tailored pulse lengths and magnitudes of protein expression. This methodology will prove useful in the functional roles of essential proteins, fine-tuning of gene-expression systems, and enabling a higher complexity in the design of synthetic biological systems in mammalian cells.

From synthetic biology to human therapy: engineered mammalian cells.

Mammalian synthetic biology has evolved to become a key driver of biomedical innovation in the area of cell therapy. Advances in receptor engineering, immunotherapy and cell implants promise new treatment options for complex diseases. Synthetic receptors have already found applications in cellular immunotherapy for cancer treatment, and are being introduced into the field of cell implants. Here, we discuss prospects for the next generation of engineered mammalian cells for human therapy, highlighting selected recent studies.

Caffeine-inducible gene switches controlling experimental diabetes.

Programming cellular behavior using trigger-inducible gene switches is integral to synthetic biology. Although significant progress has been achieved in trigger-induced transgene expression, side-effect-free remote control of transgenes continues to challenge cell-based therapies. Here, utilizing a caffeine-binding single-domain antibody we establish a caffeine-inducible protein dimerization system, enabling synthetic transcription factors and cell-surface receptors that enable transgene expression in response to physiologically relevant concentrations of caffeine generated by routine intake of beverages such as tea and coffee. Coffee containing different caffeine concentrations dose-dependently and reversibly controlled transgene expression by designer cells with this caffeine-stimulated advanced regulators (C-STAR) system. Type-2 diabetic mice implanted with microencapsulated, C-STAR-equipped cells for caffeine-sensitive expression of glucagon-like peptide 1 showed substantially improved glucose homeostasis after coffee consumption compared to untreated mice. Biopharmaceutical production control by caffeine, which is non-toxic, inexpensive and only present in specific beverages, is expected to improve patient compliance by integrating therapy with lifestyle.

Generalized extracellular molecule sensor platform for programming cellular behavior.

Strategies for expanding the sensor space of designer receptors are urgently needed to tailor cell-based therapies to respond to any type of medically relevant molecules. Here, we describe a universal approach to designing receptor scaffolds that enables antibody-specific molecular input to activate JAK/STAT, MAPK, PLCG or PI3K/Akt signaling rewired to transgene expression driven by synthetic promoters. To demonstrate its scope, we equipped the GEMS (generalized extracellular molecule sensor) platform with antibody fragments targeting a synthetic azo dye, nicotine, a peptide tag and the PSA (prostate-specific antigen) biomarker, thereby covering inputs ranging from small molecules to proteins. These four GEMS devices provided robust signaling and transgene expression with high signal-to-noise ratios in response to their specific ligands. The sensitivity of the nicotine- and PSA-specific GEMS devices matched the clinically relevant concentration ranges, and PSA-specific GEMS were able to detect pathological PSA levels in the serum of patients diagnosed with prostate cancer.

Nonimmune cells equipped with T-cell-receptor-like signaling for cancer cell ablation.

The ability to engineer custom cell-contact-sensing output devices into human nonimmune cells would be useful for extending the applicability of cell-based cancer therapies and for avoiding risks associated with engineered immune cells. Here we have developed a new class of synthetic T-cell receptor-like signal-transduction device that functions efficiently in human nonimmune cells and triggers release of output molecules specifically upon sensing contact with a target cell. This device employs an interleukin signaling cascade, whose OFF/ON switching is controlled by biophysical segregation of a transmembrane signal-inhibitory protein from the sensor cell-target cell interface. We further show that designer nonimmune cells equipped with this device driving expression of a membrane-penetrator/prodrug-activating enzyme construct could specifically kill target cells in the presence of the prodrug, indicating its potential usefulness for target-cell-specific, cell-based enzyme-prodrug cancer therapy. Our study also contributes to the advancement of synthetic biology by extending available design principles to transmit extracellular information to cells.