De novo design of potent and resilient hACE2 decoys to neutralize SARS-CoV-2

We developed a de novo protein design strategy to swiftly engineer decoys for neutralizing pathogens that exploit extracellular host proteins to infect the cell. Our pipeline allowed the design, validation, and optimization of de novo hACE2 decoys to neutralize SARS-CoV-2. The best decoy, CTC-445.2, binds with low nanomolar affinity and high specificity to the RBD of the spike protein. Cryo-EM shows that the design is accurate and can simultaneously bind to all three RBDs of a single spike protein. Because the decoy replicates the spike protein target interface in hACE2, it is intrinsically resilient to viral mutational escape. A bivalent decoy, CTC-445.2d, shows ~10-fold improvement in binding. CTC-445.2d potently neutralizes SARS-CoV-2 infection of cells in vitro and a single intranasal prophylactic dose of decoy protected Syrian hamsters from a subsequent lethal SARS-CoV-2 challenge

De novo design of potent and resilient hACE2 decoys to neutralize SARS-CoV-2

We developed a de novo protein design strategy to swiftly engineer decoys for neutralizing pathogens that exploit extracellular host proteins to infect the cell. Our pipeline allowed the design, validation, and optimization of de novo hACE2 decoys to neutralize SARS-CoV-2. The best decoy, CTC-445.2, binds with low nanomolar affinity and high specificity to the RBD of the spike protein. Cryo-EM shows that the design is accurate and can simultaneously bind to all three RBDs of a single spike protein. Because the decoy replicates the spike protein target interface in hACE2, it is intrinsically resilient to viral mutational escape. A bivalent decoy, CTC-445.2d, shows ~10-fold improvement in binding. CTC-445.2d potently neutralizes SARS-CoV-2 infection of cells in vitro and a single intranasal prophylactic dose of decoy protected Syrian hamsters from a subsequent lethal SARS-CoV-2 challenge

Trehalose Glycopolymers for Protein and Cell Stabilization

Proteins, biomolecules, and cells play an important role for their use as therapeutics and reagents for research applications. However, one main limitation is their inherent instability. Proteins can easily denature and lose activity under a variety of stresses, and cells can easily lose viability due to cytotoxic compounds and conditions. This has led to the investigation of excipients that can aid in preserving activity, including polymers, osmolytes, proteins, and sugars. Among the sugars, trehalose has been shown to have remarkable properties for general protein and cell stabilization. This dissertation presents the utilization of trehalose glycopolymers for the stabilization of cells and proteins in three different applications. The polymers were used for protein stabilization during protein micro- and nano-patterning, stabilization of a therapeutic-relevant protein, and for cell preservation.Direct write patterning of multiple biological molecules on surfaces has tremendous potential in applications of tissue engineering, diagnostics, proteomics and biosensors. Precise control over the position and arrangement of proteins, especially on the micro- and nano- scale level is a particular challenge towards the improvement of miniaturized devices. A trehalose glycopolymer was utilized as a resist material that protects proteins during electron beam lithography, and thus enables direct write multiplexed protein patterns with micro- and nano- scale features and alignment capabilities. The trehalose glycopolymers behave as negative resists, crosslinking to the substrate upon electron beam irradiation, and provide stabilization to proteins against the harsh processing conditions. Patterns with user-defined shapes were generated with a variety of protein types, such as antibodies, enzymes, and growth factors, and were shown to retain their activity. To further demonstrate the technique, fabrication of antibody patterns for multiplexed cytokine detection from live cells was achieved. A sandwich immunoassay was developed for the detection of interleukin-6 (IL-6) and tumor necrosis factor alpha (TNFα) secreted directly from stimulated macrophage cells. Multiplexing with both IL-6 and TNFα on a single chip was demonstrated successfully with high specificity and in relevant cell culture conditions. The ability to monitor cytokine release over time following cell stimulation was also demonstrated. Simultaneous detection of these extracellular signaling markers demonstrates the potential application towards disease profiling and diagnostics.Next, the trehalose glycopolymers were used for the stabilization and enhancement of the pharmacokinetic properties of a therapeutic protein, granulocyte colony-stimulating factor (G-CSF). G-CSF was designed with a polyhistidine-tag, maltose binding protein for solubility, and an enzyme cleavage site, and was expressed in E. coli and purified for the studies. Trehalose polymers with polystyrene or polymethacrylate backbones (P1, P2, and P3), as well as a degradable trehalose polymer (p(BMDO-co-trehalose)) were synthesized to investigate their potential for therapeutic use. The trehalose polymers were shown to be non-cytotoxic in mouse and human cell lines up to a concentration of 8 mg/mL. However, the degraded products of p(BMDO-co-trehalose) exhibited cytotoxicity at a lower concentration of 1 mg/mL. Stability screening with the trehalose glycopolymers on G-CSF showed that styrenyl acetal linked trehalose polymer, P1, best stabilized G-CSF compared to the other polymers and was selected for G-CSF conjugation. Therefore, P1 with a benzaldehyde end group was synthesized and conjugated to G-CSF via reductive amination. The resulting G-CSF-P1 conjugate retained higher bioactivity compared to native G-CSF when subjected to heat and lyophilization stressors.Finally, the trehalose glycopolymers were investigated for the stabilization and preservation of cells. Three different approaches were evaluated. First, extracellular protection by the covalent attachment of trehalose polymers to cells using modified sugars was tested. To evaluate the effectiveness of extracellular trehalose on cell stabilization, the trehalose polymers were added as excipients. Jurkat cells were heated at 50 ?C and the added trehalose polymer did not significantly stabilize the cells at longer stress times. Cryopreservation with a combination of the polymer and dimethylsulfoxide (DMSO) showed slight increase in cell viabilities (65.7% ? 3.1 with polymer and DMSO compared to 55.6% ? 2.1 with DMSO only), but the results suggest that trehalose polymers are not likely to fully replace DMSO as a cryopreservative agent. The final approach utilized a bacterial pore-forming toxin, streptolysin O (SLO), to permeabilize human dermal fibroblast (HDF) cells for loading trehalose polymers. Permeabilization and loading was characterized by fluorescence with a fluorophore-labeled trehalose polymer and by the anthrone assay. No significant improvements in cell preservation were observed through trehalose polymer loading by this approach. Other techniques for cytosolic uptake of trehalose polymers should be considered for future work

Trehalose glycopolymer resists allow direct writing of protein patterns by electron-beam lithography

Direct-write patterning of multiple proteins on surfaces is of tremendous interest for a myriad of applications. Precise arrangement of different proteins at increasingly smaller dimensions is a fundamental challenge to apply the materials in tissue engineering, diagnostics, proteomics and biosensors. Herein, we present a new resist that protects proteins during electron-beam exposure and its application in direct-write patterning of multiple proteins. Polymers with pendant trehalose units are shown to effectively crosslink to surfaces as negative resists, while at the same time providing stabilization to proteins during the vacuum and electron-beam irradiation steps. In this manner, arbitrary patterns of several different classes of proteins such as enzymes, growth factors and immunoglobulins are realized. Utilizing the high-precision alignment capability of electron-beam lithography, surfaces with complex patterns of multiple proteins are successfully generated at the micrometre and nanometre scale without requiring cleanroom conditions

Trehalose Glycopolymers as Excipients for Protein Stabilization

Herein, the synthesis of four different trehalose glycopolymers and investigation of their ability to stabilize proteins to heat and lyophilization stress are described. The disaccharide, alpha,alpha-trehalose, was modified with a styrenyl acetal, methacrylate acetal, styrenyl ether, or methacrylate moiety resulting in four different monomers. These monomers were then separately polymerized using free radical polymerization with azobisisobutyronitrile (AIBN) as an initiator to synthesize the glycopolymers. Horseradish peroxidase and glucose oxidase were incubated at 70 and 50 degrees C, respectively, and beta-galactosidase was lyophilized multiple times in the presence of various ratios of the polymers or trehalose. The protein activities were subsequently tested and found to be significantly higher when the polymers were present during the stress compared to no additive and to equivalent amounts of trehalose. Different molecular weights (10 kDa, 20 kDa, and 40 kDa) were tested, and all were equivalent in their stabilization ability. However, some subtle differences were observed regarding stabilization ability between the different polymer samples, depending on the stress. Small molecules such as benzyl ether trehalose were not better stabilizers than trehalose, and the trehalose monomer decreased protein activity, suggesting that hydrophobized trehalose was not sufficient and that the polymeric structure was required. In addition, cytotoxicity studies with NIH 3T3 mouse embryonic fibroblast cells, RAW 264.7 murine macrophages, human dermal fibroblasts (HDFs), and human umbilical vein endothelial cells (HUVECs) were conducted with polymer concentrations up to 8 mg/mL. The data showed that all four polymers were noncytotoxic for all tested concentrations. The results together suggest that trehalose glycopolymers are promising as additives to protect proteins from a variety of stressors

Direct Write Protein Patterns for Multiplexed Cytokine Detection from Live Cells Using Electron Beam Lithography

Simultaneous detection of multiple biomarkers, such as extracellular signaling molecules, is a critical aspect in disease profiling and diagnostics. Precise positioning of antibodies on surfaces, especially at the micro- and nanoscale, is important for the improvement of assays, biosensors, and diagnostics on the molecular level, and therefore, the pursuit of device miniaturization for parallel, fast, low-volume assays is a continuing challenge. Here, we describe a multiplexed cytokine immunoassay utilizing electron beam lithography and a trehalose glycopolymer as a resist for the direct writing of antibodies on silicon substrates, allowing for micro- and nanoscale precision of protein immobilization. Specifically, anti-interleukin 6 (IL-6) and antitumor necrosis factor alpha (TNFα) antibodies were directly patterned. Retention of the specific binding properties of the patterned antibodies was shown by the capture of secreted cytokines from stimulated RAW 264.7 macrophages. A sandwich immunoassay was employed using gold nanoparticles and enhancement with silver for the detection and visualization of bound cytokines to the patterns by localized surface plasmon resonance detected with dark-field microscopy. Multiplexing with both IL-6 and TNFα on a single chip was also successfully demonstrated with high specificity and in relevant cell culture conditions and at different times after cell stimulation. The direct fabrication of capture antibody patterns for cytokine detection described here could be useful for biosensing applications

Trehalose Glycopolymers as Excipients for Protein Stabilization

Herein, the synthesis of four different trehalose glycopolymers and investigation of their ability to stabilize proteins to heat and lyophilization stress are described. The disaccharide, α,α-trehalose, was modified with a styrenyl acetal, methacrylate acetal, styrenyl ether, or methacrylate moiety resulting in four different monomers. These monomers were then separately polymerized using free radical polymerization with azobisisobutyronitrile (AIBN) as an initiator to synthesize the glycopolymers. Horseradish peroxidase and glucose oxidase were incubated at 70 and 50 °C, respectively, and β-galactosidase was lyophilized multiple times in the presence of various ratios of the polymers or trehalose. The protein activities were subsequently tested and found to be significantly higher when the polymers were present during the stress compared to no additive and to equivalent amounts of trehalose. Different molecular weights (10 kDa, 20 kDa, and 40 kDa) were tested, and all were equivalent in their stabilization ability. However, some subtle differences were observed regarding stabilization ability between the different polymer samples, depending on the stress. Small molecules such as benzyl ether trehalose were not better stabilizers than trehalose, and the trehalose monomer decreased protein activity, suggesting that hydrophobized trehalose was not sufficient and that the polymeric structure was required. In addition, cytotoxicity studies with NIH 3T3 mouse embryonic fibroblast cells, RAW 264.7 murine macrophages, human dermal fibroblasts (HDFs), and human umbilical vein endothelial cells (HUVECs) were conducted with polymer concentrations up to 8 mg/mL. The data showed that all four polymers were noncytotoxic for all tested concentrations. The results together suggest that trehalose glycopolymers are promising as additives to protect proteins from a variety of stressors

Correction to Trehalose Glycopolymers as Excipients for Protein Stabilization

Correction to Trehalose Glycopolymers as Excipients for Protein Stabilizatio