Photoacoustic imaging of the dynamics of a dye-labeled IgG4 monoclonal antibody in subcutaneous tissue reveals a transient decrease in murine blood oxygenation under anesthesia.

Significance: Over 100 monoclonal antibodies have been approved by the U.S. Food and Drug Administration (FDA) for clinical use; however, a paucity of knowledge exists regarding the injection site behavior of these formulated therapeutics, particularly the effect of antibody, formulation, and tissue at the injection site. A deeper understanding of antibody behavior at the injection site, especially on blood oxygenation through imaging, will help design improved versions of the therapeutics for a wide range of diseases. Aim: The aim of this research is to understand the dynamics of monoclonal antibodies at the injection site as well as how the antibody itself affects the functional characteristics of the injection site [e.g., blood oxygen saturation (sO2)]. Approach: We employed triple-wavelength equipped functional photoacoustic imaging to study the dynamics of dye-labeled and unlabeled monoclonal antibodies at the site of injection in a mouse ear. We injected a near-infrared dye-labeled (and unlabeled) human IgG4 isotype control antibody into the subcutaneous space in mouse ears to analyze the injection site dynamics and quantify molecular movement, as well as its effect on local hemodynamics. Results: We performed pharmacokinetic studies of the antibody in different regions of the mouse body to show that dye labeling does not alter the pharmacokinetic characteristics of the antibody and that mouse ear is a viable model for these initial studies. We explored the movement of the antibody in the interstitial space to show that the bolus area grows by ∼300% over 24 h. We discovered that injection of the antibody transiently reduces the local sO2 levels in mice after prolonged anesthesia without affecting the total hemoglobin content and oxygen extraction fraction. Conclusions: This finding on local oxygen saturation opens a new avenue of study on the functional effects of monoclonal antibody injections. We also show the suitability of the mouse ear model to study antibody dynamics through high-resolution imaging techniques. We quantified the movement of antibodies at the injection site caused by the interstitial fluid, which could be helpful for designing antibodies with tailored absorption speeds in the future.

Development of a long-acting relaxin analogue, LY3540378, for treatment of chronic heart failure.

BACKGROUND AND PURPOSE: Chronic heart failure, a progressive disease with limited treatment options currently available, especially in heart failure with preserved ejection fraction (HFpEF), represents an unmet medical need as well as an economic burden. The development of a novel therapeutic to slow or reverse disease progression would be highly impactful to patients and society. Relaxin-2 (relaxin) is a human hormone regulating cardiovascular, renal, and pulmonary adaptations during pregnancy. A short-acting recombinant relaxin, Serelaxin, demonstrated short-term heart failure symptom relief and biomarker improvement in acute heart failure trials. Here, we present the development of a long-acting relaxin analogue to be tested in the treatment of chronic heart failure. EXPERIMENTAL APPROACH: LY3540378 is a long-acting protein therapeutic composed of a human relaxin analogue and a serum albumin-binding VHH domain. KEY RESULTS: LY3540378 is a potent agonist of the relaxin family peptide receptor 1 (RXFP1) and maintains selectivity against RXFP2/3/4 comparable to native relaxin. The half-life of LY3540378 in preclinical species is extended through high affinity binding of the albumin-binding VHH domain to serum albumin. When tested in a single dose administration, LY3540378 elicited relaxin-mediated pharmacodynamic responses, such as reduced serum osmolality and increased renal blood flow in rats. In an isoproterenol-induced cardiac hypertrophy mouse model, treatment with LY3540378 significantly reduced cardiac hypertrophy and improved isovolumetric relaxation time. In a monkey cardiovascular safety study, there were no adverse observations from administration of LY3540378. CONCLUSION AND IMPLICATIONS: LY3540378 demonstrated to be a suitable clinical development candidate, and is progressing in clinical trials.

Long-Duration and Non-Invasive Photoacoustic Imaging of Multiple Anatomical Structures in a Live Mouse Using a Single Contrast Agent.

Long-duration in vivo simultaneous imaging of multiple anatomical structures is useful for understanding physiological aspects of diseases, informative for molecular optimization in preclinical models, and has potential applications in surgical settings to improve clinical outcomes. Previous studies involving simultaneous imaging of multiple anatomical structures, for example, blood and lymphatic vessels as well as peripheral nerves and sebaceous glands, have used genetically engineered mice, which require expensive and time-consuming methods. Here, an IgG4 isotype control antibody is labeled with a near-infrared dye and injected into a mouse ear to enable simultaneous visualization of blood and lymphatic vessels, peripheral nerves, and sebaceous glands for up to 3 h using photoacoustic microscopy. For multiple anatomical structure imaging, peripheral nerves and sebaceous glands are imaged inside the injected dye-labeled antibody mass while the lymphatic vessels are visualized outside the mass. The efficacy of the contrast agent to label and localize deep medial lymphatic vessels and lymph nodes using photoacoustic computed tomography is demonstrated. The capability of a single injectable contrast agent to image multiple structures for several hours will potentially improve preclinical therapeutic optimization, shorten discovery timelines, and enable clinical treatments.

Photoacoustic imaging reveals mechanisms of rapid-acting insulin formulations dynamics at the injection site.

OBJECTIVE: Ultra-rapid insulin formulations control postprandial hyperglycemia; however, inadequate understanding of injection site absorption mechanisms is limiting further advancement. We used photoacoustic imaging to investigate the injection site dynamics of dye-labeled insulin lispro in the Humalog® and Lyumjev® formulations using the murine ear cutaneous model and correlated it with results from unlabeled insulin lispro in pig subcutaneous injection model. METHODS: We employed dual-wavelength optical-resolution photoacoustic microscopy to study the absorption and diffusion of the near-infrared dye-labeled insulin lispro in the Humalog and Lyumjev formulations in mouse ears. We mathematically modeled the experimental data to calculate the absorption rate constants and diffusion coefficients. We studied the pharmacokinetics of the unlabeled insulin lispro in both the Humalog and Lyumjev formulations as well as a formulation lacking both the zinc and phenolic preservative in pigs. The association state of insulin lispro in each of the formulations was characterized using SV-AUC and NMR spectroscopy. RESULTS: Through experiments using murine and swine models, we show that the hexamer dissociation rate of insulin lispro is not the absorption rate-limiting step. We demonstrated that the excipients in the Lyumjev formulation produce local tissue expansion and speed both insulin diffusion and microvascular absorption. We also show that the diffusion of insulin lispro at the injection site drives its initial absorption; however, the rate at which the insulin lispro crosses the blood vessels is its overall absorption rate-limiting step. CONCLUSIONS: This study provides insights into injection site dynamics of insulin lispro and the impact of formulation excipients. It also demonstrates photoacoustic microscopy as a promising tool for studying protein therapeutics. The results from this study address critical questions around the subcutaneous behavior of insulin lispro and the formulation excipients, which could be useful to make faster and better controlled insulin formulations in the future.

Production of D-Xylonic Acid from Hemicellulose Using Artificial Enzyme Complexes.

Lignocellulosic biomass represents a potentially large resource to supply the world's fuel and chemical feedstocks. Enzymatic bioconversion of this substrate offers a reliable strategy for accessing this material under mild reaction conditions. Owing to the complex nature of lignocellulose, many different enzymatic activities are required to function in concert to perform efficient transformation. In nature, large multienzyme complexes are known to effectively hydrolyze lignocellulose into constituent monomeric sugars. We created artificial complexes of enzymes, called rosettazymes, in order to hydrolyze glucuronoxylan, a common lignocellulose component, into its cognate sugar D-xylose and then further convert the D-xylose into D-xylonic acid, a Department of Energy top-30 platform chemical. Four different types of enzymes (endoxylanase, α-glucuronidase, ß-xylosidase, and xylose dehydrogenase) were incorporated into the artificial complexes. We demonstrated that tethering our enzymes in a complex resulted in significantly more activity (up to 71%) than the same amount of enzymes free in solution. We also determined that varying the enzyme composition affected the level of complex-related activity enhancement as well as overall yield.

Beyond Helper Phage: Using "Helper Cells" to Select Peptide Affinity Ligands.

Peptides are important affinity ligands for microscopy, biosensing, and targeted delivery. However, because they can have low affinity for their targets, their selection from large naïve libraries can be challenging. When selecting peptidic ligands from display libraries, it is important to: 1) ensure efficient display; 2) maximize the ability to select high affinity ligands; and 3) minimize the effect of the display context on binding. The "helper cell" packaging system has been described as a tool to produce filamentous phage particles based on phagemid constructs with varying display levels, while remaining free of helper phage contamination. Here we report on the first use of this system for peptide display, including the systematic characterization and optimization of helper cells, their inefficient use in antibody display and their use in creating and selecting from a set of phage display peptide libraries. Our libraries were analyzed with unprecedented precision by standard or deep sequencing, and shown to be superior in quality than commercial gold standards. Using our helper cell libraries, we have obtained ligands recognizing Yersinia pestis surface antigen F1V and L-glutamine-binding periplasmic protein QBP. In the latter case, unlike any of the peptide library selections described so far, we used a combination of phage and yeast display to select intriguing peptide ligands. Based on the success of our selections we believe that peptide libraries obtained with helper cells are not only suitable, but preferable to traditional phage display libraries for selection of peptidic ligands.

Production of Glucaric Acid from Hemicellulose Substrate by Rosettasome Enzyme Assemblies.

Hemicellulose biomass is a complex polymer with many different chemical constituents that can be utilized as industrial feedstocks. These molecules can be released from the polymer and transformed into value-added chemicals through multistep enzymatic pathways. Some bacteria produce cellulosomes which are assemblies composed of lignocellulolytic enzymes tethered to a large protein scaffold. Rosettasomes are artificial engineered ring scaffolds designed to mimic the bacterial cellulosome. Both cellulosomes and rosettasomes have been shown to facilitate much higher rates of biomass hydrolysis compared to the same enzymes free in solution. We investigated whether tethering enzymes involved in both biomass hydrolysis and oxidative transformation to glucaric acid onto a rosettasome scaffold would result in an analogous production enhancement in a combined hydrolysis and bioconversion metabolic pathway. Three different enzymes were used to hydrolyze birchwood hemicellulose and convert the substituents to glucaric acid, a top-12 DOE value added chemical feedstock derived from biomass. It was demonstrated that colocalizing the three different enzymes to the synthetic scaffold resulted in up to 40 % higher levels of product compared to uncomplexed enzymes.

Site-selective dual modification of periplasmic binding proteins for sensing applications.

We have developed three sensitive and specific amino acid sensors based on bacterial periplasmic solute binding proteins. A site-specific amino-terminal transamination reaction provides a useful complement to cysteine chemistry for the covalent modification of biomolecules in this application. We demonstrate this combination to attach two different chromophores to a single biomolecule in two locations. The periplasmic glutamine binding protein from E. coli was modified with a pair of dyes suitable for fluorescence resonance energy transfer, and this conjugate exhibited an l-glutamine dependent optical response. Two periplasmic binding proteins from the thermophilic organism Thermotoga maritima, for arginine and aliphatic amino acids, were modified and evaluated similarly. All three conjugates manifested signal changes mediated by resonant energy transfer upon binding their respective ligands, with nanomolar dissociation constants and stereochemical specificity. This represents a readily generalizable method for construction of reagentless biosensors. The double-labeling strategy was also exploited for the surface attachment of a dye-labeled glutamine binding protein via a biotin-streptavidin interaction.

The rosettazyme: a synthetic cellulosome.

Cellulose is an attractive feedstock for biofuel production because of its abundance, but the cellulose polymer is extremely stable and its constituent sugars are difficult to access. In nature, extracellular multi-enzyme complexes known as cellulosomes are among the most effective ways to transform cellulose to useable sugars. Cellulosomes consist of a diversity of secreted cellulases and other plant cell-wall degrading enzymes bound to a protein scaffold. These scaffold proteins have cohesin modules that bind conserved dockerin modules on the enzymes. It is thought that the localization of these diverse enzymes on the scaffold allows them to function synergistically. In order to understand and harness this synergy smaller, simplified cellulosomes have been constructed, expressed, and reconstituted using truncated cohesin-containing scaffolds. Here we show that an 18-subunit protein complex called a rosettasome can be genetically engineered to bind dockerin-containing enzymes and function like a cellulosome. Rosettasomes are thermostable, group II chaperonins from the hyperthermo-acidophilic archaeon Sulfolobus shibatae, which in the presence of ATP/Mg(2+) assemble into 18-subunit, double-ring structures. We fused a cohesin module from Clostridium thermocellum to a circular permutant of a rosettasome subunit, and we demonstrate that the cohesin-rosettasomes: (1) bind dockerin-containing endo- and exo-gluconases, (2) the bound enzymes have increased cellulose-degrading activity compared to their activity free in solution, and (3) this increased activity depends on the number and ratio of the bound glucanases. We call these engineered multi-enzyme structures rosettazymes.

A self-assembling protein template for constrained synthesis and patterning of nanoparticle arrays.

Self-assembling biomolecules that form highly ordered structures have attracted interest as potential alternatives to conventional lithographic processes for patterning materials. Here, we introduce a general technique for patterning nanoparticle arrays using two-dimensional crystals of genetically modified hollow protein structures called chaperonins. Constrained chemical synthesis of transition metal nanoparticles is initiated using templates functionalized with polyhistidine sequences. These nanoparticles are ordered into arrays because the template-driven synthesis is constrained by the nanoscale structure of the crystallized protein. We anticipate that this system may be used to pattern different classes of nanoparticles based on the growing library of sequences shown to specifically bind or direct the growth of materials.

Identification of the glycosaminoglycan binding site of the CC chemokine, MCP-1: implications for structure and function in vivo.

In a recent study, we demonstrated that glycosaminoglycan (GAG) binding and oligomerization are essential for the in vivo function of the chemokines MCP-1/CCL2, RANTES/CCL5, and MIP-1beta/CCL4 (1). Binding to the GAG chains of cell surface proteoglycans is thought to facilitate the formation of high localized concentrations of chemokines, which in turn provide directional signals for leukocyte migration. To understand the molecular details of the chemokine-GAG interaction, in the present study we identified the GAG binding epitopes of MCP-1/CCL2 by characterizing a panel of surface alanine mutants in a series of heparin-binding assays. Using sedimentation equilibrium and cross-linking methods, we also observed that addition of heparin octasaccharide induces tetramer formation of MCP-1/CCL2. Although MCP-1/CCL2 forms a dimer in solution, both a dimer and tetramer have been observed by x-ray crystallography, providing a glimpse of the putative heparin-bound state. When the GAG binding residues are mapped onto the surface of the tetramer, the pattern that emerges is a continuous ring of basic residues encircling the tetramer, creating a positively charged surface well suited for binding GAGs. The structure also suggests several possible functional roles for GAG-induced oligomerization beyond retention of chemokines at the site of production.

Intracellular localization of a group II chaperonin indicates a membrane-related function.

Chaperonins are protein complexes that are believed to function as part of a protein folding system in the cytoplasm of the cell. We observed, however, that the group II chaperonins known as rosettasomes in the hyperthermophilic archaeon Sulfolobus shibatae, are not cytoplasmic but membrane associated. This association was observed in cultures grown at 60 degrees C and 76 degrees C or heat-shocked at 85 degrees C by using immunofluorescence microscopy and in thick sections of rapidly frozen cells grown at 76 degrees C by using immunogold electron microscopy. We observed that increased abundance of rosettasomes after heat shock correlated with decreased membrane permeability at lethal temperature (92 degrees C). This change in permeability was not seen in cells heat-shocked in the presence of the amino acid analogue azetidine 2-carboxylic acid, indicating functional protein synthesis influences permeability. Azetidine experiments also indicated that observed heat-induced changes in lipid composition in S. shibatae could not account for changes in membrane permeability. Rosettasomes purified from cultures grown at 60 degrees C and 76 degrees C or heat-shocked at 85 degrees C bind to liposomes made from either the bipolar tetraether lipids of Sulfolobus or a variety of artificial lipid mixtures. The presence of rosettasomes did not significantly change the transition temperature of liposomes, as indicated by differential scanning calorimetry, or the proton permeability of liposomes, as indicated by pyranine fluorescence. We propose that these group II chaperonins function as a structural element in the natural membrane based on their intracellular location, the correlation between their functional abundance and membrane permeability, and their potential distribution on the membrane surface.

Construction of a fluorescent biosensor family.

Bacterial periplasmic binding proteins (bPBPs) are specific for a wide variety of small molecule ligands. bPBPs undergo a large, ligand-mediated conformational change that can be linked to reporter functions to monitor ligand concentrations. This mechanism provides the basis of a general system for engineering families of reagentless biosensors that share a common physical signal transduction functionality and detect many different analytes. We demonstrate the facility of designing optical biosensors based on fluorophore conjugates using 8 environmentally sensitive fluorophores and 11 bPBPs specific for diverse ligands, including sugars, amino acids, anions, cations, and dipeptides. Construction of reagentless fluorescent biosensors relies on identification of sites that undergo a local conformational change in concert with the global, ligand-mediated hinge-bending motion. Construction of cysteine mutations at these locations then permits site-specific coupling of environmentally sensitive fluorophores that report ligand binding as changes in fluorescence intensity. For 10 of the bPBPs presented in this study, the three-dimensional receptor structure was used to predict the location of reporter sites. In one case, a bPBP sensor specific for glutamic and aspartic acid was designed starting from genome sequence information and illustrates the potential for discovering novel binding functions in the microbial genosphere using bioinformatics.

Ordered nanoparticle arrays formed on engineered chaperonin protein templates.

Traditional methods for fabricating nanoscale arrays are usually based on lithographic techniques. Alternative new approaches rely on the use of nanoscale templates made of synthetic or biological materials. Some proteins, for example, have been used to form ordered two-dimensional arrays. Here, we fabricated nanoscale ordered arrays of metal and semiconductor quantum dots by binding preformed nanoparticles onto crystalline protein templates made from genetically engineered hollow double-ring structures called chaperonins. Using structural information as a guide, a thermostable recombinant chaperonin subunit was modified to assemble into chaperonins with either 3 nm or 9 nm apical pores surrounded by chemically reactive thiols. These engineered chaperonins were crystallized into two-dimensional templates up to 20 microm in diameter. The periodic solvent-exposed thiols within these crystalline templates were used to size-selectively bind and organize either gold (1.4, 5 or 10nm) or CdSe-ZnS semiconductor (4.5 nm) quantum dots into arrays. The order within the arrays was defined by the lattice of the underlying protein crystal. By combining the self-assembling properties of chaperonins with mutations guided by structural modelling, we demonstrate that quantum dots can be manipulated using modified chaperonins and organized into arrays for use in next-generation electronic and photonic devices.