Codelivery of TGFß and Cox2 siRNA inhibits HCC by promoting T-cell penetration into the tumor and improves response to Immune Checkpoint Inhibitors.

Upregulation of TGFß and Cox2 in the tumor microenvironment results in blockade of T-cell penetration into the tumor. Without access to tumor antigens, the T-cell response will not benefit from administration of the immune checkpoint antibodies. We created an intravenous polypeptide nanoparticle that can deliver two siRNAs (silencing TGFß and Cox2). Systemic administration in mice, bearing a syngeneic orthotopic hepatocellular carcinoma (HCC), delivers the siRNAs to various cells in the liver, and significantly reduces the tumor. At 2 mg/kg (BIW) the nanoparticle demonstrated a single agent action and induced tumor growth inhibition to undetectable levels after five doses. Reducing the siRNAs to 1mg/kg BIW demonstrated greater inhibition in the presence of PD-L1 mAbs. After only three doses BIW, we could still recover a smaller tumor and, in tumor sections, showed an increase in penetration of CD4+ and CD8+ T-cells deeper into the remaining tumor that was not evident in animals treated with non-silencing siRNA. The combination of TGFß and Cox2 siRNA co-administered in a polypeptide nanoparticle can act as a novel therapeutic alone against HCC and may augment the activity of the immune checkpoint antibodies. Silencing TGFß and Cox2 converts an immune excluded (cold) tumor into a T-cell inflamed (hot) tumor.

In situ regeneration of bioactive coatings enabled by an evolved Staphylococcus aureus sortase A.

Surface immobilization of bioactive molecules is a central paradigm in the design of implantable devices and biosensors with improved clinical performance capabilities. However, in vivo degradation or denaturation of surface constituents often limits the long-term performance of bioactive films. Here we demonstrate the capacity to repeatedly regenerate a covalently immobilized monomolecular thin film of bioactive molecules through a two-step stripping and recharging cycle. Reversible transpeptidation by a laboratory evolved Staphylococcus aureus sortase A (eSrtA) enabled the rapid immobilization of an anti-thrombogenic film in the presence of whole blood and permitted multiple cycles of film regeneration in vitro that preserved its biological activity. Moreover, eSrtA transpeptidation facilitated surface re-engineering of medical devices in situ after in vivo implantation through removal and restoration film constituents. These studies establish a rapid, orthogonal and reversible biochemical scheme to regenerate selective molecular constituents with the potential to extend the lifetime of bioactive films.

Targeted antithrombotic protein micelles.

Activated platelets provide a promising target for imaging inflammatory and thrombotic events along with site-specific delivery of a variety of therapeutic agents. Multifunctional protein micelles bearing targeting and therapeutic proteins were now obtained by one-pot transpeptidation using an evolved sortase A. Conjugation to the corona of a single-chain antibody (scFv), which binds to the ligand-induced binding site (LIBS) of activated GPIIb/IIIa receptors, enabled the efficient detection of thrombi. The inhibition of thrombus formation was subsequently accomplished by incorporating the catalytically active domain of thrombomodulin (TM) onto the micelle corona for the local generation of activated protein C, which inhibits the formation of thrombin. An effective strategy has been developed for the preparation of protein micelles that can be targeted to sites of activated platelets with broad potential for treatment of acute thrombotic events.

Designing protein-based biomaterials for medical applications.

Biomaterials produced by nature have been honed through billions of years, evolving exquisitely precise structure-function relationships that scientists strive to emulate. Advances in genetic engineering have facilitated extensive investigations to determine how changes in even a single peptide within a protein sequence can produce biomaterials with unique thermal, mechanical and biological properties. Elastin, a naturally occurring protein polymer, serves as a model protein to determine the relationship between specific structural elements and desirable material characteristics. The modular, repetitive nature of the protein facilitates the formation of well-defined secondary structures with the ability to self-assemble into complex three-dimensional architectures on a variety of length scales. Furthermore, many opportunities exist to incorporate other protein-based motifs and inorganic materials into recombinant protein-based materials, extending the range and usefulness of these materials in potential biomedical applications. Elastin-like polypeptides (ELPs) can be assembled into 3-D architectures with precise control over payload encapsulation, mechanical and thermal properties, as well as unique functionalization opportunities through both genetic and enzymatic means. An overview of current protein-based materials, their properties and uses in biomedicine will be provided, with a focus on the advantages of ELPs. Applications of these biomaterials as imaging and therapeutic delivery agents will be discussed. Finally, broader implications and future directions of these materials as diagnostic and therapeutic systems will be explored.

Recombinant protein polymers in biomaterials.

Naturally occurring protein-based materials have been found that function as critical components in biomechanical response, fibers and adhesives. A relatively small but growing number of recombinant protein-based materials that mimic the desired features of their natural sources, such as collagens, elastins and silks, are considered as an alternative to conventional synthetic polymers. Advances in genetic engineering have facilitated the synthesis of repetitive protein polymers with precise control of molecular weights which are designed by using synthetic genes encoding tandem repeats of oligopeptide originating from a modular domain of natural proteins. Many repeat sequences as protein polymer building blocks adopt a well-defined secondary structure and undergo self-assembly to result in physically cross-linked networks or with chemical cross-linking so that further form three-dimensional architectures similar to natural counterparts. In this review, recombinant protein polymers currently developed will be presented that have emerged as promising class of next generation biomaterials.

Amphiphilic protein micelles for targeted in vivo imaging.

A variety of polymeric nanoparticles have been developed for bioimaging applications. This study reports on the use of a 50 nm recombinant protein nanoparticle with a multivalent surface as a vehicle for functionalization with a model imaging agent. Multiple fluorescent probes were covalently conjugated to surface amines of crosslinked amphiphilic elastin-mimetic protein micelles using N-hydroxysuccinimide ester chemistry. In vivo fluorescence imaging confirmed that protein micelles selectively accumulated at sites of angioplasty induced vessel wall injury, presumably via an enhanced permeability and retention effect. This investigation demonstrates the potential of amphiphilic protein micelles to be used as a vehicle for selective imaging of sites associated with a disrupted or leaky endothelium.

Recombinant amphiphilic protein micelles for drug delivery.

Amphiphilic block polypeptides can self-assemble into a range of nanostructures in solution, including micelles and vesicles. Our group has recently described the capacity of recombinant amphiphilic diblock copolypeptides to form highly stable micelles. In this report, we demonstrate the utility of protein nanoparticles to serve as a vehicle for controlled drug delivery. Drug-loaded micelles were produced by encapsulating dipyridamole as a model hydrophobic drug with anti-inflammatory activity. Murine studies confirmed the capacity of drug-loaded protein micelles to limit the in vivo recruitment of neutrophils in response to an inflammatory stimulus.

An activation-specific platelet inhibitor that can be turned on/off by medically used hypothermia.

OBJECTIVE: Therapeutic hypothermia is successfully used, for example, in cardiac surgery to protect organs from ischemia. Cardiosurgical procedures, especially in combination with extracorporeal circulation, and hypothermia itself are potentially prothrombotic. Despite the obvious need, the long half-life of antiplatelet drugs and thus the risk of postoperative bleedings have restricted their use in cardiac surgery. We describe here the design and testing of a unique recombinant hypothermia-controlled antiplatelet fusion protein with the aim of providing increased safety of hypothermia, as well as cardiac surgery. METHODS AND RESULTS: An elastin-mimetic polypeptide was fused to an activation-specific glycoprotein (GP) IIb/IIIa-blocking single-chain antibody. In silico modeling illustrated the sterical hindrance of a ß-spiral conformation of elastin-mimetic polypeptide preventing the single-chain antibody from inhibiting GPIIb/IIIa at 37°C. Circular dichroism spectra demonstrated reverse temperature transition, and flow cytometry showed binding to and blocking of GPIIb/IIIa at hypothermic body temperature (≤32°C) but not at normal body temperature. In vivo thrombosis in mice was selectively inhibited at hypothermia but not at 37°C. CONCLUSIONS: This is the first description of a broadly applicable pharmacological strategy by which the activity of a potential drug can be controlled by temperature. In particular, this drug steerability may provide substantial benefits for antiplatelet therapy.

Recombinant elastin-mimetic biomaterials: Emerging applications in medicine.

Biomaterials derived from protein-based block copolymers are increasingly investigated for potential application in medicine. In particular, recombinant elastin block copolymers provide significant opportunities to modulate material microstructure and can be processed in various forms, including particles, films, gels, and fiber networks. As a consequence, biological and mechanical responses of elastin-based biomaterials are tunable through precise control of block size and amino acid sequence. In this review, the synthesis of a set of elastin-mimetic triblock copolymers and their diverse processing methods for generating material platforms currently applied in medicine will be discussed.

A stereoelectronic effect on turn formation due to proline substitution in elastin-mimetic polypeptides.

Stereoelectronic effects have been identified as contributing factors to the conformational stability of collagen-mimetic peptide sequences. To assess the relevance of these factors within other protein structural contexts, three polypeptide sequences were prepared in which the sequences were derived from the canonical repeat unit (Val-Pro-Gly-Val-Gly) of the protein material elastin. These elastin-mimetic polypeptides, elastin-1, elastin-2, and elastin-3, incorporate (2S)-proline, (2S,4S)-4-fluoroproline, and (2S,4R)-4-fluoroproline, respectively, at the second position of the elastin repeat. Calorimetric and spectroscopic investigations of these three polypeptides indicate that the incorporation of the substituted proline residues had a dramatic effect upon the self-assembly of the corresponding elastin peptide. The presence of (2S,4R)-4-fluoroproline in elastin-3 lowered the temperature of the phase transition and increased the type II beta-turn population with respect to the parent polypeptide, while the presence of (2S,4S)-4-fluoroproline in elastin-2 had the opposite effect. These results suggest that stereoelectronic effects could either enhance or hinder the self-assembly of elastin-mimetic polypeptides, depending on the influence of the proline analogue on the energetics of the beta-turn conformation that develops within the pentapeptide structural repeats above the phase transition. Density functional theory (DFT) was employed to model three possible turn types (betaI-, betaII-, and inverse gamma-turns) derived from model peptide segments (MeCO-Xaa-Gly-NHMe) (Xaa = Pro, 4S-F-Pro, or 4R-F-Pro) corresponding to the turn-forming residues of the elastin repeat unit (Val-Pro-Gly-Val-Gly). The results of the these calculations suggested a similar outcome to the experimental data for the elastin-mimetic polypeptides, in that type II beta-turn structures were stabilized for peptide segments containing (2S,4R)-fluoroproline and destabilized for segments containing (2S,4S)-fluoroproline relative to the canonical proline residue.

Cotranslational incorporation of a structurally diverse series of proline analogues in an Escherichia coli expression system.

A set of Escherichia coli expression strains have been defined that are competent for the incorporation of a structurally diverse series of proline analogues under culture conditions that are compatible with high levels of analogue substitution within a proline-rich protein substrate. These bacterial strains have been employed to assay the efficacy of incorporation of noncanonical amino acids into a recombinant-protein test substrate and to create variant polypeptides in which native protein sequences have been globally substituted with imino acid analogues in response to proline codons. We envision that these methods may be used to interrogate the effect of imino acid substitution on protein structure and function and may be particularly informative in the context of structural comparison of a series of modified proteins with respect to the stereoelectronic differences between the incorporated proline analogues.