Polypeptide Scaffolds Research Articles

Non-immunoglobulin synthetic binding proteins for oncology

Extensive application of technologies like phage display in screening peptide and protein combinatorial libraries has not only facilitated creation of new recombinant antibodies but has also significantly enriched repertoire of the protein binders that have polypeptide scaffolds without homology to immunoglobulins. These innovative synthetic binding protein (SBP) platforms have grown in number and now encompass monobodies/adnectins, DARPins, lipocalins/anticalins, and a variety of miniproteins such as affibodies and knottins, among others. They serve as versatile modules for developing complex affinity tools that hold promise in both diagnostic and therapeutic settings. An optimal scaffold typically has low molecular weight, minimal immunogenicity, and demonstrates resistance against various challenging conditions, including proteolysis - making it potentially suitable for peroral administration. Retaining functionality under reducing intracellular milieu is also advantageous. However, paramount to its functionality is the scaffold’s ability to tolerate mutations across numerous positions, allowing for the formation of a sufficiently large target binding region. This is achieved through the library construction, screening, and subsequent expression in an appropriate system. Scaffolds that exhibit high thermodynamic stability are especially coveted by the developers of new SBPs. These are steadily making their way into clinical settings, notably as antagonists of oncoproteins in signaling pathways. This review surveys the diverse landscape of SBPs, placing particular emphasis on the inhibitors targeting the oncoprotein KRAS, and highlights groundbreaking opportunities for SBPs in oncology.

Fibrous polypeptide based bioscaffold delivery of minocycline hydrochloride for nerve regeneration

Neural tissue engineering is a technology with the potential to treat irreversible neurodegenerative diseases. We chose poly (γ-benzyl-l-glutamate) (PBG) and its hydrolyzed copolymer (poly (γ-benzyl-glutamate)80-r-(γ –glutamic acid)20) (PBGA) as the raw materials to make fibrous scaffolds by electrospinning process. These raw materials are biocompatible polypeptides which contain the neurotransmitter glutamate. Morphologically, the scaffolds consist of aligned fibers, which is important for directionality. Minocycline hydrochloride (MH) is a neuroprotective antibiotic obviously shown to enhance neurite growth, but is unstable in phosphate buffer solution. By incorporating and immobilizing MH into either PBG or PBGA polypeptide scaffolds, we created a stable drug delivery system. After 7-day release, the cumulative release of MH from PBGA scaffold was 42.65%. Nerve growth factor (NGF) responsive rat pheochromocytoma (PC12) cells were cultured on the scaffold to assess cell biocompatibility and proliferation. We found cell viability and differentiation improved when cultured on scaffolds bearing 2 wt% and 4 wt% of MH. PC12 cells cultured on PBGA incorporating 4 wt% MH exhibited the longest neurite outgrowth of 190.29 μm, the average is 33% increase over that of pristine PBGA after 5-days of NGF mediated differentiation. These polypeptide-based scaffolds loaded with MH are a promising drug delivery system for neural tissue engineering.

Bioactive Anti-Inflammatory Thermocatalytic Nanometal-Polyphenol Polypeptide Scaffolds for MRSA-Infection/Tumor Postsurgical Tissue Repair.

Postsurgical tumor recurrence, infection, and tissue defect are still the challenges in clinical medicine. The development of multifunctional biomaterial scaffolds with a microenvironment-responsive tumor-infection therapy-tissue repair is highly desirable. Herein, we report a bioactive, injectable, adhesive, self-healing, antibacterial, and anti-inflammatory metal-polyphenol polypeptide nanocomposite scaffold (PEAPF) with temporal-spatial-controlled inflammation-triggered therapeutic properties for efficient infection and postsurgical tumor therapy and skin repair. PEAPF scaffolds showed sustained and inherent inflammation-triggered Fenton catalysis and mild thermochemical effect for specifically inhibiting tumor recurrence in vitro and in vivo. The PEAPF scaffolds significantly facilitated skin tissue regeneration in MRSA-infected chronic wounds and postsurgical tissue defects after tumor resection. This study presents the multifunctional scaffold-based safe and efficient therapeutic strategy to prevent local tumor recurrence and enhance postsurgical tissue regeneration.

Comparative Evaluation of Engineered Polypeptide Scaffolds in HER2-Targeting Magnetic Nanocarrier Delivery.

Targeted drug delivery is one of the most intriguing and challenging issues in modern biomedicine. For active targeting, full-size IgG molecules (150 kDa) are usually used. Recent studies have revealed that small artificial polypeptide scaffolds such as DARPins (14 kDa) and affibodies (8 kDa) are much more promising tools for drug delivery due to their small size, artificial nature, low immunogenicity, and many other properties. However, there is no comparative information on the targeting abilities of scaffold polypeptides, which should be taken into account when developing drug delivery systems (DDSs). The present work is the first comprehensive study on the comparison of the effectiveness of different HER2-targeting proteins within the architecture of nanoparticles. Namely, we synthesized trimodal nanoparticles: magnetic, fluorescent, and directed toward HER2 oncomarker on cancer cells. The magnetic particles (MPs) were covalently modified with (i) full-size IgG, 150 kDa, (ii) DARPin_G3, 14 kDa, and (iii) affibody ZHER2:342, 8 kDa. We showed that the number of DARPin_G3 and affibody ZHER2:342 molecules conjugated to the nanoparticle surface are 10 and 40 times higher, respectively, than the corresponding value for trastuzumab. Using the methods of magnetic particle quantification (MPQ)-cytometry and confocal microscopy, we showed that all types of the obtained magnetic conjugates specifically labeled HER2-overexpressing cells. Namely, we demonstrated that particle binding to HER2-positive cells is 1113 ± 39 fg/cell for MP*trastuzumab, 1431 ± 186 fg/cell for MP*ZHER2:342, and 625±21 fg/cell for MP*DARPin_G3, which are 2.77, 2.75, and 2.30 times higher than the corresponding values for control HER2-negative cells. Thus, we showed that the smallest HER2-recognizing polypeptide affibody ZHER2:342 is more effective in terms of specificity and selectivity in nanoparticle-mediated cell labeling.

A Nanoscaffolded Spike-RBD Vaccine Provides Protection against SARS-CoV-2 with Minimal Anti-Scaffold Response.

The response of the adaptive immune system is augmented by multimeric presentation of a specific antigen, resembling viral particles. Several vaccines have been designed based on natural or designed protein scaffolds, which exhibited a potent adaptive immune response to antigens; however, antibodies are also generated against the scaffold, which may impair subsequent vaccination. In order to compare polypeptide scaffolds of different size and oligomerization state with respect to their efficiency, including anti-scaffold immunity, we compared several strategies of presentation of the RBD domain of the SARS-CoV-2 spike protein, an antigen aiming to generate neutralizing antibodies. A comparison of several genetic fusions of RBD to different nanoscaffolding domains (foldon, ferritin, lumazine synthase, and β-annulus peptide) delivered as DNA plasmids demonstrated a strongly augmented immune response, with high titers of neutralizing antibodies and a robust T-cell response in mice. Antibody titers and virus neutralization were most potently enhanced by fusion to the small β-annulus peptide scaffold, which itself triggered a minimal response in contrast to larger scaffolds. The β-annulus fused RBD protein increased residence in lymph nodes and triggered the most potent viral neutralization in immunization by a recombinant protein. Results of the study support the use of a nanoscaffolding platform using the β-annulus peptide for vaccine design.

Emulsion-templated synthetic polypeptide scaffolds prepared by ring-opening polymerization ofN-carboxyanhydrides

Ring-opening polymerization ofN-carboxyanhydrides was performed in oil-in-oil high internal phase emulsion to obtain well-defined macroporous synthetic polypeptides.

Versatile N-Methylaminooxy-Functionalized Polypeptides for Preparation of Neoglycoconjugates.

The preparation and characterization of a new set of well-defined polypeptides containing N-methylaminooxy side-chain functionality is described. These functional groups enabled the direct coupling of polypeptides with a variety of unmodified reducing saccharides in water to give neoglycopolypeptides in high yields. The use of different polypeptide scaffolds resulted in neoglycoconjugates with tunable chain conformations, hydrophobicity, and charge. These new neoglycopolypeptides were also found to be stable in aqueous media at pH 7.4 and 37 °C for 1 week. The combination of straightforward synthesis using unmodified saccharides, high yields of saccharide conjugation, and conjugate stability makes these polypeptides attractive candidates for development of degradable glycoprotein mimics.

Bioorthogonal Elastin-like Polypeptide Scaffolds for Immunoassay Enhancement.

Artificial multiprotein complexes are sought after reagents for biomolecular engineering. A current limiting factor is the paucity of molecular scaffolds which allow for site-specific multicomponent assembly. Here, we address this limitation by synthesizing bioorthogonal elastin-like polypeptide (ELP) scaffolds containing periodic noncanonical l-azidohomoalanine amino acids in the guest residue position. The nine azide ELP guest residues served as conjugation sites for site-specific modification with dibenzocyclooctyne (DBCO)-functionalized single-domain antibodies (SdAbs) through strain-promoted alkyne-azide cycloaddition (SPAAC). Sortase A and ybbR tags at the C- and N-termini of the ELP scaffold provided two additional sites for derivatization with small molecules and peptides by Sortase A and 4'-phosphopantetheinyl transferase (Sfp), respectively. These functional groups are chemically bioorthogonal, mutually compatible, and highly efficient, thereby enabling synthesis of multi-antibody ELP complexes in a one-pot reaction. We demonstrate application of this material for enhancing the performance of sandwich immunoassays of the recombinant protein mCherry. In undiluted human plasma, surfaces modified with multi-antibody ELP complexes showed between 2.3- and 14.3-fold improvement in sensitivity and ∼30-40% lower limits of detection as compared with nonspecifically adsorbed antibodies. Dual-labeled multi-antibody ELP complexes were further used for cytometric labeling and analysis of live eukaryotic cells. These results demonstrate how multiple antibodies complexed onto bioorthogonal protein-based polymers can be used to enhance immunospecific binding interactions through multivalency effects.

ChemInform Abstract: Designing the Structure and Folding Pathway of Modular Topological Bionanostructures

AbstractReview: 61 refs.

Preparation and characterization of bimodal porous poly(γ-benzyl-L-glutamate) scaffolds for bone tissue engineering

An ideal scaffold in bone tissue-engineering strategy should provide biomimetic extracellular matrix-like architecture and biological properties. Poly(γ-benzyl-L-glutamate) (PBLG) has been a popular model polypeptide for various potential biomedical applications due to its good biocompatibility and biodegradability. This study developed novel bimodal porous PBLG polypeptide scaffolds via a combination of biotemplating method and in situ ring-opening polymerization of γ-benzyl-L-gIutamate N-carboxyanhydride (BLG-NCA). The PBLG scaffolds were characterized by proton nuclear magnetic resonance spectroscopy, X-ray diffraction, differential scanning calorimetry, scanning electron microscope (SEM) and mechanical test. The results showed that the semi-crystalline PBLG scaffolds exhibited an anisotropic porous structure composed of honeycomb-like channels (100–200μm in diameter) and micropores (5–20μm), with a very high porosity of 97.4±1.6%. The compressive modulus and glass transition temperature were 402.8±20.6 kPa and 20.2°C, respectively. The in vitro biocompatibility evaluation with MC3T3-E1 cells using SEM, fluorescent staining and MTT assay revealed that the PBLG scaffolds had good biocompatibility and favored cell attachment, spread and proliferation. Therefore, the bimodal porous polypeptide scaffolds are promising for bone tissue engineering.

Rapid mass spectrometric determination of disulfide connectivity in peptides and proteins

Disulfide crosslinks are ubiquitous in natural peptides and proteins, providing rigidity to polypeptide scaffolds. The assignment of disulfide connectivity in multiple crosslinked systems is often difficult to achieve. Here, we show that rapid unambiguous characterisation of disulfide connectivity can be achieved through direct mass spectrometric CID fragmentation of the disulfide intact polypeptides. The method requires a direct mass spectrometric fragmentation of the native disulfide bonded polypeptides and subsequent analysis using a newly developed program, DisConnect. Technical difficulties involving direct fragmentation of proteins are surmounted by an initial proteolytic nick and subsequent determination of the structures of these proteolytic peptides through DisConnect. While the connectivity in proteolytic fragments containing one cystine is evident from the MS profile alone, those with multiple cystines are subjected to subsequent mass spectrometric fragmentation. The wide applicability of this method is illustrated using examples of peptide hormones, peptide toxins, proteins, and disulfide foldamers of a synthetic analogue of a marine peptide toxin. The method, coupled with DisConnect, provides an unambiguous, straightforward approach, especially useful for the rapid screening of the disulfide crosslink fidelity in recombinant proteins, determination of disulfide linkages in natural peptide toxins and characterization of folding intermediates encountered in oxidative folding pathways.

Photochemical thiol–yne functionalization of polypeptide scaffolds

Copolypeptides with propargyl side chains were functionalized with different thiols (1-thio-β-D-glucose tetraacetate, 7-mercapto-4-methylcoumarin, and methyl-3-mercaptopropionate) by initiator/catalyst-free thiol–yne photochemistry. No more than 1.5 instead of ideally 2 equivalents of thiol could be added to the propargyl units even when the thiol was used in excess, attributable to steric constraints (close proximity of the reaction site to the copolypeptide backbone) or conjugation (extended aromatic system). Nevertheless, this procedure enables a facile and modular synthesis of highly functionalized homo- and hetero-functional copolypeptides.

A synthetic polypeptide conjugate from a 42‐residue polypeptide and salicylhydroxamic acid binds human myeloperoxidase with high affinity

Myeloperoxidase (MPO) is a 150 kD tetrameric heme protein consisting of two heavy chains and two light chains, which is present in neutrophils, white blood cells, at concentrations between 2% and 5% and plays an important role in the innate immune system. The MPO concentration in serum or plasma has been shown to be linked to the risk for cardiovascular diseases, and MPO is considered to be a high potential diagnostic biomarker. To develop a molecule that binds MPO, salicylhydroxamic acid (SHA), a substrate analog inhibitor of MPO with a KD=2 μM, was conjugated to a designed set of 42-residue polypeptide scaffolds via 9- and 11-carbon atom aliphatic spacers to form 20 different protein binder candidates, and their interactions with MPO were evaluated by surface plasmon resonance analysis. The polypeptide conjugate 4C37L34C11SHA was found to bind to MPO with an affinity that could be estimated to have a dissociation constant of around 400 pM, nearly four orders of magnitude higher than that of SHA. Inhibition of binding to MPO by free SHA was observed in competition experiments demonstrating that the binding of the polypeptide conjugate is dominated by the interactions of SHA with the heme cavity. Although still in the future, the discovery of these new synthetic binders for MPO suggests a route to clinical diagnostic tests in vivo or in vitro, independent of antibodies.

Preparation and characterization of elastin-like polypeptide scaffolds for local delivery of antibiotics and proteins

Tissue engineering applications could benefit from simultaneous release of growth factors, signaling molecules, and antibiotics to obtain optimal healing of tissues. Elastin-like polypeptides (ELPs) are genetically engineered polymers that possess good biocompatibility, are biodegradable, and exhibit mechanical properties similar to natural elastin. In addition, ELPs exhibit a characteristic inverse phase transition temperature (T(t)). This T(t) behavior is widely exploited in hyperthermia mediated drug delivery. The objectives of this research were to prepare ELP hydrogel scaffolds using a novel ultrasonication method and to investigate the release of a model protein (bovine serum albumin, BSA) and a commonly used antibiotic in periodontal therapy (doxycycline) from the scaffolds at two different temperatures (25 °C <T(t) vs. 37 °C >T(t)). Both BSA and doxycycline showed a gradual time dependent release and showed a trend of higher release fractions with higher loading doses. Based on the comparison between the release profiles at the two selected temperatures, the release was higher at 37 °C compared to that at 25 °C for both the loading concentrations of doxycycline (0.05 and 0.1 % v/v) and only one of the loading concentrations of BSA (0.5 % v/v) studied, while the release was higher at 25 °C compared to that at 37 °C only for the other loading concentration of BSA (1 % v/v) studied. These results suggested that the drug molecular weight and loading concentration were significant factors that affected the release kinetics. The experiments in this study demonstrated that the ELP hydrogel scaffolds can successfully release proteins and antibiotics critical to tissue engineering.

Architecture effects on L-selectin shedding induced by polypeptide-based multivalent ligands

Multivalent interactions between selectins and their ligands play key roles in mediating the rolling and tethering of leukocytes in the early steps of the inflammatory response, as well as in lymphocyte circulation. L-selectin shedding, which is the proteolytic cleavage of L-selectin, can be induced by L-selectin clustering through the binding of multivalent ligands to multiple L-selectin molecules, and it has been shown to regulate leukocyte rolling and subsequent integrin activation for firm adhesion. In this paper, we report the production of homogenous glycopolypeptides modified with a 3,6-disulfo-galactopyranoside equipped with a caproyl linker. The saccharide residue was chemically attached to various polypeptide backbones of differing architectures; the composition and purity of the sulfated glycopolypeptides was confirmed via1H-NMR spectroscopy, amino acid analysis (AAA), and electrophoretic analysis. The retention of the conformation of the polypeptide backbone was confirmed via circular dichroic spectroscopy. The shedding of l-selectin from the surface of Jurkat cells induced by these sulfated glycopolypeptides, determined via ELISA-based methods, varied based on differences in the architectures of the polypeptide scaffolds, suggesting opportunities for these strategies in probing cell-surface receptor arrays and directing cell signaling events.

Amyloid Fibril Recognition with the Conformational B10 Antibody Fragment Depends on Electrostatic Interactions

Amyloid fibrils are naturally occurring polypeptide scaffolds with considerable importance for human health and disease. These supermolecular assemblies are β-sheet rich and characterized by a high structural order. Clinical diagnosis and emerging therapeutic strategies of amyloid-dependent diseases, such as Alzheimer's, rely on the specific recognition of amyloid structures by other molecules. Recently, we generated the B10 antibody fragment, which selectively binds to Alzheimer's Aβ(1–40) amyloid fibrils but does not explicitly recognize other protein conformers, such as oligomers and disaggregated Aβ peptide. B10 presents poly-amyloid specific binding and interacts with fibrillar structures consisting of different polypeptide chains. To determine the molecular basis behind its specificity, we have analyzed the molecular properties of B10 with a battery of biochemical and biophysical techniques, ranging from X-ray crystallography to chemical modification studies. We find that fibril recognition depends on positively charged residues within the B10 antigen binding site. Mutation of these basic residues into alanine potently impairs fibril binding, and reduced B10–fibril interactions are also observed when the fibril carboxyl groups are covalently masked by a chemical modification approach. These data imply that the B10 conformational specificity for amyloid fibrils depends upon specific electrostatic interactions with an acidic moiety, which is common to different amyloid fibrils.

A new assay design for clinical diagnostics based on alternative recognition elements

Herein, we present a new sandwich assay design containing a high affinity polypeptide scaffold as immobilized capture element and an antibody for detection. These polypeptide scaffolds provide a good affinity towards one antigen and can be linked to biosensor surfaces without affecting their binding capabilities. Furthermore, the small peptides are very stable, which allows for regenerating the surface several hundreds of times and thus for reuse of the biosensor. Moreover, these receptors can be synthesized with different affinities towards one antigen, which has been proven by characterizing them using a label-free detection method RIfS (reflectometric interference spectroscopy) for collecting kinetic data. Polypeptide scaffolds with different affinities have been chosen and characterized. Upon these results, sandwich-type assays have been set-up using a fluorescently labelled antibody as detection element. Thereby could be shown, that the working range of the assay can be shifted according to the affinity of the used capturing polypeptide scaffold. The scaffolds with a higher affinity towards the antigen can detect lower concentration, and in contrary, scaffolds with lower affinities can detect higher concentrations. In consequence, using this new sandwich-type assay, we avoid the complex procedure to immobilize antibodies in correct orientation, but simultaneously keep this well-known recognition element in the assay for detection. Furthermore, in addition to all the acknowledged properties of immunoassays, we add the possibility of tuning the working range of assays in distinct manner according to request.

Use of modular, synthetic scaffolds for improved production of glucaric acid in engineered E. coli

The field of metabolic engineering has the potential to produce a wide variety of chemicals in both an inexpensive and ecologically-friendly manner. Heterologous expression of novel combinations of enzymes promises to provide new or improved synthetic routes towards a substantially increased diversity of small molecules. Recently, we constructed a synthetic pathway to produce d-glucaric acid, a molecule that has been deemed a “top-value added chemical” from biomass, starting from glucose. Limiting flux through the pathway is the second recombinant step, catalyzed by myo-inositol oxygenase (MIOX), whose activity is strongly influenced by the concentration of the myo-inositol substrate. To synthetically increase the effective concentration of myo-inositol, polypeptide scaffolds were built from protein–protein interaction domains to co-localize all three pathway enzymes in a designable complex as previously described ( Dueber et al., 2009). Glucaric acid titer was found to be strongly affected by the number of scaffold interaction domains targeting upstream Ino1 enzymes, whereas the effect of increased numbers of MIOX-targeted domains was much less significant. We determined that the scaffolds directly increased the specific MIOX activity and that glucaric acid titers were strongly correlated with MIOX activity. Overall, we observed an approximately 5-fold improvement in product titers over the non-scaffolded control, and a 50% improvement over the previously reported highest titers. These results further validate the utility of these synthetic scaffolds as a tool for metabolic engineering.

Transplantation of Mesenchymal Stem Cells with Self-Assembling Polypeptide Scaffolds Is Conducive to Treating Myocardial Infarction in Rats

The poor survival and differentiation of the donor cells in the infarcted myocardium has hampered the therapeutic efficacy of cell transplantation. A self-assembling polypeptide RAD16-II (Ac-RARADADARARADADA-CONH₂) spontaneously assembles into stable nanofiber scaffolds, which mimic natural extracellular matrix at 0.1-1% peptide concentrations in the myocardium. We isolated mesenchymal stem cells from the bone marrow of adult male rats that express both c-kit and Nkx2.5, a cardiac transcription factor, yielding selected mesenchymal stem cells (SMSCs). We initially confirmed that the self-assembling polypeptide scaffolds are conducive to growth, survival and differentiation of SMSCs in vitro. Subsequently, SMSCs mixed with the self-assembling polypeptide were injected into the infarcted area at 30 min after the establishment of myocardial infarction in female rats. The donor cells were tracked with Y chromosome in the myocardium. The changes of cardiac function, myocardial structure and capillary density were detected at 4 weeks after cell transplantation. The hearts transplanted with SMSCs incorporated into the nanofiber scaffolds showed smaller infarction size (19.55 ± 2.1%) than the hearts injected with SMSCs (27.37 ± 4.8%). Importantly, the systolic function indices, left ventricle ejection fraction and left ventricle fractional shortening, were significantly improved in the animals transplanted with SMSCs mixed with the nanofiber scaffolds (59.31 ± 4.9% and 31.91 ± 8.1%), compared to those with SMSCs alone (48.31 ± 9.2% and 23.58 ± 8.5%). In conclusion, transplantation of SMSCs mixed with the self-assembling polypeptide RAD16-II is more effective to promote myocardial regeneration and attenuate cardiac injury in a rat model of myocardial infarction.

Generation of Surface‐Bound Multicomponent Protein Gradients

Spatial control of bioactive ligands is achieved by integrating microfluidics and protein engineering. The proteins of interest are mixed in a gradient generator and immobilized on artificial polypeptide scaffolds through the strong association of heterodimeric ZE/ZR leucine zipper pairs. Protein densities and gradient shapes are easily controlled and varied in this method.